Review ArticleIntravesical Liposome and Antisense Treatment for DetrusorOveractivity and Interstitial CystitisPainful Bladder Syndrome

Pradeep Tyagi1 Mahendra P Kashyap1 Naoki Kawamorita1 Tsuyoshi Yoshizawa1

Michael Chancellor2 and Naoki Yoshimura1

1 Department of Urology University of Pittsburgh PA 15213 USA2Department of Urology William Beaumont School of Medicine Royal oak MI 48073 USA

Correspondence should be addressed to Pradeep Tyagi prt18pittedu

Received 18 July 2013 Accepted 24 October 2013 Published 16 January 2014

Academic Editors S Mingmalairak T W Stone and S-N Wu

Copyright copy 2014 Pradeep Tyagi et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Purpose The following review focuses on the recent advancements in intravesical drug delivery which brings added benefitto the therapy of detrusor overactivity and interstitial cystitispainful bladder syndrome (ICPBS) Results Intravesical route isa preferred route of administration for restricting the action of extremely potent drugs like DMSO for patients of interstitialcystitispainful bladder syndrome (ICPBS) and botulinum toxin for detrusor overactivity Patients who are either refractory tooral treatment or need to mitigate the adverse effects encountered with conventional routes of administration also chose this routeIts usefulness in some cases can be limited by vehicle (carrier) toxicity or short duration of action Efforts have been underwayto overcome these limitations by developing liposome platform for intravesical delivery of biotechnological products includingantisense oligonucleotides Conclusions Adoption of forward-thinking approaches can achieve advancements in drug deliverysystems targeted to future improvement in pharmacotherapy of bladder diseases Latest developments in the field of nanotechnologycan bring this mode of therapy from second line of treatment for refractory cases to the forefront of disease management

1 Introduction

Intravesical therapies have demonstrated varying degrees ofefficacy and safety in treatment of interstitial cystitispainfulbladder syndrome (ICPBS) [1] and overactive bladder OAB[2] Pharmacotherapy by this route provides high local drugconcentrations in the bladder with low risk of systemic sideeffects [3] Conventional therapies for OAB and detrusoroveractivity (DO) either neurogenic or idiopathic have lim-ited efficacy and acceptability Anticholinergic medicationswhich are currently the mainstay of the treatment of OABare not always effective and often have undesirable side effectssuch as dry mouth and constipation [4]Therefore search foralternative therapies directed against local targets with fewerside effects is encouraged

The ICPBS syndrome is characterized by pelvic painand urinary storage symptoms (eg urinary urgency andfrequency) The OrsquoLeary-Sant symptom and problem score

(interstitial cystitis symptom index (ICSI) and problem index(ICPI)) is recognized as one of the most reliable and validinstruments to identify the extent of bothersome symptomsand the most prominent voiding and painful symptomsin ICPBS patients [5 6] Pentosan polysulfate PPS is asynthetic sulphated polysaccharide and is the only approvedoral drug for ICPBS but it requires daily administrationfor 6 months before any benefit accrues for the patients[7 8] Less than one-tenth of oral dose is excreted into theurine of ICPBS patients which is considered to replenishthe damaged glycosaminoglycan GAG layer and reduce theinflux of potassium back into bladder from urine

Orally administered agents are often unable to cre-ate effective luminal drug concentration due to low uri-nary excretion of drugs which justifies bladder instillationReplacement of GAG layer by intravesical administration ofhyaluronic acid has been successfully tried in ICPBS patients[9] Intravesical therapy also holds the potential to facilitate

Hindawi Publishing CorporationISRN PharmacologyVolume 2014 Article ID 601653 12 pageshttpdxdoiorg1011552014601653

2 ISRN Pharmacology

the separation of therapeutic actions from side effects byinvolving a diverse array of novel chemical pharmacolog-ical and formulation strategies However drug delivery byintravesical route is constrained by the impermeability ofthe urothelium short duration of action and the need forfrequent administration

The urinary bladder lining is the most impermeable bar-rier in the human body [10 11]Therefore therapies delivereddirectly to the bladder lumen have limited opportunity forsystemic distribution which typically leads to fewer sideeffects In addition the mechanisms for locally deliveredtherapies can be independent of existing oral therapies andin some cases an additive effect can provide compellingcomarketing opportunities [12] We will review the currentunderstanding of urothelium structure and role of intravesi-cal drug delivery in unmasking the pharmacological functionof different receptors expressed on its luminal surface

2 Urothelium

Recent investigations have revealed that the urothelium isnot just a physical barrier between blood and urine but canexpress a host of receptors having a functional significance inmicturition reflexThe recent identification of a cannabinoidnicotinic neurokinin receptors and potassium ion channelsin urothelium [13ndash16] have revealed the role of urothelium asan excitable cell layer in bladder that responds to stretch andconvey messages to underlying afferents in bladder There isalso mounting evidence to demonstrate expression of adren-ergic bradykinin and transient receptor potential (TRP)receptors in urothelium and in proximity of afferent nerves[17 18] Urothelium is the primary nonneuronal source forthe release of molecules such as adenosine triphosphate(ATP) acetylcholine and nitric oxide which are known toaffect micturition [19 20] Intravesical therapy can be used tounravel the pharmacology of these receptors and paracrinemessengers released from urothelium [21 22]

3 Muscarinic Receptors

It is widely accepted that oral antimuscarinics act on mus-carinic receptors in the detrusor for managing the symptomsof DO and OAB Conventional wisdom largely ignores therole of muscarinic receptors expressed on urothelium [23]Muscarinic receptors expressed on urothelium are believedto be involved in afferent signaling for micturition [23] Theafferent signals are believed to be generated from the basalnonneuronal acetylcholine released during the storage phasefromurothelium to enhance themyogenic contractile activityof the detrusor [19 20]

Theoretically speaking not only can receptors expressedon the urothelium be influenced by antimuscarinics via thebloodstream but also few selected antimuscarinics and theiractive metabolites can affect the muscarinic receptors fromthe luminal side following their excretion into urine [12]Alternative mode of action for two antimuscarinic drugstrospium and solifenacin was demonstrated by our group[21 22] DO in the rat wasmimicked by intravesical carbachol

[21 22] Urine collected from human volunteers who tooktrospium and solifenacinwas then instilled into rat bladder todetermine the effect of the drug fraction excreted into urineTherefore intravesical therapy can assist in elucidating the yetunexploredmechanisms for improvement of OAB symptomsby antimuscarinics

4 Liposomes

Liposomes were earliest prototype of nanoparticles (particleswith one of the dimensions in nanometers) that are describedas lipid vesicles composed of concentric phospholipid bilay-ers enclosing an aqueous interior [24 25] The lipid vesiclescomprise either one or several aqueous compartments delin-eated by either one (unilamellar) or several (multilamellar)phospholipid bilayers [26] Liposomes have been widelystudied as drug carriers for a variety of chemotherapeuticagents (approximately 40000 scientific articles have beenpublished on the liposomes use so far) [25 27] Liposomesimproved the delivery of chemotherapeutic agents by alteringpharmacokinetics and reducing toxicity [26 28 29] Thesuccess of liposomes in the clinic has been attributed to thenontoxic nature of the lipids used in their formulation

5 Empty Liposomes

Empty liposomes itself can act as a topical healing agentand same has been demonstrated in treatment of dry eye[30 31] Either empty or with entrapped drugs liposomeshave also been used in ophthalmology to ameliorate keratitiscorneal transplant rejection uveitis endophthalmitis andproliferative vitreoretinopathy [32] These reports encour-aged investigation of empty liposomes as a therapeutic agentfor bladder injury Interaction of liposomes with culturedurothelium cells suggested that liposomes can be adsorbedand be endocytosed [33] Previous studies showed thatbinding of large multilamellar liposomes to the bladder cellswas stronger than of sonicated small size liposomes [34 35]

51 Preclinical Studies A ratmodel of bladder injury inducedby protamine sulfate [36] was used to asses efficacy ofempty liposomes Instillation of liposomes protected bladderirritation induced by protamine sulfate instillation into ratbladder [37] (Figure 1) In earlier study empty liposomeswere used as controls against capsaicin delivery study whichreported tolerance of empty liposomes in uninjured bladder[38] Bladder tolerance was investigated by cystometry andhistology [38]

Physiological effect of liposomes on bladder irritationmodel induced by protamine sulfate was studied in separatestudies [39 40] Cystometric studies involved bladder injuryinduced by infusion of protamine sulfate for an hour fol-lowed by irritation caused by infusion of high concentrationof potassium chloride solution [39 40] Post-treatment ofliposomes demonstrated the protective effect in this model[39 40] which involved coadministration of liposomes withpotassium chloride to mimic the clinical disease conditionThe comparative efficacy of liposomes was evaluated against

ISRN Pharmacology 3

(a) (b)

Figure 1 Protective effect of liposomes evaluated by histology in rat bladder injured by protamine sulfate Focal loss of apical layer andulcerated areas were noted in saline treated rats (a) Liposome post-treatment protected rats from ulceration induced by PS as evident fromphotograph in (b) taken on bladder harvest at 8 h after liposome instillation

FDAapproved therapies of dimethyl sulphoxide (DMSO) andintravesical instillation of PPS [1]

Clinically DMSO (RIMSO-50) is the only FDA approvedintravesical treatment for PBSIC [41] but off-label instil-lation of PPS has also been pursued [42] The efficacy ofvarious treatments was evaluated in chemically inducedbladder hyperactivity in rats by sequential infusion of pro-tamine sulfate and potassium chloride Bladder reflex activityof female Sprague-Dawley rats before and after treatmentwas evaluated by continuous cystometry under urethaneanaesthesia (10 gkg) Intravesical liposomes were effectivein doubling the intercontractile interval (ICI) compared withPPS while acute instillation of DMSO failed to produce anyprotective effect in this animal model [43]

Recently Lee et al [44] further improved this modelof DO induced by intravesical infusion by combining itwith systemic metabolic alteration through fructose feedingMetabolic syndrome created by feeding of fructose to ratscan lead to DO and urination frequency [44] Cystometricbladder capacity of fructose fed rats can be further decreasedby instillation of acidic ATP solution which provokesreflex micturition via afferent noise Evidence suggests thatincreased expression or release of neurotransmitters in themucosal layer of the bladder can generate afferent noise viaC-fiber pathway and result in DO [45] Compared to infusionwith normal saline ATP solution decreased bladder capacityand increased phasic contractions Addition of liposomesto the ATP solution partially reversed the ATP solution-induced response [46] Capsaicin induced desensitizationalso blunted the provocation of ATP in this model todemonstrate the role of afferent noise via C-fibers

52 Clinical Studies Encouraged by the exciting preclin-ical efficacy of empty liposomes as a therapeutic agentfor intravesical therapy of ICPBS Chuang et al recentlypublished the clinical safety and efficacy of liposomes inICPBS patients [47] In an open label prospective study on24 ICPBS patients the effect of intravesical liposomes wascompared against oral PPS Patients were equally dividedinto the two treatment arms administered either intravesical

liposomes (80mg40 cc distilled water) once weekly or oralPPS (100mg) 3 times daily for 4 weeks each Ten possibleresponses to treatment were monitored at 3 time pointsincluding baseline and at weeks 4 and 8

Comparable efficacy of liposomes to oral PPS wasdemonstrated by statistically significant decreases in urinaryfrequency and nocturia in both treatment arms Liposometreated patients showed statistically significant decreases inpain urgency and the OrsquoLeary-Sant symptom score withthe effect being most profound on urgency None of thetreated patients in the study reported urinary incontinenceretention or infection due to liposome instillation and therewere no unanticipated adverse events and no significantworsening of symptoms during followup Intravesical instilla-tion of liposomes in ICPBS patients was found to be safe withpotential improvement after 1 course of therapy for up to 8weeksThe study designwas suboptimal with lack of blindingand two treatment arms assigned to different routes of drugadministration and regimen Still once weekly liposometherapywas able to demonstrate efficacy comparable to thricedaily oral administration of PPS

Subsequently Lee et al assessed the safety and efficacy oftwice weekly administration of liposomes on ICPBS symp-toms [48] Five patients were given twice a week treatment ofliposomes for 4 weeks The primary outcome was the changein the OrsquoLeary-Sant symptomproblem score and OrsquoLeary-Sant total score from baseline to week 4 and week 8 TheOrsquoLeary-Sant symptomproblem score OrsquoLeary-Sant totalscore and pain score at the 4-week followup showed sig-nificantly greater improvement from baseline with biweeklyinstillation than once a week instillation Tolerability ofliposomes remains unchanged from once a week regimento twice a week regimen The followup at 8 weeks was alsosimilar for both treatment regimensThe incidence of urinaryincontinence retention or unanticipated adverse changeswas not noted with any regimen Intravesical liposomesappear to be a promising new treatment for ICPBS and futurelarge-scale placebo controlled studies are needed to verifythese results from a pilot study

4 ISRN Pharmacology

(a) (b)

Liposomes

Afferent nervesAfferent nerves

Leaky urothelium

(c)

Figure 2 Ex vivo images of rat bladder taken in visible and near-infrared light indicate the coating formed by instilled liposomeson bladder surface Liposomes carry a trace amount of near-infrared (NIR) lipophilic carbocyanine dye 111015840-dioctadecyl-333101584031015840-tetramethylindotricarbocyanine iodide (DiR) that fluoresces on exposure to NIR light Rat bladder filled with urine was tied at the basewith thread prior to harvest Liposomes coating the bladder surface are invisible in visible light photograph (a) but is indicated by bluecolored coating on the bladder luminal surface in NIR light (b) NIR imaging in vivo can allow noninvasive repeat objective measurement forbladder residence time of instilled treatments (c) Schematic illustration of liposome coating the bladder surface Given the chemical affinityof phospholipids of instilled liposomes with lipids in cells lining the bladder surface the liposomes form a protective film coating on theinjured bladder lumen surface and assist in the repair of leaky and inflamed uroepithelium

The exact mechanism of action for liposomes in ICPBSremains to be established but protective coating effect basedon preclinical studies cannot be ruled out as illustrated inFigure 2

6 Liposomes as a Delivery Platform

Not only is the lining of the urinary bladder the most im-permeable tissue in the human body but it is also the mostcompliant As a bladder lining expands additionalmembranematerial is added to its cells to help retain impermeability[49] Therefore vesicular trafficking may provide a favorableenvironment for drug delivery and therefore it is worthinvestigating whether vesicle nature of liposomes can aid in

improving the delivery of cargo across the bladder permeabil-ity barrier

In the field of neurourology instillation of neurotoxinsinto bladder is an accepted approach to achieve chemicalneuromodulation of afferent neurotransmission underlyingneurogenic bladder and ICPBS [50] Existing approachesof chemical neuromodulation by intravesical neurotoxinsare suboptimal due to vehicle toxicity for capsaicin [51] ordegradation of botulinum toxin (BoNT) in urine Possiblereasons underlying the lack of efficacy fromBoNT instillationin bladder can be protein degradation by proteases andproteinases in urine dilution in urine or poor uptake of theBoNT solution into the urothelium

Liposomes have been previously studied as a carrier oftoxins to enhance efficacy at lower doses [52] In the context of

ISRN Pharmacology 5

Lipotoxin

Intravesical instillation

Liposomes

+

BoNT-A

Figure 3 Scheme for formulation of BoNT into liposomes for blad-der instillation Freeze dried BoNT is resuspended in distilled waterand added to vials containing a desiccated liposomal preparationmade of phospholipids This reconstituted suspension is allowed tohydrate at room temperature for 30ndash60min before instillation

toxins instilled in the bladder fat-soluble neurotoxin such ascapsaicin can be integrated into the phospholipid bilayer [38]and water-soluble neurotoxin such as BoNT can be protectedinside the aqueous compartment(s) of liposomes delimitedby the phospholipid bilayer(s) [53] (Figure 3) Cystoscopeguided injections are the current standard practice in theclinic for administering BoNT to the bladder But in recentyears studies have also assessed the potential for intravesicalinstillation of BoNT alone in animal models of bladderirritation [54]

Development of instillation as a mode for administeringBoNT in patients will drastically bring down the cost oftreatment for patients with refractory overactive bladderOther groups have recently reported the use of DMSO forinstillation of BoNT instead of injection [55] DMSO doesnot afford the natural state of the BoNT as a protein and needto be formulated immediately before instillation DissolvingBoNT in DMSO may not be advisable owing to concernsof BoNT uptake into the systemic circulation of patientMoreover biochemical studies have demonstrated that met-alloproteolytic activity of the BoNT is strongly enhanced bythe presence of lipid membranes [56]

Recent studies reaffirmed the potential of liposomesas a promising vehicle for delivery of neurotoxins to thebladder [38 53] The transport of BoNT into urotheliumfrom liposomes was confirmed by detection of its uniqueeffect on neurotransmitters and proteolysis of synaptosomal-associated protein SNAP-25 through immunohistochemistry[53] The protection of BoNT entrapped inside liposomesfrom degradation in urine without compromising efficacywas demonstrated by attenuation of acetic acid induced blad-der irritation in rats [53] Similar results were obtained in pre-clinical studies with liposomes encapsulating capsaicin [38]

Liposomes have proven themselves as biocompatible deliveryagents in the bladder

7 Intravesical Antisense Therapeutics

The term ldquoantisenserdquo therapeutics emerged from seminalstudies done 4 decades using a short synthetic oligonu-cleotide for sequence-specific gene silencing [57] Genesilencing involves introduction of short strands of DNA(termed as antisense) with sequences complementary to themRNA encoding a particular gene inside the cell with theintent to block gene expression through either translationalinhibition or enzymatic cleavage of the mRNA target [3]

Oligonucleotide ODN binds specifically and stronglyto the mRNA target through Watson-Crick base pairingODN can be basically categorized into those that directcleavage of the target mRNA as caused by small interferingRNAs (siRNAs) and those that alter mRNA translationwithout causing mRNA cleavage Recent discovery of smallinterfering RNAs (siRNAs) and the elucidation of the RNAinterference (RNAi) pathway has also brought a sea changein the control of posttranscriptional gene expression siRNAtakes advantage of endogenous cellular pathways to potentlysilence the expression of specific genes (Figure 4)

Antisense mechanism is a promising approach for devel-oping therapeutics based on rational gene-based drug designAntisense therapeutics have been under clinical investigationfor more than 30 years [58] However drug development ofthis approach has been hampered by inefficient intracellulardelivery and cellular uptake of the ODN The translation ofbasic antisense research into therapeutics is also impeded byintracellular stability of ODN and potential for ldquooff-targetrdquogene silencing immunostimulation and other side effects

A vast array of chemical modifications to ODN has beendeveloped to overcome the therapeutically limiting featuresby altering internucleotide phosphate linkages backbonesugars or nucleobases One such modified ODN is peptidenucleic acid which replaces phosphodiester bond betweennucleobases with a peptide bond Replacement of phosphodi-ester bond by a phosphorothioate linkage is another methodto improve the stability of ODN against nucleases

Great progress has been made in translating antisenseresearch into clinical therapies based on local injection intoeye [58]The field recently progressed further with a systemicinjection therapy for treating familial hypercholesterolemia[59] Antisense therapeutics have been used for exon skippingto optimize the functionality of a truncated dystrophinprotein in dog model of Duchenne muscular dystrophy [60]

Nevertheless applied research for bladder diseases haslagged behind considering that the anatomical architec-ture of bladder provides ease of local administration withrestricted systemic side effects due to lower serum uptakeof antisense ODN Bladder instillation of antisense ODNor their chemically modified mimics can therefore be anefficient means to control the expression of therapeuticallyrelevant genes Antisense agents can also be used to elucidatethe role of newly discovered genes in bladder function

6 ISRN Pharmacology

Liposomes with antisense

Transcription

Genomic DNA RNA Duplex

Antisense DNA

Activated PI 3-kinase

Activated receptortyrosine kinase

NGF

Anti-NGFantibodies

mRNAdegradation

PIP2 PIP3

PP P P P P

oligonucleotides

Figure 4 Illustration of mechanisms for blocking NGF expression by either NGF antibodies or downregulation of NGF mRNA expressionNGF antibody sequesters the freely available NGF and prevents it from binding with the cognate TrkA receptors NGF antisense selectivelybinds with NGF mRNA inside the cells to block the translation of corresponding mRNA siRNA takes advantage of endogenous cellularpathways to degrade NGF mRNA and control the posttranscriptional NGF gene expression

8 NGF Expression in Bladder

Several studies have reported that patients with OAB orICPBS excrete more nerve growth factor (NGF) in theirurine relative to asymptomatic controls [61] Higher expres-sion of NGF in bladder of patients [62] with correspondinglower serum levels of NGFmakes the bladder tissue the likelysource for the elevated NGF in urine [63] Previous studieshave indicated that increased levels of NGF in the bladderand bladder afferent pathways are directly involved in theemergence of hyperexcitability of C-fiber bladder sensorypathways leading to the pathology of DO andOAB (Figure 4)[64]

In addition intrathecal application of NGF antibodiesreducedNGF levels in bladder afferent pathways and normal-ized bladderurethral function in spinal cord injured (SCI)rats [65] Because it is likely that the major pathology of OABis driven by NGF targeting the intracellular synthesis of NGFmolecule in bladder is a promising therapeutic alternative

9 NGF Expression a Drug Target

Overexpression of NGF can be blocked either directly byantibodies [66] or by blocking the synthesis of NGF proteinfrom mRNA [67] Systemic administration of monoclonalhuman NGF antibodies (tanezumab) has been explored fortherapeutic outcomes in ICPBS patients but not withoutencountering safety concerns such as paresthesia hypoesthe-sia and arthralgia [66] Generalized blockade ofNGF activityat sites other than bladder by anti-NGF antibodies maynot be the preferred outcome because NGF is an essentialhousekeeping growth factor necessary for the survival andgrowth of neurons [68] The physiological necessity of NGF

action at those sites may explain the incidence of paresthesiahypoesthesia and arthralgia in patients treated with systemicanti-NGF antibodies [66]

91 Peptide Nucleic Acid (PNA) Therefore to reduce thetoxicity of systemic blockade of NGF we sought to developa novel intravesical therapy of OAB by targeting the intra-cellular synthesis of NGF in the urothelium Antisense ODNneeded to be able to cross cell membrane to act as a drugand negatively charged ODN will not pass through a lipidlayer such as cell membranes Bladder uptake of ODN islimited by the anionic charge and size of the ODN as wellas anionic glycosaminoglycan layer of the inner bladdersurface Therefore primary impediment to be overcome inthe development of intravesical antisense therapeutics isinefficient bladder uptake of the ODN across urotheliumPrevious studies showed that heparan sulfate proteoglycansexpressed on cell surface act as receptors for extracellularTAT uptake [69] Therefore it was reasoned that GAG layeron bladder surface can facilitate bladder uptake of peptidenucleic acid PNA if it could be conjugatedwith synthetic TATpeptide

Water insoluble peptide nucleic acid targeting NGF wasconjugated with cell penetrating cationic peptide TAT forintracellular delivery of antisense moiety to demonstrate effi-cacy in animal models Studies showed that PNA conjugatedwith TAT suppressed cyclophosphamide cystitis followinglocal instillation of antisense against NGF [67] There wasnegligible uptake of peptide nucleic acid in absence of TATconjugation Prior to determination of in vivo efficacy ofconjugate suitable target sequence on NGF mRNA wasdetermined by predicted folding structure and cell transfec-tion experiments Successful intravesical delivery of peptide

ISRN Pharmacology 7

nucleic acid in bladder at the same time of cyclophosphamideinjection protected against the cystitis by blocking the rise inbladder contraction frequency and inflammation

92 Phosphorothioate-Linked Analogues Considering thedifficulty and possible nonspecific toxicity of peptide nucleicacid and TAT recent developments focused on simplifyingthe approach using water-soluble phosphorothioated ODNwhich have an increased resistance to exo- and endonucleasesfor improved stability [70] As alluded to in the above textsuccess of antisense therapeutics is largely dependent on thedevelopment of a delivery vehicle that can efficiently deliverantisense ODN in bladder Preliminary studies showed thatliposomes can be far better biocompatible effective carriersfor local gene silencing of NGF gene in bladder cells

Cationic liposomes and mimics have emerged as themost popular nonviral method to deliver nucleic acids intherapeutic applications Easy and reversible complex forma-tion of cationic liposomes with ODN at room temperatureallows their use as carriers Electrostatic attraction betweenthe cationic lipid DOTAP and the polyanionic antisenseODN is responsible for the complex formation The efficacyof liposome delivered siRNA by intravesical route has beenpreviously demonstrated in preclinical models of bladdercancer [71] Residence of ODN in the rat bladder after intrav-esical instillation was demonstrated to be longer than 24 husing fluorescent tagged ODN complexed with liposomes[72] Fluorescent probe was localized in bladder urotheliumcells 24 h following instillation The 24 h residence time wasalso demonstrated for siRNA in mouse bladder [73] Bladderuptake of fluorescent ODN without liposomes in normalrat bladder is poor Previous studies have shown that veryhigh concentration of phosphorothioated ODN can deliverODN without liposomes to bladder cells of mice havingbladder cancer [74] The urothelium barrier may be slightlycompromised in cancerous condition and the strategy ofloading bladder with high dose of ODN may not workin noncancerous diseased condition of bladder with intactbarrier

In order to evaluate the efficacy of NGF antisense ODNacetic acid infusion was used to cause a rapid rise of NGFprotein levels A single dose instillation of OND complexedwith liposomes protected against bladder overactivity (BO)induced by acetic acid Together with data of bladder uptakestudies using fluorescent ODN it is demonstrated that ONDis readily available to the bladder after intravesical instillationIt has been previously reported that within 2 h of exposureto irritants such as turpentine and acetic acid there occursa rapid rise in bladder content of NGF [75] Later studiesfound that bladder responds to insults with upregulation inthe genes for NGF sE-Selectin and receptor for monocytechemoattractant protein-1 (MCP-1) within 30min of expo-sure to lipopolysaccharide (Figure 5) [76] The acetic acidinduced overexpression of NGF was blunted by pretreatmentwith NGF antisense OND with phosphorothioate linkageprior to exposure of acetic acid The downregulation of NGFmRNA expression was in agreement with reduced proteinlevels and suppressed NGF-like immunoreactivity in theurothelium

93 Antisense and Downstream Signalling of NGF Drugdevelopment of intravesical antisense for NGF was also ableto unmask the downstream signaling [77] involving NGFfollowing exposure to acetic acid Experiments supportedthe role of NGF as a paracrine messenger [77] which isknown to activate several downstream effectors to manifestphysiological and pathological signaling changes linked to it[76 78ndash80] The bladder injury set off by acetic acid initiatesthe signalling cascades that upregulate the expression ofNGF and other chemokines MCP-1 CXCL-1 and CXCL-10 and prostaglandins [76 78ndash80] As reported elsewherelocalization of chemokines within synaptic vesicles in neu-rons [81] is consistent with their ability to act as excitatoryneurotransmitters following AA exposure

Chemokines are one of several downstream effectorsactivated by NGF [76 78ndash80] and interestingly chemokinereceptors are widely expressed in neural and nonneuralelements of the nociceptive pathways that are responsible forvisceral and somatic pain sensation [82] MCP-1 [83] andCXCL-10 [84] are constitutively expressed in neurons wherethey participate in excitability of primary afferent neuronsthrough transactivation of transient receptor channels andnociceptor sensitization [84] Overexpression ofNGF is likelyto drive the expression of VEGF fromneurons and leptin [78]from adipocytes covering neurons [85]

Cooperative expression of NGF and MCP-1 is able toinduce hyperexcitability in neurons by activating TRPV1receptor In addition MCP-1 CXCL-1 and CXCL-10 causechemoattraction of monocytes neutrophils and lympho-cytes respectively to mediate the bladder injury set offby acetic acid Extravasation is an essential prerequisitefor infiltration of monocytes neutrophils and lymphocyteswhich requires the expression of adhesion molecules likeE-selectin and intracellular adhesion molecule ICAM-1 [7686] E-selectin is a cytokine-inducible adhesion moleculethat supports the rolling and stable arrest of leukocyteson activated vascular endothelium Expression of E-selectinhas been linked to adherence of neutrophils to bladdermicrovascular endothelial cells and to cyclophosphamidecystitis [87 88] E-selectin gene expression is activated bythe NF-120581B and MAP kinase signal transduction pathways[76 86]

Activation and recruitment of leukocytes to acetic acidinduced bladder injury also require expression of intra-cellular adhesion molecule ICAM-1 [89] ICAM-1 interactsspecifically with its receptors of the integrin family to inducereversible cell-cell interactions involving adhesion Expres-sion of ICAM-1 was found to be increased in patients withICPBS and reduced in patients responding to instillation ofhyaluronic acid [90] Expression of ICAM-1 and VCAM-1was also noted in a recent study on biopsy tissue of ICPBSpatients [91] Increased expression of ICAM-1 subsequentto exposure with acetic acid in animal model is thereforeclinically relevant

Antisense experiments supported the earlier report thatinhibition of NGF expression significantly downregulates theexpression of ICAM-1 [80] It is reported that binding ofNGF to its high-affinity TrkA receptor controls the sICAM-1expression on target cells [80] Expression of ICAM-1 was

8 ISRN Pharmacology

Neutrophil

Monocytes

Acetic acid

Integrin

NGF

MCP-1

Release of cytokinesVanilloid

receptor

Trk receptors NGF

Membrane damage

VEGF

CXCL1

Activated leucocyte

Startrolling

Extravasation

CXCL10

sICAMssE-selectin

ActivationAdhesion

MCP-1

NGF

PKC

DAG

PLC

IP3Kinases

Ras

AKT

PI3K

CXCL10

sICAMs

LymphocyteAfferents

Figure 5 Signalling cascades induced by NGF overexpression following exposure to acetic acid Injury from exposure to acetic sets off thesignalling cascades that upregulate the expression of NGF MCP-1 prostaglandins CXCL-1 and CXCL-10 Overexpression of NGF drives theexpression of VEGF from neurons and leptin from adipocytes covering neurons NGF and MCP-1 also induce hyperexcitability in neuronsby activating TRPV1 receptor MCP-1 CXCL-1 and CXCL-10 cause chemoattraction of monocytes neutrophils and lymphocytes whichrequires extravasation across endothelium from blood through the cooperation of adhesion molecules E-selectin and ICAM-1

negatively associated with NGF expression in antisenseexperiments Ultimately expression of ICAM-1 is regulated atthe level of transcription by one of several signaling cascadesNF-120581B JAKSTAT IFN-g (CXCL-10) AP MAP kinase andPKC [92]

Intravesical route can allow selective exposure of anti-sense ODN to the NGF producing cells in urothelium andavoid systemic side effects from genetic manipulation ofNGF expression Intravesical route can also prove to be costeffective given the cost of ODN or siRNA Contrary to thegoal of loss of function in bladder with antisense ODN inrecent studies liposomes have also been used to deliver ODNfor gain of function in bladder [93]

10 Imaging of Intravesical Therapy

Drug development for DO and ICPBS relies heavily onsubjective outcomes for predicting efficacy and safety in earlyclinical development Although there are many validatedmeasurement tools used in DO and ICPBS research theyare usually burdensome and do not capture all symptomsrelated to the lower urinary tract Moreover improvement insymptoms scores following an intervention does not alwayscorrelate with patient expectations satisfaction and goalachievement which are critically important for successfulmanagement of DO and ICPBS

There is underdevelopment and underutilization of urinebiomarkers and imaging methods for investigational drugsgiven by intravesical route Overreliance on subjectiveimpressions of patients for therapeutic response limits clini-cal relevance due to weak correlation with patient satisfactionand can also impede continued scientific progress in assessing

study outcomes relative to symptom bother Symptom scoresmay also be related to the cause of high heterogeneity inclinical response in DO and ICPBS patients

Imaging can offer the possibility to provide region-specific information in situations where serum and urineassays may not reflect the true state of bladder [94ndash96]Urine chemokines have been reported to be associated withsymptom severity of ICPBS patients [97] Application offluorescent microscopy for imaging of pelvic floor has beenlimited due to the issues pertaining to light absorption bytissues scattering and autofluorescence [98]

Recent studies have shown that dyes fluorescing inthe near-infrared (NIR) band can overcome this handi-cap in deep-tissue imaging of experimental animals [99]Selected NIR fluorochromes emit light with tissue pen-etration approaching 10ndash15 cm [100] The light emissionfrom probes in this spectrum encounters low backgroundautofluorescence and minimal attenuation of signal due tolight absorption by tissue components [98] Therefore NIRimaging yields high signal-to-noise ratios and is well suitedfor studying the distribution of instilled treatments in bladder(Figure 2(b))

Preliminary studies suggest that imaging of mouse pelvicfloor in NIR spectrum can be a viable option [101] NIRimaging easily allows visualization and quantification ofbladder distribution by tracing themigration of fluorescence-labeled liposomes in anaesthetized mouse Compared withother biological assays imaging could provide objectiveendpoint for patients with ulcerative cystitis to indicate itshigh translation potential Other benefits of optical imaginginclude its relatively low cost compared withmore traditionalimaging systems such as MRI and PET The instrument

ISRN Pharmacology 9

consists of two near-infrared lasers emitting at 685 nm and785 nm camera and novel optics

Furthermore preferential binding of liposomes andnanoparticles to bladder lesions of ICPBS after instillationcan be assessed by NIR imaging of bladder where it cantheoretically provide anatomically specific information aboutthe real time status of ulcerlesion in bladder surface or tissueabnormality [102] Therefore future studies can substitute aresponse variable that is continuous in nature instead of asubjective clinical outcome

11 Conclusions

Adoption of forward-thinking approaches can achieveadvancements in drug delivery systems targeted to improvepharmacotherapy of bladder diseases in the future Latestdevelopments in the field of nanotechnology can bring thismode of therapy from second line of treatment for refractorycases to the forefront of disease management Liposomesare an attractive drug delivery platform by virtue of theirbiodegradability biocompatibility low toxicity and simpleand mild preparation methods

Conflict of Interests

All authors except Dr Michael Chancellor declare that thereis no conflict of interests regarding the publication of thispaper Dr Michael Chancellor owns stocks in the start-upliposome company Lipella

Acknowledgments

This work was supported by the National Institutes of Health(DK057267 and DK088836) and the Department of Defense(W81XWH-11-1-0763 and W81XWH-12-1-0565)

References

[1] J Parkin C Shea and G R Sant ldquoIntravesical dimethyl sul-foxide (DMSO) for interstitial cystitismdasha practical approachrdquoUrology vol 49 no 5 pp 105ndash107 1997

[2] C Dowson J Watkins M S Khan P Dasgupta and A SahaildquoRepeated botulinum toxin type A injections for refractoryoveractive bladder medium-term outcomes safety profile anddiscontinuation ratesrdquo European Urology vol 61 no 4 pp 834ndash839 2012

[3] J Kaufman V Tyagi M Anthony M B Chancellor and PTyagi ldquoState of the art in intravesical therapy for lower urinarytract symptomsrdquo Reviews in Urology vol 12 pp e181ndashe189 2010

[4] N Yoshimura and M B Chancellor ldquoCurrent and futurepharmacological treatment for overactive bladderrdquo Journal ofUrology vol 168 no 5 pp 1897ndash1913 2002

[5] K A Killinger J A Boura and K M Peters ldquoPain ininterstitial cystitisbladder pain syndrome do characteristicsdiffer in ulcerative and non-ulcerative subtypesrdquo InternationalUrogynecology Journal vol 24 no 8 pp 1295ndash1301 2013

[6] J C Nickel P Hanno K Kumar and HThomas ldquoSecond mul-ticenter randomized double-blind parallel-group evaluationof effectiveness and safety of intravesical sodium chondroitin

sulfate compared with inactive vehicle control in subjects withinterstitial cystitisbladder pain syndromerdquoUrology vol 79 no6 pp 1220ndash1225 2012

[7] P M Hanno ldquoAnalysis of long-term elmiron therapy forinterstitial cystitisrdquo Urology vol 49 no 5 pp 93ndash99 1997

[8] A Giannantoni V Bini R Dmochowski et al ldquoReply to ClausRiedlrsquos letter to the editor re antonella Giannantoni VittorioBini Roger Dmochowski et al Contemporary management ofthe painful bladder a systematic reviewrdquo EuropeanUrology vol61 no 5 pp 29ndash53 2012

[9] MC Lai Y CKuo andHCKuo ldquoIntravesical hyaluronic acidfor interstitial cystitispainful bladder syndrome a comparativerandomized assessment of different regimensrdquo InternationalJournal of Urology vol 20 pp 203ndash207 2013

[10] J Eldrup J Thorup and S L Nielsen ldquoPermeability andultrastructure of human bladder epitheliumrdquo British Journal ofUrology vol 55 no 5 pp 488ndash492 1983

[11] C L Parsons D Boychuk S Jones R Hurst and H CallahanldquoBladder surface glycosaminoglycans an epithelial permeabil-ity barrierrdquo Journal of Urology vol 143 no 1 pp 139ndash142 1990

[12] Y Kim N Yoshimura H Masuda F De Miguel and MB Chancellor ldquoAntimuscarinic agents exhibit local inhibitoryeffects on muscarinic receptors in bladder-afferent pathwaysrdquoUrology vol 65 no 2 pp 238ndash242 2005

[13] V Tyagi B J Philips R Su et al ldquoDifferential expression offunctional cannabinoid receptors in human bladder detrusorand urotheliumrdquo Journal of Urology vol 181 no 4 pp 1932ndash1938 2009

[14] B J Philips M C Smaldone V L Erickson N YoshimuraM B Chancellor and P Tyagi ldquoDifferential expression ofneurokinin receptor subtypes in urothelium and detrusor ofhuman bladderrdquo Journal of Urology vol 179 article 354 2008

[15] S A Baker G W Hennig J Han F C Britton T K Smithand S D Koh ldquoMethionine and its derivatives increase blad-der excitability by inhibiting stretch-dependent K+ channelsrdquoBritish Journal of Pharmacology vol 153 no 6 pp 1259ndash12712008

[16] J M Beckel A Kanai S-J Lee W C De Groat and LA Birder ldquoExpression of functional nicotinic acetylcholinereceptors in rat urinary bladder epithelial cellsrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 290 no 1 ppF103ndashF110 2006

[17] LA Birder A J KanaiWCDeGroat et al ldquoVanilloid receptorexpression suggests a sensory role for urinary bladder epithelialcellsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 23 pp 13396ndash13401 2001

[18] P Tyagi C A Thomas N Yoshimura and M B ChancellorldquoInvestigations into the presence of functional szlig1 szlig2 and szlig3-adrenoceptors in urothelium and detrusor of human bladderrdquoInternational Brazilian Journal of Urology vol 35 no 1 pp 76ndash83 2009

[19] A T Hanna-Mitchell J M Beckel S Barbadora A J Kanai WC de Groat and L A Birder ldquoNon-neuronal acetylcholine andurinary bladder urotheliumrdquo Life Sciences vol 80 no 24-25 pp2298ndash2302 2007

[20] C P Smith D A Gangitano A Munoz et al ldquoBotulinumtoxin type A normalizes alterations in urothelial ATP and NOrelease induced by chronic spinal cord injuryrdquo NeurochemistryInternational vol 52 no 6 pp 1068ndash1075 2008

[21] Y-C Chuang C A Thomas S Tyagi N Yoshimura PTyagi and M B Chancellor ldquoHuman urine with solifenacin

10 ISRN Pharmacology

intake but not tolterodine or darifenacin intake blocks detrusoroveractivityrdquo International Urogynecology Journal and PelvicFloor Dysfunction vol 19 no 10 pp 1353ndash1357 2008

[22] Y Kim N Yoshimura H Masuda F D Miguel and M BChancellor ldquoIntravesical instillation of human urine after oraladministration of trospium tolterodine and oxybutynin in a ratmodel of detrusor overactivityrdquo BJU International vol 97 no 2pp 400ndash403 2006

[23] S Tyagi P Tyagi S Van-le N YoshimuraM B Chancellor andF de Miguel ldquoQualitative and quantitative expression profileof muscarinic receptors in human urothelium and detrusorrdquoJournal of Urology vol 176 no 4 pp 1673ndash1678 2006

[24] G Gregoriadis and B E Ryman ldquoLiposomes as carriers ofenzymes or drugs a new approach to the treatment of storagediseasesrdquo Biochemical Journal vol 124 no 5 p 58 1971

[25] G Gregoriadis S Jain I Papaioannou and P Laing ldquoImprov-ing the therapeutic efficacy of peptides and proteins a role forpolysialic acidsrdquo International Journal of Pharmaceutics vol300 no 1-2 pp 125ndash130 2005

[26] P Sapra P Tyagi and T M Allen ldquoLigand-targeted liposomesfor cancer treatmentrdquo Current Drug Delivery vol 2 no 4 pp369ndash381 2005

[27] G Gregoriadis G Dapergolas and E D Neerunjun ldquoPene-tration of target areas in the rat by liposome associated agentsadministered parenterally and intragastricallyrdquo BiochemicalSociety Transactions vol 4 no 2 pp 256ndash259 1976

[28] G Gregoriadis ldquoEngineering liposomes for drug deliveryprogress and problemsrdquo Trends in Biotechnology vol 13 no 12pp 527ndash537 1995

[29] G Gregoriadis and A C Allison ldquoEntrapment of proteins inliposomes prevents allergic reactions in pre immunised micerdquoFEBS Letters vol 45 no 1 pp 71ndash74 1974

[30] S Lee S Dausch CMaierhofer andD Dausch ldquoA new therapyconcept with a liposome eye spray for the treatment of the lsquodryeyersquordquo Klinische Monatsblatter fur Augenheilkunde vol 221 no10 pp 825ndash836 2004

[31] D Dausch S Lee S Dausch J C Kim G Schwert and WMichelson ldquoComparative study of treatment of the dry eyesyndrome due to disturbances of the tear film lipid layer withlipid-containing tear substitutes efficacy of lipid-containingtear substitutesrdquo Klinische Monatsblatter fur Augenheilkundevol 223 no 12 pp 974ndash983 2006

[32] S Ebrahim G A Peyman and P J Lee ldquoApplications ofliposomes in ophthalmologyrdquo Survey of Ophthalmology vol 50no 2 pp 167ndash182 2005

[33] J Nirmal P Tyagi L Dang et al ldquoEndocytosis uptake ofliposomes in urothelium cells detected by transmission electronmicroscopyrdquoThe Journal of Urology vol 187 article e15 2012

[34] D N Frangos J J Killion D Fan R Fishbeck A C VonEschenbach and I J Fidler ldquoThe development of liposomescontaining interferon alpha for the intravesical therapy ofhuman superficial bladder cancerrdquo Journal of Urology vol 143no 6 pp 1252ndash1266 1990

[35] J W Johnson R Nayar J J Killion A C Von Eschenbachand I J Fidler ldquoBinding of liposomes to human bladdertumor epithelial cell lines implications for an intravesical drugdelivery system for the treatment of bladder cancerrdquo SelectiveCancer Therapeutics vol 5 no 4 pp 147ndash155 1989

[36] Y-C Chuang M B Chancellor S Seki et al ldquoIntravesicalprotamine sulfate and potassium chloride as a model forbladder hyperactivityrdquoUrology vol 61 no 3 pp 664ndash670 2003

[37] J Kaufman P Tyagi and M B Chancellor ldquoIntravesicalliposomal (Lp08) instillation protects bladder urothelium fromchemical irritationrdquoThe Journal of Urology vol 181 article 5392009

[38] P Tyagi M B Chancellor Z Li et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical capsaicindelivery using thermosensitive hydrogel and liposomesrdquo Jour-nal of Urology vol 171 no 1 pp 483ndash489 2004

[39] MO Fraser Y-CChuang P Tyagi et al ldquoIntravesical liposomeadministrationmdasha novel treatment for hyperactive bladder inthe ratrdquo Urology vol 61 no 3 pp 656ndash663 2003

[40] P Tyagi M Chancellor N Yoshimura and L Huang ldquoActivityof different phospholipids in attenuating hyperactivity in blad-der irritationrdquo BJU International vol 101 no 5 pp 627ndash6322008

[41] Y Sun and T C Chai ldquoEffects of dimethyl sulphoxide andheparin on stretch-activated ATP release by bladder urothelialcells from patients with interstitial cystitisrdquo BJU Internationalvol 90 no 4 pp 381ndash385 2002

[42] J J Bade ldquoA placebo-controlled study of intravesical pentosan-polysulphate for the treatment of interstitial cystitisrdquo BritishJournal of Urology vol 79 no 2 pp 168ndash171 1997

[43] P Tyagi V C Hsieh N Yoshimura J Kaufman and M BChancellor ldquoInstillation of liposomes vs dimethyl sulphoxide orpentosan polysulphate for reducing bladder hyperactivityrdquo BJUInternational vol 104 no 11 pp 1689ndash1692 2009

[44] W-C Lee C-T Chien H-J Yu and S-W Lee ldquoBladderdysfunction in rats with metabolic syndrome induced by long-term fructose feedingrdquo Journal of Urology vol 179 no 6 pp2470ndash2476 2008

[45] J I Gillespie G A Van Koeveringe S G DeWachter and J DeVente ldquoOn the origins of the sensory output from the bladderthe concept of afferent noiserdquo BJU International vol 103 no 10pp 1324ndash1333 2009

[46] W C Lee P H Chiang Y L Tain C C Wu and Y CChuang ldquoSensory dysfunction of bladder mucosa and bladderoversensitivity in a rat model of metabolic syndromerdquo PloSONE vol 7 Article ID e45578 2012

[47] Y-C Chuang W-C Lee W-C Lee and P-H Chiang ldquoIntrav-esical liposome versus oral pentosan polysulfate for interstitialcystitispainful bladder syndromerdquo Journal of Urology vol 182no 4 pp 1393ndash1400 2009

[48] W-C Lee Y-C Chuang W-C Lee and P-H Chiang ldquoSafetyand dose flexibility clinical evaluation of intravesical liposomein patients with interstitial cystitis or painful bladder syn-dromerdquo Kaohsiung Journal of Medical Sciences vol 27 no 10pp 437ndash440 2011

[49] S T Truschel E Wang W G Ruiz et al ldquoStretch-regulatedexocytosisendocytosis in bladder umbrella cellsrdquo MolecularBiology of the Cell vol 13 no 3 pp 830ndash846 2002

[50] M B Chancellor and W C De Groat ldquoIntravesical capsaicinand resiniferatoxin therapy spicing up the ways to treat theoveractive bladderrdquo Journal of Urology vol 162 no 1 pp 3ndash111999

[51] D S Byrne A Das J Sedor et al ldquoEffect of intravesicalcapsaicin and vehicle on bladder integrity in control and spinalcord injured ratsrdquo Journal of Urology vol 159 no 3 pp 1074ndash1078 1998

[52] M Mandal and K-D Lee ldquoListeriolysin O-liposome-mediatedcytosolic delivery of macromolecule antigen in vivo enhance-ment of antigen-specific cytotoxic T lymphocyte frequency

ISRN Pharmacology 11

activity and tumor protectionrdquo Biochimica et Biophysica Actavol 1563 no 1-2 pp 7ndash17 2002

[53] Y-C Chuang P Tyagi C-C Huang et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical botulinumtoxin a delivery using liposomesrdquo Journal of Urology vol 182no 2 pp 786ndash792 2009

[54] Y-C Chuang N Yoshimura C-C Huang P-H Chiang andM B Chancellor ldquoIntravesical botulinum toxin a administra-tion produces analgesia against acetic acid induced bladder painresponses in ratsrdquo Journal of Urology vol 172 no 4 pp 1529ndash1532 2004

[55] S P Petrou A S Parker J E Crook A Rogers D Metz-Kudashick and D D Thiel ldquoBotulinum A toxindimethylsulfoxide bladder instillations for women with refractory idio-pathic detrusor overactivity a phase 12 studyrdquo Mayo ClinicProceedings vol 84 no 8 pp 702ndash706 2009

[56] P Caccin O Rossetto M Rigoni E Johnson G Schiavoand C Montecucco ldquoVAMPsynaptobrevin cleavage by tetanusand botulinum neurotoxins is strongly enhanced by acidicliposomesrdquo FEBS Letters vol 542 no 1ndash3 pp 132ndash136 2003

[57] M L Stephenson and P C Zamecnik ldquoInhibition of Roussarcoma viral RNA translation by a specific oligodeoxyribonu-cleotiderdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 75 no 1 pp 285ndash288 1978

[58] H Dadgostar and N Waheed ldquoThe evolving role of vascularendothelial growth factor inhibitors in the treatment of neovas-cular age-related macular degenerationrdquo Eye vol 22 no 6 pp761ndash767 2008

[59] C Gebhard G Huard E A Kritikou and J C TardifldquoApolipoprotein B antisense inhibitionmdashupdate on mipom-ersenrdquo Current Pharmaceutical Design vol 919 no 17 pp 3132ndash3142 2013

[60] T Yokota E Hoffman and S Takeda ldquoAntisense oligo-mediated multiple exon skipping in a dog model of duchennemuscular dystrophyrdquoMethods inMolecular Biology vol 709 pp299ndash312 2011

[61] B L Jacobs M C Smaldone V Tyagi et al ldquoIncreased nervegrowth factor in neurogenic overactive bladder and interstitialcystitis patientsrdquoThe Canadian Journal of Urology vol 17 no 1pp 4989ndash4994 2010

[62] H-T Liu and H-C Kuo ldquoIntravesical botulinum toxin Ainjections plus hydrodistension can reduce nerve growth factorproduction and control bladder pain in interstitial cystitisrdquoUrology vol 70 no 3 pp 463ndash468 2007

[63] H T Liu and H C Kuo ldquoIncreased urine and serum nervegrowth factor levels in interstitial cystitis suggest chronicinflammation is involved in the pathogenesis of diseaserdquo PloSONE vol 7 Article ID e44687 2012

[64] N Yoshimura N E Bennett Y Hayashi et al ldquoBladderoveractivity and hyperexcitability of bladder afferent neuronsafter intrathecal delivery of nerve growth factor in ratsrdquo Journalof Neuroscience vol 26 no 42 pp 10847ndash10855 2006

[65] S Seki K Sasaki Y Igawa et al ldquoSuppression of detrusor-sphincter dyssynergia by immunoneutralization of nervegrowth factor in lumbosacral spinal cord in spinal cord injuredratsrdquo Journal of Urology vol 171 no 1 pp 478ndash482 2004

[66] R J Evans R M Moldwin N Cossons A Darekar I WMills andD Scholfield ldquoProof of concept trial of tanezumab forthe treatment of symptoms associated with interstitial cystitisrdquoJournal of Urology vol 185 no 5 pp 1716ndash1721 2011

[67] P Tyagi R Banerjee S Basu N YoshimuraM Chancellor andL Huang ldquoIntravesical antisense therapy for cystitis using TAT-peptide nucleic acid conjugatesrdquoMolecular Pharmaceutics vol3 no 4 pp 398ndash406 2006

[68] D C Molliver D E Wright M L Leitner et al ldquoIB4-bindingDRG neurons switch from NGF to GDNF dependence in earlypostnatal liferdquo Neuron vol 19 no 4 pp 849ndash861 1997

[69] M TyagiM RusnatiM Presta andMGiacca ldquoInternalizationof HIV-1 tat requires cell surface heparan sulfate proteoglycansrdquoJournal of Biological Chemistry vol 276 no 5 pp 3254ndash32612001

[70] E Uhlmann A Ryte and A Peyman ldquoStudies on the mech-anism of stabilization of partially phosphorothioated oligonu-cleotides against nucleolytic degradationrdquoAntisense andNucleicAcid Drug Development vol 7 no 4 pp 345ndash350 1997

[71] M Nogawa T Yuasa S Kimura et al ldquoIntravesical adminis-tration of small interfering RNA targeting PLK-1 successfullyprevents the growth of bladder cancerrdquo Journal of ClinicalInvestigation vol 115 no 4 pp 978ndash985 2005

[72] M Kashyap N Kawamorita V Tyagi et al ldquoDownregulation ofNGF expression in the bladder by antisense oligoucleotides asnew treatment for overactive bladderrdquo Journal of Urology vol27190 no 2 pp 757ndash764 2013

[73] C-J Arum Y Kodama N Rolim et al ldquoA rat model ofintravesical delivery of small interfering RNA for studyingurinary carcinomardquoWorld Journal of Urology vol 28 no 4 pp479ndash485 2010

[74] C E Blietz B Thode M Hauses et al ldquoIn vivo studies on theavailability and toxicity of antisense oligonucleotides in bladdercancerrdquo In Vivo vol 23 no 1 pp 13ndash19 2009

[75] D Oddiah P Anand S B McMahon and M Rattray ldquoRapidincrease of NGF BDNF andNT-3mRNAs in inflamed bladderrdquoNeuroReport vol 9 no 7 pp 1455ndash1458 1998

[76] M R Saban H Hellmich N B Nguyen J Winston T GHammond and R Saban ldquoTime course of LPS-induced geneexpression in a mouse model of genitourinary inflammationrdquoPhysiol Genomics vol 5 no 3 pp 147ndash160 2001

[77] B Schnegelsberg T T Sun G Cain et al ldquoOverexpression ofNGF in mouse urothelium leads to neuronal hyperinnervationpelvic sensitivity and changes in urinary bladder functionrdquoTheAmerican Journal of PhysiologymdashRegulatory Integrative andComparative Physiology vol 298 no 5 pp R534ndashR547 2010

[78] S Cardenas M Scuri L Samsell et al ldquoNeurotrophic andneuroimmune responses to early-life Pseudomonas aeruginosainfection in rat lungsrdquo American Journal of PhysiologymdashLungCellular and Molecular Physiology vol 299 no 3 pp L334ndashL344 2010

[79] K Nakamura F Tan Z Li and C J Thiele ldquoNGF activationof TrkA induces vascular endothelial growth factor expressionvia induction of hypoxia-inducible factor-1120572rdquo Molecular andCellular Neuroscience vol 46 no 2 pp 498ndash506 2011

[80] S Othumpangat M Regier and G Piedimonte ldquoNerve growthfactor modulates human rhinovirus infection in airway epithe-lial cells by controlling ICAM-1 expressionrdquo American Journalof Physiology Lung Cellular andMolecular Physiology vol 15302pp L1057ndashL1066 2012

[81] H Jung P T Toth F A White and R J Miller ldquoMonocytechemoattractant protein-1 functions as a neuromodulator indorsal root ganglia neuronsrdquo Journal of Neurochemistry vol104 no 1 pp 254ndash263 2008

[82] F A White H Jung and R J Miller ldquoChemokines andthe pathophysiology of neuropathic painrdquo Proceedings of the

12 ISRN Pharmacology

National Academy of Sciences of the United States of Americavol 104 no 51 pp 20151ndash20158 2007

[83] G Banisadr R-D Gosselin P Mechighel W Rostene PKitabgi and S M Parsadaniantz ldquoConstitutive neuronalexpression of CCR2 chemokine receptor and its colocalizationwith neurotransmitters in normal rat brain functional effectof MCP-1CCL2 on calcium mobilization in primary culturedneuronsrdquo Journal of Comparative Neurology vol 492 no 2 pp178ndash192 2005

[84] S Bhangoo D Ren R J Miller et al ldquoDelayed functionalexpression of neuronal chemokine receptors following focalnerve demyelination in the rat a mechanism for the devel-opment of chronic sensitization of peripheral nociceptorsrdquoMolecular Pain vol 3 article 38 2007

[85] T Maeda N Kiguchi Y Kobayashi T Ikuta M Ozaki and SKishioka ldquoLeptin derived from adipocytes in injured periph-eral nerves facilitates development of neuropathic pain viamacrophage stimulationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 106 no 31 pp13076ndash13081 2009

[86] H Kajiwara M Yasuda N Kumaki T Shibayama and YOsamura ldquoExpression of carbohydrate antigens (SSEA-1 Sialyl-Lewis X DU-PAN-2 and CA19-9) and E-selectin in urothelialcarcinoma of the renal pelvis ureter and urinary bladderrdquoTokaiJournal of Experimental and Clinical Medicine vol 30 no 3 pp177ndash182 2005

[87] A M S Assreuy G J Martins M E F Moreira et al ldquoPre-vention of cyclophosphamide-induced hemorrhagic cystitis byglucose-mannose binding plant lectinsrdquo Journal of Urology vol161 no 6 pp 1988ndash1993 1999

[88] S M Vinson A Rickard J S Ryerse and J McHowat ldquoNeu-trophil adherence to bladder microvascular endothelial cellsfollowing platelet-activating factor acetylhydrolase inhibitionrdquoJournal of Pharmacology and Experimental Therapeutics vol314 no 3 pp 1241ndash1247 2005

[89] M Green A Filippou G Sant and T CTheoharides ldquoExpres-sion of intercellular adhesion molecules in the bladder ofpatients with interstitial cystitisrdquoUrology vol 63 no 4 pp 688ndash693 2004

[90] M Leppilahti P Hellstrom and T L J Tammela ldquoEffect ofdiagnostic hydrodistension and four intravesical hyaluronicacid instillations on bladder ICAM-1 intensity and associationof ICAM-1 intensity with clinical response in patients withinterstitial cystitisrdquo Urology vol 60 no 1 pp 46ndash51 2002

[91] A T Corcoran N Yoshimura V Tyagi B Jacobs W Lengand P Tyagi ldquoMapping the cytokine profile of painful bladdersyndromeinterstitial cystitis in human bladder and urinespecimensrdquoWorld Journal of Urology vol 31 no 1 pp 241ndash2462013

[92] SGarrean X-PGaoV Brovkovych et al ldquoCaveolin-1 regulatesNF-120581B activation and lung inflammatory response to sepsisinduced by lipopolysacchariderdquo Journal of Immunology vol 177no 7 pp 4853ndash4860 2006

[93] M R Kang G Yang R F Place et al ldquoIntravesical deliveryof small activating RNA formulated into lipid nanoparticlesinhibits orthotopic bladder tumor growthrdquo Cancer Researchvol 172 pp 5069ndash5079 2012

[94] P Tyagi D Barclay R Zamora et al ldquoUrine cytokines suggestan inflammatory response in the overactive bladder a pilotstudyrdquo International Urology and Nephrology vol 42 no 3 pp629ndash635 2010

[95] P Tyagi B L Jacobs D Barcaly et al ldquoUrine levels ofinflammatory chemokines can be novel biomarkers for theoveractive bladderrdquoThe Journal of Urology vol 181 article 5882009

[96] P Tyagi V Tyagi D Bui et al ldquoDiscrimination Of OAB fromICPBS by multivariate data modeling of urinary proteinsrdquoTheJournal of Urology vol 183 pp e614ndashe6e5 2010

[97] P Tyagi D Nikolavsky Y Vodovotz et al ldquoUrine levels ofselected chemokines positively correlate with lower bladdercapacity and psychometric scores in IcPbs patientsrdquo TheJournal of Urology vol 181 article 21 2009

[98] R Weissleder and V Ntziachristos ldquoShedding light onto livemolecular targetsrdquo Nature Medicine vol 9 no 1 pp 123ndash1282003

[99] C Bremer V Ntziachristos and R Weissleder ldquoOptical-basedmolecular imaging contrast agents and potentialmedical appli-cationsrdquo European Radiology vol 13 no 2 pp 231ndash243 2003

[100] UMahmood andRWeissleder ldquoNear-infrared optical imagingof proteases in cancerrdquoMolecular CancerTherapeutics vol 2 pp489ndash496 2003

[101] J Kaufman H Hensley J Jacobs et al ldquoNon-invasive imagingof near infrafred dye labeled liposomes facilitates evaluation ofbioresidence timerdquoThe Journal of Urology vol 183 article e6282010

[102] Y-X J Wang ldquoMedical imaging in pharmaceutical clinicaltrials what radiologists should knowrdquo Clinical Radiology vol60 no 10 pp 1051ndash1057 2005

ISRN Pharmacology 3

(a) (b)

Figure 1 Protective effect of liposomes evaluated by histology in rat bladder injured by protamine sulfate Focal loss of apical layer andulcerated areas were noted in saline treated rats (a) Liposome post-treatment protected rats from ulceration induced by PS as evident fromphotograph in (b) taken on bladder harvest at 8 h after liposome instillation

FDAapproved therapies of dimethyl sulphoxide (DMSO) andintravesical instillation of PPS [1]

Clinically DMSO (RIMSO-50) is the only FDA approvedintravesical treatment for PBSIC [41] but off-label instil-lation of PPS has also been pursued [42] The efficacy ofvarious treatments was evaluated in chemically inducedbladder hyperactivity in rats by sequential infusion of pro-tamine sulfate and potassium chloride Bladder reflex activityof female Sprague-Dawley rats before and after treatmentwas evaluated by continuous cystometry under urethaneanaesthesia (10 gkg) Intravesical liposomes were effectivein doubling the intercontractile interval (ICI) compared withPPS while acute instillation of DMSO failed to produce anyprotective effect in this animal model [43]

Recently Lee et al [44] further improved this modelof DO induced by intravesical infusion by combining itwith systemic metabolic alteration through fructose feedingMetabolic syndrome created by feeding of fructose to ratscan lead to DO and urination frequency [44] Cystometricbladder capacity of fructose fed rats can be further decreasedby instillation of acidic ATP solution which provokesreflex micturition via afferent noise Evidence suggests thatincreased expression or release of neurotransmitters in themucosal layer of the bladder can generate afferent noise viaC-fiber pathway and result in DO [45] Compared to infusionwith normal saline ATP solution decreased bladder capacityand increased phasic contractions Addition of liposomesto the ATP solution partially reversed the ATP solution-induced response [46] Capsaicin induced desensitizationalso blunted the provocation of ATP in this model todemonstrate the role of afferent noise via C-fibers

52 Clinical Studies Encouraged by the exciting preclin-ical efficacy of empty liposomes as a therapeutic agentfor intravesical therapy of ICPBS Chuang et al recentlypublished the clinical safety and efficacy of liposomes inICPBS patients [47] In an open label prospective study on24 ICPBS patients the effect of intravesical liposomes wascompared against oral PPS Patients were equally dividedinto the two treatment arms administered either intravesical

liposomes (80mg40 cc distilled water) once weekly or oralPPS (100mg) 3 times daily for 4 weeks each Ten possibleresponses to treatment were monitored at 3 time pointsincluding baseline and at weeks 4 and 8

Comparable efficacy of liposomes to oral PPS wasdemonstrated by statistically significant decreases in urinaryfrequency and nocturia in both treatment arms Liposometreated patients showed statistically significant decreases inpain urgency and the OrsquoLeary-Sant symptom score withthe effect being most profound on urgency None of thetreated patients in the study reported urinary incontinenceretention or infection due to liposome instillation and therewere no unanticipated adverse events and no significantworsening of symptoms during followup Intravesical instilla-tion of liposomes in ICPBS patients was found to be safe withpotential improvement after 1 course of therapy for up to 8weeksThe study designwas suboptimal with lack of blindingand two treatment arms assigned to different routes of drugadministration and regimen Still once weekly liposometherapywas able to demonstrate efficacy comparable to thricedaily oral administration of PPS

Subsequently Lee et al assessed the safety and efficacy oftwice weekly administration of liposomes on ICPBS symp-toms [48] Five patients were given twice a week treatment ofliposomes for 4 weeks The primary outcome was the changein the OrsquoLeary-Sant symptomproblem score and OrsquoLeary-Sant total score from baseline to week 4 and week 8 TheOrsquoLeary-Sant symptomproblem score OrsquoLeary-Sant totalscore and pain score at the 4-week followup showed sig-nificantly greater improvement from baseline with biweeklyinstillation than once a week instillation Tolerability ofliposomes remains unchanged from once a week regimento twice a week regimen The followup at 8 weeks was alsosimilar for both treatment regimensThe incidence of urinaryincontinence retention or unanticipated adverse changeswas not noted with any regimen Intravesical liposomesappear to be a promising new treatment for ICPBS and futurelarge-scale placebo controlled studies are needed to verifythese results from a pilot study

4 ISRN Pharmacology

(a) (b)

Liposomes

Afferent nervesAfferent nerves

Leaky urothelium

(c)

Figure 2 Ex vivo images of rat bladder taken in visible and near-infrared light indicate the coating formed by instilled liposomeson bladder surface Liposomes carry a trace amount of near-infrared (NIR) lipophilic carbocyanine dye 111015840-dioctadecyl-333101584031015840-tetramethylindotricarbocyanine iodide (DiR) that fluoresces on exposure to NIR light Rat bladder filled with urine was tied at the basewith thread prior to harvest Liposomes coating the bladder surface are invisible in visible light photograph (a) but is indicated by bluecolored coating on the bladder luminal surface in NIR light (b) NIR imaging in vivo can allow noninvasive repeat objective measurement forbladder residence time of instilled treatments (c) Schematic illustration of liposome coating the bladder surface Given the chemical affinityof phospholipids of instilled liposomes with lipids in cells lining the bladder surface the liposomes form a protective film coating on theinjured bladder lumen surface and assist in the repair of leaky and inflamed uroepithelium

The exact mechanism of action for liposomes in ICPBSremains to be established but protective coating effect basedon preclinical studies cannot be ruled out as illustrated inFigure 2

6 Liposomes as a Delivery Platform

Not only is the lining of the urinary bladder the most im-permeable tissue in the human body but it is also the mostcompliant As a bladder lining expands additionalmembranematerial is added to its cells to help retain impermeability[49] Therefore vesicular trafficking may provide a favorableenvironment for drug delivery and therefore it is worthinvestigating whether vesicle nature of liposomes can aid in

improving the delivery of cargo across the bladder permeabil-ity barrier

In the field of neurourology instillation of neurotoxinsinto bladder is an accepted approach to achieve chemicalneuromodulation of afferent neurotransmission underlyingneurogenic bladder and ICPBS [50] Existing approachesof chemical neuromodulation by intravesical neurotoxinsare suboptimal due to vehicle toxicity for capsaicin [51] ordegradation of botulinum toxin (BoNT) in urine Possiblereasons underlying the lack of efficacy fromBoNT instillationin bladder can be protein degradation by proteases andproteinases in urine dilution in urine or poor uptake of theBoNT solution into the urothelium

Liposomes have been previously studied as a carrier oftoxins to enhance efficacy at lower doses [52] In the context of

ISRN Pharmacology 5

Lipotoxin

Intravesical instillation

Liposomes

+

BoNT-A

Figure 3 Scheme for formulation of BoNT into liposomes for blad-der instillation Freeze dried BoNT is resuspended in distilled waterand added to vials containing a desiccated liposomal preparationmade of phospholipids This reconstituted suspension is allowed tohydrate at room temperature for 30ndash60min before instillation

toxins instilled in the bladder fat-soluble neurotoxin such ascapsaicin can be integrated into the phospholipid bilayer [38]and water-soluble neurotoxin such as BoNT can be protectedinside the aqueous compartment(s) of liposomes delimitedby the phospholipid bilayer(s) [53] (Figure 3) Cystoscopeguided injections are the current standard practice in theclinic for administering BoNT to the bladder But in recentyears studies have also assessed the potential for intravesicalinstillation of BoNT alone in animal models of bladderirritation [54]

Development of instillation as a mode for administeringBoNT in patients will drastically bring down the cost oftreatment for patients with refractory overactive bladderOther groups have recently reported the use of DMSO forinstillation of BoNT instead of injection [55] DMSO doesnot afford the natural state of the BoNT as a protein and needto be formulated immediately before instillation DissolvingBoNT in DMSO may not be advisable owing to concernsof BoNT uptake into the systemic circulation of patientMoreover biochemical studies have demonstrated that met-alloproteolytic activity of the BoNT is strongly enhanced bythe presence of lipid membranes [56]

Recent studies reaffirmed the potential of liposomesas a promising vehicle for delivery of neurotoxins to thebladder [38 53] The transport of BoNT into urotheliumfrom liposomes was confirmed by detection of its uniqueeffect on neurotransmitters and proteolysis of synaptosomal-associated protein SNAP-25 through immunohistochemistry[53] The protection of BoNT entrapped inside liposomesfrom degradation in urine without compromising efficacywas demonstrated by attenuation of acetic acid induced blad-der irritation in rats [53] Similar results were obtained in pre-clinical studies with liposomes encapsulating capsaicin [38]

Liposomes have proven themselves as biocompatible deliveryagents in the bladder

7 Intravesical Antisense Therapeutics

The term ldquoantisenserdquo therapeutics emerged from seminalstudies done 4 decades using a short synthetic oligonu-cleotide for sequence-specific gene silencing [57] Genesilencing involves introduction of short strands of DNA(termed as antisense) with sequences complementary to themRNA encoding a particular gene inside the cell with theintent to block gene expression through either translationalinhibition or enzymatic cleavage of the mRNA target [3]

Oligonucleotide ODN binds specifically and stronglyto the mRNA target through Watson-Crick base pairingODN can be basically categorized into those that directcleavage of the target mRNA as caused by small interferingRNAs (siRNAs) and those that alter mRNA translationwithout causing mRNA cleavage Recent discovery of smallinterfering RNAs (siRNAs) and the elucidation of the RNAinterference (RNAi) pathway has also brought a sea changein the control of posttranscriptional gene expression siRNAtakes advantage of endogenous cellular pathways to potentlysilence the expression of specific genes (Figure 4)

Antisense mechanism is a promising approach for devel-oping therapeutics based on rational gene-based drug designAntisense therapeutics have been under clinical investigationfor more than 30 years [58] However drug development ofthis approach has been hampered by inefficient intracellulardelivery and cellular uptake of the ODN The translation ofbasic antisense research into therapeutics is also impeded byintracellular stability of ODN and potential for ldquooff-targetrdquogene silencing immunostimulation and other side effects

A vast array of chemical modifications to ODN has beendeveloped to overcome the therapeutically limiting featuresby altering internucleotide phosphate linkages backbonesugars or nucleobases One such modified ODN is peptidenucleic acid which replaces phosphodiester bond betweennucleobases with a peptide bond Replacement of phosphodi-ester bond by a phosphorothioate linkage is another methodto improve the stability of ODN against nucleases

Great progress has been made in translating antisenseresearch into clinical therapies based on local injection intoeye [58]The field recently progressed further with a systemicinjection therapy for treating familial hypercholesterolemia[59] Antisense therapeutics have been used for exon skippingto optimize the functionality of a truncated dystrophinprotein in dog model of Duchenne muscular dystrophy [60]

Nevertheless applied research for bladder diseases haslagged behind considering that the anatomical architec-ture of bladder provides ease of local administration withrestricted systemic side effects due to lower serum uptakeof antisense ODN Bladder instillation of antisense ODNor their chemically modified mimics can therefore be anefficient means to control the expression of therapeuticallyrelevant genes Antisense agents can also be used to elucidatethe role of newly discovered genes in bladder function

6 ISRN Pharmacology

Liposomes with antisense

Transcription

Genomic DNA RNA Duplex

Antisense DNA

Activated PI 3-kinase

Activated receptortyrosine kinase

NGF

Anti-NGFantibodies

mRNAdegradation

PIP2 PIP3

PP P P P P

oligonucleotides

Figure 4 Illustration of mechanisms for blocking NGF expression by either NGF antibodies or downregulation of NGF mRNA expressionNGF antibody sequesters the freely available NGF and prevents it from binding with the cognate TrkA receptors NGF antisense selectivelybinds with NGF mRNA inside the cells to block the translation of corresponding mRNA siRNA takes advantage of endogenous cellularpathways to degrade NGF mRNA and control the posttranscriptional NGF gene expression

8 NGF Expression in Bladder

Several studies have reported that patients with OAB orICPBS excrete more nerve growth factor (NGF) in theirurine relative to asymptomatic controls [61] Higher expres-sion of NGF in bladder of patients [62] with correspondinglower serum levels of NGFmakes the bladder tissue the likelysource for the elevated NGF in urine [63] Previous studieshave indicated that increased levels of NGF in the bladderand bladder afferent pathways are directly involved in theemergence of hyperexcitability of C-fiber bladder sensorypathways leading to the pathology of DO andOAB (Figure 4)[64]

In addition intrathecal application of NGF antibodiesreducedNGF levels in bladder afferent pathways and normal-ized bladderurethral function in spinal cord injured (SCI)rats [65] Because it is likely that the major pathology of OABis driven by NGF targeting the intracellular synthesis of NGFmolecule in bladder is a promising therapeutic alternative

9 NGF Expression a Drug Target

Overexpression of NGF can be blocked either directly byantibodies [66] or by blocking the synthesis of NGF proteinfrom mRNA [67] Systemic administration of monoclonalhuman NGF antibodies (tanezumab) has been explored fortherapeutic outcomes in ICPBS patients but not withoutencountering safety concerns such as paresthesia hypoesthe-sia and arthralgia [66] Generalized blockade ofNGF activityat sites other than bladder by anti-NGF antibodies maynot be the preferred outcome because NGF is an essentialhousekeeping growth factor necessary for the survival andgrowth of neurons [68] The physiological necessity of NGF

action at those sites may explain the incidence of paresthesiahypoesthesia and arthralgia in patients treated with systemicanti-NGF antibodies [66]

91 Peptide Nucleic Acid (PNA) Therefore to reduce thetoxicity of systemic blockade of NGF we sought to developa novel intravesical therapy of OAB by targeting the intra-cellular synthesis of NGF in the urothelium Antisense ODNneeded to be able to cross cell membrane to act as a drugand negatively charged ODN will not pass through a lipidlayer such as cell membranes Bladder uptake of ODN islimited by the anionic charge and size of the ODN as wellas anionic glycosaminoglycan layer of the inner bladdersurface Therefore primary impediment to be overcome inthe development of intravesical antisense therapeutics isinefficient bladder uptake of the ODN across urotheliumPrevious studies showed that heparan sulfate proteoglycansexpressed on cell surface act as receptors for extracellularTAT uptake [69] Therefore it was reasoned that GAG layeron bladder surface can facilitate bladder uptake of peptidenucleic acid PNA if it could be conjugatedwith synthetic TATpeptide

Water insoluble peptide nucleic acid targeting NGF wasconjugated with cell penetrating cationic peptide TAT forintracellular delivery of antisense moiety to demonstrate effi-cacy in animal models Studies showed that PNA conjugatedwith TAT suppressed cyclophosphamide cystitis followinglocal instillation of antisense against NGF [67] There wasnegligible uptake of peptide nucleic acid in absence of TATconjugation Prior to determination of in vivo efficacy ofconjugate suitable target sequence on NGF mRNA wasdetermined by predicted folding structure and cell transfec-tion experiments Successful intravesical delivery of peptide

ISRN Pharmacology 7

nucleic acid in bladder at the same time of cyclophosphamideinjection protected against the cystitis by blocking the rise inbladder contraction frequency and inflammation

92 Phosphorothioate-Linked Analogues Considering thedifficulty and possible nonspecific toxicity of peptide nucleicacid and TAT recent developments focused on simplifyingthe approach using water-soluble phosphorothioated ODNwhich have an increased resistance to exo- and endonucleasesfor improved stability [70] As alluded to in the above textsuccess of antisense therapeutics is largely dependent on thedevelopment of a delivery vehicle that can efficiently deliverantisense ODN in bladder Preliminary studies showed thatliposomes can be far better biocompatible effective carriersfor local gene silencing of NGF gene in bladder cells

Cationic liposomes and mimics have emerged as themost popular nonviral method to deliver nucleic acids intherapeutic applications Easy and reversible complex forma-tion of cationic liposomes with ODN at room temperatureallows their use as carriers Electrostatic attraction betweenthe cationic lipid DOTAP and the polyanionic antisenseODN is responsible for the complex formation The efficacyof liposome delivered siRNA by intravesical route has beenpreviously demonstrated in preclinical models of bladdercancer [71] Residence of ODN in the rat bladder after intrav-esical instillation was demonstrated to be longer than 24 husing fluorescent tagged ODN complexed with liposomes[72] Fluorescent probe was localized in bladder urotheliumcells 24 h following instillation The 24 h residence time wasalso demonstrated for siRNA in mouse bladder [73] Bladderuptake of fluorescent ODN without liposomes in normalrat bladder is poor Previous studies have shown that veryhigh concentration of phosphorothioated ODN can deliverODN without liposomes to bladder cells of mice havingbladder cancer [74] The urothelium barrier may be slightlycompromised in cancerous condition and the strategy ofloading bladder with high dose of ODN may not workin noncancerous diseased condition of bladder with intactbarrier

In order to evaluate the efficacy of NGF antisense ODNacetic acid infusion was used to cause a rapid rise of NGFprotein levels A single dose instillation of OND complexedwith liposomes protected against bladder overactivity (BO)induced by acetic acid Together with data of bladder uptakestudies using fluorescent ODN it is demonstrated that ONDis readily available to the bladder after intravesical instillationIt has been previously reported that within 2 h of exposureto irritants such as turpentine and acetic acid there occursa rapid rise in bladder content of NGF [75] Later studiesfound that bladder responds to insults with upregulation inthe genes for NGF sE-Selectin and receptor for monocytechemoattractant protein-1 (MCP-1) within 30min of expo-sure to lipopolysaccharide (Figure 5) [76] The acetic acidinduced overexpression of NGF was blunted by pretreatmentwith NGF antisense OND with phosphorothioate linkageprior to exposure of acetic acid The downregulation of NGFmRNA expression was in agreement with reduced proteinlevels and suppressed NGF-like immunoreactivity in theurothelium

93 Antisense and Downstream Signalling of NGF Drugdevelopment of intravesical antisense for NGF was also ableto unmask the downstream signaling [77] involving NGFfollowing exposure to acetic acid Experiments supportedthe role of NGF as a paracrine messenger [77] which isknown to activate several downstream effectors to manifestphysiological and pathological signaling changes linked to it[76 78ndash80] The bladder injury set off by acetic acid initiatesthe signalling cascades that upregulate the expression ofNGF and other chemokines MCP-1 CXCL-1 and CXCL-10 and prostaglandins [76 78ndash80] As reported elsewherelocalization of chemokines within synaptic vesicles in neu-rons [81] is consistent with their ability to act as excitatoryneurotransmitters following AA exposure

Chemokines are one of several downstream effectorsactivated by NGF [76 78ndash80] and interestingly chemokinereceptors are widely expressed in neural and nonneuralelements of the nociceptive pathways that are responsible forvisceral and somatic pain sensation [82] MCP-1 [83] andCXCL-10 [84] are constitutively expressed in neurons wherethey participate in excitability of primary afferent neuronsthrough transactivation of transient receptor channels andnociceptor sensitization [84] Overexpression ofNGF is likelyto drive the expression of VEGF fromneurons and leptin [78]from adipocytes covering neurons [85]

Cooperative expression of NGF and MCP-1 is able toinduce hyperexcitability in neurons by activating TRPV1receptor In addition MCP-1 CXCL-1 and CXCL-10 causechemoattraction of monocytes neutrophils and lympho-cytes respectively to mediate the bladder injury set offby acetic acid Extravasation is an essential prerequisitefor infiltration of monocytes neutrophils and lymphocyteswhich requires the expression of adhesion molecules likeE-selectin and intracellular adhesion molecule ICAM-1 [7686] E-selectin is a cytokine-inducible adhesion moleculethat supports the rolling and stable arrest of leukocyteson activated vascular endothelium Expression of E-selectinhas been linked to adherence of neutrophils to bladdermicrovascular endothelial cells and to cyclophosphamidecystitis [87 88] E-selectin gene expression is activated bythe NF-120581B and MAP kinase signal transduction pathways[76 86]

Activation and recruitment of leukocytes to acetic acidinduced bladder injury also require expression of intra-cellular adhesion molecule ICAM-1 [89] ICAM-1 interactsspecifically with its receptors of the integrin family to inducereversible cell-cell interactions involving adhesion Expres-sion of ICAM-1 was found to be increased in patients withICPBS and reduced in patients responding to instillation ofhyaluronic acid [90] Expression of ICAM-1 and VCAM-1was also noted in a recent study on biopsy tissue of ICPBSpatients [91] Increased expression of ICAM-1 subsequentto exposure with acetic acid in animal model is thereforeclinically relevant

Antisense experiments supported the earlier report thatinhibition of NGF expression significantly downregulates theexpression of ICAM-1 [80] It is reported that binding ofNGF to its high-affinity TrkA receptor controls the sICAM-1expression on target cells [80] Expression of ICAM-1 was

8 ISRN Pharmacology

Neutrophil

Monocytes

Acetic acid

Integrin

NGF

MCP-1

Release of cytokinesVanilloid

receptor

Trk receptors NGF

Membrane damage

VEGF

CXCL1

Activated leucocyte

Startrolling

Extravasation

CXCL10

sICAMssE-selectin

ActivationAdhesion

MCP-1

NGF

PKC

DAG

PLC

IP3Kinases

Ras

AKT

PI3K

CXCL10

sICAMs

LymphocyteAfferents

Figure 5 Signalling cascades induced by NGF overexpression following exposure to acetic acid Injury from exposure to acetic sets off thesignalling cascades that upregulate the expression of NGF MCP-1 prostaglandins CXCL-1 and CXCL-10 Overexpression of NGF drives theexpression of VEGF from neurons and leptin from adipocytes covering neurons NGF and MCP-1 also induce hyperexcitability in neuronsby activating TRPV1 receptor MCP-1 CXCL-1 and CXCL-10 cause chemoattraction of monocytes neutrophils and lymphocytes whichrequires extravasation across endothelium from blood through the cooperation of adhesion molecules E-selectin and ICAM-1

negatively associated with NGF expression in antisenseexperiments Ultimately expression of ICAM-1 is regulated atthe level of transcription by one of several signaling cascadesNF-120581B JAKSTAT IFN-g (CXCL-10) AP MAP kinase andPKC [92]

Intravesical route can allow selective exposure of anti-sense ODN to the NGF producing cells in urothelium andavoid systemic side effects from genetic manipulation ofNGF expression Intravesical route can also prove to be costeffective given the cost of ODN or siRNA Contrary to thegoal of loss of function in bladder with antisense ODN inrecent studies liposomes have also been used to deliver ODNfor gain of function in bladder [93]

10 Imaging of Intravesical Therapy

Drug development for DO and ICPBS relies heavily onsubjective outcomes for predicting efficacy and safety in earlyclinical development Although there are many validatedmeasurement tools used in DO and ICPBS research theyare usually burdensome and do not capture all symptomsrelated to the lower urinary tract Moreover improvement insymptoms scores following an intervention does not alwayscorrelate with patient expectations satisfaction and goalachievement which are critically important for successfulmanagement of DO and ICPBS

There is underdevelopment and underutilization of urinebiomarkers and imaging methods for investigational drugsgiven by intravesical route Overreliance on subjectiveimpressions of patients for therapeutic response limits clini-cal relevance due to weak correlation with patient satisfactionand can also impede continued scientific progress in assessing

study outcomes relative to symptom bother Symptom scoresmay also be related to the cause of high heterogeneity inclinical response in DO and ICPBS patients

Imaging can offer the possibility to provide region-specific information in situations where serum and urineassays may not reflect the true state of bladder [94ndash96]Urine chemokines have been reported to be associated withsymptom severity of ICPBS patients [97] Application offluorescent microscopy for imaging of pelvic floor has beenlimited due to the issues pertaining to light absorption bytissues scattering and autofluorescence [98]

Recent studies have shown that dyes fluorescing inthe near-infrared (NIR) band can overcome this handi-cap in deep-tissue imaging of experimental animals [99]Selected NIR fluorochromes emit light with tissue pen-etration approaching 10ndash15 cm [100] The light emissionfrom probes in this spectrum encounters low backgroundautofluorescence and minimal attenuation of signal due tolight absorption by tissue components [98] Therefore NIRimaging yields high signal-to-noise ratios and is well suitedfor studying the distribution of instilled treatments in bladder(Figure 2(b))

Preliminary studies suggest that imaging of mouse pelvicfloor in NIR spectrum can be a viable option [101] NIRimaging easily allows visualization and quantification ofbladder distribution by tracing themigration of fluorescence-labeled liposomes in anaesthetized mouse Compared withother biological assays imaging could provide objectiveendpoint for patients with ulcerative cystitis to indicate itshigh translation potential Other benefits of optical imaginginclude its relatively low cost compared withmore traditionalimaging systems such as MRI and PET The instrument

ISRN Pharmacology 9

consists of two near-infrared lasers emitting at 685 nm and785 nm camera and novel optics

Furthermore preferential binding of liposomes andnanoparticles to bladder lesions of ICPBS after instillationcan be assessed by NIR imaging of bladder where it cantheoretically provide anatomically specific information aboutthe real time status of ulcerlesion in bladder surface or tissueabnormality [102] Therefore future studies can substitute aresponse variable that is continuous in nature instead of asubjective clinical outcome

11 Conclusions

Adoption of forward-thinking approaches can achieveadvancements in drug delivery systems targeted to improvepharmacotherapy of bladder diseases in the future Latestdevelopments in the field of nanotechnology can bring thismode of therapy from second line of treatment for refractorycases to the forefront of disease management Liposomesare an attractive drug delivery platform by virtue of theirbiodegradability biocompatibility low toxicity and simpleand mild preparation methods

Conflict of Interests

All authors except Dr Michael Chancellor declare that thereis no conflict of interests regarding the publication of thispaper Dr Michael Chancellor owns stocks in the start-upliposome company Lipella

Acknowledgments

This work was supported by the National Institutes of Health(DK057267 and DK088836) and the Department of Defense(W81XWH-11-1-0763 and W81XWH-12-1-0565)

References

[1] J Parkin C Shea and G R Sant ldquoIntravesical dimethyl sul-foxide (DMSO) for interstitial cystitismdasha practical approachrdquoUrology vol 49 no 5 pp 105ndash107 1997

[2] C Dowson J Watkins M S Khan P Dasgupta and A SahaildquoRepeated botulinum toxin type A injections for refractoryoveractive bladder medium-term outcomes safety profile anddiscontinuation ratesrdquo European Urology vol 61 no 4 pp 834ndash839 2012

[3] J Kaufman V Tyagi M Anthony M B Chancellor and PTyagi ldquoState of the art in intravesical therapy for lower urinarytract symptomsrdquo Reviews in Urology vol 12 pp e181ndashe189 2010

[4] N Yoshimura and M B Chancellor ldquoCurrent and futurepharmacological treatment for overactive bladderrdquo Journal ofUrology vol 168 no 5 pp 1897ndash1913 2002

[5] K A Killinger J A Boura and K M Peters ldquoPain ininterstitial cystitisbladder pain syndrome do characteristicsdiffer in ulcerative and non-ulcerative subtypesrdquo InternationalUrogynecology Journal vol 24 no 8 pp 1295ndash1301 2013

[6] J C Nickel P Hanno K Kumar and HThomas ldquoSecond mul-ticenter randomized double-blind parallel-group evaluationof effectiveness and safety of intravesical sodium chondroitin

sulfate compared with inactive vehicle control in subjects withinterstitial cystitisbladder pain syndromerdquoUrology vol 79 no6 pp 1220ndash1225 2012

[7] P M Hanno ldquoAnalysis of long-term elmiron therapy forinterstitial cystitisrdquo Urology vol 49 no 5 pp 93ndash99 1997

[8] A Giannantoni V Bini R Dmochowski et al ldquoReply to ClausRiedlrsquos letter to the editor re antonella Giannantoni VittorioBini Roger Dmochowski et al Contemporary management ofthe painful bladder a systematic reviewrdquo EuropeanUrology vol61 no 5 pp 29ndash53 2012

[9] MC Lai Y CKuo andHCKuo ldquoIntravesical hyaluronic acidfor interstitial cystitispainful bladder syndrome a comparativerandomized assessment of different regimensrdquo InternationalJournal of Urology vol 20 pp 203ndash207 2013

[10] J Eldrup J Thorup and S L Nielsen ldquoPermeability andultrastructure of human bladder epitheliumrdquo British Journal ofUrology vol 55 no 5 pp 488ndash492 1983

[11] C L Parsons D Boychuk S Jones R Hurst and H CallahanldquoBladder surface glycosaminoglycans an epithelial permeabil-ity barrierrdquo Journal of Urology vol 143 no 1 pp 139ndash142 1990

[12] Y Kim N Yoshimura H Masuda F De Miguel and MB Chancellor ldquoAntimuscarinic agents exhibit local inhibitoryeffects on muscarinic receptors in bladder-afferent pathwaysrdquoUrology vol 65 no 2 pp 238ndash242 2005

[13] V Tyagi B J Philips R Su et al ldquoDifferential expression offunctional cannabinoid receptors in human bladder detrusorand urotheliumrdquo Journal of Urology vol 181 no 4 pp 1932ndash1938 2009

[14] B J Philips M C Smaldone V L Erickson N YoshimuraM B Chancellor and P Tyagi ldquoDifferential expression ofneurokinin receptor subtypes in urothelium and detrusor ofhuman bladderrdquo Journal of Urology vol 179 article 354 2008

[15] S A Baker G W Hennig J Han F C Britton T K Smithand S D Koh ldquoMethionine and its derivatives increase blad-der excitability by inhibiting stretch-dependent K+ channelsrdquoBritish Journal of Pharmacology vol 153 no 6 pp 1259ndash12712008

[16] J M Beckel A Kanai S-J Lee W C De Groat and LA Birder ldquoExpression of functional nicotinic acetylcholinereceptors in rat urinary bladder epithelial cellsrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 290 no 1 ppF103ndashF110 2006

[17] LA Birder A J KanaiWCDeGroat et al ldquoVanilloid receptorexpression suggests a sensory role for urinary bladder epithelialcellsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 23 pp 13396ndash13401 2001

[18] P Tyagi C A Thomas N Yoshimura and M B ChancellorldquoInvestigations into the presence of functional szlig1 szlig2 and szlig3-adrenoceptors in urothelium and detrusor of human bladderrdquoInternational Brazilian Journal of Urology vol 35 no 1 pp 76ndash83 2009

[19] A T Hanna-Mitchell J M Beckel S Barbadora A J Kanai WC de Groat and L A Birder ldquoNon-neuronal acetylcholine andurinary bladder urotheliumrdquo Life Sciences vol 80 no 24-25 pp2298ndash2302 2007

[20] C P Smith D A Gangitano A Munoz et al ldquoBotulinumtoxin type A normalizes alterations in urothelial ATP and NOrelease induced by chronic spinal cord injuryrdquo NeurochemistryInternational vol 52 no 6 pp 1068ndash1075 2008

[21] Y-C Chuang C A Thomas S Tyagi N Yoshimura PTyagi and M B Chancellor ldquoHuman urine with solifenacin

10 ISRN Pharmacology

intake but not tolterodine or darifenacin intake blocks detrusoroveractivityrdquo International Urogynecology Journal and PelvicFloor Dysfunction vol 19 no 10 pp 1353ndash1357 2008

[22] Y Kim N Yoshimura H Masuda F D Miguel and M BChancellor ldquoIntravesical instillation of human urine after oraladministration of trospium tolterodine and oxybutynin in a ratmodel of detrusor overactivityrdquo BJU International vol 97 no 2pp 400ndash403 2006

[23] S Tyagi P Tyagi S Van-le N YoshimuraM B Chancellor andF de Miguel ldquoQualitative and quantitative expression profileof muscarinic receptors in human urothelium and detrusorrdquoJournal of Urology vol 176 no 4 pp 1673ndash1678 2006

[24] G Gregoriadis and B E Ryman ldquoLiposomes as carriers ofenzymes or drugs a new approach to the treatment of storagediseasesrdquo Biochemical Journal vol 124 no 5 p 58 1971

[25] G Gregoriadis S Jain I Papaioannou and P Laing ldquoImprov-ing the therapeutic efficacy of peptides and proteins a role forpolysialic acidsrdquo International Journal of Pharmaceutics vol300 no 1-2 pp 125ndash130 2005

[26] P Sapra P Tyagi and T M Allen ldquoLigand-targeted liposomesfor cancer treatmentrdquo Current Drug Delivery vol 2 no 4 pp369ndash381 2005

[27] G Gregoriadis G Dapergolas and E D Neerunjun ldquoPene-tration of target areas in the rat by liposome associated agentsadministered parenterally and intragastricallyrdquo BiochemicalSociety Transactions vol 4 no 2 pp 256ndash259 1976

[28] G Gregoriadis ldquoEngineering liposomes for drug deliveryprogress and problemsrdquo Trends in Biotechnology vol 13 no 12pp 527ndash537 1995

[29] G Gregoriadis and A C Allison ldquoEntrapment of proteins inliposomes prevents allergic reactions in pre immunised micerdquoFEBS Letters vol 45 no 1 pp 71ndash74 1974

[30] S Lee S Dausch CMaierhofer andD Dausch ldquoA new therapyconcept with a liposome eye spray for the treatment of the lsquodryeyersquordquo Klinische Monatsblatter fur Augenheilkunde vol 221 no10 pp 825ndash836 2004

[31] D Dausch S Lee S Dausch J C Kim G Schwert and WMichelson ldquoComparative study of treatment of the dry eyesyndrome due to disturbances of the tear film lipid layer withlipid-containing tear substitutes efficacy of lipid-containingtear substitutesrdquo Klinische Monatsblatter fur Augenheilkundevol 223 no 12 pp 974ndash983 2006

[32] S Ebrahim G A Peyman and P J Lee ldquoApplications ofliposomes in ophthalmologyrdquo Survey of Ophthalmology vol 50no 2 pp 167ndash182 2005

[33] J Nirmal P Tyagi L Dang et al ldquoEndocytosis uptake ofliposomes in urothelium cells detected by transmission electronmicroscopyrdquoThe Journal of Urology vol 187 article e15 2012

[34] D N Frangos J J Killion D Fan R Fishbeck A C VonEschenbach and I J Fidler ldquoThe development of liposomescontaining interferon alpha for the intravesical therapy ofhuman superficial bladder cancerrdquo Journal of Urology vol 143no 6 pp 1252ndash1266 1990

[35] J W Johnson R Nayar J J Killion A C Von Eschenbachand I J Fidler ldquoBinding of liposomes to human bladdertumor epithelial cell lines implications for an intravesical drugdelivery system for the treatment of bladder cancerrdquo SelectiveCancer Therapeutics vol 5 no 4 pp 147ndash155 1989

[36] Y-C Chuang M B Chancellor S Seki et al ldquoIntravesicalprotamine sulfate and potassium chloride as a model forbladder hyperactivityrdquoUrology vol 61 no 3 pp 664ndash670 2003

[37] J Kaufman P Tyagi and M B Chancellor ldquoIntravesicalliposomal (Lp08) instillation protects bladder urothelium fromchemical irritationrdquoThe Journal of Urology vol 181 article 5392009

[38] P Tyagi M B Chancellor Z Li et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical capsaicindelivery using thermosensitive hydrogel and liposomesrdquo Jour-nal of Urology vol 171 no 1 pp 483ndash489 2004

[39] MO Fraser Y-CChuang P Tyagi et al ldquoIntravesical liposomeadministrationmdasha novel treatment for hyperactive bladder inthe ratrdquo Urology vol 61 no 3 pp 656ndash663 2003

[40] P Tyagi M Chancellor N Yoshimura and L Huang ldquoActivityof different phospholipids in attenuating hyperactivity in blad-der irritationrdquo BJU International vol 101 no 5 pp 627ndash6322008

[41] Y Sun and T C Chai ldquoEffects of dimethyl sulphoxide andheparin on stretch-activated ATP release by bladder urothelialcells from patients with interstitial cystitisrdquo BJU Internationalvol 90 no 4 pp 381ndash385 2002

[42] J J Bade ldquoA placebo-controlled study of intravesical pentosan-polysulphate for the treatment of interstitial cystitisrdquo BritishJournal of Urology vol 79 no 2 pp 168ndash171 1997

[43] P Tyagi V C Hsieh N Yoshimura J Kaufman and M BChancellor ldquoInstillation of liposomes vs dimethyl sulphoxide orpentosan polysulphate for reducing bladder hyperactivityrdquo BJUInternational vol 104 no 11 pp 1689ndash1692 2009

[44] W-C Lee C-T Chien H-J Yu and S-W Lee ldquoBladderdysfunction in rats with metabolic syndrome induced by long-term fructose feedingrdquo Journal of Urology vol 179 no 6 pp2470ndash2476 2008

[45] J I Gillespie G A Van Koeveringe S G DeWachter and J DeVente ldquoOn the origins of the sensory output from the bladderthe concept of afferent noiserdquo BJU International vol 103 no 10pp 1324ndash1333 2009

[46] W C Lee P H Chiang Y L Tain C C Wu and Y CChuang ldquoSensory dysfunction of bladder mucosa and bladderoversensitivity in a rat model of metabolic syndromerdquo PloSONE vol 7 Article ID e45578 2012

[47] Y-C Chuang W-C Lee W-C Lee and P-H Chiang ldquoIntrav-esical liposome versus oral pentosan polysulfate for interstitialcystitispainful bladder syndromerdquo Journal of Urology vol 182no 4 pp 1393ndash1400 2009

[48] W-C Lee Y-C Chuang W-C Lee and P-H Chiang ldquoSafetyand dose flexibility clinical evaluation of intravesical liposomein patients with interstitial cystitis or painful bladder syn-dromerdquo Kaohsiung Journal of Medical Sciences vol 27 no 10pp 437ndash440 2011

[49] S T Truschel E Wang W G Ruiz et al ldquoStretch-regulatedexocytosisendocytosis in bladder umbrella cellsrdquo MolecularBiology of the Cell vol 13 no 3 pp 830ndash846 2002

[50] M B Chancellor and W C De Groat ldquoIntravesical capsaicinand resiniferatoxin therapy spicing up the ways to treat theoveractive bladderrdquo Journal of Urology vol 162 no 1 pp 3ndash111999

[51] D S Byrne A Das J Sedor et al ldquoEffect of intravesicalcapsaicin and vehicle on bladder integrity in control and spinalcord injured ratsrdquo Journal of Urology vol 159 no 3 pp 1074ndash1078 1998

[52] M Mandal and K-D Lee ldquoListeriolysin O-liposome-mediatedcytosolic delivery of macromolecule antigen in vivo enhance-ment of antigen-specific cytotoxic T lymphocyte frequency

ISRN Pharmacology 11

activity and tumor protectionrdquo Biochimica et Biophysica Actavol 1563 no 1-2 pp 7ndash17 2002

[53] Y-C Chuang P Tyagi C-C Huang et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical botulinumtoxin a delivery using liposomesrdquo Journal of Urology vol 182no 2 pp 786ndash792 2009

[54] Y-C Chuang N Yoshimura C-C Huang P-H Chiang andM B Chancellor ldquoIntravesical botulinum toxin a administra-tion produces analgesia against acetic acid induced bladder painresponses in ratsrdquo Journal of Urology vol 172 no 4 pp 1529ndash1532 2004

[55] S P Petrou A S Parker J E Crook A Rogers D Metz-Kudashick and D D Thiel ldquoBotulinum A toxindimethylsulfoxide bladder instillations for women with refractory idio-pathic detrusor overactivity a phase 12 studyrdquo Mayo ClinicProceedings vol 84 no 8 pp 702ndash706 2009

[56] P Caccin O Rossetto M Rigoni E Johnson G Schiavoand C Montecucco ldquoVAMPsynaptobrevin cleavage by tetanusand botulinum neurotoxins is strongly enhanced by acidicliposomesrdquo FEBS Letters vol 542 no 1ndash3 pp 132ndash136 2003

[57] M L Stephenson and P C Zamecnik ldquoInhibition of Roussarcoma viral RNA translation by a specific oligodeoxyribonu-cleotiderdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 75 no 1 pp 285ndash288 1978

[58] H Dadgostar and N Waheed ldquoThe evolving role of vascularendothelial growth factor inhibitors in the treatment of neovas-cular age-related macular degenerationrdquo Eye vol 22 no 6 pp761ndash767 2008

[59] C Gebhard G Huard E A Kritikou and J C TardifldquoApolipoprotein B antisense inhibitionmdashupdate on mipom-ersenrdquo Current Pharmaceutical Design vol 919 no 17 pp 3132ndash3142 2013

[60] T Yokota E Hoffman and S Takeda ldquoAntisense oligo-mediated multiple exon skipping in a dog model of duchennemuscular dystrophyrdquoMethods inMolecular Biology vol 709 pp299ndash312 2011

[61] B L Jacobs M C Smaldone V Tyagi et al ldquoIncreased nervegrowth factor in neurogenic overactive bladder and interstitialcystitis patientsrdquoThe Canadian Journal of Urology vol 17 no 1pp 4989ndash4994 2010

[62] H-T Liu and H-C Kuo ldquoIntravesical botulinum toxin Ainjections plus hydrodistension can reduce nerve growth factorproduction and control bladder pain in interstitial cystitisrdquoUrology vol 70 no 3 pp 463ndash468 2007

[63] H T Liu and H C Kuo ldquoIncreased urine and serum nervegrowth factor levels in interstitial cystitis suggest chronicinflammation is involved in the pathogenesis of diseaserdquo PloSONE vol 7 Article ID e44687 2012

[64] N Yoshimura N E Bennett Y Hayashi et al ldquoBladderoveractivity and hyperexcitability of bladder afferent neuronsafter intrathecal delivery of nerve growth factor in ratsrdquo Journalof Neuroscience vol 26 no 42 pp 10847ndash10855 2006

[65] S Seki K Sasaki Y Igawa et al ldquoSuppression of detrusor-sphincter dyssynergia by immunoneutralization of nervegrowth factor in lumbosacral spinal cord in spinal cord injuredratsrdquo Journal of Urology vol 171 no 1 pp 478ndash482 2004

[66] R J Evans R M Moldwin N Cossons A Darekar I WMills andD Scholfield ldquoProof of concept trial of tanezumab forthe treatment of symptoms associated with interstitial cystitisrdquoJournal of Urology vol 185 no 5 pp 1716ndash1721 2011

[67] P Tyagi R Banerjee S Basu N YoshimuraM Chancellor andL Huang ldquoIntravesical antisense therapy for cystitis using TAT-peptide nucleic acid conjugatesrdquoMolecular Pharmaceutics vol3 no 4 pp 398ndash406 2006

[68] D C Molliver D E Wright M L Leitner et al ldquoIB4-bindingDRG neurons switch from NGF to GDNF dependence in earlypostnatal liferdquo Neuron vol 19 no 4 pp 849ndash861 1997

[69] M TyagiM RusnatiM Presta andMGiacca ldquoInternalizationof HIV-1 tat requires cell surface heparan sulfate proteoglycansrdquoJournal of Biological Chemistry vol 276 no 5 pp 3254ndash32612001

[70] E Uhlmann A Ryte and A Peyman ldquoStudies on the mech-anism of stabilization of partially phosphorothioated oligonu-cleotides against nucleolytic degradationrdquoAntisense andNucleicAcid Drug Development vol 7 no 4 pp 345ndash350 1997

[71] M Nogawa T Yuasa S Kimura et al ldquoIntravesical adminis-tration of small interfering RNA targeting PLK-1 successfullyprevents the growth of bladder cancerrdquo Journal of ClinicalInvestigation vol 115 no 4 pp 978ndash985 2005

[72] M Kashyap N Kawamorita V Tyagi et al ldquoDownregulation ofNGF expression in the bladder by antisense oligoucleotides asnew treatment for overactive bladderrdquo Journal of Urology vol27190 no 2 pp 757ndash764 2013

[73] C-J Arum Y Kodama N Rolim et al ldquoA rat model ofintravesical delivery of small interfering RNA for studyingurinary carcinomardquoWorld Journal of Urology vol 28 no 4 pp479ndash485 2010

[74] C E Blietz B Thode M Hauses et al ldquoIn vivo studies on theavailability and toxicity of antisense oligonucleotides in bladdercancerrdquo In Vivo vol 23 no 1 pp 13ndash19 2009

[75] D Oddiah P Anand S B McMahon and M Rattray ldquoRapidincrease of NGF BDNF andNT-3mRNAs in inflamed bladderrdquoNeuroReport vol 9 no 7 pp 1455ndash1458 1998

[76] M R Saban H Hellmich N B Nguyen J Winston T GHammond and R Saban ldquoTime course of LPS-induced geneexpression in a mouse model of genitourinary inflammationrdquoPhysiol Genomics vol 5 no 3 pp 147ndash160 2001

[77] B Schnegelsberg T T Sun G Cain et al ldquoOverexpression ofNGF in mouse urothelium leads to neuronal hyperinnervationpelvic sensitivity and changes in urinary bladder functionrdquoTheAmerican Journal of PhysiologymdashRegulatory Integrative andComparative Physiology vol 298 no 5 pp R534ndashR547 2010

[78] S Cardenas M Scuri L Samsell et al ldquoNeurotrophic andneuroimmune responses to early-life Pseudomonas aeruginosainfection in rat lungsrdquo American Journal of PhysiologymdashLungCellular and Molecular Physiology vol 299 no 3 pp L334ndashL344 2010

[79] K Nakamura F Tan Z Li and C J Thiele ldquoNGF activationof TrkA induces vascular endothelial growth factor expressionvia induction of hypoxia-inducible factor-1120572rdquo Molecular andCellular Neuroscience vol 46 no 2 pp 498ndash506 2011

[80] S Othumpangat M Regier and G Piedimonte ldquoNerve growthfactor modulates human rhinovirus infection in airway epithe-lial cells by controlling ICAM-1 expressionrdquo American Journalof Physiology Lung Cellular andMolecular Physiology vol 15302pp L1057ndashL1066 2012

[81] H Jung P T Toth F A White and R J Miller ldquoMonocytechemoattractant protein-1 functions as a neuromodulator indorsal root ganglia neuronsrdquo Journal of Neurochemistry vol104 no 1 pp 254ndash263 2008

[82] F A White H Jung and R J Miller ldquoChemokines andthe pathophysiology of neuropathic painrdquo Proceedings of the

12 ISRN Pharmacology

National Academy of Sciences of the United States of Americavol 104 no 51 pp 20151ndash20158 2007

[83] G Banisadr R-D Gosselin P Mechighel W Rostene PKitabgi and S M Parsadaniantz ldquoConstitutive neuronalexpression of CCR2 chemokine receptor and its colocalizationwith neurotransmitters in normal rat brain functional effectof MCP-1CCL2 on calcium mobilization in primary culturedneuronsrdquo Journal of Comparative Neurology vol 492 no 2 pp178ndash192 2005

[84] S Bhangoo D Ren R J Miller et al ldquoDelayed functionalexpression of neuronal chemokine receptors following focalnerve demyelination in the rat a mechanism for the devel-opment of chronic sensitization of peripheral nociceptorsrdquoMolecular Pain vol 3 article 38 2007

[85] T Maeda N Kiguchi Y Kobayashi T Ikuta M Ozaki and SKishioka ldquoLeptin derived from adipocytes in injured periph-eral nerves facilitates development of neuropathic pain viamacrophage stimulationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 106 no 31 pp13076ndash13081 2009

[86] H Kajiwara M Yasuda N Kumaki T Shibayama and YOsamura ldquoExpression of carbohydrate antigens (SSEA-1 Sialyl-Lewis X DU-PAN-2 and CA19-9) and E-selectin in urothelialcarcinoma of the renal pelvis ureter and urinary bladderrdquoTokaiJournal of Experimental and Clinical Medicine vol 30 no 3 pp177ndash182 2005

[87] A M S Assreuy G J Martins M E F Moreira et al ldquoPre-vention of cyclophosphamide-induced hemorrhagic cystitis byglucose-mannose binding plant lectinsrdquo Journal of Urology vol161 no 6 pp 1988ndash1993 1999

[88] S M Vinson A Rickard J S Ryerse and J McHowat ldquoNeu-trophil adherence to bladder microvascular endothelial cellsfollowing platelet-activating factor acetylhydrolase inhibitionrdquoJournal of Pharmacology and Experimental Therapeutics vol314 no 3 pp 1241ndash1247 2005

[89] M Green A Filippou G Sant and T CTheoharides ldquoExpres-sion of intercellular adhesion molecules in the bladder ofpatients with interstitial cystitisrdquoUrology vol 63 no 4 pp 688ndash693 2004

[90] M Leppilahti P Hellstrom and T L J Tammela ldquoEffect ofdiagnostic hydrodistension and four intravesical hyaluronicacid instillations on bladder ICAM-1 intensity and associationof ICAM-1 intensity with clinical response in patients withinterstitial cystitisrdquo Urology vol 60 no 1 pp 46ndash51 2002

[91] A T Corcoran N Yoshimura V Tyagi B Jacobs W Lengand P Tyagi ldquoMapping the cytokine profile of painful bladdersyndromeinterstitial cystitis in human bladder and urinespecimensrdquoWorld Journal of Urology vol 31 no 1 pp 241ndash2462013

[92] SGarrean X-PGaoV Brovkovych et al ldquoCaveolin-1 regulatesNF-120581B activation and lung inflammatory response to sepsisinduced by lipopolysacchariderdquo Journal of Immunology vol 177no 7 pp 4853ndash4860 2006

[93] M R Kang G Yang R F Place et al ldquoIntravesical deliveryof small activating RNA formulated into lipid nanoparticlesinhibits orthotopic bladder tumor growthrdquo Cancer Researchvol 172 pp 5069ndash5079 2012

[94] P Tyagi D Barclay R Zamora et al ldquoUrine cytokines suggestan inflammatory response in the overactive bladder a pilotstudyrdquo International Urology and Nephrology vol 42 no 3 pp629ndash635 2010

[95] P Tyagi B L Jacobs D Barcaly et al ldquoUrine levels ofinflammatory chemokines can be novel biomarkers for theoveractive bladderrdquoThe Journal of Urology vol 181 article 5882009

[96] P Tyagi V Tyagi D Bui et al ldquoDiscrimination Of OAB fromICPBS by multivariate data modeling of urinary proteinsrdquoTheJournal of Urology vol 183 pp e614ndashe6e5 2010

[97] P Tyagi D Nikolavsky Y Vodovotz et al ldquoUrine levels ofselected chemokines positively correlate with lower bladdercapacity and psychometric scores in IcPbs patientsrdquo TheJournal of Urology vol 181 article 21 2009

[98] R Weissleder and V Ntziachristos ldquoShedding light onto livemolecular targetsrdquo Nature Medicine vol 9 no 1 pp 123ndash1282003

[99] C Bremer V Ntziachristos and R Weissleder ldquoOptical-basedmolecular imaging contrast agents and potentialmedical appli-cationsrdquo European Radiology vol 13 no 2 pp 231ndash243 2003

[100] UMahmood andRWeissleder ldquoNear-infrared optical imagingof proteases in cancerrdquoMolecular CancerTherapeutics vol 2 pp489ndash496 2003

[101] J Kaufman H Hensley J Jacobs et al ldquoNon-invasive imagingof near infrafred dye labeled liposomes facilitates evaluation ofbioresidence timerdquoThe Journal of Urology vol 183 article e6282010

[102] Y-X J Wang ldquoMedical imaging in pharmaceutical clinicaltrials what radiologists should knowrdquo Clinical Radiology vol60 no 10 pp 1051ndash1057 2005

4 ISRN Pharmacology

(a) (b)

Liposomes

Afferent nervesAfferent nerves

Leaky urothelium

(c)

Figure 2 Ex vivo images of rat bladder taken in visible and near-infrared light indicate the coating formed by instilled liposomeson bladder surface Liposomes carry a trace amount of near-infrared (NIR) lipophilic carbocyanine dye 111015840-dioctadecyl-333101584031015840-tetramethylindotricarbocyanine iodide (DiR) that fluoresces on exposure to NIR light Rat bladder filled with urine was tied at the basewith thread prior to harvest Liposomes coating the bladder surface are invisible in visible light photograph (a) but is indicated by bluecolored coating on the bladder luminal surface in NIR light (b) NIR imaging in vivo can allow noninvasive repeat objective measurement forbladder residence time of instilled treatments (c) Schematic illustration of liposome coating the bladder surface Given the chemical affinityof phospholipids of instilled liposomes with lipids in cells lining the bladder surface the liposomes form a protective film coating on theinjured bladder lumen surface and assist in the repair of leaky and inflamed uroepithelium

The exact mechanism of action for liposomes in ICPBSremains to be established but protective coating effect basedon preclinical studies cannot be ruled out as illustrated inFigure 2

6 Liposomes as a Delivery Platform

Not only is the lining of the urinary bladder the most im-permeable tissue in the human body but it is also the mostcompliant As a bladder lining expands additionalmembranematerial is added to its cells to help retain impermeability[49] Therefore vesicular trafficking may provide a favorableenvironment for drug delivery and therefore it is worthinvestigating whether vesicle nature of liposomes can aid in

improving the delivery of cargo across the bladder permeabil-ity barrier

In the field of neurourology instillation of neurotoxinsinto bladder is an accepted approach to achieve chemicalneuromodulation of afferent neurotransmission underlyingneurogenic bladder and ICPBS [50] Existing approachesof chemical neuromodulation by intravesical neurotoxinsare suboptimal due to vehicle toxicity for capsaicin [51] ordegradation of botulinum toxin (BoNT) in urine Possiblereasons underlying the lack of efficacy fromBoNT instillationin bladder can be protein degradation by proteases andproteinases in urine dilution in urine or poor uptake of theBoNT solution into the urothelium

Liposomes have been previously studied as a carrier oftoxins to enhance efficacy at lower doses [52] In the context of

ISRN Pharmacology 5

Lipotoxin

Intravesical instillation

Liposomes

+

BoNT-A

Figure 3 Scheme for formulation of BoNT into liposomes for blad-der instillation Freeze dried BoNT is resuspended in distilled waterand added to vials containing a desiccated liposomal preparationmade of phospholipids This reconstituted suspension is allowed tohydrate at room temperature for 30ndash60min before instillation

toxins instilled in the bladder fat-soluble neurotoxin such ascapsaicin can be integrated into the phospholipid bilayer [38]and water-soluble neurotoxin such as BoNT can be protectedinside the aqueous compartment(s) of liposomes delimitedby the phospholipid bilayer(s) [53] (Figure 3) Cystoscopeguided injections are the current standard practice in theclinic for administering BoNT to the bladder But in recentyears studies have also assessed the potential for intravesicalinstillation of BoNT alone in animal models of bladderirritation [54]

Development of instillation as a mode for administeringBoNT in patients will drastically bring down the cost oftreatment for patients with refractory overactive bladderOther groups have recently reported the use of DMSO forinstillation of BoNT instead of injection [55] DMSO doesnot afford the natural state of the BoNT as a protein and needto be formulated immediately before instillation DissolvingBoNT in DMSO may not be advisable owing to concernsof BoNT uptake into the systemic circulation of patientMoreover biochemical studies have demonstrated that met-alloproteolytic activity of the BoNT is strongly enhanced bythe presence of lipid membranes [56]

Recent studies reaffirmed the potential of liposomesas a promising vehicle for delivery of neurotoxins to thebladder [38 53] The transport of BoNT into urotheliumfrom liposomes was confirmed by detection of its uniqueeffect on neurotransmitters and proteolysis of synaptosomal-associated protein SNAP-25 through immunohistochemistry[53] The protection of BoNT entrapped inside liposomesfrom degradation in urine without compromising efficacywas demonstrated by attenuation of acetic acid induced blad-der irritation in rats [53] Similar results were obtained in pre-clinical studies with liposomes encapsulating capsaicin [38]

Liposomes have proven themselves as biocompatible deliveryagents in the bladder

7 Intravesical Antisense Therapeutics

The term ldquoantisenserdquo therapeutics emerged from seminalstudies done 4 decades using a short synthetic oligonu-cleotide for sequence-specific gene silencing [57] Genesilencing involves introduction of short strands of DNA(termed as antisense) with sequences complementary to themRNA encoding a particular gene inside the cell with theintent to block gene expression through either translationalinhibition or enzymatic cleavage of the mRNA target [3]

Oligonucleotide ODN binds specifically and stronglyto the mRNA target through Watson-Crick base pairingODN can be basically categorized into those that directcleavage of the target mRNA as caused by small interferingRNAs (siRNAs) and those that alter mRNA translationwithout causing mRNA cleavage Recent discovery of smallinterfering RNAs (siRNAs) and the elucidation of the RNAinterference (RNAi) pathway has also brought a sea changein the control of posttranscriptional gene expression siRNAtakes advantage of endogenous cellular pathways to potentlysilence the expression of specific genes (Figure 4)

Antisense mechanism is a promising approach for devel-oping therapeutics based on rational gene-based drug designAntisense therapeutics have been under clinical investigationfor more than 30 years [58] However drug development ofthis approach has been hampered by inefficient intracellulardelivery and cellular uptake of the ODN The translation ofbasic antisense research into therapeutics is also impeded byintracellular stability of ODN and potential for ldquooff-targetrdquogene silencing immunostimulation and other side effects

A vast array of chemical modifications to ODN has beendeveloped to overcome the therapeutically limiting featuresby altering internucleotide phosphate linkages backbonesugars or nucleobases One such modified ODN is peptidenucleic acid which replaces phosphodiester bond betweennucleobases with a peptide bond Replacement of phosphodi-ester bond by a phosphorothioate linkage is another methodto improve the stability of ODN against nucleases

Great progress has been made in translating antisenseresearch into clinical therapies based on local injection intoeye [58]The field recently progressed further with a systemicinjection therapy for treating familial hypercholesterolemia[59] Antisense therapeutics have been used for exon skippingto optimize the functionality of a truncated dystrophinprotein in dog model of Duchenne muscular dystrophy [60]

Nevertheless applied research for bladder diseases haslagged behind considering that the anatomical architec-ture of bladder provides ease of local administration withrestricted systemic side effects due to lower serum uptakeof antisense ODN Bladder instillation of antisense ODNor their chemically modified mimics can therefore be anefficient means to control the expression of therapeuticallyrelevant genes Antisense agents can also be used to elucidatethe role of newly discovered genes in bladder function

6 ISRN Pharmacology

Liposomes with antisense

Transcription

Genomic DNA RNA Duplex

Antisense DNA

Activated PI 3-kinase

Activated receptortyrosine kinase

NGF

Anti-NGFantibodies

mRNAdegradation

PIP2 PIP3

PP P P P P

oligonucleotides

Figure 4 Illustration of mechanisms for blocking NGF expression by either NGF antibodies or downregulation of NGF mRNA expressionNGF antibody sequesters the freely available NGF and prevents it from binding with the cognate TrkA receptors NGF antisense selectivelybinds with NGF mRNA inside the cells to block the translation of corresponding mRNA siRNA takes advantage of endogenous cellularpathways to degrade NGF mRNA and control the posttranscriptional NGF gene expression

8 NGF Expression in Bladder

Several studies have reported that patients with OAB orICPBS excrete more nerve growth factor (NGF) in theirurine relative to asymptomatic controls [61] Higher expres-sion of NGF in bladder of patients [62] with correspondinglower serum levels of NGFmakes the bladder tissue the likelysource for the elevated NGF in urine [63] Previous studieshave indicated that increased levels of NGF in the bladderand bladder afferent pathways are directly involved in theemergence of hyperexcitability of C-fiber bladder sensorypathways leading to the pathology of DO andOAB (Figure 4)[64]

In addition intrathecal application of NGF antibodiesreducedNGF levels in bladder afferent pathways and normal-ized bladderurethral function in spinal cord injured (SCI)rats [65] Because it is likely that the major pathology of OABis driven by NGF targeting the intracellular synthesis of NGFmolecule in bladder is a promising therapeutic alternative

9 NGF Expression a Drug Target

Overexpression of NGF can be blocked either directly byantibodies [66] or by blocking the synthesis of NGF proteinfrom mRNA [67] Systemic administration of monoclonalhuman NGF antibodies (tanezumab) has been explored fortherapeutic outcomes in ICPBS patients but not withoutencountering safety concerns such as paresthesia hypoesthe-sia and arthralgia [66] Generalized blockade ofNGF activityat sites other than bladder by anti-NGF antibodies maynot be the preferred outcome because NGF is an essentialhousekeeping growth factor necessary for the survival andgrowth of neurons [68] The physiological necessity of NGF

action at those sites may explain the incidence of paresthesiahypoesthesia and arthralgia in patients treated with systemicanti-NGF antibodies [66]

91 Peptide Nucleic Acid (PNA) Therefore to reduce thetoxicity of systemic blockade of NGF we sought to developa novel intravesical therapy of OAB by targeting the intra-cellular synthesis of NGF in the urothelium Antisense ODNneeded to be able to cross cell membrane to act as a drugand negatively charged ODN will not pass through a lipidlayer such as cell membranes Bladder uptake of ODN islimited by the anionic charge and size of the ODN as wellas anionic glycosaminoglycan layer of the inner bladdersurface Therefore primary impediment to be overcome inthe development of intravesical antisense therapeutics isinefficient bladder uptake of the ODN across urotheliumPrevious studies showed that heparan sulfate proteoglycansexpressed on cell surface act as receptors for extracellularTAT uptake [69] Therefore it was reasoned that GAG layeron bladder surface can facilitate bladder uptake of peptidenucleic acid PNA if it could be conjugatedwith synthetic TATpeptide

Water insoluble peptide nucleic acid targeting NGF wasconjugated with cell penetrating cationic peptide TAT forintracellular delivery of antisense moiety to demonstrate effi-cacy in animal models Studies showed that PNA conjugatedwith TAT suppressed cyclophosphamide cystitis followinglocal instillation of antisense against NGF [67] There wasnegligible uptake of peptide nucleic acid in absence of TATconjugation Prior to determination of in vivo efficacy ofconjugate suitable target sequence on NGF mRNA wasdetermined by predicted folding structure and cell transfec-tion experiments Successful intravesical delivery of peptide

ISRN Pharmacology 7

nucleic acid in bladder at the same time of cyclophosphamideinjection protected against the cystitis by blocking the rise inbladder contraction frequency and inflammation

92 Phosphorothioate-Linked Analogues Considering thedifficulty and possible nonspecific toxicity of peptide nucleicacid and TAT recent developments focused on simplifyingthe approach using water-soluble phosphorothioated ODNwhich have an increased resistance to exo- and endonucleasesfor improved stability [70] As alluded to in the above textsuccess of antisense therapeutics is largely dependent on thedevelopment of a delivery vehicle that can efficiently deliverantisense ODN in bladder Preliminary studies showed thatliposomes can be far better biocompatible effective carriersfor local gene silencing of NGF gene in bladder cells

Cationic liposomes and mimics have emerged as themost popular nonviral method to deliver nucleic acids intherapeutic applications Easy and reversible complex forma-tion of cationic liposomes with ODN at room temperatureallows their use as carriers Electrostatic attraction betweenthe cationic lipid DOTAP and the polyanionic antisenseODN is responsible for the complex formation The efficacyof liposome delivered siRNA by intravesical route has beenpreviously demonstrated in preclinical models of bladdercancer [71] Residence of ODN in the rat bladder after intrav-esical instillation was demonstrated to be longer than 24 husing fluorescent tagged ODN complexed with liposomes[72] Fluorescent probe was localized in bladder urotheliumcells 24 h following instillation The 24 h residence time wasalso demonstrated for siRNA in mouse bladder [73] Bladderuptake of fluorescent ODN without liposomes in normalrat bladder is poor Previous studies have shown that veryhigh concentration of phosphorothioated ODN can deliverODN without liposomes to bladder cells of mice havingbladder cancer [74] The urothelium barrier may be slightlycompromised in cancerous condition and the strategy ofloading bladder with high dose of ODN may not workin noncancerous diseased condition of bladder with intactbarrier

In order to evaluate the efficacy of NGF antisense ODNacetic acid infusion was used to cause a rapid rise of NGFprotein levels A single dose instillation of OND complexedwith liposomes protected against bladder overactivity (BO)induced by acetic acid Together with data of bladder uptakestudies using fluorescent ODN it is demonstrated that ONDis readily available to the bladder after intravesical instillationIt has been previously reported that within 2 h of exposureto irritants such as turpentine and acetic acid there occursa rapid rise in bladder content of NGF [75] Later studiesfound that bladder responds to insults with upregulation inthe genes for NGF sE-Selectin and receptor for monocytechemoattractant protein-1 (MCP-1) within 30min of expo-sure to lipopolysaccharide (Figure 5) [76] The acetic acidinduced overexpression of NGF was blunted by pretreatmentwith NGF antisense OND with phosphorothioate linkageprior to exposure of acetic acid The downregulation of NGFmRNA expression was in agreement with reduced proteinlevels and suppressed NGF-like immunoreactivity in theurothelium

93 Antisense and Downstream Signalling of NGF Drugdevelopment of intravesical antisense for NGF was also ableto unmask the downstream signaling [77] involving NGFfollowing exposure to acetic acid Experiments supportedthe role of NGF as a paracrine messenger [77] which isknown to activate several downstream effectors to manifestphysiological and pathological signaling changes linked to it[76 78ndash80] The bladder injury set off by acetic acid initiatesthe signalling cascades that upregulate the expression ofNGF and other chemokines MCP-1 CXCL-1 and CXCL-10 and prostaglandins [76 78ndash80] As reported elsewherelocalization of chemokines within synaptic vesicles in neu-rons [81] is consistent with their ability to act as excitatoryneurotransmitters following AA exposure

Chemokines are one of several downstream effectorsactivated by NGF [76 78ndash80] and interestingly chemokinereceptors are widely expressed in neural and nonneuralelements of the nociceptive pathways that are responsible forvisceral and somatic pain sensation [82] MCP-1 [83] andCXCL-10 [84] are constitutively expressed in neurons wherethey participate in excitability of primary afferent neuronsthrough transactivation of transient receptor channels andnociceptor sensitization [84] Overexpression ofNGF is likelyto drive the expression of VEGF fromneurons and leptin [78]from adipocytes covering neurons [85]

Cooperative expression of NGF and MCP-1 is able toinduce hyperexcitability in neurons by activating TRPV1receptor In addition MCP-1 CXCL-1 and CXCL-10 causechemoattraction of monocytes neutrophils and lympho-cytes respectively to mediate the bladder injury set offby acetic acid Extravasation is an essential prerequisitefor infiltration of monocytes neutrophils and lymphocyteswhich requires the expression of adhesion molecules likeE-selectin and intracellular adhesion molecule ICAM-1 [7686] E-selectin is a cytokine-inducible adhesion moleculethat supports the rolling and stable arrest of leukocyteson activated vascular endothelium Expression of E-selectinhas been linked to adherence of neutrophils to bladdermicrovascular endothelial cells and to cyclophosphamidecystitis [87 88] E-selectin gene expression is activated bythe NF-120581B and MAP kinase signal transduction pathways[76 86]

Activation and recruitment of leukocytes to acetic acidinduced bladder injury also require expression of intra-cellular adhesion molecule ICAM-1 [89] ICAM-1 interactsspecifically with its receptors of the integrin family to inducereversible cell-cell interactions involving adhesion Expres-sion of ICAM-1 was found to be increased in patients withICPBS and reduced in patients responding to instillation ofhyaluronic acid [90] Expression of ICAM-1 and VCAM-1was also noted in a recent study on biopsy tissue of ICPBSpatients [91] Increased expression of ICAM-1 subsequentto exposure with acetic acid in animal model is thereforeclinically relevant

Antisense experiments supported the earlier report thatinhibition of NGF expression significantly downregulates theexpression of ICAM-1 [80] It is reported that binding ofNGF to its high-affinity TrkA receptor controls the sICAM-1expression on target cells [80] Expression of ICAM-1 was

8 ISRN Pharmacology

Neutrophil

Monocytes

Acetic acid

Integrin

NGF

MCP-1

Release of cytokinesVanilloid

receptor

Trk receptors NGF

Membrane damage

VEGF

CXCL1

Activated leucocyte

Startrolling

Extravasation

CXCL10

sICAMssE-selectin

ActivationAdhesion

MCP-1

NGF

PKC

DAG

PLC

IP3Kinases

Ras

AKT

PI3K

CXCL10

sICAMs

LymphocyteAfferents

Figure 5 Signalling cascades induced by NGF overexpression following exposure to acetic acid Injury from exposure to acetic sets off thesignalling cascades that upregulate the expression of NGF MCP-1 prostaglandins CXCL-1 and CXCL-10 Overexpression of NGF drives theexpression of VEGF from neurons and leptin from adipocytes covering neurons NGF and MCP-1 also induce hyperexcitability in neuronsby activating TRPV1 receptor MCP-1 CXCL-1 and CXCL-10 cause chemoattraction of monocytes neutrophils and lymphocytes whichrequires extravasation across endothelium from blood through the cooperation of adhesion molecules E-selectin and ICAM-1

negatively associated with NGF expression in antisenseexperiments Ultimately expression of ICAM-1 is regulated atthe level of transcription by one of several signaling cascadesNF-120581B JAKSTAT IFN-g (CXCL-10) AP MAP kinase andPKC [92]

Intravesical route can allow selective exposure of anti-sense ODN to the NGF producing cells in urothelium andavoid systemic side effects from genetic manipulation ofNGF expression Intravesical route can also prove to be costeffective given the cost of ODN or siRNA Contrary to thegoal of loss of function in bladder with antisense ODN inrecent studies liposomes have also been used to deliver ODNfor gain of function in bladder [93]

10 Imaging of Intravesical Therapy

Drug development for DO and ICPBS relies heavily onsubjective outcomes for predicting efficacy and safety in earlyclinical development Although there are many validatedmeasurement tools used in DO and ICPBS research theyare usually burdensome and do not capture all symptomsrelated to the lower urinary tract Moreover improvement insymptoms scores following an intervention does not alwayscorrelate with patient expectations satisfaction and goalachievement which are critically important for successfulmanagement of DO and ICPBS

There is underdevelopment and underutilization of urinebiomarkers and imaging methods for investigational drugsgiven by intravesical route Overreliance on subjectiveimpressions of patients for therapeutic response limits clini-cal relevance due to weak correlation with patient satisfactionand can also impede continued scientific progress in assessing

study outcomes relative to symptom bother Symptom scoresmay also be related to the cause of high heterogeneity inclinical response in DO and ICPBS patients

Imaging can offer the possibility to provide region-specific information in situations where serum and urineassays may not reflect the true state of bladder [94ndash96]Urine chemokines have been reported to be associated withsymptom severity of ICPBS patients [97] Application offluorescent microscopy for imaging of pelvic floor has beenlimited due to the issues pertaining to light absorption bytissues scattering and autofluorescence [98]

Recent studies have shown that dyes fluorescing inthe near-infrared (NIR) band can overcome this handi-cap in deep-tissue imaging of experimental animals [99]Selected NIR fluorochromes emit light with tissue pen-etration approaching 10ndash15 cm [100] The light emissionfrom probes in this spectrum encounters low backgroundautofluorescence and minimal attenuation of signal due tolight absorption by tissue components [98] Therefore NIRimaging yields high signal-to-noise ratios and is well suitedfor studying the distribution of instilled treatments in bladder(Figure 2(b))

Preliminary studies suggest that imaging of mouse pelvicfloor in NIR spectrum can be a viable option [101] NIRimaging easily allows visualization and quantification ofbladder distribution by tracing themigration of fluorescence-labeled liposomes in anaesthetized mouse Compared withother biological assays imaging could provide objectiveendpoint for patients with ulcerative cystitis to indicate itshigh translation potential Other benefits of optical imaginginclude its relatively low cost compared withmore traditionalimaging systems such as MRI and PET The instrument

ISRN Pharmacology 9

consists of two near-infrared lasers emitting at 685 nm and785 nm camera and novel optics

Furthermore preferential binding of liposomes andnanoparticles to bladder lesions of ICPBS after instillationcan be assessed by NIR imaging of bladder where it cantheoretically provide anatomically specific information aboutthe real time status of ulcerlesion in bladder surface or tissueabnormality [102] Therefore future studies can substitute aresponse variable that is continuous in nature instead of asubjective clinical outcome

11 Conclusions

Adoption of forward-thinking approaches can achieveadvancements in drug delivery systems targeted to improvepharmacotherapy of bladder diseases in the future Latestdevelopments in the field of nanotechnology can bring thismode of therapy from second line of treatment for refractorycases to the forefront of disease management Liposomesare an attractive drug delivery platform by virtue of theirbiodegradability biocompatibility low toxicity and simpleand mild preparation methods

Conflict of Interests

All authors except Dr Michael Chancellor declare that thereis no conflict of interests regarding the publication of thispaper Dr Michael Chancellor owns stocks in the start-upliposome company Lipella

Acknowledgments

This work was supported by the National Institutes of Health(DK057267 and DK088836) and the Department of Defense(W81XWH-11-1-0763 and W81XWH-12-1-0565)

References

[1] J Parkin C Shea and G R Sant ldquoIntravesical dimethyl sul-foxide (DMSO) for interstitial cystitismdasha practical approachrdquoUrology vol 49 no 5 pp 105ndash107 1997

[2] C Dowson J Watkins M S Khan P Dasgupta and A SahaildquoRepeated botulinum toxin type A injections for refractoryoveractive bladder medium-term outcomes safety profile anddiscontinuation ratesrdquo European Urology vol 61 no 4 pp 834ndash839 2012

[3] J Kaufman V Tyagi M Anthony M B Chancellor and PTyagi ldquoState of the art in intravesical therapy for lower urinarytract symptomsrdquo Reviews in Urology vol 12 pp e181ndashe189 2010

[4] N Yoshimura and M B Chancellor ldquoCurrent and futurepharmacological treatment for overactive bladderrdquo Journal ofUrology vol 168 no 5 pp 1897ndash1913 2002

[5] K A Killinger J A Boura and K M Peters ldquoPain ininterstitial cystitisbladder pain syndrome do characteristicsdiffer in ulcerative and non-ulcerative subtypesrdquo InternationalUrogynecology Journal vol 24 no 8 pp 1295ndash1301 2013

[6] J C Nickel P Hanno K Kumar and HThomas ldquoSecond mul-ticenter randomized double-blind parallel-group evaluationof effectiveness and safety of intravesical sodium chondroitin

sulfate compared with inactive vehicle control in subjects withinterstitial cystitisbladder pain syndromerdquoUrology vol 79 no6 pp 1220ndash1225 2012

[7] P M Hanno ldquoAnalysis of long-term elmiron therapy forinterstitial cystitisrdquo Urology vol 49 no 5 pp 93ndash99 1997

[8] A Giannantoni V Bini R Dmochowski et al ldquoReply to ClausRiedlrsquos letter to the editor re antonella Giannantoni VittorioBini Roger Dmochowski et al Contemporary management ofthe painful bladder a systematic reviewrdquo EuropeanUrology vol61 no 5 pp 29ndash53 2012

[9] MC Lai Y CKuo andHCKuo ldquoIntravesical hyaluronic acidfor interstitial cystitispainful bladder syndrome a comparativerandomized assessment of different regimensrdquo InternationalJournal of Urology vol 20 pp 203ndash207 2013

[10] J Eldrup J Thorup and S L Nielsen ldquoPermeability andultrastructure of human bladder epitheliumrdquo British Journal ofUrology vol 55 no 5 pp 488ndash492 1983

[11] C L Parsons D Boychuk S Jones R Hurst and H CallahanldquoBladder surface glycosaminoglycans an epithelial permeabil-ity barrierrdquo Journal of Urology vol 143 no 1 pp 139ndash142 1990

[12] Y Kim N Yoshimura H Masuda F De Miguel and MB Chancellor ldquoAntimuscarinic agents exhibit local inhibitoryeffects on muscarinic receptors in bladder-afferent pathwaysrdquoUrology vol 65 no 2 pp 238ndash242 2005

[13] V Tyagi B J Philips R Su et al ldquoDifferential expression offunctional cannabinoid receptors in human bladder detrusorand urotheliumrdquo Journal of Urology vol 181 no 4 pp 1932ndash1938 2009

[14] B J Philips M C Smaldone V L Erickson N YoshimuraM B Chancellor and P Tyagi ldquoDifferential expression ofneurokinin receptor subtypes in urothelium and detrusor ofhuman bladderrdquo Journal of Urology vol 179 article 354 2008

[15] S A Baker G W Hennig J Han F C Britton T K Smithand S D Koh ldquoMethionine and its derivatives increase blad-der excitability by inhibiting stretch-dependent K+ channelsrdquoBritish Journal of Pharmacology vol 153 no 6 pp 1259ndash12712008

[16] J M Beckel A Kanai S-J Lee W C De Groat and LA Birder ldquoExpression of functional nicotinic acetylcholinereceptors in rat urinary bladder epithelial cellsrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 290 no 1 ppF103ndashF110 2006

[17] LA Birder A J KanaiWCDeGroat et al ldquoVanilloid receptorexpression suggests a sensory role for urinary bladder epithelialcellsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 23 pp 13396ndash13401 2001

[18] P Tyagi C A Thomas N Yoshimura and M B ChancellorldquoInvestigations into the presence of functional szlig1 szlig2 and szlig3-adrenoceptors in urothelium and detrusor of human bladderrdquoInternational Brazilian Journal of Urology vol 35 no 1 pp 76ndash83 2009

[19] A T Hanna-Mitchell J M Beckel S Barbadora A J Kanai WC de Groat and L A Birder ldquoNon-neuronal acetylcholine andurinary bladder urotheliumrdquo Life Sciences vol 80 no 24-25 pp2298ndash2302 2007

[20] C P Smith D A Gangitano A Munoz et al ldquoBotulinumtoxin type A normalizes alterations in urothelial ATP and NOrelease induced by chronic spinal cord injuryrdquo NeurochemistryInternational vol 52 no 6 pp 1068ndash1075 2008

[21] Y-C Chuang C A Thomas S Tyagi N Yoshimura PTyagi and M B Chancellor ldquoHuman urine with solifenacin

10 ISRN Pharmacology

intake but not tolterodine or darifenacin intake blocks detrusoroveractivityrdquo International Urogynecology Journal and PelvicFloor Dysfunction vol 19 no 10 pp 1353ndash1357 2008

[22] Y Kim N Yoshimura H Masuda F D Miguel and M BChancellor ldquoIntravesical instillation of human urine after oraladministration of trospium tolterodine and oxybutynin in a ratmodel of detrusor overactivityrdquo BJU International vol 97 no 2pp 400ndash403 2006

[23] S Tyagi P Tyagi S Van-le N YoshimuraM B Chancellor andF de Miguel ldquoQualitative and quantitative expression profileof muscarinic receptors in human urothelium and detrusorrdquoJournal of Urology vol 176 no 4 pp 1673ndash1678 2006

[24] G Gregoriadis and B E Ryman ldquoLiposomes as carriers ofenzymes or drugs a new approach to the treatment of storagediseasesrdquo Biochemical Journal vol 124 no 5 p 58 1971

[25] G Gregoriadis S Jain I Papaioannou and P Laing ldquoImprov-ing the therapeutic efficacy of peptides and proteins a role forpolysialic acidsrdquo International Journal of Pharmaceutics vol300 no 1-2 pp 125ndash130 2005

[26] P Sapra P Tyagi and T M Allen ldquoLigand-targeted liposomesfor cancer treatmentrdquo Current Drug Delivery vol 2 no 4 pp369ndash381 2005

[27] G Gregoriadis G Dapergolas and E D Neerunjun ldquoPene-tration of target areas in the rat by liposome associated agentsadministered parenterally and intragastricallyrdquo BiochemicalSociety Transactions vol 4 no 2 pp 256ndash259 1976

[28] G Gregoriadis ldquoEngineering liposomes for drug deliveryprogress and problemsrdquo Trends in Biotechnology vol 13 no 12pp 527ndash537 1995

[29] G Gregoriadis and A C Allison ldquoEntrapment of proteins inliposomes prevents allergic reactions in pre immunised micerdquoFEBS Letters vol 45 no 1 pp 71ndash74 1974

[30] S Lee S Dausch CMaierhofer andD Dausch ldquoA new therapyconcept with a liposome eye spray for the treatment of the lsquodryeyersquordquo Klinische Monatsblatter fur Augenheilkunde vol 221 no10 pp 825ndash836 2004

[31] D Dausch S Lee S Dausch J C Kim G Schwert and WMichelson ldquoComparative study of treatment of the dry eyesyndrome due to disturbances of the tear film lipid layer withlipid-containing tear substitutes efficacy of lipid-containingtear substitutesrdquo Klinische Monatsblatter fur Augenheilkundevol 223 no 12 pp 974ndash983 2006

[32] S Ebrahim G A Peyman and P J Lee ldquoApplications ofliposomes in ophthalmologyrdquo Survey of Ophthalmology vol 50no 2 pp 167ndash182 2005

[33] J Nirmal P Tyagi L Dang et al ldquoEndocytosis uptake ofliposomes in urothelium cells detected by transmission electronmicroscopyrdquoThe Journal of Urology vol 187 article e15 2012

[34] D N Frangos J J Killion D Fan R Fishbeck A C VonEschenbach and I J Fidler ldquoThe development of liposomescontaining interferon alpha for the intravesical therapy ofhuman superficial bladder cancerrdquo Journal of Urology vol 143no 6 pp 1252ndash1266 1990

[35] J W Johnson R Nayar J J Killion A C Von Eschenbachand I J Fidler ldquoBinding of liposomes to human bladdertumor epithelial cell lines implications for an intravesical drugdelivery system for the treatment of bladder cancerrdquo SelectiveCancer Therapeutics vol 5 no 4 pp 147ndash155 1989

[36] Y-C Chuang M B Chancellor S Seki et al ldquoIntravesicalprotamine sulfate and potassium chloride as a model forbladder hyperactivityrdquoUrology vol 61 no 3 pp 664ndash670 2003

[37] J Kaufman P Tyagi and M B Chancellor ldquoIntravesicalliposomal (Lp08) instillation protects bladder urothelium fromchemical irritationrdquoThe Journal of Urology vol 181 article 5392009

[38] P Tyagi M B Chancellor Z Li et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical capsaicindelivery using thermosensitive hydrogel and liposomesrdquo Jour-nal of Urology vol 171 no 1 pp 483ndash489 2004

[39] MO Fraser Y-CChuang P Tyagi et al ldquoIntravesical liposomeadministrationmdasha novel treatment for hyperactive bladder inthe ratrdquo Urology vol 61 no 3 pp 656ndash663 2003

[40] P Tyagi M Chancellor N Yoshimura and L Huang ldquoActivityof different phospholipids in attenuating hyperactivity in blad-der irritationrdquo BJU International vol 101 no 5 pp 627ndash6322008

[41] Y Sun and T C Chai ldquoEffects of dimethyl sulphoxide andheparin on stretch-activated ATP release by bladder urothelialcells from patients with interstitial cystitisrdquo BJU Internationalvol 90 no 4 pp 381ndash385 2002

[42] J J Bade ldquoA placebo-controlled study of intravesical pentosan-polysulphate for the treatment of interstitial cystitisrdquo BritishJournal of Urology vol 79 no 2 pp 168ndash171 1997

[43] P Tyagi V C Hsieh N Yoshimura J Kaufman and M BChancellor ldquoInstillation of liposomes vs dimethyl sulphoxide orpentosan polysulphate for reducing bladder hyperactivityrdquo BJUInternational vol 104 no 11 pp 1689ndash1692 2009

[44] W-C Lee C-T Chien H-J Yu and S-W Lee ldquoBladderdysfunction in rats with metabolic syndrome induced by long-term fructose feedingrdquo Journal of Urology vol 179 no 6 pp2470ndash2476 2008

[45] J I Gillespie G A Van Koeveringe S G DeWachter and J DeVente ldquoOn the origins of the sensory output from the bladderthe concept of afferent noiserdquo BJU International vol 103 no 10pp 1324ndash1333 2009

[46] W C Lee P H Chiang Y L Tain C C Wu and Y CChuang ldquoSensory dysfunction of bladder mucosa and bladderoversensitivity in a rat model of metabolic syndromerdquo PloSONE vol 7 Article ID e45578 2012

[47] Y-C Chuang W-C Lee W-C Lee and P-H Chiang ldquoIntrav-esical liposome versus oral pentosan polysulfate for interstitialcystitispainful bladder syndromerdquo Journal of Urology vol 182no 4 pp 1393ndash1400 2009

[48] W-C Lee Y-C Chuang W-C Lee and P-H Chiang ldquoSafetyand dose flexibility clinical evaluation of intravesical liposomein patients with interstitial cystitis or painful bladder syn-dromerdquo Kaohsiung Journal of Medical Sciences vol 27 no 10pp 437ndash440 2011

[49] S T Truschel E Wang W G Ruiz et al ldquoStretch-regulatedexocytosisendocytosis in bladder umbrella cellsrdquo MolecularBiology of the Cell vol 13 no 3 pp 830ndash846 2002

[50] M B Chancellor and W C De Groat ldquoIntravesical capsaicinand resiniferatoxin therapy spicing up the ways to treat theoveractive bladderrdquo Journal of Urology vol 162 no 1 pp 3ndash111999

[51] D S Byrne A Das J Sedor et al ldquoEffect of intravesicalcapsaicin and vehicle on bladder integrity in control and spinalcord injured ratsrdquo Journal of Urology vol 159 no 3 pp 1074ndash1078 1998

[52] M Mandal and K-D Lee ldquoListeriolysin O-liposome-mediatedcytosolic delivery of macromolecule antigen in vivo enhance-ment of antigen-specific cytotoxic T lymphocyte frequency

ISRN Pharmacology 11

activity and tumor protectionrdquo Biochimica et Biophysica Actavol 1563 no 1-2 pp 7ndash17 2002

[53] Y-C Chuang P Tyagi C-C Huang et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical botulinumtoxin a delivery using liposomesrdquo Journal of Urology vol 182no 2 pp 786ndash792 2009

[54] Y-C Chuang N Yoshimura C-C Huang P-H Chiang andM B Chancellor ldquoIntravesical botulinum toxin a administra-tion produces analgesia against acetic acid induced bladder painresponses in ratsrdquo Journal of Urology vol 172 no 4 pp 1529ndash1532 2004

[55] S P Petrou A S Parker J E Crook A Rogers D Metz-Kudashick and D D Thiel ldquoBotulinum A toxindimethylsulfoxide bladder instillations for women with refractory idio-pathic detrusor overactivity a phase 12 studyrdquo Mayo ClinicProceedings vol 84 no 8 pp 702ndash706 2009

[56] P Caccin O Rossetto M Rigoni E Johnson G Schiavoand C Montecucco ldquoVAMPsynaptobrevin cleavage by tetanusand botulinum neurotoxins is strongly enhanced by acidicliposomesrdquo FEBS Letters vol 542 no 1ndash3 pp 132ndash136 2003

[57] M L Stephenson and P C Zamecnik ldquoInhibition of Roussarcoma viral RNA translation by a specific oligodeoxyribonu-cleotiderdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 75 no 1 pp 285ndash288 1978

[58] H Dadgostar and N Waheed ldquoThe evolving role of vascularendothelial growth factor inhibitors in the treatment of neovas-cular age-related macular degenerationrdquo Eye vol 22 no 6 pp761ndash767 2008

[59] C Gebhard G Huard E A Kritikou and J C TardifldquoApolipoprotein B antisense inhibitionmdashupdate on mipom-ersenrdquo Current Pharmaceutical Design vol 919 no 17 pp 3132ndash3142 2013

[60] T Yokota E Hoffman and S Takeda ldquoAntisense oligo-mediated multiple exon skipping in a dog model of duchennemuscular dystrophyrdquoMethods inMolecular Biology vol 709 pp299ndash312 2011

[61] B L Jacobs M C Smaldone V Tyagi et al ldquoIncreased nervegrowth factor in neurogenic overactive bladder and interstitialcystitis patientsrdquoThe Canadian Journal of Urology vol 17 no 1pp 4989ndash4994 2010

[62] H-T Liu and H-C Kuo ldquoIntravesical botulinum toxin Ainjections plus hydrodistension can reduce nerve growth factorproduction and control bladder pain in interstitial cystitisrdquoUrology vol 70 no 3 pp 463ndash468 2007

[63] H T Liu and H C Kuo ldquoIncreased urine and serum nervegrowth factor levels in interstitial cystitis suggest chronicinflammation is involved in the pathogenesis of diseaserdquo PloSONE vol 7 Article ID e44687 2012

[64] N Yoshimura N E Bennett Y Hayashi et al ldquoBladderoveractivity and hyperexcitability of bladder afferent neuronsafter intrathecal delivery of nerve growth factor in ratsrdquo Journalof Neuroscience vol 26 no 42 pp 10847ndash10855 2006

[65] S Seki K Sasaki Y Igawa et al ldquoSuppression of detrusor-sphincter dyssynergia by immunoneutralization of nervegrowth factor in lumbosacral spinal cord in spinal cord injuredratsrdquo Journal of Urology vol 171 no 1 pp 478ndash482 2004

[66] R J Evans R M Moldwin N Cossons A Darekar I WMills andD Scholfield ldquoProof of concept trial of tanezumab forthe treatment of symptoms associated with interstitial cystitisrdquoJournal of Urology vol 185 no 5 pp 1716ndash1721 2011

[67] P Tyagi R Banerjee S Basu N YoshimuraM Chancellor andL Huang ldquoIntravesical antisense therapy for cystitis using TAT-peptide nucleic acid conjugatesrdquoMolecular Pharmaceutics vol3 no 4 pp 398ndash406 2006

[68] D C Molliver D E Wright M L Leitner et al ldquoIB4-bindingDRG neurons switch from NGF to GDNF dependence in earlypostnatal liferdquo Neuron vol 19 no 4 pp 849ndash861 1997

[69] M TyagiM RusnatiM Presta andMGiacca ldquoInternalizationof HIV-1 tat requires cell surface heparan sulfate proteoglycansrdquoJournal of Biological Chemistry vol 276 no 5 pp 3254ndash32612001

[70] E Uhlmann A Ryte and A Peyman ldquoStudies on the mech-anism of stabilization of partially phosphorothioated oligonu-cleotides against nucleolytic degradationrdquoAntisense andNucleicAcid Drug Development vol 7 no 4 pp 345ndash350 1997

[71] M Nogawa T Yuasa S Kimura et al ldquoIntravesical adminis-tration of small interfering RNA targeting PLK-1 successfullyprevents the growth of bladder cancerrdquo Journal of ClinicalInvestigation vol 115 no 4 pp 978ndash985 2005

[72] M Kashyap N Kawamorita V Tyagi et al ldquoDownregulation ofNGF expression in the bladder by antisense oligoucleotides asnew treatment for overactive bladderrdquo Journal of Urology vol27190 no 2 pp 757ndash764 2013

[73] C-J Arum Y Kodama N Rolim et al ldquoA rat model ofintravesical delivery of small interfering RNA for studyingurinary carcinomardquoWorld Journal of Urology vol 28 no 4 pp479ndash485 2010

[74] C E Blietz B Thode M Hauses et al ldquoIn vivo studies on theavailability and toxicity of antisense oligonucleotides in bladdercancerrdquo In Vivo vol 23 no 1 pp 13ndash19 2009

[75] D Oddiah P Anand S B McMahon and M Rattray ldquoRapidincrease of NGF BDNF andNT-3mRNAs in inflamed bladderrdquoNeuroReport vol 9 no 7 pp 1455ndash1458 1998

[76] M R Saban H Hellmich N B Nguyen J Winston T GHammond and R Saban ldquoTime course of LPS-induced geneexpression in a mouse model of genitourinary inflammationrdquoPhysiol Genomics vol 5 no 3 pp 147ndash160 2001

[77] B Schnegelsberg T T Sun G Cain et al ldquoOverexpression ofNGF in mouse urothelium leads to neuronal hyperinnervationpelvic sensitivity and changes in urinary bladder functionrdquoTheAmerican Journal of PhysiologymdashRegulatory Integrative andComparative Physiology vol 298 no 5 pp R534ndashR547 2010

[78] S Cardenas M Scuri L Samsell et al ldquoNeurotrophic andneuroimmune responses to early-life Pseudomonas aeruginosainfection in rat lungsrdquo American Journal of PhysiologymdashLungCellular and Molecular Physiology vol 299 no 3 pp L334ndashL344 2010

[79] K Nakamura F Tan Z Li and C J Thiele ldquoNGF activationof TrkA induces vascular endothelial growth factor expressionvia induction of hypoxia-inducible factor-1120572rdquo Molecular andCellular Neuroscience vol 46 no 2 pp 498ndash506 2011

[80] S Othumpangat M Regier and G Piedimonte ldquoNerve growthfactor modulates human rhinovirus infection in airway epithe-lial cells by controlling ICAM-1 expressionrdquo American Journalof Physiology Lung Cellular andMolecular Physiology vol 15302pp L1057ndashL1066 2012

[81] H Jung P T Toth F A White and R J Miller ldquoMonocytechemoattractant protein-1 functions as a neuromodulator indorsal root ganglia neuronsrdquo Journal of Neurochemistry vol104 no 1 pp 254ndash263 2008

[82] F A White H Jung and R J Miller ldquoChemokines andthe pathophysiology of neuropathic painrdquo Proceedings of the

12 ISRN Pharmacology

National Academy of Sciences of the United States of Americavol 104 no 51 pp 20151ndash20158 2007

[83] G Banisadr R-D Gosselin P Mechighel W Rostene PKitabgi and S M Parsadaniantz ldquoConstitutive neuronalexpression of CCR2 chemokine receptor and its colocalizationwith neurotransmitters in normal rat brain functional effectof MCP-1CCL2 on calcium mobilization in primary culturedneuronsrdquo Journal of Comparative Neurology vol 492 no 2 pp178ndash192 2005

[84] S Bhangoo D Ren R J Miller et al ldquoDelayed functionalexpression of neuronal chemokine receptors following focalnerve demyelination in the rat a mechanism for the devel-opment of chronic sensitization of peripheral nociceptorsrdquoMolecular Pain vol 3 article 38 2007

[85] T Maeda N Kiguchi Y Kobayashi T Ikuta M Ozaki and SKishioka ldquoLeptin derived from adipocytes in injured periph-eral nerves facilitates development of neuropathic pain viamacrophage stimulationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 106 no 31 pp13076ndash13081 2009

[86] H Kajiwara M Yasuda N Kumaki T Shibayama and YOsamura ldquoExpression of carbohydrate antigens (SSEA-1 Sialyl-Lewis X DU-PAN-2 and CA19-9) and E-selectin in urothelialcarcinoma of the renal pelvis ureter and urinary bladderrdquoTokaiJournal of Experimental and Clinical Medicine vol 30 no 3 pp177ndash182 2005

[87] A M S Assreuy G J Martins M E F Moreira et al ldquoPre-vention of cyclophosphamide-induced hemorrhagic cystitis byglucose-mannose binding plant lectinsrdquo Journal of Urology vol161 no 6 pp 1988ndash1993 1999

[88] S M Vinson A Rickard J S Ryerse and J McHowat ldquoNeu-trophil adherence to bladder microvascular endothelial cellsfollowing platelet-activating factor acetylhydrolase inhibitionrdquoJournal of Pharmacology and Experimental Therapeutics vol314 no 3 pp 1241ndash1247 2005

[89] M Green A Filippou G Sant and T CTheoharides ldquoExpres-sion of intercellular adhesion molecules in the bladder ofpatients with interstitial cystitisrdquoUrology vol 63 no 4 pp 688ndash693 2004

[90] M Leppilahti P Hellstrom and T L J Tammela ldquoEffect ofdiagnostic hydrodistension and four intravesical hyaluronicacid instillations on bladder ICAM-1 intensity and associationof ICAM-1 intensity with clinical response in patients withinterstitial cystitisrdquo Urology vol 60 no 1 pp 46ndash51 2002

[91] A T Corcoran N Yoshimura V Tyagi B Jacobs W Lengand P Tyagi ldquoMapping the cytokine profile of painful bladdersyndromeinterstitial cystitis in human bladder and urinespecimensrdquoWorld Journal of Urology vol 31 no 1 pp 241ndash2462013

[92] SGarrean X-PGaoV Brovkovych et al ldquoCaveolin-1 regulatesNF-120581B activation and lung inflammatory response to sepsisinduced by lipopolysacchariderdquo Journal of Immunology vol 177no 7 pp 4853ndash4860 2006

[93] M R Kang G Yang R F Place et al ldquoIntravesical deliveryof small activating RNA formulated into lipid nanoparticlesinhibits orthotopic bladder tumor growthrdquo Cancer Researchvol 172 pp 5069ndash5079 2012

[94] P Tyagi D Barclay R Zamora et al ldquoUrine cytokines suggestan inflammatory response in the overactive bladder a pilotstudyrdquo International Urology and Nephrology vol 42 no 3 pp629ndash635 2010

[95] P Tyagi B L Jacobs D Barcaly et al ldquoUrine levels ofinflammatory chemokines can be novel biomarkers for theoveractive bladderrdquoThe Journal of Urology vol 181 article 5882009

[96] P Tyagi V Tyagi D Bui et al ldquoDiscrimination Of OAB fromICPBS by multivariate data modeling of urinary proteinsrdquoTheJournal of Urology vol 183 pp e614ndashe6e5 2010

[97] P Tyagi D Nikolavsky Y Vodovotz et al ldquoUrine levels ofselected chemokines positively correlate with lower bladdercapacity and psychometric scores in IcPbs patientsrdquo TheJournal of Urology vol 181 article 21 2009

[98] R Weissleder and V Ntziachristos ldquoShedding light onto livemolecular targetsrdquo Nature Medicine vol 9 no 1 pp 123ndash1282003

[99] C Bremer V Ntziachristos and R Weissleder ldquoOptical-basedmolecular imaging contrast agents and potentialmedical appli-cationsrdquo European Radiology vol 13 no 2 pp 231ndash243 2003

[100] UMahmood andRWeissleder ldquoNear-infrared optical imagingof proteases in cancerrdquoMolecular CancerTherapeutics vol 2 pp489ndash496 2003

[101] J Kaufman H Hensley J Jacobs et al ldquoNon-invasive imagingof near infrafred dye labeled liposomes facilitates evaluation ofbioresidence timerdquoThe Journal of Urology vol 183 article e6282010

[102] Y-X J Wang ldquoMedical imaging in pharmaceutical clinicaltrials what radiologists should knowrdquo Clinical Radiology vol60 no 10 pp 1051ndash1057 2005

ISRN Pharmacology 5

Lipotoxin

Intravesical instillation

Liposomes

+

BoNT-A

Figure 3 Scheme for formulation of BoNT into liposomes for blad-der instillation Freeze dried BoNT is resuspended in distilled waterand added to vials containing a desiccated liposomal preparationmade of phospholipids This reconstituted suspension is allowed tohydrate at room temperature for 30ndash60min before instillation

toxins instilled in the bladder fat-soluble neurotoxin such ascapsaicin can be integrated into the phospholipid bilayer [38]and water-soluble neurotoxin such as BoNT can be protectedinside the aqueous compartment(s) of liposomes delimitedby the phospholipid bilayer(s) [53] (Figure 3) Cystoscopeguided injections are the current standard practice in theclinic for administering BoNT to the bladder But in recentyears studies have also assessed the potential for intravesicalinstillation of BoNT alone in animal models of bladderirritation [54]

Development of instillation as a mode for administeringBoNT in patients will drastically bring down the cost oftreatment for patients with refractory overactive bladderOther groups have recently reported the use of DMSO forinstillation of BoNT instead of injection [55] DMSO doesnot afford the natural state of the BoNT as a protein and needto be formulated immediately before instillation DissolvingBoNT in DMSO may not be advisable owing to concernsof BoNT uptake into the systemic circulation of patientMoreover biochemical studies have demonstrated that met-alloproteolytic activity of the BoNT is strongly enhanced bythe presence of lipid membranes [56]

Recent studies reaffirmed the potential of liposomesas a promising vehicle for delivery of neurotoxins to thebladder [38 53] The transport of BoNT into urotheliumfrom liposomes was confirmed by detection of its uniqueeffect on neurotransmitters and proteolysis of synaptosomal-associated protein SNAP-25 through immunohistochemistry[53] The protection of BoNT entrapped inside liposomesfrom degradation in urine without compromising efficacywas demonstrated by attenuation of acetic acid induced blad-der irritation in rats [53] Similar results were obtained in pre-clinical studies with liposomes encapsulating capsaicin [38]

Liposomes have proven themselves as biocompatible deliveryagents in the bladder

7 Intravesical Antisense Therapeutics

The term ldquoantisenserdquo therapeutics emerged from seminalstudies done 4 decades using a short synthetic oligonu-cleotide for sequence-specific gene silencing [57] Genesilencing involves introduction of short strands of DNA(termed as antisense) with sequences complementary to themRNA encoding a particular gene inside the cell with theintent to block gene expression through either translationalinhibition or enzymatic cleavage of the mRNA target [3]

Oligonucleotide ODN binds specifically and stronglyto the mRNA target through Watson-Crick base pairingODN can be basically categorized into those that directcleavage of the target mRNA as caused by small interferingRNAs (siRNAs) and those that alter mRNA translationwithout causing mRNA cleavage Recent discovery of smallinterfering RNAs (siRNAs) and the elucidation of the RNAinterference (RNAi) pathway has also brought a sea changein the control of posttranscriptional gene expression siRNAtakes advantage of endogenous cellular pathways to potentlysilence the expression of specific genes (Figure 4)

Antisense mechanism is a promising approach for devel-oping therapeutics based on rational gene-based drug designAntisense therapeutics have been under clinical investigationfor more than 30 years [58] However drug development ofthis approach has been hampered by inefficient intracellulardelivery and cellular uptake of the ODN The translation ofbasic antisense research into therapeutics is also impeded byintracellular stability of ODN and potential for ldquooff-targetrdquogene silencing immunostimulation and other side effects

A vast array of chemical modifications to ODN has beendeveloped to overcome the therapeutically limiting featuresby altering internucleotide phosphate linkages backbonesugars or nucleobases One such modified ODN is peptidenucleic acid which replaces phosphodiester bond betweennucleobases with a peptide bond Replacement of phosphodi-ester bond by a phosphorothioate linkage is another methodto improve the stability of ODN against nucleases

Great progress has been made in translating antisenseresearch into clinical therapies based on local injection intoeye [58]The field recently progressed further with a systemicinjection therapy for treating familial hypercholesterolemia[59] Antisense therapeutics have been used for exon skippingto optimize the functionality of a truncated dystrophinprotein in dog model of Duchenne muscular dystrophy [60]

Nevertheless applied research for bladder diseases haslagged behind considering that the anatomical architec-ture of bladder provides ease of local administration withrestricted systemic side effects due to lower serum uptakeof antisense ODN Bladder instillation of antisense ODNor their chemically modified mimics can therefore be anefficient means to control the expression of therapeuticallyrelevant genes Antisense agents can also be used to elucidatethe role of newly discovered genes in bladder function

6 ISRN Pharmacology

Liposomes with antisense

Transcription

Genomic DNA RNA Duplex

Antisense DNA

Activated PI 3-kinase

Activated receptortyrosine kinase

NGF

Anti-NGFantibodies

mRNAdegradation

PIP2 PIP3

PP P P P P

oligonucleotides

Figure 4 Illustration of mechanisms for blocking NGF expression by either NGF antibodies or downregulation of NGF mRNA expressionNGF antibody sequesters the freely available NGF and prevents it from binding with the cognate TrkA receptors NGF antisense selectivelybinds with NGF mRNA inside the cells to block the translation of corresponding mRNA siRNA takes advantage of endogenous cellularpathways to degrade NGF mRNA and control the posttranscriptional NGF gene expression

8 NGF Expression in Bladder

Several studies have reported that patients with OAB orICPBS excrete more nerve growth factor (NGF) in theirurine relative to asymptomatic controls [61] Higher expres-sion of NGF in bladder of patients [62] with correspondinglower serum levels of NGFmakes the bladder tissue the likelysource for the elevated NGF in urine [63] Previous studieshave indicated that increased levels of NGF in the bladderand bladder afferent pathways are directly involved in theemergence of hyperexcitability of C-fiber bladder sensorypathways leading to the pathology of DO andOAB (Figure 4)[64]

In addition intrathecal application of NGF antibodiesreducedNGF levels in bladder afferent pathways and normal-ized bladderurethral function in spinal cord injured (SCI)rats [65] Because it is likely that the major pathology of OABis driven by NGF targeting the intracellular synthesis of NGFmolecule in bladder is a promising therapeutic alternative

9 NGF Expression a Drug Target

Overexpression of NGF can be blocked either directly byantibodies [66] or by blocking the synthesis of NGF proteinfrom mRNA [67] Systemic administration of monoclonalhuman NGF antibodies (tanezumab) has been explored fortherapeutic outcomes in ICPBS patients but not withoutencountering safety concerns such as paresthesia hypoesthe-sia and arthralgia [66] Generalized blockade ofNGF activityat sites other than bladder by anti-NGF antibodies maynot be the preferred outcome because NGF is an essentialhousekeeping growth factor necessary for the survival andgrowth of neurons [68] The physiological necessity of NGF

action at those sites may explain the incidence of paresthesiahypoesthesia and arthralgia in patients treated with systemicanti-NGF antibodies [66]

91 Peptide Nucleic Acid (PNA) Therefore to reduce thetoxicity of systemic blockade of NGF we sought to developa novel intravesical therapy of OAB by targeting the intra-cellular synthesis of NGF in the urothelium Antisense ODNneeded to be able to cross cell membrane to act as a drugand negatively charged ODN will not pass through a lipidlayer such as cell membranes Bladder uptake of ODN islimited by the anionic charge and size of the ODN as wellas anionic glycosaminoglycan layer of the inner bladdersurface Therefore primary impediment to be overcome inthe development of intravesical antisense therapeutics isinefficient bladder uptake of the ODN across urotheliumPrevious studies showed that heparan sulfate proteoglycansexpressed on cell surface act as receptors for extracellularTAT uptake [69] Therefore it was reasoned that GAG layeron bladder surface can facilitate bladder uptake of peptidenucleic acid PNA if it could be conjugatedwith synthetic TATpeptide

Water insoluble peptide nucleic acid targeting NGF wasconjugated with cell penetrating cationic peptide TAT forintracellular delivery of antisense moiety to demonstrate effi-cacy in animal models Studies showed that PNA conjugatedwith TAT suppressed cyclophosphamide cystitis followinglocal instillation of antisense against NGF [67] There wasnegligible uptake of peptide nucleic acid in absence of TATconjugation Prior to determination of in vivo efficacy ofconjugate suitable target sequence on NGF mRNA wasdetermined by predicted folding structure and cell transfec-tion experiments Successful intravesical delivery of peptide

ISRN Pharmacology 7

nucleic acid in bladder at the same time of cyclophosphamideinjection protected against the cystitis by blocking the rise inbladder contraction frequency and inflammation

92 Phosphorothioate-Linked Analogues Considering thedifficulty and possible nonspecific toxicity of peptide nucleicacid and TAT recent developments focused on simplifyingthe approach using water-soluble phosphorothioated ODNwhich have an increased resistance to exo- and endonucleasesfor improved stability [70] As alluded to in the above textsuccess of antisense therapeutics is largely dependent on thedevelopment of a delivery vehicle that can efficiently deliverantisense ODN in bladder Preliminary studies showed thatliposomes can be far better biocompatible effective carriersfor local gene silencing of NGF gene in bladder cells

Cationic liposomes and mimics have emerged as themost popular nonviral method to deliver nucleic acids intherapeutic applications Easy and reversible complex forma-tion of cationic liposomes with ODN at room temperatureallows their use as carriers Electrostatic attraction betweenthe cationic lipid DOTAP and the polyanionic antisenseODN is responsible for the complex formation The efficacyof liposome delivered siRNA by intravesical route has beenpreviously demonstrated in preclinical models of bladdercancer [71] Residence of ODN in the rat bladder after intrav-esical instillation was demonstrated to be longer than 24 husing fluorescent tagged ODN complexed with liposomes[72] Fluorescent probe was localized in bladder urotheliumcells 24 h following instillation The 24 h residence time wasalso demonstrated for siRNA in mouse bladder [73] Bladderuptake of fluorescent ODN without liposomes in normalrat bladder is poor Previous studies have shown that veryhigh concentration of phosphorothioated ODN can deliverODN without liposomes to bladder cells of mice havingbladder cancer [74] The urothelium barrier may be slightlycompromised in cancerous condition and the strategy ofloading bladder with high dose of ODN may not workin noncancerous diseased condition of bladder with intactbarrier

In order to evaluate the efficacy of NGF antisense ODNacetic acid infusion was used to cause a rapid rise of NGFprotein levels A single dose instillation of OND complexedwith liposomes protected against bladder overactivity (BO)induced by acetic acid Together with data of bladder uptakestudies using fluorescent ODN it is demonstrated that ONDis readily available to the bladder after intravesical instillationIt has been previously reported that within 2 h of exposureto irritants such as turpentine and acetic acid there occursa rapid rise in bladder content of NGF [75] Later studiesfound that bladder responds to insults with upregulation inthe genes for NGF sE-Selectin and receptor for monocytechemoattractant protein-1 (MCP-1) within 30min of expo-sure to lipopolysaccharide (Figure 5) [76] The acetic acidinduced overexpression of NGF was blunted by pretreatmentwith NGF antisense OND with phosphorothioate linkageprior to exposure of acetic acid The downregulation of NGFmRNA expression was in agreement with reduced proteinlevels and suppressed NGF-like immunoreactivity in theurothelium

93 Antisense and Downstream Signalling of NGF Drugdevelopment of intravesical antisense for NGF was also ableto unmask the downstream signaling [77] involving NGFfollowing exposure to acetic acid Experiments supportedthe role of NGF as a paracrine messenger [77] which isknown to activate several downstream effectors to manifestphysiological and pathological signaling changes linked to it[76 78ndash80] The bladder injury set off by acetic acid initiatesthe signalling cascades that upregulate the expression ofNGF and other chemokines MCP-1 CXCL-1 and CXCL-10 and prostaglandins [76 78ndash80] As reported elsewherelocalization of chemokines within synaptic vesicles in neu-rons [81] is consistent with their ability to act as excitatoryneurotransmitters following AA exposure

Chemokines are one of several downstream effectorsactivated by NGF [76 78ndash80] and interestingly chemokinereceptors are widely expressed in neural and nonneuralelements of the nociceptive pathways that are responsible forvisceral and somatic pain sensation [82] MCP-1 [83] andCXCL-10 [84] are constitutively expressed in neurons wherethey participate in excitability of primary afferent neuronsthrough transactivation of transient receptor channels andnociceptor sensitization [84] Overexpression ofNGF is likelyto drive the expression of VEGF fromneurons and leptin [78]from adipocytes covering neurons [85]

Cooperative expression of NGF and MCP-1 is able toinduce hyperexcitability in neurons by activating TRPV1receptor In addition MCP-1 CXCL-1 and CXCL-10 causechemoattraction of monocytes neutrophils and lympho-cytes respectively to mediate the bladder injury set offby acetic acid Extravasation is an essential prerequisitefor infiltration of monocytes neutrophils and lymphocyteswhich requires the expression of adhesion molecules likeE-selectin and intracellular adhesion molecule ICAM-1 [7686] E-selectin is a cytokine-inducible adhesion moleculethat supports the rolling and stable arrest of leukocyteson activated vascular endothelium Expression of E-selectinhas been linked to adherence of neutrophils to bladdermicrovascular endothelial cells and to cyclophosphamidecystitis [87 88] E-selectin gene expression is activated bythe NF-120581B and MAP kinase signal transduction pathways[76 86]

Activation and recruitment of leukocytes to acetic acidinduced bladder injury also require expression of intra-cellular adhesion molecule ICAM-1 [89] ICAM-1 interactsspecifically with its receptors of the integrin family to inducereversible cell-cell interactions involving adhesion Expres-sion of ICAM-1 was found to be increased in patients withICPBS and reduced in patients responding to instillation ofhyaluronic acid [90] Expression of ICAM-1 and VCAM-1was also noted in a recent study on biopsy tissue of ICPBSpatients [91] Increased expression of ICAM-1 subsequentto exposure with acetic acid in animal model is thereforeclinically relevant

Antisense experiments supported the earlier report thatinhibition of NGF expression significantly downregulates theexpression of ICAM-1 [80] It is reported that binding ofNGF to its high-affinity TrkA receptor controls the sICAM-1expression on target cells [80] Expression of ICAM-1 was

8 ISRN Pharmacology

Neutrophil

Monocytes

Acetic acid

Integrin

NGF

MCP-1

Release of cytokinesVanilloid

receptor

Trk receptors NGF

Membrane damage

VEGF

CXCL1

Activated leucocyte

Startrolling

Extravasation

CXCL10

sICAMssE-selectin

ActivationAdhesion

MCP-1

NGF

PKC

DAG

PLC

IP3Kinases

Ras

AKT

PI3K

CXCL10

sICAMs

LymphocyteAfferents

Figure 5 Signalling cascades induced by NGF overexpression following exposure to acetic acid Injury from exposure to acetic sets off thesignalling cascades that upregulate the expression of NGF MCP-1 prostaglandins CXCL-1 and CXCL-10 Overexpression of NGF drives theexpression of VEGF from neurons and leptin from adipocytes covering neurons NGF and MCP-1 also induce hyperexcitability in neuronsby activating TRPV1 receptor MCP-1 CXCL-1 and CXCL-10 cause chemoattraction of monocytes neutrophils and lymphocytes whichrequires extravasation across endothelium from blood through the cooperation of adhesion molecules E-selectin and ICAM-1

negatively associated with NGF expression in antisenseexperiments Ultimately expression of ICAM-1 is regulated atthe level of transcription by one of several signaling cascadesNF-120581B JAKSTAT IFN-g (CXCL-10) AP MAP kinase andPKC [92]

Intravesical route can allow selective exposure of anti-sense ODN to the NGF producing cells in urothelium andavoid systemic side effects from genetic manipulation ofNGF expression Intravesical route can also prove to be costeffective given the cost of ODN or siRNA Contrary to thegoal of loss of function in bladder with antisense ODN inrecent studies liposomes have also been used to deliver ODNfor gain of function in bladder [93]

10 Imaging of Intravesical Therapy

Drug development for DO and ICPBS relies heavily onsubjective outcomes for predicting efficacy and safety in earlyclinical development Although there are many validatedmeasurement tools used in DO and ICPBS research theyare usually burdensome and do not capture all symptomsrelated to the lower urinary tract Moreover improvement insymptoms scores following an intervention does not alwayscorrelate with patient expectations satisfaction and goalachievement which are critically important for successfulmanagement of DO and ICPBS

There is underdevelopment and underutilization of urinebiomarkers and imaging methods for investigational drugsgiven by intravesical route Overreliance on subjectiveimpressions of patients for therapeutic response limits clini-cal relevance due to weak correlation with patient satisfactionand can also impede continued scientific progress in assessing

study outcomes relative to symptom bother Symptom scoresmay also be related to the cause of high heterogeneity inclinical response in DO and ICPBS patients

Imaging can offer the possibility to provide region-specific information in situations where serum and urineassays may not reflect the true state of bladder [94ndash96]Urine chemokines have been reported to be associated withsymptom severity of ICPBS patients [97] Application offluorescent microscopy for imaging of pelvic floor has beenlimited due to the issues pertaining to light absorption bytissues scattering and autofluorescence [98]

Recent studies have shown that dyes fluorescing inthe near-infrared (NIR) band can overcome this handi-cap in deep-tissue imaging of experimental animals [99]Selected NIR fluorochromes emit light with tissue pen-etration approaching 10ndash15 cm [100] The light emissionfrom probes in this spectrum encounters low backgroundautofluorescence and minimal attenuation of signal due tolight absorption by tissue components [98] Therefore NIRimaging yields high signal-to-noise ratios and is well suitedfor studying the distribution of instilled treatments in bladder(Figure 2(b))

Preliminary studies suggest that imaging of mouse pelvicfloor in NIR spectrum can be a viable option [101] NIRimaging easily allows visualization and quantification ofbladder distribution by tracing themigration of fluorescence-labeled liposomes in anaesthetized mouse Compared withother biological assays imaging could provide objectiveendpoint for patients with ulcerative cystitis to indicate itshigh translation potential Other benefits of optical imaginginclude its relatively low cost compared withmore traditionalimaging systems such as MRI and PET The instrument

ISRN Pharmacology 9

consists of two near-infrared lasers emitting at 685 nm and785 nm camera and novel optics

Furthermore preferential binding of liposomes andnanoparticles to bladder lesions of ICPBS after instillationcan be assessed by NIR imaging of bladder where it cantheoretically provide anatomically specific information aboutthe real time status of ulcerlesion in bladder surface or tissueabnormality [102] Therefore future studies can substitute aresponse variable that is continuous in nature instead of asubjective clinical outcome

11 Conclusions

Adoption of forward-thinking approaches can achieveadvancements in drug delivery systems targeted to improvepharmacotherapy of bladder diseases in the future Latestdevelopments in the field of nanotechnology can bring thismode of therapy from second line of treatment for refractorycases to the forefront of disease management Liposomesare an attractive drug delivery platform by virtue of theirbiodegradability biocompatibility low toxicity and simpleand mild preparation methods

Conflict of Interests

All authors except Dr Michael Chancellor declare that thereis no conflict of interests regarding the publication of thispaper Dr Michael Chancellor owns stocks in the start-upliposome company Lipella

Acknowledgments

This work was supported by the National Institutes of Health(DK057267 and DK088836) and the Department of Defense(W81XWH-11-1-0763 and W81XWH-12-1-0565)

References

[1] J Parkin C Shea and G R Sant ldquoIntravesical dimethyl sul-foxide (DMSO) for interstitial cystitismdasha practical approachrdquoUrology vol 49 no 5 pp 105ndash107 1997

[2] C Dowson J Watkins M S Khan P Dasgupta and A SahaildquoRepeated botulinum toxin type A injections for refractoryoveractive bladder medium-term outcomes safety profile anddiscontinuation ratesrdquo European Urology vol 61 no 4 pp 834ndash839 2012

[3] J Kaufman V Tyagi M Anthony M B Chancellor and PTyagi ldquoState of the art in intravesical therapy for lower urinarytract symptomsrdquo Reviews in Urology vol 12 pp e181ndashe189 2010

[4] N Yoshimura and M B Chancellor ldquoCurrent and futurepharmacological treatment for overactive bladderrdquo Journal ofUrology vol 168 no 5 pp 1897ndash1913 2002

[5] K A Killinger J A Boura and K M Peters ldquoPain ininterstitial cystitisbladder pain syndrome do characteristicsdiffer in ulcerative and non-ulcerative subtypesrdquo InternationalUrogynecology Journal vol 24 no 8 pp 1295ndash1301 2013

[6] J C Nickel P Hanno K Kumar and HThomas ldquoSecond mul-ticenter randomized double-blind parallel-group evaluationof effectiveness and safety of intravesical sodium chondroitin

sulfate compared with inactive vehicle control in subjects withinterstitial cystitisbladder pain syndromerdquoUrology vol 79 no6 pp 1220ndash1225 2012

[7] P M Hanno ldquoAnalysis of long-term elmiron therapy forinterstitial cystitisrdquo Urology vol 49 no 5 pp 93ndash99 1997

[8] A Giannantoni V Bini R Dmochowski et al ldquoReply to ClausRiedlrsquos letter to the editor re antonella Giannantoni VittorioBini Roger Dmochowski et al Contemporary management ofthe painful bladder a systematic reviewrdquo EuropeanUrology vol61 no 5 pp 29ndash53 2012

[9] MC Lai Y CKuo andHCKuo ldquoIntravesical hyaluronic acidfor interstitial cystitispainful bladder syndrome a comparativerandomized assessment of different regimensrdquo InternationalJournal of Urology vol 20 pp 203ndash207 2013

[10] J Eldrup J Thorup and S L Nielsen ldquoPermeability andultrastructure of human bladder epitheliumrdquo British Journal ofUrology vol 55 no 5 pp 488ndash492 1983

[11] C L Parsons D Boychuk S Jones R Hurst and H CallahanldquoBladder surface glycosaminoglycans an epithelial permeabil-ity barrierrdquo Journal of Urology vol 143 no 1 pp 139ndash142 1990

[12] Y Kim N Yoshimura H Masuda F De Miguel and MB Chancellor ldquoAntimuscarinic agents exhibit local inhibitoryeffects on muscarinic receptors in bladder-afferent pathwaysrdquoUrology vol 65 no 2 pp 238ndash242 2005

[13] V Tyagi B J Philips R Su et al ldquoDifferential expression offunctional cannabinoid receptors in human bladder detrusorand urotheliumrdquo Journal of Urology vol 181 no 4 pp 1932ndash1938 2009

[14] B J Philips M C Smaldone V L Erickson N YoshimuraM B Chancellor and P Tyagi ldquoDifferential expression ofneurokinin receptor subtypes in urothelium and detrusor ofhuman bladderrdquo Journal of Urology vol 179 article 354 2008

[15] S A Baker G W Hennig J Han F C Britton T K Smithand S D Koh ldquoMethionine and its derivatives increase blad-der excitability by inhibiting stretch-dependent K+ channelsrdquoBritish Journal of Pharmacology vol 153 no 6 pp 1259ndash12712008

[16] J M Beckel A Kanai S-J Lee W C De Groat and LA Birder ldquoExpression of functional nicotinic acetylcholinereceptors in rat urinary bladder epithelial cellsrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 290 no 1 ppF103ndashF110 2006

[17] LA Birder A J KanaiWCDeGroat et al ldquoVanilloid receptorexpression suggests a sensory role for urinary bladder epithelialcellsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 23 pp 13396ndash13401 2001

[18] P Tyagi C A Thomas N Yoshimura and M B ChancellorldquoInvestigations into the presence of functional szlig1 szlig2 and szlig3-adrenoceptors in urothelium and detrusor of human bladderrdquoInternational Brazilian Journal of Urology vol 35 no 1 pp 76ndash83 2009

[19] A T Hanna-Mitchell J M Beckel S Barbadora A J Kanai WC de Groat and L A Birder ldquoNon-neuronal acetylcholine andurinary bladder urotheliumrdquo Life Sciences vol 80 no 24-25 pp2298ndash2302 2007

[20] C P Smith D A Gangitano A Munoz et al ldquoBotulinumtoxin type A normalizes alterations in urothelial ATP and NOrelease induced by chronic spinal cord injuryrdquo NeurochemistryInternational vol 52 no 6 pp 1068ndash1075 2008

[21] Y-C Chuang C A Thomas S Tyagi N Yoshimura PTyagi and M B Chancellor ldquoHuman urine with solifenacin

10 ISRN Pharmacology

intake but not tolterodine or darifenacin intake blocks detrusoroveractivityrdquo International Urogynecology Journal and PelvicFloor Dysfunction vol 19 no 10 pp 1353ndash1357 2008

[22] Y Kim N Yoshimura H Masuda F D Miguel and M BChancellor ldquoIntravesical instillation of human urine after oraladministration of trospium tolterodine and oxybutynin in a ratmodel of detrusor overactivityrdquo BJU International vol 97 no 2pp 400ndash403 2006

[23] S Tyagi P Tyagi S Van-le N YoshimuraM B Chancellor andF de Miguel ldquoQualitative and quantitative expression profileof muscarinic receptors in human urothelium and detrusorrdquoJournal of Urology vol 176 no 4 pp 1673ndash1678 2006

[24] G Gregoriadis and B E Ryman ldquoLiposomes as carriers ofenzymes or drugs a new approach to the treatment of storagediseasesrdquo Biochemical Journal vol 124 no 5 p 58 1971

[25] G Gregoriadis S Jain I Papaioannou and P Laing ldquoImprov-ing the therapeutic efficacy of peptides and proteins a role forpolysialic acidsrdquo International Journal of Pharmaceutics vol300 no 1-2 pp 125ndash130 2005

[26] P Sapra P Tyagi and T M Allen ldquoLigand-targeted liposomesfor cancer treatmentrdquo Current Drug Delivery vol 2 no 4 pp369ndash381 2005

[27] G Gregoriadis G Dapergolas and E D Neerunjun ldquoPene-tration of target areas in the rat by liposome associated agentsadministered parenterally and intragastricallyrdquo BiochemicalSociety Transactions vol 4 no 2 pp 256ndash259 1976

[28] G Gregoriadis ldquoEngineering liposomes for drug deliveryprogress and problemsrdquo Trends in Biotechnology vol 13 no 12pp 527ndash537 1995

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[30] S Lee S Dausch CMaierhofer andD Dausch ldquoA new therapyconcept with a liposome eye spray for the treatment of the lsquodryeyersquordquo Klinische Monatsblatter fur Augenheilkunde vol 221 no10 pp 825ndash836 2004

[31] D Dausch S Lee S Dausch J C Kim G Schwert and WMichelson ldquoComparative study of treatment of the dry eyesyndrome due to disturbances of the tear film lipid layer withlipid-containing tear substitutes efficacy of lipid-containingtear substitutesrdquo Klinische Monatsblatter fur Augenheilkundevol 223 no 12 pp 974ndash983 2006

[32] S Ebrahim G A Peyman and P J Lee ldquoApplications ofliposomes in ophthalmologyrdquo Survey of Ophthalmology vol 50no 2 pp 167ndash182 2005

[33] J Nirmal P Tyagi L Dang et al ldquoEndocytosis uptake ofliposomes in urothelium cells detected by transmission electronmicroscopyrdquoThe Journal of Urology vol 187 article e15 2012

[34] D N Frangos J J Killion D Fan R Fishbeck A C VonEschenbach and I J Fidler ldquoThe development of liposomescontaining interferon alpha for the intravesical therapy ofhuman superficial bladder cancerrdquo Journal of Urology vol 143no 6 pp 1252ndash1266 1990

[35] J W Johnson R Nayar J J Killion A C Von Eschenbachand I J Fidler ldquoBinding of liposomes to human bladdertumor epithelial cell lines implications for an intravesical drugdelivery system for the treatment of bladder cancerrdquo SelectiveCancer Therapeutics vol 5 no 4 pp 147ndash155 1989

[36] Y-C Chuang M B Chancellor S Seki et al ldquoIntravesicalprotamine sulfate and potassium chloride as a model forbladder hyperactivityrdquoUrology vol 61 no 3 pp 664ndash670 2003

[37] J Kaufman P Tyagi and M B Chancellor ldquoIntravesicalliposomal (Lp08) instillation protects bladder urothelium fromchemical irritationrdquoThe Journal of Urology vol 181 article 5392009

[38] P Tyagi M B Chancellor Z Li et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical capsaicindelivery using thermosensitive hydrogel and liposomesrdquo Jour-nal of Urology vol 171 no 1 pp 483ndash489 2004

[39] MO Fraser Y-CChuang P Tyagi et al ldquoIntravesical liposomeadministrationmdasha novel treatment for hyperactive bladder inthe ratrdquo Urology vol 61 no 3 pp 656ndash663 2003

[40] P Tyagi M Chancellor N Yoshimura and L Huang ldquoActivityof different phospholipids in attenuating hyperactivity in blad-der irritationrdquo BJU International vol 101 no 5 pp 627ndash6322008

[41] Y Sun and T C Chai ldquoEffects of dimethyl sulphoxide andheparin on stretch-activated ATP release by bladder urothelialcells from patients with interstitial cystitisrdquo BJU Internationalvol 90 no 4 pp 381ndash385 2002

[42] J J Bade ldquoA placebo-controlled study of intravesical pentosan-polysulphate for the treatment of interstitial cystitisrdquo BritishJournal of Urology vol 79 no 2 pp 168ndash171 1997

[43] P Tyagi V C Hsieh N Yoshimura J Kaufman and M BChancellor ldquoInstillation of liposomes vs dimethyl sulphoxide orpentosan polysulphate for reducing bladder hyperactivityrdquo BJUInternational vol 104 no 11 pp 1689ndash1692 2009

[44] W-C Lee C-T Chien H-J Yu and S-W Lee ldquoBladderdysfunction in rats with metabolic syndrome induced by long-term fructose feedingrdquo Journal of Urology vol 179 no 6 pp2470ndash2476 2008

[45] J I Gillespie G A Van Koeveringe S G DeWachter and J DeVente ldquoOn the origins of the sensory output from the bladderthe concept of afferent noiserdquo BJU International vol 103 no 10pp 1324ndash1333 2009

[46] W C Lee P H Chiang Y L Tain C C Wu and Y CChuang ldquoSensory dysfunction of bladder mucosa and bladderoversensitivity in a rat model of metabolic syndromerdquo PloSONE vol 7 Article ID e45578 2012

[47] Y-C Chuang W-C Lee W-C Lee and P-H Chiang ldquoIntrav-esical liposome versus oral pentosan polysulfate for interstitialcystitispainful bladder syndromerdquo Journal of Urology vol 182no 4 pp 1393ndash1400 2009

[48] W-C Lee Y-C Chuang W-C Lee and P-H Chiang ldquoSafetyand dose flexibility clinical evaluation of intravesical liposomein patients with interstitial cystitis or painful bladder syn-dromerdquo Kaohsiung Journal of Medical Sciences vol 27 no 10pp 437ndash440 2011

[49] S T Truschel E Wang W G Ruiz et al ldquoStretch-regulatedexocytosisendocytosis in bladder umbrella cellsrdquo MolecularBiology of the Cell vol 13 no 3 pp 830ndash846 2002

[50] M B Chancellor and W C De Groat ldquoIntravesical capsaicinand resiniferatoxin therapy spicing up the ways to treat theoveractive bladderrdquo Journal of Urology vol 162 no 1 pp 3ndash111999

[51] D S Byrne A Das J Sedor et al ldquoEffect of intravesicalcapsaicin and vehicle on bladder integrity in control and spinalcord injured ratsrdquo Journal of Urology vol 159 no 3 pp 1074ndash1078 1998

[52] M Mandal and K-D Lee ldquoListeriolysin O-liposome-mediatedcytosolic delivery of macromolecule antigen in vivo enhance-ment of antigen-specific cytotoxic T lymphocyte frequency

ISRN Pharmacology 11

activity and tumor protectionrdquo Biochimica et Biophysica Actavol 1563 no 1-2 pp 7ndash17 2002

[53] Y-C Chuang P Tyagi C-C Huang et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical botulinumtoxin a delivery using liposomesrdquo Journal of Urology vol 182no 2 pp 786ndash792 2009

[54] Y-C Chuang N Yoshimura C-C Huang P-H Chiang andM B Chancellor ldquoIntravesical botulinum toxin a administra-tion produces analgesia against acetic acid induced bladder painresponses in ratsrdquo Journal of Urology vol 172 no 4 pp 1529ndash1532 2004

[55] S P Petrou A S Parker J E Crook A Rogers D Metz-Kudashick and D D Thiel ldquoBotulinum A toxindimethylsulfoxide bladder instillations for women with refractory idio-pathic detrusor overactivity a phase 12 studyrdquo Mayo ClinicProceedings vol 84 no 8 pp 702ndash706 2009

[56] P Caccin O Rossetto M Rigoni E Johnson G Schiavoand C Montecucco ldquoVAMPsynaptobrevin cleavage by tetanusand botulinum neurotoxins is strongly enhanced by acidicliposomesrdquo FEBS Letters vol 542 no 1ndash3 pp 132ndash136 2003

[57] M L Stephenson and P C Zamecnik ldquoInhibition of Roussarcoma viral RNA translation by a specific oligodeoxyribonu-cleotiderdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 75 no 1 pp 285ndash288 1978

[58] H Dadgostar and N Waheed ldquoThe evolving role of vascularendothelial growth factor inhibitors in the treatment of neovas-cular age-related macular degenerationrdquo Eye vol 22 no 6 pp761ndash767 2008

[59] C Gebhard G Huard E A Kritikou and J C TardifldquoApolipoprotein B antisense inhibitionmdashupdate on mipom-ersenrdquo Current Pharmaceutical Design vol 919 no 17 pp 3132ndash3142 2013

[60] T Yokota E Hoffman and S Takeda ldquoAntisense oligo-mediated multiple exon skipping in a dog model of duchennemuscular dystrophyrdquoMethods inMolecular Biology vol 709 pp299ndash312 2011

[61] B L Jacobs M C Smaldone V Tyagi et al ldquoIncreased nervegrowth factor in neurogenic overactive bladder and interstitialcystitis patientsrdquoThe Canadian Journal of Urology vol 17 no 1pp 4989ndash4994 2010

[62] H-T Liu and H-C Kuo ldquoIntravesical botulinum toxin Ainjections plus hydrodistension can reduce nerve growth factorproduction and control bladder pain in interstitial cystitisrdquoUrology vol 70 no 3 pp 463ndash468 2007

[63] H T Liu and H C Kuo ldquoIncreased urine and serum nervegrowth factor levels in interstitial cystitis suggest chronicinflammation is involved in the pathogenesis of diseaserdquo PloSONE vol 7 Article ID e44687 2012

[64] N Yoshimura N E Bennett Y Hayashi et al ldquoBladderoveractivity and hyperexcitability of bladder afferent neuronsafter intrathecal delivery of nerve growth factor in ratsrdquo Journalof Neuroscience vol 26 no 42 pp 10847ndash10855 2006

[65] S Seki K Sasaki Y Igawa et al ldquoSuppression of detrusor-sphincter dyssynergia by immunoneutralization of nervegrowth factor in lumbosacral spinal cord in spinal cord injuredratsrdquo Journal of Urology vol 171 no 1 pp 478ndash482 2004

[66] R J Evans R M Moldwin N Cossons A Darekar I WMills andD Scholfield ldquoProof of concept trial of tanezumab forthe treatment of symptoms associated with interstitial cystitisrdquoJournal of Urology vol 185 no 5 pp 1716ndash1721 2011

[67] P Tyagi R Banerjee S Basu N YoshimuraM Chancellor andL Huang ldquoIntravesical antisense therapy for cystitis using TAT-peptide nucleic acid conjugatesrdquoMolecular Pharmaceutics vol3 no 4 pp 398ndash406 2006

[68] D C Molliver D E Wright M L Leitner et al ldquoIB4-bindingDRG neurons switch from NGF to GDNF dependence in earlypostnatal liferdquo Neuron vol 19 no 4 pp 849ndash861 1997

[69] M TyagiM RusnatiM Presta andMGiacca ldquoInternalizationof HIV-1 tat requires cell surface heparan sulfate proteoglycansrdquoJournal of Biological Chemistry vol 276 no 5 pp 3254ndash32612001

[70] E Uhlmann A Ryte and A Peyman ldquoStudies on the mech-anism of stabilization of partially phosphorothioated oligonu-cleotides against nucleolytic degradationrdquoAntisense andNucleicAcid Drug Development vol 7 no 4 pp 345ndash350 1997

[71] M Nogawa T Yuasa S Kimura et al ldquoIntravesical adminis-tration of small interfering RNA targeting PLK-1 successfullyprevents the growth of bladder cancerrdquo Journal of ClinicalInvestigation vol 115 no 4 pp 978ndash985 2005

[72] M Kashyap N Kawamorita V Tyagi et al ldquoDownregulation ofNGF expression in the bladder by antisense oligoucleotides asnew treatment for overactive bladderrdquo Journal of Urology vol27190 no 2 pp 757ndash764 2013

[73] C-J Arum Y Kodama N Rolim et al ldquoA rat model ofintravesical delivery of small interfering RNA for studyingurinary carcinomardquoWorld Journal of Urology vol 28 no 4 pp479ndash485 2010

[74] C E Blietz B Thode M Hauses et al ldquoIn vivo studies on theavailability and toxicity of antisense oligonucleotides in bladdercancerrdquo In Vivo vol 23 no 1 pp 13ndash19 2009

[75] D Oddiah P Anand S B McMahon and M Rattray ldquoRapidincrease of NGF BDNF andNT-3mRNAs in inflamed bladderrdquoNeuroReport vol 9 no 7 pp 1455ndash1458 1998

[76] M R Saban H Hellmich N B Nguyen J Winston T GHammond and R Saban ldquoTime course of LPS-induced geneexpression in a mouse model of genitourinary inflammationrdquoPhysiol Genomics vol 5 no 3 pp 147ndash160 2001

[77] B Schnegelsberg T T Sun G Cain et al ldquoOverexpression ofNGF in mouse urothelium leads to neuronal hyperinnervationpelvic sensitivity and changes in urinary bladder functionrdquoTheAmerican Journal of PhysiologymdashRegulatory Integrative andComparative Physiology vol 298 no 5 pp R534ndashR547 2010

[78] S Cardenas M Scuri L Samsell et al ldquoNeurotrophic andneuroimmune responses to early-life Pseudomonas aeruginosainfection in rat lungsrdquo American Journal of PhysiologymdashLungCellular and Molecular Physiology vol 299 no 3 pp L334ndashL344 2010

[79] K Nakamura F Tan Z Li and C J Thiele ldquoNGF activationof TrkA induces vascular endothelial growth factor expressionvia induction of hypoxia-inducible factor-1120572rdquo Molecular andCellular Neuroscience vol 46 no 2 pp 498ndash506 2011

[80] S Othumpangat M Regier and G Piedimonte ldquoNerve growthfactor modulates human rhinovirus infection in airway epithe-lial cells by controlling ICAM-1 expressionrdquo American Journalof Physiology Lung Cellular andMolecular Physiology vol 15302pp L1057ndashL1066 2012

[81] H Jung P T Toth F A White and R J Miller ldquoMonocytechemoattractant protein-1 functions as a neuromodulator indorsal root ganglia neuronsrdquo Journal of Neurochemistry vol104 no 1 pp 254ndash263 2008

[82] F A White H Jung and R J Miller ldquoChemokines andthe pathophysiology of neuropathic painrdquo Proceedings of the

12 ISRN Pharmacology

National Academy of Sciences of the United States of Americavol 104 no 51 pp 20151ndash20158 2007

[83] G Banisadr R-D Gosselin P Mechighel W Rostene PKitabgi and S M Parsadaniantz ldquoConstitutive neuronalexpression of CCR2 chemokine receptor and its colocalizationwith neurotransmitters in normal rat brain functional effectof MCP-1CCL2 on calcium mobilization in primary culturedneuronsrdquo Journal of Comparative Neurology vol 492 no 2 pp178ndash192 2005

[84] S Bhangoo D Ren R J Miller et al ldquoDelayed functionalexpression of neuronal chemokine receptors following focalnerve demyelination in the rat a mechanism for the devel-opment of chronic sensitization of peripheral nociceptorsrdquoMolecular Pain vol 3 article 38 2007

[85] T Maeda N Kiguchi Y Kobayashi T Ikuta M Ozaki and SKishioka ldquoLeptin derived from adipocytes in injured periph-eral nerves facilitates development of neuropathic pain viamacrophage stimulationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 106 no 31 pp13076ndash13081 2009

[86] H Kajiwara M Yasuda N Kumaki T Shibayama and YOsamura ldquoExpression of carbohydrate antigens (SSEA-1 Sialyl-Lewis X DU-PAN-2 and CA19-9) and E-selectin in urothelialcarcinoma of the renal pelvis ureter and urinary bladderrdquoTokaiJournal of Experimental and Clinical Medicine vol 30 no 3 pp177ndash182 2005

[87] A M S Assreuy G J Martins M E F Moreira et al ldquoPre-vention of cyclophosphamide-induced hemorrhagic cystitis byglucose-mannose binding plant lectinsrdquo Journal of Urology vol161 no 6 pp 1988ndash1993 1999

[88] S M Vinson A Rickard J S Ryerse and J McHowat ldquoNeu-trophil adherence to bladder microvascular endothelial cellsfollowing platelet-activating factor acetylhydrolase inhibitionrdquoJournal of Pharmacology and Experimental Therapeutics vol314 no 3 pp 1241ndash1247 2005

[89] M Green A Filippou G Sant and T CTheoharides ldquoExpres-sion of intercellular adhesion molecules in the bladder ofpatients with interstitial cystitisrdquoUrology vol 63 no 4 pp 688ndash693 2004

[90] M Leppilahti P Hellstrom and T L J Tammela ldquoEffect ofdiagnostic hydrodistension and four intravesical hyaluronicacid instillations on bladder ICAM-1 intensity and associationof ICAM-1 intensity with clinical response in patients withinterstitial cystitisrdquo Urology vol 60 no 1 pp 46ndash51 2002

[91] A T Corcoran N Yoshimura V Tyagi B Jacobs W Lengand P Tyagi ldquoMapping the cytokine profile of painful bladdersyndromeinterstitial cystitis in human bladder and urinespecimensrdquoWorld Journal of Urology vol 31 no 1 pp 241ndash2462013

[92] SGarrean X-PGaoV Brovkovych et al ldquoCaveolin-1 regulatesNF-120581B activation and lung inflammatory response to sepsisinduced by lipopolysacchariderdquo Journal of Immunology vol 177no 7 pp 4853ndash4860 2006

[93] M R Kang G Yang R F Place et al ldquoIntravesical deliveryof small activating RNA formulated into lipid nanoparticlesinhibits orthotopic bladder tumor growthrdquo Cancer Researchvol 172 pp 5069ndash5079 2012

[94] P Tyagi D Barclay R Zamora et al ldquoUrine cytokines suggestan inflammatory response in the overactive bladder a pilotstudyrdquo International Urology and Nephrology vol 42 no 3 pp629ndash635 2010

[95] P Tyagi B L Jacobs D Barcaly et al ldquoUrine levels ofinflammatory chemokines can be novel biomarkers for theoveractive bladderrdquoThe Journal of Urology vol 181 article 5882009

[96] P Tyagi V Tyagi D Bui et al ldquoDiscrimination Of OAB fromICPBS by multivariate data modeling of urinary proteinsrdquoTheJournal of Urology vol 183 pp e614ndashe6e5 2010

[97] P Tyagi D Nikolavsky Y Vodovotz et al ldquoUrine levels ofselected chemokines positively correlate with lower bladdercapacity and psychometric scores in IcPbs patientsrdquo TheJournal of Urology vol 181 article 21 2009

[98] R Weissleder and V Ntziachristos ldquoShedding light onto livemolecular targetsrdquo Nature Medicine vol 9 no 1 pp 123ndash1282003

[99] C Bremer V Ntziachristos and R Weissleder ldquoOptical-basedmolecular imaging contrast agents and potentialmedical appli-cationsrdquo European Radiology vol 13 no 2 pp 231ndash243 2003

[100] UMahmood andRWeissleder ldquoNear-infrared optical imagingof proteases in cancerrdquoMolecular CancerTherapeutics vol 2 pp489ndash496 2003

[101] J Kaufman H Hensley J Jacobs et al ldquoNon-invasive imagingof near infrafred dye labeled liposomes facilitates evaluation ofbioresidence timerdquoThe Journal of Urology vol 183 article e6282010

[102] Y-X J Wang ldquoMedical imaging in pharmaceutical clinicaltrials what radiologists should knowrdquo Clinical Radiology vol60 no 10 pp 1051ndash1057 2005

6 ISRN Pharmacology

Liposomes with antisense

Transcription

Genomic DNA RNA Duplex

Antisense DNA

Activated PI 3-kinase

Activated receptortyrosine kinase

NGF

Anti-NGFantibodies

mRNAdegradation

PIP2 PIP3

PP P P P P

oligonucleotides

Figure 4 Illustration of mechanisms for blocking NGF expression by either NGF antibodies or downregulation of NGF mRNA expressionNGF antibody sequesters the freely available NGF and prevents it from binding with the cognate TrkA receptors NGF antisense selectivelybinds with NGF mRNA inside the cells to block the translation of corresponding mRNA siRNA takes advantage of endogenous cellularpathways to degrade NGF mRNA and control the posttranscriptional NGF gene expression

8 NGF Expression in Bladder

Several studies have reported that patients with OAB orICPBS excrete more nerve growth factor (NGF) in theirurine relative to asymptomatic controls [61] Higher expres-sion of NGF in bladder of patients [62] with correspondinglower serum levels of NGFmakes the bladder tissue the likelysource for the elevated NGF in urine [63] Previous studieshave indicated that increased levels of NGF in the bladderand bladder afferent pathways are directly involved in theemergence of hyperexcitability of C-fiber bladder sensorypathways leading to the pathology of DO andOAB (Figure 4)[64]

In addition intrathecal application of NGF antibodiesreducedNGF levels in bladder afferent pathways and normal-ized bladderurethral function in spinal cord injured (SCI)rats [65] Because it is likely that the major pathology of OABis driven by NGF targeting the intracellular synthesis of NGFmolecule in bladder is a promising therapeutic alternative

9 NGF Expression a Drug Target

Overexpression of NGF can be blocked either directly byantibodies [66] or by blocking the synthesis of NGF proteinfrom mRNA [67] Systemic administration of monoclonalhuman NGF antibodies (tanezumab) has been explored fortherapeutic outcomes in ICPBS patients but not withoutencountering safety concerns such as paresthesia hypoesthe-sia and arthralgia [66] Generalized blockade ofNGF activityat sites other than bladder by anti-NGF antibodies maynot be the preferred outcome because NGF is an essentialhousekeeping growth factor necessary for the survival andgrowth of neurons [68] The physiological necessity of NGF

action at those sites may explain the incidence of paresthesiahypoesthesia and arthralgia in patients treated with systemicanti-NGF antibodies [66]

91 Peptide Nucleic Acid (PNA) Therefore to reduce thetoxicity of systemic blockade of NGF we sought to developa novel intravesical therapy of OAB by targeting the intra-cellular synthesis of NGF in the urothelium Antisense ODNneeded to be able to cross cell membrane to act as a drugand negatively charged ODN will not pass through a lipidlayer such as cell membranes Bladder uptake of ODN islimited by the anionic charge and size of the ODN as wellas anionic glycosaminoglycan layer of the inner bladdersurface Therefore primary impediment to be overcome inthe development of intravesical antisense therapeutics isinefficient bladder uptake of the ODN across urotheliumPrevious studies showed that heparan sulfate proteoglycansexpressed on cell surface act as receptors for extracellularTAT uptake [69] Therefore it was reasoned that GAG layeron bladder surface can facilitate bladder uptake of peptidenucleic acid PNA if it could be conjugatedwith synthetic TATpeptide

Water insoluble peptide nucleic acid targeting NGF wasconjugated with cell penetrating cationic peptide TAT forintracellular delivery of antisense moiety to demonstrate effi-cacy in animal models Studies showed that PNA conjugatedwith TAT suppressed cyclophosphamide cystitis followinglocal instillation of antisense against NGF [67] There wasnegligible uptake of peptide nucleic acid in absence of TATconjugation Prior to determination of in vivo efficacy ofconjugate suitable target sequence on NGF mRNA wasdetermined by predicted folding structure and cell transfec-tion experiments Successful intravesical delivery of peptide

ISRN Pharmacology 7

nucleic acid in bladder at the same time of cyclophosphamideinjection protected against the cystitis by blocking the rise inbladder contraction frequency and inflammation

92 Phosphorothioate-Linked Analogues Considering thedifficulty and possible nonspecific toxicity of peptide nucleicacid and TAT recent developments focused on simplifyingthe approach using water-soluble phosphorothioated ODNwhich have an increased resistance to exo- and endonucleasesfor improved stability [70] As alluded to in the above textsuccess of antisense therapeutics is largely dependent on thedevelopment of a delivery vehicle that can efficiently deliverantisense ODN in bladder Preliminary studies showed thatliposomes can be far better biocompatible effective carriersfor local gene silencing of NGF gene in bladder cells

Cationic liposomes and mimics have emerged as themost popular nonviral method to deliver nucleic acids intherapeutic applications Easy and reversible complex forma-tion of cationic liposomes with ODN at room temperatureallows their use as carriers Electrostatic attraction betweenthe cationic lipid DOTAP and the polyanionic antisenseODN is responsible for the complex formation The efficacyof liposome delivered siRNA by intravesical route has beenpreviously demonstrated in preclinical models of bladdercancer [71] Residence of ODN in the rat bladder after intrav-esical instillation was demonstrated to be longer than 24 husing fluorescent tagged ODN complexed with liposomes[72] Fluorescent probe was localized in bladder urotheliumcells 24 h following instillation The 24 h residence time wasalso demonstrated for siRNA in mouse bladder [73] Bladderuptake of fluorescent ODN without liposomes in normalrat bladder is poor Previous studies have shown that veryhigh concentration of phosphorothioated ODN can deliverODN without liposomes to bladder cells of mice havingbladder cancer [74] The urothelium barrier may be slightlycompromised in cancerous condition and the strategy ofloading bladder with high dose of ODN may not workin noncancerous diseased condition of bladder with intactbarrier

In order to evaluate the efficacy of NGF antisense ODNacetic acid infusion was used to cause a rapid rise of NGFprotein levels A single dose instillation of OND complexedwith liposomes protected against bladder overactivity (BO)induced by acetic acid Together with data of bladder uptakestudies using fluorescent ODN it is demonstrated that ONDis readily available to the bladder after intravesical instillationIt has been previously reported that within 2 h of exposureto irritants such as turpentine and acetic acid there occursa rapid rise in bladder content of NGF [75] Later studiesfound that bladder responds to insults with upregulation inthe genes for NGF sE-Selectin and receptor for monocytechemoattractant protein-1 (MCP-1) within 30min of expo-sure to lipopolysaccharide (Figure 5) [76] The acetic acidinduced overexpression of NGF was blunted by pretreatmentwith NGF antisense OND with phosphorothioate linkageprior to exposure of acetic acid The downregulation of NGFmRNA expression was in agreement with reduced proteinlevels and suppressed NGF-like immunoreactivity in theurothelium

93 Antisense and Downstream Signalling of NGF Drugdevelopment of intravesical antisense for NGF was also ableto unmask the downstream signaling [77] involving NGFfollowing exposure to acetic acid Experiments supportedthe role of NGF as a paracrine messenger [77] which isknown to activate several downstream effectors to manifestphysiological and pathological signaling changes linked to it[76 78ndash80] The bladder injury set off by acetic acid initiatesthe signalling cascades that upregulate the expression ofNGF and other chemokines MCP-1 CXCL-1 and CXCL-10 and prostaglandins [76 78ndash80] As reported elsewherelocalization of chemokines within synaptic vesicles in neu-rons [81] is consistent with their ability to act as excitatoryneurotransmitters following AA exposure

Chemokines are one of several downstream effectorsactivated by NGF [76 78ndash80] and interestingly chemokinereceptors are widely expressed in neural and nonneuralelements of the nociceptive pathways that are responsible forvisceral and somatic pain sensation [82] MCP-1 [83] andCXCL-10 [84] are constitutively expressed in neurons wherethey participate in excitability of primary afferent neuronsthrough transactivation of transient receptor channels andnociceptor sensitization [84] Overexpression ofNGF is likelyto drive the expression of VEGF fromneurons and leptin [78]from adipocytes covering neurons [85]

Cooperative expression of NGF and MCP-1 is able toinduce hyperexcitability in neurons by activating TRPV1receptor In addition MCP-1 CXCL-1 and CXCL-10 causechemoattraction of monocytes neutrophils and lympho-cytes respectively to mediate the bladder injury set offby acetic acid Extravasation is an essential prerequisitefor infiltration of monocytes neutrophils and lymphocyteswhich requires the expression of adhesion molecules likeE-selectin and intracellular adhesion molecule ICAM-1 [7686] E-selectin is a cytokine-inducible adhesion moleculethat supports the rolling and stable arrest of leukocyteson activated vascular endothelium Expression of E-selectinhas been linked to adherence of neutrophils to bladdermicrovascular endothelial cells and to cyclophosphamidecystitis [87 88] E-selectin gene expression is activated bythe NF-120581B and MAP kinase signal transduction pathways[76 86]

Activation and recruitment of leukocytes to acetic acidinduced bladder injury also require expression of intra-cellular adhesion molecule ICAM-1 [89] ICAM-1 interactsspecifically with its receptors of the integrin family to inducereversible cell-cell interactions involving adhesion Expres-sion of ICAM-1 was found to be increased in patients withICPBS and reduced in patients responding to instillation ofhyaluronic acid [90] Expression of ICAM-1 and VCAM-1was also noted in a recent study on biopsy tissue of ICPBSpatients [91] Increased expression of ICAM-1 subsequentto exposure with acetic acid in animal model is thereforeclinically relevant

Antisense experiments supported the earlier report thatinhibition of NGF expression significantly downregulates theexpression of ICAM-1 [80] It is reported that binding ofNGF to its high-affinity TrkA receptor controls the sICAM-1expression on target cells [80] Expression of ICAM-1 was

8 ISRN Pharmacology

Neutrophil

Monocytes

Acetic acid

Integrin

NGF

MCP-1

Release of cytokinesVanilloid

receptor

Trk receptors NGF

Membrane damage

VEGF

CXCL1

Activated leucocyte

Startrolling

Extravasation

CXCL10

sICAMssE-selectin

ActivationAdhesion

MCP-1

NGF

PKC

DAG

PLC

IP3Kinases

Ras

AKT

PI3K

CXCL10

sICAMs

LymphocyteAfferents

Figure 5 Signalling cascades induced by NGF overexpression following exposure to acetic acid Injury from exposure to acetic sets off thesignalling cascades that upregulate the expression of NGF MCP-1 prostaglandins CXCL-1 and CXCL-10 Overexpression of NGF drives theexpression of VEGF from neurons and leptin from adipocytes covering neurons NGF and MCP-1 also induce hyperexcitability in neuronsby activating TRPV1 receptor MCP-1 CXCL-1 and CXCL-10 cause chemoattraction of monocytes neutrophils and lymphocytes whichrequires extravasation across endothelium from blood through the cooperation of adhesion molecules E-selectin and ICAM-1

negatively associated with NGF expression in antisenseexperiments Ultimately expression of ICAM-1 is regulated atthe level of transcription by one of several signaling cascadesNF-120581B JAKSTAT IFN-g (CXCL-10) AP MAP kinase andPKC [92]

Intravesical route can allow selective exposure of anti-sense ODN to the NGF producing cells in urothelium andavoid systemic side effects from genetic manipulation ofNGF expression Intravesical route can also prove to be costeffective given the cost of ODN or siRNA Contrary to thegoal of loss of function in bladder with antisense ODN inrecent studies liposomes have also been used to deliver ODNfor gain of function in bladder [93]

10 Imaging of Intravesical Therapy

Drug development for DO and ICPBS relies heavily onsubjective outcomes for predicting efficacy and safety in earlyclinical development Although there are many validatedmeasurement tools used in DO and ICPBS research theyare usually burdensome and do not capture all symptomsrelated to the lower urinary tract Moreover improvement insymptoms scores following an intervention does not alwayscorrelate with patient expectations satisfaction and goalachievement which are critically important for successfulmanagement of DO and ICPBS

There is underdevelopment and underutilization of urinebiomarkers and imaging methods for investigational drugsgiven by intravesical route Overreliance on subjectiveimpressions of patients for therapeutic response limits clini-cal relevance due to weak correlation with patient satisfactionand can also impede continued scientific progress in assessing

study outcomes relative to symptom bother Symptom scoresmay also be related to the cause of high heterogeneity inclinical response in DO and ICPBS patients

Imaging can offer the possibility to provide region-specific information in situations where serum and urineassays may not reflect the true state of bladder [94ndash96]Urine chemokines have been reported to be associated withsymptom severity of ICPBS patients [97] Application offluorescent microscopy for imaging of pelvic floor has beenlimited due to the issues pertaining to light absorption bytissues scattering and autofluorescence [98]

Recent studies have shown that dyes fluorescing inthe near-infrared (NIR) band can overcome this handi-cap in deep-tissue imaging of experimental animals [99]Selected NIR fluorochromes emit light with tissue pen-etration approaching 10ndash15 cm [100] The light emissionfrom probes in this spectrum encounters low backgroundautofluorescence and minimal attenuation of signal due tolight absorption by tissue components [98] Therefore NIRimaging yields high signal-to-noise ratios and is well suitedfor studying the distribution of instilled treatments in bladder(Figure 2(b))

Preliminary studies suggest that imaging of mouse pelvicfloor in NIR spectrum can be a viable option [101] NIRimaging easily allows visualization and quantification ofbladder distribution by tracing themigration of fluorescence-labeled liposomes in anaesthetized mouse Compared withother biological assays imaging could provide objectiveendpoint for patients with ulcerative cystitis to indicate itshigh translation potential Other benefits of optical imaginginclude its relatively low cost compared withmore traditionalimaging systems such as MRI and PET The instrument

ISRN Pharmacology 9

consists of two near-infrared lasers emitting at 685 nm and785 nm camera and novel optics

Furthermore preferential binding of liposomes andnanoparticles to bladder lesions of ICPBS after instillationcan be assessed by NIR imaging of bladder where it cantheoretically provide anatomically specific information aboutthe real time status of ulcerlesion in bladder surface or tissueabnormality [102] Therefore future studies can substitute aresponse variable that is continuous in nature instead of asubjective clinical outcome

11 Conclusions

Adoption of forward-thinking approaches can achieveadvancements in drug delivery systems targeted to improvepharmacotherapy of bladder diseases in the future Latestdevelopments in the field of nanotechnology can bring thismode of therapy from second line of treatment for refractorycases to the forefront of disease management Liposomesare an attractive drug delivery platform by virtue of theirbiodegradability biocompatibility low toxicity and simpleand mild preparation methods

Conflict of Interests

All authors except Dr Michael Chancellor declare that thereis no conflict of interests regarding the publication of thispaper Dr Michael Chancellor owns stocks in the start-upliposome company Lipella

Acknowledgments

This work was supported by the National Institutes of Health(DK057267 and DK088836) and the Department of Defense(W81XWH-11-1-0763 and W81XWH-12-1-0565)

References

[1] J Parkin C Shea and G R Sant ldquoIntravesical dimethyl sul-foxide (DMSO) for interstitial cystitismdasha practical approachrdquoUrology vol 49 no 5 pp 105ndash107 1997

[2] C Dowson J Watkins M S Khan P Dasgupta and A SahaildquoRepeated botulinum toxin type A injections for refractoryoveractive bladder medium-term outcomes safety profile anddiscontinuation ratesrdquo European Urology vol 61 no 4 pp 834ndash839 2012

[3] J Kaufman V Tyagi M Anthony M B Chancellor and PTyagi ldquoState of the art in intravesical therapy for lower urinarytract symptomsrdquo Reviews in Urology vol 12 pp e181ndashe189 2010

[4] N Yoshimura and M B Chancellor ldquoCurrent and futurepharmacological treatment for overactive bladderrdquo Journal ofUrology vol 168 no 5 pp 1897ndash1913 2002

[5] K A Killinger J A Boura and K M Peters ldquoPain ininterstitial cystitisbladder pain syndrome do characteristicsdiffer in ulcerative and non-ulcerative subtypesrdquo InternationalUrogynecology Journal vol 24 no 8 pp 1295ndash1301 2013

[6] J C Nickel P Hanno K Kumar and HThomas ldquoSecond mul-ticenter randomized double-blind parallel-group evaluationof effectiveness and safety of intravesical sodium chondroitin

sulfate compared with inactive vehicle control in subjects withinterstitial cystitisbladder pain syndromerdquoUrology vol 79 no6 pp 1220ndash1225 2012

[7] P M Hanno ldquoAnalysis of long-term elmiron therapy forinterstitial cystitisrdquo Urology vol 49 no 5 pp 93ndash99 1997

[8] A Giannantoni V Bini R Dmochowski et al ldquoReply to ClausRiedlrsquos letter to the editor re antonella Giannantoni VittorioBini Roger Dmochowski et al Contemporary management ofthe painful bladder a systematic reviewrdquo EuropeanUrology vol61 no 5 pp 29ndash53 2012

[9] MC Lai Y CKuo andHCKuo ldquoIntravesical hyaluronic acidfor interstitial cystitispainful bladder syndrome a comparativerandomized assessment of different regimensrdquo InternationalJournal of Urology vol 20 pp 203ndash207 2013

[10] J Eldrup J Thorup and S L Nielsen ldquoPermeability andultrastructure of human bladder epitheliumrdquo British Journal ofUrology vol 55 no 5 pp 488ndash492 1983

[11] C L Parsons D Boychuk S Jones R Hurst and H CallahanldquoBladder surface glycosaminoglycans an epithelial permeabil-ity barrierrdquo Journal of Urology vol 143 no 1 pp 139ndash142 1990

[12] Y Kim N Yoshimura H Masuda F De Miguel and MB Chancellor ldquoAntimuscarinic agents exhibit local inhibitoryeffects on muscarinic receptors in bladder-afferent pathwaysrdquoUrology vol 65 no 2 pp 238ndash242 2005

[13] V Tyagi B J Philips R Su et al ldquoDifferential expression offunctional cannabinoid receptors in human bladder detrusorand urotheliumrdquo Journal of Urology vol 181 no 4 pp 1932ndash1938 2009

[14] B J Philips M C Smaldone V L Erickson N YoshimuraM B Chancellor and P Tyagi ldquoDifferential expression ofneurokinin receptor subtypes in urothelium and detrusor ofhuman bladderrdquo Journal of Urology vol 179 article 354 2008

[15] S A Baker G W Hennig J Han F C Britton T K Smithand S D Koh ldquoMethionine and its derivatives increase blad-der excitability by inhibiting stretch-dependent K+ channelsrdquoBritish Journal of Pharmacology vol 153 no 6 pp 1259ndash12712008

[16] J M Beckel A Kanai S-J Lee W C De Groat and LA Birder ldquoExpression of functional nicotinic acetylcholinereceptors in rat urinary bladder epithelial cellsrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 290 no 1 ppF103ndashF110 2006

[17] LA Birder A J KanaiWCDeGroat et al ldquoVanilloid receptorexpression suggests a sensory role for urinary bladder epithelialcellsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 23 pp 13396ndash13401 2001

[18] P Tyagi C A Thomas N Yoshimura and M B ChancellorldquoInvestigations into the presence of functional szlig1 szlig2 and szlig3-adrenoceptors in urothelium and detrusor of human bladderrdquoInternational Brazilian Journal of Urology vol 35 no 1 pp 76ndash83 2009

[19] A T Hanna-Mitchell J M Beckel S Barbadora A J Kanai WC de Groat and L A Birder ldquoNon-neuronal acetylcholine andurinary bladder urotheliumrdquo Life Sciences vol 80 no 24-25 pp2298ndash2302 2007

[20] C P Smith D A Gangitano A Munoz et al ldquoBotulinumtoxin type A normalizes alterations in urothelial ATP and NOrelease induced by chronic spinal cord injuryrdquo NeurochemistryInternational vol 52 no 6 pp 1068ndash1075 2008

[21] Y-C Chuang C A Thomas S Tyagi N Yoshimura PTyagi and M B Chancellor ldquoHuman urine with solifenacin

10 ISRN Pharmacology

intake but not tolterodine or darifenacin intake blocks detrusoroveractivityrdquo International Urogynecology Journal and PelvicFloor Dysfunction vol 19 no 10 pp 1353ndash1357 2008

[22] Y Kim N Yoshimura H Masuda F D Miguel and M BChancellor ldquoIntravesical instillation of human urine after oraladministration of trospium tolterodine and oxybutynin in a ratmodel of detrusor overactivityrdquo BJU International vol 97 no 2pp 400ndash403 2006

[23] S Tyagi P Tyagi S Van-le N YoshimuraM B Chancellor andF de Miguel ldquoQualitative and quantitative expression profileof muscarinic receptors in human urothelium and detrusorrdquoJournal of Urology vol 176 no 4 pp 1673ndash1678 2006

[24] G Gregoriadis and B E Ryman ldquoLiposomes as carriers ofenzymes or drugs a new approach to the treatment of storagediseasesrdquo Biochemical Journal vol 124 no 5 p 58 1971

[25] G Gregoriadis S Jain I Papaioannou and P Laing ldquoImprov-ing the therapeutic efficacy of peptides and proteins a role forpolysialic acidsrdquo International Journal of Pharmaceutics vol300 no 1-2 pp 125ndash130 2005

[26] P Sapra P Tyagi and T M Allen ldquoLigand-targeted liposomesfor cancer treatmentrdquo Current Drug Delivery vol 2 no 4 pp369ndash381 2005

[27] G Gregoriadis G Dapergolas and E D Neerunjun ldquoPene-tration of target areas in the rat by liposome associated agentsadministered parenterally and intragastricallyrdquo BiochemicalSociety Transactions vol 4 no 2 pp 256ndash259 1976

[28] G Gregoriadis ldquoEngineering liposomes for drug deliveryprogress and problemsrdquo Trends in Biotechnology vol 13 no 12pp 527ndash537 1995

[29] G Gregoriadis and A C Allison ldquoEntrapment of proteins inliposomes prevents allergic reactions in pre immunised micerdquoFEBS Letters vol 45 no 1 pp 71ndash74 1974

[30] S Lee S Dausch CMaierhofer andD Dausch ldquoA new therapyconcept with a liposome eye spray for the treatment of the lsquodryeyersquordquo Klinische Monatsblatter fur Augenheilkunde vol 221 no10 pp 825ndash836 2004

[31] D Dausch S Lee S Dausch J C Kim G Schwert and WMichelson ldquoComparative study of treatment of the dry eyesyndrome due to disturbances of the tear film lipid layer withlipid-containing tear substitutes efficacy of lipid-containingtear substitutesrdquo Klinische Monatsblatter fur Augenheilkundevol 223 no 12 pp 974ndash983 2006

[32] S Ebrahim G A Peyman and P J Lee ldquoApplications ofliposomes in ophthalmologyrdquo Survey of Ophthalmology vol 50no 2 pp 167ndash182 2005

[33] J Nirmal P Tyagi L Dang et al ldquoEndocytosis uptake ofliposomes in urothelium cells detected by transmission electronmicroscopyrdquoThe Journal of Urology vol 187 article e15 2012

[34] D N Frangos J J Killion D Fan R Fishbeck A C VonEschenbach and I J Fidler ldquoThe development of liposomescontaining interferon alpha for the intravesical therapy ofhuman superficial bladder cancerrdquo Journal of Urology vol 143no 6 pp 1252ndash1266 1990

[35] J W Johnson R Nayar J J Killion A C Von Eschenbachand I J Fidler ldquoBinding of liposomes to human bladdertumor epithelial cell lines implications for an intravesical drugdelivery system for the treatment of bladder cancerrdquo SelectiveCancer Therapeutics vol 5 no 4 pp 147ndash155 1989

[36] Y-C Chuang M B Chancellor S Seki et al ldquoIntravesicalprotamine sulfate and potassium chloride as a model forbladder hyperactivityrdquoUrology vol 61 no 3 pp 664ndash670 2003

[37] J Kaufman P Tyagi and M B Chancellor ldquoIntravesicalliposomal (Lp08) instillation protects bladder urothelium fromchemical irritationrdquoThe Journal of Urology vol 181 article 5392009

[38] P Tyagi M B Chancellor Z Li et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical capsaicindelivery using thermosensitive hydrogel and liposomesrdquo Jour-nal of Urology vol 171 no 1 pp 483ndash489 2004

[39] MO Fraser Y-CChuang P Tyagi et al ldquoIntravesical liposomeadministrationmdasha novel treatment for hyperactive bladder inthe ratrdquo Urology vol 61 no 3 pp 656ndash663 2003

[40] P Tyagi M Chancellor N Yoshimura and L Huang ldquoActivityof different phospholipids in attenuating hyperactivity in blad-der irritationrdquo BJU International vol 101 no 5 pp 627ndash6322008

[41] Y Sun and T C Chai ldquoEffects of dimethyl sulphoxide andheparin on stretch-activated ATP release by bladder urothelialcells from patients with interstitial cystitisrdquo BJU Internationalvol 90 no 4 pp 381ndash385 2002

[42] J J Bade ldquoA placebo-controlled study of intravesical pentosan-polysulphate for the treatment of interstitial cystitisrdquo BritishJournal of Urology vol 79 no 2 pp 168ndash171 1997

[43] P Tyagi V C Hsieh N Yoshimura J Kaufman and M BChancellor ldquoInstillation of liposomes vs dimethyl sulphoxide orpentosan polysulphate for reducing bladder hyperactivityrdquo BJUInternational vol 104 no 11 pp 1689ndash1692 2009

[44] W-C Lee C-T Chien H-J Yu and S-W Lee ldquoBladderdysfunction in rats with metabolic syndrome induced by long-term fructose feedingrdquo Journal of Urology vol 179 no 6 pp2470ndash2476 2008

[45] J I Gillespie G A Van Koeveringe S G DeWachter and J DeVente ldquoOn the origins of the sensory output from the bladderthe concept of afferent noiserdquo BJU International vol 103 no 10pp 1324ndash1333 2009

[46] W C Lee P H Chiang Y L Tain C C Wu and Y CChuang ldquoSensory dysfunction of bladder mucosa and bladderoversensitivity in a rat model of metabolic syndromerdquo PloSONE vol 7 Article ID e45578 2012

[47] Y-C Chuang W-C Lee W-C Lee and P-H Chiang ldquoIntrav-esical liposome versus oral pentosan polysulfate for interstitialcystitispainful bladder syndromerdquo Journal of Urology vol 182no 4 pp 1393ndash1400 2009

[48] W-C Lee Y-C Chuang W-C Lee and P-H Chiang ldquoSafetyand dose flexibility clinical evaluation of intravesical liposomein patients with interstitial cystitis or painful bladder syn-dromerdquo Kaohsiung Journal of Medical Sciences vol 27 no 10pp 437ndash440 2011

[49] S T Truschel E Wang W G Ruiz et al ldquoStretch-regulatedexocytosisendocytosis in bladder umbrella cellsrdquo MolecularBiology of the Cell vol 13 no 3 pp 830ndash846 2002

[50] M B Chancellor and W C De Groat ldquoIntravesical capsaicinand resiniferatoxin therapy spicing up the ways to treat theoveractive bladderrdquo Journal of Urology vol 162 no 1 pp 3ndash111999

[51] D S Byrne A Das J Sedor et al ldquoEffect of intravesicalcapsaicin and vehicle on bladder integrity in control and spinalcord injured ratsrdquo Journal of Urology vol 159 no 3 pp 1074ndash1078 1998

[52] M Mandal and K-D Lee ldquoListeriolysin O-liposome-mediatedcytosolic delivery of macromolecule antigen in vivo enhance-ment of antigen-specific cytotoxic T lymphocyte frequency

ISRN Pharmacology 11

activity and tumor protectionrdquo Biochimica et Biophysica Actavol 1563 no 1-2 pp 7ndash17 2002

[53] Y-C Chuang P Tyagi C-C Huang et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical botulinumtoxin a delivery using liposomesrdquo Journal of Urology vol 182no 2 pp 786ndash792 2009

[54] Y-C Chuang N Yoshimura C-C Huang P-H Chiang andM B Chancellor ldquoIntravesical botulinum toxin a administra-tion produces analgesia against acetic acid induced bladder painresponses in ratsrdquo Journal of Urology vol 172 no 4 pp 1529ndash1532 2004

[55] S P Petrou A S Parker J E Crook A Rogers D Metz-Kudashick and D D Thiel ldquoBotulinum A toxindimethylsulfoxide bladder instillations for women with refractory idio-pathic detrusor overactivity a phase 12 studyrdquo Mayo ClinicProceedings vol 84 no 8 pp 702ndash706 2009

[56] P Caccin O Rossetto M Rigoni E Johnson G Schiavoand C Montecucco ldquoVAMPsynaptobrevin cleavage by tetanusand botulinum neurotoxins is strongly enhanced by acidicliposomesrdquo FEBS Letters vol 542 no 1ndash3 pp 132ndash136 2003

[57] M L Stephenson and P C Zamecnik ldquoInhibition of Roussarcoma viral RNA translation by a specific oligodeoxyribonu-cleotiderdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 75 no 1 pp 285ndash288 1978

[58] H Dadgostar and N Waheed ldquoThe evolving role of vascularendothelial growth factor inhibitors in the treatment of neovas-cular age-related macular degenerationrdquo Eye vol 22 no 6 pp761ndash767 2008

[59] C Gebhard G Huard E A Kritikou and J C TardifldquoApolipoprotein B antisense inhibitionmdashupdate on mipom-ersenrdquo Current Pharmaceutical Design vol 919 no 17 pp 3132ndash3142 2013

[60] T Yokota E Hoffman and S Takeda ldquoAntisense oligo-mediated multiple exon skipping in a dog model of duchennemuscular dystrophyrdquoMethods inMolecular Biology vol 709 pp299ndash312 2011

[61] B L Jacobs M C Smaldone V Tyagi et al ldquoIncreased nervegrowth factor in neurogenic overactive bladder and interstitialcystitis patientsrdquoThe Canadian Journal of Urology vol 17 no 1pp 4989ndash4994 2010

[62] H-T Liu and H-C Kuo ldquoIntravesical botulinum toxin Ainjections plus hydrodistension can reduce nerve growth factorproduction and control bladder pain in interstitial cystitisrdquoUrology vol 70 no 3 pp 463ndash468 2007

[63] H T Liu and H C Kuo ldquoIncreased urine and serum nervegrowth factor levels in interstitial cystitis suggest chronicinflammation is involved in the pathogenesis of diseaserdquo PloSONE vol 7 Article ID e44687 2012

[64] N Yoshimura N E Bennett Y Hayashi et al ldquoBladderoveractivity and hyperexcitability of bladder afferent neuronsafter intrathecal delivery of nerve growth factor in ratsrdquo Journalof Neuroscience vol 26 no 42 pp 10847ndash10855 2006

[65] S Seki K Sasaki Y Igawa et al ldquoSuppression of detrusor-sphincter dyssynergia by immunoneutralization of nervegrowth factor in lumbosacral spinal cord in spinal cord injuredratsrdquo Journal of Urology vol 171 no 1 pp 478ndash482 2004

[66] R J Evans R M Moldwin N Cossons A Darekar I WMills andD Scholfield ldquoProof of concept trial of tanezumab forthe treatment of symptoms associated with interstitial cystitisrdquoJournal of Urology vol 185 no 5 pp 1716ndash1721 2011

[67] P Tyagi R Banerjee S Basu N YoshimuraM Chancellor andL Huang ldquoIntravesical antisense therapy for cystitis using TAT-peptide nucleic acid conjugatesrdquoMolecular Pharmaceutics vol3 no 4 pp 398ndash406 2006

[68] D C Molliver D E Wright M L Leitner et al ldquoIB4-bindingDRG neurons switch from NGF to GDNF dependence in earlypostnatal liferdquo Neuron vol 19 no 4 pp 849ndash861 1997

[69] M TyagiM RusnatiM Presta andMGiacca ldquoInternalizationof HIV-1 tat requires cell surface heparan sulfate proteoglycansrdquoJournal of Biological Chemistry vol 276 no 5 pp 3254ndash32612001

[70] E Uhlmann A Ryte and A Peyman ldquoStudies on the mech-anism of stabilization of partially phosphorothioated oligonu-cleotides against nucleolytic degradationrdquoAntisense andNucleicAcid Drug Development vol 7 no 4 pp 345ndash350 1997

[71] M Nogawa T Yuasa S Kimura et al ldquoIntravesical adminis-tration of small interfering RNA targeting PLK-1 successfullyprevents the growth of bladder cancerrdquo Journal of ClinicalInvestigation vol 115 no 4 pp 978ndash985 2005

[72] M Kashyap N Kawamorita V Tyagi et al ldquoDownregulation ofNGF expression in the bladder by antisense oligoucleotides asnew treatment for overactive bladderrdquo Journal of Urology vol27190 no 2 pp 757ndash764 2013

[73] C-J Arum Y Kodama N Rolim et al ldquoA rat model ofintravesical delivery of small interfering RNA for studyingurinary carcinomardquoWorld Journal of Urology vol 28 no 4 pp479ndash485 2010

[74] C E Blietz B Thode M Hauses et al ldquoIn vivo studies on theavailability and toxicity of antisense oligonucleotides in bladdercancerrdquo In Vivo vol 23 no 1 pp 13ndash19 2009

[75] D Oddiah P Anand S B McMahon and M Rattray ldquoRapidincrease of NGF BDNF andNT-3mRNAs in inflamed bladderrdquoNeuroReport vol 9 no 7 pp 1455ndash1458 1998

[76] M R Saban H Hellmich N B Nguyen J Winston T GHammond and R Saban ldquoTime course of LPS-induced geneexpression in a mouse model of genitourinary inflammationrdquoPhysiol Genomics vol 5 no 3 pp 147ndash160 2001

[77] B Schnegelsberg T T Sun G Cain et al ldquoOverexpression ofNGF in mouse urothelium leads to neuronal hyperinnervationpelvic sensitivity and changes in urinary bladder functionrdquoTheAmerican Journal of PhysiologymdashRegulatory Integrative andComparative Physiology vol 298 no 5 pp R534ndashR547 2010

[78] S Cardenas M Scuri L Samsell et al ldquoNeurotrophic andneuroimmune responses to early-life Pseudomonas aeruginosainfection in rat lungsrdquo American Journal of PhysiologymdashLungCellular and Molecular Physiology vol 299 no 3 pp L334ndashL344 2010

[79] K Nakamura F Tan Z Li and C J Thiele ldquoNGF activationof TrkA induces vascular endothelial growth factor expressionvia induction of hypoxia-inducible factor-1120572rdquo Molecular andCellular Neuroscience vol 46 no 2 pp 498ndash506 2011

[80] S Othumpangat M Regier and G Piedimonte ldquoNerve growthfactor modulates human rhinovirus infection in airway epithe-lial cells by controlling ICAM-1 expressionrdquo American Journalof Physiology Lung Cellular andMolecular Physiology vol 15302pp L1057ndashL1066 2012

[81] H Jung P T Toth F A White and R J Miller ldquoMonocytechemoattractant protein-1 functions as a neuromodulator indorsal root ganglia neuronsrdquo Journal of Neurochemistry vol104 no 1 pp 254ndash263 2008

[82] F A White H Jung and R J Miller ldquoChemokines andthe pathophysiology of neuropathic painrdquo Proceedings of the

12 ISRN Pharmacology

National Academy of Sciences of the United States of Americavol 104 no 51 pp 20151ndash20158 2007

[83] G Banisadr R-D Gosselin P Mechighel W Rostene PKitabgi and S M Parsadaniantz ldquoConstitutive neuronalexpression of CCR2 chemokine receptor and its colocalizationwith neurotransmitters in normal rat brain functional effectof MCP-1CCL2 on calcium mobilization in primary culturedneuronsrdquo Journal of Comparative Neurology vol 492 no 2 pp178ndash192 2005

[84] S Bhangoo D Ren R J Miller et al ldquoDelayed functionalexpression of neuronal chemokine receptors following focalnerve demyelination in the rat a mechanism for the devel-opment of chronic sensitization of peripheral nociceptorsrdquoMolecular Pain vol 3 article 38 2007

[85] T Maeda N Kiguchi Y Kobayashi T Ikuta M Ozaki and SKishioka ldquoLeptin derived from adipocytes in injured periph-eral nerves facilitates development of neuropathic pain viamacrophage stimulationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 106 no 31 pp13076ndash13081 2009

[86] H Kajiwara M Yasuda N Kumaki T Shibayama and YOsamura ldquoExpression of carbohydrate antigens (SSEA-1 Sialyl-Lewis X DU-PAN-2 and CA19-9) and E-selectin in urothelialcarcinoma of the renal pelvis ureter and urinary bladderrdquoTokaiJournal of Experimental and Clinical Medicine vol 30 no 3 pp177ndash182 2005

[87] A M S Assreuy G J Martins M E F Moreira et al ldquoPre-vention of cyclophosphamide-induced hemorrhagic cystitis byglucose-mannose binding plant lectinsrdquo Journal of Urology vol161 no 6 pp 1988ndash1993 1999

[88] S M Vinson A Rickard J S Ryerse and J McHowat ldquoNeu-trophil adherence to bladder microvascular endothelial cellsfollowing platelet-activating factor acetylhydrolase inhibitionrdquoJournal of Pharmacology and Experimental Therapeutics vol314 no 3 pp 1241ndash1247 2005

[89] M Green A Filippou G Sant and T CTheoharides ldquoExpres-sion of intercellular adhesion molecules in the bladder ofpatients with interstitial cystitisrdquoUrology vol 63 no 4 pp 688ndash693 2004

[90] M Leppilahti P Hellstrom and T L J Tammela ldquoEffect ofdiagnostic hydrodistension and four intravesical hyaluronicacid instillations on bladder ICAM-1 intensity and associationof ICAM-1 intensity with clinical response in patients withinterstitial cystitisrdquo Urology vol 60 no 1 pp 46ndash51 2002

[91] A T Corcoran N Yoshimura V Tyagi B Jacobs W Lengand P Tyagi ldquoMapping the cytokine profile of painful bladdersyndromeinterstitial cystitis in human bladder and urinespecimensrdquoWorld Journal of Urology vol 31 no 1 pp 241ndash2462013

[92] SGarrean X-PGaoV Brovkovych et al ldquoCaveolin-1 regulatesNF-120581B activation and lung inflammatory response to sepsisinduced by lipopolysacchariderdquo Journal of Immunology vol 177no 7 pp 4853ndash4860 2006

[93] M R Kang G Yang R F Place et al ldquoIntravesical deliveryof small activating RNA formulated into lipid nanoparticlesinhibits orthotopic bladder tumor growthrdquo Cancer Researchvol 172 pp 5069ndash5079 2012

[94] P Tyagi D Barclay R Zamora et al ldquoUrine cytokines suggestan inflammatory response in the overactive bladder a pilotstudyrdquo International Urology and Nephrology vol 42 no 3 pp629ndash635 2010

[95] P Tyagi B L Jacobs D Barcaly et al ldquoUrine levels ofinflammatory chemokines can be novel biomarkers for theoveractive bladderrdquoThe Journal of Urology vol 181 article 5882009

[96] P Tyagi V Tyagi D Bui et al ldquoDiscrimination Of OAB fromICPBS by multivariate data modeling of urinary proteinsrdquoTheJournal of Urology vol 183 pp e614ndashe6e5 2010

[97] P Tyagi D Nikolavsky Y Vodovotz et al ldquoUrine levels ofselected chemokines positively correlate with lower bladdercapacity and psychometric scores in IcPbs patientsrdquo TheJournal of Urology vol 181 article 21 2009

[98] R Weissleder and V Ntziachristos ldquoShedding light onto livemolecular targetsrdquo Nature Medicine vol 9 no 1 pp 123ndash1282003

[99] C Bremer V Ntziachristos and R Weissleder ldquoOptical-basedmolecular imaging contrast agents and potentialmedical appli-cationsrdquo European Radiology vol 13 no 2 pp 231ndash243 2003

[100] UMahmood andRWeissleder ldquoNear-infrared optical imagingof proteases in cancerrdquoMolecular CancerTherapeutics vol 2 pp489ndash496 2003

[101] J Kaufman H Hensley J Jacobs et al ldquoNon-invasive imagingof near infrafred dye labeled liposomes facilitates evaluation ofbioresidence timerdquoThe Journal of Urology vol 183 article e6282010

[102] Y-X J Wang ldquoMedical imaging in pharmaceutical clinicaltrials what radiologists should knowrdquo Clinical Radiology vol60 no 10 pp 1051ndash1057 2005

ISRN Pharmacology 7

nucleic acid in bladder at the same time of cyclophosphamideinjection protected against the cystitis by blocking the rise inbladder contraction frequency and inflammation

92 Phosphorothioate-Linked Analogues Considering thedifficulty and possible nonspecific toxicity of peptide nucleicacid and TAT recent developments focused on simplifyingthe approach using water-soluble phosphorothioated ODNwhich have an increased resistance to exo- and endonucleasesfor improved stability [70] As alluded to in the above textsuccess of antisense therapeutics is largely dependent on thedevelopment of a delivery vehicle that can efficiently deliverantisense ODN in bladder Preliminary studies showed thatliposomes can be far better biocompatible effective carriersfor local gene silencing of NGF gene in bladder cells

Cationic liposomes and mimics have emerged as themost popular nonviral method to deliver nucleic acids intherapeutic applications Easy and reversible complex forma-tion of cationic liposomes with ODN at room temperatureallows their use as carriers Electrostatic attraction betweenthe cationic lipid DOTAP and the polyanionic antisenseODN is responsible for the complex formation The efficacyof liposome delivered siRNA by intravesical route has beenpreviously demonstrated in preclinical models of bladdercancer [71] Residence of ODN in the rat bladder after intrav-esical instillation was demonstrated to be longer than 24 husing fluorescent tagged ODN complexed with liposomes[72] Fluorescent probe was localized in bladder urotheliumcells 24 h following instillation The 24 h residence time wasalso demonstrated for siRNA in mouse bladder [73] Bladderuptake of fluorescent ODN without liposomes in normalrat bladder is poor Previous studies have shown that veryhigh concentration of phosphorothioated ODN can deliverODN without liposomes to bladder cells of mice havingbladder cancer [74] The urothelium barrier may be slightlycompromised in cancerous condition and the strategy ofloading bladder with high dose of ODN may not workin noncancerous diseased condition of bladder with intactbarrier

In order to evaluate the efficacy of NGF antisense ODNacetic acid infusion was used to cause a rapid rise of NGFprotein levels A single dose instillation of OND complexedwith liposomes protected against bladder overactivity (BO)induced by acetic acid Together with data of bladder uptakestudies using fluorescent ODN it is demonstrated that ONDis readily available to the bladder after intravesical instillationIt has been previously reported that within 2 h of exposureto irritants such as turpentine and acetic acid there occursa rapid rise in bladder content of NGF [75] Later studiesfound that bladder responds to insults with upregulation inthe genes for NGF sE-Selectin and receptor for monocytechemoattractant protein-1 (MCP-1) within 30min of expo-sure to lipopolysaccharide (Figure 5) [76] The acetic acidinduced overexpression of NGF was blunted by pretreatmentwith NGF antisense OND with phosphorothioate linkageprior to exposure of acetic acid The downregulation of NGFmRNA expression was in agreement with reduced proteinlevels and suppressed NGF-like immunoreactivity in theurothelium

93 Antisense and Downstream Signalling of NGF Drugdevelopment of intravesical antisense for NGF was also ableto unmask the downstream signaling [77] involving NGFfollowing exposure to acetic acid Experiments supportedthe role of NGF as a paracrine messenger [77] which isknown to activate several downstream effectors to manifestphysiological and pathological signaling changes linked to it[76 78ndash80] The bladder injury set off by acetic acid initiatesthe signalling cascades that upregulate the expression ofNGF and other chemokines MCP-1 CXCL-1 and CXCL-10 and prostaglandins [76 78ndash80] As reported elsewherelocalization of chemokines within synaptic vesicles in neu-rons [81] is consistent with their ability to act as excitatoryneurotransmitters following AA exposure

Chemokines are one of several downstream effectorsactivated by NGF [76 78ndash80] and interestingly chemokinereceptors are widely expressed in neural and nonneuralelements of the nociceptive pathways that are responsible forvisceral and somatic pain sensation [82] MCP-1 [83] andCXCL-10 [84] are constitutively expressed in neurons wherethey participate in excitability of primary afferent neuronsthrough transactivation of transient receptor channels andnociceptor sensitization [84] Overexpression ofNGF is likelyto drive the expression of VEGF fromneurons and leptin [78]from adipocytes covering neurons [85]

Cooperative expression of NGF and MCP-1 is able toinduce hyperexcitability in neurons by activating TRPV1receptor In addition MCP-1 CXCL-1 and CXCL-10 causechemoattraction of monocytes neutrophils and lympho-cytes respectively to mediate the bladder injury set offby acetic acid Extravasation is an essential prerequisitefor infiltration of monocytes neutrophils and lymphocyteswhich requires the expression of adhesion molecules likeE-selectin and intracellular adhesion molecule ICAM-1 [7686] E-selectin is a cytokine-inducible adhesion moleculethat supports the rolling and stable arrest of leukocyteson activated vascular endothelium Expression of E-selectinhas been linked to adherence of neutrophils to bladdermicrovascular endothelial cells and to cyclophosphamidecystitis [87 88] E-selectin gene expression is activated bythe NF-120581B and MAP kinase signal transduction pathways[76 86]

Activation and recruitment of leukocytes to acetic acidinduced bladder injury also require expression of intra-cellular adhesion molecule ICAM-1 [89] ICAM-1 interactsspecifically with its receptors of the integrin family to inducereversible cell-cell interactions involving adhesion Expres-sion of ICAM-1 was found to be increased in patients withICPBS and reduced in patients responding to instillation ofhyaluronic acid [90] Expression of ICAM-1 and VCAM-1was also noted in a recent study on biopsy tissue of ICPBSpatients [91] Increased expression of ICAM-1 subsequentto exposure with acetic acid in animal model is thereforeclinically relevant

Antisense experiments supported the earlier report thatinhibition of NGF expression significantly downregulates theexpression of ICAM-1 [80] It is reported that binding ofNGF to its high-affinity TrkA receptor controls the sICAM-1expression on target cells [80] Expression of ICAM-1 was

8 ISRN Pharmacology

Neutrophil

Monocytes

Acetic acid

Integrin

NGF

MCP-1

Release of cytokinesVanilloid

receptor

Trk receptors NGF

Membrane damage

VEGF

CXCL1

Activated leucocyte

Startrolling

Extravasation

CXCL10

sICAMssE-selectin

ActivationAdhesion

MCP-1

NGF

PKC

DAG

PLC

IP3Kinases

Ras

AKT

PI3K

CXCL10

sICAMs

LymphocyteAfferents

Figure 5 Signalling cascades induced by NGF overexpression following exposure to acetic acid Injury from exposure to acetic sets off thesignalling cascades that upregulate the expression of NGF MCP-1 prostaglandins CXCL-1 and CXCL-10 Overexpression of NGF drives theexpression of VEGF from neurons and leptin from adipocytes covering neurons NGF and MCP-1 also induce hyperexcitability in neuronsby activating TRPV1 receptor MCP-1 CXCL-1 and CXCL-10 cause chemoattraction of monocytes neutrophils and lymphocytes whichrequires extravasation across endothelium from blood through the cooperation of adhesion molecules E-selectin and ICAM-1

negatively associated with NGF expression in antisenseexperiments Ultimately expression of ICAM-1 is regulated atthe level of transcription by one of several signaling cascadesNF-120581B JAKSTAT IFN-g (CXCL-10) AP MAP kinase andPKC [92]

Intravesical route can allow selective exposure of anti-sense ODN to the NGF producing cells in urothelium andavoid systemic side effects from genetic manipulation ofNGF expression Intravesical route can also prove to be costeffective given the cost of ODN or siRNA Contrary to thegoal of loss of function in bladder with antisense ODN inrecent studies liposomes have also been used to deliver ODNfor gain of function in bladder [93]

10 Imaging of Intravesical Therapy

Drug development for DO and ICPBS relies heavily onsubjective outcomes for predicting efficacy and safety in earlyclinical development Although there are many validatedmeasurement tools used in DO and ICPBS research theyare usually burdensome and do not capture all symptomsrelated to the lower urinary tract Moreover improvement insymptoms scores following an intervention does not alwayscorrelate with patient expectations satisfaction and goalachievement which are critically important for successfulmanagement of DO and ICPBS

There is underdevelopment and underutilization of urinebiomarkers and imaging methods for investigational drugsgiven by intravesical route Overreliance on subjectiveimpressions of patients for therapeutic response limits clini-cal relevance due to weak correlation with patient satisfactionand can also impede continued scientific progress in assessing

study outcomes relative to symptom bother Symptom scoresmay also be related to the cause of high heterogeneity inclinical response in DO and ICPBS patients

Imaging can offer the possibility to provide region-specific information in situations where serum and urineassays may not reflect the true state of bladder [94ndash96]Urine chemokines have been reported to be associated withsymptom severity of ICPBS patients [97] Application offluorescent microscopy for imaging of pelvic floor has beenlimited due to the issues pertaining to light absorption bytissues scattering and autofluorescence [98]

Recent studies have shown that dyes fluorescing inthe near-infrared (NIR) band can overcome this handi-cap in deep-tissue imaging of experimental animals [99]Selected NIR fluorochromes emit light with tissue pen-etration approaching 10ndash15 cm [100] The light emissionfrom probes in this spectrum encounters low backgroundautofluorescence and minimal attenuation of signal due tolight absorption by tissue components [98] Therefore NIRimaging yields high signal-to-noise ratios and is well suitedfor studying the distribution of instilled treatments in bladder(Figure 2(b))

Preliminary studies suggest that imaging of mouse pelvicfloor in NIR spectrum can be a viable option [101] NIRimaging easily allows visualization and quantification ofbladder distribution by tracing themigration of fluorescence-labeled liposomes in anaesthetized mouse Compared withother biological assays imaging could provide objectiveendpoint for patients with ulcerative cystitis to indicate itshigh translation potential Other benefits of optical imaginginclude its relatively low cost compared withmore traditionalimaging systems such as MRI and PET The instrument

ISRN Pharmacology 9

consists of two near-infrared lasers emitting at 685 nm and785 nm camera and novel optics

Furthermore preferential binding of liposomes andnanoparticles to bladder lesions of ICPBS after instillationcan be assessed by NIR imaging of bladder where it cantheoretically provide anatomically specific information aboutthe real time status of ulcerlesion in bladder surface or tissueabnormality [102] Therefore future studies can substitute aresponse variable that is continuous in nature instead of asubjective clinical outcome

11 Conclusions

Adoption of forward-thinking approaches can achieveadvancements in drug delivery systems targeted to improvepharmacotherapy of bladder diseases in the future Latestdevelopments in the field of nanotechnology can bring thismode of therapy from second line of treatment for refractorycases to the forefront of disease management Liposomesare an attractive drug delivery platform by virtue of theirbiodegradability biocompatibility low toxicity and simpleand mild preparation methods

Conflict of Interests

All authors except Dr Michael Chancellor declare that thereis no conflict of interests regarding the publication of thispaper Dr Michael Chancellor owns stocks in the start-upliposome company Lipella

Acknowledgments

This work was supported by the National Institutes of Health(DK057267 and DK088836) and the Department of Defense(W81XWH-11-1-0763 and W81XWH-12-1-0565)

References

[1] J Parkin C Shea and G R Sant ldquoIntravesical dimethyl sul-foxide (DMSO) for interstitial cystitismdasha practical approachrdquoUrology vol 49 no 5 pp 105ndash107 1997

[2] C Dowson J Watkins M S Khan P Dasgupta and A SahaildquoRepeated botulinum toxin type A injections for refractoryoveractive bladder medium-term outcomes safety profile anddiscontinuation ratesrdquo European Urology vol 61 no 4 pp 834ndash839 2012

[3] J Kaufman V Tyagi M Anthony M B Chancellor and PTyagi ldquoState of the art in intravesical therapy for lower urinarytract symptomsrdquo Reviews in Urology vol 12 pp e181ndashe189 2010

[4] N Yoshimura and M B Chancellor ldquoCurrent and futurepharmacological treatment for overactive bladderrdquo Journal ofUrology vol 168 no 5 pp 1897ndash1913 2002

[5] K A Killinger J A Boura and K M Peters ldquoPain ininterstitial cystitisbladder pain syndrome do characteristicsdiffer in ulcerative and non-ulcerative subtypesrdquo InternationalUrogynecology Journal vol 24 no 8 pp 1295ndash1301 2013

[6] J C Nickel P Hanno K Kumar and HThomas ldquoSecond mul-ticenter randomized double-blind parallel-group evaluationof effectiveness and safety of intravesical sodium chondroitin

sulfate compared with inactive vehicle control in subjects withinterstitial cystitisbladder pain syndromerdquoUrology vol 79 no6 pp 1220ndash1225 2012

[7] P M Hanno ldquoAnalysis of long-term elmiron therapy forinterstitial cystitisrdquo Urology vol 49 no 5 pp 93ndash99 1997

[8] A Giannantoni V Bini R Dmochowski et al ldquoReply to ClausRiedlrsquos letter to the editor re antonella Giannantoni VittorioBini Roger Dmochowski et al Contemporary management ofthe painful bladder a systematic reviewrdquo EuropeanUrology vol61 no 5 pp 29ndash53 2012

[9] MC Lai Y CKuo andHCKuo ldquoIntravesical hyaluronic acidfor interstitial cystitispainful bladder syndrome a comparativerandomized assessment of different regimensrdquo InternationalJournal of Urology vol 20 pp 203ndash207 2013

[10] J Eldrup J Thorup and S L Nielsen ldquoPermeability andultrastructure of human bladder epitheliumrdquo British Journal ofUrology vol 55 no 5 pp 488ndash492 1983

[11] C L Parsons D Boychuk S Jones R Hurst and H CallahanldquoBladder surface glycosaminoglycans an epithelial permeabil-ity barrierrdquo Journal of Urology vol 143 no 1 pp 139ndash142 1990

[12] Y Kim N Yoshimura H Masuda F De Miguel and MB Chancellor ldquoAntimuscarinic agents exhibit local inhibitoryeffects on muscarinic receptors in bladder-afferent pathwaysrdquoUrology vol 65 no 2 pp 238ndash242 2005

[13] V Tyagi B J Philips R Su et al ldquoDifferential expression offunctional cannabinoid receptors in human bladder detrusorand urotheliumrdquo Journal of Urology vol 181 no 4 pp 1932ndash1938 2009

[14] B J Philips M C Smaldone V L Erickson N YoshimuraM B Chancellor and P Tyagi ldquoDifferential expression ofneurokinin receptor subtypes in urothelium and detrusor ofhuman bladderrdquo Journal of Urology vol 179 article 354 2008

[15] S A Baker G W Hennig J Han F C Britton T K Smithand S D Koh ldquoMethionine and its derivatives increase blad-der excitability by inhibiting stretch-dependent K+ channelsrdquoBritish Journal of Pharmacology vol 153 no 6 pp 1259ndash12712008

[16] J M Beckel A Kanai S-J Lee W C De Groat and LA Birder ldquoExpression of functional nicotinic acetylcholinereceptors in rat urinary bladder epithelial cellsrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 290 no 1 ppF103ndashF110 2006

[17] LA Birder A J KanaiWCDeGroat et al ldquoVanilloid receptorexpression suggests a sensory role for urinary bladder epithelialcellsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 23 pp 13396ndash13401 2001

[18] P Tyagi C A Thomas N Yoshimura and M B ChancellorldquoInvestigations into the presence of functional szlig1 szlig2 and szlig3-adrenoceptors in urothelium and detrusor of human bladderrdquoInternational Brazilian Journal of Urology vol 35 no 1 pp 76ndash83 2009

[19] A T Hanna-Mitchell J M Beckel S Barbadora A J Kanai WC de Groat and L A Birder ldquoNon-neuronal acetylcholine andurinary bladder urotheliumrdquo Life Sciences vol 80 no 24-25 pp2298ndash2302 2007

[20] C P Smith D A Gangitano A Munoz et al ldquoBotulinumtoxin type A normalizes alterations in urothelial ATP and NOrelease induced by chronic spinal cord injuryrdquo NeurochemistryInternational vol 52 no 6 pp 1068ndash1075 2008

[21] Y-C Chuang C A Thomas S Tyagi N Yoshimura PTyagi and M B Chancellor ldquoHuman urine with solifenacin

10 ISRN Pharmacology

intake but not tolterodine or darifenacin intake blocks detrusoroveractivityrdquo International Urogynecology Journal and PelvicFloor Dysfunction vol 19 no 10 pp 1353ndash1357 2008

[22] Y Kim N Yoshimura H Masuda F D Miguel and M BChancellor ldquoIntravesical instillation of human urine after oraladministration of trospium tolterodine and oxybutynin in a ratmodel of detrusor overactivityrdquo BJU International vol 97 no 2pp 400ndash403 2006

[23] S Tyagi P Tyagi S Van-le N YoshimuraM B Chancellor andF de Miguel ldquoQualitative and quantitative expression profileof muscarinic receptors in human urothelium and detrusorrdquoJournal of Urology vol 176 no 4 pp 1673ndash1678 2006

[24] G Gregoriadis and B E Ryman ldquoLiposomes as carriers ofenzymes or drugs a new approach to the treatment of storagediseasesrdquo Biochemical Journal vol 124 no 5 p 58 1971

[25] G Gregoriadis S Jain I Papaioannou and P Laing ldquoImprov-ing the therapeutic efficacy of peptides and proteins a role forpolysialic acidsrdquo International Journal of Pharmaceutics vol300 no 1-2 pp 125ndash130 2005

[26] P Sapra P Tyagi and T M Allen ldquoLigand-targeted liposomesfor cancer treatmentrdquo Current Drug Delivery vol 2 no 4 pp369ndash381 2005

[27] G Gregoriadis G Dapergolas and E D Neerunjun ldquoPene-tration of target areas in the rat by liposome associated agentsadministered parenterally and intragastricallyrdquo BiochemicalSociety Transactions vol 4 no 2 pp 256ndash259 1976

[28] G Gregoriadis ldquoEngineering liposomes for drug deliveryprogress and problemsrdquo Trends in Biotechnology vol 13 no 12pp 527ndash537 1995

[29] G Gregoriadis and A C Allison ldquoEntrapment of proteins inliposomes prevents allergic reactions in pre immunised micerdquoFEBS Letters vol 45 no 1 pp 71ndash74 1974

[30] S Lee S Dausch CMaierhofer andD Dausch ldquoA new therapyconcept with a liposome eye spray for the treatment of the lsquodryeyersquordquo Klinische Monatsblatter fur Augenheilkunde vol 221 no10 pp 825ndash836 2004

[31] D Dausch S Lee S Dausch J C Kim G Schwert and WMichelson ldquoComparative study of treatment of the dry eyesyndrome due to disturbances of the tear film lipid layer withlipid-containing tear substitutes efficacy of lipid-containingtear substitutesrdquo Klinische Monatsblatter fur Augenheilkundevol 223 no 12 pp 974ndash983 2006

[32] S Ebrahim G A Peyman and P J Lee ldquoApplications ofliposomes in ophthalmologyrdquo Survey of Ophthalmology vol 50no 2 pp 167ndash182 2005

[33] J Nirmal P Tyagi L Dang et al ldquoEndocytosis uptake ofliposomes in urothelium cells detected by transmission electronmicroscopyrdquoThe Journal of Urology vol 187 article e15 2012

[34] D N Frangos J J Killion D Fan R Fishbeck A C VonEschenbach and I J Fidler ldquoThe development of liposomescontaining interferon alpha for the intravesical therapy ofhuman superficial bladder cancerrdquo Journal of Urology vol 143no 6 pp 1252ndash1266 1990

[35] J W Johnson R Nayar J J Killion A C Von Eschenbachand I J Fidler ldquoBinding of liposomes to human bladdertumor epithelial cell lines implications for an intravesical drugdelivery system for the treatment of bladder cancerrdquo SelectiveCancer Therapeutics vol 5 no 4 pp 147ndash155 1989

[36] Y-C Chuang M B Chancellor S Seki et al ldquoIntravesicalprotamine sulfate and potassium chloride as a model forbladder hyperactivityrdquoUrology vol 61 no 3 pp 664ndash670 2003

[37] J Kaufman P Tyagi and M B Chancellor ldquoIntravesicalliposomal (Lp08) instillation protects bladder urothelium fromchemical irritationrdquoThe Journal of Urology vol 181 article 5392009

[38] P Tyagi M B Chancellor Z Li et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical capsaicindelivery using thermosensitive hydrogel and liposomesrdquo Jour-nal of Urology vol 171 no 1 pp 483ndash489 2004

[39] MO Fraser Y-CChuang P Tyagi et al ldquoIntravesical liposomeadministrationmdasha novel treatment for hyperactive bladder inthe ratrdquo Urology vol 61 no 3 pp 656ndash663 2003

[40] P Tyagi M Chancellor N Yoshimura and L Huang ldquoActivityof different phospholipids in attenuating hyperactivity in blad-der irritationrdquo BJU International vol 101 no 5 pp 627ndash6322008

[41] Y Sun and T C Chai ldquoEffects of dimethyl sulphoxide andheparin on stretch-activated ATP release by bladder urothelialcells from patients with interstitial cystitisrdquo BJU Internationalvol 90 no 4 pp 381ndash385 2002

[42] J J Bade ldquoA placebo-controlled study of intravesical pentosan-polysulphate for the treatment of interstitial cystitisrdquo BritishJournal of Urology vol 79 no 2 pp 168ndash171 1997

[43] P Tyagi V C Hsieh N Yoshimura J Kaufman and M BChancellor ldquoInstillation of liposomes vs dimethyl sulphoxide orpentosan polysulphate for reducing bladder hyperactivityrdquo BJUInternational vol 104 no 11 pp 1689ndash1692 2009

[44] W-C Lee C-T Chien H-J Yu and S-W Lee ldquoBladderdysfunction in rats with metabolic syndrome induced by long-term fructose feedingrdquo Journal of Urology vol 179 no 6 pp2470ndash2476 2008

[45] J I Gillespie G A Van Koeveringe S G DeWachter and J DeVente ldquoOn the origins of the sensory output from the bladderthe concept of afferent noiserdquo BJU International vol 103 no 10pp 1324ndash1333 2009

[46] W C Lee P H Chiang Y L Tain C C Wu and Y CChuang ldquoSensory dysfunction of bladder mucosa and bladderoversensitivity in a rat model of metabolic syndromerdquo PloSONE vol 7 Article ID e45578 2012

[47] Y-C Chuang W-C Lee W-C Lee and P-H Chiang ldquoIntrav-esical liposome versus oral pentosan polysulfate for interstitialcystitispainful bladder syndromerdquo Journal of Urology vol 182no 4 pp 1393ndash1400 2009

[48] W-C Lee Y-C Chuang W-C Lee and P-H Chiang ldquoSafetyand dose flexibility clinical evaluation of intravesical liposomein patients with interstitial cystitis or painful bladder syn-dromerdquo Kaohsiung Journal of Medical Sciences vol 27 no 10pp 437ndash440 2011

[49] S T Truschel E Wang W G Ruiz et al ldquoStretch-regulatedexocytosisendocytosis in bladder umbrella cellsrdquo MolecularBiology of the Cell vol 13 no 3 pp 830ndash846 2002

[50] M B Chancellor and W C De Groat ldquoIntravesical capsaicinand resiniferatoxin therapy spicing up the ways to treat theoveractive bladderrdquo Journal of Urology vol 162 no 1 pp 3ndash111999

[51] D S Byrne A Das J Sedor et al ldquoEffect of intravesicalcapsaicin and vehicle on bladder integrity in control and spinalcord injured ratsrdquo Journal of Urology vol 159 no 3 pp 1074ndash1078 1998

[52] M Mandal and K-D Lee ldquoListeriolysin O-liposome-mediatedcytosolic delivery of macromolecule antigen in vivo enhance-ment of antigen-specific cytotoxic T lymphocyte frequency

ISRN Pharmacology 11

activity and tumor protectionrdquo Biochimica et Biophysica Actavol 1563 no 1-2 pp 7ndash17 2002

[53] Y-C Chuang P Tyagi C-C Huang et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical botulinumtoxin a delivery using liposomesrdquo Journal of Urology vol 182no 2 pp 786ndash792 2009

[54] Y-C Chuang N Yoshimura C-C Huang P-H Chiang andM B Chancellor ldquoIntravesical botulinum toxin a administra-tion produces analgesia against acetic acid induced bladder painresponses in ratsrdquo Journal of Urology vol 172 no 4 pp 1529ndash1532 2004

[55] S P Petrou A S Parker J E Crook A Rogers D Metz-Kudashick and D D Thiel ldquoBotulinum A toxindimethylsulfoxide bladder instillations for women with refractory idio-pathic detrusor overactivity a phase 12 studyrdquo Mayo ClinicProceedings vol 84 no 8 pp 702ndash706 2009

[56] P Caccin O Rossetto M Rigoni E Johnson G Schiavoand C Montecucco ldquoVAMPsynaptobrevin cleavage by tetanusand botulinum neurotoxins is strongly enhanced by acidicliposomesrdquo FEBS Letters vol 542 no 1ndash3 pp 132ndash136 2003

[57] M L Stephenson and P C Zamecnik ldquoInhibition of Roussarcoma viral RNA translation by a specific oligodeoxyribonu-cleotiderdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 75 no 1 pp 285ndash288 1978

[58] H Dadgostar and N Waheed ldquoThe evolving role of vascularendothelial growth factor inhibitors in the treatment of neovas-cular age-related macular degenerationrdquo Eye vol 22 no 6 pp761ndash767 2008

[59] C Gebhard G Huard E A Kritikou and J C TardifldquoApolipoprotein B antisense inhibitionmdashupdate on mipom-ersenrdquo Current Pharmaceutical Design vol 919 no 17 pp 3132ndash3142 2013

[60] T Yokota E Hoffman and S Takeda ldquoAntisense oligo-mediated multiple exon skipping in a dog model of duchennemuscular dystrophyrdquoMethods inMolecular Biology vol 709 pp299ndash312 2011

[61] B L Jacobs M C Smaldone V Tyagi et al ldquoIncreased nervegrowth factor in neurogenic overactive bladder and interstitialcystitis patientsrdquoThe Canadian Journal of Urology vol 17 no 1pp 4989ndash4994 2010

[62] H-T Liu and H-C Kuo ldquoIntravesical botulinum toxin Ainjections plus hydrodistension can reduce nerve growth factorproduction and control bladder pain in interstitial cystitisrdquoUrology vol 70 no 3 pp 463ndash468 2007

[63] H T Liu and H C Kuo ldquoIncreased urine and serum nervegrowth factor levels in interstitial cystitis suggest chronicinflammation is involved in the pathogenesis of diseaserdquo PloSONE vol 7 Article ID e44687 2012

[64] N Yoshimura N E Bennett Y Hayashi et al ldquoBladderoveractivity and hyperexcitability of bladder afferent neuronsafter intrathecal delivery of nerve growth factor in ratsrdquo Journalof Neuroscience vol 26 no 42 pp 10847ndash10855 2006

[65] S Seki K Sasaki Y Igawa et al ldquoSuppression of detrusor-sphincter dyssynergia by immunoneutralization of nervegrowth factor in lumbosacral spinal cord in spinal cord injuredratsrdquo Journal of Urology vol 171 no 1 pp 478ndash482 2004

[66] R J Evans R M Moldwin N Cossons A Darekar I WMills andD Scholfield ldquoProof of concept trial of tanezumab forthe treatment of symptoms associated with interstitial cystitisrdquoJournal of Urology vol 185 no 5 pp 1716ndash1721 2011

[67] P Tyagi R Banerjee S Basu N YoshimuraM Chancellor andL Huang ldquoIntravesical antisense therapy for cystitis using TAT-peptide nucleic acid conjugatesrdquoMolecular Pharmaceutics vol3 no 4 pp 398ndash406 2006

[68] D C Molliver D E Wright M L Leitner et al ldquoIB4-bindingDRG neurons switch from NGF to GDNF dependence in earlypostnatal liferdquo Neuron vol 19 no 4 pp 849ndash861 1997

[69] M TyagiM RusnatiM Presta andMGiacca ldquoInternalizationof HIV-1 tat requires cell surface heparan sulfate proteoglycansrdquoJournal of Biological Chemistry vol 276 no 5 pp 3254ndash32612001

[70] E Uhlmann A Ryte and A Peyman ldquoStudies on the mech-anism of stabilization of partially phosphorothioated oligonu-cleotides against nucleolytic degradationrdquoAntisense andNucleicAcid Drug Development vol 7 no 4 pp 345ndash350 1997

[71] M Nogawa T Yuasa S Kimura et al ldquoIntravesical adminis-tration of small interfering RNA targeting PLK-1 successfullyprevents the growth of bladder cancerrdquo Journal of ClinicalInvestigation vol 115 no 4 pp 978ndash985 2005

[72] M Kashyap N Kawamorita V Tyagi et al ldquoDownregulation ofNGF expression in the bladder by antisense oligoucleotides asnew treatment for overactive bladderrdquo Journal of Urology vol27190 no 2 pp 757ndash764 2013

[73] C-J Arum Y Kodama N Rolim et al ldquoA rat model ofintravesical delivery of small interfering RNA for studyingurinary carcinomardquoWorld Journal of Urology vol 28 no 4 pp479ndash485 2010

[74] C E Blietz B Thode M Hauses et al ldquoIn vivo studies on theavailability and toxicity of antisense oligonucleotides in bladdercancerrdquo In Vivo vol 23 no 1 pp 13ndash19 2009

[75] D Oddiah P Anand S B McMahon and M Rattray ldquoRapidincrease of NGF BDNF andNT-3mRNAs in inflamed bladderrdquoNeuroReport vol 9 no 7 pp 1455ndash1458 1998

[76] M R Saban H Hellmich N B Nguyen J Winston T GHammond and R Saban ldquoTime course of LPS-induced geneexpression in a mouse model of genitourinary inflammationrdquoPhysiol Genomics vol 5 no 3 pp 147ndash160 2001

[77] B Schnegelsberg T T Sun G Cain et al ldquoOverexpression ofNGF in mouse urothelium leads to neuronal hyperinnervationpelvic sensitivity and changes in urinary bladder functionrdquoTheAmerican Journal of PhysiologymdashRegulatory Integrative andComparative Physiology vol 298 no 5 pp R534ndashR547 2010

[78] S Cardenas M Scuri L Samsell et al ldquoNeurotrophic andneuroimmune responses to early-life Pseudomonas aeruginosainfection in rat lungsrdquo American Journal of PhysiologymdashLungCellular and Molecular Physiology vol 299 no 3 pp L334ndashL344 2010

[79] K Nakamura F Tan Z Li and C J Thiele ldquoNGF activationof TrkA induces vascular endothelial growth factor expressionvia induction of hypoxia-inducible factor-1120572rdquo Molecular andCellular Neuroscience vol 46 no 2 pp 498ndash506 2011

[80] S Othumpangat M Regier and G Piedimonte ldquoNerve growthfactor modulates human rhinovirus infection in airway epithe-lial cells by controlling ICAM-1 expressionrdquo American Journalof Physiology Lung Cellular andMolecular Physiology vol 15302pp L1057ndashL1066 2012

[81] H Jung P T Toth F A White and R J Miller ldquoMonocytechemoattractant protein-1 functions as a neuromodulator indorsal root ganglia neuronsrdquo Journal of Neurochemistry vol104 no 1 pp 254ndash263 2008

[82] F A White H Jung and R J Miller ldquoChemokines andthe pathophysiology of neuropathic painrdquo Proceedings of the

12 ISRN Pharmacology

National Academy of Sciences of the United States of Americavol 104 no 51 pp 20151ndash20158 2007

[83] G Banisadr R-D Gosselin P Mechighel W Rostene PKitabgi and S M Parsadaniantz ldquoConstitutive neuronalexpression of CCR2 chemokine receptor and its colocalizationwith neurotransmitters in normal rat brain functional effectof MCP-1CCL2 on calcium mobilization in primary culturedneuronsrdquo Journal of Comparative Neurology vol 492 no 2 pp178ndash192 2005

[84] S Bhangoo D Ren R J Miller et al ldquoDelayed functionalexpression of neuronal chemokine receptors following focalnerve demyelination in the rat a mechanism for the devel-opment of chronic sensitization of peripheral nociceptorsrdquoMolecular Pain vol 3 article 38 2007

[85] T Maeda N Kiguchi Y Kobayashi T Ikuta M Ozaki and SKishioka ldquoLeptin derived from adipocytes in injured periph-eral nerves facilitates development of neuropathic pain viamacrophage stimulationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 106 no 31 pp13076ndash13081 2009

[86] H Kajiwara M Yasuda N Kumaki T Shibayama and YOsamura ldquoExpression of carbohydrate antigens (SSEA-1 Sialyl-Lewis X DU-PAN-2 and CA19-9) and E-selectin in urothelialcarcinoma of the renal pelvis ureter and urinary bladderrdquoTokaiJournal of Experimental and Clinical Medicine vol 30 no 3 pp177ndash182 2005

[87] A M S Assreuy G J Martins M E F Moreira et al ldquoPre-vention of cyclophosphamide-induced hemorrhagic cystitis byglucose-mannose binding plant lectinsrdquo Journal of Urology vol161 no 6 pp 1988ndash1993 1999

[88] S M Vinson A Rickard J S Ryerse and J McHowat ldquoNeu-trophil adherence to bladder microvascular endothelial cellsfollowing platelet-activating factor acetylhydrolase inhibitionrdquoJournal of Pharmacology and Experimental Therapeutics vol314 no 3 pp 1241ndash1247 2005

[89] M Green A Filippou G Sant and T CTheoharides ldquoExpres-sion of intercellular adhesion molecules in the bladder ofpatients with interstitial cystitisrdquoUrology vol 63 no 4 pp 688ndash693 2004

[90] M Leppilahti P Hellstrom and T L J Tammela ldquoEffect ofdiagnostic hydrodistension and four intravesical hyaluronicacid instillations on bladder ICAM-1 intensity and associationof ICAM-1 intensity with clinical response in patients withinterstitial cystitisrdquo Urology vol 60 no 1 pp 46ndash51 2002

[91] A T Corcoran N Yoshimura V Tyagi B Jacobs W Lengand P Tyagi ldquoMapping the cytokine profile of painful bladdersyndromeinterstitial cystitis in human bladder and urinespecimensrdquoWorld Journal of Urology vol 31 no 1 pp 241ndash2462013

[92] SGarrean X-PGaoV Brovkovych et al ldquoCaveolin-1 regulatesNF-120581B activation and lung inflammatory response to sepsisinduced by lipopolysacchariderdquo Journal of Immunology vol 177no 7 pp 4853ndash4860 2006

[93] M R Kang G Yang R F Place et al ldquoIntravesical deliveryof small activating RNA formulated into lipid nanoparticlesinhibits orthotopic bladder tumor growthrdquo Cancer Researchvol 172 pp 5069ndash5079 2012

[94] P Tyagi D Barclay R Zamora et al ldquoUrine cytokines suggestan inflammatory response in the overactive bladder a pilotstudyrdquo International Urology and Nephrology vol 42 no 3 pp629ndash635 2010

[95] P Tyagi B L Jacobs D Barcaly et al ldquoUrine levels ofinflammatory chemokines can be novel biomarkers for theoveractive bladderrdquoThe Journal of Urology vol 181 article 5882009

[96] P Tyagi V Tyagi D Bui et al ldquoDiscrimination Of OAB fromICPBS by multivariate data modeling of urinary proteinsrdquoTheJournal of Urology vol 183 pp e614ndashe6e5 2010

[97] P Tyagi D Nikolavsky Y Vodovotz et al ldquoUrine levels ofselected chemokines positively correlate with lower bladdercapacity and psychometric scores in IcPbs patientsrdquo TheJournal of Urology vol 181 article 21 2009

[98] R Weissleder and V Ntziachristos ldquoShedding light onto livemolecular targetsrdquo Nature Medicine vol 9 no 1 pp 123ndash1282003

[99] C Bremer V Ntziachristos and R Weissleder ldquoOptical-basedmolecular imaging contrast agents and potentialmedical appli-cationsrdquo European Radiology vol 13 no 2 pp 231ndash243 2003

[100] UMahmood andRWeissleder ldquoNear-infrared optical imagingof proteases in cancerrdquoMolecular CancerTherapeutics vol 2 pp489ndash496 2003

[101] J Kaufman H Hensley J Jacobs et al ldquoNon-invasive imagingof near infrafred dye labeled liposomes facilitates evaluation ofbioresidence timerdquoThe Journal of Urology vol 183 article e6282010

[102] Y-X J Wang ldquoMedical imaging in pharmaceutical clinicaltrials what radiologists should knowrdquo Clinical Radiology vol60 no 10 pp 1051ndash1057 2005

8 ISRN Pharmacology

Neutrophil

Monocytes

Acetic acid

Integrin

NGF

MCP-1

Release of cytokinesVanilloid

receptor

Trk receptors NGF

Membrane damage

VEGF

CXCL1

Activated leucocyte

Startrolling

Extravasation

CXCL10

sICAMssE-selectin

ActivationAdhesion

MCP-1

NGF

PKC

DAG

PLC

IP3Kinases

Ras

AKT

PI3K

CXCL10

sICAMs

LymphocyteAfferents

Figure 5 Signalling cascades induced by NGF overexpression following exposure to acetic acid Injury from exposure to acetic sets off thesignalling cascades that upregulate the expression of NGF MCP-1 prostaglandins CXCL-1 and CXCL-10 Overexpression of NGF drives theexpression of VEGF from neurons and leptin from adipocytes covering neurons NGF and MCP-1 also induce hyperexcitability in neuronsby activating TRPV1 receptor MCP-1 CXCL-1 and CXCL-10 cause chemoattraction of monocytes neutrophils and lymphocytes whichrequires extravasation across endothelium from blood through the cooperation of adhesion molecules E-selectin and ICAM-1

negatively associated with NGF expression in antisenseexperiments Ultimately expression of ICAM-1 is regulated atthe level of transcription by one of several signaling cascadesNF-120581B JAKSTAT IFN-g (CXCL-10) AP MAP kinase andPKC [92]

Intravesical route can allow selective exposure of anti-sense ODN to the NGF producing cells in urothelium andavoid systemic side effects from genetic manipulation ofNGF expression Intravesical route can also prove to be costeffective given the cost of ODN or siRNA Contrary to thegoal of loss of function in bladder with antisense ODN inrecent studies liposomes have also been used to deliver ODNfor gain of function in bladder [93]

10 Imaging of Intravesical Therapy

Drug development for DO and ICPBS relies heavily onsubjective outcomes for predicting efficacy and safety in earlyclinical development Although there are many validatedmeasurement tools used in DO and ICPBS research theyare usually burdensome and do not capture all symptomsrelated to the lower urinary tract Moreover improvement insymptoms scores following an intervention does not alwayscorrelate with patient expectations satisfaction and goalachievement which are critically important for successfulmanagement of DO and ICPBS

There is underdevelopment and underutilization of urinebiomarkers and imaging methods for investigational drugsgiven by intravesical route Overreliance on subjectiveimpressions of patients for therapeutic response limits clini-cal relevance due to weak correlation with patient satisfactionand can also impede continued scientific progress in assessing

study outcomes relative to symptom bother Symptom scoresmay also be related to the cause of high heterogeneity inclinical response in DO and ICPBS patients

Imaging can offer the possibility to provide region-specific information in situations where serum and urineassays may not reflect the true state of bladder [94ndash96]Urine chemokines have been reported to be associated withsymptom severity of ICPBS patients [97] Application offluorescent microscopy for imaging of pelvic floor has beenlimited due to the issues pertaining to light absorption bytissues scattering and autofluorescence [98]

Recent studies have shown that dyes fluorescing inthe near-infrared (NIR) band can overcome this handi-cap in deep-tissue imaging of experimental animals [99]Selected NIR fluorochromes emit light with tissue pen-etration approaching 10ndash15 cm [100] The light emissionfrom probes in this spectrum encounters low backgroundautofluorescence and minimal attenuation of signal due tolight absorption by tissue components [98] Therefore NIRimaging yields high signal-to-noise ratios and is well suitedfor studying the distribution of instilled treatments in bladder(Figure 2(b))

Preliminary studies suggest that imaging of mouse pelvicfloor in NIR spectrum can be a viable option [101] NIRimaging easily allows visualization and quantification ofbladder distribution by tracing themigration of fluorescence-labeled liposomes in anaesthetized mouse Compared withother biological assays imaging could provide objectiveendpoint for patients with ulcerative cystitis to indicate itshigh translation potential Other benefits of optical imaginginclude its relatively low cost compared withmore traditionalimaging systems such as MRI and PET The instrument

ISRN Pharmacology 9

consists of two near-infrared lasers emitting at 685 nm and785 nm camera and novel optics

Furthermore preferential binding of liposomes andnanoparticles to bladder lesions of ICPBS after instillationcan be assessed by NIR imaging of bladder where it cantheoretically provide anatomically specific information aboutthe real time status of ulcerlesion in bladder surface or tissueabnormality [102] Therefore future studies can substitute aresponse variable that is continuous in nature instead of asubjective clinical outcome

11 Conclusions

Adoption of forward-thinking approaches can achieveadvancements in drug delivery systems targeted to improvepharmacotherapy of bladder diseases in the future Latestdevelopments in the field of nanotechnology can bring thismode of therapy from second line of treatment for refractorycases to the forefront of disease management Liposomesare an attractive drug delivery platform by virtue of theirbiodegradability biocompatibility low toxicity and simpleand mild preparation methods

Conflict of Interests

All authors except Dr Michael Chancellor declare that thereis no conflict of interests regarding the publication of thispaper Dr Michael Chancellor owns stocks in the start-upliposome company Lipella

Acknowledgments

This work was supported by the National Institutes of Health(DK057267 and DK088836) and the Department of Defense(W81XWH-11-1-0763 and W81XWH-12-1-0565)

References

[1] J Parkin C Shea and G R Sant ldquoIntravesical dimethyl sul-foxide (DMSO) for interstitial cystitismdasha practical approachrdquoUrology vol 49 no 5 pp 105ndash107 1997

[2] C Dowson J Watkins M S Khan P Dasgupta and A SahaildquoRepeated botulinum toxin type A injections for refractoryoveractive bladder medium-term outcomes safety profile anddiscontinuation ratesrdquo European Urology vol 61 no 4 pp 834ndash839 2012

[3] J Kaufman V Tyagi M Anthony M B Chancellor and PTyagi ldquoState of the art in intravesical therapy for lower urinarytract symptomsrdquo Reviews in Urology vol 12 pp e181ndashe189 2010

[4] N Yoshimura and M B Chancellor ldquoCurrent and futurepharmacological treatment for overactive bladderrdquo Journal ofUrology vol 168 no 5 pp 1897ndash1913 2002

[5] K A Killinger J A Boura and K M Peters ldquoPain ininterstitial cystitisbladder pain syndrome do characteristicsdiffer in ulcerative and non-ulcerative subtypesrdquo InternationalUrogynecology Journal vol 24 no 8 pp 1295ndash1301 2013

[6] J C Nickel P Hanno K Kumar and HThomas ldquoSecond mul-ticenter randomized double-blind parallel-group evaluationof effectiveness and safety of intravesical sodium chondroitin

sulfate compared with inactive vehicle control in subjects withinterstitial cystitisbladder pain syndromerdquoUrology vol 79 no6 pp 1220ndash1225 2012

[7] P M Hanno ldquoAnalysis of long-term elmiron therapy forinterstitial cystitisrdquo Urology vol 49 no 5 pp 93ndash99 1997

[8] A Giannantoni V Bini R Dmochowski et al ldquoReply to ClausRiedlrsquos letter to the editor re antonella Giannantoni VittorioBini Roger Dmochowski et al Contemporary management ofthe painful bladder a systematic reviewrdquo EuropeanUrology vol61 no 5 pp 29ndash53 2012

[9] MC Lai Y CKuo andHCKuo ldquoIntravesical hyaluronic acidfor interstitial cystitispainful bladder syndrome a comparativerandomized assessment of different regimensrdquo InternationalJournal of Urology vol 20 pp 203ndash207 2013

[10] J Eldrup J Thorup and S L Nielsen ldquoPermeability andultrastructure of human bladder epitheliumrdquo British Journal ofUrology vol 55 no 5 pp 488ndash492 1983

[11] C L Parsons D Boychuk S Jones R Hurst and H CallahanldquoBladder surface glycosaminoglycans an epithelial permeabil-ity barrierrdquo Journal of Urology vol 143 no 1 pp 139ndash142 1990

[12] Y Kim N Yoshimura H Masuda F De Miguel and MB Chancellor ldquoAntimuscarinic agents exhibit local inhibitoryeffects on muscarinic receptors in bladder-afferent pathwaysrdquoUrology vol 65 no 2 pp 238ndash242 2005

[13] V Tyagi B J Philips R Su et al ldquoDifferential expression offunctional cannabinoid receptors in human bladder detrusorand urotheliumrdquo Journal of Urology vol 181 no 4 pp 1932ndash1938 2009

[14] B J Philips M C Smaldone V L Erickson N YoshimuraM B Chancellor and P Tyagi ldquoDifferential expression ofneurokinin receptor subtypes in urothelium and detrusor ofhuman bladderrdquo Journal of Urology vol 179 article 354 2008

[15] S A Baker G W Hennig J Han F C Britton T K Smithand S D Koh ldquoMethionine and its derivatives increase blad-der excitability by inhibiting stretch-dependent K+ channelsrdquoBritish Journal of Pharmacology vol 153 no 6 pp 1259ndash12712008

[16] J M Beckel A Kanai S-J Lee W C De Groat and LA Birder ldquoExpression of functional nicotinic acetylcholinereceptors in rat urinary bladder epithelial cellsrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 290 no 1 ppF103ndashF110 2006

[17] LA Birder A J KanaiWCDeGroat et al ldquoVanilloid receptorexpression suggests a sensory role for urinary bladder epithelialcellsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 23 pp 13396ndash13401 2001

[18] P Tyagi C A Thomas N Yoshimura and M B ChancellorldquoInvestigations into the presence of functional szlig1 szlig2 and szlig3-adrenoceptors in urothelium and detrusor of human bladderrdquoInternational Brazilian Journal of Urology vol 35 no 1 pp 76ndash83 2009

[19] A T Hanna-Mitchell J M Beckel S Barbadora A J Kanai WC de Groat and L A Birder ldquoNon-neuronal acetylcholine andurinary bladder urotheliumrdquo Life Sciences vol 80 no 24-25 pp2298ndash2302 2007

[20] C P Smith D A Gangitano A Munoz et al ldquoBotulinumtoxin type A normalizes alterations in urothelial ATP and NOrelease induced by chronic spinal cord injuryrdquo NeurochemistryInternational vol 52 no 6 pp 1068ndash1075 2008

[21] Y-C Chuang C A Thomas S Tyagi N Yoshimura PTyagi and M B Chancellor ldquoHuman urine with solifenacin

10 ISRN Pharmacology

intake but not tolterodine or darifenacin intake blocks detrusoroveractivityrdquo International Urogynecology Journal and PelvicFloor Dysfunction vol 19 no 10 pp 1353ndash1357 2008

[22] Y Kim N Yoshimura H Masuda F D Miguel and M BChancellor ldquoIntravesical instillation of human urine after oraladministration of trospium tolterodine and oxybutynin in a ratmodel of detrusor overactivityrdquo BJU International vol 97 no 2pp 400ndash403 2006

[23] S Tyagi P Tyagi S Van-le N YoshimuraM B Chancellor andF de Miguel ldquoQualitative and quantitative expression profileof muscarinic receptors in human urothelium and detrusorrdquoJournal of Urology vol 176 no 4 pp 1673ndash1678 2006

[24] G Gregoriadis and B E Ryman ldquoLiposomes as carriers ofenzymes or drugs a new approach to the treatment of storagediseasesrdquo Biochemical Journal vol 124 no 5 p 58 1971

[25] G Gregoriadis S Jain I Papaioannou and P Laing ldquoImprov-ing the therapeutic efficacy of peptides and proteins a role forpolysialic acidsrdquo International Journal of Pharmaceutics vol300 no 1-2 pp 125ndash130 2005

[26] P Sapra P Tyagi and T M Allen ldquoLigand-targeted liposomesfor cancer treatmentrdquo Current Drug Delivery vol 2 no 4 pp369ndash381 2005

[27] G Gregoriadis G Dapergolas and E D Neerunjun ldquoPene-tration of target areas in the rat by liposome associated agentsadministered parenterally and intragastricallyrdquo BiochemicalSociety Transactions vol 4 no 2 pp 256ndash259 1976

[28] G Gregoriadis ldquoEngineering liposomes for drug deliveryprogress and problemsrdquo Trends in Biotechnology vol 13 no 12pp 527ndash537 1995

[29] G Gregoriadis and A C Allison ldquoEntrapment of proteins inliposomes prevents allergic reactions in pre immunised micerdquoFEBS Letters vol 45 no 1 pp 71ndash74 1974

[30] S Lee S Dausch CMaierhofer andD Dausch ldquoA new therapyconcept with a liposome eye spray for the treatment of the lsquodryeyersquordquo Klinische Monatsblatter fur Augenheilkunde vol 221 no10 pp 825ndash836 2004

[31] D Dausch S Lee S Dausch J C Kim G Schwert and WMichelson ldquoComparative study of treatment of the dry eyesyndrome due to disturbances of the tear film lipid layer withlipid-containing tear substitutes efficacy of lipid-containingtear substitutesrdquo Klinische Monatsblatter fur Augenheilkundevol 223 no 12 pp 974ndash983 2006

[32] S Ebrahim G A Peyman and P J Lee ldquoApplications ofliposomes in ophthalmologyrdquo Survey of Ophthalmology vol 50no 2 pp 167ndash182 2005

[33] J Nirmal P Tyagi L Dang et al ldquoEndocytosis uptake ofliposomes in urothelium cells detected by transmission electronmicroscopyrdquoThe Journal of Urology vol 187 article e15 2012

[34] D N Frangos J J Killion D Fan R Fishbeck A C VonEschenbach and I J Fidler ldquoThe development of liposomescontaining interferon alpha for the intravesical therapy ofhuman superficial bladder cancerrdquo Journal of Urology vol 143no 6 pp 1252ndash1266 1990

[35] J W Johnson R Nayar J J Killion A C Von Eschenbachand I J Fidler ldquoBinding of liposomes to human bladdertumor epithelial cell lines implications for an intravesical drugdelivery system for the treatment of bladder cancerrdquo SelectiveCancer Therapeutics vol 5 no 4 pp 147ndash155 1989

[36] Y-C Chuang M B Chancellor S Seki et al ldquoIntravesicalprotamine sulfate and potassium chloride as a model forbladder hyperactivityrdquoUrology vol 61 no 3 pp 664ndash670 2003

[37] J Kaufman P Tyagi and M B Chancellor ldquoIntravesicalliposomal (Lp08) instillation protects bladder urothelium fromchemical irritationrdquoThe Journal of Urology vol 181 article 5392009

[38] P Tyagi M B Chancellor Z Li et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical capsaicindelivery using thermosensitive hydrogel and liposomesrdquo Jour-nal of Urology vol 171 no 1 pp 483ndash489 2004

[39] MO Fraser Y-CChuang P Tyagi et al ldquoIntravesical liposomeadministrationmdasha novel treatment for hyperactive bladder inthe ratrdquo Urology vol 61 no 3 pp 656ndash663 2003

[40] P Tyagi M Chancellor N Yoshimura and L Huang ldquoActivityof different phospholipids in attenuating hyperactivity in blad-der irritationrdquo BJU International vol 101 no 5 pp 627ndash6322008

[41] Y Sun and T C Chai ldquoEffects of dimethyl sulphoxide andheparin on stretch-activated ATP release by bladder urothelialcells from patients with interstitial cystitisrdquo BJU Internationalvol 90 no 4 pp 381ndash385 2002

[42] J J Bade ldquoA placebo-controlled study of intravesical pentosan-polysulphate for the treatment of interstitial cystitisrdquo BritishJournal of Urology vol 79 no 2 pp 168ndash171 1997

[43] P Tyagi V C Hsieh N Yoshimura J Kaufman and M BChancellor ldquoInstillation of liposomes vs dimethyl sulphoxide orpentosan polysulphate for reducing bladder hyperactivityrdquo BJUInternational vol 104 no 11 pp 1689ndash1692 2009

[44] W-C Lee C-T Chien H-J Yu and S-W Lee ldquoBladderdysfunction in rats with metabolic syndrome induced by long-term fructose feedingrdquo Journal of Urology vol 179 no 6 pp2470ndash2476 2008

[45] J I Gillespie G A Van Koeveringe S G DeWachter and J DeVente ldquoOn the origins of the sensory output from the bladderthe concept of afferent noiserdquo BJU International vol 103 no 10pp 1324ndash1333 2009

[46] W C Lee P H Chiang Y L Tain C C Wu and Y CChuang ldquoSensory dysfunction of bladder mucosa and bladderoversensitivity in a rat model of metabolic syndromerdquo PloSONE vol 7 Article ID e45578 2012

[47] Y-C Chuang W-C Lee W-C Lee and P-H Chiang ldquoIntrav-esical liposome versus oral pentosan polysulfate for interstitialcystitispainful bladder syndromerdquo Journal of Urology vol 182no 4 pp 1393ndash1400 2009

[48] W-C Lee Y-C Chuang W-C Lee and P-H Chiang ldquoSafetyand dose flexibility clinical evaluation of intravesical liposomein patients with interstitial cystitis or painful bladder syn-dromerdquo Kaohsiung Journal of Medical Sciences vol 27 no 10pp 437ndash440 2011

[49] S T Truschel E Wang W G Ruiz et al ldquoStretch-regulatedexocytosisendocytosis in bladder umbrella cellsrdquo MolecularBiology of the Cell vol 13 no 3 pp 830ndash846 2002

[50] M B Chancellor and W C De Groat ldquoIntravesical capsaicinand resiniferatoxin therapy spicing up the ways to treat theoveractive bladderrdquo Journal of Urology vol 162 no 1 pp 3ndash111999

[51] D S Byrne A Das J Sedor et al ldquoEffect of intravesicalcapsaicin and vehicle on bladder integrity in control and spinalcord injured ratsrdquo Journal of Urology vol 159 no 3 pp 1074ndash1078 1998

[52] M Mandal and K-D Lee ldquoListeriolysin O-liposome-mediatedcytosolic delivery of macromolecule antigen in vivo enhance-ment of antigen-specific cytotoxic T lymphocyte frequency

ISRN Pharmacology 11

activity and tumor protectionrdquo Biochimica et Biophysica Actavol 1563 no 1-2 pp 7ndash17 2002

[53] Y-C Chuang P Tyagi C-C Huang et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical botulinumtoxin a delivery using liposomesrdquo Journal of Urology vol 182no 2 pp 786ndash792 2009

[54] Y-C Chuang N Yoshimura C-C Huang P-H Chiang andM B Chancellor ldquoIntravesical botulinum toxin a administra-tion produces analgesia against acetic acid induced bladder painresponses in ratsrdquo Journal of Urology vol 172 no 4 pp 1529ndash1532 2004

[55] S P Petrou A S Parker J E Crook A Rogers D Metz-Kudashick and D D Thiel ldquoBotulinum A toxindimethylsulfoxide bladder instillations for women with refractory idio-pathic detrusor overactivity a phase 12 studyrdquo Mayo ClinicProceedings vol 84 no 8 pp 702ndash706 2009

[56] P Caccin O Rossetto M Rigoni E Johnson G Schiavoand C Montecucco ldquoVAMPsynaptobrevin cleavage by tetanusand botulinum neurotoxins is strongly enhanced by acidicliposomesrdquo FEBS Letters vol 542 no 1ndash3 pp 132ndash136 2003

[57] M L Stephenson and P C Zamecnik ldquoInhibition of Roussarcoma viral RNA translation by a specific oligodeoxyribonu-cleotiderdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 75 no 1 pp 285ndash288 1978

[58] H Dadgostar and N Waheed ldquoThe evolving role of vascularendothelial growth factor inhibitors in the treatment of neovas-cular age-related macular degenerationrdquo Eye vol 22 no 6 pp761ndash767 2008

[59] C Gebhard G Huard E A Kritikou and J C TardifldquoApolipoprotein B antisense inhibitionmdashupdate on mipom-ersenrdquo Current Pharmaceutical Design vol 919 no 17 pp 3132ndash3142 2013

[60] T Yokota E Hoffman and S Takeda ldquoAntisense oligo-mediated multiple exon skipping in a dog model of duchennemuscular dystrophyrdquoMethods inMolecular Biology vol 709 pp299ndash312 2011

[61] B L Jacobs M C Smaldone V Tyagi et al ldquoIncreased nervegrowth factor in neurogenic overactive bladder and interstitialcystitis patientsrdquoThe Canadian Journal of Urology vol 17 no 1pp 4989ndash4994 2010

[62] H-T Liu and H-C Kuo ldquoIntravesical botulinum toxin Ainjections plus hydrodistension can reduce nerve growth factorproduction and control bladder pain in interstitial cystitisrdquoUrology vol 70 no 3 pp 463ndash468 2007

[63] H T Liu and H C Kuo ldquoIncreased urine and serum nervegrowth factor levels in interstitial cystitis suggest chronicinflammation is involved in the pathogenesis of diseaserdquo PloSONE vol 7 Article ID e44687 2012

[64] N Yoshimura N E Bennett Y Hayashi et al ldquoBladderoveractivity and hyperexcitability of bladder afferent neuronsafter intrathecal delivery of nerve growth factor in ratsrdquo Journalof Neuroscience vol 26 no 42 pp 10847ndash10855 2006

[65] S Seki K Sasaki Y Igawa et al ldquoSuppression of detrusor-sphincter dyssynergia by immunoneutralization of nervegrowth factor in lumbosacral spinal cord in spinal cord injuredratsrdquo Journal of Urology vol 171 no 1 pp 478ndash482 2004

[66] R J Evans R M Moldwin N Cossons A Darekar I WMills andD Scholfield ldquoProof of concept trial of tanezumab forthe treatment of symptoms associated with interstitial cystitisrdquoJournal of Urology vol 185 no 5 pp 1716ndash1721 2011

[67] P Tyagi R Banerjee S Basu N YoshimuraM Chancellor andL Huang ldquoIntravesical antisense therapy for cystitis using TAT-peptide nucleic acid conjugatesrdquoMolecular Pharmaceutics vol3 no 4 pp 398ndash406 2006

[68] D C Molliver D E Wright M L Leitner et al ldquoIB4-bindingDRG neurons switch from NGF to GDNF dependence in earlypostnatal liferdquo Neuron vol 19 no 4 pp 849ndash861 1997

[69] M TyagiM RusnatiM Presta andMGiacca ldquoInternalizationof HIV-1 tat requires cell surface heparan sulfate proteoglycansrdquoJournal of Biological Chemistry vol 276 no 5 pp 3254ndash32612001

[70] E Uhlmann A Ryte and A Peyman ldquoStudies on the mech-anism of stabilization of partially phosphorothioated oligonu-cleotides against nucleolytic degradationrdquoAntisense andNucleicAcid Drug Development vol 7 no 4 pp 345ndash350 1997

[71] M Nogawa T Yuasa S Kimura et al ldquoIntravesical adminis-tration of small interfering RNA targeting PLK-1 successfullyprevents the growth of bladder cancerrdquo Journal of ClinicalInvestigation vol 115 no 4 pp 978ndash985 2005

[72] M Kashyap N Kawamorita V Tyagi et al ldquoDownregulation ofNGF expression in the bladder by antisense oligoucleotides asnew treatment for overactive bladderrdquo Journal of Urology vol27190 no 2 pp 757ndash764 2013

[73] C-J Arum Y Kodama N Rolim et al ldquoA rat model ofintravesical delivery of small interfering RNA for studyingurinary carcinomardquoWorld Journal of Urology vol 28 no 4 pp479ndash485 2010

[74] C E Blietz B Thode M Hauses et al ldquoIn vivo studies on theavailability and toxicity of antisense oligonucleotides in bladdercancerrdquo In Vivo vol 23 no 1 pp 13ndash19 2009

[75] D Oddiah P Anand S B McMahon and M Rattray ldquoRapidincrease of NGF BDNF andNT-3mRNAs in inflamed bladderrdquoNeuroReport vol 9 no 7 pp 1455ndash1458 1998

[76] M R Saban H Hellmich N B Nguyen J Winston T GHammond and R Saban ldquoTime course of LPS-induced geneexpression in a mouse model of genitourinary inflammationrdquoPhysiol Genomics vol 5 no 3 pp 147ndash160 2001

[77] B Schnegelsberg T T Sun G Cain et al ldquoOverexpression ofNGF in mouse urothelium leads to neuronal hyperinnervationpelvic sensitivity and changes in urinary bladder functionrdquoTheAmerican Journal of PhysiologymdashRegulatory Integrative andComparative Physiology vol 298 no 5 pp R534ndashR547 2010

[78] S Cardenas M Scuri L Samsell et al ldquoNeurotrophic andneuroimmune responses to early-life Pseudomonas aeruginosainfection in rat lungsrdquo American Journal of PhysiologymdashLungCellular and Molecular Physiology vol 299 no 3 pp L334ndashL344 2010

[79] K Nakamura F Tan Z Li and C J Thiele ldquoNGF activationof TrkA induces vascular endothelial growth factor expressionvia induction of hypoxia-inducible factor-1120572rdquo Molecular andCellular Neuroscience vol 46 no 2 pp 498ndash506 2011

[80] S Othumpangat M Regier and G Piedimonte ldquoNerve growthfactor modulates human rhinovirus infection in airway epithe-lial cells by controlling ICAM-1 expressionrdquo American Journalof Physiology Lung Cellular andMolecular Physiology vol 15302pp L1057ndashL1066 2012

[81] H Jung P T Toth F A White and R J Miller ldquoMonocytechemoattractant protein-1 functions as a neuromodulator indorsal root ganglia neuronsrdquo Journal of Neurochemistry vol104 no 1 pp 254ndash263 2008

[82] F A White H Jung and R J Miller ldquoChemokines andthe pathophysiology of neuropathic painrdquo Proceedings of the

12 ISRN Pharmacology

National Academy of Sciences of the United States of Americavol 104 no 51 pp 20151ndash20158 2007

[83] G Banisadr R-D Gosselin P Mechighel W Rostene PKitabgi and S M Parsadaniantz ldquoConstitutive neuronalexpression of CCR2 chemokine receptor and its colocalizationwith neurotransmitters in normal rat brain functional effectof MCP-1CCL2 on calcium mobilization in primary culturedneuronsrdquo Journal of Comparative Neurology vol 492 no 2 pp178ndash192 2005

[84] S Bhangoo D Ren R J Miller et al ldquoDelayed functionalexpression of neuronal chemokine receptors following focalnerve demyelination in the rat a mechanism for the devel-opment of chronic sensitization of peripheral nociceptorsrdquoMolecular Pain vol 3 article 38 2007

[85] T Maeda N Kiguchi Y Kobayashi T Ikuta M Ozaki and SKishioka ldquoLeptin derived from adipocytes in injured periph-eral nerves facilitates development of neuropathic pain viamacrophage stimulationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 106 no 31 pp13076ndash13081 2009

[86] H Kajiwara M Yasuda N Kumaki T Shibayama and YOsamura ldquoExpression of carbohydrate antigens (SSEA-1 Sialyl-Lewis X DU-PAN-2 and CA19-9) and E-selectin in urothelialcarcinoma of the renal pelvis ureter and urinary bladderrdquoTokaiJournal of Experimental and Clinical Medicine vol 30 no 3 pp177ndash182 2005

[87] A M S Assreuy G J Martins M E F Moreira et al ldquoPre-vention of cyclophosphamide-induced hemorrhagic cystitis byglucose-mannose binding plant lectinsrdquo Journal of Urology vol161 no 6 pp 1988ndash1993 1999

[88] S M Vinson A Rickard J S Ryerse and J McHowat ldquoNeu-trophil adherence to bladder microvascular endothelial cellsfollowing platelet-activating factor acetylhydrolase inhibitionrdquoJournal of Pharmacology and Experimental Therapeutics vol314 no 3 pp 1241ndash1247 2005

[89] M Green A Filippou G Sant and T CTheoharides ldquoExpres-sion of intercellular adhesion molecules in the bladder ofpatients with interstitial cystitisrdquoUrology vol 63 no 4 pp 688ndash693 2004

[90] M Leppilahti P Hellstrom and T L J Tammela ldquoEffect ofdiagnostic hydrodistension and four intravesical hyaluronicacid instillations on bladder ICAM-1 intensity and associationof ICAM-1 intensity with clinical response in patients withinterstitial cystitisrdquo Urology vol 60 no 1 pp 46ndash51 2002

[91] A T Corcoran N Yoshimura V Tyagi B Jacobs W Lengand P Tyagi ldquoMapping the cytokine profile of painful bladdersyndromeinterstitial cystitis in human bladder and urinespecimensrdquoWorld Journal of Urology vol 31 no 1 pp 241ndash2462013

[92] SGarrean X-PGaoV Brovkovych et al ldquoCaveolin-1 regulatesNF-120581B activation and lung inflammatory response to sepsisinduced by lipopolysacchariderdquo Journal of Immunology vol 177no 7 pp 4853ndash4860 2006

[93] M R Kang G Yang R F Place et al ldquoIntravesical deliveryof small activating RNA formulated into lipid nanoparticlesinhibits orthotopic bladder tumor growthrdquo Cancer Researchvol 172 pp 5069ndash5079 2012

[94] P Tyagi D Barclay R Zamora et al ldquoUrine cytokines suggestan inflammatory response in the overactive bladder a pilotstudyrdquo International Urology and Nephrology vol 42 no 3 pp629ndash635 2010

[95] P Tyagi B L Jacobs D Barcaly et al ldquoUrine levels ofinflammatory chemokines can be novel biomarkers for theoveractive bladderrdquoThe Journal of Urology vol 181 article 5882009

[96] P Tyagi V Tyagi D Bui et al ldquoDiscrimination Of OAB fromICPBS by multivariate data modeling of urinary proteinsrdquoTheJournal of Urology vol 183 pp e614ndashe6e5 2010

[97] P Tyagi D Nikolavsky Y Vodovotz et al ldquoUrine levels ofselected chemokines positively correlate with lower bladdercapacity and psychometric scores in IcPbs patientsrdquo TheJournal of Urology vol 181 article 21 2009

[98] R Weissleder and V Ntziachristos ldquoShedding light onto livemolecular targetsrdquo Nature Medicine vol 9 no 1 pp 123ndash1282003

[99] C Bremer V Ntziachristos and R Weissleder ldquoOptical-basedmolecular imaging contrast agents and potentialmedical appli-cationsrdquo European Radiology vol 13 no 2 pp 231ndash243 2003

[100] UMahmood andRWeissleder ldquoNear-infrared optical imagingof proteases in cancerrdquoMolecular CancerTherapeutics vol 2 pp489ndash496 2003

[101] J Kaufman H Hensley J Jacobs et al ldquoNon-invasive imagingof near infrafred dye labeled liposomes facilitates evaluation ofbioresidence timerdquoThe Journal of Urology vol 183 article e6282010

[102] Y-X J Wang ldquoMedical imaging in pharmaceutical clinicaltrials what radiologists should knowrdquo Clinical Radiology vol60 no 10 pp 1051ndash1057 2005

ISRN Pharmacology 9

consists of two near-infrared lasers emitting at 685 nm and785 nm camera and novel optics

Furthermore preferential binding of liposomes andnanoparticles to bladder lesions of ICPBS after instillationcan be assessed by NIR imaging of bladder where it cantheoretically provide anatomically specific information aboutthe real time status of ulcerlesion in bladder surface or tissueabnormality [102] Therefore future studies can substitute aresponse variable that is continuous in nature instead of asubjective clinical outcome

11 Conclusions

Adoption of forward-thinking approaches can achieveadvancements in drug delivery systems targeted to improvepharmacotherapy of bladder diseases in the future Latestdevelopments in the field of nanotechnology can bring thismode of therapy from second line of treatment for refractorycases to the forefront of disease management Liposomesare an attractive drug delivery platform by virtue of theirbiodegradability biocompatibility low toxicity and simpleand mild preparation methods

Conflict of Interests

All authors except Dr Michael Chancellor declare that thereis no conflict of interests regarding the publication of thispaper Dr Michael Chancellor owns stocks in the start-upliposome company Lipella

Acknowledgments

This work was supported by the National Institutes of Health(DK057267 and DK088836) and the Department of Defense(W81XWH-11-1-0763 and W81XWH-12-1-0565)

References

[1] J Parkin C Shea and G R Sant ldquoIntravesical dimethyl sul-foxide (DMSO) for interstitial cystitismdasha practical approachrdquoUrology vol 49 no 5 pp 105ndash107 1997

[2] C Dowson J Watkins M S Khan P Dasgupta and A SahaildquoRepeated botulinum toxin type A injections for refractoryoveractive bladder medium-term outcomes safety profile anddiscontinuation ratesrdquo European Urology vol 61 no 4 pp 834ndash839 2012

[3] J Kaufman V Tyagi M Anthony M B Chancellor and PTyagi ldquoState of the art in intravesical therapy for lower urinarytract symptomsrdquo Reviews in Urology vol 12 pp e181ndashe189 2010

[4] N Yoshimura and M B Chancellor ldquoCurrent and futurepharmacological treatment for overactive bladderrdquo Journal ofUrology vol 168 no 5 pp 1897ndash1913 2002

[5] K A Killinger J A Boura and K M Peters ldquoPain ininterstitial cystitisbladder pain syndrome do characteristicsdiffer in ulcerative and non-ulcerative subtypesrdquo InternationalUrogynecology Journal vol 24 no 8 pp 1295ndash1301 2013

[6] J C Nickel P Hanno K Kumar and HThomas ldquoSecond mul-ticenter randomized double-blind parallel-group evaluationof effectiveness and safety of intravesical sodium chondroitin

sulfate compared with inactive vehicle control in subjects withinterstitial cystitisbladder pain syndromerdquoUrology vol 79 no6 pp 1220ndash1225 2012

[7] P M Hanno ldquoAnalysis of long-term elmiron therapy forinterstitial cystitisrdquo Urology vol 49 no 5 pp 93ndash99 1997

[8] A Giannantoni V Bini R Dmochowski et al ldquoReply to ClausRiedlrsquos letter to the editor re antonella Giannantoni VittorioBini Roger Dmochowski et al Contemporary management ofthe painful bladder a systematic reviewrdquo EuropeanUrology vol61 no 5 pp 29ndash53 2012

[9] MC Lai Y CKuo andHCKuo ldquoIntravesical hyaluronic acidfor interstitial cystitispainful bladder syndrome a comparativerandomized assessment of different regimensrdquo InternationalJournal of Urology vol 20 pp 203ndash207 2013

[10] J Eldrup J Thorup and S L Nielsen ldquoPermeability andultrastructure of human bladder epitheliumrdquo British Journal ofUrology vol 55 no 5 pp 488ndash492 1983

[11] C L Parsons D Boychuk S Jones R Hurst and H CallahanldquoBladder surface glycosaminoglycans an epithelial permeabil-ity barrierrdquo Journal of Urology vol 143 no 1 pp 139ndash142 1990

[12] Y Kim N Yoshimura H Masuda F De Miguel and MB Chancellor ldquoAntimuscarinic agents exhibit local inhibitoryeffects on muscarinic receptors in bladder-afferent pathwaysrdquoUrology vol 65 no 2 pp 238ndash242 2005

[13] V Tyagi B J Philips R Su et al ldquoDifferential expression offunctional cannabinoid receptors in human bladder detrusorand urotheliumrdquo Journal of Urology vol 181 no 4 pp 1932ndash1938 2009

[14] B J Philips M C Smaldone V L Erickson N YoshimuraM B Chancellor and P Tyagi ldquoDifferential expression ofneurokinin receptor subtypes in urothelium and detrusor ofhuman bladderrdquo Journal of Urology vol 179 article 354 2008

[15] S A Baker G W Hennig J Han F C Britton T K Smithand S D Koh ldquoMethionine and its derivatives increase blad-der excitability by inhibiting stretch-dependent K+ channelsrdquoBritish Journal of Pharmacology vol 153 no 6 pp 1259ndash12712008

[16] J M Beckel A Kanai S-J Lee W C De Groat and LA Birder ldquoExpression of functional nicotinic acetylcholinereceptors in rat urinary bladder epithelial cellsrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 290 no 1 ppF103ndashF110 2006

[17] LA Birder A J KanaiWCDeGroat et al ldquoVanilloid receptorexpression suggests a sensory role for urinary bladder epithelialcellsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 23 pp 13396ndash13401 2001

[18] P Tyagi C A Thomas N Yoshimura and M B ChancellorldquoInvestigations into the presence of functional szlig1 szlig2 and szlig3-adrenoceptors in urothelium and detrusor of human bladderrdquoInternational Brazilian Journal of Urology vol 35 no 1 pp 76ndash83 2009

[19] A T Hanna-Mitchell J M Beckel S Barbadora A J Kanai WC de Groat and L A Birder ldquoNon-neuronal acetylcholine andurinary bladder urotheliumrdquo Life Sciences vol 80 no 24-25 pp2298ndash2302 2007

[20] C P Smith D A Gangitano A Munoz et al ldquoBotulinumtoxin type A normalizes alterations in urothelial ATP and NOrelease induced by chronic spinal cord injuryrdquo NeurochemistryInternational vol 52 no 6 pp 1068ndash1075 2008

[21] Y-C Chuang C A Thomas S Tyagi N Yoshimura PTyagi and M B Chancellor ldquoHuman urine with solifenacin

10 ISRN Pharmacology

intake but not tolterodine or darifenacin intake blocks detrusoroveractivityrdquo International Urogynecology Journal and PelvicFloor Dysfunction vol 19 no 10 pp 1353ndash1357 2008

[22] Y Kim N Yoshimura H Masuda F D Miguel and M BChancellor ldquoIntravesical instillation of human urine after oraladministration of trospium tolterodine and oxybutynin in a ratmodel of detrusor overactivityrdquo BJU International vol 97 no 2pp 400ndash403 2006

[23] S Tyagi P Tyagi S Van-le N YoshimuraM B Chancellor andF de Miguel ldquoQualitative and quantitative expression profileof muscarinic receptors in human urothelium and detrusorrdquoJournal of Urology vol 176 no 4 pp 1673ndash1678 2006

[24] G Gregoriadis and B E Ryman ldquoLiposomes as carriers ofenzymes or drugs a new approach to the treatment of storagediseasesrdquo Biochemical Journal vol 124 no 5 p 58 1971

[25] G Gregoriadis S Jain I Papaioannou and P Laing ldquoImprov-ing the therapeutic efficacy of peptides and proteins a role forpolysialic acidsrdquo International Journal of Pharmaceutics vol300 no 1-2 pp 125ndash130 2005

[26] P Sapra P Tyagi and T M Allen ldquoLigand-targeted liposomesfor cancer treatmentrdquo Current Drug Delivery vol 2 no 4 pp369ndash381 2005

[27] G Gregoriadis G Dapergolas and E D Neerunjun ldquoPene-tration of target areas in the rat by liposome associated agentsadministered parenterally and intragastricallyrdquo BiochemicalSociety Transactions vol 4 no 2 pp 256ndash259 1976

[28] G Gregoriadis ldquoEngineering liposomes for drug deliveryprogress and problemsrdquo Trends in Biotechnology vol 13 no 12pp 527ndash537 1995

[29] G Gregoriadis and A C Allison ldquoEntrapment of proteins inliposomes prevents allergic reactions in pre immunised micerdquoFEBS Letters vol 45 no 1 pp 71ndash74 1974

[30] S Lee S Dausch CMaierhofer andD Dausch ldquoA new therapyconcept with a liposome eye spray for the treatment of the lsquodryeyersquordquo Klinische Monatsblatter fur Augenheilkunde vol 221 no10 pp 825ndash836 2004

[31] D Dausch S Lee S Dausch J C Kim G Schwert and WMichelson ldquoComparative study of treatment of the dry eyesyndrome due to disturbances of the tear film lipid layer withlipid-containing tear substitutes efficacy of lipid-containingtear substitutesrdquo Klinische Monatsblatter fur Augenheilkundevol 223 no 12 pp 974ndash983 2006

[32] S Ebrahim G A Peyman and P J Lee ldquoApplications ofliposomes in ophthalmologyrdquo Survey of Ophthalmology vol 50no 2 pp 167ndash182 2005

[33] J Nirmal P Tyagi L Dang et al ldquoEndocytosis uptake ofliposomes in urothelium cells detected by transmission electronmicroscopyrdquoThe Journal of Urology vol 187 article e15 2012

[34] D N Frangos J J Killion D Fan R Fishbeck A C VonEschenbach and I J Fidler ldquoThe development of liposomescontaining interferon alpha for the intravesical therapy ofhuman superficial bladder cancerrdquo Journal of Urology vol 143no 6 pp 1252ndash1266 1990

[35] J W Johnson R Nayar J J Killion A C Von Eschenbachand I J Fidler ldquoBinding of liposomes to human bladdertumor epithelial cell lines implications for an intravesical drugdelivery system for the treatment of bladder cancerrdquo SelectiveCancer Therapeutics vol 5 no 4 pp 147ndash155 1989

[36] Y-C Chuang M B Chancellor S Seki et al ldquoIntravesicalprotamine sulfate and potassium chloride as a model forbladder hyperactivityrdquoUrology vol 61 no 3 pp 664ndash670 2003

[37] J Kaufman P Tyagi and M B Chancellor ldquoIntravesicalliposomal (Lp08) instillation protects bladder urothelium fromchemical irritationrdquoThe Journal of Urology vol 181 article 5392009

[38] P Tyagi M B Chancellor Z Li et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical capsaicindelivery using thermosensitive hydrogel and liposomesrdquo Jour-nal of Urology vol 171 no 1 pp 483ndash489 2004

[39] MO Fraser Y-CChuang P Tyagi et al ldquoIntravesical liposomeadministrationmdasha novel treatment for hyperactive bladder inthe ratrdquo Urology vol 61 no 3 pp 656ndash663 2003

[40] P Tyagi M Chancellor N Yoshimura and L Huang ldquoActivityof different phospholipids in attenuating hyperactivity in blad-der irritationrdquo BJU International vol 101 no 5 pp 627ndash6322008

[41] Y Sun and T C Chai ldquoEffects of dimethyl sulphoxide andheparin on stretch-activated ATP release by bladder urothelialcells from patients with interstitial cystitisrdquo BJU Internationalvol 90 no 4 pp 381ndash385 2002

[42] J J Bade ldquoA placebo-controlled study of intravesical pentosan-polysulphate for the treatment of interstitial cystitisrdquo BritishJournal of Urology vol 79 no 2 pp 168ndash171 1997

[43] P Tyagi V C Hsieh N Yoshimura J Kaufman and M BChancellor ldquoInstillation of liposomes vs dimethyl sulphoxide orpentosan polysulphate for reducing bladder hyperactivityrdquo BJUInternational vol 104 no 11 pp 1689ndash1692 2009

[44] W-C Lee C-T Chien H-J Yu and S-W Lee ldquoBladderdysfunction in rats with metabolic syndrome induced by long-term fructose feedingrdquo Journal of Urology vol 179 no 6 pp2470ndash2476 2008

[45] J I Gillespie G A Van Koeveringe S G DeWachter and J DeVente ldquoOn the origins of the sensory output from the bladderthe concept of afferent noiserdquo BJU International vol 103 no 10pp 1324ndash1333 2009

[46] W C Lee P H Chiang Y L Tain C C Wu and Y CChuang ldquoSensory dysfunction of bladder mucosa and bladderoversensitivity in a rat model of metabolic syndromerdquo PloSONE vol 7 Article ID e45578 2012

[47] Y-C Chuang W-C Lee W-C Lee and P-H Chiang ldquoIntrav-esical liposome versus oral pentosan polysulfate for interstitialcystitispainful bladder syndromerdquo Journal of Urology vol 182no 4 pp 1393ndash1400 2009

[48] W-C Lee Y-C Chuang W-C Lee and P-H Chiang ldquoSafetyand dose flexibility clinical evaluation of intravesical liposomein patients with interstitial cystitis or painful bladder syn-dromerdquo Kaohsiung Journal of Medical Sciences vol 27 no 10pp 437ndash440 2011

[49] S T Truschel E Wang W G Ruiz et al ldquoStretch-regulatedexocytosisendocytosis in bladder umbrella cellsrdquo MolecularBiology of the Cell vol 13 no 3 pp 830ndash846 2002

[50] M B Chancellor and W C De Groat ldquoIntravesical capsaicinand resiniferatoxin therapy spicing up the ways to treat theoveractive bladderrdquo Journal of Urology vol 162 no 1 pp 3ndash111999

[51] D S Byrne A Das J Sedor et al ldquoEffect of intravesicalcapsaicin and vehicle on bladder integrity in control and spinalcord injured ratsrdquo Journal of Urology vol 159 no 3 pp 1074ndash1078 1998

[52] M Mandal and K-D Lee ldquoListeriolysin O-liposome-mediatedcytosolic delivery of macromolecule antigen in vivo enhance-ment of antigen-specific cytotoxic T lymphocyte frequency

ISRN Pharmacology 11

activity and tumor protectionrdquo Biochimica et Biophysica Actavol 1563 no 1-2 pp 7ndash17 2002

[53] Y-C Chuang P Tyagi C-C Huang et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical botulinumtoxin a delivery using liposomesrdquo Journal of Urology vol 182no 2 pp 786ndash792 2009

[54] Y-C Chuang N Yoshimura C-C Huang P-H Chiang andM B Chancellor ldquoIntravesical botulinum toxin a administra-tion produces analgesia against acetic acid induced bladder painresponses in ratsrdquo Journal of Urology vol 172 no 4 pp 1529ndash1532 2004

[55] S P Petrou A S Parker J E Crook A Rogers D Metz-Kudashick and D D Thiel ldquoBotulinum A toxindimethylsulfoxide bladder instillations for women with refractory idio-pathic detrusor overactivity a phase 12 studyrdquo Mayo ClinicProceedings vol 84 no 8 pp 702ndash706 2009

[56] P Caccin O Rossetto M Rigoni E Johnson G Schiavoand C Montecucco ldquoVAMPsynaptobrevin cleavage by tetanusand botulinum neurotoxins is strongly enhanced by acidicliposomesrdquo FEBS Letters vol 542 no 1ndash3 pp 132ndash136 2003

[57] M L Stephenson and P C Zamecnik ldquoInhibition of Roussarcoma viral RNA translation by a specific oligodeoxyribonu-cleotiderdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 75 no 1 pp 285ndash288 1978

[58] H Dadgostar and N Waheed ldquoThe evolving role of vascularendothelial growth factor inhibitors in the treatment of neovas-cular age-related macular degenerationrdquo Eye vol 22 no 6 pp761ndash767 2008

[59] C Gebhard G Huard E A Kritikou and J C TardifldquoApolipoprotein B antisense inhibitionmdashupdate on mipom-ersenrdquo Current Pharmaceutical Design vol 919 no 17 pp 3132ndash3142 2013

[60] T Yokota E Hoffman and S Takeda ldquoAntisense oligo-mediated multiple exon skipping in a dog model of duchennemuscular dystrophyrdquoMethods inMolecular Biology vol 709 pp299ndash312 2011

[61] B L Jacobs M C Smaldone V Tyagi et al ldquoIncreased nervegrowth factor in neurogenic overactive bladder and interstitialcystitis patientsrdquoThe Canadian Journal of Urology vol 17 no 1pp 4989ndash4994 2010

[62] H-T Liu and H-C Kuo ldquoIntravesical botulinum toxin Ainjections plus hydrodistension can reduce nerve growth factorproduction and control bladder pain in interstitial cystitisrdquoUrology vol 70 no 3 pp 463ndash468 2007

[63] H T Liu and H C Kuo ldquoIncreased urine and serum nervegrowth factor levels in interstitial cystitis suggest chronicinflammation is involved in the pathogenesis of diseaserdquo PloSONE vol 7 Article ID e44687 2012

[64] N Yoshimura N E Bennett Y Hayashi et al ldquoBladderoveractivity and hyperexcitability of bladder afferent neuronsafter intrathecal delivery of nerve growth factor in ratsrdquo Journalof Neuroscience vol 26 no 42 pp 10847ndash10855 2006

[65] S Seki K Sasaki Y Igawa et al ldquoSuppression of detrusor-sphincter dyssynergia by immunoneutralization of nervegrowth factor in lumbosacral spinal cord in spinal cord injuredratsrdquo Journal of Urology vol 171 no 1 pp 478ndash482 2004

[66] R J Evans R M Moldwin N Cossons A Darekar I WMills andD Scholfield ldquoProof of concept trial of tanezumab forthe treatment of symptoms associated with interstitial cystitisrdquoJournal of Urology vol 185 no 5 pp 1716ndash1721 2011

[67] P Tyagi R Banerjee S Basu N YoshimuraM Chancellor andL Huang ldquoIntravesical antisense therapy for cystitis using TAT-peptide nucleic acid conjugatesrdquoMolecular Pharmaceutics vol3 no 4 pp 398ndash406 2006

[68] D C Molliver D E Wright M L Leitner et al ldquoIB4-bindingDRG neurons switch from NGF to GDNF dependence in earlypostnatal liferdquo Neuron vol 19 no 4 pp 849ndash861 1997

[69] M TyagiM RusnatiM Presta andMGiacca ldquoInternalizationof HIV-1 tat requires cell surface heparan sulfate proteoglycansrdquoJournal of Biological Chemistry vol 276 no 5 pp 3254ndash32612001

[70] E Uhlmann A Ryte and A Peyman ldquoStudies on the mech-anism of stabilization of partially phosphorothioated oligonu-cleotides against nucleolytic degradationrdquoAntisense andNucleicAcid Drug Development vol 7 no 4 pp 345ndash350 1997

[71] M Nogawa T Yuasa S Kimura et al ldquoIntravesical adminis-tration of small interfering RNA targeting PLK-1 successfullyprevents the growth of bladder cancerrdquo Journal of ClinicalInvestigation vol 115 no 4 pp 978ndash985 2005

[72] M Kashyap N Kawamorita V Tyagi et al ldquoDownregulation ofNGF expression in the bladder by antisense oligoucleotides asnew treatment for overactive bladderrdquo Journal of Urology vol27190 no 2 pp 757ndash764 2013

[73] C-J Arum Y Kodama N Rolim et al ldquoA rat model ofintravesical delivery of small interfering RNA for studyingurinary carcinomardquoWorld Journal of Urology vol 28 no 4 pp479ndash485 2010

[74] C E Blietz B Thode M Hauses et al ldquoIn vivo studies on theavailability and toxicity of antisense oligonucleotides in bladdercancerrdquo In Vivo vol 23 no 1 pp 13ndash19 2009

[75] D Oddiah P Anand S B McMahon and M Rattray ldquoRapidincrease of NGF BDNF andNT-3mRNAs in inflamed bladderrdquoNeuroReport vol 9 no 7 pp 1455ndash1458 1998

[76] M R Saban H Hellmich N B Nguyen J Winston T GHammond and R Saban ldquoTime course of LPS-induced geneexpression in a mouse model of genitourinary inflammationrdquoPhysiol Genomics vol 5 no 3 pp 147ndash160 2001

[77] B Schnegelsberg T T Sun G Cain et al ldquoOverexpression ofNGF in mouse urothelium leads to neuronal hyperinnervationpelvic sensitivity and changes in urinary bladder functionrdquoTheAmerican Journal of PhysiologymdashRegulatory Integrative andComparative Physiology vol 298 no 5 pp R534ndashR547 2010

[78] S Cardenas M Scuri L Samsell et al ldquoNeurotrophic andneuroimmune responses to early-life Pseudomonas aeruginosainfection in rat lungsrdquo American Journal of PhysiologymdashLungCellular and Molecular Physiology vol 299 no 3 pp L334ndashL344 2010

[79] K Nakamura F Tan Z Li and C J Thiele ldquoNGF activationof TrkA induces vascular endothelial growth factor expressionvia induction of hypoxia-inducible factor-1120572rdquo Molecular andCellular Neuroscience vol 46 no 2 pp 498ndash506 2011

[80] S Othumpangat M Regier and G Piedimonte ldquoNerve growthfactor modulates human rhinovirus infection in airway epithe-lial cells by controlling ICAM-1 expressionrdquo American Journalof Physiology Lung Cellular andMolecular Physiology vol 15302pp L1057ndashL1066 2012

[81] H Jung P T Toth F A White and R J Miller ldquoMonocytechemoattractant protein-1 functions as a neuromodulator indorsal root ganglia neuronsrdquo Journal of Neurochemistry vol104 no 1 pp 254ndash263 2008

[82] F A White H Jung and R J Miller ldquoChemokines andthe pathophysiology of neuropathic painrdquo Proceedings of the

12 ISRN Pharmacology

National Academy of Sciences of the United States of Americavol 104 no 51 pp 20151ndash20158 2007

[83] G Banisadr R-D Gosselin P Mechighel W Rostene PKitabgi and S M Parsadaniantz ldquoConstitutive neuronalexpression of CCR2 chemokine receptor and its colocalizationwith neurotransmitters in normal rat brain functional effectof MCP-1CCL2 on calcium mobilization in primary culturedneuronsrdquo Journal of Comparative Neurology vol 492 no 2 pp178ndash192 2005

[84] S Bhangoo D Ren R J Miller et al ldquoDelayed functionalexpression of neuronal chemokine receptors following focalnerve demyelination in the rat a mechanism for the devel-opment of chronic sensitization of peripheral nociceptorsrdquoMolecular Pain vol 3 article 38 2007

[85] T Maeda N Kiguchi Y Kobayashi T Ikuta M Ozaki and SKishioka ldquoLeptin derived from adipocytes in injured periph-eral nerves facilitates development of neuropathic pain viamacrophage stimulationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 106 no 31 pp13076ndash13081 2009

[86] H Kajiwara M Yasuda N Kumaki T Shibayama and YOsamura ldquoExpression of carbohydrate antigens (SSEA-1 Sialyl-Lewis X DU-PAN-2 and CA19-9) and E-selectin in urothelialcarcinoma of the renal pelvis ureter and urinary bladderrdquoTokaiJournal of Experimental and Clinical Medicine vol 30 no 3 pp177ndash182 2005

[87] A M S Assreuy G J Martins M E F Moreira et al ldquoPre-vention of cyclophosphamide-induced hemorrhagic cystitis byglucose-mannose binding plant lectinsrdquo Journal of Urology vol161 no 6 pp 1988ndash1993 1999

[88] S M Vinson A Rickard J S Ryerse and J McHowat ldquoNeu-trophil adherence to bladder microvascular endothelial cellsfollowing platelet-activating factor acetylhydrolase inhibitionrdquoJournal of Pharmacology and Experimental Therapeutics vol314 no 3 pp 1241ndash1247 2005

[89] M Green A Filippou G Sant and T CTheoharides ldquoExpres-sion of intercellular adhesion molecules in the bladder ofpatients with interstitial cystitisrdquoUrology vol 63 no 4 pp 688ndash693 2004

[90] M Leppilahti P Hellstrom and T L J Tammela ldquoEffect ofdiagnostic hydrodistension and four intravesical hyaluronicacid instillations on bladder ICAM-1 intensity and associationof ICAM-1 intensity with clinical response in patients withinterstitial cystitisrdquo Urology vol 60 no 1 pp 46ndash51 2002

[91] A T Corcoran N Yoshimura V Tyagi B Jacobs W Lengand P Tyagi ldquoMapping the cytokine profile of painful bladdersyndromeinterstitial cystitis in human bladder and urinespecimensrdquoWorld Journal of Urology vol 31 no 1 pp 241ndash2462013

[92] SGarrean X-PGaoV Brovkovych et al ldquoCaveolin-1 regulatesNF-120581B activation and lung inflammatory response to sepsisinduced by lipopolysacchariderdquo Journal of Immunology vol 177no 7 pp 4853ndash4860 2006

[93] M R Kang G Yang R F Place et al ldquoIntravesical deliveryof small activating RNA formulated into lipid nanoparticlesinhibits orthotopic bladder tumor growthrdquo Cancer Researchvol 172 pp 5069ndash5079 2012

[94] P Tyagi D Barclay R Zamora et al ldquoUrine cytokines suggestan inflammatory response in the overactive bladder a pilotstudyrdquo International Urology and Nephrology vol 42 no 3 pp629ndash635 2010

[95] P Tyagi B L Jacobs D Barcaly et al ldquoUrine levels ofinflammatory chemokines can be novel biomarkers for theoveractive bladderrdquoThe Journal of Urology vol 181 article 5882009

[96] P Tyagi V Tyagi D Bui et al ldquoDiscrimination Of OAB fromICPBS by multivariate data modeling of urinary proteinsrdquoTheJournal of Urology vol 183 pp e614ndashe6e5 2010

[97] P Tyagi D Nikolavsky Y Vodovotz et al ldquoUrine levels ofselected chemokines positively correlate with lower bladdercapacity and psychometric scores in IcPbs patientsrdquo TheJournal of Urology vol 181 article 21 2009

[98] R Weissleder and V Ntziachristos ldquoShedding light onto livemolecular targetsrdquo Nature Medicine vol 9 no 1 pp 123ndash1282003

[99] C Bremer V Ntziachristos and R Weissleder ldquoOptical-basedmolecular imaging contrast agents and potentialmedical appli-cationsrdquo European Radiology vol 13 no 2 pp 231ndash243 2003

[100] UMahmood andRWeissleder ldquoNear-infrared optical imagingof proteases in cancerrdquoMolecular CancerTherapeutics vol 2 pp489ndash496 2003

[101] J Kaufman H Hensley J Jacobs et al ldquoNon-invasive imagingof near infrafred dye labeled liposomes facilitates evaluation ofbioresidence timerdquoThe Journal of Urology vol 183 article e6282010

[102] Y-X J Wang ldquoMedical imaging in pharmaceutical clinicaltrials what radiologists should knowrdquo Clinical Radiology vol60 no 10 pp 1051ndash1057 2005

10 ISRN Pharmacology

intake but not tolterodine or darifenacin intake blocks detrusoroveractivityrdquo International Urogynecology Journal and PelvicFloor Dysfunction vol 19 no 10 pp 1353ndash1357 2008

[22] Y Kim N Yoshimura H Masuda F D Miguel and M BChancellor ldquoIntravesical instillation of human urine after oraladministration of trospium tolterodine and oxybutynin in a ratmodel of detrusor overactivityrdquo BJU International vol 97 no 2pp 400ndash403 2006

[23] S Tyagi P Tyagi S Van-le N YoshimuraM B Chancellor andF de Miguel ldquoQualitative and quantitative expression profileof muscarinic receptors in human urothelium and detrusorrdquoJournal of Urology vol 176 no 4 pp 1673ndash1678 2006

[24] G Gregoriadis and B E Ryman ldquoLiposomes as carriers ofenzymes or drugs a new approach to the treatment of storagediseasesrdquo Biochemical Journal vol 124 no 5 p 58 1971

[25] G Gregoriadis S Jain I Papaioannou and P Laing ldquoImprov-ing the therapeutic efficacy of peptides and proteins a role forpolysialic acidsrdquo International Journal of Pharmaceutics vol300 no 1-2 pp 125ndash130 2005

[26] P Sapra P Tyagi and T M Allen ldquoLigand-targeted liposomesfor cancer treatmentrdquo Current Drug Delivery vol 2 no 4 pp369ndash381 2005

[27] G Gregoriadis G Dapergolas and E D Neerunjun ldquoPene-tration of target areas in the rat by liposome associated agentsadministered parenterally and intragastricallyrdquo BiochemicalSociety Transactions vol 4 no 2 pp 256ndash259 1976

[28] G Gregoriadis ldquoEngineering liposomes for drug deliveryprogress and problemsrdquo Trends in Biotechnology vol 13 no 12pp 527ndash537 1995

[29] G Gregoriadis and A C Allison ldquoEntrapment of proteins inliposomes prevents allergic reactions in pre immunised micerdquoFEBS Letters vol 45 no 1 pp 71ndash74 1974

[30] S Lee S Dausch CMaierhofer andD Dausch ldquoA new therapyconcept with a liposome eye spray for the treatment of the lsquodryeyersquordquo Klinische Monatsblatter fur Augenheilkunde vol 221 no10 pp 825ndash836 2004

[31] D Dausch S Lee S Dausch J C Kim G Schwert and WMichelson ldquoComparative study of treatment of the dry eyesyndrome due to disturbances of the tear film lipid layer withlipid-containing tear substitutes efficacy of lipid-containingtear substitutesrdquo Klinische Monatsblatter fur Augenheilkundevol 223 no 12 pp 974ndash983 2006

[32] S Ebrahim G A Peyman and P J Lee ldquoApplications ofliposomes in ophthalmologyrdquo Survey of Ophthalmology vol 50no 2 pp 167ndash182 2005

[33] J Nirmal P Tyagi L Dang et al ldquoEndocytosis uptake ofliposomes in urothelium cells detected by transmission electronmicroscopyrdquoThe Journal of Urology vol 187 article e15 2012

[34] D N Frangos J J Killion D Fan R Fishbeck A C VonEschenbach and I J Fidler ldquoThe development of liposomescontaining interferon alpha for the intravesical therapy ofhuman superficial bladder cancerrdquo Journal of Urology vol 143no 6 pp 1252ndash1266 1990

[35] J W Johnson R Nayar J J Killion A C Von Eschenbachand I J Fidler ldquoBinding of liposomes to human bladdertumor epithelial cell lines implications for an intravesical drugdelivery system for the treatment of bladder cancerrdquo SelectiveCancer Therapeutics vol 5 no 4 pp 147ndash155 1989

[36] Y-C Chuang M B Chancellor S Seki et al ldquoIntravesicalprotamine sulfate and potassium chloride as a model forbladder hyperactivityrdquoUrology vol 61 no 3 pp 664ndash670 2003

[37] J Kaufman P Tyagi and M B Chancellor ldquoIntravesicalliposomal (Lp08) instillation protects bladder urothelium fromchemical irritationrdquoThe Journal of Urology vol 181 article 5392009

[38] P Tyagi M B Chancellor Z Li et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical capsaicindelivery using thermosensitive hydrogel and liposomesrdquo Jour-nal of Urology vol 171 no 1 pp 483ndash489 2004

[39] MO Fraser Y-CChuang P Tyagi et al ldquoIntravesical liposomeadministrationmdasha novel treatment for hyperactive bladder inthe ratrdquo Urology vol 61 no 3 pp 656ndash663 2003

[40] P Tyagi M Chancellor N Yoshimura and L Huang ldquoActivityof different phospholipids in attenuating hyperactivity in blad-der irritationrdquo BJU International vol 101 no 5 pp 627ndash6322008

[41] Y Sun and T C Chai ldquoEffects of dimethyl sulphoxide andheparin on stretch-activated ATP release by bladder urothelialcells from patients with interstitial cystitisrdquo BJU Internationalvol 90 no 4 pp 381ndash385 2002

[42] J J Bade ldquoA placebo-controlled study of intravesical pentosan-polysulphate for the treatment of interstitial cystitisrdquo BritishJournal of Urology vol 79 no 2 pp 168ndash171 1997

[43] P Tyagi V C Hsieh N Yoshimura J Kaufman and M BChancellor ldquoInstillation of liposomes vs dimethyl sulphoxide orpentosan polysulphate for reducing bladder hyperactivityrdquo BJUInternational vol 104 no 11 pp 1689ndash1692 2009

[44] W-C Lee C-T Chien H-J Yu and S-W Lee ldquoBladderdysfunction in rats with metabolic syndrome induced by long-term fructose feedingrdquo Journal of Urology vol 179 no 6 pp2470ndash2476 2008

[45] J I Gillespie G A Van Koeveringe S G DeWachter and J DeVente ldquoOn the origins of the sensory output from the bladderthe concept of afferent noiserdquo BJU International vol 103 no 10pp 1324ndash1333 2009

[46] W C Lee P H Chiang Y L Tain C C Wu and Y CChuang ldquoSensory dysfunction of bladder mucosa and bladderoversensitivity in a rat model of metabolic syndromerdquo PloSONE vol 7 Article ID e45578 2012

[47] Y-C Chuang W-C Lee W-C Lee and P-H Chiang ldquoIntrav-esical liposome versus oral pentosan polysulfate for interstitialcystitispainful bladder syndromerdquo Journal of Urology vol 182no 4 pp 1393ndash1400 2009

[48] W-C Lee Y-C Chuang W-C Lee and P-H Chiang ldquoSafetyand dose flexibility clinical evaluation of intravesical liposomein patients with interstitial cystitis or painful bladder syn-dromerdquo Kaohsiung Journal of Medical Sciences vol 27 no 10pp 437ndash440 2011

[49] S T Truschel E Wang W G Ruiz et al ldquoStretch-regulatedexocytosisendocytosis in bladder umbrella cellsrdquo MolecularBiology of the Cell vol 13 no 3 pp 830ndash846 2002

[50] M B Chancellor and W C De Groat ldquoIntravesical capsaicinand resiniferatoxin therapy spicing up the ways to treat theoveractive bladderrdquo Journal of Urology vol 162 no 1 pp 3ndash111999

[51] D S Byrne A Das J Sedor et al ldquoEffect of intravesicalcapsaicin and vehicle on bladder integrity in control and spinalcord injured ratsrdquo Journal of Urology vol 159 no 3 pp 1074ndash1078 1998

[52] M Mandal and K-D Lee ldquoListeriolysin O-liposome-mediatedcytosolic delivery of macromolecule antigen in vivo enhance-ment of antigen-specific cytotoxic T lymphocyte frequency

ISRN Pharmacology 11

activity and tumor protectionrdquo Biochimica et Biophysica Actavol 1563 no 1-2 pp 7ndash17 2002

[53] Y-C Chuang P Tyagi C-C Huang et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical botulinumtoxin a delivery using liposomesrdquo Journal of Urology vol 182no 2 pp 786ndash792 2009

[54] Y-C Chuang N Yoshimura C-C Huang P-H Chiang andM B Chancellor ldquoIntravesical botulinum toxin a administra-tion produces analgesia against acetic acid induced bladder painresponses in ratsrdquo Journal of Urology vol 172 no 4 pp 1529ndash1532 2004

[55] S P Petrou A S Parker J E Crook A Rogers D Metz-Kudashick and D D Thiel ldquoBotulinum A toxindimethylsulfoxide bladder instillations for women with refractory idio-pathic detrusor overactivity a phase 12 studyrdquo Mayo ClinicProceedings vol 84 no 8 pp 702ndash706 2009

[56] P Caccin O Rossetto M Rigoni E Johnson G Schiavoand C Montecucco ldquoVAMPsynaptobrevin cleavage by tetanusand botulinum neurotoxins is strongly enhanced by acidicliposomesrdquo FEBS Letters vol 542 no 1ndash3 pp 132ndash136 2003

[57] M L Stephenson and P C Zamecnik ldquoInhibition of Roussarcoma viral RNA translation by a specific oligodeoxyribonu-cleotiderdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 75 no 1 pp 285ndash288 1978

[58] H Dadgostar and N Waheed ldquoThe evolving role of vascularendothelial growth factor inhibitors in the treatment of neovas-cular age-related macular degenerationrdquo Eye vol 22 no 6 pp761ndash767 2008

[59] C Gebhard G Huard E A Kritikou and J C TardifldquoApolipoprotein B antisense inhibitionmdashupdate on mipom-ersenrdquo Current Pharmaceutical Design vol 919 no 17 pp 3132ndash3142 2013

[60] T Yokota E Hoffman and S Takeda ldquoAntisense oligo-mediated multiple exon skipping in a dog model of duchennemuscular dystrophyrdquoMethods inMolecular Biology vol 709 pp299ndash312 2011

[61] B L Jacobs M C Smaldone V Tyagi et al ldquoIncreased nervegrowth factor in neurogenic overactive bladder and interstitialcystitis patientsrdquoThe Canadian Journal of Urology vol 17 no 1pp 4989ndash4994 2010

[62] H-T Liu and H-C Kuo ldquoIntravesical botulinum toxin Ainjections plus hydrodistension can reduce nerve growth factorproduction and control bladder pain in interstitial cystitisrdquoUrology vol 70 no 3 pp 463ndash468 2007

[63] H T Liu and H C Kuo ldquoIncreased urine and serum nervegrowth factor levels in interstitial cystitis suggest chronicinflammation is involved in the pathogenesis of diseaserdquo PloSONE vol 7 Article ID e44687 2012

[64] N Yoshimura N E Bennett Y Hayashi et al ldquoBladderoveractivity and hyperexcitability of bladder afferent neuronsafter intrathecal delivery of nerve growth factor in ratsrdquo Journalof Neuroscience vol 26 no 42 pp 10847ndash10855 2006

[65] S Seki K Sasaki Y Igawa et al ldquoSuppression of detrusor-sphincter dyssynergia by immunoneutralization of nervegrowth factor in lumbosacral spinal cord in spinal cord injuredratsrdquo Journal of Urology vol 171 no 1 pp 478ndash482 2004

[66] R J Evans R M Moldwin N Cossons A Darekar I WMills andD Scholfield ldquoProof of concept trial of tanezumab forthe treatment of symptoms associated with interstitial cystitisrdquoJournal of Urology vol 185 no 5 pp 1716ndash1721 2011

[67] P Tyagi R Banerjee S Basu N YoshimuraM Chancellor andL Huang ldquoIntravesical antisense therapy for cystitis using TAT-peptide nucleic acid conjugatesrdquoMolecular Pharmaceutics vol3 no 4 pp 398ndash406 2006

[68] D C Molliver D E Wright M L Leitner et al ldquoIB4-bindingDRG neurons switch from NGF to GDNF dependence in earlypostnatal liferdquo Neuron vol 19 no 4 pp 849ndash861 1997

[69] M TyagiM RusnatiM Presta andMGiacca ldquoInternalizationof HIV-1 tat requires cell surface heparan sulfate proteoglycansrdquoJournal of Biological Chemistry vol 276 no 5 pp 3254ndash32612001

[70] E Uhlmann A Ryte and A Peyman ldquoStudies on the mech-anism of stabilization of partially phosphorothioated oligonu-cleotides against nucleolytic degradationrdquoAntisense andNucleicAcid Drug Development vol 7 no 4 pp 345ndash350 1997

[71] M Nogawa T Yuasa S Kimura et al ldquoIntravesical adminis-tration of small interfering RNA targeting PLK-1 successfullyprevents the growth of bladder cancerrdquo Journal of ClinicalInvestigation vol 115 no 4 pp 978ndash985 2005

[72] M Kashyap N Kawamorita V Tyagi et al ldquoDownregulation ofNGF expression in the bladder by antisense oligoucleotides asnew treatment for overactive bladderrdquo Journal of Urology vol27190 no 2 pp 757ndash764 2013

[73] C-J Arum Y Kodama N Rolim et al ldquoA rat model ofintravesical delivery of small interfering RNA for studyingurinary carcinomardquoWorld Journal of Urology vol 28 no 4 pp479ndash485 2010

[74] C E Blietz B Thode M Hauses et al ldquoIn vivo studies on theavailability and toxicity of antisense oligonucleotides in bladdercancerrdquo In Vivo vol 23 no 1 pp 13ndash19 2009

[75] D Oddiah P Anand S B McMahon and M Rattray ldquoRapidincrease of NGF BDNF andNT-3mRNAs in inflamed bladderrdquoNeuroReport vol 9 no 7 pp 1455ndash1458 1998

[76] M R Saban H Hellmich N B Nguyen J Winston T GHammond and R Saban ldquoTime course of LPS-induced geneexpression in a mouse model of genitourinary inflammationrdquoPhysiol Genomics vol 5 no 3 pp 147ndash160 2001

[77] B Schnegelsberg T T Sun G Cain et al ldquoOverexpression ofNGF in mouse urothelium leads to neuronal hyperinnervationpelvic sensitivity and changes in urinary bladder functionrdquoTheAmerican Journal of PhysiologymdashRegulatory Integrative andComparative Physiology vol 298 no 5 pp R534ndashR547 2010

[78] S Cardenas M Scuri L Samsell et al ldquoNeurotrophic andneuroimmune responses to early-life Pseudomonas aeruginosainfection in rat lungsrdquo American Journal of PhysiologymdashLungCellular and Molecular Physiology vol 299 no 3 pp L334ndashL344 2010

[79] K Nakamura F Tan Z Li and C J Thiele ldquoNGF activationof TrkA induces vascular endothelial growth factor expressionvia induction of hypoxia-inducible factor-1120572rdquo Molecular andCellular Neuroscience vol 46 no 2 pp 498ndash506 2011

[80] S Othumpangat M Regier and G Piedimonte ldquoNerve growthfactor modulates human rhinovirus infection in airway epithe-lial cells by controlling ICAM-1 expressionrdquo American Journalof Physiology Lung Cellular andMolecular Physiology vol 15302pp L1057ndashL1066 2012

[81] H Jung P T Toth F A White and R J Miller ldquoMonocytechemoattractant protein-1 functions as a neuromodulator indorsal root ganglia neuronsrdquo Journal of Neurochemistry vol104 no 1 pp 254ndash263 2008

[82] F A White H Jung and R J Miller ldquoChemokines andthe pathophysiology of neuropathic painrdquo Proceedings of the

12 ISRN Pharmacology

National Academy of Sciences of the United States of Americavol 104 no 51 pp 20151ndash20158 2007

[83] G Banisadr R-D Gosselin P Mechighel W Rostene PKitabgi and S M Parsadaniantz ldquoConstitutive neuronalexpression of CCR2 chemokine receptor and its colocalizationwith neurotransmitters in normal rat brain functional effectof MCP-1CCL2 on calcium mobilization in primary culturedneuronsrdquo Journal of Comparative Neurology vol 492 no 2 pp178ndash192 2005

[84] S Bhangoo D Ren R J Miller et al ldquoDelayed functionalexpression of neuronal chemokine receptors following focalnerve demyelination in the rat a mechanism for the devel-opment of chronic sensitization of peripheral nociceptorsrdquoMolecular Pain vol 3 article 38 2007

[85] T Maeda N Kiguchi Y Kobayashi T Ikuta M Ozaki and SKishioka ldquoLeptin derived from adipocytes in injured periph-eral nerves facilitates development of neuropathic pain viamacrophage stimulationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 106 no 31 pp13076ndash13081 2009

[86] H Kajiwara M Yasuda N Kumaki T Shibayama and YOsamura ldquoExpression of carbohydrate antigens (SSEA-1 Sialyl-Lewis X DU-PAN-2 and CA19-9) and E-selectin in urothelialcarcinoma of the renal pelvis ureter and urinary bladderrdquoTokaiJournal of Experimental and Clinical Medicine vol 30 no 3 pp177ndash182 2005

[87] A M S Assreuy G J Martins M E F Moreira et al ldquoPre-vention of cyclophosphamide-induced hemorrhagic cystitis byglucose-mannose binding plant lectinsrdquo Journal of Urology vol161 no 6 pp 1988ndash1993 1999

[88] S M Vinson A Rickard J S Ryerse and J McHowat ldquoNeu-trophil adherence to bladder microvascular endothelial cellsfollowing platelet-activating factor acetylhydrolase inhibitionrdquoJournal of Pharmacology and Experimental Therapeutics vol314 no 3 pp 1241ndash1247 2005

[89] M Green A Filippou G Sant and T CTheoharides ldquoExpres-sion of intercellular adhesion molecules in the bladder ofpatients with interstitial cystitisrdquoUrology vol 63 no 4 pp 688ndash693 2004

[90] M Leppilahti P Hellstrom and T L J Tammela ldquoEffect ofdiagnostic hydrodistension and four intravesical hyaluronicacid instillations on bladder ICAM-1 intensity and associationof ICAM-1 intensity with clinical response in patients withinterstitial cystitisrdquo Urology vol 60 no 1 pp 46ndash51 2002

[91] A T Corcoran N Yoshimura V Tyagi B Jacobs W Lengand P Tyagi ldquoMapping the cytokine profile of painful bladdersyndromeinterstitial cystitis in human bladder and urinespecimensrdquoWorld Journal of Urology vol 31 no 1 pp 241ndash2462013

[92] SGarrean X-PGaoV Brovkovych et al ldquoCaveolin-1 regulatesNF-120581B activation and lung inflammatory response to sepsisinduced by lipopolysacchariderdquo Journal of Immunology vol 177no 7 pp 4853ndash4860 2006

[93] M R Kang G Yang R F Place et al ldquoIntravesical deliveryof small activating RNA formulated into lipid nanoparticlesinhibits orthotopic bladder tumor growthrdquo Cancer Researchvol 172 pp 5069ndash5079 2012

[94] P Tyagi D Barclay R Zamora et al ldquoUrine cytokines suggestan inflammatory response in the overactive bladder a pilotstudyrdquo International Urology and Nephrology vol 42 no 3 pp629ndash635 2010

[95] P Tyagi B L Jacobs D Barcaly et al ldquoUrine levels ofinflammatory chemokines can be novel biomarkers for theoveractive bladderrdquoThe Journal of Urology vol 181 article 5882009

[96] P Tyagi V Tyagi D Bui et al ldquoDiscrimination Of OAB fromICPBS by multivariate data modeling of urinary proteinsrdquoTheJournal of Urology vol 183 pp e614ndashe6e5 2010

[97] P Tyagi D Nikolavsky Y Vodovotz et al ldquoUrine levels ofselected chemokines positively correlate with lower bladdercapacity and psychometric scores in IcPbs patientsrdquo TheJournal of Urology vol 181 article 21 2009

[98] R Weissleder and V Ntziachristos ldquoShedding light onto livemolecular targetsrdquo Nature Medicine vol 9 no 1 pp 123ndash1282003

[99] C Bremer V Ntziachristos and R Weissleder ldquoOptical-basedmolecular imaging contrast agents and potentialmedical appli-cationsrdquo European Radiology vol 13 no 2 pp 231ndash243 2003

[100] UMahmood andRWeissleder ldquoNear-infrared optical imagingof proteases in cancerrdquoMolecular CancerTherapeutics vol 2 pp489ndash496 2003

[101] J Kaufman H Hensley J Jacobs et al ldquoNon-invasive imagingof near infrafred dye labeled liposomes facilitates evaluation ofbioresidence timerdquoThe Journal of Urology vol 183 article e6282010

[102] Y-X J Wang ldquoMedical imaging in pharmaceutical clinicaltrials what radiologists should knowrdquo Clinical Radiology vol60 no 10 pp 1051ndash1057 2005

ISRN Pharmacology 11

activity and tumor protectionrdquo Biochimica et Biophysica Actavol 1563 no 1-2 pp 7ndash17 2002

[53] Y-C Chuang P Tyagi C-C Huang et al ldquoUrodynamic andimmunohistochemical evaluation of intravesical botulinumtoxin a delivery using liposomesrdquo Journal of Urology vol 182no 2 pp 786ndash792 2009

[54] Y-C Chuang N Yoshimura C-C Huang P-H Chiang andM B Chancellor ldquoIntravesical botulinum toxin a administra-tion produces analgesia against acetic acid induced bladder painresponses in ratsrdquo Journal of Urology vol 172 no 4 pp 1529ndash1532 2004

[55] S P Petrou A S Parker J E Crook A Rogers D Metz-Kudashick and D D Thiel ldquoBotulinum A toxindimethylsulfoxide bladder instillations for women with refractory idio-pathic detrusor overactivity a phase 12 studyrdquo Mayo ClinicProceedings vol 84 no 8 pp 702ndash706 2009

[56] P Caccin O Rossetto M Rigoni E Johnson G Schiavoand C Montecucco ldquoVAMPsynaptobrevin cleavage by tetanusand botulinum neurotoxins is strongly enhanced by acidicliposomesrdquo FEBS Letters vol 542 no 1ndash3 pp 132ndash136 2003

[57] M L Stephenson and P C Zamecnik ldquoInhibition of Roussarcoma viral RNA translation by a specific oligodeoxyribonu-cleotiderdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 75 no 1 pp 285ndash288 1978

[58] H Dadgostar and N Waheed ldquoThe evolving role of vascularendothelial growth factor inhibitors in the treatment of neovas-cular age-related macular degenerationrdquo Eye vol 22 no 6 pp761ndash767 2008

[59] C Gebhard G Huard E A Kritikou and J C TardifldquoApolipoprotein B antisense inhibitionmdashupdate on mipom-ersenrdquo Current Pharmaceutical Design vol 919 no 17 pp 3132ndash3142 2013

[60] T Yokota E Hoffman and S Takeda ldquoAntisense oligo-mediated multiple exon skipping in a dog model of duchennemuscular dystrophyrdquoMethods inMolecular Biology vol 709 pp299ndash312 2011

[61] B L Jacobs M C Smaldone V Tyagi et al ldquoIncreased nervegrowth factor in neurogenic overactive bladder and interstitialcystitis patientsrdquoThe Canadian Journal of Urology vol 17 no 1pp 4989ndash4994 2010

[62] H-T Liu and H-C Kuo ldquoIntravesical botulinum toxin Ainjections plus hydrodistension can reduce nerve growth factorproduction and control bladder pain in interstitial cystitisrdquoUrology vol 70 no 3 pp 463ndash468 2007

[63] H T Liu and H C Kuo ldquoIncreased urine and serum nervegrowth factor levels in interstitial cystitis suggest chronicinflammation is involved in the pathogenesis of diseaserdquo PloSONE vol 7 Article ID e44687 2012

[64] N Yoshimura N E Bennett Y Hayashi et al ldquoBladderoveractivity and hyperexcitability of bladder afferent neuronsafter intrathecal delivery of nerve growth factor in ratsrdquo Journalof Neuroscience vol 26 no 42 pp 10847ndash10855 2006

[65] S Seki K Sasaki Y Igawa et al ldquoSuppression of detrusor-sphincter dyssynergia by immunoneutralization of nervegrowth factor in lumbosacral spinal cord in spinal cord injuredratsrdquo Journal of Urology vol 171 no 1 pp 478ndash482 2004

[66] R J Evans R M Moldwin N Cossons A Darekar I WMills andD Scholfield ldquoProof of concept trial of tanezumab forthe treatment of symptoms associated with interstitial cystitisrdquoJournal of Urology vol 185 no 5 pp 1716ndash1721 2011

[67] P Tyagi R Banerjee S Basu N YoshimuraM Chancellor andL Huang ldquoIntravesical antisense therapy for cystitis using TAT-peptide nucleic acid conjugatesrdquoMolecular Pharmaceutics vol3 no 4 pp 398ndash406 2006

[68] D C Molliver D E Wright M L Leitner et al ldquoIB4-bindingDRG neurons switch from NGF to GDNF dependence in earlypostnatal liferdquo Neuron vol 19 no 4 pp 849ndash861 1997

[69] M TyagiM RusnatiM Presta andMGiacca ldquoInternalizationof HIV-1 tat requires cell surface heparan sulfate proteoglycansrdquoJournal of Biological Chemistry vol 276 no 5 pp 3254ndash32612001

[70] E Uhlmann A Ryte and A Peyman ldquoStudies on the mech-anism of stabilization of partially phosphorothioated oligonu-cleotides against nucleolytic degradationrdquoAntisense andNucleicAcid Drug Development vol 7 no 4 pp 345ndash350 1997

[71] M Nogawa T Yuasa S Kimura et al ldquoIntravesical adminis-tration of small interfering RNA targeting PLK-1 successfullyprevents the growth of bladder cancerrdquo Journal of ClinicalInvestigation vol 115 no 4 pp 978ndash985 2005

[72] M Kashyap N Kawamorita V Tyagi et al ldquoDownregulation ofNGF expression in the bladder by antisense oligoucleotides asnew treatment for overactive bladderrdquo Journal of Urology vol27190 no 2 pp 757ndash764 2013

[73] C-J Arum Y Kodama N Rolim et al ldquoA rat model ofintravesical delivery of small interfering RNA for studyingurinary carcinomardquoWorld Journal of Urology vol 28 no 4 pp479ndash485 2010

[74] C E Blietz B Thode M Hauses et al ldquoIn vivo studies on theavailability and toxicity of antisense oligonucleotides in bladdercancerrdquo In Vivo vol 23 no 1 pp 13ndash19 2009

[75] D Oddiah P Anand S B McMahon and M Rattray ldquoRapidincrease of NGF BDNF andNT-3mRNAs in inflamed bladderrdquoNeuroReport vol 9 no 7 pp 1455ndash1458 1998

[76] M R Saban H Hellmich N B Nguyen J Winston T GHammond and R Saban ldquoTime course of LPS-induced geneexpression in a mouse model of genitourinary inflammationrdquoPhysiol Genomics vol 5 no 3 pp 147ndash160 2001

[77] B Schnegelsberg T T Sun G Cain et al ldquoOverexpression ofNGF in mouse urothelium leads to neuronal hyperinnervationpelvic sensitivity and changes in urinary bladder functionrdquoTheAmerican Journal of PhysiologymdashRegulatory Integrative andComparative Physiology vol 298 no 5 pp R534ndashR547 2010

[78] S Cardenas M Scuri L Samsell et al ldquoNeurotrophic andneuroimmune responses to early-life Pseudomonas aeruginosainfection in rat lungsrdquo American Journal of PhysiologymdashLungCellular and Molecular Physiology vol 299 no 3 pp L334ndashL344 2010

[79] K Nakamura F Tan Z Li and C J Thiele ldquoNGF activationof TrkA induces vascular endothelial growth factor expressionvia induction of hypoxia-inducible factor-1120572rdquo Molecular andCellular Neuroscience vol 46 no 2 pp 498ndash506 2011

[80] S Othumpangat M Regier and G Piedimonte ldquoNerve growthfactor modulates human rhinovirus infection in airway epithe-lial cells by controlling ICAM-1 expressionrdquo American Journalof Physiology Lung Cellular andMolecular Physiology vol 15302pp L1057ndashL1066 2012

[81] H Jung P T Toth F A White and R J Miller ldquoMonocytechemoattractant protein-1 functions as a neuromodulator indorsal root ganglia neuronsrdquo Journal of Neurochemistry vol104 no 1 pp 254ndash263 2008

[82] F A White H Jung and R J Miller ldquoChemokines andthe pathophysiology of neuropathic painrdquo Proceedings of the

12 ISRN Pharmacology

National Academy of Sciences of the United States of Americavol 104 no 51 pp 20151ndash20158 2007

[83] G Banisadr R-D Gosselin P Mechighel W Rostene PKitabgi and S M Parsadaniantz ldquoConstitutive neuronalexpression of CCR2 chemokine receptor and its colocalizationwith neurotransmitters in normal rat brain functional effectof MCP-1CCL2 on calcium mobilization in primary culturedneuronsrdquo Journal of Comparative Neurology vol 492 no 2 pp178ndash192 2005

[84] S Bhangoo D Ren R J Miller et al ldquoDelayed functionalexpression of neuronal chemokine receptors following focalnerve demyelination in the rat a mechanism for the devel-opment of chronic sensitization of peripheral nociceptorsrdquoMolecular Pain vol 3 article 38 2007

[85] T Maeda N Kiguchi Y Kobayashi T Ikuta M Ozaki and SKishioka ldquoLeptin derived from adipocytes in injured periph-eral nerves facilitates development of neuropathic pain viamacrophage stimulationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 106 no 31 pp13076ndash13081 2009

[86] H Kajiwara M Yasuda N Kumaki T Shibayama and YOsamura ldquoExpression of carbohydrate antigens (SSEA-1 Sialyl-Lewis X DU-PAN-2 and CA19-9) and E-selectin in urothelialcarcinoma of the renal pelvis ureter and urinary bladderrdquoTokaiJournal of Experimental and Clinical Medicine vol 30 no 3 pp177ndash182 2005

[87] A M S Assreuy G J Martins M E F Moreira et al ldquoPre-vention of cyclophosphamide-induced hemorrhagic cystitis byglucose-mannose binding plant lectinsrdquo Journal of Urology vol161 no 6 pp 1988ndash1993 1999

[88] S M Vinson A Rickard J S Ryerse and J McHowat ldquoNeu-trophil adherence to bladder microvascular endothelial cellsfollowing platelet-activating factor acetylhydrolase inhibitionrdquoJournal of Pharmacology and Experimental Therapeutics vol314 no 3 pp 1241ndash1247 2005

[89] M Green A Filippou G Sant and T CTheoharides ldquoExpres-sion of intercellular adhesion molecules in the bladder ofpatients with interstitial cystitisrdquoUrology vol 63 no 4 pp 688ndash693 2004

[90] M Leppilahti P Hellstrom and T L J Tammela ldquoEffect ofdiagnostic hydrodistension and four intravesical hyaluronicacid instillations on bladder ICAM-1 intensity and associationof ICAM-1 intensity with clinical response in patients withinterstitial cystitisrdquo Urology vol 60 no 1 pp 46ndash51 2002

[91] A T Corcoran N Yoshimura V Tyagi B Jacobs W Lengand P Tyagi ldquoMapping the cytokine profile of painful bladdersyndromeinterstitial cystitis in human bladder and urinespecimensrdquoWorld Journal of Urology vol 31 no 1 pp 241ndash2462013

[92] SGarrean X-PGaoV Brovkovych et al ldquoCaveolin-1 regulatesNF-120581B activation and lung inflammatory response to sepsisinduced by lipopolysacchariderdquo Journal of Immunology vol 177no 7 pp 4853ndash4860 2006

[93] M R Kang G Yang R F Place et al ldquoIntravesical deliveryof small activating RNA formulated into lipid nanoparticlesinhibits orthotopic bladder tumor growthrdquo Cancer Researchvol 172 pp 5069ndash5079 2012

[94] P Tyagi D Barclay R Zamora et al ldquoUrine cytokines suggestan inflammatory response in the overactive bladder a pilotstudyrdquo International Urology and Nephrology vol 42 no 3 pp629ndash635 2010

[95] P Tyagi B L Jacobs D Barcaly et al ldquoUrine levels ofinflammatory chemokines can be novel biomarkers for theoveractive bladderrdquoThe Journal of Urology vol 181 article 5882009

[96] P Tyagi V Tyagi D Bui et al ldquoDiscrimination Of OAB fromICPBS by multivariate data modeling of urinary proteinsrdquoTheJournal of Urology vol 183 pp e614ndashe6e5 2010

[97] P Tyagi D Nikolavsky Y Vodovotz et al ldquoUrine levels ofselected chemokines positively correlate with lower bladdercapacity and psychometric scores in IcPbs patientsrdquo TheJournal of Urology vol 181 article 21 2009

[98] R Weissleder and V Ntziachristos ldquoShedding light onto livemolecular targetsrdquo Nature Medicine vol 9 no 1 pp 123ndash1282003

[99] C Bremer V Ntziachristos and R Weissleder ldquoOptical-basedmolecular imaging contrast agents and potentialmedical appli-cationsrdquo European Radiology vol 13 no 2 pp 231ndash243 2003

[100] UMahmood andRWeissleder ldquoNear-infrared optical imagingof proteases in cancerrdquoMolecular CancerTherapeutics vol 2 pp489ndash496 2003

[101] J Kaufman H Hensley J Jacobs et al ldquoNon-invasive imagingof near infrafred dye labeled liposomes facilitates evaluation ofbioresidence timerdquoThe Journal of Urology vol 183 article e6282010

[102] Y-X J Wang ldquoMedical imaging in pharmaceutical clinicaltrials what radiologists should knowrdquo Clinical Radiology vol60 no 10 pp 1051ndash1057 2005

12 ISRN Pharmacology

National Academy of Sciences of the United States of Americavol 104 no 51 pp 20151ndash20158 2007

[83] G Banisadr R-D Gosselin P Mechighel W Rostene PKitabgi and S M Parsadaniantz ldquoConstitutive neuronalexpression of CCR2 chemokine receptor and its colocalizationwith neurotransmitters in normal rat brain functional effectof MCP-1CCL2 on calcium mobilization in primary culturedneuronsrdquo Journal of Comparative Neurology vol 492 no 2 pp178ndash192 2005

[84] S Bhangoo D Ren R J Miller et al ldquoDelayed functionalexpression of neuronal chemokine receptors following focalnerve demyelination in the rat a mechanism for the devel-opment of chronic sensitization of peripheral nociceptorsrdquoMolecular Pain vol 3 article 38 2007

[85] T Maeda N Kiguchi Y Kobayashi T Ikuta M Ozaki and SKishioka ldquoLeptin derived from adipocytes in injured periph-eral nerves facilitates development of neuropathic pain viamacrophage stimulationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 106 no 31 pp13076ndash13081 2009

[86] H Kajiwara M Yasuda N Kumaki T Shibayama and YOsamura ldquoExpression of carbohydrate antigens (SSEA-1 Sialyl-Lewis X DU-PAN-2 and CA19-9) and E-selectin in urothelialcarcinoma of the renal pelvis ureter and urinary bladderrdquoTokaiJournal of Experimental and Clinical Medicine vol 30 no 3 pp177ndash182 2005

[87] A M S Assreuy G J Martins M E F Moreira et al ldquoPre-vention of cyclophosphamide-induced hemorrhagic cystitis byglucose-mannose binding plant lectinsrdquo Journal of Urology vol161 no 6 pp 1988ndash1993 1999

[88] S M Vinson A Rickard J S Ryerse and J McHowat ldquoNeu-trophil adherence to bladder microvascular endothelial cellsfollowing platelet-activating factor acetylhydrolase inhibitionrdquoJournal of Pharmacology and Experimental Therapeutics vol314 no 3 pp 1241ndash1247 2005

[89] M Green A Filippou G Sant and T CTheoharides ldquoExpres-sion of intercellular adhesion molecules in the bladder ofpatients with interstitial cystitisrdquoUrology vol 63 no 4 pp 688ndash693 2004

[90] M Leppilahti P Hellstrom and T L J Tammela ldquoEffect ofdiagnostic hydrodistension and four intravesical hyaluronicacid instillations on bladder ICAM-1 intensity and associationof ICAM-1 intensity with clinical response in patients withinterstitial cystitisrdquo Urology vol 60 no 1 pp 46ndash51 2002

[91] A T Corcoran N Yoshimura V Tyagi B Jacobs W Lengand P Tyagi ldquoMapping the cytokine profile of painful bladdersyndromeinterstitial cystitis in human bladder and urinespecimensrdquoWorld Journal of Urology vol 31 no 1 pp 241ndash2462013

[92] SGarrean X-PGaoV Brovkovych et al ldquoCaveolin-1 regulatesNF-120581B activation and lung inflammatory response to sepsisinduced by lipopolysacchariderdquo Journal of Immunology vol 177no 7 pp 4853ndash4860 2006

[93] M R Kang G Yang R F Place et al ldquoIntravesical deliveryof small activating RNA formulated into lipid nanoparticlesinhibits orthotopic bladder tumor growthrdquo Cancer Researchvol 172 pp 5069ndash5079 2012

[94] P Tyagi D Barclay R Zamora et al ldquoUrine cytokines suggestan inflammatory response in the overactive bladder a pilotstudyrdquo International Urology and Nephrology vol 42 no 3 pp629ndash635 2010

[95] P Tyagi B L Jacobs D Barcaly et al ldquoUrine levels ofinflammatory chemokines can be novel biomarkers for theoveractive bladderrdquoThe Journal of Urology vol 181 article 5882009

[96] P Tyagi V Tyagi D Bui et al ldquoDiscrimination Of OAB fromICPBS by multivariate data modeling of urinary proteinsrdquoTheJournal of Urology vol 183 pp e614ndashe6e5 2010

[97] P Tyagi D Nikolavsky Y Vodovotz et al ldquoUrine levels ofselected chemokines positively correlate with lower bladdercapacity and psychometric scores in IcPbs patientsrdquo TheJournal of Urology vol 181 article 21 2009

[98] R Weissleder and V Ntziachristos ldquoShedding light onto livemolecular targetsrdquo Nature Medicine vol 9 no 1 pp 123ndash1282003

[99] C Bremer V Ntziachristos and R Weissleder ldquoOptical-basedmolecular imaging contrast agents and potentialmedical appli-cationsrdquo European Radiology vol 13 no 2 pp 231ndash243 2003

[100] UMahmood andRWeissleder ldquoNear-infrared optical imagingof proteases in cancerrdquoMolecular CancerTherapeutics vol 2 pp489ndash496 2003

[101] J Kaufman H Hensley J Jacobs et al ldquoNon-invasive imagingof near infrafred dye labeled liposomes facilitates evaluation ofbioresidence timerdquoThe Journal of Urology vol 183 article e6282010

[102] Y-X J Wang ldquoMedical imaging in pharmaceutical clinicaltrials what radiologists should knowrdquo Clinical Radiology vol60 no 10 pp 1051ndash1057 2005