Effects of Various Protease Inhibitors on the Stability and Permeabilityof [D-Ala2,D-Leu5]enkephalin in the Rat Intestine: Comparison withLeucine Enkephalin

TOMOMI UCHIYAMA, ATSUSHI KOTANI, TAKESHI KISHIDA, HIROYUKI TATSUMI, AYA OKAMOTO, TAKUYA FUJITA,MASAHIRO MURAKAMI, SHOZO MURANISHI, AND AKIRA YAMAMOTO*

Contribution from Department of Biopharmaceutics, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto, 607, Japan.

Received September 8, 1997. Accepted for publication January 9, 1998.

Abstract 0 The effects of various protease inhibitors on the stabilityof leucine enkephalin (Leu-Enk) and [D-Ala2,D-Leu5] enkephalin(DADLE) were investigated, and the permeability of these peptideswas also examined in an in vitro Ussing chamber. Captopril, thiorphan,bacitracin, bestatin, puromycin, amastatin, and sodium glycocholate(Na-GC) were chosen as protease inhibitors. Regional differencesin the stability of Leu-Enk and DADLE were observed, and the rankorder of the stability of these peptides was colon > duodenum > ileum> jejunum. Na-GC, amastatin, and puromycin were effective proteaseinhibitors for improving the stability of these peptides, although captopriland thiorphan did not improve the stability of Leu-Enk. In the transportstudies, Leu-Enk did not cross the intestinal membrane in the absenceof protease inhibitors, but its transport was improved in the presenceof Na-GC. In addition, Na-GC, amastatin, and puromycin improvedthe permeability of DADLE in both jejunum and colon, while thepermeability of DADLE was not improved by the addition of captopril,thiorphan, and bestatin. Furthermore, the permeability of 6-carboxy-fluorescein, a poorly absorbable and stable compound, was alsoimproved in the presence of Na-GC and bacitracin at a concentrationof 10 mM. These findings indicated that amastatin, puromycin, andNa-GC at a concentration of 0.5 mM might increase the permeabilityof DADLE due to the improved stability of DADLE in the donor site.However, Na-GC and bacitracin at a concentration of 10 mM hadabsorption-enhancing activities which might be also related to theenhanced permeability of DADLE across the intestinal membrane.

IntroductionPeptides and proteins are usually administered parenter-

ally because, when administered orally, they were degradedby the proteolytic enzymes in the gastrointestinal tract orwere impermeable to the intestinal mucosa due to theirhydrophilic characteristics and large molecular size. Vari-ous approaches such as alternative routes, absorptionenhancers, protease inhibitors, chemical modification, anddosage forms have been examined to increase the intestinalabsorption.1,2 Of these approaches, the use of proteaseinhibitors has been shown to improve both the small andlarge intestinal absorption of peptides. We have previouslyreported that the intestinal absorption of insulin wasenhanced in the presence of various protease inhibitors,especially sodium glycocholate, and suggested the coad-ministration of protease inhibitors would be useful forimproving the large intestinal absorption of insulin.3Furthermore, we also found that the degradation of ebi-ratide was markedly inhibited by aminopeptidase inhibi-tors such as sodium glycocholate, puromycin, bestatin, andbacitracin.4 These results suggested that coadministration

of certain protease inhibitors is useful to improve thestability and absorption of these peptide and protein drugs.Leucine-enkephalin (Leu-Enk) and methionine-enkepha-

lin (Met-Enk), the naturally occurring analgesic pentapep-tides, are known to act as neurotransmitters or neuromod-ulators in pain transmission. Their analgesic activities are,however, rather short in duration. This is because of theirrapid inactivation by the enzymes in various organsincluding the gastrointestinal tract. Thus, one of the majorproblems in the delivery of enkephalins is their rapiddegradation by various peptidases at the site of adminis-tration. Consequently, many enzyme inhibition studiesusing various protease inhibitors have been examined toimprove the stability and absorption of enkephalins.5-8

However, it remained to be determined whether theimproving effects of some protease inhibitors on the stabil-ity and absorption of these peptides were better than othersin a single study, and whether the improving effects ofsome protease inhibitors were site-dependent.In the present study, therefore, Leu-Enk and [D-Ala2,D-

Leu5] enkephalin (DADLE) were chosen as model enkepha-lins and regional differences in the intestinal stability ofthese peptides were investigated in rats. We also examinedthe effects of protease inhibitors on the stability andpermeability of Leu-Enk and DADLE in the intestine.Furthermore, the absorption enhancing activities of theseprotease inhibitors were examined using 6-carboxyfluores-cein, a poorly absorbable and stable compound in rats.

Experimental SectionMaterialssLeucine enkephalin, sodium glycocholate (Na-GC),

amastatin, bestatin, captopril, puromycin, and thiorphan werepurchased from Sigma Chemical Co. (St. Louis, MO). DADLE waspurchased from the Peptide Institute (Osaka, Japan). Bacitracinwas purchased fromWako Pure Chemical Industries, Ltd. (Osaka,Japan). 6-Carboxyfluorescein was kindly supplied by EastmanKodak Co. (Rochester, NY).Preparation of Mucosal Tissue HomogenatessMucosal

tissue homogenates were prepared as previously described.9Briefly, Male Wistar rats, weighing 200-250 g, were anesthetizedwith sodium pentobarbital (32 mg/kg body weight, ip). Animalswere fasted for about 16 h prior to the experiments but allowedwater ad libitum. After washing the luminal surface with salinesolution, duodenal, jejunal, ileal, and the colonic mucosae wereremoved by scraping the epithelial cell layers. These specimenswere pooled by tissue type and stored at -80 °C. Immediatelybefore each experiment, specimens were thawed at room temper-ature and then homogenized in 1-2 mL of isotonic phosphatebuffer (pH 7.4) at 4 °C using a Polytron homogenizer. Thehomogenate was centrifuged at 5000g in a refrigerated (4 °C)centrifuge for 10 min to remove cellular and nuclear debris. Theresulting supernatant was diluted with isotonic phosphate bufferto a protein concentration of 10 mg/mL, as determined by theLowry method with bovine serum albumin as the standard.10Degradation of Leu-Enk and DADLE in Intestinal Mu-

cosal HomogenatessThe degradation of Leu-Enk and DADLE* Tel: +81-75-595-4662. Fax: +81-75-595-4761.

S0022-3549(97)00357-2 CCC: $15.00448 / Journal of Pharmaceutical Sciences © 1998, American Chemical Society andVol. 87, No. 4, April 1998 American Pharmaceutical AssociationPublished on Web 03/04/1998

was studied by incubating 300 µL of tissue supernatant (1 mgprotein/mL), which had been preincubated at 37 °C for 15 min,and 300 µL of 0.6 mM Leu-Enk or DADLE solution in the presenceor absence of 1 mM various protease inhibitors. At predeterminedtimes up until a maximum of 120 min, 50 µL of solution wassampled and then 100 µL of 50% acetic acid was added, toterminate the reaction. The resulting mixture was centrifugedat 10 000 rpm for 5 min to remove the precipitated protein. Thesesamples were analyzed by HPLC.Absorption ExperimentssAbsorption experiments were per-

formed in a modified Ussing chamber (surface area, 0.3 cm2) usingstripped rat intestine for 3 h. Male Wistar rats, weighing 200-250 g, were used. The intestine was excised and rinsed inphosphate buffer solution. The experimental segments wereobtained and the underlying muscularis was removed beforemounting in a modified Ussing chamber. Each site was definedas described below. The duodenum segment was the first 10 cmportion from the stomach. The ileum segment was the final 10cm portion of the small intestine. The residual intestine was usedas the jejunum. Two and a half milliliters of modified Ringer’ssolution was added to the serosal side. An equal volume of drugsolution was added to the mucosal side. Mixing was performedby bubbling with 95% O2 - 5% CO2 gas and the temperaturewithin the chamber was maintained at 37 °C by a circulating waterbath. At predetermined times, 200 µL aliquots were taken fromthe serosal side and assayed by UV detection HPLC. The apparentpermeability coefficients (Papp) were calculated by the relationshipPapp ) dXR/dT 1/A C0, where Papp is the apparent permeabilitycoefficient in centimeters per second, XR is the amount of the drugsin moles in the receptor side, A is the diffusion area (i.e., in squarecentimeters), and C0 is the initial concentration of drugs in thedonor side in moles per milliliter. The viability of intestinalmembrane during the test period was monitored by measuringthe transport of trypan blue dye. There was no transport of dyeduring the incubation, suggesting the maintenance of viability ofthe intestinal membrane.Analytical MethodssAliquots were assayed in a reversed-

phase HPLC system containing 5 µM Cosmosil (4.6 mm × 15 cm)particles in an analytical column from Nacalai tesque, a HitachiLC-10 pump system, a Hitachi LC-10 autoinjector, a Hitachi LC-10 detector, and a Hitachi CR-6A integrator. The mobile phasewas a binary mixture of varying proportions of acetonitrile andwater containing 0.1% H3PO4 and 0.005 M NaClO4. The composi-tion of acetonitrile in the mobile phase was increased linearly from5 to 17% for the first 12 min and from 17 to 50% for the next 20min at a flow rat of 1.0 mL/min. Leu-Enk and DADLE weremonitored spectrophotometrically at 214 nm. Concentrations weredetermined using external standards.Statistical AnalysessResults are expressed as the mean (

SE and the statistical significance was assessed by the Student’st-test.

Results

Stability of Leu-Enk and DADLE in Various Intes-tinal Mucosal HomogenatessThe stability of Leu-Enkand DADLE are shown in Figure 1. Leu-Enk and DADLEdisappearance followed first-order kinetics. In both cases,the regional differences in proteolysis were observed. Thehalf-live values were calculated from the first-order rateconstants obtained from semilogarithmic plots. The half-lives for enzymatic hydrolysis in rank order were colon >duodenum > ileum > jejunum. The half-live of DADLEwas at least 120 min, indicating that DADLE is moreresistant for enzymatic hydrolysis than Leu-Enk. In thenext experiment, the jejunum and colon were selected tocompare the effects of various protease inhibitors.Effects of Various Protease Inhibitors on the Sta-

bility of Leu-Enk and DADLE in Jejunal and ColonicHomogenatessTable 1 summarizes effects of variousprotease inhibitors on the half-life of the enzymatic hy-drolysis of Leu-Enk in jejunal and colonic homogenates.The rank order of the effectiveness of decreasing Leu-Enkhydrolysis in homogenates of jejunal membrane was Na-

GC > amastatin > puromycin > bestatin ) bacitracin, allat a concentration of 0.5 mM. Captopril and thiorphan,enkephalinase inhibitors, did not reduce the degradationof Leu-Enk. In the colonic homogenate, with the exceptionof captopril and thiorphan, they effectively decreasedenzymatic hydrolysis, and the maximum reduction in theproteolysis of Leu-Enk was seen in the presence of Na-GC.On the other hand, all protease inhibitors were effectivein decreasing the hydrolysis of DADLE, as shown in Table2. Puromycin, amastatin, and Na-GC remarkably reducedthe hydrolysis of DADLE in jejunal mucosal homogenates,while its hydrolysis was moderately inhibited in thepresence of captopril, thiorphan, bacitracin, and bestatin.In the case of colonic membranes, a similar result was alsoobserved. Bestatin, puromycin, amastatin, and Na-GCconsiderably reduced DADLE hydrolysis. Overall, theeffectiveness of these protease inhibitors on hydrolysis was

Figure 1sStability of Leu-Enk and DADLE in various intestinal mucosalhomogenates at a concentration of 0.3 mM; duodenum (2), jejunum (b),ileum (9), colon ([). Each value represents the mean ± SE of at least threeexperiments.

Table 1sEffects of Various Protease Inhibitors on the Half-Life ofLeu-Enk Proteolysis in Jejunal and Colonic Mucosal Homogenatesa

jejunum colon

T1/2 (min) ratio T1/2 (min) ratio

control 1.09 ± 0.03 1.0 5.23 ± 0.10 1.0captopril (0.5 mM) 1.01 ± 0.03 0.9 5.57 ± 0.32 1.1thiorphan (0.5 mM) 1.22 ± 0.02 1.1 5.42 ± 0.18 1.0bacitracin (0.5 mM) 1.60 ± 0.01** 1.5 6.05 ± 0.51* 1.2bestatin (0.5 mM) 1.67 ± 0.03** 1.5 8.05 ± 0.38** 1.5puromycin (0.5 mM) 2.29 ± 0.06** 2.1 16.00 ± 0.76** 3.1amastatin (0.5 mM) 10.05 ± 0.65** 9.2 20.05 ± 0.65** 3.8Na-GC (0.5 mM) 22.11 ± 1.95** 20.3 72.17 ± 1.96** 13.8

a Each value represents the mean ± SE of three experiments. *p < 0.05,**p < 0.01, compared with the control.

Journal of Pharmaceutical Sciences / 449Vol. 87, No. 4, April 1998

greater in the jejunum than in the colon, as evaluated bythe stability-enhancing ratios (T1/2 protease inhibitor/T1/2control).Effects of Protease Inhibitors on the Permeability

of Leu-Enk across Intestinal MembranessTable 3shows the apparent permeability coefficients of Leu-Enkcoadministered with protease inhibitors across the intes-tinal mucosae. Puromycin, amastatin, and Na-GC wereselected in this experiment because these protease inhibi-tors were especially effective in Leu-Enk hydrolysis, asshown in Table 1. In the case of the control, Leu-Enk didnot cross the jejunal or colonic membrane. Na-GC onlyimproved the permeability of Leu-Enk, however, puromycinand amastatin were not effective in the jejunum. On theother hand, these three protease inhibitors significantlyincreased Leu-Enk permeability in the colon. Of theseprotease inhibitors, Na-GC was most effective in both cases.Effects of Protease Inhibitors on the Permeability

of DADLE across Intestinal MembranessApparentpermeability coefficients of DADLE across the jejunal andcolonic membranes in the presence of various proteaseinhibitors are shown in Table 4. Puromycin, amastatin,and Na-GC improved the permeability of DADLE acrossthe jejunal and colonic membranes. However, captopril,thiorphan, and bestatin had no effect on the permeabilityof DADLE across the jejunal membrane all at a concentra-tion of 0.5 mM, whereas bestatin slightly improved thepermeability of DADLE across the colonic membrane. Onthe other hand, bacitracin at 10 mM increased the perme-ability of DADLE in both these membranes, but its perme-ability was not enhanced by the bacitracin at 0.5 mM.Assessment of the Absorption Enhancing Effect of

Various Protease InhibitorssWe examined the absorp-tion-enhancing actions of these protease inhibitors. 6-Car-boxyfluorescein (6-CF), a poorly absorbable and stablecompound, was chosen as a model compound in thisexperiment. Figure 2 shows the permeability of 6-CFacross the jejunal and colonic membranes in the presenceof various protease inhibitors. Puromycin and amastatinhad no absorption-enhancing effect at both concentrations.

Bacitracin and Na-GC did not enhance the absorption of6-CF across the intestinal membranes at a concentrationof 0.5 mM. However, 10 mM Na-GC and bacitracinimproved the transport of CF.

DiscussionIn this present study, we demonstrated the regional

differences in the stability of Leu-Enk and DADLE inhomogenates of rat intestinal mucosae. That is, thesepeptides were more stable in colonic homogenates than inother homogenates of small intestinal mucosae. Previ-ously, we reported that the half-life of tetragastrin wasapproximately 6.8 times longer in colonic homogenatesthan in jejunal homogenates.11 Furthermore, we also foundthat insulin was approximately 13 times more stable incolonic homogenates than in jejunal homogenates.12 There-fore, these previous results were consistent with ourpresent finding. With the respect of enzymatic activitiesof enkephalins, Bai reported that the activities of enkepha-linases and aminopeptidases, which are known to beresponsible for enkephalin hydrolysis, were highest injejunum among various intestinal regions.13 Therefore, itmay be considered that the low stability of Leu-Enk andDADLE in jejunum is attributed to the high activities ofthese enzymes in this region.The present study indicated that the degradation of Leu-

Enk was markedly inhibited by the addition of Na-GC,amastatin, puromycin, bestain, and bacitracin. This find-ing is in good agreement with the previous result of Dodda-Kashi et al. that Leu-Enk was mainly degraded by ami-

Table 2sEffects of Various Protease Inhibitors on the Half-Life ofDADLE Proteolysis in Jejunal and Colonic Mucosal Homogenatesa

jejunum colon

T1/2 (min) ratio T1/2 (min) ratio

control 126 ± 3.2 1.0 240 ± 5.8 1.0captopril (0.5 mM) 184 ± 4.7** 1.5 295 ± 9.8* 1.2thiorphan (0.5 mM) 236 ± 6.1** 1.9 329 ± 8.1** 1.4bacitracin (0.5 mM) 367 ± 9.2** 2.9 435 ± 12** 1.8bestatin (0.5 mM) 396 ± 10** 3.1 >500 >2.1puromycin (0.5 mM) >500 >4.0 >500 >2.1amastatin (0.5 mM) >500 >4.0 >500 >2.1Na-GC (0.5 mM) >500 >4.0 >500 >2.1

a Each value represents the mean ± SE of three experiments. *p < 0.05,**p < 0.01, compared with the control.

Table 3sApparent Permeability Coefficients of Leu-Enk (1 mM)Coadministered with Various Protease Inhibitors in Jejunal andColonic Membranesa

Papp (× 10-7 cm/s)

jejunum colon

control ND NDpuromycin (0.5 mM) ND 2.53 ± 0.25amastatin (0.5 mM) ND 2.88 ± 0.27Na-GC (0.5 mM) 4.83 ± 0.64 4.36 ± 0.56

a Each value represents the mean ± SE of three experiments. ND: notdetected.

Table 4sEffects of Various Protease Inhibitors on ApparentPermeability Coefficients of DADLE (1 mM) in the Jejunal and ColonicMembranesa

Papp (× 10-6 cm/s)

jejunum colon

control 3.01 ± 0.39 2.11 ± 0.35captopril (0.5 mM) 3.16 ± 0.31 2.16 ± 0.24thiorphan (0.5 mM) 3.23 ± 0.48 2.22 ± 0.38bacitracin (0.5 mM) 3.30 ± 0.26 2.27 ± 0.44bacitracin (10 mM) 4.51 ± 0.43* 6.86 ± 0.63**bestatin (0.5 mM) 3.48 ± 0.45 4.60 ± 0.39*puromycin (0.5 mM) 6.01 ± 0.51** 5.35 ± 0.64**puromycin (10 mM) 6.25 ± 0.49** 5.44 ± 0.75**amastatin (0.5 mM) 8.63 ± 0.78** 5.35 ± 0.64**amastatin (10 mM) 8.61 ± 0.98** 5.12 ± 0.63*Na-GC (0.5 mM) 8.77 ± 0.40** 5.69 ± 0.69**Na-GC (10 mM) 9.01 ± 0.51** 8.54 ± 0.53**

a Each value represents the mean ± SE of at least three experiments. *p< 0.05, **p < 0.01, compared with the control.

Figure 2sPermeability coefficients of 6-CF across jejunal and colonicmembranes in the presence of various protease inhibitors. Each valuerepresents the mean ± SE of at least three experiments.

450 / Journal of Pharmaceutical SciencesVol. 87, No. 4, April 1998

nopeptidases in homogenates of various mucosae.14 As forNa-GC, Hirai et al. reported that this bile salt reduced theactivity of Leu-aminopeptidase, thereby improving thestability of insulin in nasal mucosal homogenates.15 There-fore, this enzymatic inhibitory action of Na-GC may berelated to the enhanced stability of Leu-Enk in the presentstudy. On the other hand, our present study demonstratedthat amastatin was more effective for improving thestability of Leu-Enk than bestatin. This finding wasinconsistent with the previous result of Kerchner et al.16The reason of this discrepancy was not fully understood.However, we may consider the contribution of carboxypep-tidases to the hydrolysis of Leu-Enk when coadministeredwith amastatin, since amastatin also had an inhibitoryaction on carboxypeptidases in addition to its inhibitoryaction on aminopeptidases.We found that there existed a regional differences in the

stability of DADLE as in the case of Leu-Enk. However,overall, the stability of DADLE in jejunal or colonic mucosalhomogenates was much higher than that of Leu-Enk. Thisfinding concurs with the previous result of Kerchner et al.16and this higher stability of DADLE may be due to thesubstitution of L-Gly to D-Ala at the second amino acid andL-Leu to D-Leu at C-terminal amino acid. The presentstudy also demonstrated that the stability of DADLE wasimproved by the addition of all types of protease inhibitors.That is, it was observed that captopril and thiorphan,which are known to be inhibitors of enkephalinases andangiotensin converting enzymes, effectively reduced thedegradation of DADLE in jejunal and colonic homogenates,although we found no significant effect on the stability ofLeu-Enk in the presence of these protease inhibitors. Thedifferent effects of these protease inhibitors on the stabilityof DADLE and Leu-Enk are not clearly understood. Pre-sumably, the contribution of enkephalinases for the hy-drolysis of DADLE is more remarkably observed than theother enzymes, since DADLE is relatively stable and isresistant to aminopeptidases, carboxypeptidases, and dipep-tidylaminopeptidases, as compared with Leu-Enk.In the transport studies, we found that Leu-Enk did not

cross the jejunal or colonic membranes in the absence ofprotease inhibitors. This negative transport of Leu-Enkmay be attributed to its low stability in the donor side ofthe chamber and most of Leu-Enk may be degraded beforereaching to the receptor side of the chamber. However,we observed the transport of Leu-Enk in the presence ofvarious protease inhibitors, especially Na-GC. This resultmay be due to the enhanced stability of Leu-Enk upon theaddition of these protease inhibitors. This speculation wasalso supported by the fact that there existed a goodcorrelation between the remaining percentage of Leu-Enkin the donor side and its transport percentage in thepresence of various protease inhibitors (data not shown).However, we may also consider the contribution of absorp-tion-enhancing action of Na-GC, since bile salts are gener-ally known to be one of the typical absorption enhancers.2

Unlike Leu-Enk, we observed the transport of DADLEeven in the absence of protease inhibitors, which may berelated to its high stability in the donor side of the chamber.Moreover, the permeability of DADLE was improved by theaddition of various protease inhibitors such as Na-GC,amastatin, puromycin, and bacitracin (10 mM). In thecolonic membrane, bestatin was also effective for improvingthe permeability of DADLE. We also found that theeffectiveness of puromycin, an inhibitor of dipeptidyl-aminopeptidases, was more predominant for improving thepermeability of DADLE than that of bestatin, an inhibitorof aminopeptidases. This finding concurs with the previousresult of Taki et al. that puromycin remarkably enhancedthe intestinal absorption of metokephamid, an enkephalin

analogue, rather than bestatin in isolated vascular per-fused intestinal loops.17As we indicated, protease inhibitors enhanced the stabil-

ity of peptides by reducing the activities of proteaseinhibitors. However, it may be possible that these proteaseinhibitors may also have absorption enhancing activitiesin addition to their actions on proteases, like Na-GC. Inthis study, therefore, 6-CF was chosen as a poorly absorb-able and stable model compound and the permeability of6-CF was examined in the presence of various proteaseinhibitors. Our present study demonstrated that thepermeability of 6-CF across the intestinal membranes wasenhanced by the addition of bacitracin and Na-GC at aconcentration of 10 mM, although amastatin and puromy-cin did not improve its permeability. This finding is well-correlated with our previous result that bacitracin in-creased the absorption of phenol red and fluoresceinisothiocyanate-labeled dextran with average molecularweight of 4000 (FD-4) from the rat intestine by an in situloop method.18 Therefore, we indicated that some proteaseinhibitors including bacitracin at higher concentrations hadabsorption-enhancing activities in addition to their pro-tease-inhibiting actions. On the other hand, at a concen-tration of 0.5 mM of these protease inhibitors, theyenhanced the intestinal permeability of Leu-Enk andDADLE by their proteolytic-inhibiting activities ratherthan their absorption-enhancing actions.In conclusion, it was indicated that the stability and

permeability of Leu-Enk and DADLE were improved bythe coadministration of various protease inhibitors, espe-cially Na-GC. Furthermore, we also found that bacitracinas well as Na-GC had absorption-enhancing activities inaddition to their protease-inhibiting actions. However, aconcentration of 0.5 mM might increase the permeabilityof DADLE due to the improved stability of DADLE in donorsite. These findings give us basic information to improvethe stability and permeability of peptide and protein drugsincluding enkephalins in the gastrointestinal tract.

References and Notes1. Lee, V. H. L.; Yamamoto A. Penetration and enzymatic

barriers to peptide and protein absorption. Adv. DrugDelivery Rev. 1990, 4, 171-207.

2. Lee, V. H. L.; Yamamoto A.; Kompella, U. B., Mucosalpenetration enhancers for facilitation of peptide and proteindrug absorption. Crit. Rev. Ther. Drug. Carrier Syst. 1991,8, 91-192.

3. Yamamoto, A.; Taniguchi, T.; Rikyuu, K.; Tsuji, T.; Fujita,T.; Murakami, M.; Muranishi, S. Effects of various proteaseinhibitors on the intestinal absorption and degradation ofinsulin in rats. Pharm. Res. 1994, 11, 1496-1500.

4. Okagawa, T.; Fujita, T.; Murakami, M.; Yamamoto, A.;Shimura, T.; Tabata, S.; Kondo, S.; Muranishi, S. Suscepti-bility of ebiratide to proteolysis in rat intestinal fluid andhomogenates and its protection by various protease inhibi-tors. Life Sci. 1994, 55, 677-683.

5. Sayani, A. P.; Chun, I. K.; Chien, Y. W. Transmucosaldelivery of leucine enkephalin: stabilization in rabbit enzymeextracts and enhancement of permeation through mucosae.J. Pharm. Sci. 1993, 82, 1179-1185.

6. Bouboutou R.; Waksman, J.; Devin, J.; Zalski, M. F.; Roques,B. P. Highly potent new inhibitors of enkephalin degradingenzymes. Life Sci. 1984, 35, 1023-1030.

7. Chipkin, R. E.; Berger, J. G.; Billard, W.; Iorio, L. C.Chapman, R.; Barnett, A. Pharmacology of SCH 34826, anorally active enkephalinase inhibitor analgesic. J. Pharma-col. Exp. Ther. 1988, 245, 829-838.

8. Konkoy, C. S.; Davis, T. P. Regional metabolism of Met-enkephalin and cholecystokinin on intact rat brain slices:characterization of specific peptidases. J. Neurochem. 1995,65, 2773-2783.

9. Yamamoto, A.; Hayakawa, E.; Lee, V. H. L. Insulin andproinsulin proteolysis in mucosal homogenates of the albinorabbit: Implications in peptide delivery from nonoral routes.Life Sci. 1990, 47, 2465-2474.

Journal of Pharmaceutical Sciences / 451Vol. 87, No. 4, April 1998

10. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J.Protein measurement with the folin phenol reagent. J. Biol.Chem. 1951, 193, 265-275.

11. Yodoya, E.; Uemura, K.; Tenma, T.; Fujita, T.; Murakami,M.; Yamamoto, A.; Muranishi, S. Enhanced permeability oftetragastrin across the rat intestinal membrane and itsreduced degradation by acylation with various fatty acids.J. Pharmacol. Exp. Ther. 1994, 271, 1509-1513.

12. Asada, H.; Douen, T.; Waki, M.; Adachi, S.; Fujita, T.;Yamamoto, A.; Muranishi, S. Absorption characteristics ofchemically modified-insulin derivatives with various fattyacids in the small and large intestine. J. Pharm. Sci. 1995,84, 682-687.

13. Bai, J. P. F. Distribution of brush-border membrane pepti-dases along the intestine of rabbits and rats: implication forsite-specific delivery of peptide drugs. J. Drug Targeting1993, 1, 231-236.

14. Dodda-Kashi, S.; Lee, V. H. L. Enkephalin hydrolysis inhomogenates of various absorptive mucosae of the albinorabbit: similarities in rates and involvement of aminopep-tidases. Life Sci. 1986, 38, 2019-2028.

15. Hirai, S.; Yashiki, T.; Mima, H. Mechanisms for the enhance-ment of the nasal absorption of insulin by surfactants. Int.J. Pharm. 1981, 9, 173-184.

16. Kerchner, G. A.; Geary, L. E. Studies on the transport ofenkephalin-like oligopeptides in rat intestinal mucosa. J.Pharmacol. Exp. Ther. 1983, 226, 33-38.

17. Taki, Y.; Sakane, T.; Nadai, T.; Sezaki G.; Amidon, G. L.;Langguth, P.; Yamashita S. Gastrointestinal absorption ofpeptide drug: quantitative evaluation of the degradation andthe permeation of metkephamid in rat small intestine. J.Pharmacol. Exp. Ther. 1995, 274, 373-377.

18. Gotoh, S.; Nakamura, R.; Nishiyama, M.; Quan, Y. S.; Fujita,T.; Yamamoto, A.; Muranishi, S. Effects of protease inhibitorson the absorption of phenol red and fluorescein isothiocyan-ate dextrans from the rat intestine. J. Pharm. Sci. 1996, 85,858-862.

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