EUROPEAN JOURNAL OF DRUG METABOLISM AND PHARMACOKINETICS, 1989, Vol. 14, No.3, pp. 229-234

Des-Enkephalin-y-Endorphin (DEyE) : pharmacokinetics indogs after intravenous and subcutaneous administration

J. VERHOEF, and H.M. van den WILDENBERG

Scientific Development Group, Organon International B.V., Oss, The Netherlands

Receivedfor publication: January 21,1988

Keywords: y-endorphins, DEyE, in vivo fate, i.v. administration, s.c. administration, half-life, bioavailability

SUMMARY

After intravenous dosing in dogs eH-Lys9]-DEyE (Org 5878) was very rapidly eliminated from the circulation. Disappearance ofthe neuropeptide from blood followed a biphasic decay with half-lives of 0.6 ± 0.1 min (± S.D.; a-phase) and 2.4 ± 1.0 min(Il-phase). The central volume of distribution ranged between 0.05 and 0.23I.kg-1

• The mean blood clearance rate amounted to 0.15l.min-l.kg-I, which is indicative of extensive hepatic and extrahepatic metabolism of DE-yE. In contrast to intravenous dosing,subcutaneous injection of eH]-DEyE in dogs resulted in low but relatively long-lasting peptide levels in blood. Peak: values werefound at 5-10 min, whereafter they declined to the limit of detection at 1.5-2 h. The bioavailability of DEyE for this route ofadministration was shown to be 20-23%.

INTRODUCTION

The non-opioid peptide des-enkephalin-y-endorphin(DEyE; Org 5878; ~-endorphin-(6-17» has beenshown to display neuroleptic-like effects in rats (1-3)and antipsychotic activity in schizophrenic patients (4,5). Recent in vivo fate studies with tritiated DEyE inrats demonstrated extensive distribution andbiotransformation of this neuropeptide (6, 7). Afterintravenous injection the elimination half-life wasfound to be 6.3 min. Subcutaneous or intramuscularadministration, however, resulted in longer-lastingblood peptide levels. The bioavailability of DEyE forthe subcutaneous route of administration wasapproximately 31%, whereas the bioavailability afterintramuscular injection appeared to be 3.5 times lower(7).

The aim of the present study was to extend theserat phannacokinetic data on DEyE with those in dogs.

Send reprint requests to : Dr J. Verhoef, Center forBio-Pharmaceutical Sciences, P.O. Box 9502, NL-2300 RALeiden, The Netherlands

To that end, the in vivo behaviour of eHl-DEyE indogs was investigated following a single intravenousinjection. In addition, the time course profile ofeHJ-DEyE in dog blood was measured aftersubcutaneous injection in order to detennine thebioavailability for this route.

MATERIALS AND METHODS

Animals

Male and female beagle dogs, weighing10-12kg, wereused in this study. The animals were housed in steelmetabolism cages. They received standard laboratorydog food and had free access to tap water.

Peptides and chemicals

eH-Lys9]-DEyE was prepared at AmershamInternational p.l.c. (Cardiff, UK) by catalytic reductionwith tritium gas of the L-2,6-diamino-4-hexynoic acid

230 Eur. J. DrugMetab. Pharmacokinet., 1989, No.3

containing-precursor as described previously (8).Purification of the reaction mixture was carried out byhigh performance liquid chromatography (HPLC).The final radiochemical purity of eH]-DEyE exceeded95%. The specific activity as determined by FABmass spectrometry (9), was 43 Ci/mmol.

Reference DEyE and the ~-endorphin sequences7-17,8-17,9-17,10-17,14-17,6--16,6--9 and 10-13were synthesized, purified and kindly donated by DrJ.W. van Nispen (Organon, Oss, The Netherlands).

Uvasol-quality acetonitrile (CH3CN), ammoniumacetate (NH40Ac), sodium dihydrogen phosphate(NaH:zPOdf20), sodium l-octanesulfonate andtrichloroacetic acid (TCA) were purchased fromMerck (Darmstadt, FRG).

r3H]-Peptide administration and bloodsampling

All eX~riments were performed on conscious dogsand [H]-DEyE was dosed as a solution inphysiological saline. Intravenous (i.v.) administrationwas performed by injection of the peptide into theright forepaw vein (dose volume 1 ml); one dogreceived a radioactive dose of 0.9 mCi (27 ug), whiletwo animals were injected. with 2.5 mCi (76 ug).

Subcutaneous (s.c.) administration in two dogs wascarried out under the neck skin with a solution of 2.6mCi (78 J,Lg) eH]-DEyE in 3 ml.

Following i.v. or s.c. administration ofeHJ-peptide, blood samples of approx. 2.5 ml weretaken from the jugular vein with Venoject evacuated.blood-collecting tubes (Terumo Europe, Louvain,Belgium). These tubes contained lithium heparin (45USP Units) as an anticoagulant

Extraction of r3H]-DE'YE from blood

Blood samples of 2 ml were immediately mixed with3 ml of an ice-cold 6% (w/v) TCA solution in watercontaining 25 J,Lg reference DEyE. The mixtures werecentrifuged for 10 min at 5000 gay. From thesupernatants 50 III aliquots were added to 5 mlPicofluor-30 (Packard Instruments, Brussels, Belgium)and counted for radioactivity.

Subsequently, the TCA supernatants wereprepurified on Seppak-Cis cartridges (Waters,Milford, USA) as described elsewhere (7). The DEyEcontaining fractions were evaporated to dryness at400C in a Speed Vac Concentrator (Dumee, Soest, TheNetherlands) and stored at -20°C until HPLC analysis.

From previous experiments it was verified that nobreakdown of DEyE occurred during the extractionand purification procedures and that the recovery ofthe peptide was almost complete (7).

High performance liquid chromatography

All experiments were carried out on a HewlettPackard liquid chromatograph (Model 1090) equipped.with a ternary solvent delivery system, variablevolume injector (1-25 J.Ll) and diode-array detectoroperating at 210 om. Chromatography was performedon a reverse-phase Ultrasphere 3 IlID-ODS column(0.46 x 7.5 em; Beckman Instruments. Berkeley, USA)at 50°C with a flow rate of 0.5 mlImin. The elutionpattern was recorded on a Hewlett Packard 5880 Aseries printer/plotter; determination of the retentiontimes and integration of the peakareas were done withthe built-in data system Chromatographic conditionswere adapted to those reported. by Janssen et al. (10).In the mobile phase the following solvents A, B and Cwere used: 0.25 M NaH:zP04 with 0.025 M sodiuml-octanesulfonate in water adjusted. to pH 2.1 withconcentrated. H3P04 (A), water (B) and acetonitrile(C). Blood extracts were dissolved in 30 J.Ll distilledwater and aliquots of 25 J.Ll were injected.. Lineargradient elution was carried out for 15 min from A : B: C =69 : 10 : 21 to A : B : C = 65 : 10 : 25 (by vol).The [3HJ-DEyE area was collected. based on theretention time of reference DEyE as fractions of 0.25min in counting vials containing 4.5 ml Picofluor@-30.Radioactivity in the fractions obtained was measured.b~ liquid scintillation counting. The amount of[HJ-DEyE was calculated. by summation of theradioactivity in the individual fractions of the DEyEarea and corrected. for the mean background in thatsame area. The recoveries were determined byintegration of the UV peak area of reference DByE(which had been previously added to the TCAextraction medium).

Pharmacokinetic evaluationI

The disappearance curves of eHJ-DEyE from bloodafter i.v. dosing to dQgs were analyzed bycomputerized non-linear, extended least squares fittingusing an open two-compartmental model withelimination from the central compartment (11).Following s.c. administration, the areas under theblood concentration versus time curves (AUC) ofeHl-DEyE were determined with the trapezoidal rule

J. Verhoef& H.M. van denWildenberg, DE-gamma-E in dog 231

6·9~

6.16~

10.13--

14·17 7.17

10-17 - ~ Org 18.175878 ~

~

9.17

1

o 5 10 15minutes

2lI1510

minute.5o

Fig. 2 Disappearance of total radioactivity (*) andnon-metabolized DEyE (0) from blood afterintravenous administration of 2.5 mCi (76 J.Lg)eH]-DEyE to dog C

In fact, the amounts of the peptide were found to bebelow the limit of detection at 13.5 min, 19 min and25 min for dogs A, B and C respectively. Theseobservations demonstrate that extensive in vivobiotransformation of DEyE already occurs within afew minutes after i.v, dosing to dogs. For all animalsthe eH]-DEyE disappearance from blood followed abiphasic decay. The best fitting curves were

10-3+- ---,,-- -.- ~---_,

liPFigure 1 shows the elution profile of a mixture ofDEyE (f3-endorphin-(6-17); ~E 6-17) and eight of itspeptide fragments using an Ultrasphere-30DS columnand octanesulfonate as an ion-pairing reagent in themobile phase. Baseline separation was accomplishedbetween the parent compound DEyE and itsC-terrninal fragments ~E 7-17, ~E 8-17 and ~E 9-17with retention times of 10.3 min, 10.9 min, 11.5 minand 12.8 min respectively. Partial resolution wasobtained between BE 10-17 (retention time 9.3 min)and ~E 14-17 (retention time 9.7 min), whereas thetetrapeptides BE 6-9 and BE 10-13 comigrated justbehind the void volume of the column. Thus theHPLC system used in this study appears to beselective for measurement of DEyE in biologicalsamples. Following a single i.v. dose of eH]-DEyE tothree dogs, 12-14 blood samples were taken from thejugular vein for up to 25 min (dogs A and B) and forup to 90 min (dog C). A typical example of the bloodconcentration versus time course of total radioactivityand non-metabolized [3H]-DEyE is given in Figure 2(dog C).. As compared to total radioactivity, the bloodlevels of eH]-DEyE decreased much faster with time.

Fig. 1 : HPLC chromatogram of a mixture of DEyE (Org 5878) and the IJ-endorphin fragments 10--13, 6-9, 6-16, 10--17, 14-17,7-17,8-17 and 9-17 (3 J.1g of each peptide) on an Ultrasphere-30DS column. UV absorption was measured at 210 nm.Chromatographic conditions are given in detail in Materials and Methods

and the absolute bioavailabilities for this route ofadministration were calculated from the ratios % Dose/' blood

AUCs.clAUe.v. (12). III Dog C

RESULTS

232 Eur.J. DrugMetab. Pharmacokinet., 1989, No.3

Table I : Pharmacokinetic parameters of DElE as determined after intravenous administration to dogs

Parameter Units Dog A DogB Doge

(female) (male) (female)

Dose (D) I.Lg 27 76 76

A %D.11 176.0 63.6 46.1

B %Dr1 8.25 0.60 0.21

a. . -11.46 0.99 1.01min

~min-I 0.52 0.27 0.22

tY2 (o) min 0.5 0.7 0.7

t~ (~) min 1.3 2.6 3.2

VI l.kg-1 0.05 0.16 0.23

V2 l.kg-1 0.006 0.010 0.013

AUC(Q-2h) %Dr1.min 166 71 58

AUC(Q-oo) %Dr1.min 136 66 47

BCR l.min-1.kg-l 0.07 0.16 0.22

Mean

95.2

3.02

1.15

0.34

0.6

2.4

0.15

0.01

98

83

0.15

VI - central volume (Dosel(A+B»; V2 - peripheral volume [(k12lk21).VI]; AUC(o-2h) - area under blood concentration-time CUlVe from 0 to 2 h(trapezoidal rule); AUC«()-co) - Ala.+B/P;BCR - blood clearance rate [DoseIAUC«()-co)]

200

009 C

11lOlOll

mlnut••1lO

Fig. 3 : Blood concentration-time course of totalradioactivity (*) and non-metabolized DE-yE (0)after subcutaneous administration of 2.6 mCi (78J..L8) em-DElE to dog C

(dog D) of the dose.rl.min. These data correspond toabsolute bioavailability values of 23% (dog C) and20% (dog D), assuming 100% bioavailability after i.v,administration (AUC (0-2 h) = 98% dose.rl.min;Table I).

lO-3-+- --r ..-- --r_~::L_____.

liP

% 00••/1 blood

.,}

determined by computerized non-linear regressionanalysis, assuming a two-compartment model and theexperimental data being fitted to the equation[DEyE]blood = Ae-<1J. + Be-Pt. Parameter estimates forthe individual dogs are summarized in Table LConsequently, half-lives for the initial a-phase andterminal ~phase were 0.6 min (range 0.5-0.7 min)and 2.4 min (range 1.3-3.2 min) respectively. Thevolume of distribution for the central compartment(VI) was high, ranging from 0.05 to 0.23 l.kg-l. Onthe other hand, the volume of the peripheralcompartment (V2) was very small and amounted to6-12% of the central volume. The mean bloodclearance rate of DEyE was calculated to be 0.15l.min-l.kg-l (range 0.07-0.22).

Following s.c. injection of 2.6 mCi (78 J,Ig)eH]-DEyE in dog C (female) and dog D (male) bloodsamples were withdrawn for up to 3 h after dosing.The time course of the blood concentrations of totalradioactivity and [3H]-DEyE is presented in Figure 3(dog C). Levels of total radioactivity were foundmaximal at 15-20 min, whereafter they decreased veryslowly. Levels of non metabolized eHl-DEyE,however, were low compared to those of totalradioactivity. Peak values were measured at 5-10min, being 2.2% and 1.0% of the dosell blood for dogC and dog D respectively. Thereafter, they declinedto below the limit of detection at 1.5-2 h. TheAUC's, as determined by the trapezoidal role up to 2h after s.c. injection, were 22.2% (dog C) and 19.7%

J. Verhoef& H.M. van den Wildenberg, DE-gamma-E in dog 233

DISCUSSION

The present data obtained after a single i.v. injectionof [3H]-DEyE in dogs show extremely fastdisappearance of the peptide from the circulation. Theblood levels of non-metabolized DEyE were alreadybelow the limit of detection 14-25 min after injection.The disappearance curves were best characterized byan open two-compartment model, despite the fact thatonly slight differences were observed between the a.­and ~-phases. The mean half-lives were estimated tobe 0.6 min for the initial a.-phase (distribution phase)and 2.4 min for the terminal ~phase (eliminationphase). Previous phannacokinetic studies haverevealed that the elimination half-life of DEyE in ratsis 6.3 min (7). Thus it is apparent that theneuropeptide is eliminated more rapidly in dogs thanin rats. This is also in agreement with the observedhigher rate of in vitro metabolic degradation of DEyEin dog plasma than in rat plasma (13). Comparablespecies-dependent differences in the in vivoelimination of peptides have been noticed for thevasopressin derivative DGAVP, which showshalf-lives of about 8 min in dogs (14) and 15 min inrats (15). Due to the remarkably short half-life values,the physiological relevance of the phannacokineticparameters of DEyE in dogs as summarized in Table Ishould be interpreted with caution. Nevertheless, therapid decay of eH]-DEyE from blood appears to bereflected by high values for the blood clearance rate(0.15 l.min- l.k.g- l). Such a high clearance isindicative of extensive hepatic as well as extrahepaticmetabolism of DEyE, for instance by peptidaseactivities in plasma. All these data favour theconclusion that, upon i.v, administration in dogs,DEyE is rapidly distributed and eliminated bymetabolic degradation both in plasma and in tissues.Most likely the peptide and its degradation products(smaller peptide fragments) are taken up by a varietyof well-vascularized organs and tissues, in which theyare further converted enzymatically into theirconstituent amino acid residues. Subsequently theamino acids are released into the blood circulationfrom which they are excreted very slowly (6).

Since CNS effects of DEyE have been reportedfollowing the s.c. route of administration (1, 2), theblood concentrations of eH]-DEyE were alsomeasured after s.c. dosing in two dogs. In contrast tothe i.v. route of administration, s.c. injection results inlow but longer-lasting DEyE levels in blood. Theselevels appear to persist for a period of 1.5-2 h,probably due to sustained release from a depot at thes.c. injection site. Pharmacokinetically, the blood

concentration-time curves after s.c. administrattoncould not be described accurately in terms of'absorption phase' and 'elimination phase'. On thecontrary, the absolute bioavailability of DEyE for thes.c. route of administration to dogs could be reliablyestimated from the ratios AUCs.c}AUCiv., being20-23%. These values are of the same older ofmagnitude as established for DEyE in rats (30.9 ±16.3%; ref. 7). Bioavailability data for otheroligopeptides in dogs following s.c, injection are notyet available. In rats, however, they appear to varyexcessively, ranging from only 2-3% for a.-MSH (16)to almost 100% for DGAVP (15).

In conclusion, upon i,v. administration to dogsDEyE disappears very rapidly from the bloodcirculation. The elimination half-life of the peptide inthis species amounts to 2.4 ± 1.0 min, which is about2.5 times lower than previously observed in rats (7).Moreover, s,c. dosing of DEyE leads to remarkablylonger-lasting DEyE levels in dog blood than i.v,dosing, comparable to those obtained in rats (7).

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