EUROPEAN JOURNAL OF DRUG METABOLISM AND PHARMACOKINETICS, 1986, Vol. II, No 4, pp. 291-302

Des -enkephalin -y- endorphinrat, dog and human plasma

(DEyE): Biotransformation .In

J. COOS VERHOEF*, HENK M. VAN DEN WILDENBERG and JAN W. VAN NISPENScientific Development Group. Organon International B. v.. The Netherlands

Received for publication: May 27, 1986

Key words: y- Type-endorphins, DEyE, in vitro metabolism, in vivo metabolism, plasma peptidases.

SUMMARY

Biotransformation of eH- Lys"] DEyE was investigated after in vitro incubation of the tritiated peptide with rat, dog and humanplasma. In addition, its metabolite profile in blood was studied following intravenous administration to rats and dogs. Half-lives forthe in vitro disappearance of DEyE in plasma were 13.0± 0.8 min (dog), 15.7± 1.2 min (rat) and 19.2± 0.9 min (human), indicatingvery rapid degradation of the peptide by proteolytic enzymes. Biotransformation products were identified on the basis of co-chromato­graphy on HPLC with synthetic reference peptides. The six principal fragments appeared to be p-endorphin (PE) sequences 7-17, 8- 17,9-17, 6 -15, 7-15 and 8-15. The abundance of PE6-15, PE7-15 and PE8 -15 in rat and human plasma suggests preferential, subsequentcarboxypeptidase and aminopeptidase mechanisms, whereas in dog plasma DEyE is predominantly degraded by aminopeptidaseactivities (major peptide metabolites: PE7 -17 and PE8-17). In the in vivo studies with rats and dogs the same radioactive peptidefragments were detected in blood as found in the in vitro experiments with plasma. In both species their blood levels were alreadymaximal within a minute after intravenous administration of the parent peptide, thereafter they declined ra pidly. 3H - Lysine was themain radioactive metabolite in vivo. exceeding 70% of total radioactivity in rat and dog blood 10 min after 3H-DEyE dosing.

INTRODUCTION

The non -opiate dodecapeptide des -enkephalin­y -endorphin (DEy E; Org 5878) is identical to ~­

endorphin -(6-17). In a variety of behavioural testsituations in rats it has been shown to possessneuroleptic-like properties with probably a morespecific profile than that of classical, currently usedneuroleptic drugs (1-3). In addition, this neuro­peptide has been reported to elicit beneficial effectsin patients suffering from schizophrenic psychoses(4,5). From these animal and clinical studies it is sug-

* Present address: Center for Bio - Pharmaceutical Sciences,Gorlaeus Laboratories, P.O. Box 9502, 2300 RA LEIDEN,The Netherlands

Send reprint requests to: Dr J. Coos Verhoef, GorlaeusLaboratories on the above address.

gested that DEyE is a potential therapeutic drug incertain areas of schizophrenia.

Recent pharmacokinetic data obtained with tri­tiated DEyE in rats (6,7) and dogs (8) have revealedextensive distribution and in vivo metabolic de­gradation, resulting in very short elimination half­lives in the order of a few minutes (6.3 min and 2.4min in rats and dogs, respectively). Thus these ob­servations indicate that one should be careful incorrelating blood concentrations of DEyE with itsbiological effectiveness. Factors like penetration intothe brain (the putative target tissue) and brainreceptor interactions might also be relevant for theultimate biological response of DEyE (6).

The present experiments were conducted to gainmore information on the stability and biotrans­formation pathway of DEyE in rat, dog and humanplasma, using the in vitro approach and tritiatedDEyE.

In addition, the radioactive metabolite profilewas investigated in rat and dog blood following asingle intravenous administration of 3 H -DEyE.

292 European Journal of Drug Metabolism and Pharmacokinetics. 1986. No 4

MATERIALS AND METHODS

Radioactive material

eH-Lys9] DEyE was prepared and purified asdescribed elsewhere (9). Two batches were used inthe present study. The final radiochemical purity ofthe tritiated peptides exceeded 85%. The specificactivity, as determined by FAB mass spectrometry(10), amounted to 31 Ci/mmol and 43 Ci/mmol,respectively.

Peptides and chemicals

The synthesis and properties of the following~-endorphin (~E) fragments have previously beenreported: ~E6-17 (DEyE), ~E7-17, ~E8-17 and~E9-17 (II); ~EIO-17 (12); ~E6-16 and ~E6-9 (13).The same approach of fragment condensations asapplied in the synthesis of those peptides has beenused to obtain ~E6-15, ~E7-15, ~E8-15 and ~E6-13.

Thus, Z-Ser-Gln-Thr-Pro-Leu- Val-OtBu (13) washydrogenated and acylated with Boc-Thr-Ser-Glu(OtBu)- Lys(Boc)-, Boc-Ser -Glu (OtBu)- Lys(Boc)­(both via an azide reaction) and Z-Glu(OtBu)­Lys(Boc)-OH using N, N' -dicyclohexylcarbodiimideand I-hydroxybenzotriazole, to give the protectedfragments ~E6-15, ~E7-15 and ~E8-15. Boc-Thr­Ser-GlutOtbuj-Lysflsocj-N, was also coupled inan azide reaction with H - Ser-Gin -Thr - Pro -OtBu(12) to give the protected peptide ~E6-13. Removal

of the protecting groups was performed at roomtemperature with 90% aqueous trifluoroacetic acid(methanesulfonic acid was added to protected ~E8-15

to remove simultaneously the Z-group), and thepeptides were isolated by precipitation with ether.After exchange for acetate ions and lyophilization,final purification was performed on a silica gelcolumn for ~E7 -17 (solvent system; I-bytanol:pyridine: actice acid: water = 7: 3: I :4, by volume)and by counter current distribution for ~E8-15,

while ~E6-13 and ~E6-15 were used without furtherpurification. The available partially protected ~­

endorphin fragments 10- 13 and 14-17 were treatedwith trifluoroacetic acid and isolated as describedabove. All fragments were analyzed by thin-layerchromatography (TLC), HPLC, amino acid analysis,isotachophoresis and optical rotation (12,13); thedata are presented in Table I.

Methanol (MeOH) was obtained from a localcommercial source and distilled in glass before use.Uvasol-quality acetonitrile (CH 3CN), ammoniumacetate (NH 40Ac), sodium dihydrogen phosphate(NaHzP04 ' Hz 0), sodium I-octanesulfonate, tri­chloroacetic acid (TCA) and trifluoroacetic acidwere purchased from Merck (Darmstadt, G.F.R.).

Animals

Male Wistar rats (Cpb: WU, TNO, Zeist, TheNetherlands), weighing 230-250 g, were used. Theywere housed in plastic cages and received standard

Table I: Analysis data on several ~ -endorphin -(6-17) fragments.

Fragment Amino acid analysis Peptide HPLC (main Isotacho- TLC [a]~1content component; phoresis Rj-value (c= I,

Thr Ser Glu Lys Pro Leu Val (%) (%) (% HOAc) (system) 10% HOAc)

6-15 1.90 1.85 1.98 1.05 0.96 0.99 1.03 86.0 90.7* 4.1 0.10 (a) - 97.7°7-15 0.96 1.93 2.06 0.98 1.01 0.95 1.02 86.4 97.1* 1.9 0.10 (a) -10 1.9°8-15 0.95 0.92 2.02 1.06 0.98 0.95 1.00 85.8 92.4* 2.3 0.08 (a) - 98.5°6-13 1.85 1.80 1.99 1.05 0.96 91.7 94.8* 4.4 0.11 (b) 86.4°

10-13 1.01 0.93 1.06 0.95 83.2 97.4** 11.3 0.28 (c) 92.7°14-17 1.03 1.96 1.05 80.1 95.2** 3.1 0.67 (a) 54.2°

For HPLC analysis the mobile phase and chromatographic conditions used were adapted to those described by Bijl et al. (2).* Stationary phase: Supelcosil LC-18- DB (Supelchem, Hilversum, The Netherlands).** Stationary phase: Nucleosil-IOC-18 (Chrompack, Middelburg, The Netherlands).

TLC on Merck silicagel plates (F.254, 0.25 mm) using the following solvents (ratios are by volume): (a) I-butanol: pyridine:acetic acid: water = 8: 3: I :4; (b) l-butanol :acetic acid: water = 2: I: I; (c) I-butanol: pyridine :acetic acid: water = 4: I: I: 2.Optical rotation as measured with a Perkin - Elmer 241 polarimeter (I).

H. Coos Verhoef et al.. Biotransformation of DEyE 293

laboratory rat food and water ad libitum. In addition,male Beagle dogs (TNO; body weight approx. 10 kg)were used; they were housed in steel metabolismcages, received standard laboratory food (300g/day)and had free access to tap water.

In vitro incubation

Freshly obtained rat, dog and human plasma wasused. Amounts of 25 IlCi (1.0 ug) 3H-DEyE,previously dissolved in 0.1 ml water, were added to 2ml of plasma (final peptide concentration in theincubation mixture, 3.8 x 10-7 M). After vigorousshaking, the mixtures were incubated under at­mospheric conditions in a constantly shaking waterbath at 37°C. At pre-set time periods 0.1 mlsamples were taken from the incubates and immediatelyprecipitated with 2 ml of an ice-cold 4% (w/v)aqueous TCA solution. For each species duplicateexperiments were carried out with plasma fromdifferent males. Incubations of 25 IlCi 3H-DEyEwith rat plasma containing a thousand-fold excessof non -Iabelled DEyE (final peptide concentration,3.8 x 1O-4M) were carried out in the same way asdescribed above.

Intravenous administration and bloodsampling

Conscious rats received a single intravenous (i.v.)dose of 0.47 mCi (19 ug) 3H-DEyE, dissolved in0.15 ml 0.9% NaC!. Administration was performedvia the right cannulated jugular vein, immediatelyfollowed by 0.15 ml 0.9% NaC! to wash out thecannula. After various time intervals (0.6-15 min)blood samples of I ml were withdrawn from the leftcannulated carotid artery and mixed with 3 ml of a6% (w/v) TCA solution. Cannulation procedureshave been described elsewhere (7).

One male dog was injected i.v. into the rightforepaw vein with a solution of 2.5 mCi (76 ug)3H-DEyE in 1 ml 0.9% NaCI. Blood samples of2 ml were taken from the jugular vein with Venojectevacuated blood collecting tubes (8) and extractedwith 6% TCA.

Extraction of 3 H-DEyE and metabolites

TCA extraction as described above results ininstantaneous denaturation and precipitation of pro­teins (e.g. peptidases), preventing possible metabolic

breakdown of DEyE during sample preparation (6).The TCA mixtures were centrifuged for 10 min at5000 gav' From the supernatants 0.1 ml aliquotswere added to 4.5 ml Picofluor-30 (Packard In­struments, Brussels, Belgium) to determine totalradioactivity. The remaining supernatants were pre­purified on Seppak-Cjg cartridges (Waters, Milford,USA), which were previously washed with 5 mlmethanol and 5 ml water. To remove TCA andinterfering blood constituents, the columns wereeluted with 4 ml 0.01 M NH40Ac (adjusted topH = 4.2 with glacial acetic acid). These fractionswere combined with their column effluents (totalvolume approx. 6 ml) and 0.5-1 ml aliquots werecounted for radioactivity. Finally, 3H-DEyEand itspeptide metabolites were washed from the cartridgeswith 3 ml of 60% methanol in 0.0 I M NH40Ac

buffer (pH = 4.2). The eluates were evaporated todryness, either at 60°C under a stream of nitrogenor at 40°C in a Speed Vac Concentrator (Dumee,Soest, The Netherlands), and stored at -20°C untilHPLC analysis.

Combined Seppak -Cjg effluents and 0.01 MNH40Ac eluates were also analyzed by HPLC.Therefore, 1.5-2 ml of these fractions were ex­tracted four times with equal volumes of diethyletherto remove most of the TCA (14). The remainingaqueous solutions were dried and stored as de­scribed for the 60% methanol eluates.

HPLC analysis and radioactiVity monitoring

All experiments were done with a Hewlett Packardliquid chromatograph (Model 1090), equipped witha ternary solvent delivery system, variable volumeinjector (1-25 Ill) and diode-array detector operatingat 210 nm. Two different reversed-phase Ultraspherecolumns (5 11m-ODS and 311m-ODS; BeckmanInstruments, Berkeley, USA) were used. The elutionpattern was recorded on a Hewlett Packard 5880Aseries printer/plotter. Chromatographic conditionswere adapted to those described by Janssen et al.(15). The mobile phase consisted of the followingsolvents: 0.25 M NaH zP04 with 0.025 M sodiumI-octanesulfonate in water, adjusted to pH 2.1 withconcentrated H3P04 (A), water (B) and acetonitrile(C). The column temperature was 50°C. Using theUltrasphere-5 ODS column (0.46 x 25 em), lineargradient elution was conducted for I hour goingfrom A:B:C=79:7:14 to A:B:C=55:15:30(numbers in percentages) with a flow rate of Iml/min (System I). Using the 3 ODS column(0.46 x 7.5 em), linear gradient elution was performed

294 European Journal of Drug Metabolism and Pharmacokinetics. 1986. No 4

7 -15

over 15 minutes from A:B:C=69: 10:21 toA:B:C = 65: 10:25 with a flow rate of 0.5 mllmin(System II).

The dried 60% MeOH fractions, obtained afterprepurification of the extracts on Seppak-Cj , car­tridges, were dissolved in 50 III distilled water. Thesolutions were cleared by centrifugation and 25 IIIaliquots were fractionated by HPLC (System II).For the samples obtained from in vitro incubationsof 3H-DEyE in rat, dog and human plasma as wellas from in vivo experiments in rats, radioactivity wasmeasured by direct interfacing the HPLC effluentthrough a flow-through radioactivity detector(Ramona; Isomess, G.F.R.). For the blood extractsobtained after i.v. injection of 3H-DEyE in dogs,the HPLC effluent was colIected as fractions of 0.25min in counting vials, mixed with 4.5 ml Picofluor­30 and measured for radioactivity by liquid scin­tillation counting.

The combined effluents and 0.0 I M NH40Ac

eluates from the Seppak-C 18cartridges were fractionatedby HPLC using the Ultrasphere-30DS column, butstarting at a lower acetonitrile concentration (SystemIII). The dried extracts were dissolved in 50 III waterand 10- 25 III aliquots were loaded on the column.Linear gradient elution was performed over 15minutes from A:B:C =80: 10: 10 to A:B:C == 65: 10: 25. Radioactivity was measured with theon -line method as described above.

RESULTS

HPLC separation of CErE and metabolites

absorbance, co-eluting on~E6-15, ~E7-15, ~E8-15,

respectively.

10-13-6-13 8-15

6-9

6.16 6-15

*

HPLC with reference~E7-17 and ~E8-17,

A

7-17 /-17

6-17 9-1714-17

10-17

B

Fig. 1: HPLC chromatograms of a mixture of DEyE(p-endorphin-(6-17» and a variety of its syntheticpeptide fragments (A) and a digest of DEyE withrat plasma (B). The synthetic peptides used areindicated by numbers corresponding to their re­spective P-endorphin sequence and the arrowsshow their elution position (5 -10 ug of eachpeptide; A). DEyE was incubated for I hour withrat plasma (final peptide concentration 3.8 l< 10-4 M;B). UV absorption was monitored at 210 nm; theasterisks indicate endogenous constituents of ratplasma. Chromatographic conditions and experi­mental details a re described in the text.

Figure IA gives the elution profile of a mixtureof DEyE WE6-17) and 12 of its peptide fragmentsusing an Ultrasphere-50DS column and octane­sulfonate as an ion-pairing reagent in the mobilephase (System I). Most of the peptide fragmentsinvestigated (the ~-endorphin sequences 6-9,6-13,6-16, 6-15, 7-15, 8-15, 7-17, 8-17 and 9-17)represent peptides that might arise as radioactivelylabelIed metabolites upon enzymatic digestion ofeH-Lys9] DEyE. Baseline resolution betweenDEyE (retention time 37.8 min) and all thesefragments was achieved within 45 minutes. ThusHPLC system I appears to be highly selective formeasurement of DEyE and its peptide metabolites.This is also illustrated in Fig. IB, showing theDEyE metabolite profile after I hour incubation ofthe peptide with rat plasma (final DEyE con­centration, 3.8 x 10-4 M). In this incubation mixture5 peptide fragments could be detected by UV

*

r~.J·v j.r

*J.. _

H. Coos Verhoef et aJ.. Biotransformation of DEyE 295

With HPLC System II (3 ODS-column) com­parable peptide separations were observed, althoughthe selectivity was less than that with System I (8).System II, however, has the advantage of shortanalysis times (total separation within 13 minutes)and has been established to be suitable enough forstudying the metabolite profile of 3H-DEyE inbiological samples. Therefore, the second system(3 ODS) was chosen for routine assays and the firstone (5 ODS) for isolation and identification pur­poses.

In vitro biotransformation of 3 H -DEyE inrat, dog and human plasma

Extraction with 4% TCA resulted in almostcomplete recovery of radioactivity from the plasmaincubation mixtures, viz. 96 ± 3% (± S.D., n =68).The final recovery of total radioactivity after pre­purification on Seppak C-18 cartridges was 80± 6%.For all three species HPLC analysis of the 60%MeOH fractions showed 5 major radioactive de­gradation products of 3H-DEyE. These radioactivepeaks appeared to co-migrate with the synthetic~-endorphin fragments 6-15, 7-15, 8-15, 7-17 and8-17. In the incubates with dog plasma low amountsof radioactivity were found at the position of re­ference ~E9-17, whereas in rat and human plasmathis peptide fragment was not detected.

HPLC fractionation of the combined Seppakeffluents and 0.0 I M NH40Ac eluates (System III)revealed that the major part of radioactivity ('> 90%)co-eluted with reference 3H-Iysine. Control in­cubations of 3H-DEyE with heat-denaturated rat,dog and human plasma did not show any de­gradation of the peptide for up to 3 hours.

Typical examples of the time course profiles of3H-DEyE and its radioactively labeIled metabolitesfoIlowing in vitro incubation of [3H_Lys9] DEyEwith rat, dog and human plasma are presented inFigures 2, 3 and 4, respectively. It is evident thatDEyE is rapidly metabolized in plasma of all threespecies. Curve -fitting of the disappearance of DEy E(2-60 min) was performed by linear regressionanalysis, using a monoexponential function andusing the least squares method. The in vitro half-lifevalues were found to be 15.7 ± 1.2 min, 13.0± 0.8min and 19.2± 0.9 min in rat, dog and humanplasma respectively (± S.D., n = 2 for each species).Species-dependent differences were observed withrespect to the quantity of generated radioactivemetabolites. On the one hand, accumulation of

~E7- 17 and ~E8- 17 was more pronounced in dogplasma than in rat or human plasma. For all speciesfragment ~E7-17 reached peak values 15-20 min,and fragment ~E8-17 30-45 min after in vitroincubation. Then they declined rapidly in rat andhuman plasma, but very slowly in dog plasma. Onthe other hand, formation of the ~ -endorphinsequences 6-15, 7-15 and 8-15 occurred to a largerextent in rat and human plasma than in dog plasma.In fact, the ~E6-15 levels in dog plasma were belowthe limit of detection. For all species 3H -lysine wasshown to be the main radioactive metabolite, in­creasing from 3% of total radioactivity 5 minutesafter incubation to 43% after 3 hours.

Incubations with high concentrations of DEyE inrat plasma (3.8 x 1O-4M) revealed the generation ofthe same 5 peptide fragments as observed afterincubation with low 3H -peptide concentrations(3.8 x 1O-7M). In addition, the in vitro half-lifevalues did not differ essentially whether high or lowDEyE concentrations were used (13.7 min versus15.7 min).

In vivo biotransformation of 3 H- DEyE in ratand dog blood

FoIlowing i.v. administration of 0.47 mCi (19 Ilg)3H-DEyE to rats (Fig. 5) the same peptide meta­bolites were detected in blood as shown in the invitro studies with rat plasma (~-endorphin sequences6-15, 7-15, 8-15, 7-17 and 8-17). Peak levels for allthe fragments were already observed within a minuteafter i.v, dosing of the parent compound; thereafterthey decreased rapidly. Tritiated lysine was themajor radioactive metabolite, reaching maximallevels after 2.3 min and increasing from 21% to 83%of total radioactivity during the time period in­vestigated.

The blood concentration - time curves of 3H­DEyE and metabolites upon i.v. dosing of 2.5 mCi(76 ug) to the dog are given in Fig. 6. Similar to theresults obtained after in vitro incubation with dogplasma, three peptide metabolites were detectedwhich co-eluted on HPLC with ~E7-17, ~E8-17

and ~E9-17. These C-terminal fragments of DEyEwere eliminated very rapidly from the blood cir­culation. No radioactivity could be detected co­migrating on HPLC with references ~E6-15, ~E7-15

and ~E8- 15. Also in the dog 3H -lysine was themain labelled metabolite, being 72% of total radio­activity in dog blood IO min after peptide dosing.

296 European Journal of Drug Metabolism and Pharmacokinetics. 1986. No 4

% of initialradioactivity

102 Rat

Lys

8·17

100eo60

minutes4020o

10-1-t--.,---.--,-,....-.,-,-..,.--,---.---.--,-.....-.,-..,......,.--,---.---.--,---,r--r--..,....-r-,

120

(a)

% of initialradioactivity

102 Rat

8-15

6-15

Org 5878

o(b)

20 40 110

minutes

I80 100

I120

Fig. 2: Time course profiles of 3 H - DEyE (3 H - Org 5878; a, b) and its radioactive metabolites PE7-17, PE8-17, 3H -lysine (a),PE6-15, PE7-15 and PE8-15 (b) following in vitro incubation of [3 H _Lys9] DEyE with rat plasma. Incubationconditions are described in the text. Data were obtained from a typical experiment.

% of initialradioactivity

102

H. Coos Verhoef et al.. Biotransformation of DEyE

Org 5878

Dog

297

o(a)

% of initialradioactivity

102

30 60 90 120 150

minutes

Lys

8·15

7.15

Org 5878

180 210 240

Dog

o(b)

30 1IO 90 120

minutesISO 180 210 240

Fig. 3: Time course profiles of 3H- D EyE eH-Org 5878; a.b) and its radioactive metabolites /3E7-17, /3E8-17, /3E9-17 (a),/3E7-15, /3E8-15 and 3H-lysine (b) following in vitro incubation of [3H_ Lys9] DEyE with dog plasma. Incubationconditions are described in the text. Data were obtained from a typical experiment.

298 European Journal of Drug Metabolism and Pharmacokinetics, 1986, No 4

% 01 initialradioactivity

102

lys

Human

10- 1-)

7-17

I I I I I I I0 30 60 90 120 150 180 210 240

(a) minutes

% of initialradioactivity

Ilf Human

o(b)

30 go 120 150

minutes180 210

Fig. 4: Time course profiles of 3H-DEyE (3 H_ Org 5878; a, b) and its radioactive metabolites ~E7-17, ~E8-l7, 3H-lysine (a),~E6-15, ~E7-l5 and ~E8-15 (b) following in vitro incubation of [3 H_ Lys9] DEyE with human plasma. Incubationconditions are described in the text. Data were obtained from a typical experiment.

H Coos Verhoef et 01.. Biotransformation of DEyE 299

Lys

Rat

% Do s e y l O ml blood

102

10- 1

7.17

10-28.17

10- 3

0 5 10 IS

(a) minutes

% DoseflO ml blood

102

Rat

101

100

Org 5878-I

10

-210

10- 3

0 5 10 IS

(b) minutes

Fig. 5: Blood concentration-time course profile of 3H-DEyE eH-Org 5878: a,b) and its radioactive metabolites ~E7-17,pE8-17, 3H-Iysine (a), PE6-15 PE7-15 and pE8-15 (b) after i.v. administration of 0.47 mCi (19Ilg) [3 H_ Lys9] DEyE inthe rat.

300 European Journal of Drug Metabolism and Pharmacokinetics. 1986. No 4

10BII2

10- 34-...#-- - - --.-- - - - -,-- - - - -,- - - - -,--- - - - -,o

% Dose/I blood

102

minutes

Fig. 6: Blood concentration-time course profiles of 3 H -DyE eH - Org 5878) and its radioactive metabolites ~E7 - 17,~E8-17, ~E9-l7 and 3H -lysine after i.v. administration of 2.5 mCi (76 ug) [3 H _Lys9] DEyE in the dog.

DISCUSSION

From the present in vitro experiments it isevident that DEyE is rapidly degraded by peptidaseactivities in plasma. Half -lives, based on the di­sappearance of 3 H-DEyE, were measured to be 13.0min (dog), 15.7 min (rat) and 19.2 min (human).These results indicate that DEyE is slightly morestable in human plasma than in rat or dog plasma,and that the highest rate of metabolic breakdownoccurs in dog plasma. This is in agreement withrecent in vivo pharmacokinetic studies of DEyE,showing elimination half-Iives of 6.3 min and 2.4min in rats and dogs, respectively (7,8). Species­dependent differences in the degradation rates inplasma (more pronounced than observed for DEyE)have been reported for the neuropeptides prolyl­leucyl- glycinamide (16,17) and 9-desglycinamidearginine vasopressin (18).

The products of biotransformation of DEyE inplasma were identified on the basis of co-chromato­graphy on HPLC with well-defined synthetic re­ference peptides. The profile of products is suggestiveof aminopeptidase and dipeptidyl carboxypeptidase­like actions on DEyE. Firstly, the occurrence oftritiated ~E7 -17, ~E8-17 and ~E9-17 points to

sequential removal of the NH2-terminal amino acidresidues from DEyE by the sole action of plasmaaminopeptidases. Further processing by these en­zymes results in the formation of non-labelled~EIO-17 and concomitant release of 3 H-Iysine,which is in agreement with the observed highaccumulation of 3 H -lysine in the plasma incubates.Secondly, the presence of radioactively labelled ~E6­

15 but not of ~E6-16 strongly suggests dipeptidylcarboxypeptidase action on DEyE. Further de­grada tion by plasma aminopeptidases then leads tosubsequent generation of the ~ -endorphin fragments7- 15 and 8-15. The route of biotransformation ofDEyE in plasma, as outlined above, is mainlysimilar to that reported for the proteolytic con­version of y -endorphin and des-Tyr I -y -endorphin(DTyE) by peptidases associated with rat brainmembranes (19,20).

It is noteworthy that remarkable species-de­pendent differences have been found concerning thepredominant pathway of DEyE biotransformation.This is schematically presented in Fig. 7. In rat andparticularly in human plasma the abundance of~E6-15, ~E7-15 and ~E8-15 is indicative for di­peptidyl carboxypeptidase and subsequent amino­peptidase mechanisms in these two species, whereas

H. Coos Verhoef et al.. Biotransformation of DEyE

6 17H-THR.SER-GlU- [3H] l YS-SER-GlN-THR.PRO-lEU- VAl.THR-lEU-OH

LLL-l_~L____ Lj'

!AP1d 0

9 1!cp (rat/humanl

(6 16)

301

7----------178 179---------17

[3H] lysine

6---------15!API,at/human!

7-------158------15

(9 15)

[3H]lysine

Fig. 7: Predominant pathway of DEyE biotransformation in rat, dog and human plasma, as derived from the present in vitroand in vivo studies with r)H- Lys9]-DEyE. The marked peptide fragments were identified on basis of co-migration onHPLC with synthetic ~-endorphin sequences, whereas the fragments shown in parentheses were below the limit ofdetection. Vertical arrows indicate cleavage sites in DEyE. Involved types of enzymes are aminopeptidases (AP) andcarboxypeptidases (CP).

in dog plasma DEyE is preferentially degraded byaminopeptidase activities (major peptide metabolitesin dog plasma: PE7-17 and PE8- 17).

The results obtained from the in vivo studies of3H-DEyE in rats and dogs are very similar to thoseobtained from the in vitro incubations with rat anddog plasma, including the identity of degradationproducts and proposed pathway of biotransformation.Radioactive PE6-IS, PE7-IS and PE8-IS were belowthe limit of detection in dog blood after i.v. dosingof 3H-DEyE. The fragment of PE9-17 was found inlow amounts in dog blood, but not in rat blood. Incontrast to the in vitro data, none of the peptidefragments accumulated in vivo. Both in rats and dogsblood levels were already maximal within a minuteafter i.v. administration of the parent peptide, the­reafter decreasing very rapidly. Moreover, 3H-Iysinewas shown to be the main metabolite in vivo,exceeding 70% of total radioactivity in rat and dogblood 10 min after peptide dosing. As has beenpreviously suggested by pharmacokinetic studies inrats and dogs (6-8), the present data unequivocallyconfirm that DEyE is extensively metabolized invivo. Most likely, besides biotransformation in theblood circulation, DEyE and its peptide fragmentsare rapidly taken up in well-vascularized organs andtissues in which they are further converted en­zymatically into their constituent amino acid re-

sidues. Subsequently, the amino acids are releasedinto the circulation and excreted by the kidneys veryslowly. This is confirmed by the observed rapid andhigh accumulation of radioactivity in kidneys, liver,pancreas and salivary gland after i.v. injection of3H-DEyE in rats, and by the observed slowness ofurinary excretion of radioactivity following i.v. ad­ministration of the labelled peptide in rats and dogs(21).

ACKNOWLEDGEMENTS

The authors wish to express their gratitude to W.A.A.J.Bijl, F.W.M. Bogaers, A.M.M. Hendrix, W.P.A. Janssen andA.H.P.M. Tiemissen for expert technical assistance.

REFERENCES

I. De Wied, D., Van Ree, J.M. and Greven, H.M. (1980~:

Neuroleptic -Iike activity of peptides related to des -Tyr ­y-endorphin: structure activity studies. Life Sci., 26.1575-1579.

2. Gaffori, O. and De Wied, D. (1982): Effects of des-Tyr'­y-endorphin and des-enkephalin-v-endorphin on activeand passive avoidance behavior in rats: a dose-responserelationship study. Eur, J. Pharrnacol., 85. 115-119.

302 European Journal of Drug Metabolism and Pharmacokinetics, 1986, No 4

3. Kovacs, G.L., Telegdy, G. and De Wied, D. (1982):Selective attenuation of passive avoidance behaviour bymicro-injection of P-LPH 62-77 and P-LPH 66-77 intothe nucleus accumbens in rats. NeuropharmacoI., 21,451-454.

4. Van Ree, J.M., Verhoeven, W.M.A., Van Praag, H.M.and De Wied, D. (1981): Neuroleptic-like and anti­psychotic effects of y-type endorphins. In: ModernProblems of Pharmacopsychiatry, Vol. 17, The Role ofEndorphins in Neuropsychiatry, edited by H.M. Emrich.Karger, Basel, pp. 226-278.

5. Verhoeven, W.M.A., Van Ree, J.M., Heezius-van Bentum,A., De Wied, D. and Van Praag, H.M. (1982): Anti­psychotic properties of des-enkephalin -y-endorphin intreatment of schizophrenic patients. Arch. Gen. Psychiat.,39, 648-654.

6. Verhoef, J., Scholtens, H., Vergeer, E.G. and Witter, A.(1985): Des -Tyr l -y -endorphin (DTyE) and des-enke­phalin-j -endorphin (DEyE): plasma profile and brainuptake after systemic administration in the rat. Peptides,6, 467-474.

7. Verhoef, J. and Van den Wildenberg, H.M. (1986):Des-enkephalin-y-endorphin (DEyE): bioavailability inrats following the subcutaneous and intramuscular routeof administration. Regul. Peptides, 14, 113-124.

8. Verhoef, J. and Van den Wildenberg, H.M. (1987):Des-enkephalin -y-endorphin (DEy E): pharmacokineticsin dogs after intravenous and subcutaneous admini­stration. Pharmacology, in press.

9. Kaspersen, F.M., Van Rooy, A.A.M., Wallaart, J. andFunke, C.W. (1983): Synthesis and analysis of tritiatedneuropeptides. Reel. Trav. Chim. Pay-Bas, 102,450-453.

10. Lehmann, W.D. and Kaspersen, F.M. (1984): Specificradioactivity determinations of ionic organic compoundsof high specific activity by fast atom bombardment andfield desorption mass spectrometry. J. Label. CompoundsRadiopharm., 2/. 455-469.

I I. Greven, H.M., Van Nispen, J.W. and Bijl, W.A.A.J.(1980): Synthesis of fragments of human p-lipotropin,Ph- LPH. Part IV. The synthesis of shortened peptidesrelated to des-I-tyrosine-y-endorphin [P-LPH-(62-77)J.Reel. Trav. Chim. Pays-Bas, 99, 284-286.

12. Bijl, W.A.A.J., Van Nispen, J.W. and Greven, H.M.(1979): Synthesis of fragments of human P-lipotropin,Ph- LPH. Part I. The synthesis of P- LPH-(61 -76) andP- LPH -(61- 77), i.e. a- and y-endorphin, respectively.Reel. Trav. Chim. Pays-Bas, 98, 571-576.

13. Bijl, W.A.A.J., Van Nispen, J.W. and Greven, H.M.(1983): Synthesis of fragments of human P-lipotropin,Ph-LPH. Part VI. The synthesis of des-J-tyrosine-u­endorphin and shortened peptides. Reel. Trav. Chim.Pays-Bas, /02, 469-474.

14. Verhoef, J. and Witter, A. (1976): In vivo fate of abehaviorally active ACTH 4-9 analog in rats aftersystemic administration. Pharmac. Biochem. Behav., 4,583-590.

15. Janssen, P.S.L., Van Nispen, J.W., Melgers, P.A.T.A.and Hamelinck, R.L.A.E. (1986): Ion-pair HPLC se­paration of P-endorphin-(6-17) from fourteen fragments.Chromatographia, 2/,461-466.

16. Walter, R., Neidle, A. and Marks, N. (1975): Significantdifferences in the degradation of Pro-Leu-Gly-Nlf, byhuman serum and that of other species. Proc. Soc. Exp.BioI. Med., /48, 98-103.

17. Witter, A., Scholtens, H. and Verhoef, J. (1980): H-Pro­[3 H] Leu - Gly -NH 2: Metabolism in human and rat plasmainvestigated by high-pressure liquid chromatography.Neuroendocrinology, 30, 377- 381.

18. Verhoef, J., Van den Wildenberg, HM. and Van Nispen,J.W. (1986): eH]9-Desglycinamide, 8-arginine vaso­pressin: metabolism and in vivo fate. J. Endocrinol., //0.557-562.

19. Burbach, J.P.H., De Kloet, E.R., Schotman, P. and DeWied, D. (1981): Proteolytic conversion of p-endorphinby brain synaptic membranes: characterization of ge­nerated p-endorphin fragments and proposed metabolicpathway. J. BioI. Chern., 256, 12463-12469.

20. Burbach, J.P.H., Schotman , P., Verhoef, J., De Kloet,E.R. and De Wied, D. (1980): Conversion of des­tyrosine-j -endorphin by brain synaptic membraneassociated peptidases: identification of generated peptidefragments. Biochem. Biophys. Res. Cornmun., 97, 995­1004.

21. Paanakker J.E. and Peters H.A.M., Organon Inter­national B.V., unpublished observations.