Antidiuretic agonism and antagonism of some O-alkylated analogues of vasopressin containing 2-O-alkylated tyrosine

European Journal of Pharmacology, 67 (1980) 173--177 © Elsevier/North-Holland Biomedical Press

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A N T I D I U R E T I C A G O N I S M A N D A N T A G O N I S M O F SOME O - A L K Y L A T E D A N A L O G U E S OF VASOPRESSIN CONTAINING 2-O-ALKYLATED TYROSINE PER MELIN, STEFAN LUNDIN, HANS VILHARDT*, GUNNAR LINDEBERG, LARS-ERIK LARSSON and VLADIMIR PLI~KA Ferring Pharmaceuticals, 200 62 MalmiS, Sweden, Institute of Biochemistry, Biomedical Center, Uppsala University, 751 23 Uppsala, Sweden and Institute of Molecular Biology and Biophysics, ETH-HSnggerberg, 8093 Z~rich, Switzerland Received 9 January 1980, revised MS received 11 March 1980, accepted 30 June 1980

P. MELIN, S. LUNDIN, H. VILHARDT, G. LINDEBEI~G, L.E. LARSSON and V. PLI~KA, Antidiuretic agonism and antagonism of some O-alkylated analogues of vasopressin containing 2-O-alkylated tyrosine, European J. Pharmacol. 67 (1980) 173-177. A series of 2-O-alkylated tyrosine analogues of lysine-vasopressin and desamino-lysine and arginine-vasopressin were synthesized and tested for antidiuretic activity in the water-loaded anaesthetized rat. The analogues displayed only weak antidiuretic activities. When they were infused in the rats together with lysine vasopressin it was found that 1-deamino-[2-O-ethyltyrosine]-lysine-vasopressin inhibited the vasopressin-induced antidiuretic response. The antagonistic properties were further evaluated in long-term experiments on conscious non-hydrated rats with implanted minipumps. The analogues inhibited the vasopressin-induced antidiuresis at antagonist:agonist ratios of 0.5 and 1.0. Antidiuresis

O-Alkylated vasopressins

Agonism

1. I n t r o d u c t i o n In a previous s t u d y (Larsson et al., 1 9 7 8 ) a series o f lysine-vasopressin analogues (alkyla t e d at t h e t y r o s i n e residue) were s y n t h e s i z e d and t e s t e d f o r a n t i d i u r e t i c activity in t h e w a t e r - l o a d e d a n a e s t h e t i z e d rat. T h e analogues all possessed w e a k a n t i d i u r e t i c activity. U n d e r certain c o n d i t i o n s it was f o u n d t h a t s o m e o f the analogues d i s p l a y e d antagonistic e f f e c t s against vasopressin-induced antidiuresis. In the p r e s e n t s t u d y t h e original m e t h o d was m o d i f i e d in o r d e r t o o b t a i n m o r e r e p r o d u c i b l e results. In a d d i t i o n the e f f e c t o f t h e m o s t p o t e n t a n t a g o n i s t was studied b y l o n g - t e r m a d m i n i s t r a t i o n in c o n s c i o u s rats k e p t in

* To whom correspondence should be addressed: Ferring Pharmaceuticals, Box 30561, S-200 62 MaimS, Sweden.

Antagonism

m e t a b o l i c cages w h e r e t h e urine o u t p u t c o u l d be m o n i t o r e d over a p e r i o d o f several days. M o r e o v e r , t h e series o f O - a l k y l a t e d vasopressins was e x t e n d e d b y synthesizing some n e w analogues. T h u s t h e steric e f f e c t s were further explored by means of a branched alkyl s u b s t i t u e n t and the i m p o r t a n c e o f the n a t u r e and c o n f i g u r a t i o n o f t h e basic a m i n o acid was investigated b y e x c h a n g i n g lysine with arginine a n d D-arginine.

2. Materials and m e t h o d s 2.1. P e p t i d e s y n t h e s i s

T h e synthesis o f t h e O - n - a l k y l a t e d lysinevasopressins, 1- d e a m i n o - [ 2-O- e t h y l t y r o s i n e ] lysine-vasopressin ( d - E t h y l - L V P ) , 1 - d e s a m i n o [2- O - p r o p y l t y r o s i n e ] - lysine -vasopressin {dP r o p y l - LVP), 1 -desamino - [ 2 - O - b u t y l t y r o -

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sine]-lysine-vasopressin (d-Butyl-LVP), 1-desamino - [2 - O-hexyltyrosine] -lysine-vasopressin (d- Hexylo LVP }, [ 2- O- ethyltyrosine ]- lysine-vasopressin (Ethyl-LVP) and [2-O-butyltyrosine]-lysine-vasopressin {Butyl-LVP) was recently described {Larsson et al., 1978). The new analogues, 1-desamino-[2-O-t-butyltyrosine] -lysine -vasopressin (d- t-Butyl- LVP), 1desamino- [ 2- O - butyltyrosine ] - arginine- vaso pressin (d-Butyl-AVP) and 1-desamino-[2-Obutyltyrosine] - D - arginine- vasopressin (d- ButyI-D-AVP) were prepared according to a previously outlined scheme for convenient synthesis of 4, 8-disubstituted vasopressins by fragment condensation on a solid support (Larsson et al., 1976) with some modifications. The removal, by transesterification and subsequent hydrolysis, of the benzyl group used for carboxyl protection of the N-terminal tripeptide fragment in the earlier work was not entirely free from racemization {3--6%). We therefore abandoned this procedure and chose to block the carboxyl group as its phenacyl ester (Taylor-Padamitriou et al., 1967). The phenacyl group can be selectively removed by sodium thiopenolate (Sheehan and Daves, 1964) but is not stable during catalytic hydrogenation. The phenacyl ester approach could therefore n o t be used in the synthesis of benzylmercapto-propionyl-O-tbutyltyrosylphenylalanine, since the wellknown acid lability of the t-butyl ether necessitated the hydrogenolytic removal of the carbobenzoxy-group in the protected dipeptide. In this case esterification with methanol afforded the desired stability. The xanthyl group used for amide protection in Asn and Gin (Dorman et al., 1972) was shown by amino acid analysis to be completely removed after a 10 min exposure to 33% (v/v) trifluoroacetic acid--CH2 C12. The protected nonapeptides were liberated from the resin by ammonolysis (Manning, 1968). Subsequent deblocking and purification methods were essentially the same as those described earlier (Larsson et al., 1978). Elemental analysis and amino acid analysis of the final products and the protected inter-

P. M E L I N E T AL.

mediates were in agreement with theoretical values.

2.2. Antidiuretic tests in hydrated anaesthetized rats Antidiuretic tests were carried o u t as previously described (Larsson et al., 1978). The antagonistic properties of the analogues were evaluated by means of two additional methods. (1) Dose-response curves for vasopressin were obtained by injecting LVP during and without simultaneous infusion of an analogue. (2) Urine flow was measured during periods of infusion of vasopressin alone or in combination with an analogue. Agonistic properties of the peptides were evaluated by means of the four-point dosage schedule. Synthetic lysine vasopressin (LVP) or arginine vasopressin {AVP, Ferring AB) were used as standards. In antagonistic studies AVP analogues and LVP analogues were tested on antidiuresis induced by their respective parent c o m p o u n d s (AVP and LVP).

2.3. Antidiuretic experiments in non-hydrated unanaesthetized rats using osmotic minipump infusion Sprague-Dawley male rats, 120 g b.w. were placed separately in metabolic cages and had free access to f o o d {rat pellets, R o s t o c k mixture, KSK /~rhus, Denmark) and water. The daffy urine o u t p u t was stabilised during a four day adaption period. The rats were grouped, then implanted subcutaneously under light Barbital ® anaesthesia with osmotic minipumps (Alza ® ), delivery rate 1 pl/h. The animals were infused with 10 pg/h per 100 g b.w. of LVP alone or the same concentration of LVP combined with different amounts of d-Ethyl-LVP in the dose range 1--100 pg/h per 100 g b.w. One group of animals served as control and received isotonic saline only. Daffy urine volumes were measured from each individual during four consecutive days starting from the implantation of the minipumps. The effects of the different analogue:

RENAL EFFECTS OF O-ALKYLATED VASOPRESSIONS

LVP ratios were related to the antidiuretic effect of LVP infused alone.

3. Results

175

abolished at these dose ratios (fig.3). On the other hand the effect of LVP was enhanced at the higher dose ratios of 2.0 and 10.0 reflecting the partial agonism of the vasopressin analogue.

3.1. Anaesthetized rats ].0

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All of the O-alkylated analogues showed only weak antidiuretic activities in the range 1.0--4.0 IU/mg (table 1). There was a tendency for the activity to decrease with increasing length of the alkyl chain. None of the new compounds showed any clear antagonistic properties in any of the infusion methods used. In addition the previously synthesized O-alkylated lysine vasopressins were investigated for antagonistic properties using the present animal test system. When LVP was injected during simultaneous infusion of d-Ethyl-LVP the antidiuretic response was slightly inhibited (fig.l). Infused d-ButylLVP displayed weak antagonistic action against the antidiuretic effect of infused LVP but failed to inhibit the effect of injected LVP. None of the other analogues showed inhibition of the response to LVP.

3.2. Minipump infusion As d-Ethyl-LVP turned out to be the most potent antagonist in anaesthetized rats this analogue was chosen for further testing in the minipump experiments. During the first 24 h period after minipump implantation the infused amount of LVP reduced urine flow by about 35% as compared to the controls (fig. 2). The antidiuretic effect decreased somewhat over the next days. It is not likely that the decrease with time of the antidiuretic effect of LVP as seen in the minipump experiments was due to the inactivation of the hormone in the minipump since the peptide is stable at 37°C for at least some months in the concentration used here (unpublished results). The inhibitory effect of the analogue was quite evident at an antagonist : angonist molar ratio of 0.5 and 1.0, the effect of LVP being almost

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Fig. 1. Anaesthetized water-loaded rats were infused with d-Ethyl-LVP (100 pg/min per 100 g) or isotonic saline (controls). W h e n urine flow (ml/min) and urine osmolality (mOsm/kg) had reached .steady state conditions (V0 and Osm0) different doses of L V P were injected and the effect on urine flow rate (Vmin) and osmolality (Osmmax) was measured. Pooled values from 4 experiments. Bars indicate S.E. The inhibition by the analogue of the effect on urine osmolality of 0.4 ng LVP/100 g b.w. was statisticallysignificant (P < 0.005). X Control; © d-Ethyl-LVP.

TABLE 1 Antidiuretic activity of O-alkylated vasopressins. Analogue

Antidiuretic activity IU/mg

Ethyl-LVP Butyl-LVP d-Ethyl-LVP d-Propyl-LVP d-Butyl -LVP d-Hexyl-LVP d-t-Butyl-LVP d-Butyl-AVP d-Butyl-D-AVP

2.5 1.8 4.1 3.6 3.8 2.1 1.6 1.8 1.0

176

P. MELIN ET AL. 4. Discussion

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Fig. 2. Effect of d-Ethyl-LVP on LVP-induced antidiuresis in conscious rats. The arrow indicates implantation of the minipumps containing A: isotonic saline, B: LVP (10 pg/h per 100 g b.w.) and C: LVP (10 pg/h per 100 g b.w.) and d-Ethyl-LVP (10/~g/h per 100 g b.w.). The columns represent means of 24 h urine volumes per rat, bars indicate S.E. The number of rats in each group is given in the columns. Ordinate : urine (ml/24 h). (7) (7)

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Fig. 3. Effects of different amounts of infused d-EthylLVP on the antidiuretic response to a fixed infusion rate of LVP in rats implanted with minipumps. The percentage reversal (hatched columns) or amplification (open columns) of the LVP effect (reduction in urine volume) was calculated on the basis of 24 h mean urine volumes collected over a 3 day period. Bars give S.E. and the figures in parentheses the number of experiments.

T he results of the present study permit certain tentative conclusions to be drawn a b o u t structure-activity relationships among O-alkylated vasopressin analogues. O t her reports have shown that m e t h y l a t i o n of the tyrosine residue in LVP leads to only m i nor reduct i on in antidiuretic activity (79 IU/mg, Zaoral et al., 1965). However, when the length of the alkyl substituent is increased the antidiuretic effect decreases dramatically. Thus Ethyl-LVP has an antidiuretic activity of only 2.5 IU/mg. F u r t h e r the ethyl, propyl, butyl and hexyl derivatives of desamino-LVP show low specific activities, 4.1, 3.6, 3.8 and 2.1 IU/mg respectively (table 1). It should be noted, however, that these figures are rather rough estimates of the antidiuretic activity since in some cases the dose-response curves for the standard and the test substance were n o t parallel. While alkylation of vasopressin at the t yrosine residue thus significantly impairs the biological activity, the o c c u p a n c y at the receptors in the kidney m ay under certain circumstances still be quite high as expressed by the p r o n o u n c e d antagonistic properties displayed by some of these analogues. Thus vasopressin-induced antidiuresis was inhibited at an antagonist : agonist molar ratio of 1 : 2 in the m i ni pum p experiments. This high r e c e p t o r o c c u p a n c y could be due to high affinity of the analogues to the receptors or to high c o n c e n t r a t i o n of the analogues at the r e c e p t o r c o m p a r t m e n t . It is possible that the increased h y d r o p h o b i c i t y introduced by alkylation of the molecule could facilitate the access of the analogue to the receptor c o m p a r t m e n t . The use of preparations of isolated kidney membranes to study the kinetics of the antagonism m a y clarify these points. It should be born in mind, however, t hat nothing is known a b o u t the metabolism of these analogues and the alkylated compounds might well be m ore resistant to enzymic breakdown than is LVP, in which case the analogue : LVP ratio in the circulation

RENAL EFFECTS OF O-ALKYLATED VASOPRESSINS

would be higher than the same ratio in the infusion solutions. When the molar concentration of infused dEthyl-LVP exceeded that of LVP no inhibition of the antidiuretic response could be demonstrated. This may indicate that mechanisms other than purely receptor-mediated reactions could be involved in the kidney response. It is thus possible that the analogues may cause changes in renal blood pressure and hemodynamics or in solute excretion which might influence the response to infused LVP. The choice of agonist was dictated by the desire to minimize the difference in chemical structure between the agonistic and antagonistic component, thus permitting the possible effects to be attributed to distinct structural features of the molecule. When testing c o m p o u n d s for possible antagonistic effects against the antidiuretic action of vasopressin the water-loaded, anaesthetized rat is n o t an ideal experimental model since hydration per se leads to decreased extracellular fluid osmolality. Furthermore, anaesthesia may change kidney responsiveness as well as endogenous vasopressin secretion. Infusion of low, physiological doses of vasopressin and of antagonists over a long period of time to conscious animals therefore seems advantageous. The use of minipumps constitutes a simple way of achieving a constant rate of release of the c o m p o u n d s over several days under conditions which are n o t stressing to the animals.

177

Acknowledgement Financial support was given by The Swedish National Board for Technical Development (77-6623), and by the Swiss National Science Foundation, grant No. 3.040-1.76.

References Dorman, L.C., D.A. Nelson and R.C.L. Chow, 1972, Solid phase synthesis of glutamine-containing peptides, in: Progress in Peptide Research, ed. S. Lande, Vol. II (Gordon and Breach Science Publishers, Inc., New York). Larsson, L.-E., P. Melin and U. Ragnarsson, 1976, Synthesis of peptides by fragment condensation on a solid support, Int. J. Peptide Res. 8, 39. Larsson, L.-E., G. Lindeberg, P. Melin and V. Pli~ka, 1978, Synthesis of O-alkylated lysine-vasopressins, inhibitors of the aatidiuretic response to lysinevasopressin, J. Med. Chem. 21,352. Manning, M., 1968, Synthesis by the Merrifield method of a protracted nonapeptide amide with the amino acid sequence of oxytocin, J. Am. Chem. Soc. 90, 1348.. Sh~ehan, J.C. and G.D. Davies, Jr., 1964, Facile alkyloxygen ester cleavage, J. Org. Chem. 29, 2006. Taylor-Padamitriou, J., C. Yovanidis, A. Paganou and L. Zervas, 1967, New methods in peptide synthesis, part V. On the 0~- and 7-diphenylmethyl and phenacyl esters of L-glutamic acid, J. Chem. Soc. C, 1830. Zaoral, M., E. Kasafirek, J. Rudinger and F. ~orm, 1965, Synthesis of 2-O-methyl-tyrosine and 2-0ethyl-tyrosine-lysine-vasopressin, Collect. Czech. Chem. Commun. 30, 1869.