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In-Vivo Studies on Kappa Opioid Receptors

In-Vivo Studies on Kappa Opioid Receptors

TIPS - February 1986 69 action is deemed to be spinal, In-vivo studies on kappa supraspinal, or in the periphery. A pharmaceutical company search- receptors ing for its own or U- 50488 might well choose mouse abdominal constriction and hot Alan Cowan and Debra E. Gmerek* plate tests to establish levels of antinociception and reversibility The actions of are mediated by multiple types of . As a by 3. Differences between result, 'obtaining the right balance' is the catchphrase most frequently heard test compounds may be empha- these days in research laboratories throughout the pharmaceutical sized by using supraspinal (i.c.v.) industry. Tomorrow's will feature a prominent K component, a touch and spinal (intrathecal) routes of of 6, a tickle of ~, but not even a wisp of o. New compounds are fashioned largely administration 4-6. For example, U- from structure-activity relationships involving bioassays and radioligand 50088, and receptor binding. These in-vitro approaches have become well established over are all active after intrathecal the past decade since they help to link receptor type to the analgesic under administration in the mouse (0.6% investigation. What about the complementary preclinical tests in-vivo? acetic acid) writhing test but only Specifically, how can the animal pharmacologist assist in characterizing K morphine is active by the i.c.v. opioid activity? In this article, Alan Cowan and Debra E. Gmerek present a route. It seems that K-receptors survey of tests that are being used to detect and define K activity in-vivo. modulate visceral pain at spinal, Special emphasis is placed on the rat bombesin-scratch test, a new procedure in but not at supraspinal, levels in which several K-preferring agents are selectively active. this procedure (Porreca, Mosberg, Burks and Cowan, unpublished Compounds that are classified as K- ant, given that this compound was data). Note, however, that the (e.g. tifluadom, U-50488; introduced as the prototype agon- choice of noxious stimulus is U-50088 is trans3,4-dichloro-N- ist at K receptors 1. Cross-reaction critical. Indeed, the quality of the methyl-N- [2-(1-pyrrolidinyl)-cyclo- between K and o opioid receptors stimulus used has long been hexyl 1]-benzeneacetamide); usually is clearly a major worry in the considered a key variable in the on the basis of bioassay and commercial development of K- evaluation of novel analgesics. binding activity profile, are gener- agonists as potential analgesics. This point is obvious when results ally perceived to be safer than the Another concern is the possibility from the mouse hot plate (55°C) traditional morphine-like or ~-agon- that i< receptors per se (also) test are compared. In this test, U- ists. This view derives from the mediate some of the disagreeable 50088 has unimpressive efficacy differentpharmacologies of ~- and K- subjective effects associated with and potency when given by i.c.v. agonists, a topic that has been acute administration of certain K- or intrathecal administration analysed at length 1. In short, K- agonists to humans. whereas morphine is still active by agonists are of interest because they A key objective of analgesic drug either route s . offer diversity in chemical structure, development throughout K- The next level of testing includes antinociception, a milder form of research has been to manoeuvre procedures that help to further physical dependence, and limited between and beyond the twin distinguish tifluadom and U-50488 actions on respiration and gastro- threats of physical dependence from ~-agonists such as morphine. intestinal transit. Unfortunately, and negative central effects. Many Several approaches are outlined in with many of the older K-agonists, groups accepted the challenge, Table I. There is no common the price paid for the pleasing believing that the ideal K analgesic, consensus on the best mix of profile was the emergence of un- although elusive, can ultimately be methods. The flurothyl test 7 con- wanted dysphoric and psychoto- designed. At the moment, a clini- trasts markedly with the bomb- mimetic (i.e. o) side-effects. Nalor- cal opinion on full, selective K- esin-scratch test in that ethyl- phine, the historic antag- agonists of the tifluadom and U- ketazocine, tifluadom and U-50088 onist analgesic, was clinically aban- 50488 type has still to be formed. are active in the latter and inactive doned for this reason. in the former. Morphine is not and altered perception have been Pharmacological profiles of active against bombesin but raises associated with some of the more K-agonists in rodents the seizure threshold (along with recently described I<-agonists (e.g. A screening cascade for K act- several other ~-agonists) in rats the , ketazocine ivity conventionally starts with exposed to flurothyl, a volatile and MR 2034, i.e. (--)-(1R,5R,9R,2"S), bioassays and radioligand recep- convulsant. 5,9-dimethyl-2-tetrahydrofurfuryl- tor binding. Next, the subtle twists 2'-hydroxy-6,7-). of antinociceptive testing are tackl- The rat bombesin-scratch test The finding that ketazocine can ed. The selection of particular tests The bombesin-scratch test (Ref. cause unpleasant symptoms in is dependent on the philosophy 8; see Fig. 1) is a recent addition to humans 2 is conceptually import- behind the analgesic project e.g. those in-vivo methods that can be the relative balance of receptor used in the evaluation of com- Alan Cowan is Associate Professor of Pharma- activities that is perceived to be pounds with activity at i< recep- cology at the Department of Pharmacology, ideal for the target pain state in tors. Bombesin, a tetradecapeptide Temple University School of Medicine, Phila- man. Selection of antinociceptive originally isolated from frog skin delphia, PA 19140, USA and Debra E. Gmerek is Research Investigator in the *Department of method and route of admini- in 1971, is one of a surprisingly Pharmacology, University of Michigan Medi- stration is also dependent on large number of well-known en- cal School, Ann Arbor, MI 48109, USA. whether the primary focus of dogenous substances which cause

1986, Elsevier Science Publishers B.V., Amsterdam 0165 - 6147186/$02.00 70 TIPS -February1986

blocked with , still antagonized bombesin-induced scratching. One puzzling result was the inclusion of (gener- ally regarded as a ~t in vivo) in the list of active agents (Table II). Since all compounds showing antibombesin effects were benzo- morphans (including phenazo- cine), the question of K- v. benzo- morphan-selective binding sites needed to be addressed. Recent experiments with nonbenzomor- phan K-agonists (tifluadom and U- 50488H) showed that these agents are active in the procedure (Table II) and can be antagonized in a dose-related manner by naloxone. It may be concluded that the bombesin-scratch test is a useful addition to the list of ic evaluative methods outlined in Table I. As a postscript to the test, note that there is a link between this less common measure - scratching Fig. 1. Scratching caused by bombesin O. 10 p,g, Lc.v. - and K compounds, that stretches back to the early 1970s. At that excessive scratching and/or against bombesin when tested time, it was noticed that monkeys, grooming when given centrally to at behaviourally nondepressant receiving it-directed mixed agonist- antagonists such as rodents (e.g. prolactin, SP and doses. Examples of inactive com- and for a month, ). Reasons for these pounds were buprenorphine, scratched excessively when they behaviours have yet to be es- , meperidine, metha- were withdrawn from these agents tablished. Neuropeptide-induced done, metkephamid and ~-endor- grooming/scratching in rats can phin. The scratching was essen- or were challenged with nalox- one z2. Scratching was also obser- be altered in different ways tially unaffected by behaviouraUy by subcutaneously administered nondepressant doses of haloperi- ved during withdrawal from cer- morphine and naloxone9-11. Thus, dol, , , tain bridged oripavines, com- pounds that were classified as K- the excessive grooming and/or neurotensin and several antihista- preferring almost a decade later 13. scratching associated with ACTHl_24 minic/antiserotoninergic agents. Recent work from the University of is attenuated by both mor- Suppression of bombesin-indu- Michigan has shown that rhesus phine and naloxone; that asso- ced scratching by ethylketazocine monkeys undergoing abrupt with- ciated with thyrotropin releasing seems to involve stereospecific drawal from U-50488 (but not from hormone (TRH) is suppressed by opioid binding sites since (-) nal- morphine) also display an unusu- morphine but not by naloxone; oxone, but not (+)naloxone, ally high incidence of scratching 1~. while that associated with bomb- attenuated the antibombesin effect Interestingly, this behaviour is esin is unaffected by both mor- of ethylketazocines. The following suppressed by fifluadom but not phine and naloxone. The resist- observations implicate Jc, rather by morphine. ance of bombesin towards mor- than ~, binding sites: (1) multiple phine prompted testing of many injections of morphine did not Other tests opioids and opioid peptides in an influence the ability of ethyl- Other tests show that morphine- attempt to suppress the robust ketazocine to antagonize bomb- like agonists have antidiuretic scratching elicited by bombesin,' esin, and (2) when ~-receptors were effects. K-Agonists are completely in rats. The initial results revealed that several mixed agonist--antago- TABLE I. Pharmacological profiles of test agents after peripheral administration nists/K-agonists attenuated bomb- Procedure Ethylketazocine Tifluaclom U-50488 Morphine esin-induced scratching in a ste- reoselective and dose-related man- Rata Bombesin-scratch test + + + - ner. Examples of these agents (all Increased flurothyl thresholds - - - + benzomorphans) are , Diuresis + + + - cyclazocine, ethylketazocine, ket- Increased food intake + + + --- azocine and (-); they Inhibition of gastrointestinal seem to share pharmacological transit + - - + effects in rats that dispel Rheetal monkeys the animals' preoccupation with Nalorphine discrimination + + + - scratching. Other commonly used Morphine-withdrawal opioids (mainly tt-agonists) and suppression - - - + opioid peptides were ineffective + = positive or dose-related effect. _+ = equivocal. - = no consistent effect. TIPS - February 1986 71

TABLE II. Effects of representative opioids and opioid peptides On bombesin-induced fingers and toes, and frequent scratching in rats" grooming of other monkeys 14. Test agent Aso (mg kg -~ s.c.) Ethylketazocine 0.36 (0.3,3--0.40)b Future trends U-50488 6.61 (5.37-8.13) Discovering new and better Tifluadom 6.80 (5.52-8.36) Phenazocine 0.29 (0.19-0.43) analgesics is no easy matter. We Morphine >10 ¢ may have to settle for less than Meperidine >25 ideal opioids, and only modest Metkephamid >30 gains, at least in the short term. I~-endorphin > 10 I~] i.c.v. Perhaps the growing emphasis on • The standard dose of bombesin was 0.10 p,g i.c.v, t'95% Confidence limits. ¢Highest 'nonopioid' pain modulating sys- behaviourally nondepressant dose tested. tems, both central and peripheral, will foster a rate of drug intro- different. Water diuresis seems to delays the passage of charcoal duction more in keeping with be an inherent property of virtu- along the GI tract in a dose-related academic advances. In the mean- ally all known *:-agonists, at least in manner but tifluadom and U-50488 time, opioids with various activi- rats and monkeys (ketazocine and are without major effects 2°. Ethyl- ties at ~, K and 6 receptors are MR 2033 are in man; MR ketazocine resembles morphine undergoing clinical trials. The evalua- 2033 is (+)-(1R,5R,9R,2"S)-5,9-di- rather than tifluadom and U-50488 tion of key compounds from chemical methyl-2-tetrahydrofurfuryl-2'-hy- in this test; the antitransit action of classes as disparate as benzene- droxy-6,7benzomorphan). One ethylketazocine is probably medi- acetamide (U-62066; 5(x,7a,8~-(+)-3, current theory is that *:-agonists ated by ~t-receptors. 4-dichlom-N)-methyl-N(7-(1-pyrro- suppress vasopressin release from lidinyl)- 1-oxaspiro-(4,5)dec-8-yl) the neurohypophysis and this Studies with K-agonists in benzene + acetamide) and mor- leads to increased urine output. monkeys phinan () should pro- There may be a peripheral site of Knowledge that several opioids vide the missing and vital links action in the rat since bilateral can function as discriminative between clinical analgesia, subjec- adrenal demeduUation abolishes stimuli to control the behaviour of tive effects, antinociception and K-mediated diuresis ~s. At a practi- animals is utilized in more ad- balance of receptor activities. For cal level, diuresis is undoubtedly vanced levels of testing. For the better or for worse, results from the most popular approach in vivo evaluation of *: activity, drug studies such as these will have a for comparing new and standard *: discrimination experiments use profound influence on the way~ directed ligands. Reasons for this animals that are trained to respond analgesic researchers pursue elu- are quite clear. Increased urine in either one way or another sive anodyne. output represents a simple, distin- depending on the presence or guishing feature of K-agonists. absence of the interoceptive cues Acknowledgements Examination of dose-response associated with injection of *:- Generous amounts of tifluadom curves, along with interactional agonists. The work of Tang and from Kali-Chemie and U-50488H studies with morphine, bremazo- Code 21 provides a good example. from Upjohn are gratefully ack- cine and ~-funaltrexamine (i.e. the They found that rhesus monkeys, nowledged. Our research was ~-fumaramate methyl ester of nal- trained to discriminate the phar- supported by Grants DA 03681 and trexone) (an irreversible ~t- macological effects of nalorphine DA 00254 from the National Insti- antagonist in vitro), allows differ- (a K-agonist in this species) from tute on Drug Abuse. entiation within the K class e.g. saline, generalized completely to ethylketazocine from U-50488 and ethylketazocine, tifluadom and U- References from nalorphine 16. Parenthetically, 50488 but (critically) not to three ~- 1 Martin, W. R., Eades, C. G., Thompson, ,:-preferring opioid peptides also agonists - morphine, J.A., Huppler, R.E. and Gilbert, P.E. seem to be active in the diuresis (1976) J. Pharmacol. Exp. Ther. 197, 517- and meperidine. 532 test since -A, the endo- Monkeys have also been used in 2 lasinski, D. R., Johnson, R. E., Hickey, genous K ligand, increases urina- morphine-withdrawal suppres- J. E., Haertzen, C. A., Henningfield, J. E. tion within 1-2 h of i.c.v, adminis- sion studies (at the University of and Kumor, K. (1985) NIDA Res. Monogr. tration to normally hydrated rats 17. Michigan) to characterize K-direc- Ser. 55, 59--65 3 Tyers, M. B. (1980) Br. J. Pharmacol. 69, [D-Pen2, D-PenS], prob- ted ligands. Behaviourally nonde- 503-512 ably the most selective agonist at pressant doses of ethylketazocine, 4 Przewlocki, R., Stala, L., Greczek, M., 5-opioid receptors that is currently tifluadom and U-50488 (in contrast Shearman, G.T., Przewlocka, B. and availableis, has no marked in- to morphine) do not suppress Herz, A. (1983) Life Sci. 33 Suppl. 1, 649- fluence on urine output 17. 652 signs of withdrawal in 14h 5 Porreca, F., Mosberg, H. I., Humt, R., Dynorphin-A (i.c.v.) initiates withdrawn, morphine-dependent Hruby, V.J. and Burks, T. F. (1984) J. feeding in rats. This action is antago- monkeys 14. In these animals, a Pharraacol. Exp. Ther. 230, 341--348 nized by naloxone. Ethylketazo- well-known behavioural syn- 6 Schmauss, C. and YakBh, T.L. (1984) cine, tifluadom and U-50488 like- drome emerges (e.g. excessive J. Pharraacol. Exp. Ther. 228, 1-12 7 Cowan, A., Geller, E.B. and Adler, wise induce feeding (after s,c. vocalization, abdominal defense M. W. (1979) Science 206, 465-467 Injection) but morphine is often reactions, vomiting) 22. The syn- 8 Gmerek, D.E. and Cowan, A. (1984) ineffective in drug-naive rats 19. drome associated with depriva- J. Pharraacol. Exp. Ther. 230, 110-115 Morphine can be more dearly tion-induced withdrawal from U- 9 Gmerek, D. E. and Cowan, A. (1983) Eur. distinguished from tifluadom and J. Pharmacol. 88, 339--346 50488 is qualitatively different; as 10 Cowan, A., Khunawat, P., Zhu, X.Z. U-50488 in the rat charcoal meal already indicated, scratching is and Gmerek, D.E. (1985) Life Sci. 37, test. In this procedure, morphine prominent, along with picking at 135-145 72 TIPS - February 1986

11 Gispen,W. H. and Wiegant,V. M. (1976) Friend, J. (1985) Br. J. Pharmacol. 85, Sci. USA 80, 5871-5874 Neurosci. Lett. 2, 159--164 382P 19 Morley, J.E. and Levine, A.S. (1985) 12 Cowan,A. (1973) Adv. Biochem. Psycho- 16 Leander, J.D. (1985) in Behavioral Med. J. Aust. 142, $11-$13 pharmacol. 8, 427-438 Pharmacology: The Current Status 20 Petrillo, P., Amato, M. and Tavani, A. 13 Katz, J. L., Woods, J. H., Winger, G. D. (Selden, L. S. and Balster, R. L., eds), pp. (1985) Neuropeptides 5, 403--406 and Jacobson, A. E. (1982) Life Sci. 31, 93--110, A. R. Liss, New York 2375-2378 17 Cowan, A. and Khunawat,P. Ann. N.Y. 21 Tang, A. H. and Code, R.A. (1983) ~" 14 Gmerek, D. E., Dykstra, L.A. and Acad. Sci. (in press) J. Pharmacol. Exp. Ther. 227, 563-569 Woods, J. H. J. Pharmacol. Exp. Ther. (in 18 Mosberg, H. I., Hurst, R., Hruby, V. J., 22 Villarreal, J.E. and Karbowski, M.G. press) Gee, K., Yamamura, H. I., Galligan,J. J. (1973) Adv. Biochem. Psychopharmacol. 8, 15 Blackburn,T. P., Borkowski, K. R. and and Burks, T. F. (1983) Proc. Natl Acad. 273--289

betically. A further subdivision allows the interactions of particu- lar drug-drug pairs to be dealt with in summary form under the helpful and down-to-earth style headings of 'Mechanism', 'Clinical Short and concise which can be used when drugs are Significance' and 'Management'. handling of drug being prescribed or dispensed. So The summaries are for the most it is essentially a practical reference part short and concise, and are interactions book. If you want a discursive and referenced with superscript num- Drug interactions, 5th edn detailed treatise on the pharmaco- bers which refer to bibliographies logical mechanisms which underly at the end of each chapter. Gener- by Philip D. Hansten, Lea & Febiger, the interactions between drugs, ally speaking, the size of the 1985. $24.75 (x + 460 pages) ISBN then this book is not for you summary is some indication of the 0 8121 0944 9 (although you might find it very extent of the knowledge of the There are so many drugs available helpful in pointing your nose in interaction in question. And, of that it is quite impossible for the right direction). But if, on the course, as is to be expected of a anyone to be able to remember other hand, you are looking for a reference book, there is a good with any certainty more than a practical manual which offers index at the back of the book which handful of drugs which can, and direct guidance, then this book uses for the most part generic which cannot, be safely admini- will fit the bill very nicely. names, but with a sprinkling of stered together. The philosophy The author has divided the book proprietary names as well. Those behind the writing of the first into a number of chapters which familiar with previous editions edition of this book, published in neatly categorize drugs into thera- will note that the section which 1971, and behind the writing of all peutic or chemical groupings was given over to effects on clinical of the subsequent editions, has ('Anticoagulants', 'Anticonvul- laboratory results is missing. This, been to offer a solution to the sants', 'Phenothiazines', etc.) and we are told in the introduction, is problem of drug interactions by then subdivided the drugs which to be published separately later. providing concise information in a fall into these categories - Professor Hansten is one of the

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