TIPS - February 1986 69 action is deemed to be spinal, In-vivo studies on kappa supraspinal, or in the periphery. A pharmaceutical company search- opioid receptors ing for its own tifluadom 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 opioids are mediated by multiple types of opioid receptor. As a by naloxone 3. Differences between result, 'obtaining the right balance' is the catchphrase most frequently heard test compounds may be empha- these days in analgesic research laboratories throughout the pharmaceutical sized by using supraspinal (i.c.v.) industry. Tomorrow's analgesics 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, ketazocine and morphine 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 this procedure (Porreca, Mosberg, Burks and Cowan, unpublished Compounds that are classified as K- ant, given that this compound was data).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 agonists (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 narcotic 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. Dysphoria 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 benzomorphans, 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-benzomorphan). 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 buprenorphine, ethylketazocine still antagonized bombesin-induced scratching. One puzzling result was the inclusion of phenazocine (gener- ally regarded as a ~t agonist 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 cyclazocine rodents (e.g. prolactin, SP and doses. Examples of inactive com- and nalorphine for a month, vasopressin). Reasons for these pounds were buprenorphine, scratched excessively when they behaviours have yet to be es- levorphanol, 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, indometacin, lidocaine, 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