Pharmacological Study of 1 4fl.-(Thioglycolamido).-7,8-Dihydro- N(Cyclopropylmethyl)-Normorphinone

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1 /93/ $3.OO/O THE JORNAL OP PHARMACOLOGY AND EXPERIMENTAL THERAPETICS Copyright 1993 by The American Society for Pharmacology and Experimental Therapeutics Vol. 264, No. 3 Printed in SA. Pharmacological Study of 1 4fl.-(Thioglycolamido).-7,8-Dihydro- N(Cyclopropylmethyl)-Normorphinone (N-CPM-TAMO) 1 JIANG, AHMAD SEYED-MOZAFFARI, SYDNEY ARCHER and JEAN M. BIDLACK Department of Pharmacology, The niversity of Rochester, School of Medicine and Dentistry, Rochester, New York (Q.J., J.M.B.) and Department of Chemistry, Rensselaer Polytechnic Institute, Troy, New York (A.S.-M., S.A.) Accepted for publication September 25, Selective irreversible opioids have been useful in investigating the mechanism of opioid action and the properties of opioid receptors (Caruso et al, 1979; Portoghese et al., 198; Hallermayer et al., 1983; Rice et al, 1983; Burke et a!., 1986; Pasternak and Wood, 1986). Studies of a number of alkylating derivatives of morphinone both in vitro (Bidlack et al., 19) and in vivo (Jiang et a!., 1992) have demonstrated that this group of opioid ligands has high affinity for opioid receptors and can alkylate these receptors selectively. The apparent irreversible inhibition of opioid binding and antinociceptive properties of TAMO, one of the thiol-containing alkylating derivatives of morphinone, have been characteried. TAMO covalently bound to opioid receptors by forming a mixed disulfide bond between TAMO and the opioid receptor, as determined by the irreversible inhibition of the binding of the pt-selective peptide [3H] [D-A1a2,(Me)Phe4,Gly(ol)6]enkephalin (Bidlack et a!., 1991; Jiang et al, submitted). This wash-resistant inhibition of bind- Thia work was aupported by.s. Public Health Service Grants DA3742, DA1674 and DA6786 from the National Institute on Drug Abuse, and in part by the Aaron Diamond Foundation. Received for publication July 23, ABSTRACT Opioid effects of 143-(thioglycoIamido)-7,8-dihydro-N(cyclopropylmethyl)-normorphinone (N-CPM-TAMO) were studied in the mouse tail-flick and acetic acid writhing assays. In the tailflick test, N-CPM-TAMO failed to produce any antinociception after i.c.v. administration of up to 3 nmol. However, pretreatment of mice with N-CPM-TAMO produced a time- and dosedependent antagonism of morphine-induced antinociception. The antagonism by N-CPM-TAMO lasted up to 48 hr, with a maximal effect at 24 hr after i.c.v. administration. Similarly, pretreatment of mice with N-CPM-TAMO at 24 hr also produced a dosedependent antagonism of K-mediated antinociception, induced by 5,488 However, the antagonistic potency of N-CPM-TAMC) against 5,488 was 1 -fold less than against morphine. Pretreatment with N-CPM-TAMO had no effect on #{244} opioid receptormediated antinociception, as measured with [D-Pen2,D-Pen5]enkephalin. In the writhing assay, N-CPM-TAMO produced a timeand dose-dependent antinociception after i.c.v. administration, with a value of the dose producing 5% analgesia of 18.4( ) nmol. The antinociceptive effect lasted up to 3 hr after administration. N-CPM-TAMO-induced antinociception was antagonied by coadministration of the K-selective antagonist, norbinaltorphimine. Pretreatment of mice with N-CPM-TAMO also produced a time -and dose-dependent antagonism of 5,488 - induced antinociception, which lasted up to 72 hr, with a maximal effect at 24 hr after administration. These data indicate that N- CPM-TAMO is a s-selective, long-term antagonist. In addition, higher doses of N-CPM-TAMO produced both short-term K- mediated antinociception and long-term antagonism at K opiold receptors. ing was reversed by the addition of the disulfide bond-reducing agent, dithiothreitol (Jiang et a!., submitted). In the mouse warm water tail-flick antinociceptive study, TAMO acted as a IL agonist to produce short-term antinociception and also acted as a long-lasting, a-selective antagonist after i.c.v. administration (Jiang et a!., 1992). Our studies indicate that the thiol group in these opioid affinity ligands is an important constituent for the affinity ligand to alkylate opioid receptors. After the development of TAMO, we synthesied another sulfhydrylalkylating derivative of morphinone, N.CPM-TAMO, which is identical to TAMO with the exception of a N-cyclopropylmethyl group replacing the N-methyl group in TAMO. In previous studies, the substitution of a N-cyclopropylmethyl group for the N-methyl group resulted in compounds displaying antago. fistic properties, as first shown by Archer et al (1964) and Gates and Montka (1964). In binding studies, N-CPM-TAMO irreversibly alkylated t preferentially, followed by K and to a lesser degree t5 opioid binding sites in bovine striatal membranes, by forming a disulfide bond with the receptor (Jiang et a!., submitted). In the present study, we characteried the longterm actions and receptor selectivity of N-CPM-TAMO on supraspinal antinociception in the mouse. ABBREVIATIONS: N-CPM-TAMO, 14f3-(thioglycolamido)-7,8-dihydro-N(cyclopropylmethyl)-normorphinone; TAMO, 14fl-(thioglycolamido)-7,8-dihydromorphinone; (3-FNA, $-funaltrexamine; lcl 174,864, N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH; nor-bni, nor-binaltorphimine; DPDPE, [D-Pen2,D-Pen5] enkephalin; 5,488, (trans)-3,4-dichloro-n-methyl-n-[2-(1-pyrrolidinyl)-cyclohexyl]beneneacetamide methanesulfonate hydrate. 121

2 122 Jlang at al. Vol. 263 Animals. Male, ICR mice (2-25 g, Harlen, IN) were used for all experimenta. Mice were kept in groups of six in a temperature-controlled room with a 12-hr light-dark cycle (lights on, 7: AM. to 7: P.M.). Food and water were available ad libitum until the time of the experiment. Injection techniques. Intracerebroventricular injections were made directly into the lateral ventricle according to the modified method of Haley and McCormick (1957). The mouse was anesthetied lightly with ether, an incision was made in the scalp and the injection was made 2 mm lateral and 2 mm caudal to bregma at a depth of 3 mm using a lo-ll Hamilton microliter syringe. The volume of all i.c.v. injections was 5 Ml. Tail-flick aasay. The thermal nociceptive stimulus was 55 C water with the latency to tail flick or withdrawal taken as the endpoint (Vaught and Takemori, 1979). After determining control latencies, the mice received graded i.c.v. doses of opioid agonists or antagonists at various times. Morphine, DPDPE and 5,488 were given as a single i.c.v. injection with testing taking place 1 mm after the injection, which was the maximal response for these compounds as established in our previous study (Jiang et al, 1992). In the antagonistic study, various doses of N-CPM-TAMO were given as a single pretreatment at 2, 4, 8, 16, 24, 48, 72 and 96 hr before testing. A cut-off time of 15 sec was used; if the mouse failed to display a tail flick, the tail was removed from the water and that animal was assigned a maximal antinociceptive score of %. Mice showing no response within 5 sec in the initial control test were eliminated from the experiment. Antinociception at each time point was calculated according to the following formula: % antinociception = Acetic acid writhing assay. After receiving graded i.c.v. doses of opioid agonists and antagonists at various times, an i.p. injection of.6% acetic acid (1 ml/kg) was administered to each mouse. Five mm after administration, the number of writhing signs displayed by each mouse was counted for an additional further 5 mm. Antinociception for each tested mouse was calculated by comparing the test group to a control group in which mice were treated with i.c.v. saline. Data were calculated according to the following formula: % antinociception = Materials and Methods x (test latency - control latency) (15 - control latency) x (mean number of writhes in control group - number of writhes by each mouse) mean number of writhes in control group Chemicals. Morphine sulfate was purchased from Mallinckrodt Chemical Company (St. Louis, MO). N-CPM-TAMO was synthesied as described elsewhere (Jiang et al, submitted). 5,488 and nor-bni were obtained from Research Biochemicals Inc. (Natick, MA). DPDPE was purchased from Bachem Inc. (Torrance, CA). All compounds were dissolved in distilled water just before use, except N-CPM-TAMO, wh!ch Was dissofved in 2% dimcthybsulfotide, a coricentration that did not produce any dbtectble effct in either mouse tail flick or writhing assays. 4sics. All dose-response lines werc arialyed using regrcssiori methods a described by Tallarida and Murray (1986) Regression lines values of the doscs producing 5% analgesia and 95% confidence limits were determined with each inditridual data point using the computer program by Tallarid and Murray (1988). Statistical signiflc#{226}ricewris determined using the StudCnt s t test All data points shown are the mean of 8 to 12 mice, and eo brirs eprcscnt the SE. ouse ail;hlk #{225}R#{225}. N-CPM-TAMO did not produce any antnociception in the mouse tail flick assay after 1 C V administration of up to 3 nmol (data not shown). However, pretreatment of mice with N-CPM-TAMO produced a timeand dose-dependent antagonism of morphine-induced antinociception (figs. 1 and 2). The antagonism of N-CPM-TAMO appeared at 16 hr and lasted for 48 hr after i.c.v. administration, with a maximal effect at 16 to 24 hr. The antagonistic effect of N-CPM-TAMO was observed with an i.c.v. dose from.3 to 1 nmol against a submaximal dose (3 nmol) of morphine. Pretreatment of mice with N-CPM-TAMO at 24 hr also produced a dose-dependent antagonism of the antinociception mediated by the K agonist, 5,488 (fig. 3). The dose range for K-mediated antagonism was from 1 to nmol when antagoniing a submaximal dose ( nmol) of 5,488. N-CPM- TAMO failed to antagonie antinociception induced by the #{244}- selective agonist, DPDPE, in either coadministration with DPDPE or in mice pretreated with N-CPM-TAMO at 24 hr (fig. 4). Mouse writhing assay. Antinociception produced by K opioid agonists has been difficult to determine in many tests, especially with thermal stimuli such as the warm water tailflick assay (Porreca et a!., 1987). Therefore, we also investigated the actions of N-CPM-TAMO in the mouse acetic acid writhing test. nlike the warm water tail-flick assay, % antinociception can be obtained with K agonists in the writhing test. N- CPM-TAMO produced a dose-dependent antinociception after i.c.v. administration in the mouse writhing assay (fig. 5). The antinociceptive value of the dose producing 5% analgesia (and 95% CL) for i.c.v. N-CPM-TAMO was 18.4 ( ) nmol. The antinociception of 3 nmol N-CPM-TAMO lasted up to at least 3 hr after i.c.v. administration (fig. 6). The antinociception produced by N-CPM-TAMO was blocked by coadministration of nor-bni, the K-selective antagonist, in a dose-dependent manner (fig. 7). Pretreatment of mice with N-CPM-TAMO produced a timeand dose-dependent antagonism of 5,488-induced antinociception in the writhing assay (figs. 8 and 9). The antagonistic effect of N-CPM-TAMO was observed from 16 to 72 hr, with a maximal effect at 24 hr after i.c.v. administration. The antagonistic dose range of N-CPM-TAMO after 24-hr pretreatment was 3 to 3 nmol against 1 nmol 5,488, which produced approximately 6% antinociception. Opioid agonistic and antagonistic effects of N-CPM-TAMO were investigated in both mouse tail-flick and writhing assays. Our results demonstrated that N-CPM-TAMO produced a time- and dose-dependent antinociception in the mouse aceticacid *rithing saa. N-CPM-TAME-iriduc#{235}d aritinocicejtkth iii the *rithikig aaa *as bkked by the k-se1ctfve anthgonist, htjfsil (T#{225}knibri et a!., 1988), iridkatiiig that N-CtM- *#{225}s a#{235}tliig at suaspihal K opiold #{235}tejtoa to ifdd#{252}te tamii Discussion is antindcieption In addition to its agrnnstic efft a single jie#{128}eattherit with I-CPM-TAMO also produced briglastirig #{225}ritagbriisms of p- arid K-niediated antinoriceptiomi as insuied with thbhiri#{235} thud 5,48, esectily. Hti*evei, a -fbld greater dose as needed to oduce a bohg-teim antagonism of ahtiriociceptioh mediated by K teceptors In coinprisori with opboid retetos. the same highef dose was needed to detect ic-induced antinoicetion iri the writhing test. It should be rioted that thefe is a diffeence bet*een the tailflick and acetic acid writhing antinocicetie assays for k oibld

3 1993 Pharmacology of N-CPM-TAMO 123 vi C.) 3#{176} 2 K 1 * Fig. 1. Antinociceptive effects of i.c.v. morphine (3 nmol, 1 mm) in mice pretreated with a single icy. dose of N- CPM-TAMO(.3,.1,.3 or I nmol, 24 hr before testing) in the mouse tail-flick test. <.5, significantly different from the morphine group. 3 orphtn. -1mm 1 N-CPM-TAMO -24hr DOSE (nmol, l.c.v.) ) I- C.) 1- K V) 8 7 g :: 3 2 K 1 Korphin. lonain 2 4 S umol T Lill Fig. 2. Antinociceptive effects of cv. morphine (3 nmol, 1 mm) in mice pretreated with a single icy, dose of N- CPM-TAMO (1 nmol, 1 mm, 2, 4, 8, 16, 24, 48, 72 and 96 hr before testing) in the mouse tail-flick test. *).5, significantly different from the morphine group. N-CPM-TAMO PRETREATMENT TIME (hours, 1 ninol) * momma Fig. 3. Antinociceptive effects of cv. 5,488 ( nmol, 1 mm) in mice pretreated with a single icy, dose of N-CPM-TAMO (3, 1, 3 or nmol, 24 hr before testing) in the mouse tail-flick test. #{176}P.5, significantly different from the 5,488 group. N-CPM-TAMO -Z4hr DOSE (ninol, i.c.v.) agonists (e.g., N-CPM-TAMO) to produce antinociception. N- CPM-TAMO produced antinociception in the writhing assay but not in the tail-flick assay. It has been reported that supraspinal and spinal antinociceptions produced by K opioid agofists can be difficult to determine in many tests, especially to thermal stimulus such as warm water (55#{176}C)tail-flick assay (Porreca et al., 1987). Some K agonists fail to display antinociception in the tail-flick assay. For example, fl-fna, which is a reversible K agonist and usually used as an irreversible M antagonist (Portoghese et al., 198; Takemori et a!., 1981; Ward et

4 124 Jiangetal. Vol So j 2! Fig. 4. Antinociceptive effects of i.c.v. DPDPE (1 nmol, 1 mm) in mice given or pretreated with a single i.c.v. dose of N-CPM-TAMO (3 nmol, 1 mm or 24 hr, respectively, before testing) in the mouse tail-flick test. -1 mm -1 mm -1 mm -1 win -1 mm DPDPI 1 nmol -24 hr N-CPM-TAKO 3 amol ru L- a4 5 ẕ4 1-2 K w DOSE N-CPM--TAMO (nmol, i.c.v.) 3 Fig. 5. Dose-response line for icy. N-CPM-TAMO (1 mm) in the mouse writhing test. Fig. 6. Time-response line for icy. N-CPM-TAMO (3 nmol) in the mouse writhing test. K 1 I.. I.. I.. I.. I.. I.. I.. I TIME (minutes) cii., 1982,a,b,c), failed to show any antinociception after i.t. or i.c.v. administration in the tail-flick assay. However, in the writhing assay, fi-fna produced antinociception after i.t. administration (Jiang et a!., 1989), and this agonistic effect of fi- FNA was antagonied by the K-selective opioid antagonist, nor- BNI (Takemori et a!., 1988), confirming that fl-fna indeed produces antinociception through K opioid receptors (Jiang et a!., 1989). Our previous study showed that TAMO produced short-term antinociception and long-lasting antagonism in the mouse tailflick assay. Both agonistic and antagonistic actions of TAMO are mediated through j opioid receptors (Jiang et a!., 1992). N- CPM-TAMO, a derivative of TAMO, that has substituted the N-methyl group in TAMO with a N-cycbopropylmethyl group, lost the M agonistic effect of TAMO. Other studies also have

5 1993 Pharmacology of N-CPM-TAMO ) 7 eo 5 K So 2 1 * Fig. 7. Antinociceptive effects of cv. N-CPM-TAMO (3 nmol, 1 mm) in the absence and presence of i.c.v. nor- BNI (.3,.1,.3 and 1 nmol, 1 mm) in the mouse writhing test..5, signthcandy different from the N- CPM-TAMO group. SO N-CP-TAMO Xor-BNI ) 1w C.) 1w K So DOSE (nmol, Lc.v.) 1 5o.488-1mm 3 )t-cp-tam -S4hr DOSE (nmol, i.c.v.) Fig. 8. Antinociceptive effects of icy. 5,488 (1 nmol, 1 mm) in mice pretreated with a single icy, dose of N- CPM-TAMO (3, 1, 3, or 3 nrnol, 24 hr before testing) in the mouse writhing test. #{176}P.5, significantly different from the 1)5,488 group. LI nmol I Fig. 9. Antinociceptive effects of icy. 5,488 (1 nmol, 1 mm) in mice pretreated with a single cv. dose of N- CPM-TAMO (3 nmol, 4, 8, 16, 24, 48, 72, 96 and 12 hr before testing) in the mouse writhing test. P.5, significantly different from 1)5,488 group. N-CPM--TAMO PRETREATMENT TIME (hours, 3 nmol) reported that the substitution of the N-methyl group with either an N-cyclopropylmethyl or N-allyl moiety can convert an opioid agonist to an antagonist. For example, two c opioid antagonists, naltrexone and naltrexaone (Pasternak and Hahn, 198; Hahn et al, 1985), were derived from the substitution of a N-cyclopropylmethyl moiety in the opioid agonists, oxymorphone and oxymorphaone, respectively (Pasternak and Hahn, 198; Ling et al, 1984; Williams et a!., 1987). Although

6 126 Jlangetal. Vol. 263 N-CPM-TAMO has a N-cycbopropylmethyl moiety in comparison to the N-methyl-containing TAMO, their antagonistic potencies remained identical. Both compounds at the same dose, 1 nmol, administered by i.c.v. injection 24 hr before the tail-flick test, totally antagonied antinociception induced by a submaximal dose (3 nmol, i.c.v.) of morphine (Jiang et al, 1992). In addition to the long-lasting antagonism of N-CPM- TAMO, our study also showed that N-CPM-TAMO had an antagonistic effect on K opioid receptors. This long-lasting K antagonistic effect of N-CPM-TAMO started at 16 hr and lasted up to 72 hr after a single i.c.v. administration. Many studies have shown that ic opioid agonists can functionally antagonie some agonistic effects (Sheldon et a!., 1989). For example, the K-selective opioid peptide, dynorphin A (1-13), antagonied morphine-induced antinociception in the mouse (Friedman et al, 1981). Because N-CPM-TAMO also produced a ic agonistic effect, it could be questioned whether this antagonism by N-CPM-TAMO might be due not to a real i antagonistic effect, but instead to N-CPM-TAMO acting as a K agonist. This possibility is unlikely, however, because the N- CPM-TAMO time courses of K agonistic and and K antagonistic actions are very different. The time course for the K agonistic effect by N-CPM-TAMO lasted only 3 hr, however, the i and K antagonistic effects began to appear significantly at 16 hr after i.c.v. pretreatment when antagoniing a submaximal dose of morphine or 5,488. Therefore, these two different time courses of ic agonistic and and K antagonistic actions for N-CPM-TAMO indicate that N-CPM-TAMO indeed did produce a long-lasting antagonism at K opioid receptors. The presence of sulfhydryl groups near or in opioid binding sites (Bowen et a!., 1987; Gioannini et al, 1989; Bidlack et al, 19) has been reported, and the sulfhydryl group is an important functional group for irreversible opioid ligands to covalently bind to opioid receptors (Bowen et al, 1987; Calcagnetti et al, 1989; Jiang et al, 19, submitted; Mattia et a!., 1991). As TAMO covalently bound to opioid receptors (Jiang et al, 1992), N-CPM-TAMO showed similar long-lasting antagonism. Our in vitro study (Jiang et al, submitted) demonstrated that N-CPM-TAMO irreversibly bound to and K opioid binding sites by forming a disulfide bond between the ligand and the receptor. This disulfide bond was reduced by the disulfide bond-reducing agent, dithiothreitol. Thiols can be converted to disulfides by oxidants, and many thiols will spontaneously oxidie to disulfides merely on standing in air. Because the level of molecular oxygen is low in vivo, this fact may explain the relatively slow rate of reactivity of N-CPM-TAMO and TAMO in vivo (e.g., hr) (Jiang et at., 1992) in comparison to in vitro experiments in which the reaction was complete within 2 mm (Jiang et al, submitted). Another explanation for the different rates of reactivity between in vivo and in vitro experiments may be due to different pharmacokinetic effects in the two systems. Although N-CPM-TAMO can alkylate both and K opioid receptors to produce long-term antagonism, N-CPM-TAMO was more selective toward i than K opioid receptors. N-CPM- TAMO completely antagonied u opioid receptor-mediated antinociception in the tail-flick assay at a dose of only 1 nmol, whereas this dose produced neither antagonism of K-mediated antinociception nor did it display any agonistic effects in either tail-flick or writhing tests. 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