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Home , Codeine, Enkephalinase inhibitor, Thiorphan

Antinociceptive and -Like Profiles of BL-2401, a Novel Inhibitor, in Mice and Rats

Atsuko Kita1, Kiyomi Imano1, Yasuhiro Seto1, Ikuhisa Yakuo2, Takashi Deguchi3 and Hideo Nakamura4

1 Department of I, Discovery Research Laboratories I, 2Department of Toxicology and Pharmacology, Developmental Research Laboratories,3Department of Chemistry II, Discovery Research Laboratories II, Dainippon Pharmaceuticalof RegulatoryCo., Ltd., 33-94 Affairs, Enoki, Suita/Osaka 564, Japan4Division Dainippon Pharmaceutical Co., Ltd., 33-94 Enoki, Suita/Osaka 564, Japan

Received May 26, 1997 Accepted August 29, 1997

ABSTRACT-To clarify the properties of BL-2401 ((•})-3-[2-benzyl-3-(propionylthio) propionyl]amino-5 methylbenzoic acid), a novel , we examined its antinociceptive and antidepressant

like activities after , along with their association with endogenous systems. BL 2401 produced an antinociceptive effect after oral administration in the mouse phenylbenzoquinone writh ing test (ED50: 12.4 mg/kg) and the rat acetic acid writhing test (ED50: 55.8 mg/kg), the antinociceptive effect being antagonized by hydrochloride. BL-2401 also relieved arthritis-induced hyperalgesia in rats. In the mouse hot-plate and tail pressure tests, BL-2401 showed significant but modest antinociception at higher doses (200 and 400 mg/kg). In addition, BL-2401 (100 mg/kg) produced a naloxone-reversible antidepressant-like effect in the mouse forced swimming test. As for the mechanism of the action, the of BL-2401, BL-2240 ((•})-3-(2-benzyl-3-mercaptopropionyl) amino-5-methylbenzoic acid), selectively inhibited enkephalinase in vitro (IC50: 5.2 nM). Oral administration of BL-2401 to mice sig nificantly inhibited the enkephalinase activity in the striatum and also potentiated the antinociceptive effect of (D-A1a2,Met5)- given intracisternally. These findings indicate that BL-2401 is an orally active enkephalinase inhibitor and may produce antinociceptive and antidepressant-like effects in association with endogenous opioid systems.

Keywords: BL-2401, Enkephalinase inhibitor, Antinociception, Antidepressant-like action, Opioid

The discovery of in 1975 (1) accelerated the opioid receptors. Moreover, enkephalinase inhibitors and research on endogenous opioid systems, leading to suc exogenous enkephalins have been reported to relieve cessive isolation of various opioid peptides and fruitful forced swimming-induced immobility and conditioned investigation of their physiological, pathological and suppression of motility (9-12), supporting the physio pharmacological roles (2, 3). More recently, several logical roles of endogenous enkephalins in stress groups have succeeded in molecular cloning of a-, B response, mood or emotion. Various enkephalinase in and x-opioid receptors (4). These findings have been stimu hibitors have been reported in the literature (6), and they lating the development of novel acting via show characteristic profiles; e.g., , the proto endogenous opioid systems, which has progressed along type of enkephalinase inhibitor, produces an antinoci two strategies: first, direct ligands to opioid receptors, ceptive effect after i.v. and i.c.v. administration (13). such as selective x-opioid (5), and sec Acetorphan, a of thiorphan, shows antinocicep ond, modulators of endogenous , such as enkepha tive and antidepressant-like effects after i.v. administra linase inhibitors (6). tion (11). In addition, RB101 (N-[(R,S)-2-benzyl-3[(S)(2 Enkephalinase (neutral endopeptidase, EC. 3.4.24.11), amino-4-methylthio)butyl dithio]-1-oxopropyl]-L-phenyl as well as aminopeptidase, has been shown to play an benzyl ester) produces potent antinociceptive and important role in degrading enkephalins in vivo (7). The antidepressant-like effects through its activity to inhibit inhibitors of enkephalinase have been expected to have both enkephalinase and aminopeptidase (12, 14). an antinociceptive effect (6, 8), but not to elicit However, few compounds have been demonstrated to like side effects because they do not bind directly to show an antinociceptive effect after oral administration; that is, SCH34826 ((S)-N-[N-[1-[[(2,2-dimethyl-l,3 according to the method of Ueda et al. (22). dioxolan-4y1) methoxy]carbonyl]-2-phenylethyl]-L-phenyl Acetic acid writhing test in rats (20, 23): Male Wistar alanine]-,3-alanine) (15) and ONO9902 (16). rats (90-130 g) were used. Writhing was induced by i.p. We have aimed to develop an orally active enkepha injection of 1 ml of 1 % aqueous acetic acid. Rats showing linase inhibitor as an drug that does not cause writhing responses within 15 min after the acetic acid in morphine-like side effects such as drug dependency (17). A jection were used for the experiment. BL-2401 and other novel compound, BL-2240 ((±)-3-(2-benzyl-3-mercapto test were orally administered 15 min after the acetic propionyl) amino-5-methylbenzoic acid), was found to acid injection, and the number of writhes was counted for inhibit enkephalinase potently, and its prodrug, BL-2401 a 20-min period commencing 45 min after the oral ad ((±)-3-[2-benzyl-3-(propionylthio) propionyl]amino-5 ministration (1 hr after acetic acid injection). The anti methylbenzoic acid), was synthesized as an orally active writhing ED50 was calculated from the effective rates as antinociceptive compound. BL-2401 was metabolized to BL cited above. 2240; a pharmacokinetic study demonstrated that BL-2240 Tail pressure test in mice (20): Male ddY mice (22-30 was detected in the plasma and brain of mice and rats g) were used. The root of the tail was gradually pressed after oral administration of BL-2401 (K. Hayashi and with the apparatus of Nakamura and Shimizu (24). The M. Miyamoto, unpublished data). Thus, the present study pressure to elicit biting behavior was measured as a was carried out to characterize the antinociceptive profile index (1 mm =12.5 g) at 30-min intervals for 3 hr after of BL-2401 in comparison with and . We oral administration of BL-2401 or other test drugs. The also investigated its antidepressant-like effect that might pressure was not over 50 mm to avoid injury. In the be of benefit for relief of pain; patients with pain often Results section, the scale of the pressure threshold was have depressive disorders (18). In addition, the represented by g. of BL-2401 in inhibiting enkephalinase was evaluated Hot plate test in mice (15, 25): Male ddY mice (20-26 using in vitro, ex vivo and in vivo assays to ascertain the g) were treated with BL-2401 or other test drugs 30 min mechanism of its effects. before testing. The mouse was put onto a heated surface (501C), and the latencies of the paw licking and the jump MATERIALS AND METHODS were measured. A plastic cylinder (20-cm-high and 15 cm in diameter) was used to keep the mouse on the heated Animals plate. Cut-off time was set at 400 sec. ddY Mice and Wistar rats were purchased from Japan Paw pressure test in adjuvant-induced arthritic rats: SLC (Hamamatsu) and SD rats from Clea Japan (Osaka). A 10 mg/ml suspension of adjuvant (Mycobacterium Animals were housed in groups (except for arthritis test) butyricum; Difco, Detroit, MI, USA) was prepared using under conditions of controlled temperature (23±21C), light mineral oil (Bayol F). A 0.06 ml volume of this sus humidity (55-L10%) and light (06:00-18:00 hr). Food pension was injected intradermally into the root of the and water were available ad libitum. Each animal was tail of female SD rats (8-weeks-old). Two weeks later (day used only once in all experiments. 14), rats showing arthritis in their hind paws were select ed, and they were orally administered BL-2401 or codeine Nociceptive tests phosphate once a day during the following 7 days (days Phenylbenzoquinone writhing test in mice (19, 20): 14-20). The nociceptive threshold of the hind paw for Female ddY mice (18-22 g) were used. Writhing was mechanical stimulation was measured with the apparatus induced by i.p. injection of 10 ml/kg of 0.03% phenyl cited in the section on the tail pressure test, and the pres p-benzoquinone in a 5% ethanol aqueous solution. The sure to elicit either a struggle response or paw withdrawal number of writhes was counted for a 15-min period com was determined. The cut-off pressure was 50 mm. The mencing 5 min after the phenylbenzoquinone injection. pressure test was done before and 1 hr after drug ad When the writhes were 50% or less the mean number in ministration on day 14 and at 1 hr after drug administra the vehicle control group, the was considered effec tion on days 16, 18 and 20. On days 21 and 28 (1 and 8 tive. The anti-writhing ED50 and 95076 confidence limits days after the last administration), only the pressure test were calculated from the effective rates (% of number of was done. In the Results section, the scale of the pressure mice in which the dose was effective/number of mice test threshold was represented by g. ed) according to the method of Litchfield and Wilcoxon (21). BL-2401 and other test drugs were orally admin Antidepressant tests istered 30 min before the injection of phenylbenzoqui Forced-swimming test in mice (26): Male ddY mice none. Intracisternal (i.c.) administration of naloxone (25 32 g) were dropped into a vertical cylinder contain hydrochloride was performed in a volume of 10 p1/mouse ing 15 cm of water at 21-231C and left there for 6 min. The total duration of immobility during the last 4 min clude from the brain. The striata were dissected and was measured. Mice were treated orally with BL-2401 1 hr homogenized with 1 ml of 50 mM Tris-HCl buffer (pH before the test. 7.4). The homogenate (50 p1) was incubated for 30 min at 25V with [3H](D-A1a2,Leu5)-enkephalin (25 pl, final con In vitro enkephalinase inhibition assay centration 20 nM), bestatin (10 pM) and captopril (1 pM) Enzyme preparation: Enkephalin-degrading enzymes and with or without thiorphan (1 pM) (final volume: 100 were prepared from rat striata according to the method pl). The reaction was stopped with 25 pl of 0.2 N HCI, of Gorenstein and Snyder (27) with minor modification. and then a 50-pl aliquot of the reaction mixture was The striata from male Wistar rats (200-300g) were applied to a Porapak Q column. The metabolite homogenized in 30 vol. 50 mM Tris-HC1 buffer (pH 7.7) ([3H]Tyr-D-Ala-Gly) was eluted with 3 x 1 ml of water. with a glass-Teflon homogenizer. The homogenate was Enkephalinase activity was calculated as the difference centrifuged at 1,000 x g for 15 min, and then the super of metabolite production between the reaction in the natant was centrifuged at 50,000 x g for 15 min. The presence of thiorphan and that in its absence. Protein was pellet was washed three times according to the above assayed with Lowry's method (29). described procedure. The resulting pellet was resuspended in 15 vol. 50 mM Tris-HC1 buffer (pH 7.7) containing 1 % (D-Ala2,Met5)-enkephalin (DAME) potentiation test (in Triton X-100 and then incubated at 37V for 45 min, vivo assay for enkephalinase inhibition) (13) followed by centrifugation at 100,000 x g for 1 hr. The DAME administered into the brain is degraded by supernatant containing solubilized enzymes was applied enkephalinase, but not by aminopeptidase; therefore, to a DEAE-cellulose column previously equilibrated with enkephalinase inhibitors are able to potentiate the anti 50 mM Tris-HCl buffer (pH 7.7) containing I % Triton X nociceptive activity of DAME. This test has been used 100. Elution was done with two bed volumes of 50 mM for an in vivo enkephalinase inhibition assay. Male ddY Tris-HCl buffer (pH 7.7) containing 1010 Triton X-100 mice (20-26 g) were administered intracisternally with followed by a linear salt gradient from 0 to 0.4 M NaCI. DAME (1 pg/mouse) at various intervals after oral ad These procedures were carried out at 41C except for the ministration of BL-2401. Antinociceptive effect was as incubation. Under these conditions, enkephalinase activ sessed by the tail pressure test. In the time-course study, ity was detected in the void volume, and aminopeptidase DAME was given only once to each mouse pre-treated and dipeptidylaminopeptidase were eluted within the with BL-2401. NaCI gradient. These fractions were used as enzyme preparations. Materials Enzyme assay: [3H]Leucine-enkephalin was used as a BL-2401, BL-2240, thiorphan and captopril were syn substrate. After pre-incubation of enzyme (50 pl) and test thesized in our laboratory. Naloxone hydrochloride and compound solution (25 p1) at 371C for 5 min, [3H]leucine phenyl p-benzoquinone (Sigma Chemical Co., St. Louis, enkephalin (25 pl, final concentration 20 nM) was added. MO, USA), codeine phosphate (Dainippon Pharmaceuti The reaction was performed at 371C for 1 hr (10 min for cal Co., Osaka), bestatin and DAME (Peptide Institute, dipeptidylaminopeptidase) and stopped with 25 pl of 0.2 Minoh), DEAE-cellulose (Chisso, Chiba), Porapak Q N HCI. A 50 pl aliquot of reaction mixture was applied to (100-120 mesh; Waters Associates, Milford, MA, USA), a Pasteur pipette containing polystyrene beads (Porapak [3H]leucine-enkephalin (New England Nuclear, Boston, Q) (28). The substrate, but not its metabolites, was MA, USA), [3H](D-Ala2,Leu5)-enkephalin (CEA, Gif retained on the beads. The metabolites were eluted with sur-Yvette, ), aspirin (Nacalai Tesque, Kyoto) and 3 x 1 ml of water from the column. The reactions other reagents (Nacalai Tesque, and Wako, Osaka) were progressed linearly at the initial velocity. Test compounds used. BL-2401, codeine phosphate and aspirin were sus were dissolved in ethanol and diluted with 50 mM Tris pended in 0.5010 tragacanth gum for oral administration, HC1 buffer (pH 7.7). IC50 values were calculated from and DAME and naloxone hydrochloride were dissolved inhibitory rates at three to four concentrations according in saline. These compounds were administered at volumes to logit log analysis. The inhibitory rate was estimated of 10 ml/kg (mice; p.o., s.c., i.v.), 5 ml/kg (rat acetic from two or three experiments. acid writhing test; p.o., s.c.) or 3 ml/kg (rat arthritis test; p.o.). Ex vivo enkephalinase inhibition assay (11) Female ddY mice (17-25 g) were treated orally with Statistical analyses BL-2401. The mice were sacrificed with cervical disloca Results are presented as the mean with S.E. Statistical tion; the right ventricle of the heart was then incised, and difference was estimated by the Dunnett's test or Bonfer saline (20 ml) was injected into the left ventricle to ex roni multiple comparison test (for naloxone antagonism). Appropriate groups were compared by Student's t-test. Table 1. Anti-writhing activity of BL-2401 in the phenylbenzoqui none writhing test in mice and in the acetic acid writhing test in rats RESULTS

Antinociceptive effect In the mouse phenylbenzoquinone writhing test, BL 2401 inhibited the writhes at doses of 10-100 mg/kg, p.o. (Table 1), with an ED50 value (95% confidence limits: C.L.) of 12.4 (8.4-18.4) mg/kg. ED50s of codeine phos phate and aspirin were 15.6 (8.7 28.1) (n = 31) and 126.5 (77.1-207.5) (n=18) mg/kg, respectively. To estimate the duration of the action, BL-2401 was orally admin istered 3 and 6 hr before the injection of phenylbenzo quinone. The anti-writhing activity of BL-2401 (50 and 100 mg/kg) was still significant 6 hr after administration; the inhibitory rates were 38.3010 (P<0.01) and 54.6% (P < 0.01), respectively. The ED50s at 3 and 6 hr after ad a)( ): number of mice or rats showing writhing counts 50% or less of ministration were 21.4 (11.4-40.0) and 75.4 (52.6-108) each vehicle control. b)NLX: naloxone hydrochloride (0.5 mg/kg) was administered s.c. 45 min after the injection of acetic acid. mg/kg, respectively. To clarify the involvement of opioid **P<0 .01, significantly different from each vehicle control group. receptors in the action of BL-2401, naloxone antago ##P<0 .01, significantly different from BL-2401 (160 mg/kg) alone. nism was tested. As shown in Fig. 1, the anti-writhing activity produced by BL-2401 (100 mg/kg, p.o.) was sig nificantly reduced by naloxone hydrochloride at doses of rat acetic acid writhing test, BL-2401 produced a dose 0.1-0.5 mg/kg, i.v. To further test the involvement of related inhibition of writhes with an ED50 (95% C.L.) central opioid receptors, naloxone hydrochloride was in of 55.8 (29.0-107) mg/kg (Table 1). ED50s of codeine jected i.c. As shown in Fig. 1, the anti-writhing activity of phosphate and aspirin were 14.2 (8.6 23.5) (n = 32) and BL-2401 (100 mg/kg) was also reduced by i.c. injected 16.9 (8.8 32.5) (n =18) mg/kg, respectively. Naloxone naloxone hydrochloride at a dose of 0.5 pg/mouse (ca. hydrochloride (0.5 mg/kg, s.c.) significantly reduced the 0.025 mg/kg), a dose of naloxone hydrochloride which anti-writhing activity of BL-2401 (160 mg/kg, p.o.). did not show antagonism after i.v. administration. In the In the mouse tail pressure test, BL-2401 (400 mg/kg,

Fig. 1. Antagonism of BL-2401-induced anti-writhing activity by naloxone hydrochloride (NLX) given i.v. or i.c. in the phenylbenzoquinone writhing test in mice. BL-2401 (100 mg/kg) was orally administered 30 min before i.p. injection of phenylbenzoquinone. NLX was given i.v. (left panel) or i.c. (right panel) 5 min before phenylbenzoquinone injection. Eight mice were used for each group (6 for NLX alone). *P<0.05, **P<0.01, significantly different from each vehicle control group. Fig. 2. Antinociceptive activities of BL-2401 and codeine phosphate in the tail pressure test in mice. Left panel: 0, vehicle; 0, BL-2401 400 mg/kg. Right panel: 0, vehicle; A, codeine phosphate, 20 mg/kg; 0, 40 mg/kg; 0, 80 mg/kg. Each dose was administered orally. n=6-9. *P<0.05, **P<0.01, significantly different from each vehicle control group. Data were pre viously analyzed by repeated measures analysis of variance, indicating significant (P<0.01) interaction between treatment and time in both experiments.

p.o.) produced a modest antinociceptive effect (Fig. 2), (Fig. 3). Aspirin had no effect; the mean and S.E. for 12 while codeine phosphate produced a marked antinocicep mice were 147.8± 14.8 and 163.0± 15.8 (sec) for vehicle tive effect at doses of 20-80 mg/kg. Aspirin (800 mg/kg) and 800 mg/kg, respectively. The licking latency was not had no significant effect. In the mouse hot-plate test, BL affected by any doses. 2401 significantly prolonged the jump latency at doses of In the adjuvant-induced arthritic rats, hyperalgesia was 200 and 400 mg/kg (Fig. 3). Codeine phosphate showed observed throughout the test period; the pressure significant prolongation at doses of 20 and 40 mg/kg threshold for arthritic rats (119± 10 g, n=20, day 14) was

Fig. 3. Antinociceptive effects of BL-2401 and codeine phosphate in the hot-plate test in mice. Each dose was administered orally 30 min before testing. n=11-12. *P<0.05, **P<0.01, significantly different from each vehicle control group. Fig. 4. Antinociceptive effects of BL-2401 and codeine phosphate on the paw pressure threshold in adjuvant-induced arthritic rats. BL-2401 and codeine phosphate were orally administered once a day for 7 days commencing day 14. Left panel: 0, vehi cle; Is, BL-2401, 30 mg/kg; 0, BL-2401, 100 mg/kg. Right panel: 0, vehicle; 0, codeine phosphate, 100 mg/kg. Seventeen rats were used for BL-2401 and codeine phosphate and 20 rats for the vehicle. *P <0.05, **P <0.01, significantly different from the vehicle control group on each day. "Pre" in the abscissa indicates the threshold measured before administration on day 14.

significantly lower than that for normal rats (212± 11 g, inhibitory effect on only enkephalinase with an IC50 of 74 n=20, day 14). BL-2401 partially but significantly nM. Thiorphan, a prototype enkephalinase inhibitor, in relieved the hyperalgesia at doses of 30 and 100 mg/kg hibited enkephalinase with an IC50 of 2.6 nM. In the ex (Fig. 4). However, its dose-response relation was not vivo experiment, enkephalinase activity in the striata was clear. Codeine phosphate (100 mg/kg) markedly in measured 30 min after oral administration of BL-2401 at creased the pressure threshold, which rose over the nor doses of 5, 25 and 100 mg/kg. The enkephalinase activ mal level. One and 8 days after the last administration, the thresholds in all treated groups returned to the level for the vehicle control group. BL-2401 and codeine phos phate did not affect the hind paw volume.

Antidepressant-like effect In the mouse forced-swimming test, BL-2401 sig nificantly reduced the duration of immobility at a dose of 100 mg/kg (Fig. 5). The decrease induced by BL-2401 (100 mg/kg) was reversed by s.c. administered naloxone hydrochloride (0.5 mg/kg).

Enkephalinase inhibitory activity BL-2240 was used as an active compound of BL-2401 in the in vitro experiment. BL-2240 inhibited enkephalinase in a concentration-dependent manner with an IC50 of 5.2 nM. The kinetic experiment using various concentrations of substrate and BL-2240 demonstrated that the inhibition by BL-2240 was competitive. BL-2240 Fig. 5. Antidepressant-like effect of BL-2401 in the forced swim also inhibited dipeptidylaminopeptidase with an IC50 of ming test in mice. BL-2401 was administered orally 1 hr before test 8.3 riM, although the potency was 1/1000 times weaker ing. Naloxone hydrochloride was administered s.c. 15 min before than that against enkephalinase. BL-2240 did not inhibit testing at a dose of 0.5 mg/kg. n = 5 9. **P < 0.01, significantly aminopeptidase up to 100 eM. BL-2401 showed weak different from the vehicle control group. Fig. 6. Inhibitory effect of orally administered BL-2401 on the enkephalinase activity in the striata in mice. BL-2401 was ad ministered orally 30 min before dissection of the striata. n=7-10. **P<0 .01, significantly different from the vehicle control group.

Fig. 8. Time course of the DAME potentiation induced by BL 2401 in the tail pressure test in mice. DAME (1 pg/mouse) was given ity in the striata was dose-dependently inhibited (Fig. 6); i.c. at various intervals after the oral administration of BL-2401 (50 the inhibitory rates were 24.0%, 41.107oand 63.6% for 5, and 100 mg/kg), and pressure threshold was measured 5 min after 25 and 100 mg/kg, respectively. Furthermore, the time DAME injection. El, vehicle (p.o.) + DAME; ®, BL-2401 (50 course study showed that BL-2401 (100 mg/kg) produced mg/kg, p.o.) + DAME; ®, BL-2401 (100 mg/kg, p.o.) + DAME. n=4-8. **P<0.01, significantly different from DAME alone. potent inhibition throughout 8 hr after oral administra tion; the inhibitory rates were 80.5%, 76.6%, 67.2% and

48.4% at 1, 2, 4 and 8 hr after administration, respec tively. In the DAME potentiation test, BL-2401, when given 30 min before DAME, dose-relatedly potentiated the antinociceptive effect of DAME (Fig. 7). In addition, time-course studies of the potentiation induced by BL 2401 were performed at doses of 50 and 100 mg/kg (p.o.). As shown in Fig. 8, significant potentiation was still detected 8 hr after the administration of either dose of BL-2401. BL-2401 alone (up to 100 mg/kg, p.o.) did not significantly affect the pressure threshold.

DISCUSSION

In the nociceptive tests using acute experimental pain models, BL-2401 produced an antinociceptive effect after oral administration, and its antinociceptive profile was different from that of codeine phosphate, an opioid , and aspirin, a non-steroidal anti-inflammatory Fig. 7. Potentiation of antinociceptive activity of (D-Ala2,Met5) drug (NSAID). The minimum effective dose (MED)s of enkephalin (DAME) by BL-2401 in the tail pressure test in mice. BL-2401 was orally administered 30 min before i.c. injection of BL-2401 were 10 mg/kg (phenylbenzoquinone writhing DAME (1 ,ug/mouse). [:1, vehicle (p.o.) + saline (i.c.); /, vehicle test), 40 mg/kg (acetic acid writhing test), 200 mg/kg (hot (p.o.) + DAME; A, BL-2401 (10 mg/kg) + DAME; 0, BL-2401 plate test) and 400 mg/kg (tail pressure test). The differ (50 mg/kg) + DAME; 0, BL-2401 (100 mg/kg) + DAME. ence in effective doses among tests was larger in compari n = 7 8. **P < 0.01, significantly different from DAME alone. Data were previously analyzed by repeated measures analysis of variance, son with that of codeine phosphate; the MEDs of codeine indicating significant (P<0.01) interaction between treatment and were 5, 10, 20 and 20 mg/kg, respectively. Furthermore, time. in the hot plate and the tail pressure tests, the effect elic ited by BL-2401 occurred to a small degree compared to bulbectomized rats (Y. Seto, unpublished data). These that elicited by codeine phosphate. On the other hand, results indicate that BL-2401 possess an antidepressant aspirin showed antinociceptive effects in the writhing tests like effect after oral administration. We are interested in only, and its anti-writhing potency in rats (acetic acid the emotional influence on the pain suppression and the writhing test) was more potent than that in mice (phenyl roles of endogenous opioids in emotion, and further stud benzoquinone writhing test), similar to the potency ratio ies are necessary to clarify these points. for other NSAIDs (30). Thus, BL-2401 was demonstrated In the present study, the antinociceptive and anti to possess a characteristic antinociceptive profile that was -like effects of BL-2401 were antagonized by different from those of opioid agonists and NSAIDs. naloxone hydrochloride, suggesting the involvement of Chronic pain is demonstrated to differ from acute pain endogenous opioid systems in the effects of BL-2401. in certain aspects such as the nervous route of the per Since BL-2401 and BL-2240 did not show direct binding ception (31). It is conceivable that a novel compound activity in an assay using isolated guinea shows different antinociceptive potencies against chronic pig ileum longitudinal muscle (A. Kita, unpublished and acute pain. Thus, the antinociceptive activity of BL data), the actions of BL-2401 may be mediated by en 2401 was tested in adjuvant-induced arthritic rats as a dogenous opioids, indicating the difference between BL model of chronic pain. BL-2401 relieved hyperalgesia ob 2401 and codeine phosphate as to the mode of the anti served in the arthritic rats, and this effect was not at nociceptive action. Chipkin (8) has postulated that tenuated by repeated administration. The reason why the enkephalinase inhibitors produce an antinociceptive significant effect appeared in the latter part of dosing term effect, only when endogenous enkephalinergic systems is unclear. At least, it is unlikely caused by the accumula are active in response to noxious stimuli. The activation tion of BL-2240 or BL-2401 in the body after repeated of endogenous opioid systems is estimated by certain dosing because on day 21 (one day after the last admin features, i.e., hyperalgesia induced by naloxone, and istration), the pressure thresholds in the BL-2401-treated increase in the release of endogenous opioids elicited groups reduced to the level in the vehicle group. The effect by noxious stimuli. It has been previously reported that induced by BL-2401 was slight, unlike the effect of naloxone hydrochloride (1 mg/kg, i.p.) shows an algesic codeine phosphate, which elicited an increase over the effect in the writhing test and the hot plate test (only jump threshold for normal rats. This different potency may be latency) (34), and acetic acid stimulus increases the release related to the distinct mechanism of actions of BL-2401 of enkephalin (35). The positive effects of BL-2401 in the and opioid analgesics. BL-2401 did not show an anti above-mentioned tests suggest that the antinociception inflammatory effect, unlike the known action of NSAIDs, elicited by BL-2401 depends on the activation of the en which decrease the edema of the arthritic paws. NSAIDs dogenous opioid systems. produce their antinociceptive and anti-inflammatory ac To further clarify the mechanism responsible for tion via inhibiting cyclooxygenase and often cause gastro the actions of BL-2401, its inhibitory activity on intestinal side-effects. BL-2401 may be without such enkephalinase was investigated using in vitro, ex vivo and side-effects because of the different mechanism of action. in vivo assays. In the in vitro study, BL-2240 inhibited These findings suggest that BL-2401 may have an anti enkephalinase selectively among the enkephalin-de nociceptive effect against chronic pain. grading enzymes. The ex vivo study indicated that BL Pain, especially a chronic one, and emotions are 2401 given orally reached the brain and inhibited thought to form a vicious circle (32). Emotions such as enkephalinase there. In addition, based on the ability of anxiety, fear and depression often amplify pain; mean BL-2401 to potentiate DAME-induced antinociception, it while, pain may serve as a stressor that causes such emo is considered that BL-2401 may inhibit the degradation of tions. This is supported by the application of antianxiety DAME, and that of endogenous enkephalins as well. The and antidepressant drugs as adjuvants for clinical chronic effective doses and the time course for ex vivo and in vivo pain (33). The antidepressant-like activity of BL-2401 was enkephalinase inhibition elicited by BL-2401 were similar examined in the forced swimming test which is useful for to those for its anti-writhing activity, suggesting that the screening of . In this test, BL-2401 antinociceptive effect of BL-2401 is due to its inhibitory decreased the duration of immobility. It is reported, action on enkephalinase. however, that some compounds with locomotor As already reported, RB101, which inhibits both activity might cause false-positive effect in this enkephalinase and aminopeptidase, shows a potent anti test (26). BL-2401 slightly increased locomotor activity in nociceptive effect against thermal or electrical nocicep mice (Y. Seto et al., unpublished data), though not inten tive stimuli (14) after parenteral administration. In con sively like morphine. In addition, BL-2401 inhibited the trast, the antinociceptive potency of BL-2401 on the acute incidence of muricide behavior observed in olfactory thermal or mechanical stimuli was modest, similar to a selective enkephalinase inhibitor such as SCH34826 (15). 937-944 (1986) However, BL-2401 showed both antinociception and anti 12 Baamonde A, Dauge V, Ruiz-Gayo M, Fulga IG, Turcaud S, depressant-like effects after oral administration. Fournie-Zaluski M-C and Roques BP: Antidepressant-type effects of endogenous enkephalins protected by systemic RB101 In conclusion, the property of BL-2401 as a potent and are mediated by opioid 5 and dopamine D1 receptor stimula selective enkephalinase inhibitor was demonstrated in the tion. Eur J Pharmacol 216, 157-166 (1992) in vitro, the ex vivo and the in vivo studies. When ad 13 Roques BP, Fournie-Zaluski M-C, Soroca E, Lecomte JM, ministered orally, BL-2401 produced an antinociceptive Malfloy B, Llorens C and Schwartz J-C: The enkephalinase in effect mediated by opioid systems, although the effect was hibitor thiorphan shows antinociceptive activity in mice. Nature modest against acute mechanical and physical stimuli. 288, 286-288 (1980) The different efficiency of BL-2401 from opioid analgesics 14 Noble F, Soleilhac JM, Soroca-Lucas E, Turcaud S, Fournie Zaluski MC and Roques BP: Inhibition of the enkephalin- and NSAIDs in various antinociceptive tests may be metabolizing enzymes by the first systemically active mixed based on the different mechanism of action. Furthermore, inhibitor prodrug RB 101 induces potent analgesic responses in BL-2401 produced antidepressant-like effects via opioid mice and rats. J Pharmacol Exp Ther 261, 181-190 (1992) systems after oral administration to mice. 15 Chipkin RE, Berger JG, Billard W, Iorio LC, Chapman R and Barnett A: Pharmacology of SCH 34826, an orally active Acknowledgments enkephalinase inhibitor analgesic. J Pharmacol Exp Ther 245, We thank Mr. Y. Nakamura, Mr. T. Sasagawa, Ms. J. Nishino 829-838 (1988) and Dr. T. Mimura for synthesis of compounds; Mr. K. Hayashi 16 Foong FW, Nakagawa N, Katsube N, Aishita H, Okegawa T and Ms. M. Miyamoto for pharmacokinetic study; and Drs. M. and Kawasaki A: A novel enkephalinase inhibitor as an orally Oka, K. Hosoki, K. Takeyama, T. Sawayama and T. Karasawa for active analgesic. Pain Suppl 5, S198 (1990) helpful advice. 17 Mimura T, Nakamura Y, Nishino J, Sawayama T, Komiya T, Deguchi T, Kita A, Nakamura H and Matsumoto J: A novel REFERENCES class of enkephalinase inhibitors containing a C-terminal sulfo group. J Med Chem 35, 602-608 (1992) 1 Hughes J, Smith TW, Kosterlitz HW, Fothergill LA, Morgan 18 Dworkin RH and Gitlin MJ: Clinical aspects of depression in BA and Morris HR: Identificationof two related pentapeptides chronic pain patients Clin J Pain 7, 79-94 (1991) from the brain with potent agonist activity. Nature 258, 19 Siegmund E, Cadmus R and Lu G: A method for evaluating 577-579 (1975) both non- and narcotic analgesics. Proc Soc Exp Biol 2 Martin WR: Pharmacology of opioids. Pharmacol Rev 35, Med 95, 729-731 (1957) 283-323 (1983) 20 Nakamura H, Ishii K, Yokoyama Y, Imazu C, Shimoda A, 3 Olson GA, Olson RD and Kastin AJ: Endogenous : Kadokawa T and Shimizu M: Central actions of AD-1211, an 1994. Peptides 16 1517-1555 (1995) analgesic lacking common opiate features. Eur J Pharmacol 4 Minami M and Satoh M: Molecular biology of the opioid 106, 345-356 (1984) receptors: structures, functions and distributions. Neurosci Res 21 Litchfield JT and Wilcoxon F: A simplified method of evalu 23, 121-145 (1995) ating dose-effect experiments. J Pharmacol Exp Ther 96, 99 5 Horwell DC: Kappa opioid analgesics.Drugs of the Future 13, 113(1949) - 1061-1071 (1988) 22 Ueda H, Amano H, Shiomi H and Takagi H: Comparison of 6 Roques BP, Beaumont A, Dauge V and Fournie-Zaluski MC: the analgesic effects of various opioid peptides by a newly Peptidase inactivation of enkephalins: Design of inhibitors and devised intracisternal injection technique in conscious mice. Eur biochemical,pharmacological and clinicalapplication. In Hand J Pharmacol 56, 265-268 (1979) book of Experimental Pharmacology, Vol104/I, Opioids I, 23 Niemegeers CJE, Van Bruggen JAA and Janssen PAJ: Edited by Herz A, pp 547-584, Springer-Verlag,Berlin (1993) , a potent antagonist of acetic acid-induced writhing 7 Schwartz JC: Metabolism of enkephalins and the inactivating in rats. Arzneimittelforschung 25, 1505-1509 (1975) neuropeptidaseconcept. Trends Neurosci 6, 45-48 (1983) 24 Nakamura H and Shimizu M: Comparative study of 1-cyclo 8 Chipkin RE: Inhibitors of enkephalinase:the next generation of hexyl-4-(1,2-diphenylethyl)-piperazine and its enantiomorphs analgesics.Drugs of the Future 11, 593-606 (1986) on analgesic and other pharmacological activities in experimen 9 Nabeshima T, Katoh A and Kameyama T: Inhibition of tal animals. Arch Int Pharmacodyn Ther 221, 105-121 (1976) enkephalin degradation attenuated stress-inducedmotorsupres 25 Eddy NB and Leimbach D: Synthetic analgesics. II. Dithienyl sion (conditioned suppression of motility). J Pharmacol Exp butenyl and dithienylbutylamines. J Pharmacol Exp Ther 107, Ther 244, 303-309 (1988) 385-393 (1953) 10 Katoh A, Nabeshima T and Kameyama T: Behavioral changes 26 Porsolt RD, Bertin A and Jalfre M: Behavioural despair in induced by stressful situations: Effects of enkephalins, dynor mice: a primary screening test for antidepressants. Arch Int phin, and their interactions. J Pharmacol Exp Ther 253, Pharmacodyn 229, 327-336 (1977) 600-607 (1990) 27 Gorenstein C and Snyder SH: Two distinct : 11 Lecomte JM, Costentin J, VlaiculescuA, Chaillet P, Marcais solubilization, partial purification and separation from angio Collado H, Llorens-Cortes C, Leboyer M and Schwartz- J-C: tensin converting enzyme. Life Sci 25, 2065-2070 (1979) Pharmacologicalproperties of acetorphan, a parenterally active 28 Vogel Z and Alstein M: The adsorption of enkephalin to porous "enkephalinase" inhibitor . J Pharmacol Exp Ther 237, polystyrene beads: a simple assay for enkephalin hydrolysis. FEBS Lett 80, 332-336 (1977) 32 Markenson JA: Mechanisms of chronic pain. Am J Med 101, 29 Lowry OH, Rosebrough NJ, Farr AL and Randall RJ: Protein Suppl 1A, 6S-18S (1996) measurement with the Folin phenol reagent. J Bio Chem 193, 33 Katz WA: Approach to the management of nonmalignant pain. 265-275 (1951) Am J Med 101, Suppl 1A, 54S-63S (1996) 30 Nakamura H, Yokoyama Y, Motoyoshi S, Ishii K, Imazu C, 34 Costentin J, Vlaiculescu A, Chaillet P, Natan LB, Aveaux D Seto Y, Kadokawa T and Shimizu M: The pharmacological and Schwartz J-C: Dissociated effects of inhibitors of profile of 2-(8-methyl-10,11-dihydro-l1-oxidibenz[b,f]oxepin enkephalin-metabolizing peptidase or naloxone on various 2-yl)propionic- acid (AD-1590), a new non-steroidal anti-inflam nociceptive responses. Eur J Pharmacol 123, 37-44 (1986) matory agent with potent antipyretic activity. Arzneimittelfor 35 Cesselin F, Le Bars D, Bourgoin S, Artaud F, Gozlan H, Clot schung 33, 1555 -1569 (1983) AM, Besson JM and Hamon M: Spontaneous and evoked re 31 Dray A, Urban L and Dickenson A: Pharmacology of chronic lease of methionine-enkephalin-like material from the rat spinal pain. Trends Pharmacol Sci 15, 190-197 (1994) cord in vivo. Brain Res 339, 305-313 (1985)