Synthesis and receptor binding activity of elephant β-endorphin,a β-endorphin homolog with highly potent analgesic activity#

Elephant β-endorphin and its analog, elephant β-endorphin(6-31) were synthesized by standard solid phase method. Receptor binding activity showed that elephant β-endorphin was five to six times more potent than human β-endorphin in its ability to bind to opiate receptors on rat brain membrane. In a previous study (Wong, C.-L., Wai, M.-K., Cheng, H.-C., Chung, D. & Yamashiro, D (1990) Clinical and Experimental Pharmacology and Physiology 16 , 33–37), tail flick test for intracerebroventricularly administered β-endor-phin showed that the antinociceptive potency of elephant β-endorphin was seven to eight times higher than that of human β-endorphin in mice. Results from both studies suggest that elephant β-endorphin was a much more potent antinociceptive agent than human β-endorphin in tail flick test and its higher analgesic activity might be due to its higher affinity for opiate receptors in the brain.     

Agonist and antagonist properties of buprenorphine, a new antinociceptive agent.

1. Buprenorphine is a highly lipophilic derivative of oripavine. In rodent antinociceptive assays (writhing, tail pressure), buprenorphine had an action which was rapid in onset and of long duration; it was 25-40 times more potent than morphine after parenteral injection and 7-10 times more potent after oral administration. 2. The log dose-response relationship for buprenorphine was curvilinear in mouse and rat tail flick tests with the antinociceptive effect decreasing at higher, non-toxic doses. 3. Tolerance developed to the antinociceptive activity of buprenorphine in mice. 4. No signs of abstinence were observed on naloxone challenge or after abrupt withdrawal in monkeys receiving buprenorphine chronically for one month. 5. Buprenorphine antagonized the antinociceptive actions of morphine in mouse and rat tail flick tests but was an ineffective antagonist in the rat tail pressure test. 6. Buprenorphine precipitated signs of abstinence in morphine-dependent mice and monkeys but not in morphine-dependent rats. 7. Buprenorphine produced Straub tails in mice. This effect was not antagonized when the animals were pretreated with naloxone. However, in the rat tail pressure test high doses of diprenorphine antagonized established antinociceptive effects of buprenorphine. 8. It is concluded that buprenorphine represents a definite advance in the search for a narcotic antagonist analgesic of low physical dependence potential.     

Central components in the antinociceptive activity of dipyrone in mice

Dipyrone is classified as a nonsteroidal anti-inflammatory drug. It has analgesic, antipyretic and anti-inflammatory properties and exerts its analgesic effect via both peripheral and central action. Dipyrone at the dose of 250 and 500 mg/kg showed dose-dependent antinociceptive activity in the hot plate, tail flick tests to radiant heat and tail clip test and the writhing test induced by acetic acid in mice. The antinociceptive effects of dipyrone (500 mg/kg) were antagonized by naloxone (1, 2, 5 mg/kg) in the tail flick test to radiant heat and tail clip test and hot plate tests but not in the writhing test. Cyproheptadine (100 g/kg) decreased the antinociceptive effect of dipyrone. There was an increase in the antinociceptive effects of dipyrone (500 mg/kg) when combined with buspiron (0.5 mg/kg) in the tail flick test to radiant heat and tail clip test. The results provide evidence for a central antinociceptive effect of dipyrone antagonized by naloxone which suggests that its activity may also involve the serotoninergic system.     

Antinociceptive property of intrathecal and intraperitoneal administration of a novel water-soluble isoindolin-1-one derivative, JM 1232 (-) in rats

Antinociceptive effects of a new water-soluble sedative agent, an isoindolin-1-one derivative, (-)-3-[2-(4-methyl-1-piperazinyl)-2-oxoethyl]-2-phenyl-3,5,6,7-tetrahydrocyclopenta[f]isoindol-1-(2H)-one (JM 1232 (-)) were studied in two different nociceptive models of rats. Sprague-Dawley rats with lumbar intrathecal catheters were tested for their thermal tail withdrawal response and for their paw flinches by formalin injection after intrathecal administration of various doses of JM 1232 (-). The effects of intraperitoneal administration were also investigated. In addition, behavioral side effects were examined. When antinociceptive effects were observed, the effects of pretreatment with flumazenil or naloxone for the maximum effective dose were evaluated. Eight rats were used in each dose of each group. Intrathecal JM 1232 (-) increased the tail flick latency and decreased the number of flinches in both phases 1 and 2 of the formalin test. These changes were antagonized by intrathecal flumazenil but not by naloxone. Intraperitoneal JM 1232 (-) had no effects on the tail flick latency, but decreased the number of flinches in both phases 1 and 2 of the formalin test. The latter was antagonized by intraperitoneal flumazenil and naloxone with bigger effects of flumazenil. Intraperitoneal JM 1232 (-) 3000 microg induced reversible motor disturbance. In conclusion, intrathecal JM 1232 (-) exerts antinociceptive effects on acute thermal and inflammatory stimuli through benzodiazepine-GABA(A) receptors in the spinal cord. Intraperitoneal JM 1232 (-) was antinociceptive only against inflammatory stimulus and this is mediated mainly by benzodiazepine-GABA(A) receptors, but partially by micro-opioid receptors in the brain.     

Studies on o-methylflavinantine

The antinociceptive effects of O-methylflavinantine (OMF), a morphinandienone alkaloid, were investigated in the mouse hot plate and abdominal constriction tests (nociceptive agents: 5-Hydroxytryptamine, acetylcholine, bradykinin, prostaglandin, E (1) (PGE (1), formic acid and phenylquinone). The potency of OMF in the hot plate test was approximately 10 times less than that of morphine and the effect was naloxone reversible. In the abdominal constriction test, morphine was 78-650 times more potent than OMF, depending on the nociceptive agent used, but a higher dose of naloxone was necessary to reverse the response to formic acid. Pretreatment of mice with reserpine (1 mg/kg, s.c., 24 h) reduced and alpha-methyl-p-tyrosine (200 mg/kg, i.p., 3 h) potentiated the antinociceptive effects of both morphine and OMF in the hot plate test. The results are considered to indicate that OMF possesses centrally mediated antinociceptive activity which is similar to that of morphine.     

SHORT COMMUNICATION: EFFECT OF OESTRADIOL REPLACEMENT ON SWIM-INDUCED ANTINOCICEPTION IN OVARIECTOMIZED MICE

1. A 30 s swim in water at 30 degrees C reduced the number of abdominal constrictions produced in female mice by intraperitoneally administered acetic acid and this antinociceptive effect was antagonized by naloxone. 2. Oophorectomy abolished the development of antinociceptive activity in response to swim. 3. The development of antinociceptive activity and naloxone antagonism were restored in oophorectomized mice maintained on daily oestradiol injection. 4. The present study indicates that the increase in abdominal constriction response to acetic acid in oophorectomized mice was due to the removal of the source of female sex hormone.     

Spinal antinociception by Tyr-D-Ser(otbu)-Gly-Phe-Leu-Thr, a selective delta-opioid receptor agonist.

The spinal antinociceptive potency of the delta-opioid receptor agonist, Tyr-D-Ser(otbu)-Gly-Phe-Leu-Thr (DSTBULET), was studied in rats. The tail flick test was used as nociceptive stimulus and the rotarod test was used to detect any motor or sedative effects. A dose-response curve was also made for the mu-opioid receptor agonist, morphine. The ED50 for DSTBULET was 0.3 micrograms (0.4 nmol) and a near 100% maximum effect was achieved with 5 micrograms (7.5 nmol). No motor or sedative effects were detected. Antinociception by DSTBULET was antagonized by s.c. naltrindole (1 mg/kg), a selective delta-opioid receptor antagonist, and naloxone (1 mg/kg), a non-selective opioid receptor antagonist. The ED50 for morphine was 0.5 micrograms (1.0 nmol) and the antinociceptive effects were not antagonized by naltrindole (1 mg/kg). The results evidence further the important role of the delta-opioid receptor in spinal nociceptive processing.     

Implication of endogenous opioid mechanism in the production of the antinociceptive effect induced by psychological stress in mice

Psychological (PSY) stress using the communication box produced a short-lasting antinociceptive effect which was less potent than that induced by physical stress such as footshock (FS) and forced swimming (SW) in mice. Naloxone completely antagonized PSY-stress induced analgesia (SIA) when the analgesia was measured by the tail pinch (TP) method; however, the antagonist did not reverse the effect in the tail flick (TF) assay. On the other hand, FS-SIA was antagonized by naloxone in both methods, while naloxone failed to reverse SW-SIA in either TF or TP assessment. Daily exposure to psychological stress developed tolerance to the analgesia. One-way cross-tolerance between PSY-SIA and morphine and the naloxone antagonism of PSY-SIA by the tail pinch method lead to the suggestion that an endogenous opioid system may be involved in the underlying mechanism for its production. On the contrary, from the findings of cross-tolerance between SW- or FS-SIA and the lack of naloxone antagonism in the TF method, the involvement of a more complicated mechanism is suggested in PSY-SIA. In both tests, U-50488H, a selective kappa-agonist, produced profound analgesia; however, no appreciable antagonism of naloxone was found in the TF test, whereas the effect was completely blocked by naloxone in the TP test. From the similarity in naloxone antagonism of PSY-stress and U-50488H induced analgesia, the participation of a common mechanism which may be mediated by kappa-opioid receptors, is suggested in the production of PSY-SIA.     

Differential sensitivity of antinociceptive tests to opioid agonists and partial agonists.

1. The antinociceptive activity of a range of opioid agonists and agonist-antagonist analgesics was determined in mice by use of the 55 degrees C hot plate and abdominal constriction assays. 2. Opioid agonists were approximately 10 times more effective in the abdominal constriction assay. 3. The agonist-antagonists produced analgesia only in the abdominal constriction assay, and antagonized the antinociceptive action of opioid agonists in the 55 degrees C hot plate test. 4. These differences were shown to be attributable to the different levels of stimulus employed in the two tests. 5. By comparing the antagonist potencies of the agonist-antagonists in the 55 degrees C hot plate test with their antinociceptive ED50 values in the abdominal constriction assay, an index of intrinsic activity was calculated.     

Sex difference in naloxone antagonism of swim stress-induced antinociception in mice

A 30 sec swim in water at 30 degrees C reduced the number of abdominal constrictions produced in mice by the intraperitoneal injection of acetic acid. In male mice this reduction in abdominal constrictions induced by swim stress was not affected by prior subcutaneous administration of naloxone hydrochloride. However, in the female mice naloxone hydrochloride administered 5 min, 10 min, or 15 min before the 30 sec swim dose-dependently antagonized the effect of swim stress on the abdominal constriction response to i.p. acetic acid. In view of the possibility that female mice may perceive the stress at different intensity from those of the male, the effects of different swimming durations on female mice were also studied. It was found that a 15 sec swim in water at 30 degrees C was sufficient to induce antinociceptive response to i.p. acetic acid. The antinociceptive effect was greater if the duration of swim was extended to 60 sec. In both instances, prior administration of naloxone dose-dependently reduced the antinociceptive effect induced by swimming. These results suggest sex difference in the involvement of endogenous opioid system in swim stress-induced antinociception in mice.     

Tazadolene: A centrally-acting,non-narcotic analgesic

Intravenous tazadolene elevated the threshold stimulus for rabbits to respond to electrical tooth-pulp stimulation. Systemic tazadolene also increased the latency for mice to lick their paws in the hot-plate analgesic test. However, tazadolene did not alter the mouse tail flick, a spinal reflex most sensitive to analgesics acting at spinal sites. Tazadolene analgesia was not antagonized by naloxone. Intracranial (i.c.), but not intraspinal (i.s.), injections of tazadolene were more effective than systemic injections in producing analgesic effects in the hot-plate test. It is concluded that tazadolene is a centrally-acting, non-narcotic analgesic.     

Dual action mechanisms of KK-3, a newly synthesized leu-enkephalin derivative, in the production of spinal analgesic effects

The action mechanism for the production of spinal analgesia of KK-3, tyrosyl-N-methyl-gamma-aminobutylyl-phenylalaninol, was examined by the tail pinch and tail flick methods. Intrathecal KK-3, 2.5, 5 and 10 nmol/mouse, dose-dependently produced an analgesic effect in both methods. In the tail pinch method, the analgesia was suppressed by 2 mg/kg but not by 1 mg/kg of naloxone; however, the analgesic effect was significantly antagonized by 1 and 2 mg/kg Mr2266, a kappa-antagonist. Meanwhile, both naloxone and Mr2266 failed to block the analgesic effect of KK-3 in the tail flick test. Intrathecal capsaicin, 0.3, 3 and 15 nmol/mouse, also produced a dose-dependent analgesic effect in the tail flick test, whereas no appreciable analgesia could be found in the tail pinch test. Neither naloxone nor Mr2266 blocked the analgesic effect of capsaicin. The results indicate that KK-3 may possess two separate pharmacological mechanisms for the production of analgesic effects on the spinal level: one is the depletion of substance P following its release from the spinal cord, and the other is the mediation through kappa-opioid receptors.     

INCREASED NALOXONE POTENCY INDUCED BY PRETREATMENT WITH MORPHINE AND NALBUPHINE IN MICE

1. Both morphine and nalbuphine were effective in suppressing the abdominal constriction response induced by intraperitoneal injection of acetic acid in mice. On a weight to weight basis, nalbuphine was more potent than morphine in this test. However, the effect of nalbuphine was more effectively blocked by naloxone. 2. Pretreatment with morphine 2.0 mg/kg subcutaneously did not alter the antinociceptive effect of either morphine or nalbuphine measured 3 h later. However, naloxone was about 1.4-fold more effective in antagonizing the antinociceptive effect of both drugs in morphine-pretreated mice than in saline-pretreated animals. 3. Pretreatment with nalbuphine (1.0–2.0 mg/kg s.c.) did not alter the antinociceptive effect of either morphine or nalbuphine measured 3 h later, while naloxone was more effective in antagonizing the antinociceptive actions of morphine and nalbuphine. 4. The increases in naloxone potency in antagonizing morphine after nalbuphine pretreatment were not dose-dependent on the amount of nalbuphine in the pretreatment and they were only marginally significant. In addition, these increases were much lower than that induced by morphine pretreatment. 5. On the other hand, the naloxone effectiveness against nalbuphine itself was enhanced to a greater extent than that induced by morphine pretreatment. Furthermore, these increases in naloxone potency showed a dose-dependent relationship to the amount of nalbuphine used in the pretreatment. 6. Based on these results, it was concluded that nalbuphine is an analgesic drug with properties in between those of the full agonist morphine and the partial agonist pentazocine.     

The antinociceptive effect of vaginal stimulation in the rat is reduced by naloxone

Mechanical stimulation of the vagina produces a powerful antinociceptive effect against a variety of noxious stimuli. In rats tested in either the radiant heat tail flick or the warm water tail immersion test the antinociceptive effect of vaginal stimulation was found to be significantly reduced, but not abolished, following the administration of 10 mg/kg of naloxone. These results are in contrast to those of an earlier study in which naloxone was ineffective against the antinociceptive action of vaginal stimulation in a tailshock vocalization paradigm. It therefore appears that the nature of the noxious stimulus used may influence the type of antinociceptive mechanism triggered by vaginal stimulation.     

Role of nitric oxide in benzodiazepines-induced antinociception in mice

The influence of nitric oxide (NO) on antinociceptive activity of diazepam (DZ), chlordiazepoxide (CDP) and clonazepam (CZ) was examined using the writhing test in mice. The effect of DZ was also studied in mice using hot plate and tail flick tests. DZ (1.25, 2.5 and 5 mg/kg), CDP (1.25, 2.5, 5, 10 and 20 mg/kg) and CZ (0.075, 0.3125, 0.625, 1.25 and 2.5 mg/kg) produced significant, dose-dependent (DZ, CDP) antinociception in mice. The benzodiazepines (BZs)-induced antinociception was antagonized by flumazenil (5 mg/kg) and was not changed by naloxone (2.5, 5 and 10 mg/kg), except that of CZ, which was reversed by 5 mg/kg of naloxone. NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) as well as 7-nitroindazole (7-NI) intensified antinociceptive activity of BZs. The antinociceptive effect resulting from co-administration of L-NAME with CZ and 7-NI with CDP was reversed by L-arginine. Methylene blue (MB) increased, whereas L-arginine (but not D-arginine) decreased antinociceptive effects of the studied BZs. These results suggest that the NO-cGMP pathway is involved in the mechanism of BZs-induced antinociception in the writhing test in mice.     

Alpha 2-adrenoceptor- and D2-dopamine receptor-mediated analgesic response of B-HT 920.

The involvement of D2-dopamine receptors in the antinociceptive action of B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-(4H) thiazolo-(4,5d)-azepine) has been investigated in mice. B-HT 920 (0.1-2.0 mg kg-1) and apomorphine (0.1-2.0 mg kg-1) produced a dose-dependent increase in tail flick latency. Analgesia induced by apomorphine was blocked by the D2-antagonist, haloperidol (1 mg kg-1) but not by the opioid antagonist, naloxone (1 mg kg-1). The antinociceptive action of B-HT 920 was potentiated by the D1-agonist SKF 38393 (5 mg kg-1), an action antagonized by haloperidol. The selective alpha 2-adrenoceptor blocking drug yohimbine (1 mg kg-1) and naloxone (1 mg kg-1) blocked the antinociceptive action of B-HT 920 (1 mg kg-1). Haloperidol, however, failed to modify the B-HT 920-induced increase in tail flick latency. B-HT 920 and apomorphine reversed reserpine (2 mg kg-1) 4 h-induced hyperalgesia. The reversing action of apomorphine was blocked by haloperidol but not by yohimbine. Thus, a role of alpha 2-adrenoceptors and D2-dopamine receptors is postulated in the antinociceptive action of B-HT 920.     

Further investigations on the antinociceptive activity of tuftsin analogs.

C-Terminal dipeptide fragment of tuftsin, Pro-Arg, substituted by D-amino acids, and tuftsin analogs with n-hexyl- and n-heptylamine coupled to their C-termini were synthesized by a classical method in solution and their antinociceptive activity was measured by tail flick immersion test (0.4 microM/icv). D-Pro-D-Arg and D-Pro-L-Arg showed an analgesic activity, with the duration of 60 and 40 min, respectively. The strong behavioral effects observed after injection of D-Pro-D-Arg were decreased by naloxone. L-Pro-D-Arg and Thr-Lys-Pro-Arg-HxA display no antinociceptive effect; the tetrapeptide amide showed some toxicity effects. Thr-Lys-Pro-Arg-HpA was very toxic and caused death of all experimental animals. This effect was not influenced by previous injection of naloxone.     

1,6-二(4-苯乙基-1-甲基-1-哌嗪基)己烷二溴化物的镇痛活性研究

目的　研究哌嗪类新化合物 1,6 二 (4 苯乙基 1 甲基 1 哌嗪基 )己烷二溴化物 (97 9 G4 )的镇痛作用及机制。方法　通过醋酸扭体模型、热板、甩尾、纳洛酮拮抗试验及豚鼠回肠离体标本研究样品的镇痛活性及机制。结果　sc 97 9 G4 5mg·kg-1即可有效抑制小鼠扭体反应 (P <0 0 5 ) ,ID50为 8 8mg·kg-1;sc 4 0mg·kg-1、icv 2 5 μg·kg-1均可延长热板实验的舔足阈 (P <0 0 5 ) ;sc 2 0mg·kg-1可延长甩尾试验的潜伏期 (P <0 0 1) ;纳洛酮可拮抗其镇痛活性 ;97 9 G4可激动豚鼠回肠离体标本上的阿片受体。结论　 97 9 G4具有的镇痛活性 ,主要作用部位在中枢 ,其镇痛活性可被纳洛酮拮抗 ,但作用特点有别于吗啡     

THE ACTIONS OF SOME α-ADRENORECEPTOR AGONISTS AND ANTAGONISTS IN AN ANTINOCICEPTIVE TEST IN MICE

1. It has been shown that a number of sympathomimetic drugs, administered subcutaneously, have potent antinociceptive activity in the mouse abdominal constriction test. These drugs were found to be equieffective antagonists of the nociceptive action of acetic acid and acetylcholine. Of the drugs tested, clonidine was the most potent, being almost 60 times more active than morphine, whilst noradrenaline and oxymetazoline were approximately three and five times less active respectively than clonidine. Phenylephrine was without effect after doses of 2 mg/kg. 2. The regression lines relating the magnitude of the antinociceptive effect of noradrenaline and oxymetazoline to log dose were moved to the right in an approximately parallel manner after subcutaneous administration of piperoxane, 8 and 16 mg/kg. The antinociceptive action of clonidine was also antagonised by piperoxane, but to a lesser extent. Phentolamine had no antagonistic effect on any of these three a-agonists after subcutaneous doses of 16 mg/kg. 3. Oxymetazoline and clonidine, when given by intracisternal injection, were approximately eight-three and fifty times more potent respectively than by subcutaneous administration, and their antinociceptive action was not antagonized by piperoxane, 16 mg/kg subcutaneously, or 50 μg/kg intracisternally. 4. It is postulated that the antinociceptive action of the a-agonists is due to an effect on α-adrenoreceptors located on sensory nerve endings in the peritoneum, and that the affinities of these receptors for the α-agonists and antagonists is different from that shown by either pre- or postsynaptic α-adrenoreceptors. It also appears likely that the antinociceptive action of clonidine and oxymetazoline when given by intracisternal injection involves different mechanisms from their peripheral effects.     

The turnover rate of acetylcholine in brain nuclei of rats injected intraventricularly and intraseptally with alpha and beta-endorphin

The effects of intraventricular injections of alpha-endorphin, beta-endorphin and morphine have been studied on the tail flick test, on catalepsy and on acetylcholine turnover rate in selected nuclei of the rat brain.Intraventricular beta-endorphin was able to induce analgesia and catalepsy as well as a dose-dependent decrease of the turnover rate of acetylcholine in the cortex, hippocampus N. accumbens and globus pallidus. No change has been observed in the N. caudatus. These effects are qualitatively similar to those induced by intraventricular morphine which was approximately 100 times less active. Alphaendorphin at a dose 30 times higher than the minimal effective dose of beta-endorphin was completely inactive. None of the drugs cited above modified acetylcholine or choline content. Pretreatment with naltrexone completely antagonized all the effects induced by beta-endorphin or by morphine. Intraseptal injection of beta-endorphin and morphine selectively decreased the acetylcholine turnover rate in the hippocampus without causing analgesia. This finding supports the idea that opiate receptors and their endogenous ligands regulate neuronal activity which is unrelated to analgesia.These studies also suggest that not all of the brain cholinergic neurones are regulated by opiate receptors and they further indicate that the opioid peptides are inhibitory neuromodulators.