A long-form α-neurotoxin from cobra venom produces potent opioidindependent analgesia

AIM: In light of the antinociceptive activity of the short-chain neurotoxin, cobrotoxin, and other acetylcholine antagonists, the antinociceptive activity and mechanisms of cobratoxin (CTX), a long-chain postsynaptic alpha-neurotoxin, was investigated in rodent pain models. METHODS: CTX was administered intraperitoneally (30, 45, 68 microg/kg), intra-cerebral ventricularly (4.5 microg/kg) or microinjected into periaqueductal gray (PAG; 4.5 microg/kg). The antinociceptive action was tested using the hot-plate and acetic acid writhing tests in mice and rats. The involvement of the cholinergic system and opioid system in CTX-induced analgesia was examined by pretreatment of animals with atropine (0.5 mg/kg, im; or 10 mg/kg, ip) or naloxone (1 and 5 mg/kg, ip). The effect of CTX on motor activity was tested using the Animex test. RESULTS: CTX exhibited a dose-dependent analgesic action in mice as determined by both the hot-plate and acetic acid writhing tests. The peak effect of analgesia was seen 3 h after administration. In the mouse acetic acid writhing test, the intra-cerebral ventricular administration of CTX at 4.5 microg/kg (1/12th of a systemic dose) produced marked analgesic effects. Microinjection of CTX (4.5 microg/kg) into the PAG region did not elicit an analgesic action in rats in the hot-plate test. Atropine at 0.5 mg/kg (im) and naloxone at 1 and 5 mg/kg (ip) both failed to block the analgesic effects of CTX, but atropine at 10 mg/kg (ip) did antagonize the analgesia mediated by CTX in the mouse acetic acid writhing test. Acetylsalicylic acid (300 mg/kg) did not enhance the analgesic effects of CTX. At the highest effective dose of 68 microg/kg the neurotoxin did not change the spontaneous mobility of mice. CONCLUSION: CTX has analgesic effects, which are mediated in the central nervous system though not through the PAG. The central cholinergic system but not opioid system appears to be involved in the antinociceptive action of CTX.     

Autophagy and cathepsin L are involved in the antinociceptive effect of DMBC in a mouse acetic acid-writhing model

Aim:

2-(3′,5′-Dimethoxybenzylidene) cyclopentanone (DMBC) is a novel synthetic compound with antinociceptive activities. The aim of this study was to investigate the roles of the autophagic-lysosomal pathway in the antinociceptive effect of DMBC in a mouse acetic acid-writhing model.

Methods:

Mouse acetic acid-writhing test and hotplate test were used to assess the antinociceptive effects of DMBC, 3-MA (autophagy inhibitor) and Clik148 (cathepsin L inhibitor). The drugs were administered peripherally (ip) or centrally (icv).

Results:

Peripheral administration of 3-MA (7.5–30 mg/kg) or Clik148 (10–80 mg/kg) produced potent antinociceptive effect in acetic acid-writhing test. Central administration of 3-MA or Clik148 (12.5–50 nmol/L) produced comparable antinociceptive effect in acetic acid-writhing test. Peripheral administration of DMBC (25–50 mg/kg) produced potent antinociceptive effects in both acetic acid-writhing and hotplate tests. Furthermore, the antinociceptive effect produced by peripheral administration of DMBC (50 mg/kg) in acetic acid-writhing test was antagonized by low doses of 3-MA (3.75 mg/kg) or Clik148 (20 mg/kg) peripherally administered, but was not affected by 3-MA or Clik148 (25 nmol/L) centrally administered.

Conclusion:

Activation of central autophagy and cathepsin L is involved in nociception in mice, whereas peripheral autophagy and cathepsin L contributes, at least in part, to the antinociceptive effect of DMBC in mice.     

马来酸三甲氧苯丁氨酯的镇痛作用

研究马来酸三甲氧苯丁氨酯（ＴＭ）的镇痛作用，结果显示，腹腔注射ＴＭ２４５、３５０、５００ｍｇ／ｋｇ能显著抑制小鼠扭体反应，５０、７０ｍｇ／ｋｇ能显著提高小鼠热板反应痛阈值，３５、５０、７０ｍｇ／ｋｇ则能够显著提高小鼠压尾痛阈值并减少甲醛致痛评分值，３７５、７００ｍｇ／ｋｇ可显著提高大鼠电刺激痛阈值．连续给小鼠腹腔注射ＴＭ７０ｍｇ／ｋｇ７天，每天用热板法测定小鼠痛阈值，表明其镇痛作用无耐受性．     

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.     

Studies on the mode of action of ambrein as a new antinociceptive compound.

The compound ambrein was isolated from ambergris, which is commonly used as an analgesic in the Saudi folklore medicine. The LD50 of ambrein, given intraperitoneally (i.p.) in mice, was found to be high (7.5 g/kg), and ambrein proved to be a safe compound in this species. In the hotplate test, ambrein was found to possess antinociceptive activity in mice at doses which did not sedate or incapacitate the animals. By the i.p. administration route, ambrein produced antinociception in mice at a dose as low as 10 mg/kg. The antinociceptive activity of ambrein (250 mg/kg i.p.) was inhibited by a noradrenergic neurotoxin (DSP-4) and by naloxone, methysergide or prazosin. It was not influenced by a serotonin depletor, p-chlorophenylalanine. The possible mechanism of ambrein antinociception is discussed.     

去氧乌头碱的抗炎、镇痛和解热作用

本文报告了新近以伏毛铁棒锤中提得的去氧乌头碱(DAC)的抗炎、镇痛和解热作用实验结果,并与已报道过的3-乙酰乌头碱(3AAC)进行比较。DAC0.8mg/kg (ip)显著抑制巴豆油所致小鼠耳廓肿;0.2mg/kg(ip或sc)显著抑制角叉菜胶或甲醛所致大鼠足爪肿、组胺所致大鼠皮肤渗出及醋酸所致小鼠腹腔渗出。于大鼠角叉菜胶足爪肿模型,测得DAC(ip)的抗炎治疗指数(6.38)略高于3AAC(5.92)。醋酸扭体法测得DAC抑制小鼠扭体反应50％的剂量为0.22±0.06mg/kg(sc);热板法测得DAC的小鼠镇痛ED_(50)为0.41±0.10mg/kg(ip),其镇痛治疗指数(6.37)高于3AAC(4.60)。DAC0.24mg/kg(ip)对伤寒副伤寒混合菌苗所致家兔发热有显著解热作用。     

Involvement of serotoninergic mechanism in analgesia by castration and flutamide, a testosterone antagonist, in the rat formalin test

Several studies have suggested that testosterone has a role in nociception. Recently, we have shown that castration and flutamide, a testosterone antagonist, induce analgesia in the late phase of formalin test, which is related to increase of 5-HT levels in the dorsal horn of the lumbar spinal cord. The aim of the present study was to investigate the effect of fluoxetine, a selective serotonin reuptake inhibitor, on castration and flutamide-induced analgesia in order to further explore the role of 5-HT systems in such analgesia. Four weeks after castration, there was an analgesia in the late phase of formalin test, and this was potentiated by acute (0.32 mg kg(-1) ip) treatment of fluoxetine. Furthermore, coadministration of fluoxetine (0.32 mg kg(-1) ip) and flutamide (10 mg kg(-1) ip) produced more antinociceptive effect than those animals receiving fluoxetine and flutamide alone. The analgesic effect of fluoxetine (0.32 mg kg(-1) ip) and flutamide (10 mg kg(-1) ip) was abolished by pretreatment with 5,7-DHT (100 microg/rat it) and naloxone (2 mg kg(-1) ip). In summary, our data suggest that fluoxetine and flutamide have antinociceptive effects in tonic inflammatory pain through functional alteration of serotonergic systems, and their effects are potentiated by coadministration. The possible role of opioidergic system in their antinociceptive effect cannot be neglected.     

Paradoxical effects of opioid antagonist naloxone on SSRI-induced analgesia and tolerance in mice

Selective serotonin reuptake inhibitors (SSRIs) have been used clinically as co-analgesics in various devastating painful conditions. Upon chronic treatment tolerance develops to their analgesic effect, which is often refractory to increasing dose. Although modulation of serotonergic pathways considerably explains their clinical efficacy, numerous reports nevertheless indicate the direct/indirect role of the opioidergic pathway in SSRI-induced analgesia. The present study was designed to investigate the effect, if any, of the opioid antagonist naloxone on SSRIs-induced analgesia and tolerance employing acetic acid-induced writhing assay. Two SSRIs, fluoxetine (FLX), and citalopram (CTP) were used in the study. Acute systemic (5-40 mg kg(-1) i.p.), or intrathecal (5-40 microg per mouse, i.t.) administration of fluoxetine or citalopram exhibited a dose-dependent and significant (p < 0.05) antinociceptive effect. Single systemic (2-5 mg kg(-1) i.p.) or intrathecal (1 microg per mouse, i.t.) administration of opioid antagonist naloxone blocked where as systemic ultra-low dose (10 ng/kg) or intrathecal (0.05 ng) naloxone potentiated the acute antinociceptive effect of both SSRIs (10 mg kg(-1) i.p. and 10 microg i.t.). Animals treated chronically over a 7-day period with SSRIs developed tolerance to their antinociceptive effect. Further, chronic administration of ultra-low dose of naloxone intrathecal (0.05 ng per mouse, i.t.) or systemic (10 ng kg(-1) i.p.) with fluoxetine or citalopram (10 microg i.t.; 5 mg kg(-1) i.p.) over a 7-day period reversed the tolerance to the antinociceptive effect of SSRIs. Thus, in ultra-low doses, naloxone paradoxically enhances SSRIs-induced analgesia and reverse tolerance through spinal and peripheral action. These effects of opioid antagonist naloxone on SSRIs-induced antinociception may have an implication in refractory cases upon chronic use of SSRIs as co-analgesics.     

Antinociceptive profile of the new nicotinic agonist DBO-83

The antinociceptive effect of DBO-83 (3-[6-Cl-pyridazin-3-yl]-diazabicyclo[3.2.1]octane) was examined in mice and rats by using the hot-plate, abdominal constriction, and paw-pressure tests. In both species, DBO-83 (10–20 mg/kg ip in the mouse; 10–20 mg/kg ip in the rat) produced significant antinociception, an effect that was prevented by mecamylamine (2–5 mg/kg ip) but not by atropine (5 mg/kg ip), naloxone (1 mg/kg ip), and CGP 35348 (100 mg/kg ip). DBO-83 antinociception is mediated by a central mechanism of action since it is also effective after icv injection (5–7.5 μg per mouse). By comparing the areas under the curve of several well-known analgesic drugs such as morphine, diphenhydramine, clomipramine, and ketorolac at the highest doses that did not produce overt behavioral side effects, the antinociceptive efficacy of DBO-83 (20 mg/kg ip) was found to be greater than that exerted by the reference drugs. In the antinociceptive dose range, DBO-83 did not impair mouse motor coordination, spontaneous motility, or inspection activity. In vitro experiments showed the ability of DBO-83 to evoke the contractions of non-stimulated guinea pig ileum and the shift to the right of the DBO-83 concentration-response curve by mecamylamine (10^–5 M). On the basis of the above data, it can be postulated that DBO-83 exerted an antinociceptive effect mediated by a central nicotinic activation. Drug Dev. Res. 40:251–258, 1997. © 1997 Wiley-Liss, Inc.     

Antinociceptive activity of Vitex-negundo Linn leaf extract

Tail flick test in rats and acetic acid induced writhing in mice were employed to study the antinociceptive activity of ethanolic leaf extract of Vitex-negundo (VN) (100, 250 and 500 mg/kg, p.o). The effect was compared with meperidine (40 mg/kg, sc) in tail flick method and aspirin (50 mg/kg, p.o) in writhing test as a standard control respectively. An interaction with naloxone hydrochloride was also studied in tail flick method for its mechanism of central analgesic action. The test drug showed significant analgesic activity in dose dependant manner in both the experimental models. In comparison to standard drug (meperidine), more than ten times dose of VN extract was required to produce comparable significant antinociceptive activity. The sub-effective dose (5 mg/kg, po) of VN potentiated the analgesic activity of meperidine (4 mg/kg, sc) and aspirin (25 mg/kg, po). Naloxone (1 mg/kg, sc) did not reverse the analgesic effect of VN extract. Our observations suggest that VN possesses both central and peripheral analgesic activity. The central analgesic action does not seem to be mediated through opioid receptors. It, may prove to be a useful adjuvant therapy along with standard analgesic drug.     

Analgesic effect of benzodiazepines and flumazenil.

1. In the present work the analgesic effect of benzodiazepines (BZD) and flumazenil (FLU) using the writhing test in mice was studied. 2. Intracerebroventricular administration of BZD exhibited a dose-dependent antinociceptive effect when compared to control value. 3. Intracerebroventricular administration of FLU induced a dose-dependent antinociceptive action that was not antagonized by naloxone (NX). 4. BZD administered as subcutaneous pellets produced an antinociceptive action in the writhing test, when compared to control mice, only at relative high doses and was partially antagonized by naloxone. 5. These findings could be explained assuming that NX and/or FLU have partial agonist properties in a common receptor which mediates the antinociceptive action. 6. The antinociceptive action of BZD could be related to an increased release of adenosine, which by itself has analgesic effects.     

Vinpocetine and piracetam exert antinociceptive effect in visceral pain model in mice

The effect of vinpocetine or piracetam on thermal and visceral pain was studied in mice. In the hot plate test, vinpocetine (0.9 and 1.8 mg/kg), but not piracetam, produced a reduction in nociceptive response. Vinpocetine (0.45-1.8 mg/kg, ip) or piracetam (75-300 mg/kg, ip) caused dose-dependent inhibition of the abdominal constrictions evoked by ip injection of acetic acid. The effect of vinpocetine or piracetam was markedly potentiated by co-administration of propranolol, guanethidine, atropine, naloxone, yohimbine or prazosin. The marked potentiation of antinociception occurred upon a co-administration of vinpocetine and baclofen (5 or 10 mg/kg). In contrast, piracetam antagonized antinociception caused by the low (5 mg/kg), but not the high (10 mg/kg) dose of baclofen. The antinociception caused by vinpocetine was reduced by sulpiride; while that of piracetam was enhanced by haloperidol or sulpiride. Either vinpocetine or piracetam enhanced antinociception caused by imipramine. The antinociceptive effects of vinpocetine or piracetam were blocked by prior administration of theophylline. Low doses of either vinpocetine or piracetam reduced immobility time in the Porsolt's forced-swimming test. This study indicates that vinpocetine and piracetam possess visceral antinociceptive properties. This effect depends on activation of adenosine receptors. Piracetam in addition inhibits GABA-mediated antinociception.     

The analgesic effects of peripheral and central administration of melatonin in rats

To explore the site and mechanism of the analgesic action of melatonin, the present study was designed to evaluate the analgesic effects of intraperitoneal (i.p.) and intracerebroventricular (i.c.v. ) administration of melatonin, and to investigate the effect of i.c. v. naloxone on the analgesic effect induced by i.p. melatonin in rats. Antinociception was determined by tail-flick latency to hot water at 50 degrees C. On i.p. administration, melatonin (30, 60 and 120 mg/kg) produced the antinociceptive effect in a dose-dependent manner, with an A(50) of 72.8 mg/kg. Administered i.c.v., melatonin (0.25, 0.5 and 1 mg/kg) also resulted in dose-dependent antinociception, with an A(50) of only 0.693 mg/kg. Injected i.c.v. to rats, 10 microg of naloxone antagonized significantly the antinociceptive effect induced by i.p. melatonin. It is concluded that melatonin has an analgesic effect in rats and the central nervous system (CNS) may be the primary site for melatonin to elicit the response, and the effect of melatonin is related to the central opioid system.     

Effect of chronic and acute administration of fluoxetine and its additive effect with morphine on the behavioural response in the formalin test in rats

Serotonergic systems are involved in the central regulation of nociceptive sensitivity. Fluoxetine, a selective inhibitor of the reuptake of serotonin (5-hydroxytryptamine, 5-HT), was administered orally (0.16, 0.32, 0.8 mg kg(-1) daily for 7 days), intraperitoneally (0.04, 0.08, 0.16 mg kg(-1) day(-1) for 7 days and a single dose of 0.32 mg kg(-1)) and intracerebroventricularly (10 microg/rat) to rats and nociceptive sensitivity was evaluated using the formalin test (50 microL of 2.5% formalin injected subcutaneously). The effect of fluoxetine was also studied in the presence of 5,7-dihydroxytryptamine creatinine sulfate (5,7-DHT) and after co-administration with morphine. Oral (0.8 mg kg(-1)), intraperitoneal (0.16 and 0.32 mg kg(-1)) and intracerebroventricular (10 microg/rat) fluoxetine induced antinociception in the late phase of the formalin test. Furthermore, intrathecal administration of 5-HT (100 microg/rat) induced an analgesic effect. The analgesic effect of fluoxetine (0.16 and 0.32 mg kg(-1), i.p.) and 5-HT (100 microg/rat, i.t.) was abolished by pre-treatment with 5,7-DHT (100 microg/rat, i.t.). In addition, the analgesic effect of 5-HT (100 microg/rat, i.t.) was decreased by pre-treatment with naloxone (2 mg kg(-1), i.p.). Morphine (5 mg kg(-1), i.p.) induced analgesia that was increased by fluoxetine (0.32 mg kg(-1), i.p.). These results suggest that fluoxetine has an antinociceptive effect in tonic inflammatory pain through functional alteration of the serotonergic system and also potentiates the analgesic effect of morphine.     

Antinociceptive and anti-inflammatory effects of the monoterpene α,β-epoxy-carvone in mice

α,β-Epoxy-carvone (EC) is a monoterpene found in the essential oils of many species of plants. It can also be obtained by organic synthesis. EC exerts a depressant effect on the central nervous system and is also known to have anticonvulsant, antimicrobial and antioxidant effects. The present study investigated the antinociceptive and anti-inflammatory effects of EC. Intraperitoneal administration of EC at doses of 100, 200 or 300 mg/kg promoted a significant antinociceptive effect, as shown in the acetic acid-induced abdominal writhing test. EC also provoked a reduction in formalin-induced nociception in the first (300 mg/kg) and second phases (200 and 300 mg/kg). In the hot-plate test, an increase in response latency was found at 30 min (at 100, 200 and 300 mg/kg), and at 60 and 120 min (at 300 mg/kg) following administration of EC, an effect that was reversed by naloxone. Intraperitoneal administration of EC (300 mg/kg) inhibited the increased vascular permeability provoked by acetic acid. These findings suggest that EC inhibited the acute inflammatory reaction, with a pronounced peripheral and central antinociceptive effect in mice that is probably associated with activation of the opioidergic system, which appears to play a role in the antinociceptive activity induced by EC.     

Purification and characterization of a novel antinociceptive toxin from Cobra venom (Naja naja atra)

Snake venoms have demonstrated antinociceptive activity, and certain isolated neurotoxins have demonstrated significant analgesia in animal models. Here we report a novel analgesic toxin which was isolated from Naja naja atra and was given the name ‘najanalgesin’. The LD₅₀ of the crude venom and najanalgesin were 0.89 mg/kg and 2.69 mg/kg, respectively. We used the writhing test and hot plate test to evaluate the antinociceptive properties of the crude venom and najanalgesin after intraperitoneal (ip) administration. The analgesic mechanism of najanalgesin was also studied. The response latency time was significantly prolonged in the hot plate test after ip administration of the crude venom of Naja naja atra (0.111-0.445 mg/kg) in a dose-dependent manner. Najanalgesin (1 mg/kg) elicited almost the same antinociceptive effect as that of the crude venom of Naja naja atra at the dose of 0.445 mg/kg and remained for 6 h after intraperitoneal injection, shown by hot plate test. The percentage of increase in the latency time for the venom and the najanalgesin 3 h after drug administration was 96.2% and 112%, respectively. The number of writhes decreased to almost 1/3, 1/6, and 1/12 of the NS (physiological saline) group after intraperitoneal administration of najanalgesin at 0.25, 0.5, and 1.0 mg/kg, respectively. Pretreatment with atropine (1 mg/kg) or naloxone (3 mg/kg) blocked the antinociception of najanalgesin in the hot plate test. Based on the sequence information, najanalgesin is found to be highly homologous with the conventional CTXs (cardiotoxins). To our knowledge, no study had previously reported that a toxin which was homologous with CTXs possessed the antinociceptive activity. Thus, this is the first report that the antinociceptive effect induced by najanalgesin is mediated by cholinergic and opioidergic mechanisms.     

The Antinociceptive Effects of Midazolam on Three Different Types of Nociception in Mice

Antinociceptive effects of systemically administered midazolam remain controversial. The present study was performed to investigate its antinociceptive effects on different types of nociception in mice. Four different doses of midazolam (1, 3, 10, and 30 mg/kg) were administered intraperitoneally (i.p.). Saline was used as a control. The hot plate test, tail pressure test, acetic acid writhing test, the running wheel test, and the balance beam test were performed following the drug administration. In the hot plate test and tail pressure test, i.p. midazolam produced significant antinociceptive effects with the 50% effective dose (ED₅₀) of 3.46 mg/kg [confidence interval (CI), 1.99 – 6.01 mg/kg] and 3.52 mg/kg (CI, 2.77 – 4.47 mg/kg), respectively. In the acetic acid writhing test, i.p. midazolam also produced significant antinociceptive effects. In the running wheel test, no mice stopped running after saline or midazolam at 1, 3, or 10 mg/kg, but all mice stopped running 30 and 45 min after i.p. administration of midazolam at 30 mg/kg. In the balance beam test, 30 min after i.p. administration of saline or midazolam at 1, 3, and 10 mg/kg, all mice were able to stay on the beam for 90 s, none of them could with midazolam at 30 mg/kg. In conclusion, systemically administered midazolam had antinociceptive effects on acute thermal, acute mechanical, and acute inflammatory-induced nociception in mice. The antinociceptive potency of midazolam was the same for both acute thermal-induced nociception and mechanical-induced nociception.     

利血平与优降宁对动物痛阈和吗啡镇痛作用影响因素探讨

采用三种测痛方法,观察了利血平、优降宁对小鼠、大鼠正常痛阈和吗啡镇痛作用的影响,结果表明:ip利血平2 mg/kg,优降宁100 mg/kg均能明显抑制小鼠扭体反应;ip利血平1 mg/kg能明显提高小鼠热板反应时间,但ip优降宁75 mg/kg无明显影响;ip利血平6 mg/kg,优降宁75 mg/kg对大鼠甩尾反应时间均无明显影响;利血平(小鼠0.5～1.0 mg/kg,大鼠2 mg/kg ip)能明显对抗吗啡镇痛作用;优降宁(小鼠35 mg/kg,大鼠50 mg/kg ip)能明显增强吗啡镇痛作用,并能“逆转”利血平对抗吗啡镇痛作用。其“逆转”作用的强弱取决于利血平、优降宁给药的先后次序。     

利血平与优降宁对动物痛阈和吗啡镇痛作用影响因素探讨

采用三种测痛方法,观察了利血平、优降宁对小鼠、大鼠正常痛阈和吗啡镇痛作用的影响,结果表明:ip利血平2 mg/kg,优降宁100 mg/kg均能明显抑制小鼠扭体反应;ip利血平1 mg/kg能明显提高小鼠热板反应时间,但ip优降宁75 mg/kg无明显影响;ip利血平6 mg/kg,优降宁75 mg/kg对大鼠甩尾反应时间均无明显影响;利血平(小鼠0.5～1.0 mg/kg,大鼠2 mg/kg ip)能明显对抗吗啡镇痛作用;优降宁(小鼠35 mg/kg,大鼠50 mg/kg ip)能明显增强吗啡镇痛作用,并能“逆转”利血平对抗吗啡镇痛作用。其“逆转”作用的强弱取决于利血平、优降宁给药的先后次序。