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Non-opioid analgesics

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ANALGESIC EFFICACITY OF SOME DRUG COMBINATIONS TO FORMALIN TEST

D.G.Ionescu. R.L.Gorduza, A.Negru, O.C.Mungiu - Univ of Medicine and Pharmacy "Gr.T.Popa" lasi

Aim of Investigation: Clinical observations suggest a potentiation of the analgesic effects of beta-blocker or anxiolitic drugs. We made the experimental trials to confirm it.

Methods: MaleWistar rats were used (groups with 6 rats) for intra-plantary injection of 0.1 ml formalin 5% in right hind paw (Du-buisson & Dennis, 1977). Were monitored some behavior aspects: flinching of the paw, licking of the paw, beating of the paw. Experimental groups were tested for: I-metamizol, II-clorazepate, III-propranolol, IV-clorazepate +metamizol,V-propranoIo]+ metam-izol, VI-control group.

Results: Formalin administration at the group VI produces a bipha-sic curve of the algesic behavior. That curve is statistical significant decreased (60%) for flinching of the paw at group I. For groups II and III we obtained a decreased curve too but not so pronounced like for group I. The results for group V have not statistical significance.

Conclusions: 1. Separate administration ofpropranolol, clorazepate and metamizol produces a statistical significant decrease of algesic behavior in both phases. 2. The association of metamizol with pro-pranolol or clorazepate doesn't produce a statistical significant decrease of the algesic behavior. 3. Clinical observations that were the aim of that investigation are due probably to the human psychic influences, which lack in animals.

GASTRIC ULCEROGENICITY AND PLATELET ANTIAGGREGATION ACTIVITY OF ANALGESIC EXTRACT OF TRIGONELLA FOENUM-GRAECUM (TFG)

Parvizpur*. A. Ahmadiani, (SPON: MA Jafary) Dept. Pharmacology, Shaheed Beheshti Univ. Med. Sci., Tehran, Iran

Aim of Investigation: To study more about the similarity of TFG extract and NSAIDs, it has been tried to investigate the effects of TFG extract on gastric ulcerogenicity and platelet aggregation.

Methods: Gastric ulcerogenicity was studied by oral administration of the extract and aspirin (as a positive control) in NMRI male rats. Platelets antiaggregation activity of the TFG extract was investigated by ADP-induced platelet aggregation model in rabbit.

Results: Gastric ulcerogenicity effects of both TFG extract 1000 mg/kg and aspirin 300 mg/kg were significant (p<0.01 and p<0.0001 respectively) and the effect of aspirin was more potent than TFG extract (pO.0001). TFG extract dose dependently inhibited the ADP-induced rabbit platelet aggregation.

Conclusions: TFG extract exert NSAID-like effects such as gastric ulcerogenicity and platelet aggregation inhibitory effect. NSAIDs that have a low ratio ofCoxI/CoxII inhibitory activity, usually produce potent side effects and those have a low ratio ofCoxI/CoxII inhibitory effect are more safe. So because of weak ulcerogenicity effect (comparing with aspirin), TFG may be more safe than aspirin. ADP induces reversible platelet aggregation in rabbit that is mediated by purinoceptors. So antiaggregation effect of TFG in this model suggest that TFG extract may be able to act as a purinoceptors antagonists. Because the antinociceptive effects of purinoceptors antagonists in i.t. administration, it is advisable to study the role ofpurinergic system in analgesic effects of TFG.

THE ROLE OF SEROTONERGIC AND OPIOID SYSTEMS IN ANALGESIA

M. Javan*. A. Parvizpur*, A. Ahmadiani, (SPON: MA Kherzi) Dept. Pharmacology, Shaheed Beheshti Univ. Med. Sci., Tehran, Iran

Aim of Investigation: Trigonella foenum-graecum (TFG) extract exert analgesic effect in tail flick and both phase of formalin test, and also produce anti-inflammatory and antipyretic effects. Because of some similar effects of TFG extract and NSAIDs, and the role of5HT system in analgesic effects of NSAIDs, we tried to investigate the role of spinal 5HT system in analgesic effects of TFG. Role ofopioid system was also studied. Methods: Nociception was assessed by formalin test in male NMRI rats. 5HTergic system was lesioned by 5,7-dihydroxytriptamine, and naloxone was used for opioid system inhibition.

Results: TFG dose-dependently showed analgesic effects in i.p. and i.t. but not in i.c.v. administration. Analgesic effect of TFG in second phase of formalin test reversed after lesioning of spinal 5HT system. Naloxone (2mg/ kg, i.p.) did not affect on TFG extract analgesia.

Conclusions: Analgesic effects of TFG extract in i.p. and i.t. but not in i.c.v. administration suggest that spinal cord is the site of action of TFG extract. It was shown that the analgesic effect of TFG in second phase of formalin test was dependent to 5HT system. NSAIDs also produce analgesia in the second phase of formalin test. So the analgesic effect of TFG in second phase of formalin test is similar to that of NSAIDs. It was also shown that analgesic effect of TFG is not dependent on opioid system. On the other hand 5HT system lesion inhibited only the second phase of formalin pain but not the first phase, therefore study the role of another systems in analgesic effect of TFG extract is recommended.

ETOMIDATE: A NEW CLASS OF POTENT ANALGESIC?

Jun Chen. Hui-Li Li*, Yan-Yan Sun*', Ceng Luo*, Dept of Anatomy & K.K. Leung Brain Research Centre, Anesthesiology, The Fourth Military Medical Univ, Van 710032, P.R.China

Aim of Investigation: To investigate whether etomidate, a nonbar-biturate intravenous hypnotic drug, has suppressive effects upon persistent nociception induced by chemical tissue injury.

Methods: Two models of tonic pain: the formalin test and the bee venom test were used to assess i.p., s.c. and i.t. effects ofsubanes-thetic doses of etomidate on the persistent nociception induced by s.c. formalin or bee venom injection into the plantar surface of one hindpaw in the conscious rat.

Results: (1) i.p. administration ofsubanesthetic doses (3, 6, and 10 mg/kg) of etomidate, but not the vehicle, produced a dose-dependent suppression of formalin-induced persistent nociceptive behaviors. (2) i.t. etomidate (40, 80 and 120 ug) also produced a dose-dependent suppression of bee venom-induced persistent nociception, while s.c. etomidate did not have any effect. (3) transec-tion of the dorsolateral fasciculus, where the descending antinociceptive system (DANS) passes through, or i.t. pretreatment with bicuculline or strychnine, the antagonist ofGABAA or glycine receptor, resulted in a complete or partial elimination of the suppressive effects of i.t. etomidate.

Conclusions: (1) Etomidate, in addition to the well characterized general anesthetic action, has a new, potent antinociceptive (analgesic) action in rats. (2) The sites of antinociceptive action of etomidate are located, to a large extent, in the spinal cord. (3) The antinociceptive action of spinal etomidate is dependent upon tonic activation of the DANS. (4) The tonic release ofGABA and gly-cine is involved in the process of (3), which is mediated by the activation of both GABAA and glycine receptors in the spinal dorsal horn.

Acknowledgments: Supported in part by NSFC fund 39870791 and XuZhou Third Pharmaceutical Factory, Jiangsu, People's Republic of China.

CONTRIBUTION OF DESCENDING FIBERS TO OPIOID ANALGESIA BUT NOT TO NOMIFENSINE ANALGESIA IN MODELS OF ACUTE AND PERSISTENT PAIN IN RATS.

Annie-Kim Gilbert, Keith B.J. Franklin*. Dept. of Psychology, McGill Univ, Montreal, PQ, Canada, H3A 1B1.

Aim of Investigation: To examine the role of descending inhibitory fibers in nomifensine-, buprenorphine-, and morphine-induced analgesia in models of acute and persistent pain.

Methods: Rats were implanted with a permanent indwelling can-nulae aimed at the nucleus raphe magnus (NRM). Saline or mus-cimol (50 ng) was microinjected in the NRM and animals were tested in the tail-immersion (54C), hot plate (54C) and formalin (2%) tests. Analgesia was induced by subcutaneous administration of nomifensine maleate (0.625-5 mg/ml/kg), morphine sulfate (1.25-20 mg/ml/kg), or buprenorphine hydrochloride (12.5-800 ug/ml/kg). Data were analysed by comparing dose-response curves for saline- and muscimol-treated animals.

Results: Compared to saline, muscimol microinjected in the NRM dose-dependently decreased the analgesic effects of systemic morphine and buprenorphine. Nomifensine-induced analgesia was not affected by microinjection of muscimol in the NRM.

Conclusions: These studies indicate an important role of descending fibers from the medulla in opioid analgesia in acute and persistent pain. However, the data suggest that nomifensine analgesia is not dependent on the functional integrity of these fibers. Nomifensine analgesia is likely to be mediated by forebrain dopamine. The present findings suggest that forebrain-mediated analgesia may be independent of descending systems. Acknowledgments: Supported by the Canadian agencies NSERC (A6303), FCAR and MRC studentship to A.K.G.

THE CAPSAICIN ANALOGUE EC665 PREVENTS THE HYPERREFLEXIA AND REFERRED MECHANICAL HYPERALGESIA ASSOCIATED WITH INFLAMMATION OF THE RAT URINARY BLADDER.

S.I. Jaggar*, H.C.F. Scott*, I.F. James* & A.S.C. Rice*, * Pain Research, Imperial College London, W2, U.K. Novartis Inst for Medical Research, London WC1, U.K.

Aim: To assess the effects of prophylactic administration of the systemically active capsaicin analogue EC665 on responses to inflammation of the rat urinary bladder.

Methods: Female Wistar rats were anaesthetised and the urinary bladder inflamed via a trans-urethral cannula (50% turpentine, 0.5ml, Ihr). Two experimental paradigms were used: 1. Vis-cero-visceral hyper-reflexia (VVH). The micturition threshold (Vmic) was derived from the cystometrogram (CMC). Inflammation is associated with reduction in V^.c indicating VVH. 2. Referred viscero-somatic hyperalgesia (VSH) after recovery from anaesthesia. The outcome measure was the area under the curve (AUC) produced by plotting the difference in fore and hind paw withdrawal thresholds from a mechanical stimulus (von Frey hairs) against time (for 24hrs). A negative value indicates relative hyperalgesia of the hind paw. Comparisons made using ANOVA [Dun-nett's], significance level p < 0.05.

Results: 1. VVH - Solvent control treated animals (n = 6) exhibited inflammation associated VVH (Vn,,c 43.7% (+/- 6.3) of baseline). EC665 (0.05 - Img/kg, n = 6 each group) prevented this VVH.

2. VSH - Control animals developed a VSH [AUC (-5.2 (+/-1.7) x 103) (n = 8) in comparison to uninflamed controls (n =11) (AUC -0.02 +/- 0.6 x 103)]. EC665 (0.1 - Img/kg) prevented this VSH in a dose related manner (threshold 0.1 mg/kg). Conclusions: Administration of EC665 prevents both the VVH and VSH associated with bladder inflammation, supporting work suggesting capsaicin sensitive neurons are important in pathophysi-ological events occurring during visceral inflammatory pain.

Acknowledgments: AAGBI, JSCR St. Mary's Hospital

SDZ249-665: A NON-EXCITATORY CAPSAICIN ANALOGUE WITH ANTIHYPERALGESIC AND ANTINOCICEPTIVE PROPERTIES.

lain F. James, Elizabeth Campbell, Laszlo Urban, Gillian Burgess, Nicholas Wilmott, Janet Winter and Stuart Bevan. Novartis Inst for Medical Sciences, Gower Place, London WC1E 6BN.

Aim of Investigation: To characterise the pharmacological profile of the capsaicin analogue SDZ249-665 in vivo and in vitro.

Methods: Activity of SDZ249-665 was compared to that of capsaicin and standard analgesics in models of acute pain (tail flick, writhing) and prolonged inflammatory pain (induced by injection of turpentine or carrageenan into rat or guinea pig paws). Excitatory properties of capsaicin and SDZ249-665 were assessed in assays ofbronchoconstriction, blood pressure and pungency. In vitro SDZ249-665 was compared to capsaicin in binding, calcium uptake and intracellular calcium assays on cultured sensory neurones, as well measures of nerve activity in the tail/cord preparation.

Results: SDZ249-665 was antinociceptive and antihyperalgesic in the pain models, but unlike capsaicin showed no excitatory activity in vivo. In vitro SDZ249-665 was a full agonist at capsaicin receptors and activated sensory nerves, but was about 3-fold less potent than capsaicin. Both capsaicin and SDZ 249-665 evoked increases in intracellular calcium in sensory neurones. The response latency, the rate of rise in calcium concentration and the recovery time for responses to SDZ 249-665 were all slower than for responses to capsaicin. In the tail/cord preparation, treatment of the tail with SDZ 249-665 caused inhibition of responses to other noxious chemical stimuli such as capsaicin, acidic pH and bradykinin. Concentrations required to obtain inhibition (0.1-0.5u.M) were lower than those required to produce ventral root depolarisation (EC5o = 3.4 u.m). This difference along with the difference in kinetics may underlie the separation of analgesia from excitatory side effects seen in vivo.

EVALUATION OF AN INHIBITOR OF P38 MITOGEN ACTIVATED PROTEIN KINASE (MAPK) IN MODELS OF ESTABLISHED HYPERALGESIA.

Pam Ganju. Ximena Nunez, Andy Davis, Sadhana Patel and Aly-son Fox (SPON: Humphrey Rang). Novartis Inst for Medical Sciences, 5 Gower Place, London WC1 6BN, England, U.K.

Aim of Investigation: To evaluate the effect ofSB203580, a p38 MAPK inhibitor in animal models of chronic pain.

Methods: Animal models of inflammation (Freund's complete adjuvant [FCA] induced) and neuropathic pain (partial ligation of the sciatic nerve) were treated with SB203580 after hyperalgesia was established. Nociceptive mechanical thresholds were measured either with modified force transducers or an analgesymeter. Super-natants prepared from homogenates of untreated and inflamed paws were evaluated by ELISA and Western blot analysis.

Results: Oral but not intrathecal administration ofSB203580 was effective in reversing inflammatory hyperalgesia in the joint (D50 of2.4mg/kg; peak reversal of 55 9% at 25mg/kg) and the paw (maximal reversal of69 11%; no strict dose-response relationship). In the neuropathic pain model, oral but not intrathecal application ofSB203580 produced a slight (37%) but significant reversal of mechanical hyperalgesia at 25mg/kg. Tumour Necrosis Factor a (TNF oc), Interleukin-1 (3 (IL-1 P) and Cycloxygenase (COX-2) protein levels were not significantly altered after p38 inhibitor treatment compared with the vehicle group.

Conclusions: p38 inhibitors may be orally effective agents against inflammatory mechanical hyperalgesia with a peripheral locus of action, p38 MAPK. is a key component in signal transduction pathways induced by proinflammatory cytokines. However, in these models the analgesic action of the p38 inhibitor cannot directly be attributed to changes in TNF a or IL-1 (3 protein levels but may involve mediators downstream of them.

POTENT ANTINOCICEPTIVE EFFECT OF (S)-N-DESMETHYL TRIMEBUTINE IN A RAT MODEL OF PERIPHERAL NEUROPATHY.

G. Guilbaud', D. Christensen'*, J. Idanpaan-Heikkila'*, F. Roman2, V. Kayser'. 'INSERM U161, 2 rue d'Alesia, 75014 Pans, ^nstitut de Recherche Jouveinal/Parke Davis, 94265 Fresnes, France.

Aim of Investigation: Trimebutine (2-dimethyl-amino-2-phenylbutyl 3,4,5-trimethoxybenzoate hydrogen maleate) has been shown to relieve abdominal pain in humans. In the present study, the antinociceptive action of systemic (S)-N-desmethyl-trimebutine, a stereoisomer ofN-monodesmethyl-trimebutine, the main metabolite oftrimebutine in humans, was studied in a rat model ofneuropathic pain produced by chronic constriction injury to the sciatic nerve.

Methods: Chronic constriction injury was produced by four ligatures around one sciatic nerve. Experiments were performed two weeks after surgery when the pain-related behaviour has fully developed. Mechanical (vocalization threshold to hindpaw pressure) test was used.

Results: (S)-N-desmethyl-trimebutine (1,3, 10, 30 mg/kg s.c.) produced significant antinociceptive effects, that peaked at the dose 10 mg/kg and were more potent in the nerve-injured hindpaw than in the contralateral hindpaw. The effect of the dose 10 mg/kg lasted for 100 min and peaked between 30 and 50 min in the nerve-injured hindpaw. The mean vocalization threshold to paw pressure at 40 min was 57060 g (253% of the pre-injection value, P< 0.01). The effects of(S)-N-desmethyl-trimebutine (1 mg/kg) were not reversed by naloxone at a dose (0.1 mg/kg i.v.) that prevents the effect of I mg/kg of i.v. morphine in neuropathic rats (Kayser et al, 1995).

Conclusion: The result suggests that systemic (S)-N-desmethyl-trimebutine may be useful in the treatment of some aspects ofneuropathic pain.

ROLE OF ENDOGENOUS OPIOIDS OF THE ROSTRAL VENTROMEDIAL MEDULLA IN THE ANALGESIA PRODUCED BY DIPYRONE MICROINJECTION INTO P.A.G.

Enrique Vasquez*. Dilia Hemandez, Horacio Vanegas. Institute Venezolano de Investigaciones Cientificas (IVIC), Caracas 1020A, Venezuela (E-mail: hvanegas@ivic.ivic.ve)

Aim of Investigation: In addition to their well-known effect upon peripheral tissues, non-opioid analgesics such as aspirin and dipy-rone (metamizol) decrease nociception by acting upon central nervous system structures such as the periaqueductal gray matter (PAG). This latter action involves endogenous opioids ofPAG and engages pain-modulating neurons of the rostral ventromedial medulla (RVM), but it is unknown whether endogenous opioids of RVM are also involved. This question was addressed herein.

Methods: In deeply anesthetized rats, microinjection cannulae were inserted into PAG and RVM (nucleus raphe magnus and adjacent structures); 10 s mechanical stimuli were then applied every 6 min to the hindpaw by means of two standardized clamps of noxious and innocuous strength, and the responses of wide-dynamic-range (WDR) neurons in the spinal dorsal hom were extracellularly recorded. After three baseline responses were obtained, dipyrone (lOOug in 0.5u.l saline) was microinjected into the ventrolateral PAG. Twenty minutes later, naloxone (0.5u.g in 0.5u.l saline) was microinjected into RVM.

Results: Baseline spinal neuronal responses to the noxious and the innocuous stimulus were respectively (meanSEM) 94677 and 50070 spikes/lOs. Dipyrone microinjection into PAG produced a decrease in the response to the noxious stimulus to 58786 spikes/ 10s (down 38%, p<0.004) in 18 min, without affecting the response to the innocuous stimulus. Naloxone microinjection into RVM completely reversed the dipyrone effect, that is, within 2 min the response to noxious stimulation returned to 984127 spikes/lOs (no statistically significant difference to baseline); responses to innocuous stimulation remained unchanged. Microinjection of saline alone into RVM did not modify the dipyrone effect. Microinjection of naloxone into RVM without previous dipyrone microinjection into PAG had no effect upon responses to noxious or innocuous stimulation.

Conclusions: The present results confirm that dipyrone has an antinociceptive effect when acting directly upon PAG and further show that this effect involves endogenous opioids in RVM. Since the involvement of endogenous opioids of PAG in this effect had been previously demonstrated, these findings suggest that the PAG effect of dipyrone gives rise to an activation ofbrainstem descending opioidergic systems involved in pain modulation.

Acknowledgments: Supported by CONICIT grant Sl-97000106.

PROGRESSIVE DECREASE OF THE ANTINOCICEPTIVE EFFECT OF DIPYRONE FOLLOWING REPEATED MICROINJECTION INTO THE VENTROLATERAL PAG OF THE RAT.

Victor Tortorici. Francisco Rodriguez*, Horacio Vanegas. Institute Venezolano de Investigaciones Cientificas (1V1C), Caracas 1020-A, Venezuela.

Aim of Investigation: Dipyrone (DIP), a non-opioid analgesic, can exert an anti-nociceptive effect when microinjected into the periaqueductal gray matter (PAG). Based on the fact that the anti- no-ciceptive effect of morphine administered into the ventrolateral PAG is attenuated with repeated microinjection, we decided to evaluate if the anti-nociceptive effect of DIP is also attenuated after repeated microinjection into PAG.

Methods: Male Sprague-Dawley rats anesthetized with pentobar-bital (55 mg/kg i.p.) were implanted with a guide cannula aimed at the ventrolateral PAG. One week after surgery, DIP (150 u.g/0.5 u.1) or saline (SAL) was microinjected into the PAG twice a day for two days. Antinociception was evaluated with the hot plate (HP) and the immersion tail flick test (ITF) 20 min after each microinjection. On the third day, all the animals were tested again following DIP microinjection into the same PAG site.

Results: As expected, PAG microinjection of DIP caused a lenghtening of ITF and HP latencies. This effect progressively decreased with each subsequent microinjection. On the third day, rats that only received DIP showed ITF and HP latencies similar to the rats receiving only SAL, i.e., a 52% decrease of the initial antinociceptive effect was observed. The rats that received SAL were also microinjected with DIP on the third day and showed an increase of their ITF and HP latencies of 65.72% and 79.60% respectively. When microinjections were located at the dorsal PAG, only a marginal anti-nociception was obtained.

Conclusions: The anti-nociceptive effect of DIP is attenuated after repeated microinjection into the ventrolateral PAG. The decrease of DIP efficacy might be due to the development of tolerance to endogenous opioids which have been previously associated with the anti-nociceptive effect of DIP microinjected into the PAG.

BUTYLTHIO[2,2,2](LY297802/NNC11-1053) ON MUS-CARINIC M2 AND M4 KNOCKOUTS MICE: ANTINOCI-CEPTION AND SOME CLASSIC MUSCARINIC AGONIST SIDE EFFECTS

Lu Zhang*, Jesus Gomeza*, Jurgen Wess* and Harlan Shannon (SPON: B. D. Goldstein), Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, IN 46285 USA and Laboratory ofBioorganic Chemistry, NIDDKD, Bethesda, MD 20892

Aim of Investigation: It has been well documented that muscarinic agonists and acetylcholinesterase inhibitors produce antinocicep-tion. LY297802 tartrate (Butylthio[2,2,2]) is a mixed muscarinic M2/M4 muscarinic receptor agonist and M1/M3/M5 muscarinic receptor antagonist having potent antinociceptive properties. Moreover, LY297802 did not produce salivation and tremor at antinociceptive doses. Although it has been speculated that LY297802 produces antinociception by agonist actions at the M4 muscarinic receptor, there is presently no direct evidence to support the relative involvement of M2 vs. M4 receptors. In order to address this question, we studied LY297802 in muscarinic M2 and M4 receptor knockout mice.

Methods: Wild type (WT), heterozygotes (Hz), and muscarinic receptor knockout (K.0) mice (M2 and M4) were used. Dose-response curves for LY297802 were determined in the tail-flick test and for the typical muscarinic agonist side effects of salivation, tremor and hypothermia.

Results: In the tail flick test, LY297802 produced significantly less antinociception in M2 K.0 mice relative to WT and HZ mice. In contrast, LY297802 did not produce significantly different antinociceptive effects among the three M4 genotypes. In addition, LY297802 produced significantly less hypothermia in M2 K.0 mice than in M2 WT or Hz. LY297802 produced only mild salivation and no tremor in any of the M2 or M4 genotypes.

Conclusions: The present data suggest that the M2 muscarinic receptor, rather than the M4 muscarinic receptor, at least in part mediates the antinociceptive effects of LY297802 in the tail-flick test as well as for the hypothermic effects of LY297802.

PHARMACOGENETIC VARIABILITY IN NEURONAL NICOTINIC RECEPTOR-MEDIATED ANTINOCICEPTION.

Sonya G. Wilson*', Jeffrey S. Mogil' and Christopher Flores2, 'Dept of Psychology and Neuroscience Program, Univ of Illinois at Urbana-Champaign, Champaign, IIL 61820 USA and Depts of Endodontics and Pharmacology, The Univ of Texas Health Science Center, San Antonio, TX 78284 USA

Aim of Investigation: The purpose of the present studies is to test the hypothesis that there is heritable variability in the magnitude and duration of antinociception produced via activation ofneuronal nicotinic receptors.

Methods: Antinociception in response to epibatidine, a neuronal nicotinic receptor agonist, was measured in adult mice of eight inbred strains using the 49C tail-immersion/ withdrawal (TW) assay. TW latency was determined immediately before, and every 10 min after drug injection. Group (n = 6-13/strain) TW latency data (s) were analyzed by ANOVA followed where appropriate by Tukey's post hoc test.

Results: Epibatidine (50 ug/kg, i.p.) produced significant antinociception in all mouse strains, exhibiting peak effects at 10-20 min with an approximate rank order sensitivity ofDBA/2 = BALB/c = A C57BL/6 = C57BL/10 = SM = C3H/He = AKR. Narrow-sense heritability ofepibatidine-induced antinociception was estimated as h2 = 0.68. TW latencies returned to baseline values by 30 min in all but the A strain, which manifested antinociception in response to epibatidine lasting for >3 h. Effects were dose-dependent (10-100 ug/kg), and blocked by the neuronal nicotinic receptor blocker, mecamylamine.

Conclusions: These data provide strong support for the hypothesis that neuronal nicotinic receptor-mediated antinociception is subject to significant genetic variability, although the identification of the relevant genes and allelic variants will require further study. Furthermore, these results suggest that an assessment of potential pharmacogenetic variability of any future nicotinic analgesics to be used in humans is warranted.

Acknowledgments: This work was supported by NIH grants DE 12735 (JSM) and DA 10510 (CMF).

EFFECTS OF ACETYL-L-CARNITINE ON THERMAL NOCICEPTIVE THRESHOLD OF MICE.

F. Ambrosio, G. Andaloro, M. Dan, G. Scutari, D. Franceschini, P. Giusti. Research Center for Biochemistry, Pathology, Pharmacology and Pain Therapy. Univ of Padua, Largo Colombo, 2, 35100 Padova, Italy.

Aim of Investigation: Cholinergic system is involved in central pain processing. Acetyl-L-Camitine (ALC) increases central choli-nergic activities. The effects of ALC on antinociception was investigated.

Methods: CDI mice (n=10 animals per group) were used. Animals received daily an administration of ALC (15 mg/kg, i.v.) or saline for 21 days. Two hours later a hot plate test procedure was applied to the mice. Animals were placed individually on a plate maintained at 55 0.5C by feedback from a surface-mounted thermocouple. The response latency was evaluated on the basis of either hind paw lick or jump reaction, following contact with the plate. Animals were discharged from the study after the hot plate test.

Results: Mice chronically treated with ALC were devoid of gross behavioral abnormalities. Significant (PO.01) increases in response latency was observed in mice after 12 administrations of ALC. Mice receiving 21 administrations of ALC were treated the day after with atropine (0.1 mg/kg, s.c.). The alkaloid reversed the antinociceptive effects of ALC.

Conclusions: Cholinergic system is reported to be involved in central pain antinociception. However, cholinomimetic drugs cannot be used as analgesics because their side-effects. Our data suggested that the chronic administration of ALC in mice may produce antinociception through an involvement of Cholinergic system. Furthermore, these antinociceptive actions were devoid of major side-effects.

ANTINOCICEPTIVE EFFECTS OF EPIBATIDINE IN THE FORMALIN MODEL OF OROFACIAL PAIN.

Shawn Gilbert*', TracyClark*' and Christopher M. Flores'^Depts of 'Endodontics and Pharmacology, The Univ of Texas Health Science Center, San Antonio, TX 78284 USA

Aim of Investigation: A growing body of evidence indicates that nicotine and other nicotinic Cholinergic agents possess broad spectrum antinociceptive activity across animal models of both inflammatory and neuropathic pain. However, such effects have yet to be demonstrated in the trigeminal field ofinnervation. The purpose of the present studies is to evaluate the antinociceptive efficacy of a neuronal nicotinic receptor agonist in the first and second phases of the formalin test applied to the orofacial region.

Methods: Adult, male Sprague-Dawley rats (n = 3-5/group) weighing 250-350 gm received a 1 ml/kg subcutaneous injection of either saline vehicle or epibatidine (1-10 ug/kg). Five minutes later, all animals received a 50 )il injection of a 5% formalin solution into the vibrissal pad. Total time (sees) spent grooming the injected area during the first (0-12 min) and second (12-45 min) phases was recorded for each animal. Between group comparisons were made using a one-way analysis of variance for repeated measures. Dose-response data were analyzed by linear regression. Statistical significance was accepted at p<0.05.

Results: Epibatidine produced profound antinociception in both phases of the formalin test reaching statistical significance in the second phase (p< 0.001). Moreover, this effect of epibatidine appeared to be dose-dependent. The involvement ofneuronal nicotinic receptors in mediating the effects of epibatidine was assessed in animals pretreatcd with mecamylamine, a non-competitive antagonist at this receptor class.

Conclusions: Taken together these data suggest that neuronal nico-tinic receptor agonists such as epibatidine possess antinociceptive efficacy in the formalin model oforofacial pain.

Acknowledgments: This work was supported by NIH grant DA 10510 from the National Inst on Drug Abuse.

ANTINOCICEPTIVE EFFECTS OF NICOTINIC RECEPTOR AGONISTS, (+)-EPIBATIDINE AND ABT-594, IN RAT MODELS OF ACUTE AND CHRONIC PAIN

Adam Kesingland*, Clive Gentry*, Michael Bowes*, Mohanjit Panesar*, Jeffrey McKeIvy* ' Jean-Michel Vernier*', Laszlo Urban & Katharine Walker. Novartis Inst for Medical Sciences, 5 Gower Place, London WC1E 6BN, UK and 'Sibia Neurosciences, Inc, La Jolla.Ca 92037-4641.

Aims of Investigation: (+)-epibatidinc and ABT-594 were tested in rat models of acute and chronic pain, and locomotor function. Methods:) ABT-594 (3-300 ug/kg, s.c) or (+)-epibatidine (0.3-10 ug/kg, s.c.) were tested in male Wistar rats (180-220 g). Acute nociceptive responses were measured using the tail-flick test. Inflammatory hyperalgesia was measured as the decrease in paw withdrawal threshold (PWT) to mechanical stimulation 24 hours following intraplantar injection ofFreund's complete adjuvant. Neuropathic hyperalgesia was measured as the decrease in PWT to mechanical stimulation, 10-15 days following partial ligation of the sciatic nerve. Locomotor function was measured using an accelerating rotarod.

Results: (+)-Epibatidine and ABT-594 dose-dependently reversed inflammatory and neuropathic hyperalgesia (Epibatidine: D5o at 1 h of 1.9 and 0.44 ug/kg, respectively; ABT-594:D;o at 1 hot 33.7 and 27 ug/kg, respectively). Both (+)-epibatidine and ABT-594 produced dose-dependent increases in tail-flick latency (D5o at 1 h of 2.49 and >300 u.g/kg, respectively) at doses that also disrupted locomotor performance (D<,o at 1 h of 3.46 and 231 ug/kg, respectively).

Conclusions: (+)-Epibatidine and ABT-594 produce potent anti-hyperalgesia in models of acute and chronic pain. Both drugs produced acute antinociceptive effects at doses that disrupt locomotor function.

INTRATHECAL NEOSTIGMINE DIMINISHES, BUT DOES NOT ABOLISH, HYPOTENSION FROM SPINAL BUPIVACAINE IN DOGS

E.F. Ismail. L.M. El-Zorkany*, G.L. Gad*, A.A. Abou-Saife*, S.M. Hassan*, Depts ofAnesthesiology, Pharmacology, and Anatomy, Faculty of Medicine, Al-Azhar Univ, Cairo, 11727, Egypt

Aim of Investigation: To study the hemodynamic effects of spinal neostigmine and to test whether neostigmine would prevent spinal bupivacaine-induced hypotension in dogs.

Methods: Ten dogs were included in this study. They were anesthetized with intravenous (iv) pentobarbital. Polyvinyl cannulas were inserted into a femoral artery and vein. The femoral artery cannula was connected to the Super Speed Kymograph via hepa-rinized tubing for arterial blood pressure (BP) monitoring. ECG electrodes were connected to the dog via special needles for heart rate (HR) recording. In the lateral decubitus, a polyvinyl (epidural) catheter was inserted intrathecally (IT) via a Tuohy needle inserted in L4-5 space. After a period of 30 min, baseline values for HR and BP were recorded. In five animals, 1 ml 0.5% bupivacaine in 8.33% dextrose was injected via the IT catheter. In another five animals, 1 mg (1-ml) neostigmine was injected via the IT catheter and followed by 1 ml 0.5% bupivacaine in 8.33% dextrose after 30 min. HR and BP were recorded at 10-min intervals. Transverse sections were taken from the spinal cord at the lumbar region from neostigmine-treated dogs and from control ones for histopathologi-cal examination by light and electron microscopes.

Results: Spinal neostigmine alone increased (non significantly) arterial BP by 5% with a latency of 15 min, but it had no effect on HR. Compared with spinal bupivacaine alone, addition of neostigmine resulted in hypotension of slower onset (15 vs 5 min), shorter duration (60 vs 90 min) and smaller magnitude (-11% vs -19%). Light and electron microscope examination did not reveal any change between control and neostigmine-treated dogs.

Conclusion: Spinal bupivacaine-induced hypotension was partially counteracted by spinal neostigmine in dogs.

DRUG INTERACTION BETWEEN INTRATHECAL NEOSTIGMINE AND MK-801

Jai-Hyun Hwang, Dong-Myung Lee, Dept ofAnesthesiology, Asan Medical Center, 388-1 Pungnap Dong, Songpa-Ku, Seoul 138-736, S. Korea

Background: Peripheral nerve injury may generate symptoms like allodynia and hyperalgesia. Spinally administered neostigmine and MK-801 have been shown to have actions attenuating the allodynia in the rat model. Using an isobolographic analysis, we examined the spinal interaction between neostigmine and MK-801 in a rat model ofneuropathy.

Methods: Following approval from the Animal Care Committee, male SD rats were prepared with tight ligation of left lumbar 5th and 6th spinal nerves and chronic lumbar intrathecal (i.t.) catheters. I.t. dose response curves were established for the antiallodynic effect of neostigmine (0.3, 1,3, and 10 ug) and MK-801 (1,3, 10, and 30 ug) alone to obtain the ED50 for each agent. ED50 fractions (1/2, 1/4, 1/8, and 1/16) of drug combination ofneostigmme-MK-801 were administered. Allodynic thresholds for left hindpaw withdrawal to von Frey hair application were assessed and converted to %MPE. The log dose responses were plotted from the peak effect of %MPE in each group. The ED50 ofneostigmine-MK-801 combinations was established and isobolographic analysis of the drug interaction was carried out.

Results: I.t. neostigmine, MK-801, and combination groups produced dose dependent reductions of tactile allodynia. ED50 values are 0.3 ug (0.11-0.83 ug) for neostigmine, 4.33 ug (2.11 -8.91 u,g) for MK-801, and 0.147 ug (0.023-0.921 ug) for the combination. The experimental ED50 value for combination group was found to be significantly below the theoretical additive line ( p < 0.05 ).

Conclusion: The results suggest that i.t. neostigmine and MK-801 alone produce a dose related antagonism on touch evoked allodynia and intrathecal MK-801 is synergistic at the spinal level when combined with intrathecal neostigmine in a rat model ofneuropathy.

ANTINOCICEPTIVE EFFECTS OF THE COMBINATIONS OF MORPHINE AND CLONIDINE WITH PHYSOSTIGMINE OR NEOSTIGMINE AFTER INTRATHECAL INJECTION IN THE RAT.

A. Nemirovsky'. Y. Zhang*', S. Steen*''2, J. Szenohradszky*', V. Zeiman*'. 'Dept ofAnesthesiology, Univ of Southern California; Dept ofAnesthesiology, Charles R. Drew Univ of Medicine and Science, 1200 N. State St., Los Angeles, CA 90033, USA

Aim of Investigation: The aim of the present study was to investigate the antinociceptive effect produced by a combination ofphar-macological agents from three different classes.

Methods: Nociccption was assessed in male Sprague Dawley rats (300-350 g) by means of "plantar stimulation" test. Experimental substances, morphine (1 ug), clonidine (1 ug), neostigmine (1 ug) and physostigmine (10 u.g), were administered intrathecally. The specificity of the effect was investigated with intravenous antagonists, naloxone (1 mg/kg), atropinc (1 mg/kg) or yohimbine (1 mg/kg).

Results: All agonists injected alone produced a slight increase in the nociceptive threshold. The percentage of maximal possible effect (%MPE) was 22.5 1.7; 13.7 2.8; 25.9 2.1 and 19.0 1.6 for morphine, clonidine, neostigmine and physostigmine, re-spectively. Simultaneous administration of morphine and clonidine with physostigmine or neostigmine resulted in a dramatic increase in the latencies ofnociceptive response with %MPE rising to 83.2 7.3 or 98.1 1.2, respectively. The duration of the effect was also significantly increased. Administration of specific antagonists demonstrated the involvement ofu-opioid, Ot^-adrenergic and mus-carinic receptors in the antinociceptive effect of the combination.

Conclusions: The results of the experiments demonstrate that simultaneous administration ofu.-opioid agonist morphine, (x;-adrenergic agonist clonidine, and cholinesterase inhibitors physostigmine or neostigmine, in the threshold doses, which do not produce any notable side effects, results in a profound antinocicep-tion. The present study might be of a significant clinical value, since it demonstrates a way of enhancing analgesia without increasing the risk of undesirable side effects.

THE NSAID DEXKETOPROFEN TROMETAMOL IS A MORE POTENT ANALGESIC THAN EITHER THE R(-) OR S(+) ENANTIOMERS OF FLURBIPROFEN TROMETAMOL.

Javier Mazario*. Ramon E. Solano*, Juan F. Herrero, Dept de Physiology. Univ ofAlcala. Madrid, Spain.

Aim of Investigation: To investigate and compare the analgesic potency of S(+) ketoprofen trometamol (dexketoprofen) and R(-) and S(+)-flurbiprofen trometamol in rat hind limb withdrawal reflexes.

Methods: Male Wistar rats were used for the recording of Single Motor Units (SMU) under a-chloralose anesthesia. The units were activated by either noxious mechanical stimulation (pinch) or repetitive high intensity electrical stimulation. All the drugs were dissolved in saline and injected i.v., in cumulative doses, either in normal rats or in rats with carrageenan-induced mono-arthritic.

Results: Neither of the enantiomers offlurbiprofen depressed SMU activity evoked by noxious mechanical or electrical stimulation in normal animals at the doses used (0.8 to 26umol/kg). In arthritic rats however, both enantiomers inhibited responses to mechanical stimulation, although they showed different potency (ID50s of 8 and 27umol/kg for S(+) and R(-)-flurbiprofen respectively). Responses to electrical stimulation were little affected by flurbiprofen and wind-up remained unchanged. The effect produced by the administration offlurbiprofen was not reversed by naloxone (Img/kg). Dexketoprofen was very potent in normal animals (ID50s of 0.1 and 0.76 umol/kg for mechanical and electrical stimulation respectively) and in animals with inflammation (ID50s of 1.8 and 2umol/kg). It was also effective in reducing SMU wind-up either in normal (MED:50nmol/kg) or arthritic rats (MED: lOOnmol/kg).

Conclusions: Dexketoprofen trometamol is more potent depressor of withdrawal reflexes than either of the enantiomers of flurbiprofen trometamol, both in normal and arthritic rats. The effect of dexketoprofen is probably produced at both peripheral and central sites, since it inhibited responses to electrical stimulation and wind-up. Flurbiprofen however was only active in the periphery since it did not affect responses to electrical stimulation.

Acknowledgments: Supported by Laboratories Menarini (Spain) and Ministry of Education and Culture, Spain (grant SAF97-0104).

ANTINOCICEPTIVE "SELF-SYNERGY" BETWEEN SPINAL AND SUPRASPINAL ACETAMINOPHEN (PARACETAMOL)

Robert B. Raffa. Dennis J. Stone*, Ronald J. Tallarida*, Temple Univ School of Pharmacy and School of Medicine, Philadelphia, PA 19140 USA

Aim of Investigation: To examine a possible site-site antinociceptive interactive effect between acetaminophen administered in-tracerebroventricularly (i.c.v.) and intrathecally (i.th.).

Methods: Pathogen-free albino ICR male mice, 18-24 g (Cri:CD-1& ; Charles River Laboratories, Wilmington, MA) were administered 5 uL acetaminophen (in a 5% EtOH vehicle) i.c.v. (Ha-ley&McCormick,Br.J.Pharmacol., 12:12-15, 1957), i.th. (Hylden & Wilcox, Eur. J. Pharmacol. 67: 313-316, 1980) or combined i.c.v./i.th. in fixed ratios and antinociception was assessed using the abdominal irritant test of Collier el al. (Br. J. Pharmacol. 32:295-310, 1968) with an i.p. injection ofacetylcholine.

Results: I.th. acetaminophen produced a dose-related antinociception, with ED50 value of 828 205 nmol. I.c.v. acetaminophen had no effect. Therefore, the expected total amount additive ED50add (1:l)was 1656 410. Combined (1:1) i.c.v./i.th. administration produced a dose-related antinociception with an ED50mix value of 483 107. Log(ED50n,i,) was significantly less (PO.05; Student t-test) than log (ED50add), indicative ofsynergism between sites of administration.

Conclusions: Acetaminophen induces spin ally-mediated antinociception and displays antinociceptive "self-synergy" between spinal and supraspinal sites. These results suggest the novel hypothesis that acetaminophen-induced analgesia derives in part from a "self-synergistic" interaction between brain and spinal cord. The less activity at either site alone could account for prior difficulty identifying a mechanism of acetaminophen action. In addition, the results suggest that acetaminophen has two central analgesic mechanisms.

Acknowledgment: The laboratory work was conducted at the R.W. Johnson Pharmaceutical Research Inst in Spring House, PA

DISTRIBUTION OF ACETAMINOPHEN IN RAT CENTRAL NERVOUS SYSTEM.

D. Besse'. J-P. Courade*''3, C. Delchambre*', M. Hamon2, F. Caussade', A. Eschalier3, A. Cloarec', 'Biology Dept., UPSA La-boratoires, 92500 Rueil-Malmaison, France. 1NSERM U288, Fac-ulte de Medecine Pitie-Salpetriere, 75013 Paris, France 'Laboratoire de Pharmacologie Medicale, Equipe NPPUA, Faculte de Medecine, BP 38, 63001 Clermont-Ferrand, France

Aim of Investigation: Recent experimental and clinical data ('i'b) suggest that central mechanisms of action may underlie the analgesic effect of acetaminophen (APAP). Based on this assumption, the possible accumulation of APAP in the central nervous system (CNS) was investigated in adult rats treated acutely with this drug. Methods: Two different protocols were used: a) 43 uCi of ^HJAPAP (50Ci/mmol) were injected i.v. to male Sprague-Dawley (SD) rats. Forty five minutes later, blood (lOOu.1) was sampled and animals sacrificed. Whole brain or striatum, hypothalamus, hippocampus, brain stem, cortex and spinal cord were collected. ['HJAPAP levels were measured by liquid scintillation, b) APAP (400 mg/kg) was administered orally to male SD rats. At 45 min, APAP accumulated in the same CNS structures was determined by HPLC coupled to electrochemical detection.

Results: At 45 min after i.v. injection, ['HJAPAP blood concentration was 90 nCi/ml, which corresponded to 3% of the injected dose. In the whole brain, radioactivity levels reached 38 nCi/g tissue indicating that ['HJAPAP largely impregnated the CNS. Comparison between CNS structures revealed no significant variations in ['HJAPAP levels (35 3 to 39 5 nCi/g tissue), suggesting that ['HJAPAP was homogeneously distributed in the CNS. Similarly, in rats treated orally with the non-radioactive drug, high APAP levels were found by HPLC in all CNS structures, with no significant regional variations (72 6 to 108 8 ug/g tissue).

Conclusion: Using two different protocols, APAP was found to accumulate throughout the CNS in rats. These data strengthen the hypothesis that antinociceptive effects of APAP may be the consequence of its direct action within the CNS

a) Pelissier et al., J. Pharmacol. Exp. Ther. 1996, 278, 8-14
b) Pilettae/Clin.Pharmacol.Ther., 1991,49,350-354

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