Experimental data regarding the implications of certain minimum structure enkephalin-like peptides in nociceptive processing.

The aim of this study was to investigate the importance of the amino acidic sequence at N-terminal end of certain minimum structure enkephalin-like peptides for the analgesic activity. Different groups of mice or rats were treated with 1) L-tyrosine (i.p. 200 mg/kg), 2) Tyr-Phe (i.t. 0.5 mg/rat), 3) Tyr-Pro-Phe (i.t. 0.5 mg/rat), 4) Gly-Tyr (i.t. 0.5 mg/rat), 5) Tyr-Gly-Gly (i.t. 0.5 mg/rat). Different tests were utilized to evaluate the antinociceptive effect of the substances tested: thermal nociception (hot plate test, plantar test), mechanical nociception (analgesymeter test). Tyr-Pro-Phe, Tyr-Gly-Gly, Tyr-Phe, but not Gly-Tyr, elicited analgesic activity. So, the presumption made in the case of atypical opioid peptides that opioid-like activity in case of peptides presumes a tyrosine residue at the N-terminal sequence, applies for shorter peptides. It appears also that minimal structure brain peptides with an N-terminal Tyr-Pro, rather than the Tyr-Gly-Gly-Phe sequence typical of other endogenous opioids, can provide better affinity for the opioid receptors and stronger analgesic activity. The inhibition of their analgesic effect by previous administration of naloxone proves that this effect is mediated through the endogenous opioid system.     

Antidepressant-like effects of tramadol and other central analgesics with activity on monoamines reuptake,in helpless rats

Affective states are regulated mainly by serotonin and noradrenaline. However the opioid system has been also related to antidepressant-induced mood improvement, and the mu-opioid receptor has been involved in affective responses to a sustained painful stimulus. Similarly, antidepressant drugs induce an antinociceptive effect via both the monoaminergic and opioid systems, probably involving sensorial and affective dimensions of pain. The aim of this study was to test three opiate analgesics, which also inhibit monoamine reuptake, in the learned helplessness model of depression in rats. Helpless rats receiving (+/-)tramadol (10, 20 mg/Kg) or (-)methadone (2, 4 mg/Kg) showed a decreased number of failures to avoid or escape aversive stimulus (shock) in both the second and the third daily sessions, compared with controls. Rats receiving levorphanol (0.5, 1 mg/Kg) showed a decreased number of such failures in the third session. The number of crossings in the intertrial interval (ITI) was not significantly modified by (+/-)tramadol or (-)methadone. Levorphanol enhanced ITI crosses at 1 mg/Kg. These results, together with other clinical and experimental data, suggest that analgesics with monoaminergic properties improve mood and that this effect may account for their analgesic effect in regulating the affective dimension of pain. From this, it seems probable that the analgesic effect of opiates could be induced by adding together the attenuation produced of both the sensorial and the affective dimensions of pain.     

The blockade of serotonin uptake into synaptosomes:relationship to an interaction with monoamine oxidase inhibitors.

To test the hypothesis that the hyperpyrexia produced by meperidine and detromethorphan in rabbits pretreated with a monoamine oxidase inhibitor is related to inhibition of neuronal uptake of serotonin (5-hydroxytryptamine (5-HT)), fluoxetine (Lilly 110140) was studied. This potent and specific 5-HT neuronal uptake blocker was administered to phenelzine-pretreated rabbits and found to produce a lethal hyperpyrexia in doses equal to or greater than 2.5 mg/kg. The order of potency in blocking 5-[14C]HT uptake into synaptosomes prepared from rabbits was: fluoxetine greater than meperidine = dextromethorphan = levorphanol greater than anileridine greater than alphaprodine greater than morphine. Since fluoxetine, meperidine, and dextromethorphan produce hyperpyrexia in phenelzine-pretreated rabbits, whereas anileridine, alphaprodine, and morphine do not, there appears to be some correlation between the hyperpyrexic response and inhibition of 5-HT uptake. The exception is levorphanol, which is not hyperpyrexic despite being equipotent with meperidine and dextromethorphan in inhibiting 5-HT uptake. The ineffectiveness of levorphanol in producing hyperpyrexia may be due to its marked depressant properties, since the addition of another depressant drug (pentobarbital) antagonized the hyperpyrexic effect of meperidine.     

Stereoselective effect of morphine on antinociception and endogenous opioid peptide levels in plasma but not cerebrospinal fluid of dogs.

Morphine releases endogenous opioids into the circulation of dogs. To test the stereospecificity of this effect, as well as to determine whether morphine also releases endogenous opioids centrally, which might be involved in its antinociceptive action, the effects of (-)-morphine sulfate (10 mg/kg, sc) or (+)-morphine hydrobromide on antinociception in a dog tail-flick test, on semi-quantified morphine-induced signs of salivation, emesis, defecation and ataxia, and on the plasma and cerebrospinal fluid (CSF) levels of endogenous opioid peptides were studied. Plasma and CSF levels of immunoreactive beta-endorphin (i-BE), met-enkephalin (i-ME), leu-enkephalin (i-LE), and dynorphin (i-DY) were quantified by radioimmunoassay in octadecylsilyl-silica cartridge extracts. Immunoreactive morphine (i-M) levels were measured in unextracted samples. (-)-Morphine treatment significantly increased antinociception, morphine-induced signs, i-M levels in plasma and CSF, and i-BE, i-ME, and i-LE levels in plasma, but not CSF. Levels of i-DY remained constant in plasma and CSF. (+)-Morphine treatment did not alter any of these parameters, indicating that the effects of morphine on nociception, behavioral signs, and plasma endogenous opioids in dogs were stereoselective. It is concluded that morphine does not cause an increase in immunoreactive endogenous opioid peptides in the CSF at the time of its peak antinociceptive effect.     

Antagonism of the morphine-induced locomotor activation of mice by fructose: comparison with other opiates and sugars, and sugar effects on brain morphine

The mouse locomotor activation test of opiate action in a 2+2 dose parallel line assay was used in a repeated testing paradigm to determine the test, opiate and hexose specificities of a previously reported antagonism of morphine-induced antinocociception by hyperglycemia. In opiate specificity studies, fructose (5 g/kg, i.p.) significantly reduced the potency ratio for morphine and methadone, but not for levorphanol, meperidine or phenazocine when intragroup comparisons were made. In intergroup comparisons, fructose significantly reduced the potencies of levorphanol and phenazocine, but not methadone or meperidine. In hexose/polyol specificity studies, tagatose and fructose significantly reduced the potency ratio for morphine, whereas glucose, galactose, mannose and the polyols, sorbitol and xylitol, caused no significant decrease in potency. Fructose, tagatose, glucose and mannose (5 g/kg, i.p.) were tested for effects on brain morphine levels 30 min after morphine (60 min after sugar), and all four sugars significantly increased brain morphine relative to saline-pretreated controls. It is concluded that the antagonism of morphine by acute sugar administration shows specificity for certain sugars and occurs despite sugar-induced increases in the distribution of morphine to the brain. Furthermore, the effects of fructose show an opiate specificity similar to that of glucose on antinociception observed previously in our laboratory, except that methadone was also significantly inhibited in the present study, when a repeated-testing experimental design was used.     

Determination of heroin and its metabolites by high-performance liquid chromatography

A method is described for the simultaneous determination of heroin (3, 6-diacetylmorphine, DAM) and its two active metabolites 6-acetylmorphine and morphine in blood by high-performance liquid chromatography using a normal-phase column and a UV detector at 218 nm. The compounds are stabilized in blood by rapid freezing and recovered by a multistep liquid—liquid extraction. The mobile phase is acetonitrile—methanol (75:25, v/v) buffered to apparent pH 7 with ammonium hydroxide and acetic acid. Usingl--acetylmethadol as an internal standard, UV detection and a 1-ml biofluid sample, the lower limit of sensitivity is 12.5 ng/ml. Commonly used narcotic analgesics including codeine, propoxyphene, meperidine, methadone and levorphanol do not interfere with the analysis. The method has been applied to blood samples from humans and rats. Extracts of blood from a patient who had received an intravenous dose of 14 mg of DAM contained DAM and both of its active metabolites.     

Significance of GABAergic systems in the action of improgan, a non-opioid analgesic

Improgan is the prototype drug from a new class of non-opioid analgesics chemically related to histamine and histamine antagonists, but the mechanism of action of these compounds has not been identified. Because several classes of analgesics act in the brain by reducing GABAergic inhibition of endogenous pain-relieving circuits, and because the activity of these substances is abolished by the GABA(A) agonist muscimol, the present study assessed the effects of muscimol on improgan antinociception in rats. Intracerebroventricular (icv) improgan (80 microg) and morphine (20 microg) both induced 80-100% of maximal analgesic responses on the tail flick test 10 to 30 min later. However, muscimol pretreatment (0.5 microg, icv) completely eliminated the antinociceptive activity of both compounds. Since improgan in vitro lacks activity at opioid and GABA(A) receptors, these findings: 1) confirm earlier literature showing that muscimol inhibits morphine analgesia, and 2) suggest that improgan activates a supraspinal, descending analgesic pathway, possibly via inhibition of GABAergic transmission. Since muscimol is the first compound discovered which inhibits improgan analgesia, muscimol will be a useful tool for the further characterization of this new class of pain-relieving substances.     

Electroacupuncture Effects in a Rat Model of Complete Freund’s Adjuvant-Induced Inflammatory Pain: Antinociceptive Effects Enhanced and Tolerance Development Accelerated

We have previously shown that electroacupuncture (EA) produced antinociception through the release of endogenous opioid peptides to activate opioid receptors during acute nociception. EA produced tolerance after its prolonged application. It has reported that 100 Hz EA could reduce mechanical hyperalgesia in complete Freund’s adjuvant (CFA)-induced inflammatory nociception rats. The present study aims to investigate the antinociceptive effect of EA and the development of EA tolerance in chronic inflammatory nociception rats with CFA injection into the hind paw plantar. The results showed that the antinociceptive effect of 100 Hz EA was significantly enhanced in CFA-induced inflammatory nociception rats. Naloxone at 20 mg/kg could significantly block this antinociceptive effect. Chronic tolerance to EA was developed faster in CFA-induced inflammatory nociception rats than in normal rats. Therefore, 100 Hz EA could enhance antinociceptive effects and accelerate tolerance development in CFA-induced inflammatory nociception rats. The enhancement of EA antinociceptive effect in CFA-induced inflammatory nociception rats might involve the endogenous opioid peptides such as dynorphin. Special issue article in honor of Dr. Ji-Sheng Han.     

Antinociceptive effect of 7-hydroxymitragynine in mice: Discovery of an orally active opioid analgesic from the Thai medicinal herb Mitragyna speciosa

Mitragynine is an indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa. We previously reported the morphine-like action of mitragynine and its related compounds in the in vitro assays. In the present study, we investigated the opioid effects of 7-hydroxymitragynine, which is isolated as its novel constituent, on contraction of isolated ileum, binding of the specific ligands to opioid receptors and nociceptive stimuli in mice. In guinea-pig ileum, 7-hydroxymitragynine inhibited electrically induced contraction through the opioid receptors. Receptor-binding assays revealed that 7-hydroxymitragynine has a higher affinity for micro-opioid receptors relative to the other opioid receptors. Administration of 7-hydroxymitragynine (2.5-10 mg/kg, s.c.) induced dose-dependent antinociceptive effects in tail-flick and hot-plate tests in mice. Its effect was more potent than that of morphine in both tests. When orally administered, 7-hydroxymitragynine (5-10 mg/kg) showed potent antinociceptive activities in tail-flick and hot-plate tests. In contrast, only weak antinociception was observed in the case of oral administration of morphine at a dose of 20 mg/kg. It was found that 7-hydroxymitragynine is a novel opioid agonist that is structurally different from the other opioid agonists, and has potent analgesic activity when orally administered.     

Mechanisms of the antinociceptive action of (?) Epicatechin obtained from the hydroalcoholic fraction of Combretum leprosum Mart & Eic in rodents

ABSTRACT: BACKGROUND: The mechanisms of the antinociceptive activity of () epicatechin (EPI), a compound isolated from the hydroalcoholic fraction of Combreum leprosum Mart & Eicher. METHODS: were assessed in the model of chemical nociception induced by glutamate (20 mumol/paw). To evaluate the mechanisms involved, the animals , male Swiss mice (25-30 g), received EPI (50 mg/kg p.o.) after pretreatment with naloxone (2 mg/kg s.c. opioid antagonist), glibenclamide (2 mg/kg s.c. antagonist K + channels sensitive to ATP), ketanserin (0.3 mg/kg s.c. antagonist of receptor 5-HT2A), yoimbine (0.15 mg/kg s.c. alpha2 adrenergic receptor antagonist), pindolol (1 mg/kg s.c. 5-HT1a/1b receptor antagonist), atropine (0.1 mg/kg s.c. muscarinic antagonist) and caffeine (3 mg/kg s.c. adenosine receptor antagonist), ondansetron (0.5 mg/kg s.c. for 5-HT3 receptor) and L-arginine (600 mg/kg i.p.). RESULTS: The antinociceptive effect of EPI was reversed by pretreatment with naloxone and glibenclamide, ketanserin, yoimbine, atropine and pindolol, which demonstrates the involvement of opioid receptors and potassium channels sensitive to ATP, the serotoninergic (receptor 5HT1A and 5HT2A), adrenergic (receptor alpha 2) and cholinergic (muscarinic receptor) systems in the activities that were observed. The effects of EPI, however, were not reversed by pretreatment with caffeine, L-arginine or ondansetron, which shows that there is no involvement of 5HT3 receptors or the purinergic and nitrergic systems in the antinociceptive effect of EPI. In the Open Field and Rotarod test, EPI had no significant effect, which shows that there was no central nervous system depressant or muscle relaxant effect on the results. CONCLUSIONS: This study demonstrates that the antinociceptive activity of EPI in the glutamate model involves the participation of the opioid system, serotonin, adrenergic and cholinergic.     

Antinociceptive activity of Sesbania sesban (Linn) wood extracts, a preliminary study

The wood of the plant Sesbania sesban, is reported to have antinociceptive activity. To validate its folk use in the treatment of pain, wood was extracted successively with petroleum ether, chloroform, ethyl acetate, ethanol, and water to produce respective extracts. The extracts (50 and 100 mg/kg, ip) were screened for antinociceptive activity using hot plate test and acetic acid-induced writhing test in mice. Petroleum ether, chloroform, and ethyl acetate extracts showed significant and dose-dependent activity in both the tests. In order to find out the involvement of opioid receptors, effect of naloxone (1 mg/kg, sc) on the action of extracts was checked in hot plate test. Petroleum ether, chloroform, and ethyl acetate extracts showed significant and dose dependant antinociceptive activity. The antinociceptive action of the extracts was blocked by naloxone, suggesting involvement of opioid receptors in the action.     

Modulation of acute morphine tolerance by corticotropin-releasing factor and dynorphin A in the mouse spinal cord.

Previously, we have demonstrated that intrathecally (i.t.) administered corticotropin-releasing factor (CRF) in mice produces stimulus-specific antinociception and modulation of morphine-induced antinociception by mechanisms involving spinal kappa opioid receptors. Recently, we also have found that CRF releases immunoreactive dynorphin A, a putative endogenous kappa opioid receptor agonist, from superfused mice spinal cords in vitro. Dynorphin A administered intracerebroventricularlly (i.c.v.) to mice has been shown to modulate the expression of morphine tolerance. In the present study, the possible modulatory effects of i.t. administered CRF as well as dynorphin A on morphine tolerance were studied in an acute tolerance model. Subcutaneous administration of 100 mg/kg of morphine sulfate (MS) to mice caused an acute tolerance to morphine-induced antinociception. The antinociceptive ED50 of MS was increased from 4.4 mg/kg (naive mice) to 17.9 mg/kg (4 hours after the injection of 100 mg/kg MS). To study the modulatory effects of spinally administered CRF and dynorphin A on the expression of morphine tolerance, CRF and dynorphin A were injected i.t. at 15 min and 5 min, respectively, before testing the tolerant mice by the tail-flick assay. The antinociceptive ED50 of MS in tolerant mice was decreased to 8.8 mg/kg and 7.1 mg/kg, respectively, after i.t. administration of CRF (0.1 nmol) and dynorphin A (0.2 nmol). In contrast, 0.5 nmol of alpha-helical CRF (9-41), a CRF antagonist and 0.4 nmol of norbinaltorphimine, a highly selective kappa opioid receptor antagonist, when administered i.t. at 15 min before the tail-flick test in tolerant mice, increased the antinociceptive ED50 of MS to 56.6 mg/kg and 88.8 mg/kg, respectively. These data confirmed the modulatory effect of dynorphin A on morphine tolerance and suggested that CRF, which releases dynorphin A in several central nervous system regions, also plays a modulatory role in the expression of morphine tolerance.     

Anticonvulsant properties of the cerebrospinal fluid during activation of the antiepileptic system of the brain

Cerebrospinal fluid (CSF) was obtained after 30-40 sessions of daily electrical stimulation of the cat cerebellum vermis. The intraventricular injection of CSF (10 microliters) to Wistar rats increased the latent period of initial seizure manifestations, significantly reduced the number of animals with seizures and reduced the severity of seizures induced by korazol injection (40 mg/kg). Analogous seizure changes were observed in rats after intraventricular injection of CSF (10 microliters) from cats subject to 3-10 electroshock seizure fits. Intraventricular injection of CSF (250 microliters) obtained from cats after electroshock to cats with strychnine-induced epileptic foci in the brain cortex led to the suppression of the epileptic activity. The conclusion was made that different ways of antiepileptic system activation cause the accumulation of endogenous antiepileptic substances in CSF.     

Reversal of morphine, methadone and heroin induced effects in mice by naloxone methiodide

Opioid overdose, which is commonly associated with opioid induced respiratory depression, is a problem with both therapeutic and illicit opioid use. While the central mechanisms involved in the effects of opioids are well described, it has also been suggested that a peripheral component may contribute to the effects observed. This study aimed to further characterise the effects of the peripherally acting naloxone methiodide on the respiratory, analgesic and withdrawal effects produced by various opioid agonists. A comparison of the respiratory and analgesic effects of morphine, methadone and heroin in male Swiss-Albino mice was conducted and respiratory depressive ED(80) doses of each opioid determined. These doses (morphine 9 mg/kg i.p., methadone 7 mg/kg i.p., and heroin 17 mg/kg i.p.) were then used to show that both naloxone (3 mg/kg i.p.) and naloxone methiodide (30-100 mg/kg i.p.) could reverse the respiratory and analgesic effects of these opioid agonists, but only naloxone precipitated withdrawal. Further investigation in female C57BL/6J mice using barometric plethysmography found that both opioid antagonists could reverse methadone induced decreases in respiratory rate and increases in tidal volume. Its effects do not appear to be strain or sex dependent. It was concluded that naloxone methiodide can reverse the respiratory and analgesic actions of a variety of opioid agonists, without inducing opioid withdrawal.     

The effect of the cerebrospinal fluid from rats subjected to picrotoxin-induced kindling on the motor activity and seizure susceptibility of recipient animals

Experiments on rats have shown that repeated administration of primarily subthreshold dose of picrotoxin leads to the occurrence and progressive enhancement of seizure manifestations. During picrotoxin kindling the decrease of locomotor activity in interictal periods was recorded. Microinjection of cerebrospinal fluid (CSF) of kindled rats into lateral brain ventriculi of recipients resulted in decrease of locomotor activity and acute primarily generalized picrotoxin induced seizures. These effects of CSF were blocked by naloxone administration and were observed only if injecting CSF which was preliminarily treated with protease inhibitors. It is concluded that endogenous opioid substances accumulate in CNS during kindling and evoke a decrease in the locomotor activity. These substances act as anticonvulsant factors which control the development of epileptic activity.     

Evidence for a supraspinal analgesic effect with cyclazocine and pentazocine

The antinociceptive effect of the benzomorphan class of opioid analgesics have been difficult to measure utilizing some of the standard animal pain models. This may be due, in part, to the sedative and/or motor effects associated with these drugs. In addition, it has been proposed that the major site of action for drugs with agonist activity at the kappa opiate receptor is exclusively at the spinal level opposed to both spinal and supraspinal as with the mu receptor agonists such as morphine. The present study examines the antinociceptive effect of the mixed agonist-antagonists cyclazocine and pentazocine utilizing electrical stimulation of the midbrain reticular formation (MRF) as the aversive stimulus in the rat. Animals were trained to escape MRF stimulation by turning a cylindrical manipulandum. An escape threshold was determined by varying the current intensity according to a modification of the psychophysical method of limits. In addition to the determination of the escape threshold the response latency and strength of response was also measured. Both cyclazocine (0.25-1.0 mg/kg) and pentazocine (2.5-12.5 mg/kg) raised the escape threshold in a dose-dependent manner without any concomitant change in the response latency or strength of response. These data suggest that the observed threshold elevation is due to a specific antinociceptive effect. Since the aversive stimulation was delivered supraspinally, the data also suggest that there are supraspinal mechanisms mediated by kappa receptors responsible for this analgesic effect.     

Morphine induces aggression but not analgesia in the naked mole-rat (Heterocephalus glaber)

1. The antinociceptive effect in the mole-rat of morphine (1, 10, 20 or 30 mg/kg) and nefopam (10 or 20 mg/kg) was studied. 2. In the hotplate test, morphine had no analgesic effect. A reduced response latency after morphine (10 and 20 mg/kg) could possibly be explained by hyperactivity and excited behaviour. 3. After morphine (10, 20 and 30 mg/kg) most of the animals died after fighting when kept in colony cages. Aggressive behaviour and death was prevented by naloxone, or by keeping the animals in single cages. 4. Nefopam (20 mg/kg) significantly increased the latency for the nociceptive response. 5. It was concluded that in the mole-rat, opioid systems in the CNS may not be involved in the regulation of nociception, but in the regulation of agonistic and motor behaviour.     

Antinociceptive activity of salsolinol (racemate, R(+)-, S(-)-enantiomers): evidence of a peripheral mechanism

The existence and the characteristics of the antinociceptive action of salsolinol (racemate) and its two R(+)- and S(-)-enantiomers were studied using different pain tests in mice. None of these drugs possessed a significant activity on the tests sensitive to central acting analgesics (hot-plate and tail-flick tests), either after systemic (i.p.) or central (i.c.v.) injections. However, injected i.p., they reduced the number of writhes induced by phenylbenzoquinone; the ED50 was 79 +/- 2, 73 +/- 2 and 61 +/- 2 mg/kg for racemate, R(+)- and S(-)-enantiomer respectively. This activity was not antagonized by naloxone. Moreover, racemate and S(-) reduced, only for the highest used active dose on the PBQ test (128 mg/kg, i.p.), the edema induced by an intraplantar injection of carrageenin. These results provide evidence of an analgesic activity independent of the endogenous opiate systems and involving a peripheral mechanism.     

Antinociceptive activity of a neurosteroid tetrahydrodeoxycorticosterone (5alpha-pregnan-3alpha-21-diol-20-one) and its possible mechanism(s) of action

The present study investigates the effects of a neurosteroid tetrahydrodeoxycorticosterone (5alpha-pregnan-3alpha-21-diol-20-one) in two experimental models of pain sensitivity in mice. Tetrahydrodeoxycorticosterone (2.5, 5 mg/kg, i.p.) dose dependently decreased the licking response in formalin test and increased the tail flick latency (TFL) in tail flick test. Bicuculline (2 mg/kg, i.p.), a GABA(A) receptor antagonist blocked the antinociceptive effect of tetrahydrodeoxycorticosterone in TFL test but failed to modulate licking response in formalin test. Naloxone (1 mg/kg, i.p.), an opioid antagonist effectively attenuated the analgesic effect of tetrahydrodeoxycorticosterone in both the models. Tetrahydrodeoxycorticosterone pretreatment potentiated the antinociceptive response of morphine, an opioid compound and nimodipine, a calcium channel blocker in formalin as well as TFL test. Thus, tetrahydrodeoxycorticosterone exerts an analgesic effect, which may be mediated by modulating GABA-ergic and/or opioid-ergic mechanisms and voltage-gated calcium channels.     

Opioids and efflux transporters. Part 3: P-glycoprotein substrate activity of 3-hydroxyl addition to meperidine analogs

Numerous studies have shown that many clinically employed opioid analgesics are substrates for P-glycoprotein (P-gp), suggesting that up-regulation of P-gp may contribute to the development of central tolerance to opioids. The studies herein focus on the development of SAR for P-gp substrate activity in the meperidine series of opioids. Addition of a 3-OH to meperidine and the ketone analog of meperidine yielding bemidone and ketobemidone, respectively, significantly increased P-gp substrate affinity. The results of this study have implications in the development of novel analgesics to be utilized as tools to study the contribution of P-gp on the development of central tolerance to opioids.