Interleukin-1beta in synergism gabapentin with tramadol in murine model of diabetic neuropathy.

Neuropathic pain is a complication of cancer and diabetes mellitus and the most commonly used drugs in the treatment of the diabetic neuropathic pain have only limited efficacy. The aim of this study was to evaluate the role of the biomarker interleukin-1beta (IL-1ß) in the pharmacological interaction of gabapentin with tramadol in a model of diabetic neuropathic pain. CF-1 male mice, pretreated with 200 mg/kg i.p. of streptozocin (STZ), were used and at day 3 and 7 were evaluated by the hot plate test and the spinal cord level of IL-1ß was determined. Antinociceptive interaction of the coadministration i.p. of gabapentin with tramadol, in basic of the fixed the ratio 1:1 of their ED50 values alone, was ascertained by isobolographic analysis. Tramadol was 1.13 times more potent than gabapentin in saline control mice, 1.40 times in STZ mice at 3 days and 1.28 times in STZ at 7 days. The interaction between gabapentin and tramadol was synergic, with an interaction index of 0.30 and 0.22 for mice pretreated with STZ at 3 and 7 days. The combination of gabapentin with tramadol reversed the increased concentration of IL-1ß induced by STZ in diabetic neuropathic mice. These findings could help clarify the mechanism of diabetic neuropathy.

Antinociceptive interaction of gabapentin with minocycline in murine diabetic neuropathy.

OBJECTIVE: Diabetic neuropathy (DN) is the most common complication of diabetes and pain is one of the main symptoms of diabetic neuropathy, however, currently available drugs are often ineffective and complicated by adverse events. The purpose of this research was to evaluate the antinociceptive interaction between gabapentin and minocycline in a mice experimental model of DN by streptozocin (STZ). METHODS: The interaction of gabapentin with minocycline was evaluated by the writhing and hot plate tests at 3 and 7 days after STZ injection or vehicle in male CF1 mice. RESULTS: STZ (150 mg/kg, i.p.) produced a marked increase in plasma glucose levels on day 7 (397.46 ± 29.65 mg/dL) than on day 3 (341.12 ± 35.50 mg/dL) and also developed neuropathic pain measured by algesiometric assays. Gabapentin produced similar antinociceptive activity in both writhing and hot plate tests in mice pretreated with STZ. However, minocycline was more potent in the writhing than in the hot plate test in the same type of mice. The combination of gabapentin with minocycline produced synergistic interaction in both test. CONCLUSION: The combination of gabapentin with minocycline in a 1:1 proportion fulfills all the criteria of multimodal analgesia and this finding suggests that the combination provide a therapeutic alternative that could be used for human neuropathic pain management.

Synergism between COX-3 inhibitors in two animal models of pain.

OBJECTIVE AND DESIGN: The antinociception induced by the intraperitoneal coadministration in mice of combinations of metamizol and paracetamol was evaluated in the tail flick test and orofacial formalin test. METHODS: The antinociception of each drugs alone and the interaction of the combinations was evaluated by isobolographic analysis in the tail-flick and in the formalin orofacial assay of mice. RESULTS: Mice pretreated with the drugs demonstrated that the antinociception of metamizol and paracetamol is dose-dependent. The potency range on the antinocifensive responses for metamizol or paracetamol was as follows: orofacial (Phase II) > orofacial (Phase I) > tail flick. In addition, the coadministration of metamizol with paracetamol induced a strong synergistic antinociception in the algesiometer assays. Both drugs showed effectiveness in inflammatory pain. CONCLUSION: These actions can be related to the differential selectivity of the drugs for inhibition of COX isoforms and also to the several additional antinociception mechanisms and pathways initiated by the analgesic drugs on pain transmission. Since the efficacy of the combination of metamizol with paracetamol has been demonstrated in the present study, this association could have a potential beneficial effect on the pharmacological treatment of clinical pain.

Receptors involved in dexketoprofen analgesia in murine visceral pain.

Various animal models, especially rodents, are used to study pain, due to the difficulty of studying it in humans. Many drugs that produce analgesia have been studied and there is evidence among which NSAIDs deserve to be highlighted. Dexketoprofen (DEX) provides a broad antinociceptive profile in different types of pain; therefore, this study was designed to evaluate the profile of antinociceptive potency in mice. Analgesic activity was evaluated using the acetic acid abdominal constriction test (writhing test), a chemical model of visceral pain. Dose-response curves for i.p. DEX administration (1, 3, 10, 30 and 100 mg/kg), using at least six mice in each of at least five doses, was obtained before and 30 min after pre-treatment with different pharmacological agents. Pretreatment of the mice with opioid receptor antagonists was not effective; however, the serotonin receptor antagonist and nitric oxide synthase inhibitor produce a significant increase in DEX-induced antinociception. The data from the present study shows that DEX produces antinociception in the chemical twisting test of mice, which is explained with difficulty by the simple inhibition of COX. This effect appears to be mediated by other mechanisms in which the contribution of the NO and 5-HT pathways has an important effect on DEXinduced antinociception.

The effect of opioid antagonists on synergism between dexketoprofen and tramadol.

The antinociceptive activity of dexketoprofen was studied in mice using the formalin assay for orofacial pain. The interaction between dexketoprofen and co-administered tramadol was studied using isobolographic analysis. The intraperitoneal administration of dexketoprofen or tramadol, showed dose-dependent antinociceptive activity in both phases of the assay. When administered together, the interaction was mildly synergistic during the first phase, and antagonistic in the second phase. Selective opioid receptor antagonists where used in order to measure the analgesic activity of tramadol in other regions of the CNS. The co-administration of dexketoprofen and tramadol, with previous administration of naltrexone, showed high synergistic activity during the first phase, and less but still synergistic during the second. When using naltrindole, the interaction was mildly more synergistic than the mixture dexketoprofen+tramadol during both phases. Using norbinaltorphimine, the interaction was synergistic in both phases, more marked in the second. These results suggest that the opioid activity of tramadol has an inhibiting effect in antinociceptive activity of the interaction between dexketoprofen and tramadol during the inflammatory (late) stages of pain.

Synergism between NSAIDs in the orofacial formalin test in mice.

Opioids and non-steroidal anti-inflammatory drugs (NSAIDs) are used to relieve acute and chronic pain. The purpose of this study was to determine the degree of interaction between dexketoprofen and NSAID examples of COXs inhibitors using the isobolographic analysis in the formalin orofacial test in mice. The drugs, i.p., induced a dose-dependent antinociception with different potencies in both test phases. Combinations of dexketoprofen with naproxen, nimesulide, ibuprofen or paracetamol on the basis of the fixed ratio (1:1) of their ED(50)'s values alone demonstrated synergism in both phases. This is important since the orofacial pain is a test not currently used in mice; the drugs are all analgesic for humans and phase II is representative of inflammatory pain. The synergism was: COX-3>COX-2>COX-1 inhibitors, this is particularly interesting since the inhibitor of COX-3, paracetamol, displayed a robust anti-inflammatory activity in an assay of acute and inflammatory pain that mimics inflammatory pain in humans. In conclusion, the synergism of the dexketoprofen/NSAID combinations may improve this type of therapeutic profile, since with low doses of the components, side effects are not likely to occur, and they may be used in long-term treatments.

Analgesic activity of Ugni molinae (murtilla) in mice models of acute pain.

Leaf extracts of Ugni molinae Turcz. (Myrtaceae) are used in Chilean folk medicine as analgesic and anti-inflammatory. The antinociceptive effect of dichloromethane (DCM), ethyl acetate (EA) and methanol (ME) leaf extracts was assessed by intraperitoneal, oral and topical administration in writhing, tail flick, and tail formalin tests in mice. The extracts showed a dose-dependent antinociceptive activity in all the assays under different administration routes. The ED(50) values for the different tests for the DCM, EA, ME extract and reference drug (ibuprofen) were as follows. Writhing test in acetic acid (i.p. administration): 0.21, 0.37, 1.37 and 0.85mg/kg, respectively; tail flick test (oral administration): 199, 189, 120 and 45.9mg/kg. The EC(50) values for tail flick test were (topical administration): 2.0, 0.35, 1.4 and 8.2% (w/v), respectively; and the topical analgesic effects were (formalin assay) 75.5, 77.5, 31.6 and 76.5%, respectively. Ugni molinae extracts produce antinociception in chemical and thermal pain models through a mechanism partially linked to either lipooxygenase and/or cyclooxygenase via the arachidonic acid cascade and/or opioid receptors. Flavonoid glycosides and triterpenoids have been isolated from the plant and can be associated with the observed effect. Our results corroborate the analgesic effects of Ugni molinae, and justify its traditional use for treating pain.

Receptors involved in dexketoprofen analgesia in murine visceral pain

Various animal models, especially rodents, are used to study pain, due to the difficulty of studying it inhumans. Many drugs that produce analgesia have been studied and there is evidence among whichNSAIDs deserve to be highlighted. Dexketoprofen (DEX) provides a broad antinociceptive profile indifferent types of pain; therefore, this study was designed to evaluate the profile of antinociceptivepotency in mice. Analgesic activity was evaluated using the acetic acid abdominal constriction test(writhing test), a chemical model of visceral pain. Dose-response curves for i.p. DEX administration (1,3, 10, 30 and 100 mg/kg), using at least six mice in each of at least five doses, was obtained before and30 min after pre-treatment with different pharmacological agents. Pretreatment of the mice with opioidreceptor antagonists was not effective; however, the serotonin receptor antagonist and nitric oxidesynthase inhibitor produce a significant increase in DEX-induced antinociception. The data from thepresent study shows that DEX produces antinociception in the chemical twisting test of mice, which isexplained with difficulty by the simple inhibition of COX. This effect appears to be mediated by othermechanisms in which the contribution of the NO and 5-HT pathways has an important effect on DEXinduced antinociception.

Castration and tolerance induces changes in the levels of the activity of acetylcholinesterase in the isolated vas deferens of the rat.

Changes in the activity of acetylcholinesterase (AChE) of the isolated vas deferens from normal, castrated, morphine and ethanol-tolerant rats were studied. Three days after the termination of treatment with morphine and on the last day of treatment with ethanol, a significant inhibition of the activity of AChE was detected. This reduction in the enzymatic activity persisted in morphine-tolerant rats for 15 days, but not for 30 days, at which time the levels of AChE were determined to be normal. However, in ethanol-tolerant rats, there were no significant changes found at days 15 or 30. The activity of AChE was decreased significantly in castrated rats, but this effect was reversed by treatment with testosterone. During withdrawal from morphine or ethanol, the levels of AChE were significantly increased. The results indicate that morphine and ethanol may be inducing changes in the feedback mechanism which regulates the levels of AChE at post-synaptic sites, and these changes could play an important role in the development of tolerance to morphine and to ethanol.

alpha-Adrenoceptor and opioid receptor modulation of clonidine-induced antinociception.

1. The antinociceptive action of clonidine (Clon) and the interactions with alpha 1, alpha 2 adrenoceptor and opioid receptor antagonists was evaluated in mice by use of chemical algesiometric test (acetic acid writhing test). 2. Clon produced a dose-dependent antinociceptive action and the ED50 for intracerebroventricular (i.c.v.) was lower than for intraperitoneal (i.p.) administration (1 ng kg-1 vs 300 ng kg-1). The parallelism of the dose-response curves indicates activation of a common receptor subtype. 3. Systemic administration of prazosin and terazosin displayed antinociceptive activity. Pretreatment with prazosin produced a dual action: i.c.v. Clon effect did not change, and i.p. Clon effect was enhanced. Yohimbine i.c.v. or i.p. did not induce antinonciception, but antagonized Clon-induced activity. These results suggest that alpha 1- and alpha 2-adrenoceptors, either located at the pre- and/or post-synaptic level, are involved in the control of spinal antinociception. 4. Naloxone (NX) and naltrexone (NTX) induced antinociceptive effects at low doses (microgram kg-1 range) and a lower antinociceptive effect at higher doses (mg kg-1 range). Low doses of NX or NTX antagonized Clon antinociception, possibly in relation to a preferential mu opioid receptor antagonism. In contrast, high doses of NX or NTX increased the antinociceptive activity of Clon, which could be due to an enhanced inhibition of the release of substance P. 5. The results obtained in the present work suggest the involvement of alpha 1-, alpha 2-adrenoceptor and opioid receptors in the modulation of the antinociceptive activity of clonidine, which seems to be exerted either at spinal and/or supraspinal level.

Interaction of opioids with antidepressant-induced antinociception.

The antinociceptive activity of antidepressant drugs is poorly understood. In this study, using the acetic acid writhing test in mice, the antinociception produced by clomipramine (CLO), maprotiline (MAP), imipramine (IMI), and zimelidine (ZIM) was tested and correlated with opioid drugs. All the compounds displayed a significant dose-dependent antinociception, which was not antagonized by naloxone (NX) or naltrexone (NTX). The administration of morphine (M) plus CLO, MAP, IMI or ZIM resulted in a significant additive effect that was antagonized by 1 or 10 mg/kg NX or NTX, except in the case of IMI. This finding suggests that the additive effect seems to be partially due to activation of opioid receptors, except for the case of imipramine. However, aminophylline, a non-selective blocker of A1/A2 adenosine receptors, significantly antagonized the antinociceptive activity of CLO, IMI, MAP and ZIM, demonstrating an interaction at the level of adenosine receptors. This work suggests that the antinociceptive activity of antidepressants could be dependent on critical levels of free 5-HT and NE at receptor(s) site(s) in CNS and on their interaction with opioid and adenosine receptors.

Neuromodulatory actions of substance P on the muscarinic receptors of the vas deferens of the rat.

The response of post-synaptic neurokinin receptors to SP were not changed by pirenzepine or N-methyl-scopolamine. Atropine led to a slight increase in the EC50 of SP for its post-synaptic neurokinin (NK-A) receptor. In the presence of neostigmine no changes in the Emax and EC50 values of SP for its post- and pre-synaptic receptor site were observed. Only the muscarinic receptor site were observed. Only the muscarinic receptor antagonists, atropine and NMS, elicited statistically significant increases in the Emax of SP at its presynaptic receptor (NK-A). Addition of 7.4-740 nM SP resulted in a decrease in the EC50 and Emax values of ACh for its post-synaptic muscarinic receptor (M1). Conversely, 740 nM SP produced an increase in the EC50 and Emax values of ACh at its pre-synaptic muscarinic receptor (M2). Concentrations of 7.4 and 74 nM SP did not produce statistically significant changes in the Emax of ACh for its pre-synaptic M2 receptor.

Pharmacological actions of substance P in the rat vas deferens.

SP produces two different muscular responses in the isolated vas deferens of the rat. 1) A myotropic, post-synaptic effect that results from stimulation of one subtype of SP receptor, located on the membrane of the smooth muscle (NK-3 receptor). 2) A neurogenic effect at the pre-synaptic level that results from stimulation of another subtype of SP receptor, located on the nerve terminals (NK-1 receptor).

Antinociceptive effects of Ca2+ channel blockers.

The antinociceptive action of four Ca2+ channel blockers, nifedipine, nimodipine, verapamil and diltiazem, was evaluated and compared to that of morphine using three algesiometric tests in mice and rats, namely, formalin, writhing and modified hot-plate test. Dose-response curves for all the drugs tested were similar and a significant dose-dependent antinociceptive action was evident in the formalin and writhing tests. However, in the hot-plate test, only nimodipine exhibited a significant analgesic effect, confirming the misleading results previously reported for this test. The findings suggest a pharmacological role of Ca2+ channel blockers in the modulation of antinociception under acute conditions. The analgesic action of Ca2+ channel blockers could be mediated by an increase in the nociceptive threshold resulting from interference with Ca2+ influx at opioid receptors, because Ca2+ influx is critical for the release of neurotransmitters and other substances implicated in nociception and inflammation. It is suggested that if a substance has a Ca2+ channel blocking effect, it should probably have some antinociceptive properties.

Effect of the inhibition of serotonin biosynthesis on the antinociception induced by nonsteroidal anti-inflammatory drugs.

The antinociceptive activity of nonsteroidal anti-inflammatory drugs (NSAIDs) has been explained mainly on the basis of their inhibition of the enzyme cyclooxygenase (COX); however, this inhibition is not enough to completely explain the analgesic efficacy of these drugs. The modulation exerted by serotonergic systems on antinociception is well known. The purpose of the present work was to further explore the role of serotonin in the antinociceptive activity of NSAIDs using the writhing test and the tail-flick test of the mice after the inhibition of serotonin biosynthesis with intraperitoneal p-chlorophenylalanine (p-CPA). Pretreatment with p-CPA produced a significant decrease in the antinociceptive activity of NSAIDs administered either by the intraperitoneal or intrathecal routes, in both algesiometric tests. These results suggest a complementary mechanism of antinociception for NSAIDs, independent of their ability to inhibit the activity of COX, involving the activation of descending serotonergic pathways. By the pharmacological nature of the study, one limitation was the absence of biochemical measurement of the synthesis of 5-HT, since the reduction of the brain 5-HT synthesis by pretreatment with p-CPA will be expressed as a diminished antinociceptive activity of NSAIDs, which would be a new argument to consider NSAIDs acting as central analgesic agents.

Removal of the endothelial layer in perfused mesenteric vascular bed of the rat.

The endothelial layer was removed from the isolated mesenteric vascular bed of the rat by perfusion with hypotonic Tyrode solution for 12.5 min. This procedure damaged more than 95% of the endothelial cells. After endothelial removal, the response to norepinephrine was significantly enhanced, whereas the relaxation induced by acetylcholine (ACh) was completely abolished. The results of this work show that perfusion with hypotonic solutions provides a reliable method of endothelial removal in isolated perfused vascular beds, allowing the study of endothelial-dependent vascular responses.

Antinociception, tolerance, and physical dependence comparison between morphine and tramadol.

The mechanism of action of tramadol includes the activation of opioid receptors, and the potential ability of the drug to induce tolerance and physical dependence has been evaluated in different animal species and humans. This work was designed to study the involvement of opioid receptors in the antinociceptive activity and the potential ability to develop tolerance, crosstolerance, and/or physical dependence of tramadol. The writhes induced by acetic acid administration was used as algesiometric test. After chronic administration of tramadol, tolerance was evaluated by measuring the antinociceptive activity, and physical dependence was measured by naloxone administration. Morphine was used as drug of comparison. The i.p. administration of tramadol produced a dose-dependent antinociception with an ED50 value of 7.82 +/- 1.16 mg/kg, which was unchanged after chronic administration of either tramadol (39.1 or 100 mg/kg) or morphine (1.05 or 100 mg/kg). By contrast, the ED50 for morphine (0.21 +/- 0.08 mg/kg) was significantly reduced only by chronic pretreatment with both doses of morphine (tolerance). Physical dependence was developed only in mice pretreated with morphine, as evidenced by the presence of jumps, wet-dog shakes, tachypnea, piloerection, seizures, diarrhea, and urination after the administration of naloxone (1 mg/kg). These findings suggest that the antinociceptive activity of tramadol in mice is due to activation of opioid and nonopioid mechanisms, and as opposed to morphine, is not likely to induce tolerance and physical dependence.

Atropine reverses the antinociception of nonsteroidal anti-inflammatory drugs in the tail-flick test of mice.

The nonsteroidal anti-inflammatory drugs (NSAIDs) clonixin, diclofenac, piroxicam, ketoprofen, meloxicam, and paracetamol induced antinociception after intraperitoneal or intrathecal administration in mice submitted to an acute thermal algesiometric test without inflammation (tail-flick). Antinociception was evaluated by the increase in reaction time difference (Delta latency), between readings obtained before and after the administration of drugs. The antinociception induced by doses of NSAIDs producing between 20% and 30% of the maximum possible effect (MPE) 30 min after intraperitoneal and 15 min after intrathecal injections was compared with the antinociception obtained after pretreatment with 1 mg/kg atropine ip, 30 min before. Systemic atropine (1 mg/kg) significantly antagonized NSAID-induced antinociception in all cases, both after intraperitoneal and intrathecal administration. Cholinergic depletion by intracerebroventricular hemicholinium-3 (HC-3, 5 microg) 5 h before prevented the antinociceptive effect of all NSAIDs. These observations suggest that intrinsic muscarinic cholinergic facilitatory pathways represent an important modulating system in pain perception in this animal model of acute thermal pain. The results of the present work support the increasingly accepted notion that NSAIDs are effective analgesics even when inflammation is not present, acting by mechanisms that involve actions on spinal and supraspinal nociceptive transmission. It is suggested that, similar to morphine and clonidine, the active mechanism of NSAIDs may involve the release of acetylcholine (ACh) in the spinal cord.

Interaction between the antinociceptive effect of ketoprofen and adrenergic modulatory systems.

The interaction between the antinociceptive activity of ketoprofen and adrenergic agents was evaluated in the writhing test of mice. Dose-response curves were obtained for systemic and intrathecal antinociceptive effects of ketoprofen, phenylephrine, clonidine, desipramine, and prazosin; and ED50 were calculated. The interactions were evaluated by isobolographic analysis of the systemic or intrathecal co-administration of fixed-ratio combinations of ketoprofen with each adrenergic agent. The intraperitoneal combinations of ketoprofen with phenylephrine, clonidine, and prazosin showed supra-additivity, indicating that activation of alpha1 and alpha2 adrenoceptors play a role in nociceptive transmission at supraspinal levels. The same combinations given intrathecal were only additive. Desipramine intraperitoneal was also supra-additive: however, when ketoprofen was administered intrathecally with desipramine, only an additive interaction was obtained. The supra-additive interactions suggest that complementary mechanisms of antinociception have been activated, related with interference with the multiplicity of receptors and systems involved in the transmission of the nociceptive information. Racemic ketoprofen has an antinociceptive activity which is probably not only due to COX inhibition but also involves noradrenergic systems at spinal and supraspinal levels.