Morphine-6-glucuronide: analgesic effects and receptor binding profile in rats

The antinociceptive effects of morphine-6-glucuronide (M6G) were examined in two animal models of pain, the tail immersion test (reflex withdrawal to noxious heat) and the formalin test (behavioral response to minor tissue injury). In the tail immersion test, M6G produced an increase in withdrawal latency that rose rapidly between 0.01 and 0.025 ug ICV or 1 and 2 mg/kg SC. A further increase occurred at doses greater than 0.2 ug ICV or 4 mg/kg SC and was associated with marked catalepsy and cyanosis. Naloxone, 0.1 mg/kg SC, shifted the lower component of the dose-effect relation by a factor of 24. In the formalin test, 0.01 ug M6G ICV produced hyperalgesia, while between 0.05 and 0.2 ug ICV, antinociception increased rapidly without toxicity. The dose effect relations for hyperalgesia and antinociception were shifted to the right by factors of 20- and 3-fold, respectively. By comparison, ICV morphine was 60 (formalin test) to 145-200 (tail immersion test) times less potent than M6G. At sub-nanomolar concentrations, M6G enhanced the binding of [3H]-etorphine, [3H]-dihydromorphine and [3H]-naloxone to rat brain membrane receptors by 20-40%. At higher concentrations, M6G displaced each ligand from binding sites, with Ki values of about 30 nM, as compared to morphine Ki values of about 3 nM. The data indicate that the in vivo and in vitro effects of M6G are complex and that M6G may play an important role in analgesia in experimental animals, and by implication, in man.     

Increased CNS uptake and enhanced antinociception of morphine-6-glucuronide in rats after inhibition of P-glycoprotein

Morphine-6-glucuronide (M6G) is a substrate of P-glycoprotein (P-gp), which forms an outward transporter at the blood-brain barrier. Inhibition of P-gp may therefore be expected to cause increased CNS uptake of M6G. We directly assessed the spinal concentrations of M6G and its antinociceptive effects in rats following pharmacological inhibition of P-gp. Spinal cord tissue concentrations of M6G were assessed by microdialysis with probes transversally implanted through the dorsal horns of the spinal cord at level L4. Ten rats received M6G intravenously (0.018 mg/kg loading dose plus 0.00115 mg/kg/min for an 8-h infusion), five of them together with PSC833 to inhibit P-gp (32-h infusion, starting 24 h before the addition of M6G). Antinociceptive effects were explored by means of formalin tests. After having obtained evidence for enhanced CNS uptake and antinociception of M6G in the presence of PSC833, additional behavioural experiments were performed in another 32 rats to assess the dose dependency of the antinociceptive effects of M6G either with or without PSC833 in comparison with both PSC833 alone and placebo. Inhibition of P-gp increased the M6G concentrations in the spinal cord approximately three-fold whereas the plasma concentrations were increased only by a factor of 1.4, which resulted in a more than doubled spinal cord/plasma concentration ratio (from 0.08 +/- 0.03 for M6G alone to 0.17 +/- 0.08 for M6G plus PSC833). Antinociceptive effects of M6G were significantly enhanced by inhibition of P-gp. Inhibition of P-gp alters the transport of M6G across the blood-brain barrier, resulting in enhanced spinal cord uptake and enhanced antinociception.     

Antinociceptive effects of morphine-6-glucuronide in homozygous MDR1a P-glycoprotein knockout and in wildtype mice in the hotplate test

Morphine-6-glucuronide (M6G), a major metabolite of morphine with agonist opioid-receptor activity, was reported to be a substrate of P-glycoprotein (P-gp). Inhibition of P-gp may thus result in higher brain uptake of M6G. The goal of this observer-blinded, placebo controlled study, was to compare the antinociceptive effects of M6G in homozygous P-gp knockout (mdr1a(-/-)) and wildtype (mdr1a(+/+)) mice. M6G was injected intraperitoneally as a single dose of 0, 0.5, 1, 2.5, 5, and 10 mg/kg. Eight P-gp knockout and eight wildtype mice were studied per dose. A hot plate test was performed before and 5, 15, 30, 60, 90, 120, and 150 min after M6G administration. Plasma-concentrations of M6G, morphine, and morphine-3-glucuronide (M3G) were measured after intraperitoneal injection of 5 mg/kg M6G in another 14 P-gp knockout and 14 wildtype mice. No difference neither in the dose response relationship, nor in the time course of response latency times were observed between P-gp knockout and wildtype mice. However, latency times increased with higher doses of M6G, with antinociception significantly different from placebo at a M6G dose of 5 and 10 mg/kg. P-gp knockout mice tended to have higher plasma concentrations than the wildtype. However, plasma concentrations widely overlapped between groups and therefore no statistical significant group difference could be detected. We conclude that despite reported doubling of M6G brain uptake, absence of mdr1a coded P-gp does not enhance antinociceptive effects of M6G in the hotplate test after acute single-dose administration in mdr1a(-/-) knockout mice.     

Identification and synthesis of norhydromorphone, and determination of antinociceptive activities in the rat formalin test

The main objective of this paper is to report the identification and synthesis of norhydromorphone, a novel metabolite of hydromorphone, and its antinociceptive activities when tested in the formalin test as compared to other known analgesics. In addition, we are reporting for the first time the lack of antinociceptive activities of hydromorphone-3-glucuronide, dihydromorphine-3-glucuronide and dihydroisomorphine-3-glucuronide in the rat formalin test. Norhydromorphone was isolated and identified as a metabolite of hydromorphone in a cancer patient's urine. An authentic standard of norhydromorphone was synthesized. The identity of norhydromorphone in the urine sample was confirmed by comparing the LC retention time and MS ion fragmentation with the synthetic standard using a liquid chromatographic-mass spectrometric-mass spectrometric (LC-MS-MS) assay. Norhydromorphone was found to be a minor metabolite of hydromorphone in the urine. Additionally, the antinociceptive activities of norhydromorphone, hydromorphone, morphine, dihydromorphine, dihydroisomorphine, hydromorphone-3-glucuronide, dihydromorphine-3-glucuronide and dihydroisomorphine-3-glucuronide were determined in the rat formalin test following intraperitoneal (i.p.) administration. Only limited antinociception was observed and no significant increase in antinociception was detected at the three doses tested. The increased polarity of norhydromorphone as compared to hydromorphone due to the primary piperidine nitrogen may make it less favorable to cross the blood-brain-barrier (BBB), which may be partly responsible. In addition, lower intrinsic antinociceptive activity, which remains to be determined, could also contribute to the low antinociception. Our results also show that hydromorphone was five times as potent as morphine in the formalin test, while dihydromorphine and dihydroisomorphine were equipotent to and 36% as potent as morphine, respectively. Hydromorphone-3-glucuronide, dihydromorphine-3-glucuronide and dihydroisomorphine-3-glucuronide did not exhibit any antinociceptive effect at the doses tested. The results further underscore the importance of a free C3-OH to the analgesic effect of morphine alkaloids.     

Intrathecal endomorphin-1 produces antinociceptive activities modulated by alpha 2-adrenoceptors in the rat tail flick, tail pressure and formalin tests

It is known that spinal morphine produces antinociception that is modulated by alpha 2-adrenoceptors. Endomorphin-1, a newly-isolated endogenous opioid ligand, shows the greatest selectivity and affinity for the mu-opiate receptor of any endogenous substance found to date and may serve as a natural ligand for the mu-opiate receptor. We examined the antinociceptive effects of endomorphin-1 administered intrathecally (i.t.) in the rat tail flick, tail pressure and formalin tests. Intrathecal endomorphin-1 produced dose-dependent antinociceptive effects in the three tests. ED50 (CI95) values for antinociception of i.t. endomorphin-1 in the tail flick test and tail pressure test were 1.9 (0.96-3.76) nmol and 1.8 (0.8-4.2) nmol, respectively. ED50 (CI95) values for phase 1 and phase 2 in the formalin test were 12.5 (7.9-19.8) nmol and 17.5 (10.2-30) nmol, respectively. Pretreatment with i.t. beta-funaltrexamine (a mu-opioid receptor selective antagonist) significantly antagonized the antinociceptive effects of endomorphin-1 in the three tests. Beta-funaltrexamine alone had not effects on the three tests. The antinociceptive effects of endomorphin-1 were also antagonized by i.t. yohimbine (an alpha 2-adrenoceptor selective antagonist). The combination of ineffective doses of i.t. clonidine (an alpha 2-adrenoceptor agonist) and endomorphin-1 produced a significant antinociception in the three tests. The results showed that intrathecal endomorphin-1 produced antinociception in a dose-dependent manner in the rat tail flick, tail pressure and formalin tests, which was mediated by spinal mu-opioid receptors and modulated by alpha 2-adrenoceptors.     

Involvement of spinal kappa opioid receptors in the antagonistic effect of dynorphins on morphine antinociception.

The modulatory effects of intrathecally (i.t.) administered dynorphin A(1-17) and dynorphin A(1-13) on morphine antinociception have been studied previously in rats by other investigators. However, both potentiating and attenuating effects have been reported. In this study, the modulatory effects of i.t. administered dynorphin A(1-17) as well as the smaller fragment, dynorphin A(1-8), were studied in mice. In addition, nor-binaltorphimine (nor-BNI), a highly selective kappa opioid receptor antagonist, and naltrindole (NTI), a highly selective delta opioid receptor antagonist, were used to characterize the possible involvement of spinal kappa and delta opioid receptors in the modulatory effects of the dynorphins. Dynorphin A(1-17) and dynorphin A(1-8) administered i.t. at doses that did not alter tail-flick latencies, were both able to antagonize in a dose-dependent manner, the antinociceptive action of s.c. administered morphine sulfate. The antinociceptive ED50 of morphine sulfate was increased 3.9- and 5.3-fold by 0.4 nmol/mouse of dynorphin A(1-17) and dynorphin A(1-8), respectively. Injections of 0.4 and 0.8 nmol/mouse of nor-BNI i.t., but not its inactive enantiomer (+)-1-nor-BNI, inhibited dose-dependently the antagonistic effects of the dynorphins. These doses of nor-BNI alone did not affect the antinociceptive action of morphine sulfate. Intrathecal administration of 5 nmol/mouse of NTI also did not affect the modulatory effects of dynorphins. These observations that dynorphins exert their antagonistic effects on morphine-induced antinociception stereoselectively through spinal kappa opioid receptors may suggest a coupling between spinal kappa and mu opioid receptors.     

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.     

Morphine antinociception: evidence for the release of endogenous substance(s).

Complete blockade of both the neural and the humoral pathways by spinal ligations at a low thoracic level (T₁₁–T₁₃) abolished morphine induced antinociception in mice. However, when only the humoral pathway was maintained, i.e. spinalization at T₁₂ with the dura mater intact, the antinociceptive effect of morphine was not reduced. Furthermore, blocking only the humoral pathway, i.e. removal of the dura mater at T¹¹–T₁₃ without damaging spinal neurons, or making a leakage of CSF through an opening in the cisterna magna reduced morphine antinociception. These findings indicate that morphine antinociceptive effects in mice are mainly mediated by substance(s) released from supraspinal structures and transported in the CSF.     

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.     

The Peripheral Versus Central Antinociception of a Novel Opioid Agonist: Acute Inflammatory Pain in Rats

Opioid analgesics devoid of central side effects are unmet medical need in the treatment of acute pain (e.g. post-operative pain). Recently, we have reported on 14-O-methylmorphine-6-O-sulfate (14-O-MeM6SU), a novel opioid agonist of high efficacy producing peripheral antinociception in subchronic inflammatory pain in certain doses. The present study focused on the antinociceptive effect of 14-O-MeM6SU compared to morphine in formalin test of an early/acute (Phase I) and late/tonic (Phase II) pain phases. Subcutaneous 14-O-MeM6SU (253–1012 nmol/kg) and morphine (3884–31075 nmol/kg) dose dependently reduced the pain behaviors of both phases. Co-administered naloxone methiodide (NAL-M), a peripherally acting opioid antagonist, abolished the antinociceptive effect of 506 nmol/kg 14-O-MeM6SU. On the other hand, the effects of 14-O-MeM6SU (1012 nmol/kg) and morphine (15538 nmol/kg) were only partially affected by NAL-M, indicating the contribution of CNS to antinociception. Locally injected test compounds into formalin treated paws caused antinociception in both phases. Locally effective doses of test compounds were also injected into contralateral paws. Morphine showed effects in both phases, 14-O-MeM6SU in certain doses failed to produce antinociception in either phase. A NAL-M reversible systemic dose of 14-O-MeM6SU and the lowest systemic effective dose of morphine were evaluated for their sedative effects following isoflurane-induced sleeping (righting reflex). In contrast to morphine, 14-O-MeM6SU in certain antinociceptive doses showed no impact on sleeping time. These data highlight that high efficacy opioids of limited CNS penetration in certain doses mitigate somatic and inflammatory pain by targeting MOR at the periphery.     

Carrier-mediated processes at several rat brain interfaces determine the neuropharmacokinetics of morphine and morphine-6-beta-D-glucuronide

We investigated whether capacity-limited transport processes were involved in morphine and morphine-6-beta-D-glucuronide (M6G) neuropharmacokinetics, at the level of the blood-brain barrier (BBB), the brain extra- and intra-cellular fluids (bECF/bICF), and the bECF/cerebrospinal fluid (CSF) interfaces. We performed transcortical retrodialysis in the rat, by perfusing morphine or M6G through the microdialysis probe in the presence or absence of probenecid. We measured for each compound the in vitro and in vivo (R(D)) probe recoveries. The in vivo R(D), which takes into account the permeability of the tissue surrounding the probe, informs about the morphine and M6G distribution capabilities from bECF to adjacent fluids (bICF, CSF, plasma). We also measured plasma and CSF concentrations at three time points after having added probenecid or not. Finally, we tested several pharmacokinetic models, assuming first-order or capacity-limited processes at each brain interface, to describe experimental morphine and M6G concentrations previously obtained in rat plasma and brain fluids. We found that morphine distributes more easily outside bECF than M6G. Adding probenecid caused a 2-fold decrease and a 1.3-fold increase in morphine and M6G R(D), respectively, and 30 min after adding probenecid, plasma and CSF concentrations increased for M6G but not for morphine. The pharmacokinetic model that gave the best fit included capacity-limited processes at the BBB and bECF/bICF interface for morphine and at the BBB and bECF/CSF interface for M6G. In conclusion, morphine accumulates into brain cells thanks to a probenecid-sensitive transporter located at the bECF/bICF interface, whereas M6G is trapped in bECF thanks to transporters located at the BBB and the bECF/CSF interface.     

The involvement of nitric oxide in the analgesic effects of ketamine

We investigated the contribution of NO-cyclic GMP (cGMP) pathway to the antinociceptive effects of ketamine in mice by using the nitric oxide synthase inhibitor, nitro(g)- L-arginine methyl ester (L-NAME). Intraperitoneal (i.p.) (1, 5 or 10 mg/kg) or intrathecal (i.th.) (10, 30 or 60 microg/mouse) administration of ketamine produced dose-dependent antinociceptive effects in the acetic acid-induced writhing and formalin tests but not in the tail-flick nor in hot-plate tests. Pretreatment of mice with L-NAME (10 mg/kg, i.p.) which produced no antinociception on its own, significantly inhibited the antinociceptive effect of ketamine (1, 5 or 10 mg/kg, i.p.). However, L-NAME (30 microg/mouse) was given intrathecally, it neither modified the antinociceptive effect of i.th. ketamine (10, 30 or 60 microg/mouse) nor did it produce an antinociceptive effect alone. These data suggest that the activation of the NO-cGMP pathway probably at the supraspinal level, but not spinal level, contributes to the antinociceptive effects of ketamine.     

Comparison of the antinociceptive effect of celecoxib, diclofenac and resveratrol in the formalin test

The peripheral antinociceptive effect of the selective COX-2 inhibitor celecoxib in the formalin-induced inflammatory pain was compared with that of resveratrol (COX-1 inhibitor) and diclofenac (non-selective COX inhibitor). Rats received local pretreatment with saline, celecoxib, diclofenac or resveratrol followed by 50 microl of either 1% or 5% formalin. Peripheral administration of celecoxib did not produce antinociception at either formalin concentration. In contrast, diclofenac and resveratrol produced a dose-dependent antinociceptive effect in the second phase of both 1% and 5% formalin test. The peripheral antinociception produced by diclofenac or resveratrol was due to a local action, as drug administration in the contralateral paw was ineffective. Results indicate that the selective COX-2 inhibitor celecoxib does not produce peripheral antinociception in formalin-induced inflammatory pain. In contrast, selective COX-1 and non-selective COX inhibitors (resveratrol and diclofenac, respectively) are effective drugs in this model of pain.     

Pharmacological Characterization of Morphine-Induced In Vivo Release of Cholecystokinin in Rat Dorsal Horn

Previous studies indicate that an increased release of cholecystokinin (CCK) in response to morphine administration may counteract opioid-induced analgesia at the spinal level. In the present study we used in vivo microdialysis to demonstrate that systemic administration of antinociceptive doses of morphine (1-5 mg/kg, s.c.) induces a dose-dependent and naloxone-reversible release of CCK-like immunoreactivity (CCK-LI) in the dorsal horn of the spinal cord. A similar response could also be observed following perfusion of the dialysis probe for 60 min with 100 microM but not with 1 microM morphine. The CCK-LI release induced by morphine (5 mg/kg, s.c.) was found to be calcium-dependent and tetrodotoxin-sensitive (1 microM in the perfusion medium). Topical application of either the L-type calcium channel blocker verapamil (50 microg) or the N-type calcium channel blocker omega-conotoxin GVIA (0.4 microg) onto the dorsal spinal cord completely prevented the CCK-LI release induced by morphine (5 mg/kg, s.c.). Our data indicate that activation of L- and N-type calcium channels is of importance for morphine-induced CCK release, even though the precise site of action of morphine in the dorsal horn remains unclear. The present findings also suggest a mechanism for the potentiation of opioid analgesia by L- and N-type calcium channel blocking agents.     

Centrally-mediated antinociceptive action of RWJ-22757 (formerly McN-5195): involvement of spinal descending inhibitory pathways (an hypothesis)

The present studies were an attempt to examine the mechanism of action of the novel antinociceptive compound RWJ-22757, (+/-)-trans-3-(2-bromophenyl)-octahydroindolizine (McN-5195). Intracerebroventricular (i.c.v.) administration of RWJ-22757 produced dose-related antinociception in the mouse tail-flick (48 degrees C) and rat hot-plate (51 degrees C) tests (ED50 = 243.3 and 261.3 micrograms, respectively). In contrast, intrathecal (i.t.) administration was without effect. The antinociception produced by peripherally (i.p.) or centrally (i.c.v.) administered RWJ-22757 was attenuated by i.t. administration of 2 micrograms phentolamine, 5 micrograms yohimbine, or 10 micrograms methysergide. I.t. administration of naloxone, at a dose (0.5 micrograms) that significantly attenuated the antinociceptive effects of peripherally or centrally administered morphine, had no effect on RWJ-22757-induced antinociception. We conclude from these results, coupled with the overall pharmacological and neurochemical profile of RWJ-22757, that the data are consistent with the hypothesis that RWJ-22757 produces antinociception predominantly at a site or sites located supraspinally with little or no activity at the spinal level and that RWJ-22757 activates adrenergic and serotonergic descending inhibitory pathways, increasing the tonic activity of endogenous antinociceptive systems.     

Nimodipine is more effective than nifedipine in attenuating morphine tolerance on chronic co-administration in the rat tail-flick test

Opioids, when co-administered with L-type calcium channel blockers (L-CCBs) show morphine like higher antinociceptive effect. This antinociceptive effect has been further investigated using a different experimental paradigm. The effect of two different L-CCBs (nifedipine and nimodipine) on morphine-induced antinociception was studied by the tail-flick test (40 min after morphine administration) in adult Wistar rats. A fixed-dose of nimodipine or nifedipine (2 mg/kg, once daily) was combined with a fixed dose of morphine (10 mg/kg, twice daily) for 10 days. Co-administration of L-CCBs significantly increased the antinociceptive effect of morphine, even 12 hr after administration. Also, nimodipine was more effective than nifedipine. Nimodipine was further studied using a higher and escalating doses of morphine (20-30 mg/kg twice daily for 14 days). Nimodipine increased the antinociceptive effect of morphine in the latter part of the study (days nine to fourteen) though significant difference was observed on 11th evening and 12th morning. No obvious adverse effects were observed in the present study. The results show for the first time that nimodipine is more effective than nifedipine and that these L-CCBs continue to be effective, even 12 hr after administration in the tail-flick test.     

Analgesic responses to intrathecal morphine in relation to CSF concentrations of morphine-3,beta-glucuronide and morphine-6,beta-glucuronide.

This study was performed to determine whether variations in analgesic responses to intrathecal morphine could be explained by cerebrospinal fluid (CSF) concentrations of morphine metabolites. Twenty-four CSF samples were collected at the beginning, middle and end of treatment periods in seven cancer patients with pain of malignant origin. CSF concentrations of morphine-3,beta-glucuronide (M3G) and morphine-6,beta-glucuronide (M6G) metabolites were measured by gas chromatography/mass spectrometry. Analgesic responses to morphine were estimated concurrent with CSF collection using a visual analog scale representing percentages of pain relief. Effective analgesia was defined as > or = 75% pain relief. CSF concentration of M3G and M6G in the 24 samples were 722 +/- 116 ng/ml and 699 +/- 158 ng/ml, respectively. CSF samples were categorized into two groups: (1) those collected during effective analgesia (N=14), and (2) those collected during ineffective analgesia (N=10). M6G levels detected in group 1 samples (effective analgesia) were significantly greater than those found in group 2 samples (ineffective analgesia) (978 +/- 243 ng/ml vs 309 +/- 68 ng/ml, P<0.05). Intergroup differences in CSF M3G concentrations and M3G/M6G ratios were not significant. It is concluded that CSF M6G may be indicative of effectiveness of analgesia in cancer patients subjected to intrathecal morphine.     

Environmental and intraperitoneal ethanol influences morphine antinociceptive effect in mice

The ability of acute environmental or intraperitoneal (i.p.) ethanol to influence morphine antinociceptive effect was studied in mice. In order to induce tolerance to morphine analgesia, mice received daily injections of 10 mg/Kg morphine over a period of 10 days. Mice were divided into three groups: i.p. ethanol (E), environmental ethanol (E*), and control saline (M). During the induction of tolerance these groups were treated identically except on days 1 and 11. On these days, 10 minutes prior to morphine injection, mice received either i.p. ethanol (1g/Kg), environmental ethanol (a bottle of 10% ethanol placed next to the animals cage during the experiments), or an equivalent volume of saline. Analgesia was assessed using a standard hot plate protocol and dose-response cumulative curves for morphine analgesia were obtained on days 1 and 11. On day 1, both the i.p. and environmental administration of ethanol showed similar morphine-potentiation effects [Mean Effective Dose: ED50 (M1)=4.5 mg/kg; ED50 (E1)=2.4 mg/kg; ED50 (E*1)=2.1 mg/kg]. On day 11, control group mice showed a reduction of morphine analgesia at test [ED50 (M11)=14.1 mg/kg]. Mice receiving i.p. and environmental ethanol again showed a leftward shift in dose-response cumulative curves for morphine antinociception with respect to controls [ED50 (E11)=9.1 mg/kg; ED50 (E*11)=4.7 mg/kg]. I.p. ethanol administration at non-antinociceptive doses enhances the morphine antinociception effect similarly in tolerant and non-tolerant (naive) mice. The presence of environmental ethanol can also induce a similar pattern of increase in morphine antinociception effect.     

Spinal NTS1 receptors regulate nociceptive signaling in a rat formalin tonic pain model

Central administration of the neuropeptide neurotensin (NT) was shown to induce antinociceptive responses both spinally and supraspinally. Although NTS2 receptors play an important role in modulating the activity of spinal neurons, we have recently implicated NTS1 receptors in NT's analgesic effects in acute spinal pain paradigms. The current experiments were thus designed to examine the antinociceptive effects of intrathecal administration of NTS1 agonists in formalin-induced tonic pain in rats. We first established, using immunoblotting and immunohistochemical approaches, that NTS1 receptors were present in small- and medium-sized dorsal root ganglion cells and localized in the superficial layers of the dorsal horn of the spinal cord. We then examined the effects of intrathecal injection of NT (1–15 μg/kg) or NTS1 preferring agonists on the nocifensive response to intraplantar formalin. Both NTS1-agonists, PD149163 (10–120 μg/kg) and NT69L (1–100 μg/kg), dose-dependently attenuated the formalin-induced behaviors. Accordingly, NTS1 agonists markedly suppressed pain-evoked c- fos expression in the superficial, nucleus proprius and neck regions of the spinal dorsal horn. The concomitant administration of PD149163 with the NTS1 antagonist SR48692 (3 μg/kg) significantly reversed PD149163-induced antinociception, confirming the implication of NTS1 in tonic pain. In contrast, NT69L's analgesic effects were partly abolished by co-administration of SR48692, indicating that NT69L-induced effects may also be exerted through interaction with NTS2. These results demonstrate that NTS1 receptors play a key role in the mediation of the analgesic effects of NT in persistent pain and suggest that NTS1-selective agonists may represent a new line of analgesic compounds.     

Antinociceptive effect of chrysin in diabetic neuropathy and formalin-induced pain models

ABSTRACT

Chrysin, a natural flavonoid, is the main ingredient of many medicinal plants, which shows potent pharmacological properties. In the present study, the antinociceptive effects of chrysin were examined in ICR mice. Chrysin orally administered at the doses of from 10 to 100?mg/kg exerted the reductions of formalin-induced pain behaviors observed during the second phase in the formalin test in a dose-dependent manner. In addition, the antinociceptive effect of chrysin was further characterized in streptozotocin-induced diabetic neuropathy model. Oral administration chrysin caused reversals of decreased pain threshold observed in diabetic-induced peripheral neuropathy model. Intraperitoneally (i.p.) pretreatment with naloxone (a classic opioid receptor antagonist), but not yohimbine (an antagonist of α2-adrenergic receptors) or methysergide (an antagonist of serotonergic receptors), effectively reversed chrysin-induced antinociceptive effect in the formalin test. Moreover, chrysin caused a reduction of formalin-induced up-regulated spinal p-CREB level, which was also reversed by i.t. pretreated naloxone. Finally, chrysin also suppressed the increase of the spinal p-CREB level induced by diabetic neuropathy. Our results suggest that chrysin shows an antinociceptive property in formalin-induced pain and diabetic neuropathy models. In addition, spinal opioid receptors and CREB protein appear to mediate chrysin-induced antinociception in the formalin-induced pain model.