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.     

Antagonism of nitrous oxide antinociception in mice by intrathecally administered antisera to endogenous opioid peptides

Previously it was demonstrated that nitrous oxide antinociception in the mouse abdominal constriction test is mediated by kappa-opioid receptors. Since nitrous oxide is thought to cause the neuronal release of endogenous opioid peptide to stimulate opioid receptors, this study was designed to identify the opioid peptides involved, especially in the spinal cord, by determining whether nitrous oxide antinociception can be differentially inhibited by intrathecally (i. t.) administered antisera to different opioid peptides. Male NIH Swiss mice were pretreated i.t. with rabbit antisera to opioid peptides then exposed 24 h later to one of three different concentrations of nitrous oxide in oxygen. Dose-response curves constructed from the data indicated that the antinociceptive effect of nitrous oxide was significantly antagonized by antisera to various dynorphins (DYNs) and methionine-enkephalin (ME), but not by antiserum to beta-endorphin (beta-EP). The AD(50) values for nitrous oxide antinociception were significantly elevated by antisera to DYNs and ME but not beta-EP. These findings of this study support the hypothesis that nitrous oxide antinociception in the mouse abdominal constriction test involves the neuronal release of DYN and ME in the spinal cord.     

Synergistic interactions between the antinociceptive effect of Rhodiola rosea extract and B vitamins in the mouse formalin test

AimIn this study, the pharmacological interactions between a Rhodiola rosea ethanol extract and B-vitamins such as thiamine (B₁), riboflavine (B₂), pyridoxine (B₆), cyanocobalamin (B₁₂) and a mixture of vitamins B₁ + B₆ + B₁₂ was investigated in the mouse formalin test.MethodsIndividual dose response curves of the Rhodiola rosea ethanol extract, as well as B-vitamins alone or in a mixture were evaluated in mice in which nociception was induced with 2% formalin intraplantarly. The antinociceptive mechanisms of the Rhodiola rosea were investigated by exploring the role of the opioid and serotonin receptors and the nitric oxide pathway. Isobolographic analysis was used to evaluate the pharmacological interactions between the Rhodiola rosea ethanol extract and each B-vitamin individually or the mixture of vitamins B₁ + B₆ +B₁₂ by using the ED₃₀ and a fixed 1:1 ratio combination.ResultsAdministration of the Rhodiola rosea extract alone or in combination with all of the vitamins produced a significant and dose-dependent antinociceptive response. The antinociceptive effect of the Rhodiola rosea extract (ED₅₀ = 81 mg/kg, p.o.) was significant and reverted in the presence of antagonists of the 5-HT_1A, GABA/BDZs and opioid receptors and by blocking mediators of the nitric oxide/cGMP/K⁺ channels pathway. Isobolograms demonstrate that all of the combinations investigated in this study produced a synergistic interaction experimental ED₃₀ values were significantly smaller than those calculated theoretically.ConclusionsThese results provide evidence that a Rhodiola rosea ethanol extract in combination with B-vitamins produces a significant diminution in the nociceptive response in a synergistic manner, which is controlled by various mechanisms. These findings could aid in the design of clinical studies and suggest that these combinations could be applied for pain therapy.     

Naturally Occurring Compounds Affect Glutamatergic Neurotransmission in Rat Brain

Natural products, including those derived from plants, have largely contributed to the development of therapeutic drugs. Glutamate is the main excitatory neurotransmitter in the central nervous system and it is also considered a nociceptive neurotransmitter, by acting on peripheral nervous system. For this reason, in this study we investigated the effects of the hydroalcooholic extracts from Drymis winteri (polygodial and drimanial), Phyllanthus (rutin and quercetine), Jathopha elliptica (jatrophone), Hedyosmum brasiliense (13HDS), Ocotea suaveolens (Tormentic acid), Protium kleinii (αβ-amyrin), Citrus paradise (naringin), soybean (genistein) and Crataeva nurvala (lupeol), described as having antinociceptive effects, on glutamatergic transmission parameters, such as [³H]glutamate binding, [³H]glutamate uptake by synaptic vesicles and astrocyte cultures, and synaptosomal [³H]glutamate release. All the glutamatergic parameters were affected by one or more of these compounds. Specifically, drimanial and polygodial presented more broad and profound effects, requiring more investigation on their mechanisms. The putative central side effects of these compounds, via the glutamatergic system, are discussed.     

Involvement of orexin-2 receptors in the ventral tegmental area and nucleus accumbens in the antinociception induced by the lateral hypothalamus stimulation in rats

Orexin, which is mainly produced by orexin-expressing neurons in the lateral hypothalamus (LH), plays an important role in pain modulation. Both kinds of orexin-1 (Ox1) and orexin-2 (Ox2) receptors have been found at high density in the ventral tegmental area (VTA) and nucleus accumbens (NAc). However, the quantity of Ox1 receptors in the VTA is more than that in the NAc. Additionally, it seems that the functional interaction between the LH, VTA and NAc implicates pain processing and modulation. In this study, we tried to examine the involvement of Ox2 receptors in the NAc and VTA using tail-flick test as an animal model of acute pain following microinjection of effective dose of carbachol (125 nmol/0.5 μl saline) into the LH. In this set of experiments, different doses of TCS OX2 29 as an Ox2 receptor antagonist were microinjected into the VTA (1, 7 and 20 nmol/0.3 μl DMSO) and the NAc (2, 10, 20 and 40 nmol/0.5 μl DMSO) 5 min prior to carbachol administration. Administration of TCS OX2 29 into the VTA and NAc dose-dependently blocked intra-LH carbachol-induced antinociception. However, the inhibitory effect of TCS OX2 29 as an Ox2 receptor antagonist was more potent in the VTA than that in the NAc. It seems that VTA orexinergic receptors are more effective on LH stimulation-induced antinociception and the modulation of pain descending inhibitory system originated from the LH than those of the same receptors in the nucleus accumbens in rats.     

Antinociceptive action of intrathecal neurotensin in mice

Neurotensin has been demonstrated to be analgesic in rodents. This study used intrathecal injection of neurotensin in unanesthetized mice to evaluate the effect of the peptide at the spinal level on unconditioned behavior. Intrathecal administration of neurotensin produced dose-related inhibition of locomotor activity and of the response elicited by subcutaneous hypertonic saline. The effects of the peptide in the tail flick assay were variable and it produced no inhibition of the behavioral response to intrathecal substance P. The results indicate that neurotensin antinociception at the spinal level does not result from locomotor impairment, may be somewhat selective for chemically induced pain, and may be mediated by a presynaptic action on primary afferent fibers.     

Involvement of spinal release of α-neo-endorphin on the antinociceptive effect of TAPA

The antinociceptive effect of i.t.-administered Tyr-d-Arg-Phe-β-Ala (TAPA), an N-terminal tetrapeptide analog of dermorphin, was characterized in ddY mice. In the mouse tail-flick test, TAPA administered i.t. produced a potent antinociception. The antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with the κ-opioid receptor antagonist nor-binaltorphimine, as well as by the μ-opioid receptor antagonist β-funaltrexamine and the μ₁-opioid receptor antagonist naloxonazine. TAPA-induced antinociception was also significantly suppressed by co-administration of the μ₁-opioid receptor antagonist Tyr-d-Pro-Phe-Phe-NH₂ (d-Pro²-endomorphin-2) but not by co-administration of the μ₂-opioid receptor antagonists Tyr-d-Pro-Trp-Phe-NH₂ (d-Pro²-endomorphin-1) and Tyr-d-Pro-Trp-Gly-NH₂ (d-Pro²-Tyr-W-MIF-1). In CXBK mice whose μ₁-opioid receptors were naturally reduced, the antinociceptive effect of TAPA was markedly suppressed compared to the parental strain C57BL/6ByJ mice. Moreover, the antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with antiserum against the endogenous κ-opioid peptide α-neo-endorphin but not antisera against other endogenous opioid peptides. In prodynorphin-deficient mice, the antinociceptive effect of TAPA was significantly reduced compared to wild-type mice. These results suggest that the spinal antinociception induced by TAPA is mediated in part through the release of α-neo-endorphin in the spinal cord via activation of spinal μ₁-opioid receptors.