Epigenetic modulation of mGlu2 receptors by histone deacetylase inhibitors in the treatment of inflammatory pain.

Knowing that expression of metabotropic glutamate 2 (mGlu2) receptors in the dorsal root ganglia is regulated by acetylation mechanisms, we examined the effect of two selective and chemically unrelated histone deacetylase (HDAC) inhibitors, N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide (MS-275) and suberoylanilide hydroamic acid (SAHA), in a mouse model of persistent inflammatory pain. Although a single subcutaneous injection of MS-275 (3 mg/kg) or SAHA (5-50 mg/kg) was ineffective, a 5-day treatment with either of the two HDAC inhibitors substantially reduced the nociceptive response in the second phase of the formalin test, which reflects the development of central sensitization in the dorsal horn of the spinal cord. Analgesia was abrogated by a single injection of the mGlu2/3 receptor antagonist (alphaS)-alpha-amino-alpha-[(1S,2S)-2-carboxycyclopropyl]-9H-xantine-9-propanoic acid (LY341495; 1 mg/kg, i.p.), which was inactive per se. Both MS-275 and SAHA up-regulated the expression of mGlu2 receptors in the dorsal root ganglion (DRG) and spinal cord under conditions in which they caused analgesia, without changing the expression of mGlu1a, mGlu4, or mGlu5 receptors. Induction of DRG mGlu2 receptors in response to SAHA was associated with increased acetylation of p65/RelA on lysine 310, a process that enhances the transcriptional activity of p65/RelA at nuclear factor-kappaB-regulated genes. Transcription of the mGlu2 receptor gene is known to be activated by p65/RelA in DRG neurons. We conclude that HDAC inhibition produces analgesia by up-regulating mGlu2 receptor expression in the DRG, an effect that results from the amplification of NF-kappaB transcriptional activity. These data provide the first evidence that HDAC inhibitors cause analgesia and suggest that HDACs are potential targets for the epigenetic treatment of pain.

Pregabalin in the treatment of chronic pain: an overview.

Chronic 'pathological' pain is sustained by mechanisms of peripheral and central sensitization, which are being increasingly investigated at the molecular and cellular levels. The molecular determinants of nociceptive sensitization are natural targets for potential analgesic drugs used in the treatment of different forms of pain. Most of these determinants are common to all forms of chronic pain, and it is therefore not surprising that drugs specifically targeted for the treatment of neuropathic pain are effective in relieving nociceptive inflammatory pain and vice versa. The molecular mechanisms of sensitization that occur in peripheral nociceptors and the dorsal horns of the spinal cord are putative targets for context-dependent drugs, i.e. drugs that are able to discriminate between 'normal' and 'pathological' pain transmission. Among these, pregabalin and gabapentin bind to the alpha(2)delta subunit of voltage-sensitive Ca2+ channels, which sustain the enhanced release of pain transmitters at the synapses between primary afferent fibres and second-order sensory neurons under conditions of chronic pain. Pregabalin in particular represents a remarkable example of a context-dependent analgesic drug that acts at a critical step of nociceptive sensitization. Preclinical and clinical data suggest that pregabalin is more than a structural and functional analogue of gabapentin and may be effective in the treatment of nociceptive inflammatory pain that is resistant to gabapentin.

L-acetylcarnitine: a proposed therapeutic agent for painful peripheral neuropathies.

During the past two decades, many pharmacological strategies have been investigated for the management of painful neuropathies. However, neuropathic pain still remains a clinical challenge. A combination of therapies is often required, but unfortunately in most cases adequate pain relief is not achieved. Recently, attention has been focused on the physiological and pharmacological effects of L-acetylcarnitine in neurological disorders. There are a number of reports indicating that L-acetylcarnitine can be considered as a therapeutic agent in neuropathic disorders including painful peripheral neuropathies. In this review article, we will examine the antinociceptive and the neuroprotective effects of Lacetylcarnitine as tested in clinical studies and in animal models of nerve injury.

Novel neuronal targets for the acetylcholinesterase inhibitor donepezil.

The effects of the acetylcholinesterase inhibitor donepezil on cell viability and proliferation events have been analysed in SH-SY5Y human neuroblastoma cells. Short- (48 h) or long-term (7 days) exposure of SH-SY5Y cells to donepezil (100 nM-10 microM) induced a concentration-dependent inhibition of cell proliferation that was not modified by muscarinic and nicotinic receptor antagonists, or mimicked by galantamine, and was not related to induction of apoptosis. By analysing the distribution profile of cell populations within the cell cycle following treatment with 10 microM donepezil, a reduction of cells in the S-G2/M phases of the cycle and a parallel increase of the G0/G1 population were observed. In addition, the expression of two cyclins of the G1/S and G2/M transitions, cyclin E and cyclin B, was significantly reduced in donepezil-treated cells. In contrast, the expression of the cell cycle inhibitor p21 rapidly (6 h) increased following exposure to the drug. Finally, donepezil increased the expression of the neuronal marker MAP-2 in selected subpopulations of SH-SY5Y cells, suggesting that the effect on cell proliferation by donepezil may correlate to a trend to neuronal differentiation.

Metabotropic receptors as targets for drugs of potential use in the treatment of neuropathic pain.

Glutamate is the major neurotransmitter in the mammalian central nervous system and plays a pivotal role in both acute and chronic pain. The actions of glutamate are mediated by two receptor families: ionotropic glutamate receptors (iGluRs), and metabotropic glutamate receptors (mGluRs). Activation of glutamate receptor can elicit both hyperalgesic and analgesic effects. Eight mGluRs subtypes (mGluR1-mGluR8) have been identified and classified into three groups. Among these, group I mGluRs (mGlu1 and -5) have been implicated in the processes of central sensitization and persistent nociception, whereas activation of group II mGluRs (mGlu2/3) is effective against neuropathic or inflammatory pain. In this review we focus on the role of mGlu2/3 in the modulation of persistent pain, and on their potential use as drug targets in pain management.

An increased expression of the mGlu5 receptor protein following LTP induction at the perforant path-dentate gyrus synapse in freely moving rats.

The involvement of metabotropic glutamate (mGlu) receptors in the induction of long-term potentiation (LTP) in vivo has been consistently documented. We have investigated whether LTP induction in the dentate gyrus of rats leads to changes in expression of mGlu2/3 or -5 receptor subtypes in the hippocampus. LTP was induced at the medial perforant path-dentate gyrus synapses, and mGlu receptor expression was examined by Western blot or in situ hybridization. An up-regulation of mGlu5 receptors was observed in the hippocampus both 24 and 48 h following LTP induction. This effect was restricted to the dentate gyrus and CA1 region, whereas no changes in mGlu5 receptor protein (but an increase in mRNA levels) were observed in the CA3 region. The increased expression of mGlu5 receptors was directly related to the induction of LTP, because it was not observed when tetanic stimulation was carried out in animals treated with the NMDA receptor antagonist, 2-amino-5-phosphonopentanoate (AP5). Western blot analysis also showed a reduced expression of mGlu2/3 receptors in the whole hippocampus 24 h after LTP induction, indicating that the increased expression of mGlu5 receptors was specific. These data suggest that an up-regulation of mGlu5 receptors is a component of the plastic changes that follow the induction of LTP at the perforant path-dentate gyrus synapse.

Erratic expression of DNA polymerases by beta-amyloid causes neuronal death.

An ectopic reentrance into the cell cycle with ensuing DNA replication is required for neuronal apoptosis induced by beta-amyloid. Here, we investigate the repertoire of DNA polymerases expressed in beta-amyloid-treated neurons, and their specific role in DNA synthesis and apoptosis. We show that exposure of cultured cortical neurons to beta-amyloid induces the expression of DNA polymerase-beta, proliferating cell nuclear antigen, and the p49 and p58 subunits of DNA primase. Induction requires the activity of cyclin-dependent kinases. The knockdown of the p49 primase subunit prevents beta-amyloid-induced neuronal DNA synthesis and apoptosis. Similar effects are observed by knocking down DNA polymerase-beta or by using dideoxycytidine, a preferential inhibitor of this enzyme. Thus, the reparative enzyme DNA polymerase-beta unexpectedly mediates a large component of de novo DNA synthesis and apoptotic death in neurons exposed to beta-amyloid. These data indicate that DNA polymerases become death signals when erratically expressed by differentiated neurons.

L-Acetylcarnitine induces analgesia by selectively up-regulating mGlu2 metabotropic glutamate receptors.

L-Acetylcarnitine (LAC, 100 mg/kg, s.c.), a drug commonly used for the treatment of painful neuropathies, substantially reduced mechanical allodynia in rats subjected to monolateral chronic constriction injury (CCI) of the sciatic nerve and also attenuated acute thermal pain in intact rats. In both cases, induction of analgesia required repeated injections of LAC, suggesting that the drug induces plastic changes within the nociceptive pathway. In both CCI- and sham-operated rats, a 24-day treatment with LAC increased the expression of metabotropic glutamate (mGlu) receptors 2 and 3 in the lumbar segment of the spinal cord, without changing the expression of mGlu1a or -5 receptors. A similar up-regulation of mGlu2/3 receptors was detected in the dorsal horns and dorsal root ganglia of intact rats treated with LAC for 5-7 days, a time sufficient for the induction of thermal analgesia. Immunohistochemical analysis showed that LAC treatment enhanced mGlu2/3 immunoreactivity in the inner part of lamina II and in laminae III and IV of the spinal cord. An increased mGlu2/3 receptor expression was also observed in the cerebral cortex but not in the hippocampus or cerebellum of LAC-treated animals. Reverse transcription-polymerase chain reaction combined with Northern blot analysis showed that repeated LAC injections selectively induced mGlu2 mRNA in the dorsal horns and cerebral cortex (but not in the hippocampus). mGlu3 mRNA levels did not change in any brain region of LAC-treated animals. To examine whether the selective up-regulation of mGlu2 receptors had any role in LAC-induced analgesia, we have used the novel compound LY 341495, which is a potent and systemically active mGlu2/3 receptor antagonist. LAC-induced analgesia was largely reduced 45 to 75 min after a single injection of LY 341495 (1 mg/kg, i.p.) in both CCI rats tested for mechanical allodynia and intact rats tested for thermal pain. We conclude that LAC produces analgesia against chronic pain produced not only by peripheral nerve injury but also by acute pain in intact animals and that LAC-induced analgesia is associated with and causally related to a selective up-regulation of mGlu2 receptors. This offers the first example of a selective induction of mGlu2 receptors and discloses a novel mechanism for drug-induced analgesia.

Preparation and analgesic activity of Eudragit RS100 microparticles containing diflunisal.

Two different techniques, the quasi-emulsion solvent diffusion method and spray drying that provide polar and nonpolar preparation environments, were used to prepare microspheres from Eudragit RS100 (RS) (acrylic/methacrylic copolymer) incorporating the nonsteroidal anti-inflammatory drug diflunisal. The effects of pH on the preparation medium and drug/polymer ratio on production yield and drug incorporation, as well as on the in vitro drug release at pH 1.2 and 6.8 from tabletted microparticles, were evaluated. The drug-polymer interactions and the effect of diflunisal incorporation in the polymer matrix on drug crystallinity have been evaluated by using differential scanning calorimetry, IR and ultraviolet spectroscopy, x-ray diffraction, and microscopy analysis. A preliminary biological assay confirmed that diflunisal maintains its analgesic activity after intraperitoneal administration to rats.

Evidence of a key-role for histamine from mast cells in the analgesic effect of clomipramine in rats.

OBJECTIVE AND DESIGN: We investigated the role of neuronal and mast cell histamine in the analgesic effect of clomipramine. SUBJECTS: Male Sprague-Dawley rats (4-6 weeks old) were used (n = 228). TREATMENT: Clomipramine (10, 20 or 40 mg/kg i.p.) was injected in rats pretreated with [a] saline i.cv. or i.p., [b] alpha-fluoromethylhistidine (alpha-FMH, 200 microg i.cv.), [c] compound 48/80 (C48/80, 1 mg/kg i.p.). Other rats were pretreated with clomipramine, before C48/80. METHODS: Antinociceptive responses were determined before and 30, 60, 90, 120 min after drug injection by tail-flick (TFT) and hot-plate (HPT) tests. Results for each treatment group are given as mean %MPE +/- SEM (Student's t-test, ANOVA). RESULTS: Clomipramine produced no significant changes in TFT and HPT in saline- or alpha-FMH-pretreated rats. Following C48/80, clomipramine (10 and 20 mg/kg) produced a dose-related significant increase in latencies, between 30 and 120 min: 28.5 +/- 5.7 vs 8 +/- 1.6 (p < 0.05), 56 +/- 5 vs 9.2 +/- 1.9 (p < 0.01) in TFT; 31 +/- 4.3 vs 12 +/- 2.5 (p < 0.05), 46.2 +/- 6 vs 11.5 +/- 1.9 (p < 0.01) in HPT. Clomipramine (40 mg/kg, after C48/80) produced marked and persistent increase in latencies 83.2 +/- 4.2 vs 10.5 +/- 3 (p < 0.01) in TFT and 91.2 +/- 4.6 vs 10.5 +/- 3 (p < 0.01) in HPT, followed by symptoms of toxicity and death of some animals. In rats pretreated with clomipramine, C48/80 was unable to show antinociceptive effects on TFT and HPT. CONCLUSIONS: Results suggest that the antinociceptive effect of clomipramine may depend on mast cell histamine levels.

Histaminergic mechanisms in clonidine induced analgesia in rat tail-flick test.

The role of neural histamine in clonidine-analgesia and in clonidine-induced potentiation of stress analgesia was studied. Pretreatment of rats with alpha-fluoromethylhistidine (FMH) (200 ug icv/rat; daily for five days) increased the analgesic effect of the alpha 2-agonist clonidine on the spinal reflex of the tail-flick test. Rats subjected to cold-restraint stress (30 min at 4 degrees C) showed increased latency compared to the unstressed rats. The analgesic efficacy of clonidine was significantly greater in rats subjected to cold-restraint with respect to unstressed rats. However, the inhibition of histamine biosynthesis by FMH significantly reduced cold-restraint analgesia in saline-controls, and consistently increased the analgesic efficacy of the alpha 2-agonist, showing a maximum latency. Yohimbine exhibited high affinity as an antagonist for alpha 2-receptors, inducing hyperalgesic effects and antagonizing clonidine analgesia and clonidine-induced potentiation of cold stress analgesia. In FMH-pretreated rats, yohimbine failed to reverse clonidine analgesia and did not block the increased analgesic efficacy of clonidine in cold-restrained FMH-pretreated rats. Results of this study suggest that inhibition of histamine release through alpha 2-adrenoceptors on histaminergic axons may contribute to the analgesic efficacy of systemically injected clonidine, also confirming that neural histaminergic pathways are implicated in the mediation of pain response in particular conditions of stress.

Synthesis of carboxyalkylthio-triazoles and bicyclic derivatives with antiinflammatory and analgesic activity.

3-Carboxyalkylthio derivatives of 5-alkoxyphenyl-1,2,4-triazole were prepared, performing some substitutions both on carboxyalkyl chain, by lengthening it or introducing substituents with increasing molecular weight in alpha- at the carboxy group, and on N-4 atom in the triazole ring, by introducing an amino or methyl group, so that to vary steric conformation along with the lipophilicity of molecules. The corresponding bicyclic thiazolo-triazolone and triazolo-thiazinone derivatives, which represent the rigid models of carboxymethylthio- and carboxyethylthio- open structures, were also obtained. All the compounds show "in vivo" antiinflammatory activity, while only carboxyalkylthio derivatives of 4-amino- and 4-methyltriazole display an appreciable analgesic one. From the relief of some data on substituent present in the synthesized compounds, a structure-activity relationship is also suggested.