Arafat Nasser

P2X7 receptor-deficient mice are susceptible to bone cancer pain.

&NA; The purinergic P2X7 receptor is implicated in both neuropathic and inflammatory pain, and has been suggested as a possible target in pain treatment. However, the specific role of the P2X7 receptor in bone cancer pain is unknown. We demonstrated that BALB/cJ P2X7 receptor knockout (P2X7R KO) mice were susceptible to bone cancer pain and moreover had an earlier onset of pain‐related behaviours compared with cancer‐bearing, wild‐type mice. Furthermore, acute treatment with the selective P2X7 receptor antagonist, A‐438079, failed to alleviate pain‐related behaviours in models of bone cancer pain with and without astrocyte activation (BALB/cJ or C3H mice inoculated with 4T1 mammary cancer cells or NCTC 2472 osteosarcoma cells, respectively), suggesting that astrocytic P2X7 receptors play a negligible role in bone cancer pain. The results support the hypothesis that bone cancer pain is a separate pain state compared with those of neuropathic and inflammatory pain. However, the recent discovery of a P2X7 receptor splice variant expressed in the knockout mice used for this study complicates the interpretation of the results. The P2X7 splice variant receptor was detected in the spinal cord but not in osteoclasts of the P2X7R KO mouse. Further experiments are needed to elucidate the exact role of the P2X7 receptors in bone cancer pain. Pain‐related behaviours had an earlier onset in bone cancer‐bearing, P2X7 receptor‐deficient mice, and treatment with A‐438079 failed to alleviate pain‐related behaviours.

Chronic administration of the selective P2X3, P2X2/3 receptor antagonist, A-317491, transiently attenuates cancer-induced bone pain in mice

The purinergic P2X3 and P2X2/3 receptors are in the peripheral nervous system almost exclusively confined to afferent sensory neurons, where they are found both at peripheral and central synapses. The P2X3 receptor is implicated in both neuropathic and inflammatory pain. However, the role of the P2X3 receptor in chronic cancer-induced bone pain is less known. Here we investigated the effect of systemic acute and chronic administration of the selective P2X3, P2X2/3 receptor antagonist (5-[[[(3-Phenoxyphenyl)methyl][(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl]-1,2,4-benzenetricarboxylic acid sodium salt hydrate) (A-317491) in a murine model of cancer-induced bone pain. Chronic administration of A-317491 (30 μmol/kgs.c., b.i.d.) resulted in a transient attenuation of pain related behaviours in the early stage of the bone cancer model, but had no effect in the late and more progressed stage of bone cancer. Also, acute administration of A-317491 (100 μmol/kgs.c.) had no effect in the progressed stage of the bone cancer pain model. Thus, systemically administered A-317491 did not demonstrate a robust effect in the present mouse model of cancer-induced bone pain.

Impaired behavioural pain responses in hph-1 mice with inherited deficiency in GTP cyclohydrolase 1 in models of inflammatory pain

BackgroundGTP cyclohydrolase 1 (GTP-CH1), the rate-limiting enzyme in the synthesis of tetrahydrobiopterin (BH4), encoded by the GCH1 gene, has been implicated in the development and maintenance of inflammatory pain in rats. In humans, homozygous carriers of a “pain-protective” (PP) haplotype of the GCH1 gene have been identified exhibiting lower pain sensitivity, but only following pain sensitisation. Ex vivo, the PP GCH1 haplotype is associated with decreased induction of GCH1 after stimulation, whereas the baseline BH4 production is not affected. Contrary, loss of function mutations in the GCH1 gene results in decreased basal GCH1 expression, and is associated with DOPA-responsive dystonia (DRD). So far it is unknown if such mutations affect acute and inflammatory pain.ResultsIn the current study, we examined the involvement of the GCH1 gene in pain models using the hyperphenylalaninemia 1 (hph-1) mouse, a genetic model for DRD, with only 10% basal GTP-CH1 activity compared to wild type mice. The study included assays for determination of acute nociception as well as models for pain after sensitisation. Pain behavioural analysis of the hph-1 mice showed reduced pain-like responses following intraplantar injection of CFA, formalin and capsaicin; whereas decreased basal level of GTP-CH1 activity had no influence in naïve hph-1 mice on acute mechanical and heat pain thresholds. Moreover, the hph-1 mice showed no signs of motor impairment or dystonia-like symptoms.ConclusionsIn this study, we demonstrate novel evidence that genetic mutations in the GCH1 gene modulate pain-like hypersensitivity. Together, the present data suggest that BH4 is not important for basal heat and mechanical pain, but they support the hypothesis that BH4 plays a role in inflammation-induced hypersensitivity. Our studies suggest that the BH4 pathway could be a therapeutic target for the treatment of inflammatory pain conditions. Moreover, the hph-1 mice provide a valid model to study the consequence of congenital deficiency of GCH1 in painful conditions.

Anxiety- and depression-like phenotype of hph-1 mice deficient in tetrahydrobiopterin

Decreased tetrahydrobiopterin (BH4) biosynthesis has been implicated in the pathophysiology of anxiety and depression. The aim of this study was therefore to characterise the phenotype of homozygous hph-1 (hph) mice, a model of BH4 deficiency, in behavioural tests of anxiety and depression as well as determine hippocampal monoamine and plasma nitric oxide levels. In the elevated zero maze test, hph mice displayed increased anxiety-like responses compared to wild-type mice, while the marble burying test revealed decreased anxiety-like behaviour. This was particularly observed in male mice. In the tail suspension test, hph mice of both sexes displayed increased depression-like behaviours compared to wild-type counterparts, whereas the forced swim test showed a trend towards increased depression-like behaviours in male hph mice, but significant decrease in depression-like behaviours in female mice. This study provides the first evidence that congenital BH4 deficiency regulates anxiety- and depression-like behaviours. The altered responses observed possibly reflect decreased hippocampal serotonin and dopamine found in hph mice compared to wild-type mice, but also reduced nitric oxide formation. We propose that the hph-1 mouse may be a novel tool to investigate the role of BH4 deficiency in anxiety and depression.

Intraplantar injection of tetrahydrobiopterin induces nociception in mice

Tetrahydrobiopterin (BH4) is implicated in the development and maintenance of chronic pain. After injury/inflammation, the biosynthesis of BH4 is markedly increased in sensory neurons, and the pharmacological and genetic inhibition of BH4 shows analgesic effects in pre-clinical animal pain models. Intrathecal injections of BH4 have been shown to induce and enhance pain-like behaviours in rats, suggesting that under chronic pain conditions BH4 may act by facilitating central sensitisation. So far it is unknown whether BH4 acts on peripheral sites of the somatosensory system or whether BH4 per se provokes nociceptive pain behaviours. The purpose of this study was therefore to investigate the acute nociceptive effects of intraplantar injection of BH4. BH4 was found to induce dose-dependent licking/biting of the paw lasting 5 min, which was not observed following an injection of biopterin (inactive BH4 metabolite). Paw swelling, measured as paw thickness and weight, was not observed after BH4 injection. To explore possible mechanisms of action of BH4, the effect of local pre-treatment with indomethacin, Nω-nitro-L-arginine methyl ester, Nω-nitro-L-arginine, capsazepine and ruthenium red was tested. Morphine served as a positive control. Intraplantar pre-injection of morphine dose-dependently inhibited BH4-induced nociception, while none of the other compounds showed any statistical significant antinociception. These results suggest that BH4 exhibits nociceptive properties at peripheral sites of the somatosensory system, proposing an as yet unexplored involvement of BH4 in peripheral nociceptive processes. However, this appears not to be mediated through nitric oxide and prostaglandin release or by activation of the transient receptor potential vanilloid 1.

GCH1 variants, tetrahydrobiopterin and their effects on pain sensitivity

Abstract Background A great proportion of the variation in pain experience and chronicity is caused by heritable factors. Within the last decades several candidate genes have been discovered either increasing or decreasing pain sensitivity or the risk of chronic pain in humans. One of the most studied genes is the GCH1 gene coding for the enzyme GTP cyclohydrolase 1 (GCH1). GCH1 catalyses the initial and rate-limiting step in the biosynthesis of tetrahydrobiopterin (BH4). The main function of BH4 is regulation of monoamine and nitric oxide biosynthesis, all involved in nociceptive signalling. Methods In this topical review we focus on the implication of the GCH1 gene and BH4 in painful conditions. We discuss experimental evidence from our group in relation to relevant research publications evaluating the BH4 pathway in pain. Studies assessing the role of GCH1 and BH4 in pain consist of human and animal studies, including DOPA-responsive dystonia (DRD) patients and hph-1 mice (a genetic mouse model of DRD) having mutations in the GCH1 gene as well as preclinical studies with the GCH1 inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP). The hypothesis is that genetic and pharmacological reduction of GCH1 would result in lower pain sensitivity. Results Previous studies have demonstrated that a particular “pain protective” GCH1 haplotype, found in 15% of the general human population, is linked to decreased pain sensitivity. We further support these findings in DRD patients, showing normal thresholds to mechanical and thermal stimuli, whereas a trend towards lower pain sensitivity is seen following chemical pain sensitisation. Consistent with these observations, non-injured hph-1 mice displayed normal mechano- and thermosensation compared to wild-type mice. After peripheral inflammation with Complete Freund’ Adjuvant or sensitisation with capsaicin the mutant mice exhibited lower sensitivity to mechanical and heat stimuli. Moreover, hph-1 mice showed decreased nociception in the first phase of the formalin test. Several studies report analgesic effects of GCH1 inhibition with 90–270 mg/kg DAHP in rat models of inflammatory and neuropathic pain. However, we could not completely replicate these findings in mice. Fairly higher doses of DAHP (≥270 mg/kg) were needed to reduce inflammatory pain in mice, but the window between antinociception and toxic effects was small, since 400 mg/kg DAHP affected motor performance and general appearance. Also, the analgesic effects were marginal in mice compared to that observed in rats. Conclusions Variations in the GCH1 gene in both humans and mice appear to regulate pain sensitivity and pain behaviours, particularly after pain sensitisation, whereas pain sensitivity to phasic mechanical and thermal stimuli is normal. Moreover, pharmacological inhibition of GCH1 shows antinociceptive effects in preclinical pain studies, though our studies imply that GCH1 inhibition may have a small therapeutic index. Implications The implication of the GCH1 gene in pain may increase our understanding of the risk factors of chronic pain development and improve current pain therapy by personalised medicine. In addition, inhibition of GCH1 provides a potential target for analgesic drug development, though GCH1 inhibitors should possess local or partial effects to avoid serious side-effects to the central nervous system and cardiovascular system.

Effects of the dimeric PSD-95 inhibitor UCCB01-144 in mouse models of pain, cognition and motor function

NMDAR antagonism shows analgesic action in humans and animal pain models, but disrupts cognitive and motor functions. NMDAR-dependent NO production requires tethering of the NMDAR to neuronal NO synthase (nNOS) by the postsynaptic density protein-95 (PSD-95). Perturbing the NMDAR/PSD-95/nNOS interaction has therefore been proposed as an alternative analgesic mechanism. We recently reported that UCCB01-125, a dimeric PSD-95 inhibitor with limited blood-brain-barrier permeability, reduced mechanical hypersensitivity in the complete Freunds adjuvant (CFA) inflammatory pain model, without disrupting cognitive or motor functions. Here, we investigated the analgesic efficacy in the CFA model of UCCB01-144, a PSD-95 inhibitor with improved blood-brain-barrier permeability. To extend the comparison of UCCB01-125 and UCCB01-144, we also tested both compounds in the spared nerve injury (SNI) model of neuropathic pain. Potential cognitive effects of UCCB01-144 were examined using the social transmission of food preference (STFP) test and the V-maze test, and motor coordination was assessed with the rotarod test. UCCB01-144 (10mg/kg) reversed CFA-induced mechanical hypersensitivity after 1h, and completely normalised sensitivity after 24h. In the SNI model, UCCB01-144 (30mg/kg) partially reversed hypersensitivity after 1h, but no effect was observed after 24h. UCCB01-125 did not affect SNI-induced hypersensitivity. Rotarod performance was unaffected by UCCB01-144, but 30mg/kg UCCB01-144 impaired performance in the STFP test. Collectively, UCCB01-144 reversed both CFA and SNI-induced hypersensitivity, but the efficacy in the SNI model was only transient. This suggests that enhanced BBB permeability of PSD-95 inhibitors improves the analgesic action in neuropathic pain states.

Heterozygous Mutations in Gtp-Cyclohydrolase-1 Reduce Bh4 Biosynthesis but Not Pain Sensitivity

Abstract Human studies have demonstrated a correlation between noncoding polymorphisms of “the pain protective” haplotype in the GCH1 gene that encodes for GTP cyclohydrolase I (GTPCH1)—which leads to reduced tetrahydrobiopterin (BH4) production in cell systems—and a diminished perception of experimental and clinical pain. Here, we investigate whether heterozygous mutations in the GCH1 gene which lead to a profound BH4 reduction in patients with dopa-responsive dystonia (DRD) have any effect on pain sensitivity. The study includes an investigation of GCH1-associated biomarkers and pain sensitivity in a cohort of 22 patients with DRD and 36 controls. The patients with DRD had, when compared with controls, significantly reduced levels of BH4, neopterin, biopterin, and GTPCH1 in their urine, blood, or cytokine-stimulated fibroblasts, but their pain response with respect to non-painful stimulation, (acute) stimulus-evoked pain, or pain response after capsaicin-induced sensitization was not significantly different. A family-specific cohort of 11 patients with DRD and 11 controls were included in this study. The patients with DRD were heterozygous for the pain protective haplotype in cis with the GCH1 disease-causing mutation, c.899T>C. No effect on pain perception was observed for this combined haplotype. In conclusion, a reduced concentration of BH4 is not sufficient to alter ongoing pain sensitivity or evoked pain responses.