1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea and Disease-Models--Animal

Ongoing, spontaneous pain is characteristic of inflammatory joint pain and reduces an individual's quality of life. To understand the neural basis of inflammatory joint pain, we made a unilateral knee injection of complete Freund's adjuvant (CFA) in mice, which reduced their natural digging behavior. We hypothesized that sensitization of knee-innervating dorsal root ganglion (DRG) neurons underlies this altered behavior. To test this hypothesis, we performed electrophysiological recordings on retrograde labeled knee-innervating primary DRG neuron cultures and measured their responses to a number of electrical and chemical stimuli. We found that 24-h after CFA-induced knee inflammation, knee neurons show a decreased action potential generation threshold, as well as increased GABA and capsaicin sensitivity, but have unaltered acid sensitivity. The inflammation-induced sensitization of knee neurons persisted for 24-h in culture, but was not observed after 48-h in culture. Through immunohistochemistry, we showed that the increased knee neuron capsaicin sensitivity correlated with enhanced expression of the capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1) in knee-innervating neurons of the CFA-injected side. We also observed an increase in the co-expression of TRPV1 with tropomyosin receptor kinase A (TrkA), which is the receptor for nerve growth factor (NGF), suggesting that NGF partially induces the increased TRPV1 expression. Lastly, we found that systemic administration of the TRPV1 antagonist, A-425619, reversed the decrease in digging behavior induced by CFA injection, further confirming the role of TRPV1, expressed by knee neurons, in acute inflammatory joint pain.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arthralgia; Capsaicin; Cells, Cultured; Disease Models, Animal; Female; Freund's Adjuvant; gamma-Aminobutyric Acid; Ganglia, Spinal; Hindlimb; Inflammation; Isoquinolines; Mice, Inbred C57BL; Motor Activity; Receptor, trkA; Sensory Receptor Cells; TRPV Cation Channels; Urea

Pain is the leading cause of emergency department visits, hospitalizations, and daily suffering in individuals with sickle cell disease (SCD). The pathologic mechanisms leading to the perception of pain during acute RBC sickling episodes and development of chronic pain remain poorly understood and ineffectively treated. We provide the first study that explores nociceptor sensitization mechanisms that contribute to pain behavior in mice with severe SCD. Sickle mice exhibit robust behavioral hypersensitivity to mechanical, cold, and heat stimuli. Mechanical hypersensitivity is further exacerbated when hypoxia is used to induce acute sickling. Behavioral mechanical hypersensitivity is mediated in part by enhanced excitability to mechanical stimuli at both primary afferent peripheral terminal and sensory membrane levels. In the present study, inhibition of the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1) with the selective antagonist A-425619 reversed the mechanical sensitization at both primary afferent terminals and isolated somata, and markedly attenuated mechanical behavioral hypersensitivity. In contrast, inhibition of TRPA1 with HC-030031 had no effect on mechanical sensitivity. These results suggest that the TRPV1 receptor contributes to primary afferent mechanical sensitization and a substantial portion of behavioral mechanical hypersensitivity in SCD mice. Therefore, TRPV1-targeted compounds that lack thermoregulatory side effects may provide relief from pain in patients with SCD.

Topics: Action Potentials; Anemia, Sickle Cell; Animals; Capsaicin; Disease Models, Animal; Female; Humans; Hyperalgesia; Hypoxia; Isoquinolines; Male; Mice; Mice, Inbred Strains; Microelectrodes; Nociceptors; Pain; Pain Measurement; Patch-Clamp Techniques; TRPV Cation Channels; Urea

Peripheral nerve injury leads to neuropathic pain, but the underlying mechanisms are not clear. The TRPV1 channel expressed by nociceptors is one receptor for noxious heat and inflammatory molecules. Lumbar 4 (L4) spinal nerve ligation (SNL) in mice induced persistent heat hyperalgesia 4 to 10 days after injury. The heat hypersensitivity was completely reversed by the TRPV1 antagonist A-425619. Furthermore, DRG neurons were isolated from the injured L4 ganglia or adjacent L3 ganglia 4 to 10 days after L4 SNL. Whole-cell patch-clamp recordings were performed and heat stimuli (22 degrees C to 50 degrees C/3 s) were applied to the soma. Neurons were classified by soma size and isolectin-B4 (IB4) binding. Among directly injured L4 neurons, SNL increased the percentage of small-diameter IB4-positive neurons that were heat-sensitive from 13% (naive controls) to 56% and conversely decreased the proportion of small IB4-negative neurons that were heat-sensitive from 66% (naive controls) to 34%. There was no change in IB4 binding in neurons from the injured ganglia. Surprisingly, in neurons from the adjacent L3 ganglia, SNL had no effect on the heat responsiveness of either IB4-positive or negative small neurons. Also, SNL had no effect on heat responses in medium-large-diameter neurons from either the injured or adjacent ganglia.. TRPV1 function is upregulated in IB4-positive sensory neurons, and TRPV1 is responsible for the behavioral heat hypersensitivity in the spinal nerve ligation model. Because IB4-positive neurons may contribute to the emotional perception of pain, TRPV1 antagonists, targeting both sensory and affective pain components, could have broad analgesic effects.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Ganglia, Spinal; Hot Temperature; Isoquinolines; Lectins; Lumbar Vertebrae; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neuralgia; Nociceptors; Peripheral Nervous System Agents; Random Allocation; Spinal Nerves; TRPV Cation Channels; Urea

Evidence implicating Nav1.8 and TRPV1 ion channels in various chronic pain states is extensive. In this study, we used isobolographic analysis to examine the in vivo effects of the combination of the Nav1.8 blocker A-803467 [5-(4-Chloro-phenyl)-furan-2-carboxylic acid (3,5-dimethoxy-phenyl)-amide] with 2 structurally distinct TRPV1 antagonists, A-840257 [1-(1H-Indazol-4-yl)-3-([R]-4-piperidin-1-yl-indan-1-yl)-urea] or A-425619 [1-Isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea]. The antinociceptive effects of the Nav1.8 blocker alone and in combination with each TRPV1 antagonist were examined in an inflammatory (complete Freund's adjuvant, CFA) and a neuropathic (spinal nerve ligation, SNL) pain model after systemic (intraperitoneal) administration. Alone, A-803467 was efficacious in both CFA and SNL models with ED(50) values of 70 (54.2 to 95.8) mg/kg and 70 (38.1 to 111.9) mg/kg, respectively. The ED(50) values of the TRPV1 antagonists A-840257 and A-425619 alone in the CFA model were 10 (3.6 to 14.9) mg/kg and 43 (24.1 to 62.2) mg/kg, respectively; both were without significant effect in the SNL model. A series of experiments incorporating 1:1, 3:1, or 0.3:1 ED(50) dose-ratio combinations of A-840257 and A-803467, or A-425619 and A-803467 were performed in both pain models, and the effective doses of mixtures that produced 50% antinociception (ED(50, mix)) were determined by isobolographic analysis. The ED(50, mix) in each case was not found to be statistically different than ED(50, add), the theoretical ED(50) calculated assuming additive effects. These data demonstrate that Nav1.8 blockers and TRPV1 antagonists administered in combination produce an additive effect in rat pain models. Using such a combination strategy to produce analgesia may potentially provide an improved therapeutic separation from unwanted in vivo side effects associated with blockade of either Nav1.8 or TRPV1 alone.. In this report, effects of coadministration of TRPV1 antagonists and A-803467, a Nav1.8 blocker, were investigated in preclinical rodent models of neuropathic and inflammatory pain. The 2 classes of novel antinociceptive agents produced an additive interaction in attenuating CFA-induced thermal hyperalgesia, providing a rationale for their use as a combination strategy in the clinic for treating inflammatory pain.

Topics: Analgesics; Aniline Compounds; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Freund's Adjuvant; Furans; Inflammation; Isoquinolines; Male; NAV1.8 Voltage-Gated Sodium Channel; Nerve Tissue Proteins; Pain; Pain Measurement; Pain Threshold; Rats; Rats, Sprague-Dawley; Sodium Channels; Spinal Nerves; Substance-Related Disorders; Treatment Outcome; TRPV Cation Channels; Urea

Novel transient receptor potential vanilloid 1 (TRPV1) receptor antagonists with various bicyclic heteroaromatic pharmacophores were synthesized, and their in vitro activity in blocking capsaicin activation of TRPV1 was assessed. On the basis of the contribution of these pharmacophores to the in vitro potency, they were ranked in the order of 5-isoquinoline > 8-quinoline = 8-quinazoline > 8-isoquinoline > or = cinnoline approximately phthalazine approximately quinoxaline approximately 5-quinoline. The 5-isoquinoline-containing compound 14a (hTRPV1 IC50 = 4 nM) exhibited 46% oral bioavailability and in vivo activity in animal models of visceral and inflammatory pain. Pharmacokinetic and pharmacological properties of 14a are substantial improvements over the profile of the high-throughput screening hit 1 (hTRPV1 IC50 = 22 nM), which was not efficacious in animal pain models and was not orally bioavailable.

Topics: Abdominal Pain; Administration, Oral; Analgesics; Animals; Biological Availability; Calcium; Cells, Cultured; Disease Models, Animal; Heterocyclic Compounds, 2-Ring; Humans; Hyperalgesia; Isoquinolines; Models, Molecular; Pain; Quinazolines; Quinolines; Rats; Receptors, Drug; Static Electricity; Structure-Activity Relationship; Urea