Yoshihito Kanai

Involvement of an increased spinal TRPV1 sensitization through its up-regulation in mechanical allodynia of CCI rats

Vanilloid receptor 1 (TRPV1) antagonists are known to attenuate the neuropathic pain symptoms in peripheral nerve injury models, but the mechanism(s) of their effect remains unclear. At the same time, the role of spinal TRPV1 in pain transduction system has not been fully understood. In this study, the role of spinal TRPV1 in mechanical allodynia in rat chronic constriction injury (CCI) model was investigated. Intrathecal administration of a selective TRPV1 antagonist, N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropryazine-1(2H)-carbox-amide (BCTC) significantly attenuated mechanical allodynia in CCI rats at 100 and 300 nmol. In vitro, BCTC inhibited capsaicin (300 nM)-induced releases of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) and substance P-like immunoreactivity (SP-LI) from the rat spinal cord slice preparations with IC(50)s of 37.0 and 36.0 nM, respectively, confirming that BCTC potently inhibits TRPV1 function in the rat spinal cord. TRPV1 expression levels in the spinal cord following CCI were quantified in by Western blot analysis. TRPV1 protein levels were significantly increased in the ipsilateral side of the lumbar spinal cord at 7 and 14 days following CCI surgery, but not in the contralateral side. Furthermore, capsaicin (300 nM)-evoked release of CGRP-LI was significantly higher in the ipsilateral spinal cord of CCI rats (14 days after surgery) than that of sham-operated rats. These findings suggest that an increased sensitization of the spinal TRPV1 through its up-regulation is involved in the development and/or maintenance of mechanical allodynia in rat CCI model.

Antiinflammatory and analgesic activity of a non-peptide substance P receptor antagonist

CP-96,345, a potent non-peptide antagonist of the substance P (SP) receptor, inhibited SP-, neurokinin A (NKA)- and neurokinin B-induced plasma extravasation in guinea pig dorsal skin. The inhibition was specific for the three tachykinins; CP-96,345 was not active against plasma leakage caused by histamine, bradykinin, platelet-activating factor or leukotriene D4. CP-96,345 inhibited capsaicin-induced plasma extravasation in the ureter, an inflammatory response caused by neuropeptides released from afferent C-fibers. Thus, the NK1 receptor appears to play a major role in vascular permeability increases induced by exogenous and endogenous tachykinins. In contrast, CP-96,345 was inactive against SP- and NKA-induced contraction of guinea pig ureter, suggesting that the smooth muscle contraction is not NK1-mediated. CP-96,345 exhibited analgesic activity in acetic acid-induced abdominal stretching in mice, indicating for the first time that SP plays a critical role in this model. The results of these studies support a pathophysiological role of SP and NK1 receptor under acute neurogenic inflammatory conditions and in pain.

Non-specific activity of (+/-)-CP-96,345 in models of pain and inflammation.

The non‐peptide NK1 receptor antagonist, CP‐96,345, and its 2R,3R enantiomer CP‐96,344, which is not an NK1 receptor antagonist (IC50 > 10 μm), were evaluated for antinociceptive and anti‐inflammatory activities in several classical models of pain and inflammation in the rat. Both CP‐96,345 and CP‐96,344 reduced carrageenin‐induced paw oedema and hyperalgesia, and attenuated the second phase of formalin‐induced paw licking with equal potency. These results indicate that NK1 antagonism is not responsible for the activity of (±)‐CP‐96,345 in the above animal models.

Differential involvement of TRPV1 receptors at the central and peripheral nerves in CFA‐induced mechanical and thermal hyperalgesia

Transient receptor potential vanilloid 1 (TRPV1) antagonists are known to attenuate two typical symptoms of inflammatory hyperalgesia: thermal and mechanical. However, it is not clear whether the sites of participation of TRPV1 for each symptom are different. In this study, we clarified the difference between the site of TRPV1 involvement in both symptoms by analysing the anti‐hyperalgesic activity of two kinds of TRPV1 antagonists given locally (i.e. intraplantarly and intrathecally) in rats with CFA (complete Freunds adjuvant)‐induced inflammation. TRPV1 antagonists BCTC (N‐(4‐tertiarybutylphenyl)‐4‐(3‐cholorphyridin‐2‐yl) tetrahydropyrazine‐1 (2H)‐carbox‐amide, 1–300 μg) and SB‐366791 (N‐(3‐methoxyphenyl)‐4‐chlorocinnamide, 30–300 μg) administered intraplantarly in a dose‐dependent manner inhibited CFA‐induced thermal hyperalgesia. In addition, CFA‐induced thermal hyperalgesia was significantly reversed by intrathecal administration of 1–100μg of BCTC and SB‐366791. While intraplantar BCTC (1–300 μg) and SB‐366791 (30–300 μg) did not reverse CFA‐induced mechanical hyperalgesia, 1–100 μg of intrathecally administered BCTC and SB‐366791 dose‐dependently reduced mechanical hyperalgesia. Regression analysis showed that a correlation exists between the inhibitory effects on thermal hyperalgesia and mechanical hyperalgesia after intrathecal administration (correlation factor = 0.6521), but not after intraplantar administration (correlation factor = 0.0215). These data suggest that TRPV1 in the peripheral endings of the primary afferents plays a key role in thermal hyperalgesia, but it makes only a minor contribution in CFA‐induced mechanical hyperalgesia. Furthermore, it is suggested that the spinal TRPV1 is critical in the development of both types of hyperalgesia.

Participation of the spinal TRPV1 receptors in formalin-evoked pain transduction: a study using a selective TRPV1 antagonist, iodo-resiniferatoxin.

The involvement of spinal transient receptor potential vanilloid 1 (TRPV1) in formalin‐evoked pain has remained unclear, because investigation of this kind of pain with selective antagonists has not been conducted. The purpose of this study is to investigate the participation of spinal TRPV1 in formalin‐evoked pain with iodo‐resiniferatoxin (I‐RTX), a potent TRPV1‐selective antagonist. I‐RTX given intrathecally dose‐dependently and significantly decreased the number of flinching responses in the formalin‐evoked 1st and 2nd phase with ID50 values (drug dose producing 50% inhibition of response) of 1.0 and 3.8 μg, respectively, and concentration‐dependently suppressed capsaicin‐evoked calcitonin gene‐related peptide‐like immunoreactivity (CGRP‐LI) release from rat spinal cord slices with an IC50 value (drug concentration producing 50% inhibition of response) of 86 nm. Capsazepine, a classical non‐selective TRPV1 antagonist, given intrathecally also inhibited formalin‐evoked flinching in both the 1st and 2nd phase with ID50s of 420 and 200 μg, respectively, and CGRP‐LI release from rat spinal cord slices with an IC50 of 7.8 μm. Ratios of in‐vivo analgesic potencies of I‐RTX and capsazepine well reflected their intrinsic in‐vitro activity. These findings suggest that spinal TRPV1 participates in the transduction system of formalin‐evoked pain.

Anti-Emetic Activity of the Novel Nonpeptide Tachykinin NK1 Receptor Antagonist Ezlopitant (CJ-11,974) against Acute and Delayed Cisplatin-Induced Emesis in the Ferret

The anti-emetic effects of a novel tachykinin NK1 receptor antagonist, ezlopitant ((2S,3S-cis)-2-diphenylmethyl)- N--1-azabicyclo- [2.2.2]octan-3-amine), were investigated in ferrets. Ezlopitant inhibited [3H]substance P ([3H]SP) binding to the human, guinea pig, ferret and gerbil NK1 receptors (Ki = 0.2, 0.9. 0.6 and 0.5 nmol/l, respectively), but had no affinity to NK2 and NK3 receptors up to 1 µmol/l. Ezlopitant also inhibited SP-induced contraction of guinea pig trachea with a pA2 value of 7.8, but had no effects on the baseline tension and maximum contractile response. In ferrets, ezlopitant, either orally (0.03–3 mg/kg) or subcutaneously (0.3–3 mg/kg), prevented acute retching and vomiting responses induced by intraperitoneal injection of cisplatin (10 mg/kg). In addition, repeated subcutaneous injection of ezlopitant significantly inhibited delayed retching and vomiting responses that occurred in ferrets treated with the lower dose of cisplatin (5 mg/kg, i.p.). Ezlopitant (0.1–1 mg/kg, s.c.) also produced a dose-dependent inhibition of hindpaw tapping induced by intracerebroventricular injection of [Sar9,Met(O2)11]SP in gerbils, which is known to be mediated by NK1 receptors in the brain. These findings indicate that ezlopitant is a potent and selective NK1 receptor antagonist, and that it inhibits both acute and delayed emetic reactions induced by cisplatin in ferrets via acting on NK1 receptors in the central nervous system.

Discovery of CP-96,345 and its characterization in disease models involving substance P.

Studies with CP-96,345, a potent, selective, orally active, nonpeptide NK1 receptor antagonist, have provided considerable insight into SP pharmacology. Rather than being a primary neurotransmitter, SP prolongs the nociception produced by other neurotransmitters. By controlling endothelial permeability, SP plays a major role in inflammation and inflammatory aspects of asthma, possibly by regulating the access of neutrophils to an inflammatory site. These results indicate potential therapeutic applications for SP antagonists in the treatment of chronic pain, inflammation, and inflammatory aspects of asthma, and signal a new era in the clinical management of these important diseases.

Involvement of NGF in the induction of increased noradrenergic innervation of the ureter in neonatally capsaicin-treated rats

Neonatal denervation of primary afferents with capsaicin leads to increased sympathetic innervation of the rat ureter. In the present study the development and the immunohistochemical characterization of this sympathetic hyperinnervation as well as the specific involvement of nerve growth factor (NGF) was investigated. Noradrenaline levels were found elevated in neonatally capsaicin-treated rats by 2 weeks of age and remained at that high level into adulthood. Injections of an anti-NGF antiserum during postnatal days (PN) PN 8-14, PN 13-19 or during PN 17-23 counteracted the capsaicin effect and reduced noradrenaline towards control levels. Immunohistochemical localization of tyrosine hydroxylase (TH), a marker for sympathetic nerve fibres, revealed that the capsaicin-induced hyperinnervation was mainly represented by fibres in deeper muscle layers and to a smaller extent by fibres in the submucosa. In control animals and in rats treated with capsaicin and anti-NGF antiserum fibres were mainly distributed in the adventitia and in the outer part of the smooth muscle layer. These results show that NGF is responsible for the development of an increased noradrenergic innervation in the rat ureter after neonatal capsaicin treatment.

Non-specific activity of (±)CP-96,345 in models of pain and inflammation

The non-peptide NK1 receptor antagonist, CP-96,345, and its 2R,3R enantiomer CP-96,344, which is not an NK1 receptor antagonist (IC50 > 10 microM), were evaluated for antinociceptive and anti-inflammatory activities in several classical models of pain and inflammation in the rat. Both CP-96,345 and CP-96,344 reduced carrageenin-induced paw oedema and hyperalgesia, and attenuated the second phase of formalin-induced paw licking with equal potency. These results indicate that NK1 antagonism is not responsible for the activity of (+/-)-CP-96,345 in the above animal models.