1 Capsazepine is a synthetic analogue of the sensory neurone excitotoxin, capsaicin. The present study shows the capsazepine acts as a competitive antagonist of capsaicin.2 Capsazepine (10 JAM) reversibly reduced or abolished the current response to capsaicin (500 nM) of voltage-clamped dorsal root ganglion (DRG) neurones from rats. In contrast, the responses to 50 JM y-aminobutyric acid (GABA) and S JM adenosine 5'-triphosphate (ATP) were unaffected. 3 The effects of capsazepine were examined quantitatively with radioactive ion flux experiments.Capsazepine inhibited the capsaicin (500 nM)-induced 45Ca2" uptake in cultures of rat DRG neurones with an IC50 of 420 ± 46 nM (mean ± s.e.mean, n = 6). The 45Ca2" uptake evoked by resiniferatoxin (RTX), a potent capsaicin-like agonist was also inhibited. (Log concentration)-effect curves for RTX (0.3 nM-1 JAM) were shifted in a competitive manner by capsazepine. The Schild plot of the data had a slope of 1.08 ± 0.15 (s.e.) and gave an apparent Kd estimate for capsazepine of 220 nM (95% confidence limits, 57-400 nM). 4 Capsazepine also inhibited the capsaicin-and RTX-evoked efflux of 86Rb+ from cultured DRG capsazepine the inhibition by Ruthenium Red (10-500nM in DRG and 0.5-10AM in vagus nerve experiments) was not consistent with a competitive antagonism, but rather suggested a more complex, non-competitive inhibition.

Capsaicin and resiniferatoxin are natural products which act specifically on a subset of primary afferent sensory neurons to open a novel cation-selective ion channel in the plasma membrane. These sensory neurons are involved in nociception, and so, these agents are targets for the design of a novel class of analgesics. Although synthetic agonists at the capsaicin receptor have been described previously, competitive antagonists at this receptor would be interesting and novel pharmacological agents. Structure-activity relationships for capsaicin agonists have previously been rationalized, by ourselves and others, by dividing the capsaicin molecule into three regions--the A (aromatic ring)-, B (amide bond)-, and C (hydrophobic side chain)-regions. In this study, the effects on biological activity of conformational constraint of the A-region with respect to the B-region are discussed. Conformational constraint was achieved by the introduction of saturated ring systems of different sizes. The resulting compounds provided agonists of comparable potency to unconstrained analogues as well as a moderately potent antagonist, capsazepine. This compound is the first competitive antagonist of capsaicin and resiniferatoxin to be described and is active in various systems, in vitro and in vivo. It has recently attracted considerable interest as a tool for dissecting the mechanisms by which capsaicin analogues evoke their effects. NMR spectroscopy and X-ray crystallography experiments, as well as molecular modeling techniques, were used to study the conformational behavior of a representative constrained agonist and antagonist. The conformation of the saturated ring contraint in the two cases was found to differ markedly, dramatically affecting the relative disposition of the A-ring and B-region pharmacophores. In agonist structures, the A- and B-regions were virtually coplanar in contrast to those in the antagonist, in which they were approximately orthogonal. A rationale for agonist and antagonist activity at the capsaicin receptor is proposed, based on the consideration of these conformational differences.

1 Bradyzide is from a novel class of rodent-selective non-peptide B 2 bradykinin antagonists (1-(2-Nitrophenyl)thiosemicarbazides). 7 Bradyzide is orally available and blocks bradykinin-induced hypotension and plasma extravasation. 8 Bradyzide shows long-lasting oral activity in rodent models of in¯ammatory hyperalgesia, reversing Freund's complete adjuvant (FCA)-induced mechanical hyperalgesia in the rat knee joint (ED 50 , 0.84 mmol kg 71 ; duration of action 44 h). It is equipotent with morphine and diclofenac, and 1000 times more potent than paracetamol, its maximal eect exceeding that of the non-steroidal anti-in¯ammatory drugs (NSAIDs). Bradyzide does not exhibit tolerance when administered over 6 days. 9 In summary, bradyzide is a potent, orally active, antagonist of the B 2 bradykinin receptor, with selectivity for the rodent over the human receptor.

Structural features of three regions of the capsaicin molecule necessary for agonist properties were delineated by a previously reported modular approach. These in vitro agonist effects were shown to correlate with analgesic potency in rodent models. Combination of optimal structural features from each of these regions of the capsaicin molecule have led to highly potent agonists (eg., 1b). Evaluation in vivo established that 1b had analgesic properties but poor oral activity, short duration of action, and excitatory side effects which precluded further development of this compound. Preliminary metabolism studies had shown that the phenol moiety of 1b was rapidly glucuronidated in vivo, providing a possible explanation for the poor pharmacokinetic profile. Subsequent specific modification of the phenol group led to compounds 2a-j, which retained in vitro potency. The in vivo profiles of two representatives of this series, 2a,h, were much improved over the "parent" phenol series, and they are candidates for development as analgesic agents.

Vanilloid receptor 1 (VR1, TRPV1) is a cation-selective ion channel that is expressed on primary afferent neurons and is upregulated following inflammation and nerve damage. Blockers of this channel may have utility in the treatment of chronic nociceptive and neuropathic pain. Here, we describe the optimization from a high throughput screening hit, of a series of 6-aryl-7-isopropylquinazolinones that are TRPV1 antagonists in vitro. We also demonstrate that one compound is active in vivo against capsaicin-induced hyperalgesia and in models of neuropathic and nociceptive pain in the rat.