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.

A series of analogues of capsaicin, the pungent principle of chilli peppers, was synthesized and tested in assays for capsaicin-like agonism in vitro. The results of these assays were compared with activities in an acute nociceptive model and a correlation was observed which established that the results of these in vitro assays were predictive of analgesia. Using a modular approach the structure-activity profile of specific regions of capsaicin congeners was established using an in vitro assay measuring 45Ca2+ uptake into neonatal rat dorsal root ganglia neurones. Substituted benzylnonanamides 2a-z and N-octyl-substituted phenylacetamides 4a-v were made to test the requirements for activity in the aromatic "A-region" of the molecule. Compounds with the natural substitution pattern (2b and 4c) and the corresponding catechols (2i and 4g) were the most potent, although the catechols were less potent in vivo. Other substitution patterns have reduced activity. These results have established stringent structural requirements for capsaicin-like activity in this part of the molecule.

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.

Structural variants of the hydrophobic side chain ("C region") of the capsaicin molecule have been incorporated into a series of vanillylamides and vanillylthioureas. These compounds have been tested in an in vitro assay for agonism (45Ca2+ influx into dorsal root ganglia neurones), previously shown to be predictive of analgesic activity. The results of this study have established the requirement for a hydrophobic substituent of limited size (molar refractivity, MR, < 55) in order to obtain high potency. Combination of the information gained here about the "C-region" of the capsaicin molecule with the studies described in the preceding two papers provides a rational basis for the design of compounds of increased potency.

A series of compounds incorporating replacements for the amide bond "B-region" moiety of capsaicin have been synthesized, including vanillylamides and esters, homovanillic acid amides and esters, ureas, and thioureas. These have been tested in an in vitro assay for agonism (45Ca2+ influx into dorsal root ganglia neurones), which is predictive of analgesic activity, to investigate the requirements in this region of capsaicin for activity. N-(4-Hydroxy-3-methoxybenzyl)-N'-octylthiourea (14a) emerged as the most potent analogue (EC50 = 0.06 microM). An operational model based on multiple hydrogen-bonding interactions is proposed to explain the structure-activity profile observed. In combination with studies on the other regions of the capsaicin molecule these results describe a picture of the molecular interactions of capsaicin with its putative receptor.