The effect of the forage-to-concentrate ratio of the partial mixed ration and the quantity of concentrate in an automatic milking system for lactating Holstein cows.

This study was conducted to evaluate the effects of the forage-to-concentrate ratio of the partial mixed ration (PMR) and the quantity of concentrate offered in an automated milking system (AMS), in a feed-first guided-flow barn, on the behavior and performance of dairy cows. Eight ruminally cannulated multiparous Holstein cows were used in a replicated 4 × 4 Latin square balanced for carry-over effects. Treatments were arranged in a 2 × 2 factorial consisting of a PMR that contained (dry matter basis) either a low (54:46; L-FOR) or a high (64:36; H-FOR) forage-to-concentrate ratio and AMS concentrate provision to achieve low (2 kg/d; L-AMS) or high (6 kg/d; H-AMS) intake. Each period consisted of 28 d with 6 d for dietary transition, 13 d for adaptation, and 9 d of collection. The first 4 d of data and sample collection were used to evaluate behavioral data (milking frequency, feeding behavior, and standing and lying behavior) and ruminal pH. Subsequently, a sampling device removal day was provided, and the last 4 d were used to evaluate ruminal fermentation and apparent total-tract digestibility. All 9 d were used for milk yield measurement, and the 8 d were used for dry matter intake measurement. Cows fed the H-AMS consumed 3.5 kg/d less PMR while consuming 4.2 kg/d more AMS concentrate, but total dry matter intake (PMR+AMS) was not affected by treatments averaging 27.3 kg/d. Although cows fed H-AMS had greater concentrate intake, they also had greater variability for AMS concentrate intake among days (0.85 vs. 0.25 kg/d, respectively). The number of PMR meals and PMR eating behavior were not affected by the PMR or AMS treatments. Feeding H-AMS did not affect milking frequency averaging 3.63 milkings/d, but tended to increase milk yield by 1.25 kg/d relative to L-AMS. Likewise, cows fed the L-FOR tended to have greater milk yield relative to H-FOR (39.3 vs 37.9 kg/d, respectively), but had greater holding area time. Minimum ruminal pH tended to be lower for cows fed L-FOR compared with cows fed H-FOR but was not affected by the AMS concentrate treatment. When fed the L-FOR, feeding the H-AMS increased total short-chain fatty acid concentration in the rumen relative to cows fed L-AMS, whereas the response for H-FOR was not affected by the AMS concentrate. These data suggest that feeding H-AMS may improve milk yield, but also increases the day-to-day variability in AMS concentrate consumption. Feeding a L-FOR PMR may increase milk yield without affecting variability in AMS concentrate consumption; however, it may reduce ruminal pH and increase the time spent in the holding area compared with cows fed a H-FOR PMR.

UT-B Urea Transporter Localization in the Bovine Gastrointestinal Tract.

Facilitative UT-B urea transporters play an important role in the urea nitrogen salvaging process that occurs in the gastrointestinal tract of mammals, particularly ruminants. Gastrointestinal UT-B transporters have previously been reported in various ruminant species-including cow, sheep and goat. In this present study, UT-B transporter localization was investigated in tissues throughout the bovine gastrointestinal tract. RT-PCR analysis showed that UT-B2 was the predominant UT-B mRNA transcript expressed in dorsal, ventral and cranial ruminal sacs, while alternative UT-B transcripts were present in other gastrointestinal tissues. Immunoblotting analysis detected a strong, glycosylated ~50 kDa UT-B2 protein in all three ruminal sacs. Immunolocalization studies showed that UT-B2 protein was predominantly localized to the plasma membrane of cells in the stratum basale layer of all ruminal sac papillae. In contrast, other UT-B protein staining was detected in the basolateral membranes of the surface epithelial cells lining the abomasum, colon and rectum. Overall, these findings confirm that UT-B2 cellular localization is similar in all ruminal sacs and that other UT-B proteins are located in epithelial cells lining various tissues in the bovine gastrointestinal tract.

Expression and localization of a UT-B urea transporter in the human bladder.

Facilitative UT-B urea transporters have been shown to play an important role in the urinary concentrating mechanism. Recent studies have now suggested a link between UT-B allelic variation and human bladder cancer risk. UT-B1 protein has been previously identified in the bladder of various mammalian species, but not yet in humans. The aim of the present study was to investigate whether any UT-B protein was present in the human bladder. First, RT-PCR results confirmed that UT-B1 was strongly expressed at the RNA level in the human bladder, whereas UT-B2 was only weakly present. Initial Western blot analysis confirmed that a novel UT-B COOH-terminal antibody detected human UT-B proteins. Importantly, this antibody detected a specific 40- to 45-kDa UT-B signal in human bladder protein. Using a peptide-N-glycosidase F enzyme, this bladder UT-B signal was deglycosylated to a core 30-kDa protein, which is smaller than the predicted size for UT-B1 but similar to many proteins reported to be UT-B1. Finally, immunolocalization experiments confirmed that UT-B protein was strongly expressed throughout all urothelium layers except for the apical membrane of the outermost umbrella cells. In conclusion, these data confirm the presence of UT-B protein within the human bladder. Further studies are now required to determine the precise nature, regulation, and physiological role of this UT-B.

Turn structures in CGRP C-terminal analogues promote stable arrangements of key residue side chains.

The 37-amino acid calcitonin gene-related peptide (CGRP) is a potent endogenous vasodilator thought to be implicated in the genesis of migraine attack. CGRP antagonists may thus have therapeutic value for the treatment of migraine. The CGRP C-terminally derived peptide [D(31),P(34),F(35)]CGRP(27-37)-NH(2) was recently identified as a high-affinity hCGRP(1) receptor selective antagonist. Reasonable CGRP(1) affinity has also been demonstrated for several related analogues, including [D(31),A(34),F(35)]CGRP(27-37)-NH(2). In the study presented here, conformational and structural features in CGRP(27-37)-NH(2) analogues that are important for hCGRP(1) receptor binding were explored. Structure-activity studies carried out on [D(31),P(34),F(35)]CGRP(27-37)-NH(2) resulted in [D(31),P(34),F(35)]CGRP(30-37)-NH(2), the shortest reported CGRP C-terminal peptide analogue exhibiting reasonable hCGRP(1) receptor affinity (K(i) = 29.6 nM). Further removal of T(30) from the peptide's N-terminus greatly reduced receptor affinity from the nanomolar to micromolar range. Additional residues deemed critical for hCGRP(1) receptor binding were identified from an alanine scan of [A(34),F(35)]CGRP(28-37)-NH(2) and included V(32) and F(37). Replacement of the C-terminal amide in this same peptide with a carboxyl, furthermore, resulted in a greater than 50-fold reduction in hCGRP(1) affinity, thus suggesting a direct role for the amide moiety in receptor binding. The conformational properties of two classes of CGRP(27-37)-NH(2) peptides, [D(31),X(34),F(35)]CGRP(27-37)-NH(2) (X is A or P), were examined by NMR spectroscopy and molecular modeling. A beta-turn centered on P(29) was a notable feature consistently observed among active peptides in both series. This turn led to exposure of the critical T(30) residue to the surrounding environment. Peptides in the A(34) series were additionally characterized by a stable C-terminal helical turn that resulted in the three important residues (T(30), V(32), and F(37)) adopting consistent interspatial positions with respect to one another. Peptides in the P(34) series were comparatively more flexible at the C-terminus, although a large proportion of the [D(31),P(34),F(35)]CGRP(27-37)-NH(2) calculated conformers contained a gamma-turn centered on P(34). These results collectively suggest that turn structures at both the C-terminus and N-terminus of CGRP(27-37)-NH(2) analogues may help to appropriately orient critical residues (T(30), V(32), and F(37)) for hCGRP(1) receptor binding.

Design of non-peptide CCK2 and NK1 peptidomimetics using 1-(2-nitrophenyl)thiosemicarbazide as a novel common scaffold.

A beta-turn overlay hypothesis has been used to transform the core scaffold of a selective non-peptide bradykinin B2 receptor antagonist into ligands specifically recognized by the CCK2 or NK1 receptors.

New diarylmethylpiperazines as potent and selective nonpeptidic delta opioid receptor agonists with increased In vitro metabolic stability.

Nonpeptide delta opioid agonists are analgesics with a potentially improved side-effect and abuse liability profile, compared to classical opioids. Andrews analysis of the NIH nonpeptide lead SNC-80 suggested the removal of substituents not predicted to contribute to binding. This approach led to a simplified lead, N, N-diethyl-4-[phenyl(1-piperazinyl)methyl]benzamide (1), which retained potent binding affinity and selectivity to the human delta receptor (IC(50) = 11 nM, mu/delta = 740, kappa/delta > 900) and potency as a full agonist (EC(50) = 36 nM) but had a markedly reduced molecular weight, only one chiral center, and increased in vitro metabolic stability. From this lead, the key pharmacophore groups for delta receptor affinity and activation were more clearly defined by SAR and mutagenesis studies. Further structural modifications on the basis of 1 confirmed the importance of the N, N-diethylbenzamide group and the piperazine lower basic nitrogen for delta binding, in agreement with mutagenesis data. A number of piperazine N-alkyl substituents were tolerated. In contrast, modifications of the phenyl group led to the discovery of a series of diarylmethylpiperazines exemplified by N, N-diethyl-4-[1-piperazinyl(8-quinolinyl)methyl]benzamide (56) which had an improved in vitro binding profile (IC(50) = 0.5 nM, mu/delta = 1239, EC(50) = 3.6 nM) and increased in vitro metabolic stability compared to SNC-80.

N,N-Diethyl-4-(phenylpiperidin-4-ylidenemethyl)benzamide: a novel, exceptionally selective, potent delta opioid receptor agonist with oral bioavailability and its analogues.

The design, synthesis, and pharmacological evaluation of a novel class of delta opioid receptor agonists, N, N-diethyl-4-(phenylpiperidin-4-ylidenemethyl)benzamide (6a) and its analogues, are described. These compounds, formally derived from SNC-80 (2) by replacing the piperazine ring with a piperidine ring containing an exocyclic carbon carbon double bond, were found to bind with high affinity and exhibit excellent selectivity for the delta opioid receptor as full agonists. 6a, the simplest structure in the class, exhibited an IC(50) = 0.87 nM for the delta opioid receptors and extremely high selectivity over the mu receptors (mu/delta = 4370) and the kappa receptors (kappa/delta = 8590). Rat liver microsome studies on a selected number of compounds show these olefinic piperidine compounds (6) to be considerably more stable than SNC-80. This novel series of compounds appear to interact with delta opioid receptors in a similar way to SNC-80 since they demonstrate similar SAR. Two general approaches have been established for the synthesis of these compounds, based on dehydration of benzhydryl alcohols (7) and Suzuki coupling reactions of vinyl bromide (8), and are herewith reported.

Selective neurokinin-1 receptor antagonists are anti-hyperalgesic in a model of neuropathic pain in the guinea-pig.

Neuropathic pain is poorly managed by conventional analgesic therapy, such as non-steroidal anti-inflammatory drugs and opiates. The development of animal models of peripheral neural damage has aided in our understanding of the pathology and pharmacology of neuropathic pain. This report is the first clear demonstration using selective neurokinin-1 receptor antagonists of a potentially novel therapeutic approach to the treatment of neuropathic pain resulting from peripheral nerve damage in a guinea-pig model. The neurokinin-1 receptor antagonists, SDZ NKT 343 and LY 303,870 significantly reduced mechanical hyperalgesia following oral and intrathecal administration. (R,R)-SDZ NK T343, the enantiomer of SDZ NKT 343 did not show anti-hyperalgesic activity. RPR 100,893 showed significant anti-hyperalgesic activity only following intrathecal administration suggesting poor absorption or low level penetration of the blood-brain barrier. These results imply that neurokinin-1 receptor antagonists offer a new class of anti-hyperalgesic drugs with a largely central site of action in neuropathic pain.

The effects of SDZ NKT 343, a potent NK1 receptor antagonist, on cutaneous responses of primate spinothalamic tract neurones sensitized by intradermal capsaicin injection.

Substance P, acting through neurokinin I receptors, is involved in the processing of nociceptive information in the spinal cord. Sensitization of spinothalamic tract neurons occurs to low-intensity stimuli following capsaicin injection. The current study tested the effects of the novel neurokinin I receptor antagonist, SDZ NKT 343, on the sensitization of spinothalamic tract cells by capsaicin in monkeys. Spinothalamic tract cells from the lumbar enlargement with receptive fields in the hindpaw were isolated and recorded before and after intradermal injection of capsaicin. The background activity and responses to brushing, pressing and pinching the skin were assessed. Thirty minutes after capsaicin injection there was an increase in background activity and responses to brush and pressure applied to the receptive field. Infusion of SDZ NKT 343 (for 30-45 min) significantly reversed the increased response to brushing without affecting the increased background activity or the increased response to pressure. Thus, blockade of neurokinin 1 receptors reduces the sensitized responses to innocuous mechanical stimuli but not to noxious mechanical stimuli.

2-Nitrophenylcarbamoyl-(S)-prolyl-(S)-3-(2-naphthyl)alanyl-N-benzyl-N - methylamide (SDZ NKT 343), a potent human NK1 tachykinin receptor antagonist with good oral analgesic activity in chronic pain models.

A lead compound which had sub-micromolar affinity for the rabbit NK1 receptor but negligible affinity for rat NK1 receptors, 3a, was discovered by directed screening. 2-Substitution in the ring of the benzylthiourea substituent in the initial lead was found to be important, and halogens (Cl, Br) in this position were found to improve affinity for the human receptor. The activity of a series of 2-halo-substituted benzylthioureas was then optimized by modification of the proline diphenylmethyl amide, guided by a simple conceptual model based on structural overlay between these early antagonists and NK1 selective peptides. In this way, aromatic amino acid amides were identified which had improved affinity with respect to the starting diphenylmethyl (DPM) amides. The first sub-nanomolar ligand for the human NK1 receptor which arose from this series, 4af, combined a 2-chlorobenzylthiourea unit with a 2-naphthylalanine amide. Contemporaneously it was discovered that the benzylthiourea unit could be simplified to a phenylthiourea providing that an appropriate 2-substituent was also incorporated. Combination of these two series gave 2-NO2 phenylthiourea analogues which led directly to the analogous urea, 5f (2-nitrophenylcarbamoyl-(S)-prolyl-(S)-3-(2-naphthyl)alanyl-N-benz yl- N-methylamide, SDZ NKT 343), a highly potent ligand for the human NK1 receptor (Ki = 0.16 nM). In addition to its high in vitro potency, 5f proved to be a potent orally active analgesic in guinea pig models of chronic inflammatory and neuropathic pain. The nature of the 2-aryl substituent was found to be critical for oral activity in this series. Clinical evaluation of 5f as a novel analgesic agent is currently underway.

Comparative, general pharmacology of SDZ NKT 343, a novel, selective NK1 receptor antagonist.

1. The in vitro and in vivo pharmacology of SDZ NKT 343 (2-nitrophenyl-carbamoyl-(S)-prolyl-(S)-3-(2-naphthyl)alanyl-N-benzyl-N- methylamide), a novel tachykinin NK1 receptor antagonist was investigated. 2. SDZ NKT 343 inhibited [3H]-substance P binding to the human NK1 receptor in transfected Cos-7 cell membranes (IC50 = 0.62+/-0.11 nM). In comparison, in the same assay Ki values for FK888, CP 99,994, SR 140,333 and RPR 100,893 were 2.13+/-0.04 nM, 0.96+/-0.20 nM, 0.15+/-0.06 nM and 1.77+/-0.41 nM, respectively. SDZ NKT 343 showed a markedly lower affinity at rat NK1 receptors in whole forebrain membranes (IC50 = 451+/-139 nM). 3. SDZ NKT 343 caused an increase in EC50 as well as reduction in the number of binding sites (Bmax) determined for [3H]-substance P, suggesting a non-competitive interaction at the human NK1 receptor. SDZ NKT 343 also caused a reduction in the maximum elevation of [Ca2+]i evoked by substance P (SP) in human U373MG cells and depressed the maximum [Sar9]SP sulphone-induced contraction of the guinea-pig isolated ileum. The antagonism of SP effects on U373MG cells by SDZ NKT 343 was reversible. 4. SDZ NKT 343 showed weak affinity to human NK2 and NK3 receptors in transfected Cos-7 cells (Ki of 0.52+/-0.04 microM and 3.4+/-1.2 microM, respectively). SDZ NKT 343 was inactive in a broad array of binding assays including the bradykinin B2 receptor the histamine H1 receptor, opiate receptors and adrenoceptors. SDZ NKT 343 only weakly inhibited the voltage-activated Ca2+ and Na+ currents in guinea-pig dorsal root ganglion neurones. The enantiomer of SDZ NKT 343, (R,R)-SDZ NKT 343 was about 1000 times less active at human NK1 receptors expressed in Cos-7 cell membranes. 5. Contractions of the guinea-pig ileum by [Sar9]SP sulphone were inhibited by SDZ NKT 343 in a concentration-dependent manner, with an IC50 = 1.60+/-0.94 nM, while the enantiomer (R,R)-SDZ NKT 343 was 100 times less active (IC50 = 162+/-26 nM). In comparison, in the same assay IC50 values for other NK1 receptor antagonists CP 99,994, SR 140,333, RPR 100,893 and FK 888 were 2.90+/-07 nM, 0.14+/-0.02 nM, 11.4+/-2.9 nM and 2.4+/-0.83 nM, respectively. 6. In anaesthetized guinea-pigs i.v. administered SDZ NKT 343 antagonized [Sar9]SP sulphone-evoked bronchoconstriction (70% reduction at 0.4 mg kg(-1), i.v.). Basal airway resistance, mean arterial blood pressure and heart rate were not affected. 7. In conclusion, SDZ NKT 343 is a highly selective NK1 receptor antagonist with high potency at the human and guinea-pig receptors. SDZ NKT 343 may be used as a potential novel therapeutic agent in human diseases where NK1 receptor hyperfunction is involved.

Analogues of capsaicin with agonist activity as novel analgesic agents: structure-activity studies. 4. Potent, orally active analgesics.

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.

Similarities and differences in the structure-activity relationships of capsaicin and resiniferatoxin analogues.

Structure-activity relationships in analogues of the irritant natural product capsaicin have previously been rationalized by subdivision of the molecule into three structural regions (A,B, and C). The hypothesis that resiniferatoxin (RTX), which is a high-potency ligand for the same receptor and which has superficial structural similarities with capsaicin, could be analogously subdivided has been investigated. The effects of making parallel changes in the two structural series have been studied in a cellular functional assay which is predictive of analgesic activity. Parallel structural changes in the two series lead to markedly different consequences on biological activity; the 3- and 4-position aryl substituents (corresponding to the capsaicin 'A-region') which are strictly required for activity in capsaicin analogues are not important in RTX analogues. The homovanillyl C-20 ester group in RTX (corresponding to the capsaicin 'B-region') is more potent than the corresponding amide, in contrast to the capsaicin analogues. Structural variations to the diterpene moiety suggest that the functionalized 5-membered diterpene ring of RTX is an important structural determinant for high potency. Modeling studies indicate that the 3D position of the alpha-hydroxy ketone moiety in the 5-membered ring is markedly different in the phorbol (inactive) analogues and RTX (active) series. This difference appears to be due to the influence of the strained ortho ester group in RTX, which acts as a local conformational constraint. The reduced activity of an analogue substituted in this region and the inactivity of a simplified analogue in which this unit is entirely removed support this conclusion.

Receptors mediating tachykinin-evoked depolarisations of neurons in the neonatal rat spinal cord.

We have examined the contribution of NK1, NK2 and NK3 receptors to the depolarisation of the neonatal rat spinal cord in vitro evoked by exogenously applied tachykinine. Potential changes were recorded extracellularly from a lumbar ventral root. The NK1 receptor selective agonists substance P methly ester (SPOMs), septide and/Sar9/-substance P-sulphone (/Sar9/-P-sulphone), perfused onto the cord for 20 s, evoked ventral root potentials (VRPs) with similar EC50 values of 7.2 nM (95% confidence limits, 4.4-10.9 nM), 4.6 nM (1.8-9.3 nM) and 3.1 nM (1.6-5.1 nM), respectively. The NK3 receptor selective agonist senktide also evoked VRPs with an EC50 of 12.0 nM (5.1-23.4 nM), whilst the NK2 receptor selective agonist/beta-AlaB/-neurokinin A(4-10) (/beta-AlaB/-NKA(4-10)) was much less potent (EC50 = 228.3 nM, 95% confidence limits 138.0-350.0 nM). The non-peptide NK1 receptor selective antagonist RP67580 inhibited responses to SPOMe, septide and/Sar9/-SP-sulphone to varying degrees with IC50 values against each of 16.0 nM (10.7-23.4 nM), 19.8 nM (8.9-37 nM) and 58.0 nM (41-89 nM), respectively. The NK1 receptor antagonist CP-96,345 similarly inhibited responses to these agonists, although with higher IC50 estimates of 0.84 microM (0.51-1.40 microM) against SPOMe, 0.79 microM (0.50-1.17 microM) against/Sar9/-SP-sulphone and 0.37 microM (0.27-0.51 microM) against septide. Further analysis of the activity of RP67580 yielded a significantly higher pKB estimate for antagonism of responses to septide (7.67 +/- 0.04) than of responses to/Sar9/-SP-sulphone (7.18 +/- 0.05). In both cases Schild analysis indicated competitive antagonism. RP67580 also reversibly inhibited responses to /beta-AlaB/-NKA(4-10). However, in this case the Schild alope was significantly different from unity (0.44 +/- 0.55; P < 0.001), although the potency of the antagonist appeared similar to that seen with the NK1 receptor agonists (pA2 = 7.52). There was no effect of RP67580 against responses to senktide or of the inactive isomer RP67581 against septide-evoked VRPs. The NK2 receptor antagonist MEN 10,376 at concentrations up to 1 microM produced a partial but reversible inhibition of responses to a submaximal concentration of /beta-AlaB/-NKA(4-10) (0.3 microM) with a maximum reduction in VRP amplitude of 25.6 +/- 7.3%. A similar inhibitory effect was seen against septide-evoked VRPs (30.2 +/- 5.6% inhibition), although there was no effect against responses to submaximal concentrations of /Sar9/-SP-sulphone or senktide. In contrast, the non-peptide NK2 receptor antagonist SR 48,968 (1 microM) produced a maximal 48.0 +/- 7.7% inhibition of/beta-AlaB/-NKA(4-10)-evoked VRP's with no effect against responses to a submaximal concentration of septide. These data show that NK1 and NK3 receptor activation mediates depolarisation of the neonatal rat spinal cord, and suggest the presence of two NK1 receptor populations showing preference for septide and/Sar9/-SP-sulphone. Depolarisations mediated by /beta-AlaB/-NKA(4-10), previously described as a selective NK2 receptor ligand, are mediated predominantly via an action at NK1 receptors with a lesser involvement of NK2 receptors.

The discovery of capsazepine, the first competitive antagonist of the sensory neuron excitants capsaicin and resiniferatoxin.

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.

Characterization of resiniferatoxin binding sites on sensory neurons: co-regulation of resiniferatoxin binding and capsaicin sensitivity in adult rat dorsal root ganglia.

Binding of [3H]resiniferatoxin was seen by autoradiography in sections of rat dorsal root ganglia and the superficial dorsal horn of the spinal cord. Membranes from rat dorsal root ganglia and spinal cord, but not other tissues, had saturable high-affinity binding sites for [3H]resiniferatoxin. A series of capsaicin analogues competed for these sites. The sites probably correspond to capsaicin receptors. Systemic pretreatment of rats with capsaicin caused loss of capsaicin sensitivity in sensory neurons and a reduction in binding of resiniferatoxin to rat dorsal root ganglia, measured by binding assays and autoradiography. Adult rat dorsal root ganglion neurons cultured without nerve growth factor also lost their capsaicin-sensitivity and showed reduced resiniferatoxin binding. Therefore, capsaicin responses in sensory neurons may be regulated by nerve growth factor through control of the number of capsaicin receptors.

Analogues of capsaicin with agonist activity as novel analgesic agents; structure-activity studies. 2. The amide bond "B-region".

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.

Analogues of capsaicin with agonist activity as novel analgesic agents; structure-activity studies. 1. The aromatic "A-region".

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.

Analogues of capsaicin with agonist activity as novel analgesic agents; structure-activity studies. 3. The hydrophobic side-chain "C-region".

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 comparison of capsazepine and ruthenium red as capsaicin antagonists in the rat isolated urinary bladder and vas deferens.

1. The ability of capsazepine, a recently developed capsaicin receptor antagonist, to prevent the effects of capsaicin on the rat isolated urinary bladder (contraction) and vas deferens (inhibition of electrically-evoked twitches) was compared to that of ruthenium red, a dye which behaves as a functional antagonist of capsaicin. 2. In the rat bladder, capsazepine (3-30 microM) produced a concentration-dependent rightward shift of the curve to capsaicin without any significant depression of the maximal response to the agonist. By contrast, ruthenium red (10-30 microM) produced a non-competitive type of antagonism, characterized by marked depression of the maximal response attainable. Similar findings were obtained in the rat isolated vas deferens in which capsazepine (10 microM) produced a rightward shift of the curve to capsaicin while ruthenium red (3 microM) depressed the maximal response to the agonist. 3. At the concentrations used to block the effect of capsaicin, neither capsazepine nor ruthenium red affected the contractile response of the rat urinary bladder produced by either neurokinin A or electrical field stimulation or the twitch inhibition produced by rat alpha-calcitonin gene-related peptide (alpha CGRP) in the vas deferens. 4. These findings provide additional evidence that both capsazepine and ruthenium red are valuable tools for exploration of the function of capsaicin-sensitive primary afferent neurones. The antagonism of the action of capsaicin by capsazepine is entirely consistent with the proposed interaction of this substance with a vanilloid receptor located on primary afferents, while the action of ruthenium red apparently involves a more complex, non-competitive antagonism.