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.

Immunochemical detection of photoaffinity-labelled capsaicin-binding proteins from sensory neurons.

Capsaicin is a plant neurotoxin which depolarises a subset of mammalian sensory neurons. A photoaffinity probe (4-azidophenylpropionamide) with capsaicin-like agonist activity (EC50 5 microM) has been used to covalently label rat and chick sensory neurons in culture, as well as membrane preparations from both neurons and other tissues. Dorsal root ganglion cell specific capsaicin-binding proteins, including a major band of apparent molecular mass 58,000, have been identified by means of Western blotting, using a specific anti-capsaicin antiserum characterised by radioimmunoassay with a large range of capsaicin congeners. Using the same radioimmunoassay, no endogenous capsaicin-like immunoreactive material in normal or inflamed tissue has, however, been detected.

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.

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.

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.

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.

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.

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.

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.

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.

Capsazepine: a competitive antagonist of the sensory neurone excitant capsaicin.

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 microM) 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 microM gamma-aminobutyric acid (GABA) and 5 microM 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 microM) 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 neurones. The inhibition appeared to be competitive and Schild plots yielded apparent Kd estimates of 148 nM (95% confidence limits, 30-332 nM) with capsaicin as the agonist and 107 nM (95% confidence limits, 49-162 nM) with RTX as agonist. 5. A similar competitive inhibition by capsazepine was seen for capsaicin-induced [14C]-guanidinium efflux from segments of adult rat vagus nerves (apparent Kd = 690 nM; 95% confidence limits, 63 nM-1.45 microM). No significant difference was noted in the apparent Kd estimates for capsazepine in assays on cultured DRG neurones and vagus nerve as shown by the overlap in the 95% confidence limits.6. Capsazepine, at concentrations up to 1O microM, had no significant effects on the efflux of 86Rb+ from cultured DRG neurones evoked either by depolarization with high (50 mM) K' solutions or by acidification of the external medium to pH 5.0-5.6. Similarly capsazepine had no significant effect on he depolarization (50 mM KCl)-induced efflux of [14C]-guanidinium from vagus nerve preparations.7. Ruthenium Red was also tested for antagonism against capsaicin evoked ['4C]-guanidinium release from vague nerves and capsaicin induced 45Ca2" uptake in cultures of DRG neurones. In contrast to capsazepine the inhibition by Ruthenium Red (10-500nM in DRG and 0.5-10microM in vagus nerve experiments) was not consistent with a competitive antagonism, but rather suggested a more complex,non-competitive inhibition.

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.

NE-19550 and NE-21610, antinociceptive capsaicin analogues: studies on nociceptive fibres of the neonatal rat tail in vitro.

When applied to peripheral fibres in a neonatal rat tail/spinal cord preparation in vitro, capsaicin (0.2-50 microM) induced an activation, selective desensitization and reduced responses to other noxious stimuli (heat, bradykinin). Similar concentrations of the antinociceptive analogues NE-19550 and NE-21610, did not affect peripheral fibre responsiveness but induced cross desensitization to capsaicin. At 500 microM both analogues produced similar effects to capsaicin. Capsaicin analogues may induce analgesia without initial activation of nociceptors.

Effective sterilization of a plastic film rack isolator with 'Alcide'.

A 'use challenge' test of the sterilizing ability of 'Alcide' was devised against a 'cocktail' of 8 selected bacteria and 2 fungi. The organisms were used to coat the inside of an isolator prior to sterilization with 'Alcide' solution. After exposure to the solution for 24 h, no organisms could be recovered from the surfaces of the isolator. No operator discomfort was observed during preparation of the 'Alcide' solution.