Therapeutic opportunities from muscarinic receptor research.

Muscarinic acetylcholine receptor subtypes have been the subjects of research for at least a quarter of a century. Nonetheless, there are few selective muscarinic receptor ligands presently used as therapeutics. The extensive development of muscarinic M(1) receptor agonists for the treatment of cognitive dysfunction has culminated in a series of unsuccessful drug candidates, which reflects a lack of understanding of the disease and the role played by muscarinic cholinergic transmission. Paradoxically, the most successful antagonist approved for use in urinary incontinence is the nonselective muscarinic receptor antagonist tolterodine. This deficit in subtype-selective ligands could be circumvented by the development of transgenic mice, each lacking functional M(1), M(2), M(3), M(4) or M(5) receptors. In this article, the current status of muscarinic receptor research is critically assessed.

Muscarinic receptor ligands and their therapeutic potential.

Over the past year, the introduction of novel ligands has accelerated the classification of muscarinic receptor subtypes and has led to a better understanding of their physiological role. Important in this respect is the recent recognition of the exquisite selectivity of a series of snake toxins, enabling better definition of the muscarinic subtype 4 receptor. Moreover, several compounds, both agonists and antagonists, are progressing in advanced clinical trials for the treatment of several conditions, including Alzheimer's disease, pain, urinary incontinence and chronic obstructive pulmonary disease.

Muscarinic receptors in isolated urinary bladder smooth muscle from different mouse strains.

1. The pharmacological characteristics of muscarinic receptors in male and female mouse urinary bladder smooth muscle from different strains (C57Bl/6, 129/SvJ and hybrid backcross N1F2) were studied. 2. (+)-Cis-dioxolane, oxotremorine-M, acetylcholine, carbachol and pilocarpine induced concentration-dependent contractions of the urinary bladder smooth muscle (range for pEC(50)=6.4-6.6, 6.2-6.7, 6.2-6.4, 5.4-6.0 and 0.0-5.1, T(max)=1.9-4.7 g, 1.3-3.4 g, 1.0-3.0 g, 1.4-2.4 and 0.0-0.3 g, respectively, n=4-6 depending on the gender and the strain). In females, these contractions were competitively antagonized by a range of muscarinic receptor antagonists (pK(B) value range, depending on the strain): atropine (8.0-8.9), pirenzepine (6.1-6.4), 4-DAMP (7.6-8.4), methoctramine (5.6-6.1), p-F-HHSiD (7.5-7.7), zamifenacin (7.7-8.4) and darifenacin (8.2-8.7). 3. In recontraction studies, in which the muscarinic M(3) receptor population was decreased, and conditions optimized to study M(2) receptor activation, methoctramine exhibited an affinity estimate consistent with muscarinic M(3) receptors (pK(B)=6.26+/-0.08, pA(2)=6.31+/-0.07; pK(B)=6.09+/-0.22, pA(2)=6.08+/-0.01 for female inbred strain 129/SvJ and hybrid backcross N1F2, respectively) or intermediate between the one expected for this compound at M(2) and M(3) receptors, (pK(B)=6.66+/-0.08, pA(2)=7.00+/-0.27 for female inbred strain C57BL/6). 4. These data study suggest that muscarinic M(3) receptors are the predominant, if not the exclusive, subtype mediating contractile responses to muscarinic agonists in female mouse urinary bladder smooth muscle, with strain differences.

Pharmacological characterization of muscarinic receptors in mouse isolated urinary bladder smooth muscle.

The pharmacological characteristics of muscarinic receptors in the male mice urinary bladder smooth muscle were studied. (+)-Cis-dioxolane, oxotremorine-M, acetylcholine, carbachol and pilocarpine induced concentration-dependent contractions of the urinary bladder smooth muscle (pEC(50)=6.6+/-0.1, 6.9+/-0.1, 6.7+/-0.1, 5.8+/-0.1 and 5.8+/-0.1, E(Max)=3.2+/-0.8 g, 2.7+/-0.4 g, 1.0+/-0.1 g, 2.7+/-0.3 and 0.9+/-0.2 g, respectively, n=4). These contractions were competitively antagonized by a range of muscarinic receptor antagonists (pK(B) values): atropine (9.22+/-0.09), pirenzepine (6.85+/-0.08), 4-DAMP (8.42+/-0.14), methoctramine (5.96+/-0.05), p-F-HHSiD (7.48+/-0.09), tolterodine (8.89+/-0.13), AQ-RA 741 (7.04+/-0.12), s-secoverine (8.21+/-0.09), zamifenacin (8.30+/-0.17) and darifenacin (8.70+/-0.09). In this tissue, the pK(B) values correlated most favourably with pK(i) values for these compounds at human recombinant muscarinic M(3) receptors. A significant correlation was also noted at human recombinant muscarinic m5 receptors given the poor discriminative ability of ligands between M(3) and m5 receptors. In recontraction studies, in which the muscarinic M(3) receptor population was decreased, and conditions optimized to study M(2) receptor activation, methoctramine exhibited an affinity estimate consistent with muscarinic M(3) receptors (pK(B)=6.23+/-0.14; pA(2)=6.16+/-0.03). Overall, these data study suggest that muscarinic M(3) receptors are the predominant, if not the exclusive, subtype mediating contractile responses to muscarinic agonists in male mouse urinary bladder smooth muscle.

Presence of vasoconstrictor 5HT1-like receptors revealed by precontraction of rabbit isolated mesenteric artery.

1. A series of 5-hydroxytryptamine (5-HT) receptor agonists including 5-HT, 5-carboxamidotryptamine (5-CT) and sumatriptan produced little or no contraction of rabbit isolated mesenteric arteries under resting tone conditions, even at concentrations up to 10(-4) M. 2. When the same agonists were retested in mesenteric artery preparations pre-contracted with the thromboxane-mimetic, U46619, each demonstrated concentration-related vasoconstrictor activity. 5-CT and 5-HT were especially potent and effective in this model giving EC50 values of 4.3 x 10(-9) M and 1.6 x 10(-8) M respectively and maximum effects equivalent to those of KCl 80 mM. In preparations precontracted by U46619 (conditions retained throughout the rest of the study) the order of agonist potency was 5-CT > 5-HT > RU 24969 = sumatriptan > 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT) > cisapride. 3. The vasoconstrictor effects of 5-CT were competitively antagonized by methiothepin (pA2 8.20) but resistant to antagonism by a range of other 5-HT receptor antagonists, i.e. pindolol (5-HT1A/5-HT1B), propranolol (5-HT1B), spiperone (5-HT2A), ondansetron (5-HT3), ICS 205930 (5-HT3/5-HT4) and SDZ 205557 (5-HT4). 5-CT responses were slightly antagonized by a high concentration of ritanserin (5-HT2A/5-HT2C). Responses to 5-HT and sumatriptan were also antagonized by methiothepin with similar affinity (pA2/pKB values congruent to 8.0). 4. Metergoline and rauwolscine (10(-7)-10(-6) M) antagonized the effects of 5-CT in a non-competitive fashion giving pKBapp values of 7.13 (metergoline) and 6.86 (rauwolscine). 5. Vasoconstrictor responses to 5-HT were not modified in the presence of ritanserin (3 x 10-7 M) orspiperone (3 x 10-7 M) and only modestly antagonized by ketanserin (10-6 M) suggesting that 5-HT2Areceptors do not make a significant contribution in this model.6. Hence, precontraction of rabbit mesenteric arteries reveals potent vasoconstrictor effects of 5-HT and related agonists. Based on the agonist potency order and the antagonist studies performed, the receptor subtype responsible has the characteristics of a 5-HT1-like (probably 5-HTlD) receptor. This study therefore demonstrates a particularly striking example of vasoconstrictor synergy involving 5-HT1-like receptors.

Pharmacological characterization of muscarinic receptors in dog isolated ciliary and urinary bladder smooth muscle.

1. The pharmacological characteristics of muscarinic receptors mediating contraction of dog isolated ciliary muscle were determined and compared to those mediating contraction of dog urinary bladder smooth muscle. 2. (+)-Cis-dioxolane induced concentration-dependent contractions of ciliary muscle (pEC50=7.18+/-0.07, Emax=453+/-64 mg, n=19) and urinary bladder isolated smooth muscle (pEC50=6.55+/-0.07, Emax=11+/-1 g, n=19). These responses were antagonized by several muscarinic receptor antagonists (pKb values for the ciliary muscle and the bladder smooth muscle, respectively): atropine (8.25+/-0.14 and 9.21+/-0.09), pirenzepine (6.31+/-0.13 and 6.70+/-0.25), tolterodine (7.97+/-0.14 and 8.68+/-0.12), oxybutynin (7.40+/-0.08 and 7.88+/-0.12), zamifenacin (6.46+/-0.19 and 7.69+/-0.11), S-secoverine (6.66+/-0.14 and 8.13+/-0.07), AQ-RA 741 (6.16+/-0.15 and 7.08+/-0.23), p-F-HHSiD (7.10+/-0.27 and 7.35+/-0.07) and responses were not antagonized by PD 102807 (up to 100 nM). 3. In urinary bladder smooth muscle, the profile of antagonist pKB values correlated significantly with pK(i) values at human recombinant m3 muscarinic receptors, suggesting that M3 muscarinic receptors mediated the response. In the ciliary muscle, a significant (P<0.01) correlation was obtained with human recombinant m3 and m5 receptors. 4. Darifenacin displayed insurmountable antagonism at receptors in the bladder. At receptors in the ciliary muscle, it exhibited two phases of antagonism, comprising an initial low affinity (pKB<6) component and a high affinity phase (pKB>8). 5. The role of pigmentation in the atypical behaviour of darifenacin was examined. In blue coloured eyes, darifenacin produced apparent surmountable, competitive antagonism of the responses to (+)-cis-dioxolane (pKB=8.76+/-0.07). The antagonist profile obtained in this tissue suggested the involvement of a site which has the pharmacological attributes of the M5 receptor. 6. We suggest that the dog urinary bladder contracts in response to M3 muscarinic receptor activation. Contraction of the brown-eyed dog ciliary muscle is more complex and may include involvement of at least two receptors, possibly the M5 and M3 receptor, whereas blue-eyed dog ciliary muscle may involve a single population of M5 muscarinic receptors.

Pharmacological characterization of muscarinic receptors in rabbit isolated iris sphincter muscle and urinary bladder smooth muscle.

1. The pharmacological characteristics of muscarinic receptors in the rabbit iris sphincter muscle were studied and compared to M3 receptors in rabbit urinary bladder smooth muscle. 2. (+/-)-Cis-dioxolane induced concentration-dependent contractions of the iris sphincter muscle (pEC50 = 6.41+/-0.10, Emax = 181+/-17 mg, n = 38) and urinary bladder smooth muscle (pEC50 = 6.97+/-0.04, Emax = 4.28+/-0.25 g, n = 54). These contractions were competitively antagonized by a range of muscarinic receptor antagonists (pK(B) values are given for the iris sphincter muscle and the bladder smooth muscle, respectively): atropine (9.30+/-0.07 and 9.40+/-0.04), AQ-RA 741 (6.35+/-0.04 and 6.88+/-0.03), darifenacin (9.56+/-0.05 and 9.12+/-0.05), methoctramine (5.75+/-0.07 and 5.81+/-0.06), oxybutynin (8.10+/-0.09 and 8.59+/-0.06), pirenzepine (6.79+/-0.05 and 6.89+/-0.04), secoverine (7.54+/-0.05 and 7.66+/-0.05), p-F-HHSiD (7.55+/-0.09 and 7.50+/-0.05) and zamifenacin (8.69+/-0.10 and 8.36+/-0.06). A significant correlation between the pK(B) values in the bladder and the pK(B) values in the iris was obtained. 3. In both tissues, the pK(B) values correlated most favorably with pKi values for these compounds at human recombinant muscarinic m3 receptors. A reasonable correlation was also noted at human recombinant muscarinic m5 receptors given the poor discriminative ability of ligands between m3 and m5 receptors. 4. Overall, the data from this study suggest that the muscarinic receptors mediating contraction of the rabbit iris sphincter muscle and urinary bladder smooth muscle are similar and equate most closely with the pharmacologically-defined muscarinic M3 receptor.

Characterization of the muscarinic receptor in isolated uterus of sham operated and ovariectomized rats.

1. The pharmacological characteristics of muscarinic receptors in rat isolated uterus were studied in ovariectomized (ov.) and sham operated (sh.) animals. 2. Competition radioligand binding studies, using uterine membranes and [3H]-NMS, were undertaken with several muscarinic receptor antagonists. Most of the antagonists indicated a one-site fit with apparent affinity estimates (pKi) unchanged by ovariectomy. The selective M2 antagonist, tripitramine revealed high (representing 33+/-8 and 38+/-2%) and low (67+/-8 and 62+/-2%) affinity binding sites in both sh. and ov. rat uterus, respectively. These sites likely represented muscarinic M2 and M3 receptors and the proportions were not significantly different in the two conditions. 3. Carbachol induced concentration-dependent contractions which were surmountably antagonized by several muscarinic receptor antagonists (pKB, sh.; ov.): zamifenacin (9.19; 9.18), p-F-HHSiD (8. 50; 9.06), tripitramine (7.23; 7.54), himbacine (7.21; 7.41), methoctramine (6.79; 7.49), pirenzepine (6.48; 7.21), AF DX 116 (6. 26; 6.61), MTx 3 (<7.00; <7.00) and PD 102807 (<7.00; <7.00). 4. The apparent affinity values obtained in functional studies using the uteri from both sh. and ov. animals correlated most closely with values reported at human recombinant muscarinic M3 receptors. This suggests that the muscarinic M3 receptor mediates contraction under both conditions. 5. Radioligand binding experiments indicate the presence of M2 receptors, in addition to M3 receptors, which probably explains the discrepancies between functional and binding affinities. These data further suggest that the pharmacological profile and proportions of the two populations of muscarinic receptors are unaffected by ovariectomy.

Characterization of an atypical muscarinic cholinoceptor mediating contraction of the guinea-pig isolated uterus.

In many smooth muscle tissues a minor M3-muscarinic acetylcholine (mACh) receptor population mediates contraction, despite the presence of a larger M2-mACh receptor population. However, this is not the case for guinea-pig uterus where radioligand binding and functional studies exclude a dominant role for M3-mACh receptors. Using tissue from animals pre-treated with diethylstilboestrol, estimates of antagonist affinity were made before and after selective alkylation procedures, together with estimates of agonist affinity to characterise the mACh receptor population mediating carbachol-induced contraction of guinea-pig isolated uterus. Antagonist affinity estimates made at 'protected' receptors were not significantly different from those made in untreated tissues. However all estimations were significantly different from those reported in guinea-pig ileum and atria. The rank order of affinities were atropine>zamifenacin=tripitramine> methoctramine. Carbachol-induced contractions were insensitive to the M4-selective muscarinic toxin MTx-3, or PD102807 (0.1 microM) ruling out a role for M4-mACh receptors. The agonist affinity value for L-660,863, a putative 'M2-selective' agonist of 5.44+/-0.30 (n=6) was significantly different from that reported in guinea-pig atria. In contrast, the pKA value for carbachol (4.22+/-0.17 n = 8) agrees with that reported for guinea-pig ileum. Carbachol-induced contractions were insensitive to pertussis toxin although carbachol-induced inhibition of forskolin-stimulated cyclic AMP production was attenuated, ruling out the involvement of Gi-proteins in contraction. Radioligand binding studies revealed a KD for N-[3H]-methylscopolamine of 0.12+/-0.05 nM and a Bmax of 147+/-18 fmol mg protein(-1). Antagonist affinity estimates made using competition binding studies supported previous data suggesting the presence of a homogenous population of M2-mACh receptors. These data suggest a small population of mACh receptors with an atypical operational profile which can not be distinguished using radioligand binding studies may mediate carbachol-induced contraction of guinea-pig isolated uterus.

Functional role of M2 and M3 muscarinic receptors in the urinary bladder of rats in vitro and in vivo.

1. Urinary bladder smooth muscle is enriched with muscarinic receptors, the majority of which are of the M2 subtype whereas the remaining minority belong to the M3 subtype. The objective of the present study was to assess the functional role of M2 and M3 receptors in the urinary bladder of rat in vitro and in vivo by use of key discriminatory antagonists. 2. In the isolated bladder of rat, (+)-cis-dioxolane produced concentration-dependent contractions (pEC50 = 6.3) which were unaffected by tetrodotoxin (0.1 microM). These contractions were antagonized by muscarinic antagonists with the following rank order of affinity (pA2) estimates: atropine (9.1) > 4-diphenyl acetoxy-methyl piperidine methiodide (4-DAMP) (8.9) > darifenacin (8.5) > para fluoro hexahydrosiladifenidol (p-F-HHSiD) (7.4) > pirenzepine (6.8) > methoctramine (5.9). These pA2 estimates correlated most favourably (r = 0.99, P < 0.001) with the binding affinity (pKi) estimates of these compounds at human recombinant muscarinic m3 receptors expressed in Chinese hamster ovary cells, suggesting that the receptor mediating the direct contractile responses to (+)-cis-dioxolane equates with the pharmacologically defined M3 receptor. 3. As M2 receptors in smooth muscle are negatively coupled to adenylyl cyclase, we sought to determine whether a functional role of M2 receptors could be unmasked under conditions of elevated adenylyl cyclase activity (i.e., isoprenaline-induced relaxation of KCl pre-contracted tissues). Muscarinic M3 receptors were preferentially alkylated by exposing tissues to 4-DAMP mustard (40 nM, 1 h) in the presence of methoctramine (0.3 microM) to protect M2 receptors. Under these conditions, (+)-cis-dioxolane produced concentration-dependent reversal (re-contraction) of isoprenaline-induced relaxation (pEC50 = 5.8) but had marginal effects on pinacidil-induced, adenosine 3':5'-cyclic monophosphate (cyclic AMP)-independent, relaxation. The re-contractions were antagonized by methoctramine and darifenacin, yielding pA2 estimates of 6.8 and 7.6, respectively. These values are intermediate between those expected for these compounds at M2 and M3 receptors and were consistent with the involvement of both of these subtypes. 4. In urethane-anaesthetized rats, the cholinergic component (approximately 55%) of volume-induced bladder contractions was inhibited by muscarinic antagonists with the following rank order of potency (ID35%inh, nmol kg-1, i.v.): 4-DAMP (8.1) > atropine (20.7) > methoctramine (119.9) > darifenacin (283.3) > pirenzepine (369.1) > p-F-HHSiD (1053.8). These potency estimates correlated most favourably (r = 0.89, P = 0.04) with the pKi estimates of these compounds at human recombinant muscarinic m2 receptors. This is consistent with a major contribution of M2 receptors in the generation of volume-induced bladder contractions, although the modest potency of darifenacin does not exclude a role of M3 receptors. Pretreatment with propranolol (1 mg kg-1, i.v.) increased the ID35%inh of methoctramine significantly from 95.9 to 404.5 nmol kg-1 but had no significant effects on the inhibitory responses to darifenacin. These data suggest an obligatory role of beta-adrenoceptors in M2 receptor-mediated bladder contractions in vivo. 5. The findings of the present study suggest that both M2 and M3 receptors can cause contraction of the rat bladder in vitro and may also mediate reflex bladder contractions in vivo. It is proposed that muscarinic M3 receptor activation primarily causes direct contraction of the detrusor whereas M2 receptor activation can contract the bladder indirectly by reversing sympathetically (i.e. beta-adrenoceptor)-mediated relaxation. This dual mechanism may allow the parasympathetic nervous system, which is activated during voiding, to cause more efficient and complete emptying of the bladder.

Influence of vascular tone on vasoconstrictor responses to the 5-HT 1-like receptor agonist sumatriptan in anaesthetised rabbits.

The cardiovascular profile of the 5-HT1-like receptor agonist sumatriptan has been studied in anaesthetised rabbits pretreated with chlorisondamine (0.5 mg kg-1 i.v.) and enalapril (0.3 mg kg-1 i.v.) to eliminate autonomic reflexes and minimise endogenous vasoconstrictor tone. Under these conditions sumatriptan (2-100 micrograms kg-1 i.v.) produced modest increases in carotid vascular resistance but had no significant influence on heart rate, blood pressure or mesenteric vascular resistance. In a similarly pretreated group of animals in which vasoconstrictor tone was elevated by infusion of angiotensin (100 ng kg-1 min-1 i.v.) sumatriptan caused moderate increases in blood pressure (55 +/- 5 to 65 +/- 5 mm Hg after 25 micrograms kg-1 i.v.) and mesenteric vascular resistance (1.4 +/- 0.2 to 1.6 +/- 0.2 mm Hg min ml-1 after 25 micrograms kg-1 i.v.) and tended to produce a greater carotid vasoconstriction (3.6 +/- 0.5 to 4.7 +/- 0.7 mm Hg min ml-1 after 25 micrograms kg-1). These effects were antagonised by methiothepin 0.3 mg kg-1 i.v. implying the involvement of 5-HT1-like receptor stimulation. Hence, the presence of angiotensin produces a modest amplification of the vasoconstrictor effects of sumatriptan and, in particular, unmasks a constriction of the mesenteric vascular bed. The degree of synergy observed between these two vasoconstrictors was, however, less marked than might have been expected on the basis of previous isolated tissue studies.

Pre-contraction with histamine and U46619 unmasks a 5-HT1-like receptor in rabbit renal artery.

Rabbit renal arteries were virtually unresponsive to 5-hydroxytryptamine (5-HT) and 5-carboxamidotryptamine (5-CT) under control conditions (including the presence of ketanserin 10(-6) M). However, both agents produced prominent contractions over the range 10(-9)-10(-5) M in tissues pre-contracted with threshold concentrations of either histamine or U46619. These responses were considered to be mediated by activation of 5-HT1-like receptors based on the potency order 5-CT > or = 5-HT and the sensitivity of agonist responses to antagonism by methiothepin (10(-7) M). Pre-contraction with different types of vasoconstrictor can therefore unmask functional 5-HT1-like receptors in rabbit renal artery.

Evaluation of oral ro70-0004/003, an alpha1A-adrenoceptor antagonist, in the treatment of male erectile dysfunction.

Alpha-adrenoceptor antagonists have been used for the treatment of male erectile dysfunction (MED). Ro70-0004/003 (Ro70-0004) is a selective and orally active alpha1A-adrenoceptor antagonist. The objective of this study was to: (1) pharmacologically elucidate the alpha1-adrenoceptor subtype mediating norepinephrine-induced contraction of human isolated corpus cavernosal tissue and (2) conduct a clinical proof-of-concept study with Ro70-0004 to test the hypothesis that selective alpha1A-adrenoceptor blockade would improve erectile function in patients with MED. In vitro organ bath studies were conducted with strips of human isolated corpus cavernosal tissue obtained from patients undergoing penile prosthesis implantation. Prazosin, cyclazosin, RS-100329 and Ro70-0004/003 antagonized norepinephrine-induced contractile responses with affinity estimates (pK(B) or pA2) of 8.4, 7.3, 9.2 and 8.8, respectively, consistent with the singular involvement of alpha1A-adrenoceptor subtype. A clinical study (single center, observer-blind, randomized, placebo-controlled, extended period Latin-Square crossover design) was conducted in 24 male patients (mean age 44 y) with MED of no established organic cause to evaluate the efficacy of a 5-mg oral dose of Ro70-0004. The primary efficacy endpoint was the duration of rigidity > 60% at the base of the penis measured between 0.5 and 2.5 h post-dose. Rigidity was assessed by penile plethysmography using the RigiScan Plus device during visual sexual stimulation. The safety and efficacy of Ro70-0004 was also assessed. A 50-mg dose of sildenafil was included as a positive control. For the primary efficacy endpoint, the mean duration of erection was 9.69 min following administration of placebo, 8.28 min following Ro70-0004, and 22.64 min following sildenafil. Only the difference between sildenafil and placebo reached statistical significance (P < 0.05). A similar pattern was observed when measuring a duration of rigidity > 80% at the base of the penis (secondary endpoint). Ro70-0004 was safe and generally well tolerated (only two out of 20 patients reported at least one adverse event). The highly selective alpha1A-adrenoceptor antagonist, Ro70-0004, given at a single dose of 5 mg, did not improve erectile function when compared to placebo.

Peripheral benzodiazepine ligands inhibit aggregation and thromboxane synthesis induced by arachidonic acid in rabbit platelets in vitro.

Seven compounds having varying affinities for peripheral benzodiazepine (p sites) were evaluated in vitro as inhibitors of arachidonic acid-induced rabbit platelet aggregation and thromboxane B2 (TXB2) synthesis. With the exception of flumazenil, all compounds inhibited both parameters with IC50 values ranging from 0.19 to 3.5 microM, with a significant correlation between the inhibition of aggregation and the synthesis of thromboxane B2. The antiaggregatory effect was stereoselective, and inhibition was increased when cellular penetration was favoured by increasing the volume of the organic solvent. The order of potency these compounds is consistent with an effect at intracellular p sites (Ro5-4864 > PK14067 > PK 11195 >> PK 14068 > or = clonazepam > or = diazepam >>> flumazenil). However, the correlation between the aggregation inhibition and the synthesis of TXB2 suggests that microsomal cyclooxygenase may be the intracellular target of these ligands. We therefore propose that this enzyme possesses a site for benzodiazepine ligands which may share certain characteristics with the peripheral benzodiazepine receptor.

Effect of tecarfarin, a novel vitamin K epoxide reductase inhibitor, on coagulation in beagle dogs.

BACKGROUND AND PURPOSE: Tecarfarin (ATI-5923) is a novel vitamin K epoxide reductase inhibitor that is metabolized by esterase (mainly human carboxylesterase 2) to a single major metabolite, ATI-5900, in rats, dogs and humans. Tecarfarin is not significantly metabolized by CYP450 enzymes. The objective of this study was to test and compare the efficacy of tecarfarin with that of warfarin, when administered either intravenously or once a day orally, to produce stable anticoagulation in beagle dogs. EXPERIMENTAL APPROACH: Effects on coagulation were assessed by measuring the activity levels of Factor VII and Factor X and thromboplastin-induced coagulation times, reported as prothrombin time (PT). KEY RESULTS: Continuous intravenous infusions and oral administration of tecarfarin and warfarin caused a dose-dependent decrease in activity of Factor VII and Factor X, and associated increase in PT. Intravenous fresh frozen canine plasma or subcutaneous vitamin K(1) treatment reversed the anticoagulant effects of orally administered tecarfarin. Consistent with the inhibitory effects of amiodarone on CYP2C9, co-administration of amiodarone significantly increased the anticoagulation effect of warfarin and plasma warfarin concentrations. In contrast, amiodarone had no effect on the anticoagulation induced by tecarfarin or tecarfarin plasma concentrations in this model. CONCLUSIONS AND IMPLICATIONS: Overall, the data presented herein indicate that tecarfarin, via a vitamin K-dependent mechanism, causes changes in key parameters of haemostasis in beagle dogs that are consistent with effective anticoagulation. Compared to warfarin it has a decreased potential to interact metabolically with drugs that inhibit CYP450 enzymes and, therefore, may offer an improved safety profile for patients.

5-Hydroxytryptamine1D-like receptors mediate contraction of partially depolarized rabbit renal arteries.

This study characterizes the 5-hydroxytryptamine (5-HT) receptors involved in the contraction of rabbit renal arteries which had been previously precontracted submaximally by partial depolarization. In the presence of ketanserin (10(-6) M) to block 5-HT2 receptors, 5-HT, 5-carboxamido-tryptamine (5-CT) and other 5-HT receptor agonists caused contraction of partially depolarized (22 mM KCl) rabbit renal arteries whereas they were without effect in quiescent tissues (4.7 mM KCl). 5-CT was the most potent agent tested, producing concentration-related increases in tension over the range of 10(-9) to 10(-6) M, attaining 44 +/- 2% of the tissue maximum with a EC50 of 4.8 +/- 0.9 x 10(-8) M. The relative order of agonist potency in partially depolarized tissues was 5-CT > 5-HT > 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H indole > or = sumatriptan > 8-hydroxy-2-(di-n-propyl-amino)tetralin > cisapride. Responses to 5-CT and sumatriptan were antagonized in a concentration-dependent fashion by methiothepin (3 x 10(-8)-3 x 10(-7) M). In the case of 5-CT, Schild analysis showed that this antagonism was competitive, giving a pA2 value of 7.72. Rauwolsine (3 x 10(-7)-10(-6) M) and metergoline (10(-6) M) also produced modest antagonism of 5-CT and sumatriptan-induced responses. With sumatriptan as agonist, rauwolscine gave a pKB of 6.43, however its antagonism of 5-CT appeared noncompetitive because it was associated with a reduction in maximum response (pKB' = 6.54).(ABSTRACT TRUNCATED AT 250 WORDS)