In vivo pharmacological characterization of TD-4208, a novel lung-selective inhaled muscarinic antagonist with sustained bronchoprotective effect in experimental animal models.

Tiotropium is currently the only once-daily, long-acting muscarinic antagonist (LAMA) approved in the United States and other countries for the treatment of chronic obstructive pulmonary disease (COPD). Glycopyrronium has shown promise as a LAMA and was recently approved for once-daily maintenance treatment of COPD in the European Union. Here, we describe the in vivo preclinical efficacy and lung selectivity of a novel inhaled muscarinic antagonist, TD-4208 (biphenyl-2-ylcarbamic acid 1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-yl ester) and compare its profile to tiotropium and glycopyrronium. In anesthetized dogs, TD-4208, along with tiotropium and glycopyrronium, produced sustained inhibition of acetylcholine-induced bronchoconstriction for up to 24 hours. In anesthetized rats, inhaled TD-4208 exhibited dose-dependent 24-hour bronchoprotection against methacholine-induced bronchoconstriction. The estimated 24-hour potency (expressed as concentration of dosing solution) was 45.0 µg/ml. The bronchoprotective potencies of TD-4208 and tiotropium were maintained after 7 days of once-daily dosing, whereas glycopyrronium showed a 6-fold loss in potency after repeat dosing. To assess systemic functional activity using a clinically relevant readout, the antisialagogue effect of compounds was also evaluated. The calculated lung selectivity index (i.e., ratio of antisialagogue and bronchoprotective potency) of TD-4208 was superior to glycopyrronium after both single and repeat dosing regimens and was superior to tiotropium after repeat dosing. In conclusion, the in vivo preclinical profile suggests that TD-4208 has the potential to be a long-acting bronchodilator for once-daily treatment of respiratory diseases. Its greater functional selectivity for the lung in preclinical models may translate to an improved tolerability profile compared with marketed muscarinic receptor antagonists.

Telavancin pharmacokinetics and pharmacodynamics in patients with complicated skin and skin structure infections and various degrees of renal function.

This study characterized the pharmacokinetic/pharmacodynamic profiles of the Food and Drug Administration (FDA)-approved telavancin renal dose adjustment schemes. A previously published two-compartment open model with first-order elimination and a combined additive and proportional residual error model derived from 749 adult subjects in 11 clinical trials was used to simulate the individual concentration-time profiles for 10,260 subjects (NONMEM). The dosing regimens simulated were 10 mg/kg of body weight once daily for individuals with creatinine clearances (CL(CR)s) of >50 ml/min, 7.5 mg/kg once daily for individuals with CL(CR)s of 30 to 50 ml/min, and 10 mg/kg every 2 days for those with CL(CR)s of <30 ml/min. The area under the concentration-time curve (AUC) under one dosing interval (AUC(τ)) was computed as dose/CL. The probability of achieving an AUC(τ)/MIC ratio of ≥ 219 was evaluated separately for each renal dosing scheme. Evaluation of the dosing regimens demonstrated similar AUC values across the different renal function groups. For all renal dosing strata, >90% of the simulated subjects achieved an AUC(τ)/MIC ratio of ≥ 219 for MIC values as high as 2 mg/liter. For patients with CL(CR)s of <30 ml/min, the probability of target attainment (PTA) exceeded 90% for both the AUC0₋24 (AUC from 0 to 24 h) and AUC24₋48 intervals for MICs of ≤ 1 mg/liter. At a MIC of 2 mg/liter, the PTAs were 89.3% and 23.6% for the AUC0₋24 and AUC24₋48 intervals, respectively. The comparable PTA profiles for the three dosing regimens across their respective dosing intervals indicate that the dose adjustments employed in phase III trials for complicated skin and skin structure infections were appropriate.

Thermodynamics of ligand (substrate/end product) binding to endoxylanase from Chainia sp. (NCL-82-5-1): isothermal calorimetry and fluorescence titration studies.

The binding of xylo-oligosaccharides to Chainia endoxylanase resulted in a decrease in fluorescence intensity of the enzyme with the formation of 1:1 complex. Equilibrium and thermodynamic parameters of ligand binding were determined by fluorescence titrations and titration calorimetry. The affinity of xylanase for the oligosaccharides increases in the order X2

Role of kidney dopamine in the natriuretic response to volume expansion in rats.

It has been postulated that endogenously produced dopamine (DA) may play a role in the regulation of renal sodium excretion. In the present study, experiments were designed to test the hypothesis that acute volume expansion with isotonic sodium chloride stimulates the production of DA within the kidney, which in turn acts on specific DA1 receptors to promote sodium excretion. In pentobarbital-anesthetized rats, acute volume expansion over a period of 1 hour evoked a pronounced increase in urine output and urinary sodium excretion. These diuretic and natriuretic effects were not accompanied by any significant changes in blood pressure or heart rate. However, there was a significant elevation in central venous pressure and a transient rise in glomerular filtration rate. The natriuretic and diuretic response was accompanied by a significant increase in urinary DA excretion, and this effect was clearly dissociated from the rise in glomerular filtration rate. In a separate group of rats, the effects of acute volume expansion were studied in the presence of selective DA1 receptor antagonist SCH-23390 (50 micrograms/kg i.v. bolus; 10 micrograms/kg/min). During DA1 receptor blockade, there was a marked attenuation in the diuretic and natriuretic response throughout the period of volume expansion, when compared with that in the control group. The changes in central venous pressure and glomerular filtration rate were identical in the two groups. In another group of rats, the renal effects of exogenously administered DA were studied. DA (0.5 micrograms/kg/min) produced significant increases in urine output and urinary sodium excretion, without causing any alterations in blood pressure or glomerular filtration rate, suggesting a tubular site of action.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopamine-beta-hydroxylase inhibition: a novel sympatho-modulatory approach for the treatment of congestive heart failure.

Pre-clinical and clinical studies suggest that chronic sympathetic activation in congestive heart failure (CHF) is a maladaptive response which accelerates the progressive worsening of the disease. Consequently, therapeutic interventions which inhibit sympathetic nerve function are likely to favorably alter the natural course of the disease. Indeed, recent clinical studies have shown that treatment with carvedilol, a beta-blocker, reduces mortality and the risk of death and hospitalization. The therapeutic value of beta-blockers, however, may be limited by their propensity to cause acute hemodynamic deterioration which results from abrupt withdrawal of sympathetic support. Thus, although the introduction of beta-blockers represents an important advance in the treatment of CHF, a better tolerated means of modulating the sympathetic nervous system would be highly desirable. An alternative strategy for directly modulating sympathetic nerve function is to inhibit the biosynthesis of norepinephrine (NE) via inhibition of dopamine-beta-hydroxylase (DBH), the enzyme which catalyzes the conversion of dopamine (DA) to NE in sympathetic nerves. This approach may have the following three merits over beta-blockade. First, this class of drugs would be expected to produce gradual modulation, as opposed to abrupt blockade, of sympathetic nerve function and, consequently, would not be associated with acute hemodynamic worsening thereby obviating the need for dose-titration. Second, from a theoretical standpoint, DBH inhibitors, at low doses, would preferentially inhibit NE release in the heart since the storage pool of NE in this organ is selectively depleted in CHF. Lastly, inhibition of DBH would augment the levels of DA which, via agonism of dopamine receptors, could have beneficial effects on renal function. Nepicastat is a novel, selective and potent (IC50 = 9 nM) inhibitor of DBH. Preclinical studies have shown that nepicastal produces gradual modulation of catecholamine levels (reduction in NE and elevation of DA and DA/NE ratio) in cardiovascular tissues and plasma, attenuates sympathetically-mediated cardiovascular responses and also has salutary effects on renal function. In a canine heart failure model, normalization of transmyocardial norepinephrine balance with nepicastat retards the process of ventricular dilation and prevents progressive worsening of cardiac function. Early short-term clinical studies in CHF patients have shown that nepicastat is well tolerated and produces significant and dose-dependent increases in plasma DA/NE concentrations.

Cardiovascular pharmacology of adrenergic and dopaminergic receptors: therapeutic significance in congestive heart failure.

This review discusses the localization of adrenergic- and dopaminergic-adrenoceptors within the cardiovascular system and describes the cardiovascular and renal changes produced following the activation of these receptors by appropriate agonists. Whereas the role of alpha- and beta-adrenergic agents in the treatment of heart failure is well recognized, recent studies with dopamine (DA)-receptor agonists indicate that they offer a novel approach in the therapy of congestive heart failure. DA-adrenoceptor agonists reduce afterload by causing vasodilation and promote sodium excretion via direct activation of DA1-adrenoceptors located on renal tubules. Fenoldopam is a selective DA1-adrenoceptor agonist found to be effective in heart failure. It reduces afterload by causing peripheral vasodilation and produces natriuresis and diuresis. Dopexamine is a DA1- and beta 2-adrenoceptor agonist, and its efficacy in heart failure is due to its ability to provide mild inotropic support and cause a reduction in afterload. Ibopamine is a prodrug that is converted into its active metabolite, epinine. This compound activates primarily DA1- and DA2-adrenoceptors. It is effective in heart failure, and the mechanism progresses via DA1- and DA2-adrenoceptor-mediated reduction in afterload. Agonists of DA2-adrenoceptors reduce afterload by decreasing the release of norepinephrine and by reducing the levels of renin-angiotensin-aldosterone system. Since both of these systems are active in heart failure, ibopamine offers a rational approach for therapy. The present review addresses the concept of pharmacologic intervention in adrenergic and dopaminergic influence in the cardiovascular and renal systems to produce changes that are desirable for the pharmacotherapy of congestive heart failure.

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.

Functional characterization of rat submaxillary gland muscarinic receptors using microphysiometry.

1. Muscarinic cholinoceptors (MChR) in freshly dispersed rat salivary gland (RSG) cells were characterized using microphysiometry to measure changes in acidification rates. Several non-selective and selective muscarinic antagonists were used to elucidate the nature of the subtypes mediating the response to carbachol. 2. The effects of carbachol (pEC(50) = 5.74 +/- 0.02 s.e.mean; n = 53) were highly reproducible and most antagonists acted in a surmountable, reversible fashion. The following antagonist rank order, with apparent affinity constants in parentheses, was noted: 4-DAMP (8.9)= atropine (8.9) > tolterodine (8.5) > oxybutynin (7.9) > S-secoverine (7.2) > pirenzepine (6.9) > himbacine (6.8) > AQ-RA 741 (6.6) > methoctramine (5.9). 3. These studies validate the use of primary isolated RSG cells in microphysiometry for pharmacological analysis. These data are consistent with, and extend, previous studies using alternative functional methods, which reported a lack of differential receptor pharmacology between bladder and salivary gland tissue. 4. The antagonist affinity profile significantly correlated with the profile at human recombinant muscarinic M(3) and M(5) receptors. Given a lack of antagonists that discriminate between M(3) and M(5), definitive conclusion of which subtype(s) is present within RSG cells cannot be determined.

Comparative pharmacology of recombinant human M3 and M5 muscarinic receptors expressed in CHO-K1 cells.

1. Affinity estimates were obtained for several muscarinic antagonists against carbachol-stimulated [3H]-inositol phosphates accumulation in Chinese hamster ovary (CHO-KI) cells stably expressing either human muscarinic M3 or M5 receptor subtypes. The rationale for these studies was to generate a functional antagonist affinity profile for the M5 receptor subtype and compare this with that of the M3 receptor, in order to identify compounds which discriminate between these two subtypes. 2. The rank order of antagonist apparent affinities (pK(B)) at the muscarinic M5 receptor was atropine (8.7) > or =tolterodine (8.6) = 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 8.6)> darifenacin (7.7) > or =zamifenacin (7.6)>oxybutynin (6.6)= para-fluorohexahydrosiladifenidol (p-F-HHSiD, 6.6)>pirenzepine (6.4) > or = methoctramine (6.3)=himbacine (6.3)>AQ-RA 741 (6.1). 3. Antagonist apparent affinities for both receptor subtypes compare well with published binding affinity estimates. No antagonist displayed greater selectivity for the muscarinic M5 subtype over the M3 subtype, but himbacine, AQ-RA 741, p-F-HHSiD, darifenacin and oxybutynin displayed between 9- and 60 fold greater selectivity for the muscarinic M3 over the M5 subtype. 4. This study highlights the similarity in pharmacological profiles of M3 and M5 receptor subtypes and identifies five antagonists that may represent useful tools for discriminating between these two subtypes. Collectively, these data show that in the absence of a high affinity M5 selective antagonist, affinity data for a large range of antagonists is critical to define operationally the M5 receptor subtype.

RS 23597-190: a potent and selective 5-HT4 receptor antagonist.

1. The pharmacological properties of RS 23597-190 (3-(piperdine-1-yl)-propyl-4-amino-5-chloro-2-methoxy benzoate hydrochloride) have been studied in vitro and in vivo. 2. RS 23597-190 competitively antagonized 5-HT4 receptor-mediated relaxations of rat, carbachol precontracted oesophageal muscularis mucosae, (pA2 = 7.8 +/- 0.1; Schild slope = 1.2 +/- 0.2). Affinity estimates (-log KB) at 5-HT4 receptors using either renzapride or SC-53116 as agonists yielded a -log KB value of 8.0 +/- 0.01. In contrast, RS 23597-190 failed to antagonize contractile responses to 5-HT of guinea-pig ileal 5-HT3 receptors, even at concentrations up to 10 microM. 3. Increases in short-circuit current, induced by 5-HT, were studied in guinea-pig ileal mucosal sheets. Concentration-response curves to 5-HT were biphasic, with the high potency phase to 5-HT inhibited by RS 23597-190 and mimicked by 5-methoxytryptamine. The -log KB value for RS 23597-190 at the high potency phase was 7.3 confirming that 5-HT4 receptors mediated the high potency phase. 4. In rat isolated vagus nerve, 5-HT elicited a slow, maintained depolarization at low concentrations and a rapid, transient depolarization at higher concentrations. The high potency, slow depolarizing phase to 5-HT was abolished selectively in the presence of 1 microM RS 23597-190 and the low potency phase was abolished selectively in the presence of 1 microM ondansetron. These data confirm that 5-HT4 and 5-HT3 receptors mediated slow and fast depolarization responses, respectively. 5. At 5-HT3 binding sites in membranes from NG 108-15 cells, labelled by [3H]-quipazine, RS 23597-190 exhibited an apparent affinity (- log Ki) of 5.7 +/- 0.1. At 5-HT3 receptors in membranes from rat cerebral cortex, labelled by [3H]-RS 42358-197, the apparent affinity (- log Ki) of RS 23597-190 was also 5.7 +/- 0.1. In both studies, Hill coefficients were not significantly different from unity. At 5-HT1A, 5-HT2,muscarinic M1, M2, M3, M4 and dopamine D1 and D2 receptors, RS 23597-190 exhibited low apparent affinities, with all - log Ki values less than 5.5.6. Intravenous infusion of RS 23597-190 in the conscious, restrained rat antagonized the von Bezold Jarisch reflex induced by 2-methyl 5-HT, with an ID50 of 300 microg kg-1 min-1, i.v. In the anaesthetized,bilaterally vagotomized micropig, RS 23597-190 (6 mg kg-1, i.v.) antagonized 5-HT-induced tachycardia with a half-life of 77 (63-99) min. Transient arrhythmic effects were noted after administration of the compound.7. In conclusion, RS 23597-190 acts as a high affinity, selective competitive antagonist at 5-HT4 receptors. Thus, the compound appears to be a useful tool for 5-HT4 receptor identification in vitro. In vivo, the compound is rapidly metabolized in pigs such that 5-HT4 blockade is not maintained. However,in the rat, when given by infusion, RS 23597-190 antagonizes 5-HT3 mediated responses, at doses consistent with a low affinity 5-HT3 receptor. These data suggest that, under appropriate experimental conditions, RS 23597-190 may also be used in vivo to characterize further 5-HT4 receptor function.

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.

RS 39604: a potent, selective and orally active 5-HT4 receptor antagonist.

1. Selective antagonism of 5-HT4 receptors may provide therapeutic benefit in certain disorders of the myocardium, alimentary and lower urinary tract. We now report on RS 39604, a novel and selective 5-HT4 receptor antagonist and compare its pharmacological properties with those of SB 204070. 2. In guinea-pig striatal membranes, both RS 39604 and SB 204070 inhibited specific binding of [3H]-GR 113808 in a concentration-dependent manner yielding pKi estimates of 9.1 and 10.9, respectively. RS 39604 displayed a low affinity (pKi < 6.5) for 5-HT1A, 5-HT2C, 5-HT3, alpha 1c, D1, D2, M1, M2, AT1, B1 and opioid mu receptors and moderate affinity for sigma 1, (pKi = 6.8) and sigma 2 (pKi = 7.8) sites. 3. In the rat isolated oesophagus, precontracted with carbachol, RS 39604 (30-300 nM) behaved as a competitive antagonist towards 5-HT-induced relaxation (pA2 = 9.3; Schild slope = 1.0). We and others have shown previously that SB 204070 behaves as an unsurmountable antagonist in this preparation (pA2 approximately 10.5). In the guinea-pig isolated ileal mucosa, RS 39604 (30 nM) antagonized 5-MeOT-induced increase in short-circuit current (pA2 = 9.1). 4. In anaesthetized vagotomized micropigs, RS 39604, administered by the i.v. or intraduodenal (i.duod.) route, produced dose-dependent inhibition of 5-HT-induced tachycardia (ID50 = 4.7 micrograms kg-1, i.v. and 254.5 micrograms kg-1, i.duod). At maximal doses of 30 micrograms kg-1, i.v. and 6 mg kg-1, i.duod., the inhibitory effects of RS 39604 lasted for more than 6 h. In this preparation, SB 204070 was as potent as RS 39604by the i.v. route but was inactive by the intraduodenal route at doses up to 3 mg kg-1.5. In conscious mice, RS 39604, administered by the i.p. or p.o. route, produced dose-depend entinhibition of 5-hydroxytryptophan (5-HTP)-induced diarrhoea (ID50= 81.3 microg kg-1, i.p. and 1.1 mg kg-1,p.o.). In this assay, SB 204070 was inactive by the oral route at doses up to 30 mg kg-1.6. In anaesthetized guinea-pigs, RS 39604 antagonized the contractile effect of 5-HT in the proximal colon by producing parallel, dextral displacement of the dose-response curve to 5-HT. The mean dose ratios to 5-HT at 0.1 mg kg-1, i.v., 1 mg kg-1, i.v. and 10 mg kg-1, i.duod. were 4.6, 30.7 and 10.8,respectively. SB 204070 behaved as an unsurmountable antagonist in this assay.7. In a model of visceral pain in conscious rats, RS 39604 (0.01-1 mg kg-1, i.v.) did not affect colorectal distension-induced increases in arterial pressure whereas morphine (1 mg kg-1, i.v.) produced significant inhibition of the response, implying that 5-HT4 receptors are not involved in nociception in this model.8. The data suggest that RS 39604 is a high affinity and selective 5-HT4 receptor antagonist that is orally active and long-lasting in vivo. It is concluded that RS 39604 may be the preferable probe to use for investigating the physiological and pathophysiological role of 5-HT4 receptors in vivo.

Pharmacological characterization of RS 25259-197, a novel and selective 5-HT3 receptor antagonist, in vivo.

1. The pharmacological effects in vivo, of RS 25259-197, a selective 5-HT3 receptor antagonist, have been investigated. 2. In anaesthetized rats, RS 25259-197, administered by the intravenous, intraduodenal or transdermal route, dose-dependently inhibited the von Bezold-Jarisch reflex induced by 2-methyl 5-HT (ID50 = 0.04 micrograms kg-1, i.v., 3.2 micrograms kg-1, i.d. and 32.8 micrograms per chamber, respectively). In this regard, when administered intraduodenally, RS 25259-197 was more potent and exhibited a longer duration of action than either ondansetron or granisetron. 3. In conscious ferrets, RS 25259-197, administered intravenously or orally, dose-dependently inhibited emesis induced by cisplatin. The ID50 estimates of RS 25259-197 were 1.1 micrograms kg-1, i.v. and 3.2 micrograms kg-1, p.o. In this respect, RS 25259-197 was more potent than ondansetron and equipotent with granisetron. 4. In conscious dogs, RS 25259-197, administered intravenously or orally, dose-dependently inhibited emesis induced by cisplatin (ID50 = 1.9 micrograms kg-1, i.v. and 8.5 micrograms kg-1, p.o.), dacarbazine (ID50 = 4.1 micrograms kg-1, i.v. and 9.7 micrograms kg-1, p.o.), actinomycin D (ID50 = 4.9 micrograms kg-1, i.v. and 2.5 micrograms kg-1, p.o.) and mechlorethamine (ID50 = 4.4 micrograms kg-1, i.v. and 3.0 micrograms kg-1, p.o.). Against each of the emetogenic agents, RS 25259-197 was very much more potent than ondansetron. When tested at equi-effective intravenous doses against cisplatin-induced emesis in dogs, RS 25259-197 had a longer duration of anti-emetic activity (7 h) than ondansetron (4 h). At doses up to and including 1000 microg kg-1, p.o., neither RS25259-197 nor ondansetron was capable of inhibiting apomorphine-induced emesis.5. At doses up to 1000 microg kg-1, i.v., RS 25259-197 produced no meaningful haemodynamic changes in anaesthetized dogs.6. In summary, RS 25259-197 is a novel, highly potent and orally active 5-HT3 receptor antagonist in vivo. With respect to its anti-emetic activity, RS 25259-197 appears to be a significant improvement over ondansetron in terms of potency and duration of action.

Catecholamine modulatory effects of nepicastat (RS-25560-197), a novel, potent and selective inhibitor of dopamine-beta-hydroxylase.

1. Inhibitory modulation of sympathetic nerve function may have a favourable impact on the progression of congestive heart failure. Nepicastat is a novel inhibitor of dopamine-beta-hydroxylase, the enzyme which catalyses the conversion of dopamine to noradrenaline in sympathetic nerves. The in vitro pharmacology and in vivo catecholamine modulatory effects of nepicastat were investigated in the present study. 2. Nepicastat produced concentration-dependent inhibition of bovine (IC50 = 8.5 +/- 0.8 nM) and human (IC50 = 9.0 +/- 0.8 nM) dopamine-beta-hydroxylase. The corresponding R-enantiomer (RS-25560-198) was approximately 2-3 fold less potent than nepicastat. Nepicastat had negligible affinity (> 10 microM) for twelve other enzymes and thirteen neurotransmitter receptors. 3. Administration of nepicastat to spontaneously hypertensive rats (SHRs) (three consecutive doses of either 3, 10, 30 or 100 mg kg-1, p.o.; 12 h apart) or beagle dogs (0.05, 0.5, 1.5 or 5 mg kg-1, p.o.; b.i.d., for 5 days) produced dose-dependent decreases in noradrenaline content, increases in dopamine content and increases in dopamine/noradrenaline ratio in the artery (mesenteric or renal), left ventricle and cerebral cortex. At the highest dose studied, the decreases in tissue noadrenaline were 47%, 35% and 42% (in SHRs) and 88%, 91% and 96% (in dogs) in the artery, left ventricle and cerebral cortex, respectively. When tested at 30 mg kg-1, p.o., in SHRs, nepicastat produced significantly greater changes in noradrenaline and dopamine content, as compared to the R-enantiomer (RS-25560-198), in the mesenteric artery and left ventricle. 4. Administration of nepicastat (2 mg kg-1, b.i.d, p.o.) to beagle dogs for 15 days produced significant decreases in plasma concentrations of noradrenaline and increases in plasma concentrations of dopamine and dopamine/noradrenaline ratio. The peak reduction (52%) in plasma concentration of noradrenaline and the peak increase (646%) in plasma concentration of dopamine were observed on day-6 and day-7 of dosing, respectively. 5. The findings of this study suggest that nepicastat is a potent, selective and orally active inhibitor of dopamine-beta-hydroxylase which produces gradual modulation of the sympathetic nervous system by inhibiting the biosynthesis of noradrenaline. This drug may, therefore, be of value in the treatment of cardiovascular disorders associated with over-activation of the sympathetic nervous system, such as congestive heart failure.

Renal dopamine and sodium excretion.

In the present study, we examined the renal effects of a low dose of fenoldopam in pentobarbital anesthetized rats in an attempt to unmask a direct tubular DA-1 receptor mediated diuresis and natriuresis. We also performed experiments to determine the possible contribution of renal dopamine (DA) and DA receptors in the natriuretic response to acute sodium loading. Fenoldopam (0.5 microgram/kg/min) produced significant increases in urine output (UV) and urinary sodium excretion (UNaV) without altering mean blood pressure (MBP), renal blood flow (RBF) or heart rate (HR), suggesting a direct tubular site of action. This renal response to fenoldopam was completely abolished in the presence of the selective DA-1 receptor antagonist, SCH 23390. Isotonic NaCl loading (5% body weight for 1 h) enhanced urinary DA excretion (UDA V) which correlated significantly with the increase in UNa V. Pretreatment with selective DA-1 receptor antagonist SCH 23390 resulted in significant attenuation of the increase in UNa V whereas the increase in UDA V was unaltered. Pretreatment with selective DA-2 receptor antagonist domperidone did not alter the increase in UNa V during isotonic NaCl loading. These results suggest that renal tubules are endowed with DA-1 receptors activation which results in diuresis and natriuresis. Furthermore, activation of these DA-1 receptors but not DA-2 receptors by endogenous DA contributes to the natriuretic response to isotonic saline loading.

Pulmonary mucormycosis: results of medical and surgical therapy.

Mucormycosis is an opportunistic fungal infection that commonly begins by invading the respiratory tract. The purpose of the present study was to define the clinical presentation of pulmonary mucormycosis and to evaluate current treatment regimens. Thirty patients treated at our institution and 225 cases reported in the literature were reviewed. For the combined groups, the mean age at presentation was 41 +/- 21 years and associated medical conditions included leukemia or lymphoma (37%), diabetes mellitus (32%), chronic renal failure (18%), history of organ transplantation (7.6%), or a known solid tumor (5.6%). The in-hospital mortality was 65% for patients with isolated pulmonary mucormycosis, 96% for those with disseminated disease, and 80% overall. The mortality in patients treated surgically was 11%, significantly lower than the 68% mortality in those treated medically (p = 0.0004). The most common causes of death were fungal sepsis (42%), respiratory insufficiency (27%), and hemoptysis (13%). Pulmonary mucormycosis has a high mortality; however, antifungal agents appear to improve survival. In addition, surgical resection may provide additional benefit to patients with pulmonary mucormycosis confined to one lung.

Pulsatile versus nonpulsatile reperfusion improves cerebral blood flow after cardiac arrest.

Cardiopulmonary bypass using nonpulsatile flow (NF) is currently advocated for treating refractory cardiac arrest. Although the heart can be revived using cardiopulmonary bypass support, the brain must recover if such therapy is to be considered successful. Previous studies have demonstrated that pulsatile flow (PF) reperfusion can improve neurologic outcome compared with NF reperfusion after cardiac arrest. The purpose of this study was to assess cerebral perfusion and oxygen consumption during either PF or NF reperfusion after cardiac arrest. Dogs (n = 22) underwent a 15-minute cardiac arrest followed by 1 hour of either PF or NF reperfusion. Microsphere techniques were used to assess cerebral perfusion and oxygen consumption at 3, 15, and 60 minutes of reperfusion. Mean arteriovenous gradients and total brain flows were similar in both groups. However, cerebral oxygen consumption was significantly improved at 3 minutes of reperfusion with PF versus NF (1.8 +/- 0.3 versus 0.9 +/- 0.3 mL O2.dL-1.min-1, respectively; p < 0.05). These results were coincident with improved gray-to-white flow ratios at 3 minutes of PF versus NF reperfusion (5.2 +/- 1.0 versus 2.0 +/- 0.3, respectively; p < 0.05). There were no statistically significant differences in brain perfusion variables by 15 minutes of reperfusion. However, a relative hyperemia was exhibited at 15 minutes of NF versus PF reperfusion, which suggests nutrient flow was insufficient during early NF versus PF reperfusion. In conclusion, PF reperfusion can better restore cerebral blood flow and oxygen consumption than can NF reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiovascular effects of R-alpha-methylhistamine, a selective histamine H3 receptor agonist, in rats: lack of involvement of histamine H3 receptors.

Selective histamine H3 receptor agonists, such as R-alpha-methylhistamine, have been shown to inhibit neurogenic sympathetic pressor responses in vivo. The objective of the present study was to test the hypothesis that R-alpha-methylhistamine would evoke an histamine H3 receptor mediated hypotensive response in an animal with intact sympathetic vascular tone. In pentobarbital anaesthetized rats, administration of R-alpha-methylhistamine (0.1-3 mg.kg-1, i.v.) had a biphasic effect on arterial pressure, consisting of a transient depressor response followed by a more long-lasting pressor response. The depressor response was antagonized by chlorpheniramine (selective histamine H1 receptor antagonist, 3 mg.kg-1, i.v.), but was unaffected by cimetidine (selective histamine H2 receptor antagonist, 3 mg.kg-1, i.v.) or thioperamide (selective histamine H3 receptor antagonist, 3 mg.kg-1, i.v.). The pressor response was unaltered by chlorpheniramine, cimetidine or thioperamide. In pithed rats, R-alpha-methylhistamine had a biphasic effect on arterial pressure which was qualitatively similar to that seen in anaesthetized rats with the exception that the pressor responses were much greater in magnitude and duration and were accompanied by significant increases in heart rate. On a pharmacological basis, the biphasic response in pithed rats was identical to that seen in anaesthetized rats inasmuch as the depressor response was antagonized by chlorpheniramine whereas the pressor response was resistant to histamine H1, H2 and H3 receptor antagonists. Combined alpha- and beta-adrenoceptor blockade (with phentolamine and nadolol) produced significant attenuation of the pressor and tachycardic responses to R-alpha-methylhistamine in pithed rats.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Evidence for purinergic neurotransmission in the urinary bladder of pithed rats.

The purpose of this study was to investigate the contribution of adenosine-5'-triphosphate (ATP) to segmental (L6-S2) spinal electrical stimulation evoked increases in intra-vesical pressure in pithed rats. Exogenous ATP and substance P produced dose-dependent increases in intra-vesical pressure (ED10 mmHg (dose required to elicit 10 mmHg increase in intra-vesical pressure)= 1.7 mg/kg and 1.1 microg/kg, i.v., respectively). Desensitisation (or antagonism) of P2x purinoceptors with alpha,beta-methylene ATP (alpha,beta-meATP; 30 microg/kg per min, i.v.) or pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 10 mg/kg, i.v.) significantly (p < 0.05) antagonized the intra-vesical pressure responses to ATP (> 8 and 3.6-fold increase in ED10 mmHg, respectively) but had no significant effect on intra-vesical pressure responses to substance P. Spinal stimulation evoked frequency-dependent increases in intra-vesical pressure (EF20 mmHg (frequency required to produce 20 mmHg increase in intra-vesical pressure) = 3.4 Hz). Blockade of muscarinic cholinoceptors and adrenoceptors with atropine (3 mg/kg, i.v.), propranolol (3 mg/kg, i.v.) and phentolamine (10 mg/kg, i.v.) produced marginal attenuation of the intra-vesical pressure responses to spinal stimulation indicating a major non-adrenergic non-cholinergic (NANC) component in the overall response. The NANC responses were significantly (p < 0.05) antagonized by alpha,beta-meATP (30 microg/kg per min, i.v.) and PPADS (10 mg/kg, i.v.) (> 2.6-fold increase in EF20 mmHg), consistent with involvement of a purinergic neurotransmitter, presumably ATP. Comparative studies in young (4-6 months) and old (21-23 months) Fischer rats revealed no age-dependent changes in the relative contribution of the cholinergic and purinergic systems, with the latter being the dominant one. These findings suggest that purinergic neurotransmission, presumably mediated by ATP acting via P2x purinoceptors, represents a major component of excitatory innervation to the urinary bladder in pithed rats.