Quantitative Evaluation of Reactivity and Toxicity of Acyl Glucuronides by [35S]Cysteine Trapping.

Acyl glucuronides (AGs) are reactive metabolites of carboxylic acid-containing drugs, which are associated with idiosyncratic toxicity (IDT) such as anaphylaxis, drug-induced liver injury, and so on. In this study, we developed a new in vitro approach for the quantitative assessment of the reactivity of AGs and their toxicity risk. Thirteen test drugs were incubated with human liver microsomes and uridine 5'-diphospho-glucuronic acid in the presence of cysteine (Cys) as a trapping agent. Both acylation and glycation Cys adducts formed from the AGs of the test drugs and were analyzed by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry. Acylation Cys adduct formation can closely reflect the reactivity of AGs to predict their IDT risk. Subsequently, we performed a quantitative trapping assay using radiometric analysis, with [35S]-labeled Cys ([35S]Cys) as the trapping agent, and the results showed that the test drugs associated with IDT resulted in a high product formation of [35S]Cys adducts. In conclusion, this approach can be used for the easy and quantitative evaluation of the reactivity of AGs without the need for authentic AG standards and to screen the potential IDT of new chemical entities during the early drug discovery phase.

Non-clinical pharmacokinetic profiles of rovatirelin, an orally available thyrotropin-releasing hormone analogue.

1. The non-clinical pharmacokinetic profiles of rovatirelin, a novel thyrotropin-releasing hormone (TRH) analogue, were investigated in vivo and in vitro. 2. Rovatirelin orally administered to rats and dogs was rapidly absorbed and bioavailability was estimated to be 7.3 and 41.3%, respectively. The extent of plasma protein binding of rovatirelin in rats, dogs, and humans was low in all species (∼15%). The permeability of rovatirelin from blood to brain (permeability-surface area) ranged from 1.04 ± 0.14 to 1.29 ± 0.28 µL/min/g in rats, and rovatirelin was stable in rat plasma and brain homogenates. 3. The metabolite pattern was qualitatively similar in vitro and in vivo. In animals, rovatirelin aminopentanoic acid (rovatirelin-acid), rovatirelin aminopentanone (rovatirelin-ketone), rovatirelin pyrrolidine (4S)-hydroxy (rovatirelin-OH), (thiazoylalanyl)methylpyrrolidine (TAMP), 3-(4-thiazoyl)-l-alanine (TA), and unknown metabolites were observed. In human hepatocytes, TAMP was mainly formed and no unique human metabolite was observed. 4. The radioactivity from administered [14C]rovatirelin was predominantly excreted in faeces in rats and dogs, and almost all radioactivity was recovered 168 h after administration. Absorption, brain penetration, and stability of rovatirelin in the brain were greater than for taltirelin. 5. Thus, orally administered rovatirelin is a potentially improved treatment for spinocerebellar degeneration compared with taltirelin.

Human mass balance, pharmacokinetics and metabolism of rovatirelin and identification of its metabolic enzymes in vitro.

The mass balance, pharmacokinetics and metabolism of rovatirelin were characterised in healthy male subjects after a single oral dose of [14C]rovatirelin. [14C]Rovatirelin was steadily absorbed, and the peak concentrations of radioactivity and rovatirelin were observed in plasma at 5-6 h after administration. The AUCinf of radioactivity was 4.9-fold greater than that of rovatirelin. Rovatirelin and its metabolite (thiazoylalanyl)methylpyrrolidine (TAMP) circulated in plasma as the major components. The total radioactivity recovered in urine and faeces was 89.0% of the administered dose. The principal route of elimination was excretion into faeces (50.1% of the dose), and urinary excretion was the secondary route (36.8%). Rovatirelin was extensively metabolised to 20 metabolites, and TAMP was identified as the major metabolite in plasma and excreta among its metabolites. To identify the metabolic enzymes responsible for TAMP formation, the in vitro activity was determined in human liver microsomes. The enzymatic activity depended on NADPH, and it was inhibited by ketoconazole. Furthermore, recombinant human cytochrome P450 (CYP) 3A4 and CYP3A5 displayed enzymatic activity in the assay. Therefore, CYP3A4/5 are the most important enzymes responsible for TAMP formation.

A comparison between the combined effect of calcium carbonate with sucroferric oxyhydroxide and other phosphate binders: an in vitro and in vivo experimental study.

BACKGROUND: Approximately 30% of patients on dialysis received combination therapy for their phosphate binder prescription; however, few studies for combined effects of phosphate binders are reported. For the purpose of evaluating the efficacy of combination therapy, we compared the efficacy of sucroferric oxyhydroxide (PA21) combined with calcium carbonate with that of lanthanum carbonate hydrate, sevelamer hydrochloride, and ferric citrate hydrate combined with calcium carbonate. METHODS: For in vitro studies, calcium carbonate and the other phosphate binders alone or in combination were stirred in phosphate solution at pH 2-8 for 2 h. After centrifuging the suspension, the phosphorus level in the supernatant was determined. For in vivo studies, rats were orally administered calcium carbonate and the other phosphate binders (except for sevelamer hydrochloride) alone or in combination, followed by oral administration of phosphate solution adjusted to pH 2 or 7. Serum samples were collected from the rats at predetermined timepoints and the serum phosphorus levels were determined and analyzed using a two-way analysis of variance. RESULTS: In the in vitro study, the measured phosphate-binding capacity of combining sevelamer hydrochloride, PA21, and lanthanum carbonate hydrate with calcium carbonate was approximately equal to or greater than the theoretical values under most conditions. Furthermore, these combined effects were insensitive to pH in that order. The measured phosphate-binding capacity of ferric citrate hydrate combined with calcium carbonate was smaller than the theoretical values, and the combination did not exhibit efficacy under any of the tested conditions. In the in vivo study, the combined effect of PA21 and calcium carbonate at both pH values and that of lanthanum carbonate hydrate and calcium carbonate at pH 2 were additive. In contrast, the combined effect of lanthanum carbonate hydrate and calcium carbonate at pH 7 and that of ferric citrate hydrate and calcium carbonate at pH 2 were antagonistic. CONCLUSIONS: These results suggest that coadministration of PA21 and calcium carbonate showed good and relatively stable efficacy throughout the range of the gastrointestinal pH and that combining lanthanum carbonate hydrate and ferric citrate hydrate with calcium carbonate may not produce the expected efficacy under certain conditions.

Synthesis and optimization of novel α-phenylglycinamides as selective TRPM8 antagonists.

Transient receptor potential melastatin 8 (TRPM8) is activated by innocuous cold and chemical substances, and antagonists of this channel have been considered to be effective for pain and urinary diseases. N-(3-aminopropyl)-2-{[(3-methylphenyl)methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride (AMTB), a TRPM8 antagonist, was proposed to be effective for overactive bladder and painful bladder syndrome; however, there is a potential risk of low blood pressure. We report herein the synthesis and structure-activity relationships of novel phenylglycine derivatives that led to the identification of KPR-2579 (20l), a TRPM8 selective antagonist. KPR-2579 reduced the number of icilin-induced wet-dog shakes and rhythmic bladder contraction in rats, with no negative cardiovascular effects at the effective dose.

Effects of combination of mitiglinide with various oral antidiabetic drugs in streptozotocin-nicotinamide-induced type 2 diabetic rats and Zucker fatty rats.

We examined the effects of combining the rapid insulin secretagogue, mitiglinide, with various oral hypoglycaemic drugs including biguanides, pioglitazone, α-glucosidase inhibitors, and sodium-glucose co-transporter 2 inhibitors in a rat model of type 2 diabetes. The oral glucose tolerance test (OGTT) using glucose, sucrose, or a liquid meal was used to compare the effects of mitiglinide with those of the four oral hypoglycaemic drugs and examine their combined effects on blood glucose levels and insulin secretion in the rat model. The combination of mitiglinide with other oral hypoglycaemic drugs suppressed the plasma glucose levels more than either agent did alone. Furthermore, the combination of these agents decreased insulin secretion more than mitiglinide did alone. These results indicate that mitiglinide is suitable for use in combination with other hypoglycaemic drugs because it inhibits postprandial hyperglycaemia by rapidly stimulating insulin secretion.

Therapeutic effects of silodosin and urapidil on underactive bladder associated with diabetic cystopathy.

OBJECTIVES: Pharmacological treatment options for underactive bladder (UAB) syndrome are limited. Urapidil is the only alpha1 -adrenoceptor (AR) antagonist that can be used for urinary disorders in women in some countries. However, no studies have directly verified the effects of alpha1 -AR antagonists on the female urethra and UAB-like dysfunctions. We investigated the effects of silodosin (alpha1A -AR antagonist) and urapidil (nonselective alpha1 -AR antagonist) on the voiding function in female rats with diabetes mellitus (DM). METHODS: Changes in intraurethral pressure (IUP) induced by midodrine (alpha1 -AR agonist) and mean blood pressure (MBP) were continuously measured in normal female rats to verify the pharmacological profiles of the drugs. To establish a DM model, rats were administered streptozotocin (STZ; 50 mg/kg, intravenous). Eight weeks after STZ administration, drugs were subcutaneously delivered through an osmotic pump. Four weeks after drug administration, emptied bladder blood flow (BBF), intravesical pressure, and the micturition volume were measured. RESULTS: Both silodosin and urapidil inhibited the midodrine-induced increase in IUP and decreased MBP in a dose-dependent manner. Silodosin had a more substantial effect on the lower urinary tract than on MBP. Twelve weeks after STZ administration, DM rats exhibited UAB-like dysfunction (increased bladder capacity/bladder weight and residual volume and decreased bladder voided efficiency) and decreased BBF. Both drug treatments controlled this dysfunction. CONCLUSIONS: Alpha1 -AR antagonists induced dose-dependent urethral relaxation in female rats. These drugs ameliorated UAB-like dysfunction in STZ-induced DM rats. In addition, alpha1A -AR antagonists such as silodosin, which have limited effects on blood pressure, appear to be useful for treating UAB.

KTO-7924, a Beta3-adrenergic receptor agonist, reduces hyperglycemia, and protects beta-cells in the islets of langerhans of db/db mice.

INTRODUCTION: The effect of beta3-adrenergic receptor agonists on beta cells in the islets of Langerhans is not yet clear. This study examined the beta3-adrenergic receptor agonist on beta cells in the islets of Langerhans. METHODS: Obese diabetic C57BL/KsJ-db/db mice were treated with KTO-7924, a newly-developed beta3-adrenergic receptor agonist for 28-day. We analyzed plasma parameters, insulin resistance, and insulin-positive areas among beta-cells in the islets of Langerhans. RESULTS AND CONCLUSION: After a 28-day oral administration period, plasma levels of hemoglobin (Hb) A1c, glucose, triglyceride (TG), and free fatty acid (FFA) were all significantly reduced in KTO-7924 treatment groups compared with controls. Plasma adiponectin levels decreased with age in the control group, but were significantly higher in a treatment group throughout the study period. Furthermore, sequential administration of KTO-7924 led to an improvement in insulin resistance in the OGTT (Oral glucose tolerance test (OGTT)), and an increase in the percentage of insulin-positive areas among beta-cells in the islets of Langerhans compared with controls. This is the first study to show islet histology after treatment of a beta3-adrenergic receptor agonist, and reveals that KTO-7924 reduces hyperglycemia, and protects beta-cells in the islets of Langerhans of db/db mice.

Pharmacokinetic Drug Interactions of an Orally Available TRH Analog (Rovatirelin) With a CYP3A4/5 and P-Glycoprotein Inhibitor (Itraconazole).

The effects of itraconazole on the pharmacokinetics of rovatirelin were investigated in an open-label, single-sequence drug-drug interaction study in 16 healthy subjects. Subjects were administered a single oral dose of rovatirelin (1.6 mg) on day 1 and day 15. From day 8 through 16, subjects received daily oral doses of itraconazole (200 mg/day). Concentrations of rovatirelin and (thiazolylalanyl)methylpyrrolidine (TAMP), the major metabolite of rovatirelin formed by cytochrome P450 (CYP) 3A4/5, were determined in plasma and urine. Pharmacokinetic parameters were used to evaluate the drug-drug interaction potential of rovatirelin as a victim. With coadministration, maximum concentration (Cmax ) and area under the concentration-time curve extrapolated to infinity (AUCinf ) of rovatirelin increased 3.05-fold and 2.82-fold, respectively, and the 90% confidence intervals of the ratios for Cmax (2.64-3.52) and AUCinf (2.47-3.23) did not fall within the 0.8-1.25 boundaries. Urinary excretion of rovatirelin increased at almost the same ratio as the AUCinf ratio with coadministration; however, renal clearance did not change. Cmax , AUCinf , and urinary excretion of TAMP were decreased by coadministration. Itraconazole has the potential to inhibit drug transport via intestinal P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP); therefore, substrate assessments of rovatirelin for the 2 transporters were evaluated using Caco-2 cell monolayers. In vitro studies showed that rovatirelin is a substrate for P-gp but not for BCRP. The current study shows that itraconazole's effect on rovatirelin pharmacokinetics is mediated through inhibition of CYP3A4/5 and intestinal P-gp.

PA21, a novel phosphate binder, improves renal osteodystrophy in rats with chronic renal failure.

The effects of PA21, a novel iron-based and non-calcium-based phosphate binder, on hyperphosphatemia and its accompanying bone abnormality in chronic kidney disease-mineral and bone disorder (CKD-MBD) were evaluated. Rats with adenine-induced chronic renal failure (CRF) were prepared by feeding them an adenine-containing diet for four weeks. They were also freely fed a diet that contained PA21 (0.5, 1.5, and 5%), sevelamer hydrochloride (0.6 and 2%) or lanthanum carbonate hydrate (0.6 and 2%) for four weeks. Blood biochemical parameters were measured and bone histomorphometry was performed for femurs, which were isolated after drug treatment. Serum phosphorus and parathyroid hormone (PTH) levels were higher in the CRF rats. Administration of phosphate binders for four weeks decreased serum phosphorus and PTH levels in a dose-dependent manner and there were significant decreases in the AUC0-28 day of these parameters in 5% PA21, 2% sevelamer hydrochloride, and 2% lanthanum carbonate hydrate groups compared with that in the CRF control group. Moreover, osteoid volume improved significantly in 5% of the PA21 group, and fibrosis volume and cortical porosity were ameliorated in 5% PA21, 2% sevelamer hydrochloride, and 2% lanthanum carbonate hydrate groups. These results suggest that PA21 is effective against hyperphosphatemia, secondary hyperparathyroidism, and bone abnormalities in CKD-MBD as sevelamer hydrochloride and lanthanum carbonate hydrate are, and that PA21 is a new potential alternative to phosphate binders.

Longitudinal analysis of motor symptoms and histopathology in woozy mice, a model of cerebellar ataxia.

Woozy (wz) mice develop ataxia and carry a mutation in the Sil1 gene. Homozygous wz mice have been characterized histopathologically, but no details of their motor function have been reported. In the present study, to comprehensively understand the relationship between symptomatic progression and pathological feature, we evaluated motor function and neurodegeneration with age from presymptomatic to terminal stages. We evaluated the motor function of homozygous and heterozygous wz mice aged from 5 to 71 weeks. Motor function was evaluated using the rotarod test, the footprint test, and the parallel rod floor test. Furthermore, we carried out a histopathological analysis of the mice at several ages. Impairment of motor function in homozygous wz mice began at around 11 weeks of age and became markedly worse until around 14 weeks. Heterozygous wz mice did not show motor dysfunction until 71 weeks of age. Features of cerebellar ataxia were evaluated using the footprint test and the parallel rod floor test. In addition to the observation of ubiquitin-positive aggregates at 6 weeks of age, Purkinje cell loss at 9 weeks of age and cerebellar atrophy were confirmed by histopathology. Apart from the cerebellar changes, we detected no other pathology that could contribute toward ataxia. In heterozygous wz mice, only minimal formation of ubiquitin-positive aggregates was observed. Homozygous wz mice showed adult-onset ataxia with progressive neurodegeneration of the cerebellum. Homozygous wz mice might be useful as an animal model of diseases showing adult-onset ataxia because of cerebellar neurodegeneration.

Comparison of the Effects of Mitiglinide and Glibenclamide Administered in Combination with the Dipeptidyl Peptidase-IV Inhibitor Sitagliptin in Rats with Streptozotocin-Nicotinamide-Induced Type 2 Diabetes.

We compared the individual effects of mitiglinide and glibenclamide administered in combination with the dipeptidyl peptidase-IV (DPP-IV) inhibitor sitagliptin on plasma DPP-IV activity and blood glucose levels in rats with streptozotocin-nicotinamide-induced type 2 diabetes (STZ-NA rats). We examined the inhibitory activity of mitiglinide and glibenclamide as well as their combination with sitagliptin on plasma DPP-IV activity in STZ-NA rats. The oral glucose tolerance test (OGTT) was used to compare effects of mitiglinide, glibenclamide, and their combination with sitagliptin on blood glucose levels in STZ-NA rats. Mitiglinide and glibenclamide did not inhibit rat DPP-IV and did not influence the inhibitory effect of sitagliptin on rat plasma DPP-IV activity. In STZ-NA rats, plasma glucose levels were stronger suppressed by a combination of mitiglinide and sitagliptin than by either drug used alone. However, no clear effect of the combination of glibenclamide and sitagliptin was observed. These results indicate that the combination of mitiglinide and sitagliptin has a lower risk of hypoglycemia in the rats with induced type 2 diabetes compared with the combination of glibenclamide and sitagliptin. The combination of mitiglinide and sitagliptin can be a promising combination for the treatment of diabetic patients.

Mizagliflozin, a novel selective SGLT1 inhibitor, exhibits potential in the amelioration of chronic constipation.

Chronic constipation is a highly common functional gastrointestinal disorder that adversely affects patient quality of life. At present, limited therapeutic options are available for the treatment of chronic constipation, which indicates the need for new therapeutic agents. Herein, we report the potential of mizagliflozin, a novel selective sodium glucose co-transporter 1 (SGLT1) inhibitor, for the amelioration of chronic constipation. Mizagliflozin's inhibitory activity against SGLTs was evaluated by an in vitro assay of cells transiently expressing SGLTs. The safety profile of an initial single dose (2-160mg, orally) and multiple doses (2-20mg, orally, once daily immediately prior to breakfast on Days 1 and 13, and three times daily immediately prior to every meal on Days 3-12) of mizagliflozin was determined by performing a phase I study in healthy male subjects. In addition, the effect of mizagliflozin and lubiprostone on fecal wet weight was compared using a dog model of loperamide-induced constipation and rat model of low-fiber-diet-induced constipation. Mizagliflozin potently inhibited human SGLT1 in a highly selective manner. The results of the phase I study showed mizagliflozin increased stool frequency and loosened stool consistency; these effects increased progressively with an increase in the dosage and the number of doses of mizagliflozin. In addition, the oral administration of mizagliflozin increased fecal wet weight in a dog model of loperamide-induced constipation and a rat model of low-fiber-diet-induced constipation, similar to lubiprostone. These results suggest the potential use of a novel selective SGLT1 inhibitor, mizagliflozin, for the amelioration of chronic constipation.

Physiological difference between obese (fa/fa) Zucker rats and lean Zucker rats concerning adiponectin.

Obese (fa/fa) Zucker rat is a spontaneous genetic obesity model and, by comparison with lean Zucker rat, exhibits hyperphagia, hyperinsulinemia, and hyperlipidemia. The aim of this study was to examine the physiological difference concerning adiponectin between obese (fa/fa) Zucker rats and control lean Zucker rats. We therefore measured plasma adiponectin level and analyzed adiponectin and adiponectin receptor 1 mRNA expression in retroperitoneal white adipose tissue (RT WAT), brown adipose tissue (BAT), liver, and soleus muscle. We also examined the tissue mRNA expression of peroxisome proliferator-activated receptor alpha (PPAR alpha), PPAR delta, and PPAR gamma, which regulate adiponectin expression sensitivity to a PPAR gamma agonist shown by brown adipocytes from obese (fa/fa) Zucker rats and lean Zucker rats, by measuring adiponectin release from these cells. Plasma adiponectin levels of obese (fa/fa) Zucker rats were significantly higher than those of lean Zucker rats. Adiponectin mRNA expression levels in RT WAT were lower in obese (fa/fa) Zucker rats than in lean Zucker rats, but those in BAT were higher. Adiponectin receptor 1 expression levels in RT WAT, BAT, and liver of obese (fa/fa) Zucker rats were lower than in lean Zucker rats. The expression level of PPAR alpha, PPAR delta, and PPAR gamma in BAT was lower in obese (fa/fa) Zucker rats than in lean Zucker rats. Moreover, the PPAR gamma agonist increased adiponectin release only from the brown adipocytes isolated from lean Zucker rats. It is the conclusive difference between obese (fa/fa) Zucker rats and lean Zucker rats that plasma adiponectin levels of obese (fa/fa) Zucker rats are significantly higher than those of lean Zucker rats. Moreover, we clarified that mRNA expression level of adiponectin receptor 1 in RT WAT, BAT, and liver of obese (fa/fa) Zucker rats is low despite high plasma adiponectin level, and low expression of PPARs in BAT leads to less sensibility of adiponectin release from brown adipocytes to a PPAR gamma agonist in obese (fa/fa) Zucker rats.

Adiponectin receptor 2 expression in liver and insulin resistance in db/db mice given a beta3-adrenoceptor agonist.

Our aim was to determine the effect of a beta3-adrenoceptor agonist on plasma adiponectin levels and on the level of expression of mRNA for adiponectin, adiponectin receptor 1, and adiponectin receptor 2 in db/db mice. Two weeks' oral administration of CL-316,243 led to decreased plasma levels of hemoglobin A1c, glucose, insulin, triglyceride and free fatty acid, and to an increased plasma adiponectin levels. It also improved insulin resistance in the oral glucose tolerance test. Adiponectin mRNA expression was significantly higher in the CL-316,243-treatment group than in the control group in epididymal white adipose tissue but not in brown adipose tissue, soleus muscle or liver. Adiponectin receptor 2 mRNA expression was significantly lower only in the liver of the CL-316,243-treatment group (versus the control group). These results suggest that the increased plasma adiponectin levels seen in db/db mice treated with this beta3-adrenoceptor agonist induce a down-regulation of adiponectin receptor 2 mRNA expression specifically in the liver.

DNA microarray analysis of white adipose tissue from obese (fa/fa) Zucker rats treated with a beta3-adrenoceptor agonist, KTO-7924.

We aimed to examine the effects of KTO-7924 (beta3-adrenoceptor agonist) on lipid metabolism and mRNA expressions in retroperitoneal white adipose tissue (RP WAT) in obese (fa/fa) Zucker rats using DNA microarray. Oral KTO-7924 for 28 days significantly decreased RP WAT weight, plasma triglyceride, free fatty acid, and insulin, and improved insulin resistance in oral glucose tolerance tests. In RP WAT of KTO-7924-treated rats, DNA microarray analysis revealed specifically enhanced mRNA expressions of uncoupling protein 1 (UCP1) and cytochrome c oxidase subunit VIII-H (COX8H), which are reportedly highly expressed in brown adipose tissue (BAT). Since these mRNA expression levels in RP WAT were significantly lower in obese (fa/fa) Zucker rats than in lean Zucker rats, these genes may be important in lipid metabolism. Our results imply that in obese (fa/fa) Zucker rats, continuous stimulation of beta3-adrenoceptors by KTO-7924 causes BAT-like adipocytes to appear in RP WAT, and improves lipid metabolism.

Characterization of beta-adrenoceptor subtype in bladder smooth muscle in cynomolgus monkey.

We first investigated the relaxations of the urinary bladder induced by beta-adrenoceptor agonists in anesthetized cynomolgus monkeys and then employed a variety of beta-adrenoceptor agonists and antagonists in vitro to identify the beta-adrenoceptor subtype responsible for the relaxation (using isolated monkey detrusors). Isoprenaline reduced bladder pressure in a dose-dependent manner. Isoprenaline, noradrenaline and adrenaline each produced a concentration-dependent relaxation of isolated detrusor strips, the rank order of relaxing potencies being isoprenaline > noradrenaline > adrenaline. Subtype-selective beta-adrenoceptor agonists also relaxed isolated detrusor strips, the rank order of potencies being CGP-12177 > BRL 37344 > dobutamine, salbutamol, procaterol > xamoterol. In the antagonist experiment, bupranolol (beta-antagonist, 10(-6) to 10(-5) M) and SR 58894A (beta3-antagonist, 10(-7) to 10(-5) M) caused a rightward shift of the concentration-relaxation curve for isoprenaline, but CGP-20712A (beta1-antagonist, 10(-9) to 10(-7) M) and ICI-118551 (beta2-antagonist, 10(-9) to 10(-7) M) did not. The present functional study provides the first evidence that relaxation of the monkey detrusor by beta-adrenoceptor activation is mediated via the beta3-subtype.

Functional characterization of beta-adrenoceptor subtypes in the canine and rat lower urinary tract.

PURPOSE: We compared the effect of a beta 3-adrenoceptor (AR) agonist with that of beta 1 and beta 2-AR agonists on the urethra and bladder in the dog and rat. MATERIALS AND METHODS: In an in vitro experiment we studied the relaxant effect of subtype selective beta-AR agonists in canine and rat urethral and bladder smooth muscle using an organ bath method. In addition, in urethane anesthetized rats we measured urethral pressure and bladder pressure simultaneously in the presence of the beta 3-agonist CL316243 and the beta 2-agonist procaterol in 4 or 5 animals. RESULTS: In the dog the relaxing effects of isoprenaline in the distal urethra were about half those seen in the detrusor and trigone. The rank order of relaxing potency was CL316243 > dobutamine (beta 1-agonist) = procaterol in detrusor and trigone but procaterol > dobutamine = CL316243 in the prostatic and distal urethra. In rat urethral smooth muscle in vitro the corresponding order was procaterol > CL316243 > dobutamine and the maximal relaxation to each agonist was about half that seen in the bladder. In the anesthetized rat procaterol clearly decreased urethral pressure but CL316243 produced only a slight decrease at its maximal dose, although each agonists clearly reduced bladder pressure. The beta 2-antagonist ICI-118551 counteracted the decrease in urethral and bladder pressure induced by procaterol. CONCLUSIONS: In rats and dogs a selective beta 3-AR agonist can decrease bladder pressure without affecting urethral pressure.

Effects of beta(3)-adrenoceptor stimulation on prostaglandin E(2)-induced bladder hyperactivity and on the cardiovascular system in conscious rats.

AIMS: To investigate the effects of selective beta(2)- and selective beta(3)-adrenoceptor (AR) agonists on prostaglandin (PG) E(2)-induced bladder hyperactivity in conscious free-moving rats. METHODS: Female Sprague-Dawley rats were anesthetized for implantation of bladder, intravenous, and intra-arterial catheters. The effects of a beta(3)-AR agonist (CL316,243) on cystometric and cardiovascular parameters were assessed in conscious rats. Intravesical instillation of PGE(2) (20-60 microM, 6 mL/hr) in conscious rats produced a concentration-dependent increase in voiding frequency. RESULTS: In this model i.v. CL316,243 (beta(3)-AR agonist) reduced basal bladder pressure, increased micturition volume, and prolonged micturition interval in a dose-dependent manner, without affecting threshold pressure or micturition pressure. On the other hand, i.v. procaterol (beta(2)-AR agonist) did not counteract the bladder hyperactivity. Atropine (muscarinic antagonist) reduced micturition pressure and micturition volume, and shortened micturition interval. CL316,243 slightly decreased mean blood pressure and increased heart rate only when given at high doses (10 and 100 microg/kg, i.v.). In contrast, procaterol caused a significant decrease in mean blood pressure and a significant increase in heart rate. Atropine significantly increased heart rate. CONCLUSIONS: The present results clearly demonstrated that the beta(3)-AR agonist prolonged the micturition interval without producing significant cardiovascular side effects. The human detrusor, like the rat detrusor, relaxes on beta(3)-AR stimulation. Provided that these results are valid in humans, selective beta(3)-AR agonists might be clinically useful for controlling a certain type of bladder overactivity.

Effects of selective beta2 and beta3-adrenoceptor agonists on detrusor hyperreflexia in conscious cerebral infarcted rats.

PURPOSE: We evaluated the effects of beta-adrenoceptor agonists on detrusor hyperreflexia in cerebral infarcted rats. MATERIALS AND METHODS: To produce cerebral infarction in Sprague-Dawley rats the left middle cerebral artery was occluded by introducing a monofilament nylon thread into the artery. In sham operated rats the same artery was exposed but not occluded. After these operations cystometric and cardiovascular experiments were performed with no anesthesia or restraint. RESULTS: After the operation bladder capacity was significantly decreased and voiding pressure was significantly increased in cerebral infarcted but not in sham operated animals. The difference in cerebral infarcted and sham operated rats was significant for each parameter (p <0.01). Post-void residual urine volume was not affected in either group. In the cerebral infarction group intravenous administration of CL316243 ([R,R]-5-2-[[2-(3-chlorophenyl-2-hydroxyethyl]-amino]propyl] -1,3-benzodioxole-2,2-dicarboxylate) (Kissei Central Laboratories, Hotaka, Japan) a selective beta3-adrenoceptor agonist, significantly increased bladder capacity at 10 and 100 microgram./kg. without affecting voiding pressure or post-void residual urine volume. Procaterol, a selective beta2-adrenoceptor agonist, significantly increased bladder capacity and post-void residual urine volume at 10 microgram/kg. intravenously without affecting voiding pressure. In separate experiments procaterol (1 to 100 microgram./kg. intravenously) decreased mean blood pressure and increased heart rate in a dose dependent manner. In contrast, the effects of CL316243 (0.1 to 100 microgram./kg. intravenously) on mean blood pressure and heart rate were minimal. CONCLUSIONS: These results indicate that in cerebral infarcted rats detrusor hyperreflexia can be suppressed by the selective beta3-adrenoceptor agonist CL316243 without increasing post-void residual volume and without significant cardiovascular side effects. If the current results hold true in humans, selective beta3-adrenoceptor agonists may prove useful for treating detrusor hyperreflexia associated with cerebral infarction.