Potentiation of Muscarinic M3 Receptor Activation through a New Allosteric Site with a Novel Positive Allosteric Modulator ASP8302.

Muscarinic M3 (M3) receptors mediate a wide range of acetylcholine (ACh)-induced functions, including visceral smooth-muscle contraction and glandular secretion. Positive allosteric modulators (PAMs) can avoid various side effects of muscarinic agonists with their spatiotemporal receptor activation control and potentially better subtype selectivity. However, the mechanism of allosteric modulation of M3 receptors is not fully understood, presumably because of the lack of a potent and selective PAM. In this study, we investigated the pharmacological profile of ASP8302, a novel PAM of M3 receptors, and explored the principal site of amino-acid sequences in the human M3 receptor required for the potentiation of receptor activation. In cells expressing human M3 and M5 receptors, ASP8302 shifted the concentration-response curve (CRC) for carbachol to the lower concentrations with no significant effects on other subtypes. In a binding study with M3 receptor-expressing membrane, ASP8302 also shifted the CRC for ACh without affecting the binding of orthosteric agonists. Similar shifts in the CRC of contractions by multiple stimulants were also confirmed in isolated human bladder strips. Mutagenesis analysis indicated no interaction between ASP8302 and previously reported allosteric sites; however, it identified threonine 230 as the amino acid essential for the PAM effect of ASP8302. These results demonstrate that ASP8302 enhances the activation of human M3 receptors by interacting with a single amino acid distinct from the reported allosteric sites. Our findings suggest not only a novel allosteric site of M3 receptors but also the potential application of ASP8302 to diseases caused by insufficient M3 receptor activation. SIGNIFICANCE STATEMENT: The significance of this study is that the novel M3 receptor positive allosteric modulator ASP8302 enhances the activation of human M3 receptor by interacting with a residue distinct from the reported allosteric sites. The finding of Thr230 as a novel amino acid involved in the allosteric modulation of M3 receptors provides significant insight into further research of the mechanism of allosteric modulation of M3 and other muscarinic receptors.

Tamsulosin potently and selectively antagonizes human recombinant α(1A/1D)-adrenoceptors: slow dissociation from the α(1A)-adrenoceptor may account for selectivity for α(1A)-adrenoceptor over α(1B)-adrenoceptor subtype.

We determined the binding affinity of tamsulosin, a selective α(1)-adrenoceptor antagonist, for human α(1)-adrenoceptor subtypes in comparison with those of other α(1)-adrenoceptor antagonists including silodosin, prazosin, 5-methylurapidil, terazosin, alfuzosin, nafopidil, urapidil and BMY7378. The association and dissociation kinetics of [(3)H]tamsulosin for recombinant human α(1)-adrenoceptor subtypes were compared with those of [(3)H]prazosin. Tamsulosin competitively inhibited [(3)H]prazosin binding to human α(1A)-, α(1B)- and α(1D)-adrenoceptors (pK(i) values were 10.38, 9.33, 9.85) indicating 11 and 3.4-fold higher affinities for human α(1A)-adrenoceptor than those for α(1B)- and α(1D)-adrenoceptors, respectively. The affinity of tamsulosin for the human α(1A)-adrenoceptor was, respectively, 5, 9.9, 38, 120, 280, 400, 1200 and 10000 fold higher than those of silodosin, prazosin, 5-methylurapidil, terazosin, alfuzosin, naftopidil, urapidil and BMY7378, respectively. [(3)H]Tamsulosin dissociated from the α(1A)-adrenoceptor slower than from the α(1B)- and α(1D)-adrenoceptors (α(1B)>α(1D)>α(1A)). Moreover, [(3)H]tamsulosin dissociated slower than [(3)H]prazosin from the α(1A)-adrenoceptor and faster from the α(1B)- and α(1D)-adrenoceptors. In conclusion, tamsulosin potently and selectively antagonized α(1A/1D)-adrenoceptor ligand binding, and slowly dissociated from the α(1A)-adrenoceptor subtype.

Muscarinic M3 positive allosteric modulator ASP8302 enhances bladder contraction and improves voiding dysfunction in rats.

OBJECTIVES: Muscarinic M3 (M3 ) receptors mediate cholinergic smooth muscle contraction of the bladder. Current drugs targeting bladder M3 receptors for micturition disorders have a risk of cholinergic side effects due to excessive receptor activation and insufficient selectivity. We investigated the effect of ASP8302, a novel positive allosteric modulator (PAM) of M3 receptors, on bladder function in rats. METHODS: Modulation of carbachol-induced increases in intracellular Ca2+ was assessed in cells expressing rat muscarinic receptors. Potentiation of bladder contractions was evaluated using isolated rat bladder strips and by measuring intravesical pressure in anesthetized rats. Conscious cystometry was performed to investigate the effects on residual urine volume and voiding efficiency in rat voiding dysfunction models induced by the α1 -adrenoceptor agonist midodrine and muscarinic receptor antagonist atropine, and bladder outlet obstruction. To assess potential side effects, the number of stools and tracheal insufflation pressure were measured in conscious and anesthetized rats, respectively. RESULTS: ASP8302 demonstrated PAM effects on the rat M3 receptor in cell assays, and augmented cholinergic bladder contractions both in vivo and in vitro. ASP8302 improved voiding efficiency and reduced residual urine volume in two voiding dysfunction models as effectively as distigmine bromide, but unlike distigmine bromide did not affect the number of stools or tracheal insufflation pressure. CONCLUSIONS: Our results in rats indicate that ASP8302 improves voiding dysfunction by potentiating bladder contraction with fewer effects on cholinergic responses in other organs, and suggest a potential advantage over current cholinomimetic drugs for treating micturition disorders caused by insufficient bladder contraction.

In vitro and in vivo effects of endothelin-1 and YM598, a selective endothelin ET A receptor antagonist, on the lower urinary tract.

We investigated the contractile response of the lower urinary tract to endothelin-1 in vitro (rabbits) and in vivo (dogs). We also assessed the effects of a selective endothelin ET A receptor antagonist, (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2, 2'-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598), on endothelin-1-induced contractile responses. In the in vitro study, endothelin-1 induced contractile responses in isolated rabbit bladder base, urethra, and prostate tissues. YM598 (10(-7)-10(-5) M) antagonized these endothelin-1-induced contractile responses without affecting the maximal responses. In the in vivo study, endothelin-1 induced the elevation of non-prostatic urethral pressure as well as prostatic urethral pressure even in the presence of tamsulosin (10 microg/kg, i.v.) in anesthetized male dogs. YM598 (0.1-3 mg/kg, i.v.) inhibited these endothelin-1-induced contractile responses in a dose-dependent fashion. These results suggest that endothelin ET A receptors play an important role in the lower urinary tract contraction, and that the selective endothelin ET A receptor antagonist YM598 has ameliorating effects on various urinary dysfunctions, including benign prostatic hyperplasia.

Intratesticular Bradykinin Involvement in Rat Testicular Pain Models.

OBJECTIVES: To clarify the role of bradykinin in urogenital pain, we investigated bradykinin involvement in rat models of testicular pain. METHODS: Bradykinin (0.1, 0.3, 1, 3 and 10 mmol/L) or distilled water was injected into the testes of male Wistar rats, and induced pain behaviors in conscious rats were evaluated. The effect of pretreatment with bradykinin B2 receptor antagonist FK3657 on bradykinin-induced pain behavior was then examined. We also evaluated the analgesic effect of FK3657 in a rat acetic acid-induced testicular pain as well as changes in the intratesticular bradykinin concentration after testicular injection of acetic acid. RESULTS: An injection of bradykinin into the testes of conscious rats induced pain behaviors that were dose-proportionally reduced by prior administration of FK3657. In addition, FK3657 dose-dependently inhibited the pain responses induced by testicular injection of 1% acetic acid. An increase in intratesticular bradykinin concentration was detected after the testicular injection of 1% acetic acid. CONCLUSIONS: Here, we found that intratesticular bradykinin evokes pain behavior via stimulation of bradykinin B2 receptors and that intratesticular acetic acid injection induces intratesticular bradykinin synthesis, consequently leading to pain behavior. These findings suggest that the potential utility of bradykinin B2 receptor antagonists as a novel target for treating urogenital pain.

Urodynamics and bladder muscarinic receptors in rats with cerebral infarction and bladder outlet obstruction.

We characterized muscarinic receptor binding and urodynamic parameters in rats with cerebral infarction and chronic bladder outlet obstruction as models of detrusor overactivity. Bladder weight showed little significant difference between the cerebral-infarcted and sham rats, but the bladder weight was about three times greater in the bladder outlet-obstructed rats. Bladder capacity and voided volume were significantly lower (36.7 and 55.1%, respectively) in the cerebral-infarcted than in the sham rats. Involuntary contractions before micturition were seen in the bladder outlet-obstructed rats but not in sham rats. The bladder outlet-obstructed rats showed significant increases (2.65 and 2.57 times, respectively) in bladder capacity and voided volume, compared with those in sham rats. Bmax values for specific [N-methyl-3H]scopolamine ([3H]NMS) binding in the bladder were significantly (34%) increased in the cerebral-infarcted rats compared with sham rats, whereas Kd was unaffected by infarction. On the other hand, there was little significant change in Kd and Bmax for specific [3H]NMS binding in the bladder-obstructed rats compared with sham rats. In conclusion, the present study shows that cerebral infarction but not bladder outlet obstruction in rats causes up-regulation of bladder muscarinic receptors, and that such regulation of bladder muscarinic receptors may be at least partly associated with the symptoms of detrusor overactivity subsequent to cerebral infarction.

Physiological Roles of Bradykinin and Involvement of Bradykinin B2 Receptor in Urethral Function in Humans and Animals.

OBJECTIVE: We investigated the role of bradykinin in urethral function by examining contractile responses in urethral smooth muscle strips isolated from humans and the intraurethral pressure in rats and dogs. METHODS: The contractile responses of human urethral tissue for bradykinin (0.01-10 µmol/L) were examined, and changes in intraurethral pressure induced by bradykinin (0.003-10 µg/kg) in anesthetized rats or dogs were measured. In addition, the effects of pretreatment with the bradykinin B2 receptor antagonist FK3657 were also examined. RESULTS: In smooth muscle strips obtained from human urethra, bradykinin induced contraction, which was inhibited by FK3657 in a concentration-dependent manner. In anesthetized rats and dogs, intravenously administered bradykinin dose-dependently increased intraurethral pressure. FK3657 shifted the intraurethral pressure dose-response curve for bradykinin to the right in rats. The bradykinin-induced elevation of intraurethral pressure was also dose-dependently inhibited by FK3657 in dogs. CONCLUSIONS: The present study provides evidence that bradykinin elicits urethral smooth muscle contraction via the bradykinin B2 receptor, suggesting the potential utility of this receptor as a novel target for the treatment of voiding dysfunction.

Effect of YM598, a selective endothelin ETA receptor antagonist, on endothelin-1-induced bone formation.

We investigated the effect of endothelin-1 on bone formation in vitro and in vivo, and the effect of YM598, a novel selective endothelin ET(A) receptor antagonist, on endothelin-1-induced responses. In in vitro studies, the effect of endothelin-1 on cellular responses was investigated by measuring intracellular Ca(2+) levels, cell growth and alkaline phosphatase activity in the mouse osteoblast-like cell line MC3T3-E1. In in vivo studies, the effect of endothelin-1 on bone morphogenetic protein-2-induced ectopic bone formation in rats was investigated. A carrier containing bone morphogenetic protein-2 with or without endothelin-1 was subcutaneously implanted over the thorax, and the tissue (carrier) calcium content 3 weeks after implantation was evaluated. The inhibitory effect of YM598 on these responses was also investigated. In the in vitro studies, endothelin-1 (10(-13) to 10(-6) M) significantly increased intracellular Ca(2+) concentration, DNA synthesis and cell number in a concentration-dependent manner, while significantly decreasing alkaline phosphatase activity. YM598 (10(-12) to 10(-4) M) significantly inhibited these increases, as well as the decrease in alkaline phosphatase activity, in a concentration-dependent manner. In the in vivo studies, the tissue calcium content 3 weeks after carrier implantation was significantly higher in the group that received both bone morphogenetic protein-2 and endothelin-1 than in the group receiving bone morphogenetic protein-2 alone. Chronically administered YM598 (1 mg/kg/day) marginally inhibited this endothelin-1-potentiated ectopic bone formation. These results suggest that endothelin-1 may induce bone formation via endothelin ET(A) receptors in vitro and in vivo.

Effect of tamsulosin on spontaneous bladder contraction in conscious rats with bladder outlet obstruction: comparison with effect on intraurethral pressure.

We investigated the effect of tamsulosin, an alpha(1)-adrenoceptor antagonist, on bladder function, especially spontaneous bladder contractions before micturition (premicturition contraction), in conscious rats with bladder outlet obstruction induced by partial urethral ligation, and compared the results with the effect on intraurethral pressure response in anesthetized rats. In obstructed rats, the alpha(1)-adrenoceptor antagonists tamsulosin, naftopidil and urapidil and non-selective alpha-adrenoceptor antagonist phentolamine inhibited premicturition contractions in a dose-dependent fashion. In contrast, yohimbine, an alpha(2)-adrenoceptor antagonist, and atropine, a muscarinic receptor antagonist, hardly inhibited them. Tamsulosin and urapidil showed clearly inhibitory effects on increases in intraurethral pressure induced by phenylephrine, an alpha(1)-adrenoceptor agonist, in the same dose range as that at which they inhibited premicturition contractions, whereas naftopidil required somewhat higher doses to inhibit increases in intraurethral pressure than those at which it inhibited premicturition contractions. In conclusion, premicturition contractions observed in obstructed rats were sensitive to alpha(1)-adrenoceptor antagonists, but not to alpha(2)-adrenoceptor or muscarinic receptor antagonists. Tamsulosin was shown to be effective against both premicturition contraction and intraurethral pressure response in the same dose range in rats. These results partly support the fact that tamsulosin has improved storage symptoms as well as voiding symptoms in patients with lower urinary tract symptoms associated with bladder outlet obstruction by blocking alpha(1)-adrenoceptors.

In vivo studies on the effects of alpha1-adrenoceptor antagonists on pupil diameter and urethral tone in rabbits.

Alpha1-adrenoceptors mediate contraction of iris dilator smooth muscle and hence pupil dilatation. We compared the ability of i.v. bolus injections of alfuzosin, doxazosin, naftopidil, prazosin, tamsulosin and terazosin to antagonise phenylephrine-induced mydriasis relative to their potency for inhibiting phenylephrine-induced elevations of intraurethral pressure (IUP) in rabbits. Moreover, we compared the ability of these drugs to induce miosis in conscious rabbits in the absence of phenylephrine. All antagonists inhibited the effects of phenylephrine on pupil size and IUP, and the ratio of the respective ED50 values was close to unity in all cases. The doses required to induce statistically significant miosis in the absence of phenylephrine were 30- to 100-fold higher than those inhibiting phenylephrine-induced mydriasis for all antagonists, except for naftopidil. Moreover, the miotic effects of all alpha1-adrenoceptor antagonists were fully reversible within 8 h. We conclude that alfuzosin, doxazosin, naftopidil, prazosin, tamsulosin and terazosin inhibit phenylephrine-induced mydriasis in the same dose range as they inhibit elevations in IUP. Higher doses of all antagonists are required to induce miosis in the absence of an exogenous agonist, and such miosis is always reversible within hours.

Participation of endogenous endothelin and ETA receptor in premicturition contractions in rats with bladder outlet obstruction.

A relationship between endogenous endothelins and bladder overactivity has recently been suggested, but the related endothelin receptor subtype has not been identified. Here, to evaluate the involvement of endothelin-1 and its receptors in bladder overactivity, we investigated endothelin-1 levels and the expression of its receptors in the bladder of rats with bladder outlet obstruction (BOO), a model for bladder overactivity. We also investigated the effects of a selective endothelin ET(A) receptor antagonist, (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2,2'-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598), on bladder functions in conscious BOO rats. Partial obstruction of the urethra led to a progressive increase in bladder weight from weeks 1 to 6. Binding assays performed using plasma membranes prepared from these bladders to estimate endothelin receptor density from the maximum [(125)I]endothelin-1 binding showed increased endothelin receptor density (about double) at 1, 2, and 6 weeks after the operation in the BOO bladder. The densities of endothelin ET(A) receptors in the bladder of sham-operated and BOO rats at 2 weeks after operation were about 3.5 and 5 times those of endothelin ET(B) receptors respectively. Furthermore, the endothelin-1 level was also increased in the BOO bladder. Two weeks after operation, BOO rats showed an increase in maximum bladder capacity and micturition volume and the generation of premicturition contractions. The frequency of premicturition contractions was dose-dependently reduced by YM598 (0.1-3 mg/kg, i.v.) without any effect on other voiding parameters in BOO rats. These data suggest that endothelin-1 and endothelin ET(A) receptors might be involved in the generation of premicturition contractions in BOO rats, and that endothelin ET(A) receptor antagonists such as YM598 may have ameliorating effects in patients with bladder overactivity associated with BOO.

Effects of solifenacin succinate (YM905) on detrusor overactivity in conscious cerebral infarcted rats.

Solifenacin succinate [YM905, (+)-(1S,3'R)-quinuclidin-3'-yl 1-phenyl-1,2,3,4-tetrahydroisoquinoline-2-carboxylate monosuccinate] is a novel muscarinic receptor antagonist. We examined the effects of solifenacin and two other muscarinic receptor antagonists, tolterodine and propiverine, on detrusor overactivity in cerebral infarcted rats. Evaluation was done under conscious conditions using cystometry 1 day after middle cerebral artery occlusion. The cerebral infarcted rats showed decreases in bladder capacity and voided volume and an increase in residual volume, but no change in micturition pressure. Solifenacin increased bladder capacity and voided volume at doses of 0.03 mg/kg i.v. or more. Tolterodine increased bladder capacity and voided volume at 0.03 and 0.1 mg/kg i.v., while propiverine increased bladder capacity and voided volume at 1 mg/kg i.v. and at 0.3 and 1 mg/kg i.v., respectively. In contrast, none of the three drugs affected residual volume or micturition pressure. These results suggest that solifenacin may improve detrusor overactivity without causing urinary retention and may be a promising drug in the treatment of patients with overactive bladder syndrome.

A novel and selective endothelin ET(A) receptor antagonist YM598 prevents the development of chronic hypoxia-induced pulmonary hypertension in rats.

The preventive effects of the novel and selective endothelin ET(A) receptor antagonist YM598 on the development of pulmonary hypertension (PH) were investigated in chronic hypoxia-induced PH rats. Oral administration of YM598 at a dose of 1 mg/kg was started on the first day of chronic hypoxia exposure for 2 and 3 weeks to investigate the effects of this compound on hemodynamic and arterial blood gas variables, respectively. Cardiopulmonary organ weights were measured at the end of the 2-week administration period. Chronic hypoxia for 2 weeks induced a marked increase in pulmonary arterial pressure, right ventricular hypertrophy, and pulmonary and systemic congestion, and a decrease in right cardiac diastolic function. Repeated oral administration of YM598 significantly suppressed the increase in pulmonary arterial pressure, right ventricular hypertrophy, and pulmonary and systemic congestion. YM598 also improved the hypoxemia which was induced by 3 weeks of exposure to hypoxia. These results suggest that repeated oral administration of YM598 to rats with chronic hypoxia effectively prevented the development of PH. Oral administration of YM598 also improved hypoxemia in this model. These data strongly suggest that YM598 will be clinically useful in the treatment of patients with either primary or secondary pulmonary hypertension.

Pharmacological characterization of YM598, an orally active and highly potent selective endothelin ET(A) receptor antagonist.

We describe here the pharmacology of (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2,2'-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598), a novel selective endothelin ET(A) receptor antagonist synthesized through the modification of the ET(A)/ET(B) non-selective antagonist, bosentan. YM598 inhibited [125I]endothelin-1 binding to cloned human endothelin ET(A) and ET(B) receptor, with K(i) of 0.697 and 569 nM, and inhibited endothelin-1-induced increases in intracellular Ca(2+) concentration in human and rat endothelin ET(A) receptor. YM598 also inhibited endothelin-1-induced vasoconstriction in isolated rat aorta with a pA(2) value of 7.6. In vivo, YM598 inhibited the pressor response to big endothelin-1, a precursor peptide of endothelin-1. DR(2) values of YM598 in pithed rats were 0.53 mg/kg, i.v. and 0.77 mg/kg, p.o., and its antagonism in conscious rats was maintained for more than 6.5 h at 1 mg/kg, p.o. In contrast, YM598 had no effect on the sarafotoxin S6c-induced depressor or pressor responses. YM598 showed not only superior antagonistic activity and higher-selectivity for endothelin ET(A) receptor in vitro, but at least a 30-fold higher potency in vivo than bosentan. In conclusion, YM598 is a potent and orally active selective endothelin ET(A) receptor antagonist.

The orally active nonpeptide selective endothelin ETA receptor antagonist YM598 prevents and reverses the development of pulmonary hypertension in monocrotaline-treated rats.

We investigated the preventive and therapeutic effects of the selective endothelin ETA receptor antagonist potassium(E)-N-[6-methoxy-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyrimidin-4-yl]-2-phenylenthenesulfonamidate (YM598) on the development of pulmonary hypertension in monocrotaline-induced pulmonary hypertensive and hypoxemic rats. In the prevention study, oral administration of YM598 (0.1 and 1 mg/kg) or bosentan (30 mg/kg) for 4 weeks was started on the day following monocrotaline (60 mg/kg) injection. In the therapeutic study, oral administration of YM598 (0.1, 0.3 and 1 mg/kg) for 2 weeks was started 3 weeks after monocrotaline injection. In the prevention study, a marked increase in pulmonary arterial pressure and right ventricular hypertrophy, a decrease in right cardiac function and hypoxemia were observed. Histopathological examination indicated the presence of pulmonary remodeling, including medial wall thickening of the pulmonary microvasculature and alveolar disorders. YM598 suppressed the increase in pulmonary arterial pressure, right ventricular hypertrophy and systemic congestion, and improved the hypoxemia, but bosentan had only modest effects. Histopathological disorders were also ameliorated by YM598. In the therapeutic study, YM598 also ameliorated the pulmonary hypertension and hypoxemia in monocrotaline-treated rats. These results suggest that YM598 effectively prevented and reversed the development of pulmonary hypertension, and reduced the pulmonary vascular remodeling and parenchymal injury in monocrotaline-treated rats. YM598 also improved hypoxemia which accompanied with the severe pulmonary hypertension in these rats.

Effects of tamsulosin on hypogastric nerve stimulation-induced intraurethral pressure elevation in male and female dogs under anesthesia.

The aim of the present study was to investigate the effects of tamsulosin, an alpha(1)-adrenoceptor antagonist, on hypogastric nerve stimulation-induced intraurethral pressure elevation in anesthetized male and female dogs and to evaluate sex differences in these effects. Additionally, the effects of tamsulosin were also compared with those of other alpha(1)-adrenoceptor antagonists, namely prazosin, naftopidil and urapidil. Tamsulosin dose-dependently inhibited hypogastric nerve stimulation-induced intraurethral pressure elevation, with doses required to induce 50% inhibition of the elevation (ED(50) values) of 0.72 and 0.74 microg/kg i.v. in anesthetized male and female dogs, respectively. Mean arterial blood pressure slightly decreased after administration of tamsulosin at a dose which inhibited intraurethral pressure elevation almost completely. Prazosin, naftopidil and urapidil also inhibited increases in intraurethral pressure in a dose-dependent fashion, but caused decreases in mean arterial blood pressure at the same doses. The estimated rank order of inhibitory potency for urethral response was tamsulosin>prazosin>naftopidil=urapidil. In conclusion, tamsulosin dose-dependently inhibited increases in intraurethral pressure with little effect on mean arterial blood pressure in both male and female dogs, and these effects were almost equipotent. These results indicate that tamsulosin will be useful in the treatment of dysuria associated with lower urinary tract symptoms in women as well as men.

Superiority of YM598 over atrasentan as a selective endothelin ETA receptor antagonist.

The binding affinities of (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2,2'-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598) for native human endothelin ETA and ETB receptors expressed in human coronary artery smooth muscle cells (CASMC) and a human melanoma cell line, SK-Mel-28, respectively, were examined, and the results compared with those for the endothelin receptor antagonists atrasentan and bosentan. The in vivo endothelin ETA receptor inhibitory activities of YM598 and atrasentan were also compared through the suppression of the big endothelin-1-induced pressor response in pithed rats. Ki values of YM598, atrasentan, and bosentan for native human endothelin ETA receptors were 0.772, 0.0551, and 4.75 nM, while those for native human endothelin ETB receptors were 143, 4.80, and 40.9 nM, respectively. The calculated selectivity ratios of YM598, atrasentan, and bosentan for endothelin ETA versus ETB receptors were 185, 87 and 8.6, respectively. In pithed rats, YM598 and atrasentan inhibited the big endothelin-1 (1 nmol/kg)-induced pressor response in a dose-dependent manner on both intravenous and oral administration. The inhibitory effect of YM598 was less potent than that of atrasentan when these agents were intravenously administered, but closely similar on oral administration. These results suggest that YM598 has high selectivity for native human ETA against ETB receptors, and that YM598 is superior to atrasentan as an ETA receptor antagonist with regard to pharmacological bioavailability in rats.

Effects of selective endothelin ET(A) receptor antagonists on endothelin-1-induced potentiation of cancer pain.

In some diseases in which endothelin-1 production increases, e.g. prostate cancer, endothelin-1 is considered to be involved in the generation of pain. In the present study, we investigated the effects of a selective endothelin ET(A) receptor antagonist, (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2,2'-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598), on the nociception potentiated by endothelin-1 in a cancer inoculation-induced pain model in mice, induced by inoculation of the androgen-independent human prostate cancer cell line PPC-1 into the hind paws of severe combined immunodeficiency (SCID) mice. No pain responses were observed in the sham-operated mice, whereas monophasic pain responses were observed in the PPC-1-inoculated mice. Endothelin-1 (1 to 10 pmol/paw) but not sarafotoxin S6c potentiated the pain response in prostate cancer-inoculated mice. Both YM598 and atrasentan (0.3 to 3 mg/kg, p.o.) significantly inhibited the endothelin-1 (10 pmol/paw)-induced potentiation of nociception in a dose-dependent manner. These results suggest that selective endothelin ET(A) receptor antagonists might relieve pain in patients with various diseases in which endothelin-1 production is increased, e.g. prostate cancer.

Renal protective effect of YM598, a selective endothelin type A receptor antagonist.

We have investigated the protective effect of YM598, a selective endothelin type A receptor antagonist, against an endothelin-1-induced proliferation of rat mesangial cells and renal function in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of type II diabetes. YM598, but not K-8794, a selective endothelin type B receptor antagonist, inhibited the endothelin-1-induced proliferation of cultured mesangial cells derived from intact Wistar rats in a concentration-dependent manner. YM598 (0.1 or 1 mg/kg), enalapril (5 mg/kg), an angiotensin- converting enzyme inhibitor, or vehicle was administered once daily by gastric gavage to 22-week-old male OLETF rats for 32 weeks. YM598 blunted the development of albuminuria in a dose-dependent manner. A higher dose of YM598 reduced albuminuria comparable with enalapril. Urinary endothelin-1 excretion was greater in the diabetic rats than in the control rats, and was not substantially influenced by the agents. Enalapril, but not YM598, mildly lowered the blood pressure in the diabetic rats, indicating that blood pressure reduction is not involved in the major mechanism of the renoprotective effect of YM598 in OLETF rats. These data suggest that endothelin is involved in the progression of diabetic nephropathy in OLETF rats, and an endothelin type A antagonist is promising for the treatment of diabetic nephropathy.

YM598, an orally active ET(A) receptor antagonist, ameliorates the progression of cardiopulmonary changes and both-side heart failure in rats with cor pulmonale and myocardial infarction.

The effects of the novel, selective endothelin-A (ETA) receptor antagonist YM598 on both-side heart failure were investigated. Right-side heart failure secondary to pulmonary hypertension was produced by a single subcutaneous injection of 60 mg/kg monocrotaline, and post-ischemic congestive left-side heart failure (CHF) produced by surgical left coronary artery ligation. In right-side heart failure rats, oral YM598 (0.1 and 1 mg/kg for 4 weeks), but not bosentan (30 mg/kg), significantly inhibited the progression of pulmonary hypertension and the development of right ventricular hypertrophy. YM598 also improved hypoxemia and morphological pulmonary lesions in these rats. In CHF rats, moreover, long-term oral administration of YM598 (1 mg/kg/day for approximately 30 weeks) significantly ameliorated their poor survival rate (P < 0.05). In the measurement of cardio-hemodynamic parameters, YM598 improved the contractile/diastolic capacity of the left ventricle and the preload in the right ventricle to the levels seen in sham-operated rats. YM598 also markedly inhibited both ventricular hypertrophy and pulmonary congestion, as well as lowering high plasma brain natriuretic peptide levels in CHF rats. These findings suggest that YM598 may have a clinical benefit with regards to ameliorating the cardiopulmonary changes of right-side heart failure, and the cardiac dysfunction and mortality/morbidity of CHF.