Methods for assessing endothelin ETA-receptor antagonists in preclinical studies.

Endothelin-1 (ET-1) has been found to be one of the most potent vasoconstrictive peptides known, and is therefore considered to be an important factor in diseases such as hypertension, heart failure, pulmonary hypertension, renal diseases, etc. Thus, the development of ET-receptor antagonists may offer a new therapeutic strategy in these fields. In this article, we summarize the method for assessing our compound as a selective ETA-receptor antagonist. Binding assays and in vitro function assays (isolated vessels) were examined for the assessment of in vitro potency, selectivity of the ETA receptor against the ETB receptor, specificity for ET receptors, agonistic activities for ET receptors and the blocking manner of the compound on ET receptors. Chinese hamster ovary (CHO) cells expressing human ET receptors and tissue membrane preparations from both human and animals were used for the binding assays. The specificity of the compound against ET receptors was demonstrated using 116 and 9 receptor binding and enzyme assays, respectively. The agonistic activity and potency of the compound at tissue levels were examined using isolated vessels. We also demonstrated the effect of protein binding on the potency of the compound by adding a physiological concentration of serum albumin to the tissue baths. In vivo potency and features of the compound as a selective ETA-antagonist were confirmed using mice, rats and dogs exogenously treated with ET-1 or big ET-1. We also demonstrated the compound's duration of action and pharmacokinetics in animal models and intact animals, respectively. From these experiments, we found a nonpeptide, potent, orally active and long-lasting, highly selective ETA-receptor antagonist.

Pathophysiological roles of endogenous endothelin-1 in dogs with chronic heart failure produced by rapid right ventricular pacing.

This study was designed to analyze the pathophysiological role of the endogenous endothelin (ET) system and the therapeutic approach to congestive heart failure (CHF) with ET(A)/ET(B) receptor antagonists in a canine CHF model. After 3 weeks of rapid right ventricular pacing (240 beats/min), concentrations of immunoreactive ET-1 in dogs increased approximately 2-fold in plasma and in the left and right ventricles but not in the lung. There were no meaningful changes in the density and affinity of total ET receptors, or in the ratio of ET(A) to ET(B) receptors. To clarify the functional role of endogenous ET, we examined the effects of acute injection of J-104132 (1 and 3 mg/kg i.v.), an ET(A)/ET(B) receptor antagonist, on cardiovascular and renal function in dogs with CHF. Compared with vehicle, J-104132 at both doses significantly decreased pulmonary artery pressure (PAP), pulmonary capillary wedge pressure (PCWP), and mean arterial pressure (MAP), and increased cardiac output (CO) and renal blood flow. J-104132 had no effects on heart rate and cardiac contractility. In addition, we examined whether J-104132 has an additive effect in the presence of enalaprilat. J-104132 (1 mg/kg i.v.) administered after enalaprilat (0.05 mg/kg i.v.) induced further decreases in MAP, PCWP and PAP, and further increases in CO, resulting in further decreases in total peripheral resistance. These results indicate that the endogenous ET system is exaggerated in CHF and has a detrimental effect on cardiac function. Therefore, J-104132 given alone or as combination therapy may play a beneficial role in the treatment of CHF in humans.

Pharmacological properties of (2R)-N-[1-(6-aminopyridin-2-ylmethyl)piperidin-4-yl]-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamide: a novel mucarinic antagonist with M(2)-sparing antagonistic activity.

We evaluated the pharmacological profiles of (2R)-N-[1-(6- aminopyridin-2-ylmethyl)piperidin-4-yl]-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamide(compound A), which is a novel muscarinic receptor antagonist with M(2)-sparing antagonistic activity. Compound A inhibited [(3)H]NMS binding to cloned human muscarinic m1, m2, m3, m4, and m5 receptors expressed in Chinese hamster ovary cells with K(i) values (nM) of 1.5, 540, 2.8, 15, and 7.7, respectively. In isolated rat tissues, compound A inhibited carbachol-induced responses with 540-fold selectivity for trachea (K(B) = 1.2 nM) over atria (K(B) = 650 nM). In in vivo rat assays, compound A inhibited acetylcholine-induced bronchoconstriction and bradycardia with intravenous ED(50) values of 0.022 mg/kg and >/=10 mg/kg, respectively. Furthermore, in dogs, compound A (0.1-1 mg/kg p.o.) dose dependently shifted the methacholine concentration-respiratory resistance curves. In mice, compound A (10 mg/kg i.v.) did not inhibit oxotremorine-induced tremor. The brain/plasma ratio (K(p)) of compound A (3 mg/kg i.v.) was 0.13 in rats; this K(p) was less than that of scopolamine (1.7) and darifenacin (0.24). The inhibition of compound A (3 mg/kg i.v.) on ex vivo binding in rat cerebral cortex was almost similar to that of NMS. These findings demonstrate that compound A has high selectivity for M(3) receptors over M(2) receptors, displays a potent, oral M(3) antagonistic activity without inhibition of central muscarinic receptors because of low brain penetration. It is well known that central muscarinic antagonists may have diverse CNS effects, and M(2) receptors regulate cardiac pacing and act as autoreceptors in the lung and bladder. Thus, compound A may have fewer cardiac or CNS side effects than nonselective compounds.

Acute effects of E-3174, a human active metabolite of losartan, on the cardiovascular system in tachycardia-induced canine heart failure.

The aim of this study was to evaluate the acute effects of E-3174, a human active metabolite of the AT1 receptor antagonist, losartan, on hemodynamic functions in dogs with severe heart failure (HF). In dogs, insignificant plasma levels of E-3174 are present following administration of losartan, and therefore, the effects of these two drugs can be studied independently in the dog. HF was established by rapid pacing of the right ventricle (250-270 beats/min) for 4 weeks. We examined changes in cardiovascular functions after acute intravenous administration of losartan (1 mg/kg) and E-3174 (0.3 and 1 mg/kg), as well as an ACE inhibitor, enalapril (0.3 and 1 mg/kg), under condition of HF. The HF before treatment was characterized by increases in pre- and after-load of the left ventricle (LV), consequent low cardiac output, and LV dilatation. E-3174 at 0.3 and 1 mg/kg reduced pulmonary artery pressure (-13+/-6% and -22+/-3% from baseline, respectively, p<0.05), pulmonary capillary wedge pressure (-18+/-4% and -36+/-10%, p<0.05) and mean arterial pressure (-24+/-2% and -36+/-7%, p<0.05), increased stroke volume (SV: +12+/-7% p>0.05; +36 +/-19%, p<0.05), and reduced peripheral resistance (-23+/-5% and -41+/-9%, p<0.05), but had no effect on the first derivative of left ventricular pressure (dP/dt/P) or the time constant for relaxation. Effects of losartan at 1 mg/kg were similar to those of 0.3 mg/kg of E-3174. Enalapril at 1 mg/kg caused changes comparable to those seen after E-3174 administration (1 mg/kg), except that the increase in SV (+16+/-8%, p<0.05) with enalapril was not as great as that with E-3174. Both losartan at 1 mg/kg and E-3174 at 0.3 and 1 mg/kg increased fractional shortening to a similar extent (FS: +52+/-12%, +47+/-8% and +56+/-8%), while enalapril at 0.3 and 1 mg/kg had no significant effects on FS. Reflex elevation of plasma renin activity induced by 1 mg/kg of E-3174 was similar to that caused by 1 mg/kg of enalapril, suggesting that the two drugs achieved similar inhibition of the endogenous renin angiotensin system. Our study demonstrated that acute blockade of the AT1 receptor with E-3174 reduced elevated pre- and after-load and consequently increased stroke volume in a canine HF model. With the exception of changes in stroke volume, these effects of E-3174 were comparable to those produced by enalapril, and were 3 times stronger than those by losartan.

Antihypertensive effects of a mixed endothelin-A- and -B-receptor antagonist, J-104132, were augmented in the presence of an AT1 -receptor antagonist, MK-954.

The single oral administration of a mixed endothelin-A-and -B- (ETA/ETB) receptor antagonist, J-104132 (L-753,037) decreased the blood pressure in conscious spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP) and Dahl salt-sensitive hypertensive rats fed high-salt diet (DS-H) at doses of 3 and 10 mg/kg, with a duration of approximately 24 h. The magnitude of the antihypertensive effects was greater in DS-H than in SHR and SHRSP Preproendothelin-1 mRNA expression in the kidney and aorta was greater (about twofold) in DS-H than that in nonnotensive Dahl salt-resistant rats fed high-salt diet (DR-H), while there was no difference in preproendothelin-1 mRNA expression between SHR and Wistar Kyoto rats (WKY). An AT1-receptor antagonist, MK-954 (Losartan), also attenuated hypertension in SHR and SHRSP at oral doses of 3 and 10 mg/kg, but bad no effect in DS-H. The concomitant treatment with MK-954 and J-104132 showed additive antihypertensive effects in SHR and SHRSP. In DS-H, MK-954 potentiated the antihypertensive effects of J-104132. From these results, we suggest that: (1) the contribution of endothelin (ET) to the maintenance of hypertension is greater in salt-sensitive hypertensive models than in SHR and SHRSP; (2) the antihypertensive efficacy of ETA/ETB blockade is augmented by AT1-receptor blockade; (3) ET blockers alone or in combination with AT1 antagonists could be useful in the treatment of hypertension for patients who do not respond adequately to renin-angiotensin system inhibitors alone.

Antiplatelet and antithrombotic effects of a novel selective phosphodiesterase 3 inhibitor, NSP-513, in mice and rats.

We investigated the effects of NSP-513, (R)-4,5-dihydro-5-methyl-6-[4-(2-propyl-3-oxo-1-cyclohexenyl)amino] phenyl-3(2H)-pyridazinone, on phosphodiesterase (PDE) isozyme activities, in vitro platelet aggregation and in vivo thrombus formation. NSP-513 selectively inhibited human platelet PDE 3 isozyme with an IC50 value of 0.039 microM. In an in vitro human platelet aggregation assay, the IC50 values (microM) of NSP-513 for platelet aggregation induced by collagen, U-46619, arachidonic acid, adenosine diphosphate (ADP), epinephrine and thrombin were 0.31, 0.25, 0.082, 0.66, 0.23 and 0.73, respectively. In a mouse pulmonary thromboembolism model, orally administered NSP-513 showed in vivo antithrombotic effects that were 320 to 470 times more potent than those of cilostazol. In a rat carotid arterial thrombosis model, intraduodenally administered NSP-513 (0.1 mg/kg), cilostazol (30 mg/kg) and aspirin (30 mg/kg) reduced thrombus formation by 75%, 66% and 48%, respectively. However, intravenously administered dipyridamole (10 mg/kg) did not significantly prevent thrombus formation. These results demonstrate that NSP-513 has the potential to prevent not only in vitro platelet aggregation but also in vivo thrombus formation and indicate that the highly selective PDE 3 inhibitory effect of NSP-513 may make this compound useful for assessing the physiological role of PDE 3.

Antithrombotic activity of NSP-513, a novel selective phosphodiesterase 3 inhibitor, on femoral arterial thrombosis induced by physical stenosis and electrical current: comparison of antithrombotic and hemodynamic effects.

NSP-513, a novel potent and selective phosphodiesterase 3 (PDE 3) inhibitor, and cilostazol, a previously developed PDE 3 inhibitor, were compared with respect to antiplatelet, antithrombotic, and hemodynamic effects. In the in vitro antiplatelet aggregation studies, NSP-513 and cilostazol inhibited collagen-induced canine platelet aggregation with median inhibitory concentration (IC50) values of 0.093 and 3.1 miccroM, respectively, and inhibited adenosine diphosphate (ADP)-induced canine platelet aggregation with IC50 values of 0.15 and 12 microM, respectively. For ADP-induced platelet aggregation, the presence of prostaglandin E1 (PGE1; 3 and 10 nM) further decreased the IC50 values for NSP-513 to 0.11 and 0.032 microM, respectively. In ex vivo antiplatelet aggregation studies, orally administered NSP-513 (0.03-1 mg/kg) and cilostazol (50 mg/kg) inhibited collagen-induced canine platelet aggregation. In an in vivo canine femoral arterial thrombosis model, intraduodenally administered NSP-513 (0.01-0.03 mg/ kg) dose-dependently prevented thrombus formation without any changes in blood pressure, heart rate, or bleeding time. In conscious dogs, NSP-513 at oral doses of > or =0.3 mg/kg produced hemodynamic changes such as decreased blood pressure and increased heart rate and LVdP/dt(max). Thus the minimal hemodynamically effective dose of NSP-513 was 0.3 mg/kg, and the hemodynamic effects of this dose were comparable to those of 50 mg/kg of cilostazol. In conclusion, these data suggest that NSP-513 has in vivo selectivity for antiplatelet and antithrombotic activities over hemodynamic activity, and that the selectivity of NSP-513 is higher than that of cilostazol in dogs.

Marked reduction of mortality in salt-loaded Dahl salt-sensitive rats by the new, selective endothelin ETA receptor antagonist, J-105859.

OBJECTIVE: To examine the chronic effects of a newly synthesized, potent and selective endothelin (ET) ETA receptor antagonist, J-1 05859, on mortality in salt-loaded Dahl salt-sensitive (DS) rats and to confirm the potential of this compound as an ETA antagonist METHODS: Vehicle and J-105859 were administered to salt-loaded DS rats for 12 weeks. Throughout the experimental period, blood pressure was measured continuously using a telemetry system and the survival rate was determined. The surviving animals were subsequently sacrificed and autopsy was performed. Binding and functional assays were also carried out to characterize J-1 05859. RESULTS: The Ki values of J-1 05859 for cloned human ETA and ETB were 0.025 and 48 nmol/l, respectively. J-105859 inhibited ET-1-induced contractions in rabbit iliac artery (pA2 = 10.08) and BQ-3020 (ETB agonist)-induced contractions in pulmonary artery (pA2 = 7.63). The pressor response to intravenous (i.v.) ET-1 (0.5 nmol/kg) was significantly inhibited by J-1 05859 at a dose of 0.03 mg/kg i.v. Chronic treatment with J-1 05859 [0.1 and 1 mg/kg per day orally (p.o.)] from the prehypertensive stage decreased the mortality of salt-loaded DS rats and markedly inhibited the development of brain lesions. The survival rates in the control and J-1 05859 (0.1 and 1 mg/kg per day) groups were 34, 80 and 100%, respectively. Development of hypertension was markedly inhibited at a dose of 1 mg/kg per day. CONCLUSION: J-105859 is a selective, potent, orally active ETA-selective antagonist ETA antagonists may reduce morbidity as well as mortality in salt-sensitive hypertension.

[Pharmacological characteristics and clinical application of losartan, an orally active AT1 angiotensin II receptor antagonist].

Losartan is the first orally active angiotensin II receptor type 1 antagonist for a new class of cardiovascular therapeutic agent. Losartan is converted to an active metabolite (E3174) after oral administration in humans and rats. Both losartan and E3174 contribute to the net angiotensin II receptor blockade and produce anti-hypertensive effect. Losartan not only blocks the vasoconstrictive effect of angiotensin II but also inhibits its mitogenic effect; thus losartan is expected to protect against end-organ-damage-related hypertension and chronic heart failure. Unlike angiotensin-coverting-enzyme inhibitor, losartan does not elicit adverse effects of cough and angioneurotic edema by its blockade of angiotensin II receptor. It is also expected to reduce proteinuria in nephropathy. In addition to its blockade of angiotensin II receptor, losartan blocks thromboxane A2 receptor and facilitates excretion of uric acid, although therapeutic importance of these effects are under investigation. In summary, losartan, an angiotensin II type 1 receptor antagonist is a new class of antihypertensive agent and its therapeutic potentials are not merely reduction of blood pressure but total protection from end-organ damage resulting from activation of both the systemic and local renin-angiotensin system.

Pathophysiological roles of endothelin-1 in Dahl salt-sensitive hypertension.

The purpose of the present experiment was to study the pathophysiological roles of endothelin-1 (ET-1) in salt-sensitive hypertension with the use of Dahl salt-sensitive (DS) and salt-resistant (DR) rats. PreproET-1 mRNA expression was determined by reverse transcription-polymerase chain reaction. In the kidney, expression of preproET-1 mRNA was greater in DS rats on a normal salt diet compared with DR rats of the same age. In DS rats, the level of preproET-1 mRNA expression in kidney had a significant correlation with systolic blood pressure. The expression of preproET-1 mRNA in aorta and kidney was increased by 3-week high salt intake in DS rats but not in DR rats. Expression of preproET-1 mRNA and ET-1 levels in left ventricle was exaggerated by high salt intake in DS rats. However, there was no significant difference in plasma ET-1 levels between DS and DR rats regardless of salt intake. Pressor response curves for ET-1 in DS rats with or without high salt intake were significantly shifted to the left compared with those in DR rats. A single oral dose (3 to 10 mg/kg) of J-104132 (L-753 037), a potent, orally active mixed endothelin A and B (ET(A)/ET(B)) receptor antagonist, reduced blood pressure to normotensive levels in DS rats with high salt intake, and its action was maintained for >/=24 hours. In DS rats with normal salt intake, J-104132 (10 mg/kg) slightly but significantly decreased blood pressure. DR rats did not show obvious depressor responses to J-104132 (10 mg/kg) regardless of salt intake. These results suggest that ET-1 acts as one of the pathophysiological factors in the development and maintenance of salt-sensitive hypertension, and a mixed ET(A)/ET(B) receptor antagonist could be useful in the treatment for salt-sensitive hypertension.

[Pharmacological properties and its significance in clinical practice].

Losartan is a potent non-peptide, selective angiotensin II (AngII) type 1 (AT1) receptor antagonist. Losartan has been worldwide marketed as the first orally active AT1 receptor antagonist with once-daily dosing for treatment of hypertension. In a study of patients with heart failure, the mortality appeared to be lower with losartan than with the ACE inhibitor captopril. In healthy subjects, losartan produced a dose-dependent reduction in serum uric acid. The mechanism of action is considered to be the inhibition of reabsorption of uric acid in the proximal tubules of the kidney. Furthermore, it was recently reported that losartan has moderate affinity for the thromboxane (TX) A2 receptor in a competitive-inhibition manner in the platelets and vascular smooth muscle. The efficacy of losartan with regard to not only AT1 receptor blockade, but also the reduction of serum uric acid and the blockade of TXA2 receptors, may be advantageous to patients with hypertension having these cardiovascular risk factors.

Pharmacological properties of J-104132 (L-753,037), a potent, orally active, mixed ETA/ETB endothelin receptor antagonist.

J-104132 [(+)-(5S,6R, 7R)-2-butyl-7-[2-((2S)-2-carboxypropyl)-4-methoxyphenyl]-5-(3, 4-methylenedioxyphenyl)cyclopenteno[1,2-b]pyridine-6-carboxylic; also referred to as L-753,037] is a potent, selective inhibitor of ETA and ETB endothelin (ET) receptors (e.g., Ki: cloned human ETA = 0.034 nM; cloned human ETB = 0.104 nM). In both ligand-binding and isolated tissue preparation protocols, the inhibition of ET receptors with J-104132 is reversible and competitive. In vitro, J-104132 is a potent antagonist of ET-1-induced accumulation of [3H]inositol phosphates in Chinese hamster ovary cells stably expressing cloned human ETA receptors (IC50 = 0.059 nM), ET-1-induced contractions in rabbit iliac artery (pA2 = 9.70) and of BQ-3020-induced contractions in pulmonary artery (pA2 = 10.14). J-104132 is selective for ET receptors because it had no effect on contractions elicited by norepinephrine or KCl in the vascular preparations. The in vivo potency of J-104132 was assessed using challenges with exogenous ET-1. In conscious mice, 5 nmol/kg i.v. ET-1 causes death. Pretreatment with J-104132 prevents the lethal response to ET-1 when administered i.v. (ED50 = 0.045 mg/kg) or p.o. in fed animals (ED50 = 0.35 mg/kg). In conscious, normotensive rats, pressor responses to 0.5 nmol/kg i.v. ET-1 are inhibited by J-104132 after i.v. (0.1 mg/kg) or p.o. (1 mg/kg) administration. In anesthetized dogs, ET-1 was administered directly into the renal artery or brachial artery to generate dose-response (blood flow) curves, and the inhibitory potency of J-104132 (i.v. infusion) was quantified. J-104132 produced greater than 10-fold shifts in the ET-1 dose-response curves at 0.03 mg/kg/h (renal) and 0.3 mg/kg/h (brachial). Oral bioavailability of J-104132 in rats was approximately 40%. These studies indicate that J-104132 is a selective, potent, orally active antagonist of both ETA and ETB receptors and is an excellent pharmacological tool to explore the therapeutic use of a mixed ETA/ETB receptor antagonist.

Enhancement of pulmonary artery contraction induced by endothelin-B receptor antagonism.

We previously reported that endothelin (ET)A and ETB receptors are involved in ET-1-induced contraction in isolated rabbit pulmonary artery and human bronchus. However, the activity of an ETA/ETB dual antagonist was lower than expected in these tissues. In such ETA/ETB receptor composite-type tissues, low doses of the ETB antagonist BQ-788 shifted the concentration-contraction curve for ET-1 slightly to the left, implying potentiation of contraction. Therefore, we investigated the potentiation of contraction by ETB antagonism in isolated rabbit pulmonary artery. In endothelium-denuded artery segments, ET-1 (10(-10) M) elicited sustained, almost half-maximal contraction. BQ-123 up to 10(-6) M did not affect contractile tone in ET-1-precontracted artery segments. In contrast, the ETB-selective antagonist BQ-788 (10(-8) M to 10(-7) M) enhanced contraction in ET-1-precontracted artery. In addition, in endothelium-intact artery samples, BQ-788 enhanced contraction to the same extent as in endothelium-denuded artery. In the presence of 10(-6) M BQ-123, BQ-788 failed to enhance contraction, and concentrations > 10(-7) M elicited only relaxation. The present results suggest that blockade of ETB receptors increases contractile tone through an endothelium-independent mechanism, possibly by inhibition of ETB-mediated regional clearance of ET-1 and/or of an ETB-mediated inhibitory mechanism, in rabbit pulmonary artery. This action may explain the decreased potency of ETA/ETB dual antagonists in ETA/ETB composite-type tissue responses.

Hemodynamic effect of endothelin antagonists in dogs with myocardial infarction.

The hemodynamic effects of enalapril, an angiotensin-converting enzyme inhibitor, BQ-123, a selective ETA receptor antagonist, and bosentan, a nonselective ETA and ETB receptor antagonist, were studied in dogs 4 weeks after myocardial infarction (MI) produced by ligation of the left anterior descending coronary artery. Reduced arterial pressure and total peripheral resistance after administration of analapril, BQ-123, and bosentan revealed that both the renin-angiotensin system and endothelin participate in maintenance of cardiovascular function in chronic MI.

Necessity of dual blockade of endothelin ETA and ETB receptor subtypes for antagonism of endothelin-1-induced contraction in human bronchi.

1. Endothelin (ET)-1 has been postulated to be involved in the development of obstructive airway diseases in man. In the present study, we attempted to characterize ET receptor subtypes mediating ET-1-induced contraction in human isolated bronchi. The ET receptor antagonists used in the present study were BQ-123 (ETA receptor-selective), BQ-788 (ETB receptor-selective) and BQ-928 (ETA/ETB dual). Sarafotoxin S6c (S6c) was also used as an ETB receptor-selective agonist. 2. In human bronchi, ET-1 and S6c (10(-12)M to 10(-7) M) produced concentration-dependent contraction with almost equal potency (pD2: 8.88 +/- 0.16 for ET-1 and 9.42 +/- 0.15 for S6c). The contraction induced by S6c was competitively antagonized by BQ-788 alone (1 and 10 microM) with a pKB value of 7.49 +/- 0.21, suggesting that the stimulation of ETB receptors causes a contraction of human bronchi. However, contrary to expectation, the concentration-response curves for ET-1 were not affected by BQ-788. The ET-1- and S6c-induced contractions were not affected by BQ-123 (10 microM). Thus, ET-1-induced contraction of human bronchi is not antagonized by BQ-123 alone or by BQ-788 alone. 3. Combined treatment with 10 microM BQ-123 and 10 microM BQ-788 significantly antagonized the contraction induced by ET-1 with a dose-ratio of 11. BQ-928 also significantly antagonized ET-1-induced contraction with a pKB value of 6.32 +/- 0.24. 4. The specific binding of [125I]-ET-1 to human bronchial membrane preparations was inhibited by BQ-123 (100 pM to 1 microM) by approximately 40%. Combination treatment with BQ-788 (100 pM to 1 microM) completely inhibited the BQ-123-resistant component of [125I]-ET-1 specific binding. 5. In conclusion, the present study demonstrates that BQ-788 alone cannot inhibit ET-1-induced contractions in human bronchi, although human bronchial ETB receptors are BQ-788-sensitive. Furthermore, it was shown that blockade of both receptor subtypes antagonizes ET-1-induced contraction, and that both receptor subtypes co-exist in human bronchial smooth muscles. These findings suggest that ETA receptors as well as ETB receptors are involved in ET-1-induced contraction in human bronchi. If ET-1 is involved in human airway diseases, dual blockade of ETA and ETB receptors may be necessary to treat the diseases.

Structure-activity relationships of cyclic pentapeptide endothelin A receptor antagonists.

Analogues of the natural product endothelin A (ETA) receptor antagonists cyclo(-D-Trp1-D-Glu2-Ala3-D-Val4-Leu5-) (1) and cyclo(-D-Trp1-D-Glu2-Ala3-D-alloIle4-Leu5-) (2) were prepared and tested for inhibitory activity against [125I]endothelin (ET-1) binding to protein ETA receptors. The DDLDL chirality sequence of the natural products appeared to be critical for inhibitory activity because conversion of either D-Trp or D-Glu (or both) in 1 to the corresponding L-isomer(s) abolished this property. Systematic modifications at each position of the natural products clarified the structure-activity relationships and led to highly potent and selective ETA receptor antagonists. Most replacements of D-Trp1 and Leu5 with other amino acids caused a significant loss of inhibitory activity. In contrast, replacement of D-Glu2 with D-Asp2 enhanced the activity. With regard to the Ala3 position, all analogues with imino acids, independent of being cyclic or acyclic, showed higher affinities than did the amino acid analogues. In addition, most replacements with amino acids, which had various functional groups in their side chains, did not significantly modify ETA binding affinity. The D-Val4/D-alloIle4 position was very important for inhibitory activity, and a beta-position branched D-amino acid or a D-heteroarylglycine was preferable at this position. Among synthesized cyclic pentapeptides, compound 36 (BQ-518) was the most potent ETA receptor antagonist, with a pA2 of 8.1 against ET-1-induced vasoconstriction in isolated porcine coronary arteries. This compound also showed the greatest selectivity between ETA and ETB receptors (IC50 for human ETA = 1.2 nM, IC50 for human ETB = 55 microM). In contrast, compound 8 (BQ-123) is a highly soluble, potent, and selective ETA receptor antagonist (pA2 = 7.4, IC50 for human ETA = 8.3 nM, IC50 for human ETB = 61 microM). The sodium salt of 8 is practically freely soluble in saline. These compounds are useful tools for not only in vitro but also in vivo pharmacological studies.

Effects of a selective endothelin A-receptor antagonist, BQ-123, in salt-loaded stroke-prone spontaneously hypertensive rats.

1. We examined the effects of a selective endothelin A (ETA)-receptor antagonist, BQ-123, on the development of hypertension and organ damage in stroke-prone spontaneously hypertensive rats (SHRSP) given 1% NaCl for 6 weeks. 2. BQ-123 at doses of 0.7, 2.1 and 7.1 mg/day was continuously administered for 6 weeks to 8 week old salt-loaded SHRSP, who were given water containing 1% NaCl for the following 6 weeks, via a subcutaneous osmotic minipump. 3. Development of high blood pressure was accelerated in salt-loaded SHRSP compared with that in non-salt-loaded SHRSP. After 6 weeks of salt-loading, incidence of cerebral infarction, renal sclerosis and renal fibrosis were greater in salt-loaded than non-salt-loaded SHRSP. 4. BQ-123 attenuated the age-related rise in blood pressure in a dose-dependent manner. The effect coincided with reduction in the incidence of cerebral infarction and prevention of renal sclerosis and fibrosis. Kidney function was improved as observed by an increase in glomerular filtration rate and decreases in urinary protein excretion, blood urea nitrogen and fractional sodium excretion. Furthermore, BQ-123 prevented increases in the heart weight/bodyweight ratio and aortic wall thickness in salt-loaded SHRSP. 5. These results suggest that endogenous endothelin-1 (ET-1) and ETA-receptors may be, at least in part, involved in the pathogenesis of hypertension and organ damage in salt-loaded SHRSP.

The role of endothelin and nitric oxide in modulation of normal and spastic cerebral vascular tone in the dog.

To investigate the roles of endothelin and nitric oxide (NO) in the regulation of cerebral vascular tone under basal conditions and in cerebral vasospasm following subarachnoid hemorrhage in dogs, we used BQ-123 (cyclo(-D-Trp-D-Asp-L-Pro-D-Val-L-Leu-) sodium salt), an endothelin ETA receptor antagonist, L-arginine, a substrate for the formation of NO, and NG-nitro-L-arginine methyl ester, an NO synthesis inhibitor, and measured the angiographic diameter of the basilar artery in vivo. In normal dogs, intracisternal (i.c.) injection of BQ-123 (0.6 mg/kg) produced a 29.4 +/- 6.11% (P < 0.01) increase in the basal diameter 24 h after injection. NG-nitro-L-arginine methyl ester (0.6 mg/kg i.c.) produced a 19.3 +/- 2.93% (P < 0.05) decrease in the basal diameter 2 h after injection. This decrease was significantly attenuated by both BQ-123 (0.06-0.6 mg/kg i.c.) and L-arginine (6 mg/kg i.c.), but not by D-arginine. In the two-hemorrhage canine model, BQ-123 significantly inhibited the development of cerebral vasospasm (36.9 +/- 4.11% decrease on day 5 and 42.0 +/- 4.54% decrease on day 6 in controls vs 21.7 +/- 4.75% decrease (P < 0.05) on day 5 and 20.8 +/- 4.14% decrease (P < 0.05) on day 6 for 0.6 mg/kg i.c.) significantly attenuated the cerebral vasospasm on day 4 from a mg/kg i.c.). Furthermore, in this model, L-arginine (6 30.9 +/- 5.78% decrease (before)) to a 12.6 +/- 5.99% decrease (after). The immunoreactive endothelin-1 levels in the endothelial layer and the adventitia of the basilar artery were much higher on days 3 and 7 after the injection of autologous blood than on day 0 before blood injection. These results suggest that endogenous endothelin and NO both participate in regulating the basal tone of cerebral arteries, and, therefore, the development of cerebral vasospasm following subarachnoid hemorrhage may be at least partially attributed to an impairment of the balanced action of endothelin and NO. Furthermore, endothelin ETA antagonists or NO products may be useful in the treatment of cerebral vasospasm following subarachnoid hemorrhage.

Development of polyuria in Tsukuba hypertensive mice carrying human renin and angiotensinogen genes.

1. Tsukuba hypertensive mice (THM) carry both human renin and angiotensinogen genes, and develop hypertension. The animal has high levels of renin activity and angiotensin II concentration in the plasma. 2. Urinary excretion in THM was greater than in the control animal, non-transgenic C57BL/6j. THM showed a greater amount of daily water intake. The osmolality of 24 h urine was lower than that of the control animal. 3. When water was deprived for 12 h and then loaded with 0.25 mL/10 g bodyweight, the osmolality of urine at the first 0-3 h period was the same in THM and control, but significantly lower in THM at the following 3-6 h period, indicating that the urine concentrating activity is insufficient in THM compared with the control animal. 4. Urinary excretion of vasopressin was significantly higher in THM. Plasma aldosterone concentration and urinary excretion of aldosterone were also higher in THM. Plasma potassium level was significantly low. 5. The mechanism underlying the pathophysiology of polyuria is not totally explained; however, hypokalaemia, which was probably the result of hyperaldosteronism, may be at least partially involved, since hypokalaemia is considered to be a factor hampering the action of vasopressin for concentration of urine at the site of the collecting duct of the kidney.

Synergistic inhibition by BQ-123 and BQ-788 of endothelin-1-induced contractions of the rabbit pulmonary artery.

In the rabbit isolated pulmonary artery, neither the ETA receptor antagonist, BQ-123 (10 microM), nor the ETB receptor antagonist, BQ-788 (10 microM), inhibited the contractions induced by 1 nM endothelin-1 (ET-1). However, the combination of BQ-123 and BQ-788 completely inhibited the ET-1-induced contraction. In contrast, the ETB-selective agonist, sarafotoxin S6c (1 nM)-induced contraction was completely inhibited by BQ-788 but not by BQ-123. In receptor binding assays, [125I]-ET-1 specific binding to pulmonary arterial membranes was inhibited by BQ-123 (1 microM) by approximately 20% and additive treatment with BQ-788 (1 microM) completely inhibited the BQ-123-resistant component of [125I]-ET-1 specific binding. The present study demonstrates synergistic inhibition by BQ-123 and BQ-788 of ET-1-induced contraction of the rabbit pulmonary artery and the coexistence of ETA and ETB receptors, suggesting that the activation of either only ETA or only ETB receptors may be sufficient to cause complete vasoconstriction. Therefore, blockade of both receptor subtypes would be necessary for the inhibition of some ETA/ETB composite types of responses.