Comparison of electrocardiographic analysis for risk of QT interval prolongation using safety pharmacology and toxicological studies.

Testing for possible cardiovascular side effects of new drugs has been an essential part of drug development for years. A more detailed analysis of the electrocardiogram (ECG) to detect effects on ventricular repolarization (effects on the QT interval), as a marker for possible proarrhythmic potential has been added to that evaluation in recent years. State-of-the art evaluation of drug-induced effects on the QT interval have evolved, but due to the complexity of the assessment, the trend in safety pharmacology studies has been to collect large numbers of high quality ECGs to allow for a robust assessment including the influence of heart rate on the QT interval apart from possible drug-induced effects. Since an assessment of the ECG is often included in toxicological studies, one can consider making such an assessment using ECG data from routine toxicological studies. This review summarizes various aspects of both safety pharmacology and toxicology studies with regards to their impact on the quality and quantity of ECG data that one can reasonably derive. We conclude that ECG data from toxicological studies can offer complementary ECG data that can strengthen a risk assessment. However, for the great majority of standard toxicity studies conducted, the ECG data collected do not permit an adequate assessment of drug-induced effects on the QT interval with the sensitivity expected from the ICH S7B guidelines. Furthermore, sponsors should be discouraged from performing any analyses on low quality ECGs to avoid generating misleading data. Substantial improvements in ECG quality and quantity are available, thereby making a QT interval assessment within the context of a standard toxicological study feasible, but these methods may require a larger commitment of resources from the sponsor. From the viewpoint of risk mitigation and limiting the attrition of promising new therapies, a commitment of resources to insure ECG data quality in either toxicology or safety pharmacology studies may be well justified.

Torcetrapib-induced blood pressure elevation is independent of CETP inhibition and is accompanied by increased circulating levels of aldosterone.

BACKGROUND AND PURPOSE: Inhibition of cholesteryl ester transfer protein (CETP) with torcetrapib in humans increases plasma high density lipoprotein (HDL) cholesterol levels but is associated with increased blood pressure. In a phase 3 clinical study, evaluating the effects of torcetrapib in atherosclerosis, there was an excess of deaths and adverse cardiovascular events in patients taking torcetrapib. The studies reported herein sought to evaluate off-target effects of torcetrapib. EXPERIMENTAL APPROACH: Cardiovascular effects of the CETP inhibitors torcetrapib and anacetrapib were evaluated in animal models. KEY RESULTS: Torcetrapib evoked an acute increase in blood pressure in all species evaluated whereas no increase was observed with anacetrapib. The pressor effect of torcetrapib was not diminished in the presence of adrenoceptor, angiotensin II or endothelin receptor antagonists. Torcetrapib did not have a contractile effect on vascular smooth muscle suggesting its effects in vivo are via the release of a secondary mediator. Treatment with torcetrapib was associated with an increase in plasma levels of aldosterone and corticosterone and, in vitro, was shown to release aldosterone from adrenocortical cells. Increased adrenal steroid levels were not observed with anacetrapib. Inhibition of adrenal steroid synthesis did not inhibit the pressor response to torcetrapib whereas adrenalectomy prevented the ability of torcetrapib to increase blood pressure in rats. CONCLUSIONS AND IMPLICATIONS: Torcetrapib evoked an acute increase in blood pressure and an acute increase in plasma adrenal steroids. The acute pressor response to torcetrapib was not mediated by adrenal steroids but was dependent on intact adrenal glands.

Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development.

OBJECTIVE: To attempt to determine the relative value of preclinical cardiac electrophysiology data (in vitro and in vivo) for predicting risk of torsade de pointes (TdP) in clinical use. METHODS: Published data on hERG (or I(Kr)) activity, cardiac action potential duration (at 90% repolarisation; APD(90)), and QT prolongation in dogs were compared against QT effects and reports of TdP in humans for 100 drugs. These data were set against the free plasma concentrations attained during clinical use (effective therapeutic plasma concentrations; ETPC(unbound)). The drugs were divided into five categories: (1) Class Ia and III antiarrhythmics; (2) Withdrawn from market due to TdP; (3) Measurable incidence/numerous reports of TdP in humans; (4) Isolated reports of TdP in humans; (5) No reports of TdP in humans. RESULTS: Data from hERG (or I(Kr)) assays in addition to ETPC(unbound) data were available for 52 drugs. For Category 1 drugs, data for hERG/I(Kr) IC(50), APD(90), QTc in animals and QTc in humans were generally close to or superimposed on the ETPC(unbound) values. This relationship was uncoupled in the other categories, with more complex relationships between the data. In Category 1 (except amiodarone), the ratios between hERG/I(Kr) IC(50) and ETPC(unbound) (max) ranged from 0.1- to 31-fold. Similar ranges were obtained for drugs in Category 2 (0.31- to 13-fold) and Category 3 (0.03- to 35-fold). A large spread was found for Category 4 drugs (0.13- to 35700-fold); this category embraced an assortment of mechanisms ranging from drugs which may well be affecting I(Kr) currents in clinical use (e.g. sparfloxacin) to others such as nifedipine (35700-fold) where channel block is not involved. Finally, for the majority of Category 5 drugs there was a >30-fold separation between hERG/I(Kr) activity and ETPC(unbound) values, with the notable exception of verapamil (1.7-fold), which is free from QT prolongation in man; this is probably explained by its multiple interactions with cardiac ion channels. CONCLUSIONS: The dataset confirms the widely-held belief that most drugs associated with TdP in humans are also associated with hERG K(+) channel block at concentrations close to or superimposed upon the free plasma concentrations found in clinical use. A 30-fold margin between C(max) and hERG IC(50) may suffice for drugs currently undergoing clinical evaluation, but for future drug discovery programmes, pharmaceutical companies should consider increasing this margin, particularly for drugs aimed at non-debilitating diseases. However, interactions with multiple cardiac ion channels can either mitigate or exacerbate the prolongation of APD and QT that would ensue from block of I(Kr) currents alone, and delay of repolarisation per se is not necessarily torsadogenic. Clearly, an integrated assessment of in vitro and in vivo data is required in order to predict the torsadogenic risk of a new candidate drug in humans.

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.

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.

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.

Lack of beneficial effects of growth hormone treatment in conscious dogs during development of heart failure.

The effects of chronic treatment with growth hormone (porcine GH, 0.56 mg.kg-1.day-1 s.c.) were examined in dogs with heart failure induced by rapid ventricular pacing (240 beats/min) for 4 wk. Fourteen conscious dogs were studied 2-3 wk after surgical instrumentation with catheters in the descending aorta and left atrium, a pressure gauge in the left ventricle (LV), a flow probe around the ascending aorta, pacing leads on the ventricular free wall and left atrium, and ultrasonic crystals on the opposing anterior and posterior endomyocardium of the LV. GH treatment for 4 wk significantly increased both body weight and plasma insulin-like growth factor 1 (IGF-1) compared with vehicle-treated dogs (P < 0.01, +2.0 +/- 0.5 vs. +0.3 +/- 1.1 kg; 1,043 +/- 218 vs. 241 +/- 64 ng/ml, respectively). However, the changes in resting LV systolic (i.e., both isovolumic and ejection phases) and diastolic function (i.e., isovolumic relaxation time constant tau) and the systemic vascular resistance were similar for the GH- and vehicle-treated groups during the development of heart failure. LV contractile reserve, assessed with step infusion of isoproterenol or dobutamine challenge, was markedly attenuated after heart failure, but there were no differences between the GH- and vehicle-treated groups. During the progression of heart failure, the increases in plasma atrial natriuretic peptide correlated (P < 0.01) directly with left atrial pressure and inversely with LV circumferential fiber shortening. However, GH treatment did not substantially modify these relationships. In addition, renal function and myocardial ultrastructure at the advanced stage of heart failure also showed similar changes for the GH- and vehicle-treated groups. We conclude that in conscious dogs during the development of congestive heart failure produced by rapid ventricular pacing, GH at a dose that increases body weight and plasma IGF-1 levels does not affect LV performance or systemic vascular dynamics.

In vitro pharmacology of an angiotensin AT1 receptor antagonist with balanced affinity for AT2 receptors.

L-163,017 (6-[benzoylamino]-7-methyl-2-propyl-3-[[2'-(N-(3-methyl-1-butoxy) carbonylaminosulfonyl) [1,1']-biphenyl-4-yl]methyl]-3H-imidazo[4,5-b]pyridine) inhibited specific 125I-[Sar1, Ile8]angiotensin II binding to angiotensin AT1 receptor (Ki = 0.11-0.20 nM) in rabbit aorta, rat adrenal and human angiotensin AT1 receptor in CHO (Chinese hamster ovary transformed) cells and to AT2 receptor (Ki = 0.14-0.23 nM) in rat adrenal and brain receptors. L-163,017 also had a high affinity in the presence of bovine serum albumin (2 mg/ml), for angiotensin AT1 and AT2 receptors on human adrenal (Ki 3.9 and 4.3 nM), aorta (Ki 0.45 and 0.96 nM) and kidney (Ki 3.6 and 2.3 nM). The much higher Ki values in human tissues were likely due to the presence of bovine serum albumin in the binding assay buffer since L-163,017 had Ki values of 0.13 +/- 0.04 and 2.0 +/- 0.04 nM in the absence and presence of bovine serum albumin, respectively, in inhibiting 125I-[Sar1,Ile8]angiotensin II binding to angiotensin AT1 receptor in rat adrenal membranes. Scatchard analysis of 125I-[Sar1,Ile8]angiotensin II binding in the presence of bovine serum albumin (2 mg/ml) in rabbit aorta and bovine cerebellum indicated a competitive interaction of L-163,017 with angiotensin AT1 and AT2 receptors (Ki values 2.5 and 2.1 nM respectively). L-163,017 inhibited angiotensin II-induced aldosterone release in rat adrenal demonstrating that L-163,017 acted as a competitive antagonist (pA2 = 9.9) and lacked agonist activity. L-163,017 also inhibited angiotensin II responses in rat vascular tissues. The specificity of L-163,017 was shown by its lack of activity on the above functional responses produced by other agonists and in several binding assays.

In vivo pharmacology of an angiotensin AT1 receptor antagonist with balanced affinity for AT2 receptors.

L-163,017 (6-[benzoylamino]-7-methyl-2-propyl-3-[[2'-(N-(3-methyl-1-butoxy) carbonylaminosulfonyl)[1,1']-biphenyl-4-yl]methyl]-3H-imidazo[4,5- b]pyridine) is a potent, orally active, nonpeptide angiotensin II receptor antagonist. Conscious rats and dogs were dosed p.o. and i.v.; in both species the plasma bioequivalents are similar at the angiotensin AT1 and AT2 receptor sites indicating balanced activity is maintained in vivo. L-163,017 prevents the pressor response to intravenous (i.v.) angiotensin II in the conscious rat, dog, and rhesus monkey. L-163,017 also significantly reduces blood pressure in a renin-dependent model of hypertension, similar to an angiotensin converting enzyme inhibitor (Enalapril) and an angiotensin AT1 receptor-selective antagonist (L-159,282). These studies indicate that neither the angiotensin AT2 receptor nor bradykinin is important in the acute antihypertensive activity of angiotensin converting enzyme inhibitors or angiotensin II receptor antagonists.

Potent and orally active angiotensin II receptor antagonists with equal affinity for human AT1 and AT2 subtypes.

In order to block the effects induced by the interactions between angiotensin II (AII) and both AT1 and AT2 receptors, we have pursued the discovery of orally active non-peptide AII antagonists that exhibit potent and equal affinity for human AT1 and AT2 receptor subtypes. A series of previously prepared nanomolar (IC50) trisubstituted 1,2,4-triazolinone biphenyl-sulfonamide dual-acting AII antagonists has been modified at five different positions in order to increase AT2 binding affinity, maintain AT1 activity, and reduce the human adrenal AT2/AT1 potency ratio (IC50 ratio) from > or = 10. The targeted human adrenal potency ratio of < or = 1 was achieved with a number of compounds possessing an ethyl group at C5 of the triazolinone and a 3-fluoro substituent at the N4-biarylmethyl moiety. The most favored of these was compound 44 which exhibited subnanomolar potency at both the AT1 (rabbit aorta) and AT2 (rat midbrain) receptors, with a slight preference for the latter, and had a human adrenal AT2/AT1 IC50 ratio of 1. This tert-butyl sulfonylcarbamate with an N2-[2-bromo-5-(valerylamino)phenyl] substituent had excellent iv activity at 1 mg/kg (100% peak inhibition, > or = 4 h duration of action) and is orally active at 3 mg/kg with > 6 h duration of action in a conscious rat model. The present study shows that the NH of the amide on the N2-aryl moiety is not required for subnanomolar binding affinity to either receptor subtype, although a keto functionality at this position is essential for acceptable AT2 binding. Receptor-ligand binding interactions derived from the structure-activity relationships are discussed with respect to both receptor subtypes.

In vivo pharmacology of L-159,913, a new highly potent and selective nonpeptide angiotensin II receptor antagonist.

The present study was designed to characterize the in vivo pharmacology of L-159,913 (4-[[2'-(N-benzoylsulfamoyl)biphenyl-4-yl]-5butyl-2,4-dihydr o-2- [2-(trifluoromethyl)phenyl]-3H-1,2,4-triazol-3-one); a potent All receptor antagonist. In normotensive rats, dogs, rhesus monkeys, and chimpanzees, L-159,913 inhibited All-induced elevations in blood pressure. In conscious rats, the relative potencies (ED50) were 0.51 mg/kg i.v. and 0.72 mg/kg p.o. Duration of action with single i.v. or p.o. doses exceeded 6 hr in rats. L-159,913 was 3 times less potent than losartan in rats and equipotent to losartan in monkeys. All induced elevation of plasma aldosterone in rats was also inhibited by L-159,913. L-159,913 was antihypertensive in high renin hypertensive rats (aortic coarctation). The maximum hypotensive response to an acute dose of L-159,913 (10 mg/kg, po) was equal to that of enalaprilat (0.3 mg/kg, iv) in this renin dependent animal model. In conscious normotensive dogs, L-159,913 had a moderate diuretic, natriuretic and kaliuretic response with no effect on glomerular filtration rate, effective renal plasma flow or filtration fraction, suggesting a tubular site of action. L-159,913 is a selective and potent All receptor antagonist with good oral activity, long duration of action and antihypertensive efficacy.

Pharmacology of losartan, an angiotensin II receptor antagonist, in animal models of hypertension.

BACKGROUND: Clinical experience with angiotensin converting enzyme (ACE) inhibitors has shown that inhibition of the renin-angiotensin system is effective therapy for hypertension and heart failure. Losartan (DuP753, MK954, cozaar) is the first non-peptidic drug that inhibits the renin-angiotensin system by selectively blocking the interaction of angiotensin II with its receptor. DIFFERENCES BETWEEN LOSARTAN AND ACE INHIBITORS: Pharmacological differences between ACE inhibitors and losartan could affect comparative efficacy and/or safety. In addition to angiotensin I, ACE has other substrates (e.g. kinins). Blocking the metabolism of kinins with ACE inhibitors could be beneficial (e.g. vasodilation) and/or elicit side effects (e.g. cough) which will not be produced by losartan. Non-ACE pathways of angiotensin II formation have been described (e.g. angiotensin I convertase) which may limit the ability of ACE inhibitors to prevent formation of angiotensin II in all tissues. Losartan blocks angiotensin II responses irrespective of the route or site of angiotensin II formation. Two binding sites for angiotensin II are widely accepted, AT1 and AT2. Losartan blocks only AT1 sites while ACE inhibitors functionally block angiotensin II interaction with both sites. Since the physiological role for AT2 sites is unknown, the relevance of this difference between ACE inhibitors and losartan is questionable. HYPERTENSION: In animal models of hypertension, the efficacy of losartan is equivalent to the efficacy of ACE inhibitors. In animal models that reflect complications of hypertension, such as kidney dysfunction, cardiac and vascular hypertrophy and stroke, losartan and ACE inhibitors are also equally effective. From these results, kinin potentiation and lack of inhibition of angiotensin I convertase do not lead to differences in pharmacological efficacy between ACE inhibitors and losartan. Therefore, with respect to therapeutic efficacy, results in animal models indicate that losartan will display the beneficial pharmacology of ACE inhibitors without the detrimental side effects attributed to kinin potentiation.

In vivo receptor occupancy of the angiotensin II receptor by nonpeptide antagonists: relationship to in vitro affinities and in vivo pharmacologic potency.

The affinities of 13 angiotensin II antagonists for the AT1 subtype determined in vitro with tissue homogenates were shown not to correlate well with in vivo pharmacologic potency. The addition of human serum albumin to the in vitro assay to mimic in vivo plasma protein interactions reduced the measured affinity by reducing the effective free concentrations of antagonists, but the resulting affinities were not predictive of the in vivo effects. Using an in vivo radioligand competition assay, in which receptor occupancy is demonstrated via competitive blockade of the in vivo binding of [125I][Sar1,Ile8]angiotensin II to AT1 receptors in rat kidney cortex, we demonstrated that the in vivo pharmacologic potencies reflect receptor occupancy. By comparing the effects of rat plasma and bovine serum albumin on the in vitro affinity of two antagonists, we suggest that the use of plasma would alter free plasma concentrations in a manner more consistent with in vivo measures of potencies.

Triazolinone biphenylsulfonamides as angiotensin II receptor antagonists with high affinity for both the AT1 and AT2 subtypes.

Angiotensin II (AII), the endogenous peptide ligand of the AII receptor, has equivalent high affinity for both the AT1 and AT2 receptor subtypes while most of the reported nonpeptide AII antagonists are AT1-selective. In an effort to identify dual AT1/AT2 nonpeptide AII antagonists, we have pursued modifications of previously prepared trisubstituted 1,2,4-triazolinone biphenylsulfonamides which exhibited subnanomolar in vitro AT1 (rabbit aorta) AII antagonism and AT2 (rat midbrain) IC50 values of < 40 nM. Present results show that a suitable amide (or reversed amide) side chain appropriately positioned on the N2-aryl group of these compounds gave > 15-fold enhancement in AT2 binding affinity without sacrificing nanomolar AT1 potency (IC50). This added amide, combined with an appropriate choice of the N-substituent on the sulfonamide and the ortho substituent on the N2-aryl group, led to an analogue (46, L-163,-007) which exhibited subnanomolar AT1 binding affinity and an AT2/AT1 IC50 ratio of 3. This compound showed excellent iv activity at 1 mg/kg and oral efficacy at 3 mg/kg with > 6 h duration in a conscious rat model. Available data suggest that the newly introduced amide side chain, mandatory for low nanomolar binding affinity at the AT2 receptor, is well-tolerated by the AT1 receptor and has minimal effect on the in vivo properties of these molecules.

Juxtaglomerular cell hypertrophy and hyperplasia induced in rhesus monkeys by angiotensin II receptor antagonists.

BACKGROUND: Juxtaglomerular (JG) cell hypertrophy and hyperplasia were investigated in rhesus monkeys given angiotensin II (AII) AT1 receptor antagonists L-158,338 and DUP 753 (MK-0954, losartan). EXPERIMENTAL DESIGN: In 2 initial studies, L-158,338 was given orally at 10, 30, and 90 mg/kg/day for 3 or 14 weeks. To investigate the observed JG hypertrophy and hyperplasia, in a third 5-week experiment L-158,338 was given alone at 90 mg/kg/day, or with physiologic saline supplementation at 25 ml/kg/day, or coadministered with the angiotensin converting enzyme inhibitor enalapril at 10 mg/kg/day. Physiologic saline was given to attempt to suppress renin release through volume expansion and/or sodium retention. Enalapril was given to lower plasma AII levels and observe whether JG cell hypertrophy and hyperplasia were increased or decreased. For comparison, DUP 753 was given at 90 and 300 mg/kg/day. Plasma renin activity and AII concentration were measured in this study. RESULTS: Dose- and time-dependent increases in JG cell hypertrophy and hyperplasia were seen in the 2 initial experiment. In the third experiment, plasma renin activity and AII concentration were increased 3-fold and 6-fold over pretest values by L-158,338 at 90 mg/kg/day for 5 weeks. Saline supplementation had no effect on these parameters but diminished the group mean severity grade for JG hypertrophy and hyperplasia from 1.5 to 1.0. Enalapril coadministration had no effect on plasma renin activity, whereas it blunted the plasma AII increase caused by L-158,338 and increased the group mean grade to 2.5. DUP 753 at 300 mg/kg/day produced similar increases in plasma renin activity and AII concentration but only resulted in grade 1 JG cell hypertrophy and hyperplasia. CONCLUSIONS: L-158,338-induced JG cell hypertrophy and hyperplasia is an exaggerated pharmacologic response that can be modulated by saline supplementation and angiotensin converting enzyme inhibition. These results suggest that decreased renal perfusion or altered sodium homeostasis and plasma AII concentration are important variables that contribute to AT1 receptor blockade to induce JG cell hypertrophy and hyperplasia.

Effects of BQ-123 on renal function and acute cyclosporine-induced renal dysfunction.

Cyclosporin A (CsA) is widely used to suppress graft rejection following transplantation and in the treatment of a variety of autoimmune diseases. Therapy with CsA is often accompanied by adverse effects which include hepatotoxicity, hypertension, and nephrotoxicity. The role of endothelin (Et) in CsA-induced nephrotoxicity has been the subject of recent investigations. BQ-123 is a recently discovered Et receptor antagonist which is selective for the EtA receptor. In the present study, BQ-123 was used to further characterize the role of Et in CsA-induced nephrotoxicity. All experiments were performed in Inactin (100 mg/kg, i.p.) anesthetized male Munich-Wistar rats (250 to 350 g). Animals were prepared for the recording of blood pressure (MAP) and heart rate (HR) as well as the measurement of urine volume (UV), UNaV, UKV, GFR and effective renal plasma flow (ERPF). GFR and ERPF were estimated from the clearance of 14C-inulin and 3H-PAH, respectively. On the day of the experiment, animals were randomly assigned to one of three groups and treated according to the following protocols: Group 1, pretreatment with BQ-123 (1 mg/kg, i.v. bolus with 0.1 mg/kg/hr i.v. infusion) followed by treatment with vehicle (cremophor; 0.15 ml, i.v.); Group 2, pretreatment with normal saline (1.0 ml/kg; plus 25 microliters/min infusion) followed by treatment with CsA (20 mg/kg, i.v.); and Group 3, pretreatment with BQ-123 (same as group 1) followed by CsA (20 mg/kg, i.v.). BQ-123 administration alone produced transient changes in several of the measured parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-peptide angiotensin II receptor antagonists. 1. Design, synthesis, and biological activity of N-substituted indoles and dihydroindoles.

A series of N-acylated indoles (12-18), N-alkylated indoles (19-24), N-acylated dihydroindoles (26-30), and N-alkylated dihydroindoles (31-34) were synthesized and evaluated in the in vitro AT1 (rabbit aorta) and AT2 (rat midbrain) binding assay. The carboxylic acid 3-[[N-(2-carboxy-3,6-dichlorobenzoyl)-5-indolyl]methyl]-5,7-dimeth yl- 2-ethyl-3H-imidazo[4,5-b]pyridine (14b) was found to be the most potent AT1 (IC50 = 0.8 nM) antagonist in the N-acylated indole series and displayed a 25-fold higher potency than the parent unsubstituted derivative 14a (AT1 IC50 = 20 nM) and a 22-fold greater potency than the corresponding dihydroindole analog 27 (AT1 IC50 = 18 nM). Replacement of the terminal carboxyl (COOH) of 14a with the bioisostere tetrazole in 16 (AT1 IC50 = 5 nM, AT2 IC50 = 130 nM) not only improved the AT1 potency by 4-fold but also resulted in a 50-fold increase in AT2 activity. In the N-alkylated indole series, the tetrazole 3-[[N-(2-tetrazol-5-yl-6-chlorobenzyl)-5- indolyl]methyl]-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine (24) exhibited the highest AT1 (IC50 = 1 nM) activity, revealing a 230-fold increase in AT1 activity as a result of the incorporation of the isosteric tetrazole for the carboxyl (COOH) of 20 and a nearly 9-fold increase over the corresponding deschloro analog 22 (AT1 IC50 = 8.7 nM). Tetrazole 34 was identified as the most potent (AT1 IC50 = 18 nM) AT1 receptor antagonist in a structurally distinct series of compounds derived from N-alkylation of dihydroindole 25. A new class of highly potent (14b, AT1 IC50 = 0.8 nM; 24, AT1 IC50 = 1 nM) AT1-selective non-peptide AII receptor antagonists derived from N-substituted indoles and dihydroindoles is disclosed. Tetrazole 24 of the N-alkylated indole series displayed good in vivo activity by blocking the AII-induced pressor response for 5.5 h after intravenous administration in conscious normotensive rats at a 1.0 mg/kg dose level.

Non-peptide angiotensin II receptor antagonists. 2. Design, synthesis, and biological activity of N-substituted (phenylamino)phenylacetic acids and acyl sulfonamides.

The design, synthesis, and biological activity of a new class of highly potent non-peptide AII receptor antagonists derived from N-substituted (phenylamino)phenylacetic acids and acyl sulfonamides which exhibit a high selectivity for the AT1 receptor are described. A series of N-substituted (phenylamino)phenylacetic acids (9) and acyl sulfonamides (16) and a tetrazole derivative (19) were synthesized and evaluated in the in vitro AT1 (rabbit aorta) and AT2 (rat midbrain) binding assay. The (phenylamino)phenylacetic acids 9c (AT1 IC50 = 4 nM, AT2 IC50 = 0.74 microM), 9d (AT1 IC50 = 5.3 nM, AT2 IC50 = 0.49 microM), and 9e (AT1 IC50 = 5.3 nM, AT2 IC50 = 0.56 microM) were found to be the most potent AT1-selective AII antagonists in the acid series. Incorporation of various substituents in the central and bottom phenyl rings led to a decrease in the AT1 and AT2 binding affinity of the resulting compounds. Replacement of the carboxylic acid (CO2H) in 9c, 9d, and 9e with the bioisostere acyl sulfonamide (CONHSO2Ph) resulted in a (5-7)-fold increase in the AT1 potency of 16a (AT1 IC50 = 0.9 nM, AT2 IC50 = 0.2 microM), 16b (AT1 IC50 = 1 nM, AT2 IC50 = 2.9 microM), and 16c (AT1 IC50 = 0.8 nM, AT2 IC50 = 0.42 microM) and yielded acyl sulfonamides with subnanomolar AT1 activity. Incorporation of the acyl sulfonamide (CONHSO2Ph) for the CO2H of 9c not only enhanced the AT1 potency but also effected a marked increase in the AT2 potency of 16a (AT2 IC50 = 0.74 microM of 9c vs 0.2 microM of 16a) and made it the most potent AT2 antagonist in this study. Replacement of the CO2H of 9b with the bioisostere tetrazole resulted in 19 (AT1 IC50 = 15 nM) with a 2-fold loss in the AT1 and a complete loss in the AT2 binding affinity. (Phenylamino)phenylacetic acid 9c demonstrated good oral activity in AII-infused conscious normotensive rats at an oral dose of 1.0 mg/kg by inhibiting the pressor response for > 6 h. Acyl sulfonamides 16a-c displayed excellent in vivo activity by blocking the AII-induced pressor response for > 6 h after oral administration in conscious rats at a 3.0 mg/kg dose level. Both acyl sulfonamides 16a and 16c exhibited superior in vivo activity in rats compared to that of (phenylamino)phenylacetic acid 9c.