Influence of methylation of the histidine ring of [Sar1]angiotensin II on conformation and biological activity

[Sar¹His(1-Me)⁶]ANG II and [Sar¹His(3–Me)⁶]ANG II were synthesized by the solid-phase method and purified by reversed-phase HPLC. ¹H-NMR spectroscopy at 400 MHz demonstrated the presence of two major conformers for both peptides in DMSO, representing cis and trans isomers (ratio 1:3 and 1:4, respectively) due to restricted rotation at the His-Pro bond. The contractile activities of these peptides in the rat isolated uterus assay were <0.1 and 27% of that of ANG II, respectively. The bioactivities of these analogues were mirrored in their NMR spectra: the inactive analogue [Sar¹His(1-Me)⁶]ANG II showed perturbations of the Sar and His residues which were not present for [Sar¹His(3-Me)⁶]ANG II and the reference agonist [Sar¹] ANG II. The high activity of the analogue methylated at His N₃ suggests that an ionizable imidazole proton is not an absolute requirement for expression of biological activity by angotensin ligands, and that the imidazole group in the molecule may function in an ion dipole-based mechanism when an intramolecular proton transfer (charge relay) mechanism is not available. The biological activity of the ligand appears to depend on the degree of proton transfer from Tyr-OH to the imidazole acceptor, wherein complete (formal) proton transfer represents 100%, activity.     

Angiotensin II dose-effect curves and Schild regression plots for characterization of different angiotensin II AT1 receptor antagonists in clinical pharmacology

The 'Schild regression' method is based on the principle of assessing the rightward shift of agonist dose-effect curves in the presence of different doses/concentrations of the respective receptor antagonist and presenting their relationship in a double log plot (i.e. the 'Schild plot'). The original method was developed to quantitatively characterize antagonistic drugs in experimental pharmacology. The method was adopted for evaluation of various AT1 antagonists in humans utilizing (human) angiotensin II as the agonist. Angiotensin II (Ang II) in continuous intravenous dose-incremental administration resulted in a clearly dose-dependent increase in blood pressure. All AT1 antagonists tested after oral administration yielded concentration-dependent rightward shifts of those Ang II dose-effect curves that were quantified as dose ratio (DR). DR minus 1 (DR-1) enabled the assessment of antagonist time kinetics in humans and a quantitatively precise determination of the half-life of antagonism in vivo. Schild plots allowed for assessment of apparent Ki doses indicative of a twofold rightward shift of the Ang II effect, thus providing the means for a rational comparison of the pharmacological potency of many of these compounds, where the Ki doses obtained at 24 h after administration were in the range of 'therapeutic' doses. Schild plots of a variety of substances showed linear relations independent of whether the blockade was deemed surmountable or not. It is therefore assumed that this property does not play a role at clinical doses/concentrations. Slopes slightly below 1 in the Schild plots of all tested antagonists point to a second 'counterregulatory' vasodilatory mechanism of action of Ang II which becomes apparent with AT1 blockade in conditions of high doses/concentrations of Ang II. Concentration vs. effect relationships indicate that if assessed at the same degree of direct vascular antagonism, other effects, such as increase in plasma renin activity, may be present to a varying degree with different antagonists. Thus for irbesartan, the potency to stimulate renin release was found to be at least twice that of candesartan. These observations should stimulate further research into the relevance of these dynamic differences between the various compounds. Thus, methodologies relying on fundamental principles of experimental pharmacology can provide the clinical pharmacologist with powerful tools to measure accurately degree of antagonism and time kinetics and to investigate the nature of receptor antagonism in humans.     

The effects of KT3-671, a new angiotensin II (AT 1) receptor blocker in mild to moderate hypertension

AIMS: To compare the antihypertensive effect, and tolerability and safety of once daily doses of KT3-671 with that of placebo in patients with mild to moderate uncomplicated essential hypertension. METHODS: A randomised, multicentre, double blind, parallel-group comparison of KT3-671 with placebo. Hypertensive patients [Ambulatory Blood Pressure Monitoring (ABPM), mean daytime DBP > 90 mmHg, Office sitting mean DBP 95-114 after a 7-28 day washout period] entered a 2-week, single blind, run-in phase. Patients eligible for the double-blind phase were randomised to receive KT3-671 40 mg, 80 mg, 160 mg or placebo once daily over 4 weeks. The primary end-point was trough mean sitting office DBP. The study had 90% power to detect a 5 mmHg change between treatments and placebo at the 5% level of significance. The secondary end-points were 24 hour, daytime and night time mean ABPM. RESULTS: Office DBP was significantly lower with KT3-671 40 mg but not the other 2 dosage groups (-3.2; 95% CL -6.1 : -0.3 P < 0.03). Office SBP was significantly reduced with all dosage groups (40 mg -5.9, 95% CL -11 : -0.9; 80 mg -4.9, 95% CL -9.9 : 0.1 and 160 mg -5.7, 95% CL -10.8 : -0.7 P < 0.05). All doses of KT3-671 reduced systolic and diastolic ABPM. The number of patients with treatment related adverse events were comparable to placebo (38.8% KT3-671 vs 32.8% placebo). There was some evidence of a dose-response relationship with fall in nocturnal ABPM. CONCLUSIONS: Oral KT3-671 was well tolerated. KT3-671 reduced office systolic BP at all doses and diastolic BP at some of the doses. Due to greater precision and power, the falls in mean ambulatory systolic and diastolic pressure were all significantly lower than placebo.     

Postprandial changes in supine and erect heart rate,systemic blood pressure and plasma noradrenaline and renin activity in normal subjects

Summary The haemodynamic effects of a standard meal were assessed in a balanced cross-over study in eight normal fasting subjects, investigated under conditions applicable to many drug tests.Both the supine and erect diastolic blood pressure were reduced on average by 10 mmHg over the 4 h following the meal.The supine systolic pressure was increased on average by 2 mmHg, a difference of no biological relevance. Erect systolic blood pressure was not affected by eating.Supine heart rate was slightly but significantly increased, but the erect heart rate did not change.Postprandial plasma renin activity was increased. Venous plasma noradrenaline levels in the supine position were not affected by eating and after standing erect, and immobile for 5 min they were only slightly and not-significantly increased.A food-induced vasodepressor response combined with baroreceptor resetting is considered to have occurred in this population. The changes had a gradual onset, reaching their maximum about 2 h after eating and they were still evident after 3 h. Eating should be considered as an important potential source of bias in cardiovascular studies.     

Effects of once daily indapamide and pindolol on blood pressure,plasma aldosterone concentration and plasma renin activity in a general practice setting

Summary Sixteen patients with essential hypertension completed a double blind factorial trial comparing the effects of indapamide (2.5 mg daily) and pindolol (10 mg daily) on blood pressure, heart rate, plasma renin activity and plasma aldosterone concentration. There were four randomised test phases of eight weeks each during which patients received indapamide alone, pindolol alone, indapamide plus pindolol and no active treatment (placebo). Blood pressure and heart rate were measured every two weeks. Supine mean arterial pressure fell from 117 mm Hg in the placebo phase to 111 mm Hg in the indapamide phase, 106 mm Hg in the pindolol phase and 103 mm Hg in the combined indapamide plus pindolol phase. Factorial analysis confirmed that the hypotensive effects of the two drugs were additive, without evidence of potentiation or antagonism. Indapamide caused significant reductions in plasma potassium and chloride, and increases in plasma bicarbonate and urate concentrations; it also caused increases in plasma renin activity and aldosterone concentration. These changes are similar to those observed with thiazide diuretics.     

Effect on blood pressure and the renin-angiotensin system of repeated doses of the converting enzyme inhibitor CGS 14824A

Summary A new, orally active angiotensin converting enzyme (ACE) inhibitor, CGS 14824A, was evaluated in 12 healthy male volunteers. Two groups each of 6 volunteers were given 5 or 10 mg once daily p.o. for 8 days. Four hours after the first and the last morning doses, plasma angiotensin II, aldosterone and plasma converting enzyme activity had fallen, while blood angiotensin I and plasma renin activity had risen. Throughout the study, more than 90% inhibition of ACE was found immediately before giving either the 5 or 10 mg dose and 50% blockade was still present 72 h following the last dose. Based on the determination of ACE, there was no evidence of drug accumulation. No significant change in blood pressure or heart rate was observed during the course of the study. CGS 14824A was an effective, orally active, long-lasting and well tolerated converting enzyme inhibitor.     

Chronic effects of angiotensin II and at1 receptor antagonists in subfornical organ-lesioned rats

1. Angiotensin (Ang) II is known to exert some of its effects centrally via circumventricular organs. These unique central nervous system areas lack the normal blood-brain barrier and, therefore, allow peptide hormones access to the brain. Of these, the subfornical organ (SFO) has been shown to be involved in many of the acute dipsogenic and pressor effects of AngII, but much less is known about the role of the SFO in the chronic effects of AngII. We hypothesized that the SFO is a central site involved in the chronic hypotensive effects of endogenous AT(1) receptor blockade, as well as the chronic hypertensive effects of exogenously administered AngII. 2. In order to test these hypotheses, SFO-lesioned (SFOx) or sham Sprague-Dawley rats were instrumented with venous catheters and radiotelemetric pressure transducers for intravenous administration of losartan or AngII and continuous measurement of blood pressure and heart rate. Rats were given 3 days of saline control infusion (7 mL/day of 0.9% NaCl) and were then infused with either losartan (10 mg/kg per day) or AngII (10 ng/kg per min) for 10 days. 3. By day 4 of losartan treatment, arterial pressure had decreased 24 +/- 2 and 18 +/- 2 mmHg in sham (n = 9) and SFOx (n = 10) rats, respectively. Furthermore, by day 5 of AngII infusion, arterial pressure had increased 12 +/- 3 mmHg in sham rats (n = 9), but only by 4 +/- 1 mmHg in SFOx rats (n = 9). In each treatment group, these attenuated pressure responses in SFOx rats continued through day 10 of treatment. 4. These results support the hypotheses that the SFO plays a role in both the hypotensive effects of chronic AT(1) receptor blockade and the chronic hypertensive phase of exogenously administered AngII.     

Acute effects of a new vasodilator,Ro 12-4713, on blood pressure,plasma renin activity,aldosterone and catecholamine levels,and renal function in hypertensive and normal subjects

Summary Selected cardiovascular and endocrine effects of the new oral vasodilator Ro 12-4713 have been evaluated in an acute single dose study. In five patients with essential hypertension, Ro 12-4713 caused a dose-dependent decrease in supine and upright blood pressure and an increase in heart rate. Initial effects occurred one to 2 h after drug ingestion and maximal effects were noted after five hours and persisted for at least 8 h. Blood pressure was normalized, and the antihypertensive and chronotropic effects persisted for 24 h after a dose of about 300 mg/1.73 m². Plasma and urinary norepinephrine and plasma renin levels tended to be raised, whereas plasma and urinary epinephrine and plasma aldosterone did not change. Changes in supine heart rate were inversely correlated with changes in mean blood pressure (r=–0.60; P<0.02), and positively with those in plasma norepinephrine (r=0.55; P<0.05) and renin (r=0.62, P<0.01); changes in supine plasma renin level were also inversely correlated with those in mean blood pressure (r=–0.65; P<0.01), and positively with those in plasma norepinephrine (r=0.58; P<0.05). 24 h-urinary sodium excretion was significantly (P<0.001) decreased; it was positively correlated with mean blood pressure (r=0.51; P<0.05) and inversely with supine plasma renin activity (r=–0.63; P<0.01). In six normal subjects and six patients with essential hypertension, effective renal plasma flow and the renal clearance of sodium, potassium, calcium and uric acid were not significantly altered five hours after a dose of Ro 12-4713 of about 250 mg/1.73 m²; glomerular filtration rate tended to be slightly decreased, and filtration fraction was significantly (P<0.05) reduced in the hypertensive patients. At the same time blood pressure was decreased and plasma norepinephrine (P<0.01) and renin (ns) were slightly increased in both groups. Ro 12-4713 in a single oral dose of about 300 mg appeared to be a potent, long acting, hypotensive vasodilator.     

The effects of intravenous L-dopa on plasma renin activity, renal function, and blood pressure in man

Summary L-dopa 7 µg·kg^–1·min^–1 was given intravenously over 2 h to six healthy subjects, controlled by an infusion of saline on a separate occasion, with measurement of plasma renin activity (PRA), urinary sodium and potassium excretion, effective renal plasma flow (ERPF), glomerular filtration rate (GFR), blood pressure, and pulse rate.Mean PRA fell by 50% following L-dopa, which was significantly different from the slight rise which occurred after saline infusion. There was a significant increase in urinary sodium excretion and effective renal plasma flow on infusion of L-dopa. Mean diastolic blood pressure fell during L-dopa infusion, in contrast to the slight increase which occurred during the control study.These observations confirm the anticipated renal dopaminergic effects of L-dopa and also suggest a dopaminergic influence on renin release in man.     

Chronic treatment with the new potent vasodilator Ro 12-4713 in moderate to severe hypertension: Effects on blood pressure,endocrine function,sodium and plasma volume

Summary The antihypertensive efficacy and endocrine profile of the new antihypertensive agent, Ro 12-4713, were evaluated in 23 patients (17 men and 6 women) with moderate to severe arterial hypertension. Following addition of Ro 12-4713 to pre-existing therapy with diuretics and beta-blockers or sympatholytics, blood pressure in most of the patients was normalized within one month by a daily dose of 60 to 120 mg. Heart rate was only slightly increased. Orthostatic hypotension was not observed. Weight gain or oedema formation occurred in 14 patients within the first four weeks, but could be controlled satisfactorily by intensified diuretic therapy. Increased hair growth occurred in most of the patients. After a mean duration of treatment of 2.8 months, plasma volume and plasma and urine sodium were unaltered, and plasma potassium was slightly decreased. Plasma renin activity was doubled, whereas plasma aldosterone concentrations were unaltered. Plasma norepinephrine levels were high before and increased only slightly during chronic Ro 12-4713 treatment, whereas urinary norepinephrine excretion was unchanged. Plasma and urinary epinephrine were unaltered by Ro 12-4713. Ro 12-4713 appears to be a potent vasodilator for the combination treatment of hypertension in men.     

Effects of allisartan,a new AT1 receptor blocker,on blood pressure and end-organ damage in hypertensive animals

Aim:

To investigate the effects of allisartan, a new angiotensin II type 1 (AT₁) receptor antagonist, on blood pressure (BP) and end-organ damage (EOD) in hypertensive rats and dogs.

Methods:

First, a single dose of allisartan was given intragastrically to evaluate the BP reduction in spontaneously hypertensive rats (SHRs), two kidney-one clip (2K1C) renovascular hypertensive rats and dogs, and Beagle dogs with angiotensin II-induced hypertension. Second, allisartan was mixed in rat chow for long-term treatment. After 4 months of drug administration, rats were instrumented to determine BP and baroreflex sensitivity (BRS). Observation of morphologic changes was used to estimate EOD. Third, the acute toxicity of allisartan was compared with that of losartan in mice.

Results:

BP was significantly decreased after intragastric administration of allisartan in SHRs, 2K1C rats, 2K1C dogs and Beagle dogs with angiotensin II-induced hypertension. Compared with the control, SHRs that received long-term treatment with allisartan exhibited an improved BRS and organ protective effects. Mice who were administered allisartan experienced less acute toxicity than those treated with losartan.

Conclusion:

Allisartan is highly effective for BP reduction and organ protection with low toxicity.     

Effects of a new long-acting beta-blocker bopindolol (LT 31-200) on blood pressure,plasma catecholamines,renin and cholesterol in patients with arterial hypertension

Summary Bopindolol (LT 31-200), a new, long-acting, non-selective beta-blocker, was given as monotherapy to 13 patients, 12 with essential hypertension and 1 with renovascular hypertension. After a placebo period of 4–6 weeks, bopindolol was given once daily, starting with 1 mg and subsequently increasing at two-weekly intervals to 2 and 4 mg once daily until a diastolic blood pressure⩽90 mmHg was achieved. The effective dose was continued for 12 weeks. In 10 patients plasma levels of renin, noradrenaline, adrenaline and cholesterol were measured during placebo and after 3 months of therapy. Blood pressure and heart rate were lowered significantly during bopindolol treatment. The mean effective dose was 2.2 mg per day. In 10/13 patients a diastolic blood pressure⩽90 mmHg was achieved. Side effects were minimal. Changes in plasma noradrenaline and adrenaline were small and not significant, but renin and cholesterol were significantly reduced. Thus, LT 31-200 is an effective and well tolerated beta-blocker when given in a once daily dosage.     

Effects of low-dose atenolol on postural and postprandial changes in heart rate,blood pressure,venous plasma catecholamines,and plasma renin activity

Summary The administration of a single dose of atenolol 50 mg 1 h before a standard 3100 kJ cold meal in fasting healthy subjects reduced the supine preprandial heart rate and systolic blood pressure, and blunted the postural and postprandial rises in mean heart rate and systolic blood pressure relative to placebo. It did not affect the preprandial supine diastolic blood pressure, nor the postural rise and postprandial drop in diastolic blood pressure.Preprandial administration of atenolol blunted the postural and postprandial rises in mean plasma renin activity, and it enhanced the rise in plasma noradrenaline during eating in the sitting position, and the postprandial concentrations of noradrenaline.The findings do not permit the conclusion that beta₁-adrenergic stimulation was the predminant cause of these atenolol-responsive changes.     

A prospective study of the effects of prolonged timolol therapy on α- and β-adrenoceptor and angiotensin II receptor mediated responses in normal subjects

Aims Long-term treatment with β₁-selective adrenergic antagonists gives rise to cross-sensitisation of cardiac β₂-adrenoceptor responses, with no corresponding alteration in β₁-adrenoceptor responses. We performed a prospective randomised double-blind placebo-controlled cross-over study of the effects of nonselective β-blockade with timolol on α-adrenergic and angiotensin II receptor mediated responses in normal subjects. We also wished to study the time course of β₁- and β₂-adrenergic responses after withdrawal of timolol. Methods Six healthy males received timolol 10 mg twice daily or placebo for 14 days. On day 11 of treatment, vascular α₁-, α₂- and angiotensin II receptor responses were assessed by measuring the blood pressure increases in response to intravenous phenylephrine, α-methylnoradrenaline and angiotensin amide respectively, following one dose of timolol 10 mg (to block the β-adrenergic effects of phenylephrine and α-methylnoradrenaline). Both systolic and diastolic blood pressure increased in response to each of these drugs, but these increases were not different on timolol treatment or placebo. Following cessation of treatment with timolol or placebo, β₁- and β₂-adrenoceptor mediated responses were assessed by measuring the heart rate responses to treadmill exercise and intravenous salbutamol infusion respectively. Half each of the subjects underwent this 2 days and 3 days respectively, after the end of treatment. Results Both exercise-induced and salbutamol-induced tachycardia were not different following placebo or 3 days following the end of timolol treatment. However, 2 days following timolol treatment, both were attenuated; the reduction in salbutamol-induced tachycardia was significant, whilst the reduction in exercise tachycardia did not reach statistical significance. We also measured metabolic responses to exercise and to salbutamol infusion. Exercise induced a rise in plasma potassium and noradrenaline. Salbutamol produced a fall in plasma potassium, a rise in plasma glucose and insulin and also a rise in plasma noradrenaline. All of these changes were not different following placebo or 3 days after the end of timolol treatment; by contrast, 2 days following timolol treatment, all were significantly attenuated, with the exception of the rise in plasma glucose. In addition, the rise in both plasma glucose and insulin in response to an oral load of 75 g glucose were not different post-placebo, 2 or 3 days post-timolol. Conclusions These results show that, following 14 days of nonselective β-adrenoceptor blockade with timolol, there is evidence of residual β-adrenoceptor blockade 2 days after drug withdrawal; this finding is in contrast with the known plasma profile of timolol (half-life 3–6 hours), but is consistent with our previous observations of the slow speeds of association and dissociation of timolol with β-adrenoceptors in vitro. There is no evidence, in this study, of β-adrenergic sensitisation following timolol withdrawal, nor of cross-regulation of vascular α₁-, α₂- or angiotensin II receptors in response to nonselective β-adrenoceptor blockade.     

Effects of allisartan,a new AT1 receptor blocker,on blood pressure and end-organ damage in hypertensive animals

To investigate the effects of allisartan, a new angiotensin Ⅱ type 1 （AT1） receptor antagonist, on blood pressure （BP） and end-organ damage （EOD） in hypertensive rats and dogs.
Methods： First, a single dose of allisartan was given intragastrically to evaluate the BP reduction in spontaneously hypertensive rats （SHRs）, two kidney-one clip （2KIC） renovascular hypertensive rats and dogs, and Beagle dogs with angiotensin Ⅱ-induced hypertension. Second, allisartan was mixed in rat chow for long-term treatment. After 4 months of drug administration, rats were instrumented to determine BP and baroreflex sensitivity （BRS）. Observation of morphologic changes was used to estimate EOD. Third, the acute toxicity of allisartan was compared with that of losartan in mice.
Results： BP was significantly decreased after intragastric administration of allisartan in SHRs, 2KIC rats, 2KIC dogs and Beagle dogs with angiotensin Ⅱ-induced hypertension. Compared with the control, SHRs that received long-term treatment with allisartan exhibited an improved BRS and organ protective effects. Mice who were administered allisartan experienced less acute toxicity than those treated with losartan.
Conclusion： Allisartan is highly effective for BP reduction and organ protection with low toxicity.     

Endothelial cell-specific aryl hydrocarbon receptor knockout mice exhibit hypotension mediated, in part, by an attenuated angiotensin II responsiveness

Hypotension in aryl hydrocarbon receptor knockout mice (ahr^−/−) is mediated, in part, by a reduced contribution of angiotensin (Ang) II to basal blood pressure (BP). Since AHR is highly expressed in endothelial cells (EC), we hypothesized that EC-specific ahr^−/− (ECahr^−/−) mice would exhibit a similar phenotype. We generated ECahr^−/− mice by crossing AHR floxed mice (ahr^fx/fx) to mice expressing Cre recombinase driven by an EC-specific promoter. BP was assessed by radiotelemetry prior to and following an acute injection of Ang II or chronic treatment with an angiotensin converting enzyme inhibitor (ACEi). ECahr^−/− mice were hypotensive (ECahr^+/+: 116.1 ± 1.4; ECahr^−/−: 107.4 ± 2.0 mmHg, n = 11, p < 0.05) and exhibited significantly different responses to Ang II and ACEi. While Ang II increased BP in both genotypes, the increase was sustained in ECahr^+/+, whereas the increase in ECahr^−/− mice steadily declined. Area under the curve analysis showed that Ang II-induced increase in diastolic BP (DBP) over 30 min was significantly lower in ECahr^−/− mice (ECahr^+/+ 1297 ± 223 mmHg/30 min; ECahr^−/−_AUC: 504 ± 138 mmHg/30 min, p < 0.05). In contrast, while ACEi decreased BP in both genotypes, the subsequent rise in DBP after treatment was significantly delayed in the ECahr^−/− mice. ECahr^−/− mice also exhibited reduced vascular and adipose Ang II type 1 receptor (AT1R) expression, and reduced aortic Ang II-dependent vasoconstriction in the presence of vascular adipose. Taken together these data suggest that hypotension in ECahr^−/− mice results from reduced vascular responsiveness to Ang II that is influenced by AT1R expression and adipose.