The renin-angiotensin-aldosterone system (RAAS) appears to play an important role in blood pressure (BP) regulation and in the pathogenesis of hypertension and congestive heart failure (1-4). There exist at least two cellular receptors for the vasoconstrictive octapeptide angiotensin II (Ang II) (5). The AT1 receptor is involved in most of the clinical effects of Ang II (e.g., contraction of vascular smooth-muscle cells, proximal tubular uptake of sodium in the kidney, or decrease in renal blood flow). The role of the AT2 receptor, the second receptor, is not yet understood (5).
Some years ago, only peptide analogs of Ang II like saralasin could be used to block these receptors (6-8), but agonistic properties and the lack of oral bioavailability limited the clinical use of these substances. Clinically much more successful were therefore angiotensin-converting enzyme inhibitors (ACEIs), which inhibit the generation of Ang II (9-11). But ACE, a nonspecific carboxypeptidase, also known as kininase II, is not a specific enzyme that exclusively generates Ang II: some side-effects such as cough have been attributed to angiotensin-independent actions (12,13). Accordingly, more specific inhibitors of Ang II seemed desirable to further define the regulatory role of the RAAS and possibly to avoid side effects observed with ACEI treatment. The first nonpeptidic orally active specific AT1-receptor antagonist losartan became available for clinical use a few years ago (14-16), and this compound is marketed in several countries. We describe the evaluation of another orally active imidazole derivative with AT1-blocking properties in normal human subjects. Clinical and hormonal effects and pharmacokinetics of SC-52485 were assessed by repeated intravenous Ang II challenges and monitoring of finger BP, heart rate, plasma RAAS, and drug levels.
Sixteen male volunteers, average age 27 years (range, 21-34), weight 71.3 kg (range, 60-85), were included in the study. They gave written consent to participate after having received full explanation on the purpose and risks of the study. The protocol had been approved by the local ethics committee. The subjects were considered to be healthy according to medical history, physical examination, routine blood and urine analysis, BP <140/90 mm Hg (mean BP, 116/70 mm Hg in supine position), and normal electrocardiogram (ECG).
Angiotensin II subtype 1 (AT1)-receptor antagonist
SC-52458 was synthesized by Searle Laboratories (Skokie, IL, U.S.A.). Its chemical name is 5-((3,5-dibutyl-1H-1,2,4-triazol-1-yl)methyl)-2(2-(1H-tetrazol-5yl)phenyl)pyridine (Fig. 1). It is a white crystalline powder with a solubility in water of 25 mg/L and in ethanol of 275 mg/L. Study doses of SC-52458 powder were preweighed in stoppered and sealed bottles.
This drug has been tested in vitro and in vivo by using rats and dogs. The results of these unpublished data showed a high affinity to AT1 receptors (IC50 = 2.9 ± 0.1 nM) in the rat adrenal cortex membranes, competitive and fully reversible binding to vascular smooth muscle in the isolated aortic rings; this value compares well with that of compounds like losartan (IC50 = 19.1 nM), its active metabolite MK964 (IC50 = 3.7 nM), or even saralasin (IC50 = 1.5 nM) (17).
Each volunteer received on 4 study days, separated by 1-week intervals, three single doses of active drug and a placebo in single-blind fashion. The active drug was administered by increasing the dose at each subsequent study day. The day of placebo intake was randomized. The volunteers were divided into two groups. Group I (n = 8) received placebo and SC-52458 increasing from 10 to 25 and 50 mg. Group II (n = 8) followed the same protocol but received 100, 150, and 200 mg SC-52458 and placebo.
The subjects ate an unrestricted diet. They came to the hospital at 06:45 a.m. after an overnight fast: they were installed on a bed and kept this supine position until 6 h after drug and also for 1 h before blood sampling for hormone measurements at 10 and 24 h after drug. Intravenous cannulas were placed on both forearms for Ang II bolus injections and for blood sampling, respectively. After 1 h in the resting position, an individually predetermined standard dose of exogenous Ang II (Clinalfa Chemicals, Läufelfingen, Switzerland) was injected twice, providing a mean increase in systolic BP of 25-40 mm Hg. This increase in BP before drug intake was considered the day's baseline response to standard Ang II challenge. This same standard dose of Ang II (10-60 ng/kg) was used throughout the study. At 08:00, the volunteers of group I drank the dose of SC-52458 dissolved in 5 ml absolute ethanol and 150 ml apple juice or the same amount of apple juice with ethanol but without active drug (placebo). In group II, the three higher doses were dissolved in 10 ml ethanol and 300 ml apple juice to warrant solubility and to eliminate the bitter taste. The BP response to the Ang II challenge was determined at 1, 2, 3, 4, 5, 6, 8, 10, and 24 h after drug administration. Blood samples were drawn periodically for SC-52458 measurement (0, 0.5, 1, 2, 4, 6, 8, 10, and 24 h) as well as for the determination of plasma renin activity (PRA), plasma angiotensin II, and aldosterone (0, 4, 10, and 24 h). An additional blood sampling for routine laboratory tests and an ECG were performed 24 h after drug intake for safety purposes. After the measurements at 6 h after drug, the volunteers got up and received a light meal. They were asked to return to the supine position 30 min before each following Ang II challenge and 1 h before the hormone measurement. After 10 h, they left the hospital and returned the next morning for the measurements at 24 h.
Blood pressure measurement
Blood pressure in mm Hg was measured, beat-to-beat, non-invasively by photoplethysmograph at the finger (Finapres; Ohmeda, Englewood, CO, U.S.A.) (18). On each study day (i.e., before each SC-52458 dose tested), two i.v. bolus injections of a constant predetermined standard dose of Ang II were injected at 15-min intervals. Averages of these two Ang II-induced systolic and diastolic BP increases were defined as the volunteer's baseline responses (100% response). Subsequent (postdose) BP responses to the same standard Ang II dose were expressed in percentage of baseline.
PRA was measured by antibody trapping of Ang I generated during an incubation of 30 min at 37°C and subsequent radioimmunoassay by the technique of Poulsen and Jörgensen (19), as described elsewhere (20). Immunoreactive angiotensin II was extracted from plasma by reversible adsorption to phenylsilylsilica and quantitated by radioimmunoassay by using a monoclonal antibody developed in our laboratory (21,22). Plasma aldosterone concentration was determined by direct radioimmunoassay (23).
Plasma concentrations of SC-52458 were determined by a reversed-phase high-pressure liquid chromatography (HPLC) method with UV detection. The assay's sensitivity was 0.025 μg/ml (unpublished data).
Noncompartimental estimates of single-dose pharmacokinetics parameters were calculated for each subject. We assessed time to peak plasma concentration (tmax), peak plasma concentration (Cmax), elimination rate (Ke), half-life (t1/2), area under the curve from 0 h to last time point above assay's sensitivity limit (AUC0-Last). AUC integrated from 0 to infinity (AUC0-inf) was obtained from linear regression of log-transformed plasma concentration in the linear tail portion of the curve.
t Tests were used to compare the two groups at baseline. Repeated measures analysis of variance was applied for BP, heart rate, and hormone results with subjects, dose, and time as independent factors.
When the time-by-treatment interaction was significant, the treatments within each group were also compared. For the BPs at each hour, pair-wise comparisons of the mean response at each dose of SC-52458 and placebo were made by using Turkey's method.
When the time-by-treatment interaction was significant, the four treatments were compared within each group at each hour of blood sampling. Results are expressed as mean ± SEM unless stated otherwise; the significance level was set at p < 0.05.
Blood pressure, HR, and tolerability
No significant change in the underlying BP or heart rate was observed after administration of SC-52458 or placebo, despite a slight decrease in BP and a slight increase in heart rate during the first hours after drug intake.
There were no adverse events of clinical relevance or subjective side effects observed with SC-52458. Three occasional increases in the serum glutamyl oxalate transferase activities (after 10, 50, and 200 mg SC-52458, respectively) were paralleled by increased muscular creatine kinase levels and attributed to vigorous unauthorized exercise during the washout periods.
BP response to angiotensin II challenges
Figure 2A and B illustrates systolic and diastolic BP responses to intravenous bolus injections of Ang II. Both systolic and diastolic BP increases were dose-dependently blunted after SC-52458 when compared with predose baseline responses. The blockade was significant with SC-52458 doses of ≥25 mg for diastolic BP and with ≥100 mg for systolic BP responses. Maximal inhibition was apparently achieved within the first hour after drug intake and reached 89 ± 4% and 92 ± 3% for systolic and diastolic responses, respectively, at 1 h after the 200-mg dose. With the three highest doses of SC-52458 (group II), the systolic BP response remained decreased for ≤10 h (p < 0.001), and the diastolic BP response for ≤24 h (p < 0.02). There was no significant difference between the effects of the 100-, 150-, and 200-mg doses. The heart rate deceleration in response to the Ang II challenges was dose-dependently reduced after SC-52458 intake and mirrored the blunted increase in BP (results not shown).
Response of PRA, plasma angiotensin II, and aldosterone
Hormonal results are summarized in Table 1. PRA and Ang II levels showed a dose-related increase 4 h after drug intake. They remained increased 10 h after administration of SC-52458. At the same times, plasma aldosterone tended to be decreased. When compared with baseline, these hormonal changes, including the decrease in aldosterone, were significant, particularly after high doses of SC-52458.
Noncompartimental estimates were based on robust measures of central tendency and dispersion (median and interquartile range rather than mean and standard deviation). After oral administration, SC-52458 is rather rapidly absorbed, peak plasma concentration being reached within 1 h (Fig. 3) with a total amount of drug absorbed (AUC0-inf) ranging from 0.22 to 4.11 mg/h/L (Table 2). These pharmacokinetics were dose related for 10- to 150-mg doses of SC-52458 but for 200 mg, Cmax and AUC0-inf were comparable to the 150-mg dose. Half-life was estimated between 1.14 and 2.39 h.
Relations between plasma drug levels and inhibitory effect
The relation between plasma drug concentrations and percentage inhibition of the diastolic pressure response to Ang II is shown in Fig. 4. After a visual check of the individual data, no consistent hysteresis could be discerned, and therefore a Hill sigmoid equation curve was fitted to the data (24,25). Equation 1 where E is the effect (expressed in percentage inhibition), C is the drug level in the plasma, EC50 is the drug concentration that produces half the maximal effect, and Emax is the maximal inhibition of BP response to Ang II (for a hyperbolic curve, the sigmoidicity factor is 1).
Based on this model, an EC50 of 0.096 μg/ml (range, 0.025-0.400 μg/ml) and an Emax of 92% (range, 76-100%) have been calculated. The curve suggests that SC-52458 levels >0.900 μg/ml seem not to enhance the peak effect. A similar relation based on the log of dose administered rather than on plasma drug levels (Fig. 5) shows a mean ED50 of ∼50 mg, and no further advantage in terms of pressure-response inhibition is seen on average for doses >150 mg, although some individuals did improve BP blockade with 200 mg (26). There exists a clear dose effect up to the 150-mg dose.
The purpose of this single-blind, placebo-controlled, crossover study was to evaluate the tolerability and Ang II blocking properties of a range of doses of SC-52458, an orally active angiotensin-receptor antagonist, in healthy subjects. In addition, a preliminary characterization of pharmacokinetics was attempted.
There were no significant differences in underlying BP, heart rate, respiration rate, body temperature, and ECG intervals over the course of the study. Based on the small number of subjects studied, SC-52458 appears clinically safe and well tolerated.
We tested the efficacy of SC-52458 by measuring inhibition of finger BP response to i.v. Ang II injections, as we did in previous studies (27,28). This assesses directly the blockade of AT1 receptors. It allows indicative, but not definitive, comparison of peak effect and effect kinetics for different Ang II antagonists, as discussed later. SC-52458 inhibited systolic and diastolic BP response similarly. The maximal effect reached 89 ± 8% SD and 75 ± 9% SD inhibition of diastolic and systolic BP response, respectively, after the 150-mg dose. The maximal inhibition of the pressure response to Ang II was observed 1 h after oral administration, which is taken as tmax (time to reach maximal plasma drug level for each dose), although this may be overestimated because no earlier measurement was carried out. This reflects a rather rapid gastrointestinal absorption and might suggest a direct inhibitory effect of the drug rather than an action through an active metabolite. Indeed no active metabolite is known for SC-52458.
The BP effect blocked by SC-52458 was accompanied by dose-dependent increases in both PRA and plasma Ang II levels. The same kinetic was observed for both effects. By stimulating AT1 receptors in the kidney, Ang II exerts a negative-feedback action on the excretion of renin. Therefore blockade of the AT1 receptors abolishes the inhibitory effect of Ang II on renin release, leading to increased renin excretion by the kidney but also inhibits proximal tubular uptake of sodium (29). The limiting factor in Ang II formation is renin, as already seen with ACE inhibitors. So the unblocked secretion of renin results in a massive generation of Ang II, as shown in this study (10-fold increase for 200-mg dose). This amount of Ang II does not seem to be able to overcome the effect of SC-52458 after a single dose. Whether the same holds true after long-term administration has to be shown for this competitive AT1-receptor antagonist. Moreover, the continuous stimulation of the nonblocked AT2 receptors could have some beneficial or deleterious consequences that are still not understood.
Once again, as in some earlier studies of AT1-receptor antagonists, it was not possible to demonstrate a statistically significant decrease in circulating aldosterone levels when compared to placebo. This may relate to the rather short duration of close to maximal blockade of the AT1 receptors. To this must be added the repeated bolus injections of Ang II when the AT1 receptors are not fully blocked. In addition, it is well established that Ang II is only one among several stimulating factors of aldosterone secretion and may not be the most important one under conditions of a free sodium intake. Thus taken together, it should probably not be surprising that with our study design, it has repeatedly been impossible to demonstrate a consistent decrease in aldosterone levels with the single administration of an AT1-receptor antagonist.
An important question could be how SC-52458 compares with other angiotensin II-receptor antagonists that have been characterized clinically. Strict comparison obviously is possible only based on a comparative study designed with this goal in mind. Such a study is not available. However, in our laboratory, other compounds such as losartan, TCV-116 (Takeda), UP269-6 (UPSA), and several other molecules have been tested by using a similar approach, although the design of the studies has varied (30-33). Moreover, recently the approach based on challenge by exogenous angiotensin bolus injections could be compared with another model studying the hypotensive effect in salt-depleted volunteers; both study designs yielded similar results concerning potency and duration of action of the compound. Thus a preliminary comparative assessment of potency and duration of action with other existing molecules seems permitted. Overall, it would appear that SC-52458 has a shorter duration of action than losartan, TCV-116, or UP269-6. Whereas losartan has been demonstrated to be a once-a-day antihypertensive drug, this could not be assumed a priori to be the case with SC-52458. However, the galenic form of SC-52458 used in this study (solution) could contribute to its rather short duration of action. Perhaps a more sophisticated galenic form could modify the pharmacokinetic profile, attenuate the peak, and increase the duration of the inhibition. Furthermore, its potency is clearly lower than that of losartan and may be comparable to that of UP269-6. Based on BP responses to Ang II after single doses of SC-52458 and based on clinical experience with losartan, it appears that a dose of 150 mg of this galenic form of SC-52458 would be needed in hypertensive patients. SC-52458 seems to be an efficacious and so far well-tolerated angiotensin-receptor antagonist with a clear potential to become an effective drug for the treatment of hypertension and congestive heart failure.
Acknowledgment: We thank Monique Salvi, Catherine Amstutz, Christine Munoz, and Sylvie Novelli for excellent technical assistance.
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Keywords:© Lippincott-Raven Publishers
Antihypertensive; Finger blood pressure measurement; Tolerability; Renin activity; Plasma angiotensin II; Aldosterone