Introduction

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In recent years, several specific, orally active, nonpeptide angiotensin II (AngII) receptor antagonists have been developed and have become available clinically. These antagonists share a common mechanism of action, i.e., blockade of AngII subtype-1 (AT1) receptors. Yet, the various antagonists differ in their pharmacological profile and these differences might sometimes affect their efficacy and tolerability (Brunner, 1997; Mazzolai et al., 1999).

Tasosartan is an orally active nonpeptide AngII antagonist that has demonstrated specific and selective AT1 receptor antagonistic activity in vitro and experimentally (Ellingboe et al., 1998). Several studies have investigated its ability to lower blood pressure (BP) in hypertensive patients (Feldman et al., 1997; Oparil et al., 1997; Lacourcière et al., 1998; Neutel et al., 1999). Chemically, tasosartan is a biphenyl tetrazole substituted with a pyridopyrimidine ring (Ellingboe et al., 1994). This drug is sequentially metabolized by the P450 enzyme system, first to form a hydroxy-intermediate that is then biotransformed to enoltasosartan, another active compound that presents in vitro a higher affinity for the AT1 receptors than the parent compound (Fig. 1) (Hartupee et al., 1994). In humans, tasosartan is rapidly absorbed and cleared from the circulation with a relatively short terminal half-life (3-7 h). Enoltasosartan appears later in the circulation and presents a long elimination half-life (35-43 h). This results in plasma concentrations of enoltasosartan exceeding those of tasosartan for most of the dosing interval (Neefe et al., 1997). Thus, the long duration of action of tasosartan has been attributed to the generation of enoltasosartan. The objective of the present study was to compare the time course of the AngII-receptor blockade induced by tasosartan and enoltasosartan, to assess their respective contribution to the pharmacodynamic (PD) activity of tasosartan.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Structure of tasosartan and its active enol-metabolite. *, IC50 for inhibition of specific binding of 125I-AngII to rat adrenal membrane in the absence of proteins in binding buffer (Ellingboe et al., 1998). , in vitro binding of [14C]tasosartan, respectively, [14C]enoltasosartan to human plasma proteins (Wyeth-Ayerst Research, unpublished data).

	Materials and Methods

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Subjects. Twelve healthy male volunteers aged 25 ± 4.1 years (range 20-35) and weighing 75.1 ± 6.5 kg (range 67-84) were included in the study. Their written consent to participate was obtained after giving full explanation on the purpose and risks of the study. The protocol was approved by the local ethics committee. The subjects were considered to be healthy according to medical history, physical examination, and routine blood and urine analysis. They had a normal BP (mean 117.5/70.5 mm Hg) in supine position, and a normal electrocardiogram.

Study Design. The study was designed as a randomized, double blind, three-period crossover, single-dose administration study. Three doses were studied: a 100-mg oral dose of tasosartan, a 50-mg i.v. dose of tasosartan, and a 2.5-mg i.v. dose of enoltasosartan. A 2-wk washout separated each test period. To ensure a double blind administration of the study medication, a double-dummy technique was used. Thus, in each phase, subjects received one oral capsule and a short i.v. infusion.

Interaction of Tasosartan and Enoltasosartan with Plasma Proteins. To investigate in the in vitro AT1 receptor binding assay the influence of plasma protein on the displacement of labeled AngII by the pharmacological compounds, 6.25% of human plasma was added to the buffer to obtain a protein final concentration of 0.4%. In subsequent studies, compounds with known strong protein binding, e.g., digitoxin, warfarin, diazepam, and disopyramide, were added to the mixture in increasing concentration to detect a possible displacement of the AT1 receptor antagonists from the proteins and a resulting enhanced receptor binding.

Data Analysis. Results are expressed as mean ± S.E. unless stated otherwise; one-way ANOVA was used to evaluate the difference between groups. A P value of <.05 was considered statistically significant. The values for the maximum concentration (C_max) and the time to reach it (T_max) were derived directly from the measured plasma drug concentrations. Individual plots of the concentration-time curve were done and the elimination rate constant (_z) was estimated by the linear regression of at least three points that were in terminal phase. The terminal half-life (t_1/2) was then calculated as 0.693/_z. The relationship between plasma drug concentrations and the percentage of inhibition of the pressor response to AngII were modeled using the Hill sigmoid E_max equation (Hill, 1910) and with a log/linear model. Same modelizations were also performed with in vitro data.

	Results

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Tasosartan and enoltasosartan were well tolerated and none of the subjects were excluded from the study. No significant clinical and biochemical adverse reaction was observed in any of the volunteers. Neither tasosartan nor enoltasosartan had any effect on baseline BP and heart rate in these normotensive subjects.

Pharmacokinetics. The mean concentration-time profiles of both active species for each treatment are shown in Fig. 2. Tasosartan was rapidly absorbed after oral administration and T_max 30 min (with C_max of 1596 ± 250 ng/ml) was observed in all subjects (n = 12). The disposition of tasosartan was biexponential with a rapid elimination phase. The mean terminal t_1/2 values estimated within the 12 volunteers were 5.3 ± 0.6 h (range 2.5-8.0 h) in the dose group 100 mg p.o. and 5.2 ± 0.5 h (range 1.7-8.0 h) in the dose group 50 mg i.v. There was no significant difference between both groups. The bioavailability of tasosartan does not seem to be dependent on the mode of administration because plasma drug levels measured 1 to 14 h after 100 mg p.o. are almost twice as high as those measured after injection of tasosartan 50 mg i.v.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Time profile of the mean ± S.E. (n = 12) plasma concentration of tasosartan () and enoltasosartan (). A, administration of a single dose of tasosartan 100 mg p.o. B, administration of a single dose of tasosartan 50 mg i.v. C, administration of a single dose of enoltasosartan 2.5 mg i.v.

Effect of Tasosartan and Enoltasosartan on BP Response to Exogenous AngII. The mean percentage changes in SBP responses to exogenous AngII after the oral administration of tasosartan and i.v. infusion of tasosartan and enoltasosartan are illustrated in Fig. 3, top. Both SBP and diastolic BP (DBP) (data not shown) responses to exogenous AngII were blunted after administration of tasosartan up to 32 h after dosing, confirming the long-lasting activity of the drug. The time courses of the AngII-receptor blockade induced by tasosartan 50 mg i.v. and 100 mg p.o. were comparable throughout the 32-h study without significant differences. The peak effects appeared at 1 to 2 h after drug intake with maximal inhibition of AngII responses reaching 82.2 ± 3.9 and 81.7 ± 6.0% (SBP), respectively, and 83.7 ± 3.2 and 86.4 ± 3.8% (DBP), respectively. At time point 32 h, receptors were still blocked by 24.6 ± 3.2 and 38.1 ± 5.3% (SBP), respectively, and 32.4 ± 4.9 and 50.0 ± 3.9% (DBP), respectively. Again no significant difference was found between both treatment modes.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Top, mean ± S.E. (n = 12) PD time profile effect of tasosartan 100 mg p.o. (), tasosartan 50 mg i.v. (), and enoltasosartan 2.5 mg i.v. () characterized in vivo by the measure of the inhibition of the pressor response (SBP) to AngII. Bottom, mean AT1 blockade time-profile effect induced by single doses of tasosartan ( 100 mg p.o. and  50 mg i.v.) or enoltasosartan 2.5 mg i.v. () measured in vitro.

Effect of Tasosartan and Enoltasosartan on In Vitro Binding of AngII. When the plasma of subjects treated with tasosartan was tested in vitro for its ability to inhibit the binding of radiolabeled AngII to AT1 receptors, the displacement of ¹²⁵I-AngII was fully achieved within a 1-h incubation time. In contrast, the displacement was much slower with plasma from enoltasosartan-treated subjects. Between 4 and 6 h of incubation was needed before the binding reached a state of equilibrium (see below). These in vitro time course profiles of AT1 blockade are depicted in Fig. 3, bottom. These curves paralleled those observed in vivo with no significant differences between the effects of tasosartan 100 mg p.o. and 50 mg i.v. (maximal in vitro blockade 90.6 ± 1.1 and 87.4 ± 0.9%, respectively).

Evaluation of Interaction of Tasosartan and Enoltasosartan with Plasma Proteins. To study the mechanism(s) that could potentially explain the discrepancy between the PK (i.e., high plasma levels) and the PD (i.e., delayed in vivo blockade effect) characteristic of enoltasosartan, we evaluated the effect of human plasma on the antagonistic activity of tasosartan and enoltasosartan.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Displacement of specific 125I-AngII binding in rat vascular smooth muscle cell membrane preparation (Maillard et al., 1999) by tasosartan or enoltasosartan in the absence () or in the presence () of 6.5% human plasma in binding buffer.

View larger version (59K): [in this window] [in a new window]   Fig. 5.   Influence of increasing concentrations of digitoxin, warfarin, diazepam, or disopyramide on blockade of the AT1 receptor by tasosartan and enoltasosartan in the presence of plasma proteins. A, 10 nM tasosartan (T) reduced bound labeled AngII by 60%; digitoxin, warfarin, diazepam, or disopyramide have no influence on this displacement. B, 10 nM enoltasosartan (E) had little effect on the bound labeled AngII. However, addition of warfarin enhanced the displacement of bound labeled AngII in dose-dependent manner.

	Discussion

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Besides tasosartan itself, different active metabolites, of which enoltasosartan appears as the most important, have demonstrated high affinity for the AT1 receptor (Ellingboe et al., 1998). Comparing the parent drug with its enol-metabolite as to their effect on the AT1 receptor enabled us to assess the respective contribution of these two different active components to the overall drug effect.

The time courses of the AngII-receptor blockade induced by tasosartan 50 mg i.v. and 100 mg p.o. were similar with a peak effect at T_max 1 to 2 h and a significant residual blockade at 24 h. Bioavailability of tasosartan does not seem to be dependent on the mode of administration because its plasma drug levels measured 1 to 14 h after 100 mg p.o. were double those measured after injection of tasosartan 50 mg i.v. PK/PD relationships between effect and plasma drug concentration were the same whatever the mode of administration. The PD parameters (E_max and EC₅₀) measured in this study were also in good agreement with those published previously (Steinhäuslin et al., 1993).

In contrast, the blockade induced by i.v. enoltasosartan was markedly delayed with a peak inhibition measured 3 to 6 h after injection despite the fact that very high plasma enoltasosartan concentrations were measured between 1 and 32 h. Thus, no direct PK/PD correlation was noticed, and a counterclockwise hysteresis loop was observed.

This peculiar behavior led us to investigate the interaction of the drugs with their receptor and plasma proteins. In the radioreceptor assay, the AT1 antagonistic effects of tasosartan and enoltasosartan were differently affected by the presence of human plasma. The presence of proteins shifted the concentration-response curves to the right, thus increasing the IC₅₀. This rightward shift was rather small for tasosartan, whereas the presence of plasma drastically increased the IC₅₀ of enoltasosartan almost 1000-fold. Because both products, as it is the case for all drugs belonging to the "sartan" class (Csajka et al., 1997), are claimed to be protein bound >99% (Wyeth-Ayerst Research, unpublished data), these data suggest that the binding of enoltasosartan is qualitatively different. The displacement of labeled ligand from receptor by the plasma of volunteers having received tasosartan was fully achieved within a 1-h incubation time. In contrast, the displacement induced by the plasma of volunteers after i.v. enoltasosartan administration needed between 4 and 6 h of incubation. These results suggest either a slow dissociation from the plasma carrier or a slow association rate to the AT1 receptor. However, the affinity of enoltasosartan for these receptors is very high. Consequently, the association/dissociation of the product to the receptor does not seem to represent the determining factor. Thus, we hypothesize that enoltasosartan is not only abundantly bound to plasma proteins but also tightly fixed to specific protein binding sites. This kind of behavior has already been observed with UR-7247, an AT1 receptor antagonist with an elimination half-time higher than 50 h (Maillard et al., 2000) and to the extreme with DUP 532, which exhibited no blockade despite high circulating concentration of the drug (Goldberg et al., 1997).

In a further experiment it was attempted to increase the "free-drug" level in the protein-containing buffer using other protein-bound substances. The AT1 receptor binding of enoltasosartan increased markedly and dose-dependently in the presence of compounds known to interfere with different protein binding sites such as warfarin (site I of albumin), diazepam (site II of albumin), digitoxin (site III of albumin), and disopyramide (binding site of ₁-acid glycoprotein). In contrast, tasosartan binding to AT1 receptor was not significantly affected by the presence of such compounds even at suprapharmacological concentrations. These observations led to the conclusion that the interaction between proteins and enoltasosartan is of crucial importance in the comprehension of the peculiar PD behavior of enoltasosartan.

Our results also demonstrate that enoltasosartan produced by the rapid metabolization of tasosartan has little pharmacological effect in the early phase of the drug action. However, the delayed action of enoltasosartan, as well as its long elimination half-life explain the long-lasting antagonistic effect of the drug. Indeed, our study confirmed the very long duration of enoltasartan, with an important in vivo blockade being still measurable 32 h after drug intake, which is not the case with losartan 50 mg and valsartan 80 mg (Mazzolai et al., 1999). This observation corresponds to the long terminal half-life of enoltasosartan that in these experiments was estimated to be about 40 h but might be even longer. Indeed, the sampling time of 32 h in our study represents only a single half-life period and thus the terminal phase of elimination is truncated. Accordingly, calculations on half-life from our data might be underestimated. In comparison, the elimination half-life of the other AT1 antagonists currently on the market ranged from 2 to >20 h (i.e., losartan, 2 h; EXP3174, its metabolite, 6-9 h; valsartan, 6 h; candesartan, 3-11 h; irbesartan, 12-15 h; and telmisartan, 20-24 h) (Brunner, 1997).

As expected from previous studies conducted in normotensive subjects investigated on a free-sodium diet, no significant change in systemic BP and heart rate was found upon administration of tasosartan or enoltasosartan. In addition, no clinical or biochemical adverse reaction was noted during the course of the study.

In conclusion, tasosartan is a potent, long-acting AT1 receptor blocker with an excellent bioavailability. Enoltasosartan does not appear to contribute to the early blockade induced by tasosartan. This seems to be due to a high protein binding and a slow dissociation process from the carrier. Our in vitro AngII-receptor binding assay was shown to be a useful tool to study in vitro the mechanisms whereby AT1 receptor antagonists interact with their receptor and other components of the plasma.

Our data suggest that molecules with AngII receptor antagonistic activity may have a comparable overall plasma protein binding but different kinetics of dissociation from these proteins. The way the molecule dissociates from plasma proteins may affect significantly the free-drug concentration and hence the biological activity of the compound.