Eur J Clin Pharmacol (1991) 40:95-99

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© Springer-Verlag 1991

Pharmacokinetics and converting enzyme inhibition after morning and evening administration of oral enalapril to healthy subjects* K. Weisser 1, J. Schloos 1, K. L e h m a n n 2, R. D a s i n g 3, H. Vetter 3 and E. Mutschler 4 1 Zentrum der Pharmakologie, Klinikum der Universit~it, Frankfurt, 2 MSD Sharp & Dohme, Mtinchen, 3 Medizinische Poliklinik der Universitfit, Bonn, 4 Pharmakologisches Institut ffir Naturwissenschaftler der Universit~it, Frankfurt, Federal Republic of Germany Received: May 10, 1990/Accepted in revised form: August 4, 1990

Summary. Possible circadian changes in the pharmacokinetics and effect on serum angiotensin-converting enzyme ( A C E ) activity of the A C E inhibitor enalapril have been studied in 8 healthy subjects after oral ingestion of 10 mg enalapril maleate either at 08.00 h or 20.00 h. The time to peak serum concentration (tmax) of enalapril was increased after administration at 20.00 h compared to 08.00 h (2.4 h versus 1.3 h), whereas other kinetic parameters were not significantly altered. The 24 h-kinetics of the active metabolite enalaprilat did not differ significantly between the two treatments, but the area under the curve ( A U C (0-24)) and the peak serum concentration (Cm~/ were slightly higher after intake at 20.00 h. The relationship between the measured serum enalaprilat level and the degree of inhibition of serum A C E was the same after both treatments. Overall, the evening and morning administration of enalapril did not differ markedly in the pharmacokinetics and the time course of A C E inhibition. K e y words: Enalapril, circadian pharmacokinetics, A C E inhibition

For many antihypertensive drugs, such as beta-adrenoceptor blockers, calcium-antagonists and diuretics, circadian dependency of the pharmacokinetics and the drug effect on the cardiovascular system in man have been reported [Lemmer 1989]. Some components of the reninangiotensin-aldosterone system (RAAS), an important regulator within the cardiovascular system, show physiological circadian rhythms in man [Gordon et al. 1966; Breuer et al. 1974; Armbruster et al. 1975; Beilin et al. 1983; Takeda et al. 1984]. It is feasible that inhibitors of the angiotensin converting enzyme ( A C E ) might cause a different time course of effects and/or influence circadian rhythms of the R A A S in a different manner, dependent on the time of drug intake. * This report is part of the thesis to be presented by K. Weisser in partial fulfillment of the requirements for the degree of Doctor of Natural Science.

No data appear to be available about any possible circadian-phase-dependency of the pharmacokinetics and the pharmacodynamic effects of A C E inhibitors administered at different times of day. The purpose of the present study was to investigate whether the pharmacokinetic profiles of enalapril and its active metabolite enalaprilat were different after administration of 10 mg enalapril at either 08.00 h or 20.00 h. The relationships between serum enalaprilat and serum A C E inhibition were also compared.

Materials and methods

Study design Eight healthy, normotensive volunteers of both sexes, aged 27-41 y, weighing 52-80 kg, were included in a single dose cross-over study. The study was approved by the Ethical Committee at the _A.rztekammer Nordrhein-Westfalen, and each participant gave written informed consent to it. During a one-week run-in-phase before each study period the subjects were equilibrated on a salt diet of approximately 150 mmol/d sodium and 80 mmol/d potassium. On the study day, the subjects received a single oral dose of 10 mg enalapril maleate (tablets supplied by MSD Sharp & Dohme GmbH) either at 08.00 h or at 20.00 h. There was a 6 week-interval between each study period. In both cases the ingestion followed 2 h after a light meal. On the study days they were allowed to be normally active during the day and at 20.00 h they went to bed. Blood samples were taken at 0.5 hintervals from 0-4 h and at i h-intervals from 5-24 h after drug intake, from 08.00 h to 20.00 h in the sitting position, and from 20.00 h to 08.00 h in the supine position. Serum was obtained and frozen at - 20°Cuntil analysed.

Drug analysis Serum concentrations of enalaprilat were determined by radioimmunoassay (RIA) [Hichens et al. 1981].The assay employs a radioligand and a rabbit-antiserum, which is sensitive to enalaprilat but not to enalapril. The radioligand precursor (351 A) and antiserum were provided by MSD Sharp & Dohme GmbH. Compound 351 A, a phydroxy-benzamidine derivative of lisinopril, was radioiodinated

96

(125I; Du Pont de Nemours GmbH, Dreieich, FRG), to a specific activity of 1894 Ci/mmol. For the assay, dilutions of the tracer (1:400) and the antiserum (1:16000) in 0.05 M phosphate buffer, containing 0.05 M EDTA and i mg/ml bovine serum albumin (Cat. No. 11922, Serva GmbH, Heidelberg, FRG), were chosen which resulted in-30% binding of the label (Bo), with a signal of = 5.000 cpm. Separation between bound and free ligand was performed by the double antibody technique. The second antibody (goat anti-rabbit IgG, Sigma Chemie GmbH, Mtinchen, FRG) was titrated to ensure optimum precipitation of the specific antibody. After incubation of all components (15 ~tlsample or standard per assay tube; total volume 815 gl) for 18-24 h at room temperature, the precipitate was separated by centrifugation (800 x g, 40 min) and decantation of the supernatant, and the radioactivity was measured. Blanks were prepared replacing antiserum by buffer. Standard dilutions of enalaprilat (0.4-200 ng. ml- l) were prepared for each assay in the drug-free serum from each subject in order to exclude serum effects in the RIA. Samples were assayed in triplicate. The detection limit was 0.2 ng. ml- 1. The intra-assay precision was 7.6% and the inter-assay precision 10.7% at an enalaprilat concentration of 25 ng. ml- 1. Serum concentrations of intact enalapril were determined indirectly. Each sample was incubated over night with a crude enzyme preparation from rat liver in order to hydrolyse enalapril to enalaprilat. Afterwards the samples were analysed by the same RIA. The resulting "total drug" value is the sum of enalaprilat before and after hydrolysis in terms of enalaprilat equivalents, and the amount of enalapril was calculated by subtraction. Because of the indirect method, it is not possible to give a definite detection limit for the determination of enalapril. The sensitivity of the measurement is strictly dependent on the amount of enalaprilat present in the sample prior to hydrolysis and on the precision of its measurement. Therefore, the standard deviation (SD) of each direct enalaprilat measurement was estimated and three-times the SD was taken as the detection limit for the subsequent determination of the difference between the total and direct measurements. Values below these limits and negative values were considered to be zero.

K. Weisser et al.: Enalapril kinetics rically at 228 nm. One unit of enzyme activity (U/ml) represents 1 nmol hippuric acid generated per rain per ml serum. Postdose values were estimated as percentage inhibition of the predose level at 08.00 h or 20.00 h, respectively.

Relationship between serum enalaprilat and serum A C E inhibition For each treatment the individual serum enalaprilat concentrations were correlated with the corresponding percentage ACE inhibition independent of time after drug intake. For comparison of the resulting concentration-effect curves, classes of enalaprilat concentrations (0-1, 1-2, 2-3, 3-5, 5-7, 7-10, 10-15, 15-20, 20-30, 30-40 and 40-50 ng.m1-1) were formed, and the corresponding mean inhibition values from all subjects receiving each treatment were correlated. Regression analysis was performed by the least squares method.

Results P harma co kinetics T h e m e a n s e r u m c o n c e n t r a t i o n - t i m e p r o f i l e s of e n a l a p r i l a n d its active m e t a b o l i t e e n a l a p r i l a t after t h e m o r n i n g a n d e v e n i n g doses are s h o w n in Figs. 1 a a n d b. T h e c a l c u l a t e d

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Pharmacokinetic evaluation The individual serum concentration data of enalapril and enalaprilat were analysed model-independently. Cm,xis the observed maximum concentration and tmaxthe corresponding time value. Areas under the serum concentration-time curve were calculated by the linear trapezoidal rule, for enalaprilat from 0-24 h, A U C (0-24), and for enalapril from 0 h to the time of the last detectable serum concentration, A U C (0-last), with extrapolation to infinity by addition of C~a~]k~,resulting in AUC. Elimination rate constants (ke) were determined by log-linear regression of the terminal log-linear phase of the serum concentration-time curves (in case of enalaprilat up to 24 h). In addition, the parameters of the Bateman function were fitted to the individual enalapril serum concentration data using a computer program (GIR GieBener Iterationsprogramm; developed by D. H. Brockmeier and H. M. yon Hattingberg). Typical kinetic behaviour (no flip-flop kinetics) was assumed. Statistical evaluation of all parameters was done using the Wilcoxon matched pairs-signedRank test, two-tailed, with (z = 0.05.

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Serum A C E activity Serum ACE activity was measured by the method of Lieberman (1975). Serum (75 gl) was incubated for 1 h at 37°C with 175 gl HipHis-Leu in phosphate-sodium chloride buffer (pH 8.3; final assay concentrations: 5 mM Hip-His-Leu, 300 mM NaC1). The reaction was stopped with 250 gl i N HC1 and the hippuric acid generated was extracted with 1.5 ml ethyl acetate, After evaporation the residue was dissolved in I M NaC1 (1.5 ml) and measured spectrophotomet-

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Fig.1. Time course of serum concentrations of enalapril (-o-) and enalaprilat (-.-) following administration of 10 mg enalapril maleate to 8 healthy subjects at 08.00 h (Panel a) and at 20.00 h (Panel b). Mean (SEM)

97

K. Weisser et al.: Enalapril kinetics Table 1. Pharmacokinetic parameters after oral administration of 10 mg enalapril maleate at 08.00 h and 20.00 h in 8 healthy subjects. Mean (SD). Unless otherwise indicated the parameters were analysed model-independently Parameter Enalapril Cmax(ng. ml-~) [max(h) AUC (ng. ml-1. h) ke (h-i) ke (h-1)a k~ (h-~)~ lag time (h) ~ Enalaprilat Cma x (ng-ml 1) t~x (h) AUC (0-24) (ng-ml Z-h) ke ( h

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Time of drug intake (h) 08.00 20.00

Comparison

72.1 (42.5) 1.3 (0.5) 160 (91) 0.84 (0.38) 0.94 (0.47) 12.0 (15.1) 0.5 (0.3)

65.4 (25.2) 2.4 (1.4) 148 (67) 1.30 (1.27) 1.32 (1.34) 6.4 (9.4) 0.7 (0.4)

NS P < 0.05 NS NS NS P < 0.05 NS

29.3 (11.6) 3.9 (1) 217 (83)

37.3 (8.2) 4.3 (0.8) 260 (75)

NS NS NS

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a Parameters derived by computer analysis (see Methods)

kinetic parameters and their statistical evaluation are presented in Table 1. After drug intake at 20.00 h, a significant increase in the tmaxof enalapril (2.4 h vs 1.3 h; P < 0.05) was observed. No significant difference was found on comparing C .... A U C and ke, although the mean serum level profiles suggested slightly reduced Cm~ and A U C of enalapril after the evening administration. Only 2 out of 8 subjects showed this phenomenon. The computer-fitted values for ke were in good agreement with those derived by graphical evaluation. The fitted absorption rate constant (ks) was significantly smaller for the evening administration, whereas the lag times of absorption were similar in both cases. For the active metabolite enalaprilat no significant difference was found on comparing any of the kinetic parameters, but Cmaxand A U C (0-24) were slightly increased after drug intake at 20.00 h. That was also observed comparing the A U C (0-24) of total drug. The time period between the tmax of enalapril and of enalaprilat was shortened in the evening.

mean percentage A C E inhibition after drug intake at 08.00 h and 20.00 h when related to the same classes of enalaprilat concentrations (Fig.3). The regression line (r =0.96, y = 0.99x+ 1.1) resembles the line of identity (y=x).

Discussion

The study has demonstrated that the pharmacokinetics of enalapril maleate is in part influenced by the time of day of drug intake. The Cm~ and tm~xof enalapril and enalaprilat after drug administration at 08.00 h were in good agreement with those reported by Ulm et al. (1982) and by Dickstein (1986) in healthy volunteers. The increase in the tm~xof enalapril after administration in the evening compared to the morning is a p h e n o m e n o n which has been observed with many other drugs, e.g. nifedipine [Lemmer et al. 1989], indomethacin [Reinberg 1978], propranolol [Langher and L e m m e r 1988], and isosorbide-5-mononitrate [ISMN, immediate release formulation, Scheidel et al. 1987], with or without a decrease in AUC. Computer analysis of the present data indicates that the increase in the tmax after drug intake at 20.00 h was probably due to a lower rate of absorption rather than to an increased lag I00 90

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Predose values of A C E activity were 28.9 (2.5) and 29.3 (2.4) U/ml mean with (SD), at 08.00 h and 20.00 h, respectively. After the dose at 08.00 h the maximum inhibition was 80.9 (7.0) % and it occurred 4.3 (1.1) h post dose. After the dose at 20.00 h the maximum inhibition was 84.2 (4.6) % at 5.7 (1.3) h post dose. After 24 h A C E was still inhibited by 44.9 (10.2) and 33.9 (5.7) % after the morning and evening doses, respectively. The relationships between individual serum enalaprilat levels and the corresponding percentage A C E inhibition after both treatments are displayed in Figs. 2 a and b. Comparing both figures shows that the concentration-effect relationships for serum A C E inhibition were similar. This was confirmed by the excellent correlation between

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K. Weisser et al.: Enalapril kinetics 1O0

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time of absorption or a slower rate of elimination. As suggested by those authors, daily variations in gastrointestinal perfusion and/or motility might participate in this effect. Since, in contrast to the prodrug, tmax of the metabolite was almost unchanged and its Cm,~ and A U C (0-24) were even slightly increased after the evening administration, it can be suggested that enalapril underwent a larger first-pass effect in the evening. The first-pass effect of enalapril is said to amount to about 10-18% [Dickstein 1986; Irvin et al. 1984], suggesting that its hepatic clearance is rather extraction-limited and therefore probably independent of the hepatic blood flow. A higher firstpass effect (extraction rate) of enalapril in the evening might be caused by increased activity of liver enzymes during the resting period, as observed in rats [Lemmer 1989; Radzialowski and Bousquet 1968]. However, the increase in Cm~ and A U C (0-24) of enalaprilat was of no statistical significance here. Thus, the data show that drug intake at 20.00 h did not markedly (at least not negatively) influence the bioavailability of enalapril maleate as estimated from t .... Cmaxand A U C (0-24) of the active drug enalaprilat. Some authors have shown that there is a strong correlation between the serum concentration of A C E inhibitors and in vitro serum A C E activity [Francis et al. 1987; Swanson et al. 1981]. In that case the kinetic results should reveal a similar time course of A C E inhibition after both treatments, unless the enzyme activity or the affinity of the inhibitor were to show circadian variation. In fact, there was an excellent correlation between the percentage A C E inhibition after each treatment based on comparable serum concentrations of inhibitor. A circadian rhythm of A C E activity has been observed in rats, with a p e a k at the beginning of the activity period [Nahmod et al. 1982]. This would be in agreement with observed rhythms of other components of the R A A S in m a n [Gordon et al. 1966; Breuer et al. 1974; A r m b r u s t e r et al. 1975; Beilin et

al. 1983; Takeda et al. 1984]. However, in controlled studies in m a n constant values of A C E activity were found over 12 h during the day [Waeber et al. 1989; Thuillez et al. 1987]. The similar predose values obtained here at 08.00 h and 20.00 h are in line with that observation. Possible nocturnal variation in A C E activity and therefore a possible error in referring the values from 20.00 h until 08.00 h to the predose value must be considered. It should be noted, however, that even a 24 h drug-free baseline as a reference level cannot exclude errors, since, for example, any possible induction of enzyme activity as a result of inhibition [Jackson and Johnston 1984; Fyhrquist et al. 1980] would not be recognized. Nevertheless, because of the excellent correlation (Fig. 3), it is concluded that the concentration-effect relationship of A C E inhibition is the same after both treatments, and that in m a n it is unlikely that A C E activity varies markedly over a 24 h period. The present study does not exclude the possibility that the effect on blood pressure in hypertensives may differ in a circadian-stage dependent manner, since the relationship between A C E inhibition in serum (or tissues) and the effect on blood pressure is still unknown.

Acknowledgement." We thank MSD Sharp & Dohme GmbH, MiJnchen, FRG, for supporting the study and for supplying the reagents and standards for the radioimmunoassay.

References

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K. Weisser Zentrum der Pharmakologie Klinikum der Universit/~t, Haus 25 D Theodor-Stern-Kai 7 W-6000 Frankfurt/M. 70 FRG