Fundam Clin Pharmacol(1991) 5, 193-201 0 Elsevier. Paris

193

Lack of effect by nifedipine on hepatic mixed function oxidase in man

Y Horsmans I , JP Desager ', S Pauwels 2, C Harvengt '' I Universitt! Catholique de Louvain, Laboratoire de Pharmacothkrapie;

Universitt! Catholique de Louvain, Centre de Medecine Nucleaire, 53, A venue E Mounier, B-I200 Brussels, Belgium

(Received 9 March 1990; accepted 16 November 1990)

Summary - Nifedipine (NF), a calcium channel blocker, is often prescribed in association with other drugs. Therefore, it was interesting to know whether or not, nifedipine, which is metabolized by the cytochrome P-450,,, was able to induce or to inhibit in vivo the activity of the hepatic mixed function oxidase sys- tem. The study was conducted in ten young healthy male volunteers receiving 20 mg NF slow release bid for 15 days. Due to the small number of subjects, comparison of the NF pharmacokinetics at dose 1 and 26 failed to show a bimodality in the frequency distribution of its area under the plasma concentration- time curve (AUC 274.5 to 317.1 ngml-I h, NS). Hepatic microsomal autoinduction (t,,, 2.87 to 3.06 h, NS) was not found. No statistically significant effect was seen on the aminopyrine breath test and on the debrisoquine metabolic molar ratio performed before and at the end of the treatment. Unlike what has been suggested by in vitro studies, NF treatment did not modify significantly the urinary excretion of 6 beta-hydroxycortisol(318 to 265 pg/d, NS). After the last dose, the total oral clearance of NF was highly correlated with the metabolic clearance to 4-hydroxyantipyrine (r = 0.88; P = 0.005) but the other parameters of antipyrine biotransformation remained unchanged. We conclude that repeated nifedipine oral intake does not modify enzymatic activities of hepatic P-450 cytochromes involved in the biotransfor- mation of antypirine, aminopyrine, debrisoquine and cortisol.

nifedipine I hepatic MFO system I man

Introduction

Nifedipine (NF) is a calcium channel blocker commonly prescribed in the treatment of angina pectoris and hypertension (Braunwald, 1982 ; Echizen and Eichelbaum, 1986; Henry, 1980). In vifro, nifedipine has been shown to be oxidized by a particular cyt P-450, namely cyt P-450 NF which also catalyzes steroid 6p hydroxylation (Guen-

* Correspondence and reprints

194 Y Horsmans et al

A 0 1

000 6-pOHF

lgerich et al, 1986; Waxman et al, 1988; Aoyama et al, 1989). A possible oxidation polymorphism regulating his biotransformation has been first evoked (Kleinbloesem et al, 1984) and not confirmed thereafter (Schellens et al, 1988).

Verapamil and diltiazem, two other calcium channel blockers, undergo oxidative metabolism by the hepatic cytochrome P-450 mixed function oxidase (MFO) and may inhibit the cyt P-450-dependent biotransformation of drugs like aminopyrine or an- tipyrine (Bauer et al, 1986; Renton, 1985). Unlike these drugs, nifedipine does not impair antipyrine oxidation (Bauer et al, 1986; Dickinson et al, 1988).

In order to assess in vivo a possible nifedipine oxidation polymorphism and the influence of nifedipine on the cyt P-450 system, the effect of a standard NF 15 days treatment on various markers of some activities has been investigated in ten healthy volunteers. These markers are : detection of a microsomal autoinduction by compa- rison of NF pharmacokinetic parameters after the first and the 26th dose, influence of NF treatment on debrisoquine metabolic molar ratio, aminopyrine breath test, an- tipyrine biotransformation and cortisol 6 0-hydroxylation.

1 AC

ABT ' NlFEDlPlM

0 BO 6 pOHF

KINETICS

[FIRST DOSE1 20 mq v

NFEDlPlNE KINETICS

Materials and Methods

Subjects

Ten male volunteers, aged 23-34 years, weighing 72-92 kg, with normal ECG and healthy on the basis of medical history and physical examination, took part in the study. They were,non- smoking subjects and alcohol-abstinent from 2 weeks before and during the whole study period. None of the subjects was receiving other drug throughout the study.

All of them gave written informed consent for the study which was approved by the Ethics Committee of the Cliniques Universitaires St Luc (Brussels, Belgium).

STUDY DESIGN

NlFEDlPlNE SLOW RELEASE 20 mg b.1.d. - ABT : AHINOPYRINE BREATH TEST I+OHF ' 6-beta- HYOROXYCORTISOL DBO : DEBRISWUINE TEST AC : LNTlPYRlNE CLEARANCE I SALIVA AND URINE I

Fig 1. Study design.

Nifedipine and hepatic MFO in man 195

Study design

The design of the study was explained in figure 1. The pre-treatment values of the different investigated markers, aminopyrine breath test (ABT), debrisoquine metabolic molar ratio (DBQ- MMR), urinary excretion of 6 P-hydroxycortisol (6 /3 OHF), antipyrine clearance test (AC), were evaluated the week before. The pharmacokinetics of a single 20 mg dose of nifedipine slow release were estimated in the fasting volunteers for the next 24 h. From day 1 to day 15, 20 mg nifedipine slow release was given bid to the subjects at 8 am and 8 pm. Nifedipine as sIow-release preparation was a generous gift from Bayer (Belgium).

The DBQ-MMR and 6 beta O H F urinary excretion were measured during the 20th and after the 21th dose. The ABT and the nifedipine pharmacokinetics were repeated after the 24th and 26th dose, respectively. Finally, the AC test was performed from dose 29 up to 31.

Methods

ABT was performed in the fasting subjects (Pauwels et al, 1982). Half of the regular dose was used for each test (2 x 0.5 pCi). Debrisoquine and 4-hydroxydebrisoquine were determined according to Lennard et al (1977) after a single dose of debrisoquine taken at bedtime.

The urinary elimination of 6 beta-hydroxycortisol was measured by radioimmunoassay (Park, 1978), and it was corrected by the determination of the urinary excretion of 17-hydroxysteroids based on the colorimetric method of Silber and Porter (1954). The oral intake of 1 g antipyrine was followed by a 48 h-urine collection and 33 h-salivary sampling. Determination of antipyrine and its urinary metabolites was performed by the HPLC method of Teunissen et al (1983). The metabolic clearance for production of the antipyrine metabolites (3-hydroxymethylantipyrine HMA, norantipyrine NORA, 4-hydroxyantipyrine OHA) was calculated as proposed by Danhof et al (1982).

Nifedipine in plasma was measured according to Lesko et al (1983) and values processed by the ELSFIT program (Sheiner, 1983). Blood was collected from an antecubital vein into tubes containing EDTA: before drug intake and 11 times thereafter (in h : 0.5, 1, 1.5, 2, 2.5, 3 ,4 , 6, 8, 12, 24). Plasma was separated from blood within 30 min, protected from light and stored at -20°C.

Statistical analysis

Statistical significance for difference between baseline values and treatment values was evalu- ated by Student’s paired t test (significant difference at P < 0.05).

The non parametric WiIcoxon test was used for statistical evaluation of the pharmacokine- tic parameters (dose 1 versus 26).

Results

A wide variability in the nifedipine plasma pharmacokinetics (AUC, T,,2, T,,, CmU, total clearance) was observed among the volunteers after the first as well as after the 26th dose but, for each volunteer, no significant difference in AUC and other phar-

196 Y Horsmans et al

macokinetic parameters was observed between these two periods. A slight increase of C,,, was found between the first and the 26th dose induced by the steady state (fig 2, table I). Despite this wide variability in AUC, no bimodal distribution was observed. A good correlation was found between the nifedipine plasma levels mea- sured 12 h post drug after the first and the 26th tablet (r = 0.9117).

Before first nifedipine intake, salivary antipyrine clearance was 54.61 & 1.28 mI/min and T,,, was 13.97 & 0.92 h. On nifedipine, no modification was found; clearance: 53.01 k 5.2 ml/min and T,,,: 14.50 k 0.96 h (table I).

Table 1. Antipyrine and nifedipine mean pharmacokinetic parameters ( f SEM and range) at baseline and after repeated nifedipine administration.

A ntipyrine Baseline Nifedipine (dose 29-31)

half-life (h) 13.97 ? 0.92 14.5 f 0.96 (10.12- 19.26) (9.32- 18.63)

oral total clearance (mllmin)

area under the curve hg/ml-’-h)

Nifedipine

half life (h)

oral total clearance (ml/min)

area under the curve (ng ml-I-h)

54.12 k 4.73 53.01 f 5.27

323.01 2 24.4 337.75 f 27.44

(38.10- 75.32) (34.40-91.43)

(198.28 -437.47) (182.27 -484.45)

Dose I Dose 26

2.87 f 0.37 3.06 ? 0.37

1401.6 +_ 183 1221.8 f 162

274.5 f 36.9 317.07 k 42.7

(1.32 -4.79) (1.89-5.81)

(603.2 - 2796.4) (527.1 -2367.5)

( 1 19.2 - 552.6) (140.8 - 631.6)

1 I 1 I I l I , , 1 2 3 L 5 6 7 8 9 l o l l 1 2 h

Fig 2. Mean nifedipine plasma levels after the first dose (0) and after the 26th dose (a) of 20 mg nifedipine slow release in ten healthy subjects.

Nifedipine and hepatic MFO in man 197

In figure 3 were shown the individual variations of the metabolic clearance for production of the three main antipyrine metabolites (HMA from 4.9 to 4.6, NS; NORA from 10.9 to 10.4, NS; OHA from 21 to 19.4, NS, units: ml min-’). Corre- lation coefficients between nifedipine oral clearance and metabolic clearance to each antipyrine metabolite were calculated (table 11).

All the volunteers were extensive metabolizers of debrisoquine and no significant change in debrisoquine metabolic molar ratio was observed on nifedipine (0.46 to 0.50, NS) (fig 4).

Similarly, aminopyrine breath test (2.20 to 2.32% h- l , NS) and the 6 P- hydroxycortisol urinary excretion (3 18 to 265 pg/d, NS) were not significantly modi- fied nor the ratio 6 P-hydroxycortisol/l7 hydroxysteroids (36.8 to 33.7, NS) (fig 4).

Moreover, no correlation was found between the level of urinary 6 P-hydroxycortisol and the clearance of nifedipine in each volunteer. Tiredness was mentioned by 8 out of 10 volunteers during the 2 or 3 first days on nifedipine and disappeared thereafter.

Table 11. Correlation coefficients between nifedipine oral clearance and metabolic cleacance to antipyrine metabolites.

CI,, (dose 1)

CI,, (dose 26)

0.57 0.43 0.75* 0.58 [0.67]* [0.49]* [0.69]* [0.37] *** 0.76’ 0.43 0.77* 0.88**

* P < 0.02; ** P < 0.005; P < 0.05; [ ] Values from Schellens el a1 (1989).

nu* NORA OH*

’ O l 75 -

5-

2.5 -

4 0 1

’“1 Fig 3. Urinary clearance (ml min-I) for production of the antipyrine metabolites before (B) and after (A) the 28th dose of 20 mg nifedipine slow release in ten healthy subjects.

198 Y Horsmans ef a1

A

\ 3

- 1.5

B A

2 0 7 5

/ i

0 25

I , B A

IP-OHF l7OHC AMINOPYRINE BREATH TEST DEBRISOOUINE HETABOLIC / RATIO

Fig 4. Individual variations of 6 /3-OHF/17 OHC, ABT and DBQ-MMR in 10 subjects before (B) and after (A) at least 10 days on nifedipine 20 mg (see text).

Discussion

The nifedipine oxidative metabolism has been shown to be catalyzed by a particular cytochrome P-450, namely cyt P-450,, (Guengerich et a], 1986; Waxman et al, 1988; Aoyama et al, 1989). The rate of this oxidation shows a wide interindividual variabil- ity in all studies conducted either with the standard capsule or with the slow release tablets (Lobo et al, 1986; Kleinbloesem et al, 1987; Beerahee et a/ , 1987).

In the present study, this wide variability is also found after the first and the 26th dose. The comparison of these values between the two periods allows to exclude a microsomal autoinduction at this dosage. Although only a small number of volun- teers participated in this study, a bimodal distribution of nifedipine AUC was not observed. This confirms the results of Schellens et a1 (1988) considering a more elevated number of volunteers, which have invalidated those of the first study published by Kleinbloesem et a1 (1984) for a possible bimodal distribution of NF oxidation.

Lack of nifedipine effect on plasma antipyrine clearance has been demonstrated previously by Bauer et a1 (1986). Thereafter, Dickinson et a1 (1988) have also shown unchanged urinary antipyrine metabolites excretion on nifedipine.

The results of our study are fully in agreement with ineffectiveness of nifedipine on antipyrine biotransformation and excretion. However, a change in the statistical signification of the correlation coefficients calculated between the total clearance of nifedipine and the clearances to the 3 antipyrine metabolites is observed between the first and the last doses of nifedipine. For the first dose, our data for r are similar to those reported by Schellens et a1 (1989) although in the latter the statistical significance is reached by pooling data from baseline, after induction and inhibition

Nifedipine and hepatic MFO in man 199

of cyt P-450. After regular intake of nifedipine, the r value is greatly improved for 4-hydroxyantipyrine with a P value better than 0.005. This would suggest a selective effect on the cyt P-450 responsible for an increased or decreased 4-hydroxyantipyrine production.

The present results confirm the lack of nifedipine effect on some cyt P-450 activi- ties unlike two other calcium channel antagonists, verapamil and diltiazem (Bauer et al, 1986; Egan et al, 1986). The mechanism of verapamil effect on antipyrine bio- transformation is attributed in vitro to a competitive inhibition for the formation of two major antipyrine metabolites, 4-hydroxyantipyrine and 3-hydroxymethylantipyrine (Egan et al, 1986). Nifedipine at dosage of 20 mg bid in our study and 30 mg tid in the study of Dickinson et a1 (1988) does not impair antipyrine biotransformation in man. The antipyrine test reflects activities from at least 3 discrete mixed-function oxidases (Vesell and Penno, 1983). Our results confirm the lack of nifedipine effect on some cyt P-450 activities linked to hepatic antipyrine biotransformation. It is thus also important to investigate whether nifedipine influences other markers of the cyt P-450 activities like debrisoquine metabolic molar ratio, aminopyrine breath test and 6 P-hydroxycortisol/ 17 hydroxysteroids ratio. After nifedipine treatment, the DBQ- MMR is unaffected and unlike another study using the same dose of nifedipine (20 mg bid), no relationship between 12 h NF plasma levels and debrisoquine hydroxyla- tion phenotype or DBQ-MMR value is found (Beerahee el al, 1987).

Some studies (Guengerich el al, 1986; Waxman et al, 1988; Aoyama et al, 1989), have shown on rat and human microsomes that cyt P-450N, is the enzyme which catalyzes the biotransformation of nifedipine but also of substrates like quinidine, other 1, 4-dihydropyridines, testosterone, androstenedione, progesterone, erythromy- cin. These studies have also suggested that cyt P-450NF could be responsible for cor- tisol 6 P-hydroxylation. More recently, Ged et al (1989) has shown that urinary 6 P-hydroxycortisol is an excellent marker of P-45oNF induction. Conversely, in absence of hepatic microsomal induction, 6 beta-hydroxycortisol excretion is not a good parameter of cyt P-450NF activity (Park, 1981 ; Desager et al, 1987; Ged et al, 1989). The absence of modification in 6 P-hydroxycortisol urinary excretion and of correla- tion between 6 /3-hydroxycortisol excretion and total clearance of nifedipine in our study are 2 arguments for the lack of cyt P-45oNF induction by a chronic nifedipine treatment.

In conclusion, the main finding of our study is that chronic nifedipine oral intake fails to modify the enzymatic activity of hepatic cyt P-450s catalyzing the biotrans- formation of substrates like antipyrine, aminopyrine, debrisoquine and cortisol.

Acknowledgments

We wish to thank Dr B Vanderelst for the physical examination of the volunteers. The techni- cal assistance of Mr J Costermans and Y Van Nieuwenhuyze is acknowledged. We are indebt- ed to Mrs N Bouve for typing the manuscript.

200 Y Horsmans et al

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