The effect of ketoconazole and diltiazem on oestrogen metabolism in postmenopausal women after single dose oestradiol treatment

Abstract

Aims

The effect of the CYP3A4 inhibitors ketoconazole and diltiazem on the pharmacokinetics of oestrone was studied in six healthy postmenopausal women after treatment with a single oral dose of oestradiol.

Methods

Plasma oestrone concentrations were measured following the administration of 1) oestradiol, 2) oestradiol and ketoconazole and 3) oestradiol and diltiazem.

Results

Treatment with ketoconazole increased the AUC of oestrone (+ 4029 nmol l⁻¹ h; 95% CI on the difference: 1588, 6471) and its C_max (+ 306 nmol l⁻¹; 95% CI on the difference: 117, 496). The AUC and C_max of oestrone tended to increase on treatment with diltiazem although this did not reach the level of statistical significance.

Conclusions

The small increase in the plasma concentrations of oestrone formed from 17β-oestradiol during co-administration with ketoconazole is unlikely to be clinically significant.

Introduction

The metabolism of endogenous and exogenous oestradiol involves a number of enzymatic reactions. Oestradiol is converted to oestrone by 17β-hydroxysteroid dehydrogenase. Oestrone undergoes extensive oxidative metabolism by cytochrome P450 enzymes [1]. A major route is hydroxylation at positions C-2 and C-16 yielding 2-hydroxyoestrone and 16α-hydoxyoestrone, respectively. Hydroxylation can also occur at other positions, such as C-4, 6, 14, and 15. However, the significance of these last reactions is unclear.

The 2- and 4-hydroxylation of oestrone are reported to be catalysed primarily by CYP3A4 [2, 3], the activity of which is inhibited by a number of drugs, including fungicides (ketoconazole, fluconazole, itraconazole), macrolide antibiotics as well as calcium channel blockers (diltiazem, verapamil, nifedepine) [4, 5]. Thus inhibition of CYP3A4 activity could lead to elevated concentrations of oestrone. This might also shift the equilibrium towards more oestradiol formation if a feedback mechanism was in operation.

Many postmenopausal women are treated with oestrogen. These women are also potential users of antihy–pertensive agents, such as calcium channel blockers. Long-term use of oestrogen together with calcium blockers or other CYP3A4 inhibiting drugs could lead to a higher exposure to this hormone and therefore an increased risk of oestrogen-related adverse effects.

In the present study, we have examined the pharmacokinetics of oestrone in healthy postmenopausal women after cotreatment with a single oral dose of oestradiol and multiple doses of either ketoconazole, a probe CYP3A4 inhibitor or diltiazem, which is commonly used to treat angina pectoris and hypertension, and which also inhibits CYP3A4 activity.

Methods

Study population and design

Six healthy postmenopausal women (age 55–64 years, body mass index 23–31 kg m⁻²) who were not being treated with oestrogen participated in the study, which was approved by the Ethics Committee at Huddinge University Hospital.

Study part I

The subjects received a single oral dose of 2 mg oestradiol (Femanest®, Tika Läkemedel AB, Lund, Sweden) in the morning. Venous blood samples (15 ml) were obtained immediately before and 2, 4, 6, 8, 10, 24, 32, 48 and 56 h after drug administration.

Study part II

Ten to 16 days following the first oestradiol dose, oral treatment with ketoconazole was commenced. The subjects received 100 mg ketoconazole (Fungoral®, Janssen-Cilag, Stockholm, Sweden) twice a day on day 1. On day 2, subjects received 100 mg ketoconazole and 2 mg oestradiol 1 h apart in the morning and 100 mg ketoconazole in the evening. Blood samples were obtained immediately before and 2, 4, 6, 8, 10, 24, 32, 48 and 56 h after the administration of oestradiol. Treatment with ketoconazole 100 mg twice daily continued for 2 more days after oestradiol treatment.

Study part III

Two to 3 months after study part II, subjects received oral diltiazem (Cardizem®, Pharmacia & Upjohn, Stockholm, Sweden) in a dose of 30 mg twice daily for 4 days. On day 2, the subjects also received 2 mg of oestradiol in the morning 1 h after the diltiazem dose. Blood samples were collected as in study part II.

Safety assessment

Subjects tolerated treatment well and apart from mild gastrointestinal upset during ketoconazole treatment no adverse effects were observed. One subject (ML) participated in study part I and II but was excluded from part III because of an abnormal ECG observed on enrolment.

Analysis of oestrone in plasma

Plasma was separated by centrifugation and stored at −20 °C until analysed. Plasma concentrations of oestrone were determined by radioimmunoassay after extraction with diethyl ether, according to Lindberg et al. [6] with certain modifications. The sample volume was increased to 400 µl, the original antibody was substituted by antioestrone from Diagnostic Systems Inc, Webster, TEX, USA, and the incubation volume and amount of dextrane-coated charcoal were changed. The detection limit and within and between assay coefficients of variation were 30 pmol l⁻¹, 7% and 10%, respectively.

Pharmacokinetic analysis

Area under the plasma concentration-time curve (AUC(0,56 h)), peak concentration (C_max), time to peak (t_max) as well as terminal half-life (t_1/2) were calculated by noncompartmental analysis (NCA) using WinNonlin™ software [7].

Statistics

The data were compared using paired t-tests with the level of statistical significance set at P < 0.05. The values for measured parameters were logarithmically transformed and oestradiol treatment alone was compared with combined treatment (oestradiol + ketoconazole) and (oestradiol + diltiazem), respectively.

Results

Study part I

Pharmacokinetic measurements for oestrone in plasma following oral administration of oestradiol is presented in Table 1. As shown, there was a wide interindividual variation in AUC (7726–33003 nmol l⁻¹ h) and in C_max (374–1208 nmol l⁻¹). The mean for t_1/2 was 34 h (individual values 22, 24, 23, 38, 74, 24 h) and the mean for t_max was 6 h (range 4–8 h).

Table 1.

Effect of ketoconazole and diltiazem on the AUC and C_max of oestrone following administration of 17β-oestradiol to postmenopausal women

	AUC (nmol l−1h)			Cmax(nmol l−1)
Subject	Oestradiol	oestradiol + ketoconazole	Oestradiol + diltiazem	Oestradiol	Oestradiol + ketoconazole	Oestradiol + diltiazem
GM	21 324	24 248	22 744	1079	1315	1059
IM	23 085	32 753	23 433	1208	1972	1197
AC	33 003	35 177	38 132	1066	1239	1473
KL	24 485	28 517	22 845	856	1001	871
ML	7 726	8 610	n.d.	374	538	n.d.
RM	24 050	28 545	27 255	1031	1387	1267
Mean	22 279	26 308	25 190	936	1242	1173
SD	8 202	9 461	4 534	297	472	226
P value		0.010	0.22		0.004	0.20
95% CI for relative differences* (%)		8, 28	−2, 14		18, 48	−4, 29

^*Oestradiol alone vs oestradiol + ketoconazole or diltiazem. n.d: not determined.

Study part II

As shown in Table 1, oral treatment with ketoconazole increased AUC of oestrone by 16% (range 7–42) and its C_max by 30% (range 16–63). The mean for t_1/2 in the subjects was 29 h (individual values 26, 30, 26, 25, 45, 22 h) and the mean for t_max was 5 h (range 4–6 h).

Study part III

As shown in Table 1, there was a tendency towards higher AUC and C_max of oestrone following oral treatment with diltiazem, although this did not reach the level of statistical significance. The mean for t_1/2 in the subjects was 24 h (individual values 21, 24, 27, 22, 26 h) and the mean for t_max was 6 h (range 4–8 h).

Discussion

In the present study, treatment with the CYP3A4 inhibitors ketoconazole and diltiazem led to slight increases in AUC and C_max of oestrone. The effect of ketoconazole was more pronounced than that of diltiazem, probably because ketoconazole is a more powerful inhibitor of CYP3A4 activity. Previous studies on the CYP3A4-dependent metabolism of omeprazole have shown that a marked inhibition of CYP3A4 activity by ketoconazole is reached at a dose of 100 mg and that further dose escalation does not significantly increase the inhibition of the enzyme [8]. The dose of diltiazem was chosen so that an inhibitory effect on the enzyme could be achieved with a minimal risk of cardiovascular adverse effects to the subjects. Although the dose of diltiazem used was relatively low compared with those used clinically, studies have shown a larger effect of ketoconazole on the metabolism and dose requirement of cyclosporin (also a CYP3A4 substrate) compared with diltiazem even when the latter was used in clinical doses [9, 10].

Plasma concentrations of oestrone following oral administration of 17β-oestradiol are far higher than those of 17β-oestradiol itself, but the two are closely correlated [11, 12]. Orally administered 17β-oestradiol is converted mainly to oestrone by 17β-hydroxysteroid dehydrogenase. Thus, selective inhibitors of CYP3A4 activity would not be expected to increase concentrations of 17β-oestradiol. In support of this, it has been reported that grapefruit juice, another inhibitor of CYP3A4 activity, increases the AUC of oestrone but not 17β-oestradiol. [13]. However, we were able to measure 17β-oestradiol in three of our subjects and found that its concentration increased after ketoconazole administration to the same degree as did that of oestrone.

To summarize, the slight increase observed in plasma oestrone concentrations upon cotreatment with ketoconazole or diltiazem suggests that postmenopausal women can be treated with oestrogen and CYP3A4 inhibiting drugs, such as calcium channel blockers, without a significant risk of increased exposure to oestrogen and its adverse effects.