- Email: [email protected]
Pharmacokinetics of cyproterone acetate and ethinylestradiol in 15 women who received a combination oral contraceptive during three treatment cycles
Pharmacokinetics of cyproterone acetate and ethinylestradiol in 15 women who received a combination oral contraceptive during three treatment cycles W. Kuhnz,”
T. Staks” and G. Jiittingf
lSchering Aktiengesellschaft, Abteilung, Kreiskrankenhaus Germany
13342 Berlin, Germany and tGeb. -gyn. Eutin, Janusstrape 22, 2420 Eutin/Holst.,
The pharmacokinetics of cyproterone acetate (CPA) and ethinylestradiol (EEJ were determined in 15 healthy women (age 19 to 34 years), following single dose administration of a combination oral contraceptive, containing 2.0 mg CPA together with 0.035 mg EE, (Diane-35R). After a wash-out period of one week, the same preparation was administered during a treatment period of three months. After single dose administration, maximum concentrations of CPA in the serum were 15.2 _t 6.6 nglml. Post maximum drug levels declined biphasically with half-lives of 0.8 ? 0.4 h and 54.0 & 26.0 h, respectively. The apparent clearance was calculated to be 3.6 + 0.9 ml x min-l x kg-’ and the volume of distribution (V,) was 986 2 437 1. The free fraction of CPA was 3.5 + 1.9% and the fractions bound to heat labile proteins and albumin were 4.6 & 2.2 % and 92.0 & 3.5%, respectively. Trough levels of CPA in the serum increased during a treatment cycle, reaching a steady-state around day 16. An about two-fold accumulation of CPA was observed, which was less than expected theoretically. SHBG concentrations in the serum increased by a factor of three during a cycle, without having any effect on the protein binding of CPA. At the end of treatment cycle three, the terminal half-life of CPA had increased to a mean value of 78.6 + 16.0 h and the volume of distribution to a value of 1304 -+ 427
Submitted for publication September 7, 1993; accepted for publication October 27, 1993. Address correspondence to Dr. W. Kuhnz, Institut fiir Pharmakokinetik, Schering Aktiengesellschaft, 13342 Berlin, Germany.
0 1993 Butterworth-Heinemann
Contraception
1993:48,
December
557
Pharmacokinetics
of cyproterone
acetate and ethinylestradiol:
Kuhnz et al.
1. The apparent clearance showed a small, although significant decrease to a value of 3.0 ? 0.4 ml x min-’ x kg’. The observed changes V, and t,,2 during the treatment period were attributed to the distribution of CPA into a deep compartment and the slow release of the drug from this compartment. The AUC(O-4h) values of EE, following single dose administration of the combination oral contraceptive were found to be 187.5 2 79.7 pg x ml-- I x h. On the last day of cycles one and three, the AUC(O-4h) values were 311.2 +- 109.3 and 304.8 ? 121.5 pg x ml- i x h, respectively, which corresponds to an about 60% increase as compared to single dose administration. Total and free testosterone concentrations decreased during treatment cycles one and three by about 39 % and 62%, respectively, compared with the corresponding values measured prior to treatment. Keywords: Pharmacokinetics; cyproterone acetate; ethinylestradiol; binding; testosterone; combination oral contraceptive.
protein
Introduction Cyproterone acetate (CPA) is a potent steroidal antiandrogen with progestational activity and is used alone and in combination with ethinylestradiol (EE,) in the treatment of women who suffer from disorders associated with androgenization, like severe acne or hirsutism.’ At a dose of 2 mg, CPA is used in combination with 0.035 mg EE, as an oral contraceptive preparation for women with androgen-dependent skin problems. The pharmacokinetics of CPA in humans have been investigated in several studies.24 In one of them, a combination oral contraceptive containing 2 mg of CPA and 0.035 mg EE2, was administered to two groups of smoking and non-smoking women over a period of one treatment cycle. The drug levels in plasma were determined radioimmunologically and were used to derive the basic pharmacokinetic parameters of both steroids.5 No differences were seen in the pharmacokinetics of CPA in both groups of women. The aim of the present study was to examine in more detail the pharmacokinetics of CPA and EE, in women who received the same CPAcontaining contraceptive formulation during a treatment period of three months. In addition to the already available information obtained for a period of one treatment cycle, it was of interest to examine whether prolonged treatment with this preparation was associated with further changes in the pharmacokinetics of CPA, EE, or other parameters, like the concentration of serum binding proteins, CBG and SHBG and total and free testosterone levels in the serum. A highly specific and sensitive gas chromatographic/mass spectrometric (GC/MS) method was used for the analysis of CPA in the present study, in order to allow for a comparison
558
Contraception
1993:48, December
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
with the corresponding results of the previously performed study, where CPA levels were measured radioimmunologically.
Material and Methods Study Design The study was performed as an open, intraindividual comparison in 15 healthy young women. In the first part of the study, the women received a single oral dose of 2.0 mg CPA together with 0.035 mg EE, (Diane-35R, Schering AG) and, after a wash-out period of 1 week, the same women received the contraceptive formulation over a treatment period of three cycles. Each treatment cycle was separated by a drug-free interval of 1 week. The drug was administered in the morning. On those days where blood was sampled repeatedly for pharmacokinetic purposes, the drug was administered after an overnight fast. A standardized breakfast was served one hour after treatment. Blood samples were collected at the following time points: Single dose: Immediately prior to drug intake (0 h) and 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24, 48, 72, 96 and 120 h after administration. Treatment cycles one and three: day 1: 0, 0.5, 1, 1.5, 2, 4, 6, 8, 12 h; days 2, 3, 5, 8, 10, 14, 16, 18: immediately prior to drug intake; day 21: 0, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24, 48, 72, 96 and 120 h after administration. All blood samples were kept at 4°C until coagulation. The serum was separated by centrifugation and stored at - 20°C until analysis. Study Population Fifteen healthy, young women participated in the study. The women had normal biphasic menstrual cycles and showed low variation (mean +S.D.) in body weight (63 * 7 kg), height (167 + 6 cm) and age (26 ? 6 years). All women had not taken oral contraceptives for at least two months prior to their participation in the study. The subjects underwent a laboratory screening and a thorough medical and gynecological examination before entering the study. Excluded from participation were subjects who had any contraindication to the use of contraceptive steroids or a concomitant medication which might interfere with the pharmacokinetits of the contraceptive steroids CPA and EE,. Also excluded were women who smoked. The nature and purpose of the study was explained and
Contraception
1993:48, December
559
Pharmacokinetics
of cyproterone
acetate and ethinylestradiol:
written informed consent was given by each participant. approved by the local ethics committee.
Kuhnz et al. The study was
Analytical Methods The concentrations of CPA in the serum samples were measured in duplicate by a specific CC/MS method available at LAB (Neu-Ulm, FRG). After the addition of the internal standard (10 ng chlormadinone acetate] and 0.2 ml 0.1 N NaOH, 1 ml of serum sample was extracted with 6 ml of cyclohexane/i-butanol (98.5/1.5; v/v). After centrifugation (10 min, 5000 rpm), the organic phase was separated and stored in a refrigerator at 4°C until analysis. The GC/MS analysis of CPA was performed on a Finnigan MAT 4021 GC/MS system. A fused silica open tubular capillary column of cross-linked methyl silicone type was used for GC-separation. The MS was operated in the negative ion chemical ionization mode with methane as reagent gas. Selected ion monitoring was performed for m/z 356, the base peak of CPA and m/z 344, the base peak of the internal standard. The standard curve was linear over a range of 0.1 to 50 ng CPA/ml serum and the lower limit of quantification was 0.1 ng/ml. All samples were measured in sequences. One sequence consisted of the samples from one subject, one set of calibration samples and 8 quality control samples (0.15, 0.3, 3.0 and 30.0 ng/ml in duplicate). The coefficient of inter-assay variation was between 9 and 13%. The deviation of measured from nominal concentration values of quality control samples was less than 3%. The analysis of EE, has been described in detail before.7 Analyses were performed in duplicate by a radioimmunoassay using an antiserum raised in rabbits against EE,-b@CH,CO-bovine serum albumin (Schering AG) and [6,7-“HI-EE, (specific activity 2.2 TBq/mmol, NEN Products, Boston, MA, USA) as tracer. Diethylether extracts obtained from 0.3 ml serum were taken for analysis. Assay quality was assessed by the inclusion of quality control samples (50, 125 and 250 pg/ml) in each assay. Inter-assay precision was between 11 and 24%, and intra-assay precision was between 7 and 25%. The deviation of measured concentrations of EE, from nominal values was between 5 and 16%. The lower limit of quantification was 20 pglml. SHBG, CBG, total and free testosterone samples were each measured in duplicate immunoassays (SHBG: Diagnostic Products Medgenix, Fleurus, Belgium; total and free ucts Corp.). Two or three quality control these assays. The nominal concentrations of 20-28 and 56-82 nmol/l, respectively, measured in a total of 7 assays were 24.0
560
concentrations in the serum using commercially available Corp., Los Angeles, CA; CBG: testosterone: Diagnostic Prodsera were included in each of of SHBG were in the range and the mean concentrations t 2.5 and 63.2 + 4.4 nmol/l,
Contraception
1993:48,
December
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
respectively. Inter-assay precision (C.V.) was between 7 and 11%. CBG control sera contained mean nominal concentrations of 28 + 12 and 115 + 37 kg/ml, respectively, and the measured concentrations were found to be 26.7 k 0.7 and 83.7 * 1.6 kg/ml, respectively. Inter-assay precision was between 2 and 3%. For total and free testosterone determinations, three quality controls each were included in each assay. The nominal concentrations of total testosterone controls were 0.7, 2.7 and 6.9 ng/ml and the corresponding measured concentrations (n = 8 assays) were 0.65 ? 0.08 ng/ml, 2.47 -t 0.20 ng/ml and 6.02 + 0.48 ng/ml, respectively. Inter-assay precision was between 8 and 12%. The nominal concentrations of free testosterone controls were 2.5, 8.5 and 29.6 pg/ml and the corresponding measured concentrations (n = 8 assays) were 2.7 rf: 0.1 pg/ ml, 9.5 -+ 0.7 pg/ml and 34.5 t 2.9 pg/ml, respectively. Inter-assay precision was between 5 and 9%. Serum
Protein Binding
of CPA
The serum protein binding of CPA was performed by equilibrium dialysis because a high nonspecific binding of the drug to the membrane and the filtration unit precluded the use of the ultrafiltration method. In a pre-test, the optimum dialysis time was determined by successively increasing the time until a constant value of the free fraction was obtained. A dialysis time of 2 hours was sufficient to reach equilibrium. Recovery after dialysis was found to be between 76 and 83%. In the following experiments, the recovery was calculated for each individual sample by measuring the total radioactivity in aliquots of buffer and serum at equilibrium. Individual recovery values were ca. 70 to 100%. Serum aliquots of 500 ~1 were spiked with 3H-CPA (10,000 cpm) and equilibrated for 0.5 h at room temperature. Dialysis was carried out in self-designed Teflon cells with a volume of ca. 120 ~1 per half-cell. One hundred p,l of serum were dialysed against 100 p,l of dialysis buffer, separated by a high-permeability membrane (Diachema, molecular weight cut-off 5 000). The cells were rotated at 12 rpm for a period of 2 h at a temperature of 37°C. When dialysis was terminated, 50 ~1 aliquots of both, the buffer and the serum side, were taken and after addition of a scintillation cocktail (Atomlight, NEN), total radioactivity was counted in a liquid scintillation counter. The unbound fraction (fU)of CPA was calculated according to equations (I) and (II), with corrections for volume shift and losses of drug due to nonspecific adsorption.*
f”= (A, -
C” x vs &oss- C” x v,
A loss= A, - A,
Contraception 1993:48, December
(11 (111 561
Pharmacokinetics
of cyproterone
f, = fraction
acetate and ethinylestradiol:
Kuhnz et al.
unbound
C,, = unbound dialysis
concentration
or drug concentration
V, = serum volume
before dialysis
V,, = buffer volume
before dialysis
in buffer after
A,, = amount
of drug in the serum before dialysis
A, = amount
of drug in serum and buffer at equilibrium
A IDSS= drug lost by nonspecific
binding to membrane
and cell
For the determination of the fraction of CPA bound to heat labile proteins, 500 ~1 of each sample were kept for 1 h at 60°C. Subsequently, the samples were spiked with “H-CPA (10,000 cpm), equilibrated for 0.5 h at room temperature and dialysed as described above. The calculation of the fractions of CPA bound to heat labile proteins and albumin, respectively, was performed according to a published procedure.”
Pharmacokinetic Evaluation The serum concentrations of CPA, which were obtained after single oral administration and on the last treatment day of cycles one and three, were evaluated by model-fitting (TOPFIT 2.0, Goedecke, Schering AG, Thomae GmbH, Germany) on the basis of an open two-compartment model. The data obtained by model-fitting from the single dose administration were used for the simulation of total CPA concentrations in serum during one treatment cycle. Accumulation and mass balance factors of CPA were calculated according to: AUC:\ R’ = AUC(0 -&o (Cg,,)exp mentally;
= minimun
(C:,,)sim = minimum simulation;
(CzJexp ’ R* = (C:,,)sim
concentration concentration
. R** ’
at steady-state
zz- AUQ AUC,,,
;
determined
at steady-state
experi-
determined
by
AUC? = area under the serum level-time curve during one dosing interval at steady-state (7 = dosing interval); AUC,, = area under the serum level-time dose administration
562
curve obtained
Contraception
1993~48,
after single
December
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
Clearance at steady-state and the volume of distribution V, (assuming a constant CL) were calculated according to: CL
=
DMx f AUC$
v
’
=cL
A,
DM = maintenance dose f = bioavailable fraction A, = disposition constant of the terminal phase The AUC was calculated according to: AUC = AUC(0 - t”) + 2 AUC(0 - t,,) = area under the serum concentration-time curve up to the last concentration value (C,) measured at the time point (tJ The mean residence time (MRT} gives the average time necessary for the drug molecules to leave the body from when the dosage form is administered: MRT = e AUMC = area under the moment curve Those concentrations of CPA which were obtained on day 1 of cycles one and three, respectively, as well as the concentrations of EE, obtained after single dose administration and on days 1 and 21 of both cycles were evaluated model independent. Statistical
Evaluation
Pharmacokinetic parameters of CPA, like AUC, tlj2, CL and V, which were calculated for each subject after single dose administration and following the last drug intake on cycles one and three, were compared by the two-sided paired Student t-test (significance level ct = 0.05). The same test was applied for the following comparisons: SHBG (CBG) concentrations which were measured on day 2 1 of cycles one and three, respectively; simulated and measured (cycles one and three] trough levels of CPA at
Contraception 1993:48, December
563
Pharmacokinetics
of cyproterone
acetate and ethinylestradiol:
Kuhnz et al.
steady-state; C,,,, AUC(O-4h) and AUC(O-24h) values of EE, on day 21 of cycles one and three, respectively.
Results The treatment was well tolerated by the women. No positive results were obtained during the drug screening and the pregnancy tests. Pharmacokinetics of CPA and EE, After Single Dose Administration The mean concentration values of CPA in the serum of 15 women at various time points following single oral administration of 2 mg CPA together with 0.035 mg EE, and after the last drug intake during treatment cycles one and three, respectively, are presented in Table 1. A summary of the pharmacokinetic parameters of CPA is given in Table 2. After single oral administration, CPA was rapidly absorbed and maximum concentrations of 15.2 + 6.6 ng/ml were measured in the serum. Post maximum drug levels declined biphasically with half-lives of 0.8 rt 0.4 h and 54.0 + 26.0 h, respectively (Figure 1). The AUC was 160.2 & 44.8 ng x ml-l x h. The apparent volume of distribution (V,) and the apparent total clearance were calculated to be 986 +- 437 1 and 3.6 ? 0.9 ml x mini x kg’, respectively (Table 2). TABLE
1. Concentrations (mean _’ SD.) of CPA in the serum of 15 women at various time points following single oral administration of 2.0 mg CPA together with 0.035 mg EE,, and after the last drug intake during treatment cycles one and three, respectively
Time (h) 0 0.5 1 1.5 2 4 6 8 12 24 48 72 96 120
Single Dose
Cycle One
Cycle Three
(ng/mf)
(ngjmf)
(nglmf)
* 1.6 9.9 14.8 12.5 5.2 3.2 2.4 1.6 1.2 0.8 0.6 0.5 0.3
+? & + + + 2 ? * ++ t *
1.2 6.3 7.0 4.6 2.1 1.2 0.7 0.5 0.3 0.2 0.2 0.2 0.2
4.8 7.1 17.0 19.8 16.8 9.4 7.3 6.7 5.5 4.7 3.7 3.1 2.5 2.2
+ ‘lr + + & * -t * * * + + f
0.8 1.9 6.6 5.1 4.1 2.3 1.5 1.6 1.1 1.0 0.9 0.7 0.8 0.6
5.9 8.5 17.9 22.7 17.7 10.1 7.7 6.6 5.9 5.2 4.6 3.4 2.6 2.3
-t + t + + f ? + * _t * ‘f %
1.9 3.7 7.1 8.1 4.0 2.1 1.6 1.5 1.2 0.9 1.0 0.8 0.6 0.5
“Below lower limit of quantification.
564
Contraception
1993:48, December
Pharmacokinetics
of cyproterone
acetate and ethinyfestradiol:
Kuhnz et al.
TABLE 2. Pharmacokinetic parameters of CPA (mean + SD.) calculated from total concentrations in the serum of 15 healthy young women after a single oral administration of 2.0 mg CPA together with 0.035 mg EE, and afler one and three cycles, respectively, with the same contraceptive formulation
Single Dose
Parameter
C max
OWml)
t max
(h) (ng x ml-l (ng x ml-’ (I) (f) (ml x min’ (h) (h) (h)
AUC(O-24h) AUC V,, VZ CL MRT t?, A, t’,
,oo
x h) x h)
x kg-‘)
15.2 1.6 75.0 160.2 740 986 3.6 59.9 0.8 54.0
? f f ? & f 5 f ? 2
6.6 0.3 21.0 44.8 339 437 0.9 28.9 0.4 26.0
Cycle One
Cycle Three
21.0 1.4 169.3 665.6
+ + + f -
4.8 0.4 30.8 175.6
23.9 1.4 182.6 782.2
It 4 t :t
7.7 0.4 34.4 146.8
1211 3.3 94.4 0.8 68.8
+ ? % ? ‘-
397 0.6 27.5 0.2 19.1
1304 3.0 107.9 0.7 78.6
:t :t re :? :?
427 0.4 22.8 0.3 16.0
iCPAh/ml1
0
20
-_
40
80
60
100
120
140
time [h] FIGURE 1. Mean concentrations (2 SD.) of CPA in the serum of 15 women after single administration (squares) of a combination of 2 mg CPA + 0.035 mg EE, and following multiple administration on day 21 of treatment cycles one (diamonds) and three (crosses).
Contraception
1993:48, December
565
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
Twenty-four hours after single dose administration, the free fraction of CPA in the serum was 3.5 + 1.9% and the fractions bound to heat labile proteins were 4.6 k 2.2% and 92.0 k 3.5%, respectively. SHBG and CBG concentrations in the serum were 99.6 +- 23.3 nmol/l and 49.5 + 8.3 p,g/ml, respectively. These results are summarized in Table 3. The individual EE, concentrations in the serum samples are presented in Table 4. Since in several cases, the amount of serum was not sufficient for the analysis of all parameters, EE, levels could only be analysed in 11 women. The maximum concentrations were observed after 1.6 + 0.2 h and amounted to 70.8 k 27.6 pg/ml. The AUC(O-24h) was 374.4 k 255.1 pg x ml-l x h (Table 5).
TABLE 3. Protein binding of CPA in the serum samples collected prior to single dose administration and prior to the last drug intake in cycles one and three, respectively; the concentration of the binding proteins SHBG and CBG in the same samples (mean t SD.) is also presented; hl = heat labile
Single Dose
Parameter free fraction hl protein-bound albumin-bound CBG SHBG
W) (“M W) (kg/ml) (nmol/l)
3.5 4.6 92.0 49.5 99.6
Cycle One
i 1.9 ? 2.2 !I 3.5 +- 8.3 + 23.3
3.4 3.8 92.8 86.4 326.0
?z 2.0 ? 1.7 ? 3.0 + 9.7 ? 96.4
Cycle Three 3.9 3.5 92.1 91.6 314.1
f t + rt f
2.0 1.2 4.0 12.5 95.1
Concentrations of EE, (mean i: SD.) in the serum of 11 women after single dose administration of 0.033 mg EE, in combination with 2.0 mg CPA, and on the last day of one and three treatment cycles with the combined oral contraceptive, respectively TABLE 4.
Time (h) 0
0.5 1 1.5 2 4 6 8 12 24
566
Single Dose (pg/mt) 12 2 18 + 42 i70 + 65 + 39 + 26 2 10 ? 6 + 2t7
16 18 28 29 25 15 17 16 11
Cycle One (pglmf)
Cycle Three (pg/mt)
10 44 77 105 99 53 43 29 21 5
14 54 95 115 102 58 43 33 27 20
?z 19 i 35 ? 35 I? 37 t 36 t 32 2 28 ‘- 20 +- 16 t 11
+ + 2 + t c + + t ?
30 34 44 42 34 24 33 29 27 28
Contraception 1993:48, December
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
TABLE 5. Pharmacokinetic parameters of EE, (mean + SD.) obtained after single dose administration of 0.035 mg EE, in combination with 2.0 mg CPA, and after the last drug intake during treatment cycles one and three, respectively
Parameter
Single Dose
C max
@g/ml)
71 I
28
Cycle One
Cycle Three
115 2 31
118 t
311 1.9 f+ 0.8 109 787 + 342
305 1.6 +* 0.3 122 903 ? 586
42
t (pg O-4 x ml-’ (pg x ml-’
&C(O-4h) AUC(O-24h)
x h) x h)
188 1.6 5 ? 80 0.2 374 + 255
Pharmacokinetics of CPA and EE, After Multiple Dose Administration Mean trough concentrations of CPA increased during treatment and reached a steady-state on about day 16 of cycles one and three, respectively (Figure 2). On the last day (day 21) of cycles one and three, maximum concentrations of CPA in the serum were observed 1.4 k 0.4 h post administration and amounted to 21 .O + 4.8 and 23.9 -t 7.7 ng/ml, respecCPA [ng/ml] 6
,
0
2
4
6
6
10
12
day of I_esimulated]
14
16
20
22
24
cycle
j+cyclel
FIGURE 2. Comparison between simulated and measured mean of 15 women during treatment cycles one and three, respectively.
Contraception
16
1993:48, December
(-t SD.) trough levels of CPA in the serum
567
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
tively. Post maximum drug levels declined biphasically with mean halflives of 0.8 k 0.2 h and 68.8 & 19.1 h during the first cycle, and 0.7 ? 0.3 h and 78.6 t 16.0 h during the third cycle, respectively. The AUC values were 665.6 ? 175.6 and 782.2 t 146.8 ng x ml-’ x h, respectively. Total clearance and V, were 3.3 -e 0.6 ml x min -I x kg-1 and 12 11 ? 397 1 during the first cycle and the corresponding values calculated during the third cycle were 3.0 + 0.4 ml x min’ x kg-’ and 1304 + 427 1, respectively (Table 2). The free fraction of CPA on the last treatment day of cycles one and three was 3.4 + 2.0% and 3.9 ? 2.0%, respectively. The fractions of CPA bound to heat labile proteins and albumin were 3.8 t 1.7% and 92.8 f 3.0% during cycle one and 3.5 t 1.2% and 92.1 ? 4.0% during cycle three, respectively. The mean SHBG and CBG levels on day 21 of cycles one and three were 326.0 ? 96.4 nmol/l and 86.4 & 9.7 pg/ml, and 314.1 IL 95.1 nmol/l and 91.6 i 12.5 kg/ml, respectively, (Table 3). significant differences For the parameters t1/2,V, and AUC, statistically were found when single dose administration was compared to the last treatment days of cycles one and three, respectively. The clearance was not different at the end of cycle one as compared to single dose administration; only at the end of cycle three, a small, although significant difference was observed. None of the pharmacokinetic parameters except clearance was different when the last days of treatment cycles one and three were compared. No difference was found for the corresponding concentrations of CBG and SHBG. The pharmacokinetic parameters of EE, obtained on day 21 of cycles one and three, respectively, are presented in Table 5. Maximum concentrations of EE, of 115 t 31 pg/ml and 118 2 42 pg/ml were reached 1.9 ? 0.8 h and 1.6 -t 0.3 h post administration at the end (day 21) of cycles one and three, respectively. The corresponding AUC(O-24h) values were found to be 787 ? 342 and 903 t 586 pg x ml-’ XX h, respectively. No statistically significant differences were observed for either the C,,, or the AUC values on day 21 of cycles one and three, respectively. However, as compared to single dose administration, a statistically significant increase in the AUC values of about 60% was found at the end of a treatment cycle. Simulation of CPA Trough Levels in the Serum During One Month of Treatment Based on Single Dose Pharmacokinetics; Accumulation CPA in the Serum
of
A computer simulation of CPA trough levels in the serum during a complete treatment cycle was performed on the basis of the data obtained after single dose administration. A comparison of the simulated curve
568
Contraception 1993:48, December
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
with the trough levels determined experimentally during the two treatment cycles one and three is shown in Figure 2. Simulated trough levels increased to steady-state concentrations of 4.0 + 1.0 ng/ml, which were reached on day 16. The experimentally measured CPA trough levels during cycle one were practically superimposable with the simulated curve. The corresponding trough level time course measured during cycle three was in parallel to the simulated curve but slightly shifted towards higher concentration values. Steady-state concentrations of CPA reached during cycle three were statistically significantly different from those observed during cycle one and also from the corresponding simulated curve. A similar time course was seen for the corresponding SHBG levels during cycles one and three, respectively (Figure 3). An accumulation ratio (R,) of 2.2 and 2.5 was calculated for CPA in cycles one and three, respectively. Mean values calculated for the mass balance factors R* were 1.2 2. 0.2 and 1.3 + 0.2 for cycles one and three, respectively. Based on the AUC values determined after single dose administration and during steadystate, mass balance factors R* * were 1.l k 0.2 and 1.2 k 0.3 for cycles one and three, respectively (Table 6).
SHBG [nmoljl] 5wI-
1 0
2
4
6
6
10
12
14
16
16
20
22
24
day of cycle
FIGURE 3. Mean (k SD.) SHBG trough concentrations respectively.
Contraception 1993:48, December
in the serum of 15 women during cycles one and three,
569
Pharmacokinetics
of cyproterone
acetate and ethinylestradiol:
Kuhnz et al.
TABLE6. Accumulation and mass balance factors of CPA (mean ? SD.) Parameter
Cycle One
Cycle Three
RI R’ R”
2.2 + 0.3 1.2 + 0.2 1.1 t 0.2
2.5 _t 0.6 1.3 ‘- 0.2 1.2 + 0.3
Effect of Treatment on Total and Free Testosterone Concentrations in the Serum Pre-treatment concentrations of total and free testosterone in the serum were 0.41 i 0.22 ng/ml and 2.2 I 1.1 pg/ml, respectively. At the end of the first treatment cycle, these levels dropped to mean values of 0.25 ? 0.12 ng/ml for total testosterone and 0.84 -t 0.38 pg/ml for free testosterone, respectively. This corresponds to a drop of 39 % and 62 % from the corresponding pre-treatment levels, respectively. On the first day of cycle three, both total (0.47 +- 0.21 ng/ml) and free (1.7 ? 0.8 pg/ml) were not different from those measured prior to treatment. Thus, the pill-free interval of one week was sufficient to restore pre-treatment values for testosterone. At the end of cycles one and three, the same concentrations of total and free testosterone were measured, which revealed that no additional suppression of testosterone production occurred during a treatment period of three cycles as compared to the first cycle (Table 7).
Discussion In the present study, the pharmacokinetics of CPA and EE, have been investigated following single and multiple oral administration of a combination oral contraceptive to 15 women. After single oral administration, CPA was rapidly absorbed and post-maximum drug levels declined biphasically with a terminal half-life of 54 h. A large volume of distribution of about 1000 1 was observed and the apparent total serum clearance of 3.6 ml x min I x kg ’ was considerably less than the total serum liver flow of about 12 ml x minm ’ x kg- I. The apparent clearance observed in the present study was similar to the total serum clearance determined during a previous study after parenteral administration of CPA (2.3 & 0.4 ml x min ’ x kg l). This is in keeping with the finding that the absolute bioavailability of CPA was found to be almost complete.” The present results are also in good agreement with the results of a previously performed study with the same contraceptive formulation in a smaller group of women.’ As compared to single dose administration, an about two-fold accumulation of CPA in the serum occurred during treatment cycle one. Steady-
570
Contraception
1993:48,
December
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
TABLE 7. Total and free testosterone concentrations (mean + SD.) in the serum of 15 women before and during treatment with the combination oral contraceptive; pre = pre-treatment cycle
Time (cycle/day)
Total Testosterone
Free Testosterone
(ng/ml)
(pglml)
0.41 0.36 0.38 0.29 0.31 0.25 0.24 0.23 0.22 0.18 0.22 0.25 0.23 0.47 0.39 0.39 0.36 0.27 0.29 0.30 0.27 0.27 0.27 0.25
prel21 prel22 i/l l/2 l/3 l/5 118 l/10 l/l4 l/16 l/16 1121 1122 3/l 312 313 315 318 3/l 0 3114 3116 3118 3121 3122
k 5 f k + ” + + t k * k k 5 + + + + * f 5 k + f
0.22 0.17 0.15 0.15 0.14 0.13 0.11 0.13 0.13 0.10 0.12 0.12 0.11 0.21 0.18 0.18 0.35 0.13 0.15 0.27 0.15 0.17 0.16 0.17
2.2 1.7 1.8 1.5 1.5 1.1 0.9 0.9 0.8 0.6 0.8 0.8 0.7 1.7 1.4 1.2 1.2 0.9 0.9 0.9 0.8 0.8 0.7 0.7
k k k 2 t ? k k + k + + k i f k k k * 5 k t i -t
1.1 0.6 0.7 0.7 0.6 0.5 0.5 0.5 0.4 0.4 0.6 0.4 0.5 0.8 0.6 0.5 0.7 0.5 0.4 0.5 0.4 0.5 0.4 0.5
state drug levels in the serum were reached around day 16 of the cycle. The time courses of measured trough levels of CPA and those obtained by simulation of a treatment cycle based on single dose pharmacokinetics were almost superimposable, indicating linear and time-independent pharmacokinetics of CPA. The same time course was seen with the trough levels measured during cycle three; however, CPA concentrations were slightly elevated as compared to cycle one. This difference existed already on day 1 of cycle three and is very likely due to the fact that a pill-free period of 7 days between two cycles is not long enough for CPA to reach pre-treatment levels, when a terminal half-life of about 70 to 80 h is taken into account. The observed accumulation of CPA during a cycle was considerably less than one would expect from single dose administration, since based on a mean terminal half-life of 54 h and a dosing interval of 24 h, an accumulation factor of 3.8 can be calculated. If a half-life value of 70 to 80 h was considered, the accumulation factor would be about 5.
Contraception
1993:48, December
571
Pharmacokinetics
of cyproterone
acetate and ethinylestradiol:
Kuhnz et al.
The fact that the observed accumulation factor was only about 2 to 2.5, already indicates that only a small fraction of the dose is represented by this half-life value. During a treatment cycle, an increase in the SHBG concentration in the serum by a factor of 3.3 occurred which was due to the concomitant administration of EE,. Since CPA has only a minimal binding affinity to SHBG, this change in SHBG levels should not affect the distribution of CPA with respect to the binding proteins. Further, it should not contribute to the accumulation of the drug in the serum nor should it affect the Therefore, no changes in the total pharmacokinetics of the progestin. 1om12 clearance of CPA should occur and accordingly, the mass balance factors R* and R* should be equal to unity. This was indeed the case, since R* was found to be between 1.2 and 1.3 and R* * was between 1.1 and 1.2 at the end of cycles one and three, respectively, and thus very close to unity. If one compared the pharmacokinetic parameters of CPA following single dose administration with those obtained at the end of treatment cycles one and three, however, some changes became obvious. Although clearance remained practically unchanged, there was a marked increase in the volume of distribution from about 1000 1 (single dose) to about 1200-1300 1 (multiple dose] and a prolongation of the terminal half-life from 54 h to about 70-80 h. Since terminal half-life is dependent on the two independent variables CL and Vr), an increase in V,, invariably leads to an increase in tljz, if both CL and the binding of CPA in the tissue remain unchanged. It has to be mentioned that the mean terminal halflife at the end of the treatment period may be somewhat underestimated, because the observation period of 120 h after the last drug intake allowed an accurate determination only for tljz values up to about 60 h. There were, however, a number of cases with half-life values > 90 h, which could not be assessed accurately. A large volume of distribution which exceeds by far all physiologic compartments, is usually an indication of high tissue binding of the drug. From animal experiments, it is known that CPA preferentially distributes into fatty tissue. It has been suggested before that fatty tissue and the skin represent a deep compartment with limited capacity into which the drug distributes slowly. 13 This would explain the time to reach steadystate conditions as well as the prolongation of the terminal half-life after cessation of treatment. The pharmacokinetic parameters of CPA determined after single and multiple oral administration in the present study were similar to those obtained with the same preparation in a previous study, where the CPA levels were measured radioimmunologically. Only the corresponding values obtained for AUC and tX12were found to be considerably higher in the previous study. A possible explanation could be the presence of lSBl
572
Contraception
1993:48,
December
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
hydroxy-CPA in the serum samples and the known interference of this metabolite with the radioimmunological analysis of CPA.6J4 If there was a substantial contribution of this metabolite, particularly at late sampling points, this could explain the higher values of tliz and AUC, respectively. Another interesting result of the present study is the fact that the total apparent clearance of CPA was almost unaffected during treatment with the combination oral contraceptive (only at the end of cycle three was there a small, although significant decrease in the clearance]. In similar studies with preparations containing progestogens of the 19-nor testosterone series, like levonorgestrel or gestodene, in combination with EE2, both total and free clearance of the progestins decreased markedly during treatment. It was assumed that this was due to the presence of EE,, since no such changes in the clearance were seen when these progestogens were administered alone.15J6 The underlying mechanism of this reduction in clearance, however, remained unknown. Inhibition of hepatic cytochrome I?-450-dependent isozymes by EE, or an influence of EE, on the hormonal regulation of the production of these enzymes were proposed as hypothetical explanations. Obviously, in the case of CPA, none of these possible alternatives seems to hold true. The pharmacokinetic parameters of EE, in the present study were similar to those observed in previously performed studies with this contraceptive preparation.” Since the lower limit of quantification was already reached 8 to 12 hours post administration in many cases, C,,, and AUC(O-4h) values can be regarded as more reliable than the corresponding AUC(O-24h) values. An increase in the EE, serum levels during a treatment cycle by about 60 % has been found and this can probably be ascribed to the relation between dosing interval and terminal half-life of EE2, which has been discussed in detail earlier.17 The induction of hepatic CBG synthesis is an effect of the estrogenic component of the contraceptive formulation administered. An about 90 % increase over pretreatment levels was observed at the end of cycle one and there was no further increase in the CBG levels at the end of cycle three. This increase is concordant with the available information on the dose-dependency of EE,-induced CBG levels in the serum.‘” Total and free testosterone concentrations decreased during treatment cycles one and three by about 39% and 62%, respectively, compared with the corresponding values measured prior to treatment. Similar values had been observed by others with the same contraceptive preparation during a treatment period of 36 cycles. l9 In the present study, the unbound fraction of testosterone decreased by about 40% from a mean value of 0.5% on the first day of cycle one to about 0.3% at the end of treatment cycles one and three, respectively. Since there was a concomitant rise in the SHBG levels in the serum, a decrease in the free fraction of testosterone could be expected. No further changes were seen in these parameters
Contraception
1993:48, December
573
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
during cycle three when compared with cycle one. In that respect, treatment with the CPA-containing formulation had effects on free and total testosterone levels in the serum which were very similar to those observed with other combination oral contraceptives containing different progestogens. ’ 720 In conclusion, the present study confirmed and extended the already existing information on the pharmacokinetics of CPA following single dose administration and during a treatment period of one month obtained in an earlier study with the same oral contraceptive. After three months of treatment, no further changes in the pharmacokinetic parameters of CPA and EE, are to be expected, except for the steady-state trough levels of CPA, which might show a minor further increase during subsequent treatment cycles. No further changes are expected for SHBG and CBG and for total and free testosterone levels in the serum during long-term treatment.
References 1. 2.
3.
4.
5.
6.
7.
8.
574
Hammerstein J, Moltz L, Schwartz U. Antiandrogens in the treatment of acne and hirsutism. J Steroid Biochem 1983; 19: 591-7. Hiimpel M, Wendt H, Schultze P-E, Dogs, Weiss C, Speck U. Bioavailability and pharmacokinetics of cyproterone acetate after oral administration of 2.0 mg cyproterone acetate in combination with 50 pg ethinyloestradiol to 6 young women. Contraception 1977; 15: 579-88. Hiimpel M, Dogs C, Wendt H, Speck U. Plasmaspiegel und Pharmakokinetik von Cyproteronacetat nach oraler Applikation als 50-mg-Tablette bei 5 Mannern. Arzneim-Forsch/Drug Res 1978; 28: 319-22. Dusterberg B, Hiimpel M, Wendt H. Plasma levels of active ingredients after single and repeated administration of a new oral contraceptive containing 2 mg of cyproterone acetate and 50 ug of ethinyl estradiol (DianeR) to five young women. Acta Obstet Gynecol Stand Suppl 1979; 88: 2731. Zurth C, Tauber U. Pharmakokinetik von Cyproteronacetat und Ethinylestradiol wahrend der Behandlung mit DianeR-35: Vergleich zwischen Raucherinnen und Nichtraucherinnen. In: Schindler AE, ed. New Developments in Biosciences vol 3: Antiandrogen-estrogen therapy for signs of androgenization. Berlin, New York: de Gruyter, 1987: 43-52. Huber J, Zeillinger R, Schmidt J, Tauber U, Kuhnz W, Spona J. Pharmacokinetits of cyproterone acetate and its main metabolite I5B-hydroxy-cyproterone acetate in young healthy women. Int J Clin Pharmacol Ther Toxic01 1988; 26: 355-61. Kuhnz W, Louton T, Back DJ, Michaelis K. Radioimmunological analysis of ethinylestradiol in human serum: Validation of the method and comparison with a gas chromatographic/mass spectrometric assay. Arzneim-Forsch/Drug Res 1993; 43: 16-21. Hu OY-P, Curry SH. Calculation of fraction bound in equilibrium dialysis
Contraception 1993:48, December
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
9.
10.
11.
12. 13.
14. 15.
16.
17.
18.
19.
20.
Kuhnz et al.
with special reference to drug losses by decomposition and adsorption. Biopharm Drug Disp 1986; 7: 21 l-4. Hammond GL, Lahteenmaki PLA, Lahteenmiki P, Luukkainen T. Distribution and percentages of non-protein bound contraceptive steroids in human serum. J Steroid Biochem 1982; 17: 375-80. Pugeat MM, Dunn JF, Nisula BC. Transport of steroid hormones: Interaction of 70 drugs with testosterone-binding globulin and corticosteroid-binding globulin in human plasma. J Clin Endocr Metab 1981; 53: 69-75. Hammond GL, Langley MS, Robinson PA, Nummi S, Lund L. Serum steroid binding protein concentrations, distribution of progestogens, and bioavailability of testosterone during treatment with contraceptives containing desogestrel or levonorgestrel. Fertil Steril 1984; 42: 44 51. Juchem M, Pollow K. Binding of oral contraceptive progestogens to serum proteins and cytoplasmic receptor. Am J Obstet Gynecol 1990; 163: 2171-82. Hiimpel M, Nieuweboer B, Dusterberg B, Wendt H. Die Pharmakokinetik von Cyproteronacetat beim Menschen. In: Hammerstein J, Lachnit-Fixson U, Neumann F, Plewig G, ed. Androgenisierungs-erscheinungen bei der Frau. Berlin: Excerpta Medica 1979: 212-23. Nieuweboer B, Lubke K. Radioimmunological determination of cyproterone acetate. Hormone Res 1977; 8: 210-8. Kuhnz W, Al-Yacoub G, Fuhrmeister A. Pharmacokinetics of levonorgestrel in 12 women who received a single oral dose of 0.15 mg levonorgestrel and, after a wash-out phase, the same dose during one treatment cycle. Contraception 1992; 46: 44-54. Kuhnz W, Gansau C, Fuhrmeister A. Pharmacokinetics of gestodene in 12 women who received a single oral dose of 0.075 mg gestodene and, after a wash-out phase, the same dose during one treatment cycle. Contraception 1992; 46: 2940. Kuhnz W, Sostarek D, Gansau C, Louton T, Mahler M. Single and multiple administration of a new oral contraceptive to women: Pharmacokinetics of ethinylestradiol and free and total testosterone levels in serum. Am J Obstet Gynecol 1991; 165: 596-602. Schwartz U, Hammerstein J. The oestrogenic potency of various contraceptive steroids as determined by their effects on transcortin-binding capacity. Acta Endocr (Copenh.) 1974; 76: 159-71. Falsetti L, Galbignani E. Long-term treatment with the combination ethinylestradiol and cyproterone acetate in polycystic ovary syndrome. Contraception 1990; 42: 611-9. Jung-Hoffmann C, Kuhl H. Divergent effects of two low-dose oral contraceptives on sex hormone-binding globulin and free testosterone. Am J Obstet Gynecol 1987; 156: 199-203.
Contraception 1993:48,
December
575
T. Staks” and G. Jiittingf
lSchering Aktiengesellschaft, Abteilung, Kreiskrankenhaus Germany
13342 Berlin, Germany and tGeb. -gyn. Eutin, Janusstrape 22, 2420 Eutin/Holst.,
The pharmacokinetics of cyproterone acetate (CPA) and ethinylestradiol (EEJ were determined in 15 healthy women (age 19 to 34 years), following single dose administration of a combination oral contraceptive, containing 2.0 mg CPA together with 0.035 mg EE, (Diane-35R). After a wash-out period of one week, the same preparation was administered during a treatment period of three months. After single dose administration, maximum concentrations of CPA in the serum were 15.2 _t 6.6 nglml. Post maximum drug levels declined biphasically with half-lives of 0.8 ? 0.4 h and 54.0 & 26.0 h, respectively. The apparent clearance was calculated to be 3.6 + 0.9 ml x min-l x kg-’ and the volume of distribution (V,) was 986 2 437 1. The free fraction of CPA was 3.5 + 1.9% and the fractions bound to heat labile proteins and albumin were 4.6 & 2.2 % and 92.0 & 3.5%, respectively. Trough levels of CPA in the serum increased during a treatment cycle, reaching a steady-state around day 16. An about two-fold accumulation of CPA was observed, which was less than expected theoretically. SHBG concentrations in the serum increased by a factor of three during a cycle, without having any effect on the protein binding of CPA. At the end of treatment cycle three, the terminal half-life of CPA had increased to a mean value of 78.6 + 16.0 h and the volume of distribution to a value of 1304 -+ 427
Submitted for publication September 7, 1993; accepted for publication October 27, 1993. Address correspondence to Dr. W. Kuhnz, Institut fiir Pharmakokinetik, Schering Aktiengesellschaft, 13342 Berlin, Germany.
0 1993 Butterworth-Heinemann
Contraception
1993:48,
December
557
Pharmacokinetics
of cyproterone
acetate and ethinylestradiol:
Kuhnz et al.
1. The apparent clearance showed a small, although significant decrease to a value of 3.0 ? 0.4 ml x min-’ x kg’. The observed changes V, and t,,2 during the treatment period were attributed to the distribution of CPA into a deep compartment and the slow release of the drug from this compartment. The AUC(O-4h) values of EE, following single dose administration of the combination oral contraceptive were found to be 187.5 2 79.7 pg x ml-- I x h. On the last day of cycles one and three, the AUC(O-4h) values were 311.2 +- 109.3 and 304.8 ? 121.5 pg x ml- i x h, respectively, which corresponds to an about 60% increase as compared to single dose administration. Total and free testosterone concentrations decreased during treatment cycles one and three by about 39 % and 62%, respectively, compared with the corresponding values measured prior to treatment. Keywords: Pharmacokinetics; cyproterone acetate; ethinylestradiol; binding; testosterone; combination oral contraceptive.
protein
Introduction Cyproterone acetate (CPA) is a potent steroidal antiandrogen with progestational activity and is used alone and in combination with ethinylestradiol (EE,) in the treatment of women who suffer from disorders associated with androgenization, like severe acne or hirsutism.’ At a dose of 2 mg, CPA is used in combination with 0.035 mg EE, as an oral contraceptive preparation for women with androgen-dependent skin problems. The pharmacokinetics of CPA in humans have been investigated in several studies.24 In one of them, a combination oral contraceptive containing 2 mg of CPA and 0.035 mg EE2, was administered to two groups of smoking and non-smoking women over a period of one treatment cycle. The drug levels in plasma were determined radioimmunologically and were used to derive the basic pharmacokinetic parameters of both steroids.5 No differences were seen in the pharmacokinetics of CPA in both groups of women. The aim of the present study was to examine in more detail the pharmacokinetics of CPA and EE, in women who received the same CPAcontaining contraceptive formulation during a treatment period of three months. In addition to the already available information obtained for a period of one treatment cycle, it was of interest to examine whether prolonged treatment with this preparation was associated with further changes in the pharmacokinetics of CPA, EE, or other parameters, like the concentration of serum binding proteins, CBG and SHBG and total and free testosterone levels in the serum. A highly specific and sensitive gas chromatographic/mass spectrometric (GC/MS) method was used for the analysis of CPA in the present study, in order to allow for a comparison
558
Contraception
1993:48, December
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
with the corresponding results of the previously performed study, where CPA levels were measured radioimmunologically.
Material and Methods Study Design The study was performed as an open, intraindividual comparison in 15 healthy young women. In the first part of the study, the women received a single oral dose of 2.0 mg CPA together with 0.035 mg EE, (Diane-35R, Schering AG) and, after a wash-out period of 1 week, the same women received the contraceptive formulation over a treatment period of three cycles. Each treatment cycle was separated by a drug-free interval of 1 week. The drug was administered in the morning. On those days where blood was sampled repeatedly for pharmacokinetic purposes, the drug was administered after an overnight fast. A standardized breakfast was served one hour after treatment. Blood samples were collected at the following time points: Single dose: Immediately prior to drug intake (0 h) and 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24, 48, 72, 96 and 120 h after administration. Treatment cycles one and three: day 1: 0, 0.5, 1, 1.5, 2, 4, 6, 8, 12 h; days 2, 3, 5, 8, 10, 14, 16, 18: immediately prior to drug intake; day 21: 0, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24, 48, 72, 96 and 120 h after administration. All blood samples were kept at 4°C until coagulation. The serum was separated by centrifugation and stored at - 20°C until analysis. Study Population Fifteen healthy, young women participated in the study. The women had normal biphasic menstrual cycles and showed low variation (mean +S.D.) in body weight (63 * 7 kg), height (167 + 6 cm) and age (26 ? 6 years). All women had not taken oral contraceptives for at least two months prior to their participation in the study. The subjects underwent a laboratory screening and a thorough medical and gynecological examination before entering the study. Excluded from participation were subjects who had any contraindication to the use of contraceptive steroids or a concomitant medication which might interfere with the pharmacokinetits of the contraceptive steroids CPA and EE,. Also excluded were women who smoked. The nature and purpose of the study was explained and
Contraception
1993:48, December
559
Pharmacokinetics
of cyproterone
acetate and ethinylestradiol:
written informed consent was given by each participant. approved by the local ethics committee.
Kuhnz et al. The study was
Analytical Methods The concentrations of CPA in the serum samples were measured in duplicate by a specific CC/MS method available at LAB (Neu-Ulm, FRG). After the addition of the internal standard (10 ng chlormadinone acetate] and 0.2 ml 0.1 N NaOH, 1 ml of serum sample was extracted with 6 ml of cyclohexane/i-butanol (98.5/1.5; v/v). After centrifugation (10 min, 5000 rpm), the organic phase was separated and stored in a refrigerator at 4°C until analysis. The GC/MS analysis of CPA was performed on a Finnigan MAT 4021 GC/MS system. A fused silica open tubular capillary column of cross-linked methyl silicone type was used for GC-separation. The MS was operated in the negative ion chemical ionization mode with methane as reagent gas. Selected ion monitoring was performed for m/z 356, the base peak of CPA and m/z 344, the base peak of the internal standard. The standard curve was linear over a range of 0.1 to 50 ng CPA/ml serum and the lower limit of quantification was 0.1 ng/ml. All samples were measured in sequences. One sequence consisted of the samples from one subject, one set of calibration samples and 8 quality control samples (0.15, 0.3, 3.0 and 30.0 ng/ml in duplicate). The coefficient of inter-assay variation was between 9 and 13%. The deviation of measured from nominal concentration values of quality control samples was less than 3%. The analysis of EE, has been described in detail before.7 Analyses were performed in duplicate by a radioimmunoassay using an antiserum raised in rabbits against EE,-b@CH,CO-bovine serum albumin (Schering AG) and [6,7-“HI-EE, (specific activity 2.2 TBq/mmol, NEN Products, Boston, MA, USA) as tracer. Diethylether extracts obtained from 0.3 ml serum were taken for analysis. Assay quality was assessed by the inclusion of quality control samples (50, 125 and 250 pg/ml) in each assay. Inter-assay precision was between 11 and 24%, and intra-assay precision was between 7 and 25%. The deviation of measured concentrations of EE, from nominal values was between 5 and 16%. The lower limit of quantification was 20 pglml. SHBG, CBG, total and free testosterone samples were each measured in duplicate immunoassays (SHBG: Diagnostic Products Medgenix, Fleurus, Belgium; total and free ucts Corp.). Two or three quality control these assays. The nominal concentrations of 20-28 and 56-82 nmol/l, respectively, measured in a total of 7 assays were 24.0
560
concentrations in the serum using commercially available Corp., Los Angeles, CA; CBG: testosterone: Diagnostic Prodsera were included in each of of SHBG were in the range and the mean concentrations t 2.5 and 63.2 + 4.4 nmol/l,
Contraception
1993:48,
December
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
respectively. Inter-assay precision (C.V.) was between 7 and 11%. CBG control sera contained mean nominal concentrations of 28 + 12 and 115 + 37 kg/ml, respectively, and the measured concentrations were found to be 26.7 k 0.7 and 83.7 * 1.6 kg/ml, respectively. Inter-assay precision was between 2 and 3%. For total and free testosterone determinations, three quality controls each were included in each assay. The nominal concentrations of total testosterone controls were 0.7, 2.7 and 6.9 ng/ml and the corresponding measured concentrations (n = 8 assays) were 0.65 ? 0.08 ng/ml, 2.47 -t 0.20 ng/ml and 6.02 + 0.48 ng/ml, respectively. Inter-assay precision was between 8 and 12%. The nominal concentrations of free testosterone controls were 2.5, 8.5 and 29.6 pg/ml and the corresponding measured concentrations (n = 8 assays) were 2.7 rf: 0.1 pg/ ml, 9.5 -+ 0.7 pg/ml and 34.5 t 2.9 pg/ml, respectively. Inter-assay precision was between 5 and 9%. Serum
Protein Binding
of CPA
The serum protein binding of CPA was performed by equilibrium dialysis because a high nonspecific binding of the drug to the membrane and the filtration unit precluded the use of the ultrafiltration method. In a pre-test, the optimum dialysis time was determined by successively increasing the time until a constant value of the free fraction was obtained. A dialysis time of 2 hours was sufficient to reach equilibrium. Recovery after dialysis was found to be between 76 and 83%. In the following experiments, the recovery was calculated for each individual sample by measuring the total radioactivity in aliquots of buffer and serum at equilibrium. Individual recovery values were ca. 70 to 100%. Serum aliquots of 500 ~1 were spiked with 3H-CPA (10,000 cpm) and equilibrated for 0.5 h at room temperature. Dialysis was carried out in self-designed Teflon cells with a volume of ca. 120 ~1 per half-cell. One hundred p,l of serum were dialysed against 100 p,l of dialysis buffer, separated by a high-permeability membrane (Diachema, molecular weight cut-off 5 000). The cells were rotated at 12 rpm for a period of 2 h at a temperature of 37°C. When dialysis was terminated, 50 ~1 aliquots of both, the buffer and the serum side, were taken and after addition of a scintillation cocktail (Atomlight, NEN), total radioactivity was counted in a liquid scintillation counter. The unbound fraction (fU)of CPA was calculated according to equations (I) and (II), with corrections for volume shift and losses of drug due to nonspecific adsorption.*
f”= (A, -
C” x vs &oss- C” x v,
A loss= A, - A,
Contraception 1993:48, December
(11 (111 561
Pharmacokinetics
of cyproterone
f, = fraction
acetate and ethinylestradiol:
Kuhnz et al.
unbound
C,, = unbound dialysis
concentration
or drug concentration
V, = serum volume
before dialysis
V,, = buffer volume
before dialysis
in buffer after
A,, = amount
of drug in the serum before dialysis
A, = amount
of drug in serum and buffer at equilibrium
A IDSS= drug lost by nonspecific
binding to membrane
and cell
For the determination of the fraction of CPA bound to heat labile proteins, 500 ~1 of each sample were kept for 1 h at 60°C. Subsequently, the samples were spiked with “H-CPA (10,000 cpm), equilibrated for 0.5 h at room temperature and dialysed as described above. The calculation of the fractions of CPA bound to heat labile proteins and albumin, respectively, was performed according to a published procedure.”
Pharmacokinetic Evaluation The serum concentrations of CPA, which were obtained after single oral administration and on the last treatment day of cycles one and three, were evaluated by model-fitting (TOPFIT 2.0, Goedecke, Schering AG, Thomae GmbH, Germany) on the basis of an open two-compartment model. The data obtained by model-fitting from the single dose administration were used for the simulation of total CPA concentrations in serum during one treatment cycle. Accumulation and mass balance factors of CPA were calculated according to: AUC:\ R’ = AUC(0 -&o (Cg,,)exp mentally;
= minimun
(C:,,)sim = minimum simulation;
(CzJexp ’ R* = (C:,,)sim
concentration concentration
. R** ’
at steady-state
zz- AUQ AUC,,,
;
determined
at steady-state
experi-
determined
by
AUC? = area under the serum level-time curve during one dosing interval at steady-state (7 = dosing interval); AUC,, = area under the serum level-time dose administration
562
curve obtained
Contraception
1993~48,
after single
December
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
Clearance at steady-state and the volume of distribution V, (assuming a constant CL) were calculated according to: CL
=
DMx f AUC$
v
’
=cL
A,
DM = maintenance dose f = bioavailable fraction A, = disposition constant of the terminal phase The AUC was calculated according to: AUC = AUC(0 - t”) + 2 AUC(0 - t,,) = area under the serum concentration-time curve up to the last concentration value (C,) measured at the time point (tJ The mean residence time (MRT} gives the average time necessary for the drug molecules to leave the body from when the dosage form is administered: MRT = e AUMC = area under the moment curve Those concentrations of CPA which were obtained on day 1 of cycles one and three, respectively, as well as the concentrations of EE, obtained after single dose administration and on days 1 and 21 of both cycles were evaluated model independent. Statistical
Evaluation
Pharmacokinetic parameters of CPA, like AUC, tlj2, CL and V, which were calculated for each subject after single dose administration and following the last drug intake on cycles one and three, were compared by the two-sided paired Student t-test (significance level ct = 0.05). The same test was applied for the following comparisons: SHBG (CBG) concentrations which were measured on day 2 1 of cycles one and three, respectively; simulated and measured (cycles one and three] trough levels of CPA at
Contraception 1993:48, December
563
Pharmacokinetics
of cyproterone
acetate and ethinylestradiol:
Kuhnz et al.
steady-state; C,,,, AUC(O-4h) and AUC(O-24h) values of EE, on day 21 of cycles one and three, respectively.
Results The treatment was well tolerated by the women. No positive results were obtained during the drug screening and the pregnancy tests. Pharmacokinetics of CPA and EE, After Single Dose Administration The mean concentration values of CPA in the serum of 15 women at various time points following single oral administration of 2 mg CPA together with 0.035 mg EE, and after the last drug intake during treatment cycles one and three, respectively, are presented in Table 1. A summary of the pharmacokinetic parameters of CPA is given in Table 2. After single oral administration, CPA was rapidly absorbed and maximum concentrations of 15.2 + 6.6 ng/ml were measured in the serum. Post maximum drug levels declined biphasically with half-lives of 0.8 rt 0.4 h and 54.0 + 26.0 h, respectively (Figure 1). The AUC was 160.2 & 44.8 ng x ml-l x h. The apparent volume of distribution (V,) and the apparent total clearance were calculated to be 986 +- 437 1 and 3.6 ? 0.9 ml x mini x kg’, respectively (Table 2). TABLE
1. Concentrations (mean _’ SD.) of CPA in the serum of 15 women at various time points following single oral administration of 2.0 mg CPA together with 0.035 mg EE,, and after the last drug intake during treatment cycles one and three, respectively
Time (h) 0 0.5 1 1.5 2 4 6 8 12 24 48 72 96 120
Single Dose
Cycle One
Cycle Three
(ng/mf)
(ngjmf)
(nglmf)
* 1.6 9.9 14.8 12.5 5.2 3.2 2.4 1.6 1.2 0.8 0.6 0.5 0.3
+? & + + + 2 ? * ++ t *
1.2 6.3 7.0 4.6 2.1 1.2 0.7 0.5 0.3 0.2 0.2 0.2 0.2
4.8 7.1 17.0 19.8 16.8 9.4 7.3 6.7 5.5 4.7 3.7 3.1 2.5 2.2
+ ‘lr + + & * -t * * * + + f
0.8 1.9 6.6 5.1 4.1 2.3 1.5 1.6 1.1 1.0 0.9 0.7 0.8 0.6
5.9 8.5 17.9 22.7 17.7 10.1 7.7 6.6 5.9 5.2 4.6 3.4 2.6 2.3
-t + t + + f ? + * _t * ‘f %
1.9 3.7 7.1 8.1 4.0 2.1 1.6 1.5 1.2 0.9 1.0 0.8 0.6 0.5
“Below lower limit of quantification.
564
Contraception
1993:48, December
Pharmacokinetics
of cyproterone
acetate and ethinyfestradiol:
Kuhnz et al.
TABLE 2. Pharmacokinetic parameters of CPA (mean + SD.) calculated from total concentrations in the serum of 15 healthy young women after a single oral administration of 2.0 mg CPA together with 0.035 mg EE, and afler one and three cycles, respectively, with the same contraceptive formulation
Single Dose
Parameter
C max
OWml)
t max
(h) (ng x ml-l (ng x ml-’ (I) (f) (ml x min’ (h) (h) (h)
AUC(O-24h) AUC V,, VZ CL MRT t?, A, t’,
,oo
x h) x h)
x kg-‘)
15.2 1.6 75.0 160.2 740 986 3.6 59.9 0.8 54.0
? f f ? & f 5 f ? 2
6.6 0.3 21.0 44.8 339 437 0.9 28.9 0.4 26.0
Cycle One
Cycle Three
21.0 1.4 169.3 665.6
+ + + f -
4.8 0.4 30.8 175.6
23.9 1.4 182.6 782.2
It 4 t :t
7.7 0.4 34.4 146.8
1211 3.3 94.4 0.8 68.8
+ ? % ? ‘-
397 0.6 27.5 0.2 19.1
1304 3.0 107.9 0.7 78.6
:t :t re :? :?
427 0.4 22.8 0.3 16.0
iCPAh/ml1
0
20
-_
40
80
60
100
120
140
time [h] FIGURE 1. Mean concentrations (2 SD.) of CPA in the serum of 15 women after single administration (squares) of a combination of 2 mg CPA + 0.035 mg EE, and following multiple administration on day 21 of treatment cycles one (diamonds) and three (crosses).
Contraception
1993:48, December
565
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
Twenty-four hours after single dose administration, the free fraction of CPA in the serum was 3.5 + 1.9% and the fractions bound to heat labile proteins were 4.6 k 2.2% and 92.0 k 3.5%, respectively. SHBG and CBG concentrations in the serum were 99.6 +- 23.3 nmol/l and 49.5 + 8.3 p,g/ml, respectively. These results are summarized in Table 3. The individual EE, concentrations in the serum samples are presented in Table 4. Since in several cases, the amount of serum was not sufficient for the analysis of all parameters, EE, levels could only be analysed in 11 women. The maximum concentrations were observed after 1.6 + 0.2 h and amounted to 70.8 k 27.6 pg/ml. The AUC(O-24h) was 374.4 k 255.1 pg x ml-l x h (Table 5).
TABLE 3. Protein binding of CPA in the serum samples collected prior to single dose administration and prior to the last drug intake in cycles one and three, respectively; the concentration of the binding proteins SHBG and CBG in the same samples (mean t SD.) is also presented; hl = heat labile
Single Dose
Parameter free fraction hl protein-bound albumin-bound CBG SHBG
W) (“M W) (kg/ml) (nmol/l)
3.5 4.6 92.0 49.5 99.6
Cycle One
i 1.9 ? 2.2 !I 3.5 +- 8.3 + 23.3
3.4 3.8 92.8 86.4 326.0
?z 2.0 ? 1.7 ? 3.0 + 9.7 ? 96.4
Cycle Three 3.9 3.5 92.1 91.6 314.1
f t + rt f
2.0 1.2 4.0 12.5 95.1
Concentrations of EE, (mean i: SD.) in the serum of 11 women after single dose administration of 0.033 mg EE, in combination with 2.0 mg CPA, and on the last day of one and three treatment cycles with the combined oral contraceptive, respectively TABLE 4.
Time (h) 0
0.5 1 1.5 2 4 6 8 12 24
566
Single Dose (pg/mt) 12 2 18 + 42 i70 + 65 + 39 + 26 2 10 ? 6 + 2t7
16 18 28 29 25 15 17 16 11
Cycle One (pglmf)
Cycle Three (pg/mt)
10 44 77 105 99 53 43 29 21 5
14 54 95 115 102 58 43 33 27 20
?z 19 i 35 ? 35 I? 37 t 36 t 32 2 28 ‘- 20 +- 16 t 11
+ + 2 + t c + + t ?
30 34 44 42 34 24 33 29 27 28
Contraception 1993:48, December
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
TABLE 5. Pharmacokinetic parameters of EE, (mean + SD.) obtained after single dose administration of 0.035 mg EE, in combination with 2.0 mg CPA, and after the last drug intake during treatment cycles one and three, respectively
Parameter
Single Dose
C max
@g/ml)
71 I
28
Cycle One
Cycle Three
115 2 31
118 t
311 1.9 f+ 0.8 109 787 + 342
305 1.6 +* 0.3 122 903 ? 586
42
t (pg O-4 x ml-’ (pg x ml-’
&C(O-4h) AUC(O-24h)
x h) x h)
188 1.6 5 ? 80 0.2 374 + 255
Pharmacokinetics of CPA and EE, After Multiple Dose Administration Mean trough concentrations of CPA increased during treatment and reached a steady-state on about day 16 of cycles one and three, respectively (Figure 2). On the last day (day 21) of cycles one and three, maximum concentrations of CPA in the serum were observed 1.4 k 0.4 h post administration and amounted to 21 .O + 4.8 and 23.9 -t 7.7 ng/ml, respecCPA [ng/ml] 6
,
0
2
4
6
6
10
12
day of I_esimulated]
14
16
20
22
24
cycle
j+cyclel
FIGURE 2. Comparison between simulated and measured mean of 15 women during treatment cycles one and three, respectively.
Contraception
16
1993:48, December
(-t SD.) trough levels of CPA in the serum
567
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
tively. Post maximum drug levels declined biphasically with mean halflives of 0.8 k 0.2 h and 68.8 & 19.1 h during the first cycle, and 0.7 ? 0.3 h and 78.6 t 16.0 h during the third cycle, respectively. The AUC values were 665.6 ? 175.6 and 782.2 t 146.8 ng x ml-’ x h, respectively. Total clearance and V, were 3.3 -e 0.6 ml x min -I x kg-1 and 12 11 ? 397 1 during the first cycle and the corresponding values calculated during the third cycle were 3.0 + 0.4 ml x min’ x kg-’ and 1304 + 427 1, respectively (Table 2). The free fraction of CPA on the last treatment day of cycles one and three was 3.4 + 2.0% and 3.9 ? 2.0%, respectively. The fractions of CPA bound to heat labile proteins and albumin were 3.8 t 1.7% and 92.8 f 3.0% during cycle one and 3.5 t 1.2% and 92.1 ? 4.0% during cycle three, respectively. The mean SHBG and CBG levels on day 21 of cycles one and three were 326.0 ? 96.4 nmol/l and 86.4 & 9.7 pg/ml, and 314.1 IL 95.1 nmol/l and 91.6 i 12.5 kg/ml, respectively, (Table 3). significant differences For the parameters t1/2,V, and AUC, statistically were found when single dose administration was compared to the last treatment days of cycles one and three, respectively. The clearance was not different at the end of cycle one as compared to single dose administration; only at the end of cycle three, a small, although significant difference was observed. None of the pharmacokinetic parameters except clearance was different when the last days of treatment cycles one and three were compared. No difference was found for the corresponding concentrations of CBG and SHBG. The pharmacokinetic parameters of EE, obtained on day 21 of cycles one and three, respectively, are presented in Table 5. Maximum concentrations of EE, of 115 t 31 pg/ml and 118 2 42 pg/ml were reached 1.9 ? 0.8 h and 1.6 -t 0.3 h post administration at the end (day 21) of cycles one and three, respectively. The corresponding AUC(O-24h) values were found to be 787 ? 342 and 903 t 586 pg x ml-’ XX h, respectively. No statistically significant differences were observed for either the C,,, or the AUC values on day 21 of cycles one and three, respectively. However, as compared to single dose administration, a statistically significant increase in the AUC values of about 60% was found at the end of a treatment cycle. Simulation of CPA Trough Levels in the Serum During One Month of Treatment Based on Single Dose Pharmacokinetics; Accumulation CPA in the Serum
of
A computer simulation of CPA trough levels in the serum during a complete treatment cycle was performed on the basis of the data obtained after single dose administration. A comparison of the simulated curve
568
Contraception 1993:48, December
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
with the trough levels determined experimentally during the two treatment cycles one and three is shown in Figure 2. Simulated trough levels increased to steady-state concentrations of 4.0 + 1.0 ng/ml, which were reached on day 16. The experimentally measured CPA trough levels during cycle one were practically superimposable with the simulated curve. The corresponding trough level time course measured during cycle three was in parallel to the simulated curve but slightly shifted towards higher concentration values. Steady-state concentrations of CPA reached during cycle three were statistically significantly different from those observed during cycle one and also from the corresponding simulated curve. A similar time course was seen for the corresponding SHBG levels during cycles one and three, respectively (Figure 3). An accumulation ratio (R,) of 2.2 and 2.5 was calculated for CPA in cycles one and three, respectively. Mean values calculated for the mass balance factors R* were 1.2 2. 0.2 and 1.3 + 0.2 for cycles one and three, respectively. Based on the AUC values determined after single dose administration and during steadystate, mass balance factors R* * were 1.l k 0.2 and 1.2 k 0.3 for cycles one and three, respectively (Table 6).
SHBG [nmoljl] 5wI-
1 0
2
4
6
6
10
12
14
16
16
20
22
24
day of cycle
FIGURE 3. Mean (k SD.) SHBG trough concentrations respectively.
Contraception 1993:48, December
in the serum of 15 women during cycles one and three,
569
Pharmacokinetics
of cyproterone
acetate and ethinylestradiol:
Kuhnz et al.
TABLE6. Accumulation and mass balance factors of CPA (mean ? SD.) Parameter
Cycle One
Cycle Three
RI R’ R”
2.2 + 0.3 1.2 + 0.2 1.1 t 0.2
2.5 _t 0.6 1.3 ‘- 0.2 1.2 + 0.3
Effect of Treatment on Total and Free Testosterone Concentrations in the Serum Pre-treatment concentrations of total and free testosterone in the serum were 0.41 i 0.22 ng/ml and 2.2 I 1.1 pg/ml, respectively. At the end of the first treatment cycle, these levels dropped to mean values of 0.25 ? 0.12 ng/ml for total testosterone and 0.84 -t 0.38 pg/ml for free testosterone, respectively. This corresponds to a drop of 39 % and 62 % from the corresponding pre-treatment levels, respectively. On the first day of cycle three, both total (0.47 +- 0.21 ng/ml) and free (1.7 ? 0.8 pg/ml) were not different from those measured prior to treatment. Thus, the pill-free interval of one week was sufficient to restore pre-treatment values for testosterone. At the end of cycles one and three, the same concentrations of total and free testosterone were measured, which revealed that no additional suppression of testosterone production occurred during a treatment period of three cycles as compared to the first cycle (Table 7).
Discussion In the present study, the pharmacokinetics of CPA and EE, have been investigated following single and multiple oral administration of a combination oral contraceptive to 15 women. After single oral administration, CPA was rapidly absorbed and post-maximum drug levels declined biphasically with a terminal half-life of 54 h. A large volume of distribution of about 1000 1 was observed and the apparent total serum clearance of 3.6 ml x min I x kg ’ was considerably less than the total serum liver flow of about 12 ml x minm ’ x kg- I. The apparent clearance observed in the present study was similar to the total serum clearance determined during a previous study after parenteral administration of CPA (2.3 & 0.4 ml x min ’ x kg l). This is in keeping with the finding that the absolute bioavailability of CPA was found to be almost complete.” The present results are also in good agreement with the results of a previously performed study with the same contraceptive formulation in a smaller group of women.’ As compared to single dose administration, an about two-fold accumulation of CPA in the serum occurred during treatment cycle one. Steady-
570
Contraception
1993:48,
December
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
TABLE 7. Total and free testosterone concentrations (mean + SD.) in the serum of 15 women before and during treatment with the combination oral contraceptive; pre = pre-treatment cycle
Time (cycle/day)
Total Testosterone
Free Testosterone
(ng/ml)
(pglml)
0.41 0.36 0.38 0.29 0.31 0.25 0.24 0.23 0.22 0.18 0.22 0.25 0.23 0.47 0.39 0.39 0.36 0.27 0.29 0.30 0.27 0.27 0.27 0.25
prel21 prel22 i/l l/2 l/3 l/5 118 l/10 l/l4 l/16 l/16 1121 1122 3/l 312 313 315 318 3/l 0 3114 3116 3118 3121 3122
k 5 f k + ” + + t k * k k 5 + + + + * f 5 k + f
0.22 0.17 0.15 0.15 0.14 0.13 0.11 0.13 0.13 0.10 0.12 0.12 0.11 0.21 0.18 0.18 0.35 0.13 0.15 0.27 0.15 0.17 0.16 0.17
2.2 1.7 1.8 1.5 1.5 1.1 0.9 0.9 0.8 0.6 0.8 0.8 0.7 1.7 1.4 1.2 1.2 0.9 0.9 0.9 0.8 0.8 0.7 0.7
k k k 2 t ? k k + k + + k i f k k k * 5 k t i -t
1.1 0.6 0.7 0.7 0.6 0.5 0.5 0.5 0.4 0.4 0.6 0.4 0.5 0.8 0.6 0.5 0.7 0.5 0.4 0.5 0.4 0.5 0.4 0.5
state drug levels in the serum were reached around day 16 of the cycle. The time courses of measured trough levels of CPA and those obtained by simulation of a treatment cycle based on single dose pharmacokinetics were almost superimposable, indicating linear and time-independent pharmacokinetics of CPA. The same time course was seen with the trough levels measured during cycle three; however, CPA concentrations were slightly elevated as compared to cycle one. This difference existed already on day 1 of cycle three and is very likely due to the fact that a pill-free period of 7 days between two cycles is not long enough for CPA to reach pre-treatment levels, when a terminal half-life of about 70 to 80 h is taken into account. The observed accumulation of CPA during a cycle was considerably less than one would expect from single dose administration, since based on a mean terminal half-life of 54 h and a dosing interval of 24 h, an accumulation factor of 3.8 can be calculated. If a half-life value of 70 to 80 h was considered, the accumulation factor would be about 5.
Contraception
1993:48, December
571
Pharmacokinetics
of cyproterone
acetate and ethinylestradiol:
Kuhnz et al.
The fact that the observed accumulation factor was only about 2 to 2.5, already indicates that only a small fraction of the dose is represented by this half-life value. During a treatment cycle, an increase in the SHBG concentration in the serum by a factor of 3.3 occurred which was due to the concomitant administration of EE,. Since CPA has only a minimal binding affinity to SHBG, this change in SHBG levels should not affect the distribution of CPA with respect to the binding proteins. Further, it should not contribute to the accumulation of the drug in the serum nor should it affect the Therefore, no changes in the total pharmacokinetics of the progestin. 1om12 clearance of CPA should occur and accordingly, the mass balance factors R* and R* should be equal to unity. This was indeed the case, since R* was found to be between 1.2 and 1.3 and R* * was between 1.1 and 1.2 at the end of cycles one and three, respectively, and thus very close to unity. If one compared the pharmacokinetic parameters of CPA following single dose administration with those obtained at the end of treatment cycles one and three, however, some changes became obvious. Although clearance remained practically unchanged, there was a marked increase in the volume of distribution from about 1000 1 (single dose) to about 1200-1300 1 (multiple dose] and a prolongation of the terminal half-life from 54 h to about 70-80 h. Since terminal half-life is dependent on the two independent variables CL and Vr), an increase in V,, invariably leads to an increase in tljz, if both CL and the binding of CPA in the tissue remain unchanged. It has to be mentioned that the mean terminal halflife at the end of the treatment period may be somewhat underestimated, because the observation period of 120 h after the last drug intake allowed an accurate determination only for tljz values up to about 60 h. There were, however, a number of cases with half-life values > 90 h, which could not be assessed accurately. A large volume of distribution which exceeds by far all physiologic compartments, is usually an indication of high tissue binding of the drug. From animal experiments, it is known that CPA preferentially distributes into fatty tissue. It has been suggested before that fatty tissue and the skin represent a deep compartment with limited capacity into which the drug distributes slowly. 13 This would explain the time to reach steadystate conditions as well as the prolongation of the terminal half-life after cessation of treatment. The pharmacokinetic parameters of CPA determined after single and multiple oral administration in the present study were similar to those obtained with the same preparation in a previous study, where the CPA levels were measured radioimmunologically. Only the corresponding values obtained for AUC and tX12were found to be considerably higher in the previous study. A possible explanation could be the presence of lSBl
572
Contraception
1993:48,
December
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
hydroxy-CPA in the serum samples and the known interference of this metabolite with the radioimmunological analysis of CPA.6J4 If there was a substantial contribution of this metabolite, particularly at late sampling points, this could explain the higher values of tliz and AUC, respectively. Another interesting result of the present study is the fact that the total apparent clearance of CPA was almost unaffected during treatment with the combination oral contraceptive (only at the end of cycle three was there a small, although significant decrease in the clearance]. In similar studies with preparations containing progestogens of the 19-nor testosterone series, like levonorgestrel or gestodene, in combination with EE2, both total and free clearance of the progestins decreased markedly during treatment. It was assumed that this was due to the presence of EE,, since no such changes in the clearance were seen when these progestogens were administered alone.15J6 The underlying mechanism of this reduction in clearance, however, remained unknown. Inhibition of hepatic cytochrome I?-450-dependent isozymes by EE, or an influence of EE, on the hormonal regulation of the production of these enzymes were proposed as hypothetical explanations. Obviously, in the case of CPA, none of these possible alternatives seems to hold true. The pharmacokinetic parameters of EE, in the present study were similar to those observed in previously performed studies with this contraceptive preparation.” Since the lower limit of quantification was already reached 8 to 12 hours post administration in many cases, C,,, and AUC(O-4h) values can be regarded as more reliable than the corresponding AUC(O-24h) values. An increase in the EE, serum levels during a treatment cycle by about 60 % has been found and this can probably be ascribed to the relation between dosing interval and terminal half-life of EE2, which has been discussed in detail earlier.17 The induction of hepatic CBG synthesis is an effect of the estrogenic component of the contraceptive formulation administered. An about 90 % increase over pretreatment levels was observed at the end of cycle one and there was no further increase in the CBG levels at the end of cycle three. This increase is concordant with the available information on the dose-dependency of EE,-induced CBG levels in the serum.‘” Total and free testosterone concentrations decreased during treatment cycles one and three by about 39% and 62%, respectively, compared with the corresponding values measured prior to treatment. Similar values had been observed by others with the same contraceptive preparation during a treatment period of 36 cycles. l9 In the present study, the unbound fraction of testosterone decreased by about 40% from a mean value of 0.5% on the first day of cycle one to about 0.3% at the end of treatment cycles one and three, respectively. Since there was a concomitant rise in the SHBG levels in the serum, a decrease in the free fraction of testosterone could be expected. No further changes were seen in these parameters
Contraception
1993:48, December
573
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
Kuhnz et al.
during cycle three when compared with cycle one. In that respect, treatment with the CPA-containing formulation had effects on free and total testosterone levels in the serum which were very similar to those observed with other combination oral contraceptives containing different progestogens. ’ 720 In conclusion, the present study confirmed and extended the already existing information on the pharmacokinetics of CPA following single dose administration and during a treatment period of one month obtained in an earlier study with the same oral contraceptive. After three months of treatment, no further changes in the pharmacokinetic parameters of CPA and EE, are to be expected, except for the steady-state trough levels of CPA, which might show a minor further increase during subsequent treatment cycles. No further changes are expected for SHBG and CBG and for total and free testosterone levels in the serum during long-term treatment.
References 1. 2.
3.
4.
5.
6.
7.
8.
574
Hammerstein J, Moltz L, Schwartz U. Antiandrogens in the treatment of acne and hirsutism. J Steroid Biochem 1983; 19: 591-7. Hiimpel M, Wendt H, Schultze P-E, Dogs, Weiss C, Speck U. Bioavailability and pharmacokinetics of cyproterone acetate after oral administration of 2.0 mg cyproterone acetate in combination with 50 pg ethinyloestradiol to 6 young women. Contraception 1977; 15: 579-88. Hiimpel M, Dogs C, Wendt H, Speck U. Plasmaspiegel und Pharmakokinetik von Cyproteronacetat nach oraler Applikation als 50-mg-Tablette bei 5 Mannern. Arzneim-Forsch/Drug Res 1978; 28: 319-22. Dusterberg B, Hiimpel M, Wendt H. Plasma levels of active ingredients after single and repeated administration of a new oral contraceptive containing 2 mg of cyproterone acetate and 50 ug of ethinyl estradiol (DianeR) to five young women. Acta Obstet Gynecol Stand Suppl 1979; 88: 2731. Zurth C, Tauber U. Pharmakokinetik von Cyproteronacetat und Ethinylestradiol wahrend der Behandlung mit DianeR-35: Vergleich zwischen Raucherinnen und Nichtraucherinnen. In: Schindler AE, ed. New Developments in Biosciences vol 3: Antiandrogen-estrogen therapy for signs of androgenization. Berlin, New York: de Gruyter, 1987: 43-52. Huber J, Zeillinger R, Schmidt J, Tauber U, Kuhnz W, Spona J. Pharmacokinetits of cyproterone acetate and its main metabolite I5B-hydroxy-cyproterone acetate in young healthy women. Int J Clin Pharmacol Ther Toxic01 1988; 26: 355-61. Kuhnz W, Louton T, Back DJ, Michaelis K. Radioimmunological analysis of ethinylestradiol in human serum: Validation of the method and comparison with a gas chromatographic/mass spectrometric assay. Arzneim-Forsch/Drug Res 1993; 43: 16-21. Hu OY-P, Curry SH. Calculation of fraction bound in equilibrium dialysis
Contraception 1993:48, December
Pharmacokinetics of cyproterone acetate and ethinylestradiol:
9.
10.
11.
12. 13.
14. 15.
16.
17.
18.
19.
20.
Kuhnz et al.
with special reference to drug losses by decomposition and adsorption. Biopharm Drug Disp 1986; 7: 21 l-4. Hammond GL, Lahteenmaki PLA, Lahteenmiki P, Luukkainen T. Distribution and percentages of non-protein bound contraceptive steroids in human serum. J Steroid Biochem 1982; 17: 375-80. Pugeat MM, Dunn JF, Nisula BC. Transport of steroid hormones: Interaction of 70 drugs with testosterone-binding globulin and corticosteroid-binding globulin in human plasma. J Clin Endocr Metab 1981; 53: 69-75. Hammond GL, Langley MS, Robinson PA, Nummi S, Lund L. Serum steroid binding protein concentrations, distribution of progestogens, and bioavailability of testosterone during treatment with contraceptives containing desogestrel or levonorgestrel. Fertil Steril 1984; 42: 44 51. Juchem M, Pollow K. Binding of oral contraceptive progestogens to serum proteins and cytoplasmic receptor. Am J Obstet Gynecol 1990; 163: 2171-82. Hiimpel M, Nieuweboer B, Dusterberg B, Wendt H. Die Pharmakokinetik von Cyproteronacetat beim Menschen. In: Hammerstein J, Lachnit-Fixson U, Neumann F, Plewig G, ed. Androgenisierungs-erscheinungen bei der Frau. Berlin: Excerpta Medica 1979: 212-23. Nieuweboer B, Lubke K. Radioimmunological determination of cyproterone acetate. Hormone Res 1977; 8: 210-8. Kuhnz W, Al-Yacoub G, Fuhrmeister A. Pharmacokinetics of levonorgestrel in 12 women who received a single oral dose of 0.15 mg levonorgestrel and, after a wash-out phase, the same dose during one treatment cycle. Contraception 1992; 46: 44-54. Kuhnz W, Gansau C, Fuhrmeister A. Pharmacokinetics of gestodene in 12 women who received a single oral dose of 0.075 mg gestodene and, after a wash-out phase, the same dose during one treatment cycle. Contraception 1992; 46: 2940. Kuhnz W, Sostarek D, Gansau C, Louton T, Mahler M. Single and multiple administration of a new oral contraceptive to women: Pharmacokinetics of ethinylestradiol and free and total testosterone levels in serum. Am J Obstet Gynecol 1991; 165: 596-602. Schwartz U, Hammerstein J. The oestrogenic potency of various contraceptive steroids as determined by their effects on transcortin-binding capacity. Acta Endocr (Copenh.) 1974; 76: 159-71. Falsetti L, Galbignani E. Long-term treatment with the combination ethinylestradiol and cyproterone acetate in polycystic ovary syndrome. Contraception 1990; 42: 611-9. Jung-Hoffmann C, Kuhl H. Divergent effects of two low-dose oral contraceptives on sex hormone-binding globulin and free testosterone. Am J Obstet Gynecol 1987; 156: 199-203.
Contraception 1993:48,
December
575