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Comparative progestational and androgenic activity of norgestimate and levonorgestrel in the rat
Comparative progestational and androgenic activity of norgestimate and levonorgestrel in the rat W. Kuhnz and S. Beier Research Germany
Laboratories,
Schering
Aktiengesellschaft
13342 Berlin,
The androgenic and the progestational activity of norgestimate (NORG) and levonorgestrel (LNG) were compared in two animal studies. During a Hershberger test in immature castrated male rats and a pregnancy maintenance test in pregnant rats, NORG and LNG were administered subcutaneously at several doses, serum samples were collected from each animal during the treatment period and the concentration of LNG was measured in these samples. It could be shown in both studies that in those animals which were treated with NORG, LNG was a major metabolite present in the serum. The difference in the pharmacological response in LNG- and NORGtreated rats was equivalent to the difference in the exposure of the animals to either directly administered or metabolically derived LNG. This was true for the androgenic and the progestational activity of NORG. It is concluded that according to the present results, NORG can be described as a pro-drug of LNG and that the latter is probably mainly responsible for the pharmacological effects observed during treatment with NORG. It cannot be excluded, however, that NORG itself or other metabolites of this drug may also contribute to the pharmacodynamic response. Keywords: Pharmacokinetics; norgestimate; levonorgestrel; rat; progestational activity; androgenic activity
Submitted for publication November 16, 1993; accepted for publication January 4, 1994. Address correspondence to: Dr. Wilhelm Kuhnz, Institut Schering Aktiengesellschaft, 13342 Berlin.
0 1994 Butterworth-Heinemann
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Introduction Norgestimate (NORG) has been introduced as a novel progestogen with highly selective progestational activity and only minimal androgenicity. In that respect, NORG was claimed to possess a pharmacological profile quite different from levonorgestrel (LNG), another 19-nor-testosterone type progestin, which still has some androgenic partial activity. This claim was largely based on preclinical investigations on receptor binding and studies on the androgenic and progestogenic activity in several animal speciesm4 Studies on the biotransformation of orally administered NORG in rats, dogs, monkeys and humans revealed the presence of a number of metabolites in urine which have also been observed after oral administration of LNG? These studies indicated the possibility that metabolites of NORG, like levonorgestrel acetate, levonorgestrel oxime and LNG, might contribute to the pharmacologic response in these species. Although these compounds had been observed during in vitro metabolism studies,7fX only scant information has been provided as to the presence of NORG and its metabolites in the plasma of animals treated with NORG,” which precluded an evaluation of their possible contribution to the overall pharmacodynamic response. Since LNG is known to be a potent progestogen, it was supposed that NORG might be only a pro-drug of LNG and the latter being mainly, if not exclusively, responsible for the observed pharmacological effects. If that was the case, the administration of NORG would not offer additional advantages over a treatment with LNG itself. The aim of the present study was to assess and compare both the androgenic and the progestational activity of NORG and LNG in two studies with rats. In order to evaluate the possible contribution of LNG to the effects observed in the NORG-treated rats, serum samples were obtained from the animals treated with either LNG or NORG and the concentration of LNG was measured in these samples. The observed pharmacological response was related to the actual exposure of the animals to LNG.
Material
and Methods
The progestogens, norgestimate levonorgestrel, were synthesized Pharmacologic
(levonorgestrel-3-oxime-17-acetate) at Schering AG.
and
Tests
and Housing Conditions. The rats used in both tests were housed in temperature controlled rooms (20 2 2°C) with an artificial light (14 h) - dark (10 h) rhythm. The animals had free access to food (AltromirPpellets, Lage, FRG) and tap water. During the test period, threereated
Animals
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female animals (pregnancy maintenance test) and two male animals (Hershberger test) were kept per cage, respectively. Progestational activity. The progestational activity of LNG and NORG was assessed in a pregnancy maintenance test in ovariectomized rats. Pregnant rats were randomly allocated to 10 groups. Each group comprised 6 animals and each animal (except those belonging to the control group 10) was ovariectomized on day 8 of pregnancy. On the same day, two hours after castration as well as on the subsequent 13 days, the animals received either NORG or LNG at different doses, or vehicle. LNG was administered in doses of 0.3, 0.1, 0.03 and 0.01 mg/day, and NORG in doses of 1.0, 0.3, 0.1 and 0.03 mg/day, respectively (Table 1). The daily dose of each compound was administered subcutaneously in a total volume of 0.2 ml of castor oil/benzyl benzoate (4/l). In addition, those animals which were treated with either LNG or NORG, received an injection (s.c.) of 1 g estrone/day in a volume of 0.1 ml of the same vehicle. The body weight of the pregnant rats was determined on days 1, 8 and 22. One day after the last treatment (day 22 of pregnancy), the animals were sacrificed and the number of living fetuses per animal was determined. The degree of pregnancy maintenance was expressed in % of fetuses per animal. Ten fetuses per animal were observed in the intact control group and this value was defined as a 100% response. Blood samples (ca. 0.5 ml) were taken from each of the animals at the
TABLE 1. Efficacy of NORG and LNG to maintain pregnancy, when administered subcutaneously over a period of 14 days to ovariectomized rats. Groups 1 to 8 received an additional administration of 1 kg estrone/day. Body weights (mean k SD.) and number of living fetuses per animal (mean) are presented. Controls included castrated (group 9) and intact (group 10) animals. Group size was n = 6 in each case.
% of No. of Living Fe$za;/
Body Weight Group No. 1 2 3 4 5 6 7 8 9 10
Is1
Dose Drug
D’WWI
NORG NORG NORG NORG LNG LNG LNG LNG controls controls
1 .o 0.3 0.1 0.03 0.3 0.1 0.03 0.01 -
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* ?I k k + k f + k k
11 12 14 12 10 10 9 9 7 7
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Day 8 230 231 232 236 235 234 230 244 241 239
t k + i k + k k I! 2
16 14 16 16 11 12 10 14 9 6
Rats With Living Fetuses
[%I
Day 22 298 341 274 261 354 332 282 270 290 328
2 + ? + + k f k lr +
31 29 30 15 23 30 20 18 17 10
Degree of Pregnancy Maintenance
7.0 10.7 2.5 0 12.0 8.0 4.5 0 0 10.8
100 100 33.3 0 100 83.3 83.3 0 0 100
70 100 25 0 100 80 45 0 0 100
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following time points post administration: day 8 [prior to drug administration), day 9 at 1 h post administration, day 10 at 2 h, day 12 at 4 h, day 14 at 6 h, day 16 at 8 h and day 18 at 24 h post administration. Blood samples were centrifuged, the serum was separated and equal aliquots of each sample, obtained at corresponding times post administration, were pooled within each treatment group. Per treatment group, seven serum pools were thus obtained. The pooled samples were kept deep frozen at - 18°C until analysis. LNG was determined in the serum of the animals belonging to treatment groups l-8. Androgenic activity. The androgenic activity of LNG and NORG was assessed in a Hershberger test in immature orchidectomized rats. Male castrated juvenile rats were randomly allocated to 13 groups. Each group comprised 6 animals and each animal received a daily dose of either LNG, NORG, testosterone propionate (TP) or vehicle over a treatment period of 13 days. The daily dose of each compound was administered subcutaneously in a total volume of 0.2 ml of castor oil/benzyl benzoate (4/l). The body weight of the animals was recorded on days 1 and 13. One day after the last treatment, the animals were sacrificed and the weight of the seminal vesicles and the prostate glands were determined. A Dunnett’s t-test was used to compare the castrated control group with the other treatment groups. This test controls the type I error for comparisons of all treatments against a control. The significance level was set at (Y=0.05. The null hypothesis is that there is no difference between the treatment groups and the castrated control group with respect to the target variables (weights of seminal vesicles and prostate). The alternative hypothesis is that with a sample size of 6 animals per treatment and control group, a test power of 1-B = 0.9 and a significance level of a = 0.05, a t-test would detect a difference of A = 2.1 standard deviations. LNG was analysed in the serum of animals which were treated with LNG and NORG. Blood samples (ca. 0.2 ml) were taken from each of the animals at the following time points post administration: day 1, prior to drug administration, day 2 at 1 h post administration, day 3 at 2 h, day 5 at 4 h, day 7 at 6 h, day 9 at 8 h and day 11 at 24 h post administration. Blood samples were centrifuged, the serum was separated and equal aliquots of each sample were pooled as described above. Per treatment group, seven serum pools were thus obtained. The pooled samples were kept deep frozen at - 18°C until analysis. LNG was determined in each pool serum.
Analytical
Methods
of LNG by GC/MS. The analytical determination of LNG in serum samples obtained during the pregnancy maintenance test in the rat, was performed by a GC/MS method (LAB, Neu-Ulm, Germany). Determination
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Briefly, to 1 ml of the serum sample, the internal standard (2H,norethindrone) was added and subsequently, the sample was extracted with 5 ml of cyclohexane/2-butanol (98.5 : 1.5; v/v). The organic layer was transferred into a new vial and stored refrigerated until analysis. Prior to analysis, the extract was dried at 40 - 50°C under a stream of nitrogen and the residue was redissolved in 10 - 20 pl of toluene. An aliquot of this solution was injected into a GC/MS system (Finnigan MAT 4500) operated in the positive chemical ionization mode with selected ion monitoring. The samples were analyzed in two sequences. One sequence was composed of serum samples of treatment groups 1 - 4 and 5 - 8, respectively, as well as a complete standard curve and 4 quality controls (0.1, 0.25, 2.0 and 10.0 ng/ml). For the preparation of the standard curve, a methanolic solution of LNG (100 Fg/ml) was diluted with a pool of blank serum, to yield final concentrations between 0.1 and 20 ng/ml. The lower limit of quantification (LLQ) was 0.1 ng/ml. The recovery was between 94 and 110%. Individual sequences were accepted when at least 3 out of the 4 quality controls differed by not more than + 15% from their nominal concentrations.
of LNG By a Combination of HPLC and RIA. The analytical determination of LNG in serum samples obtained during the Hershberger test in the rat, was performed by HPLC-RIA. An aliquot of 0.05 ml of either undiluted serum, or serum which had been diluted with physiological saline was spiked with about 1500 cpm of 3H-LNG and injected into an automated HPLC-system with precolumn switching. The fraction of the eluate which contained LNG was taken to dryness under a stream of nitrogen and the remaining residue was redissolved in 1 ml of BSA-buffer. An aliquot of 0.1 ml was used for the determination of the recovery and an aliquot of 0.8 ml was subjected to radioimmunological analysis. During method development, the recovery of LNG was examined at concentrations of 0.1, 1.0 and 5.0 ng/ml and was found to be between 68 and 77%. The absence of a carry-over effect between two injections was also demonstrated. Assay quality was assessed by the inclusion of two quality control sera (1 .O and 2.0 ng/ml) in each batch. The recovery was found to be 119% for both controls. Determination
HPLC-system. precolumn:
wash-fluid: purge-time: flow rate: analytical column:
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Lichrosorb RP- 18 (Merck, Darmstadt, Germany], lo-25 pm, 20 x 4.6 mm methanol/water (20/80, v/v) 3 min 1.5 ml/min ODS-Hypersil (Shandon), 5 p.m, 125 x 4.6 mmm
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phase:
activity:
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A: phosphate
and Beier
buffer (0.01 m, pH 7)/ methanol
(90/10, v/v) B: phosphate buffer (0.01 m, pH 7)/ methanol gradient:
flow rate: column temp.: fraction
collection:
( 1O/90, v/v) O-12 min 50% A, isocratic 12-15 min 50% A to 10 % A 15-18 min 10% A to 50 % A 18-21 min 50% A, isocratic 2 ml/min 30°C for both precolumns and the analytical column between 7.5 and 10.5 min after injection on the analytical column
Radioimmunoassay of LNG. The assay has been described in detail elsewhere.‘O For the determination of LNG, an antiserum (Schering AG) raised against levonorgestrel-3-( 0-carboxymethyl)-oxime-BSA and [ 15, 16“HI-levonorgestrel with a specific radioactivity of 1.8 TBq/mmol (Schering AG) were used. An aliquot of 0.8 ml of the purified (by HPLC) sample was incubated with the antiserum (dilution: 1:80, 000) and ca. 5000 cpm of tracer; incubation time was 16 h at 4°C. The LLQ was 40 pg/ml. Assay performance was assessed by the inclusion of two quality control sera in each assay (1 .O and 3.0 ng/ml). This allowed an additional control over the assay, independent from the sample purification by HPLC. The mean coefficients of inter-assay precision were between 11 and 24% and the recovery was 98%. Pharmacokinetic Evaluation. The concentrations of LNG measured in the serum samples obtained from the animals of each treatment group in both pharmacologic tests were used to calculate the area under the serum level-time curve AUC(O-24h) according to the linear trapezoidal rule. The systemic availability (fr) of norgestimate-derived LNG relative to an administered dose of LNG was calculated according to: f = I
AUC(O-24h), = AUC(O-24h),,, = LNG D LNG= DN =
280
AUCP24h), x D,,, AUC(O-24h),,,
AUC(O-24h) AUC(O-24h)
x D,
of norgestimate-derived LNG of LNG after the administration
dose of LNG administered dose of norgestimate-derived LNG which could be released at most from the dose of norgestimate
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administered taking the different molecular weights of LNG (312) and norgestimate (369) into account.
Results Pregnancy
Maintenance
Test
The progestational potencies of NORG and LNG were compared with respect to the pregnant control animals and the results are presented in Table 1. In the intact control group, a mean of 10 fetuses were found per animal. Thus, this value was defined as 100% pregnancy maintenance and the results obtained in the treatment groups were expressed relative to this standard. The body weights were practically identical in each group at the outset of the study. On day 22, similar body weights were observed in intact controls and in treatment groups 1, 2, 5 and 6, where a high degree of pregnancy maintenance was achieved. Those animals which belonged to the castrated control group and to treatment groups 3, 4, 7 and 8, showed a reduced weight gain which was due to the smaller number of living fetuses during the treatment with lower doses of the two progestins (Table 1). A similar degree in the percentage of pregnancy maintenance was achieved at doses of NORG which were about 3- to lo-fold higher than the corresponding doses of LNG (Figure 1). Only at one point, at a dose of 0.3 mg/day, the pregnancy maintenance obseved was the same for both progestins.
Determination of LNG in Pooled Serum Samples.
The LNG concentrations which were determined in the pooled serum samples of rats belonging to treatment groups l-8 are presented in Table 2. In all groups, serum levels of LNG increased up to 2-8 h post administration and remained on a plateau up to 24 h post dose. A linear relation was found between the dose of LNG administered (groups 5-8) and the AUC(O-24h) of LNG administered. The same was true for the dose of NORG administered (groups l-4) and the AUC(O-24h) of norgestimate-derived LNG calculated. The mean bioavailability of NORG-derived LNG was 16.5 & 1.9% over the dose range of 0.03-0.3 mg NORG/day. The doses of the two progestins administered are presented together with the corresponding area under the curves of LNG in Figure 1. The difference in doses of LNG and NORG required to achieve the same pregnancy maintenance rate is equivalent to the difference in doses required to reach the same AUC values of LNG.
Hershberger
Test
The androgenic potencies of NORG, LNG and TP were compared to control groups of intact and castrated animals. The results on body weight
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maintenance [%]
i.C. 100 00 60 40 20 0 0.001
0.1
0.01
1
10
1
10
mglday
200
AUC(O-24h) of LNG [ngxh/ml]
150 100 50 0 0.001
0.01
0.1 Dose PwWl piciiq
FIGURE 1. Pregnancy maintaining potential of LNG and NORG when administered subcutaneously to ovariectomized pregnant rats together with 1 .O pg estrone over a treatment period of 14 days. Pregnancy maintenance relative to the intact (i.c.) and the castrated (cc.) control group is presented (top) together with the corresponding LNG levels in the serum of the treated animals (bottom).
gain and influence of treatment on the relative organ weights of seminal vesicles and prostate glands are presented in Table 3. The body weights were practically identical in each group at the outset of the study. On day 13, similar body weights were observed in intact controls and those animals which were treated with TP (groups 9, 10 and 11). Those animals which were under LNG treatment showed a slightly reduced weight gain as compared to the intact controls and this was even more marked for
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TABLE 2. Concentration of LNG (nglml) in pooled serum samples of rats which were treated (s.c.) with either LNG (groups 5-8) or NORG (groups l-4) at different doses. The calculated AUC (O-24h) values of LNG are also presented. Values below the lower limit of quantification (0.1 nglml) were set to zero; n.e. = not evaluable.
Group Time
&W-d
1
2
810 9/l 1012 1214 1416 1618 18124
0 1.7 2.0 2.3 2.7 3.9 3.0 73.7
AUC(O-24h) [ng x mlk’ x h]
3
4
5
6
7
8
0 0.6 0.8 0.8 1 .o 1.1 1.2
0 n.e. 0.2 0.2 0.3 0.4 0.3
0 0.3 0.1 0.1 0.1 0.1 0
0 8.2 7.1 5.1 7.1 9.1 5.9
0.5 2.9 2.9 2.5 2.6 3.3 1.8
0 1.4 0.9 0.8 0.9 0.7 0.5
0 0.3 0.2 0.4 0.3 0.3 0.1
24.5
7.6
2.4
171.9
62.1
16.0
5.6
TABLE 3. Androgenic activity of NORG, LNG and TP after daily administration to immature castrated male rats over a treatment period of 13 days. Body weights and relative organ weights (mgll00 g body weight) of seminal vesicles and prostage glands are presented (mean t S.D.) Controls included intact (group 12) and castrated (group 13) animals. Group size was n = 6 in each case.
Body Weight Group No. 1 2 3 4 5 6 7 8 9 10 11 12 13
Drug
[mgldayl
LNG LNG LNG LNG NORG NORG NORG NORG TP TP TP control control
0.1 0.3 1.0 3.0 1.0 3.0 10.0 30.0 0.01 0.03 0.1 -
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Seminal Vesicle
[gl
Dose Day 1 97.3 97.2 98.3 103.7 96.8 99.7 99.3 103.7 96.7 97.3 100.0 103.8 97.0
k k k 2 f k + + 2 t k -t 5
March
3.7 3.1 3.3 2.9 2.6 4.1 4.3 3.3 3.1 3.9 3.0 4.9 2.0
Day 13 160.2 171.3 174.8 174.7 161.0 159.8 151.2 143.2 176.3 174.8 182.0 181.8 167.2
k + k k -+ + k 5 t k 5 k k
10.9 7.2 7.3 8.0 11.7 11.6 8.7 12.6 12.8 6.1 8.2 10.8 13.8
]mg/lOO 16.3 44.6 115.0 173.8 16.1 22.6 35.4 62.9 12.0 26.5 103.1 34.0 8.2
2 k 2 + 2 -c k 5 k f k ij,
Prostate
sl
3.0 8.1 26.9 29.0 3.9 5.0 7.8 12.3 3.4 4.5 22.9 9.1 0.9
b-Ml00 91 33.0 67.7 114.5 158.6 25.2 33.9 54.2 79.4 23.0 53.0 128.2 79.1 12.4
k t t k t -+ t -+ -+ t -+ 2 k
4.3 9.1 6.3 18.2 8.0 9.2 10.6 13.6 6.0 9.2 15.4 13.7 2.8
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those animals which were treated with NORG. A similar degree in the increase of relative organ weights was achieved at doses of NORG which were about lo- to lOO-fold higher than corresponding doses of LNG (Figure 2).
Determination of LNG in Pooled Serum Samples.
The LNG concentrations which were determined in the pooled serum samples of rats belonging to treatment groups 1-8 are presented in Table 4; each value represents the mean of duplicate or triplicate analysis. In all groups, serum levels of LNG increased up to 2-8 h post administration and remained on a plateau up to 24 h post dose. AUC(O-24h) values were calculated for each treatment group and there was a linear relation to the dose of LNG administered (groups l-4). The same was true for the dose of NORG administered (groups 5-8) and the AUC(O-24h) of NORG-derived LNG calculated. The mean bioavailability of NORG-derived LNG was 8.5 + 3.6% over a dose range of l-30 mg NORG/day. The doses of the two progestins administered are presented together with the corresponding area under the curves of LNG in Figure 2. The difference in doses of LNG and NORG required to achieve the same gain in relative organ weights is almost equivalent to the difference in doses required to reach the same AUC values of LNG.
Discussion NORG has been introduced as a novel selective progestogen with minimal androgenic activity and in that respect it was claimed to be superior to LNG. This was concluded mainly from the results of preclinical studies and further support was obtained from the clinical performance of NORGll--ls On the other hand, it has containing combination oral contraceptives. been argued by others that metabolites of NORG may be the pharmacologically active principles and NORG might only be a pro-drug.y,‘6 The chemical structure of NORG on the one hand and metabolism studies in animals and man as well as in vitro studies on the other hand made it very likely that LNG could be a major metabolite of NORG.“,” This has always been disputed by the Phillips group and it was stated by them and others that another metabolite (LNG-oxime) contributed significantly to the pharmacologic response of NORG.3,ss’4 The fact that none of the published animal studies were accompanied by drug level measurements, made it impossible to follow that conclusion and to positively assign specific pharmacological effects to the presence of either NORG or one or several of its metabolites. In the present study, we have examined the androgenic activity of NORG in comparison to LNG using the same animal model which has been used before by the Phillips group.3 However, in order to investigate
284
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Prostate welghl [mg/lwg body weight]
200
* 150
t l
i.c.
100
*
I
l l
l
50
*
* C.C.
I
-22
0 0.001
0.01
0.1
1
10
Seminal vesicle weight [mg/lOOg body weight] 200 * 150
100
50
0 0.001
0.1
0.01
1
10
mg/day AUC(CL24h)of LNG [ngxh/ml]
1500 1000 500 0 0.001
, , . ,( 0.01
,
, ,,
,L!.A
0.1
1
D-
DmWwl
*LNG
+NORG
10
FIGURE 2. Dose-dependent androgenic effects (prostate, top: seminal vesicle, middle) in juvenile castrated rats and corresponding LNG levels (bottom) in serum obtained after S.C. treatment with either LNG or NORG. Values, significantly different (~~0.05) from those obtained in castrated controls are marked by an asterisk. TP (open circles); ic. = intact controls; cc. = castrated controls.
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TABLE 4. Concentration of LNG (ng/ml) in pooled serum samples of rats which were treated (s.c.) with either LNG (groups l-4) or NORG (groups 5-8) at different doses. The calculated (AUC (O-24h) values of LNG are also presented. Values below the lower limit of quantification (1 .O ng/ml) were set to zero.
Group Time
[day/W
1
2
3
110 2/l 312 514 716 918 11124
0 3.4 2.6 2.9 3.2 3.1 2.3
0 8.3 8.6 9.2 10.2 10.1 13.4
65.9
258.2
AUC(O-24h) [ng x mlk’ x h]
4
5
6
7
8
0 23.0 30.5 32.0 27.3 22.1 23.1
1.5 53.2 51.7 84.4 93.4 69.6 64.9
0 2.7 3.0 3.8 2.9 2.3 2.8
0 2.8 2.7 4.0 4.9 4.6 4.9
0 7.5 12.0 9.5 9.9 11.7 15.2
1.7 10.7 13.2 16.3 27.9 45.3 43.3
571.4
1632.6
64.0
105.4
291.4
874.0
whether LNG is indeed a major metabolite in the rat following the administration of NORG, LNG was measured in the serum of the animals during the whole treatment period.The results with respect to the relative androgenic potencies of NORG and LNG obtained in the present study are almost identical to those published earlier by Phillips and coworkers.“+ A factor of about ten in androgenic potency was observed between TP, LNG and NORG, respectively, based on the results of prostate growth and seminal vesicle weight gain. Thus, on the basis of a simple relation of dose administered and effects on the androgen-sensitive target organs measured, it would appear that NORG had an about ten-fold less androgenie potency than LNG. The concomitant drug level monitoring, however, revealed two important findings: Firstly, LNG was present in the serum of those animals which were treated with NORG. Thus, in the rat, LNG is a major metabolite after S.C. administration of NORG. Secondly, the exposure of these animals to NORG-derived LNG over the complete treatment period expressed as the area under the serum level-time curve (AUC), was about one-tenth of the exposure of those animals which were treated with LNG directly. The observed difference in androgenicity between LNG and NORG turned out to be equivalent to the difference in the exposure of the animals to LNG. In the light of these results, it seems likely that the pharmacological response observed in the NORG-treated rats can be directly related to the presence of LNG, which would support the view of NORG being basically only a pro-drug of the pharmacological active metabolite LNG. It would be important to know, whether this view is only true for the
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androgenic partial activity of NORG or whether it is also consistent with respect to the progestational activity of this drug. It has been claimed before that there was evidence of NORG possessing an inherent progestational activity. Although the possible contribution of other active metabolites was not excluded, it was thought to be unlikely that NORG could be acting mainly via LNG.” In the present study, the progestational activities of NORG and LNG were examined in a pregnancy maintenance test in rats. Again, LNG was measured in the serum of the animals during the whole treatment period. A difference by a factor of about three to ten in progestational potency was observed between LNG and NORG, based on the pregnancy maintenance potential. Only at a dose of 0.3 mg, the same degree of pregnancy maintenance was observed for both drugs. At the highest dose of 1 mg NORG, the efficacy to maintain pregnancy was even reduced. One should bear in mind, however, that during normal pregnancy, there is a balance in the estrogen/progesterone ratio which is limited to a narrow range. In the experimental model, a constant dose of 1 pg estrone was administered each day together with doses of the progestogens, covering a range of two orders of magnitude which eventually shifts this ratio extremely towards the progestogen. It has been shown before in similar experiments that this imbalance can negatively affect the outcome of pregnancy.” On the basis of a simple relation of dose administered and pharmacological effect observed, it would appear from the present results that NORG had an about three- to ten-fold less progestational potency than LNG. The concomitant analysis of LNG in the serum of the animals treated with NORG revealed an exposure of these animals to NORG-derived LNG over the complete treatment period of about one-third to one-tenth of the exposure of those animals which were treated with LNG directly. The observed difference in the progestational activity between LNG and NORG was in good agreement with the difference in the exposure of the animals to LNG. Thus, not only with respect to the androgenic potency but also with respect to the progestational efficacy, the pharmacological response observed in the NORG-treated rats can be quantitatively related to the exposure of the animals to LNG, which is consistent with the view of NORG being basically only a pro-drug of LNG. Nowadays, it is generally accepted and has been proven in numerous comparative pharmacological studies in animals that none of the synthetic progestogens equals progesterone in its entire spectrum of pharmacological action. Besides their progestogenic activity, synthetic sex hormones display additional specific partial activities, like e.g., androgenie properties, which are characteristic for sex steroids of the 19-nortestosterone series. During comparative pharmacological studies in animals examining, both qualitatively and quantitatively, the spectrum of efficacy and possible differences between drugs, it has to be taken into
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account that there are species-specific differences in intrinsic activity, pharmacokinetics and biotransformation. This is of particular importance, when different animal models are used to assess relative potencies to conclude on a possible dissociation of pharmacological effects.17,‘8 If the chemical structure of a drug suggests that it could potentially be a pro-drug and its pharmacological profile could probably be ascribed to an active metabolite, then the test conditions should take this into account. In order to exclude possible species-specific differences, all investigations with respect to various partial activities should be investigated in the same animal species with concomitant drug monitoring and pharmacokinetic evaluation. The route of administration as well as the galenic formulation of the drug should be identical in each test. Previously published animal studies with NORG have, from our point of view, not completely taken care of these prerequisites.1-3 In particular, no drug level monitoring was performed during the pivotal animal studies. Therefore, the conclusions drawn from these experiments, like NORG itself being a selective progestogen with minimal androgenic activity and not a pro-drug of LNG, seem to be questionable. In conclusion, the present study clearly revealed that following S.C. administration to rats, NORG is metabolized to a considerable extent to LNG. The pharmacological effects observed can be directly related to the exposure of the animals to NORG-derived LNG. Therefore, there is no doubt that NORG is a pro-drug of LNG; however, it cannot be excluded that either NORG itself or other metabolites of NORG may also contribute to the overall pharmacodynamic response. This, however, has yet to be demonstrated by conclusive experiments where the presence of these substances in the serum of the animals is shown and related to the pharmacological response.
References 1. 2.
3.
4.
5.
288
Hahn DW, Allen GO, McGuire JL. The pharmacological profile of norgestimate, a new orally active progestin. Contraception 1977;16:541-53. Phillips A, Hahn DW, Klimek S, McGuire JL. A comparison of the potencies and activities of progestogens used in contraceptives. Contraception 1987;36:181-92. Phillips A, Demarest K, Hahn DW, Wong F, McGuire JL. Progestational and androgenic binding affinities and in vitro activities of norgestimate and other progestins. Contraception 199Oj41:399-410. Phillips A, Hahn DW, McGuire JL. Preclinical evaluation of norgestimate, a progestin with minimal androgenic activity. Am J Obstet Gynecol 1992;167:1191-6. McGuire JL, Phillips A, Hahn DW, Tolman EL, Flor S, Kafrissen ME. Pharmacologic and pharmacokinetic characteristics of norgestimate and its metabolites. Am J Obstet Gynecol 1990;163:2127-31.
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Progestational 6. 7.
8.
9.
10.
11. 12. 13. 14.
15.
16.
17.
18.
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and androgenic
activity:
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and Beier
Alton KB, Hetyei NS, Shaw C, Patrick JE. Biotransformation of norgestimate in women. Contraception 1984;29:19-29. Madden S, Back DJ. Metabolism of norgestimate by human gastrointestinal mucosa and liver microsomes in vitro. J Steroid Biochem Molec Biol 1991;38:497-503. Wild MJ, Rudland PS, Back DJ. Metabolism of the oral contraceptive steroids ethynylestradiol and norgestimate by normal (HUMA 7) and malignant (MCF-7 and ZR-75-1) human breast cells in culture. J Steroid Biochem Molec Biol 1991;39:535-43. Sisenwine SF, Liu AL, Kimmel HB, Ruelius HW. The conversion of dnorgestrel-3-oxime-17-acetate to d-norgestrel in female rhesus monkeys. Contraception 1977;15:25-37. Hiimpel M, Wendt H, Pommerenke G, Weiss C, Speck U. Investigations of pharmacokinetics of levonorgestrel to specific consideration of a possible first pass effect in women. Contraception 1978;17:207-20. Chapdelaine A, Desmarais JL, Derman RJ. Clinical evidence of the minimal androgenic activity of norgestimate. Int J Fertil 1989;334:347-52. Becker H. Supportive European data on a new oral contraceptive containing norgestimate. Acta Obstet Gynecol Stand Suppl 1990;152:33-9. Kafrissen ME. A norgestimate-containing oral contraceptive: review of clinical studies. Am J Obstet Gynecol 1992;167:1196-202. Anderson F. Selectivity and minimal androgenicity of norgestimate in monophasic and triphasic oral contraceptives. Acta Obstet Gynecol Stand Suppl 1992;156:15-21. London RS, Chapdelaine A, Upmalis D, Olson W, Smith J. Comparative contraceptive efficacy and mechanism of action of the norgestimatecontaining triphasic oral contraceptive. Acta Obstet Gynecol Stand Suppl 1992;156:9-14. Juchem M, Pollow K, Elger W, Hoffmann G, Mobus V. Receptor binding of norgestimate-a new orally active synthetic progestational compound. Contraception 1993;47:283-94. Neumann F, Elger W. Critical considerations of the biological basis of toxicity studies with steroid (sex) hormones. In: Plotz EJ, Hazler J, eds. Methods in Steroid Toxicology. Los Altos, California, 1972: 10-91. Beier S, Dusterberg B, El Etreby MF, Elger W, Neumann F, Nishino Y. Toxicology of hormonal fertility-regulating agents. In: Benagiano G, Diczfalusy E, eds. Endocrine Mechanisms in Fertility Regulation. New York: Raven Press, 1983;261-346.
1994:49,
March
289
Laboratories,
Schering
Aktiengesellschaft
13342 Berlin,
The androgenic and the progestational activity of norgestimate (NORG) and levonorgestrel (LNG) were compared in two animal studies. During a Hershberger test in immature castrated male rats and a pregnancy maintenance test in pregnant rats, NORG and LNG were administered subcutaneously at several doses, serum samples were collected from each animal during the treatment period and the concentration of LNG was measured in these samples. It could be shown in both studies that in those animals which were treated with NORG, LNG was a major metabolite present in the serum. The difference in the pharmacological response in LNG- and NORGtreated rats was equivalent to the difference in the exposure of the animals to either directly administered or metabolically derived LNG. This was true for the androgenic and the progestational activity of NORG. It is concluded that according to the present results, NORG can be described as a pro-drug of LNG and that the latter is probably mainly responsible for the pharmacological effects observed during treatment with NORG. It cannot be excluded, however, that NORG itself or other metabolites of this drug may also contribute to the pharmacodynamic response. Keywords: Pharmacokinetics; norgestimate; levonorgestrel; rat; progestational activity; androgenic activity
Submitted for publication November 16, 1993; accepted for publication January 4, 1994. Address correspondence to: Dr. Wilhelm Kuhnz, Institut Schering Aktiengesellschaft, 13342 Berlin.
0 1994 Butterworth-Heinemann
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fiir Pharmakokinetik,
1994:49, March
275
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activity:
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and Beier
Introduction Norgestimate (NORG) has been introduced as a novel progestogen with highly selective progestational activity and only minimal androgenicity. In that respect, NORG was claimed to possess a pharmacological profile quite different from levonorgestrel (LNG), another 19-nor-testosterone type progestin, which still has some androgenic partial activity. This claim was largely based on preclinical investigations on receptor binding and studies on the androgenic and progestogenic activity in several animal speciesm4 Studies on the biotransformation of orally administered NORG in rats, dogs, monkeys and humans revealed the presence of a number of metabolites in urine which have also been observed after oral administration of LNG? These studies indicated the possibility that metabolites of NORG, like levonorgestrel acetate, levonorgestrel oxime and LNG, might contribute to the pharmacologic response in these species. Although these compounds had been observed during in vitro metabolism studies,7fX only scant information has been provided as to the presence of NORG and its metabolites in the plasma of animals treated with NORG,” which precluded an evaluation of their possible contribution to the overall pharmacodynamic response. Since LNG is known to be a potent progestogen, it was supposed that NORG might be only a pro-drug of LNG and the latter being mainly, if not exclusively, responsible for the observed pharmacological effects. If that was the case, the administration of NORG would not offer additional advantages over a treatment with LNG itself. The aim of the present study was to assess and compare both the androgenic and the progestational activity of NORG and LNG in two studies with rats. In order to evaluate the possible contribution of LNG to the effects observed in the NORG-treated rats, serum samples were obtained from the animals treated with either LNG or NORG and the concentration of LNG was measured in these samples. The observed pharmacological response was related to the actual exposure of the animals to LNG.
Material
and Methods
The progestogens, norgestimate levonorgestrel, were synthesized Pharmacologic
(levonorgestrel-3-oxime-17-acetate) at Schering AG.
and
Tests
and Housing Conditions. The rats used in both tests were housed in temperature controlled rooms (20 2 2°C) with an artificial light (14 h) - dark (10 h) rhythm. The animals had free access to food (AltromirPpellets, Lage, FRG) and tap water. During the test period, threereated
Animals
276
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female animals (pregnancy maintenance test) and two male animals (Hershberger test) were kept per cage, respectively. Progestational activity. The progestational activity of LNG and NORG was assessed in a pregnancy maintenance test in ovariectomized rats. Pregnant rats were randomly allocated to 10 groups. Each group comprised 6 animals and each animal (except those belonging to the control group 10) was ovariectomized on day 8 of pregnancy. On the same day, two hours after castration as well as on the subsequent 13 days, the animals received either NORG or LNG at different doses, or vehicle. LNG was administered in doses of 0.3, 0.1, 0.03 and 0.01 mg/day, and NORG in doses of 1.0, 0.3, 0.1 and 0.03 mg/day, respectively (Table 1). The daily dose of each compound was administered subcutaneously in a total volume of 0.2 ml of castor oil/benzyl benzoate (4/l). In addition, those animals which were treated with either LNG or NORG, received an injection (s.c.) of 1 g estrone/day in a volume of 0.1 ml of the same vehicle. The body weight of the pregnant rats was determined on days 1, 8 and 22. One day after the last treatment (day 22 of pregnancy), the animals were sacrificed and the number of living fetuses per animal was determined. The degree of pregnancy maintenance was expressed in % of fetuses per animal. Ten fetuses per animal were observed in the intact control group and this value was defined as a 100% response. Blood samples (ca. 0.5 ml) were taken from each of the animals at the
TABLE 1. Efficacy of NORG and LNG to maintain pregnancy, when administered subcutaneously over a period of 14 days to ovariectomized rats. Groups 1 to 8 received an additional administration of 1 kg estrone/day. Body weights (mean k SD.) and number of living fetuses per animal (mean) are presented. Controls included castrated (group 9) and intact (group 10) animals. Group size was n = 6 in each case.
% of No. of Living Fe$za;/
Body Weight Group No. 1 2 3 4 5 6 7 8 9 10
Is1
Dose Drug
D’WWI
NORG NORG NORG NORG LNG LNG LNG LNG controls controls
1 .o 0.3 0.1 0.03 0.3 0.1 0.03 0.01 -
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Day 1 202 205 204 203 204 203 200 209 204 206
* ?I k k + k f + k k
11 12 14 12 10 10 9 9 7 7
1994:49, March
Day 8 230 231 232 236 235 234 230 244 241 239
t k + i k + k k I! 2
16 14 16 16 11 12 10 14 9 6
Rats With Living Fetuses
[%I
Day 22 298 341 274 261 354 332 282 270 290 328
2 + ? + + k f k lr +
31 29 30 15 23 30 20 18 17 10
Degree of Pregnancy Maintenance
7.0 10.7 2.5 0 12.0 8.0 4.5 0 0 10.8
100 100 33.3 0 100 83.3 83.3 0 0 100
70 100 25 0 100 80 45 0 0 100
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following time points post administration: day 8 [prior to drug administration), day 9 at 1 h post administration, day 10 at 2 h, day 12 at 4 h, day 14 at 6 h, day 16 at 8 h and day 18 at 24 h post administration. Blood samples were centrifuged, the serum was separated and equal aliquots of each sample, obtained at corresponding times post administration, were pooled within each treatment group. Per treatment group, seven serum pools were thus obtained. The pooled samples were kept deep frozen at - 18°C until analysis. LNG was determined in the serum of the animals belonging to treatment groups l-8. Androgenic activity. The androgenic activity of LNG and NORG was assessed in a Hershberger test in immature orchidectomized rats. Male castrated juvenile rats were randomly allocated to 13 groups. Each group comprised 6 animals and each animal received a daily dose of either LNG, NORG, testosterone propionate (TP) or vehicle over a treatment period of 13 days. The daily dose of each compound was administered subcutaneously in a total volume of 0.2 ml of castor oil/benzyl benzoate (4/l). The body weight of the animals was recorded on days 1 and 13. One day after the last treatment, the animals were sacrificed and the weight of the seminal vesicles and the prostate glands were determined. A Dunnett’s t-test was used to compare the castrated control group with the other treatment groups. This test controls the type I error for comparisons of all treatments against a control. The significance level was set at (Y=0.05. The null hypothesis is that there is no difference between the treatment groups and the castrated control group with respect to the target variables (weights of seminal vesicles and prostate). The alternative hypothesis is that with a sample size of 6 animals per treatment and control group, a test power of 1-B = 0.9 and a significance level of a = 0.05, a t-test would detect a difference of A = 2.1 standard deviations. LNG was analysed in the serum of animals which were treated with LNG and NORG. Blood samples (ca. 0.2 ml) were taken from each of the animals at the following time points post administration: day 1, prior to drug administration, day 2 at 1 h post administration, day 3 at 2 h, day 5 at 4 h, day 7 at 6 h, day 9 at 8 h and day 11 at 24 h post administration. Blood samples were centrifuged, the serum was separated and equal aliquots of each sample were pooled as described above. Per treatment group, seven serum pools were thus obtained. The pooled samples were kept deep frozen at - 18°C until analysis. LNG was determined in each pool serum.
Analytical
Methods
of LNG by GC/MS. The analytical determination of LNG in serum samples obtained during the pregnancy maintenance test in the rat, was performed by a GC/MS method (LAB, Neu-Ulm, Germany). Determination
278
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and Beier
Briefly, to 1 ml of the serum sample, the internal standard (2H,norethindrone) was added and subsequently, the sample was extracted with 5 ml of cyclohexane/2-butanol (98.5 : 1.5; v/v). The organic layer was transferred into a new vial and stored refrigerated until analysis. Prior to analysis, the extract was dried at 40 - 50°C under a stream of nitrogen and the residue was redissolved in 10 - 20 pl of toluene. An aliquot of this solution was injected into a GC/MS system (Finnigan MAT 4500) operated in the positive chemical ionization mode with selected ion monitoring. The samples were analyzed in two sequences. One sequence was composed of serum samples of treatment groups 1 - 4 and 5 - 8, respectively, as well as a complete standard curve and 4 quality controls (0.1, 0.25, 2.0 and 10.0 ng/ml). For the preparation of the standard curve, a methanolic solution of LNG (100 Fg/ml) was diluted with a pool of blank serum, to yield final concentrations between 0.1 and 20 ng/ml. The lower limit of quantification (LLQ) was 0.1 ng/ml. The recovery was between 94 and 110%. Individual sequences were accepted when at least 3 out of the 4 quality controls differed by not more than + 15% from their nominal concentrations.
of LNG By a Combination of HPLC and RIA. The analytical determination of LNG in serum samples obtained during the Hershberger test in the rat, was performed by HPLC-RIA. An aliquot of 0.05 ml of either undiluted serum, or serum which had been diluted with physiological saline was spiked with about 1500 cpm of 3H-LNG and injected into an automated HPLC-system with precolumn switching. The fraction of the eluate which contained LNG was taken to dryness under a stream of nitrogen and the remaining residue was redissolved in 1 ml of BSA-buffer. An aliquot of 0.1 ml was used for the determination of the recovery and an aliquot of 0.8 ml was subjected to radioimmunological analysis. During method development, the recovery of LNG was examined at concentrations of 0.1, 1.0 and 5.0 ng/ml and was found to be between 68 and 77%. The absence of a carry-over effect between two injections was also demonstrated. Assay quality was assessed by the inclusion of two quality control sera (1 .O and 2.0 ng/ml) in each batch. The recovery was found to be 119% for both controls. Determination
HPLC-system. precolumn:
wash-fluid: purge-time: flow rate: analytical column:
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1994:49, March
Lichrosorb RP- 18 (Merck, Darmstadt, Germany], lo-25 pm, 20 x 4.6 mm methanol/water (20/80, v/v) 3 min 1.5 ml/min ODS-Hypersil (Shandon), 5 p.m, 125 x 4.6 mmm
279
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and androgenic mobile
phase:
activity:
Kuhnz
A: phosphate
and Beier
buffer (0.01 m, pH 7)/ methanol
(90/10, v/v) B: phosphate buffer (0.01 m, pH 7)/ methanol gradient:
flow rate: column temp.: fraction
collection:
( 1O/90, v/v) O-12 min 50% A, isocratic 12-15 min 50% A to 10 % A 15-18 min 10% A to 50 % A 18-21 min 50% A, isocratic 2 ml/min 30°C for both precolumns and the analytical column between 7.5 and 10.5 min after injection on the analytical column
Radioimmunoassay of LNG. The assay has been described in detail elsewhere.‘O For the determination of LNG, an antiserum (Schering AG) raised against levonorgestrel-3-( 0-carboxymethyl)-oxime-BSA and [ 15, 16“HI-levonorgestrel with a specific radioactivity of 1.8 TBq/mmol (Schering AG) were used. An aliquot of 0.8 ml of the purified (by HPLC) sample was incubated with the antiserum (dilution: 1:80, 000) and ca. 5000 cpm of tracer; incubation time was 16 h at 4°C. The LLQ was 40 pg/ml. Assay performance was assessed by the inclusion of two quality control sera in each assay (1 .O and 3.0 ng/ml). This allowed an additional control over the assay, independent from the sample purification by HPLC. The mean coefficients of inter-assay precision were between 11 and 24% and the recovery was 98%. Pharmacokinetic Evaluation. The concentrations of LNG measured in the serum samples obtained from the animals of each treatment group in both pharmacologic tests were used to calculate the area under the serum level-time curve AUC(O-24h) according to the linear trapezoidal rule. The systemic availability (fr) of norgestimate-derived LNG relative to an administered dose of LNG was calculated according to: f = I
AUC(O-24h), = AUC(O-24h),,, = LNG D LNG= DN =
280
AUCP24h), x D,,, AUC(O-24h),,,
AUC(O-24h) AUC(O-24h)
x D,
of norgestimate-derived LNG of LNG after the administration
dose of LNG administered dose of norgestimate-derived LNG which could be released at most from the dose of norgestimate
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1994:49,
March
Progestational
and androgenic
activity:
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and Beier
administered taking the different molecular weights of LNG (312) and norgestimate (369) into account.
Results Pregnancy
Maintenance
Test
The progestational potencies of NORG and LNG were compared with respect to the pregnant control animals and the results are presented in Table 1. In the intact control group, a mean of 10 fetuses were found per animal. Thus, this value was defined as 100% pregnancy maintenance and the results obtained in the treatment groups were expressed relative to this standard. The body weights were practically identical in each group at the outset of the study. On day 22, similar body weights were observed in intact controls and in treatment groups 1, 2, 5 and 6, where a high degree of pregnancy maintenance was achieved. Those animals which belonged to the castrated control group and to treatment groups 3, 4, 7 and 8, showed a reduced weight gain which was due to the smaller number of living fetuses during the treatment with lower doses of the two progestins (Table 1). A similar degree in the percentage of pregnancy maintenance was achieved at doses of NORG which were about 3- to lo-fold higher than the corresponding doses of LNG (Figure 1). Only at one point, at a dose of 0.3 mg/day, the pregnancy maintenance obseved was the same for both progestins.
Determination of LNG in Pooled Serum Samples.
The LNG concentrations which were determined in the pooled serum samples of rats belonging to treatment groups l-8 are presented in Table 2. In all groups, serum levels of LNG increased up to 2-8 h post administration and remained on a plateau up to 24 h post dose. A linear relation was found between the dose of LNG administered (groups 5-8) and the AUC(O-24h) of LNG administered. The same was true for the dose of NORG administered (groups l-4) and the AUC(O-24h) of norgestimate-derived LNG calculated. The mean bioavailability of NORG-derived LNG was 16.5 & 1.9% over the dose range of 0.03-0.3 mg NORG/day. The doses of the two progestins administered are presented together with the corresponding area under the curves of LNG in Figure 1. The difference in doses of LNG and NORG required to achieve the same pregnancy maintenance rate is equivalent to the difference in doses required to reach the same AUC values of LNG.
Hershberger
Test
The androgenic potencies of NORG, LNG and TP were compared to control groups of intact and castrated animals. The results on body weight
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1994:49,
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281
Progestational
and androgenic
120
Pregnamy
activity:
Kuhnz and Beier
maintenance [%]
i.C. 100 00 60 40 20 0 0.001
0.1
0.01
1
10
1
10
mglday
200
AUC(O-24h) of LNG [ngxh/ml]
150 100 50 0 0.001
0.01
0.1 Dose PwWl piciiq
FIGURE 1. Pregnancy maintaining potential of LNG and NORG when administered subcutaneously to ovariectomized pregnant rats together with 1 .O pg estrone over a treatment period of 14 days. Pregnancy maintenance relative to the intact (i.c.) and the castrated (cc.) control group is presented (top) together with the corresponding LNG levels in the serum of the treated animals (bottom).
gain and influence of treatment on the relative organ weights of seminal vesicles and prostate glands are presented in Table 3. The body weights were practically identical in each group at the outset of the study. On day 13, similar body weights were observed in intact controls and those animals which were treated with TP (groups 9, 10 and 11). Those animals which were under LNG treatment showed a slightly reduced weight gain as compared to the intact controls and this was even more marked for
282
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and androgenic
Kuhnz
activity:
and Beier
TABLE 2. Concentration of LNG (nglml) in pooled serum samples of rats which were treated (s.c.) with either LNG (groups 5-8) or NORG (groups l-4) at different doses. The calculated AUC (O-24h) values of LNG are also presented. Values below the lower limit of quantification (0.1 nglml) were set to zero; n.e. = not evaluable.
Group Time
&W-d
1
2
810 9/l 1012 1214 1416 1618 18124
0 1.7 2.0 2.3 2.7 3.9 3.0 73.7
AUC(O-24h) [ng x mlk’ x h]
3
4
5
6
7
8
0 0.6 0.8 0.8 1 .o 1.1 1.2
0 n.e. 0.2 0.2 0.3 0.4 0.3
0 0.3 0.1 0.1 0.1 0.1 0
0 8.2 7.1 5.1 7.1 9.1 5.9
0.5 2.9 2.9 2.5 2.6 3.3 1.8
0 1.4 0.9 0.8 0.9 0.7 0.5
0 0.3 0.2 0.4 0.3 0.3 0.1
24.5
7.6
2.4
171.9
62.1
16.0
5.6
TABLE 3. Androgenic activity of NORG, LNG and TP after daily administration to immature castrated male rats over a treatment period of 13 days. Body weights and relative organ weights (mgll00 g body weight) of seminal vesicles and prostage glands are presented (mean t S.D.) Controls included intact (group 12) and castrated (group 13) animals. Group size was n = 6 in each case.
Body Weight Group No. 1 2 3 4 5 6 7 8 9 10 11 12 13
Drug
[mgldayl
LNG LNG LNG LNG NORG NORG NORG NORG TP TP TP control control
0.1 0.3 1.0 3.0 1.0 3.0 10.0 30.0 0.01 0.03 0.1 -
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1994:49,
Seminal Vesicle
[gl
Dose Day 1 97.3 97.2 98.3 103.7 96.8 99.7 99.3 103.7 96.7 97.3 100.0 103.8 97.0
k k k 2 f k + + 2 t k -t 5
March
3.7 3.1 3.3 2.9 2.6 4.1 4.3 3.3 3.1 3.9 3.0 4.9 2.0
Day 13 160.2 171.3 174.8 174.7 161.0 159.8 151.2 143.2 176.3 174.8 182.0 181.8 167.2
k + k k -+ + k 5 t k 5 k k
10.9 7.2 7.3 8.0 11.7 11.6 8.7 12.6 12.8 6.1 8.2 10.8 13.8
]mg/lOO 16.3 44.6 115.0 173.8 16.1 22.6 35.4 62.9 12.0 26.5 103.1 34.0 8.2
2 k 2 + 2 -c k 5 k f k ij,
Prostate
sl
3.0 8.1 26.9 29.0 3.9 5.0 7.8 12.3 3.4 4.5 22.9 9.1 0.9
b-Ml00 91 33.0 67.7 114.5 158.6 25.2 33.9 54.2 79.4 23.0 53.0 128.2 79.1 12.4
k t t k t -+ t -+ -+ t -+ 2 k
4.3 9.1 6.3 18.2 8.0 9.2 10.6 13.6 6.0 9.2 15.4 13.7 2.8
283
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and androgenic
activity:
Kuhnz and Beier
those animals which were treated with NORG. A similar degree in the increase of relative organ weights was achieved at doses of NORG which were about lo- to lOO-fold higher than corresponding doses of LNG (Figure 2).
Determination of LNG in Pooled Serum Samples.
The LNG concentrations which were determined in the pooled serum samples of rats belonging to treatment groups 1-8 are presented in Table 4; each value represents the mean of duplicate or triplicate analysis. In all groups, serum levels of LNG increased up to 2-8 h post administration and remained on a plateau up to 24 h post dose. AUC(O-24h) values were calculated for each treatment group and there was a linear relation to the dose of LNG administered (groups l-4). The same was true for the dose of NORG administered (groups 5-8) and the AUC(O-24h) of NORG-derived LNG calculated. The mean bioavailability of NORG-derived LNG was 8.5 + 3.6% over a dose range of l-30 mg NORG/day. The doses of the two progestins administered are presented together with the corresponding area under the curves of LNG in Figure 2. The difference in doses of LNG and NORG required to achieve the same gain in relative organ weights is almost equivalent to the difference in doses required to reach the same AUC values of LNG.
Discussion NORG has been introduced as a novel selective progestogen with minimal androgenic activity and in that respect it was claimed to be superior to LNG. This was concluded mainly from the results of preclinical studies and further support was obtained from the clinical performance of NORGll--ls On the other hand, it has containing combination oral contraceptives. been argued by others that metabolites of NORG may be the pharmacologically active principles and NORG might only be a pro-drug.y,‘6 The chemical structure of NORG on the one hand and metabolism studies in animals and man as well as in vitro studies on the other hand made it very likely that LNG could be a major metabolite of NORG.“,” This has always been disputed by the Phillips group and it was stated by them and others that another metabolite (LNG-oxime) contributed significantly to the pharmacologic response of NORG.3,ss’4 The fact that none of the published animal studies were accompanied by drug level measurements, made it impossible to follow that conclusion and to positively assign specific pharmacological effects to the presence of either NORG or one or several of its metabolites. In the present study, we have examined the androgenic activity of NORG in comparison to LNG using the same animal model which has been used before by the Phillips group.3 However, in order to investigate
284
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Prostate welghl [mg/lwg body weight]
200
* 150
t l
i.c.
100
*
I
l l
l
50
*
* C.C.
I
-22
0 0.001
0.01
0.1
1
10
Seminal vesicle weight [mg/lOOg body weight] 200 * 150
100
50
0 0.001
0.1
0.01
1
10
mg/day AUC(CL24h)of LNG [ngxh/ml]
1500 1000 500 0 0.001
, , . ,( 0.01
,
, ,,
,L!.A
0.1
1
D-
DmWwl
*LNG
+NORG
10
FIGURE 2. Dose-dependent androgenic effects (prostate, top: seminal vesicle, middle) in juvenile castrated rats and corresponding LNG levels (bottom) in serum obtained after S.C. treatment with either LNG or NORG. Values, significantly different (~~0.05) from those obtained in castrated controls are marked by an asterisk. TP (open circles); ic. = intact controls; cc. = castrated controls.
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1994:49, March
285
Progestational
and androgenic
activity:
Kuhnz and Beier
TABLE 4. Concentration of LNG (ng/ml) in pooled serum samples of rats which were treated (s.c.) with either LNG (groups l-4) or NORG (groups 5-8) at different doses. The calculated (AUC (O-24h) values of LNG are also presented. Values below the lower limit of quantification (1 .O ng/ml) were set to zero.
Group Time
[day/W
1
2
3
110 2/l 312 514 716 918 11124
0 3.4 2.6 2.9 3.2 3.1 2.3
0 8.3 8.6 9.2 10.2 10.1 13.4
65.9
258.2
AUC(O-24h) [ng x mlk’ x h]
4
5
6
7
8
0 23.0 30.5 32.0 27.3 22.1 23.1
1.5 53.2 51.7 84.4 93.4 69.6 64.9
0 2.7 3.0 3.8 2.9 2.3 2.8
0 2.8 2.7 4.0 4.9 4.6 4.9
0 7.5 12.0 9.5 9.9 11.7 15.2
1.7 10.7 13.2 16.3 27.9 45.3 43.3
571.4
1632.6
64.0
105.4
291.4
874.0
whether LNG is indeed a major metabolite in the rat following the administration of NORG, LNG was measured in the serum of the animals during the whole treatment period.The results with respect to the relative androgenic potencies of NORG and LNG obtained in the present study are almost identical to those published earlier by Phillips and coworkers.“+ A factor of about ten in androgenic potency was observed between TP, LNG and NORG, respectively, based on the results of prostate growth and seminal vesicle weight gain. Thus, on the basis of a simple relation of dose administered and effects on the androgen-sensitive target organs measured, it would appear that NORG had an about ten-fold less androgenie potency than LNG. The concomitant drug level monitoring, however, revealed two important findings: Firstly, LNG was present in the serum of those animals which were treated with NORG. Thus, in the rat, LNG is a major metabolite after S.C. administration of NORG. Secondly, the exposure of these animals to NORG-derived LNG over the complete treatment period expressed as the area under the serum level-time curve (AUC), was about one-tenth of the exposure of those animals which were treated with LNG directly. The observed difference in androgenicity between LNG and NORG turned out to be equivalent to the difference in the exposure of the animals to LNG. In the light of these results, it seems likely that the pharmacological response observed in the NORG-treated rats can be directly related to the presence of LNG, which would support the view of NORG being basically only a pro-drug of the pharmacological active metabolite LNG. It would be important to know, whether this view is only true for the
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androgenic partial activity of NORG or whether it is also consistent with respect to the progestational activity of this drug. It has been claimed before that there was evidence of NORG possessing an inherent progestational activity. Although the possible contribution of other active metabolites was not excluded, it was thought to be unlikely that NORG could be acting mainly via LNG.” In the present study, the progestational activities of NORG and LNG were examined in a pregnancy maintenance test in rats. Again, LNG was measured in the serum of the animals during the whole treatment period. A difference by a factor of about three to ten in progestational potency was observed between LNG and NORG, based on the pregnancy maintenance potential. Only at a dose of 0.3 mg, the same degree of pregnancy maintenance was observed for both drugs. At the highest dose of 1 mg NORG, the efficacy to maintain pregnancy was even reduced. One should bear in mind, however, that during normal pregnancy, there is a balance in the estrogen/progesterone ratio which is limited to a narrow range. In the experimental model, a constant dose of 1 pg estrone was administered each day together with doses of the progestogens, covering a range of two orders of magnitude which eventually shifts this ratio extremely towards the progestogen. It has been shown before in similar experiments that this imbalance can negatively affect the outcome of pregnancy.” On the basis of a simple relation of dose administered and pharmacological effect observed, it would appear from the present results that NORG had an about three- to ten-fold less progestational potency than LNG. The concomitant analysis of LNG in the serum of the animals treated with NORG revealed an exposure of these animals to NORG-derived LNG over the complete treatment period of about one-third to one-tenth of the exposure of those animals which were treated with LNG directly. The observed difference in the progestational activity between LNG and NORG was in good agreement with the difference in the exposure of the animals to LNG. Thus, not only with respect to the androgenic potency but also with respect to the progestational efficacy, the pharmacological response observed in the NORG-treated rats can be quantitatively related to the exposure of the animals to LNG, which is consistent with the view of NORG being basically only a pro-drug of LNG. Nowadays, it is generally accepted and has been proven in numerous comparative pharmacological studies in animals that none of the synthetic progestogens equals progesterone in its entire spectrum of pharmacological action. Besides their progestogenic activity, synthetic sex hormones display additional specific partial activities, like e.g., androgenie properties, which are characteristic for sex steroids of the 19-nortestosterone series. During comparative pharmacological studies in animals examining, both qualitatively and quantitatively, the spectrum of efficacy and possible differences between drugs, it has to be taken into
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and androgenic
activity:
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and Beier
account that there are species-specific differences in intrinsic activity, pharmacokinetics and biotransformation. This is of particular importance, when different animal models are used to assess relative potencies to conclude on a possible dissociation of pharmacological effects.17,‘8 If the chemical structure of a drug suggests that it could potentially be a pro-drug and its pharmacological profile could probably be ascribed to an active metabolite, then the test conditions should take this into account. In order to exclude possible species-specific differences, all investigations with respect to various partial activities should be investigated in the same animal species with concomitant drug monitoring and pharmacokinetic evaluation. The route of administration as well as the galenic formulation of the drug should be identical in each test. Previously published animal studies with NORG have, from our point of view, not completely taken care of these prerequisites.1-3 In particular, no drug level monitoring was performed during the pivotal animal studies. Therefore, the conclusions drawn from these experiments, like NORG itself being a selective progestogen with minimal androgenic activity and not a pro-drug of LNG, seem to be questionable. In conclusion, the present study clearly revealed that following S.C. administration to rats, NORG is metabolized to a considerable extent to LNG. The pharmacological effects observed can be directly related to the exposure of the animals to NORG-derived LNG. Therefore, there is no doubt that NORG is a pro-drug of LNG; however, it cannot be excluded that either NORG itself or other metabolites of NORG may also contribute to the overall pharmacodynamic response. This, however, has yet to be demonstrated by conclusive experiments where the presence of these substances in the serum of the animals is shown and related to the pharmacological response.
References 1. 2.
3.
4.
5.
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Hahn DW, Allen GO, McGuire JL. The pharmacological profile of norgestimate, a new orally active progestin. Contraception 1977;16:541-53. Phillips A, Hahn DW, Klimek S, McGuire JL. A comparison of the potencies and activities of progestogens used in contraceptives. Contraception 1987;36:181-92. Phillips A, Demarest K, Hahn DW, Wong F, McGuire JL. Progestational and androgenic binding affinities and in vitro activities of norgestimate and other progestins. Contraception 199Oj41:399-410. Phillips A, Hahn DW, McGuire JL. Preclinical evaluation of norgestimate, a progestin with minimal androgenic activity. Am J Obstet Gynecol 1992;167:1191-6. McGuire JL, Phillips A, Hahn DW, Tolman EL, Flor S, Kafrissen ME. Pharmacologic and pharmacokinetic characteristics of norgestimate and its metabolites. Am J Obstet Gynecol 1990;163:2127-31.
Contraception
1994:49,
March
Progestational 6. 7.
8.
9.
10.
11. 12. 13. 14.
15.
16.
17.
18.
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and androgenic
activity:
Kuhnz
and Beier
Alton KB, Hetyei NS, Shaw C, Patrick JE. Biotransformation of norgestimate in women. Contraception 1984;29:19-29. Madden S, Back DJ. Metabolism of norgestimate by human gastrointestinal mucosa and liver microsomes in vitro. J Steroid Biochem Molec Biol 1991;38:497-503. Wild MJ, Rudland PS, Back DJ. Metabolism of the oral contraceptive steroids ethynylestradiol and norgestimate by normal (HUMA 7) and malignant (MCF-7 and ZR-75-1) human breast cells in culture. J Steroid Biochem Molec Biol 1991;39:535-43. Sisenwine SF, Liu AL, Kimmel HB, Ruelius HW. The conversion of dnorgestrel-3-oxime-17-acetate to d-norgestrel in female rhesus monkeys. Contraception 1977;15:25-37. Hiimpel M, Wendt H, Pommerenke G, Weiss C, Speck U. Investigations of pharmacokinetics of levonorgestrel to specific consideration of a possible first pass effect in women. Contraception 1978;17:207-20. Chapdelaine A, Desmarais JL, Derman RJ. Clinical evidence of the minimal androgenic activity of norgestimate. Int J Fertil 1989;334:347-52. Becker H. Supportive European data on a new oral contraceptive containing norgestimate. Acta Obstet Gynecol Stand Suppl 1990;152:33-9. Kafrissen ME. A norgestimate-containing oral contraceptive: review of clinical studies. Am J Obstet Gynecol 1992;167:1196-202. Anderson F. Selectivity and minimal androgenicity of norgestimate in monophasic and triphasic oral contraceptives. Acta Obstet Gynecol Stand Suppl 1992;156:15-21. London RS, Chapdelaine A, Upmalis D, Olson W, Smith J. Comparative contraceptive efficacy and mechanism of action of the norgestimatecontaining triphasic oral contraceptive. Acta Obstet Gynecol Stand Suppl 1992;156:9-14. Juchem M, Pollow K, Elger W, Hoffmann G, Mobus V. Receptor binding of norgestimate-a new orally active synthetic progestational compound. Contraception 1993;47:283-94. Neumann F, Elger W. Critical considerations of the biological basis of toxicity studies with steroid (sex) hormones. In: Plotz EJ, Hazler J, eds. Methods in Steroid Toxicology. Los Altos, California, 1972: 10-91. Beier S, Dusterberg B, El Etreby MF, Elger W, Neumann F, Nishino Y. Toxicology of hormonal fertility-regulating agents. In: Benagiano G, Diczfalusy E, eds. Endocrine Mechanisms in Fertility Regulation. New York: Raven Press, 1983;261-346.
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