Ovulation inhibition by a new low-dose progestagen

CONTRACEPTION

OVULATION

INHIBITION

BY A NEW LOW-DOSE

PROGESTAGEN

lx , E. Hirvonen2, 0. Ylikorkala3, S. Nummi', L. Viinikka P. Virkkunen5, T. Ranta5, C. hlapiessa6 and R. Vihkol 1 3 Department of Clinical Chemistry and Department of Gynaecology and Obstetrics, University of Oulu, SF-90220 Oulu 22, Finland, 2Department of Gynaecology and Obstetrics, and 5Depaitment of Physiology, University of Helsinki, Department of Gynaecology and Obstetrics, UniverFinland, sity of Kuopio, Finland and hOrganon Scientific Development Group, Organon, Oss, The Netherlands

ABSTRACT Org 2969 (13-ethyl-ll-methylene-18,19 A new synthetic progestagen, -dinor-17r-pregn-4-en-ZO-yn-l7-o1) was administered to nine healthy normally menstruating women. Three subjects ingested 0.030 mg, while six others ingested 0.015 mg of the compound daily on days l-20 of one menstrual cycle. Serum follicle stimulating hormone, luteinizing hormone, progesterone and estradiol analyzed on the days 8-23 showed that all the treatment cycles were anovulatory in the group ingesting 0.030 Two out of the six volunteers using the 0.015 mg mg of Org 2969. daily dose had ovulatory treatment cycles. Serum activities of aspartate amino transferase, alanine amino transferase, alkaline phosphatase, gamma glutamyl transpeptidase and bilirubin concentration determined on days 8, 15 and 23 did not reveal any change in liver function. Serum cortisol measured on days 8 and 23 remained unchanged.

Accepted

for

publication

JULY 1977 VOL. 16 NO. 1

April

18,

1977

51

CONTRACEPTION

INTRODUCTION

Fig.

1.

The structure

of Or-g 2969.

Org 2969 (13-ethyl-ll~ethylene-18,19-dinor-17~-pregn-4-en-2O-yn -17-01) is a new progestagen manufactured by OrganonInt.B.V., Oss, The Netherlands (Fig. 1). In animal experiments it has proved to be a very potent progestational agent (1). In ovariectomized women, its progestational effects on endometrium and cervical mucus were about 15-fold that of lynestrenol (unpublished results). In fertile women a 0.060 mg daily dose of Org 2969 inhibited ovulation in all volunteers, while 0.030 mg/day of Org 2969 inhibited ovulation in one of two volunteers without any harmful effects (2,3). These results encouraged further studies on the possible use of this compound as a contraceptive agent. In this investigation, further data were collected on the biological effects of still lower doses of Org 2969.

MATERIALS

AND METHODS

Nine healthy women between 26-33 years of age volunteered for this study. The number of previous deliveries ranged from l-3, and abortions O-l. The lengths of menstrual periods varied between 25 and 30 days and the duration of bleeding was 4-6 days. No hormonal treatment was used for at least five months before the start of this trial. The study was performed during two consecutive cycles, the first cycle serving as a control. During the treatment cycle 0.030 mg (3 volunteers) and 0.015 mg (6 volunteers) of Org 2969 were administered orally on cycle days l-20. Endometrial biopsies were taken on the 21st or 22nd day of the menstrual cycle for conventional histopathological examination. The basal body temperature was recorded daily during the control and treatment cycles.

52

JULY 1977 VOL. 16 NO. 1

CONTRACEPTION

Daily

blood

samples

were

After having 8 and 12 a.m. and the separated serum was different thawed

analyses just before

Serum

follicle

(LH) determinations

and the

frozen. start

clotted,

drawn

on days 8-23 of the cycle between the blood samples were centrifuged

divided All

into appropriate aliquots for samples were stored at -20°C and

of

stimulating were carried

the

analyses.

hormone out by

(FSH) and luteinizing hormone radioimmunoassay according tc

the instructions Institutrs of

of the Hormone Distribution Office, NIAMDD, National Health (NIH), Bethesda, Maryland, U.S.A. using LER 907 The sensitivity of the method was 0.2 mILI/ml for FSH and as standard. The intra-assay coefficient of variation for the 0.4 mILJ/ml for LH. Serum progesterone was methods was 3.2 % and interassay 11.0 1. measured on days 8-23 by the method of JZinne s &. (4) modified by Viinikka et al. (2). Estradiol was measured on days 8-23 as described -previously by Viinikka et al. (2) and serum cortisol on days 8 and 23 -by

the

method

of

Apter

The serum aspartate and alkaline phosphatase were measured on cycle (Technican and serum Boehringer

et --

al.

(5).

amino transferase, alanine amino transferase activities and serum bilirubin concentration days 8, 15 and 23 by Technicon SMAC analyzer

Instrument Corporation, Tarrytown, New York 10581, U.S.A.) gamma glutamyl transpeptidase with a kit MonotestR-yGT fron! Mannheim GmBH, Mannheim, Federal Republic of Germany.

RESULTS The results of the The control cycles were criteria: the peak value rise of the progesterone

hormone analyses are considered ovulatory of LH at least concentration

presented according

60 mIU/ml was up to at least

in to

Fig. 2 and following

followed 10 ng/ml.

by

3.

the The

FSH-peak appeared at the same time as the LH-peak in all instances, except for the volunteers LE, RC and SL (see Fig. 3), and the highest estradiol concentration was usually measured the day before. The periphernl changes recorded (biphasic basal body temperature, secretory endometrium) also indicated that ovulatory control cycles occurred in all cases. During the treatment the LH-peak was all cases and the rise of the progesterone in two volunteers from the group ingesting (LE and SL). In all others the progesterone 2 ng/ml throughout the treatment cycle.

missing or highly reduced in concentration was seen only 0.015 mg of Org 2969 daily concentration

was

under

A marked rise in estradiol concentration during treatment appeared in three volunteers (SK, AM and MA, Fig. 2 and 3) while a decrease occurred in two others (RT and RC, Fig. 3). In the remainder little effect on estradiol concentration was seen. On the temperature of volunteers anovulatory.

basis of the hormone determinations, and secretory endometrial biopsies, LE and SL were considered ovulatory,

JULY 1977 VOL. 16 NO. 1

biphasic basal body the treatment cycles and all the others

53

CONTRACEPTION

of luteinizing hormone, follicle stimuFig. 2. Serum concentrations lating hormone, progesterone and estradiol during control and treatment cycles in the patients ingesting 0.030 mg of Org 2969/day. Table

I

aspartate amino The mean values + S.D. of serum bilirubin concentration, gamma transferase, alanine amino transferase, alkaline phosphatase, glutamyl transpeptidase activities and cortisol concentration during None of the changes are statistically the control and treatment cycles. significant. Control

5.9 t 1.8

SERUM BILIRUBIN (nmol/ml) SERUM ASPARTATE (mILJ/ml) SERUM ALANINE (mIlJ/ml) SERUM ALKALINE (mIU/ml) SERUM GAMMA (mIU/ml)

54

AMINO

AMINO

TRANSFERASE

TRANSFERASE

PHOSPHATASE

GLUTAI+mL TRANSPEPTIDASE

SERUM CORTISOL (ng/ml)

Cycle

Treatment

Cycle

5.7 _ + 2.1

26.3 + 9.4

24.3 _ + 10.3

14.5 + 7.9

13.9 t 6.4

130.7 +_ 36.1

129.9 & 32.3

18.1 +_ 5.3

18.3 + 4.8

107.8 +_ 36.1

97.3 + 41.5

JULY 1977 VOL. 16 NO. I

CONTRACEPTION

. . . . . . .

ESTRADIOL

of luteinizing hormone, follicle stimuFig. 3. Serum concentrations lating hormone, progesterone and estradiol during control and treatment cycles in the patients ingesting 0.015 mg of Org 2969/day.

JULY 1977 VOL. 16 NO. 1

CONTRACEPTION

One volunteer with a 0.030 mg (RK) and one with a 0.015 mg daily dose (RC) experienced spotting on 11-20 and 17-20 days of the treatment cycle, respectively. 1n addition, two volunteers in the 0.015 mg dose group had a breakthrough bleeding during the treatment (AM, on days 19-21 and RT, on days 16-19). No other bleeding irregularities were seen during this trial. The data of the effects of Org 2969 on liver function cortisol are presented in Table I. No significant changes during the treatment.

and serum appeared

DISCUSSION The results of this paper further confirm the very high ovulation inhibiting activity of Org 2969, because all volunteers with a 0.030 mg daily dose and four out of six volunteers with a 0.015 mg daily dose had anovulatory treatment cycles. When the previous data available about this compound (2,3) and the present results are surmnarized,it can be seen that ovulation has been inhibited in all volunteers using a 0.125 and 0.060 mg daily dose, in four out of five volunteers using a 0.030 mg dose and in four of six volunteers using a 0.015 mg daily According to the data available about ovulation inhibiting propdose. erties of other progestagens, 0.500 mg of norethindrone inhibits ovulation in 70 % (6) and 0.300 mg in 77 % (7) of the cycles. Chlormadinone acetate, 0.500 mg, inhibited ovulation in O-69 % (7,8), and the same amount of lynestrenol in 85-91 % (9,lO) of the treatment With norgestrel, inhibition of ovulation was seen in 48-66 % cycles. of the cycles with a 0.050-0.075 mg dose (11,12,13). Although inaccuracies due to methodological differences in interpreting the percentage of inhibited ovulation may exist, there is no doubt that Org 2969 is one of the most potent progestagens available. A rise in estradiol concentration was observed in volunteers AM, Similar findings have been reported previously for this SK and MA. steroid (2,3), norethindrone (6) and chlormadinone acetate (8). As a possible mechanism for this phenomenon it has been suggested (i) that the drug promotes the release or production of FSH from the hypophysis, (ii) that there is an increased ovarian sensitivity for FSH,or (iii) that some alterations in the activities of ovarian steroid producing In this series an increased FSH production enzymes take place (6). RT (Fig. 3), but this did not lead to increased is evident in patient Explanation (ii) might be true in volunteers SK, serum estradiol. MA and AM (Fig. 2 and 3), but the situation is reversed in RT and RC If any enzyme activation in the ovary takes place during (Fig. 3). Org 2969 ingestion, the effect in various subjects is also far from The increased estrogen secretion often seen during treatment uniform. with low-dosage progestagens was not seen in the subjects ovulating This suggests during drug ingestion in this or a previous (2) study. that during drug treatment ovarian follicle development takes place to a variable degree leading to a considerable variation in its steroid synthetizing capacity.

56

JULY 1977 VOL. 16 NO. 1

CONTRACEPTION

Four out of the nine subjects studied had bleeding irregularities. Therefore further clinical studies with other doses of Org 2969 alone, or combined with estrogen, should be performed in order to assess its applicability to contraception.

ACKNOWLEDGMENTS We are grateful determinations.

to NIAMDD

for providing

reagents

for LH and FSH

Ford Foundation (R.V.), the Finnish Medical Society Grants: "Duodecim" and the Medical Research Foundation of Oulu (L.V.).

REFERENCES 1.

de Visser, J., de Jager, E., de Jongh, H.P., van der Vies, J. and Zeelen, F. Pharmacological profile of a new orally active progestational steroid: Org 2969. Acta endocr. (Kbh) Suppl. 199: 405 (1975)

2.

Viinikka, L., Ylikorkala, O., Nummi, S., Virkkunen, P., Ranta, T., Alapiessa, U. and Vihko, R. Biological effects of a new and A clinical study. potent progestagen. Acta endocr. (Kbh) 83: 429-438 (1976)

3.

Skouby, S.O. The influence on the pituitary-ovarian function, cervical mucus and vaginal cytology of a new progestational compound. Contraception 14:529-539 (1976)

4.

Janne, O., Apter, D. and Vihko, R. Assay of testosterone, progesterone and 17cx-hydroxyprogesterone in human plasma by radioimmunoassay after separation on hydroxyalkoxypropyl Sephadex. J. Steroid Biochem. 5:155-162 (1974)

5.

Apter, D., JBnne, 0. and Vihko, R. Lipidex chromatography in the radioimmunoassay of serum and urinary cortisol. Clin. Chim. Acta 63:139-148 (1975)

6.

Larsson-Cohn, LJ., Johansson, E.D.B., Wide, L. and Gemzell, C. Effects of continuous daily administration of 0.5 mg of norethindrone on the plasma levels of progesterone and on the urinary excretion of luteinizing hormone, pregnanediol and total oestrogens. Acta endocr. (Kbh) 63:216-224 (1970)

7.

Larsson-Cohn, U. Contraceptive treatment with gestagens. Acta endocr. (Kbh) Suppl. 144:1-46

JULY

1977

VOL. 16 NO. 1

low doses of (1970)

57

CONTRACEPTION

8.

Larsson-Cohn, U., Johansson, E.D.B., Wide, L. and Gemzell, C. Effects of continuous daily administration of 0.5 mg of chlormadinone acetate on the plasma levels of progesterone and on the urinary excretion of luteinizing hormone and total oestrogens. Acta endocr. (Kbh) 63:705-716 (1970)

9.

JBrvinen, P.A., Ylikorkala, 0. and YlBstalo, P. Low dose lynestrenol as a contraceptive. Influence on ovarian function. Ann. clin. Res. 6:109-114 (1974)

10.

Wiese, J. Ovarian function 0.5 mg daily. Acta endocr.

11.

Fotherby, K., Svendsen, E.K. and Foss, G.L. Ovarian function in women receiving low doses of a synthetic progestin, norgestrel. J. Reprod. Fertil. Suppl. 5:155-166 (1968)

12.

Eckstein, P., Whitby, M., Fotherby, K., Butler, C., Mukherjee, T.K., Burnett, J.B.C., Richards, D.J. and Whitehead, T.P. Clinical and laboratory findings in a trial of norgestrel, a lowdose progestogen-only contraceptive. Brit. med. J. '3:195-200 (1972)

13.

Mukherjee, T.K., Wright, S.W., Davidson, N.J.H. Effect of norgestrel on corpus luteum function. Brit. Cwlth 79:175-182 (1972)

58

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with lynestrenol (1975)

and Fotherby, K. J. Obstet. Gynaec.

JULY

1977

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