CONTRACEPTION
EFFECTS OF A NEW ORAL PROGESTAGEN ON PITUITARY-OVARIAN FUNCTION L. Viinikkalx, S. Nummi2, 0. Ylikorkala3, P. Virkkunen4, T. Ranta', U. Alapiessa' and R. Vihkol 1 3 Department of Clinical Chemistry and Department of Gynaecology and Obstetrics, University of Oulu, SF-90220 Oulu 22, Finland, 2De artment of Gynaecology and Obstetrics, University of Kuopio, Finland, fDepartment of Physiology, University of Helsinki, Finland and 50rganon Scientific Development Group, Organon, Oss, The Netherlands
ABSTRACT A new progestagen, Org 3236 (13-ethyl-11-methylene-18,19-dinor-l7B-pregn-4-en-20-yn-3-one), which is a 3-keto derivative of another new progestagen, Org 2969 (13-ethyl-ll-methylene-l8,l9-dinor-l7e-pregn-4-en-20-yn) was administered to seven healthy female volunteers in doses 0.025 mg/day (3 volunteers) and 0.050 mg/day (4 volunteers) to evaluate its biological properties. The function of the hypophysealovarian axis was tested by measuring serum follicle stimulating hormone, luteinizing hormone, estradiol and progesterone daily on days 7-23 of two consecutive cycles, of which the first served as a control. Org 3236 was given on days 1-21 during the second menstrual cycle. The results revealed that ovulation was inhibited in 3 out of 4 volunteers with the 0.050 mg daily dose. Also in the fourth volunteer the occurrence of ovulation is questionable. In the group with a daily dose of 0.025 mg two of the three volunteers had anovulatory treatment cycles. The ovulation inhibiting activity of Org 3236 is comparable to that reported for Org 2969.
Accepted
JANUARY
for publication
1978 VOL. 17 NO. 1
November
4, 1977
19
CONTRACEPTION
INTRODUCTION Org 2969 (13-ethyl~ll-methylene-l8,l9-dinor-l7~-pregn-4-en-ZO-yn, Fig. 1) is a new progestagen synthesized by Organon Int. B.V., Oss, The It has proved to be a potent compound both in in viva Netherlands. animal experiments (1) and in clinical studies with human volunteers (2,3,4). However, in -in vitro binding studies, its affinity to human and rabbit myometrial progesterone receptor is only 16 % and 18 % of that of progesterone, respectiveiy (2). Because the binding affinity of the 3-keto derivative of Org 2969, Org 3236 (13-ethyl-ll-methylene-18,19-dinor-17a-pregn-4-en-2O-yn-3-one, Fig. 1) is much higher, e.g. 150 % (human) and 280 % (rabbit) of that of progesterone (2), and because another 3_deoxyprogestagen, lynestrenol, has been suggested
ORC
ORG
2969
Fig. 1. The structures
3236
of Org 2969 and Org 3236.
to be bioactivated via the formation of a keto group at position 3 (5), it has been assumed that the high biological activity of Org 2969 is also exerted through bioactivation effected by the formation of a keto group at carbon 3 (2). The aim of the present study was to evaluate the ovulation inhibiting properties of Org 3236, in doses which are known to inhibit ovulation in most volunteers during daily administration of Org 2969. SUBJECTS
AND METHODS
Seven healthy
20
normally
menstruating
women between
21-36 years of age,
JANUARY
1978 VOL. 17 NO. 1
CONTRACEPTION
volunteered for the study. The number of previous deliveries were The lengths of menstrual periods varied beO-2, and abortions O-l. The tween 28-30 days and the duration of the bleeding was 3-7 days. study was performed during two consecutive cycles, the first cycle During the treatment cycle four volunteers inserving as a control. gested 0.050 mg and three 0.025 mg of Org 3236 daily, on cycle days 1-21. Endometrial biopsies for histopathological evaluation were taken on the 22nd day of the cycle and basal body temperature was recorded daily throughout the cycles. Blood samples were drawn between 8 and 12 a.m. on days 7-23 of the The cycles. The blood was allowed to clot and then centrifuged. separated serum was divided into aliquots, stored frozen at -20' and thawed just before the start of the analysis. The assays of serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) were carried out by radioimmunoassay with reagents obtained from the Hormone Distribution Office, NIAMDD, National Institutes of Health (NIH), Bethesda, Maryland, U.S.A.,using LER 907 as standard. Serum progesterone was measured by radioimmunoassay according to the method of JPnne et al. (6) modified by Viinikka -et al. (2). -Serum estradiol was also determined as described previously (2). Serum cortisol was measured according to Apter et&. (7) to evaluate possible effects on the adrenal function on the 8th and 23rd day of the cycles. Serum aspartate amino transferase, alanine amino transferase and alkaline phosphatase activities as well as bilirubin concentration were measured on the 8th, 15th and 23rd day of the cycle by a Technicon SMAC analyzer (Technicon Instrument Corporation, Tarrytown, New York 10581, U.S.A.) as well as serum gamma glutamyl transpeptidase activity with the MonotestR '(GT from Boehringer Mannheim GmbH, Mannheim, F.R.Gr., to evaluate possible liver disturbances sometimes seen in connection with the use of contraceptive steroids. RESULTS The results of the hormone measurements presented in Fig. 2 and 3, revealed all seven control cycles to be ovulatory according to the following criteria: a midcyclic LH peak was followed by a rise in serum progesterone concentration up to at least 9 ng/ml during the latter half of the cycle in six out of seven volunteers. In RS the LH peak is not clearly demonstrable, but the very sharp rise in progesterone concentration also revealed that this cycle was ovulatory. The FSH peak value was seen at the same time as the LH peak except in volunteers RS and MY. The biphasic estradiol curve, biphasic basal body temperature and secretory endometrium during the latter half of the cycle further confirmed that all the control cycles were ovulatory. During the treatment, the LH peak was missing or clearly decreased in all cases and the progesterone values remained under 2 ng/ml throughout
JANUARY
1978 VOL. 17 NO. 1
21
CONTRACEPTION
-------
LH FSH
PROGESTERONE ESTRAOIOL
Fig. 2. estradiol
The serum concentrations of LH, FSH, progesterone and in volunteers with 0.025 mg daily dose of Org 3236.
the cycle in all cases except OL and AP (Fig. 2 and 3). The FSH values were definitely lower during the treatment than during the control little or incycle in volunteers MA and SF, whereas in the others consistent effect on FSH secretion was seen. The concentration of estradiol was considerably higher during the treatment in subjects OL, SP and MA, and lower in AS, whereas in the remainder little effect on On the basis of the hormone determinationsl estradiol was observed. monophasic or irregular basal body temperature curves and samples wrth atypical secretory endometrium, the treatment cycles of volunteers AS, RS, MA, SP and MY were interpreted as anovulatory, whereas the treatment cycle of AP was regarded ovulatory. The data about the treatment cycle of OL speaks for a greatly impaired luteal function, but a definite decision, whether ovulation took place or not, cannot be made. One subject (AS) with the 0.025 mg dose experienced a breakthrough bleeding on days 13-16 of the treatment cycle, and one volunteer (WI with 0.050 mg dose had a bleeding which lasted fifteen days and started on the 11th day of the treatment cycle. III five of the volunteers the length of the cycle shortened by 1-8 days. No subjective side effects
22
JANUARY
1978 VOL. 17 NO. 1
CONTRACEPTION
-------
I_” FSN I’ROGESTERONE ESTRAOIOL
Fig. 3. The serum concentrations of LH, FSH. progesterone and estradiol in volunteers with 0.050 mg daily dose of Org 3236.
found in association volunteers.
with
oral contraceptives
were
reported
by the
The activities of alkaline phosphatase, alanine amino transferase, aspartate amino transferase and gamma glutamyl transpeptidase as well as bilirubin and cortisol concentrations were unchanged and remained within the normal range both during the control and treatment cycles in all volunteers.
DISCUSSION The purpose of this study was to evaluate the ovulation inhibiting The results revealed that a daily dose 0.050 mg activity of Org 3236. inhibited ovulation in 3 out of 4 volunteers as did 0.025 mg in 2 out When these results are compared to those of the of 3 volunteers.
JANUARY
1978 VOL. 17 NO. 1
23
CONTRACEPTION
ovulation inhibiting activity of the corresponding 3-deoxyprogestagen, Org 2969, according to which ovulation has been inhibited in all volunteers with 0.125 mg or 0.060 mg daily doses (2,3), in four out of five volunteers with 0.030 mg (3,4) and four out of six volunteers with 0.015 mg (4) daily dose, it can be seen that the minimum ovulation inhibiting dose of both Org 2969 and Org 3236 is very similar. A considerable rise in estradiol concentration in the latter half of the cycle was seen in three volunteers ingesting 0.050 mg of Org 3236/ day (MA, SP and OL). Similar findings were observed in some of the subjects ingesting Org 2969 daily (2,3,4). Although the material is small, certain relationships between the rise in estradiol concentration and dose of Org 2969 and Org 3236 can be seen. Similar increases in serum estradiol have also been observed associated with the use of norethindrone (8) and chlormadinone acetate (9), but at doses of about The reason for this phenomenon still remains to be ten times higher. investigated. In conclusion, the results of the present investigation revealed that the ovulation inhibiting potency of Org 3236 is comparable to that of Org 2969.
Acknowledgements We are grateful minations.
to NIAMDD
for providing
reagents
for LH and FSH deter-
Grants: Ford Foundation (R.V.), the Finnish Medical Society and the Medical Research Foundation of Oulu (L.V.).
Duodecim
REFERENCES 1.
2.
3.
4.
5.
24
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) Viinikka, L., Ylikorkala, O., Nummi, S., Virkkunen, P., Ranta, T., Alapiessa, LJ. and Vihko, R. Biological effects of a new and potent progestagen. A clinical study. Acta endocr. (Kbh) 83: 429-438 (1976) The influence on the pituitary-ovarian function, Skouby, S.O. cervical mucus and vaginal cytology of a new progestational compound. Contraception 14: 529-539 (1976) Viinikka, L., Hirvonen, E., Ylikorkala, O., Nummi, S., Virkkunen, P ., Ranta, T., Alapiessa, U. and Vihko, R. Ovulation inhibition by a new low-dose progestagen. Contraception 16: 51-58 (1977) Kontula, K., Jlnne, O., Vihko, R., de Jager, E., de Visser, J. and Zeelen, F. Progesterone binding proteins: in vitro binding and biological activity of different steroidal zgands. Acta endocr. (Kbh) 78: 574-592 (1975)
JANUARY
1378 VOL. 17 NO. 1
CONTRACEPTION
6.
7.
8.
9.
Jlnne, O., Apter, D. and Vihko, R. Assay of testosterone, progesterone and l:a-hydroxyprogesterone in human plasma by radioirmnunoassayafter separation on hydroxyalkoxypropyl Sephadex. J. Steroid Biochem. 5: 155-162 (1974) Apter, D., Jlnne, 0. and Vihko, R. Lipidex chromatography in the radioimmunoassay of serum and urinary control. Clin. Chim. Acta 63: 139-148 (1975) Larsson-Cohn, U., 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 secretion of luteinizing hormone, pregnanediol and total oestrogens. Acta endocr. (Kbh) 63: 216-224 (1970) 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 secretion of luteinizing hormone and total oestrogens. Acta endocr. (Kbh) 73: 705-716 (1970)
JANUARY
1978 VOL. 17 NO. 1
25
EFFECTS OF A NEW ORAL PROGESTAGEN ON PITUITARY-OVARIAN FUNCTION L. Viinikkalx, S. Nummi2, 0. Ylikorkala3, P. Virkkunen4, T. Ranta', U. Alapiessa' and R. Vihkol 1 3 Department of Clinical Chemistry and Department of Gynaecology and Obstetrics, University of Oulu, SF-90220 Oulu 22, Finland, 2De artment of Gynaecology and Obstetrics, University of Kuopio, Finland, fDepartment of Physiology, University of Helsinki, Finland and 50rganon Scientific Development Group, Organon, Oss, The Netherlands
ABSTRACT A new progestagen, Org 3236 (13-ethyl-11-methylene-18,19-dinor-l7B-pregn-4-en-20-yn-3-one), which is a 3-keto derivative of another new progestagen, Org 2969 (13-ethyl-ll-methylene-l8,l9-dinor-l7e-pregn-4-en-20-yn) was administered to seven healthy female volunteers in doses 0.025 mg/day (3 volunteers) and 0.050 mg/day (4 volunteers) to evaluate its biological properties. The function of the hypophysealovarian axis was tested by measuring serum follicle stimulating hormone, luteinizing hormone, estradiol and progesterone daily on days 7-23 of two consecutive cycles, of which the first served as a control. Org 3236 was given on days 1-21 during the second menstrual cycle. The results revealed that ovulation was inhibited in 3 out of 4 volunteers with the 0.050 mg daily dose. Also in the fourth volunteer the occurrence of ovulation is questionable. In the group with a daily dose of 0.025 mg two of the three volunteers had anovulatory treatment cycles. The ovulation inhibiting activity of Org 3236 is comparable to that reported for Org 2969.
Accepted
JANUARY
for publication
1978 VOL. 17 NO. 1
November
4, 1977
19
CONTRACEPTION
INTRODUCTION Org 2969 (13-ethyl~ll-methylene-l8,l9-dinor-l7~-pregn-4-en-ZO-yn, Fig. 1) is a new progestagen synthesized by Organon Int. B.V., Oss, The It has proved to be a potent compound both in in viva Netherlands. animal experiments (1) and in clinical studies with human volunteers (2,3,4). However, in -in vitro binding studies, its affinity to human and rabbit myometrial progesterone receptor is only 16 % and 18 % of that of progesterone, respectiveiy (2). Because the binding affinity of the 3-keto derivative of Org 2969, Org 3236 (13-ethyl-ll-methylene-18,19-dinor-17a-pregn-4-en-2O-yn-3-one, Fig. 1) is much higher, e.g. 150 % (human) and 280 % (rabbit) of that of progesterone (2), and because another 3_deoxyprogestagen, lynestrenol, has been suggested
ORC
ORG
2969
Fig. 1. The structures
3236
of Org 2969 and Org 3236.
to be bioactivated via the formation of a keto group at position 3 (5), it has been assumed that the high biological activity of Org 2969 is also exerted through bioactivation effected by the formation of a keto group at carbon 3 (2). The aim of the present study was to evaluate the ovulation inhibiting properties of Org 3236, in doses which are known to inhibit ovulation in most volunteers during daily administration of Org 2969. SUBJECTS
AND METHODS
Seven healthy
20
normally
menstruating
women between
21-36 years of age,
JANUARY
1978 VOL. 17 NO. 1
CONTRACEPTION
volunteered for the study. The number of previous deliveries were The lengths of menstrual periods varied beO-2, and abortions O-l. The tween 28-30 days and the duration of the bleeding was 3-7 days. study was performed during two consecutive cycles, the first cycle During the treatment cycle four volunteers inserving as a control. gested 0.050 mg and three 0.025 mg of Org 3236 daily, on cycle days 1-21. Endometrial biopsies for histopathological evaluation were taken on the 22nd day of the cycle and basal body temperature was recorded daily throughout the cycles. Blood samples were drawn between 8 and 12 a.m. on days 7-23 of the The cycles. The blood was allowed to clot and then centrifuged. separated serum was divided into aliquots, stored frozen at -20' and thawed just before the start of the analysis. The assays of serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) were carried out by radioimmunoassay with reagents obtained from the Hormone Distribution Office, NIAMDD, National Institutes of Health (NIH), Bethesda, Maryland, U.S.A.,using LER 907 as standard. Serum progesterone was measured by radioimmunoassay according to the method of JPnne et al. (6) modified by Viinikka -et al. (2). -Serum estradiol was also determined as described previously (2). Serum cortisol was measured according to Apter et&. (7) to evaluate possible effects on the adrenal function on the 8th and 23rd day of the cycles. Serum aspartate amino transferase, alanine amino transferase and alkaline phosphatase activities as well as bilirubin concentration were measured on the 8th, 15th and 23rd day of the cycle by a Technicon SMAC analyzer (Technicon Instrument Corporation, Tarrytown, New York 10581, U.S.A.) as well as serum gamma glutamyl transpeptidase activity with the MonotestR '(GT from Boehringer Mannheim GmbH, Mannheim, F.R.Gr., to evaluate possible liver disturbances sometimes seen in connection with the use of contraceptive steroids. RESULTS The results of the hormone measurements presented in Fig. 2 and 3, revealed all seven control cycles to be ovulatory according to the following criteria: a midcyclic LH peak was followed by a rise in serum progesterone concentration up to at least 9 ng/ml during the latter half of the cycle in six out of seven volunteers. In RS the LH peak is not clearly demonstrable, but the very sharp rise in progesterone concentration also revealed that this cycle was ovulatory. The FSH peak value was seen at the same time as the LH peak except in volunteers RS and MY. The biphasic estradiol curve, biphasic basal body temperature and secretory endometrium during the latter half of the cycle further confirmed that all the control cycles were ovulatory. During the treatment, the LH peak was missing or clearly decreased in all cases and the progesterone values remained under 2 ng/ml throughout
JANUARY
1978 VOL. 17 NO. 1
21
CONTRACEPTION
-------
LH FSH
PROGESTERONE ESTRAOIOL
Fig. 2. estradiol
The serum concentrations of LH, FSH, progesterone and in volunteers with 0.025 mg daily dose of Org 3236.
the cycle in all cases except OL and AP (Fig. 2 and 3). The FSH values were definitely lower during the treatment than during the control little or incycle in volunteers MA and SF, whereas in the others consistent effect on FSH secretion was seen. The concentration of estradiol was considerably higher during the treatment in subjects OL, SP and MA, and lower in AS, whereas in the remainder little effect on On the basis of the hormone determinationsl estradiol was observed. monophasic or irregular basal body temperature curves and samples wrth atypical secretory endometrium, the treatment cycles of volunteers AS, RS, MA, SP and MY were interpreted as anovulatory, whereas the treatment cycle of AP was regarded ovulatory. The data about the treatment cycle of OL speaks for a greatly impaired luteal function, but a definite decision, whether ovulation took place or not, cannot be made. One subject (AS) with the 0.025 mg dose experienced a breakthrough bleeding on days 13-16 of the treatment cycle, and one volunteer (WI with 0.050 mg dose had a bleeding which lasted fifteen days and started on the 11th day of the treatment cycle. III five of the volunteers the length of the cycle shortened by 1-8 days. No subjective side effects
22
JANUARY
1978 VOL. 17 NO. 1
CONTRACEPTION
-------
I_” FSN I’ROGESTERONE ESTRAOIOL
Fig. 3. The serum concentrations of LH, FSH. progesterone and estradiol in volunteers with 0.050 mg daily dose of Org 3236.
found in association volunteers.
with
oral contraceptives
were
reported
by the
The activities of alkaline phosphatase, alanine amino transferase, aspartate amino transferase and gamma glutamyl transpeptidase as well as bilirubin and cortisol concentrations were unchanged and remained within the normal range both during the control and treatment cycles in all volunteers.
DISCUSSION The purpose of this study was to evaluate the ovulation inhibiting The results revealed that a daily dose 0.050 mg activity of Org 3236. inhibited ovulation in 3 out of 4 volunteers as did 0.025 mg in 2 out When these results are compared to those of the of 3 volunteers.
JANUARY
1978 VOL. 17 NO. 1
23
CONTRACEPTION
ovulation inhibiting activity of the corresponding 3-deoxyprogestagen, Org 2969, according to which ovulation has been inhibited in all volunteers with 0.125 mg or 0.060 mg daily doses (2,3), in four out of five volunteers with 0.030 mg (3,4) and four out of six volunteers with 0.015 mg (4) daily dose, it can be seen that the minimum ovulation inhibiting dose of both Org 2969 and Org 3236 is very similar. A considerable rise in estradiol concentration in the latter half of the cycle was seen in three volunteers ingesting 0.050 mg of Org 3236/ day (MA, SP and OL). Similar findings were observed in some of the subjects ingesting Org 2969 daily (2,3,4). Although the material is small, certain relationships between the rise in estradiol concentration and dose of Org 2969 and Org 3236 can be seen. Similar increases in serum estradiol have also been observed associated with the use of norethindrone (8) and chlormadinone acetate (9), but at doses of about The reason for this phenomenon still remains to be ten times higher. investigated. In conclusion, the results of the present investigation revealed that the ovulation inhibiting potency of Org 3236 is comparable to that of Org 2969.
Acknowledgements We are grateful minations.
to NIAMDD
for providing
reagents
for LH and FSH deter-
Grants: Ford Foundation (R.V.), the Finnish Medical Society and the Medical Research Foundation of Oulu (L.V.).
Duodecim
REFERENCES 1.
2.
3.
4.
5.
24
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) Viinikka, L., Ylikorkala, O., Nummi, S., Virkkunen, P., Ranta, T., Alapiessa, LJ. and Vihko, R. Biological effects of a new and potent progestagen. A clinical study. Acta endocr. (Kbh) 83: 429-438 (1976) The influence on the pituitary-ovarian function, Skouby, S.O. cervical mucus and vaginal cytology of a new progestational compound. Contraception 14: 529-539 (1976) Viinikka, L., Hirvonen, E., Ylikorkala, O., Nummi, S., Virkkunen, P ., Ranta, T., Alapiessa, U. and Vihko, R. Ovulation inhibition by a new low-dose progestagen. Contraception 16: 51-58 (1977) Kontula, K., Jlnne, O., Vihko, R., de Jager, E., de Visser, J. and Zeelen, F. Progesterone binding proteins: in vitro binding and biological activity of different steroidal zgands. Acta endocr. (Kbh) 78: 574-592 (1975)
JANUARY
1378 VOL. 17 NO. 1
CONTRACEPTION
6.
7.
8.
9.
Jlnne, O., Apter, D. and Vihko, R. Assay of testosterone, progesterone and l:a-hydroxyprogesterone in human plasma by radioirmnunoassayafter separation on hydroxyalkoxypropyl Sephadex. J. Steroid Biochem. 5: 155-162 (1974) Apter, D., Jlnne, 0. and Vihko, R. Lipidex chromatography in the radioimmunoassay of serum and urinary control. Clin. Chim. Acta 63: 139-148 (1975) Larsson-Cohn, U., 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 secretion of luteinizing hormone, pregnanediol and total oestrogens. Acta endocr. (Kbh) 63: 216-224 (1970) 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 secretion of luteinizing hormone and total oestrogens. Acta endocr. (Kbh) 73: 705-716 (1970)
JANUARY
1978 VOL. 17 NO. 1
25
