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Uptake and disappearance of 1α-3H-chlormadinone acetate in various tissues of the rat
UPTAKE
AND DISAPPEARANCE ACETATE IN VARIOUS
OF la-3H-CHLORMADINONE TISSUES OF THE RAT
Vimla Laumas A.R. Krishnan K.R. Laumas
Department of Reproductive Biology All India Institute of Medical Sciences New Delhi-16
ABSTRACT
The uptake and disappearance of 1 03H-chlormadinone acetate (CAP) in various tissues of the rat have been studied after a single injection and a constant infusion. CAP is rapidly taken up by the neural and genital tissues. The disappearance curve showed an initial rapid fall followed by a slow disappearance: The slow disappearance part of the curve indicates some retention of the CAP and its metabolites in these tissues. The molar oncentration of e steroid and its metabolites in the uterus and ovary was 2.4 x 10‘ c/ M and 6.6 x lo- %h M, respectively, after a constant infusion for five hours. The uptake in the ovary was higher than that in the uterus indicating the possibility of direct action of CAP on the ovary. Compared with neural and genital tissues, mammary gland and fat showed a much higher upt e of CAP. The molar concentration of CAP in the mammary gland was 3.9 x lo- I! M at 120 minutes, and there was evidence of retention of CAP in the mammary gland. The significance of these findings in the action of CAP is discussed.
Accepted for Publication November 28, 1972
JANUARY
1973
VOL. 7 NO. 1
47
CONTRACEPTION
INTRODUCTION
In spite of the extensive work on the mode of action of oral progestins, very little is known about their distribution, uptake and metabolism in the different tissues. Previous studies (1) from this laboratory showed that a 17a -ethinyl-1 O-nor- steroid like norethynodrel is rapidly taken up in the reproductive tissues, disappears rapidly initially followed by a slow disappearance. Similar results (2) were found for the disappearance of norethynodrel in the plasma of women. However, it is not known whether the oral progestins belonging to the category which are derivatives of 17 a acetoxy progesterone show a similar pattern of distribution, uptake and disappearance in the different tissues. Such information should be of great aid in understanding the mechanism of action of progestational compounds. The present paper describes our results on the distribution and uptake of chlormadinone acetate in the various tissues of the rat after a single injection and a constant infusion of 1 a -3H-chlormadinone acetate. A preliminary report of the results has been presented (3).
MATERIALS
AND METHODS
Animals - Adult female rats (3-4 months old) were treated orally with radioinert chlormadinone acetate (200 Pg/O.2 ml in olive oil) daily for a period of 3 weeks before the experiment. 3H_Chlormadinone1 a-3H-chlormadinone was obtained from E. Merck, Darmstadt.
acetate (specific activity, 0.222 mc/mg)
Sinale iniection of 1 a-3H-chlormadinone acetate - A single intravenous injection of 20.0 pc of 1 a -jH-chlormadinone acetate in 0.5 ml of 10 percent ethanolic saline was given to chlormadinone pretreated rats. Groups of animals were killed by cervical dislocation at intervals of 2, 5, 10, 20,60 and 120 minutes after the injection. Uterus, vagina, ovary, mammary gland, fat, liver, adrenal, pituitary, hypothalamus and cerebral cortex were taken out and weighed. The whole brain was removed as rapidly as possible, and freed of adhering blood, Pituitary was removed from sella turcica and the hypothalamus was separated from the rest of the brain (4). Constant infusion - 1 o-3H-CAP (44.0 P c) in 4.1 ml of 10 percent ethanolic saline was infused at a constant rate of 0.0136 ml per minute for 5 hours into the femoral vein of the rat. Infusion was carried out with the aid of a microinfusion pump Model 600900 (Harvard Apparatus Co.). After the completion of the infusion, the animals were killed and tissues were taken out. Determination of radioactivitv in the tissues - The tissues were minced and extracted three times with acetone and twice with methanol, each time using 2.0 vol. of the solvent. The extracts were pooled and dried under nitrogen. Ten ml of scintillation liquid (4.0 gm of 2.5 diphenyloxazole and 0.1 gm of 1-4-bis-[2-(4methyl-5-phenyloxazolyl)] -benzene dissolved in 1 litre of redistilled toluene) was added to each sample and counted in a liquid scintillation counter (Model 3214, Packard Instrument Co., Downers Grove, Illinois).
443
JANUARY
1973
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7 NO.
I
CONTRACEPTION
RESULTS
&take
of CAP in the uterus. vaaina. ovarv and the mammarv qland:
The data on the uptake and disappearance of I 3H-CAP and its metabolites in the uterus, ovary, vagina and ammary gland are given in Figure 1. It may be seen that after an I.V. injection of 1 -F H-CAP, there is a rapid uptake in the uterus, ovary and vagina. The maximal uptake of the steroid is attained within a period of 5 minutes. The levels of uptake in the uterus and the vagina are almost the same, but the ovary showed an uptake which was much higher than that of the uterus and vagina. In the uterus, after the maximal uptake at 5 minutes, the levels at 10, 20,60 and 120 minutes were almost in the same range. In the case of the vagina, there was a decline in the levels of radioactivity between 5 and 20 minutes and subsequent to that the levels were almost in the same range for 120 minutes. However, in the case of the ovary, there was a rapid decline in the levels of the steroids between 5 and 60 minutes followed by a slower disappearance at 120 minutes. The levels of CAP and its metabolites in the ovary at all time intervals were higher than those in the uterus and vagina. The uptake in the mammary gland showed a continued increase at the different time intervals studied. At 120 minutes when the experiment was terminated, it had the maximal uptake. he molar concentration of CAP in the mammary gland at 120 minutes was 3.9 x lo- F M.
.
z
w
2 5 10
.
UTERUS OVARY
e
VAGINA
-
M.GLAND
20
60
120
TIME (MINUTES:
Figure 1. Uptake of 1 a-3H-chlormadinone acetate by the uterus, vagina, ovary and mammary gland at different time intervals.
JANUARY
1973
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7 NO. 1
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CONTRACEPTION
&take
of CAP in the fat. liver and adrenal:
These three tissues showed a higher level of uptake (Figure 2) compared with the reproductive tissues. There was a continued uptake of the steroid and its metabolites in the fat for 60 minutes when the maximal uptake was attained. Subsequent to that there was a slight decline in the levels of the steroid at 120 minutes. In the case of the adrenal, the highest uptake was noted at 2 minutes. There was a rapid decline at 5 minutes and also between 10 and 60 minutes. Subsequent to that the levels of the steroids were almost the same between 60 and 120 minutes. The maximal uptake of CAP was attained in the liver at 5 minutes followed by a rapid decline at 20 minutes. The levels of radioactivity between 20 and 120 minutes were almost the same.
L
2s
IO
20
:
\
LIVER
-
ADRENAL
I2c)
60 TIME
Figure 2. Uptake of 1 o -3H-chlormadinone different time intervals.
50
FAT
-
(MINUTE51
acetate by the fat, liver and adrenal at
JANIJARY
1973
VOL. 7 NO.
1
CONTRACEPTION
Uotake of CAP in the oituitarv.
cerebral cortex and hvoothalamus:
These three neural tissues followed an almost similar pattern of uptake and disappearance (figure 31, There was a maximal uptake at 2 minutes with a rapid decline at 5 minutes followed by a slow disappearance for 120 minutes. In the case of the cerebral cortex and hypothalamus, a slight rise in the levels of the steroids was noted at 10 minutes compared with levels at 5 minutes,
1.000 -
5
-
PlTUlTAQY
kz
-
c. CORTEX
F
-
HYPOTHALAMUS
t s-
500
-
z z D
100
-
TIME
(MINUTES)
Figure 3. Uptake of 1 a -3H-chlormadinone acetate by the pituitary, and hypothalamus at different time intervals.
JANUARY
1973
VOL. 7 NO. 1
cerebral cortex
51
CONTRACEPTION
nt infusion: The results presented in Figure 4 show that after a constant infusion of CAP, the ovary showed a much higher uptake compared with the uterus which was statistically sig ‘ficant (P < 0.01). The molar oncentration of CAP and its metabolites was 2.4 x 10 Y M in the uterus and 6.6 x lo- 7”M in the ovary. Liver showed the highest uptake of all the tissues studied, followed by fat an adrenal. The molar concentration of CAP and its metabolites in the fat was 1 x lo- g M. These three tissues were noted to have a higher uptake in the single injection studies also. Pituitary showed a higher uptake compared with hypothalamus and the cerebral cortex. The difference, however, is not statistically significant.
Figure 4. Uptake of 1 (X -31-l-chlormadinone acetate by the various tissues after a constant infusion.
52
JANUARY
1973
VOL.
7 NO.
1
CONTRACEPTION
DISCUSSION
An analysis of the uptake and disappearance of radioactivity due to CAP and its metabolites showed that it is rapidly taken up by the neural and genital tissues studied and subsequent to that the disappearance curve shows two rates--an initial rapid disappearance followed by a slow disappearance. The slow disappearance probably indicates some retention of CAP and/or its metabolites. In contrast to neural and genital tissues, the mammary gland showed a continued uptake of the steroid for 120 minutes when the experiments were terminated. Fat also showed a continued uptake for 60 minutes, and this was followed by a slow decline at 120 minutes. No analysis of the radioactivity in any of the tissues has been carried out and therefore, it is not possible to say whether it represents the parent steroid and/or its metabolites. In its pattern of uptake and disappearance, CAP shows similarities to progesterone and norethynodrel which also show a rapid uptake and rapid initial disappearance followed by a slow disappearance in the neural and genital tissues (2,5). The uptake of CAP and its metabolites in the ovary was higher than that in the uterus and va ina. The molar concentration of CAP and its metabolites in th ovary was 6.6 x IO- 9 M which was higher th the molar concentration of 2.4 x IO- 7 M in the Y uterus after constant infusion of 1 a - H-CAP. In fact, both single injection and constant infusion studies showed retention of a small amount of CAP and its metabolites in the ovary. A similar pattern of retention of norethynodrel and its metabolites in the ovary has been observed (2). These results are suggestive of a direct action of CAP on the ovary. A direct action of contraceptive steroids on the ovary has been debated (6). Norethynodrel and other contraceptive steroids have been reported to reduce ovarian sensitivity to gonadotropins (7,8). After the administration of CAP to women, increased urinary excretion of estrogens has been found in some cases suggesting an altered biosynthesis of estrogens in the ovary (9). Norethynodrel has been reported to disturb normal biochemical changes in the rabbit ovary (10). Whether CAP brings about any such changes in the ovary is not known. The evidence so far accumulated in the literature although indicative of a direct action of contraceptive steroids on the ovary, no conclusive evidence has yet been provided. An interesting feature of the results of this investigation was the high uptake of CAP and its metabolites in the mammary gland and the fat. In the mammary gland, a continued uptake of CAP and its metabolites was noted for 120 minutes, suggestive of incorporation and retention of the steroid in the mammary gland. CAP has been found to cause the development of nodules in the breast of beagle bitches ( 11 ,I 2). However, it is not yet known how CAP causes the formation of nodules in the breast. The biochemical mechanisms leading to the participation of the steroid in this process are not fully understood. It is possible that there is species variation in the effect of CAP on the mammary gland; as in beagle bitches, CAP is reported to be 45 times more potent than in women (13). On the other hand, prolonged treatment of monkeys with CAP for nearly two years did not produce any apparent nodules in their breasts (14). CAP showed an initial rapid disappearance with a half-life of 2.25 hours followed by a much slower disappearance rate with a long half-life of the order of 81 hours in the plasma of women (15). The prolonged half-life of CAP in the plasma explains the continued excretion of CAP and its metabolites in e milk of lactating women for 7 days or longer after the oral administration of 1 a- fsH-CAP (16). The present observation of a high uptake of CAP in the mammary gland indicated that this tissue has a specific affinity for concentrating CAP from the blood and its consequent excretion in the milk. It is possible that CAP may have some receptor sites in the mammary gland which need to be investigated.
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CONTRACEPTION
Equally significant was the observation of high uptake of CAP in the fat. Investigations on women have also shown that CAP is retained in the fat (17,18). In fact, storage of CAP in the fat and its slow release from there has been suggested to account for the prolonged half-life and continued excretion of the steroid in the urine of women and monkeys (18.19). The uptake and disappearance of CAP in the pituitary and hypothalamus followed a pattern almost similar to that observed in the case of genital tissues. It is well known that oral progestins bring about suppression of pituitary gonadotropirnegative feed-back mechanism on the hormone releasing centres in the anter’ halamus (20). No direct evidence of retention of CAP in the hypothalamus is avai , :nt that the later part of the disappearance curve of CAP in the hypothalarr 0 ic I a very slow disappearance which might be interpreted as retention of CAP in the hyp :halamus. On the other hand, estradiol is incorporated and retained in the hypothalamus. _ -R are reports in the literature which show that administration of CAP to women rc in suppression of the mid-cycle LH peak (21). It is possible that the small -: CAP retained in the hypothalamus produces this effect.
ACKNOWLEDGEMENT
This work was supported by grants from the Ford Foundation, New York, and the Population Council, New York. Gratef I thanks are due to Dr. Karl H. Kimble of E. Merck, Darmstadt for the supply of 1 a -3 H-chlormadinone acetate used in this investigation.
REFERENCES
1. Laumas, V., Malkani, P.K. and Laumas, K.R.: Distribution and upta of radioactivity in rat tissues after a single injection and constant infusion of 9 H-norethynodrel. Amer. J. Obstet. & Gynec. 109(3):457-462, 1971. 2. Laumas, K.R., Murugesan, K. and Hingorani, V.: Disappearyce in plasma and tissue uptake of radioactivity after an intravenous injection of 6,7- H-norethynodrel. Acta. Endocr. 66:385-400, 197 1. 3. Krishnan, A.R. and Laumas, K.R.: Uptake and disappearance of 1 a-3H-chlormadinone acetate in various tissues of rat. Presented at the XVI Annual meeting of the Association of Physio. Pharmac. India, Kanpur, Indian Journal of Physiol. and Pharmacol. 14(2):13, 1970. 4. Roy, S.K.,Jr. and Laumas, K.R.: 1,2-3H-testosterone. Distribution and uptake in neural and genital tissues of intact male, castrate male and female rats. Acta. Endocr. 61:629-640, 1969. 5. Laumas, K.R. and Farooq, A.: The uptake jn?_vivoof ( 1,2-3H) progesterone by the brain and genital tract of the rat. J. Endocr. 36:95-96, 1966. 6. Diczfalusy, E.: Mode of action of contraceptive steroids. Amer. J. Obstet. 61 Gynec. 100:136-163, 1968.
54
JANUARY
1973 VOL. 7 NO. 1
CONTRACEPTION
7. France, ES. and Pincus, G.: Biologically active substances affecting gonadotropin induced ovulation in immature rats. Endocrinology 75359-364, 1964. 8. Purshottam, N., Masson, M.M. and Pincus, G.: Induced ovulation in the mouse and the measurement of its inhibition. Fertil. & Steril. 12:346-352, 1961. 9. Larsson-Cohn, IJ., Johansson, E.D.B., Wide, L. and Gemzel, C.: Effect of continuous daily administration of 0.5 mg of chlormadinone acetate on the plasma levefs of progesterone and on the urinary excretion of luteinizing hormone and total oestrogens. Acta. Endocr. 63:705-716, 1970. 10. Yadava, H.S. and Launs, K.R.: Effect of prolonged administration of norethynodrel and mestranol on (UC) glucose metabolism in uterus, ovary and adrenal of rabbits. J. Reprod. Fertil. 20:1 l-19, 1969. 11. Goldenthal, E.I.: 1968, pp. 12-18.
Current views on safety evaluation of drugs. FDA papers, May
12. Goldenthal, E.I.: Contraceptives, estrogens and progestogens: A new FDA policy on animal studies. FDA papers, November 1969, p. 15. 13. Hill, R., Averkin, E., Brown, W., Gagne, W.E. and Segre, E.: Progestational potency of chlormadinone acetate in the immature beagle bitch. Preliminary report. Contraception 2(6):381-390, 7970. 14. Laumas, K.R.:
Long-term effect of CAP on monkeys.
Unpublished observations.
15. Dugwekar, Y.G., Narula, R.K. and Laumas, K.R.: Disappearance of 1 CZ-~Hchlormadinone acetate from the plasma of women. Contraception (in press). 16. Laumas, V., Malkani, P.K., Bhasin, K. and Lau us, K.R.: Excretion of radioactivity in the milk after administration of 1 a - H-chlormadinone acetate to women and goats. Presented at the XVI Annual Conference of the Assoc. Physiol. Pharmacol. India, Bangalore, December 1970. Indian J. Physiol. Pharmacol. 15(2):49, 1971. 17. Dugwekar, Y.G., Narula, R.K. and Laumas, K.R.: Distribution of 1 a -3H-chlormadinone acetate in the reproductive tract of women (in preparation). 18. Gallegos, A.J., Gonzalez-Diddi, M., Merino, G. and Martinez-Manautou, J.: Tissue localization of radioactive chlormadinone acetate and progesterone in the human. Contraception 1(3):151-161, 1970. 19. Laumas, V.: 20.
Unpublished observation.
Harris, G.W. and Naftolin, F.: The hypothalamus M. Bulletin 26:3-g, 1970.
and control of ovulation.
Brit.
21. Taymor, M.L. and Levesque, L.A.: Levels of serum follicle-stimulating hormone, luteinizing hormone and plasma progestin during microdose chlormadinone treatment. Fertil. & Steril. 22(1):1-22, 1971.
JANUARY
1973
VOL. 7 NO. 1
55
AND DISAPPEARANCE ACETATE IN VARIOUS
OF la-3H-CHLORMADINONE TISSUES OF THE RAT
Vimla Laumas A.R. Krishnan K.R. Laumas
Department of Reproductive Biology All India Institute of Medical Sciences New Delhi-16
ABSTRACT
The uptake and disappearance of 1 03H-chlormadinone acetate (CAP) in various tissues of the rat have been studied after a single injection and a constant infusion. CAP is rapidly taken up by the neural and genital tissues. The disappearance curve showed an initial rapid fall followed by a slow disappearance: The slow disappearance part of the curve indicates some retention of the CAP and its metabolites in these tissues. The molar oncentration of e steroid and its metabolites in the uterus and ovary was 2.4 x 10‘ c/ M and 6.6 x lo- %h M, respectively, after a constant infusion for five hours. The uptake in the ovary was higher than that in the uterus indicating the possibility of direct action of CAP on the ovary. Compared with neural and genital tissues, mammary gland and fat showed a much higher upt e of CAP. The molar concentration of CAP in the mammary gland was 3.9 x lo- I! M at 120 minutes, and there was evidence of retention of CAP in the mammary gland. The significance of these findings in the action of CAP is discussed.
Accepted for Publication November 28, 1972
JANUARY
1973
VOL. 7 NO. 1
47
CONTRACEPTION
INTRODUCTION
In spite of the extensive work on the mode of action of oral progestins, very little is known about their distribution, uptake and metabolism in the different tissues. Previous studies (1) from this laboratory showed that a 17a -ethinyl-1 O-nor- steroid like norethynodrel is rapidly taken up in the reproductive tissues, disappears rapidly initially followed by a slow disappearance. Similar results (2) were found for the disappearance of norethynodrel in the plasma of women. However, it is not known whether the oral progestins belonging to the category which are derivatives of 17 a acetoxy progesterone show a similar pattern of distribution, uptake and disappearance in the different tissues. Such information should be of great aid in understanding the mechanism of action of progestational compounds. The present paper describes our results on the distribution and uptake of chlormadinone acetate in the various tissues of the rat after a single injection and a constant infusion of 1 a -3H-chlormadinone acetate. A preliminary report of the results has been presented (3).
MATERIALS
AND METHODS
Animals - Adult female rats (3-4 months old) were treated orally with radioinert chlormadinone acetate (200 Pg/O.2 ml in olive oil) daily for a period of 3 weeks before the experiment. 3H_Chlormadinone1 a-3H-chlormadinone was obtained from E. Merck, Darmstadt.
acetate (specific activity, 0.222 mc/mg)
Sinale iniection of 1 a-3H-chlormadinone acetate - A single intravenous injection of 20.0 pc of 1 a -jH-chlormadinone acetate in 0.5 ml of 10 percent ethanolic saline was given to chlormadinone pretreated rats. Groups of animals were killed by cervical dislocation at intervals of 2, 5, 10, 20,60 and 120 minutes after the injection. Uterus, vagina, ovary, mammary gland, fat, liver, adrenal, pituitary, hypothalamus and cerebral cortex were taken out and weighed. The whole brain was removed as rapidly as possible, and freed of adhering blood, Pituitary was removed from sella turcica and the hypothalamus was separated from the rest of the brain (4). Constant infusion - 1 o-3H-CAP (44.0 P c) in 4.1 ml of 10 percent ethanolic saline was infused at a constant rate of 0.0136 ml per minute for 5 hours into the femoral vein of the rat. Infusion was carried out with the aid of a microinfusion pump Model 600900 (Harvard Apparatus Co.). After the completion of the infusion, the animals were killed and tissues were taken out. Determination of radioactivitv in the tissues - The tissues were minced and extracted three times with acetone and twice with methanol, each time using 2.0 vol. of the solvent. The extracts were pooled and dried under nitrogen. Ten ml of scintillation liquid (4.0 gm of 2.5 diphenyloxazole and 0.1 gm of 1-4-bis-[2-(4methyl-5-phenyloxazolyl)] -benzene dissolved in 1 litre of redistilled toluene) was added to each sample and counted in a liquid scintillation counter (Model 3214, Packard Instrument Co., Downers Grove, Illinois).
443
JANUARY
1973
VOL.
7 NO.
I
CONTRACEPTION
RESULTS
&take
of CAP in the uterus. vaaina. ovarv and the mammarv qland:
The data on the uptake and disappearance of I 3H-CAP and its metabolites in the uterus, ovary, vagina and ammary gland are given in Figure 1. It may be seen that after an I.V. injection of 1 -F H-CAP, there is a rapid uptake in the uterus, ovary and vagina. The maximal uptake of the steroid is attained within a period of 5 minutes. The levels of uptake in the uterus and the vagina are almost the same, but the ovary showed an uptake which was much higher than that of the uterus and vagina. In the uterus, after the maximal uptake at 5 minutes, the levels at 10, 20,60 and 120 minutes were almost in the same range. In the case of the vagina, there was a decline in the levels of radioactivity between 5 and 20 minutes and subsequent to that the levels were almost in the same range for 120 minutes. However, in the case of the ovary, there was a rapid decline in the levels of the steroids between 5 and 60 minutes followed by a slower disappearance at 120 minutes. The levels of CAP and its metabolites in the ovary at all time intervals were higher than those in the uterus and vagina. The uptake in the mammary gland showed a continued increase at the different time intervals studied. At 120 minutes when the experiment was terminated, it had the maximal uptake. he molar concentration of CAP in the mammary gland at 120 minutes was 3.9 x lo- F M.
.
z
w
2 5 10
.
UTERUS OVARY
e
VAGINA
-
M.GLAND
20
60
120
TIME (MINUTES:
Figure 1. Uptake of 1 a-3H-chlormadinone acetate by the uterus, vagina, ovary and mammary gland at different time intervals.
JANUARY
1973
VOL.
7 NO. 1
49
CONTRACEPTION
&take
of CAP in the fat. liver and adrenal:
These three tissues showed a higher level of uptake (Figure 2) compared with the reproductive tissues. There was a continued uptake of the steroid and its metabolites in the fat for 60 minutes when the maximal uptake was attained. Subsequent to that there was a slight decline in the levels of the steroid at 120 minutes. In the case of the adrenal, the highest uptake was noted at 2 minutes. There was a rapid decline at 5 minutes and also between 10 and 60 minutes. Subsequent to that the levels of the steroids were almost the same between 60 and 120 minutes. The maximal uptake of CAP was attained in the liver at 5 minutes followed by a rapid decline at 20 minutes. The levels of radioactivity between 20 and 120 minutes were almost the same.
L
2s
IO
20
:
\
LIVER
-
ADRENAL
I2c)
60 TIME
Figure 2. Uptake of 1 o -3H-chlormadinone different time intervals.
50
FAT
-
(MINUTE51
acetate by the fat, liver and adrenal at
JANIJARY
1973
VOL. 7 NO.
1
CONTRACEPTION
Uotake of CAP in the oituitarv.
cerebral cortex and hvoothalamus:
These three neural tissues followed an almost similar pattern of uptake and disappearance (figure 31, There was a maximal uptake at 2 minutes with a rapid decline at 5 minutes followed by a slow disappearance for 120 minutes. In the case of the cerebral cortex and hypothalamus, a slight rise in the levels of the steroids was noted at 10 minutes compared with levels at 5 minutes,
1.000 -
5
-
PlTUlTAQY
kz
-
c. CORTEX
F
-
HYPOTHALAMUS
t s-
500
-
z z D
100
-
TIME
(MINUTES)
Figure 3. Uptake of 1 a -3H-chlormadinone acetate by the pituitary, and hypothalamus at different time intervals.
JANUARY
1973
VOL. 7 NO. 1
cerebral cortex
51
CONTRACEPTION
nt infusion: The results presented in Figure 4 show that after a constant infusion of CAP, the ovary showed a much higher uptake compared with the uterus which was statistically sig ‘ficant (P < 0.01). The molar oncentration of CAP and its metabolites was 2.4 x 10 Y M in the uterus and 6.6 x lo- 7”M in the ovary. Liver showed the highest uptake of all the tissues studied, followed by fat an adrenal. The molar concentration of CAP and its metabolites in the fat was 1 x lo- g M. These three tissues were noted to have a higher uptake in the single injection studies also. Pituitary showed a higher uptake compared with hypothalamus and the cerebral cortex. The difference, however, is not statistically significant.
Figure 4. Uptake of 1 (X -31-l-chlormadinone acetate by the various tissues after a constant infusion.
52
JANUARY
1973
VOL.
7 NO.
1
CONTRACEPTION
DISCUSSION
An analysis of the uptake and disappearance of radioactivity due to CAP and its metabolites showed that it is rapidly taken up by the neural and genital tissues studied and subsequent to that the disappearance curve shows two rates--an initial rapid disappearance followed by a slow disappearance. The slow disappearance probably indicates some retention of CAP and/or its metabolites. In contrast to neural and genital tissues, the mammary gland showed a continued uptake of the steroid for 120 minutes when the experiments were terminated. Fat also showed a continued uptake for 60 minutes, and this was followed by a slow decline at 120 minutes. No analysis of the radioactivity in any of the tissues has been carried out and therefore, it is not possible to say whether it represents the parent steroid and/or its metabolites. In its pattern of uptake and disappearance, CAP shows similarities to progesterone and norethynodrel which also show a rapid uptake and rapid initial disappearance followed by a slow disappearance in the neural and genital tissues (2,5). The uptake of CAP and its metabolites in the ovary was higher than that in the uterus and va ina. The molar concentration of CAP and its metabolites in th ovary was 6.6 x IO- 9 M which was higher th the molar concentration of 2.4 x IO- 7 M in the Y uterus after constant infusion of 1 a - H-CAP. In fact, both single injection and constant infusion studies showed retention of a small amount of CAP and its metabolites in the ovary. A similar pattern of retention of norethynodrel and its metabolites in the ovary has been observed (2). These results are suggestive of a direct action of CAP on the ovary. A direct action of contraceptive steroids on the ovary has been debated (6). Norethynodrel and other contraceptive steroids have been reported to reduce ovarian sensitivity to gonadotropins (7,8). After the administration of CAP to women, increased urinary excretion of estrogens has been found in some cases suggesting an altered biosynthesis of estrogens in the ovary (9). Norethynodrel has been reported to disturb normal biochemical changes in the rabbit ovary (10). Whether CAP brings about any such changes in the ovary is not known. The evidence so far accumulated in the literature although indicative of a direct action of contraceptive steroids on the ovary, no conclusive evidence has yet been provided. An interesting feature of the results of this investigation was the high uptake of CAP and its metabolites in the mammary gland and the fat. In the mammary gland, a continued uptake of CAP and its metabolites was noted for 120 minutes, suggestive of incorporation and retention of the steroid in the mammary gland. CAP has been found to cause the development of nodules in the breast of beagle bitches ( 11 ,I 2). However, it is not yet known how CAP causes the formation of nodules in the breast. The biochemical mechanisms leading to the participation of the steroid in this process are not fully understood. It is possible that there is species variation in the effect of CAP on the mammary gland; as in beagle bitches, CAP is reported to be 45 times more potent than in women (13). On the other hand, prolonged treatment of monkeys with CAP for nearly two years did not produce any apparent nodules in their breasts (14). CAP showed an initial rapid disappearance with a half-life of 2.25 hours followed by a much slower disappearance rate with a long half-life of the order of 81 hours in the plasma of women (15). The prolonged half-life of CAP in the plasma explains the continued excretion of CAP and its metabolites in e milk of lactating women for 7 days or longer after the oral administration of 1 a- fsH-CAP (16). The present observation of a high uptake of CAP in the mammary gland indicated that this tissue has a specific affinity for concentrating CAP from the blood and its consequent excretion in the milk. It is possible that CAP may have some receptor sites in the mammary gland which need to be investigated.
JANUARY
1973
VOL. 7 NO. 1
53
CONTRACEPTION
Equally significant was the observation of high uptake of CAP in the fat. Investigations on women have also shown that CAP is retained in the fat (17,18). In fact, storage of CAP in the fat and its slow release from there has been suggested to account for the prolonged half-life and continued excretion of the steroid in the urine of women and monkeys (18.19). The uptake and disappearance of CAP in the pituitary and hypothalamus followed a pattern almost similar to that observed in the case of genital tissues. It is well known that oral progestins bring about suppression of pituitary gonadotropirnegative feed-back mechanism on the hormone releasing centres in the anter’ halamus (20). No direct evidence of retention of CAP in the hypothalamus is avai , :nt that the later part of the disappearance curve of CAP in the hypothalarr 0 ic I a very slow disappearance which might be interpreted as retention of CAP in the hyp :halamus. On the other hand, estradiol is incorporated and retained in the hypothalamus. _ -R are reports in the literature which show that administration of CAP to women rc in suppression of the mid-cycle LH peak (21). It is possible that the small -: CAP retained in the hypothalamus produces this effect.
ACKNOWLEDGEMENT
This work was supported by grants from the Ford Foundation, New York, and the Population Council, New York. Gratef I thanks are due to Dr. Karl H. Kimble of E. Merck, Darmstadt for the supply of 1 a -3 H-chlormadinone acetate used in this investigation.
REFERENCES
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