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ANTI-OVULATORY AND OTHER BIOLOGICAL PROPERTIES OF 17a-ACETOXY-6 METHYL PREGNA 4:6-DIENE-3:20-DIONE (B.D.H. 1298) A. DAVID, . EDWARDS, . P. FELLOWES and J. M. PLUMMER Biological Department, The British Drug Houses Limited, Godalming, Surrey {Received 5tk October 1962)

Summary. 17cc-Acetoxy-6 methyl pregna 4:6-diene-3:20-dione (B.D.H. 1298), has been given the approved name of . It appears to be the most potent anti-ovulatory compound so far reported. It also possesses high oral progestational properties, being more active in the Clauberg assay than medroxy acetate, commercial norethynodrel and given orally, and progesterone subcutaneously. It is non-toxic in mice ; prolonged admini¬ stration to rats caused no adverse effects except at high doses in females where some adrenal atrophy was observed. It has no anabolic, androgenic or oestrogenic properties and no virilizing effect on the developing foetus. INTRODUCTION Following the discovery that dimethisterone possessed well-marked oral progestational properties (David, Hartley, Millson & Petrow, 1957; David, Fellowes & Millson, 1959), a number of compounds synthesized in our Chemical Research Department were found to possess both high progest¬ ational and good anti-ovulatory properties. This paper reports some biological properties of one of these compounds 17oc-acetoxy-6 methyl pregna 4:6- diene-3:20-dione (B.D.H. 1298). B.D.H. 1298 is a tasteless, odourless, white crystalline compound with a melting point of about 215° C. It is insoluble in hot or cold water, soluble in , 1 in 55 , 1 in 11 acetone, 1 in 4-5 benzene and 1 in 3 benzyl alcohol. It has the following structural formula with a molecular weight of 384-5. COCHr

-- O.CO.CH,

CH^ 331

Downloaded from Bioscientifica.com at 09/25/2021 08:57:47PM via free access 332 A. David et al. METHODS

ACUTE Ten male and ten female mice, fasted overnight, were given, by tube, 5 g/kg of B.D.H. 1298 in an aqueous suspending medium and were kept under observation for 7 days.

CHRONIC ORAL TOXICITY Immature male and female rats, 60 to 70 g in weight, were divided into three groups each containing twenty males and twenty females. Two groups were given I and 20 mg/kg of B.D.H. 1298, respectively, whilst the remainder received the vehicle: 20% ethyl oleate in arachis oil, emulsified in water containing 20% polyethylene glycol. The compound was administered by stomach tube daily, 5 days a week for 12 weeks. Body weights were recorded weekly and the doses adjusted accordingly. Blood values were recorded initially and at the end of the experimental period. On cessation of treatment, five males and five females were selected at random from each group and killed ; sections of , lung, , stomach, spleen, , pituitary, , adrenals, , uterus, vagina, testis, epididymis and seminal vesicles were prepared and examined histologically. Dry adrenal weights were also recorded. Five males and five females were taken at random from each group for fertility studies and the remainder kept under observation for a further 3 months.

ANTI-OVULATORY PROPERTIES Rabbits To increase the incidence of mating, mature does were primed with 20 Mg oestradiol monobenzoate 48 hr prior to mating. B.D.H. 1298 was dissolved in 20% ethyl oleate in arachis oil and emulsified in water containing 20% polyethylene glycol. Serial dilutions were then carried out. Eighteen hours before mating, doses of B.D.H. 1298, ranging from 0-008 to 1-0 mg/kg, were administered by stomach tube. Twenty-four hours or II days after mating the does were killed and the incidence of ovulation and/or pregnancy recorded. Control rabbits given only the vehicle were subjected to the same experimental conditions. Cats Cats are also suitable for anti-ovulatory studies since spontaneous ovulation occurs very rarely, induced ovulation being the usual event (Dawson & Friedgood, 1940; Asdell, 1946; Knaus, 1950). They normally mate on the 3rd day of oestrus, consequently the anti-ovulatory compound may be given on the 2nd day followed by mating on the 3rd. Ten cats weighing approximately 3 kg each were given tablets of 6 or 10 mg B.D.H. 1298 on the 2nd day of oestrus for a total of fifteen cycles. On the following day, the cats were mated with a proven male and the presence of spermatozoa confirmed by a vaginal smear. The failure of pregnancy was taken as an index of ovulation inhibition.

Downloaded from Bioscientifica.com at 09/25/2021 08:57:47PM via free access Properties of megestrol acetate 333 Dogs Six spaniel bitches with a mean weight of 12 kg were given 1, 2-5 or 5 mg twice daily for 21 days, immediately they came into season. The compound was incorporated in a 454-mg tablet containing dried yeast and lactose. The tablets were placed at the back of the throat and the mouth held until successful swallowing had occurred. Each bitch was mated on three separate occasions during oestrus. The incidence of pregnancy and number of young were recorded.

PROGESTATIONAL ACTIVITY McPhail's (1934) modification of the Clauberg test was employed using dimethisterone, 1 mg/kg daily, as the reference compound. , com¬ mercial norethynodrel, medroxy progesterone acetate and progesterone were also included in the study. Immature female rabbits weighing 640 to 1220 g were sensitized with three intramuscular doses of 5 pg of oestrone in 0-2 ml of 20% ethyl oleate in arachis oil, on Days 1, 3 and 5 of the experiment. Dimethisterone 1 mg/kg was used throughout the experiment and the doses for the other compounds varied from 0-0025 to 10 mg/kg. The compounds were either suspended in 5% gum acacia or dissolved in 20% ethyl oleate in arachis oil and emulsified in water containing 20% polyethylene glycol. They were administered by stomach tube on Days 7, 8, 9 and 10 of the experiment. Twenty-four hours following the last injection, the animals were killed ; the uteri were removed and frozen sections, 20 µ thick, were prepared and stained with haematoxylin. The responses were graded according to McPhail's method.

ANABOLIC AND ANDROGENIC ACTIVITY A modification of the Eisenberg & Gordan (1950) assay was used with methyl as the reference compound. Four groups of five male rats, with a mean weight of 88 g, were given B.D.H. 1298, 4, 20 and 100 mg/kg, or methyl testosterone, 150 mg/kg, in an aqueous suspending medium. The compounds were administered by stomach tube, daily for 7 days with an interval of 72 hr between the fourth and fifth doses. Twenty-four hours following the last injection, the animals were killed and the wet weights of the prostate, seminal vesicles and levator ani recorded.

EFFECT ON ANTENATAL SEXUAL DEVELOPMENT The effect of B.D.H. 1298 on the developing foetus was investigated by a modification of the methods of Revesz, Chappel & Gaudry (1960) and Suchowsky & Junkmann (1961). For comparative purposes methyl testosterone, commercial norethynodrel containing 1-5% of ethinyl oestradiol 3 methyl ether, medroxy progesterone acetate and progesterone were included in the study. Virgin female rats weighing between 180 and 200 g were mated with mature males. The day spermatozoa were found in vaginal smears was taken as the 1st day of pregnancy. From the 15th to the 20th day of pregnancy, groups of

Downloaded from Bioscientifica.com at 09/25/2021 08:57:47PM via free access 334 A. David et al. rats were given 0-015, 0-15, 1-5 and 15 mg/kg of B.D.H. 1298; 1-5,3-0, 10-0 and 15 mg/kg of methyl testosterone; 0-15 and 1-5 mg/kg of norethynodrel and medroxy progesterone acetate, orally in aqueous suspending medium. Progeste¬ rone 0-15, 1-5 and 15 mg/kg was given subcutaneously as a solution in 20% ethyl oleate in arachis oil. On the 21st day of pregnancy, the foetuses were removed by Caesarian section. They were examined for gross abnormalities, and the number of foetuses, their weight and ano-genital distances were recorded. In control experiments with 142 foetuses, the normal ano-genital distance for females was found to be less than 1-9 mm and for males greater than 2-7 mm; approximately 10% in the intermediate range of 1-9 to 2-7 mm were called intersexuals. Histological examination, however, showed that all in this group were normal males or females. Serial or single pelvic sections of randomly selected foetuses from control and treated groups, amounting to at least 25% of the total, were examined histologically. In the ano-genital distance assay, virilization of the female is said to occur if there is a shift in the percentage distribution towards the males and feminization when the reverse occurs. Histologically, virilization of the female foetus, as

(c) Text-fig. 1. Diagrammatic cross-section of distal end of urethra of 21-day rat foetus. (a) Normal female; = preputial fold; u = urethra, (b) Virilized female shown by narrowing of preputial frenulum. (c) Normal male, urethra inside preputial fold. shown in the methyl testosterone group, is said to be present if there is incomplete development of the upper or lower vagina, an increase in amount of prostate tissue, the persistance of Wolffian ducts or the enclosure of the urethra by the preputial folds (Text-fig. 1). With progestins, there is also aplasia of the tissues surrounding the urethra and vagina. Feminization of the male is characterized by poor development of the prostate and seminal vesicles, an enlarged utriculus prostaticus or failure of the preputial folds to enclose the urethra.

OESTROGENIC ACTIVITY In the Allen & Doisy (1923) assay, groups often ovariectomized mice were primed with 0-5 Mg of oestrone, followed in 7 days by a further dose of 0-75 \xg. Three weeks later the mice were given 1 or 10 mg/kg of B.D.H. 1298 by stomach tube in two equal doses, on Days 1 and 2. Oestrone, 12-5 Mg and 25 Mg, was given subcutaneously in a similar manner. Vaginal smears were examined at 3 p.m. on Day 3, a.m. and p.m. on Day 4 and a.m. on Day 5. A positive was scored if any one of the four smears showed nucleated or cornified cells with absence of leucocytes.

Downloaded from Bioscientifica.com at 09/25/2021 08:57:47PM via free access Properties of megestrol acetate 335 In Lauson, Heller, Golden & Severingham's (1939) assay, groups of nine or ten immature female rats weighing 40 to 50 g were given 0-5 and 5 mg/kg of B.D.H. 1298 and 1 Mg/kg of stilboestrol, respectively, by stomach tube, twice daily for 3 days. The control group was given the vehicle: 20% ethyl oleate in arachis oil. Eighteen hours after the last dose, the animals were killed and the wet uterine weights recorded.

ANTI-OESTROGENIC ACTIVITY This was investigated by Dorfman, Kind & Ringold's method (1961). Groups of from ten to thirty immature female mice weighing approximately 10 g were used. A total dose of 40 Mg/kg ofoestrone in 20% ethyl oleate in arachis oil was given subcutaneously over 3 days. A control group was given the vehicle only. B.D.H. 1298 was given orally and methyl testosterone and progesterone subcutaneously in aqueous suspending medium. Twenty-four hours following the last injection, the animals were killed and the wet uterine weights recorded. The degree of inhibition of oestrone-stimulated uterine growth served as an index of anti-oestrogenic activity.

EFFECT ON OESTROUS CYCLE Daily injections of progesterone will delay oestrus in a number of animals (Christian & Casida, 1948; Ulberg, Grummer & Casida, 1951). In 1960, Nellor showed that twice-daily administration of 6 methyl-17-acetoxy progesterone, an orally active progestin, inhibits oestrus in gilts. Rats In the chronic toxicity experiments, vaginal smears were taken at intervals during and after the cessation of treatment with 1 and 20 mg/kg of B.D.H. 1298. Dogs Ten pedigree Corgi bitches were given 0-1 mg/kg of B.D.H. 1298 daily for periods ranging from 44 to 52 weeks from the age of 5 months. Vaginal smears were examined at weekly intervals.

FERTILITY STUDIES Males Two groups of five rats given 1 and 20 mg/kg of B.D.H. 1298 daily, 5 days a week for the previous 3 months, were individually caged with two females of proven fertility. Vaginal smears were examined daily for spermatozoa. The incidence of pregnancy, litter size and sex ratio at weaning were recorded. Females Two groups of five rats given 1 and 20 mg/kg of B.D.H. 1298 daily, 5 days a week for the previous 3 months, were caged with three males of proven fertility. Vaginal smears were examined daily for spermatozoa. Pregnancy, litter size and sex ratio at weaning were recorded.

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EFFECT ON WATER AND ELECTROLYTE EXCRETION Four groups of eight male albino rats were deprived of food and water overnight. The following morning, 25 ml/kg 5% acacia in isotonic saline solution was given by stomach tube to each rat. Three groups received B.D.H. 1298 at 2, 8 or 32 mg/kg, suspended in the above vehicle, whilst the fourth group acted as controls. Urine was collected over 24 hr and recorded as ml/kg/24 hr. The Na+ and K+ concentrations were estimated by means of a flame photometer and recorded as m-equiv./kg. Chlorides were estimated according to a modification of Volhard's technique described by King & Wooton 0956).

GLUCOCORTICOID ACTIVITY A liver-glycogen-deposition assay in adrenalectomized rats, with acetate as the reference compound, was carried out. Groups of five male rats were adrenalectomized and given free access to saline drinking water until the 5th day and a high protein diet for 2 hr daily up to the 4th day. Twice-daily subcutaneous injections of B.D.H. 1298 to give a dose of 16 or 32 mg/kg and 16 mg/kg of , were commenced on the 3rd day and continued until the morning of the 5th day. The compounds were given in an aqueous suspending medium and the controls were given the vehicle only. Nine hours following the last injection, hepatectomy was carried out under sodium pento- barbitone, 50 mg/kg intraperitoneally, and the liver was homogenized in 5% trichloracetic acid. The liver glycogen contents were estimated by an anthrone method as described by Van der Vies (1954) and expressed as mg of liver glycogen/100 g body weight.

ANTIGONADOTROPHIC ACTIVITY McGinty & Djerassi (1958) reported that antigonadotrophic compounds cause, in the immature male rat, inhibition ofthe growth of testes, seminal vesicles and prostate and in the adult male a reduction in the weight of these organs. Holtkamp, Greslin, Root & Lerner (1960) determined the effect of anti- gonadotrophins on both male and female rats. Immature male and female rats of 70 to 80 g were given B.D.H. 1298, 1 or 5 mg/kg by stomach tube daily for 15 days. A control group received the vehicle: 20% ethyl oleate in arachis oil. Twenty-four hours following the last injection, the animals were killed and the wet weights of testes, seminal vesicles, prostate, , uterus and pituitaries recorded. A similar experiment was carried out on intact adult animals, the females weighed 200 to 210 g and the males 300 to 350 g.

ANTI-INFLAMMATORY PROPERTIES These were estimated by a modification of the methods described by Meier, Schüler & Desaulles (1950), Cresseri & Meli (1953) and Christian and William¬ son (1958). Five per cent turpentine-agar pellets, each approximately 0-5 cm 1-0 cm and weighing 220-2 ±2-5 mg, were implanted subcutaneously under the dorsal skin, one on either side of the spine. Groups of ten young female albino rats weighing between 70 and 90 g were used. The day following

Downloaded from Bioscientifica.com at 09/25/2021 08:57:47PM via free access Properties of megestrol acetate 337 implantation, B.D.H. 1298 or acetate, the reference compound, were given orally in aqueous suspending medium, twice daily for 5 days with an interval of 72 hr between the third and fourth doses. The controls were given the vehicle only. Twenty-four hours following the last dose, the animals were killed; the granulomata were dissected out, freed from the implant and weighed wet on a torsion balance. The percentage reduction in granuloma weight, compared with the controls, gives a measure of anti- inflammatory activity.

MAINTENANCE OF PREGNANCY Vaginal smears of female rats caged with males were examined daily, and the presence of spermatozoa was taken as the 1st day of pregnancy. On the 8th day of pregnancy, the animals were ovariectomized under ether anaesthesia. They were given powdered rat diet 41 containing varying amounts of either B.D.H. 1298 or B.D.H. 1298 and ethinyl oestradiol, up to and including the 21st day of pregnancy. Medroxy progesterone acetate in the diet and progest¬ erone given subcutaneously were used as reference compounds. On the 22nd day, the animals were killed and the incidence of pregnancy and the number of live foetuses recorded.

RESULTS

ACUTE TOXICITY B.D.H. 1298 is non-toxic in mice following oral administration of 5 g/kg.

CHRONIC ORAL TOXICITY The daily administration of 1 or 20 mg/kg of B.D.H. 1298 for 3 months had no adverse effect on the growth of males or females. There was no apparent effect on haemoglobin level, or on red or white cell, or differential white-cell Table 1

adrenal weights in groups of five rats following 3 months oral treatment with B.D.H. 1298

Dose Dry adrenal wt (mg/kg) Sex (mg/100 g body wt) 10 Female 11-2 ±2-2 200 Female 8-8 ±0-8 Controls Female 14-7 ±1-4 1-0 Male 12-1 ±0-7 20-0 Male 13-3 ±0-4 Controls Male 11·5±0·8 counts, apart from a small fall in the number of circulating polymorphs in the 1-mg/kg female group. Sections of heart, stomach, spleen, pituitary, adrenal, ovary, testis, epididymis and seminal vesicles showed no abnormalities. A few sections ofliver, including the controls, showed some evidence ofparasitic infestation. All the male

Downloaded from Bioscientifica.com at 09/25/2021 08:57:47PM via free access 338 A. David et al. kidney sections were normal, but some female sections showed excess vacuolation of convoluted tubules, which is regarded as physiological. No abnormality was observed in the male thyroid sections but some increased colloid storage was seen in the female 20-mg/kg group. Lung sections from all groups showed the characteristic histology ofrat pneumonia in many of the animals. Three sections of the vagina of the 20-mg/kg group showed an increased glycogen content, which is, however, normal for certain stages of the reproductive cycle. Uterine sections showed endometrial hyperplasia in the treated groups, due to the progestational activity of B.D.H. 1298. Table 1 records the dry adrenal weights. B.D.H. 1298 had no effect at 1 or 20 mg/kg in males or at 1 mg/kg in females, but 20 mg/kg caused a statistically significant adrenal atrophy in females, as assessed by Student's t-test. In spite of this loss in weight in the 20-mg/kg female group, no thinning of the could be detected histologically.

ANTI-OVULATORY PROPERTIES Rabbits Table 2 records the incidence of ovulation in rabbits following various single doses of B.D.H. 1298. At 0-25 mg/kg, only one of the twenty rabbits ovulated, and none ovulated at 0-5 mg/kg. Table 2 number and percentage of rabbits ovulating following the oral administration of b.d.h. 1298

Table 3 incidence of pregnancy in cats following a single dose of b.d.h. 1298 on 2nd day of oestrus followed by mating on the 3rd day

Cats Table 3 records the results and shows that a single dose of 2 or 3 mg/kg can successfully prevent pregnancy in cats. All the cats have subsequently produced apparently normal litters.

Downloaded from Bioscientifica.com at 09/25/2021 08:57:47PM via free access Properties of megestrol acetate 339 Dogs One of the two bitches given 1 mg, corresponding to approximately 0-17 mg/kg, daily during season gave birth to five pups. At 0-4 and 0-8 mg/kg daily, B.D.H. 1298 was successful in preventing conception. Table 4 records the results. Table 4

number of progeny and incidence of pregnancy after three matings in bitches given various doses of b.d.h. 1298 daily for 21 days during SEASON

Total Incidence Bitch daily dose No. of (mg/kg) pups pregnancy 0-2 0-2 0-4 1/6 0-4 0-8 0-8

PROGESTATIONAL ACTIVITY Table 5 records the results. B.D.H. 1298 at 0-005 mg/kg gave a similar response to 1 mg/kg of dimethisterone. However, 0-01 mg/kg gave a McPhail score of 2, hence B.D.H. 1298 has a progestational activity between 100 and 200 times that of dimethisterone. It is more active than ethisterone, commercial nor¬ ethynodrel and medroxy progesterone acetate, given orally, and progesterone, given subcutaneously, in this assay.

ANABOLIC AND ANDROGENIC ACTIVITY B.D.H. 1298 failed to show any anabolic or androgenic properties with amounts up to 100 mg/kg.

EFFECT ON ANTENATAL SEXUAL DEVELOPMENT Table 6 records the dose, number of rats and foetuses, and the percentage distribution of sexes. B.D.H. 1298 and progesterone have no virilizing effect on the female rat foetus with amounts up to 15 mg/kg given orally and sub¬ cutaneously, respectively. In contrast, 1-5 mg/kg of methyl testosterone, norethynodrel and medroxy progesterone acetate, produce virilization of the female in the ano-genital distance assay. Histologically, norethynodrel and medroxy progesterone acetate cause an inhibition of the female development, rather than the masculinization produced by testosterone, at doses that cause a similar shift in the percentage distribution of sexes. B.D.H. 1298 at 1-5 mg/kg and above caused feminization of the male foetus. This phenomenon has previously been observed by Marois (1960) in the rat with 17-ethinyl-19-nor- testosterone, at 10 mg/day.

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OESTROGENIC ACTIVITY In the Allen-Doisy assay, 5 mg/kg orally for 2 days was inactive. In Lauson's weanling rat assay (Lauson et al., 1939), 5 mg/kg showed no activity. Satis¬ factory responses were obtained with 12-5 Mg/kg oestrone in the Allen- Doisy assay and 1 Mg/kg stilboestrol in the Lauson test.

Table 5

mean progestational responses of oestrone- primed rabbits following administration of various

Daily dose Mean Compound for 4 days No. McPhail (mg/kg) rabbits score 0-0025 6 1-0 0-005 12 2-5 001 12 2-0 0-025 12 3-5 B.D.H. 1298 0-05 12 3-5 (oral) 0-062 12 30 0-125 12 30 0-25 12 3-5 0-5 12 3-5 1-0 12 3-5 Commercial 0-1 0-6 norethynodrel 1-0 30 (oral) 10-0 2-5 Medroxy 0-02 4 1-0 progesterone 0-05 4 2-0 acetate 0-1 10 2-0 (oral) 1-0 3 4-0 Ethisterone 10-0 30 3-0 (oral) Progesterone 2-0 54 3-0 (subcutaneous) Dimethisterone 1-0 100 2-5 (oral)

ANTI-OESTROGENIC ACTIVITY Table 7 records the results. B.D.H. 1298 possesses approximately half the anti- oestrogenic activity of progesterone, and twice that of methyl testosterone, subcutaneously.

EFFECT ON OESTROUS CYCLE Rats were given daily doses of 1 or 20 mg/kg of B.D.H. 1298 for 12 weeks. After 4 weeks of treatment, all the animals were in anoestrus and remained in this condition until 3 or 4 weeks after the cessation of dosing. It appeared, however, that in the presence of a male the normal cyclic rhythm was restored somewhat earlier.

Downloaded from Bioscientifica.com at 09/25/2021 08:57:47PM via free access Properties of megestrol acetate 341 In all dogs, suppression of the first and second oestrous cycles has occurred following daily oral doses of 0-1 mg/kg.

FERTILITY STUDIES Males At 1 mg/kg, all animals mated successfully within 2 weeks. The mean litter size was seven, with a normal sex ratio. At 20 mg/kg, four out of five mated within 2 weeks and one in the 5th week, with a mean litter size of eight and a normal sex ratio.

Table 6

NUMBER OF RAT FOETUSES, PERCENTAGE OF MALE, FEMALE AND INTERSEXES AND THE EFFECT OF SEXUAL DEVELOPMENT ON 21 ST DAY OF PREGNANCY FOLLOWING ADMINISTRATION OF VARIOUS STEROIDS FROM 15 TO 20 DAY

Foetuses Dose No. Virilization Compound (mg/kg) rats Distribution (%) ( + ) Total Feminization Female Intersex Male (") 0-015 9 44 13 43 68 B.D.H. 1298 0-15 9 50 13 37 68 (oral) 1-5 10 49 23 28 65 15-0 9 55 33 12 73 Methyl 1-5 9 36 30 34 77 testosterone 3-0 10 35 15 50 99 + (oral) 10-0 10 11 32 57 85 + + 15-0 9 0 29 71 83 + + + Norethynodrel 0-15 10 50 14 36 76 (oral) 1-5 9 11 35 55 76 + + Medroxy progesterone 0-15 10 48 7 45 71 acetate 1-5 10 27 17 56 71 (oral) Progesterone 0-15 42 9 49 74 (subcutaneous) 1-5 21 32 63 Controls 19 48 10 42 142

Females All animals from both groups were mated successfully within 2 weeks. However, one from each group did not become pregnant. The average litter size of the 1- and 20-mg/kg groups was five and eight, respectively, with a normal sex ratio.

EFFECT ON WATER AND ELECTROLYTE EXCRETION The oral administration of 2, 8 and 32 mg/kg to rats loaded with saline solution had no effect on urine volume or on its Na+, K+ and Cl~ content.

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GLUCOCORTICOID ACTIVITY A total dose of 16 and 32 mg/kg of B.D.H. 1298 gave a mean liver glycogen content of 4 mg and 9 mg/100 g body weight, respectively. Cortisone acetate at 16 mg/kg gave a satisfactory response of 72 mg/100 g body weight, compared with 3 mg/100 g body weight for the controls.

ANTIGONADOTROPHIC ACTIVITY In immature animals given 1 and 5 mg/kg, there was some inhibition in the growth of the testes, seminal vesicles, prostate, uterus and ovaries. However, a Student's t-test showed that only the prostate in the 1-mg/kg group, and the ovary in the 5-mg/kg group, were significantly different from the controls.

Table 7 anti-oestrogenic effect of b.d.h. 1298, orally, and proges¬ terone and methyl testosterone, subcutaneously, on uterine growth in mice

Dose (mg/kg) No. Uterine weight Compound mice (mg/lOg Inhibition Daily Total body wt) (%) B.D.H. 1298 1-0 3-0 29 25-4 12-7 (oral) 50 15-0 20 21-9 29-1 100 30-0 29 18-2 46-5 Progesterone 0-5 1-5 10 29-8 0 (subcutaneous) 2-5 7-5 10 22-8 24-9 20-0 600 10 11-2 79-3 Methyl testosterone 100 300 10 23-3 22-5 (subcutaneous) Oestrone 0013 004 30 28-1 (subcutaneous) Controls 30 6-8 1000

In the mature animals, 1 mg/kg/day had no effect on the testis, seminal vesicle or prostate weights. Five mg/kg had no effect on the testes but caused a reduction in the weight of prostate and seminal vesicles, reduction in the latter being significant. At both dose levels, the ovarian and uterine weights were reduced but only the latter were significantly different. The pituitary weights of the immature animals were reduced but the mature groups were unaffected. Table 8 records the results.

ANTI-INFLAMMATORY PROPERTIES There is no anti-inflammatory activity following a total dose of 125 and 250 mg/kg given over 5 days, while , at 125 mg/kg, caused a 35% reduction in granuloma weight.

Downloaded from Bioscientifica.com at 09/25/2021 08:57:47PM via free access Properties of megestrol acetate 343 MAINTENANCE OF PREGNANCY The number of pregnancies and mean number of live foetuses are recorded in Table 9. At 10 mg/kg, pregnancy was maintained in 70% of the animals; the addition of 4 Mg/kg/day of ethinyl oestradiol increased the incidence to 100%. Increasing the dose of B.D.H. 1298 to 20 mg/kg did not raise the preg¬ nancy incidence, but the addition of 0-5 Mg/kg/day of ethinyl oestradiol resulted in 90% pregnancies. In all these experiments, the mean number of live foetuses in treated animals was smaller than in the controls.

Table 8

antigonadotrophic effects of b.d.h. 1298 on rat organ weights, following 15 days oral administration

Dose Organ weight (mgj 100 g body wt) (mg/kg/ Weight day) (g) Seminal Pituitary Testes vesicles Prostate Ov Uterus

10 1060 3-4 1058-2 61-9 27-4 108-0 3-6 36-6 1360 50 114-0 5-4 1089-7 560 39-5 104-0 5-6 331 126-8 Controls 124-0 6-7 11240 80-4 61-2 1100 8-3 43-5 156-8

10 3520 30 990-0 410-0 147-0 218-0 4-0 29-3 142-7 5-0 322-0 30 1020-0 370-0 1390 216-0 50 260 132-7 Controls 3420 4-0 9000 420-0 148-0 209-0 40 32-0 173-2

There were ten rats in each group.

DISCUSSION B.D.H. 1298 appears to be the most active of the synthetic anti-ovulatory steroids so far reported. In rabbits a single dose of 0-5 mg/kg completely pre¬ vented ovulation in twenty animals and at 0-25 mg/kg 95% did not ovulate. In contrast we found that 10 mg/kg orally of commercial norethynodrel was required for 100% inhibition, while Pincus, Chang, Zarrow, Hafez & Merrill (1956), with 5 mg/kg orally, only obtained an 80% inhibition; 19 nor ethiste- rone at 5 mg/kg orally was completely effective. Anti-ovulatory activity was also demonstrated in cats with the single-dose technique and in dogs given repeated doses during season. Subsequently, these anti-ovulatory properties were confirmed in women (unpublished data). anti-ovulatory agents, as suggested by Saunders & Drill (1958), probably exert action by inhibiting production or release of pituitary gonadotrophins and not by direct action on ovarian tissue. B.D.H. 1298

Downloaded from Bioscientifica.com at 09/25/2021 08:57:47PM via free access 344 A. David et al. appears to fall in this category, as it does have some anti-gonadotrophic properties but is also singularly lacking in oestrogenic activity. Its high oral progestational activity was demonstrated in the assay, 5 Mg/kg given daily for 4 days, gave a McPhail (1934) score of 2-5, which is similar to the responses obtained following 1 mg/kg of dimethisterone orally or 2 mg/kg of progesterone subcutaneously. It has no androgenic activity and this was confirmed in the antenatal sexual studies where there was a complete absence of the virilizing effects as shown by some other anti-ovulatory and progestational compounds, Table 9

pregnancy incidence and mean live litter size in rats spayed on the 8 day and given b.d.h. 1298 with and without ethinyl oestradiol, medroxy progesterone acetate, in the diet and progesterone subcutaneously

Daily Ethinyl Pregnancy incidence Mean No. Compound dose oestradiol live (mg/kg) (Mg/kg/day) No. (%) foetuses 2-5 1/10 10 1-0 2-5 1-0 2/10 20 1-5 5-0 3/10 30 1-, 50 2-0 5/10 50 2-0 100 7/10 70 2-3 B.D.H. 1298 100 0-5 7/10 70 3-4 10-0 1-0 5/10 50 2-0 100 20 8/10 80 3-0 100 4-0 100 4-1 20-0 4/10 40 2-5 20-0 0-5 9/10 90 5-3 20-0 1-0 6/8 75 40 Medroxy 50 6/9 67 4-3 progesterone 7-5 9/10 90 5-2 acetate 10-0 9/9 100 5-9 Progesterone 25-0 7/10 70 3-1 (subcutaneous) 50-0 7/9 78 3-1 Controls 0/10 (ovariectomized) Intact controls 10/10 100 9-3 (sham operated) such as commercial norethynodrel and medroxy progesterone acetate. Feminization appears to be a reversible process, as regression occurred during maturation. In addition, these animals mated without difficulty and produced normal litters. Green, Burill & Ivy (1940) showed that feminization could be produced in rats with oestrogens, and Marois (1960) has attributed the feminizing effect of 19-nor-ethisterone (Norlutin) in rats to oestrogenic activity. However, this does not appear to be the explanation for B.D.H. 1298, as no oestrogenic properties could be detected in the Allen-Doisy or Lauson assays. The feminizing effects of B.D.H. 1298 may be an indirect result of its anti-

Downloaded from Bioscientifica.com at 09/25/2021 08:57:47PM via free access Properties of megestrol acetate 345 gonadotrophic properties. This would, as suggested by Segal & Nelson (1959), prevent stimulation of the foetal testes and therefore inhibit masculine develop¬ ment in the genetic male, due to associated of testicular . Prolonged administration to rats does not have any apparent adverse effects and the fertility of both male and female rats was unimpaired after 3 months treatment. Repeated administration to females causes anoestrus in approxi¬ mately 4 weeks and this phase persists usually for 3 or 4 weeks after cessation of treatment. In the presence of a male, however, the normal cyclic rhythm is restored earlier. B.D.H. 1298 has no systemic anti-inflammatory, glucocorticoid or oestrogenic properties, or effect on water and electrolyte excretion.

ACKNOWLEDGMENT We wish to acknowledge the preliminary studies carried out by Mr P. C. Rofe and the oestrogenic, maintenance of pregnancy and anti-inflammatory assays of Miss E. J. Woodward.

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