Progestin and antiprogestin interactions with progesterone receptors in human myomas.

Progesterone receptors (PRs) in human fibromyomata (myomas) and normal myometrium were characterized by gel filtration, sucrose gradient sedimentation analysis, isoelectrofocusing and ligand specificity. The PR population in myoma tissues, 1242 +/- 505 fmol/mg protein, was 3-fold higher than normal myometrial tissues under similar hormonal milieu. The increased PR concentration in myomas appears to be of potential importance in physiopathology of fibromyoma.

Copper intravas device (IVD) and male contraception.

An alternative method of male contraception is crucial at this stage of increasing population in the developing world. A non-obstructive, non-hormonal, reversible method of male contraception would find acceptance in oriental communities. Our experience with copper IVD in small animals has been encouraging. We present here an extension of the use of the copper IVD in the rhesus monkey. Treated male rhesus ejaculate showed fifteen-to thirty-five-fold increase in copper levels up to 10 months after insertion of Cu IVD . Semen analysis showed an increase of dead sperm (23.0% - 52.3%; control 4.5% - 14.0%) and decrease in percentage of motile sperm (10-30%; control 70-80%). Fertility studies using treated male rhesus of proven fertility from the breeding colony mated with cycling females of proven fertility showed highly significant reduction of fertility up to 13 months (P less than 0.001). Reduction in fertility of the treated rhesus was also significant up to a period of 23 months (P less than 0.01). A reversal of contraceptive effect and increase in sperm count and motility is observed after 13 months post-treatment. Studies are required to achieve an increase in the effective life span of the IVD .

PIP: An alternative method of male contraception is crucial at this stage of increasing population in the developing world. A nonobstructive, nonhormonal, reversible method of male contraception would find acceptance in Oriental communities. Our experience with the copper intravas device (IVD) in small animals has been encouraging. The present study is an extension of the use of the copper IVD in the rhesus monkey. Treated male rhesus ejaculate showed 15-35 fold increase in copper levels up to 10 months after insertion of the Cu IVD. Semen analysis showed an increase of dead sperm (23.0%-52.3%; control 4.5%-14.0%) and a decrease in the percentage of motile sperm (10-30%; control 70-80%). Fertility studies using treated male rhesus of proven fertility from the breeding colony mated with cycling females of proven fertility showed highly significant reductions in fertility up to 13 months (P0.001). A reduction in fertility of the treated rhesus was also significant up to a period of 23 months (P0.01). A reversal of contraceptive effect and an increase in sperm count and motility is observed after 13 months posttreatment. Studies are required to achieve an increase in the effective span life of the IVD.

Contraceptive efficacy of norethindrone acetate (ENTA) implant: its release rate and fertility potential.

PIP: The contraceptive efficacy of norethindrone acetate (ENTA) implant was evaluated in a study of 79 healthy volunteers aged 19-30 years. The purpose of the study was to 1) relate the menstrual pattern to the average daily release rate of the hormone, 2) assess the concentration of NET in peripheral blood in cases of unintentional pregnancies and ascertain the risk to offspring in such cases, and 3) evaluate the fertility potential and menstrual pattern in the 1-year period after implant removal. In most subjects (59), the implant was removed between the 5th and 8th month. The average release rate was found to be 163.5 mcg/day in subjects with regular menstrual cycles (cycle interval of 25-32 days) compared with 190.9 mcg/day in those with an abnormal menstrual pattern (p0.05). The rate was 143.3 mcg/day in the 19 women who became pregnant during the trial, but this difference is not significant. 15 of the 19 pregnancies occurred between 4 and 8 months, suggesting that the contraceptive efficacy of this implant does not exceed 4-5 months. Mean NET concentration in the sera of the women who became pregnant was 122.5 pg/ml at the time of pregnancy diagnosis. Only 10 subjects complied with the recommendation that their pregnancy be terminated for medical indications. The remaining 9 subjects delivered 6 female and 3 male infants who appeared to clinically normal, despite the fact that 4 had been exposed to the steroid for over 6 weeks. 1 year follow-up was possible in 52 subjects. The menstrual pattern appeared to be regular during this period. 25 of the 44 women in this follow-up group who did not use contraceptive measures became pregnant during the 1st year after implant removal. Additionally, the 9 infants delivered to women during the study period were evaluated for 2-3 years. No adverse changes in growth and development have been observed. This finding suggests that reports associating teratogenicity and progestins in early pregnancy are exagerrated.^ieng

Norethindrone in serum after use of an oral contraceptive containing norethindrone acetate.

The availability of norethindrone (NET) in serum was studied in 8 women after daily administration of a combination oral contraceptive pill (MinovlarR) containing 1 mg norethindrone acetate (NETA) and 50 micrograms ethinyl estradiol (EE2) from day 5 to day 25 of the menstrual cycle. The pill was taken daily at 9:30 a.m. after a light breakfast and blood samples were collected at 3 hr on alternate days and at 24 hr on other days after ingestion of the pill. On day 12 or day 15 of the first treatment cycle, serial blood samples were also collected at 1/2, 1, 2, 4, 6, 8 and 24 hr after taking the pill. Serum NET levels were estimated by the radioimmunoassay (RIA) technique. The plot of serum NET concentration versus time (0-24 hr) profile showed a rapid absorption of steroid and a peak NET concentration (18.3 +/- 4.8 ng/ml) reached at 2 hr after ingestion of the pill. By linear regression analysis of data (y = 0.27 + 0.12x; r = 0.95), it was observed that the serum NET level was initially about 1 ng/ml. Thereafter, it increased by 0.12 ng/ml per day up to 3.5 ng/ml by the end of 21 days' treatment with oral pills. The serum NET concentration decay slope fitted a two-compartment open model with an initial rapid decay (half-life of 1.2 +/- 0.1 hr) followed by a slower beta-phase with a half-life of 8.5 +/- 1.5 hr. The study revealed that there was an accumulation of norethindrone in plasma during the treatment period and this suggests the possibility of exploring a reduction in the dose of this preparation so as to achieve the optimum NET concentration for contraception, and thereby avoiding unwanted steroid accumulation in the plasma of women.

Pharmacokinetics of norethindrone acetate in women after the insertion of a single subdermal implant releasing norethindrone acetate.

The metabolic clearance rate (MCR) of norethindrone acetate (NETA) and norethindrone (NET) levels in plasma were studied after an iv injection of [3H]NETA in three women before and at 1 week, 1, 2 and 6 months following the insertion of a single silastic subdermal implant releasing microquantities of NETA. No significant change in the MCR of NETA was observed at 1 week (459 +/- 72 1/day), 1 month (489 +/- 113 1/day) and 2 months (522 +/- 144 1/day) compared with that of control (525 +/- 108 1/day). However, MCR of NETA showed significant increase in women exposed to continuous presence of NETA for a period of 6 months (608 +/- 121 1/day; P less than 0.025). NETA was rapidly and extensively metabolized into NET. At 1 week, 1, 2 and 6 months of study, NET was observed to be present in higher amounts compared with NETA. The production rate (PR) of progesterone decreased significantly at 2 and 6 months of NETA implant insertion compared with the PR before the insertion of implant.

Screening of anti-progestins using in vitro human uterine progesterone receptor assay system.

PIP: An in vitro receptor assay system was developed to screen new antiprogestin compounds and to estimate their relative binding affinity for uterine progesterone receptors. 62 steroidal and nonsteroidal compounds including the suspected antiprogestins were screened. Normal human uterine tissues were obtained at hysterectomy operations. Premenopausal women with normal menstrual cycles and without any hormonal treatment were selected for the study. Some synthetic progestin compounds displayed high relative binding affinity (RBA) while others (metabolites of progesterone and norethisterone) showed poor or no binding affinity for human uterine progesterone receptors. Removal of the axial 19-methyl group from progesterone as in 19-norprogesterone resulted in greater binding affinity while the presence of polar groups on the molecule decreased the binding affinity. Of the 30 suspected antiprogestins tested, R2323 showed maximum competition for progesterone binding sites. Biologically, R2323 prevents implantation in women and this is attributed to its high binding affinity for the progesterone binding sites; however, the compound is associated with bleeding irregularities in women. Other compounds with high RBA are STS557 and RMI14156. The study shows that a complexity of factors (e.g., hydrophobicity, hydrophilicity, spatial requirements, optimal contact, and steric hindrances) control the ligand interactions to human uterine cytosol receptors. Screening larger numbers of antiprogestins should be done. As some of the compounds have mixed antiprogestational, antiandrogenic, and antiestrogenic properties, a comparative study on the RBA of a particular compound for the respective receptors should be carried out to better understand the biological profile and molecular interactions of the compounds.^ieng

Nuclear progestin receptors in human uterus.

The characteristics and transfer of human uterine progestin receptors from cytosol to nucleus were studied in a cell-free system using [3H]progesterone ([3H]P), [3H]norethindrone ([3H]NET), and [3H]norethindrone acetate ([3H]NETA) as progestin receptor tags. Nuclear receptor binding was observed only in the presence of uterine cytosol. Various prior treatments of uterine cytosol, including warming to 20 C, increasing the salt concentration to 150 mM KCl, or dilution of the cytosol protein concentration to 3 mg/ml, increased the affinity of the receptor for the cell nucleus and thus, facilitated more translocation. However, there was no detectable difference in the receptor sedimentation value after activation. The binding of salt-extractable (400 mM KCl) nuclear sites to [3H]P, [3H]NET, and [3H]NETA was characterized by distinct commonality in their physiochemical properties, as indexed by their coelution during gel chromatography (mol wt, 72,000), their similar sedimentation rates (4.2S), and their similar isoelectric profiles (isoelectric pH 5.3--5.6). The concentrations of specific binding sites were similar for the different 3H-labeled progestins used. However, in general, the number of binding sites was higher (3780--4480 molecules/cell) in proliferative phase tissues and significantly lower (1790--1920 molecules/cell) in secretory phase tissues. Furthermore, nuclear binding was progestin specific and of high affinity (Kd of NET, similar to or approximately 0.7--0.8 nM; Kd of P, similar to or approximately 0.9--1.2 nM; Kd of NETA, similar to or approximately 6.7--7.1 nM). Despite the similarities in the molecular properties, the nuclear bindings of NET and NETA could be distinguished from that of P by their lower dissociation rates (t 1/2 of NET, similar to or approximately 6.3 h; t 1/2 of NETA, similar to or approximately 5.5 h; t 1/2 of P, similar to or approximately 1.2 h). Moreover, NET and NETA, unlike P, did not bind to corticosteroid-binding globulin. These observations indicate that the various progestins mediate their action via a common receptor, which translocates from cytosol to nucleus. Further, because of the distinct advantages, NET could be conveniently used as a receptor tag to evaluate the progestin receptor.

Fluid-filled silastic capsules : a new approach to a more constant steroidal drug delivery system.

A new approach to a more constant steroid delivery system was made in which 7,9 mg of norethisterone acetate was shaped into a cylindrical pellet (diameter 1.47 mm, length 4 mm) and placed inside a silastic capsule (i.d. 1.57 mm, o.d. 3.18 mm) of 5 mm effective length. The extra space available around the pellet was filled with physiological saline. For the sake of comparison, the same amount of crystalline norethisterone acetate was packed up to 5 mm length inside a silastic capsule of the same dimensions. The incorporation of fluid (saline) in the system not only caused a marked decrease in the initial high release but also a more constant release. Varying the pellet diameter showed that release of the steroid increases with the increase in surface area of the pellet.

Correlation between the serum norethindrone (NET) levels attained after insertion of a silastic implant releasing norethindrone acetate and the endogenous hormones particularly progesterone.

Six normally menstruating women were inserted each with a single silastic implant-D releasing norethindrone acetate (NETA). The levels of endogenous hormones, FSH, LH, E2 and progesterone, were estimated by radioimmunoassay (RIA) procedures in the control and treatment cycles. In addition, the levels of drug in the serum as norethindrone (NET) which is a major metabolite of NETA were also estimated by RIA procedures in the treatment cycles. In all, 12 treatment cycles were studied. In the initial treatment cycles (1st/2nd or 3rd), the serum NET levels were either 1 ng/ml or above. The LH and FSH showed either normal or suppressed mid-cycle peaks, but the progesterone levels were completely suppressed. In the sixth treatment cycles, the serum NET levels were either 0.5 ng/ml or below. The FSH and LH mid-cycle peaks were lower but distinct while the luteal progesterone levels were of normal ovulatory type. These studies lead us to the conclusion that a serum level of NET of the order of 1 ng/ml is required to bring about suppression of luteal progesterone, either as a result of direct action on the ovary or through suppression of pituitary gonadotropins. When the serum level falls to 0.5 ng/ml or below, the suppressive effect is removed and ovulatory pattern of progesterone returns.

PIP: 6 normally menstruating women were each inserted with a single silastic implant-D releasing (NETA) norethindrone acetate. The levels of endogenous hormones, FSH, LH, E2, and progesterone, were estimated by (RIA) radioimmunoassay procedures in the control and treatment cycles. In addition, the levels of drug in the serum as (NET) norethindrone which is a major metabolite of NETA were also estimated by RIA procedures in the treatment cycles. In all, 12 treatment cycles were studied. In the initial treatment cycles (1st/2nd or 3rd), the serum NET levels were either 1 ng/ml or above. The LH and FSH showed either normal or suppressed midcycle peaks, but the progesterone levels were completely suppressed. In the 6th treatment cycle, the serum NET levels were either 0.5 ng/ml or below. The FSH and LH midcycle peaks were lower but distinct while the luteal progesterone levels were of normal ovulatory type. These studies lead us to the conclusion that a serum level of NET of the order of 1 ng/ml is required to bring about suppression of luteal progesterone, either as a result of direct action on the ovary or through suppression of pituitary gonadotropins. When the serum level falls to 0.5 ng/ml or below, the suppressive effect is removed and ovulatory pattern of progesterone returns.

Fate of progesterone in the plasma and various tissues of anaesthetized rabbits.

Studies on the metabolism of progesterone in rabbit plasma and tissues during constant infusion of [3H]progesterone revealed a gradual increase in the concentration of progesterone in plasma until a state of equilibrium was attained about 2 h after the start of infusion. The metabolic clearance rate of progesterone was 0.116 l/min and the turn-over time was 0.006 min/ml plasma. Under conditions of equilibrium, the concentration of radioactivity as well as that of labelled progesterone was higher in the tissues than in plasma. On a unit-weight basis, myometrium localized 6.6 nCi, Fallopian tube 5.5 nCi and vagina 6.1 nCi [3H]progesterone/g tissue. A higher concentration of progesterone, 16.5 nCi, was localized in the mammary gland tissue. Skin localized only 0.9 nCi progesterone and the lowest amount (0.34 nCi/ml) was found in plasma. The metabolism of progesterone in tissues differed from that in plasma. In plasma only 5.6% of the infused steroid was recovered as unconverted progesterone and the major amount was metabolized to polar compounds with smaller amounts of 5 alpha-pregnane-3,20-dione, 5 alpha-pregnan-3 beta-ol-20-one and 20 alpha hydroxypregn-4-en-3-one. In the myometrium and Fallopian tubes the major metabolites were 5 alpha-pregnan-3 beta-ol-20-one and 5 alpha-pregnane-3,20-dione respectively. In the vagina, like the myometrium, 5 alpha-pregnan-3 beta-ol,20-one was the major metabolite. 20 alpha-Hydroxypregn-4-en-3-one was the major identified metabolite of mammary gland tissue.

Urinary melatonin levels in human breast cancer patients.

Urinary melatonin levels were measured in 10 postmenopausal Indian women suffering from advanced stages of breast cancer and in 9 well-matched women with non-endocrine complaints, mostly uterovaginal prolapse. Urines of each patient were collected over a period of 2-3 days in four 4-hourly intervals from 6 a.m. to 10 p.m. and one 8-hourly interval from 10 p.m. to 6 a.m. Serum LH, FSH, prolactin, estradiol and cortisol levels at 11 a.m. were determined as well as estrogen and progesterone receptors of the breast tumors. It was found that 24 hour urinary melatonin excretion in cancer patients was on the average 31% decreased as compared to the controls. This change was accompanied by a 33% increase in serum cortisol levels in the cancer patients. The melatonin excretion patterns of the cancer patients were not synchronized as compared to synchronized patterns of the controls. The number of tumors tested for steroid receptors does not yet allow to conclude if melatonin is different in patients with or without hormone-dependent tumors. The data suggest that pineal melatonin secretion may be modified in quantity as well as rhythmicity in breast cancer patients.

Bioavailability of norethindrone in rabbits after administration of norethindrone acetate in single, double and quadruple doses released through subcutaneous silastic implants.

Plasma level of norethindrone (NET) and in vivo release of norethindrone acetate (NETA) were studied in three groups of albino rabbits (5 animals/group) after insertion of one, two and four subcutaneous implants, each containing 40 mg crystalline NETA over a period of 24 weeks. The ratios of the in vivo release rate of steroid were 1, 1.9 and 3.9 in the animals of group I (one implant), group II (two implants) and group III (four implants) respectively. Thus, the in vivo release rate in group II and III showed an increase which was almost twice and four times as great as that of group I. However, the mean ratios of the serum NET levels were 1, 1.2 and 2.4 in animals of group I, II and III respectively. Thus, interestingly, the serum NET level did not show the expected twofold and fourfold increase and lacked correlation with the in vivo release. Although the insertion of multiple implants gives multiple increases in the in vivo steroid release, it does not give rise to a multiple increase in the serum levels of the steroid. It is possible that there is a kind of threshold of steroid concentration in the animals when they are loaded with exogenously administered steroid. When the steroid concentration tends to cross the threshold level, pharmacokinetic or pharmacodynamic processes of the animal work maximum to hold down the steroid level in blood plasma.

Progestin receptors in human uterine cytosol.

Progestin(norethindrone and norethindrone acetate)-binding protein, exhibiting characteristics similar to uterine progesterone receptor, has been identified in human uterine cytosol. The progestin receptor was characterized by sedimentation coefficient, 4.2 S; Stokes radius, 39 A; frictional ratio, 1.29; isoelectric pH, 4.6; molecular radius, 2.7 nm; and molecular weight in the range 67 000--74 000. The ammonium-sulfate-precipitated progestin-receptor complex was eluted from a DEAE-cellulose column at 0.18 M KCl. The progestin binding was saturable and stereospecific. The sequential variation in receptor concentration (early proliferative, 3800--4300 sites/cell; late proliferative, 9500--11 200 sites/cell; early secretory, 4900--6200 sites/cell; late secretory, 1800--2300 sites/cell) was in conformity for progesterone and the progestins, when concurrently measured. Oral administration of norethindrone significantly reduced the cytoplasmic and nuclear receptor concentration for estradiol and progesterone. A significant observation was that the progestins stabilized the receptor by forming a slowly dissociating complex with a t 1/2 approximately 110--130 min as compared with the progesterone-receptor complex dissociating with t 1/2 approximately 41 min. Thus, the uterine progestin receptor recognizes progestins in general, although with a varying degree of affinity, and the altered rate constants could be of putative importance in determining the biological potency of the progestins.

Further studies of an intravasal copper device in rats.

A 1.5 cm long copper wire caused complete inhibition of fertility in male rats when placed properly in the vasal lumen. The contraceptive effectiveness of the device was directly proportional to the surface area of the device and was non-obstructive. The release rate of copper in vivo from a 1.5 cm long wire was 11.6 micrograms/day during the first month and 6.8 micrograms/day over the first 3 months. The contraceptive action of the wire starts about 20 days after insertion. Fertility was regained in 67% of the rats by 90 days after removal of the device. Similar devices of stainless steel and silver did not affect fertility.

In vitro effect of human placental lactogen on steroidogenesis in human placental tissue from early pregnancy.

Studies were carried out with human placental lactogen (hPL) to elucidate its role in regulation of steroidogenesis (progesterone and estrogens) during early pregnancy in humans. Our in vitro studies with early pregnancy placenta under different doses of hPL demonstrated that this hormone could stimulate the synthesis of progesterone as well as estrogens (estrone and estradiol) from their respective precursors.

Plasma levels of 15 (S) 15-methyl-PGF 2 alpha-methyl ester following vaginal administration for induction of abortion in women.

A single suppository containing 3.0 mg 15 (S)15-methyl-PGF2 alpha-methyl ester was administered vaginally in women to terminate mid-trimester pregnancy. Plasma levels of the drug (both methyl ester and free acid forms) were measured at different time intervals using deuterated carriers and gas chromatography-mass-spectrometry. 15(S)15-methyl PGF2 alpha (sum of methyl ester + free acid) was found to have a mean value of 1166 pg/ml plasma at 3 hrs after administration of the suppository. The mean levels were subsequently found to be maintained in the range of 1000 pg/ml. Preliminary studies showed that unlike the successfully aborted women, in those who did not abort within 30 hrs with this suppository, the plasma levels of the drug were not maintained for a sufficiently long time. The episodes of side effects, body surface area and the induction-abortion interval in relation to the plasma levels are discussed.

Pharmacokinetics of norethindrone acetate in women.

The plasma half-life, MCR and plasma metabolite levels at various time intervals have been studied in six women after an intravenous injection of 3H-norethindrone acetate. The disappearance curve due to norethindrone acetate showed an initial rapid disappearance of 3H with an average half-life of 7.5 minutes and a subsequent slow disapperance with a half-life of 51.5 hours. Norethindrone acetate was cleared from the plasma with an average MCR of 495 L/day. Norethindrone acetate is rapidly metabolised after an intravenous injection. Norethindrone, the main metabolite, disappears from the plasma with an average half-life of 34.8 hours. Norethindrone maintains a high level compared with norethindrone acetate at all time intervals up to 24 hours and an equilibrium is reached between the two at 24 to 48 hours.

PIP: 6 normally cycling females volunteered to receive radiolabeled norethindrone acetate (NA) intravenously to determine the pharmacokinetics of this steroid in women. Parameters studied were plasma half-life, metabolic clearance rate (MCR), and plasma metabolites at various times after injection. Disappearance curve due to NA showed an initial rapid disipation of the radiolabel with an average half-life of 7.5 minutes and a subsequent slow disappearance with a half-life of 51.5 hours. NA had an average MCR for blood clearance of 495 1/day. NA was rapidly metabolized after intravenous injection. Norethindrone, the main metabolite of NA, disappeared from plasma with an average half-life of 34.8 hours, and maintained a high level compared with NA at all times up to 24 hours; an equilibrium was reached between the 2 steroids at 24-48 hours postinjection.

Quantitative analysis of norethindrone in milk using deuterated carrier and gas chromatography-mass spectrometry.

PIP: A quantitative procedure using deuterated carrier and gas chromatography-mass spectrometry is described and applied to quantitate norethindrone levels in milk from 5 lactating women, aged 20-30 years, who had received 1 silastic implant of 40 mg of norethindrone. The technique is a multiple ion one (gas chromatography-mass spectrometry). The deuterated norethindrone was used as the internal standard and carrier for the detection technique, and, after its addition, the milk samples were extracted with Amberlite XAD-2 columns and purified on Sephadex columns. O-methoxime-trimethylsilyl ether derivatives of the purified samples were analyzed. The assay was precise and reproducible. The mean level of norethindrone at 3 weeks post implant insertion was 208 pg/ml of milk, after which it declined slowly and steadily.^ieng