Sex differences in the cerebroprotection by Nestorone intranasal delivery following stroke in mice.

Our previous studies showed that intranasal delivery of progesterone offers a good bioavailability and neuroprotective efficacy after experimental stroke. We have also demonstrated that progesterone receptors (PR) are essential for cerebroprotection by endogenous progesterone and by progesterone treatment. The identification of PR as a potential drug target for stroke therapy opens new therapeutic indications for selective synthetic progestins. Nestorone® (16-methylene-17α-acetoxy-19-nor-pregn-4-ene-3, 20-dione, also known as segesterone acetate) is a 19-norprogesterone derivative that more potently targets PR than progesterone. The objective of this study was to evaluate the cerebroprotective efficiency of intranasal administration of Nestorone after middle cerebral occlusion (MCAO) in mice. We show here that intranasal administration is a very efficient route to achieve a preferential delivery of Nestorone to the brain and confers a slow elimination and a sustained bioavailability. Furthermore, intranasal administration of Nestorone (at 0.08 mg/kg) improved the functional outcomes and decreased the ischemic lesion in male but not in female mice at 48 h post MCAO. Use of PRNesCre mice, selectively lacking expression of PR in neural cells, and their control PRloxP/loxP littermates showed that the cerebroprotective effects of Nestorone in male mice depended on neural PR as they were not observed in PRNesCre mice. Our findings show that intranasal delivery of Nestorone may be an efficient strategy to promote recovery after stroke in males and confirm the key role of PR in cerebroprotection. Furthermore, they point to sex differences in the response to Nestorone treatment and emphasize the necessity to include males and females in experimental studies.

Combined nestorone-testosterone gel suppresses serum gonadotropins to concentrations associated with effective hormonal contraception in men.

BACKGROUND: Novel male-based contraceptives are needed to broaden family planning choices. A progestin, Nestorone® (Nes) gel, plus a testosterone (T) gel suppresses sperm concentrations to levels associated with effective contraception in normal men. However, administration of two gels on different parts of the body daily is impractical. OBJECTIVE: Compare the effectiveness of daily application of a single, combined 8.3 mg Nes-62.5 mg T gel (Nes-T) vs. 62.7 mg T gel to suppress serum FSH and LH concentrations to ≤1.0 IU/L (a threshold associated with suppression of sperm concentrations to ≤1 million and effective contraception) and to compare the pharmacokinetics of serum Nes and T concentrations between the gel groups. DESIGN: We conducted a 28-day, double-blind, controlled trial of 44 healthy men randomized to daily Nes-T or T gel with measurement of hormones at baseline, treatment, and recovery and during 24-h pharmacokinetic studies on days 1 and 28 of treatment. RESULTS: Of the subjects who met pre-defined inclusion criteria, 84% of the Nes-T group suppressed serum gonadotropin concentrations to ≤1.0 IU/L at days 21-28 vs. 16.7% in the T group (p < 0.001). On day 1, Nes concentrations rose significantly above baseline by 2 h and continued to rise up to 24 h after Nes-T gel application. Nes concentrations were not detectable in the T group. Serum total T concentrations rose and were significantly higher in the T gel group compared to the Nes-T group at 24 h on day 1 and days 11, 14, and 21 (p < 0.01). There were no serious adverse events in either group. About 80% of the subjects reported satisfaction with both gels. CONCLUSION: Daily Nes-T gel effectively and safely suppresses serum gonadotropins and is acceptable to most men. It should be studied further in efficacy trials of hormonal male contraception.

Preventing secondary exposure to women from men applying a novel nestorone/testosterone contraceptive gel.

BACKGROUND: Testosterone (T)/Nestorone (NES) combination gel is a potential transdermal male contraceptive that suppresses gonadotropins and spermatogenesis. Transfer of transdermal T from men to women can be prevented by washing or covering application sites with clothing. OBJECTIVES: We hypothesized that showering or wearing a shirt over gel application sites would prevent secondary exposure of T and NES to a woman after close skin contact. MATERIALS AND METHODS: Twelve healthy male and 12 healthy female participants were recruited. Men applied T/NES 62 mg/8 mg gel to their shoulders and upper arms. Two hours after application, female partners rubbed the application site for 15 min. Exposure in the female partner was assessed under three conditions: a shirt covered the application site; the man showered prior to skin contact; or without intervention to reduce transfer. Serum T and NES concentrations were measured by LC-MS/MS in serial blood samples for 24 h after gel exposure. MAIN OUTCOMES: Change in female serum T and NES levels as measured by average concentration over 24 h (Cavg ). RESULTS: Median female serum T Cavg was 23.9 ng/dL (interquartile range, 19.3, 33.9) with the shirt barrier and 26.7 ng/dL (20.7, 33.9) after showering, which was higher than baseline 20.9 ng/dL (16.7, 25.0), both p < 0.03) but lower than without intervention (58.2 ng/dL [30.9, 89.1], both p < 0.01). Female serum NES Cavg and maximum concentration were below the lower limit of quantification with the shirt barrier and after showering, but increased without intervention in six of 12 women (maximum concentration <60 pg/mL). Men had lower average serum NES levels after showering (47 pg/ml [20, 94] compared to no intervention (153.3 pg/mL [51, 241], p < 0.02). CONCLUSION: Secondary transfer of T and NES occurs after intensive skin contact with the gel application site. Secondary transfer is decreased by a shirt barrier or showering before contact.

Continuous dosing of a novel contraceptive vaginal ring releasing Nestorone® and estradiol: pharmacokinetics from a dose-finding study.

BACKGROUND: As part of a program to develop a novel estradiol-releasing contraceptive vaginal ring (CVR), we evaluated the pharmacokinetic (PK) profile of CVRs releasing segesterone acetate (Nestorone® (NES)) combined with one of three different estradiol (E2) doses. STUDY DESIGN: A prospective, double-blind, randomized, multi-centered study to evaluate a 90-day CVR releasing NES [200mcg/day] plus E2, either 10mcg/day, 20mcg/day, or 40mcg/day in healthy reproductive-age women with regular cycles. Participants provided blood samples twice weekly for NES and E2 levels during the first 60 days (ring 1) and the last 30 days (ring 2) of use. A subset underwent formal PK assessments at ring initiation, ring exchange (limited PK), and study completion. RESULTS: The main study enrolled 197 women; 22 participated in the PK substudy. Baseline characteristics between the main and PK participants were comparable, with an average BMI of 25.8 kg/m2 (SD 4.3). In the PK substudy, all three rings showed similar NES PK: mean area under the curve (AUC(0-72)) 34,181 pg*day/mL; concentration maximum (Cmax) 918 pg/mL; time to maximum concentration (Tmax) 3.5 h. For E2, the Cmax occurred at 2 h, and was significantly higher with the 20 mcg/day ring (mean 390 pg/mL); 10 mcg/day, 189 pg/mL, p=.003; 40 mcg/day, 189 pg/mL, p<.001), and declined rapidly to≤50 pg/mL for all doses by 24 h. For all subjects, the median E2 levels remained under 35 pg/mL during treatment. CONCLUSION: PK parameters of NES were not affected when paired with different doses of E2, but E2 levels from all three doses were lower than anticipated and no dose response was observed. IMPLICATIONS: While these novel estradiol-releasing combination contraceptive vaginal rings provided sustained release of contraceptive levels of Nestorone over 90 days, the E2 levels achieved were not consistent with bone protection, and a dose-response was not observed.

A dose-finding, cross-over study to evaluate the effect of a Nestorone®/Estradiol transdermal gel delivery on ovulation suppression in normal ovulating women.

OBJECTIVE: This study aims to determine the lowest effective of three Nestorone (NES)/estradiol (E2) transdermal gel doses to ensure ovulation suppression in 90-95% of cycles. METHODS: This was a randomized, open-label, three-treatment-period cross-over study to evaluate the effects of NES/E2 transdermal gel on ovulation inhibition, suppression of follicular growth and pharmacokinetic parameters. The doses were low (1.5 mg NES/0.5 mg E2), medium (3.0 mg NES/1.0 mg E2) and high (4.5 mg NES/1.5 mg E2). Participants applied gel daily to a fixed area on the abdomen for 21 consecutive days. They were interviewed regarding their experiences using the gel. RESULTS: Eighteen participants were randomized; 16 completed the study. Median NES C(max) values for low, medium and high dose groups at day 21 were 318.6 pmol/L, 783.0 pmol/L and 1063.8 pmol/L, respectively. Median maximum follicular diameter was higher with the lowest dose with 16.2 mm versus 10.0 and 10.4 mm with the medium and high doses, respectively. Among adherent participants, ovulation was inhibited in all dose groups, except for one participant in the medium dose (6.7%) that had luteal activity and an ultrasound image suggestive of a luteinized unruptured follicle. There were few reports of unscheduled bleeding, with more episodes reported for the lower dose. Adverse events were mild, and no skin irritation was reported from gel application. CONCLUSION: While all three doses blocked ovulation effectively and were evaluated as safe and acceptable, the medium dose was considered the lowest effective dose based on a more adequate suppression of follicular development. Further development of this novel contraceptive delivering NES and E2 is warranted and has potential for improved safety compared to ethinyl-estradiol-based methods.

Single-dose pharmacokinetics of Nestorone, a potential female-contraceptive.

A synthetic progestin Nestorone is being developed for female-contraception. This study was conducted to determine the distribution, metabolism, and excretion of tritium-labeled Nestorone ((3)H Nestorone) in adult female rats. Rats were injected subcutaneously (S.C.) with a single dose of 400 microCi (3)H Nestorone/kg BW. Its distribution and concentrations in blood, plasma and other tissues were determined at defined times. The excreta were examined for elimination of (3)H Nestorone. Radioactivity in all samples was analyzed by liquid scintillation counter. Metabolite profiling was performed by HPLC and LC/MS analysis of the plasma, urine, and feces samples. Following subcutaneous injection of (3)H Nestorone, the mean peak concentrations of radioactivity (C(max)) in the blood and plasma were 58.1 and 95.5 ng equiv. (3)H Nestorone/g, respectively, at 2-h postdose (T(max)). Thereafter, the concentration of drug steadily declined through 96-h postdose with a terminal elimination half-life (t(1/2)) of 15.6 h. (3)H Nestorone-derived radioactivity was widely distributed in most tissues by 0.5 h and attained a mean maximal concentration by 2-h postdose. Approximately, 81.4% and 7.62% of the administered dose was excreted via feces and urine, respectively. In vivo metabolism of (3)H Nestorone resulted into a total of 19 metabolites. Among them, two metabolites viz., 17alpha-deacetyl-Nestorone (M9) and 4,5-dihydro-17alpha-deacetyl-Nestorone (M19) were identified by HPLC and LC/MS analysis. Metabolite profiling of plasma samples showed that most of the circulating radioactivity was associated with unchanged parent drug, and M19. The M19 was a major metabolite in the profiled urine and feces samples. Presence of large proportion of drug/drug-related material in feces suggested that the biliary excretion is a main elimination route of (3)H Nestorone. The distribution, metabolism, and excretion profiles of (3)H Nestorone obtained in this study provide a fairly good insight about its fate in women.

An initial pharmacokinetic study with a Metered Dose Transdermal Systemfor delivery of the progestogen Nestorone as a possible future contraceptive.

BACKGROUND: Transdermal delivery of steroids is gaining popularity for contraception and hormone replacement therapy. This study aimed to test metered spray delivery of a precise dosage of Nestorone (NES) progestogen as a possible transdermal progestogen-only contraceptive. STUDY DESIGN: Six healthy postmenopausal volunteers, not recently using any hormonal therapies, comprise the sample for this study. Each subject was studied on two occasions with multiple blood sampling for assay of NES over a 24-h period: on the first occasion, after a single dosage of 3 x 90 microL NES sprays using a specially devised, precisely metered delivery device; on the second occasion, following the fifth in a series of five daily transdermal dosages of 3 x 90 microL of NES spray. Conventional pharmacokinetic parameters were calculated. NES was assayed in serum using a specific radioimmunoassay. RESULTS: Mean serum levels of NES peaked at around 20 h following dosing, and levels plateaued at 285-290 pmol/L after 4-5 days of daily spray application. All subjects achieved satisfactory serum levels, although substantial intersubject variation was noted. The apparent elimination half-life of NES after the last dose on Day 5 was 26.8 h. No unexpected adverse events were encountered. CONCLUSION: This early pharmacokinetic trial of a new transdermal steroid delivery system has demonstrated the feasibility of achieving serum levels of NES sufficient to block ovulation and potentially provide effective contraception.

Use of a single implant of elcometrine (ST-1435), a nonorally active progestin, as a long acting contraceptive for postpartum nursing women.

Because of its unique features, the contraceptive effectiveness and tolerance during breast-feeding of 16-methylene-17 alpha-acetoxy-19-nor-4-pregnene-3,20-dione (elcometrine), delivered within a single subdermal capsule of medical grade polydimethylsiloxane, was investigated. Unlike other progestational steroids, elcometrine has no affinity for androgen and estrogen receptors and is inactive by the oral route. A total of 66 breast-feeding women receiving elcometrine by the subdermal route were enrolled in the study, and 69 women who elected to use Copper-T380 intrauterine devices (IUD) served as control subjects. The women and their infants were observed until the end of the first postpartum year. There were no significant differences in growth and development measurements among the infants in the elcometrine and control groups. The percentage of infants continuing to breast-feed at 3 and 6 months was significantly higher in the elcometrine group. There were no significant differences between the concentration of elcometrine in the mother's blood and milk. At 75 days, blood levels of elcometrine in the infants were near the undetectable and were significantly lower than the levels in maternal blood or milk (p < 0.01). In 15 of 25 infants, blood levels of elcometrine were at the limit of assay sensitivity or undetectable. Two pregnancies occurred in women using IUD, whereas none occurred in those using implants. There were menstrual bleeding irregularities in both groups. A single elcometrine capsule placed subcutaneously at 6-monthly intervals appears to be an effective method of contraception for lactating women and results in blood concentrations of nursing infants at or near undetectable levels.

PIP: Discusses the effects of the use of a single implant of elcometrine (ST-1435), a nonorally active progestin, as a long acting contraceptive for postpartum nursing women in Maternidade Climerio de Oliveira in Salvador, Bahia, Brazil. A total of 135 women aged 18-35 years having a singleton term delivery, fully breast-feeding on demand, planning to breast-feed for 6 months postpartum, and requesting effective contraception were recruited. The method was initiated for 6 weeks postpartum. Results showed that 66 breast-feeding women used elcometrine implants, while 69 women preferred an IUD insertion. The contraceptive efficacy of lactation is high for women breast-feeding on demand, particularly in those who remain in amenorrhea during the 6 months postpartum; in these women, elcometrine implant and IUD prevented pregnancy effectively. Breast-feeding was significantly higher (p 0.05) in the elcometrine group at 3 and 6 months, while at 9 and 12 months there was no statistical difference. The differences in type, frequency of supplementary feeding, and anthropometric measures between the two contraceptive groups were not significant. Menstrual irregularities were present in both groups. Thus, elcometrine could be best alternative and the most effective method of contraception for lactating women.

Synthesis and organ distribution of [18F]fluoro-Org 6141 in the rat: a potential glucocorticoid receptor ligand for positron emission tomography.

For the synthesis of [18F]Fluoro-Org 6141 via a nucleophilic substitution reaction with 18F-, the tosyl group was chosen as the leaving group because of its stability and excellent leaving group ability. The biodistribution of the high affinity and moderate lipophilicity (log P = 2.66, calculated value) ligand [18F]Fluoro-Org 6141 (specific activity 8.2 to 37 TBq/mmol, yield 10% at EOB) was examined in sham adrenalectomized (sADX) and adrenalectomized (ADX) male Wistar rats. Two days after ADX or sADX, the animals were anesthetized and 0.37 to 1.85 MBq of [18F]Fluoro-Org 6141 was administered intravenously. Kinetics of 18F activity uptake were monitored for 3 h using a stationary double-headed positron emission tomography (PET) camera, and the biodistribution was assessed by ex vivo determination of radioactivity in several tissues and different brain areas. One hour after injection of the radioligand, the bladder, kidney, liver, trachea, and bone of sADX animals contained more concentration on a wet weight basis than blood. Three hours post injection, radioactivity was retained in bladder, trachea, and bone. The accumulation of radioactivity in brain corresponded to the concentration of activity in the blood within the first hours after injection. ADX animals showed a higher uptake of 18F activity in spleen, testes, and brain areas (hippocampus and brainstem) but a lower uptake in bone than sADX rats. PET scans suggested that 18F activity uptake in the brain had not yet reached a maximum at this interval. Although [18F]Fluoro-Org 6141 is not useful for PET studies of glucocorticoid receptors (GRs), the results obtained with this compound indicate a synthetic strategy suitable for the synthesis of high-affinity radioligands for GRs.

Metabolism of a [18F]fluorine labeled progestin (21-[18F]fluoro-16 alpha-ethyl-19-norprogesterone) in humans: a clue for future investigations.

Assessment of estrogen receptors and progesterone receptors (PR) with PET may allow the determination of the hormone responsiveness of tumors without the need for multiple biopsies, and the monitoring of the effect of hormonal therapy. In spite of the favourable characteristics of 21-[18F]fluoro-16 alpha-ethyl-19-norprogesterone ([18F]FENP) found in preclinical studies, the compound failed to reveal the presence of PR in breast carcinomas and meningiomas. In view of the clinical significance of the PR assay in human breast cancer, it is worthwhile to explore mechanisms that are potentially involved in the inadequacy of [18F]FENP to image PR with PET. Our present study on the in vivo metabolism of [18F]FENP in humans demonstrates a rapid clearance and biotransformation of the compound. Results of incubation experiments suggest that the metabolic conversion of [18F]FENP is not restricted to the liver, but also occurs in blood cells (presumably the erythrocytes) and tumors (breast carcinomas and meningiomas). The predominant metabolite of [18F]FENP in plasma during the rapid distribution phase and in tumors is identified as 20-dihydro-[18F]FENP. The conversion of [18F]FENP to its 20 alpha- or 20 beta-hydroxy metabolite has a deleterious effect on the binding affinity for PR. Our findings do not justify a conclusion as to the extent of in vivo extrahepatic biotransformation of [18F]FENP, or its significance in the ineffectiveness of [18F]FENP as an imaging agent for PR. On the other hand, the ability of breast carcinomas and meningiomas to metabolize [18F]FENP avidly appears to preclude selective imaging of PR in these tumors during the time of a PET examination. It is imperative to evaluate the metabolic stability of a [18F]fluorine labeled progestin in an early stage of future screening procedures.

The progestin ST 1435--rapid metabolism in man.

Parenterally administered ST 1435 (Nestorone) is highly potent for contraception, and ovulation can be inhibited with very low serum levels of ST 1435. Orally administered ST 1435 is ineffective in various laboratory animals, presumably due to extensive first-pass metabolism. Thus, ST 1435 has been proposed for lactational contraception, to be metabolized by the suckling infant. We have studied the metabolism of ST 1435 in female volunteers following oral (10 mg), intravenous (iv) (0.1 mg) and transdermal (4.5-9.0 mg) routes of ST 1435 administration. Preliminary studies using rats were performed to develop the methodology of high performance-liquid chromatography (HPLC) fractionation and ST 1435-RIA detection. Rat portal serum revealed 4 distinct peaks of immunoreactive material with the retention times (Rt's) of 7.5, 10, 14.5 and 17.5 min (ST 1435 = 10 min). In systemic serum, only the peak with the Rt of 7.5 min could be detected. Therefore, orally administered ST 1435 is very effectively metabolized by the rat liver; this also explains the previously observed lack of biological effects of oral ST 1435. Following oral administration of ST 1435 to two women, the Rt of the major peak was 10 min. The magnitude of the ST 1435 peak decreased rapidly, and at 24h following ingestion, no ST 1435 could be detected by this method. The t1/2 of ST 1435 was approximately 1-2h. In addition, two minor peaks with Rt's of 4.5 and 16 min could be detected with the ST 1435 RIA at 1-4h following oral ingestion. Competitive receptor binding assays using the human uterine progesterone receptors (hPR) revealed that the ST 1435 fraction exhibits strong binding affinity towards the hPR; thus, in the human, a small fraction of biologically active ST 1435 seems to escape from the first-pass metabolism following oral intake. Following iv and transdermal administration of ST 1435, the only detectable peak with ST 1435-RIA was that of ST 1435. Similar magnitude of the ST 1435 peaks following oral administration of 10 mg and iv administration of 0.1 mg indicated that the bioavailability of ST 1435 is low. These data seem to confirm the suspicion that orally administered ST 1435 is also rapidly metabolized in the human, therefore encouraging further evaluation of ST 1435 during lactation. However, the rapid metabolism seen after oral intake can be successfully circumvented by sustained parenteral administration of ST 1435.

Progestin radiopharmaceuticals labeled with technetium and rhenium: synthesis, binding affinity, and in vivo distribution of a new progestin N2S2-metal conjugate.

We have prepared and evaluated three metal conjugates of a progestin-monoamine-monoamide (MAMA') bisthiol chelate system. These conjugates of rhenium and technetium-99 and -99m, are structural analogs of the bisamino-bisthiol (BAT) conjugates we have described recently, but the MAMA' chelate, being more polar than the BAT system, gives a conjugate that is much less lipophilic, having an octanol-water partition coefficient that is nearly 80-fold lower. In competitive binding assays, the Re- and 99Tc-MAMA'-progestin conjugates bind to the progesterone receptor with affinities greater than that of progesterone itself, and in a direct binding assay, the equilibrium dissociation constant (Kd) of the 99mTc-MAMA' conjugate was 0.97 nM. As is typical for 11 beta-substituted progestins, these conjugates also have substantial binding affinity for glucocorticoid receptors. In tissue distribution studies in immature female rats, the progestin-99mTc-MAMA' conjugates show selective uptake for principal target tissue (such as uterus) over that of blood and nontarget tissue (such as muscle); these uptake ratios reach maximum levels of 5 and 4, respectively. Uptake by fat, liver, and kidney is quite high; however, only the uptake in uterus is displaceable upon coinjection of the selective progestin ORG2058. Metabolism studies show that the radioactivity in the uterus is essentially unmetabolized out to 4 h, while liver activity is completely due to metabolites. Other tissues show an intermediate fraction of unmetabolized conjugates that decreases with time. The in vivo behavior of the progestin-99mTc-MAMA' conjugate is similar to that of the labeled BAT conjugate: its uptake selectivity is somewhat greater than that of the BAT conjugate, but its target tissue uptake is lower. Factors that may be responsible for limiting the target tissue uptake properties of these conjugates are their moderate affinity for progesterone receptor, their substantial binding to glucorticoid receptors, and their large overall molecular size.

Pharmacokinetics and bioavailability of ST 1435 administered by different routes.

The ovulation inhibiting potency of the synthetic progestin ST 1435 (Nestorone) is high after parenteral administration and practically nil after oral administration. The purpose of this study was to determine the pharmacokinetic parameters of ST 1435 after single oral or intravenous administration or after long-term treatment with subdermal implants in women. After administration, as a single i.v. bolus, the plasma disappearance rate of immunoreactive ST 1435 had two components with half-lives (mean +/- SE) of 3.5 +/- 0.5 and 83 +/- 14 min, respectively. The volume of distribution was 4.7 +/- 1.3 L/Kg and the metabolic clearance rate was 55 +/- 6 L/Kg/d. After oral administration, the bioavailability was about 10% of the dose. After chronic subdermal administration, the plasmatic clearance was slower than following the acute doses. These results show that ST 1435 has shorter half-lives and a faster clearance rate than progestins which bind SHBG. The large volume of distribution indicates accumulation in the extravascular space and was expected in view of the high affinity of ST 1435 for progesterone receptors. The slower plasma elimination rate after chronic administration was attributed to the re-entry of a larger mass of drug from the extravascular space, and/or accumulation of immunoreactive metabolites with slower clearance than the parent steroid.

Contraception with subdermal implants releasing the progestin ST-1435: a dose-finding study.

A new modified subdermal implant releasing the potent progestin ST-1435 was studied in eleven fertile-aged women. These implants have been developed for contraception and they have a life-time of two years. Three implant lengths of 4, 6 and 8 cm were tested to find the optimal steroid dose for inhibition of ovulation. Serum samples were collected twice per week during a six-week period every six months. The concentrations of serum ST-1435, estradiol and progesterone were determined by RIA. Ovulation was inhibited by all ST-1435 doses tested. The concentration of serum progesterone was below 6 nmol/l in all samples tested showing the absence of luteinization. The concentration of serum ST-1435 increased with increasing ST-1435 dose. Serum estradiol concentrations were quite variable, showing wide range and occasional high peak values typical of progestin treatment; the mean value of serum estradiol concentrations measured did not differ with different ST-1435 doses. The results of steroid determinations led to the conclusion that a single 4 cm subdermal implant is optimal for contraception. With this dosage level, ovulation is inhibited and side effects are minimized. Bleeding control was variable. No hormonal side effects due to the progestin ST-1435 were reported. This method, using a single 4 cm subcutaneous implant releasing the progestin ST-1435 with a life-time of two years, represents a promising alternative for inhibition of ovulation and contraception.

Transdermal absorption of the progestin ST-1435: therapeutic serum steroid concentrations and high excretion of the steroid in saliva.

The synthetic progestin ST-1435 was administered transdermally to six healthy women during the late luteal phase. The steroid was applied to the periumbilical area in a commercial gel ("Progestogel"), also containing progesterone. Single doses of 2.3, 4.5 and 9.0 mg of ST-1435 were given in three experiments and repeated doses of 2.3 mg of ST-1435 for five days were given in another three experiments. Samples of serum and saliva were collected and the concentrations of ST-1435 and progesterone were determined by radioimmunoassays. Transdermal absorption of the synthetic progestin ST-1435 was shown to result in serum steroid concentrations high enough for therapeutic purposes. The concentration of ST-1435 in serum was still high 24 hours after application, reflecting sustained release of the steroid from the skin. In the five-day experiments, relatively constant serum levels were achieved in different individuals, and a single dose per day seems sufficient. High excretion of ST-1435 in saliva was found two hours after gel application. However, the concentrations of serum and salivary ST-1435 were not directly correlated in different individuals. Transdermal application of ST-1435 might offer a good alternative for systemic progestin treatment.

Preclinical evaluation of a positron emitting progestin ([18F]fluoro-16 alpha-methyl-19-norprogesterone) for imaging progesterone receptor positive tumours with positron emission tomography.

Three 21-fluoro-progestins were investigated as potential imaging agents for the in vivo assessment of human progesterone receptor positive neoplasms with positron emission tomography. In competitive binding assays these compounds demonstrated high specificity, competing only for progesterone receptors. Binding to other steroid receptor types was negligible. Based on its high affinity binding, 21-fluoro-16 alpha-methyl-19-norprogesterone was selected for further evaluation in vivo. Tissue distribution studies in immature estrogen primed female rats revealed high uterine uptake of 21-[18F]fluoro-16 alpha-methyl-19-norprogesterone ([18F]FMNP). At 60 min after injection the ratio of uptake of radioactivity by uterine tissue to that of blood was 7. This ratio increased to 24 at 180 min. A selective decrease in uterine uptake was observed after administration of [18F]FMNP with excess unlabelled progestin. Rats bearing hormone responsive MT-W9A mammary adenocarcinomas were used to examine [18F]FMNP for tumour uptake. Animals were used irrespective of the phase of the estrous cycle. At 180 min the uterus to blood ratio and the tumour to blood ratio ranged from 3 to 20 and 3 to 17, respectively. Uterine and tumour tissue was assayed for cytosolic estrogen and progesterone receptors using a dextran-coated charcoal method and Scatchard plot analysis. The results indicate that the in vivo uptake of [18F]FMNP by uterine and mammary tumour tissue correlates well with the progesterone receptor concentration (rs = 0.98 and rs = 0.88, respectively). It is concluded that the uptake of [18F]FMNP by progesterone receptor positive tissue in vivo is primarily receptor related and that this uptake is attributable to the progesterone receptor. The study demonstrates the potential applicability of [18F]FMNP and positron emission tomography for imaging progesterone receptor positive neoplasms.

Investigations on the in vitro metabolism of five synthetic 19-norprogestins using hepatocyte suspensions isolated from five laboratory animal species.

Five synthetic progestins of the 19-nortestosterone type (norethisterone, NET; levonorgestrel, LN; gestodene, GEST; NET-3-oxime, NETO; norgestimate, NGM) were investigated in the in vitro hepatocyte model. Radiolabelled progestins were added to hepatocyte suspensions (3 x 10(6) cells/ml) freshly prepared from female rat, guinea pig, rabbit, dog (beagle) and cynomolgus monkey. Drug level decreases (NET, LN, GEST) and prodrug conversions (NETO, NGM) were followed by radiochromatography (HPLC) for 60 min. In the case of NET and NETO the conversion into ethinyl estradiol (EE2) was quantified by RIA after HPLC separation. Half-lives of drug level decreases (t1/2), areas under the curves (AUC) and metabolic clearance rates (MCR) were estimated for all progestins. For NETO and NGM the percentages of conversion into NET and LN were calculated, respectively, and levels of EE2 determined in the case of NET and NETO. Rat hepatocytes showed an extremely high metabolic activity towards NET, LN and GEST resulting in t1/2 values of below 2 min. Respective values for rabbit hepatocytes ranged from 5-8 min, whereas half-lives calculated for liver cells from guinea pig, dog and monkey were generally above 30 min. A drastic increase in t1/2 was found for NETO (as compared to NET) in hepatocytes from rat, rabbit and monkey but not from guinea pig. Dog hepatocytes degraded NETO about 3 times more rapidly than NET. NGM was degraded much faster than LN in hepatocytes from all species except the rat. Liver cells from guinea pig and dog seem to be able to metabolize the 3-oxime group much more rapidly than hepatocytes from the other animal species. The lowest degree of prodrug conversion of 4% was observed for NGM and dog hepatocytes. Elevated EE2 levels were found in all experiments with NET and NETO. Results of NET, LN and GEST were compared with published in vivo experiments. No correlations were found for t1/2, MCR, and AUC.

21-[18F]fluoro-16 alpha-ethyl-19-norprogesterone: synthesis and target tissue selective uptake of a progestin receptor based radiotracer for positron emission tomography.

We have synthesized 21-[18F]fluoro-16 alpha-ethyl-19-norprogesterone (FENP), a high affinity ligand for the progesterone receptor, labeled with the positron-emitting radionuclide fluorine-18 (t1/2 = 110 min). The synthesis proceeds in two steps from 21-hydroxy-16 alpha-ethyl-19-norprogesterone and involves [18F]fluoride ion displacement of the 21-trifluoromethanesulfonate (21-triflate). This material is purified by HPLC and is obtained in 4-30% overall yield (decay corrected) within 40 min after the end of bombardment to produce [18F]fluoride ion. The effective specific activity, determined by competitive radioreceptor binding assays, is 700-1400 Ci/mmol. In vivo, [18F]FENP demonstrates highly selective, receptor-mediated uptake by the uterus of estrogen-primed rats; the uterus to blood and uterus to muscle ratios were respectively 26 and 16 at 1 h and 71 and 41 at 3 h after injection. The high target tissue selectivity of this uptake suggests that this compound may be useful for the in vivo imaging of progestin target tissues and receptor-rich tumors (such as human breast tumors) by positron emission tomography.