Ultra-performance liquid chromatography-tandem mass spectrometry assay for determination of plasma nomegestrol acetate and estradiol in healthy postmenopausal women.

A highly sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry method is described for the simultaneous determination of nomegestrol acetate (NOMAC), a highly selective progestogen, and estradiol (E2), a natural estrogen in human plasma. NOMAC was obtained from plasma by solid-phase extraction, while E2 was first separated by liquid-liquid extraction with methyl tert-butyl ether followed by derivatization with dansyl chloride. Deuterated internal standards, NOMAC-d5 and E2-d4 were used for better control of extraction conditions and ionization efficiency. The assay recovery of the analytes was within 90-99%. The analytes were separated on UPLC BEH C18 (50 × 2.1 mm, 1.7 µm) column using a mobile phase comprising of acetonitrile and 3.0 mm ammonium trifluoroacetate in water (80:20, v/v) with a resolution factor (Rs ) of 3.21. The calibration curves were linear from 0.01 to 10.0 ng/mL for NOMAC and from 1.00 to 1000 pg/mL for E2, respectively. The intra- and inter-batch precision was ≤5.8% and the accuracy of quality control samples ranged from 96.7 to 103.4% for both analytes. The practical applicability of the method is demonstrated by analyzing samples from 18 healthy postmenopausal women after oral administration of 2.5 mg nomegestrol acetate and 1.5 mg estradiol film-coated tablets under fasting.

The sexuological impact of hormonal contraceptives based on their route of administration.

Evidence on the effects of hormonal contraceptives on female sexuality is conflicting. We enrolled 556 women, divided into six groups: two composed of subjects using a combined hormonal contraceptive (COC) containing 0.020 ("COC20") and 0.030 ("COC30") mg of ethynyl estradiol (EE), "natural", using COC containing 1.5 mg of estradiol (E2), "ring", using a vaginal ring releasing each day 0.015 mg of EE + 0.120 of etonogestrel, "subcutaneous", using a progestin only subcutaneous contraceptive implant releasing etonogestrel and "controls", using no hormonal contraceptive methods. The subjects were required to answer to the McCoy female sexuality questionnaire and were subjected to a blood test for hormonal evaluation. An ultrasound evaluation of the dorsal clitoral artery was also performed. The higher McCoy sexological value were recorded in the subdermal group; significant differences were recorded among the groups in terms of hormone distribution, with the higher levels of androstenedione in subdermal and control groups. The ultrasound evaluation of dorsal clitoral artery shows a significative correlation between pulsatility and resistance indices and orgasm parameters of McCoy questionnaire. The recorded difference in the sexual and hormonal parameters among the studied hormonal contraceptives may guide toward the personalization of contraceptive choice.

Prediction of nomegestrol acetate pharmacokinetics in healthy female adolescents and adults by whole-body physiology-based pharmacokinetic modelling and clinical validation.

OBJECTIVE: Nomegestrol acetate (NOMAC), a selective progestogen, and 17ß-estradiol (E2), which is identical to endogenous oestrogen, are components of a new monophasic combined oral contraceptive--NOMAC/E2. This study aimed to compare pharmacokinetics (PK) of NOMAC in adolescent and adult women following a single dose of NOMAC/E2. STUDY DESIGN: Healthy postmenarcheal adolescent (14-17years) and adult (18-50years) women received a single dose of NOMAC/E2 (2.5mg/1.5mg) in this single-centre, open-label, parallel-group Phase 1 study (EudraCT# 2008-002142-38). Blood samples were obtained for PK analysis, and concentrations of NOMAC, E2 and its metabolite estrone (E1) were determined for up to 129h following dosing to obtain PK data. An independent whole-body physiology-based pharmacokinetic (WB-PBPK) simulation model of NOMAC based on an independent Phase 3 dataset was used to scale NOMAC concentration-time plots to adolescents. RESULTS: Overall, 52 women were screened, of whom 30 (15 adolescents and 15 adults) were enrolled. No statistically significant differences were observed between the adolescent and adult groups for the clinically evaluated NOMAC PK parameters [maximum concentration (Cmax), area under the curve (AUC) and half-life (t1/2)]. The PK of E2 and E1 showed extensive overlap between both age groups. The WB-PBPK model accurately predicted NOMAC AUC and Cmax values in both groups. CONCLUSIONS: No differences were observed in the clinically evaluated PK parameters for NOMAC between adolescent and adult women after a single dose of NOMAC/E2. The WB-PBPK model accurately predicted NOMAC PK data (EudraCT# 2008-002142-38). IMPLICATIONS: PK studies in adolescents are challenging because of ethical considerations. The whole-body physiology-based model described here complements classic noncompartmental and population PK approaches. The utility of this method is its ability to expand to adolescent postmenarcheal girls by using virtual postmenarcheal adolescent population data and applying physiological scaling.

LC-MS/MS method for determination of megestrol in human plasma and its application in bioequivalence study.

A rapid and highly selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of megestrol in human plasma was described using medrysone as internal standard (IS). Blood samples were collected from 20 healthy volunteers after oral administration of 160 mg megestrol acetate dispersible tablets. The analytes were extracted by liquid-liquid extraction procedure and separated on a hanbon lichrospher column with the mobile phase of methanol and water containing 0.1% formic acid and 20 mmol/L ammonium acetate (5:1, v/v). Positive ion electrospray ionization with multiple reaction-monitoring mode (MRM) was employed by monitoring the transitions m/z 385.5-325.4 and m/z 387.5-327.4 for megestrol and medrysone, respectively. Under the isocratic separation conditions, the chromatographic run time was approximately 2.54 min for megestrol and 2.59 min for medrysone. The calibration curve range was from 0.5 to 200.0 ng/mL. The inter-batch and intra-batch precision and accuracy were less than 5.2% relative standard deviation (RSD) and 6.4% relative error (RE). The proposed method was successfully applied in the bioequivalence study of megestrol acetate dispersible tablets.

Pharmacokinetic profile of nomegestrol acetate and 17ß-estradiol after multiple and single dosing in healthy women.

BACKGROUND: The pharmacokinetics of the monophasic oral contraceptive nomegestrol acetate (NOMAC) plus 17ß-estradiol (E(2)) were investigated after a single dose and multiple dosing. STUDY DESIGN: NOMAC/E2 (2.5 mg/1.5 mg) was administered daily to healthy women (18-50 years, n=23) for 24 days; blood samples for pharmacokinetic analysis were obtained on Day 24 and again, after a 10-day pill-free interval, on Day 35 after a single dose. RESULTS: NOMAC reached steady state after 5 days with mean ±standard deviation (SD) trough NOMAC concentration (C(av)) of 4.4±1.4 ng/mL. On Day 24, mean±SD peak NOMAC concentration (Cmax, 12.3±3.5 ng/mL) was reached in mean 1.5 h (t(max)); the mean±SD elimination half-life (t(½)) was 45.9±15.3 h. After a single dose, NOMAC mean±SD C(max) was 7.2±2.0 ng/mL and mean±SD t(½) was 41.9±16.2 h. On Day 24, E2 mean±SD C(av) was 50.3±25.7 pg/mL; mean±SD Cmax was 86.0±51.3 pg/mL. After a single dose, mean±SD E2 Cmax was 253±179 pg/mL. CONCLUSIONS: These data demonstrate that NOMAC/E2 has a pharmacokinetic profile consistent with once-daily dosing.

Megestrol-induced Cushing syndrome.

OBJECTIVE: To report a case of Cushing syndrome associated with megestrol acetate therapy in a patient with renal insufficiency. SUMMARY: A 17-year-old boy with renal insufficiency due to unilateral renal agenesis developed Cushing syndrome and worsening of his renal function after megestrol acetate therapy. The diagnosis was based on clinical and analytical evaluation. DISCUSSION: Megestrol acetate is indicated for the treatment of cachexia associated with AIDS and malignancy. Due to its glucocorticoid activity, megestrol use has resulted in the occurrence of Cushing syndrome in both patient groups. We report the case of a young patient with renal insufficiency due to unilateral renal agenesis who developed Cushing syndrome two months after administration of high-dose (900-mg/d) megestrol acetate for an eating disorder. CONCLUSIONS: The risk of megestrol-induced Cushing syndrome, especially with high doses of the medication, should be considered as a possible adverse effect in patents with renal insufficiency.

Pharmacokinetic evaluation of two different formulations of megestrol acetate in patients with advanced malignancies.

The bioequivalence of two megestrol acetate formulations, 160-mg "tablets" and 160-mg "sachets," was investigated in a single-dose, open-label, balanced-for-sequence cross-over study involving 12 advanced-cancer patients. The observed plasma megestrol-acetate time course obtained with both formulations was consistent with the literature data. The main source of variability in the pharmacokinetic parameters was intersubject variability; drug formulation played only a minor (and nonsignificant) role. The width of the 90% confidence interval of the area-under-the-curve (AUC) ratio (sachets: tablets) computed according to Schuirmann (0.9-1.4) was mainly due to the presence of a single outlier, showing an AUC ratio of 2.7. The trend to higher bioavailability of the new formulation was not significant, especially as compared with the dose-response data reported in the literature.

Enzyme immunoassay for nomegestrol acetate in human plasma.

Currently available chromatographic assays of the progestative drug nomegestrol acetate in human plasma are not suitable for monitoring drug kinetics more than 24 h after clinical dosage. A specific and sensitive enzyme immunoassay was therefore developed. A 3(O-carboxymethyl)oxime derivative of nomegestrol acetate was synthesized and coupled to bovine serum albumin in order to raise polyclonal antibodies in rabbits. The enzymatic tracer was obtained by coupling the 3(O-carboxymethyl)oxime derivative to acetylcholinesterase (E.C.3.1.1.7.). HPLC fractionation of human plasma samples followed by enzyme immunoassay revealed the presence of cross-reacting metabolites. An automated procedure of metabolite separation was developed using silica bonded with diol groups (Diol Bakerbond column). This procedure ensured assay specificity. The quantification limit in human plasma was 0.1 ng/ml. Mean repeatability (intra-assay variation) and reproducibility (inter-assay variation) were 9 and 15%, respectively. The enzyme immunoassay allowed monitoring of the kinetics of nomegestrol acetate 144 h after oral administration of a single 5 mg dose. Values for human samples were in excellent agreement with those assayable by HPLC followed by u.v. detection.

Determination of megestrol acetate and cyproterone acetate in serum of patients with advanced breast cancer by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic method with ultraviolet detection of megestrol acetate and cyproterone acetate in human sera is described. The proposed assay is linear up to 1400 ng/ml (r = 0.999) and has a detection limit of 5 ng/ml. Recoveries of both compounds in spiked sera were ca. 95%; inter-assay coefficients of variation were 4.0 and 3.1% and intra-assay values were 1.3 and 1.4%, respectively. For validation of the method we also developed a gas chromatographic-mass spectrometric method for both steroids. The results obtained by the two methods showed good correlation: for megestrol acetate r = 0.98, n = 31, p less than 0.0001, and for cyproterone acetate r = 0.94, n = 0, p less than 0.0001. Large inter-individual differences in the serum concentrations of both substances were found in groups of patients with metastatic breast cancer receiving the same oral load of either steroid.

Influence of aminoglutethimide on the metabolism of medroxyprogesterone acetate and megestrol acetate in postmenopausal patients with advanced breast cancer.

In this study the influence of amino-glutethimide (AG) on the disposition of medroxyprogesterone acetate (MPA) and megestrol acetate (MA) was studied. When 1,000 mg AG daily was supplementally given to six patients on chronic treatment with MPA (1,000 mg/day) or MA (160 mg/day), mean serum levels of progestin were reduced by 74% as compared with control levels (P less than 0.03). AG did not change the blood clearance rate of MPA when the latter was given i.v. This discrepancy between AG's influence on oral and parenteral progestin disposition could be explained by pharmacokinetic properties of the progestins, and our results suggest that AG stimulates the metabolism of progestins. The decrease in MPA and MA serum levels was accompanied by an increase in serum cortisol, sex hormone-binding globulin (SHBG) and testosterone levels. This suggests that AG reduces the biological activity of progestins.

Oral high-dose progestins as treatment for advanced breast cancer.

Fifty-two postmenopausal, previously treated advanced breast cancer patients who received oral high-dose progestins (medroxyprogesterone acetate [MPA] and/or megestrol acetate [MA]) were retrospectively reviewed. MPA was given to 45 patients and MA to 17 (10 earlier treated with MPA); 48 were evaluable for clinical response to progestin treatment, 43 for MPA and 5 for MA. Two complete responses and 10 partial responses (25%) with median duration of 9.5 months were seen. Forty percent of the patients obtained stable disease greater than or equal to 6 months with a median duration of 8.0 months. In patients with estradiol receptor positive (n = 31) and estradiol and progesterone receptor positive (n = 19) tumors the response rates were 35% and 37% respectively. No differences in serum levels of MPA or MA were observed in the different responding groups. The serum levels of MA were twice as high as MPA in spite of a dose of 160 mg/day of MA compared to 1,000 mg/day of MPA. A long disease-free interval, and positive receptor status of primary or metastatic lesions seemed to predict response to endocrine therapy even late in a therapeutic sequence. Side effects occurred in 11/45 (24%) of MPA treated patients and in 1/15 (7%) of MA treated patients. No difference in serum levels of MPA was found between patients with side effects and patients without side effects.

Medical castration using megestrol acetate and minidose estrogen.

Sixty-two men who presented with previously untreated metastatic carcinoma of the prostate (D0: 10 patients; D1: 29 patients; D2: 23 patients) received oral megestrol acetate (80 mg twice daily) and minidose estrogen (diethylstibestrol 0.1 mg or ethinyl estradiol 0.05 mg once daily) as a means of achieving total androgen ablation (testicular and adrenal). A high incidence of feminizing side effects (70-74%), a higher than expected rate of cardiovascular complications (18%), an unexpected need for cortisone replacement (13%), and failure of patients with Stage D2 disease to obtain results better than those of standard therapy during the first year of observation suggest this regimen offers no advantage over other more conventional therapy.

Plasma concentrations of medroxyprogesterone acetate and megesterol acetate during long-term follow-up in patients treated for metastatic breast cancer.

A total of 32 patients with metastatic breast cancer responding with at least disease stabilization to treatment with two commercially available preparations of medroxyprogesterone acetate (MPA) or one preparation of megestrol acetate (MA) were followed for their plasma concentrations. The MPA and MA were measured by HPLC. MPA from Upjohn and Farmitalia was given to 12 patients (median age, 61 years; median follow-up, 20 weeks) and 8 patients (54 years, 16 weeks), respectively, on a schedule of 1000 mg daily i.m. for 10 days followed by 200 mg t.i.d.p.o. for the remainder of the treatment course. The peak concentrations (means, 163 vs 97 ng/ml), the time to peak levels (medians, 3 vs 10 weeks), and the areas under the concentration curves from time 0 to 24 weeks (means, 2400 vs 1868 ng/ml X weeks) were significantly different in the respective treatment groups (t-test; significance level, 0.05). MA from Bristol-Myers was administered orally in one daily dose of 160 mg throughout the treatment course in 12 patients (median age, 51 years; median follow-up, 20 weeks). A mean MA peak concentration of 218 ng/ml was reached after a median of 7 days. Plateau plasma levels were higher for MA than MPA.

The influence of progestins on receptor levels in breast cancer metastasis.

The influence of oral high-dose (HD) medroxyprogesterone acetate (MPA) and megestrol acetate (MA) treatment on steroid hormone receptor levels in metastatic breast tumor tissue was investigated. In ten postmenopausal women with advanced breast cancer, receptor biopsies were obtained from the same tumor before the start, and one and eight weeks after the start of oral HD progestin treatment. Endocrine treatment had been stopped in all patients at least five weeks before the start of progestin treatment. Estradiol receptor (ER) levels were reduced by 26.9% and 20.0% respectively after one and eight weeks of treatment. Progesterone receptors (PgR) were significantly reduced to undetectable levels, possibly due to MPA and MA binding to PgR. A stepwise decrease in androgen receptors (AR) was observed, indicating that the two progestins (MPA and MA) may also act through AR, and/or interfere with the replenishment of AR.

High-dose megestrol acetate therapy of ovarian carcinoma: a phase II study by the Northern California Oncology Group.

The activity of high-dose megestrol acetate was studied in 47 patients with epithelial ovarian cancers after failure of initial chemotherapy. The dose of megestrol acetate was 800 mg/d orally (PO) for 4 weeks and then 400 mg/d until tumor progression. Patients generally had far-advanced disease. Prior therapy included cisplatin, doxorubicin, and cyclophosphamide (PAC) or other cisplatin-containing regimens in 37, other combinations in eight, and single agents in only two patients. Seventeen patients (36%) developed intestinal obstructions within the first 2 months on study. Tumor histology was serous in 37, endometrioid in six, and clear-cell in two. Two thirds of the tumors were histologic grade 3, and the others were grade 2. Complete remission was obtained in one patient, with time to progression of 4 months. There were three partial remissions, with times to progression of 4, 5, and 18 months. The overall response rate (complete and partial) was 8%. Three additional patients had minor remissions (3, 5, and 8 months), and five had stable disease, for 3, 4, 5, 6, and 9 months. There was no correlation of response with grade, histologic type, or site of disease, but responding patients had a longer survival from diagnosis to protocol entry and from protocol failure to death than did nonresponding patients. The major side effect of megestrol acetate was increased appetite, which caused one patient to withdraw from the study, and resulted in a 10- to 20-kg weight gain in five patients. Plasma levels of megestrol acetate averaged 600 ng/mL in the first month of therapy and decreased to approximately 400 ng/mL at 8 and 12 weeks, after the drug dosage had been reduced. Serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were markedly lower during megestrol therapy compared with pretreatment values. Megestrol acetate at 1 microgram/mL in vitro inhibited soft agar colony formation from one of 17 specimens of ovarian carcinomas. We conclude that megestrol acetate in high doses has modest, but definite, palliative effects in some patients with advanced ovarian carcinoma in whom chemotherapy has failed. A controlled trial of megestrol plus combination chemotherapy as first-line treatment of advanced ovarian carcinoma should be considered.

Evaluation of two new megestrol acetate tablet formulations in humans.

The bioequivalence of two new investigational 160 mg tablets, one containing the regular form and the other a micronized form of megestrol acetate, was determined relative to a commercially available 40 mg tablet (Megace). The tablets were administered to 24 male subjects in a three-way cross-over study, balanced for sequence, with a week between administrations. The 40 mg tablets were administered q.i.d. at 08.00, 12.00, 18.00 and 22.00 h, while the 160 mg tablets were administered once at 08.00 h. Plasma samples were collected at appropriate times out to 96 h after administration and were analysed for megestrol acetate with a validated high performance liquid chromatographic procedure. Based on the times to maximum plasma concentrations (2.5 to 2.8 h), the absorption rate constant was the same for each of the tablets. Relative to the 40 mg q.i.d. dose, the 160 mg regular and the 160 mg micronized tablets had mean relative bioavailabilities of 97 per cent and 118 per cent, respectively.

Liquid chromatographic procedure for the quantitative analysis of megestrol acetate in human plasma.

A simple, sensitive, and reproducible high-performance liquid chromatographic (HPLC) procedure was developed for the quantitative analysis of megestrol acetate in human plasma. An internal standard, 2,3-diphenyl-1-indenone, was added to 0.5 mL of plasma followed by extraction with hexane. The residue remaining after evaporation of hexane was reconstituted in methanol and injected onto a mu-Bondapak C18 column. The column was eluted with acetonitrile:methanol:water:acetic acid (41:23:36:1), and the eluant was monitored at 280 nm. Megestrol acetate and the internal standard eluted at 6-7 and 12-14 min, respectively. The peak height ratio (megestrol acetate/internal standard) versus plasma concentration was linear over a range of 10-600 ng of megestrol acetate/mL of plasma, and the limit of detection was 5 ng/mL. The mean intra- and interassay accuracies were within 3% of the actual values. The mean intra- and interassay precision, as estimated by RSD, were 4 and 6%, respectively. Constituents in human plasma and megestrol, a possible degradation product, did not interfere in the assay. The procedure was applied to the analysis of plasma samples from subjects receiving 40 mg of Megace q.i.d.

Studies on the role of intestinal bacteria in metabolism of synthetic and natural steroid hormones.

Administration of antimicrobial agents to subjects taking oral contraceptives has been reported to lead to contraceptive failure and subsequent pregnancy. In women taking oral contraceptives antimicrobial agents could have an effect on both endogenous hormone levels and on the metabolism of the exogenously administered steroids. To investigate these possibilities, antimicrobial agents were administered for short periods to normal women taking various steroid drugs: Megestrol acetate (MA), medroxyprogesterone acetate (MPA), norethisterone (NET), a combination of NET and ethinylestradiol (EE) or a combination of lynestrenol and EE. During ampicillin administration the 24-h morning plasma concentrations of MA, MPA and NET were increased compared to the control values. In the MA and MPA experiments the afternoon values were determined and also found to be increased. In the subjects taking oral contraceptives plasma EE concentration showed a tendency to decrease during ampicillin administration on the third, fourth or fifth morning of ampicillin administration, but was never lower than the pretreatment values. In other experiments plasma estrone (E1) and estradiol (E2), urinary total E1, E2 and estriol (E3) and fecal unconjugated and conjugated E1, E2 or E3 were determined by RIA before, during and after administration of oxytetracycline (2 X 500 mg/day for 5 days) to 5 young male subjects. Furthermore urinary and fecal estrogens were determined in 1 male subject after administration of erythromycin for 6 days and in 2 normally menstruating women after tetracycline and trimethoprim administration, respectively. During treatment with antimicrobial drugs an increase in the excretion of fecal conjugated and, with the exception of the oxytetracycline experiments, also of unconjugated estrogens paralleled a decrease in urinary estrogen excretion, especially for E2 and E3. In both urine and feces the E1/E2 and E1 + E2/E3 ratios increased due to diminished reductive metabolism of estrogens in the gut. No significant effects on plasma unconjugated estrogen concentrations were observed. The results suggest that the intestinal bacterial flora plays a significant role in estrogen metabolism. However, further studies are necessary, because our results do not explain why administration of antibiotics may cause contraceptive failure.

PIP: Administration of antimicrobial agents to subjects taking oral contraceptives (OCs) has been reported to lead to contraceptive failure and subsequent pregnancy. In women taking OCs, antimicrobial agents could have an effect on both endogenous hormone levels and on the metabolism of the exogenously administered steroids. To investigate these possibilities, antimicrobial agents were administered for short periods to normal women taking various steroid drugs: megestrol acetate (MA), medroxyprogesterone acetate (MPA), norethisterone (NET), a combination of NET and ethinyl estradiol (EE), or a combination of lynestrenol and EE. During ampicillin administration, the 24 hour morning plasma concentrations of MA, MPA, and NET were increased compared to control values. In the MA and MPA experiments, the afternoon values were determined and also found to be increased. In the subjects taking OCs, plasma EE concentration showed a tendency to decrease during ampicillin administration on the 3rd, 4th, or 5th mornings of ampicillin administration, but was never lower than the pretreatment values. In other experiments, plasma estrone (E1) and estradiol (E2), urinary total E1, E2, and estriol (E3) and fecal unconjugated and conjugated E1, E2, or E3 were determined by RIA before, during, and after administration of oxytetracycline (2x500 mcg/day for 5 days) to 5 young male subjects. Furthermore, urinary and fecal estrogens were determined in 1 male subject after erythromycin administration for 6 days and in 2 normally menstruating women after tetracycline and trimethoprim administration respectively. During treatment with antimicrobial drugs, an increase in the excretion of fecal conjugated and, with the exception of oxytetracycline experiments, also of unconjugated estrogens, paralleled a decrease in urinary estrogen excretion, especially for E2 and E3. In both urine and feces, the E1/E2 and E1+E2/E3 ratios increased due to diminished reductive metabolism of estrogens in the gut. No significant effects on plasma unconjugated estrogen concentrations were observed. The results suggest that the intestinal bacterial flora plays a significant role in estrogen metabolism. However, further studies are necessary since these results do not explain why antibiotic administration causes contraceptive failure.