Quantitative determination of medroxyprogesterone acetate in plasma by liquid chromatography/electrospray ion trap mass spectrometry.

A sensitive and rapid liquid chromatography/electrospray ion trap mass spectrometry (LC/MS/MS) method has been developed for the quantitative determination of medroxyprogesterone acetate (MPA) in human plasma. Plasma samples (1.0 mL) were simply extracted with pentane and the extracts were analyzed by HPLC with the detection of the analyte in the selective reaction monitoring (SRM) mode. The determination of MPA was accurate and reproducible, with a limit of quantitation of 0.05 ng/mL in plasma. The standard calibration curve for MPA was linear (r = 0.998) over the concentration range 0.05-6.0 ng/mL in human plasma. Analysis precision over the concentration range of MPA was lower than 18.8% (relative standard deviation, RSD) and accuracy was between 96.2 and 108.7%.

Pharmacokinetics of 9alpha-fluoromedroxyprogesterone acetate in rats: comparison with medroxyprogesterone acetate.

Medroxyprogesterone acetate (MPA) is widely used in endocrine therapy for breast cancer and other diseases. Recently, it has been demonstrated that 9alpha-fluoromedroxyprogesterone acetate (FMPA) also has anti-tumour activity in chemical-induced rat mammary tumour and its activity is greater than that of MPA. In the present study, the physico-chemical properties of FMPA and MPA and their pharmacokinetics in female rats were investigated. Partition coefficients (log P) of FMPA and MPA were 3.1 and 3.8, respectively, while the solubilities of FMPA and MPA in phosphate buffer saline were 3.8 and 1.1 microg/mL, respectively. When the two agents were intravenously or orally administered into female rats, there was no significant difference between their plasma concentrations. However, unmetabolized drug excreted into urine accounted for 4.7 and 0.7% of the intravenous dose of FMPA and MPA, respectively. The free fraction of FMPA in rat plasma was approximately four times that of MPA. Assuming the well-stirred model, hepatic intrinsic clearances of FMPA and MPA were estimated to be 64 and 293 L/h per kg, respectively. In addition, the free fraction of FMPA in blood is estimated to be higher than that of MPA, which may explain the higher anti-tumour activity.

N-demethylation of ethylmorphine in pregnant and non-pregnant women and in men: an evaluation of the effects of sex steroids.

1. The effects of oestrogens, testosterone, progesterone and medroxyprogesterone acetate (MPA) on the rate of N-demethylation of ethylmorphine (EM) to norethylmorphine (NEM) were studied in human adult liver microsomes. 2. N-Demethylase activity was found to be inhibited by progesterone and MPA to a similar extent while oestrogens and testosterone had no or negligible effects. 3. These findings prompted us to measure the N-demethylation of EM in relation to serum progesterone concentration in vivo in three groups of volunteers with large physiological differences in their endogenous levels of progesterone, i.e. i) pregnant women, ii) non-pregnant ovulating women and iii) men. 4. The metabolic ratio (MRP) of EM to NEM in plasma 60 min after dosage and the corresponding ratio in urine sampled for 6 h (MRU,1), measured on two occasions 14 days apart were used to reflect intraindividual variation in the rate of N-demethylation. 5. The average difference in MRP and MRU,1 between the two occasions was similar in all groups. However, the variability in MRP between individuals within a group was significantly higher in ovulating women than in men, but this had no relation to the serum concentrations of progesterone or oestradiol. 6. The cumulative 12 h urinary excretion of EM, NEM and morphine (MO) after hydrolysis with beta-glucuronidase was about 46%. There was no difference in the metabolic ratio of EM to NEM and its conjugate(s) in the urine between the luteal and the follicular phases. Our findings suggest that the menstrual cycle does not influence the rate of N-demethylation of EM.

Bioavailability of medroxyprogesterone acetate from three oral dosage formulations.

The bioavailability of three formulations of medroxyprogesterone acetate (MPA) was assessed in 30 healthy male volunteers in a three-way, open-label, cross-over-designed trial. Each subject received one Provera 500-mg tablet, one Farlutal 500-mg tablet, and one Provera 500-mg granule packet according to a randomized schedule, with each treatment separated by a 21-day washout period. Serum MPA levels were determined using both radioimmunoassay (RIA) and high-performance liquid chromatography techniques. Based on the results of RIA analysis, Farlutal tablets produced significantly lower serum MPA concentrations compared with Provera tablets at most sampling times, resulting in statistically lower AUC0-144 for the Farlutal tablet (544 vs 768 ng.hr/ml; -29.2%). The Farlutal tablet also had a significantly lower maximum concentration than the Provera tablet (27.8 vs 47.4 ng/ml; -41.4%). However, there was no significant difference in time of maximum concentration between the tablet formulations (3.71 vs 3.41 hr), indicating that the rates of absorption of the two tablet formulations were comparable. Provera granules provided significantly higher serum MPA levels than Provera tablets at 0.5, 1, 1.5, 2, and 6 hours, and the AUC0-144 for Provera granules was higher by 5.47% (810 vs 768 ng.hr/ml). There were no differences in terminal elimination rate constants among the dosage forms. No significant adverse events were noted during the trial. The relative bioavailabilities of Provera granules and Farlutal tablets were 105% and 71.2%, respectively, compared with Provera tablets.

An enzyme linked immunosorbent assay (ELISA) for plasma medroxyprogesterone acetate (MPA).

A single extraction fixed antigen enzyme-linked immunosorbent assay (ELISA) that can be completed in less than 24 h is described for the measurement of medroxyprogesterone acetate (MPA) in plasma. MPA is covalently coupled to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to a standard 96-well microtitre plate where it competes with MPA in the extracted plasma sample for goat anti-MPA. Antibody binding to the solid phase is determined via binding of a horse-radish peroxidase second antibody which reacts colorimetrically with its substrate. The reaction is stopped by addition of 1.25 M H2SO4 and absorbance read at 492 nm. All steps except for sample addition and extraction can be performed on an automatic ELISA processing machine. The assay is sensitive, specific and precise, with intra- and inter-assay coefficients of variation of less than 10 and 15%, respectively. Assay sensitivity is 0.08 ng/ml. The assay follows established methodology for other assays in this laboratory which assists standardization, cost structure and sample throughput and thus is a useful alternative to radioimmunoassays for the determination of MPA in plasma.

Long-term effects of depot-medroxyprogesterone acetate on lipoprotein metabolism.

To assess the effects of depot-medroxyprogesterone acetate (DMPA) upon serum lipids and lipoproteins, a comparative study in chronic users and new acceptors was undertaken. Two groups of women of reproductive age were included in the study; group I (n = 8) was formed by new acceptors whereas, group II (n = 14) constituted DMPA users of more than five continuous years (7.0 + 2.1 years). Blood samples were taken on the day of injection and 15, 29, 57 and 92 days after the i.m. administration of 150 mg of DMPA for the measurement of total triglycerides (TG), cholesterol (CHOL) and phospholipids (PHL). In addition, the TG and CHOL content in the very low density (VLDL), low density (LDL) and high density (HDL) lipoprotein fractions obtained by ultracentrifugation were also determined. The results demonstrated a moderate increase in the serum total TG concentrations at the expense of the VLDL fraction in the group of chronic DMPA users. In both groups, the administration of DMPA induced a moderate, though not significant, decrease in total CHOL and HDL-chol, an effect that was noticed at the end of the treatment interval; the serum LDL-chol content remained unchanged. In addition, a decrease in the total serum phospholipids content was noticed after DMPA injection in both groups, which resembled the fluctuations observed in the luteal phase of normal ovulating women. The overall data indicate that acute and/or chronic DMPA administration at the dose currently employed for contraception does not induce major abnormalities in lipoproteins in serum.

Short-term effects of estrogen and progestin on blood pressure of normotensive postmenopausal women.

Blood pressure rises in women with increasing age, possibly related to the decrease in production of female hormones that accompanies menopause. Although estrogen or progestin administration alone consistently does not lower blood pressure in postmenopausal women, possible interactions of these two hormones in affecting blood pressure are not well understood. We studied 12 surgically postmenopausal, normotensive women, aged 51 +/- 2 years (SEM). Treatment for each subject consisted of 1 week each of placebo, estrogen (conjugated equine estrogens, 2.5 mg/day), progestin (medroxyprogesterone acetate, 60 mg/day), and combined estrogen and progestin, given in varied order. At the end of each week, auscultatory blood pressures were measured while patients were seated. Neither estrogen nor progestin alone either increased or decreased blood pressure significantly, whereas combined estrogen and progestin lowered systolic, diastolic, and mean blood pressures 6 to 7 mm Hg (P less than .05). Treatment order was unrelated to the change in blood pressure values. The authors suggest that administering progestin with estrogen may be more effective in lowering blood pressure than either hormone alone in postmenopausal women.

Mass spectrometric and high-performance liquid chromatographic studies of medroxyprogesterone acetate metabolites in human plasma.

Medroxyprogesterone acetate (MPA) treatment has been shown to exert several beneficial effects in cancer patients. It has been suggested that such effects are due in part to the metabolites derived from MPA in vivo. The first results are reported on the identification of 2 alpha-hydroxy- and 21-hydroxy-MPA, 20-dihydro-MPA, 17 alpha-acetoxy-2 alpha,3 beta-dihydroxy-6 alpha-methylpregn-1,4-dien-20-one and two X,21-dihydroxy-MPAs, one of them presumably being 6 alpha-hydroxymethyl-21-hydroxy-MPA, in patient's plasma by high-performance liquid chromatographic (HPLC), gas chromatographic-mass spectrometric and NMR methods. Additionally, the presence of other metabolites such as di- and tetrahydro-MPAs and 6,21-dihydroxy-MPA, found in urine and other samples, was demonstrated in plasma. For routine clinical examinations an HPLC method is described for determination of, e.g., the unreduced MPA metabolite group in Sep-Pak-ODS column extracts of patients' plasma.

Sequential alternate administration of tamoxifen and medroxyprogesterone acetate in advanced breast cancer: clinical-biological randomized study.

From January 1985 to September 1988, 60 women with advanced breast cancer were randomized in two arms to receive: A) tamoxifen (TAM) (20 mg/die) until progression or B) TAM (20 mg/die for 14 days) then medroxyprogesterone acetate (MPA) (1500 mg/die p.o. for 14 days) followed by 7 days of wash-out before repeating the TAM/MPA treatment. All patients were post-menopausal, previously untreated with hormone therapy, and with tumor receptor status determined immediately before randomization; all had objectively evaluable lesions. In order to verify hormone receptor variations due to the antiestrogen, when possible a second biopsy was performed after the initial 14 day cycle of TAM. Thirty-one and 29 patients were included respectively in arms A and B. Objective regression (CR + PR) was observed in 58% of group A and 75% of group B, with CR in 11% and 23%, respectively. Differences were not statistically significant. Median time to progression was 12 months for group A and 9 for group B. Overall survival has not yet been reached in group A while it was 34 months for patients of group B. Metrorrhagia was observed in two cases of group A and in 6 of group B, and thrombophlebitis in 1 and 3 cases, respectively. The second biopsy confirmed a clear increase of PgR content in 8/11 cases (75%). Plasma level variations of TAM, N-desmethyl TAM and MPA were checked at various intervals on 3 patients of group B, and confirmed that our schedule is able to produce a drug wash-out period for tumor cells. In conclusion, our study demonstrated that while the manipulation of hormone receptors seems possible, results indicating better overall survival and time to progression were not obtained with alternate sequential TAM-MPA therapy.

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.

Comparison of mass spectrometry and radioimmunoassay to measure medroxyprogesterone acetate in patients with endometrial cancer.

Serum medroxyprogesterone acetate (MPA) was measured by radioimmunoassay (RIA) and gas chromatography-mass spectrometry (GC-MS) in patients with endometrial cancer. Samples were obtained 3, 6 and 24 h after the oral administration of 100 or 200 mg MPA once a day. The levels obtained by GC-MS were lower (median 16-29%) than those obtained by RIA, which is probably attributable to the presence of metabolites interfering with the RIA. Two commercial MPA formulations gave different MPA serum levels by both RIA and GC-MS. The levels obtained by GC-MS were so low that frequently only partial saturation of the endometrial progesterone receptor may be achieved which may explain why high oral doses are needed to produce optimum therapeutic response.

Influence of aminoglutethimide on plasma levels of medroxyprogesterone acetate.

To confirm that concomitant administration of aminoglutethimide (AG) reduces plasma levels of medroxyprogesterone acetate (MPA), MPA levels were assayed in six patients with advanced breast cancer receiving the two agents. Patients had disease resistant to AG and were studied during conversion to therapy with MPA. Hydrocortisone was discontinued at the commencement of study and MPA introduced and given at a constant dose of 800 mg daily while AG was reduced in dose from 250 mg b.d. to 125 mg b.d. and then discontinued. MPA levels were measured after two weeks at each dose of AG and after two weeks on MPA alone. Mean MPA levels showed a progressive and significant (P less than 0.01) rise as the AG dose was reduced--180 ng ml-1, 250 ng ml-1 and 740 ng ml-1 respectively. MPA levels were always in excess of the accepted therapeutic level of 100 ng ml-1 and plasma cortisol levels fell in parallel with the rise in MPA levels.

Specific and sensitive quantitation of medroxyprogesterone acetate in human serum by gas chromatography-mass spectrometry.

A procedure for the quantitation of medroxyprogesterone acetate in serum using gas chromatography-quadrupole mass spectrometry is described. Medroxyprogesterone propionate, synthesized from medroxyprogesterone, was used as the internal standard. The serum samples were extracted on Bond Elut C18 cartridges, and the acetate and propionate were determined as their 3-enol heptafluorobutyrate esters by selected-ion monitoring technique. The linear range of the standard curve in serum was 0.5-30 ng/ml with a lower limit of quantitation of 0.5 ng/ml. The coefficient of variation of the method was 3.1% at 10 ng/ml and 5.5% at 1 ng/ml. The method is very rapid, and it has been applied for routine measurements of medroxyprogesterone acetate levels in human serum after oral administration of 10 or 20 mg.

Elevated serum levels of endometrial secretory protein PP14 in patients with advanced endometriosis. Suppression by treatment with danazol and high-dose medroxyprogesterone acetate.

The circulating concentration of endometrial protein PP14 varied during the menstrual cycle in patients with endometriosis. The highest levels were found on days 1 to 4 of the cycle (176 +/- 123 micrograms/L; mean +/- SD), and the lowest on days 5 to 20 (44.1 +/- 29.7 micrograms/L). Rising levels were observed on days 21 to 30 (58.3 +/- 62.6 micrograms/L). On days 5 to 20 (i.e., during period of the lowest levels) patients with advanced endometriosis had higher PP14 levels (63.9 +/- 39.0 micrograms/L) than those with mild endometriosis (29.3 +/- 18.2 micrograms/L; p less than 0.01). Patients with mild endometriosis had slightly higher serum PP14 levels than apparently healthy control subjects (p less than 0.05), but overlapping of values between the two groups is remarkable. Conservative surgical elimination of endometriosis significantly decreased the serum PP14 levels. Treatment with danazol (600 mg/day), or with medroxyprogesterone acetate (100 mg/day) after laparoscopy also resulted in significant decreases in the serum PP14 concentration. After 6 months of treatment, conservative surgery in combination with danazol or with medroxyprogesterone acetate, yielded more pronounced declines in serum PP14 level than conservative surgery plus placebo. No significant difference was observed between the effects of danazol and medroxyprogesterone acetate. We conclude that endometriosis tissue contributes to the circulating PP14 level, and the decline in PP14 level during danazol and medroxyprogesterone acetate treatments reflects regression of intrauterine and ectopic endometrial tissues.

Combination of 19-nortestosterone-hexyloxyphenylpropionate (Anadur) and depot-medroxyprogesterone-acetate (Clinovir) for male contraception.

Because monotherapy with 19-nortestosterone hexyloxyphenylpropionate (Anadur, Pharmacia Arzneimittel, Ratingen, Federal Republic of Germany) suggested improved results for male contraception compared with available testosterone esters, it was tested for induction of complete azoospermia when combined with depot-medroxyprogesterone acetate (DMPA, Clinovir, Upjohn GmbH, Heppenheim, Federal Republic of Germany). Twelve men were treated for 7 weeks with weekly intramuscular (IM) injections of 200 mg Anadur followed by 3-weekly IM injections of Anadur up to week 15. Clinovir (250 mg) IM was administered at the start of treatment and during weeks 6 and 12. Anadur and Clinovir suppressed serum gonadotropins. Although serum testosterone declined steeply, in general, libido and potency were not impaired. Sperm concentrations were reduced significantly after 3 weeks of treatment. Lowest sperm counts were seen during week 8 of follow-up, when only 2 volunteers showed measurable sperm counts of 2.1 and 3.0 X 10(6)/ml, with a declining tendency. After 43 weeks, sperm concentrations were still below pretreatment range in 2 men, but later returned to pretreatment values. Computerized sperm motion analysis revealed that motility parameters in the residual sperm were reduced. In vitro analysis excluded a direct effect of medroxyprogesterone acetate in seminal plasma on sperm motion. The data indicate that the combination of Anadur with Clinovir increases the rate of azoospermia in normal volunteers seen under Anadur monotherapy, although the goal of azoospermia in all participants was not quite achieved.

Suppression of sperm function by depot medroxyprogesterone acetate and testosterone enanthate in steroid male contraception.

Ten normal men were given three monthly intramuscular injections of 200 mg of depot medroxyprogesterone acetate (MPA) and 250 mg of testosterone (T) enanthate. Six men became azoospermic, while four remained oligozoospermic, with a mean sperm density of 1.5 +/- 0.3 standard error of the mean million/ml. Zona-free hamster oocyte penetration was abolished in all oligozoospermic samples at the end of treatment. Twenty of the 21 oligozoospermic samples yielding at least 0.6 to 5.0 million motile spermatozoa showed a complete absence of oocyte penetration. Semen parameters returned to normal, although some took up to 12 months. These findings demonstrated an antifertility action of MPA and T enanthate on the functional capacity of residual spermatozoa and support the view that extreme oligozoospermia may be a tenable target for reversible steroid male contraception.

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.

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.

Growth of human aortic smooth muscle cells cultured with human serum is retarded when serum lipids are lowered by medroxyprogesterone.

Human aortic smooth muscle cells were cultured in the presence of sera from 7 normolipidemic women before and after treatment with high-dose medroxyprogesterone acetate, which caused 16% and 25% decreases in serum cholesterol and HDL-cholesterol concentrations, respectively. As assessed by cell counting and by DNA determination the growth of the cells was retarded significantly in the presence of sera taken after the treatment. At the same time, there were no marked changes in the incorporation rate of [3H]proline into collagen or [3H]glucosamine into hyaluronic acid by the cells. The results indicate that: (1) the mitogenicity of human serum can be altered by drug treatment of serum donors, (2) simultaneously with a lowering of serum lipids in man in vivo, a decreased mitogenicity of sera occurs in vitro.