Confirmatory analysis of acetylgestagens in plasma using liquid chromatography-tandem mass spectrometry.

A confirmatory method has been developed and validated for the determination of chlormadinone acetate (CMA), megestrol acetate (MGA), melengestrol acetate (MLA) and medroxyprogesterone acetate (MPA) in bovine and porcine plasma. Analytes are extracted from plasma samples using matrix-assisted liquid-liquid extraction (LLE) on Extrelut NT columns followed by C18 solid-phase extraction (SPE). Analytes were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and quantification was performed using matrix-matched calibration standards in combination with deuterated internal standards. In accordance with Commission Decision 2002/657/EC, two ion transitions were monitored for each analyte. Decision limits (CCalpha) were estimated by analysing 20 blank plasma samples and ranged from 0.1 to 0.2 ng mL(-1). Detection capabilities (CCbeta) were estimated using 20 plasma samples fortified at 0.5 ng mL(-1) and were <0.5 ng mL(-1). In the range 0.5-2 ng mL(-1), the mean intra-laboratory reproducibility of the analytes ranged from 6 to 18% (%R.S.D.). Analytes were shown to be stable in fortified plasma samples for >8 months when stored at -20 degrees C.

Pharmacokinetics of chlormadinone acetate following single and multiple oral dosing of chlormadinone acetate (2 mg) and ethinylestradiol (0.03 mg) and elimination and clearance of a single dose of radiolabeled chlormadinone acetate.

BACKGROUND: Published data on pharmacokinetic parameters for chlormadinone acetate (CMA) are in part contradictory, especially with regard to terminal half-life (t(1/2,z)). MATERIALS AND METHODS: Single and multiple doses of CMA (2 mg) and ethinylestradiol (EE; 0.03 mg) were administered to healthy female volunteers for six menstrual cycles. Plasma concentrations of CMA and EE were determined by gas chromatography-mass spectrometry. Single-dose and steady-state pharmacokinetic parameters were calculated. In a separate study, healthy female volunteers were given a single 2-mg dose of radiolabeled CMA. Concentrations of radioactivity in fecal and urine samples were determined via liquid scintillation. Excretion of total radioactivity was calculated as percentage of administered dose. RESULTS: Eighteen women completed the repeated-dose study. Peak plasma concentrations for CMA and EE were reached within 1 and 2 h after taking the study drug. Peak plasma concentrations of CMA were approximately 1600 pg/mL after single-dose administration and 2000 pg/mL after multiple dosing. CMA and EE showed linear pharmacokinetics throughout six cycles, with constant trough values of approximately 400-500 pg/mL for CMA and 20-40 pg/mL for EE. Mass balance factors were 1.2-1.4 for CMA and 1.6-1.7 for EE, and accumulation factors were 1.7-2 for CMA and 1.7-1.8 for EE. Mean t(1/2,z) of CMA was approximately 25 h after single dosing and 36-39 h at steady state. In the excretion balance study, mean dose of CMA recovered was 87.3+/-6.4%, with urinary and fecal excretion accounting for 45% and 42%, respectively. CONCLUSIONS: The pharmacokinetics of CMA and EE is linear after multiple dosing and remains stable during long-term administration, once steady state is reached. The t(1/2,z) of CMA was 36-39 h after multiple dosing, which is considerably shorter than the 80 h often quoted in the literature.

[The relative pharmacokinetics of two oral formulations of chlormadinone acetate: Lutéran 5mg. and Lutéran tablet 10 mg]. / Etude de la biodisponibilité relative de deux formulations orales d'acétate de chlormadinone: Lutéran 5 mg et Lutéran comprimé dosé à 10 mg.

The bioavailability of a new formulation of chlormadinone acetate (one 10 mg Lutéran tablet) was compared with that of the reference formulation (two 5 mg Lutéran tablets) in a randomised crossover open trial after single oral administration of a 10 mg dose to 12 healthy female volunteers. Measurements of chlormadinone acetate plasma samples were performed by combined gas chromatography/mass spectrometry. Blood samples were collected before administration and up to 144 hours after administration. No significant difference was found between the two formulations in pharmacokinetic parameters. The bioavailability of the two formulations was equivalent in terms of time to maximum concentration (tmax [mean tmax about 2.5 h]) and area under the concentration-time curve (AUC0-infinity) [Weslake's symmetric confidence interval: 19.24%, Schuirmann two one-sided tests procedure: p < 0.05]. No difference was found between the two formulations with regard to clinical safety parameters.

Effects of combination therapy with a luteinizing hormone-releasing hormone agonist and chlormadinone acetate on rat prostate weight and plasma testosterone levels.

We investigated whether the combination of chlormadinone acetate (CMA) and a luteinizing hormone releasing hormone (LH-RH) agonist, leuprorelin acetate (leuprorelin), more markedly decreased ventral prostate and seminal vesicle weights and plasma sex hormone levels in male rats. Four weeks after administration of 0.28, 0.84 or 2.8 mg/kg of leuprorelin, ventral prostate weights significantly decreased (53.8, 54.4 and 64.1%) and the plasma testosterone levels significantly lowered, but not dose-dependently. After repetitive administrations of 3 and 30 mg/kg/day of CMA, the rates of ventral prostatic atrophy were 37.1 and 65.9%, respectively. Although there was no change in the plasma testosterone level at 3 mg/kg, 30 mg/kg of CMA significantly decreased the level. A combination of leuprorelin (0.28 mg/kg) and CMA (3 or 30 mg/kg) more potently induced ventral prostatic and seminal vesicle atrophy than leuprorelin alone. Furthermore, a combination of leuprorelin and CMA (30 mg/kg) more markedly decreased the plasma testosterone level. According to the pharmacokinetic data for CMA in male rats, the doses of CMA correspond to the clinical dose. These findings suggest that combination therapy with an LH-RH agonist and CMA is more useful than therapy with the agonist alone in the treatment of prostate cancer.

Development of a new sensitive and specific time-resolved fluoroimmunoassay (TR-FIA) of chlormadinone acetate in the serum of treated menopausal women.

We describe the development of a serum chlormadinone acetate (CMA) time-resolved fluoroimmunoassay (TR-FIA). We prepared haptens (3-CMO-chlormadinone acetate and 6-chloropregna-4,6-dien-17,20-diol-3-one-20-hemisuccinate), biotinylated tracers (3(biotinylaminopropylamido) 3-CMO-chlormadinone acetate and 3-(6-chloropregna-4,6-dien-17,20-diol-3-one-20-hemisuccinylamino)1-biotinylaminopropane), and immunogens necessary for eliciting two antibodies (anti-chlormadinone acetate 3-CMO/BSA and anti-chlormadinone 20-hemisuccinate/BSA). The specificity of the assay was rigorously studied to eliminate possible interference by polar metabolites of CMA, particularly 17 alpha-acetoxy-6-chloro-3beta-hydroxypregna-4,6-diene-20-one (3beta-hydroxy metabolite), employing an easy-to-use ethylene glycol chromatographic step prior to immunoassay, so as to separate the polar metabolites, in particular the 3beta-hydroxy-CMA metabolite, from the intact CMA. The choice of the anti-CMA antibody was guided by the high assay sensitivity obtained with the anti-CMA 3-CMO/BSA antibody. The detection limit was 51pg/ml. Interassay reproducibility CVs were between 2.6 and 4.5%. This TR-FIA thus appeared to be a sensitive, specific, precise, and consequently well-suited method for measurement of serum CMA during a pharmacokinetic study in women.

Pharmacokinetics and biliary excretion of osaterone acetate, a new steroidal antiandrogen, in dogs.

The pharmacokinetics and biliary excretion of osaterone acetate (17alpha-acetoxy-6-chloro-2-oxa-4,6-pregnadiene-3,20-dione; OA), a new steroidal antiandrogen, were investigated in intact dogs and biliary fistula dogs after bolus intravenous administration of (14)C-labeled drug. In intact dogs, OA exhibited a biexponential disposition with a very long half-life of 197.9 +/- 109.9 h. OA accounted for almost all the plasma radioactivity. The major route of excretion was in feces via the bile. One-third of the radioactivity in the bile was due to OA. The major biliary metabolite was identified as a glucuronide of 17alpha-acetoxy-6-chloro-21-hydroxy-2-oxa-4,6-pregnadiene-3,20-dione. A significant amount of biliary recycling occurs in dogs.

Regression of prostatic hypertrophy by osaterone acetate in dogs.

The prostatic regression effect of oral administration of a new steroidal anti-androgen, osaterone acetate, was investigated in dogs with prostatic hypertrophy. To dogs with prostatic hypertrophy, 0.1-1.0 mg/kg of osaterone acetate was orally administered for one week, and the regression rate was observed. It was shown that administration of osaterone acetate at 0.2 mg/kg or higher, sharply regressed prostatic hypertrophy during the early stage. Therefore, this agent may be clinically applicable as a therapeutic agent for benign prostatic hypertrophy.

Effect of chlormadinone acetate-pellet implantation on the volume of prostate, peripheral blood levels of sex hormones and semen quality in the dog.

Chlormadinone acetate (GS implant, CMA) pellet, a synthetic luteal hormone preparation, was subcutaneously implanted at 5, 10 and 20 mg/kg in four normal male dogs ranging in age from 3 to 10 years to determine the changes in the prostatic volume, peripheral plasma levels of sex hormones and semen quality. The plasma levels of CMA, LH, testosterone (T) and 5 alpha-dihydrotestosterone (DHT) were measured by radioimmunoassay. The prostatic volume was measured by computed tomography. The semen was collected by digital manipulation. The pellet was removed 26 weeks after implantation. The effects of CMA pellet implantation were examined during implantation and until 22 weeks after removal. The prostatic volume was reduced to 61 +/- 3 (mean +/- S.E., n = 4), 52 +/- 5 (n = 4) and 53 +/- 9 (n = 4)% of the preimplantation volumes in the 5, 10 and 20 mg/kg groups, respectively. The plasma CMA levels in the 10 mg/kg and 20 mg/kg groups peaked at 2 weeks, but were gradually decreased. At 22 weeks after removal of the pellet, the prostatic volume returned to 74-85% of the preimplantation volumes. The plasma LH levels tended to increase after implantation in all groups. The plasma T and DHT levels were slightly decreased in all groups. In the 10 and 20 mg/kg groups, the number of sperm and motility sperm was reduced, and the rate of abnormal sperm increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of estrus in bitches by subcutaneous implantation of chlormadinone acetate.

The efficacy and clinical safety of chlormadinone acetate (CAP) in preventing estrus were assessed in bitches on condition that CAP was subcutaneously implanted in silastic silicon rubber. To evaluate the long-term efficacy of implantation, 19 bitches were divided into 4 groups given doses of 2.5, 5, 10 and 25 mg/kg, respectively. Although estrus was observed within 13 months after implantation in all of the bitches given CAP in the 2.5 mg/kg dose, and in 13 to 15 months in 3 of the 5 bitches given the 5 mg/kg dose, it was prevented for at least 24 months in all of the bitches given doses of 10 mg/kg or more. Plasma progesterone levels remained low throughout the period of estrus prevention, indicating a close correlation with the effect of CAP. The mean body weight of the bitches in groups receiving higher doses increased slightly over the course of the experiment. Except this, no clinical, hematology or biochemistry abnormalities were found in any of the treated bitches. Another 6 bitches were given 10 to 30 mg/kg of CAP, but the implants were removed to observe the recurrence of estrus and to measure the amount of CAP in the removed implant. Estrus recurred after removal even in the bitch given 30 mg/kg. The concentrations of CAP in the plasma and the amounts of CAP remaining in the implants demonstrated the sustained release of CAP from the implants. The concentrations of CAP in the individual bitches indicated that the lowest concentration effective in preventing estrus is 0.7 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of two synthetic progesterones on ventilation in normal males: CMA vs. MPA.

We administered chlormadinone acetate (CMA), medroxyprogesterone acetate (MPA), and placebo to 16 normal male subjects using a randomized double-blind crossover study. After CMA administration, minute alveolar ventilation increased by +1.04 +/- 0.22 (SE) 1/min (P less than 0.05) accompanied by decrements of arterial PCO2 (-4.0 +/- 1.0 Torr) (P less than 0.01) and [HCO3-] (-2.1 +/- 0.05 mM/l) (P less than 0.01). On the other hand, in the MPA runs the corresponding changes of the above parameters were +0.71 +/- 0.21 l/min (P less than 0.05), -2.9 +/- 0.6 Torr (P less than 0.01), and -1.3 +/- 0.3 mM/l (P less than 0.01), respectively. The slopes of hypoxic ventilatory and occlusion pressure response lines remained unchanged in hypocapnia after CMA or MPA ingestion, but they increased when entidal PCO2 was adjusted to the predrug level. The hypercapnic ventilatory and occlusion pressure response lines merely shifted to the left without changing their slopes with these agents. No significant differences in all the above parameters were found between CMA and MPA runs. We concluded that in the normal males the effect of CMA on ventilation was similar to that of MPA, despite the fact that the luteinizing activity of CMA was reported to be approximately 10 times higher than the latter.

Disappearance of [1alpha-3H]-chlormadinone acetate in the plasma and red cells of rhesus monkey.

PIP: The half-life and metabolic clearance rate of chlormadinone acetate in 4 rhesus monkeys was computed after iv injection. Chlormadinone was analyzed as total, free, and conjugated radioactivity, and as recrystallized chlormadinone acetate. 55.0 mc Ci, 222 mc Ci/mg was injected iv in 5 ml ethanolic saline. There was an initial rapid disappearance, half-life 68 minutes, and a slower disappearance, half-life 35.1 hours. These half-lives are much longer than those of estradiol and progesterone, but are shorter in monkeys than in women. The metabolic clearance rate of chlormadinone acetate was 102.6 liters/day. The half-life in red cells of 1 monkey was similar to those seen in plasma, but the amount of chlormadinone acetate was much less.^ieng

Dissappearance of 1 alpha-3H-chlormadinone acetate from the plasma of women.

PIP: The half-life and metabolic clearance rate of chlormadinone acetate were computed after a single iv injection of 60 to 90 mcCi 1-alpha-tritiated-chlormadinone acetate (specific activity 222 mcCi/mg) into 7 women aged 34-52 years. Plasma was extracted with acetone:MeOH for total radioactivity; then extracted with water and ether for free steroid radioactivity; then with n-butanol for conjugated steroid radioactivity; and finally extracted with chloroform:MeOH and chromatographed on thin layer with ether:benzene for specific radioactivity due to chlormadinone acetate. Blood samples were taken at .25, .5, 1, 8, 24 hours and every 24 hours for 5 days. The mean half-life of radioactivity specifically indentified as chlormadinone acetate for the first 24 hours was 2.6 hours, and after 24 hours was 81.8 hours, calculated by TAIT and BURSTEIN's method. All 4 curves, total radioactivity, conjugated steroids, free steroids, and specific activity had the same biphasic form: a rapid loss for about 24 hours, and an approaching equilibrium after 24 hours. The metabolic clearance rate was 42.61 liters per day, comparable to lynestrenol (15-30 liters) and norethynodrel. The half-life is much longer than that of progesterone (.96 and 10.7 minutes), estradiol (20 and 70 minutes), and norethynodrel (76 minutes and 45 hours). These data generate an estimate of the concentration of chlormadinone acetate in plasma of women taking .5 mg daily: about .45 ng/ml, i.e. about one-thirteenth the concentration of progesterone.^ieng

[Tissue distribution of progesterone-7-H3 in the female genital tract 8 hours after intravenous administration]. / Depósito tisular de progesterona-7-H3 en el aparato reproductor femenino, ocho horas después de su administración endovenosa.

PIP: 6 patients, ages 32-55 years, were selected to establish radioactive progesterone distribution in the genital tract, and deposition in skin and fat tissue. 1/2 of the patients were in the menstrual proliferative phase, and the rest were in the secretory phase. 250 uci of progesterone-7-h cubed was given intravenously to all patients, and total hysterectomy and salpingo-oophorectomy were performed 8 hours later. A piece of skin was taken as well as some fat tissue from the anterior abdominal wall. The tissues studied were skin, fat tissue, ovary, tube, and uterus. The uterus was sectioned into 3 parts (superior, median, inferior) and the cervix was sectioned into 2 (endocervix and ectocervix). In all the patients, urine was collected during 3 consecutive days and at the time of operation; 10 ml was taken then and at the 5th postoperative day. The largest concentration of radioactive material was found in the proliferative phase patients (in fat tissue); in the secretory phase it was concentrated in the skin, uterus, and myomas. Excretion of radioactive material was 32.5% in the urine, blood radioactivity at the time of operation was 2.62%, and 0.33% of the administered total dose at the 5th postoperative day. In a previous similar study with chlormadinone acetate, urinary excretion was 17.5% and the largest radioactive material concentration occurred in fat tissue, regardless of the day of the cycle. These results may show that natural and synthetic hormones have different action mechanisms at their genital effectors, and different fat tissue deposition. (author's)^ieng