Simultaneous determination of centchroman and tamoxifen along with their metabolites in rat plasma using LC-MS/MS.

AIM: Tamoxifen and centchroman are two non-steroidal, selective estrogen receptors modulators, intended for long term therapy in the woman. Because of their wide spread use, there is a possibility of co-prescription of these agents. MATERIALS & METHODS: We studied the probable pharmacokinetic interaction between these agents in breast cancer model rats. A simple, sensitive and rapid LC-ESI-MS/MS method was developed and validated for the simultaneous determination of tamoxifen, centchroman and their active metabolites. RESULTS: The method was linear over a range of 0.2-200 ng/ml. All validation parameters met the acceptance criteria according to regulatory guidelines. CONCLUSION: LC-MS/MS method for determination of tamoxifen, centchroman and their metabolites was developed and validated. Results show the potential of drug-drug interaction upon co-administration these two marketed drugs.

Simultaneous quantification of centchroman and its 7-demethylated metabolite in rat dried blood spot samples using LC-MS/MS.

An approach has been developed for the quantitative determination of concentrations of centchroman (I), a nonsteroidal once-a-week oral contraceptive, and its major metabolite (7-desmethyl centchroman, II) using dried blood spots (DBS) on paper, rather than conventional plasma samples. The assay employed simple solvent extraction of the DBS sample circle (6 mm) requiring small blood volumes (30 µL) followed by reversed-phase HPLC separation, combined with multiple reaction monitoring mass spectrometric detection. The calibration plot in matrix using d-trans-hydroxy chroman as internal standard (IS) was linear (r² = 0.998) over ranges of 1.5-240 and 4.5-720 ng/mL for I and II, respectively. The recoveries of both I and II were always >60% with quantification limits (signal-to-noise ratio = 10) of 1.5 and 4.5 ng/mL for I and II, respectively. The intra-day and inter-day precision (%RSD) and accuracy (%bias) variations in blood spots for both I and II were better than 13%. Moreover, both I and II were stable in DBS for at least 3 months when stored at room temperature. The developed method was successfully applied to the pharmacokinetic interaction study after oral administration of centchroman with and without co-administration of carbamazepine in female Sprague-Dawley rats using serial sampling and results were comparable with the plasma concentrations reported earlier.

Development and validation of a rapid, sensitive liquid chromatography-tandem mass spectrometry method using electrospray ionization for quantitation of centchroman in rat plasma and its application to preclinical pharmacokinetic study.

A highly sensitive and specific HPLC-ESI-MS/MS method has been developed and validated for the estimation of centchroman with 100 microL rat plasma using tamoxifen as an internal standard (IS). The assay procedure involved a single-step, liquid-liquid extraction of centchroman and IS from plasma with 2.5% (v/v) isopropanol in n-hexane, which yielded consistent recoveries of 109.5 and 107.8% for centchroman and IS in rat plasma, respectively. The total chromatographic run time was 3.8 min. Peaks were resolved using 0.01 M ammonium acetate (pH 4.5):acetonitrile (10:90, v/v) mobile phase on a Supelco Discovery C(18) column. Specificity and matrix effect on ionization was determined and found that method was specific and there was no significant matrix effect. Linearity range was found to be 0.5-100.0 ng/mL with a correlation coefficient (r) of 0.9959 or better. The intra- and inter-day assay precision ranged from 3.3 to 9.0% and 5.5 to 6.8%, respectively, and intra- and inter-day assay accuracy was between 93.4-107.1% and 96.2-104.2%, respectively. Stability of centchroman in rat plasma was >89.0% in the battery of stability studies viz., bench-top, auto-sampler, freeze/thaw cycles and 30 days storage in a freezer at -80 degrees C. The assay was successfully applied to determine the pharmacokinetic parameters in Sprague-Dawley rats after an oral administration of centchroman at 20mg/kg. As a result, the plasma half-life was 29.4+/-2.3h and the AUC((0-infinity)) was 7345.1+/-21.9 ng h/mL. The maximum plasma concentration (C(max)) 117.5+/-15.7 ng/mL was achieved at 9.0+/-8.6h (t(max)).

Pharmacokinetic interaction of tetracycline with centchroman in healthy female volunteers.

OBJECTIVES: We aimed to investigate the effect of tetracycline coadministration, with and without lactic acid bacillus spores supplementation, on the pharmacokinetics of centchroman, a nonsteroidal oral contraceptive, in healthy female volunteers. PARTICIPANTS AND METHODS: The study was a single-centre, single-blinded, randomised, parallel treatment study in healthy female subjects of reproductive age randomised to two groups (11 subjects in each group). On day 1, subjects were given either a single oral dose of centchroman 30 mg with tetracycline 250 mg (group A) or a single dose of centchroman 30 mg, tetracycline 250 mg and one tablet containing 60 million lactic acid bacillus spores (group B). Tetracycline (250 mg three times daily) and lactic acid bacillus spores (one tablet three times daily) were continued for 3 days. Serial blood samples were collected and analysed by high performance liquid chromatography. The pharmacokinetic parameters were compared with the control data reported previously from this laboratory. RESULTS: Coadministration of tetracycline yielded significantly higher maximum plasma concentrations (C(max)) [35%] and a shorter time to reach C(max) (t(max)) values for centchroman (42%) than those obtained in the control group of females (p < 0.05). Inclusion of lactic acid bacillus spores in the regimen resulted in similar effects with increased C(max) (47%) and area under the concentration-time curve from time zero to infinity (34%) of centchroman (p < 0.05) with a significant decrease in t(max). Other parameters such as half-life, apparent clearance, apparent volume of distribution and mean residence time of centchroman were not affected by either of the treatments. CONCLUSIONS: The apparent effects of either of the regimens on centchroman pharmacokinetics seem to be of little clinical relevance in terms of increased rate or extent of availability. It can be concluded that this tetracycline-containing regimen is unlikely to alter the contraceptive efficacy of centchroman in humans.

Binding of centchroman with human serum as determined by charcoal adsorption method.

Protein binding of drugs is an important factor influencing both pharmacokinetic and pharmacodynamic parameters. Thus, knowing the extent of protein binding of drugs is crucial. Centchroman is a non-steroidal once a week oral contraceptive. It has been reported to be useful for the treatment of breast cancer and osteoporosis. Ample data has been generated on pharmacokinetics of centchroman in animals and humans. The extent of protein binding of centchroman has not been established so far. Non-specific adsorption of the drug limits the use of conventional methods like ultrafiltration and equilibrium dialysis. A method of charcoal adsorption as reported by Yuan et al. (method I) was used after modification (method II) to determine its binding to human serum. The extent of protein binding (%) is estimated from decline of percent drug remaining in the supernatant after adding the charcoal. Study was carried out at 1- and 10-microg/ml concentrations in drug free human serum samples and an HPLC assay was used to determine concentration-time data. The percentage of centchroman remaining in serum versus time data was analysed using non-linear fitting programs on WinNonlin software. Method II was found to give higher estimates of protein binding than the former method by preventing the dilution effect. Using this method, the extent of protein binding of centchroman was found to be 101.83+/-1.28 and 94.87+/-3.59% at 1 and 10 microg/ml, respectively. However, it was approximately 90% in the individual serum samples showing intersubject variability in protein binding of centchroman.

Characterization of centchroman binding protein in plasma of rhesus monkey (Macaca mulatta).

Centchroman, a nonsteroidal antifertility agent was studied for its binding to monkey (Macaca mulatta) plasma proteins using charcoal adsorption and electrophoretic techniques. 14C-centchroman showed a low affinity binding and did not compete for 3H-cortisol or 3H-DHT binding sites in plasma. 14C-centchroman binding protein was heat stable in nature and showed the electrophoretic pattern similar to that of albumin (Rf 0.70). Thus centchroman binds to albumin in monkey plasma which can be suggested as carrier protein for this contraceptive agent.

Pharmacokinetics of centchroman in healthy female subjects after oral administration.

The pharmacokinetics of centchroman, a non-steroidal antifertility agent, were assessed in serum of eleven healthy female subjects after a single 30 mg oral dose. Maximum serum concentration (Cmax) of 55.53 (s.d., 15.45) microgram/L was attained at 5.18 (s.d., 1.78) h after oral administration. The concentration-time profile was best described by a two-compartment open model with bi-exponential disposition functions. The mean terminal elimination half-life (t1/2) was 165 (s.d., 49) h with a clearance of 6.17 (s.d., 1.67) L/h and volume of distribution of 1420 (s.d., 478) L. Comparison of the pharmacokinetic parameters of this study with those obtained after a single 60 mg oral dose did not show statistically significant differences in the rate of absorption, distribution and elimination. The Cmax and AUC0-infinity were dose-dependent. Thus, the absorption and disposition of centchroman are of first-order, reproducible and dose-dependent.

Centchroman: a new non-steroidal oral contraceptive in human milk.

Centchroman, a non-steroidal oral contraceptive drug, was given to 13 nursing mothers comprising two groups. Each participant in group I (n = 8) received a single 30 mg dose, and in group II (n = 5) each participant received a 30 mg twice a week dose for twelve weeks. Simultaneous blood and milk samples were collected and analyzed for the parent drug by high performance liquid chromatography. In the single dose study (group I), the mean +/- peak centchroman concentrations in milk and serum were 78.7 +/- 28.4 and 63.6 +/- 23.6 ng/ml with milk-to-serum (M/S) ratio of 1.4 +/- 0.9. There was no significant increase in centchroman concentrations in milk after multiple dosing (group II). However, serum concentrations reached up to 112.5 ng/ml at 6 h after the 13th dose. Average M/S ratios were insignificantly different at trough (prior to next dose) and at peak (4-6 h after dose) centchroman levels. Additionally, the breast milk and serum centchroman concentrations showed a significant correlation (r = 0.64, P < 0.01), indicating that the amount of centchroman excreted into breast milk is dependent on serum concentrations. The weekly dose (% of the maternal dose) of centchroman ingested by the breast-fed infant at peak maternal serum and milk levels was in the range of 0.4 to 11.5%, assuming a weekly milk uptake of 1.05 l/kg. There was no significant difference in the dose ingested by the infants between the two dosing groups. These levels of centchroman passing into breast milk and subsequent exposure to the infants are unlikely to be of any physiological consequence.

Simultaneous liquid chromatographic determination of centchroman and its 7-demethylated metabolite in serum and milk.

A precise and sensitive high-performance liquid chromatographic assay was developed and validated for determination of centchroman (I) and its 7-demethylated metabolite (II) in human serum and milk. The serum, at alkaline pH, was extracted with diethyl ether. In the case of milk, after precipitation of the milk protein with acetonitrile, the supernatant was evaporated to dryness and then extracted with diethyl ether at alkaline pH. After solvent evaporation the residue was reconstituted in mobile phase. Separations were accomplished by reversed-phase liquid chromatography using a Spheri-5 cyano column. Recoveries of I and II were always > 95%. Excellent linear relationships (r > 0.999) were obtained between the measured and added concentration ratios of the corresponding serum and milk concentrations over a range of 1 to 1000 ng/ml and 2.5 to 1000 ng/ml for I and II, respectively.

High performance liquid chromatographic (HPLC) determination of centchroman in human serum and application to single-dose pharmacokinetics.

A simple and sensitive (2 ng/ml) HPLC method with fluorescence detection has been developed to measure serum concentrations of centchroman, a new nonsteroidal antifertility agent. The method was sufficiently sensitive to follow the drug over 21 days in human volunteers. Pharmacokinetic parameters of centchroman were determined after a single oral dose of 60 mg (2 x 30-mg tablets) in two healthy female volunteers. Centchroman is slowly eliminated from serum, showing a biexponential disappearance curve from serum. The terminal half-life of centchroman in the two volunteers was 168 and 175 hr, respectively.

Binding of centchroman--a nonsteroidal antifertility agent to human plasma proteins.

Centchroman, a non-steroidal antifertility agent showed a low affinity (Kd = 13.19 X 10(-6) M) and nonsaturable binding to human plasma. Centchroman did not compete either with sex hormone binding globulin or corticosteroid binding globulin. Polyacrylamide gel electrophoresis and temperature dependent binding characteristics revealed that the protein responsible for centchroman binding to human plasma resembles albumin.