Pharmacokinetic study of the novel, synthetic trioxane antimalarial compound 97-78 in rats using an LC-MS/MS method for quantification.

The present study has been designed to investigate the pharmacokinetic parameters of the novel trioxane antimalarial 97-78 (US Patent 6316493 B1, 2001) in male and female rats after single oral and intravenous administration. The pharmacokinetic profile of 97-78 was investigated in the form of its completely converted metabolite 97-63 after dose administration. Quantification of metabolite 97-63 in rat plasma was achieved using a simple and rapid LC-MS/MS method. The LC-MS/MS method has been validated in terms of accuracy, precision, sensitivity and recovery for metabolite 97-63 in rat plasma. The intra- and interday accuracy (% bias) and precision (% RSD) values of the assay were less than 10% for metabolite 97-63. The chromatographic run time was 4.0 min and the weighted (1/x2) calibration curves were linear over the range 1.56-200 ng/ml. This method was successfully applied for analysis of pharmacokinetic study samples. Maximum plasma concentrations of 97-63 at 47 mg/kg oral administration in male and female rats were 1986.6 ng/ml and 4086.7 ng/ml at time (Tmax) 0.92 h and 0.58 h, respectively. The area under the curve (AUC(0-infinity)), elimination half-life (t(1/2) beta) and mean residence time (MRT) were 4669.98 ng x h/ml, 2.8 h and 4.2 h in male and 11786.0 ng x h/ml, 4.52 h and 4.32 h in female rats respectively. After single oral and intravenous administration of 97-78 to male and female rats significant differences were observed in pharmacokinetic parameters (AUC and t (1/2) beta) for metabolite 97-63.

Preclinical pharmacokinetics, dose proportionality, gender difference and protein binding study of 16-dehydropregnenolone, an antihyperlipidemic agent, in rats.

OBJECTIVES: This manuscript addresses key pharmacokinetic issues in support of the development of a potent candidate lipid-lowering drug molecule, 16-dehydropregnenolone (DHP). METHODS: Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) assay for simultaneous estimation of DHP and its metabolites, including 5-pregnene-3ß-ol-16, 17-epoxi-20-one (M(1) ) was validated in male and female Sprague-Dawley rat plasma and applied to different studies. Pharmacokinetic studies of DHP after intravenous and oral administration were carried out to assess any gender effect. Dose-proportionality after oral administration was assessed at three dose levels. Protein binding was estimated using the modified charcoal adsorption method. KEY FINDINGS: Rapid elimination of DHP from the systemic circulation resulted in a comparatively lesser systemic exposure in male compare to female rats. The area under the curve (AUC) after oral administration in males was significantly different to females. The large volume of distribution and low degree of protein binding suggest extensive distribution of DHP. An increase in the oral dose led to a disproportionate change in peak concentration (C(max) ) and AUC, indicating variable absorption. However, the dose-normalized AUC and C(max) at two dose levels were not found to be statistically different. CONCLUSIONS: The extent of conversion of DHP to M(1) was higher after oral administration in male rats but was insignificant in female rats. DHP showed low systemic oral bioavailability and exhibited dose-independent pharmacokinetics and gender differences.

Interspecies comparison of the pharmacokinetics and oral bioavailability of 99-357, a potent synthetic trioxane antimalarial compound.

The pharmacokinetic data obtained in lower animals is of considerable importance in drug discovery and development. The objective of the present study was to generate in vitro and in vivo preclinical pharmacokinetic data of 99-357, a synthetic trioxane antimalarial, in rats and rabbits and to scale-up the data in order to apply for further studies. The pharmacokinetic profile of 99-357 was investigated after both intravenous and oral dose in rats and rabbits. Oral studies were carried out at three dose levels 6, 12 and 24mg/kg in rats while in rabbit only one dose level was selected. Both compartmental and non-compartmental approaches were used to calculate the pharmacokinetic parameters following intravenous and oral doses in both the species. The clearance in rat and rabbit was 45-57% and 60-67% respectively of hepatic blood flow. The plasma protein binding in rats was approximately 75%. In vitro studies showed high RBC partitioning and low to moderate hepatic clearance. Linearity was observed in terms of dose and AUCs suggesting linear pharmacokinetics at the dose levels studied in rats. The oral bioavailability of compound 99-357 in rat and rabbit at 12mg/kg dose level was comparable and 39% and 41% respectively.

Pharmacokinetics of the proton pump inhibitor CDRI-85/92 and its ester prodrug, a new H+/K(+)-ATPase inhibitor with anti-ulcer activities, after oral doses in rats.

5-Styryl-4,5-cis-1,3-oxazole-2-one-4-carboxylic acid (CDRI-85/92) is a new proton pump inhibitor presently in advanced stage of preclinical trials as antiulcer pharmacophore. Since proton pump inhibitors are prodrugs requiring activation in acid environment, an ester prodrug of CDRI-85/92 was also synthesized. In view of the importance, a pharmacokinetic study of CDRI-85/92 and its ester prodrug was conducted after oral doses in rats. Following a 20 mg/kg oral dose of CDRI-85/92, the compound was detectable in the serum samples up to 24 h with a maximum serum concentration (C(max)) of 1838.40 +/- 101.16 ng/ml at 1.5 h and an elimination half-life of 4.96 h, whereas, multiple C(max) values of CDRI-85/92 were observed with oral doses (equivalent to 20 mg/kg of CDRI-85/92) of the ester prodrug of the compound. All the three C(max) values of the compound were lower than that after oral dose of CDRI-85/92. The compound was eliminated slowly from serum with an elimination half-life of 5.14 h. Moreover, the systemic availability of CDRI-85/92 also decreased from 6111 to 3463 ng x h/ml after the ester prodrug administration. The decrease in systemic availability of CDRI-85/92 could be due to its higher clearance after its ester prodrug administration.

Pharmacokinetics and excretion of the novel anti-tuberculosis compound 1,2:5,6-di-O-isopropylidene-3-O-(phenyl cyclopropyl methanonyl)-alpha-D-glucofuranose (S-001-14) after oral doses in rats. Development of a sensitive and reproducible high performance liquid chromatography-UV method for the determination of the test compound in rat serum.

A sensitive and reproducible high performance liquid chromatography (HPLC)-UV method for the determination of the novel anti-tuberculosis compound 1,2:5,6-di-O-isopropylidene-3-O-(phenyl cyclopropyl methanonyl)-alpha-D-glucofuranose (S-001-14) has been developed and validated in rat serum, urine and feces. Following extraction with hexane at alkaline pH, samples were separated on a reverse phase C18 column and quantified using UV detection at 267 nm. The mobile phase was 70% acetonitrile in ammonium acetate buffer (10 mmol/L, pH 6.0) with a flow rate of 1.0 ml/min. The method was used to determine the pharmacokinetics and excretion of S-001-14 after oral doses in rats. Linearity was satisfactory over the concentration range of 5-500 ng/ml (r2, > 0.99). Recoveries were > 90% and were consistent throughout the calibration range. The precision and accuracy were acceptable as indicated by relative standard deviation ranging from 2.72 to 9.54%, bias values ranging from 1.62 to 12.05%. Moreover, S-001-14 was stable in rat serum after being subjected to three freeze-thaw cycles and for 30 days on storage at - 60 degrees C. The method was used to determine the serum concentration-time profiles for S-001-14 after oral doses of 4, 100 and 200 mg/kg in rats. A linear pharmacokinetics was found in rats at 100 and 200 mg/kg doses with a long elimination half-life (approximately 24 h), wide distribution and bioavailability of approximately 13%. The excretion study after the 100 mg/kg oral dose revealed that S-001-14 was excreted in urine (0.002 +/- 0.001%) and feces (15.6 +/- 3.5%).

Absorption, protein binding, pharmacokinetics and excretion of the anti-ischemic and anti-hypertensive arylpiperazine derivative CDRI-93/478 in rats.

CDRI-93/478 (1- [4-(4-fluorophenyl) piperazine-1-yl]-3-(2-oxopyrrolidin-1-yl) propane hydrochloride, an arylpiperazine derivative, is a potent anti-ischemic and anti-hypertensive agent and is in advanced stage of preclinical trials. In order to develop CDRI-93/478 into a clinical agent, the absorption, protein binding, pharmacokinetics, and excretion of the compound were investigated in male Sprague-Dawley rats. Oral absorption was evaluated in situ and in vivo, using the portal-venous concentration difference method. The compound showed negligible absorption (ka = 0.01 h(-1)) at pH 2.6. However, the rate of absorption of the compound at pH 7.4 was 0.6 h(-1) and was comparable to that observed in the in vivo study (ka, >0.58 h(-1)) in rats after a single 2 mg/kg oral dose. In vitro and in vivo protein binding studies using the ultrafiltration method showed that the compound was subject to low protein binding (<40%) and was independent of the substrate concentration over a range of 1-16 microg/ml. Pharmacokinetic parameters of the compound were determined after intravenous and oral administration of 0.6, 2 and 8 mg/kg doses using a model independent method. After oral administration, the compound showed the double-peak phenomenon, which could be due to the high water solubility (log P, 1.01 +/- 0.01), regional differences in the gastrointestinal absorption and enterohepatic recirculation effects. The absorption of CDRI-93/478 was rapid and showed a bioavailability of 69.9 +/- 5.1% (mean +/- S. D.) after 2 and 8 mg/kg oral dose. However, the pharmacokinetic parameters of the compound could not be determined after the 0.6 mg/kg oral dose due to insufficient data points. The studies following intravenous and oral administration demonstrated linear pharmacokinetics, low clearance and high volume of distribution over the dose range studied. The excretion studies after the 8 mg/kg oral dose indicated that the compound was not excreted through the feces and the urinary excretion was very low (<2%).

A sensitive and selective liquid chromatographic tandem mass spectrometric assay for simultaneous quantification of novel trioxane antimalarials in different biomatrices using sample-pooling approach for high throughput pharmacokinetic studies.

In the present studies, to give momentum to traditionally low throughput pharmacokinetic screening, a bioanalytical method based on the concept of sample pooling for simultaneous bioanalysis of multiple compounds is discussed. A sensitive, selective, specific and rapid HPLC/ESI-MS/MS assay method was developed and validated for the simultaneous quantitation of three novel trioxane antimalarials (99-357, 99-408 and 99-411) in rat plasma using trioxane analogue as internal standard. The suitably validated bioanalytical method was then further extrapolated to rabbit and monkey plasma by performing partial validation. Extraction from the plasma involves a simple two-step liquid-liquid extraction with n-hexane. The analytes were chromatographed on a cyano column by isocratic elution with acetonitrile:ammonium acetate buffer (pH 6) (85:15, v/v) and analyzed by mass spectrometry in multiple reaction-monitoring (MRM) mode. The chromatographic run time was 5.5 min and the weighted (1/x(2)) calibration curves were linear over a range of 1.56-200 ng/ml. The limit of detection (LOD) and lower limit of quantification (LLOQ) in rat plasma, rabbit plasma and monkey plasma were 0.78 and 1.56 ng/ml, respectively, for all three analytes. The intra- and inter-batch accuracy and precision in terms of % bias and % relative standard deviation were found to be well within the acceptable limits (< 15%). The average absolute recoveries of 99-357, 99-408 and 99-411 from spiked plasma samples were > 90%, > 70% and > 60%, respectively. The assay method described here could be applied to study the pharmacokinetics of 99-357, 99-408 and 99-411 using sample-pooling technique.

In Vitro and In Vivo pharmacokinetic studies of bulaquine (analogue of primaquine), a novel antirelapse antimalarial, in rat, rabbit and monkey--highlighting species similarities and differences.

Bulaquine (BQ) is a potent antirelapse antimalarial developed by CDRI, India. Bulaquine was rapidly absorbed in rats and rabbits with no distinct absorption phase while in monkeys a variable irregular absorption profile was observed. BQ was extensively converted to primaquine (PQ) after oral administration and the conversion was maximum in rats and minimum in rabbits, which is possibly due to the species difference. Clearance was higher in rats (3.2 l/h/kg) than in rabbits and monkeys (1.2 l/h/kg) and it was found be negligibly excreted in rat urine and feces. The elimination half-life in rats and rabbits was comparable after both oral and i.v. administration ( approximately 1.2 h). In all three species, PQ was resident in the body for a period longer than BQ. PQ, being the major active metabolite of BQ, might be responsible for the extended therapeutic effect of BQ. The oral bioavailability of BQ was 3.12%, 5.3% and 12% in rats, rabbits and monkeys, respectively, which could be mainly due to the high instability of BQ at acidic pH as demonstrated from a simulated gastric fluid stability study. Protein binding in various species was in the range 50-65% while the partition coefficient between RBCs and plasma (K(rbc/pl)) was between 0.75 and 1, indicating significant RBC uptake.

Effect of concurrently coadministered drugs on the pharmacokinetic/pharmacodynamic profile of centchroman, a nonsteroidal oral contraceptive, in rats.

INTRODUCTION: Centchroman (international nonproprietary name: ormeloxifene) is a nonsteroidal selective estrogen receptor modulator, oral contraceptive, anticancer and antiosteoporotic agent that is intended for long-term use by women. In view of the vast clinical applications and interactions of steroidal oral contraceptives with commonly used therapeutic agents, the interaction potential of certain concomitantly administered therapeutic agents was investigated in terms of postcoital contraceptive efficacy (pharmacological) and the pharmacokinetic profile of centchroman in female Sprague-Dawley rats. The coadministered drugs used in the study were ciprofloxacin, cefixime, amoxicillin, metronidazole, amlodipine, atenolol, theophylline, metformin, pioglitazone and glibenclamide. MATERIALS AND METHODS: The pharmacological activity of centchroman was evaluated in sperm-positive female rats at 1.5 mg/kg, with or without coadministered drugs. Rats were sacrificed on Day 10 postcoitus, and autopsy was performed to check for the presence or absence of implantations. The estrogenic and antiestrogenic activities of centchroman were evaluated in immature ovariectomized rats. Pharmacokinetic interaction was studied in normal female rats with or without coadministered drugs. Serum samples were taken over 120 h and analyzed using a validated high-performance liquid chromatography method to generate the pharmacokinetic profile of centchroman. Pharmacokinetic parameters were estimated using noncompartmental analysis, and the results were compared. RESULTS: In pharmacological interaction studies, centchroman alone showed a 100% success rate when given alone or in the presence of coadministered drugs. The only exception was amoxicillin coadministration, with 66% rats in the group showing resorbed implantations. Further investigation with amoxicillin in ovariectomized immature rats indicates no alteration in the estrogenic and antiestrogenic profiles of centchroman. In pharmacokinetic interaction studies, most of the therapeutic agents affected the rate and extent of absorption of centchroman. In other pharmacokinetic parameters, clearance (CL) remained unchanged; however, there was decrease in bioavailability (F) and volume of distribution (V(d)) in some situations. CONCLUSIONS: The results indicate that there is no direct link between the altered pharmacokinetics of centchroman and the failure of pharmacological effect. The pharmacological interaction with amoxicillin could not be explained on the basis of alteration in the estrogenic and antiestrogenic activities of centchroman, indicating that different mechanisms are involved. The findings, however, suggest that amoxicillin coadministration may result in pharmacological interaction with centchroman and that caution should be taken in clinical practice.

Analysis and pharmacokinetics of bulaquine and its major metabolite primaquine in rabbits using an LC-UV method--a pilot study.

A precise and reproducible HPLC assay has been developed and validated for simultaneous determination of bulaquine (BQ) and its metabolite primaquine (PQ) in rabbit plasma. The method, applicable to 0.5 ml plasma, involves double extraction of samples with n-hexane: isopropanol (98:2, v/v) containing dimethyl octylamine (DMOA) (0.1%, v/ v). Separations were accomplished by reversed-phase liquid chromatography using a Spheri-5 cyano column with a low pressure gradient with mobile phase consisting of ammonium acetate buffer (50 mM, pH 6.0) and acetonitrile with DMOA. The method was sensitive with a limit of quantitation of 20 ng ml(-1) in rabbit plasma for both BQ and PQ and the recoveries were > 85 and > 45%, respectively. Excellent linear relationships (r > 0.99) were obtained between the measured and added concentration ratios of the plasma concentrations over a range of 20-1000 ng ml(-1) for both the analytes. Precision and accuracy were acceptable as indicated by relative standard deviations from 1.8 to 15.1%, bias values ranging from -14.2 to 15.7%. Moreover, BQ was stable in rabbit plasma for 15 days of storage at -60 degrees C and after being subjected to three freeze-thaw cycles. The method was applied to determine the levels and pharmacokinetics of BQ in rabbits following a single 2.5 mg kg(-1) oral and intravenous dose. The BQ levels declined and the PQ levels increased with time. The PQ/BQ ratio after oral dose at 1 and 1.5 h were higher than that after intravenous dose. In the pilot preclinical pharmacokinetic study after a single 2.5 mg kg(-1) oral dose, BQ levels were determined up to 6 h (post-prandial) and 8 h (fasting). The plasma concentration versus time data were best fitted to a two-compartment open model with first-order absorption and elimination processes without lag time. The AUC(0-infinity) and the elimination t1/2 in fasted rabbit was higher than that in post-prandial rabbit indicating the effect of food on BQ pharmacokinetics.

LC determination of a sulphur mustard decontaminant CC-2 in rat serum.

A high-performance liquid chromatographic (HPLC) method was developed for the analysis of N,N'-Dichlorobis (2,4,6-trichlorophenyl) urea (CC-2), a potent sulphur mustard decontaminant, in rat serum. The HPLC analysis, applicable to 0.5 ml volumes of serum, involved double extraction of serum samples with diethyl ether at alkaline pH followed by separation on a RP-18 column and the use of UV detector at 230 nm. The method was sensitive with a limit of quantitation of 10 ng ml(-1) in rat serum and the recovery was always >90%. Excellent linear relationships (r>0.99) were obtained between the measured and added concentration ratios of the serum concentrations over a range of 10-200 ng ml(-1). The precision and accuracy were acceptable as indicated by relative standard deviation ranging from 2.47 to 17.49%, bias values ranging from -4.35 to 13.21%. Moreover, CC-2 was found stable in rat serum even after 3 months of storage at -60 degrees C and being subjected to three freeze-thaw cycles. The assay was found to be sensitive, specific, accurate, precise, and reliable for use in pharmacokinetic studies.

A sensitive LC assay for the simultaneous determination of centbutindole and its metabolite in rat serum using fluorescence detection.

Centbutindole (+/-) 2-gamma-[(p-fluorobenzoyl)propyl]-1, 2, 3, 4, 6, 7, 12, 12a-octahydro-pyrazino (2', 1': 6, 1) pyrido [3, 4-b] indole (I), is a new neuroleptic agent developed by Central Drug Research Institute, India. In the present study, a high performance liquid chromatography (HPLC) assay method for the simultaneous assay for I and its metabolite (II) in rat serum was developed and validated. The present method requires only 1 ml of serum with detection levels similar to that reported earlier using 4 ml serum. This assay has been found to be more suited for pre-clinical as well as phase IV studies. Linearity was observed between 1.25 and 40 ng/ml for I and 0.625 and 20 ng/ml for II in rat serum. Recoveries were consistent for both the analytes over the concentration ranges studied. Variation in intra-and inter-batch accuracy and precision were within acceptable limits of +/-20% at lowest limit of quantitation, whereas at higher concentrations it was +/-15%. The assay method was employed for the study of the pharmacokinetics and metabolism of I in rats. The parent compound and its metabolites were quantitated in serum and could be monitored up to 24 h post dose.