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.

A high throughput approach for simultaneous estimation of multiple synthetic trioxane derivatives using sample pooling for pharmacokinetic studies.

The present study describes the application of concept of sample pooling to increase the throughput of pharmacokinetic screening at drug discovery and development stage. An HPLC-UV method for the simultaneous estimation of three synthetic antimalarial compounds 99/357, 99/408 and 99/411 has been developed and validated in rat serum with internal standard for pharmacokinetic profiling. Drug compounds in serum were extracted by two-step liquid-liquid extraction with 2% isopropyl alcohol in n-hexane and quantitated using a validated gradient HPLC-UV method, which was made feasible for all compounds using gradient elution scheme. The method was validated in terms of HPLC reproducibility, linearity, specificity, recovery, accuracy and precision, freeze thaw stability and long-term storage stability. Excellent linear relationships (r>0.99) were obtained for calibration as well as analytical standards over a concentration range of 25-1000 ng/ml for three analytes. Recoveries were fond to be >85% for 99/408 and 99/357 and >70% for 99/411. The method developed for three analytes was found to be accurate and precise as bias and percent relative standard deviation (% R.S.D.) values were within limits (<20%). By employing sample pooling approach, plasma level - time profile following single intravenous dose of all three compounds were obtained in a fraction of the time required by conventional single compound dosing and analysis.

HPLC-UV method for assaying 99/357, a synthetic trioxane antimalarial derivative in rat and rabbit serum.

In the present study an accurate and precise HPLC-UV assay method in rat and rabbit serum has been developed and validated for determination of 99/357--a new synthetic analogue of artemisinin developed by Central Drug Research Institute (CDRI), of the class of trioxane derivative. Separation was achieved using a C-18 reversed phase column with a mobile phase comprising of acetonitrile and deionized water (80:20%, v/v) using a UV detector, set at a wavelength of 266 nm. The method, applicable to 200 microl serum, involved double extraction of the samples with 20% isopropyl alcohol (IPA) in n-hexane. The recovery of 99/357 in the two matrices was >90%. The method was sensitive with a limit of quantitation of 25 ng/ml in both rat and rabbit matrices. Precision and accuracy were within the acceptable limits, as indicated by relative standard deviation (accuracy) varying from -12.7 to 5.7% and bias (precision) values ranging from 0.6 to 11.8%. Moreover, 99/357 was stable in serum up to 30 days of storage at -60 degrees C and after being subjected to three freeze/thaw cycles. The method will be applied to perform pharmacokinetic studies of 99/357.

Dengue Fever with rectus sheath hematoma: a case report.

Dengue fever, also known as breakbone fever, is an infectious tropical disease caused by the Dengue virus. It is associated with a number of complications, which are well documented. However, Dengue fever associated with rectus sheath hematoma (RSH) is a very rare complication. Only one case report has been published prior supporting the association of Dengue fever with RSH. We report a case of Dengue fever who presented with RSH and was successfully treated conservatively. RSH is also an uncommon cause of acute abdominal pain. It is accumulation of blood in the sheath of the rectus abdominis, secondary to rupture of an epigastric vessel or muscle tear.

Simulation-based sodium thiosulfate dosing strategies for the treatment of calciphylaxis.

BACKGROUND AND OBJECTIVES: Calciphylaxis remains a poorly understood life-threatening disorder with limited therapeutic options. Sodium thiosulfate (STS) has reported efficacy, thought to be because solubilizing calcium deposits promote clearance by hemodialysis (HD). Lack of rigorous pharmacokinetic studies makes it problematic for determining proper STS dosing given the expanding range of dialysis prescriptions and intensities. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: The purpose of this study was to determine the dosing strategies for STS during different dialysis regimens. Given reported successes using an empiric 25 g, intravenous, 3 times per week after HD, simulations were performed to predict dosing guidelines for alternative, more or less intense dialysis to produce equivalent area under the curve drug exposure. The modeled prescriptions varied HD time from 12 to 40 h/wk over three to six sessions (Q(b) 200 to 400 ml/min, Q(d) 500 to 800 ml/min), and continuous venovenous hemodialysis at low flow rates (Q(b) 100 to 200 ml/min, Q(d) 35 to 50 ml/min), using high-flux polysulfone hemofilters. RESULTS: Simulations showed a marked variation in STS doses depending on HD frequency and duration. Blood and dialysate flows have a less prominent effect. Assuming no residual renal function, HD prescription permutations caused the dose to vary from 72 to 245 g/wk (70-kg adult), and the simulations provide specific guidelines for clinicians. CONCLUSIONS: Based on the success reported for one STS dosing regimen and assuming area under the curve exposure of STS is proportional to its effect, pharmacokinetic simulations can be used to calculate the dose for alternative, higher or lower intensity dialysis regimens. These strategies are imperative to assure adequate treatment for this mortal disease, as well as to avoid toxicity from excess dosing.

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.

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.