Nelamangala V. Nagaraja

Choosing the calibration model in assay validation.

Data transformations and weighting schemes are normally used to obtain the best-fit of standard curves in bioanalysis and the calibration model is usually selected during prevalidation. In the present study, a comparison has been made between unweighted and weighted (1/x, 1/x2, and 1/square root of x) regression models with or without an intercept in achieving the best-fit for the standard curve of CDRI compound 81/470, a new anthelmintic agent, in cow milk. Validation samples in milk at the LLOQ, medium, and high concentrations were also analysed by each of the calibration models. An unweighted regression equation with an intercept overestimated the concentrations at the LLOQ. An unweighted equation without intercept and weighted equations with or without an intercept significantly minimized the bias at the LLOQ without distorting the results at higher concentrations. Hence, an unweighted equation for a straight line passing through the origin was found to be the best model for a standard curve of 81/470 in milk. Similar results were obtained for 81/470 and UMF-078 in serum and plasma, respectively. Bioanalysts should routinely test these models to obtain the best fit model for their calibration curves as part of their assay validation not during prevalidation.

Optimization of contraceptive dosage regimen of Centchroman.

Centchroman (Ormeloxifene), a non-steroidal oral contraceptive, is used at a dose of 30 mg once a week. To prevent failures in the beginning of the therapy, it is recommended that a dose of 30 mg twice a week for 12 weeks be administered to build up adequate blood levels. The present study was undertaken to simplify the dosing schedule without sacrificing the purpose of twice a week dosing regimen, using modeling and measurement approaches. The drug was given to 60 female volunteers who were divided into seven groups: group I, 30 mg weekly; group II, 30 mg twice a week; group III, 30 mg twice a week for 12 weeks followed by 30 mg weekly; group IV, 30 mg twice a week for 6 weeks followed by 30 mg weekly; group V, 60 mg weekly; and groups VI and VII, single 60 mg loading dose followed by 30 mg weekly doses. The blood samples were collected and analyzed by HPLC. In group I, mean trough concentrations of centchroman and its active metabolite, 7-desmethyl centchroman, were comparable to the steady-state trough concentrations in groups III, IV, VI, and VII. The metabolite to parent drug ratio remained constant in all the groups. The pharmacokinetic parameters in group VII were comparable to those reported after a single 30 mg dose. Dosage regimen VI was more convenient and provided better pregnancy protection (Pearl index 1.18; unpublished report) than regimen III, which is currently on the market and, thus, could be effectively used for contraception.

Tissue distribution and excretion of CDRI-81/470 in rats.

Methyl‐N[5[[4‐(2‐pyridinyl)‐1‐piperazinyl]carbonyl]‐1H‐benzimidazol‐2‐yl] carbamate (CDRI‐81/470) is a broad spectrum anthelmintic agent, effective against both intestinal and systemic parasitism. Tissue distribution and excretion of CDRI‐81/470 were studied in rats after a single oral dose of 100 mg kg−1 CDRI‐81/470.

Sensitive high-performance liquid chromatographic method for the determination of methyl N-[5-[[4-(2-pyridinyl)-1-piperazinyl]carbonyl]-1H-benzimidazol-2-yl] carbamate in rat blood

A sensitive high-performance liquid chromatographic assay has been developed and validated for the determination of methyl N-[5-[[4-(2-pyridinyl)-1-piperazinyl]carbonyl]-1H-benzimidazol-2-y l d carbamate (CDRI compound 81/470) in normal rat blood. The method described herein is simple, with improved selectivity and sensitivity over a previously reported HPLC method. The limit of quantitation is 10 ng/ml (method 1) and 2.5 ng/ml (method 2) in blood, as compared with 40 ng/ml for the previous method. The standard curve in blood is linear over the concentration range 10-1000 ng/ml in method 1 and 2.5-1000 ng/ml in method 2 and the extraction recovery is higher than 80% for both methods.

Determination of antifilarial compound UMF-078 and its metabolites in plasma by high-performance liquid chromatography

UMF-078, methyl (+/-)-[5-(alpha-amino-4-fluorobenzyl)benzimidazol-2-yl]carba mate, is a new antifilarial compound being developed by the World Health Organization. In the present study, a HPLC method for the simultaneous estimation of UMF-078 and its metabolites (flubendazole, decarbamoylated flubendazole, UMF060 and decarbamoylated UMF-060) in plasma was developed, validated and applied to pharmacokinetic studies. Linearity was observed between 20 and 1000 ng/ml for decarbamoylated UMF-060 and between 10 and 500 ng/ml for other analytes. Recoveries were consistent over the concentration ranges studied for all the analytes. Variations in intra- and inter-batch accuracy and precision were within acceptable limits of +/-20% at the lowest limit of quantitation, whereas at higher concentrations it was +/-15%. The analytes showed stability up to two freeze-thaw cycles in plasma. No degradation was observed for any of the analytes even after 72 h of storing the dry plasma extracts at -30 degrees C. The assay method was employed to study the pharmacokinetics of hydrochloride salt of UMF-078 in rats. The parent compound and its metabolites viz: decarbamoylated UMF-060, UMF-060 and flubendazole were quantitated in serum and the compounds could be monitored up to 168 h post-dose.

Preliminary observations on the pharmacokinetics of CDRI compound 81/470 in calves

Compound 81/470 (methyl-N-[5[[4-(2-pyridinyl)-l-piperazinyl]carbonyl]-lH-benzimidazol-2-yl] carbamate, Figure 1) (AH) is a new broad-spectrum anthelmintic agent (Kumar et al., 1984) which is being developed for human and veterinary use in this institute. AH has shown efficacy against both intestinal and systemic helminths, including Ancylostoma ceylanicum, Nippostrongylus brasiliensis, HymenoIepis nana, Syphacia obvelata, Toxocara sp. and Brugia malayi, in experimentally infected animals

Estimation of anthelmintic compound 81/470 in cow's milk by high-performance liquid chromatography: method development and validation.

CDRI compound 81/470 is a new broad spectrum anthelmintic agent and is being developed for veterinary use. HPLC assay method for 81/470 in cow milk was developed and validated. The sample preparation consisted of protein precipitation, followed by extraction with ether. Separation was achieved on a C18 column using acetonitrile-buffer (pH 6, 50 mM) mobile phase and the compound was quantitated using fluorescence detector. The recovery of 81/470 was above 90% and was consistent over the calibration range of 10-1000 ng ml-1. Accuracy and precision were determined by analyzing replicate samples and were found to be within acceptable limits. Five freeze-thaw cycles of spiked milk and storage of processed dry residues at -30 degrees C for 7 days did not have any detrimental effect on the stability of 81/470.

Preliminary pharmacokinetics of a new anthelmintic agent, CDRI-81/470 in healthy subjects

Abstract Single dose pharmacokinetic study of CDRI-81/470, a new broad spectrum anthelmintic agent, was carried out in 12 healthy human subjects after a single 375-mg oral dose. The serum, saliva and urine samples were analyzed by HPLC. The compound attained peak serum levels of 15.1±4.6 μg/ml in 2.6±1.1 h and could be measured up to 5 days. Mean serum AUC was 195±69 μg per h/ml. The compound showed a mean apparent elimination half-life of 12.1±4.5 h while mean residence time (MRT) was found to be 11.1±1.7 h. Extent of urinary excretion of CDRI-81/470 (2.3±0.7%) was less than that of its decarboxylate metabolite (5.3±2.2%) up to 10 h post dose. In vivo protein binding in serum was 93.1±1.2% and remained constant over in vivo concentration range. The salivary levels of CDRI-81/470 were higher than the corresponding unbound serum levels. There was a significant correlation between serum and salivary levels of CDRI-81/470, with a mean ratio of saliva to unbound serum levels of 2.04±0.24, indicating the possibility of predicting serum concentrations of CDRI-81/470 from non-invasive salivary sampling technique.