Unsuspected poor metabolizer phenotypes of fluoxetine in bioavailability/bioequivalence studies from an indian population perspective. Retrospective pharmacokinetic data evaluation.

BACKGROUND: Fluoxetine, belonging to the class of selective serotonin uptake inhibitors, has been extensively used for the treatment of depression and other psychiatry related disorders. Fluoxetine (CAS 59333-67-4) is a substrate of polymorphic cytochrome P450 2D6 isozyme (CYP2D6) leading to the formation of norfluoxetine (CAS 83891-03-06), which is not only active but long lived than the parent in the systemic circulation. Since the parent and metabolite levels are important from a therapeutic perspective, knowledge of phenotypic distribution of the population may be an important consideration. OBJECTIVE: The aim of the work was to retrospectively evaluate the pharmacokinetic data of fluoxetine and norfluoxetine from several bioavailability/bioequivalence (BA/BE) studies to identify the poor metabolizer (PM) phenotypes from the unsuspected healthy subjects across varied protocol designs, dose sizes and differing formulations. METHODS: The pharmacokinetic data of fluoxetine and norfluoxetine were gathered from several BA/BE studies conducted at clinical facilities located at Bangalore and Chennai, India. The BA/BE studies involved open label, two-way randomized crossover designs with two periods and two treatments (reference and test). Blood samples were collected for at least 672 h after fluoxetine dosing and the plasma was analyzed using validated tandem liquid chromatography mass spectrometric assay to determine fluoxetine and norfluoxetine levels. Standard pharmacokinetic parameters were computed using noncompartmental methods. For the purpose of this paper, retrospective evaluation of pharmnacokinetic data from only the reference formulation was considered. The AUC ratio of fluoxetine/norfluoxetine was computed. The individual fluoxetine/norfluoxetine AUC(0-infinity), ratios were plotted in increasing rank order and using outlier test ('T' procedure at 5% level of significance) the subjects were categorized as extensive metabolizer (EM) and PM phenotypes. The unequivocal confirmation of PM phenotypes was obtained by performing linear regression analysis of fluoxetine vs norfluoxetine AUC(0-infinity) values. RESULTS: Each study was evaluated for the distribution of EM and PM phenotypes of fluoxetine. There were 144 subjects evaluable from four studies, 89.6% (129 out of 144) of which could be categorised as EMs and 10.4% (15 out of 144) as PMs of fluoxetine. The pharmacokinetic parameters were quite distinct between the two phenotypes: (1) PM phenotypes showed much higher exposure (approximately 2.3-fold increase in AUC(0-infinity) and much slower elimination (almost 2-fold increase in elimination half-life) for fluoxetine as compared to EM phenotypes; (2) PM phenotypes showed approximately 0.5-fold lower exposure of norfluoxetine as compared to the EM counter parts; (3) There was no change (approximately 1.2-fold) in the elimination half life of norfluoxetine in EM and PM phenotypes. CONCLUSIONS: Retrospective evaluation of fluoxetine and norfluoxetine pharmacokinetic data demonstrated existence of both PM and EM phenotypes in the Indian population. Based on the overall data (n=144 subjects) there appeared to be 10.4% of PM phenotypes for fluoxetine and/or possibly for other polymorphic CYP2D6 substrates commonly used in this region.

Influence of number of calibration standards within a defined range on pharmacokinetic disposition-case studies with omeprazole and clopidogrel carboxylic acid.

While the practice of using a smaller number of non-zero standards (typically seven to eight) has not been entertained in routine bioanalytical work, it is important to innovate and be pragmatic about minimizing the number of calibration standards to promote cost-effective and speedy assessment. In this exercise, two important compounds, omeprazole and clopidogrel carboxylic acid, were considered. Additionally, both analytes offered a 1000-fold calibration curve range with eight non-zero standards to permit a systematic evaluation. Accordingly various scenarios of post-hoc analysis of the calibration data were formulated which included step-wise reduction of the number of calibration standards from a maximum of n = 8 to a minimum of n = 3. In all the scenarios evaluated in this exercise, a calibration curve was reconstructed and both quality control samples and in vivo pharmacokinetics were calculated in each instance. Based on the data generated in this exercise, a minimum of three non-zero calibration standards were adequate to predict the quality control samples with the predefined accuracy and precision estimates for both omeprazole and clopidogrel carboxylic acid. Additionally, the in vivo pharmacokinetic characterization of the chosen compounds was not hampered by the reduction of calibration standards (from n = 8 to n = 3). Hence, consideration for reducing number of calibration standards in bioanalytical work may provide a viable alternative in several situations such as formulation screening strategies, routine therapeutic drug monitoring and sparse sample analyses.

Sensitivity enhancement and matrix effect evaluation during summation of multiple transition pairs-case studies of clopidogrel and ramiprilat.

Sensitivity enhancement via summation of multiple MRM transition pairs is gaining popularity in tandem mass spectrometric assays. Numerous validation experiments describing the assays for two model substrates, clopidogrel and ramiprilat, were performed. The quantitation of clopidogrel was achieved by the summation of transition pairs m/z 322.2 to m/z 212.0 and m/z 322.2 to m/z 184.0, while that of ramiprilat was achieved by the summation of transition pairs m/z 389.2 to m/z 206.1 and m/z 389.2 to m/z156.1. The use of summation approach achieved sensitivities of >2 fold for both compounds as compared with the reported single MRM transition pair assays. The validation experiments addressed some important assay development issues, such as: (a) lack of impact of matrix effect; (b) unequivocal verification of the percentage contribution of each MRM transition pair towards sensitivity; (c) sensitivity enhancement factor achieved by summation approach of MRM transition pairs; and (d) accurate prediction of quality control samples using summation approach vs a single MRM transition pair. In summary, the appropriateness of using two MRM transition pairs for quantitation was demonstrated for both clopidogrel and ramiprilat. Additionally, pharmacokinetic application of the MRM transition pair assays using a summation approach was established for the two compounds.