Browsing by Author "Vallie, Sarfaraaz"

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    Accuracy Optimization of anti-TB Drug Assays using Protein Evaluation in Calibration Curves during Pharmacokinetics Quality Assurance
    (Stellenbosch : Stellenbosch University, 2021-12) Vallie, Sarfaraaz; Stander, Marietjie; Reuter, Helmuth; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. Medicine: Clinical Pharmacology.
    ENGLISH ABSTRACT: Quality assurance of drug assaying is an important aspect in clinical testing. Accuracy is important to ensure correct bio-analytical results by constructing calibration curves that took blood matrix interferences into consideration. I have adhered to the United States of America Food and Drug Administration (FDA) call for improved accuracy in bio-equivalence, bio-availability and administering of narrow therapeutic indexed drugs. Significant different plasma levels were observed in clinical trials for the occasional hyperproteinemia (an increase in protein concentration in the bloodstream) and hypoproteinemia (lower-than normal levels of protein in the blood) patients this includes disease-related hyperalbuminemia (an increased concentration of albumin in the blood) and hypoalbuminemia (a deficit of albumin in the blood) patients. This research supported a modeled approach for accuracy improvements by including the patients‘ plasma protein levels using a combined calibration curve (protein evaluation calibrations curves – PROTECC-PKTM). Levels of albumin were classified as marked hypoalbuminemia (<2.5 g/dL), mild hypoalbuminemia (2.5-3.5 g/dL), normal albumin (3.5-4.5 g/dL), and hyperalbuminemia (>4.5 g/dL). This research was specifically important for drugs with a narrow therapeutic index. The rifampicin method was developed, validated and the concentration calibration curve of rifampicin with and without plasma was assessed. The limit of detection for rifampicin with and without plasma was 0.189 μg/ml ± 0.082 and 0.080 μg/ml ± 0.053 μg/ml respectively (LOD ± mean standard diviation). The limit of quantification of rifampicin with and without plasma was 0.573 μg/ml ± 0.082μg/ml and 0.243 μg/ml ± 0.053μg/ml respectively (LOQ ± mean standard diviation). The r2 for rifampicin was 0.9971 without plasma and 0.9852 with plasma present. A novel analytical method for determination of the % protein content present in blood plasma was performed using the Karl Fischer Titration process. Results indicated deviation in % protein of blood plasma for patients compared to literature values of about 8 %. Using the data obtained, the PROTECC-PKTM curves indicated that the relative accuracy differed by a minimum of 0.1% for low binding affinity drugs and a maximum of more than 20% for drugs with moderate binding affinities. The relative accuracy of the anti TB drugs was supported by computational modelling and thermodynamic analytical methods for each drug during multiple drug co-administration regimens. This study focused on the drug binding affinity that affects the extrapolation of the patient‘s sample drug concentration from the slope of LCMS calibration curve. The binding constants calculated from fluorescence spectroscopy data were as follows: rifampicin 5.379 x 102 M-1 (moderate affinity), isoniazid 9.285 M-1 (low affinity), 25-desacetyl rifampicin 3.156 M-1 (low affinity), ethambutol 3.443 M-1 (low affinity) and pyrazinamide 3.076 X 102 M-1 (moderate affinity). These drugs Gibbs free energies for these drugs indicated spontaneous binding reactions. Rifampicin, a non-polar weak acid with a higher affinity, showed the most stable complex formation with human serum albumin (HSA) compared to soluble isoniazid. This is because isoniazid in its ionized form can be easily excreted in the urine resulting in low levels of detection. This will affect the bioavailability and accuracy of the assay levels for patients experiencing hyper and hypoalbuminemia with related competition and induction processes of the enzymes. These complications are apparent where a larger number of patients are involved in clinical trials, bioequivalence and bioavailability studies with varying protein levels that may be more crucial for drugs with a narrow therapeutic index.