Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Fatemeh Akhlaghi

Abstract

The immunosuppressive agents used to prevent rejection of transplanted organs include cyclosporine (CsA), everolimus (EVE), mycophenolic acid (MPA), prednisolone (PLN), sirolimus (SIR) and tacrolimus (TAC). Because of the narrow therapeutic index and high inter- and intra-subject variability of these agents, therapeutic drug monitoring (TDM) is an integral part of immunosuppressive therapy following organ transplantation. The immunosuppressants incidence and severity of side effects correlate with the degree of exposure while under-dosed patients can be at a greater risk for allograft rejection. Currently, whole blood or plasma samples that are obtained via venipuncture are used for routine immunosuppressive monitoring. The limitations of venipuncture blood samples include (i) invasive nature associated with the sample collection and (ii) weak correlation with the drug concentration at the site of action. This thesis is consisted of the following sections written in a manuscript format.

Manuscript I provides a comprehensive review of literature published on alternative techniques that are proposed to overcome the limitation of venipuncture sampling. These methods include the use of non-conventional techniques, namely, drug monitoring in oral fluids or blood samples obtained from fingertip as well as drug concentration measurement in lymphocytes or transplanted tissue.

Drug concentration measurement in lymphocytes or transplanted tissue is primarily aimed at obtaining information on drug level at the site of action thus to facilitate prediction of clinical outcomes. However, these approaches are impractical in clinical setting because of the invasive nature of sampling as well as complicated sample preparation procedures.

The objective of finger prick sampling is to mitigate the discomfort and difficulties associated with venipuncture, especially in pediatrics and frail patients. In this approach, the fingertip blood samples are either applied onto a filter paper (dried blood spots) or are processed as a liquid. It has been reported that fingertip sampling was preferred to venipuncture by both patients and healthcare providers. Nevertheless, the main disadvantages of venipuncture whole blood sampling, which is the poor correlation with concentration at the site of action, still exist.

Finally, oral fluid sampling is a promising non-invasive method of therapeutic monitoring of immunosuppressive agents. Advances in analytical techniques have enabled measuring drug concentration in minute amount of sample. Drug concentration in oral fluids represents the free fraction which should theoretically represent drug concentration at the site of action.

Few comprehensive studies investigated the use of oral fluids as a medium for therapeutic drug monitoring. Therefore, this dissertation is focused on the development of sensitive and robust liquid chromatography tandem mass spectrometry methods for quantification of the most commonly used immunosuppressant agents, tacrolimus and mycophenolic acid. The methods are then used to quantify these agents in oral fluids samples collected from kidney transplant recipients.

Manuscript II describes, in details, the development and validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of tacrolimus in oral fluids. This method was validated in accordance with the current Food and Drug Administration (FDA) guideline. The Lower Limit of Quantification of this method is 30 pg/mL that is adequate for measuring tacrolimus concentration in oral fluid samples from transplant recipients. Full separation between tacrolimus and plasma phospholipids components was achieved in very short run time of 2.2 min. Very simple sample predations procedure was followed by extraction 50 µL of oral fluids with 100µL of acetonitrile.

Manuscript III in this manuscript, the method presented in manuscript II to quantify tacrolimus in oral fluids. It focused on investigating factors that may affect tacrolimus measurement in oral fluid, namely, sampling condition (resting, after mouth rinsing, and after give a saliva stimulant), sampling time, and blood contamination expressed as salivary transferrin level. The correlation between tacrolimus concentration in blood and oral fluids was investigated under these conditions. Correlation analysis revealed that samples collected after mouth rinse and at fasting provided better correlation in tacrolimus concentrations in blood and oral fluid.

Manuscript IV: Liquid chromatography tandem mass spectrometry methods was developed and validated according to current FDA Guidelines to quantify mycophenolic acid and its glucuronide metabolites in oral fluids, total concentration in plasma, and unbound fraction in plasma. Full separation of mycophenolic acid, metabolites, and plasma phospholipids was achieved within the total run time of 2.8 min.

Manuscript V: The assay described in manuscript IV was used to quantify mycophenolic acid and glucuronide metabolites in oral fluids. The aim was to investigate factors that may affect mycophenolic acid and glucuronide metabolites concentration in oral fluid, namely, sampling condition (resting, after mouth rinsing, and after saliva stimulation), sampling time, and blood contamination expressed as salivary transferrin level. The result of this study indicated that the blood contamination had an insignificant effect on the concentration of mycophenolic acid and metabolites in oral fluids. In addition, a good correlation was observed between AUC0-12 of MPA in OF samples and unbound and total MPA. In contrast, a weak association was observed between MPAG concentrations in oral fluids with total and unbound plasma concentration.

Manuscript VI: PF-5190457 is a ghrelin receptor inverse agonist that is currently undergoing clinical development for the treatment of alcoholism. In this manuscript, the development and validation of a simple and sensitive assay for quantitative analysis of PF-5190457 in human or rat plasma and rat brain was described using liquid chromatography-tandem mass spectrometry. Full separation was achieved between the analyte and phospholipids of the three matrices within the total chromatographic run time of 2.2 minutes. The manuscript also identified and described the abundance of phospholipids contents of the three matrices. The developed method successfully used to quantify the analytes in the three matrices as part of pre-clinical and ongoing clinical studies.

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