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Title: The clinical pharmacology of Human Immunodeficiency Virus (HIV) therapy failure
Author: Watson, Victoria
ISNI:       0000 0004 5351 083X
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2014
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The work in my thesis focuses on developing highly sensitive tests, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the measurement of antiretroviral drugs within plasma, cells, and cerebral spinal fluid from clinical studies evaluating compartmentalised antiretroviral therapy (ART) pharmacokinetics and forgiveness for missed or late dosing. Firstly I developed and validated a LC-MS/MS assay to quantify the antiretroviral (ARV) drugs, lopinavir, darunavir, ritonavir and raltegravir in peripheral blood mononuclear cells (PBMCs). This intracellular assay included optimising methodology for cell separation to minimise loss of drug (Chapter 4). All drugs eluted within an 8 minute run time. Matrix effects were minimal (-2.9%). Calibration curves were validated over a concentration range of 0.4-150ng/mL. Intra and inter assay variation ranged between 0.01-2.29% for precision and 96.76-102.32% for accuracy. Secondly I developed and validated a LC-MS/MS assay to simultaneously detect and quantify 10 ARVs; maraviroc (MVC), nevirapine (NVP), rilpivirine (RPV), raltegravir (RAL), atazanavir (ATV), darunavir (DRV), amprenavir (APV), ritonavir (RTV), lopinavir (LPV) and etravirine (ETV) in cerebral spinal fluid (CSF). All drugs eluted within a 10 minute run time. Calibration curves were validated over the following concentration ranges; LPV, MVC, RTV = 0.78-100 ng/mL, RAL, APV, ATV, RPV, ETV, DRV = 1.95-250 ng/mL and NVP = 19.5-2500 ng/mL (r2 values >0.99; quadratic 1/x). Intra and inter assay variation ranged between 1.59-15% for precision and -10.5-6.4% for accuracy. Carryover was <20% of the lower limit of quantification for all drugs. The recovery was >70% and the CV% at low, medium and high concentrations was less than 20% for all drugs. Thirdly I developed and validated a LC-MS/MS assay to quantify intracellular tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP) within PBMCs. This work was very technically challenging and something that was not being done by any other laboratory within the United Kingdom. Analytes eluted within 12 minutes run-time with adequate separation. Calibration curves were validated over the following range TFV-DP=0.35-10.91 ng/mL, FTC-TP=0.38-103.17 ng/mL (r2 values >0.99; linear 1/x). The lower limit of quantification was <20 %, signal to noise was >5% and carryover <0.1%. The precision was; TFV-DP=6.3-11% and FTC-TP=6-18.6%, and accuracy was TFV-DP=97.5-100.8%, FTC-TP=98-100.3%. Finally, all assays developed and validated were successfully applied to collaborative clinical trials (see Communications; Published Research Papers). Benefits are expected to accrue from this work in the design of more forgiving therapy regimens for HIV patients, and better drug selection to specifically target HIV replicating within sanctuary sites.
Supervisor: Khoo, Saye; Owen, Andrew; Back, David Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: RM Therapeutics. Pharmacology