A primer in strategies towards the era of precision medicine

Author: Warit Ruanglertboon

Ruanglertboon, Warit, 2021 A primer in strategies towards the era of precision medicine, Flinders University, College of Medicine and Public Health

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Precision medicine has emerged as a more refined iteration of clinical pharmacology and personalised medicine. The capacity for precision medicine to provide more nuanced and individualised estimates of drug exposure and effect has emerged due to advances in access to information and technologies. Several promising approaches have been invented to address the precision dosing, although some could not be practically usable in the real clinical setting. This thesis investigated into two distinct paradigms, including computational- and laboratory-based approach evaluating their essence in addressing the precision dosing of cancer medicines. In this thesis, sorafenib, an oral tyrosine kinase inhibitor, was selected as an exemplar drug to be extensively studied under the array of methods to address precision dosing.

Clinical epidemiology and data analysis using a big data from the pharmaceutical companies were conducted in the first two Chapters of the thesis. Notably, the current dataset was considered a "high-quality" data as it was originally derived from real patients enrolled in clinical trials. This section focussed on a current dosing guideline of sorafenib regarding dose adjustment during the therapeutic course. The prior assumption was made that dose adjustment of sorafenib due to AEs could lead to worse survival outcomes in the long term. However, the landmark analysis set at the end of the first cycle of treatment (28 days) to examine the effect of dose adjustment on the survival outcomes of sorafenib in advanced HCC patients observed no poorer outcomes. Moreover, patients experienced dose reduction due to the early AEs observed the association with an improved OS. It could be suggested that the long-term clinical benefits of sorafenib may be derived from high initial dose/plasma concentrations.

Previous studies showed a negative impact of PPI use on the clinical benefits derived from several TKIs, which sorafenib is a member of this class, such as erlotinib. Hence, concerning the effectiveness of sorafenib in patients that PPI was mandatory, a brief investigation of the effects of concomitantly used of PPI with sorafenib on the clinical outcomes was conducted. It was concluded that concomitant use of PPI with sorafenib was not associated with inferior clinical benefits. Noticeably, the finding of no association between concomitant PPI use and sorafenib outcomes was demonstrated for both OS and PFS in both univariable and adjusted analysis. This finding promotes confidence in prescribing PPI together with sorafenib in advanced HCC patients requiring PPI, leading to improving patients' quality of life.

Subsequent studies expanded on the outcomes of the clinical epidemiology analyses and involved the application of simulation and modelling-based analysis performed on Simcyp® platform to interrogate the capacity of concentration-guided sorafenib dose adjustment. The simulation was performed with and without model-informed initial dose selection (MIDS) to enhance the proportion of patients that achieve a sorafenib plasma concentration within a pre-defined optimal therapeutic. The results obtained from the simulations revealed that covariates affecting sorafenib exposure were including hepatic CYP3A4 abundance, albumin concentration, BMI, BSA, sex and weight. By accounting for these covariates, it was possible to identified subjects at risk of failing to achieve a sorafenib Cmax ≥ 4.78 μg/mL with 95.0 % specificity and 95.2 % sensitivity. Concentration-guided sorafenib dosing with MIDS facilitated a sorafenib Cmax within the range of 4.78 to 5.78 μg/mL for 38 of 52 patients who failed to achieve a Cmax ≥ 4.78 μg/mL with the standard dosing regimen. On the other hand, concentration-guided sorafenib dose adjustment without MIDS resulted in 33 of 52 patients who required dose adjustment achieving a Cmax ≥ 5.78 μg/mL.

The final section of this thesis was dedicated to laboratory-based biomarker discovery. The study aimed to verify the utility of extracellular vesicles (EVs) derived biomarkers, as part of the liquid biopsy, for the prediction of drug Absorption, Distribution, Metabolism and Excretion (ADMExosomes). Initially, a preliminary study to validate the availability of DME in EVs derived from human plasms was first conducted. Human plasma EVs was characterised before proceeding to other downstream experiments. The characterisation reported the particle size of 50-150 nm and a presentation of the small EV marker, Tsg101. EVs isolated from human plasma contained peptides and mRNAs originating from CYP (1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4 and 3A5), UGT (1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10 and 2B15), and NADPH-cytochrome P450 reductase. The number of unique peptides detected for each protein ranged between 2 and 19, with a mean of 9.65. In addition, 5 unique peptides originating from NADPH-cytochrome P450 reductase (the redox partner required for CYP activity) were also detected. While cytochrome b5 (34.5 kDa) was not detected in the current analysis as it was not contained within the window of protein bands analysed, the presence of this protein in human derived EVs has already been established.

Additional analysis following the confirmation of the availability of DME in EVs was conducted, proposing to verify the functionality aspect. In vitro hepatocyte cells line was used as the sources to produce a large amount, controllable and robust EVs. Meanwhile, the parallel experiments were conducted on human serum EVs obtained from healthy volunteers. The abundance of CYP3A4 and its catalytic activity derived from EVs samples were broadly evaluated. Targeted proteomics revealed the availability of CYP3A4 peptides derived from EVs isolated from HepaRG and human serum. Subsequently, determination of CYP3A4 catalytic activity was conducted using metabolite formation assay. LC-MS analysis detected 1OH midazolam and sorafenib N-oxide formation after incubation of human serum EVs with midazolam or sorafenib, respectively. However, no metabolite formation was observed from incubation of probe substrates with HepaRG EVs. The factors contributing to the positive metabolite formation derived from human serum EVs may be related to the integrity and conformation of CYP3A4 enzymes. However, complete attestation has not yet been identified in this thesis.

Taken together, the data presented in this thesis demonstrated a multidimensional approach to address precision dosing of sorafenib. The strengths and limitations of each technique were thoroughly investigated to explore the most appropriate method for the real clinical setting. Noticeably, the finding of DME panels and functionality provided an excellent opportunity to develop this knowledge to the real clinical diagnosis and treatment. Ultimately, this novel biomarker could serve as a bridging mediator not only to support an immediate dose monitoring and adjustment but also as a source to support model-informed precision dosing.

Keywords: precision medicine, clinical pharmacology, extracellular vesicles, precision dosing, sorafenib

Subject: Clinical Pharmacology thesis

Thesis type: Doctor of Philosophy
Completed: 2021
School: College of Medicine and Public Health
Supervisor: Associate Professor Andrew Rowland