Characterisation of hepatic uptake transporters in the minipig

Mather, Azmina

Thesis

Characterisation of hepatic uptake transporters in the minipig

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Creator

Mather, Azmina

Rights statement

Strathclyde Thesis Copyright

Awarding institution

University of Strathclyde

Date of award

2023

Thesis identifier

T17369

Person Identifier (Local)

201859359

Qualification Level

Masters (Postgraduate)

Qualification Name

Master of Philosophy (MPhil)

Department, School or Faculty

Strathclyde Institute of Pharmacy and Biomedical Sciences

Abstract

The Gottingen minipig is a popular strain (Heining and Ruysschaert, 2016, Lignet et al., 2016) and is used in toxicological studies. It is used based on its anatomical and physiological similarities to humans with respect to a variety of organs and functions (Forster et al., 2010a). Further evaluation needs to be carried out on the predictability of the minipig to human specifically on absorption, distribution, metabolism and excretion (ADME) processes. This project will aid in this predictability of the minipig to human correlation to facilitate the development and validation of mathematical models. The liver plays a major role in the ADME process of xenobiotics, and hepatic transporters can be the rate limiting step for the clearance of xenobiotics from the body, playing a critical role in their elimination (Funk, 2008, Treiber et al., 2007). The aim of this project was to characterise minipig hepatic uptake transporters by investigating the uptake of three known organic anion transporter protein (OATP) substrates, estrone sulfate (ES), estradiol glucuronide (EG), and a clinically relevant probe, rosuvastatin. The uptake of the probe substrates was assessed across three species, human, rat and minipig. This was to give an understanding on the variance in the uptake of the probe substrates across the three different species and if minipig hepatic uptake transporters can be used to predict human pharmacokinetics and be used for in vitro in vivo correlation (IVIVC). The cryopreserved hepatocytes were plated onto collagen coated plates to give a final well concentration of 0.35 x 106 cells per well. Plates were placed into a 37°C humidified incubator with 5% CO2 for approximately 4 hours to allow cells to attach. After approximately four hours the hepatocytes are incubated with the substrate at 37⁰C. Experiments were conducted to investigate time dependent carrier-mediated uptake and concentration dependent uptake to determine kinetics (Km and Vmax). To determine the amount of substrate taken up, cells were lysed with 1% triton or water and the contents measured via LC-MS/MS or liquid scintillation counting, dependent if the substrate was radioactive. To assess the amount of passive diffusion, the cells were incubated using the same conditions as above, however the pre-incubation and working solutions contained the cocktail of inhibitors, rifamycin and imipramine. As drug transporters are highly expressed in rodents, rat hepatocytes had the highest intrinsic clearance for all three substrates (Table 9). Uptake of the probe substrates was also assessed in different batches of minipig and human hepatocytes, therefore minipig hepatocyte batches GBV and IKL had lower Km values for ES compared to EG. The Intrinsic clearance (ml/min/Kg) determined for minipig and human hepatocytes was similar across both species for EG (4.78 to 10.54 ml/min/Kg). Minipig hepatocytes have a similar substrate affinity for EG and ES as human cells. Kinetics was determined in two out of the three minipig batches (RZX and IKL) for rosuvastatin and a fold change of >2 was only observed in the time linearity experiment for batches GBV and IKL which gave a fold change of just over 2, the third batch RZX gave a fold change of 1.5. There is not a huge difference in the fold change for all three minipig hepatocyte batches, which were around the acceptance threshold of a fold change of ≥2 between inhibited and uninhibited cells. This could be due to the poor affinity of rosuvastatin for the uptake transporters present in minipig hepatocytes. To assess if this was a phenomenon with just rosuvastatin, the uptake of pitavastatin was assessed and no kinetics could be determined in minipig hepatocytes. This project has highlighted further work is required to fully characterise minipig hepatocytes and the experimental design in looking at pre-incubation times, choice and concentration of inhibitors. The experimental data from this project demonstrated minipig uptake transporters, in particular OATP’s are not fully representative of human hepatocytes.

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