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Burton, Lewis (2020) Theoretical and experimental evaluation of the effects of hydrostatic pressure on hepatocyte metabolic capacity. PhD thesis, University of Nottingham.

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Abstract

Drug discovery is a lengthy and expensive process costing ~$2.6 billion per new chemical entity. To reduce this cost, potential new drugs undergo extensive testing through a series of drug metabolism and pharmacokinetic (DMPK) assays designed to ensure that only the most suitable candidates progress to clinical trials. Despite these measures, errors in predictions from in vitro data to the in vivo situation are still common and consequently attrition rates remain high.

Approximately 75% of xenobiotics are primarily eliminated through metabolism, and thus the accurate scaling of metabolic clearance is vital to successful drug development. Yet hepatic clearance, the major source of metabolism, still commonly underpredicts by 2-3-fold. Recently, several methods have been adopted to try and improve in vitro in vivo extrapolation (IVIVE), including hepatic spheroids and microfluidics devices. The latter impose a shear stress on hepatocytes that is thought to improve the production of cytochrome P450 (CYP450).

Herein, it is postulated that hepatocytes experience a static pressure in vivo imposed by the convergence of sinusoidal vessels in the liver lobule, a structural unit of the liver. Hepatocytes closer to the central vein (which express higher CYP450) experience more force than those located towards the periphery of the lobule (which express less CYP450). The aims of this study were twofold: firstly, to determine whether there was evidence to support the presence of static pressure within the liver lobule and, secondly, to determine whether the application of hydrostatic pressure improved the metabolic competency of hepatic cells. Initially, work was conducted in porcine hepatocytes. This was later transferred to cryopreserved human hepatocytes which represent the gold standard for clearance predictions.

In the first instance, immunohistochemistry techniques were used on liver slices from freshly slaughtered pigs to determine if the architecture of the liver could impose a physical pressure. It was observed that there was a significant decrease in the number of vessels and a significant increase in vessel size moving towards the central vein. This indicates that the converging sinusoidal vessels stretch to accommodate the increased blood flow per vessel. It is hypothesised that this stretching compresses the hepatocytes located around the central vessels.

To determine if CYP450 expression increased, comparative genetic, protein and activity assays were conducted on hepatocytes pre-exposed to pressure and those cultured under standard culture conditions. It was found that pressure significantly increased both the expression and activity of CYP1A2, by ~10 and ~3-fold, respectively. Furthermore, the vmax of paraxanthine formation was 2-3-fold higher in hepatocytes pre-incubated with pressure compared with their non-pressurised counterparts.

The possible involvement of the aryl hydrocarbon receptor (AhR) was investigated using known agonists and antagonists of the aryl hydrocarbon receptor (AhR) and a HepG2 derived reporter cell line. It was established that the pressure-mediated increases in both expression and activity of CYP1A2 were abolished by the introduction of an AhR antagonist. Additionally, the AhR reporter assay recorded a 4-fold increase in pressurised hepatocytes compared to the no pressure control. Taken together, these findings indicate that an AhR dependent mechanism is responsible for the pressure-mediated increases in CYP1A2 expression and activity,

Thus, this thesis presents a novel theory for the subspecialisation of hepatocytes in vivo and links it to the decreasing levels of CYP450 expression observed in hepatocytes following isolation, explaining underpredictions by isolated hepatocytes. Moreover, the proven involvement of AhR effectively extends the applicability of this work beyond phase I to phase II metabolism and transporters.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Rauch, Cyril Paine, Stuart
Keywords:	Hepatocytes; Static pressure; Liver lobule; Hydrostatic pressure; Metabolic competency; CYP450
Subjects:	Q Science > QP Physiology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Veterinary Medicine and Science
Item ID:	59708
Depositing User:	Burton, Lewis
Date Deposited:	10 Oct 2023 10:35
Last Modified:	10 Oct 2023 10:35
URI:	https://eprints.nottingham.ac.uk/id/eprint/59708

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