Innovation within Drug Metabolism and Pharmacokinetics

8HUM – A

New Mouse Model To Improve Compound Selection In Drug Discovery

Demonstration of in vivo efficacy in a mouse model of disease is a key milestone in anti-infective early drug discovery programmes Although an effective criterion by which to select the most promising preclinical candidates, differences in pathways of drug metabolism between mouse and human frequently result in medicinal chemistry resource being given to ensuring that mouse-specific metabolic liabilities are eliminated, despite these having no bearing on compound progress towards the clinic

In 2019, University of Dundee researchers introduced the “8HUM” mouse model, a line in which 33 murine cytochrome P450 (CYP) enzymes from the major xenobiotic metabolising subfamilies were replaced with 6 human enzymes, CYP1A1, CYP1A2, CYP2C9, CYP2D6, CYP3A4 and CYP3A7, and in which the transcription factors Pregnane X Receptor (Pxr) and Constitutive Androstane Receptor (Car), key regulators of CYP induction in response to xenobiotic challenge, were replaced with their human orthologues 1 This extensive genetic humanisation was shown to remove many species differences in metabolism, with the added benefit that drug exposure was typically greatly increased. Since 2020, we have been evaluating 8HUM within our anti-infective drug discovery workflows as a platform technology for the bypass of mouse-specific issues of compound metabolism. In vitro, in incubations with hepatic microsomes or primary hepatocytes from 8HUM, we have found that species differences in intrinsic metabolic clearance between mouse and human are removed for the vast majority of new chemical entities. Further, the course of infection of 8HUM with T. cruzi and L donovani matches that of the wild-type mouse strains used routinely in Dundee and, as shown by collaborators at GSK Tres Cantos, this is also the case in an acute model of M. tuberculosis infection. Combining these observations in an exemplification of the value of 8HUM within kinetoplastid drug discovery we have shown that mouse-specific metabolic liability is no longer a barrier to compound progression

(manuscript in preparation). Furthermore, in a project funded by the Tres Cantos Open Lab Foundation we have shown that, in the 8HUM mouse line, the pharmacokinetics, metabolite profiles, and magnitude of drug-drug interactions for a test set of 30 approved medicines are in much better alignment with clinical observations than equivalent data generated in wild-type mice Collectively, our results with the 8HUM line have demonstrated that the elimination of species differences in drug metabolism through the application of 8HUM, in place of wild-type mice within a drug discovery programme, has the potential to significantly increase the proportion of compounds that can be considered for progression during lead optimisation, increase speed of workflow, as well improve translational relevance of the data generated We are currently working with collaborators to validate 8HUM in additional models of infectious disease

Metabolomics

Measurement of changes in the abundance of endogenous small molecules within a biological sample in response to chemical challenge can indicate dysregulation of specific metabolic pathways. As has been demonstrated for the Medicines for Malaria Venture Malaria Box compound set 2 , hits from a screening campaign can be profiled for their effect on the endogenous metabolome and thereby classified into groups which are likely to be acting through common Modes of Action (MoA) In collaboration with the MoA team in WCAIR we are developing a medium-throughout LCMS-based metabolomics capability at the DDU which will allow us to define the metabolic fingerprint of 20–50 hit compounds in 24 hours, and thereby expedite prioritisation of those compounds with novel or preferential MoA for hit expansion To date, we have added chromatographic retention time and fragmentation spectra for approximately 400 authentic reference standards of endogenous metabolites to an in-house database This database will be used to give high confidence in metabolite annotations during sample analysis. Initially our validation work will focus on hits from screens against T cruzi but, if successful, the platform will be rolled out to other projects

1 An Extensively Humanized Mouse Model to Predict Pathways of Drug Disposition and Drug/Drug Interactions, and to Facilitate Design of Clinical Trials Henderson CJ,