Improving the predictive power of xenograft and syngeneic anti-tumour studies using mice humanised for pathways of drug metabolism.

Drug development is an expensive and time-consuming process, with only a small fraction of drugs gaining regulatory approval from the often many thousands of candidates identified during target validation. Once a lead compound has been identified and optimised, they are subject to intensive pre-clinical research to determine their pharmacodynamic, pharmacokinetic and toxicological properties, procedures which inevitably involve significant numbers of animals - mainly mice and rats, but also dogs and monkeys in much smaller numbers and for specific types of drug candidates. Many compounds that emerge from this process, having been shown to be safe and efficacious in pre-clinical studies, subsequently fail to replicate this outcome in clinical trials, therefore wasting time, money and, most importantly, animals. The poor predictive power of animal models in pre-clinical studies is predominantly due to lack of efficacy or safety reasons, which in turn can be attributed mainly to the significant species differences in drug metabolism between humans and animals. To circumvent this, we have developed a complex transgenic mouse model - 8HUM - which faithfully replicates human Phase I drug metabolism (and its regulation), and which will generate more human-relevant data [ REFINEMENT] from fewer animals [ REDUCTION] in a pre-clinical setting and reduce attrition in the clinic. One key area for the pre-clinical application of animals in an oncology setting - almost exclusively mice - is their use in anti-tumour studies. We now further demonstrate the utility of the 8HUM mouse using a murine melanoma cell line as a syngeneic tumour and also present an immunodeficient version 8HUM_Rag2 -/- - for use in xenograft studies. These models will be of significant benefit not only to Pharma for pre-clinical drug development work, but also throughout the drug efficacy, toxicology, pharmacology, and drug metabolism communities, where fewer animals will be needed to generate more human-relevant data.

Quantifying ERK activity in response to inhibition of the BRAFV600E-MEK-ERK cascade using mathematical modelling.

BACKGROUND: Simultaneous inhibition of multiple components of the BRAF-MEK-ERK cascade (vertical inhibition) has become a standard of care for treating BRAF-mutant melanoma. However, the molecular mechanism of how vertical inhibition synergistically suppresses intracellular ERK activity, and consequently cell proliferation, are yet to be fully elucidated. METHODS: We develop a mechanistic mathematical model that describes how the mutant BRAF inhibitor, dabrafenib, and the MEK inhibitor, trametinib, affect BRAFV600E-MEK-ERK signalling. The model is based on a system of chemical reactions that describes cascade signalling dynamics. Using mass action kinetics, the chemical reactions are re-expressed as ordinary differential equations that are parameterised by in vitro data and solved numerically to obtain the temporal evolution of cascade component concentrations. RESULTS: The model provides a quantitative method to compute how dabrafenib and trametinib can be used in combination to synergistically inhibit ERK activity in BRAFV600E-mutant melanoma cells. The model elucidates molecular mechanisms of vertical inhibition of the BRAFV600E-MEK-ERK cascade and delineates how elevated BRAF concentrations generate drug resistance to dabrafenib and trametinib. The computational simulations further suggest that elevated ATP levels could be a factor in drug resistance to dabrafenib. CONCLUSIONS: The model can be used to systematically motivate which dabrafenib-trametinib dose combinations, for treating BRAFV600E-mutated melanoma, warrant experimental investigation.

Application of a novel regulatable Cre recombinase system to define the role of liver and gut metabolism in drug oral bioavailability.

The relative contribution of hepatic compared with intestinal oxidative metabolism is a crucial factor in drug oral bioavailability and therapeutic efficacy. Oxidative metabolism is mediated by the cytochrome P450 mono-oxygenase system to which cytochrome P450 reductase (POR) is the essential electron donor. In order to study the relative importance of these pathways in drug disposition, we have generated a novel mouse line where Cre recombinase is driven off the endogenous Cyp1a1 gene promoter; this line was then crossed on to a floxed POR mouse. A 40 mg/kg dose of the Cyp1a1 inducer 3-methylcholanthrene (3MC) eliminated POR expression in both liver and small intestine, whereas treatment at 4 mg/kg led to a more targeted deletion in the liver. Using this approach, we have studied the pharmacokinetics of three probe drugs--paroxetine, midazolam, nelfinavir--and show that intestinal metabolism is a determinant of oral bioavailability for the two latter compounds. The Endogenous Reductase Locus (ERL) mouse represents a significant advance on previous POR deletion models as it allows direct comparison of hepatic and intestinal effects on drug and xenobiotic clearance using lower doses of a single Cre inducing agent, and in addition minimizes any cytotoxic effects, which may compromise interpretation of the experimental data.

In vivo regulation of human glutathione transferase GSTP by chemopreventive agents.

Relatively little progress has been made in determining the in vivo regulation of glutathione S-transferase P (GSTP), particularly the human enzyme hGSTP1, despite being identified as a significant factor in carcinogenesis and development of drug resistance in tumor cell lines. Here, we report the characterization of a transgenic reporter mouse that reveals how hGSTP1 is regulated in vivo by chemopreventive agents. Basal expression was found in crypts and villi of the small and large intestine, bronchiolar epithelial cells, the epidermis and hair follicles, gall bladder epithelium, choroid plexus, and biliary epithelium. Expression was induced in different tissues by the antioxidant chemopreventive agents ethoxyquin and butylated hydroxyanisole. However, genetic deletion of the Nrf2 transcription factor, which directs central genetic programs of detoxification and protection against oxidative stress, increased rather than attenuated GSTP1 expression. In vitro investigations with mouse embryonic fibroblasts revealed factors, in addition to Nrf2, that control the expression of GSTP1, offering further insights into regulation. The new reporter mouse described here provides a useful tool to gain deeper insights into the mechanisms of action of chemopreventive compounds and other environmental agents.

Increased skin papilloma formation in mice lacking glutathione transferase GSTP.

The glutathione S-transferase GSTP is overexpressed in many human cancers and chemotherapy-resistant cancer cells, where there is evidence that GSTP may have additional functions beyond its known catalytic role. On the basis of evidence that Gstp-deficient mice have a comparatively higher susceptibility to skin carcinogenesis, we investigated whether this phenotype reflected an alteration in carcinogen detoxification or not. For this study, Gstp(-/-) mice were interbred with Tg.AC mice that harbor initiating H-ras mutations in the skin. Gstp(-/-)/Tg.AC mice exposed to the proinflammatory phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) exhibited higher tumor incidence and multiplicity with a significant thickening of skin after treatment, illustrating hyperproliferative growth. Unexpectedly, we observed no difference in cellular proliferation or apoptosis or in markers of oxidative stress, although higher levels of the inflammatory marker nitrotyrosine were found in Gstp(-/-)/Tg.AC mice. Instead, gene set enrichment analysis of microarray expression data obtained from skin revealed a more proapoptotic and proinflammatory environment shortly after TPA treatment. Within 4 weeks of TPA treatment, Gstp(-/-)/Tg.AC mice displayed altered lipid/sterol metabolism and Wnt signaling along with aberrant processes of cytoskeletal control and epidermal morphogenesis at both early and late times. In extending the evidence that GSTP has a vital role in normal homeostatic control and cancer prevention, they also strongly encourage the emerging concept that GSTP is a major determinant of the proinflammatory character of the tumor microenvironment. This study shows that the GSTP plays a major role in carcinogenesis distinct from its role in detoxification and provides evidence that the enzyme is a key determinant of the proinflammatory tumor environment.

Conditional deletion of cytochrome P450 oxidoreductase in the liver and gastrointestinal tract: a new model for studying the functions of the P450 system.

We have previously described a mouse model, where hepatic cytochrome P450 oxidoreductase (POR) expression has been deleted, resulting in almost complete ablation of hepatic P450 function [Hepatic P450 Reductase Null (HRN)]. HRN mice grow normally but develop fatty livers, and they have increased cytochrome P450 levels. Associated with the hepatic lipid accumulation are significant changes in the expression of genes controlling lipid homeostasis. We have characterized this model extensively and demonstrated its value in drug efficiency testing, in toxicokinetics, and in evaluating the role of the hepatic P450 system in drug pharmacokinetics. To extend the deletion of POR, and P450 inactivation, to other tissues, and to develop the utility of this model, we have generated a mouse where POR can be deleted conditionally in the liver and gastrointestinal tract using the rat cytochrome P450 CYP1A1 promoter to drive Cre recombinase expression. Administration of the CYP1A1 inducers tetrachlorodibenzo-p-dioxin or beta-naphthoflavone resulted in both hepatic and gastrointestinal deletion of POR, whereas administration of 3-methylcholanthrene resulted specifically in loss of hepatic POR expression. In all cases, the resulting hepatic phenotype seemed identical to that of the HRN model, including increased cytochrome P450 expression. Hepatic deletion of POR and the subsequent increase in P450 expression were dependent on inducer dose, with maximal POR deletion occurring at a single dose of 3-methylcholanthrene of 40 mg/kg. This model provides a powerful approach for studying the functions of POR as well as in the evaluation of the role of hepatic and gastrointestinal P450s in drug deposition and chemical toxicity.

Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase.

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein reductase (EC, cytochrome P450 reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivo metabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.