Recent FDA guidelines “encourage the identification of differences in drug metabolism between animals used in nonclinical safety assessments and humans as early as possible during the drug development process.”[1] Metabolites formed by Phase I metabolic pathways (i.e. cytochrome P450s) are of particular concern: “Based on the nature of the chemical reactions involved, metabolites formed from Phase I reactions are more likely to be chemically reactive or pharmacologically reactive and, therefore, more likely to need safety evaluation” 1,[2]. In the worst-case scenario a full toxicity study on a human-specific metabolite (or metabolites which are produced at disproportionately higher levels in humans) may be required, delaying clinical progress. Early identification of species differences in metabolic profiles is therefore critical to avoid potential delays.
Although in vitro systems such as human hepatocytes can be used to identify drug candidates where a human-specific metabolite may be formed, until now there has been no simple way of characterising the in vivo effects of such metabolites on efficacy and/or toxicity.
The solution
Human CYP3A enzymes metabolize more than half of drugs in clinical use[3]. CYP3A represents 40% of total immunoquantified P450 in the liver[4] and 80% of total immunoquantified P450 in the intestine[5]. Mice which lack murine Cyp3a genes but which express human CYP3A4 can therefore be used as bioreactors to generate human-specific CYP3A4 metabolites, and to study their effects in an in vivo system before entering the clinic.
We offer two alternative humanized CYP3A4 approaches for assessing the in vivo effects of human CYP3A4 metabolites:
- The Humanized Liver CYP3A4 Mouse (Taconic model 9048) expresses high levels of human CYP3A4 in the liver under the control of the human ApoE promoter. The Humanized Gut CYP3A4 Mouse (Taconic model 9047) expresses high levels of human CYP3A4 in the intestine under the control of the murine villin promoter. Finally, the Humanized Liver & Gut CYP3A4 Mouse (Taconic model 9049) expresses human CYP3A4 in both the intestine and liver. All three of these models lack all 8 murine Cyp3a genes.
- The Humanized CYP3A4/3A7 Mouse (Taconic model 8842) expresses functional CYP3A4 protein in the liver and intestine, under the control of the human promoter and on a Cyp3a knockout background. This model also contains the foetally-expressed human CYP3A7, although is not expressed in adult mice. The level of CYP3A4 in the liver can be modulated by the application of CAR or PXR ligands, reflecting human variability in expression levels.
Results obtained in the Humanized Liver CYP3A4 Mouse demonstrate the high levels of CYP3A4 metabolism achievable compared to that seen in CYP3a knockout mice after IV administration:
Table 1: Drug metabolites can be generated, recovered and quantitated in these humanized mouse models. Shown are docetaxel and metabolites M1-4 (mg/g) recovered from plasma and tissues one hour after iv administration of 10 mg/kg docetaxel. *LLQ=below the lower limit of quantification. A P < 0.001, B P < 0.05, C P < 0.01 compared with Cyp3a (8-gene) Knockout mice. [6]
In addition, liver microsomes from the Humanized Liver CYP3A4 Mouse demonstrated very similar Km and Vmax values to pooled human liver microsomes.[6]
Data on the Humanized CYP3A4/3A7 Mouse is available on request from CXR Biosciences.
Using these novel models, metabolites of interest can be generated and quantified. Metabolites which may meet the criteria specified in regulatory guidance (formed at greater than 10% of parent drug exposure) can be assessed in vivo for effects on efficacy and toxicity prior to advancement of drugs into the clinic, preventing costly delays in the drug development process.
These experiments support the utility of the humanized CYP3A mouse models for use as bioreactors to generate human metabolites and to study their effects in vivo, avoiding the need to chemically synthesise the metabolite and design a dosing scheme that reflects the situation in man. We offer a variety of mice models, using either tissue-specific or human promoters.
Availability
CXR and Taconic have partnered to make the transADMETTM CYP3A Models commercially available.
Contract services: CXR are co-exclusive suppliers of contract research services using transADMETTM mice. We also offer consultancy and advice to our customers.
For more information on contract research services at CXR using the transADMETTM mice, contact us here or at transadmet@cxrbiosciences.com.
Off the shelf mice: Mice may be purchased directly from Taconic by both academic and for-profit customers. To purchase transADMETTM mice, please visit the relevant model webpages:
For questions regarding distribution of these models, please contact Dr. Megan MacBride.
[1] Guidance for Industry: Safety Testing of Drug Metabolites. US Food and Drug Administration, Center for Drug Evaluation and Research, February 2008.
[2] Powley MW, et al. (2009) Safety assessment of drug metabolites: implications of regulatory guidance and potential application of genetically engineered mouse models that express human P450s. Chem Res Toxicol 22:257-262.
[3] Guengerich FP. (1999). Cytochrome P-450 3A4: regulation and role in drug metabolism. Annu Rev Pharmacol Toxicol 39:1-17.
[4] Shimada T et al (1994) Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. JPET 270:414-423.
[5] Paine MF et al (2006) The human intestinal cytochrome P450 “pie”. Drug Metab Dispos 34(5):880-886.
[6] Van Herwaarden AE, Wagenaar E, van der Kruijssen CMM, van Waterschoot RAB, Smit JW, Song J-W, van der Valk O, van der Hoorn JWA, Rosing H, Beijnen JH, Schinkel AH. (2007) Knockout of cytochrome P450 3A yields new mouse models for understanding xenobiotic metabolism. J Clin Invest 117(11):3583-3592.
