The ability to predict the metabolism of new drugs in humans at early stages is critical for drug development, especially in light of FDA guidance issued at the end of 2008. The Guidance for Industry: For Safety Testing of Metabolites (or MIST) requires that metabolites present in greater than 10% of total drug-related systemic exposure require additional safety testing, in case they are present disproportionately in humans as compared to study animals.
“Cultures of human hepatocytes and liver preparations are two common systems for evaluation of drug metabolites,” commented Gerhard Gross, Ph.D., head of drug metabolism at Lundbeck. “However, these systems are stationary. By using these systems we cannot adequately predict the extent of systemic exposure of drug metabolites.”
Unfortunately, because of considerable differences in metabolic pathways between laboratory animals and humans, experiments with animals do not always correctly predict what happens in humans. Therefore, the recent development of a mouse with a human liver presents the best approach for studying human pharmacotoxicity.
One such model is a uPA+/+/SCID transgenic mouse. Livers of immunodeficient (SCID) mice are damaged by targeted expression of transgene (uPA, or urokinase-type plasminogen activator). When human liver cells are injected in these animals, the human cells colonize the mouse liver. The higher the replacement rates, the more predictable this model becomes for metabolism and toxicity studies. Careful examination of histology and pharmacokinetic parameters for chimeric mice supports their use as a model for evaluation of drug AME and also drug-induced hepatotoxicity.
Lundbeck’s new investigational compound is primarily metabolized by the cytosolic enzyme aldehyde oxidase, which is preferably expressed in humans and monkeys, with significant species differences in metabolism across other species. Therefore, obtaining an accurate human PK prediction was a challenge. Testing this compound in the chimeric mouse demonstrated a significantly higher clearance rate, indicating that the model may indeed be useful for accurate human PK predictions.
Lundbeck is continuing investigations with additional compounds that showed disproportionate metabolites in humans. “The logical application of this model is in studies of human pharmacokinetics/metabolism and also human-specific toxicities. However, the model can also potentially be used for ranking compounds, specifically oncology drugs, regarding maximum tolerated doses,” according to Dr. Gross.
“Chimeric mice carrying human livers have wide applications in both preclinical and clinical areas,” commented Elizabeth Wilson, senior scientist at Yecuris. “With tremendous shortages of healthy livers, human organs grown in laboratory animals may become a viable source for transplantation. Liver cells from a patient can be used to repopulate a scaffold of animal livers, resulting in an autologous organ.”
At present, Yecuris strategically focuses on preclinical applications, developing hepatocytes for drug discovery, toxicology, and metabolism studies. Yecuris technology uniquely enables controlled replacement of mouse hepatocytes. The mice lack fumarylacetoacetate hydrolase (Fah), a critical enzyme that in normal animals breaks down the byproduct of tyrosine catabolism. In the absence of the enzyme, fumarylacetoacetate accumulates in hepatocytes, causing cell death.
Yecuris mice survive when the production of fumarylacetoacetate is blocked upstream by Nitisinone, an approved clinical drug. Once the protective drug is withdrawn, the toxic buildup slowly and selectively damages mouse liver cells. The mice lack T, B, and NK cells, facilitating engraftment of human cells. Mouse cells undergo gradual injury over several induction cycles, which provides time for even slowly dividing human adult hepatocytes to expand sufficiently to sustain liver function. Therefore, cells from any donor of any age can be used for repopulation. Moreover, the ratio between human and mouse cells can be fine-tuned to support a variety of applications.
Recently, Yecuris improved upon the initial model to include simultaneous engraftment of liver and immune systems, which represents a significantly improved tool for toxicological studies. “While high replacement levels are required for drug metabolism studies, comparative toxicology studies may need a 50/50 ratio between mouse and human cells,” continued Wilson.
A Pfizer PPARa agonist Wy-14,16443 was tested in the Yecuris model, comparing its effect on mouse and human hepatocytes in the same treated animal. Consistent with the conventional assessments, the treatment resulted in different histological profiles, gene-expression, and proliferation rates between mouse and human cells. A marked increase in hypertrophy and increased proliferation was observed in mouse cells but not in the nearby human cells.
“For such studies, it is desirable to have a mixed ratio of mouse to human cells to provide elucidation and controls of species-specific toxicity. Also, in certain infectious disease applications, it is also beneficial to have fine control over a mixed cell population to avoid death in the host species,” said John Bial, CEO. Recently Yecuris engineered FAH knockout mutations into rats and pigs and has future plans to address new organ systems.