Non-human primates (NHPs) share a wide range of genetic and physiological features in common with humans. Yet, despite their widespread use in preclinical research, NHP models suffer from several limitations that can lead to a lack of translatability in clinical trials.
The poor translational potential of preclinical animal models stem from multiple factors, including:
- Species differences in genetics
- Differences in organ and tissue physiology
- Differences in metabolism, enzymes, and distribution
- Over/under prediction of xenobiotic toxicity and efficacy
Given these challenges, researchers are increasingly turning to alternative high-quality animal models that offer greater translational relevance. Cellular humanized rodent models such as hemizygous cDNA-uPA/SCID, FRG (Fahˉ‘ˉ/Rag2ˉ‘ˉ/Il2rgˉ‘ˉ), and TK-NOG (herpes simplex virus type 1 thymidine kinase NOD/Scid/IL2Rrⁿ ͧ ˡˡ) mice provide various levels of immunodeficiency with some strategy to promote liver injury of endogenous mouse hepatocytes. Not only is the genetic background important, but a high level of production quality and strict animal screening are critical for consistent and conclusive results to better predict human translation.
Superior pharmacokinetic & toxicity prediction using the PXB-mouse® model
Unlike traditional animal models, the PXB-mouse model has a humanized liver with a high proportion of human hepatocytes, making it a more accurate predictor of human physiology due to its human hepatic zonation and stable expression of human enzymes and transporters. This advantage has led the PXB-mouse model to outperform those based on monkeys and rats for predicting human pharmacokinetics.
In one study, researchers predicted clearance (CLₜ) and volume of distribution at steady state (Vdₛₛ) values using single-species allometric scaling (SSS) for 30 marketed drug compounds known to be eliminated in humans via hepatic metabolic pathways (including cytochrome P450, aldehyde oxidase (AO), and UDP-glucuronosyltransferase (UGT) pathways).¹ PXB-mouse model-determined clearance values were more accurate than those from monkeys and standard rat models, with 83.3% of compounds within a three-fold error range of actual values, compared with 70.0% for monkeys and 46.7% for rats (Figure 1). PXB-mice were particularly effective at estimating clearance for compounds metabolized via AO-mediated pathways, which are often challenging to predict in non-clinical models. PXB-mice were also highly accurate at human Vdₛₛ prediction, with 79.3% of values within a 3-fold error range of observed values. Taken together, these findings highlight the suitability of the PXB-mouse model for predicting human PK parameters.
Figure 1: Relationship between observed human and predicted clearance of 30 compounds determined by SSS of PXB-mice (left), monkeys (middle), and rats (right).
In an example of human hepatoxicity, the PXB-mouse model was used to recapitulate toxicity observed in humans. Two investigational RNAi therapeutics, ALN-HBV and VIR-2218, were evaluated. ALN-HBV had off target effects and VIR-2218 was chemically modified to reduce toxicity, as seen in in vitro data in Figure 2. Furthermore, PXB-mice were used to observe the dose-dependent h-ALT1 elevation that was also seen in the clinic. Similar to human data, PXB-mice were able to confirm dose-dependent h-ALT1 elevations with ALN-HBV administration, while VIR-2218 did not have a significant elevation of h-ALT1 from the upper limit of normal as seen in Figures 3 and 4. This data highlights the utility of human liver chimeric mice for safety research applications.
Figure 2: RNA-sequencing (RNAseq) analysis results showing differential gene expression in HepG2.2.15 cells treated with either ALN-HBV (left) or VIR-2218 (right). Fewer differentially expressed genes and a lower magnitude of gene dysregulation are seen with VIR-2218.
Figure 3: In chimeric mice with humanized livers, h-ALT1 levels were markedly lower following administration of VIR-2218 compared with ALN-HBV at equivalent dose levels up to 100 mg/kg. A dose-dependent increase in h-ALT1 levels was seen with ALN-HBV, but not VIR-2218.
Figure 4: Relative to ALN-HBV, VIR-2218 showed a substantially decreased propensity to cause alanine aminotransferase (ALT) elevations in healthy human volunteers at dose levels anticipated to be clinically relevant.
Genetic and physiological recreation of human liver biology
The PXB-mouse model represents normal human liver physiology, even at the ultrastructural level. Human hepatocytes are engrafted uniformly throughout all liver lobes, with representative zone-dependent expression of cytochrome P450 enzymes and arginase I. These structural and functional characteristics of the human liver underpin the quality translational outcomes offered by PXB-mice.
PXB-mice also express a wide range of human liver genes, making them ideal models for testing human gene therapies. Studies have shown that 82% of investigated genes were expressed at similar levels between PXB-mice and human liver hepatocytes (Figure 5). The human genes expressed in the PXB-mouse are suitable for gene editing and therapies targeting gain or loss of function studies. Other characteristics, such as the lipoprotein profile, are also similar to humans (Figure 6).
Figure 5: 82% of investigated genes were expressed at similar levels in PXB-mouse hepatocytes and human liver hepatocytes.
Figure 6: PXB-mice have a human-like lipoprotein profile. Adapted from Papazyan et al., 2018.²
Effective recapitulation of disease phenotypes
The PXB-mouse model has proven to offer effective recapitulation of a wide range of disease phenotypes.
Ornithine transcarbamylase deficiency (OTCD), for example, is a metabolic and genetic disease caused by dysfunction of the hepatocytic urea cycle. The PXB-mouse model can be transplanted with primary human hepatocytes from patients with diseases to create liver disease models that effectively translate human metabolism and pathology.³ Highly humanized liver OTCD models offer characteristics similar to OTCD patients, including increased blood ammonia levels and other important chemical markers. PXB-mice can be a valuable source of patient-derived hepatocytes that support reproducible, large-scale preclinical studies using a single donor.
Murine models of metabolic dysfunction-associated fatty liver disease and steatohepatitis (MAFLD/MASH) have also been developed from humanized liver chimeric mice.⁴ By administering a choline-deficient, L-amino-acid-defined, high-fat diet, researchers induced histological changes similar to human MASH patients, including hepatocyte ballooning, mallory-denk bodies, inflammation, apoptosis, regeneration of human hepatocytes, and fibrosis.
The PXB-mouse model is the only animal model translationally effective in preclinical research for hepatitis B and D therapies. A key strength of the PXB-mouse model is the ability to evaluate all markers of hepatitis B and D infection across the full viral lifecycle. As such, they can be used to evaluate a wide range of anti-viral therapeutics.
Improving translational success with the PXB-mouse model
Given their benefits in translatability, human liver chimeric animal models are the gold standard for evaluating preclinical lead candidates. In addition to their predictive power, human liver chimeric mouse models do not require the same intensive regulation as NHP models, making them cost-effective and a more logistically viable option for preclinical research. Additionally, PXB-mice have significantly lower body weights compared to NHPs and thus require less material to be manufactured to start your research. Overall, as we select animals with similar body weights, ages, and human liver engraftment, less PXB-mice are required for studies, moving toward compliance with the 3Rs principles.
PXB-mice are ready to use for supply shipment or study services as early as 12-weeks of age when humanization of the liver meets our strict quality control criteria. PhoenixBio produces 350–450 PXB-mice per month, ready to support your preclinical program. These animals can be serially sampled throughout the in-life, or alternatively can be sacrificed to evaluate the liver and human hepatocytes for in-depth analysis. PXB-mice can be shipped directly to researchers, or we can facilitate experiments with our contract research services.
References
- Miyamoto, M. et al. (2017) Comparison of predictability for human pharmacokinetics parameters among monkeys, rats, and chimeric mice with humanised liver. Xenobiotica, 47(12); 1052.
- Papazyan et al. (2018). FXR activation by obeticholic acid or nonsteroidal agonists induces a human-like lipoprotein cholesterol change in mice with humanized chimeric liver. Journal of Lipid Research, 59(6), 982–993.
- Sugahara, G. et al. (2020) Humanized liver mouse model with transplanted human hepatocytes from patients with ornithine transcarbamylase deficiency. J Inherit Metab Dis, 44(3); 618.
- Kiosh, K. et al. (2021) Estimating Drug Efficacy with a Diet-Induced NASH Model in Chimeric Mice with Humanized Livers. Biomedicines, 9; 1647.