Genome-based analysis of the nonhuman primate Macaca fascicularis as a model for drug safety assessment

Drug discovery and development are labor- and cost-intensive processes that can last up to 20 yr from concept to market. The most critical concerns before entry into clinical development of a novel compound are the balance between risk and benefit for the patient. International drug safety agencies like the US Food and Drug Administration (FDA) or the European Medicines Agency (EMA) apply standardized testing procedures and requirements for submission of new medicines. Animal experiments designed to predict parameters such as toxicity or pharmacokinetics are a key prerequisite of the drug approval process. Rodents, dogs, mini-pigs, and in particular, nonhuman primates are the main species used in translational drug safety research and risk assessment (Boelsterli 2003). Compared with rodents or dogs, nonhuman primates have a closer evolutionary relationship to humans, exhibit greater physiological similarity, and have the added benefit of being capable of completing memory tests originally designed for humans (Capitanio and Emborg 2008). Only a small number of species, such as macaques of the Cercopithecidae family of Old World monkeys, are well suited and established as translational models for drug testing. These primates share a common ancestor with humans that is estimated to have lived about 32 million years ago (Perelman et al. 2011). From this family, the species Macaca mulatta, also known as rhesus monkey, and Macaca fascicularis, the long-tailed macaque, are the most common and best-studied nonhuman primate animal models today (Ferguson et al. 2007; Gibbs et al. 2007). Although closely related, these two species show distinct phenotypic differences, morphology, behavior, and physiology. In 1978, India banned all rhesus monkey exports to breeding centers across the world, and since then usage of this species in drug safety testing has declined. As an alternative to rhesus, several commercial breeding centers in Indonesia, China, the Philippines, and Mauritius are able to provide sufficient numbers of captive bred long-tailed macaques originating from wild-trapped founders. The natural range of M. fascicularis monkeys spans the mainland of southern Asia, Indonesia, the Philippines, and more recently Mauritius, where a small number of founder animals was imported on a trading ship during the 15th century (Ferguson et al. 2007). Rhesus monkeys inhabit predominantly the mainland of China, Vietnam, Laos, Nepal, Thailand, northern India, and Pakistan. The wide geographic distribution of both species and considerable interspecies hybridization in shared habitats point to populations that are genetically and phenotypically quite diverse (Tosi et al. 2002). Thus, genotyping and phenotypic characterization of animals is desirable prior to studies aimed at predicting the safety of novel medicines in humans. A cornerstone of primate research with high impact for biomedical research was the publication of the first genome draft of the rhesus monkey, Macaca mulatta, in 2007 (Gibbs et al. 2007). This multicenter effort was mainly focused on highlighting differences between Old World monkeys and great apes like the chimpanzee, which differs from humans by only ∼1.23% based on genome-wide sequence identity comparison (Waterston et al. 2005). This genome draft enabled the search for macaque-specific genes related to physiology and phenotypes, as well as the assembly of signaling cascades and pathways involved in the immune response to pathogens such as simian immunodeficiency virus (SIV). In addition, the mapping of random sequence reads obtained from additional animals with different geographic origin to the genome draft allowed assessment of population diversity at single nucleotide resolution. The genome assembly also enabled the design of novel rhesus genome based mRNA expression microarrays, which were applied to the analysis of human influenza virus infection (Gibbs et al. 2007). Compared with earlier expressed sequence tag (ESTs)-based chip designs (Magness et al. 2005), availability of the rhesus draft genome allowed selection of unique probes, resulting in reduced cross-hybridization and improved performance. Drug safety studies carried out under good laboratory practice (GLP) require relatively large numbers of animals that match the experimental requirements with respect to parameters like age, weight, or gender. M. fascicularis today is the most widely used primate species for drug safety testing in pharmaceutical companies or contract research organizations (CROs). In addition, validated assays for measuring blood parameters and safety biomarkers such as liver enzymes or cytokines are available. Depending on the molecule and mode of action, repro-toxicity testing in nonhuman primates is frequently part of the required safety package. Long-tailed macaques are particularly advantageous in this area, since this species has no seasonal fertility, unlike rhesus, whose females are receptive only once a year (Weinbauer et al. 2008). We have generated the first draft genome of the long-tailed macaque M. fascicularis using a whole-genome shotgun (WGS) sequencing approach employing two independent deep sequencing technologies. We identified about 2.1 million candidate single-nucleotide polymorphisms (SNPs) and used homology-based annotation for transcript identification to design a M. fascicularis-specific gene expression microarray. With focus on drug safety, we performed a phylogenetic analysis of the SLCO drug transporter family and transcript profiling of cytokines and cytochrome p450s in liver samples from 36 naive monkeys within the context of a global gene expression analysis.