Evaluation of Potential Gastrointestinal Biomarkers in a PAK4 Inhibitor-treated Preclinical Toxicity Model to Address Unmonitorable Gastrointestinal Toxicity

Abstract

Although gastrointestinal (GI) toxicity is a significant dose-limiting safety concern noted in multiple therapeutic areas, there are no GI biomarkers that can accurately track, precede, or reliably correlate with histologic evidence of injury. While significant efforts have been made within the pharmaceutical industry, academia, and consortia to address the biomarker gaps in other target organs such as liver, kidney, and muscle (cardiac and skeletal), there have been no concerted efforts in the area of GI biomarkers. Using PAK4 inhibitor as a preclinical rat model of gastric toxicity, selected candidate biomarkers from literature were evaluated to test their usefulness as gastric injury biomarkers in this study. Biomarkers selected in this study include plasma diamino oxidase and citrulline, fecal calprotectin, bile acids, and miRNA. Based on the results, L-citrulline and miR-194 results appear to correlate well with histopathology findings. Although these biomarkers will need additional assay validation and qualification to test if they truly predict the injury prior to histopathology, the results provide promise for further testing using additional GI toxicants. In addition, this article highlights important gaps in GI biomarkers and provides substrate and rationale for additional investments either for further testing of already available biomarkers or to pursue extensive biomarker discovery approaches.

Faecal microRNAs: indicators of imbalance at the host-microbe interface?

“…In addition, recent work has demonstrated the use of faecal miRNAs as biomarkers of colorectal cancer (John-Baptiste et al, 2012), further investigation is warranted in this area but with the caveat that faecal miRNAs may be effected by antibiotic treatment as we have shown here. Further work is needed to determine the precise source of the miRNAs within the faeces, they may be exosomal or epithelial cell in origin and we have only identified alterations in four miRNAs.…”

“…No weight loss was observed in control or antibiotic treated rats. We were able to detect miRNAs in the faecal pellets of rats, as others had previously shown was possible (John-Baptiste et al, 2012). We subsequently measured the expression of the same miRNAs (let-7b-3p, miR-141-3p, miR-200a-3p and miR-1224-5p) in the faeces of rats at 8 weeks while undergoing antibiotic-mediated depletion of the gut microbiota over a 13 week period.…”

“…Secondly, it is non-invasive and is currently the source material for all faecal microbiota transplants in humans (and rodents) and finally, detection of mRNA in stool from infants has been used to monitor gastrointestinal development and maturation (Chapkin et al, 2010). We and other groups have shown that detection of miRNA in the faeces of rodents and humans is possible and quantitative (John-Baptiste et al, 2012;Liu et al, 2016;Weber et al, 2010). Furthermore, specific cells in the intestinal crypt express a distinct repertoire of miRNAs that allow each cell type to be distinguished (Peck et al, 2017).…”

Gastrointestinal Safety Pharmacology in Drug Discovery and Development

“…Citrulline is becoming a promising GI biomarker due to recent developments in its measurement by high-performance liquid chromatography (HPLC) and mass spectrometry (Crenn et al 2008). John-Baptiste et al (2012) showed that L-citrulline levels in rat plasma detected by mass spectrometry showed a significant dose-and time-dependent decrease in PAK4-inhibitor treatment groups compared with baseline consistent with an overall reduction in enterocyte mass, which manifested histologically as crypt necrosis and villus atrophy and fusion. It is important to point out that alteration in diet and gut microflora may potentially alter biochemical and metabolic pathways, leading to changes in metabolite measurements such as citrulline.…”

“…Diamine oxidase (DAO) is a highly active degradative enzyme of the polyamine metabolic pathways, catabolizes a variety of substrates including histamine and diamines, and is localized to the mature villus epithelial cells of rodent intestinal mucosa (Wolvekamp and de Bruin 1994;John-Baptiste et al 2012). Although blood DAO activity level correlates with both DAO expression in the villi of the small intestinal mucosa and the severity of small intestinal mucosal lesions induced by anticancer drugs, DAO measurement is confounded by the fact that plasma levels rise markedly upon heparin stimulation prior to blood draws, with peak elevations between 30 and 60 min (Luk et al 1980(Luk et al , 1981Tsunooka et al 2004).…”