Hepatitis A Virus Infection in Cynomolgus Monkeys Confounds the Safety Evaluation of a Drug Candidate

Abstract

In a 3-month toxicity study in cynomolgus monkeys at a European contract laboratory, animals were infected with HAV, initially resulting in hepatic injury being incorrectly attributed to the test compound. Elevated serum ALT/AST/GLDH (5- to 10-fold) were noted in individual animals from all groups including controls, with no apparent dose, exposure, or time-related relationship. Liver histopathology revealed minimal to slight inflammatory cell accumulation in periportal zones of most animals, and minimal to slight hepatocyte degeneration/necrosis in 10/42 animals from all groups. As these findings were more pronounced in 6 drug-treated animals, including 2/6 in the low dose group, the draft report concluded: "treatment-related hepatotoxicity at all dose levels precluded determination of a NOAEL." However, the unusual pattern of hepatotoxicity suggested a factor other than drug exposure might have caused the hepatic effects. Therefore, snap-frozen liver samples were tested for hepatitis viruses using a PCR method. Tests for hepatitis B, C, and E virus were negative; however, 20/42 samples were positive for hepatitis A virus (HAV). Infection was strongly associated with increased serum ALT/GLDH, and/or hepatocyte degeneration/necrosis. Re-evaluation of the study in light of these data concluded that the hepatic injury was not drug-related. A subsequent 6-month toxicology study in HAV-vaccinated cynomolgus monkeys confirmed the absence of hepatotoxicity. Identification of HAV infection supported progression of the drug candidate into later clinical trials. Although rarely investigated, subclinical HAV infection has occasionally been reported in laboratory primates, including those used for toxicology studies and it may be more prevalent than the literature indicates.

Keywords: cynomolgus monkey; drug development; hepatic pathology; hepatitis A virus; hepatotoxicity; non-human primate; safety evaluation; toxicology.

Figure 1.

(A) Individual female animals showed variable increases in serum GLDH activities during 13 weeks of treatment. The dotted line represents the upper 97.5^th percentile value (59 U/L) in 460 control animals from 2012-2018. (B) Individual male serum ALT activities showed a single control animal (M01) with a persistently high enzyme activity level throughout the study and a single animal in Group 4 (200 mg/kg - M12) which had a markedly increased enzyme activity only following a 4-week treatment-free recovery period. The dotted line represents the upper 97.5^th percentile value (92.5 U/L) in 64 control animals from 2013-2014.

Figure 2.

Hepatic histopathology (A) shows focal, predominantly periportal, mononuclear cell inflammatory infiltrates 1.5× magnification. Higher magnification (B = 5× magnification; C = 10× magnification) shows increased numbers of mononuclear cells in hepatic sinusoids (B and C) with pigment accumulation in an occasional hepatocyte or Kupffer Cell ((D) black arrow; 20× magnification). Animal M12 carriage return (200 mg/kg) following a 4-week off-treatment recovery period.

Figure 3.

Serum ALT activities in female animals, showed unusually variable activity levels in control animals and, in two low dose (50 mg/kg) individuals, a significant increase at the end of the study. The dotted line represents the upper 97.5^th percentile value (160 U/L) in 532 control animals from 2012-2018.

Figure 4.

Serum ALT and GLDH profiles for males and females, respectively, for control and all 4-dose groups at 10 different time points during a 6-month toxicology study in cynomolgus monkeys. The 2-enzyme activity levels shown (ALT and GLDH) were chosen as the most sensitive indicators of hepatic impairment in the earlier 3-month toxicology study. Plots are mean +/− SD: PD = Predose. D = Day.