Serum APOA4 Pharmacodynamically Represents Administered Recombinant Human Hepatocyte Growth Factor (E3112)

Abstract

Background: Hepatocyte growth factor (HGF) is an endogenously induced bioactive molecule that has strong anti-apoptotic and tissue repair activities. In this research, we identified APOA4 as a novel pharmacodynamic (PD) marker of the recombinant human HGF (rh-HGF), E3112.

Methods: rh-HGF was administered to mice, and their livers were investigated for the PD marker. Candidates were identified from soluble proteins and validated by using human hepatocytes in vitro and an animal disease model in vivo, in which its c-Met dependency was also ensured.

Results: Among the genes induced or highly enhanced after rh-HGF exposure in vivo, a soluble apolipoprotein, Apoa4, was found to be induced by rh-HGF in the murine liver. By using primary cultured human hepatocytes, the significant induction of human APOA4 was observed at the mRNA and protein levels, and it was inhibited in the presence of a c-Met inhibitor. Although mice constitutively expressed Apoa4 mRNA in the small intestine and the liver, the liver was the primary organ affected by administered rh-HGF to strongly induce APOA4 in a dose- and c-Met-dependent manner. Serum APOA4 levels were increased after rh-HGF administration, not only in normal mice but also in anti-Fas-induced murine acute liver failure (ALF), which confirmed the pharmacodynamic nature of APOA4.

Conclusions: APOA4 was identified as a soluble PD marker of rh-HGF with c-Met dependency. It should be worthwhile to clinically validate its utility through clinical trials with healthy subjects and ALF patients.

Keywords: ALF; APOA4; Fas; c-Met; rh-HGF.

Figure 1

Induction of human APOA4 by rh-HGF, and its c-Met dependency. (A) Relative expression of APOA4 mRNA compared to the expression of 18S rRNA in human hepatocytes, (B) Relative concentration of APOA4 protein in supernatants ** p < 0.01, *** p < 0.001, **** p < 0.0001: comparison between vehicle control (incubation medium) or rh-HGF alone-treated group (Dunnett’s test). ^# p < 0.05: comparison between rh-HGF alone-treated group and PF-04217903-rh-HGF co-culture group (unpaired t test). The data are presented as mean ± SEM (n = 3). The median value of the triplicate analyses was used for calculation. (C) Analysis of APOA4 protein levels in the supernatants of the culture using Western blot. The picture shows a representative batch of hepatocytes in triplicate.

Figure 2

Expression of Apoa4 mRNA in the liver and small intestine after rh-HGF administration in mice. (A) Liver, (B) Small intestine. Relative expression of Apoa4 mRNA against the expression of Hprt mRNA was calculated using the median value of the triplicate analyses * p < 0.05, *** p < 0.001, **** p < 0.0001: comparison between the vehicle control (PBS)- and rh-HGF-treated groups (Dunnett’s test). The data are presented as mean ± SEM (n = 3).

Figure 3

c-Met dependent induction of murine APOA4. Twenty-four hours after rh-HGF administration with or without PF-04217903 treatment, the mice were sacrificed, and their livers and bloods were collected for the analyses. (A) Relative expression of Apoa4 mRNA in the liver compared with the expression of Hprt mRNA in mice, (B) Relative value of APOA4 protein in serum * p < 0.05, ** p < 0.01: comparison between vehicle control (PBS) and rh-HGF alone-treated groups (Dunnett’s test), ^# p < 0.05, ^## p < 0.01, ^### p < 0.001: comparison between rh-HGF alone-treated group and PF-04217903- rh-HGF co-administered group (unpaired t test). The data are presented as mean ± SEM (n = 3). The median value of the triplicate analyses was used for calculation. (C) Analysis of APOA4 protein levels in serum using Western blot. The picture shows a representative image of lanes showing individual animals.

Figure 4

rh-HGF-mediated induction of APOA4 protein in anti-Fas-induced murine ALF model. rh-HGF was injected intravenously one hour prior to the anti-Fas injection. Five hours after anti-Fas administration, the mice were sacrificed and their blood collected for the analyses. (A) Relative value of APOA4 protein in serum. PBS was administered in the vehicle control group (rh-HGF, 0 mg/kg). ** p < 0.01, **** p < 0.0001: comparison between the vehicle control- and rh-HGF-treated groups (Dunnett’s test). The data are presented as mean ± SEM (n = 5–8). (B) A representative image of serum APOA4 levels by Western blot. Each lane shows individual animals.