Liver enzyme delayed clearance in rat treated by CSF1 receptor specific antagonist Sotuletinib

Abstract

Sotuletinib (BLZ945), a CSF1-R specific kinase inhibitor developed for the treatment of Amyotrophic Lateral Sclerosis, induced liver enzyme elevation in absence of hepatocellular lesions in preclinical rat and monkey studies. The monocytic cell family, including Kupffer cells, e.g., the liver-resident macrophages, are dependent upon CSF1 pathway activation for their survival, proliferation, and differentiation. Kupffer cells act as the main body compartment responsible for elimination of some blood-borne proteins, like ALT, AST, and few others. The depletion of Kupffer cells through CSF1 pathway inhibition has already been hypothesized as responsible for apparent liver enzyme elevation without detectable corresponding liver damage. However, a release of these biomarkers from unseen hepatic lesions or from other organs cannot be excluded. In order to eliminate a potential contribution of ALT elevation from an internal organ source, we injected recombinant his-Tagged ALT1 into rats pretreated with Sotuletinib. The elimination rate of the exogenous ALT1 was significantly lower in treated animals, demonstrating a delayed clearance independently of any potential organ lesions.

Keywords: ALT1, Cytoplasmic ALT; Alanine Aminotransferase (ALT); Amyotrophic Lateral Sclerosis (ALS); CSF1, Colony Stimulating Factor 1; CSF1-R, CSF1 Receptor; Colony Stimulating Factor-1 (CSF1) and CSF1-receptor; KC, Kupffer cell; Kupffer cells; Sotuletinib (BLZ945).

Graphical abstract

Fig. 1

A: Kinetic of ALT elevation upon Sotuletinib treatment and reversibility upon compound withdrawal. Open circle: continuous Sotuletinib treatment at 150 mg/kg/day (12 animals); black triangles: treatment at the same dose up to day 16 and then compound withdrawal (12 animals); black circles: vehicle controls (8 animals). Bars: standard deviation. B: miR-122 in control and treated animals at day 3, 8 and 14; black circles: vehicle controls, Open circle: Sotuletinib treatment.

Fig. 2

A. Immunohistochemical labeling of liver macrophages (Kupffer cells) with anti-CD68. In control liver numerous Kupffer cells are lining the parenchymal sinusoidal space (A). Marked reduction in the number of Kupffer cells was observed in Sotuletinib-treated animals (B). Portal space identify by asterisks. B. Number of Kupffer cells (KC) CD68 + per square mm of liver observed. ****p < 0.0001: significant difference compared to respective vehicle control using unpaired t tests.

Fig. 3

A. Plasma His-ALT1 activities of vehicle control and Sotuletinib-treated rats (day 7 to 9). Rats were treated once daily at a dose level of 150 mg/kg for 9 days (mean ± SEM, n = 5 per timepoint up to 42 h in order to respect blood volume withdrawal in live animals; n = 10 for 50 h after His-ALT1 injection). His-tagged ALT1 i.v. injection (0.2 mg/kg) was on day 7 of Sotuletinib treatment. B. Endogenous ALT values at predose, day 7 and 9 of Sotuletinib treatment, before injection of His-tag ALT1. **p < 0.01, and ****p < 0.0001: significant difference compared to respective vehicle control using the Sidak-Bonferroni multiple t tests (α = 0.05).