Numidargistat dihydrochloride

Intracellular arginase activity is determined for the arginase-expressing HepG2 and K-562 cell lines as follows. HepG2 cells are seeded at 100,000 cells per well one day prior to treatment with CB-1158. K-562 cells are seeded at 200,000 cells per well on the day of CB-1158 treatment. Cells are treated with a dose-titration of CB-1158 in SILAC RPMI-1640 media containing 5% heat-inactivated and dialyzed FBS, antibiotics/anti-mycotic, 10 mM L-arginine, 0.27 mM L-lysine, and 2 mM L-glutamine. The medium is harvested after 24 h and urea generated is determined. Wells containing media without cells are used as background controls. For assessing the effect of CB-1158 on Arg1 in primary hepatocytes, frozen human hepatocytes are thawed, allowed to adhere onto collagen-coated wells for 4 h, and then incubated for 48 h in SILAC-RPMI containing 10 mM L-ornithine, no L-arginine, and a dose-titration of CB-1158, at which time the media are analyzed for urea[1].

Mice[1]For the 4T1 tumor model, 105 cells are injected orthotopically into the mammary fat pad; for all other tumor models, 106 cells are injected subcutaneously (s.c.) in the right flank. For all studies, CB-1158 is administered by oral gavage twice per day at 100 mg/kg starting on study day 1 (1 day after tumor implant). Control groups receive vehicle (water) twice daily by gavage. Tumor volume measured by digital caliper (length × width × width/2) and body weight are recorded three times per week. Mice are euthanized when tumors necrotize or volumes reach 2000 mm3. For the CT26 model, anti-PD-L1 antibody (5 mg/kg) is injected intraperitoneally (i.p.) on days 5, 7, 9, 11, 13, and 15. For the 4T1 model, anti-CTLA-4 antibody (5 mg/kg) is injected i.p. on days 2, 5, and 8; anti-PD-1 antibody (5 mg/kg) is injected i.p. on days 3, 6, and 9. 4T1 tumors are harvested on study day 25 into Fekete’s solution and tumor nodules are enumerated visually. LY 188011 is dosed 50 mg/kg i.p. on days 10 and 16 for the CT26 model, 60 mg/kg i.p. on days 6 and 10 for the LLC model, or 30 mg/kg i.p. on day 5 for the 4T1 model. With these regimens, LY 188011 modestly reduces tumor growth and spares most tumor-infiltrating immune cells, allowing for the evaluation of combination activity with CB-1158[1].

[1]. Steggerda SM, et al. Inhibition of arginase by CB-1158 blocks myeloid cell-mediated immune suppression in the tumor microenvironment. J Immunother Cancer. 2017 Dec 19;5(1):101.