Abstract
The isoprenoid biosynthesis pathway (IBP) is responsible for the synthesis of isoprenoids used for the prenylation of proteins belonging to the Ras small GTPase superfamily. Geranylgeranyl diphosphate synthase (GGDPS) is the enzyme in the IBP pathway that catalyzes the synthesis of the 20-carbon isoprenoid GGDP. The enzymes geranylgeranyl transferase (GGTase) I and II utilize GGDP as the isoprenoid substrate for their geranylgeranylation reactions. Rab proteins mediate vesicle trafficking within the cell and their activity is dependent on geranylgeranylation. Our prior work has demonstrated that agents that disrupt Rab geranylgeranylation disrupt monoclonal protein trafficking in myeloma cells, resulting in induction of the unfolded protein response (UPR) pathway and apoptosis. Because myeloma cells express near maximal levels of UPR-associated proteins to aid in the continuous production of monoclonal proteins, they are particularly sensitive to activation of the pro-apoptotic arm of the UPR. We are therefore pursuing the development of GGDPS inhibitors as a novel therapeutic strategy for myeloma. Our lead compound VSW1198 (Figure) is the most potent GGDPS inhibitor reported to date with an IC50 of 45nM against purified enzyme, high specificity for GGDPS over the related enzyme farnesyl diphosphate synthase, and measurable cellular activity at concentrations as low as 30 nM (15 ng/mL). Due to its potent activity against myeloma cells in vitro, we were interested in evaluating the metabolic stability, pharmacokinetic (PK) profile, distribution pattern, and toxicology profile of VSW1198 in preparation for in vivo efficacy studies. Single dose testing via IV administration in CD-1 mice revealed a maximum tolerated dose of 0.5 mg/kg. Doses > 1mg/kg resulted in liver toxicity that peaked around 6-7 days post-injection. Histopathological examination of the livers demonstrated centrilobular hepatocyte necrosis and apoptosis with invasion of inflammatory cells and panlobular hepatocyte swelling. Analysis of blood samples showed elevated transaminase levels without effects on bilirubin, alkaline phosphatase, lactic dehydrogenase, or albumin, consistent with the observed pattern of hepatic injury. A control compound, RAM3059, also was tested in vivo . This compound has a similar isoprenoid triazole bisphosphonate structure (Figure) but is 380-fold less potent than VSW1198 as a GGDPS inhibitor. Mice injected with RAM3059 (1.6 mg/kg IV) were grossly and histologically normal with no signs of liver toxicity, suggesting liver injury is not due to nonspecific toxicity from the triazole bisphosphonate structure. No effects on kidney function, calcium, creatine kinase, or blood counts were observed. Histopathological examination of the brains and kidneys from the treated mice revealed no abnormalities. A PK and tissue distribution study included 11 blood samples obtained over a period from 5 minutes to 7 days post-injection of VSW1198 (0.5 mg/kg IV, n=5 per time point). Tissue samples (liver, kidney, spleen, lung, heart, brain, and bone marrow) were collected at 2, 8, 24, 48, and 72 hours post-injection (n=5 per time point). VSW1198 was extracted from plasma and tissue samples, derivatized by reaction with diazomethane, and analyzed via LC-MS/MS. The maximum concentration (C0 ) is 611.0 (+ 183.3) ng/mL, the area under curve (AUC0-∞ ) is 2355.9 (+ 160.3) hr*ng/mL, and the plasma elimination half-life (t1/2 ) is 38.0 (± 10.9) hrs. Detectable concentrations of VSW1198 at 72 hours (5.0 + 1.0 ng/mL) indicate prolonged systemic exposure. Analysis of tissues samples is underway and will be reported. The metabolic stability of VSW1198 was assessed using human liver microsomes (HLM) and mouse S9 cells for phase I and phase II metabolism. In both the HLM and mouse S9 studies, VSW1198 showed complete stability while a positive control showed rapid metabolic degradation. In summary, these studies demonstrate the metabolic stability and systemic distribution of the potent GGDPS inhibitor VSW1198. Multi-dose testing and efficacy studies in a myeloma xenograft mouse model are currently underway to assess further the clinical potential of GGDPS inhibitor therapy.
Holstein: Celgene: Consultancy; Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.
