FV-162 Is a Novel Orally Bioavailable Proteasome Inhibitor With Improved Pharmacokinetics That Displays Preclinical Efficacy In Vitro and In Vivo

Proteasome inhibitors (PIs), such as bortezomib and carfilzomib, are often associated with serious toxicities, poor pharmacokinetics (PK), and the inconvenience of intravenous administration. Therefore, there is a need for safer and more effective orally active PIs. Applying a novel fluorine-based medicinal chemistry technology, we designed, synthesized and tested novel proprietary analogs of epoxyketone-based PIs. Our initial screening identified a lead orally bioavailable PI, FV-162, which had superior potency in proteasome activity inhibition, cytotoxicity in cultured human multiple myeloma (MM) cells and metabolic stability in mouse liver microsomes, compared to an oral PI currently under clinical evaluation, ONX-0912.

FV-162 inhibited the β subunits of purified proteasomes of T. acidophilum and specifically inhibited the β5 subunit of the proteasome in 10 human myeloma cell lines with an IC50 range of 12-58 nM. In comparison, IC50 values for the β1 and β2 subunits were consistently greater than 10 µM. FV-162 also disrupted the ubiquitin-proteasome pathway in the LP-1 human myeloma cell line in a time- and dose-dependent manner, inducing intracellular accumulation of ubiquitylated proteins. FV-162 was cytotoxic to 10 human MM cell lines with an IC50 range of 16-611 nM. Furthermore, in 4 MM patient samples tested, at least 80% of primary CD138+ myeloma cells were preferentially killed by FV-162 at concentrations less than or equal to 0.5 µM after 24 hours of incubation, while more than 70% of CD138- normal hematopoietic cells remained viable.

FV-162 displayed permeability in murine gastrointestinal epithelial cells, consistent with excellent oral absorption. In addition, cytochrome P450 isozyme assays in pooled human liver microsomes using up to 3 μM FV-162 suggested minimal risk for drug-drug interaction.

The toxicity of epoxyketone-based PIs, such as carfilzomib, appears to be related to the Cmax, while efficacy is more dependent on the AUC. We hypothesized that the novel fluorine chemistry of FV-162 would enhance the stability of the drug and thereby improve its PK profile and therapeutic index. Therefore, we measured the oral PK of FV-162 and ONX-0912 in NOD/SCID mice (25 mg/kg). FV-162 reached a peak plasma concentration (Cmax) of 21 ± 5 ng/mL (mean ± SD) after 1 h (Tmax), while ONX-0912 reached a Cmax of 38 ± 46 ng/mL after a Tmax of 5 min. Despite the lower Cmax, the AUC of FV-162 was 6-fold higher compared to ONX-0912 (3117 vs. 513 min*ng/mL). In addition, the oral bioavailability and half-life of FV-162 were 3- and 4.3-fold higher than ONX-0912, respectively. Attenuated Cmax and improved AUC may improve the tolerability profile of FV-162 compared to ONX-0912 and carfilzomib. Supporting this contention, the maximum tolerated dose of ONX-0912 in NOD/SCID mice was 30 mg/kg, while mice tolerated 200 mg/kg FV-162 for 28 days without significant toxicity.

Next, we evaluated FV-162 activity in mouse models. Inhibition of β5 subunit activity reached 80% in murine red blood cells within 1 hour of oral administration of FV-162 to NOD/SCID mice (30-100 mg/kg), which was sustained for at least 24 hours. Oral dosing of 100 mg/kg FV-162 achieved similar β5 inhibition as 30 mg/kg ONX-0912. Finally, continuous daily oral administration of FV-162 at 50 or 100 mg/kg significantly inhibited tumor growth in a myeloma MM.1S mouse xenograft model without toxicity and was as effective as intravenous carfilzomib or ONX-0912 administered at their maximal doses.

Taken together, our preclinical data suggest that FV-162 has superior PK, an improved safety profile, and high oral potency, identifying it as a promising orally bioavailable PI for myeloma therapy. Further clinical investigation of this novel PI in MM is therefore warranted.