FV-162 is a novel, orally bioavailable, irreversible proteasome inhibitor with improved pharmacokinetics displaying preclinical efficacy with continuous daily dosing

Approved proteasome inhibitors have advanced the treatment of multiple myeloma but are associated with serious toxicities, poor pharmacokinetics, and most with the inconvenience of intravenous administration. We therefore sought to identify novel orally bioavailable proteasome inhibitors with a continuous daily dosing schedule and improved therapeutic window using a unique drug discovery platform. We employed a fluorine-based medicinal chemistry technology to synthesize 14 novel analogs of epoxyketone-based proteasome inhibitors and screened them for their stability, ability to inhibit the chymotrypsin-like proteasome, and antimyeloma activity in vitro. The tolerability, pharmacokinetics, pharmacodynamic activity, and antimyeloma efficacy of our lead candidate were examined in NOD/SCID mice. We identified a tripeptide epoxyketone, FV-162, as a metabolically stable, potent proteasome inhibitor cytotoxic to human myeloma cell lines and primary myeloma cells. FV-162 had limited toxicity and was well tolerated on a continuous daily dosing schedule. Compared with the benchmark oral irreversible proteasome inhibitor, ONX-0192, FV-162 had a lower peak plasma concentration and longer half-life, resulting in a larger area under the curve (AUC). Oral FV-162 treatment induced rapid, irreversible inhibition of chymotrypsin-like proteasome activity in murine red blood cells and inhibited tumor growth in a myeloma xenograft model. Our data suggest that oral FV-162 with continuous daily dosing schedule displays a favorable safety, efficacy, and pharmacokinetic profile in vivo, identifying it as a promising lead for clinical evaluation in myeloma therapy.

The ubiquitin-proteasome system is responsible for the regulation and degradation of the majority of the intracellular proteins in eukaryotic cells. 1 The 26S proteasome is a multisubunit protein complex that mediates the proteolytic degradation and turnover of damaged, misfolded or excess proteins that have been polyubiquitylated in the cytoplasm and nucleus. 1,2 The 26S proteasome consists of a 20S core particle, capped by 19S regulatory particles. 3,4 The 19S particle participates in the recognition, processing, unfolding, and translocation of ubiquitylated protein substrates into the 20S core. 5 Substrates are then degraded inside the chamber of the barrel-like 20S core particle, where the active sites of multiple β1, β2, and β5 subunits catalyze caspase-like (C-L), trypsin-like (T-L), or chymotrypsin-like (CT-L) proteolysis, respectively. 6,7 Inhibition of the 26S proteasome activity leads to disruption of the cell cycle and induction of apoptosis. 8 Cancer cells have an increased dependency on the integrity of the ubiquitin-proteasome system machinery compared with normal cells in preclinical studies. This finding is predominantly evident in hematological malignancies, identifying the 26S proteasome as a promising anticancer therapeutic target. [9][10][11][12] In particular, cells derived from multiple myeloma are notably sensitive to proteasome inhibition, at least in part, owing to their characteristically high rates of immunoglobulin protein biosynthesis and increased proteasome activity. [13][14][15] The continuous activity of the proteasome in myeloma cells makes them particularly susceptible to prolonged inhibition. 16 Bortezomib, the first proteasome inhibitor approved for clinical use, is a dipeptide boronic acid that reversibly binds to the active site of the β5 and β1 subunit to competitively inhibit proteasome function. 9,10,17 By inhibiting the proteasome, bortezomib acts through multiple cellular pathways that ultimately result in cell cycle arrest and apoptosis. 18 Bortezomib is currently approved for the treatment of newly diagnosed, relapsed or refractory multiple myeloma and mantle cell lymphoma. 18 Carfilzomib was subsequently developed as a second-generation inhibitor that belongs to the epoxyketone class and irreversibly binds to the active site of the β5 subunit of the proteasome. Carfilzomib is structurally and mechanistically distinct from bortezomib and overcomes bortezomib resistance in multiple myeloma cell lines and in primary multiple myeloma cells from patients. 17,19 Carfilzomib is currently also approved for relapsed and refractory multiple myeloma. ONX-0912 (also known as oprozomib) is another epoxyketone class oral proteasome inhibitor that is an analog of carfilzomib. 20,21 Similar to carfilzomib, ONX-0912 promotes cell death in primary myeloma cells from patients who relapsed after treatment with bortezomib. 20 ONX-0912 has advanced into phase I/II trials in hematological and solid malignancies. [22][23][24] Despite their clinical efficacy, treatment with proteasome inhibitors is associated with a number of toxicities, including neuropathy, thrombocytopenia, and cardiotoxicity. [25][26][27] The toxicity of currently available proteasome inhibitors necessitates administering the drugs in intermittent dosing schedules, typically biweekly. Although intermittent dosing permits proteasome activity in normal tissues during dose holidays, it has been shown to be sub-optimal for therapy in malignant cells. 16 Moreover, infrequent administration at relatively high exposures may give rise to undesirable and potentially unnecessary toxicities in normal cells. Potentially, by moderating exposures, an optimized oral proteasome inhibitor with continuous daily dosing could be developed that exploits the high proteasome dependency in malignant cells while sparing normal cells.
In the present study, we report the development of FV-162, a novel, metabolically stable and orally bioavailable epoxyketone-based proteasome inhibitor. FV-162 displays potent anticancer activity and maintains a wide differential activity between malignant and normal cells despite a continuous daily dosing schedule in multiple myeloma cell lines, primary patient cells, and animal models. Overall, our results show that FV-162 inhibits the proteasome, displays metabolic stability, and has a favorable toxicity profile.

Results
Synthesis and identification of a novel proteasome inhibitor, FV-162. To identify novel orally bioavailable proteasome inhibitors with improved pharmacokinetics and toxicity, we applied a unique fluorine-based medicinal chemistry platform to design and synthesize a diverse set of tripeptide and tetrapeptide epoxyketones, using the structures of ONX-0912 and carfilzomib as chemical backbones. The incorporation of difluoromethoxy moieties into the chemical scaffolds was aimed to improve metabolic stability, decrease toxicity, and increase oral bioavailability by increasing drug exposure (area under the curve (AUC)), whereas lowering the peak plasma concentration (C max ) and prolonging half-life t 1/2 . 28 Of the 14 unique structural analogs evaluated (Supplementary Figure S1), FV-162 was the lead compound based on potency in proteasome inhibition and cell viability assays (Supplementary Figure S2). Therefore, we focused on FV-162 and compared its activity with the orally bioavailable irreversible proteasome inhibitor, ONX-0912. The methodology of synthesis and the structure of FV-162 are outlined in Figure 1.
FV-162 is a metabolically stable proteasome inhibitor with potent antimyeloma activity. To assess the metabolic stability of FV-162, we incubated the compound with mouse liver microsomes. The percentage of parent compound remaining after 15 min was measured by liquid chromatography/mass spectroscopy (LC/MS) as an indicator of metabolic stability. Over 80% of FV-162 remained intact after incubation with liver microsomes. This was similar to the stability of carfilzomib at 70% (Supplementary Figure S2A) and higher than the stability of ONX-0912 at 56% (Figure 2a). Consistent with its design as a proteasome inhibitor, FV-162 inhibited the enzymatic activity of the CT-L proteasome subunit when added to total cell lysates from 10 different human myeloma cells. The IC 50 values of FV-162 were lower than 60 nM for all the cell lines tested. In 6 of the 10 cell lines, FV-162 was over five times more potent than ONX-0912 ( Figure 2b). Consistent with its ability to inhibit the proteasome, FV-162 decreased cell growth and viability in all the 10 human myeloma cell lines as measured by 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. In 5 of the 10 cell lines, FV-162 was over two times more potent than ONX-0912 Finally, we examined the preclinical efficacy of FV-162 in primary myeloma patient samples. Mononuclear cells isolated from the bone marrow of eight multiple myeloma patients were treated with FV-162 or ONX-0912 for 48 h. After treatment, the percentage of viable myeloma (CD138 + ) and normal hematopoietic (CD138 − ) cells was measured by flow cytometry by staining with Annexin V. At least 80% of primary myeloma cells were killed by FV-162 at concentrations ⩽ 0.1 μM while normal hematopoietic cells were largely insensitive (Figure 3a). At a five-fold higher concentration of 0.5 μM, FV-162 was toxic to 50% of CD138 − cells. In contrast, similar concentrations of ONX-0912 were less effective at inducing death in primary myeloma cells but had no adverse effects on CD138 − cells ( Figure 3b).
Pharmacokinetics of FV-162 in rodents. We hypothesized that stabilization of the epoxyketone backbone might improve pharmacokinetic profile and therapeutic index in vivo of FV-162. Therefore, we evaluated the pharmacokinetics of FV-162 in comparison with the benchmark inhibitor ONX-0912 in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice and Sprague-Dawley rats after oral and intravenous administration. Following oral (25 mg/kg) administration in mice, FV-162 demonstrated greater than three-fold higher oral bioavailability and a six-fold increase in drug exposure (AUC; 3117 versus 513 min × ng/ml) relative to that of oral ONX-0912. In contrast, FV-162 displayed an almost two-fold decrease in C max than that of ONX-0912 (21 versus 38 ng/ml) ( Figure 4a). Following intravenous (5 mg/kg) administration, FV-162 similarly exhibited more than a four-fold increase in its t 1/2 (95 versus 22 min) and a two-fold increase in drug exposure (AUC; 6633 versus 3248 min × ng/ml) compared with ONX-0912 ( Table 1).
The pharmacokinetic profiles of FV-162 and ONX-0912 following oral administration (40 mg/kg) in rats were similar to mice. FV-162 achieved a greater drug exposure (AUC; 27073 versus 22627 min × mg/ml) and a lower C max (615 versus 1273 mg/ml) compared with ONX-0912 ( Figure 4b). Following intravenous (5 mg/kg) administration, FV-162 exhibited an almost seven-fold increase in its t 1/2 (68 versus 10 min) and close to a three-fold decrease in C max (392 versus 1094 ng/ml), relative to ONX-0912 (Table 1).  Figure S3). All of the regimens tested were well tolerated by the mice, with no substantial effect on mouse body weight observed during treatment (Figure 5c). Thus FV-162 is well tolerated and displays preclinical efficacy with continuous daily dosing with a wide differential activity between malignant and normal cells.  . In contrast to the effects on the CT-L proteasome inhibition in murine RBCs, administration of FV-162 did not inhibit C-L or on T-L subunit activity in murine RBCs (Figure 5e). Taken together, these data indicate that that FV-162 is an orally bioavailable, selective proteasome inhibitor with a favorable ratio of efficacy to toxicity.

Discussion
The proteasome inhibitors bortezomib and carfilzomib produce clinical responses in hematological malignancies, such as multiple myeloma and mantle cell lymphoma. Although prolonged proteasome inhibition has been shown to be the most optimal antimyeloma therapy in preclinical models, 16 toxicity restricts dose intensity of these compounds in the clinical setting. Compared with bortezomib, more selective epoxyketone-based proteasome inhibitors should be capable of prolonged target inhibition by avoiding boronic-acid-based off-target toxicity. 16 However, first-generation epoxyketone inhibitors such as carfilzomib and ONX-0912 have poor pharmacokinetics, are administered at MTD, and are also associated with severe toxicities. Therefore, selective proteasome inhibitors with optimized pharmacokinetics may achieve similar or even prolonged target inhibition with reduced toxicity. Chemical optimization of this class of therapeutics may lead to an improved therapeutic window. By testing a panel of fluorinated derivatives of the epoxyketone proteasome inhibitors ONX-0912 and carfilzomib, we identified FV-162 as a novel and orally bioavailable proteasome inhibitor. FV-162 has an improved pharmacokinetic profile that was associated with comparable efficacy and improved safety profile. In studies of rodents, FV-162 had a lower C max and higher AUC than ONX-0912 after the administration of an equivalent oral dose. Despite the total greater exposure to the drug, FV-162 was better tolerated in mice xenograft models and displayed a wider differential activity between malignant and normal cells. These results are consistent with previous studies demonstrating that toxicity of irreversible proteasome inhibitors is related more closely to C max than the AUC. For example, Yang et al. 29 studied the pharmacokinetics and toxicity of bolus and 30 minute infusions of carfilzomib in rats. At a dose of 8 mg/kg, a bolus infusion of carfilzomib was lethal to 44% of rats, whereas the same dose given over 30 min was not toxic. 29 Recent human data also support the relationship between the C max of epoxyketone inhibitors and toxicity. Patients with multiple myeloma tolerated up to 56 mg/m 2 carfilzomib without adverse effects when the drug was given as an infusion over 30 min, which is more than double the MTD of carfilzomib when given as a bolus infusion. 30 Thus increasing the stability and AUC of epoxyketone-based proteasome inhibitors may reduce the need for high instantaneous exposures and concomitant C max toxicities.
In vitro metabolism studies demonstrated that FV-162 had comparable stability to carfilzomib and greater stability than ONX-0912. Furthermore, a daily dose of FV-162 reduced the tumor volume without a loss in weight in the mouse xenograft model. However, in isolated red blood cells from mice, a single low oral dose of FV-162 produced incomplete inhibition of the proteasome. Taken together, these data suggest that partial inhibition of the proteasome may be sufficient to eradicate malignant cells but spare normal cells. As such, we hypothesize that the proteasome complex operates at near maximal capacity in myeloma and partial inhibition of this complex is cytotoxic to myeloma cells. In contrast, the proteasome complex functions at submaximal capacity in normal cells and has greater reserve to tolerate partial inhibition. Future studies should test this hypothesis by examining the biological capacity of the proteasome complex.
Increased expression of the immunoproteasome subunit β5i renders leukemia and myeloma cells more sensitive to proteasome inhibitors, including bortezomib, carfilzomib, and ONX-0912. 31,32 Future studies should therefore examine the differential ability of FV-162 and ONX-0912 to inhibit components of the immunoproteasome as well as the constitutive proteasome.
Currently, ONX-0912 is being evaluated in phase 1 and 2 clinical trials, alone and in combination with dexamethasone and sorafenib for hematological malignancies and solid tumors. In a phase 1 study of once-daily orally administered ONX-0912 in patients with advanced refractory or recurrent solid tumors, the MTD was 150 mg/day. 23,24 When administered twice daily, the MTD has not been reached at cumulative doses up to 190 mg/day. The most commonly treated side effects were gastrointestinal related and thrombocytopenia. 23 24 To date, responses have not been reported.
In summary, we have identified a novel proteasome inhibitor FV-162 that has favorable pharmacokinetic and safety profiles for improved dose intensity. Continuous daily dosing of FV-162 well below its MTD appears more optimal for exploiting the proteasome dependency of malignant cells while minimizing toxicities. Thus, given the advantages of FV-162 observed in this study, clinical investigation of this promising agent is warranted. Synthesis of FV-162. FV-162 was synthesized by initially coupling fluorinated serine derivatives 1 and 2 ( Figure 1) by using HBTU to give a boc/benzyl-protected dipeptide, 3. This was hydrogenated to remove the benzyl group and the resulting carboxylic acid was coupled with an epoxyketone derivative of phenylalanine to give compound 5. The boc-protecting group was then cleaved using TFA and DCM giving compound 6 as a trifluoroacetate salt, which was, in turn, coupled with 2methylthiazole-5-carboxylic acid (using HBTU) to give the desired product, FV-162.
Cells and cell culture. Human multiple myeloma cell lines 8226, H929, JJN3, KMH11, KMS11, KMS18, LP1, MM.1 S, OPM2, and U266 were grown in Iscove's modified Dulbecco's medium (IMDM). Cell lines were authenticated with short tandem repeat method in September 2011. In addition, cell lines were continually monitored by morphological inspection. Primary mononuclear cells were isolated from peripheral blood or bone marrow samples from multiple myeloma patients at the Princess Margaret Cancer Centre (Toronto, ON, Canada) by Ficoll density gradient centrifugation and cultured in IMDM. All cell culture media were obtained from the Ontario Cancer Institute Tissue Culture Media Facility (Toronto, ON, Canada) and were supplemented with 10% fetal calf serum, 100 μg/ml penicillin, and 100 μg/ml streptomycin (Hyclone, Logan, UT, USA). All cells were grown in a humidified incubator at 37°C with 5% CO 2 .
Metabolic stability assay. Pooled liver microsomes isolated from male CD-1 mice were purchased from BD Biosciences (San Jose, CA, USA). Metabolic stability was calculated by measuring the percentage of 5 μM of each test compound remaining after 15 min incubation with 0.5 mg/ml liver microsomes at 37°C. Compounds were detected by LC/MS using a Xevo quadrupole time-of-flight mass FV-162, a novel oral proteasome inhibitor Z Wang et al spectrometer and an ACQUITY ultra-performance LC (UPLC) system (Waters Inc., Milford, MA, USA) to determine relative peak areas of each parent compound.
Cell viability assays. Cellular viability was primarily assessed by MTS assay according to the manufacturer's instructions (Promega, Madison, WI, USA). Ten thousand cells per well were seeded in triplicate in tissue culture-treated 96-well plates. Two hours after seeding, cells were treated with proteasome inhibitors for 72 h (0.01-10 μM) or DMSO vehicle control. Cell viability was independently confirmed by reading the optical density at 490 nm and by exclusion of trypan blue stain (Invitrogen, Burlington, ON, Canada). Viability of primary mononuclear cells was determined by Annexin V-fluorescein isothiocyanate and propidium iodide costaining (Biovision Research Products, Mountain View, CA, USA) using flow cytometry according to the manufacturer's instructions after 48 h of treatment with inhibitors or vehicle control.