20S proteasome β5 (IC50 = 36 nM); 20S proteasome LMP7 (IC50 = 82 nM)

Oprozomib inhibits 20S chymotrypsin-like (CT-L) at an IC50 of 55 ± 19․nM.
Oprozomib inhibits human leukemia The CT-L of molt-4 cells has an IC50 of 66 nM [1].
Oprozomib (ONX 0912; 1-1000 nM; 48 hours) reduces human multiple myeloma (MM) cell lines' viability considerably[2].
The activation of caspase-8, caspase-9, caspase-3, and PARP is linked to oprozomib's anti-MM activity[2].

Oprozomib (PR-047) has an absolute bioavailability of up to 39% in rodents and dogs[1], and it selectively inhibits the chymotrypsin-like (CT-L) activity of the constitutive proteasome (β5) and immunoproteasome (LMP7).[1]
Oprozomib, when taken orally at doses lower than the maximum tolerated dose (MTD), enhances antitumor activity in various animal models[1].
Oprozomib (30 mg/kg by oral gavage once daily for 5 consecutive days followed by 2 days of rest) treatment dreduces the amount of tumor in NOD.SCID.IL2Rγ-/- and C57Bl/6 mice[3].

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Carfilzomib has been shown to mediate antitumor responses in multiple mouse models of cancer. We evaluated the antitumor activity of the lead compounds along with carfilzomib in immunocompromised mice bearing established xenografts of the Non-Hodgkin’s lymphoma cell line RL and in BALB/c mice bearing the mouse colorectal tumor cell line CT-26. When each compound was administered on a weekly QD×2 schedule (Figure 3), compound 58/Oprozomib (po administered) promoted an equivalent antitumor response to carfilzomib (iv administered) in both models. However, 54 (po administered) was not able to achieve significant antitumor response in either model (further statistical analysis of antitumor responses are listed in Table S2 of the Supporting Information). It is noteworthy that Oprozomib/58 was administered below its maximum tolerated dose (MTD), while carfilzomib and 54 were delivered at their respective MTDs. [1]

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ONX 0912/Oprozomib inhibits human MM cell growth in vivo and prolongs survival in mouse models [2]
Having shown that ONX 0912 induced apoptosis in MM cells in vitro, we next examined the in vivo efficacy of ONX 0912 using 2 distinct mouse models. In the first study, MM.1S tumor–bearing mice were treated intravenously with carfilzomib (5 mg/kg) or orally with vehicle or ONX 0912 (30 mg/kg). Animals were treated for 2 consecutive days, and treatment was repeated weekly for 7 weeks. As seen in Figure 5A, a marked reduction (P = .002) in tumor growth was noted in ONX 0912–treated mice versus mice receiving vehicle alone. The extent of tumor growth inhibition was similar in mice treated orally with ONX 0912 or intravenously with carfilzomib.

Our previous studies have shown that the MM-host BM microenvironment confers growth, survival, and drug resistance in MM cells. We therefore next examined whether the anti-MM activity of Oprozomib/ONX 0912 is retained in the presence of the human BM microenvironment. For these studies, we used the SCID-hu model, which recapitulates the human BM milieu in vivo. In this model, INA-6 MM cells are injected directly into human bone chips that are implanted subcutaneously in SCID mice, and MM cell growth is assessed by serial measurements of circulating levels of soluble human IL-6R in mouse serum. A more robust growth inhibition of tumor occurred in mice receiving oral doses (30 mg/kg or 50 mg/kg) of ONX 0912 than in mice injected with vehicle alone (Figure 5B). Importantly, treatment of tumor-bearing mice with ONX 0912, but not vehicle alone, significantly prolonged survival (P = .03; Figure 5C).

We next examined the effect of Oprozomib/ONX 0912 on in vivo apoptosis using immunostaining of implanted human bone for caspase-3 activation. ONX 0912 dramatically increased the number of caspase-3 cleavage-positive cells compared with vehicle treatment alone (Figure 6A). Similarly, we noted a marked decrease in Factor VIII and VEGFR1 expression in bone sections from mice injected with ONX 0912 versus vehicle alone (Figure 6B-C). These in vivo IHC data confirm the apoptotic and antiangiogenic activity of ONX 0912 in MM cells. Given that ONX 0912 inhibits proteasome activity (Figure 1D) and proteasome inhibition results in accumulation of ubiquitinated proteins, we also examined human bone sections from mice for alterations in the ubiquitination pattern. Bone chips were excised 2 hours after the last dose, and IHC was performed using antiubiquitin Abs. ONX 0912 treatment markedly increased ubiquitin staining versus control (Figure 6D). These in vivo data, together with our in vitro results, confirm that the anti-MM activity of ONX 0912 is associated with inhibition of proteasome activity.

The doses of Oprozomib/ONX 0912 administered were well tolerated by mice because no significant weight loss was noted in these studies (Figure 6E). Moreover, no neurological behavioral changes were observed after ONX 0912 treatment (data not shown). Together, our findings from 2 distinct human MM xenograft models demonstrate the potent in vivo antitumor activity of ONX 0912 at doses that are well tolerated. Results from the SCID-hu model provide in vivo evidence for the ability of ONX 0912 to trigger apoptosis of tumor cells even in the presence of the BM microenvironment.

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Epoxyketone-based PIs exert bone anabolic effects on non-tumor bearing mice [3]
In vitro evidence suggests that PIs exert cell-autonomous effects on both OCs and OBs. To examine their effects on non-myelomatous bone, PIs were administered to non-tumor bearing immunocompetent C57Bl/6 mice for two weeks. Similar to bortezomib, treatment with carfilzomib or Oprozomib increased trabecular bone parameters (Figures 5a and b). All three PIs comparably inhibited OC function as measured by decreased serum levels of collagen breakdown products (carboxy-terminal telopeptide collagen crosslinks) resulting from bone resorption (Figure 5c). Furthermore, all drugs significantly increased OB activity as measured by increased serum levels of N-terminal propeptide of type I procollagen, a marker of bone formation, compared with controls (Figure 5d). Notably, carfilzomib exerted an increase in N-terminal propeptide of type I procollagen that was significantly greater than that obtained with bortezomib. In agreement, double calcein labeling demonstrated that PIs increased bone formation rate (Figure 5e). These data demonstrate that the epoxyketone-based PIs carfilzomib and oprozomib enhance bone volume in healthy mice through both anabolic and anti-catabolic properties that are equipotent to or even superior to that of bortezomib.

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Carfilzomib and Oprozomib decrease MM tumor burden and protect mice from bone destruction [3]
To examine the combined anti-tumor and bone-preserving effects of carfilzomib and oprozomib for therapeutic treatment of established myeloma, we utilized two in vivo mouse models. Intravenous injection of 5TGM1-GFP murine myeloma cells into immunocompetent, syngeneic C57Bl/KaLwRij mice yields disseminated tumors with significant bone destruction within 28 days.51,52 5TGM1 tumors were established for 14 days after which bortezomib, carfilzomib, or oprozomib were administered on schedules correlating with each drug’s clinical dosing (see Materials and Methods). All PIs significantly decreased tumor burden as measured by serum levels of the clonotypic antibody IgG2b (Figure 6a) or by percentage of BM or spleen comprised of GFP-expressing tumor cells (Figures 6b and c). Protection from tumor-induced bone loss was evident by microCT in all PI-treated groups (Figures 6d and e), with serum markers of bone turnover showing significant anti-resorptive (Figure 6f) and bone anabolic (Figure 6g) effects. Notably, although differences within PIs were not statistically significant, a trend toward increased N-terminal propeptide of type-I procollagen activity with carfilzomib and oprozomib versus bortezomib was observed.

Finally, the efficacy of Oprozomib was examined in NOD-SCID-IL2Rγ−/− mice bearing established human RPMI-8226-luc myeloma cells. Oprozomib treatment decreased tumor burden as measured by bioluminescent imaging (Figure 7a) and serum levels of human Igλ secreted by RPMI-8226-luc cells (Figure 7b). MicroCT analysis demonstrated marked tumor-associated bone loss in vehicle-treated mice. By contrast, oprozomib-treated mice presented significant increases in trabecular bone parameters (Figures 7c and d). Serum markers of bone turnover showed that oprozomib inhibited bone resorption (Figure 7e) while enhancing bone formation (Figure 7f). In summary, these data demonstrate that orally administered oprozomib exerts in vivo anti-myeloma activity along with bone anti-catabolic and anabolic effects in mice bearing human MM.

Probe PR-584 (5-15 μM) biotinylated active site is applied to samples (lysed cells or tissue homogenates) for 1 hour at room temperature. By adding 0.9% final SDS and heating the samples to 100 °C for five minutes, the samples are denatured. Following the denatured samples' transfer to a 96-or 384-well filter plate, streptavidin-sepharose beads (2.5–5 μL packed beads/well) are added, and the plate is shaken to incubate the mixture for one hour at room temperature. The ELISA buffer (PBS, 1% bovine serum albumin, 0.1% Tween-20) is vacuum-filtered five times through 100–200 μL/well to wash the beads. The following antibodies are used to incubate the beads overnight at 4 °C on a plate shaker. The antibodies are diluted into ELISA buffer and recognize the six catalytic subunits: β5, β1, and β2 at 1:3000, LMP7 and LMP2 at 1:5000, and MECL-1 at 1:1000. After washing the beads five times with 100–200 μL of ELISA buffer per well, they are incubated for two hours at room temperature on a plate shaker with a 1:5000 dilution of HRP-conjugated secondary antibody. The beads are developed for chemiluminescence signal using the supersignal ELISA pico substrate in accordance with the manufacturer's instructions after being washed five times with 100–200 μL of ELISA buffer per well. Via comparison with standard curves for untreated cell lysate or 20S proteasome, luminosity is measured on a plate reader and converted to ng of proteasome or μg/ml of lysate. Active site probe binding values for proteasome inhibitor investigations are reported as the percentage of binding in comparison to cells treated with DMSO.

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Inhibition of the CT-L activity was tested in cell free systems using purified human 20S proteasomes and in cellular lysates prepared from Molt-4 (human leukemia) cells treated with inhibitors (e.g. Oprozomib) for 1 h. In both assays, 50% inhibitory concentrations (IC50) were determined and comparison of the IC50 values using purified enzyme to intact cell exposure served as an assessment of the cell permeability of compounds. Inactivation rates (kinact/Ki) were determined for a subset of analogues using purified human 26S proteasome. Selectivity for proteasomal subunits was measured using an active site binding assay in lysates from inhibitor treated Molt-4 cells. The sensitivity of compounds to multidrug resistance transporters (MDR) was evaluated by comparing cell viability on a pair of MES (uterine sarcoma) tumor cell lines: the parental line (MDR−) and a doxorubicin resistant subline known to express MDR (MDR+). Compound stability was evaluated in simulated gastric and intestinal fluids (SGF and SIF), and the percentage of parent remaining was determined after 15 min of incubation. Metabolic stability was assessed in liver microsomes from mouse, rat, dog, and human, and the extraction ratio (Re) was calculated based on half-life to facilitate cross-species comparisons. Bioactivity was determined by pharmacodynamic (PD) measurements of residual CT-L activity in blood and tissues 1 h after oral (po) administration. Absolute bioavailability (F) was determined by pharmacokinetic (PK) assessment of areas under the plasma concentration versus time curves following iv and po administrations. Antitumor efficacy was assessed in immunocompromised mice bearing established human tumor xenografts and in normal mice bearing syngeneic tumor cells (see Supporting Information for more details on biological assays). All compounds were tested against purified human 20S proteasomes and Molt-4 cells for inhibition of the proteasome CT-L activity. Compounds with IC50 less than 100 nM and solubility ≥1.0 mg/mL in a vehicle of 10% (v/v) EtOH and 10% (v/v) PS80 in citrate buffer (pH 3.5) were then orally administered to Balb/c mice (40, 20, 10, or 5 mg/kg) for initial assessment of bioavailability by blood and tissue PD (CT-L activity 1 h postdose). A subset of compounds was subsequently chosen for further evaluation in other aforementioned assays [1].

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Immunoblotting and in vitro proteasome activity assays [2]
Western blot analysis was performed as previously described. Briefly, equal amounts of proteins were resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. Membranes were blocked by incubation in 5% nonfat dry milk in PBST (0.05% Tween-20 in phosphate-buffered saline [PBS]), and probed with specific antibodies against poly(ADP) ribose polymerase, glyceraldehyde-3-phosphate dehydrogenase, or caspase-8, caspase-9, or caspase-3. Blots were then developed by enhanced chemiluminescence. Proteasome activity assays were performed using fluorogenic peptide substrates, as previously described.

Trypan blue exclusion assays showed that ONX 0912 exhibited IC50 values in 8 different HNSCC cell lines, ranging from 58.9 to 185.7 nmol/L. Treatment with ONX 0912 caused caspase-3 to be processed into active subunits and the caspase substrate PARP to be cleaved in the four HNSCC cell lines (UMSCC-1, UMSCC-22B, 1483, and UMSCC-1) that were studied.

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In vitro migration and capillary-like tube structure formation assays [2]
Transwell Insert Assays were used to measure migration as previously described.16 In vitro angiogenesis was assessed by Matrigel capillary-like tube structure formation assay.17 For endothelial tube formation assay, human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics and maintained in endothelial cell growth medium-2 containing 5% fetal bovine serum. After 3 passages, HUVEC viability was measured using trypan blue exclusion assay, and < 5% of cell death was observed with Oprozomib/ONX 0912 treatment.

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Cell viability, proliferation, and apoptosis assays [2]
Cell viability was assessed by 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide, as previously described.15 Percent cell death in control versus treated cells was obtained using trypan blue exclusion assay. Apoptosis was quantified using annexin V/propidium iodide (PI) staining assay kit, as per manufacturer's instructions, and analyzed on a FACSCalibu.

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Viability assays [3]
A total of 5 × 104 cells/ml were plated and standard MTT assay was performed. For transient dosing experiments, cells were washed twice with phosphate-buffered saline and replaced with drug-free media after 1 h (bortezomib, carfilzomib) or 4 h (Oprozomib).

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In vitro OC differentiation and resorption [3]
Peripheral blood mononuclear cells (PBMCs) from healthy donors were differentiated as in Garcia-Gomez et al.33 Briefly, adherent cells were maintained in osteoclastogenic medium (50 ng/ml RANKL and 25 ng/ml M-CSF for 14 days (pre-OCs) or 21 days (mature OCs). TRAP + multinucleated (≥3 nuclei) OCs were enumerated. To measure resorption, PBMCs were seeded on calcium-coated wells in osteoclastogenic medium for 17 days (with 1 μM dexamethasone the first week), and resorption pit area was calculated.

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Nuclear factor-κB (NF-κB) translocation and actin ring formation [3]
Pre-OCs received a 3 h pulse of PIs followed by stimulation with 50 ng/ml RANKL for 30 min. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with a mouse anti-p65 antibody and a secondary rhodamine-conjugated antibody. Pre-OC F-actin microfilaments were stained using rhodamine-conjugated phalloidin.

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In vitro OB differentiation, alkaline phosphatase (ALP) activity and mineralization [3]
Primary mesenchymal stem cells (MSCs) from BM aspirates of healthy donors (n =6) and MM patients with (n =6) or without osteolytic bone lesions (n =3) were generated and assayed as described.33 The human MSC line (hMSC-TERT) was a generous gift from Dr D Campana. Briefly, the hMSC-TERT and primary MSCs (passage 3) were cultured in osteogenic medium (containing 5 mM β-glycerophosphate, 50 μg/ml ascorbic acid and 80 nM dexamethasone) for 11 (early OBs; ALP activity), 14 (pre-OBs) or 21 days (mature OBs; matrix mineralization). ALP activity was quantified by hydrolysis of p-nitrophenylphosphate into p-nitrophenol and mineralization assessed by alizarin red staining.

Non-Hodgkin’s lymphoma cell line RL xenograft, colorectal tumor cell line CT-26 xenograft
30 mg/kg, twice weekly on days 1 and 2
p.o.

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Human plasmacytoma xenograft and SCID-hu model [2]
The severe combined immunodeficiency (SCID)–hu model has been described previously. For SCID-hu model studies, INA-6 cells (IL-6–dependent MM cell line; 2.5 × 106) were injected directly into human bone chips implanted subcutaneously in SCID mice. Tumor growth was assessed every tenth day by measuring circulating levels of soluble interleukin-6 receptor (shIL-6R) in mouse blood using enzyme-linked immunosorbent assay. The human plasmacytoma (MM1.S) xenograft tumor model was performed as previously described with slight modifications. Female beige nude xid (BNX) mice were implanted with 3 × 107 MM1.S cells in matrigel (1:1) and randomized to treatment groups when tumors reached 250-300 mm3.

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In vivo drug treatment [3]
PIs were administered to mice on the following weekly schedules: bortezomib (1 mg/kg intravenously days 1 and 4); carfilzomib (5 mg/kg for C57Bl/6, 3 mg/kg for KaLwRij, intravenously days 1 and 2); Oprozomib (30 mg/kg by oral gavage once daily for 5 consecutive days followed by 2 days of rest). Vehicle mice were administered both oral 1% carboxy-methylcellulose (Oprozomib schedule) and intravenous 10% Captisol in 10 mM citrate buffer, pH 3.5 (carfilzomib schedule). In Figure 5f, following 14 days of drug treatment, three doses of 1 mg/kg of RANKL were given intraperitoneally at 24 h intervals as described in Tomimori et al.34 Serum was collected 90 min after the final RANKL injection.

Pharmacokinetics of Oprozomib[4]
PK parameters for the QD treatment group on days 1 and 5 of cycle 1 are shown in Table 4. PK parameters in cycle 2 are shown in Supplementary Table S1. Cycle 1 t1/2 ranged from 0.34 to 2.1 h (Table 4). Median tmax occurred between 0.6 and 2.0 h. Geometric mean peak (Cmax) and total (AUC0-last) exposures following daily treatment (QD treatment group) generally increased with increasing dose. Peak exposure, but not total exposure, was reduced in the split-dose arm. Geometric mean Cmax from the split-dose treatment group was lower than the QD treatment group while mean AUClast was similar between the two groups for the overlapping total daily dose levels of 120, 150, and 180 mg (Table 4, Supplementary Table S2). Geometric mean Cmax and AUC0-last appeared to be similar in the fasted and fed state (Supplementary Table S2). No significant accumulation of oprozomib was generally observed following administration of multiple doses in cycles 1 and 2 (data not shown).

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Compound 58/Oprozomib displayed a moderate absolute oral bioavailability (F) across multiple species by plasma PK measurement and blood PD measurement (Table 7). In mice, PD bioactivity was calculated by comparing dose response curves for proteasome inhibition in blood following po and iv administrations. Oral bioactivity measured by PK and PD was found to be comparable, which reconfirmed the rationale that measurement of PD (CT-L activity 1 h postdose) following po administration can be used as a primary screening assay to evaluate bioactivity and bioavailability (F) of this series of peptide epoxyketone analogues. [1]

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Furthermore, the kinetics of proteasome inhibition in animals following po administration of Oprozomib/58 demonstrated rapid absorption, tissue distribution, and inactivation of the proteasome (Figure 4). Within 15 min of dosing, proteasome inhibition in excess of 80% was achieved in blood and all tissues examined except the brain. This rapid onset of proteasome inhibition is comparable to that seen with iv administration of carfilzomib. Similar to carfilzomib, proteasome activity recovered through new proteasome synthesis in all tissues, with the exception of blood, within 24−72 h. [1]

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Oprozomib (formerly known as ONX 0912 and PR-047) is an orally bioavailable analog of carfilzomib, which has been reported to have anti-tumor activity equivalent to carfilzomib in xenograft models of non-Hodgkin’s lymphoma and colorectal cancer,26 and also to exert anti-MM activity in vitro and in myeloma animal models. Its favorable pharmacologic profile and tolerability supports its further clinical development and Phase I clinical trials are underway.[3]

Safety of Oprozomib [4]
Treatment-emergent AEs occurring in at least 10 % of patients are shown in Table 3. Among the 25 patients in the QD treatment cohorts, the most common nonhematologic AEs (all grades) included nausea (23 patients, 92 %), vomiting (20 patients, 80 %), fatigue (14 patients, 56 %), diarrhea (13 patients, 52 %), and decreased appetite (11 patients, 44 %). AEs of grade ≥3 included dehydration (three patients, 12 %), hyponatremia, hypophosphatemia, and vomiting (two patients each, 8 % each). There were no deaths while on treatment or within 30 days of the last dose of oprozomib in the QD treatment cohorts.

The most common nonhematologic AEs in the split-dose treatment group included vomiting (18 patients, 95 %), nausea (17 patients, 90 %), and diarrhea (14 patients, 74 %). Anemia (four patients, 21 %), fatigue, and decreased lymphocyte count (two patients each, 11 %) were the most common grade ≥3 treatment-emergent AEs. In the split-dose treatment group, AEs that occurred during fasting (cycle 1) were compared with AEs occurring in the fed state (cycle 2). Notable differences included vomiting (14 vs. 9 patients, respectively), nausea (13 vs. 4 patients), and diarrhea (10 vs. 6 patients). One of the DLTs described above was a grade 5 gastrointestinal hemorrhage that occurred in one patient while on split-dose treatment.

A protocol amendment required concomitant administration of a standard-of care antiemetic regimen prior to oprozomib. Serotonin 5HT3 antagonists were used by 23 patients (92 %) in the QD treatment group and by all 19 patients in the split-dose treatment group. Other antiemetics were taken by 19 patients (76 %) in the QD treatment group and 16 patients (84 %) in the split-dose treatment group. Two patients in the QD treatment group (150-mg and 180-mg cohorts, one patient each) and one patient in the 120-mg split-dose cohort (60 mg/60 mg) had increases in peripheral neuropathy from grade 1 at baseline to grade 2 while on treatment. There was no incidence of new-onset peripheral neuropathy. No clinically meaningful changes in corrected QT, blood pressure, or heart rate were observed. At the MTD cohorts, three of seven (43 %) patients on QD and four of seven (57 %) on split dose had at least 1 dose reduction, delay, or missed dose due to an AE. Among all patients, 1 of 25 (4 %) in the QD treatment group discontinued therapy owing to AEs, compared with 3 of 19 patients (16 %) in the split-dose treatment group.

[1]. Design and synthesis of an orally bioavailable and selective peptide epoxyketone proteasome inhibitor (PR-047). J Med Chem. 2009 May 14;52(9):3028-38.

[2]. A novel orally active proteasome inhibitor ONX 0912 triggers in vitro and in vivo cytotoxicity in multiple myeloma. Blood. 2010 Dec 2;116(23):4906-15.

[3]. The epoxyketone-based proteasome inhibitors carfilzomib and orally bioavailable oprozomib have anti-resorptive and bone-anabolic activity in addition to anti-myeloma effects. Leukemia. 2013 Feb;27(2):430-40.

[4]. A first-in-human dose-escalation study of the oral proteasome inhibitor oprozomib in patients with advanced solid tumors. Invest New Drugs. 2016 Apr;34(2):216-24.

Oprozomib has been used in trials studying the treatment of Solid Tumors, Multiple Myeloma, Waldenstrom Macroglobulinemia, Advanced Hepatocellular Carcinoma, and Advanced Non-Central Nervous System (CNS) Malignancies.
Oprozomib is an orally bioavailable proteasome inhibitor with potential antineoplastic activity. Proteasome inhibitor ONX 0912 inhibits the activity of the proteasome, thereby blocking the targeted proteolysis normally performed by the proteasome; this may result in an accumulation of unwanted or misfolded proteins. Disruption of various cell signaling pathways may follow, eventually leading to the induction of apoptosis and inhibition of tumor growth. Proteasomes are large protease complexes that degrade unneeded or damaged proteins that have been ubiquitinated.

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Proteasome inhibition has been validated as a therapeutic modality in the treatment of multiple myeloma and non-Hodgkin's lymphoma. Carfilzomib, an epoxyketone currently undergoing clinical trials in malignant diseases, is a highly selective inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. A chemistry effort was initiated to discover orally bioavailable analogues of carfilzomib, which would have potential for improved dosing flexibility and patient convenience over intravenously administered agents. The lead compound, 2-Me-5-thiazole-Ser(OMe)-Ser(OMe)-Phe-ketoepoxide (58) (Oprozomib/PR-047), selectively inhibited CT-L activity of both the constitutive proteasome (beta5) and immunoproteasome (LMP7) and demonstrated an absolute bioavailability of up to 39% in rodents and dogs. It was well tolerated with repeated oral administration at doses resulting in >80% proteasome inhibition in most tissues and elicited an antitumor response equivalent to intravenously administered carfilzomib in multiple human tumor xenograft and mouse syngeneic models. The favorable pharmacologic profile supports its further development for the treatment of malignant diseases.[1]

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Bortezomib therapy has proven successful for the treatment of relapsed, relapsed/refractory, and newly diagnosed multiple myeloma (MM). At present, bortezomib is available as an intravenous injection, and its prolonged treatment is associated with toxicity and development of drug resistance. Here we show that the novel proteasome inhibitor Oprozomib/ONX 0912, a tripeptide epoxyketone, inhibits growth and induces apoptosis in MM cells resistant to conventional and bortezomib therapies. The anti-MM activity of ONX-0912 is associated with activation of caspase-8, caspase-9, caspase-3, and poly(ADP) ribose polymerase, as well as inhibition of migration of MM cells and angiogenesis. ONX 0912, like bortezomib, predominantly inhibits chymotrypsin-like activity of the proteasome and is distinct from bortezomib in its chemical structure. Importantly, ONX 0912 is orally bioactive. In animal tumor model studies, ONX 0912 significantly reduced tumor progression and prolonged survival. Immununostaining of MM tumors from ONX 0912-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Finally, ONX 0912 enhances anti-MM activity of bortezomib, lenalidomide dexamethasone, or pan-histone deacetylase inhibitor. Taken together, our study provides the rationale for clinical protocols evaluating ONX 0912, either alone or in combination, to improve patient outcome in MM. [2]

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Proteasome inhibitors (PIs), namely bortezomib, have become a cornerstone therapy for multiple myeloma (MM), potently reducing tumor burden and inhibiting pathologic bone destruction. In clinical trials, carfilzomib, a next generation epoxyketone-based irreversible PI, has exhibited potent anti-myeloma efficacy and decreased side effects compared with bortezomib. Carfilzomib and its orally bioavailable analog Oprozomib, effectively decreased MM cell viability following continual or transient treatment mimicking in vivo pharmacokinetics. Interactions between myeloma cells and the bone marrow (BM) microenvironment augment the number and activity of bone-resorbing osteoclasts (OCs) while inhibiting bone-forming osteoblasts (OBs), resulting in increased tumor growth and osteolytic lesions. At clinically relevant concentrations, carfilzomib and oprozomib directly inhibited OC formation and bone resorption in vitro, while enhancing osteogenic differentiation and matrix mineralization. Accordingly, carfilzomib and oprozomib increased trabecular bone volume, decreased bone resorption and enhanced bone formation in non-tumor bearing mice. Finally, in mouse models of disseminated MM, the epoxyketone-based PIs decreased murine 5TGM1 and human RPMI-8226 tumor burden and prevented bone loss. These data demonstrate that, in addition to anti-myeloma properties, carfilzomib and oprozomib effectively shift the bone microenvironment from a catabolic to an anabolic state and, similar to bortezomib, may decrease skeletal complications of MM. [3]

C25H32N4O7S

532.61

532.199

C, 56.38; H, 6.06; N, 10.52; O, 21.03; S, 6.02

935888-69-0

25067547

white solid powder

1.3±0.1 g/cm3

849.9±65.0 °C at 760 mmHg

467.8±34.3 °C

0.0±3.2 mmHg at 25°C

1.573

2.79

3

9

14

37

825

4

C([C@@]1(OC1)C)(=O)[C@@H](NC(=O)[C@H](COC)NC(=O)[C@H](COC)NC(C1SC(C)=NC=1)=O)CC1C=CC=CC=1

SWZXEVABPLUDIO-WSZYKNRRSA-N

InChI=1S/C25H32N4O7S/c1-15-26-11-20(37-15)24(33)29-19(13-35-4)23(32)28-18(12-34-3)22(31)27-17(21(30)25(2)14-36-25)10-16-8-6-5-7-9-16/h5-9,11,17-19H,10,12-14H2,1-4H3,(H,27,31)(H,28,32)(H,29,33)/t17-,18-,19-,25+/m0/s1

N-[(2S)-3-methoxy-1-[[(2S)-3-methoxy-1-[[(2S)-1-[(2R)-2-methyloxiran-2-yl]-1-oxo-3-phenylpropan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]-2-methyl-1,3-thiazole-5-carboxamide

ONX 0912; ONX-0912; Oprozomib; 935888-69-0; Oprozomib (ONX 0912); N-((S)-3-methoxy-1-(((S)-3-methoxy-1-(((S)-1-((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-2-methylthiazole-5-carboxamide; PR 047; ONX0912; PR047; PR 047; PR-047

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.91 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.91 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.91 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)