OPROZOMIB (ONX-0912)   中文名称: 奥泼佐米

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

体外活性：Oprozomib 的抗 MM 活性与 caspase-8、caspase-9、caspase-3 和 PARP 的激活以及对 MM 细胞迁移和血管生成的抑制有关。激酶测定：使用生物素化活性位点探针 PR-584 (5-15 μM) 在室温下处理样品（裂解细胞或组织匀浆）1 小时。通过添加 SDS（最终浓度为 0.9%）并加热至 100 °C 5 分钟来使样品变性。将变性的样品转移至96孔或384孔过滤板中，与链霉亲和素-琼脂糖珠（2.5-5μL包装珠/孔）混合，并在板摇床上室温孵育1小时。通过真空过滤，用 100-200 μL/孔的 ELISA 缓冲液（PBS、1% 牛血清白蛋白、0.1% Tween-20）将珠子洗涤 5 次。将珠子在板摇床上于 4°C 下与以下抗体一起孵育过夜，这些抗体可识别稀释到 ELISA 缓冲液中的六个催化亚基：β5、β1 和 β2（按 1:3000 稀释）、LMP7 和 LMP2（按 1:5000 稀释）和 MECL-1稀释1：1000。用 100-200 μL/孔 ELISA 缓冲液洗涤珠子 5 次，并与在 ELISA 缓冲液中稀释 1:5000 的 HRP 偶联二抗一起孵育，并在板摇床上室温孵育 2 小时。将珠子用 100-200 μL/孔的 ELISA 缓冲液洗涤 5 次，并按照制造商的说明使用 supersignal ELISA pico 底物开发化学发光信号。在读板器上测量发光，并通过与 20S 蛋白酶体或未处理的细胞裂解物标准曲线进行比较，将其转换为 ng 蛋白酶体或 μg/ml 裂解物。对于蛋白酶体抑制剂研究，活性位点探针结合值表示为相对于 DMSO 处理细胞的结合百分比。细胞测定：在台盼蓝排除测定中，ONX 0912 在 8 种不同的 HNSCC 细胞系中表现出 IC50 值范围为 58.9 至 185.7 nmol/L。在检查的 4 种 HNSCC 细胞系（UMSCC-1、UMSCC-22B、1483 和 UMSCC-1）中，ONX 0912 处理导致 caspase-3 加工成活性亚基，并裂解 caspase 底物 PARP。

奥普罗佐米在啮齿动物和狗中的绝对生物利用度高达 39%。它具有良好的耐受性，重复口服给药剂量可在大多数组织中抑制 >80% 的蛋白酶体，并在多种人类肿瘤异种移植和小鼠同基因模型中引发抗肿瘤反应。

---

卡非佐米已被证明在多种癌症小鼠模型中介导抗肿瘤反应。我们评估了先导化合物与carfilzomib在携带已建立的非霍奇金淋巴瘤细胞系RL异种移植物的免疫功能低下小鼠和携带小鼠结直肠肿瘤细胞系CT-26的BALB/c小鼠中的抗肿瘤活性。当每种化合物按每周一次QD×2的时间表给药时（图3），化合物58/Oprozomib/奥曲佐米（口服）在两种模型中都促进了与carfilzomib（静脉注射）等效的抗肿瘤反应。然而，54（口服）在两种模型中均未达到显著的抗肿瘤反应（支持信息表S2列出了抗肿瘤反应的进一步统计分析）。值得注意的是，Oprozomib/58在低于其最大耐受剂量（MTD）的情况下给药，而carfilzomib和54则在各自的MTD下给药。[1]

---

---

ONX 0912/Oprozomib/阿曲佐米抑制人体MM细胞在体内的生长，延长小鼠模型的存活时间[2]
在证明ONX 0912在体外诱导MM细胞凋亡后，我们接下来使用2种不同的小鼠模型检查了ONX 0912中的体内功效。在第一项研究中，MM.1S荷瘤小鼠静脉注射carfilzomib（5mg/kg）或口服赋形剂或ONX 0912（30mg/kg）。动物连续治疗2天，每周重复治疗7周。如图5A所示，与仅接受载体的小鼠相比，ONX 0912治疗的小鼠肿瘤生长明显减少（P=0.002）。口服ONX 0912或静脉注射carfilzomib的小鼠肿瘤生长抑制程度相似。

我们之前的研究表明，MM宿主BM微环境赋予MM细胞生长、存活和耐药性。因此，我们接下来研究了在人类骨髓微环境存在的情况下，阿曲佐米/ONX 0912的抗MM活性是否得以保留。在这些研究中，我们使用了SCID-hu模型，该模型概括了体内人体骨髓环境。在该模型中，将INA-6 MM细胞直接注射到皮下植入SCID小鼠的人骨芯片中，并通过连续测量小鼠血清中可溶性人IL-6R的循环水平来评估MM细胞的生长。与单独注射载体的小鼠相比，口服剂量（30mg/kg或50mg/kg）ONX 0912的小鼠对肿瘤的生长抑制作用更强（图5B）。重要的是，用ONX 0912治疗荷瘤小鼠，而不是单独使用载体，显著延长了存活时间（P=0.03；图5C）。

接下来，我们使用植入的人骨的免疫染色来检测阿曲佐米/ONX 0912对体内细胞凋亡的影响，以激活半胱氨酸天冬氨酸蛋白酶-3。与单独使用载体治疗相比，ONX 0912显著增加了半胱氨酸天冬氨酸蛋白酶-3切割阳性细胞的数量（图6A）。同样，我们注意到，与单独使用载体相比，注射ONX 0912的小鼠骨切片中因子VIII和VEGFR1的表达明显降低（图6B-C）。这些体内IHC数据证实了ONX 0912在MM细胞中的凋亡和抗血管生成活性。鉴于ONX 0912抑制蛋白酶体活性（图1D），蛋白酶体抑制导致泛素化蛋白质的积累，我们还检查了小鼠的人类骨切片泛素化模式的改变。在最后一次给药后2小时切下骨片，并使用抗泛素抗体进行IHC。与对照组相比，ONX 0912治疗显著增加了泛素染色（图6D）。这些体内数据以及我们的体外结果证实，ONX 0912的抗MM活性与蛋白酶体活性的抑制有关。

小鼠对阿曲佐米/ONX 0912的剂量耐受良好，因为这些研究中没有发现明显的体重减轻（图6E）。此外，在ONX 0912治疗后没有观察到神经行为变化（数据未显示）。总之，我们对2种不同的人类MM异种移植物模型的研究结果表明，ONX 0912在耐受性良好的剂量下具有强大的体内抗肿瘤活性。SCID-hu模型的结果为ONX 0912即使在BM微环境存在的情况下也能触发肿瘤细胞凋亡提供了体内证据。

---

基于环氧酮的PI对非荷瘤小鼠具有骨合成代谢作用[3]
体外证据表明，PI对OC和OB都有细胞自主作用。为了检查它们对非骨髓瘤骨的影响，将PI给药于非荷瘤免疫活性C57Bl/6小鼠两周。与硼替佐米类似，卡氟佐米或Oprozomib/阿曲佐米治疗可增加骨小梁参数（图5a和b）。通过骨吸收导致的胶原蛋白分解产物（羧基末端端肽-胶原蛋白交联）的血清水平降低来衡量，所有三种PI都相对抑制了OC功能（图5c）。此外，与对照组相比，所有药物都显著增加了OB活性，这是通过血清中I型前胶原N-末端前肽（骨形成的标志物）水平的升高来衡量的（图5d）。值得注意的是，carfilzomib对I型前胶原N端前肽的增加明显大于硼替佐米。一致的是，双钙黄绿素标记表明PI增加了骨形成率（图5e）。这些数据表明，基于环氧酮的PIs carfilzomib和丙佐米通过合成代谢和抗分解代谢特性增强健康小鼠的骨体积，这些特性与硼替佐米相当甚至优于硼替佐密。

---

Carfilzomib和阿曲佐米/Oprozomib可降低MM肿瘤负担，保护小鼠免受骨破坏[3]
为了研究carfilzomib和丙佐米联合治疗已建立的骨髓瘤的抗肿瘤和保骨作用，我们利用了两种体内小鼠模型。将5TGM1-GFP小鼠骨髓瘤细胞静脉注射到免疫功能正常的同基因C57Bl/KaLwRij小鼠体内，在28天内产生具有明显骨破坏的播散性肿瘤。51,52个5TGM1肿瘤建立了14天，之后按照与每种药物临床剂量相关的时间表给药硼替佐米、卡氟佐米或丙佐米（见材料和方法）。根据克隆型抗体IgG2b的血清水平（图6a）或由表达GFP的肿瘤细胞组成的BM或脾脏的百分比（图6b和c），所有PI均显著降低了肿瘤负担。所有PI治疗组的microCT对肿瘤诱导的骨丢失的保护作用都很明显（图6d和e），骨转换的血清标志物显示出显著的抗吸收（图6f）和骨合成代谢（图6g）作用。值得注意的是，尽管PI内的差异没有统计学意义，但与硼替佐米相比，卡非佐米和丙佐米观察到I型前胶原活性N端前肽增加的趋势。

最后，在携带已建立的人RPMI-8226-luc骨髓瘤细胞的NOD-SCID-IL2Rγ-/-小鼠中检查了Oprozomib/阿曲佐米的疗效。通过生物发光成像（图7a）和RPMI-8226-luc细胞分泌的人Igλ血清水平（图7b）测量，阿曲佐米治疗降低了肿瘤负担。MicroCT分析显示，载体治疗的小鼠出现了明显的肿瘤相关骨丢失。相比之下，丙佐米治疗的小鼠骨小梁参数显著增加（图7c和d）。骨转换的血清标志物显示，丙佐米抑制骨吸收（图7e），同时促进骨形成（图7f）。总之，这些数据表明，口服丙佐米在携带人类MM的小鼠体内具有抗骨髓瘤活性以及骨抗分解代谢和合成代谢作用。

将探针 PR-584 (5-15 μM) 生物素化活性位点应用于样品（裂解细胞或组织匀浆），在室温下放置 1 小时。添加 0.9% 最终 SDS 并将样品加热至 100 °C 5 分钟，使样品变性。将变性样品转移至 96 或 384 孔过滤板后，添加链霉亲和素-琼脂糖珠（2.5–5 μL 填充珠/孔），并摇动板以在室温下孵育混合物一小时。将 ELISA 缓冲液（PBS、1% 牛血清白蛋白、0.1% Tween-20）通过 100–200 μL/孔真空过滤 5 次，以清洗珠子。使用以下抗体在平板摇床上 4°C 下将珠子孵育过夜。将抗体稀释到 ELISA 缓冲液中并识别六个催化亚基：1:3000 的 β5、β1 和 β2、1:5000 的 LMP7 和 LMP2 以及 1:1000 的 MECL-1。每孔用 100–200 μL ELISA 缓冲液清洗珠子 5 次后，在平板摇床上与 1:5000 稀释的 HRP 偶联二抗一起在室温下孵育 2 小时。每孔用 100–200 μL ELISA 缓冲液清洗五次后，根据制造商的说明，使用 Supersignal ELISA pico 底物开发用于化学发光信号的珠子。通过与未处理的细胞裂解物或 20S 蛋白酶体的标准曲线进行比较，在酶标仪上测量光度并转换为蛋白酶体的 ng 或裂解物的 μg/ml。蛋白酶体抑制剂研究的活性位点探针结合值以与用 DMSO 处理的细胞相比的结合百分比报告。

---

使用纯化的人20S蛋白酶体在无细胞系统中测试CT-L活性的抑制作用，并在用抑制剂（如Oprozomib/阿曲佐米）处理1小时的Molt-4（人白血病）细胞制备的细胞裂解物中测试CT-L-活性的抑制作用。在这两种测定中，确定了50%的抑制浓度（IC50），并将使用纯化酶的IC50值与完整细胞暴露的IC50值进行比较，作为化合物细胞渗透性的评估。使用纯化的人26S蛋白酶体测定一部分类似物的灭活率（kinact/Ki）。在抑制剂处理的Molt-4细胞的裂解物中，使用活性位点结合测定法测量蛋白酶体亚基的选择性。通过比较一对MES（子宫肉瘤）肿瘤细胞系的细胞存活率来评估化合物对多药耐药转运蛋白（MDR）的敏感性：亲本系（MDR-）和已知表达MDR的阿霉素耐药亚系（MDR+）。在模拟胃液和肠液（SGF和SIF）中评估化合物的稳定性，并在孵育15分钟后确定母体剩余的百分比。在小鼠、大鼠、狗和人类的肝微粒体中评估代谢稳定性，并根据半衰期计算提取率（Re），以方便跨物种比较。通过口服（po）给药后1小时血液和组织中残留CT-L活性的药效学（PD）测量来确定生物活性。绝对生物利用度（F）通过静脉和口服给药后血浆浓度与时间曲线下面积的药代动力学（PK）评估来确定。在携带已建立的人类肿瘤异种移植物的免疫功能低下小鼠和携带同基因肿瘤细胞的正常小鼠中评估了抗肿瘤疗效（有关生物测定的更多详细信息，请参阅支持信息）。 所有化合物都针对纯化的人20S蛋白酶体和Molt-4细胞进行了蛋白酶体CT-L活性抑制测试。然后将IC50小于100 nM且在柠檬酸盐缓冲液（pH 3.5）中的10%（v/v）乙醇和10%（v/v）PS80载体中的溶解度≥1.0 mg/mL的化合物口服给Balb/c小鼠（40、20、10或5 mg/kg），以通过血液和组织PD（给药后1小时的CT-L活性）初步评估生物利用度。随后，选择了一部分化合物在其他上述测定中进行进一步评估[1]。

---

免疫印迹和体外蛋白酶体活性测定[2]
如前所述进行蛋白质印迹分析。简而言之，等量的蛋白质通过10%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离，并转移到硝化纤维膜上。通过在PBST中的5%脱脂奶粉（磷酸盐缓冲盐水[PBS]中的0.05%吐温-20）中孵育来阻断膜，并用针对聚ADP核糖聚合酶、甘油醛-3-磷酸脱氢酶或胱天蛋白酶-8、胱天蛋白酶-9或胱天酶-3的特异性抗体进行探测。然后通过增强化学发光产生印迹。如前所述，使用荧光肽底物进行蛋白酶体活性测定。

台盼蓝排除试验显示 ONX 0912 在 8 种不同的 HNSCC 细胞系中表现出 IC50 值，范围为 58.9 至 185.7 nmol/L。在所研究的四种 HNSCC 细胞系（UMSCC-1、UMSCC-22B、1483 和 UMSCC-1）中，使用 ONX 0912 处理导致 caspase-3 被加工成活性亚基，并且 caspase 底物 PARP 被裂解。

---

体外迁移和毛细管结构形成试验[2]
如前所述，Transwell插入试验用于测量迁移。16通过Matrigel毛细管样管结构形成试验评估体外血管生成。17对于内皮管形成试验，人脐静脉内皮细胞（HUVEC）从Clonetics获得，并保持在含有5%胎牛血清的内皮细胞生长培养基-2中。传代3次后，使用台盼蓝排斥试验测量HUVEC的存活率，用Oprozomib/阿曲佐米/ONX 0912治疗观察到<5%的细胞死亡。

---

细胞活力、增殖和凋亡测定[2]
如前所述，通过3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑（MTT）溴化物评估细胞存活率。使用台盼蓝排斥试验获得对照细胞与处理细胞的15%细胞死亡。根据制造商的说明，使用膜联蛋白V/碘化丙啶（PI）染色试剂盒定量细胞凋亡，并在FACSCalibu上进行分析。

---

可行性分析[3]
共接种5×104个细胞/ml，进行标准MTT法。对于瞬时给药实验，用磷酸缓冲盐水洗涤细胞两次，并在1小时（硼替佐米、carfilzomib）或4小时（Oprozomib/奥曲佐米）后用无药物培养基替换。

---

体外OC分化和再吸收[3]
来自健康供体的外周血单核细胞（PBMCs）如Garcia-Gomez等人所述进行分化。33简而言之，贴壁细胞在破骨细胞培养基（50 ng/ml RANKL和25 ng/ml M-CSF）中维持14天（前OC）或21天（成熟OC）。计数TRAP+多核（≥3核）OC。为了测量再吸收，将PBMC接种在破骨细胞培养基中的钙包被孔上17天（第一周用1μM地塞米松），并计算再吸收坑面积。

---

核因子-κB（NF-κB）易位和肌动蛋白环形成[3]
预OC接受3小时的PI脉冲，然后用50 ng/ml RANKL刺激30分钟。细胞固定在4%多聚甲醛中，用0.1%Triton X-100渗透，并与小鼠抗p65抗体和二次罗丹明偶联抗体一起孵育。使用罗丹明结合的鬼笔环肽对OC前F-actin微丝进行染色。

---

体外OB分化、碱性磷酸酶（ALP）活性和矿化[3]
如所述，从健康供体（n=6）和有或没有溶骨性骨损伤（n=3）的多发性骨髓瘤患者的骨髓吸出物中产生原代间充质干细胞（MSC）并进行检测。33人类MSC系（hMSC TERT）是D Campana博士慷慨赠送的礼物。简而言之，hMSC TERT和原代MSC（第3代）在成骨培养基（含有5 mMβ-甘油磷酸、50μg/ml抗坏血酸和80 nM地塞米松）中培养11天（早期OBs；ALP活性）、14天（前OBs）或21天（成熟OBs；基质矿化）。ALP活性通过对硝基苯磷酸盐水解为对硝基苯酚进行定量，矿化度通过茜素红染色进行评估。

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.

---

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.

---

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).

---

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]

---

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]

---

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.

---

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]

---

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]

---

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

186.96

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (3.91 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

---

配方 2 中的溶解度: ≥ 2.08 mg/mL (3.91 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

---

View More

配方 3 中的溶解度: ≥ 2.08 mg/mL (3.91 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

---

请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。