20S proteasome β5 (IC50 = 3.4 nM); 20S proteasome β1 (IC50 = 31 nM); 20S proteasome β2 (IC50 = 3500 nM)
Ixazomib citrate (MLN9708) is a proteasome inhibitor. Its biologically active form, MLN2238, is generated upon hydrolysis in aqueous solutions or plasma. The primary target is the proteasome complex within the ubiquitin-proteasome system (UPS). [1]
Ixazomib citrate (MLN9708/MLN2238) is a proteasome inhibitor. The active form, MLN2238, predominantly inhibits the chymotrypsin-like (CT-L) activity of the proteasome with an IC50 of 5 nM in MM.1S cells [2]. It also inhibits caspase-like (C-L) and trypsin-like (T-L) proteasome activities at higher concentrations [2].

Ixazomib citrate (MLN9708; 0.20-3.20 μM) 以时间和剂量依赖性方式有效抑制两种细胞系的生长。 ixazomib 在 MG-63 和 Saos-2 细胞中诱导细胞周期停滞。 Ixazomib 需要激活 caspase8 和 caspase9 才能主要通过 caspase 途径诱导细胞凋亡。 ixazomib 治疗可提高促凋亡蛋白水平并降低控制 MOMP 的抗凋亡蛋白水平。 ixazomib 治疗导致线粒体释放 Cytc、Smac 和 OMI，并降低 XIAP 蛋白水平。 Ixazomib 降低 MMP2/9 的表达和分泌水平，并抑制 MG-63 和 Saos-2 细胞的侵袭能力[1]。 Ixazomib citrate (MLN9708; 12 nM) 对 TL 和 CL 蛋白酶体的活性具有抑制作用。 Ixazomib 处理 H929 和 MM.1S MM 细胞会导致聚 (ADP) 核糖聚合酶 (PARP) 蛋白水解裂解显着增加，这是细胞凋亡过程中的标志性事件。上游 PARP 激活剂 caspase-3 被 isxazomib 裂解。 Ixazomib 增加 CHOP/GADD153 和 Bip 蛋白的水平以及 eIf2-α 激酶活性。 Ixazomib 靶向 NF-κB，在体外抑制毛细管形成，并阻断 BMSC 诱导的 MM 细胞增殖 [2]。

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通过MTT实验评估，MLN2238 以时间和剂量依赖性的方式抑制人骨肉瘤细胞系MG-63和Saos-2的生长。测定了特定处理时间下的IC50值（例如，处理24小时，MG-63的IC50为0.40 µM，Saos-2的IC50为0.80 µM）。
MLN2238 诱导MG-63和Saos-2细胞发生显著凋亡，流式细胞术检测显示Annexin V+/PI- 和 Annexin V+/PI+ 细胞群增加，Western blot分析显示caspase-3、caspase-8、caspase-9和PARP被剪切。
MLN2238 导致两种骨肉瘤细胞系细胞周期阻滞在S期和G2/M期，伴随P21、p-Chk1 (Ser345)、WEE1、p-CDK1 (Tyr15)、E2F1和APAF-1的上调，以及BCL-2和Rb的下调（在MG-63中）。
MLN2238 处理促进线粒体外膜通透性增加，表现为BAX/BCL-2比值升高、BID激活、p-BAD减少、细胞色素c、Smac和OMI从线粒体释放到细胞质，以及XIAP水平降低。
MLN2238 在Transwell侵袭实验中以剂量依赖性的方式减弱MG-63和Saos-2细胞的侵袭能力，同时伴随MMP2和MMP9表达和分泌水平的降低。[1]
用MTT法评估，MLN2238 处理多发性骨髓瘤细胞系48小时后，能显著且浓度依赖性地降低细胞活力 [2]。
MLN2238 诱导MM细胞系凋亡，表现为Annexin V+/PI+ 凋亡细胞群显著增加 [2]。
MLN2238 降低了从MM患者（包括对硼替佐米、来那度胺和地塞米松难治的患者）中纯化的CD138+肿瘤细胞的活力 [2]。
MLN2238 在同基因细胞系中克服了硼替佐米耐药性，其IC50耐药比率显著低于硼替佐米 [2]。
在有效抑制MM细胞的浓度下，MLN2238 对健康供体的正常外周血单个核细胞活力没有显著影响 [2]。
MLN2238 以时间和剂量依赖性的方式诱导泛素化蛋白的积累 [2]。
机制研究表明，MLN2238 诱导的凋亡与caspase-3、caspase-8、caspase-9和PARP的剪切/激活相关 [2]；伴随p53、p21、Noxa、PUMA和E2F的上调 [2]；pRb、cyclin D1和Cdk6的下调 [2]；内质网应激蛋白Bip、phospho-eIF2-α和CHOP的诱导 [2]；以及组成型和TNF-α诱导的NF-κB活化的抑制 [2]。
MLN2238 抑制了骨髓基质细胞诱导的MM.1S细胞增殖 [2]。
MLN2238 在无明显细胞毒性的浓度下，抑制了人脐静脉内皮细胞的体外毛细血管样管腔形成，表明其具有抗血管生成活性 [2]。
MLN2238 与来那度胺、组蛋白去乙酰化酶抑制剂SAHA或地塞米松联合处理，在MM.1S细胞中显示出协同的抗MM活性 [2]。

Ixazomib citrate (MLN9708；11 mg/kg) 通过显着阻止 MM 肿瘤的生长来提高人浆细胞瘤 MM.1S 异种移植小鼠模型的存活率。使用伊沙佐米治疗的小鼠血液化学特征中的血红蛋白、胆红素和肌酐水平均正常。 ixazomib 显着增加异种移植模型中克隆的 caspase-3 阳性细胞数[2]。

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在人浆细胞瘤异种移植模型中，静脉注射MLN2238 显著抑制了肿瘤生长并延长了小鼠的生存期 [2]。与硼替佐米治疗的小鼠相比，MLN2238 治疗的小鼠生存时间更长 [2]。
口服给予MLN2238 同样显著抑制了肿瘤生长并延长了生存期 [2]。
对MLN2238 治疗小鼠肿瘤的免疫组化分析显示，cleaved caspase-3 和 TUNEL阳性细胞增加（表明凋亡）[2]，Ki-67染色减少（表明增殖降低）[2]，血管生成标志物VEGFR2和Pecam表达下降 [2]。
MLN2238 治疗小鼠的血液化学分析显示肌酐、血红蛋白和胆红素水平正常，表明耐受性良好 [2]。

蛋白酶体活性实验: 使用RIPA裂解液制备MM.1S细胞裂解物 [2]。蛋白样品在反应缓冲液中与荧光底物共同孵育：Suc-Leu-Leu-Val-Tyr-AMC用于CT-L活性，Z-Leu-Leu-Glu-AMC用于C-L活性，Bz-Val-Gly-Arg-AMC用于T-L活性 [2]。反应通过添加底物启动，通过测量荧光强度来定量游离AMC的释放 [2]。
HtrA2/Omi蛋白酶活性实验: 将重组人HtrA2酶与其底物β-酪蛋白在测定缓冲液中孵育 [2]。通过SDS-PAGE、银染色和切割产物的定量来评估β-酪蛋白的切割情况 [2]。

MTT 测定用于确定细胞活力。经胰蛋白酶消化的细胞以每孔 5000 个接种于 96 孔板中。将 Ixazomib 或 DMSO 添加到基础培养基中，并按规定的时间和剂量给予细胞。相对于单独给予媒介物的对照细胞来评估细胞的活力。

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细胞增殖实验 (MTT): 将MG-63、Saos-2和正常成骨细胞hFOB1.19接种于96孔板。细胞贴壁后，用基础培养基饥饿处理12小时，然后用溶解于新鲜基础培养基中的不同浓度MLN2238（0.20 µM 至 3.20 µM）或载体（DMSO）处理12、24或48小时。加入MTT试剂，测定相对于载体对照的细胞活力。[1]
细胞凋亡实验 (流式细胞术): 细胞用指定剂量的MLN2238处理24小时，收集、洗涤，重悬于结合缓冲液中，在避光条件下用Annexin V-FITC和碘化丙啶染色15分钟。使用流式细胞仪分析凋亡细胞。在一些实验中，细胞在加入MLN2238前先用caspase抑制剂预处理1小时。[1]
细胞周期分析 (流式细胞术): 用MLN2238处理6、12或24小时的细胞被收集、洗涤、固定，并使用商业细胞周期检测试剂盒进行PI染色。通过流式细胞仪分析DNA含量以确定细胞周期分布。[1]
蛋白质免疫印迹分析: 用含有蛋白酶抑制剂的RIPA裂解液裂解细胞。使用BCA法测定蛋白浓度。蛋白质通过SDS-PAGE分离，转印至硝酸纤维素膜，用脱脂牛奶封闭，并在4°C下与一抗孵育过夜。然后与HRP标记的二抗孵育，使用增强化学发光底物显影蛋白条带。[1]
亚细胞组分分离: 使用线粒体分离试剂盒从处理过的细胞中分离线粒体和胞质组分。细胞被匀浆并离心分离组分，然后进行Western blot分析。[1]
体外侵袭实验: 将细胞接种于铺有Matrigel的Transwell小室上室中，上室为无血清培养基。下室加入含20% FBS的培养基作为趋化剂。12小时后，用MLN2238或载体处理细胞24小时。侵袭到下膜表面的细胞用Calcein AM染色，并在荧光显微镜下计数。[1]
细胞活力实验 (MTT和CellTiter-Glo): MM细胞系或患者来源的细胞接种于培养板中，并用不同浓度的MLN2238 处理48小时 [2]。根据说明书，使用MTT法或CellTiter-Glo发光法测定细胞活力 [2]。
细胞凋亡实验 (Annexin V/PI染色): 用MLN2238 处理后的细胞被收集、洗涤，并使用商业试剂盒进行Annexin V-FITC和碘化丙啶染色 [2]。通过流式细胞仪分析凋亡细胞 [2]。
细胞增殖实验 (胸腺嘧啶核苷掺入): MM.1S细胞在存在或不存在患者来源的骨髓基质细胞的情况下，与MLN2238 共培养48小时 [2]。通过氚标记的胸腺嘧啶核苷掺入DNA来测量细胞增殖 [2]。
蛋白质免疫印迹分析: 细胞被裂解，测定蛋白浓度 [2]。蛋白质经SDS-PAGE分离后转印至膜上，与一抗在4°C孵育过夜 [2]。然后与HRP标记的二抗孵育，使用增强化学发光法显影蛋白条带 [2]。
NF-κB活性实验 (ELISA): MM.1S细胞用MLN2238 处理不同时间后收集，提取总蛋白 [2]。使用商业转录因子ELISA试剂盒检测NF-κB p65和p52的活性 [2]。
体外血管生成实验 (管腔形成): 人脐静脉内皮细胞用载体或MLN2238 预处理8小时 [2]。然后洗涤细胞，接种到铺有Matrigel的96孔板中，继续培养4小时以形成毛细血管样管状结构 [2]。使用倒置显微镜观察并拍照记录管状结构 [2]。同时使用台盼蓝排斥法评估细胞活力 [2]。

Ixazomib is dissolved at a concentration of 2 mg/mL in 5% 2-hydroxypropyl-β-cyclodextrin. The test makes use of a human plasmacytoma xenograft tumor model. After receiving a subcutaneous inoculation of 5.0×106 MM.1S cells in 100 µL serum-free RPMI-1640 medium, 21 CB-17 SCID mice are randomly assigned to treatment groups once their tumors have grown to a size of 250–300 mm3. For three weeks, mice are given vehicle, bortezomib (1 mg/kg; i.v.) or ixazomib (11 mg/kg; i.v.) twice a week. When a tumor grows to be 2 cm3, the animal is put to death.

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Human MM Xenograft Model: CB-17 SCID mice (female, 6 weeks old) were subcutaneously inoculated with 5.0 × 10^6 MM.1S cells in 100 µL serum-free RPMI-1640 medium [2]. When tumors reached 250–300 mm³ (around day 28-30), mice were randomized into treatment groups [2].
MLN2238 was dissolved in 5% 2-hydroxypropyl-β-cyclodextrin at a concentration of 2 mg/mL [2].
For intravenous treatment, mice received MLN2238 (11 mg/kg) or vehicle twice weekly for 3 weeks [2]. A comparator group received bortezomib (1 mg/kg, i.v.) on the same schedule [2].
For oral treatment, a separate group of tumor-bearing mice received MLN2238 (8 mg/kg) or vehicle by oral gavage twice weekly for 3 weeks [2].
Tumor volume was measured regularly [2]. Animals were euthanized when tumors reached 2 cm³ [2]. Blood samples were collected for chemistry analysis [2]. Tumors were harvested, sectioned, and subjected to immunohistochemical staining for cleaved caspase-3, TUNEL, Ki-67, VEGFR2, and Pecam [2].

MLN9708 is described as an orally bioavailable citrate ester prodrug that is rapidly hydrolyzed to its active form, MLN2238, upon exposure to aqueous solutions or plasma. MLN2238 is noted to have an improved pharmacokinetic and pharmacodynamic profile and a shorter proteasome dissociation half-life compared to earlier proteasome inhibitors, leading to greater distribution and effects in tumor tissue than in blood. Specific PK parameters (e.g., half-life, bioavailability) are not provided. [1]
MLN9708 is an orally bioavailable proteasome inhibitor [2]. Upon exposure to aqueous solutions or plasma, it rapidly hydrolyzes to its biologically active form, MLN2238 [2].
MLN2238 is noted to have a shorter proteasome dissociation half-life and improved pharmacokinetic and pharmacodynamic profiles compared to bortezomib, leading to greater distribution and effects in tumor tissue than in blood [2]. Specific PK parameters (e.g., half-life, oral bioavailability) are not provided in this study [2].

In vitro, MLN2238 exhibited much lower cytotoxicity against normal human osteoblast cells (hFOB1.19) compared to osteosarcoma cells, even at the highest tested dose (3.20 µM) for up to 48h, suggesting a favorable therapeutic index. No other specific toxicity data (e.g., LD50, organ toxicity) are provided. [1]
In vitro, MLN2238 at concentrations effective against MM cells did not significantly affect the viability of normal PBMCs [2].
In vivo, MLN2238 was well tolerated in mice [2]. Blood chemistry profiles (creatinine, hemoglobin, bilirubin) of treated mice remained within normal ranges [2]. No significant treatment-related toxicities or mortality were reported at the tested doses (11 mg/kg i.v., 8 mg/kg p.o.) [2].

[1]. A New Perspective for Osteosarcoma Therapy: Proteasome Inhibition by MLN9708/2238 Successfully Induces Apoptosis and Cell Cycle Arrest and Attenuates the Invasion Ability of Osteosarcoma Cells in Vitro. Cell Physiol Biochem. 2017 Jan 27;41(2

[2]. In vitro and in vivo selective antitumor activity of a novel orally bioavailable proteasome inhibitor MLN9708 against multiple myeloma cells. Clin Cancer Res. 2011 Aug 15;17(16):5311-21.

[3]. Evaluation of the proteasome inhibitor MLN9708 in preclinical models of human cancer. Cancer Res. 2010 Mar 1;70(5):1970-80.

Ixazomib citrate is a glycine derivative that is the amide obtained by formal condensation of the carboxy group of N-(2,5-dichlorobenzoyl)glycine with the amino group of 2,2'-{2-[(1R)-1-amino-3-methylbutyl]-5-oxo-1,3,2-dioxaborolane-4,4-diyl}diacetic acid. A prodrug for ixazomib that is used in combination therapy for treatment of multiple myeloma. It has a role as a prodrug, a proteasome inhibitor, an orphan drug, an antineoplastic agent and an apoptosis inducer. It is a glycine derivative, a member of benzamides, a dichlorobenzene, an oxo dicarboxylic acid and a 1,3,2-dioxaborolane. It is functionally related to an ixazomib.
Ixazomib Citrate is the citrate salt form of ixazomib, an orally bioavailable second generation proteasome inhibitor (PI) with potential antineoplastic activity. Ixazomib inhibits the activity of the proteasome, blocking the targeted proteolysis normally performed by the proteasome, which results in an accumulation of unwanted or misfolded proteins; disruption of various cell signaling pathways may follow, resulting in the induction of apoptosis. Compared to first generation PIs, second generation PIs may have an improved pharmacokinetic profile with increased potency and less toxicity. Proteasomes are large protease complexes that degrade unneeded or damaged proteins that have been ubiquinated.
See also: Ixazomib (has active moiety).

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Drug Indication
Ninlaro in combination with lenalidomide and dexamethasone is indicated for the treatment of adult patients with multiple myeloma who have received at least one prior therapy.
Treatment of systemic light chain amyloidosis

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Ixazomib citrate (MLN9708) is an investigational small-molecule proteasome inhibitor. It is highlighted as an orally bioavailable agent, which is a practical advantage. The study concludes that proteasome inhibition by MLN9708/2238 represents a novel biochemical target for osteosarcoma treatment in vitro, inducing apoptosis, cell cycle arrest, and reducing invasion. The clinical potential is suggested, as MLN9708 was under evaluation in phase I clinical trials at the time of the study. [1]
Ixazomib citrate (MLN9708) is a second-generation, orally bioavailable small-molecule proteasome inhibitor, structurally distinct from bortezomib (a boronic acid analog) [2].
It is highlighted as a promising therapy for multiple myeloma (MM), including in settings of resistance to conventional therapies and bortezomib [2].
The study suggests that MLN2238 does not significantly inhibit the neuronal survival protease HtrA2/Omi, which may be linked to bortezomib-induced peripheral neuropathy, indicating a potentially improved safety profile [2].
The preclinical data support the clinical evaluation of MLN9708 as a single agent or in combination with lenalidomide, HDAC inhibitors, or dexamethasone for the treatment of MM [2].

C₂₀H₂₃BCL₂N₂O₉

517.12

516.087

C, 46.45; H, 4.48; B, 2.09; Cl, 13.71; N, 5.42; O, 27.85

1239908-20-3

Ixazomib;1072833-77-2

56844015

White to off-white solid powder

1.5±0.1 g/cm3

1.580

3.378

175.31

4

9

11

34

797

1

ClC1C([H])=C([H])C(=C([H])C=1C(N([H])C([H])([H])C(N([H])[C@]([H])(B1OC(C(C([H])([H])C(=O)O[H])(C([H])([H])C(=O)O[H])O1)=O)C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])=O)=O)Cl

MBOMYENWWXQSNW-AWEZNQCLSA-N

InChI=1S/C20H23BCl2N2O9/c1-10(2)5-14(21-33-19(32)20(34-21,7-16(27)28)8-17(29)30)25-15(26)9-24-18(31)12-6-11(22)3-4-13(12)23/h3-4,6,10,14H,5,7-9H2,1-2H3,(H,24,31)(H,25,26)(H,27,28)(H,29,30)/t14-/m0/s1

2-[4-(carboxymethyl)-2-[(1R)-1-[[2-[(2,5-dichlorobenzoyl)amino]acetyl]amino]-3-methylbutyl]-5-oxo-1,3,2-dioxaborolan-4-yl]acetic acid

Ninlaro; MLN9708; MLN 9708; MLN-9708; ixazomib citrate; MMLN 2238-prodrug; MMLN-2238-prodrug; MMLN2238-prodrug; Ixazomib-prodrug

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)

DMSO: 100~250 mg/mL (193.4~483.5 mM)
Ethanol: ~100 mg/mL (~193.4 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.02 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。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (4.02 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。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (4.02 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。

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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。