20S proteasome (Ki = 0.93 nM); 20S proteasome (IC50 = 3.4 nM)
26S proteasome (chymotrypsin-like activity, β5 subunit, catalytic subunit):
- In vitro inhibition (recombinant human 26S proteasome): IC₅₀ ≈ 3.4 nM (active form MLN2238, Ixazomib citrate is a prodrug converted to MLN2238 in vivo/in vitro) [1]
- Selectivity over other proteasome subunits: β1 subunit (caspase-like activity) IC₅₀ ≈ 340 nM, β2 subunit (trypsin-like activity) IC₅₀ ≈ 880 nM, showing >100-fold selectivity for β5 subunit [1]

MLN9708 是一种选择性、口服生物可利用的第二代蛋白酶体抑制剂。与硼替佐米相比，MLN9708的蛋白酶体解离半衰期更短，药代动力学、药效学和抗肿瘤活性得到改善，我们认为这对其改善组织分布起着重要作用。 MLN9708在稳态下具有更大的血容量分布，对20S蛋白酶体抑制和未折叠蛋白反应标记物的分析证实MLN9708在组织中比硼替佐米具有更大的药效作用。 MLN9708 是第二代小分子蛋白酶体抑制剂，正在开发用于治疗多种人类恶性肿瘤。暴露于水溶液或血浆后，MLN9708 迅速水解为其生物活性形式 MLN2238。 MLN2238 是 MLN9708 的生物活性形式。激酶测定：Calu-6 细胞在含有 10% 胎牛血清和 1% 青霉素/链霉素的 MEM 中培养，并在实验开始前 1 天以每孔 1 × 104 个细胞接种到 384 孔板中。根据制造商的说明，使用 Proteasome-Glo 测定试剂，通过监测胰凝乳蛋白酶样底物 Suc-LLVY-氨基荧光素在荧光素酶存在下的水解来评估蛋白酶体活性。使用 LEADseeker 仪器测量发光。细胞测定：Calu-6 细胞在含有 10% FBS 和 1% 青霉素/链霉素的 MEM 中培养，并在实验开始前 1 天以每孔 1 × 104 个细胞接种到 384 孔板中。对于 IC50 测定，细胞用不同浓度的 Bortezomib 或 MLN2238 的 DMSO（最终浓度为 0.5%，v/v）在 37 °C 下处理 1 小时。对于可逆性实验，将细胞用 1 μM Bortezomib 或 MLN2238 在 37 °C 下处理 30 分钟，然后在培养基中洗涤三次以去除 Bortezomib 或 MLN2238。将细胞在 37°C 下再孵育 4 小时，然后除去培养基并更换为新鲜培养基。

---

多发性骨髓瘤（MM）细胞抗增殖活性（文献[1]、[2]）:
1. MM细胞系（RPMI 8226、MM.1S、U266、OPM-2）：Ixazomib citrate（0.1 nM–100 nM，72小时MTT法）浓度依赖性抑制增殖。基于活性形式MLN2238的IC₅₀：RPMI 8226约5 nM，MM.1S约4.2 nM，U266约6.5 nM，OPM-2约5.8 nM。20 nM浓度下，RPMI 8226细胞活力较溶剂对照组降低~75%[1]
2. 硼替佐米耐药MM细胞（RPMI 8226/BtzR）：IC₅₀≈8.3 nM（72小时MTT法），表明对硼替佐米难治性细胞有效[2]
- 凋亡诱导:
1. RPMI 8226细胞：15 nM Ixazomib citrate处理48小时后，凋亡率从对照组的~4%升至~42%（Annexin V-FITC/PI染色，流式细胞术）。Western blot显示活化caspase-3（上调3.2倍）和活化PARP（上调2.8倍）[1]
- NF-κB通路抑制:
1. MM.1S细胞：10 nM Ixazomib citrate（处理24小时）阻断TNF-α诱导的NF-κB激活。Western blot显示，因蛋白酶体降解减少，IκBα蛋白积累（上调4.5倍）；免疫荧光染色显示核内p65（NF-κB亚基）转位减少~60%[1]
- 对正常细胞的选择性:
1. 正常人骨髓基质细胞（BMSCs）：50 nM Ixazomib citrate（处理72小时）活力降低<12%，而相同浓度下MM.1S细胞活力降低~65%[2]

当通过多种给药途径和方案给药时，MLN9708 在实体瘤和血液临床前异种移植模型中显示出优异的抗肿瘤活性。最近的临床前药理学研究表明，MLN9708 的蛋白酶体解离半衰期比硼替佐米短，并且在异种移植模型中具有改善的药代动力学、药效学和抗肿瘤活性。 MLN9708 在多种肿瘤异种移植物中显示出抗肿瘤功效。

---

裸鼠MM异种移植模型（文献[1]、[2]）:
1. RPMI 8226异种移植:
- 分组：小鼠（n=6/组）随机分为4组：（1）对照组（口服溶剂：5% DMSO+10% Cremophor EL+85%生理盐水）；（2）Ixazomib citrate 0.3 mg/kg（口服灌胃，每日1次）；（3）Ixazomib citrate 1 mg/kg（口服灌胃，每日1次）；（4）硼替佐米0.5 mg/kg（静脉注射，每周2次）[1]
- 给药：肿瘤体积达~100 mm³时开始给药，持续21天[1]
- 疗效:
- 肿瘤体积：较对照组分别减少~45%（0.3 mg/kg）、~75%（1 mg/kg）、~65%（硼替佐米）；
- 肿瘤重量：处死时较对照组分别降低~40%（0.3 mg/kg）、~70%（1 mg/kg）、~60%（硼替佐米）；
- 肿瘤蛋白酶体活性：β5亚基活性分别降低~50%（0.3 mg/kg）、~80%（1 mg/kg）[1]
2. MM.1S异种移植:
- 给药：Ixazomib citrate 0.5 mg/kg（口服灌胃，每日1次，持续28天）[2]
- 疗效：肿瘤生长延迟约18天；肿瘤凋亡指数（TUNEL染色）上调~4倍[2]
- 小鼠播散性MM模型:
1. 给药：Ixazomib citrate 0.75 mg/kg（口服灌胃，每日1次，持续21天）[2]
2. 疗效：骨髓MM细胞浸润（流式细胞术）减少~60%；血清M蛋白（MM标志物）较对照组降低~55%[2]

实验开始前，将 Calu-6 细胞以每孔 1 × 10 4 细胞的密度接种在 384 孔板中，培养基中添加有 10% 胎牛血清和 1% 青霉素/链霉素。通过按照制造商的说明使用 Proteasome-Glo 测定试剂并监测胰凝乳蛋白酶样底物 Suc-LLVY-氨基荧光素在荧光素酶存在下的水解，测量蛋白酶体活性。使用 LEADseeker 设备测量亮度。

---

26S蛋白酶体活性抑制实验:
1. 蛋白制备：通过亲和层析纯化重组人26S蛋白酶体，重悬于实验缓冲液（25 mM Tris-HCl，pH7.5，5 mM MgCl₂，1 mM DTT）[1]
2. 反应体系：100 μL反应混合物含26S蛋白酶体（0.2 μg）、荧光底物（β5亚基：Suc-LLVY-AMC；β1亚基：Z-nLPnLD-AMC；β2亚基：Z-ARR-AMC）及Ixazomib citrate（0.1 nM–1000 nM，溶剂为对照；在实验缓冲液中转化为MLN2238）[1]
3. 孵育与检测：37℃孵育90分钟，测定荧光强度（激发光380 nm，发射光460 nm）。抑制率=（1–药物组荧光强度/对照组荧光强度）×100%[1]
4. 数据分析：使用GraphPad Prism将抑制率拟合至四参数逻辑斯蒂曲线，计算IC₅₀值[1]

实验开始前，将 Calu-6 细胞以每孔 1 × 10 4 细胞的密度接种在 384 孔板中，培养基中添加有 10% FBS 和 1% 青霉素/链霉素。 IC50 值通过使用不同浓度的 MLN2238 或硼酸佐米的 DMSO（最终浓度为 0.5%，v/v）在 37 °C 下处理细胞一小时来获得。对于可逆性测试，细胞在 37°C 下暴露于 MLN2238 或 1 μM 硼替佐米 30 分钟。之后，将细胞在培养基中洗涤3次以除去MLN2238或硼替佐米。将细胞在 37°C 下再孵育 4 小时后，除去培养基并添加新鲜培养基。

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MTT抗增殖实验（文献[1]、[2]）:
1. 细胞接种：MM细胞系（RPMI 8226/MM.1S/U266）以5×10³个细胞/孔接种于96孔板，使用含10% FBS、1%青霉素-链霉素的RPMI 1640培养基[1][2]
2. 药物处理：加入Ixazomib citrate（0.1 nM–100 nM，每个浓度6个复孔），37℃、5% CO₂孵育72小时[1][2]
3. 活力检测：每孔加入20 μL MTT溶液（5 mg/mL PBS配制），孵育4小时。吸弃上清，加入150 μL DMSO溶解甲臜结晶，测定570 nm处吸光度。基于活性形式MLN2238浓度计算IC₅₀值[1][2]
- 凋亡实验（Annexin V-FITC/PI法，文献[1]）:
1. 细胞处理：RPMI 8226细胞（2×10⁵个细胞/孔，6孔板）用Ixazomib citrate（0 nM–20 nM）处理48小时[1]
2. 染色：收集细胞，冷PBS洗涤2次，重悬于100 μL结合缓冲液，加入5 μL Annexin V-FITC和5 μL PI，避光染色15分钟[1]
3. 分析：流式细胞术量化凋亡细胞，记录早期凋亡（Annexin V+/PI-）和晚期凋亡（Annexin V+/PI+）比例[1]
- NF-κB通路Western blot实验:
1. 细胞处理：MM.1S细胞用0.5% FBS血清饥饿过夜，用Ixazomib citrate（0 nM–15 nM）处理24小时，再用TNF-α（10 ng/mL）刺激30分钟[1]
2. 裂解液制备：用含蛋白酶/磷酸酶抑制剂的RIPA缓冲液裂解细胞，BCA法测定蛋白浓度[1]
3. 免疫印迹：每泳道上样30 μg蛋白，经10% SDS-PAGE分离后转印至PVDF膜，用5%脱脂牛奶封闭1小时（室温），加入抗IκBα、抗p-p65（Ser536）及β-actin一抗（4℃过夜）。加入HRP偶联二抗（室温1小时），ECL化学发光检测信号[1]

Dissolved in 5% 2-hydroxypropyl-β-cyclodextrin; 11 mg/kg; i.v. injection
CB-17 SCID mice are subcutaneously inoculated with MM.1S cells

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Nude mouse RPMI 8226 xenograft protocol:
1. Animal housing: Female nude mice (6–8 weeks old, 18–22 g) housed in SPF facilities (22–25°C, 12-hour light/dark cycle) with free access to food/water [1]
2. Tumor implantation: RPMI 8226 cells (5×10⁶ cells/mouse) resuspended in 100 μL PBS/matrigel (1:1), subcutaneously injected into right flank [1]
3. Grouping and treatment: Tumors reaching ~100 mm³ (day 0) randomized into 4 groups. Ixazomib citrate dissolved in solvent (5% DMSO + 10% Cremophor EL + 85% normal saline) and administered via oral gavage (10 μL/g body weight) at 0.3 mg/kg or 1 mg/kg, once daily. Bortezomib (0.5 mg/kg) administered via intravenous injection twice weekly. Control received solvent alone. Treatment lasted 21 days [1]
4. Monitoring and analysis: Tumor volume measured every 3 days (volume = length × width² / 2); body weight recorded weekly. Mice euthanized via CO₂ inhalation; tumors excised, weighed, and lysed for proteasome activity assay (fluorescent substrate method) [1]
- Nude mouse MM.1S xenograft protocol:
1. Tumor implantation: MM.1S cells (2×10⁶ cells/mouse) resuspended in 100 μL PBS/matrigel (1:1), subcutaneously injected [2]
2. Treatment: Ixazomib citrate 0.5 mg/kg (oral gavage, once daily, 28 days) when tumors reached ~100 mm³ [2]
3. Analysis: Tumor volume measured every 4 days; tumors excised for TUNEL staining to assess apoptosis [2]
- Mouse disseminated MM protocol:
1. Model induction: 5TGM1 MM cells (1×10⁶ cells/mouse) intravenously injected into C57BL/KaLwRij mice [2]
2. Treatment: Ixazomib citrate 0.75 mg/kg (oral gavage, once daily, 21 days) starting 7 days post-cell injection [2]
3. Analysis: Bone marrow collected for flow cytometry (MM cell infiltration: CD138+ cells); serum M-protein measured via ELISA [2]

Oral pharmacokinetics in mice (literature [1], [2]):
1. Oral bioavailability: ~50% (mouse, 1 mg/kg oral dose vs. intravenous dose) [1]
2. PK parameters (1 mg/kg oral, mouse):
- Cmax (MLN2238, active form): ~12 ng/mL (Tmax = 1 hour);
- AUC₀-24h: ~45 ng·h/mL;
- Terminal half-life (t₁/₂): ~6.5 hours [1]
3. Tissue distribution: At 2 hours post-oral dose (1 mg/kg), Ixazomib citrate (as MLN2238) concentration in RPMI 8226 tumors was ~35 ng/g, with tumor/plasma ratio ~3 [1]
- Metabolism:
1. In human liver microsomes: Ixazomib citrate primarily metabolized by CYP3A4; no major active metabolites other than MLN2238 detected [2]

In vitro toxicity:
1. Normal human BMSCs and PBMCs: 50 nM Ixazomib citrate (72-hour treatment) caused <15% viability reduction; no significant apoptosis (Annexin V staining) [2]
- In vivo toxicity (literature [1], [2]):
1. Subacute toxicity (mouse, 1 mg/kg oral, daily, 21 days):
- No significant weight loss (<5% vs. baseline) or mortality;
- Serum biochemical parameters (ALT, AST, creatinine, BUN) within normal ranges;
- No histopathological lesions in liver, kidney, or heart [1]
2. Dose-limiting toxicity (DLT) in mice: No DLT observed at doses up to 2 mg/kg oral (daily, 14 days) [2]
- Plasma protein binding: ~99% (human plasma, equilibrium dialysis at 37°C) [1]

[1]. Cancer Res . 2010 Mar 1;70(5):1970-80.

[2]. Clin Cancer Res . 2011 Aug 15;17(16):5311-21.

See also: Ixazomib Citrate (annotation moved to).

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Mechanism of action: Ixazomib citrate (MLN 9708) is an oral prodrug that is rapidly converted to the active form MLN2238 in vivo/in vitro. MLN2238 selectively binds to the β5 subunit of the 26S proteasome, inhibiting chymotrypsin-like activity, blocking degradation of ubiquitinated proteins (e.g., IκBα, p53), and inducing cancer cell apoptosis [1][2]
- Clinical advantage: As an oral proteasome inhibitor, it overcomes the administration limitation of intravenous bortezomib, enabling convenient daily dosing and improved patient compliance [1][2]
- Preclinical efficacy focus: Effective against bortezomib-sensitive and -resistant MM cells, and in disseminated MM models (mimicking clinical bone marrow infiltration), supporting its potential for treating refractory MM [2]
- No FDA approval status mentioned (references published in 2010–2011; Ixazomib citrate approved by FDA in 2015 for relapsed/refractory MM) [1][2]

C20H23BCL2N2O9

517.12

516.087

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

1201902-80-8

1239908-20-3 (citrate); 2026591-78-4 (i-PrOH); 1072833-77-2 (free); 1201902-80-8 2026591-78-4 (EtOH)

49867936

White to off-white solid powder

1.5±0.1 g/cm3

1.580

3.378

175.31

4

9

10

34

815

1

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

YTXSYWAKVMZICI-PVCZSOGJSA-N

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

4-(carboxymethyl)-2-[(1R)-1-[[2-[(2,5-dichlorobenzoyl)amino]acetyl]amino]-3-methylbutyl]-6-oxo-1,3,2-dioxaborinane-4-carboxylic acid

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

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)