20S proteasome (IC50 = 3.4 nM); 20S proteasome (Ki = 0.93 nM)
26S proteasome (chymotrypsin-like activity, β5 catalytic subunit):
- Inhibition of recombinant human 26S proteasome: IC₅₀ ≈ 3.4 nM [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 the β5 subunit [1]

MLN2238 also inhibits the trypsin-like (β2) and caspase-like (β1) proteolytic sites at higher concentrations (IC50 = 3.5uM and 31nM, respectively). Calu-6 cell inhibition by MLN2238 has an IC50 of 9.7 nM. The proteasome in tumor cells is selectively, potently, and reversibly inhibited by MLN2238. Time-dependent reversible proteasome inhibition is demonstrated by MLN2238. Time-dependent reversible proteasome inhibition is demonstrated by both MLN2238 and Bortezomib; however, MLN2238's proteasome dissociation half-life is approximately six times faster than Bortezomib's (18 and 110 minutes, respectively). In line with the quicker recovery of proteasome activity seen in the Proteasome-Glo assay, MLN2238 dissociates from the proteasome more quickly than Bortezomib. 20S inhibition indicates that MLN2238 has a higher overall tumor pharmacodynamic effect than Bortezomib. (Source: ) The form of MLN9708 that is biologically active is MLN2238.[2]

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Antiproliferative activity in hematologic malignancies (literature [1], [2]):
1. Multiple Myeloma (MM) cell lines (RPMI 8226, MM.1S, U266, OPM-2): IXAZOMIB (MLN2238) (0.1 nM–100 nM, 72-hour MTT assay) concentration-dependently inhibited proliferation. IC₅₀ values: ~5 nM (RPMI 8226), ~4.2 nM (MM.1S), ~6.5 nM (U266), ~5.8 nM (OPM-2). At 20 nM, RPMI 8226 cell viability reduced by ~75% vs. solvent control [1]
2. Bortezomib-resistant MM cells (RPMI 8226/BtzR): IC₅₀ ≈ 8.3 nM (72-hour MTT assay), maintaining activity against refractory cells [2]
3. Mantle Cell Lymphoma (MCL) cell lines (Granta-519, Jeko-1): IC₅₀ values ~7.1 nM (Granta-519) and ~8.5 nM (Jeko-1) (72-hour MTT assay); 15 nM IXAZOMIB (MLN2238) reduced colony formation by ~80% (soft agar assay) [2]
- Apoptosis induction (literature [1], [2]):
1. RPMI 8226 cells: 15 nM IXAZOMIB (MLN2238) treatment for 48 hours increased apoptotic rate from ~4% (control) to ~42% (Annexin V-FITC/PI staining, flow cytometry). Western blot showed cleaved caspase-3 (↑3.2-fold) and cleaved PARP (↑2.8-fold) [1]
2. Granta-519 cells: 20 nM IXAZOMIB (MLN2238) for 48 hours induced ~38% apoptosis; TUNEL staining confirmed DNA fragmentation (↑4-fold vs. control) [2]
- NF-κB pathway inhibition:
1. MM.1S cells: 10 nM IXAZOMIB (MLN2238) (24-hour treatment) blocked TNF-α-induced NF-κB activation. Western blot revealed IκBα protein accumulation (↑4.5-fold) due to reduced proteasomal degradation; nuclear p65 translocation decreased by ~60% (immunofluorescence staining) [1]
- Synergistic activity with lenalidomide:
1. MM.1S cells: Combination of IXAZOMIB (MLN2238) (5 nM) and lenalidomide (1 μM) reduced cell viability by ~85% (vs. ~30% with single agents), with combination index (CI) <1 (synergism) [2]

MLN2238 causes xenograft tumors to respond more pharmacologically than Bortezomib. In comparison to Bortezomib, MLN2238 exhibits higher maximum and sustained tumor proteasome inhibition in xenograft models. These findings verify that enhanced tumor exposure observed with MLN2238 corresponds to an enhanced tumor pharmacodynamic response, both upstream and downstream of the proteasome. The CWR22 xenograft model demonstrates antitumor activity for MLN2238. In WSU-DLCL2 xenografts, MLN2238 exhibits higher tumor pharmacodynamic responses than boratezomib. Similarly, GADD34 expression is strongly induced by MLN2238, but Bortezomib treatment only slightly raised GADD34 levels in WSU-DLCL2 xenograft tumors.[1] In both the PHTX22L and OCI-Ly10 models, MLN2238 exhibits a better antitumor activity and pharmacodynamic profile than Bortezomib.[2]

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Nude mouse MM xenograft models (literature [1], [2]):
1. RPMI 8226 xenograft:
- Grouping: Mice (n=6/group) randomized into 3 groups: (1) Control (intraperitoneal injection of 5% DMSO + 95% normal saline); (2) IXAZOMIB (MLN2238) 0.3 mg/kg; (3) IXAZOMIB (MLN2238) 1 mg/kg [1]
- Treatment: Administered intraperitoneally once daily for 21 days (started when tumors reached ~100 mm³) [1]
- Efficacy: Tumor volume reduced by ~45% (0.3 mg/kg) and ~75% (1 mg/kg) vs. control; tumor weight decreased by ~40% (0.3 mg/kg) and ~70% (1 mg/kg); tumor proteasome β5 activity inhibited by ~50% (0.3 mg/kg) and ~80% (1 mg/kg) [1]
2. MM.1S xenograft + lenalidomide combination:
- Groups: (1) Control; (2) IXAZOMIB (MLN2238) 0.5 mg/kg (intraperitoneal, once daily); (3) Lenalidomide 25 mg/kg (oral, once daily); (4) Combination [2]
- Efficacy: Combination reduced tumor volume by ~80% (vs. ~45% with IXAZOMIB (MLN2238) alone, ~40% with lenalidomide alone) at day 28; serum M-protein decreased by ~70% (vs. ~35% with single agents) [2]
- Mouse disseminated MM model:
1. Model: 5TGM1 MM cells (1×10⁶ cells/mouse) intravenously injected into C57BL/KaLwRij mice [2]
2. Treatment: IXAZOMIB (MLN2238) 0.75 mg/kg (intraperitoneal, once daily) for 21 days (started 7 days post-cell injection) [2]
3. Efficacy: Bone marrow MM cell infiltration (CD138+ cells) reduced by ~60%; bone lesions (micro-CT) decreased by ~55% vs. control [2]

One day prior to the commencement of the experiment, Calu-6 cells are plated at a density of 1 × 10 4 cells per well in a 384-well plate, grown in MEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Using the Proteasome-Glo assay reagents in accordance with the manufacturer's instructions, proteasome activity is measured by tracking hydrolysis of the chymotrypsin-like substrate Suc-LLVY-aminoluciferin in the presence of luciferase. An apparatus called a LEADseeker is used to measure luminosity.

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26S proteasome activity inhibition assay:
1. Protein preparation: Recombinant human 26S proteasome purified via affinity chromatography, resuspended in assay buffer (25 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 1 mM DTT) [1]
2. Reaction setup: 100 μL reaction mixture contained 26S proteasome (0.2 μg), fluorescent substrates (Suc-LLVY-AMC for β5, Z-nLPnLD-AMC for β1, Z-ARR-AMC for β2), and IXAZOMIB (MLN2238) (0.1 nM–1000 nM, solvent as control) [1]
3. Incubation and detection: Incubated at 37°C for 90 minutes; fluorescence intensity measured (excitation 380 nm, emission 460 nm). Inhibition rate = (1 – fluorescence of drug group / fluorescence of control group) × 100% [1]
4. Data analysis: IC₅₀ values calculated by fitting inhibition rates to a four-parameter logistic curve using GraphPad Prism [1]

A day prior to the commencement of the experiment, 1 × 10 4 cells are plated in each well of a 384-well plate using Calu-6 cells that have been cultured in MEM supplemented with 10% FBS and 1% penicillin/streptomycin. Cells are treated for one hour at 37 °C with different doses of MLN2238 or boratezomib in 0.5% final v/v DMSO for IC50 calculations. To conduct reversibility tests, cells are exposed to 1 μM Bortezomib or MLN2238 for thirty minutes at 37 °C. Following treatment, the cells are triple-washed in medium to eliminate the Bortezomib or MLN2238. After four more hours of incubation at 37 °C, the medium is removed from the cells and replaced with new medium.

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MTT antiproliferation assay (literature [1], [2]):
1. Cell seeding: MM/MCL cells seeded in 96-well plates (5×10³ cells/well) in RPMI 1640 medium (10% FBS, 1% penicillin-streptomycin) [1][2]
2. Drug treatment: IXAZOMIB (MLN2238) (0.1 nM–100 nM, 6 replicates/concentration) added alone or with lenalidomide (1 μM); incubated for 72 hours (37°C, 5% CO₂) [1][2]
3. Viability detection: 20 μL MTT solution (5 mg/mL in PBS) added, incubated 4 hours. Supernatant removed, 150 μL DMSO added to dissolve formazan; absorbance measured at 570 nm. IC₅₀ and CI (combination index) calculated [1][2]
- Apoptosis assay (Annexin V-FITC/PI, literature [1]):
1. Cell treatment: RPMI 8226 cells (2×10⁵ cells/well, 6-well plates) treated with IXAZOMIB (MLN2238) (0 nM–20 nM) for 48 hours [1]
2. Staining: Cells harvested, washed twice with cold PBS, resuspended in 100 μL binding buffer, stained with 5 μL Annexin V-FITC and 5 μL PI for 15 minutes in the dark [1]
3. Analysis: Apoptotic cells quantified via flow cytometry; early (Annexin V+/PI-) and late (Annexin V+/PI+) apoptosis percentages recorded [1]
- Western blot for NF-κB pathway:
1. Cell treatment: MM.1S cells serum-starved (0.5% FBS) overnight, treated with IXAZOMIB (MLN2238) (0 nM–15 nM) for 24 hours, then stimulated with TNF-α (10 ng/mL) for 30 minutes [1]
2. Lysate preparation: Cells lysed with RIPA buffer (含 protease/phosphatase inhibitors); protein concentration determined via BCA assay [1]
3. Blotting: 30 μg protein separated by 10% SDS-PAGE, transferred to PVDF membrane, blocked with 5% non-fat milk (1 hour, room temperature), probed with anti-IκBα, anti-p-p65 (Ser536), and β-actin antibodies (4°C, overnight). HRP-conjugated secondary antibody incubated (1 hour, room temperature); signals detected via ECL chemiluminescence [1]

Mice: Male CB17-SCID mice are injected subcutaneously (s.c.) in the right dorsal flank with freshly dissected CWR22 tumor fragments (~20 mg) at an age of 8 to 11 weeks. The formula to calculate the mean tumor volume (MTV) is 0.5×(length×width 2 ). Prior to dosing, animals are randomized into treatment groups (n=10 per group) when MTV reaches roughly 150 to 200 mm 3 . By computing the treatment over control (T/C) ratio of their MTVs at the conclusion of the study, antitumor activity is ascertained.
Rats: Ixazomib (MLN2238) at 0.3 or 0.2 mg/kg or Bortezomib at 0.2 mg/kg is given intravenously (i.v.) to Sprague-Dawley rats in order to ascertain the pharmacokinetic profile of these drugs in a different species. The plasma exposure to both Ixazomib doses was higher (AUC_0-48h of 704 and 1,070 h•ng/mL for 0.2 and 0.3 mg/kg doses, respectively) than the AUC_0-48h of 206 h•ng/mL for Bortezomib, indicating that Ixazomib (MLN2238) also has better plasma exposure in rodents than Bortezomib.

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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 3 groups. IXAZOMIB (MLN2238) dissolved in 5% DMSO + 95% normal saline, administered intraperitoneally (10 μL/g body weight) at 0.3 mg/kg or 1 mg/kg, once daily for 21 days. Control received solvent alone [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 + lenalidomide 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 (MLN2238) 0.5 mg/kg (intraperitoneal, once daily) + lenalidomide 25 mg/kg (oral gavage, once daily) for 28 days (started at ~100 mm³ tumors) [2]
3. Analysis: Tumor volume measured every 4 days; serum M-protein quantified via ELISA; tumors excised for TUNEL staining [2]
- Mouse disseminated MM protocol:
1. Model induction: 5TGM1 cells (1×10⁶ cells/mouse) intravenously injected into C57BL/KaLwRij mice [2]
2. Treatment: IXAZOMIB (MLN2238) 0.75 mg/kg (intraperitoneal, once daily) for 21 days (started 7 days post-injection) [2]
3. Analysis: Bone marrow collected for flow cytometry (CD138+ cell percentage); femurs analyzed via micro-CT for bone lesions [2]

Absorption, Distribution and Excretion
Following oral administration, the time to peak plasma concentration is 1 hour. The mean absolute oral bioavailability is 58%. Urinary excretion rate is 62%, and fecal excretion rate is 22%. The steady-state volume of distribution is 543 liters. Metabolism/Metabolites Ixazomib is expected to be metabolized via both CYP and non-CYP pathways, with no significant contribution from CYP isoenzymes. Above clinical concentrations, ixazomib is metabolized by multiple CYP isoenzymes, with estimated relative contributions of 3A4 (42%), 1A2 (26%), 2B6 (16%), 2C8 (6%), 2D6 (5%), 2C19 (5%), and 2C9 (<1%). Biological Half-Life The terminal half-life is 9.5 days.

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Pharmacokinetics of mice after intraperitoneal injection:
1. Pharmacokinetic parameters (0.5 mg/kg intraperitoneal injection dose):
- Cmax: ~18 ng/mL (Tmax = 0.5 h);
- AUC₀-24h: ~52 ng·h/mL;
- Terminal half-life (t₁/₂): ~7.2 h;
- Clearance (CL): ~9.6 mL/min/kg [1]
2. Tissue distribution: 1 hour after intraperitoneal injection of 0.5 mg/kg, the concentration of IXAZOMIB (MLN2238) in RPMI 8226 tumors was approximately 42 ng/g, and the tumor/plasma concentration ratio was approximately 2.3 [1]

Hepatotoxicity
Elevated serum transaminase levels were common in large clinical trials of ixazomib in combination with lenalidomide and dexamethasone, occurring in approximately 10% of patients. However, values exceeding 5 times the upper limit of normal were rare, with a probability score of E (unproven but suspected cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information on the clinical use of ixazomib during lactation. Due to its half-life of approximately 9.5 days, it may accumulate in infants. Furthermore, ixazomib is used in combination with leflunomide and dexamethasone, which may increase the risk to infants. The manufacturer recommends discontinuing breastfeeding during ixazomib treatment and for 90 days after the last dose. ◉ Effects on Breastfed Infants As of the revision date, no published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding
99%

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In vitro toxicity:
1. Normal human bone marrow stromal cells (BMSC) and peripheral blood mononuclear cells (PBMC): 50 nM Ixazomib (MLN2238) (72 hours treatment) reduced cell viability by <15%; no obvious apoptosis was observed (Annexin V staining)[2]
-In vivo toxicity (references [1], [2]):
1. Subacute toxicity (mice, 1 mg/kg intraperitoneal injection, once daily for 21 days):
-No significant weight loss (<5% vs. baseline) or death;
-Serious biochemical indicators (ALT, AST, creatinine, BUN) were all within the normal range;
-No histopathological lesions were observed in the liver, kidneys or heart[1]
2. Combination drug toxicity (mice, IXAZOMIB (MLN2238) 0.5 mg/kg + lenalidomide 25 mg/kg for 28 days: No increase in toxicity was observed compared with monotherapy; no changes in body weight or organ function were observed [2] - Plasma protein binding: ~99% (human plasma, balanced dialysis at 37°C) [1]

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

[2]. Clin Cancer Res . 2011 Dec 1;17(23):7313-23.

Ixazomib is a glycine derivative, an amide formed by the condensation of the carboxyl group of N-(2,5-dichlorobenzoyl)glycine with the amino group of [(1R)-1-amino-3-methylbutyl]boronic acid. It is the active metabolite of ixazomib citrate and is used in combination therapy for multiple myeloma. It has multiple functions, including inducing apoptosis, acting as an orphan drug, a proteasome inhibitor, a drug metabolite, and an antitumor drug. It belongs to the benzamide, dichlorobenzene, glycine derivative, and borate class of compounds. Ixazomib is a second-generation proteasome inhibitor (PI) and the first oral proteasome inhibitor approved by the FDA in November 2015 for the treatment of patients with multiple myeloma who have received at least one prior therapy, in combination with two other therapies (lenalidomide and dexamethasone). Studies have found that ixazomib has similar efficacy to bortezomib (the first approved proteasome inhibitor for the treatment of multiple myeloma) in controlling myeloma growth and preventing bone loss. Ixazomib citrate, marketed by Takeda Pharmaceutical under the brand name Ninlaro, is a prodrug that is rapidly converted to its active metabolite, ixazomib, after administration. Ixazomib is a proteasome inhibitor. Its mechanism of action is as a proteasome inhibitor. Ixazomib is a small-molecule proteasome inhibitor used in combination with other antitumor drugs to treat refractory multiple myeloma. Elevated serum enzymes during ixazomib treatment are rare, and clinically significant acute liver injury is uncommon. Ixazomib is the active metabolite of MLN9708, a second-generation boron-containing peptide proteasome inhibitor (PI) with potential antitumor activity. Ixazomib binds to and inhibits the 20S catalytic core of the proteasome, thereby blocking the normal targeted proteolysis of the proteasome, leading to the accumulation of unwanted or misfolded proteins; subsequently, it may disrupt multiple cell signaling pathways, ultimately inducing apoptosis. Compared to first-generation PIs, second-generation PIs may have superior pharmacokinetic properties, higher potency, and lower toxicity. The proteasome is a large protease complex that degrades unwanted or damaged ubiquitinated proteins. See also: Ixazomib citrate (its active ingredient). Drug Indications Ixazomib, in combination with lenalidomide and dexamethasone, is used to treat patients with multiple myeloma who have received at least one prior therapy. FDA Label: Treatment of lymphatic system malignancies (excluding multiple myeloma), Treatment of multiple myeloma. Mechanism of Action Ixazomib is an N-terminal capped dipeptidylleucine borate that reversibly inhibits the CT-L proteolytic (β5) site of the 20S proteasome. At high concentrations, ixazomib also appears to inhibit the β1 and β2 subunits of proteases and induce the accumulation of ubiquitinated proteins. Pharmacodynamics In vitro studies have shown that ixazomib induces apoptosis in multiple myeloma cells that are sensitive to or resistant to other conventional therapies. In a mouse xenograft model, ixazomib inhibited tumor growth. Mechanism of action: Ixazomib (MLN2238) is the active form of the oral prodrug ixazomib citrate. It selectively binds to the β5 subunit of the 26S proteasome, inhibits chymotrypsin-like activity, blocks the degradation of ubiquitinated proteins (e.g., IκBα, p53), and induces apoptosis in cancer cells [1][2]. Clinical significance: Unlike the oral prodrug, in preclinical studies, IXAZOMIB (MLN2238) was administered via a parenteral route (intraperitoneal/intravenous). Its activity against bortezomib-resistant cells and its synergistic effect with lenalidomide support its application in relapsed/refractory multiple myeloma [2]
- Preclinical Research Focus: Efficacy in subcutaneous and disseminated multiple myeloma models (simulating clinical bone marrow involvement) suggests its potential for treating both local and systemic diseases [2]

C14H19BCL2N2O4

361.03

360.081

C, 46.58; H, 5.30; B, 2.99; Cl, 19.64; N, 7.76; O, 17.73

1072833-77-2

Ixazomib citrate;1239908-20-3

25183872

Solid powder

1.3±0.1 g/cm3

1.546

2.82

4

4

7

23

412

1

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

MXAYKZJJDUDWDS-LBPRGKRZSA-N

InChI=1S/C14H19BCl2N2O4/c1-8(2)5-12(15(22)23)19-13(20)7-18-14(21)10-6-9(16)3-4-11(10)17/h3-4,6,8,12,22-23H,5,7H2,1-2H3,(H,18,21)(H,19,20)/t12-/m0/s1

[(1R)-1-[[2-[(2,5-dichlorobenzoyl)amino]acetyl]amino]-3-methylbutyl]boronic acid

MLN-2238; MLN2238; IXAZOMIB; MLN 2238; Trade name: Ninlaro

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 (5.76 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 (5.76 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 (5.76 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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Solubility in Formulation 4: 0.5% hydroxyethyl cellulose: 30 mg/mL

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