20S proteasome (IC50 = 3.4 nM); 20S proteasome (Ki = 0.93 nM)

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]

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]

One day prior to the commencement of the experiment, Calu-6 cells are plated at a density of 1 × 10⁴ 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.

A day prior to the commencement of the experiment, 1 × 10⁴ 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.

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×width²). Prior to dosing, animals are randomized into treatment groups (n=10 per group) when MTV reaches roughly 150 to 200 mm³. 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.

Absorption, Distribution and Excretion
After oral administration, the time to reach maximum concentration in plasma was 1 hour. The mean absolute oral bioavailability is 58%.
62% in urine and 22% in feces.
The steady-state volume of distribution is 543 L.

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Metabolism / Metabolites
Metabolism of ixazomib is expected to be by CYP and non-CYP pathways, with no predominant CYP isozyme contribution. At higher than clinical concentrations, ixazomib was metabolized by multiple CYP isoforms with estimated relative contributions of 3A4 (42%), 1A2 (26%), 2B6 (16%), 2C8 (6%), 2D6 (5%), 2C19 (5%) and 2C9 (<1%).

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Biological Half-Life
Terminal half-life is 9.5 days.

Hepatotoxicity
In large clinical trials of ixazomib combined with lenalidomide and dexamethasone, elevations in serum aminotransferase levels were common, occurring in ~10% of patients. However, values greater than 5 times the upper limit of normal (ULN) were rare, occurring in
Likelihood score: E* (unproven but suspected cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of ixazomib during breastfeeding. Because its half-life is about 9.5 days, it is likely to accumulate in the infant. It is also given in combination with leflunomide and dexamethasone, which may increase the risk to the infant. The manufacturer recommends that breastfeeding be discontinued during ixazomib therapy and for 90 days after the last dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
99%

[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 that is the amide obtained by formal condensation of the carboxy group of N-(2,5-dichlorobenzoyl)glycine with the amino group of [(1R)-1-amino-3-methylbutyl]boronic acid. The active metabolite of ixazomib citrate, it is used in combination therapy for treatment of multiple myeloma. It has a role as an apoptosis inducer, an orphan drug, a proteasome inhibitor, a drug metabolite and an antineoplastic agent. It is a member of benzamides, a dichlorobenzene, a glycine derivative and a member of boronic acids.
Ixazomib a second generation proteasome inhibitor (PI) and the first oral PI approved by the FDA in November 2015 for multiple myeloma treatment in combination with 2 other therapies (lenalidomide and dexamethasone) for patients who have received at least 1 prior therapy. It was found to have similar efficacy to bortezomib (the first PI approved for multiple myeloma therapy) in the control of myeloma growth and prevention of bone loss. Ixazomib citrate is marketed by Takeda Pharmaceuticals under the brand name Ninlaro, which is a prodrug that becomes quickly converted to its active metabolite, ixazomib, after administration.
Ixazomib is a Proteasome Inhibitor. The mechanism of action of ixazomib is as a Proteasome Inhibitor.
Ixazomib is a small molecule proteasome inhibitor that is used in combination with other antineoplastic agents to treat refractory multiple myeloma. Ixazomib is associated with a low rate of serum enzyme elevations during treatment and to rare instances of clinically apparent, acute liver injury.
Ixazomib is an active metabolite of MLN9708, a second generation, boron containing peptide proteasome inhibitor (PI) with potential antineoplastic activity. Ixazomib binds to and inhibits the 20S catalytic core of the proteasome, thereby 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 Citrate (active moiety of).

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Drug Indication
Ixazomib is indicated in combination with lenalidomide and dexamethasone for the treatment of patients with multiple myeloma who have received at least one prior therapy.
FDA Label
Treatment of lymphoid malignancies (excluding multiple myeloma), Treatment of multiple myeloma

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Mechanism of Action
Ixazomib is an N-capped dipeptidyl leucine boronic acid which reversibly inhibits the CT-L proteolytic (β5) site of the 20S proteasome. At higher concentrations, ixazomib also seems to inhibit the proteolytic β1 and β2 subunits and to induce accumulation of ubiquitinated proteins.

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Pharmacodynamics
In vitro studies have shown ixazomib to induce apoptosis in multiple myeloma cells sensitive or resistant to other conventional therapies. In mouse xenograft models, ixazomib induced tumor growth inhibition.

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.)