CT-L (IC50 = 3.5 nM); CT-T-laspase-like (IC50 = 28 nM); trypsin-like (IC50 = 430 nM)
20S proteasome (chymotrypsin-like (CT-L, β5), caspase-like (C-L, β1), trypsin-like (T-L, β2) activities) [1]

Marizomib (Salinosporamide A) (0.1-10000 nM; 72 hours) effectively and dose-dependently decreases D-54 and U-251 cell survival. For D-54 and U-251, the IC50s are ≤20 nM and ～52 nM, respectively[1].
Marizomib (24 hours; 60 nM) causes apoptosis and caspase-3 activation in glioma cells[1].

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Marizomib at 60 nM reduced chymotrypsin-like proteasome activity in U-251 and D-54 glioma cells to ~15% of untreated control after 4 hours of treatment. [1]
Marizomib inhibited cell viability in a dose-dependent manner: IC50 ~52 nM for U-251 cells and ~20 nM for D-54 cells after 72 hours exposure. [1]
Marizomib (60 nM, 24 hours) robustly inhibited cell invasion in Matrigel invasion chambers (P<0.001) and reduced wound closure in U-251 cells. [1]
Marizomib induced apoptosis: 24-hour treatment increased Annexin V-positive D-54 cells; DAPI staining showed nuclear fragmentation and apoptotic bodies. [1]
Marizomib increased caspase-3 activity in a dose-dependent manner (80% increase at 60 nM, P<0.001) and induced cleaved caspase-3 and PARP by western blot in D-54 and U-251 cells. [1]
Marizomib (60 nM, 12 hours) increased ROS generation from basal 28.07% to 50.75% ROS-positive cells, and fluorescence spectroscopy showed ROS increase from ~28,000 to ~40,000 RFU. Antioxidant N-acetyl cysteine (NAC, 10 mM) blocked ROS generation, improved cell survival (P<0.0001), abolished caspase-3 activation, and repressed cleaved caspase-3 and PARP induction. [1]
Marizomib rapidly inhibited CT-L activity in all cell types tested (neural stem cells, low-grade GSCs, high-grade GSCs, meningioma-derived cultures). High-grade GSCs showed ≥40% survival reduction at 20 nM, with IC50 ranges 9.32-51.06 nM, while NSCs, meningiomas, and low-grade GSCs were insensitive. [1]

Marizomib (Salinosporamide A) (0.15 mg/kg; i.v; twice a week for three weeks) is associated with no toxicity and significantly reduces the growth of tumors[3].
Marizomib distributed into the CNS at approximately 30% of steady-state blood levels in Sprague-Dawley rats as shown by quantitative whole-body autoradiography after a single i.v. dose of [3H]-marizomib (0.1 mg/kg). [1]
In cynomolgus monkeys, marizomib administered orally (0.55-0.64 mg/m², once weekly or twice weekly) significantly inhibited CT-L (26-28% inhibition, P<0.05) and C-L (similar extent) activities in prefrontal cortex brain tissue 24 hours after final dose; T-L activity was weakly inhibited (11-16%). [1]
In an orthotopic human glioma xenograft model (D-54 MG cells implanted intracranially in athymic BALB/c nu/nu mice), marizomib at MTD (200 μg/kg, i.v., biweekly for 2.5 weeks) significantly prolonged survival (average 31 days) compared to vehicle control (18 days, P<0.05 log-rank test); 50% of MTD-treated mice survived 29-65 days, and 25% were alive beyond 50 days. The lower dose (150 μg/kg, MTD-1) showed no survival benefit. Minimal effect on body weight was observed. [1]

20S proteasome activity assay: CT-L activity in cells was measured using a fluorometric kit. Cells were treated with 60 nM marizomib for indicated times, then collected, and CT-L activity was measured as relative fluorescence units. For in vivo proteasome profiling, brain tissues from male cynomolgus monkeys were processed and evaluated using proteasome-Glo assay kits to measure CT-L, C-L, and T-L activities. Non-proteasome activity was subtracted using duplicate wells with a supramaximal concentration of proteasome inhibitor. [1]

Proteasome activity is assessed in neural stem cells and glioblastoma-derived glioma stem cells at baseline and two hours following marizomib (60 nM) treatment. Using Matrigel invasion chambers, the invading potential of D-54 MG and U-251 MG cells treated or not treated with 60 nM marizomib for a 24-hour period is examined.

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CB-17 SCID-male mice (4-6 weeks old)[3]
0.15 mg/kg
i.v; twice a week for three weeks

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In vivo nervous tissue distribution study: Male Sprague-Dawley rats (311-337 g) received a single i.v. administration of [3H]-marizomib at 0.1 mg/kg (equivalent to 0.6 mg/m²). Animals (1 per time point) were euthanized at 2, 10, 30 minutes and 1, 2, 4, 8, 24, 48, 72, and 120 hours post-dose. Carcasses were frozen, and tissue concentrations of radioactivity were determined by whole-body autoradiography. [1]
Maximum-tolerated dose determination in athymic BALB/c nu/nu mice: 6-8 week old mice (n=3 per group) were treated with control vehicle (2% DMSO in 5% Solutol) or different doses of marizomib (150, 200, 250, and 300 μg/kg) administered biweekly for 2.5 weeks (days 1,4,8,11,15) into the tail vein. The MTD was determined to be 200 μg/kg. [1]
Orthotopic xenograft implantation and treatment: D-54 cells (1×10⁵) were implanted into the right frontal lobes of athymic BALB/c nu/nu mice. Four days after implantation, mice were randomized to 3 groups (n=6-8) and treated with vehicle control, marizomib at MTD (200 μg/kg), or MTD-1 (150 μg/kg). Drug or vehicle was administered biweekly (days 1,4,8,11,15, etc.) into the tail vein. Survival was calculated from the start of treatment until moribund signs (hemiparesis, obtundation, hunchback, or >20% weight loss). [1]

Marizomib has a very short plasma half-life (2-30 minutes) and a large volume of distribution in rats and nonhuman primates. It binds irreversibly to the proteasome and is subsequently hydrolyzed, making measurement in tissue homogenates challenging. [1]
In Sprague-Dawley rats after a single i.v. dose of [3H]-marizomib (0.1 mg/kg), maximum blood levels were observed at 2 minutes post-dose and declined rapidly to a baseline plateau 10 minutes after administration. Distribution of radioactivity to the CNS was approximately 30% of steady-state blood levels. (Brain regions: cerebrum 31.33±12.77% at 2-30 min, 53.00±5.70% at 1-8 h, 51% at 24 h; cerebellum 35.00±12.86%, 56.25±3.04%, 58%; medulla 24.67±9.03%, 38.75±3.28%, 40%; all brain 30.33±6.04%, 49.33±3.17%, 49.67±5.24%). [1]

In athymic BALB/c nu/nu mice, the maximum tolerated dose of marizomib (biweekly i.v. for 2.5 weeks) was determined to be 200 μg/kg. At 250 and 300 μg/kg, higher lethality was observed. No statistical difference in animal body weight was observed between control, MTD (200 μg/kg), and MTD-1 (150 μg/kg) groups. [1]
Marizomib showed relatively little effect on normal neural stem cells compared to high-grade glioma stem-like cells, suggesting a potential for good neurologic safety profile and limited neurotoxicity. [1]

[1]. Marizomib activity as a single agent in malignant gliomas: ability to cross the blood-brainbarrier. Neuro Oncol. 2016 Jun;18(6):840-8.

[2]. Molecular mechanisms of acquired proteasome inhibitor resistance. J Med Chem. 2012 Dec 13;55(23):10317-27.

[3]. Pharmacodynamic and efficacy studies of the novel proteasome inhibitor NPI-0052 (marizomib) in a human plasmacytoma xenograft murine model. Br J Haematol. 2010 May;149(4):550-9.

Salinosporamide A is a salinomycin-like compound whose core (1R)-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione skeleton has its 1, 4, and 5 positions substituted with (1S)-cyclohexyl-2-en-1-yl(hydroxy)methyl, 2-chloroethyl, and methyl groups, respectively (1R,4R,5S diastereomers). It is a potent proteasome inhibitor and has attracted attention due to its potential applications in the treatment of various cancers. It is both an antitumor drug and a proteasome inhibitor. It is a halophilic spore amide, belonging to the organochlorine compounds, organoheterocyclic compounds, β-lactones, and γ-lactams.
Marizomib has been used in clinical trials for the treatment of various diseases, including cancer, melanoma, lymphoma, glioblastoma, and malignant glioma.
Marizomib has been reported to exist in the genus Salinispora and Salinispora tropica, and relevant data are available. Marizome is a naturally occurring halospora amide isolated from the marine actinomycete Salinospora tropica, possessing potential antitumor activity. Marizome irreversibly binds to and inhibits the activity of the threonine residue at the active site of the 20S catalytic core subunit of the proteasome by covalently modifying it. Inhibition of ubiquitin-proteasome-mediated proteolysis leads to the accumulation of polyubiquitinated proteins, which may result in cellular process disturbances, cell cycle arrest, induction of apoptosis, and inhibition of tumor growth and angiogenesis. This drug may be more effective and selective than the proteasome inhibitor bortezomib.

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Drug Indications
Treatment of malignant gliomas

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Marizomib (NPI-0052, salinosporamide A) is a second-generation irreversible proteasome inhibitor with a bicyclic β-lactone γ-lactam structure. It inhibits all three proteolytic activities (chymotrypsin-like, caspase-like, trypsin-like) of the 20S proteasome, unlike bortezomib and carfilzomib which have more limited profiles. It activates caspases 3, 8, and 9, builds up reactive oxygen species, and induces apoptosis. [1]
This study demonstrated that marizomib can cross the blood-brain barrier (BBB) in rodents and nonhuman primates, a property not shared by bortezomib and carfilzomib at efficacious doses. It shows selective growth inhibition for high-grade glioma stem-like cells over normal neural stem cells. [1]
Marizomib is being evaluated in phase 1/2 clinical trials for multiple myeloma (NCT02103335) and a phase 1 study for recurrent glioblastoma (NCT02330562). The authors suggest potential for combination therapy with temozolomide, bevacizumab, or other agents. [1]

C15H20CLNO4

313.7766

313.108

C, 57.42; H, 6.42; Cl, 11.30; N, 4.46; O, 20.40

437742-34-2

437742-34-2

11347535

Off-white to pink solid powder

1.408

2

4

4

21

508

5

O=C([C@H](CCCl)[C@]1(C)O2)N[C@@]1([C@H]([C@@H]3C=CCCC3)O)C2=O

NGWSFRIPKNWYAO-SHTIJGAHSA-N

InChI=1S/C15H20ClNO4/c1-14-10(7-8-16)12(19)17-15(14,13(20)21-14)11(18)9-5-3-2-4-6-9/h3,5,9-11,18H,2,4,6-8H2,1H3,(H,17,19)/t9-,10+,11+,14+,15+/m1/s1

(1R,4R,5S)-4-(2-chloroethyl)-1-[(S)-[(1S)-cyclohex-2-en-1-yl]-hydroxymethyl]-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione

ML 858; NPI0052; Marizomib; ML-858; NPI-0052; (-)-Salinosporamide A; ML858; NPI 0052; Salinosporamide A

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 mg/mL (~318.7 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.63 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 (6.63 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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 (Please use freshly prepared in vivo formulations for optimal results.)