MMP-1 (IC50 = 3 nM); MMP-2 (IC50 = 4 nM); MMP-9 (IC50 = 4 nM); MMP-7 (IC50 = 6 nM); MMP-7 (IC50 = 6 nM)

Batimastat (BB-94) is a strong inhibitor of matrix metalloproteinase that binds in an unusual way. Batimastat has an IC50 of 4 nM for gelatinases A and 10 nM for gelatinases B. Comparable to the results for MMP-1 (3 nM), MMP-8 (10 nM), and MMP-3 (20 nM)[2], the IC50 with the structurally identical collagenase Ht-d is 6 nM. This metalloproteinase inhibitor, Batimastat (BB-94, IC50=230 nM), is based on hydroxamic acid and successfully inhibits CD30 shedding from the cell line Karpas299[3].

In an orthotopic model of human breast cancer, intrathecal treatment of Batimastat (BB-94) significantly inhibits the growth of human ovarian carcinoma xenografts and murine melanoma metastasis while delaying the growth of primary tumors without causing cytotoxicity or altering mRNA levels[2]. Antineoplastic and antiangiogenic properties have been demonstrated by the synthetic matrix metalloproteinase inhibitor batimastat (BB) -94 in a number of tumor types. All animals are alive and well on day 200 after receiving treatment with Batimastat (60 mg/kg ip every other day, for a total of eight injections) and Cisplatin (4 mg/kg iv, every seven days, for a total of three injections), which totally stops the growth and spread of both xenografts[4]. When compared to controls (75%), animals treated with Batimastat (BB-94) had higher survival rates (95.2%) according to Kaplan-Meier analysis of survival (at 48 hours), and these changes are nearly statistically significant (p=0.064)[5]. Four hours after E2 administration—the period at which collagen density is seen to be at its lowest following hormone treatment—matrix density is assessed in animals that have been pretreated with saline or Batimastat (40 mg/kg)[6].

In vitro, batimastat IC50s are calculated using enzyme assays against various metalloproteinases.Matrix metalloproteinase enzymes have been implicated in degenerative processes like tumor cell invasion, metastasis, and arthritis. Specific metalloproteinase inhibitors have been used to block tumor cell proliferation. We have examined the interaction of batimastat (BB-94) with a metalloproteinase [atrolysin C (Ht-d), EC 3.4.24.42] active site at 2.0-angstroms resolution (R = 16.8%). The title structure exhibits an unexpected binding geometry, with the thiophene ring deeply inserted into the primary specificity site. This unprecedented binding geometry dramatizes the significance of the cavernous primary specificity site, pointing the way for the design of a new generation of potential antitumor drugs.

The IC50 was determined after incubating the cells for 22 hours at various concentrations of batimastat that had been dissolved in absolute ethanol.

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Cell growth assays
The cell lines were seeded in 24-well multidishes in growth medium and allowed to adhere for two days. When experiments were initiated (day 0), growth medium containing fulvestrant (0.1 μM), HER ligands (10 ng/ml), gefitinib, CI-1033, TAPI-2, Batimastat (BB94) or GM6001 were added at concentrations indicated in the figure. The control cells were added similar amount of vehicle as the treated cells. Growth medium was replaced on day three, and cell number was determined on day five, using a crystal violet colorimetric assay as previously described. Each experiment was performed in quadruplicate and repeated at least twice. Int J Oncol. 2014 Jul;45(1):393-400.

Mice: Female BALB/c mice six weeks of age are employed. One hour prior to and twenty-four hours following infection, mice receive intraperitoneal injections of Batimastat (BB-94, 50 mg/kg). 50 mg/mL of batimastat is suspended in DMSO and kept frozen at -20°C. It is diluted 20 times in phosphate buffered saline (PBS) before use, and 500 μL is injected into the animals. A 500 μL injection of 5% DMSO in PBS is given to control mice. 48 hours after the ic challenge, animals are sacrificed.

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Rats: Sprague-Dawley female rats receive an intraperitoneal (i.p.) injection of a single physiological dose of E2 (40 μg/kg in a 0.9% NaCl, 0.4% EtOH vehicle) at predetermined intervals before tissue is collected during necropsy. It has been demonstrated that the uterine wet weight, tissue architecture, and gene expression changes that are indicative of estrogen receptor activation are brought about by this in vivo dose level of estrogen. In each study, the animals are given a single 40 μg/kg bolus of E2 intraperitoneally four hours before tissue collection, and the control group is given only a vehicle. In vivo MMP inhibition has been demonstrated by batimastat when given intraperitoneally (i.p.) at a dose of 40 mg/kg in a 1× PBS, 0.1% Tween-20 vehicle, 4 hours before E2 or saline control.

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Dissolved in PBS containing 0.01% Tween 2O; 30 mg/kg; i.p. injection

Nude mice

[1]. Increased MMPs expression and decreased contraction in the rat myometrium during pregnancy and in response to prolonged stretch and sex hormones. Am J Physiol Endocrinol Metab. 2012 Jul 1;303(1):E55-70.

[2]. Batimastat, a potent matrix mealloproteinase inhibitor, exhibits an unexpected mode of binding. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2749-54.

[3]. Inhibition of metalloproteinases enhances the internalization of anti-CD30 antibody Ki-3 and the cytotoxic activity of Ki-3 immunotoxin. Int J Cancer. 2002 Mar 10;98(2):210-5.

[4]. Batimastat, a synthetic inhibitor of matrix metalloproteinases, potentiates the antitumor activity of cisplatin in ovarian carcinoma xenografts. Clin Cancer Res. 1998 Apr;4(4):985-92.

[5]. Inhibition of matrix metalloproteinases attenuates brain damage in experimental meningococcal meningitis. BMC Infect Dis. 2014 Dec 31;14:726.

[6]. Regulated expression of matrix metalloproteinases, inflammatory mediators, and endometrial matrix remodeling by 17beta-estradiol in the immature rat uterus. Reprod Biol Endocrinol. 2009 Nov 4;7:124.

Batimastat is a secondary carboxamide resulting from the formal condensation of the carboxy group of (2S,3R)-5-methyl-3-{[(2S)-1-(methylamino)-1-oxo-3-phenylpropan-2-yl]carbamoyl}-2-[(thiophen-2-ylsulfanyl)methyl]hexanoic acid with the amino group of hydroxylamine. It a broad-spectrum matrix metalloprotease inhibitor. It has a role as a matrix metalloproteinase inhibitor, an antineoplastic agent and an angiogenesis inhibitor. It is a L-phenylalanine derivative, a member of thiophenes, an organic sulfide, a triamide, a hydroxamic acid and a secondary carboxamide.

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Batimastat is a synthetic hydroxamate with potential antineoplastic activity. Batimastat binds covalently to the zinc ion in the active site of matrix metalloproteinases (MMPs), thereby inhibiting the action of MMPs, inducing extracellular matrix degradation, and inhibiting angiogenesis, tumor growth and invasion, and metastasis. An anticancer drug that belongs to the family of drugs called angiogenesis inhibitors. Batimastat is a matrix metalloproteinase inhibitor.

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Matrix metalloproteinases have been implicated in the growth and spread of metastatic tumors. This role was investigated in an orthotopic transplant model of human colon cancer in nude mice using the matrix metalloproteinase inhibitor BB-94 (batimastat). Fragments of human colon carcinoma (1-1.5 mm) were surgically implanted orthotopically on the colon in 40 athymic nu/nu mice. Administration of BB-94 or vehicle (phosphate buffered saline, pH 7.4, containing 0.01% Tween 80) commenced 7 days after tumor implantation (20 animals/group). Animals received 30 mg/kg BB-94 i.p. once daily for the first 60 days and then 3 times weekly. Treatment with BB-94 caused a reduction in the median weight of the primary tumor from 293 mg in the control group to 144 mg in the BB-94 treated group (P < 0.001). BB-94 treatment also reduced the incidence of local and regional invasion, from 12 of 18 mice in the control group (67%) to 7 of 20 mice in the treated group (35%). Six mice in the control group were also found to have metastases in the liver, lung, peritoneum, abdominal wall, or local lymph nodes. Only two mice in the BB-94 group had evidence of metastatic disease, in both cases confined to the abdominal wall. The reduction in tumor progression observed in the BB-94-treated group translated into an improvement in the survival of this group, from a median survival time of 110 days in the control group to a median survival time of 140 days in the treated group (P < 0.01). Treatment with BB-94 was not associated with any obvious toxic effect, and these results suggest that such agents may be effective as adjunctive cancer therapies.

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The synthetic matrix metalloproteinase inhibitor batimastat was tested for its ability to inhibit growth and metastatic spread of the B16-BL6 murine melanoma in syngeneic C57BL/6N mice. Intraperitoneal administration of batimastat resulted in a significant inhibition in the number of lung colonies produced by B16-BL6 cells injected i.v. The effect of batimastat on spontaneous metastases was examined in mice inoculated in the hind footpad with B16-BL6 melanoma. The primary tumor was removed surgically after 26-28 days. Batimastat was administered twice a day from day 14 to day 28 (pre-surgery) or from day 26 to day 44 (post-surgery). With both protocols, the median number of lung metastases was not significantly affected, but there was a significant reduction in the weight of the metastases. Finally, the effect of batimastat was examined on s.c. growth of B16-BL6 melanoma. Batimastat administered daily, starting at day of tumor transplantation, resulted in a significant growth delay, whereas treatment starting at advanced stage tumor only reduced tumor growth marginally. Our results indicate that a matrix metalloproteinase inhibitor can not only prevent the colonization of secondary organs by B16-BL6 cells but also limit the growth of solid tumors.

C23H30N3O4S2-.NA+

499.6218

499.158

130464-84-5

Batimastat;130370-60-4

59955182

Typically exists as solid at room temperature

4.378

3

6

12

33

621

3

[O-]NC([C@@H](CSC1=CC=CS1)[C@@H](CC(C)C)C(N[C@@H](CC2=CC=CC=C2)C(NC)=O)=O)=O.[Na+]

VWABIWQKZWQAKG-UHFFFAOYSA-N

InChI=1S/C23H30N3O4S2.Na/c1-15(2)12-17(18(22(28)26-30)14-32-20-10-7-11-31-20)21(27)25-19(23(29)24-3)13-16-8-5-4-6-9-16;/h4-11,15,17-19H,12-14H2,1-3H3,(H3-,24,25,26,27,28,29,30);/q-1;+1

sodium;N-[1-(methylamino)-1-oxo-3-phenylpropan-2-yl]-2-(2-methylpropyl)-N'-oxido-3-(thiophen-2-ylsulfanylmethyl)butanediamide

Batimastat (sodium salt); BB-94 sodium salt; Batimastat Sodium; 130464-84-5; sodium;(2R,3S)-N-[(2S)-1-(methylamino)-1-oxo-3-phenylpropan-2-yl]-2-(2-methylpropyl)-N'-oxido-3-(thiophen-2-ylsulfanylmethyl)butanediamide; BatimastatSodium; (2S,3R)-N-Hydroxy-N'-[(2S)-1-methylamino-1-oxo-3-phenylpropan-2-yl]-3-(2-methylpropyl)-2-(thiophen-2-ylsulfanylmethyl)butanediamide sodium salt; PD118565

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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