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 potent, broad-spectrum matrix metalloprotease (MMP) inhibitor with IC50 values of 3 nM, 4 nM, 4 nM, 6 nM, and 20 nM for MMP-1, MMP-2, MMP-9, MMP-7, and MMP-3, respectively.[1]
Batimastat has a thiophene ring that is deeply inserted into the primary specificity site, displaying an unexpected binding geometry.[2]

Batimastat has the ability to stop the growth and metastasis of B16-BL6 murine melanoma.[1]
Timastat treatment inhibits the growth of the primary tumor by 50%, the spread of the tumor locally and regionally by 35%, and the spread of the tumor distantly by 10% in an orthotopic colon tumor model in mice.[3]
Batimastat inhibits the in vivo growth of experimental hemangiomas, most likely through preventing the transformed cells from recruiting endothelial cells or by disrupting the arrangement of cells in vascular structures.[4]

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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.[1]

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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.[3]

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Daily treatment with batimastat (30, 3, and 0.3 mg/kg at the site of eEnd.1 cell injection) inhibited tumor growth, with increased doubling time. The carboxamide derivative of batimastat, BB-374, a poor inhibitor of matrix metalloproteinase activity, was less active in reducing hemangioma growth. Histologic analysis of treated tumors indicated a reduction in the size of blood-filled spaces and in hemorrhage. Batimastat also inhibited the angiogenic response induced by cultured eEnd.1 endothelioma cell supernatant embedded in a pellet of Matrigel. Batimastat significantly inhibited endothelial cell invasion in vitro through a layer of Matrigel, but it showed no direct cytotoxic activity. Conclusions: Batimastat reduces in vivo growth of experimental hemangiomas, most probably by blocking endothelial cell recruitment by the transformed cells or by interfering with cell organization in vascular structures. Implications: These results confirm the importance of matrix metalloproteinase in endothelial cell recruitment that occurs in angiogenesis and in the formation of vascular tumors and suggest a therapeutic potential for synthetic matrix metalloproteinase inhibitors.[4]

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.[2]

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 [5]
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.

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.
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.

[1]. Int J Cancer. 1994 Aug 1;58(3):460-4.

[2]. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2749-54.

[3]. Cancer Res. 1994 Sep 1;54(17):4726-8.

[4]. J Natl Cancer Inst. 1995 Feb 15;87(4):293-8.

[5]. Int J Oncol. 2014 Jul;45(1):393-400.

Pharmacodynamics
An anticancer drug that belongs to the family of drugs called angiogenesis inhibitors. Batimastat is a matrix metalloproteinase inhibitor.

C23H31N3O4S2

477.64

477.175

C, 57.84; H, 6.54; N, 8.80; O, 13.40; S, 13.43

130370-60-4

Batimastat sodium salt;130464-84-5

5362422

White solid powder

1.3±0.1 g/cm3

236-238°

1.605

3.53

4

6

12

32

614

3

O=C(N[C@@H](CC1=CC=CC=C1)C(NC)=O)[C@H](CC(C)C)[C@H](CSC2=CC=CS2)C(NO)=O

XFILPEOLDIKJHX-QYZOEREBSA-N

InChI=1S/C23H31N3O4S2/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/h4-11,15,17-19,30H,12-14H2,1-3H3,(H,24,29)(H,25,27)(H,26,28)/t17-,18+,19+/m1/s1

(2S,3R)-N-hydroxy-N'-[(2S)-1-(methylamino)-1-oxo-3-phenylpropan-2-yl]-3-(2-methylpropyl)-2-(thiophen-2-ylsulfanylmethyl)butanediamide

Batimastat; BB94; BB-94; Batimastat (BB-94); BB94; BB 94; Batimastat (MMP Inhibitor); Butanediamide, N4-hydroxy-N1-[(1S)-2-(methylamino)-2-oxo-1-(phenylmethyl)ethyl]-2-(2-methylpropyl)-3-[(2-thienylthio)methyl]-, (2R,3S)-; BB 94

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: ≥ 5 mg/mL (10.47 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 50.0 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 2: 2.5 mg/mL (5.23 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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 3: ≥ 2.5 mg/mL (5.23 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% propylene glycol, 5% Tween 80, 65% D5W: 30 mg/mL

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