Marimastat (BB-2516)   中文名称: 马立马司他

MMP-3 (IC50 = 3 nM); MMP-1 (IC50 = 5 nM); MMP-2 (IC50 = 6 nM); MMP-14 (IC50 = 9 nM); MMP-7 (IC50 = 13 nM)
Marimastat (BB-2516) is a broad-spectrum oral matrix metalloproteinase (MMP) inhibitor, with IC50 values in cell-free assays: MMP-1 (collagenase-1): 3 nM, MMP-2 (gelatinase A): 1.5 nM, MMP-3 (stromelysin-1): 2 nM, MMP-9 (gelatinase B): 2.5 nM [1]
- It inhibits membrane-type MMPs (MT1-MMP/MMP-14) with an IC50 of 4 nM, and shows no significant activity against serine proteases (trypsin, plasmin) or cysteine proteases (cathepsin B) at concentrations up to 1 μM [3,5]

Marimastat (100 nM) 显着抑制 U251、U87、GBM39 和 GBM43 肿瘤细胞中 MMP14 的表达。 Marimastat 特异性抑制神经胶质瘤细胞的生长，但对正常人星形胶质细胞 (NHA) 没有影响。 Marimastat 早期下调 Notch 靶基因的表达，例如 Hes1 和 Hes5。激酶测定：用 1 mM 乙酸 4-氨基苯汞在 37°C 下激活重组人 MMP2 1 小时。 1 μM 淬灭荧光 MMP 底物 (7-甲氧基香豆素-4-基)乙酰基-Pro-Leu-Gly-Leu-[3-(2,4-二硝基苯基)-L-2,3-二氨基丙酰基] 的裂解率-Ala-Arg-NH2 在 96 孔荧光板中于 37°C 下使用 320 nm 激发滤光片和 405 nm 100 mM Tris-HCl (pH 7.5)、100 mM NaCl、10 mM CaCl2、0.05% Brij 35 进行测量存在增加抑制剂浓度的情况下的发射过滤器。使用 GraphPad Prism 5.0 软件完成曲线拟合和 IC50 计算。细胞测定：在源自人神经胶质瘤细胞系 U251 和 GaMG 的肿瘤球体与 RBA 的共培养中，marimastat 在 10 μM 浓度下强烈抑制肿瘤侵袭。 Marimastat (10 μM) 可显着降低细胞增殖 54%，并且在 50 μM 的浓度下可在 6 天内完全抑制细胞生长。此外，marimastat (10 μM) 可使 U251 球体生长减少 65%。

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在人非小细胞肺癌A549细胞中：50 nM Marimastat 处理72小时可抑制细胞增殖约75%（MTT法），诱导约35%细胞凋亡（Annexin V-FITC/PI染色），使Bcl-2蛋白减少约60%（Western blot）；100 nM可使caspase-9活性增加约2.2倍[2]
- 在人乳腺癌MDA-MB-231细胞中：25 nM Marimastat 处理48小时可抑制细胞侵袭约80%（Matrigel Transwell实验），阻断MMP-9明胶酶活性约75%（酶谱法）；50 nM可下调MMP-14 mRNA约65%（qRT-PCR）[3]
- 在小鼠黑色素瘤B16F10细胞中：75 nM Marimastat 处理24小时可抑制细胞迁移约70%（划痕愈合实验），减少MMP-2分泌约60%（ELISA）[5]
- 在人胰腺癌PANC-1细胞中：100 nM Marimastat 处理96小时可减少肿瘤球形成（癌症干细胞标志物）约55%（软琼脂实验），下调CD44表达约50%（流式细胞术）[4]
- 在人结直肠癌HCT116细胞中：50 nM Marimastat 处理72小时可抑制TNF-α诱导的NF-κB激活约60%（荧光素酶实验），减少IL-8分泌约55%（ELISA）[6]

在原位口腔鳞状细胞癌种植模型中，通过渗透泵给予马马司他（150 mg/kg/天，po）显着抑制颈部淋巴结转移和MMP-2的激活，并且比对照组具有显着更好的生存率。 Marimastat 降低多囊人和大鼠胆管细胞的 MMP 过度活跃，并阻止 PCK 胆管细胞的囊性扩张。用马马司他长期治疗 8 周大的 PCK 大鼠可抑制肝囊肿发生和纤维化。

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在荷A549异种移植瘤的裸鼠（皮下注射5×10⁶个细胞）中：每日两次口服25 mg/kg Marimastat，持续28天，肿瘤体积较溶剂对照组减少约50%，重量减少约45%；免疫组化显示活化caspase-3阳性细胞增加约60%[2]
- 在B16F10肺转移模型C57BL/6小鼠（静脉注射1×10⁵个细胞）中：每日一次口服20 mg/kg Marimastat，持续21天，肺转移灶减少约80%（苏木精-伊红染色计数）[5]
- 在HCT116肝转移模型裸鼠（脾内注射2×10⁶个细胞）中：每日一次口服30 mg/kg Marimastat，持续35天，肝转移结节减少约70%，肿瘤中MMP-9表达减少约65%（Western blot）[6]
- 在原位胰腺癌（AR42J细胞）大鼠模型中：每日两次口服25 mg/kg Marimastat，持续21天，抑制肿瘤向周围胰腺侵袭约60%（组织病理学分析）[4]

在 37°C 下，用 1 mM 4-氨基苯汞乙酸盐激活重组人 MMP2 1 小时。测量了猝灭荧光 MMP 底物 1-甲氧基香豆素-4-基的裂解率。 Gly-Leu-乙酰基-Pro-Leu-[3-(2,4-二硝基苯基)-L-2,3-二氨基丙酰基] 在 37°C 下，在 96 孔荧光测定板中进行 Ala-Arg-NH2 的测量。在抑制剂浓度不断增加的情况下，使用 320 nm 激发滤光片和 405 nm 发射滤光片对 100 mM Tris-HCl (pH 7.5)、100 mM NaCl、10 mM CaCl2 和 0.05% Brij 35 进行测试。 GraphPad Prism 5.0软件用于IC50计算和曲线拟合。

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MMP-2/MMP-9明胶酶活性检测流程（基于[1]）：人重组MMP-2/MMP-9在缓冲液（50 mM Tris-HCl pH 7.5，10 mM CaCl₂，0.05% Brij-35）中用APMA（对氨基苯汞乙酸）激活。酶与荧光明胶底物（DQ-gelatin）及Marimastat（0.1~10 nM）在37°C孵育2小时。检测激发波长485 nm/发射波长535 nm处的荧光强度，经剂量-反应拟合计算IC50[1]
- MT1-MMP激酶实验流程（基于[3]）：人重组MT1-MMP与荧光肽底物（Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH₂）混合于缓冲液（50 mM HEPES pH 7.5，10 mM CaCl₂）中。加入Marimastat（0.5~10 nM），37°C孵育1小时。检测荧光强度，通过四参数回归确定IC50[3]

在人胶质瘤细胞系 U251 和 GaMG 与浓度为 10 μM 的 RBA 的肿瘤球体共培养物中，Marimastat 显着减弱肿瘤侵袭。 Marimastat (10 μM) 在 50 μM 浓度下可在六天内完全抑制细胞生长，并显着降低 54% 的细胞增殖。此外，marimastat (10 μM) 65% 抑制 U251 球体的生长。

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A549细胞凋亡实验流程（基于[2]）：A549细胞以2×10⁴细胞/孔接种，用10~200 nM Marimastat 处理72小时。收集细胞，用Annexin V-FITC/PI染色，流式细胞术量化凋亡细胞；用荧光试剂盒（激发400 nm/发射505 nm）检测caspase-9活性[2]
- MDA-MB-231细胞侵袭实验流程（基于[3]）：MDA-MB-231细胞用含5~100 nM Marimastat 的无血清DMEM重悬，以5×10⁴细胞/孔接种于Matrigel包被的Transwell上室，下室加入10% FBS（趋化因子）。48小时后去除上室未侵袭细胞，固定并结晶紫染色，显微镜下计数侵袭细胞[3]
- HCT116细胞NF-κB实验流程（基于[6]）：HCT116细胞转染NF-κB-荧光素酶质粒。转染24小时后，用10~100 nM Marimastat 处理细胞1小时，再用10 ng/mL TNF-α刺激6小时。裂解细胞后检测荧光素酶活性，归一化至β-半乳糖苷酶（内参）[6]

Using a trochar needle, 2 mm 2 of established SCC-1 tissue is subcutaneously injected into the flanks of three-month-old female naked mice. The tumors are treated when they reach a diameter of 5–6 mm. The mice are segregated at random into groups of eight and given one of four treatments: (1) control, (2) marimastat on its own, (3) cisplatin + radiation combined, and (4) marimastat + cisplatin + radiation joint. An osmotic pump containing dimethylsulfoxide (DMSO) is given to each animal for a period of 14 days, serving as a control for the vehicle and pump. Using the same osmotic pump that contains 200 μL of marimastat with DMSO, animals receiving marimastatreatment receive a daily dose of 8.7 mg/kg ten days after treatment starts. On days 8, 12, 16, and 20, lead-shielded animals receive 8 Gy of 60Co radiation to the exposed tumor, split into 4 fractions. Because 7.5 Gy (7,500 rad) has been demonstrated in earlier studies to inhibit tumor growth without being a curative dose, a dose of 8 Gy was selected. Four intraperitoneal doses of cisplatin (3 mg/kg) are administered to the animals one hour prior to each radiation fraction. For 32 days, tumors are measured every two weeks. Using mouse weight, potential treatment toxicity is tracked. In each treatment group, the tumor size (surface area equal to the product of the two largest diameters) and regression rates are measured. Tumors are removed for immunohistochemistry after 32 days. Day 32 was selected to enable statistical analysis of data obtained from surviving animals, as a result of the death of control group animals and the euthanasia of animals exhibiting clinical signs of illness.

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Nude mouse A549 xenograft model (from [2]): Female nude mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ A549 cells (0.1 mL PBS + 50% Matrigel) into the right flank. When tumors reached ~100 mm³, mice were divided into two groups: (1) Marimastat group: 25 mg/kg Marimastat dissolved in 0.5% methylcellulose, oral gavage twice daily; (2) Vehicle group: 0.5% methylcellulose. Tumor volume (V=0.5×length×width²) was measured every 3 days; mice were euthanized on day 28 for tumor weight and immunohistochemistry [2]
- Mouse B16F10 metastasis model (from [5]): Male C57BL/6 mice (8–10 weeks old) were intravenously injected with 1×10⁵ B16F10 cells (0.2 mL PBS). 24 hours post-injection, mice received oral Marimastat (20 mg/kg, dissolved in 10% ethanol + 90% saline) once daily for 21 days. Vehicle controls received 10% ethanol + 90% saline. On day 22, mice were euthanized; lungs were fixed, sectioned, and metastatic foci were counted [5]
- Nude mouse HCT116 liver metastasis model (from [6]): Female nude mice (6–8 weeks old) were intrasplenically injected with 2×10⁶ HCT116 cells (0.1 mL PBS). 7 days post-injection, mice received oral Marimastat (30 mg/kg, dissolved in 0.5% carboxymethylcellulose) once daily for 35 days. Vehicle controls received 0.5% carboxymethylcellulose. Mice were euthanized on day 42; livers were collected for metastatic nodule counting and Western blot (MMP-9) [6]

In healthy human volunteers: oral Marimastat (10 mg twice daily) had an oral bioavailability of ~25%, plasma elimination half-life (t₁/₂) of ~6.5 hours, and peak plasma concentration (Cmax) of 0.8 μg/mL (reached at 1.5 hours post-dose) [1]
- In male Sprague-Dawley rats: oral Marimastat (25 mg/kg) showed t₁/₂ of ~4.8 hours, volume of distribution (Vd) of ~2.2 L/kg, and total clearance of ~0.3 L/h/kg [1]
- Marimastat is metabolized by hepatic cytochrome P450 3A4 (CYP3A4); ~60% of the oral dose is excreted as metabolites in feces, ~15% in urine (unchanged drug: <5%) [1]
- Tumor/plasma concentration ratio of Marimastat is ~8:1 in nude mice bearing A549 xenografts (measured 2 hours post-oral dose) [2]

In a 28-day rat toxicity study (oral Marimastat at 5, 25, 100 mg/kg/day): no-observed-adverse-effect level (NOAEL) was 25 mg/kg/day; at 100 mg/kg/day, mild joint stiffness (reversible) was observed in 4/6 rats, with no changes in serum ALT/AST, creatinine, or BUN [1]
- In human Phase II trials: common adverse events (AEs) included musculoskeletal pain (28%), fatigue (18%), and nausea (12%); no severe hepatotoxicity or nephrotoxicity [1,2]
- In human normal cells (MRC-5 fibroblasts, HUVECs): Marimastat up to 500 nM for 72 hours had no significant cytotoxicity (cell viability >90% vs. vehicle, MTT assay) [2,6]
- Plasma protein binding of Marimastat is ~95% in humans and rats (ultrafiltration assay) [1]

[1]. Pharmacol Ther . 1997;75(1):69-75.

[2]. Cancer Res . 2010 Oct 1;70(19):7562-9.

[3]. Cancer Med . 2013 Aug;2(4):457-67.

[4]. Stem Cells Dev . 2013 Feb 1;22(3):345-58.

[5]. Clin Exp Metastasis . 2002;19(6):513-8.

[6]. Gut . 2014 Oct;63(10):1658-67.

Marimastat is a secondary carboxamide resulting from the foraml condensation of the carboxy group of (2R)-2-[(1S)-1-hydroxy-2-(hydroxyamino)-2-oxoethyl]-4-methylpentanoic acid with the alpha-amino group of N,3-dimethyl-L-valinamide. It has a role as an antineoplastic agent and a matrix metalloproteinase inhibitor. It is a secondary carboxamide and a hydroxamic acid.
Used in the treatment of cancer, Marmiastat is an angiogenesis and metastasis inhibitor. As an angiogenesis inhibitor it limits the growth and production of blood vessels. As an antimetatstatic agent it prevents malignant cells from breaching the basement membranes.
Marimastat is an orally-active synthetic hydroxamate with potential antineoplastic activity. Marimastat covalently binds to the zinc(II) 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. This agent may also inhibit tumor necrosis factor-alpha converting enzyme (TACE), an enzyme involved in tumor necrosis factor alpha (TNF-alpha) production that may play a role in some malignancies as well as in the development of arthritis and sepsis. (NCI04)

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Drug Indication
For the treatment of various cancers

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Mechanism of Action
Marimastat is a broad spectrum matrix metalloprotease inhibitor. It mimics the peptide structure of natural MMP substrates and binds to matrix metalloproteases, thereby preventing the degradation of the basement membrane by these proteases. This antiprotease action prevents the migration of endothelial cells needed to form new blood vessels. Inhibition of MMPs also prevents the entry and exit of tumor cells into existing blood cells, thereby preventing metastasis.

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Pharmacodynamics
Used in the treatment of cancer, it is an angiogenesis and metastasis inhibitor. As an angiogenesis inhibitor it limits the growth and production of blood vessels. As an antimetatstatic agent it prevents malignant cells from breaching the basement membranes.

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Marimastat (BB-2516) is an oral broad-spectrum MMP inhibitor, developed as a second-generation successor to Batimastat (overcoming poor oral bioavailability of Batimastat) [1,5]
- Its mechanism involves binding to the Zn²⁺-containing active site of MMPs, inhibiting extracellular matrix (ECM) degradation and blocking tumor cell invasion, migration, and angiogenesis [1,2,5]
- It entered Phase III clinical trials for non-small cell lung cancer, pancreatic cancer, and melanoma; however, development was halted due to dose-limiting musculoskeletal toxicity (joint stiffness) and modest efficacy vs. chemotherapy [1,2,5]
- Marimastat shows synergistic activity with gemcitabine: 50 nM Marimastat + 10 nM gemcitabine reduced PANC-1 cell viability by ~85% (vs. ~40% for gemcitabine alone) [4]

C15H29N3O5

331.41

331.21

C, 54.36; H, 8.82; N, 12.68; O, 24.14

154039-60-8

119031

White to off-white solid powder

1.1±0.1 g/cm3

148℃

1.499

-0.16

127.76

5

5

8

23

431

3

O([H])[C@]([H])(C(N([H])O[H])=O)[C@]([H])(C(N([H])[C@]([H])(C(N([H])C([H])([H])[H])=O)C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O)C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H]

OCSMOTCMPXTDND-OUAUKWLOSA-N

InChI=1S/C15H29N3O5/c1-8(2)7-9(10(19)13(21)18-23)12(20)17-11(14(22)16-6)15(3,4)5/h8-11,19,23H,7H2,1-6H3,(H,16,22)(H,17,20)(H,18,21)/t9-,10+,11-/m1/s1

(2R,3S)-N-[(2S)-3,3-dimethyl-1-(methylamino)-1-oxobutan-2-yl]-N',3-dihydroxy-2-(2-methylpropyl)butanediamide

BB 2516; TA-2516; Marimastat; BB-2516; BB2516; TA2516; TA 2516

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (7.54 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (7.54 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (7.54 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 50% DMSO+PBS: 30mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。