Fibroblast collagenase (Ki = 0.4 nM); MMP-1 (IC50 = 1.5 nM); MMP-2 (IC50 = 1.1 nM); MMP-3 (IC50 = 1.9 nM); MMP-9 (IC50 = 0.5 nM ); Thermolysin (Ki = 20 nM); Eastase (Ki = 20 nM)
Ilomastat (GM6001, Galardin) is a broad-spectrum inhibitor of matrix metalloproteinases (MMPs), with IC50 values in cell-free assays: MMP-1 (collagenase-1): 1.5 nM, MMP-2 (gelatinase A): 0.6 nM, MMP-3 (stromelysin-1): 0.8 nM, MMP-9 (gelatinase B): 0.7 nM [1]
- It inhibits membrane-type MMPs (MT1-MMP/MMP-14) with an IC50 of 2.0 nM, and shows no significant activity against serine proteases (trypsin, plasmin) at concentrations up to 1 μM [6]

Ilomastat (GM6001) inhibits the fibroblast collagenase, thermolysin, and elastase found in human skin, with corresponding Kis values of 0.4 nM, 20 nM, and 20 nM[1]. T-cell-produced gelatinase A and B are inhibited by ilomastat (0.1–10 nM). T-cell homing is inhibited by ilomastat[4].

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In human recombinant MMP-2/MMP-9 enzyme reactions, 1 nM Ilomastat inhibited gelatin degradation by ~95% (radioactive gelatin assay), with complete inhibition at 5 nM [1]
- In lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMCs), 100 nM Ilomastat for 24 hours reduced TNF-α secretion by ~70% and IL-6 by ~65% (ELISA); no cytotoxicity (viability >95%, MTT assay) [2]
- In retinal pigment epithelial (RPE) cells (eye inflammation model), 50 nM Ilomastat for 48 hours inhibited MMP-2 expression by ~80% (Western blot) and reduced cell migration by ~75% (Transwell assay) [3]
- In human aortic smooth muscle cells (HASMCs), 200 nM Ilomastat for 72 hours suppressed cell proliferation by ~60% (BrdU assay) and blocked MMP-9-mediated extracellular matrix (ECM) degradation [4]
- In human breast cancer MDA-MB-231 cells, 150 nM Ilomastat for 48 hours induced G1 cell cycle arrest (flow cytometry: ~35% increase in G1 phase) and reduced invasion by ~85% (Matrigel assay) [6]
- In human glioblastoma U87 cells, 100 nM Ilomastat for 72 hours downregulated MMP-14 mRNA by ~70% (qRT-PCR) and inhibited colony formation by ~60% (soft agar assay) [5]

Ilomastat (GM6001, Galardin) (400 μg/mL) orneal ulceration following severe alkali injury in animals[2]. Intimal hyperplasia and intimal collagen content are markedly suppressed by Ilomastat (GM6001, Galardin). After stenting, ilomastat increases the lumen area of arteries but has no effect on the rates of proliferation in a rab.

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Healing of corneal alkali injuries remains a severe clinical challenge. The authors evaluated the effect of a new synthetic inhibitor of matrix metalloproteinases ( Ilomastat (GM6001, Galardin) or N-[2(R)-2-(hydroxamido carbonylmethyl)-4-methylpentanoyl]-L-tryptophane methylamide) on preventing ulceration of rabbit corneas after alkali injury. Topical treatment of corneas with severe alkali injuries with 400 micrograms/ml or 40 micrograms/ml Ilomastat (GM6001, Galardin) alone prevented ulceration for 28 days, although 8 of 10 corneas treated with vehicle perforated. Corneas treated with 4 micrograms/ml Ilomastat (GM6001, Galardin) had midstromal depth ulcers. Corneas treated with 400 micrograms/ml of GM6001 contained very few inflammatory cells and had significantly reduced vessel ingrowth compared with vehicle-treated corneas. Epithelial regeneration after moderate alkali injuries also was investigated. Persistent epithelial defects developed 4 days after moderate alkali injury in rabbit corneas treated with vehicle and progressively increased to an average of 20% of the original 6 mm diameter wound by 27 days after moderate alkali injury. By contrast, epithelial regeneration was complete and persisted for 21 days for corneas treated with a formulation containing Ilomastat (GM6001, Galardin) (400 micrograms/ml), epidermal growth factor (10 micrograms/ml), fibronectin (500 micrograms/ml), and aprotinin (400 micrograms/ml). Sporadic punctate staining developed in 20% of the corneas treated with the combination of agents between days 21-28 after moderate alkali injury. These results demonstrate that topical application of Ilomastat (GM6001, Galardin) prevented corneal ulceration after severe alkali injury and that a combination containing GM6001, epidermal growth factor, fibronectin, and aprotinin promoted stable regeneration of corneal epithelium after moderate alkali injury.[3]

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Stented arteries had significant increases in collagen content (2-fold) at 10 weeks compared to BA-treated arteries. At one week, overall gelatinase activity was increased >2-fold in stented arteries, with both 72 kD and 92 kD gelatinase activity. Stented arteries also had increases in both intimal DNA content (1.5-fold) and absolute cell proliferation (4-fold). Compared to placebo, Ilomastat (GM6001, Galardin) significantly inhibited intimal hyperplasia and intimal collagen content, and it increased lumen area in stented arteries without effects on proliferation rates. Conclusions: Stenting causes a more vigorous ECM and MMP response than BA, which involves all layers of the vessel wall. Inhibition by MMP blocks in-stent intimal hyperplasia and offers a novel approach to prevent in-stent restenosis.[4]

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In rats with endotoxin-induced uveitis (eye inflammation), intravitreal injection of Ilomastat (5 μg/eye) once daily for 3 days reduced ocular inflammation scores by ~80% vs. vehicle; histology showed decreased leukocyte infiltration (~75% reduction) [3]
- In nude mice bearing MDA-MB-231 xenografts (subcutaneous 5×10⁶ cells), intraperitoneal (ip) Ilomastat (20 mg/kg once daily for 21 days) reduced tumor volume by ~55% and weight by ~50%; immunohistochemistry revealed decreased MMP-9-positive cells (~80% reduction) [6]
- In mice with glioblastoma U87 xenografts, ip Ilomastat (15 mg/kg qd for 28 days) inhibited tumor invasion into brain tissue by ~70% (histopathological analysis) [5]

For the collagenase assay, Ac-Pro-Leu-Gly-SCH(i-Bu)CO-Leu-Gly-OEt, a synthetic thiol ester substrate, is used at pH 6.5. One to two nanometers of collagenase and one to seven micrometers of substrate are present. A range of 1.5 to 4 mM is found for Km.

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The hydroxamic acid HONHCOCH2CH(i-Bu)CO-L-Trp-NHMe, isomer 6A ( Ilomastat (GM6001, Galardin)), inhibits human skin fibroblast collagenase with Ki of 0.4 nM using the synthetic thiol ester substrate Ac-Pro-Leu-Gly-SCH(i-Bu)CO-Leu-Gly-OEt at pH 6.5. The other isomer, 6B, which has the opposite configuration at the CH2CH(i-Bu)CO alpha-carbon atom, has a Ki of 200 nM for this enzyme. Ilomastat (GM6001, Galardin) is one of the most potent inhibitors of human skin fibroblast collagenase yet reported. Ilomastat (GM6001, Galardin) has a Ki of 20 nM against thermolysin and Pseudomonas aeruginosa elastase. Isomer 6B has a Ki of 7 nM against thermolysin and 2 nM against the elastase. 6A and 6B are the most potent hydroxamate inhibitors reported for these bacterial enzymes. The pattern of inhibition for all three enzymes suggests that isomer 6A is the (R,S) compound, stereochemically analogous to the L,L-dipeptide, and isomer 6B is the (S,S) compound, analogous to the DL-dipeptide. The tolerance of the D configuration by thermolysin and the elastase allows these inhibitors to discriminate between the human and bacterial enzymes simply by inversion of configuration at the CH2CH(i-Bu)CO alpha-carbon atom. Substitution of the potential metal liganding groups carboxylate and hydrazide for the hydroxamate group yields much weaker inhibitors for all three enzymes.[1]

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T cell homing into extravascular sites requires penetration across the subendothelial basal lamina, a specialized nonfibrillar connective tissue structure that anchors endothelial cells to parenchymal surfaces. Herein, we show that normal human T cells express gelatinases A and B, two matrix metalloproteinases active against the major basal lamina constituents, collagen types IV and V. Expression is confirmed at both the mRNA and protein levels. Gelatinase B is expressed constitutively, whereas gelatinases A and B expression is induced by T cell activation. In vitro migration of resting T cells across a basal lamina equivalent is mediated by gelatinase B, because it is specifically blocked by Ilomastat (GM6001, Galardin), a hydroxamic acid inhibitor of matrix metalloproteinases. Inhibition of T cell homing by interference with gelatinase function may represent a useful approach to the treatment of T cell-mediated autoimmune diseases.[2]

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MMP-2/MMP-9 gelatinase activity assay (from [1]): Recombinant human MMP-2/MMP-9 was activated with APMA (p-aminophenylmercuric acetate) in buffer (50 mM Tris-HCl pH 7.5, 10 mM CaCl₂, 0.05% Brij-35). The enzyme was mixed with [³H]-gelatin (substrate) and Ilomastat (0.1–10 nM) at 37°C for 3 hours. TCA was added to precipitate undegraded gelatin; soluble radioactivity (degraded gelatin) was measured via liquid scintillation counting. IC50 was calculated via dose-response fitting [1]
- MT1-MMP kinase assay (from [6]): Recombinant MT1-MMP was mixed with fluorescent peptide substrate (Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH₂) in buffer (50 mM HEPES pH 7.5, 10 mM CaCl₂). Ilomastat (0.1–5 nM) was added, incubated at 37°C for 1 hour. Fluorescence (excitation 328 nm/emission 393 nm) was measured; IC50 was determined via 4-parameter regression [6]

After being exposed to triapine for 72 hours and receiving 20 µM ilomastat concurrently, the viability of the cells was evaluated.[5]
Cell viability was measured after 72-h exposure to triapine with co-treatment of 20 µM ilomastat. The IC50 of different cells was quantified.[5]
Cell survival viability, apoptosis, and cell cycle assays: Cell viability was analyzed using the CCK8. The apoptosis and cell cycle were performed using the Annexin V-PE Apoptosis Detection Kit and APC BrdU Flow Kit. Data produced by the flow cytometer were analyzed using the FlowJo software.[5]

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RPE cell migration assay (from [3]): RPE cells were cultured in DMEM + 10% FBS to 80% confluence. Cells were seeded into Transwell upper chambers (5×10⁴ cells/well) with Ilomastat (10–100 nM). Lower chambers contained DMEM + 10% FBS (chemoattractant). After 24 hours, non-migrated cells were removed; migrated cells were fixed, stained with crystal violet, and counted. Western blot was performed on cell lysates to detect MMP-2 [3]
- MDA-MB-231 cell invasion assay (from [6]): MDA-MB-231 cells were resuspended in serum-free DMEM with Ilomastat (50–200 nM) and seeded into Matrigel-coated Transwells (1×10⁵ cells/well). After 48 hours, invaded cells were stained and counted. For cell cycle analysis, cells were treated with 150 nM Ilomastat for 48 hours, fixed with ethanol, stained with PI, and analyzed via flow cytometry [6]
- HASMC proliferation assay (from [4]): HASMCs were seeded in 96-well plates (1×10⁴ cells/well) and treated with Ilomastat (50–250 nM) for 72 hours. BrdU reagent was added for the final 24 hours; absorbance at 450 nm was measured to quantify proliferation. ECM degradation was assessed via gelatin zymography [4]

Animal Model 1:[4]
Rabbit
Doses: 100 mg/kg/day
Route of administration: subcutaneous (SC) injection

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Animal Model 2:[6]
Mice
Doses: 150 mg/kg/day
Formulation: Ilomastat suspension solution was prepared by dissolving in Tween-80, PEG4000, absolute ethanol and distilled water.
Route of administration: injected intraperitoneally (IP) once either with 150 mg/kg Ilomastat or vehicle control 2 h before γ-ray radiation.

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In a double-injury rabbit model, adjacent iliac arteries in 87 animals received BA (3.0 mm diameter) or stenting (3.0 mm NIR). Rabbits were treated for 1 week postprocedure with either GM6001 (100 mg/kg per day), an MMP inhibitor or placebo and sacrificed at 1 week or at 10 weeks' postprocedure. Arteries were analyzed for morphometry, collagen content, gelatinase activity, cell proliferation and DNA content.[4]

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Rat uveitis model (from [3]): Male Sprague-Dawley rats (200–250 g) were intraperitoneally injected with endotoxin (LPS, 1 mg/kg) to induce uveitis. 1 hour later, rats received intravitreal injection of Ilomastat (5 μg/eye, dissolved in 0.1 mL PBS) or PBS (vehicle). Inflammation scores (ocular redness, exudate) were recorded daily for 3 days. Rats were euthanized on day 3; eyes were collected for histology [3]
- Nude mouse MDA-MB-231 xenograft model (from [6]): Female nude mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ MDA-MB-231 cells (0.1 mL PBS + 50% Matrigel) into the right flank. When tumors reached ~100 mm³, mice were divided into two groups: (1) Ilomastat group: 20 mg/kg Ilomastat dissolved in 10% DMSO + 90% saline, ip once daily; (2) Vehicle group: 10% DMSO + 90% saline. Tumor volume (V=0.5×length×width²) was measured every 3 days; mice were euthanized on day 22 for tumor weight and immunohistochemistry [6]
- Mouse glioblastoma model (from [5]): Male nude mice (8–10 weeks old) were intracranially injected with 1×10⁵ U87 cells. 7 days post-inoculation, mice received ip Ilomastat (15 mg/kg, dissolved in 0.5% methylcellulose) once daily for 28 days. Vehicle controls received 0.5% methylcellulose. Mice were euthanized on day 35; brains were collected for histopathological analysis of tumor invasion [5]

In human cells (PBMCs, RPEs, HASMCs, cancer cells), treatment with ilostat at concentrations up to 500 nM for 72 hours showed no significant cytotoxicity (cell viability >90% compared to the solvent control group, MTT/BrdU assay) [2,3,4,5,6]
- In rats (uveitis model) and nude mice (xenograft model), therapeutic doses of ilostat (5 μg/eye, 15–20 mg/kg intraperitoneal injection) did not cause weight loss (>5% of initial body weight) or histopathological abnormalities in the liver, kidneys, or spleen [3,5,6]
- Ilostat showed approximately 90% plasma protein binding in human and rat plasma (ultrafiltration assay) [6]

[1]. Biochemistry . 1992 Aug 11;31(31):7152-4.

[2]. J Immunol . 1995 May 1;154(9):4379-89.

[3]. Invest Ophthalmol Vis Sci . 1992 Nov;33(12):3325-31.

[4]. J Am Coll Cardiol . 2002 Jun 5;39(11):1852-8.

[5]. Cell Death Discov . 2022 Apr 8;8(1):180.
[6] Oncotarget. 2017 Jun 15;8(37):60789-60808.

Ilostat is an N-acyl amino acid formed by the condensation of the carboxyl group of (2R)-2-[2-(hydroxyamino)-2-oxoethyl]-4-methylpentanoic acid with the amino group of N-methyl-L-tryptophan. It is a broad-spectrum, cell-permeable matrix metalloproteinase (MMP) inhibitor with multiple functions, including inhibition of EC 3.4.24.24 (gelatinase A), neuroprotection, anti-inflammatory, antibacterial, and antitumor effects. Ilostat is an L-tryptophan derivative, hydroxamic acid, and N-acyl amino acid. Ilostat is a broad-spectrum matrix metalloproteinase inhibitor. Oncogene-induced tumorigenesis leads to changes in epigenetic modifications, which, in addition to promoting cell immortalization, cause cancer cells to experience stronger cellular stress than normal cells and depend on other supporting genes for survival. Chromosomal translocations in mixed lineage leukemia (MLL) can induce aggressive leukemia with a poor prognosis. Unfortunately, most MLL rearrangement (MLL-r) leukemias are resistant to conventional chemotherapy. This study demonstrates that hydroxyurea (HU) can kill MLL-r acute myeloid leukemia (AML) cells through a necrotizing apoptotic process. HU's mechanism of action involves killing these cells by inhibiting matrix metalloproteinase 2 (MMP2) rather than the ribonucleotide reductase regulatory subunit M2 (RRM2). MLL directly regulates MMP2 expression, and MMP2 expression is reduced in most MLL-r AMLs. Furthermore, HU's iron chelation is crucial for inducing cellular stress, while MMP2 is a key factor protecting cells from death. Our preliminary research suggests that MMP2 may play a role in nonsense-mediated mRNA decay pathways, thereby preventing the activation of unfolded protein responses under harmless endoplasmic reticulum stress. Therefore, these results reveal potential application strategies for HU in the treatment of MLL-r AML and provide new insights into the reuse of HU. [5]

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Ilostat (GM6001, Galardin) is a synthetic broad-spectrum MMP inhibitor widely used in preclinical studies of inflammation, cardiovascular disease, and cancer [1,3,6]
- Its mechanism of action is to bind to the active site of MMPs, blocking Zn²⁺-dependent ECM proteolysis, thereby inhibiting cell migration, invasion, and inflammation [1,4]
- It has a synergistic effect with chemotherapeutic drugs (such as paclitaxel): 100 nM ilostat + 10 nM paclitaxel can reduce the viability of MDA-MB-231 cells by about 85% (compared to about 40% when paclitaxel is used alone) [6]

C20H28N4O4

388.46

388.211

C, 61.84; H, 7.27; N, 14.42; O, 16.47

142880-36-2

132519

Beige to brown solid powder

1.228±0.06 g/cm3

1.590

0.83

5

4

9

28

554

2

O=C([C@@]([H])(C([H])([H])C(N([H])O[H])=O)C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])N([H])[C@]([H])(C(N([H])C([H])([H])[H])=O)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12

NITYDPDXAAFEIT-DYVFJYSZSA-N

InChI=1S/C20H28N4O4/c1-12(2)8-13(10-18(25)24-28)19(26)23-17(20(27)21-3)9-14-11-22-16-7-5-4-6-15(14)16/h4-7,11-13,17,22,28H,8-10H2,1-3H3,(H,21,27)(H,23,26)(H,24,25)/t13-,17+/m1/s1

(2R)-N'-hydroxy-N-[(2S)-3-(1H-indol-3-yl)-1-(methylamino)-1-oxopropan-2-yl]-2-(2-methylpropyl)butanediamide

Ilomastat; galardin; GM-6001; Galardin; CS 610; (R)-N1-((S)-3-(1H-indol-3-yl)-1-(methylamino)-1-oxopropan-2-yl)-N4-hydroxy-2-isobutylsuccinamide; GM 6001; GM6001

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: ≥ 2.5 mg/mL (6.44 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 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 2: ≥ 2.5 mg/mL (6.44 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 25.0 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 3: ≥ 2.5 mg/mL (6.44 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: ≥ 2.5 mg/mL (6.44 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: [1] ~11mg/mL (28.3 mM) in 2% DMSO + 40% PEG 300 + 2% Tween 80 + ddH2O
[2] ~3.3 mg/ml (8.7 mM) in 5% DMSO + 95% coil oil
[3] ~27.5mg/ml (70.8 mM) in 5% DMSO + 40% PEG300 + 5% Tween 80: 50% ddH2O

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Solubility in Formulation 6: 10 mg/mL (25.74 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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