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) 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].

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]

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]

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]

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

[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.

Ilomastat is an N-acyl-amino acid obtained by formal condensation of the carboxy group of (2R)-2-[2-(hydroxyamino)-2-oxoethyl]-4-methylpentanoic acid with the amino group of N-methyl-L-tryptophanamide. A cell permeable broad-spectrum matrix metalloproteinase (MMP) inhibitor It has a role as an EC 3.4.24.24 (gelatinase A) inhibitor, a neuroprotective agent, an anti-inflammatory agent, an antibacterial agent and an antineoplastic agent. It is a L-tryptophan derivative, a hydroxamic acid and a N-acyl-amino acid.
Ilomastat is a broad-spectrum matrix metalloproteinase inhibitor.

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Oncogene-induced tumorigenesis results in the variation of epigenetic modifications, and in addition to promoting cell immortalization, cancer cells undergo more intense cellular stress than normal cells and depend on other support genes for survival. Chromosomal translocations of mixed-lineage leukemia (MLL) induce aggressive leukemias with an inferior prognosis. Unfortunately, most MLL-rearranged (MLL-r) leukemias are resistant to conventional chemotherapies. Here, we showed that hydroxyurea (HU) could kill MLL-r acute myeloid leukemia (AML) cells through the necroptosis process. HU target these cells by matrix metallopeptidase 2 (MMP2) deficiency rather than subordinate ribonucleotide reductase regulatory subunit M2 (RRM2) inhibition, where MLL directly regulates MMP2 expression and is decreased in most MLL-r AMLs. Moreover, iron chelation of HU is also indispensable for inducing cell stress, and MMP2 is the support factor to protect cells from death. Our preliminary study indicates that MMP2 might play a role in the nonsense-mediated mRNA decay pathway that prevents activation of unfolding protein response under innocuous endoplasmic reticulum stress. Hence, these results reveal a possible strategy of HU application in MLL-r AML treatment and shed new light upon HU repurposing.[5]

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.)