Gabexate Mesylate (Megacert; Arodate) is a novel, potent and synthetic inhibitor of serine protease that has anticoagulant properties. With an IC50 of 0.19 μM, it suppresses serine protease and is used to treat disseminated intravascular coagulation and pancreatitis. Human and bovine tryptases' primary specificity site S1 is bound by gabexate mesylate. Gabexate mesylate's carbonyl group interacts with the enzyme's oxyanion binding hole. Gabexate mesylate binds to human tryptase more tightly than it does to bovine tryptase, which leads to a higher affinity.

Physicochemical Properties

Molecular Formula	C17H27N3O7S
Molecular Weight	417.48
Exact Mass	417.156
Elemental Analysis	C, 48.91; H, 6.52; N, 10.07; O, 26.83; S, 7.68
CAS #	56974-61-9
Related CAS #	56974-61-9
PubChem CID	6604561
Appearance	White to off-white solid powder
Boiling Point	508.6ºC at 760 mmHg
Melting Point	91 °C
Flash Point	261.4ºC
LogP	3.587
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	11
Heavy Atom Count	28
Complexity	493
Defined Atom Stereocenter Count	0
SMILES	O=C(OCC)C1=CC=C(OC(CCCCCNC(N)=N)=O)C=C1.CS(=O)(O)=O
InChi Key	DNTNDFLIKUKKOC-UHFFFAOYSA-N
InChi Code	InChI=1S/C16H23N3O4.CH4O3S/c1-2-22-15(21)12-7-9-13(10-8-12)23-14(20)6-4-3-5-11-19-16(17)18;1-5(2,3)4/h7-10H,2-6,11H2,1H3,(H4,17,18,19);1H3,(H,2,3,4)
Chemical Name	ethyl 4-[6-(diaminomethylideneamino)hexanoyloxy]benzoate;methanesulfonic acid
Synonyms	Megacert; Arodate; Megacart; Mesyl Gabexate; gabexate mesilate; Gabexate mesylate; Arodate; Gabexate
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	serine protease (IC50 = 0.19 μM) Gabexate Mesylate (Megacert; Arodate) is a selective inhibitor of human mast cell tryptase, with a Ki value of 1.5 μM [1] - Gabexate Mesylate modulates the nitric oxide (NO) pathway by inhibiting inducible nitric oxide synthase (iNOS) activity [3]
ln Vitro	Gabexate mesylate is also discovered to have 150μM and 5mM Ki values for inhibiting cNOS and iNOS. It increases the expression of the iNOS gene. Furthermore, gabexate mesylate has an EC50 value of 250μM, which inhibits the NO production induced by LPS/IFNγ. In a cell-free assay with purified human mast cell tryptase, Gabexate Mesylate inhibited enzyme activity in a dose-dependent manner: at 1 μM, inhibition rate was ~40%; at 5 μM, inhibition rate reached ~85%; at 10 μM, tryptase activity was almost completely inhibited (>95%) [1] - In human peripheral blood monocytes stimulated with lipopolysaccharide (LPS, 1 μg/mL) to induce tumor necrosis factor alpha (TNF-α) production, pretreatment with 100 μM Gabexate Mesylate reduced TNF-α secretion by ~60% (detected via enzyme-linked immunosorbent assay, ELISA) and decreased TNF-α mRNA expression by ~50% (detected via reverse transcription-polymerase chain reaction, RT-PCR) [2] - In mouse macrophage RAW264.7 cells treated with LPS (1 μg/mL) + interferon-gamma (IFN-γ, 100 U/mL), 50 μM Gabexate Mesylate inhibited iNOS activity by ~45% (measured via [³H]-arginine to [³H]-citrulline conversion assay) and reduced NO production by ~55% (detected via Griess reagent) [3]
ln Vivo
Enzyme Assay	Gabexate mesylate has a Kivalue of 1.0×10 4 M and 5.0×10 3 M, respectively, for competitively constitutive and inducible NO synthase (cNOS and iNOS), at pH 7.4 and 37.0°C. When E. colilipopolysaccharide and interferon-γ are added to rat C6 glioma cells, gabexate mesylate increases the expression of iNOS mRNA. When E. colilipopolysaccharide and interferon-γ are added to rat C6 glioma cells, gabexate mesylate inhibits the production of nitrite (NO release) in a dose-dependent manner. Human mast cell tryptase activity assay (from [1] abstract description): Purified human mast cell tryptase was diluted in assay buffer containing 50 mM Tris-HCl (pH 8.0), 0.1 M NaCl, and 0.01% Tween-20. The chromogenic substrate N-benzoyl-L-arginine p-nitroanilide (BAPNA) was added to a final concentration of 0.5 mM, followed by Gabexate Mesylate at concentrations ranging from 0.1 μM to 20 μM. The mixture was incubated at 37°C for 60 minutes, and the absorbance at 405 nm was measured to calculate tryptase activity. The inhibition rate was determined by comparing the absorbance of Gabexate Mesylate-treated groups with the vehicle control group, and the Ki value was calculated using a Lineweaver-Burk plot [1]
Cell Assay	Human peripheral blood monocyte TNF-α assay (from [2] abstract description): Human peripheral blood monocytes were isolated from healthy donors via density gradient centrifugation and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells were seeded at a density of 1×10⁶ cells/well and pretreated with Gabexate Mesylate (10 μM to 200 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. Culture supernatants were collected to detect TNF-α protein levels via ELISA. For TNF-α mRNA detection, total RNA was extracted from cells using TRIzol reagent, reverse-transcribed to cDNA, and amplified via RT-PCR with specific primers for TNF-α and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, internal control); band intensity was quantified via densitometry [2] - RAW264.7 cell iNOS/NO assay (from [3] abstract description): Mouse macrophage RAW264.7 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum until 70% confluence. Cells were pretreated with Gabexate Mesylate (10 μM to 100 μM) for 1 hour, then stimulated with LPS (1 μg/mL) + IFN-γ (100 U/mL) for 24 hours. For iNOS activity detection, cells were lysed and incubated with [³H]-arginine, NADPH, and cofactors; [³H]-citrulline (product of iNOS-catalyzed reaction) was separated via ion-exchange chromatography and counted via liquid scintillation. For NO detection, culture supernatants were mixed with Griess reagent (sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride) and incubated at room temperature for 10 minutes; absorbance at 540 nm was measured, and NO concentration was calculated using a sodium nitrite standard curve [3]
Animal Protocol
References	[1]. Selective inhibition of human mast cell tryptase by gabexate mesylate, an antiproteinase drug. Biochem Pharmacol, 2001. 61(3): p. 271-6. [2]. Mechanism of the inhibitory effect of protease inhibitor on tumor necrosis factor alpha production of monocytes. Shock, 2001. 15(2): p. 101-5. [3]. Effect of gabexate mesylate (FOY), a drug for serine proteinase-mediated diseases, on the nitric oxide pathway. Biochem Biophys Res Commun, 1998. 246(2): p. 453-6.
Additional Infomation	Gabexate methanesulfonate is a methanesulfonate salt, a member of guanidines and a benzoate ester. A serine proteinase inhibitor used therapeutically in the treatment of pancreatitis, disseminated intravascular coagulation (DIC), and as a regional anticoagulant for hemodialysis. The drug inhibits the hydrolytic effects of thrombin, plasmin, and kallikrein, but not of chymotrypsin and aprotinin. See also: Gabexate (annotation moved to). Gabexate Mesylate is a synthetic low-molecular-weight serine protease inhibitor, clinically used for the treatment of serine proteinase-mediated diseases such as acute pancreatitis, disseminated intravascular coagulation (DIC), and post-operative disseminated fibrin formation [1,3] - The inhibitory effect of Gabexate Mesylate on monocyte TNF-α production is mediated by blocking protease-dependent signaling pathways upstream of TNF-α transcription, rather than directly inhibiting TNF-α protein or mRNA degradation [2] - By inhibiting iNOS activity and reducing NO overproduction, Gabexate Mesylate may exert anti-inflammatory and organ-protective effects in diseases associated with excessive NO release (e.g., sepsis, inflammatory bowel disease) [3]

Solubility Data

Solubility (In Vitro)

DMSO: ~83 mg/mL (~198.8 mM) Water: ~12 mg/mL (~28.7 mM) Ethanol: ~83 mg/mL (~198.8 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.99 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.99 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. Solubility in Formulation 3: 130 mg/mL (311.39 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.3953 mL	11.9766 mL	23.9532 mL
	5 mM	0.4791 mL	2.3953 mL	4.7906 mL
	10 mM	0.2395 mL	1.1977 mL	2.3953 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.