| Description |
Arginase inhibitor 1 is a potent inhibitor of human arginases I and II with IC50s of 223 and 509 nM, respectively.
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| Related Catalog |
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| Target |
IC50: 223 nM (arginases I), 509 nM (arginases II)[1]
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| In Vitro |
Arginase inhibitor 1inhibits human arginases I and II with IC50s of 223±22.3 and 509±85.1 nM, respectively, and is active in a recombinant cellular assay overexpressing human arginase I (CHO cells). Arginase inhibitor 1 is a novel second generation arginase inhibitor with significant activity in a rat model of myocardial ischemia/reperfusion injury (MI/RI). Arginase inhibitor 1 is potent against hARG I in both in vitro enzyme and cellular assays. The IC50 for Arginase inhibitor 1 is 8 μM in CHO Cells Over-Expressing hArgI[1].
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| In Vivo |
A pharmacokinetic evaluation of Arginase inhibitor 1 is conducted after intravenous (i.v.) and oral (p.o.) dosing in male Sprague-Dawley rats (n=3 per dose route). Arginase inhibitor 1 is formulated in 0.9% saline and administered intravenously at 10 mg/kg by bolus through a preimplanted cannula at a dosing volume of 1 mL/kg, and orally at 10 mg/kg via gavage at a dosing volume of 2 mL/kg. Following i.v. dosing with 10 mg/kg in fasted animals, Arginase inhibitor 1has a terminal elimination half-life (t1/2) of 3.3 h with a volume of distribution and total body clearance of 1.86 L/kg and 7.89 mL/min/kg, respectively. The oral bioavailability of Arginase inhibitor 1 (10 mg/kg, p.o.) is 28% with a Cmax of 0.45 mg/L[1].
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| Kinase Assay |
Purified recombinant full length human arginase I protein and recombinant, fully active, truncated form of human arginase II are used to develop 96-well plate human arginase I and arginase II assays. Inhibition of arginase I and arginase II by program compounds is followed spectrophotometrically at 530 nm. Arginase inhibitor 1 to be tested is dissolved in DMSO at an initial concentration that is 50-fold greater than its final concentration in the cuvette. Ten microliters of the stock solution is diluted in 90 μL of the assay buffer that comprised 0.1 M sodium phosphate buffer containing 130 mM NaCl, pH 7.4, to which is added ovalbumin (OVA) at a concentration of 1 mg/mL. Solutions of arginase I and II are prepared in 100 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of OVA to give an arginase stock solution at a final concentration of 100 ng/mL. To each well of a 96-well microtiter plate is added 40 μL of enzyme, 10 μL of the Arginase inhibitor 1 solution, and 10 μL of enzyme substrate solution (L-arginine+Manganese sulfate). For wells that are used as positive controls, only the enzyme and its substrate are added, while wells used as negative controls contained only manganese sulfate. After incubating the microtiter plate at 37°C for 60 min, 150 μL of a urea reagent obtained by combining equal proportions (1:1) of reagents A and B is added to each well of the microtiter plate to stop the reaction. The urea reagent is made just before use by combining Reagent A (10 mM o-phthaldialdehyde and 0.4% polyoxyethylene lauryl ether (w/v) in 1.8 M sulfuric acid) with Reagent B (1.3 mM primaquine diphosphate, 0.4% polyoxyethylene lauryl ether (w/v), and 130 mM boric acid in 3.6 M sulfuric acid). After quenching the reaction mixture, the microtiter plate is allowed to stand for an additional 10 min at room temperature in order to allow color development. The inhibition of arginase is computed by measuring the optical density (OD) of the reaction mixture at 530 nm and normalizing the OD value to percent inhibition observed in the control. The normalized OD is then used to generate a concentation-response curve by plotting the normalized OD values against log[concentration] and using regression analysis to compute the IC50 values[1].
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| Animal Admin |
Rats[1] Single dose pharmacokinetics are evaluated in male Sprague-Dawley rats by intravenous bolus (i.v.) and oral (p.o.) dosing. Three rats are evaluated per dose group. Arginase inhibitor 1 is freshly formulated in 0.9% saline prior to dosing, at 10 mg/mL for i.v. dosing at a dose volume of 1 mL/kg animal body weight, and at 5 mg/mL for p.o. dosing at a dose volume of 2 mL/kg. Animals are fasted overnight prior to dosing, with water given ad libitum. Arginase inhibitor 1 is administered i.v. through a preimplanted cannula or orally by gavage. Food is reintroduced to animals 4 h following dosing. Blood samples are collected through preimplanted cannulae (dual cannulated animals used for i.v. dosing; blood collection separate from dosing cannula) at 0.25 mL per draw, followed by volume replacement with 0.9% saline. Samples are collected at predose 0.083 (i.v. only), 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h following dosing. Blood samples are maintained on ice and centrifuged at 10 000g to obtain plasma. Plasma is frozen at -20 °C prior to analysis. Analysis is performed by LC/MS/MS using Agilent 1100 HPLC pumps with detection on a PESciex API 4000 Qtrap. Pharmacokinetic evaluation is performed using standard noncompartmental analyses in Phoenix WinNonlin software.
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| References |
[1]. Van Zandt MC, et al. Discovery of (R)-2-amino-6-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic acid and congeners as highly potentinhibitors of human arginases I and II for treatment of myocardial reperfusion injury. J Med Chem. 2013 Mar 28;56(6):2568-80.
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