In GSK180 primary human hepatocytes, endogenous KMO activity (IC50=2.6 μM). In GSK180 primary human hepatocytes, endogenous KMO activity (IC50=2.6 μM). In GSK180 primary human hepatocytes, endogenous KMO has an IC50 value of 7 μM, which is marginally less potent than the human enzyme [1].

In GSK180 primary human hepatocytes, endogenous KMO activity (IC50=2.6 μM). In GSK180 primary human hepatocytes, endogenous KMO activity (IC50=2.6 μM). In GSK180 primary human hepatocytes, endogenous KMO has an IC50 value of 7 μM, which is marginally less potent than the human enzyme [1].

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GSK180 shows potent inhibition of human KMO in a biochemical assay with an IC50 of approximately 6 nM (mean pIC50 8.2 ± 0.17, n=103). [1]
In a cell-based assay using HEK293 cells stably expressing human KMO, GSK180 inhibits the enzyme with an IC50 of 2.0 µM (mean pIC50 5.7 ± 0.15, n=4). [1]
In primary human hepatocytes expressing endogenous KMO activity, GSK180 inhibits the activity with an IC50 of 2.6 µM. [1]
GSK180 inhibits rat KMO expressed in HEK293 cells with an IC50 of 7 µM (mean pIC50 5.2 ± 0.09, n=5). [1]
The inhibition by GSK180 is competitive with the kynurenine substrate. [1]
GSK180 shows negligible activity against other enzymes on the tryptophan pathway (kynureninase, kynurenine aminotransferase types 1 and 2), a panel of over 50 unrelated proteins, and an additional series of acidergic proteins. [1]
GSK180 demonstrates significantly lower potency against P. fluorescens KMO (IC50 = 500 nM) compared to the human construct (IC50 = 6 nM). [1]
X-ray co-crystallography at 3.2 Å resolution shows GSK180 binding within the catalytic site of P. fluorescens KMO. The carboxylate forms a salt bridge with Arg84 and hydrogen bonds with Tyr98 and Asn369. The oxazolidinone carbonyl forms a hydrogen bond with Tyr404, and the 5-chlorine atom forms a π-interaction with Phe238. [1]
Administration of GSK180 to wild-type mice results in an increase in plasma kynurenine, confirming the effect is due to KMO inhibition. It also causes a significant reduction in circulating tryptophan levels in both wild-type and Kmo-null mice, and an increase in kynurenic acid in Kmo-null mice, suggesting additional effects unrelated to direct KMO inhibition. [1]
GSK180 shows a concentration-dependent displacement of tryptophan from plasma proteins. [1]
The passive permeability of GSK180 across an artificial membrane is extremely low (< 3 x 10^-6 cm/s, n=3), and intracellular drug levels are more than 30 times lower than extracellular concentrations. [1]

GSK180 is appropriate for injection intravenously [1].

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In Kmo-null mice, experimental acute pancreatitis (AP) induced less severe extrapancreatic organ injury (lung, kidney, liver) compared to wild-type controls, as evidenced by reduced lung histological damage, fewer apoptotic cells in lung and kidney, and a significantly smaller rise in plasma alanine aminotransferase (ALT). Pancreatic injury itself was not different between strains. [1]
In a rat model of AP, therapeutic administration of GSK180 (i.v. bolus + infusion starting 1 hour post-AP induction) resulted in biochemical changes consistent with KMO inhibition (increased plasma kynurenine and kynurenic acid, decreased plasma tryptophan and 3-hydroxykynurenine). [1]
In the same rat AP model, GSK180 treatment essentially prevented acute lung injury features: it reduced lung histopathological changes, neutrophil infiltration, bronchoalveolar lavage protein leak, serum KL-6 levels, and apoptosis. [1]
GSK180 treatment in the rat AP model also protected against acute kidney injury, significantly reducing renal tubular cell apoptosis and preventing the rise in serum creatinine and urea. [1]
GSK180 treatment did not affect the severity of pancreatic histological injury or serum amylase rise in the rat AP model. [1]
In rats, a single i.v. bolus of GSK180 (27 mg/kg) led to a rapid increase in circulating kynurenine and kynurenic acid, which returned to baseline as drug levels declined. [1]

Inhibition of human KMO activity was determined using full-length human KMO expressed as a GST fusion protein in SP9 insect cells and used as a membrane suspension. Reactions were run at saturating NADPH (200 µM) and around the KM for kynurenine (10 µM) in a buffer of 50 mM HEPES (pH 7.5), 2 mM DTT, 1 mM EDTA, 100 µM Chaps. Reactions were stopped after 2 hours by addition of trifluoroacetic acid (TFA). Product formation was quantified using a RapidFire mass spectrometry system coupled to a triple quadrupole mass spectrometer operated in positive ion mode with multiple reaction monitoring to detect kynurenine and 3-hydroxykynurenine. [1]
Assays for human kynureninase (KYNU) and kynurenine aminotransferase types 1 and 2 (KATI and KATII) were performed to assess selectivity. The enzymes were expressed in E. coli as C-terminal six-histidine tagged proteins and affinity purified. For KATI and KATII, a stopped fluorescence assay was used with specific substrates (sodium pyruvate and L-kynurenine for KATI; α-ketoglutarate, pyridoxal 5’-phosphate and L-kynurenine for KATII). Assays were incubated at 37°C and terminated with a zinc acetate/sodium acetate solution, and fluorescence intensity was measured. [1]

For the cellular KMO inhibition assay, a stable HEK293 cell line overexpressing human KMO was generated. Cells were grown in 96-well plates. Serial dilutions of GSK180 were prepared in DMSO and added to cells in duplicate. Culture medium was replaced with Opti-MEM medium containing 1% glutamine, 1% penicillin/streptomycin and 200 µM L-kynurenine, and cells were incubated for 20 hours. Following incubation, the medium was transferred to a deep well block containing acetonitrile and an internal standard (d5-tryptophan), centrifuged, dried, and reconstituted for LC-MS/MS analysis to quantify 3-hydroxykynurenine. The peak area ratio for 3-hydroxykynurenine/d5-tryptophan was used to determine percentage inhibition. [1]
For primary human hepatocyte assays, freshly prepared cells were incubated overnight with compounds in culture media containing fetal bovine serum, dexamethasone and L-tryptophan. Following incubation, the cell supernatant was analyzed by LC-MS/MS to detect and quantify metabolites. Percentage inhibition was calculated using the observed increase in kynurenine. [1]
To determine the cellular Km, HEK293 cells stably transfected with human or rat KMO were plated in 96-well plates. Cells were incubated with varying concentrations of L-kynurenine (0–2000 µM) for 8-20 hours such that substrate turnover was less than 15%. The supernatants were analyzed by LC-MS/MS to determine the amount of 3-hydroxykynurenine generated, and data were fitted to the Michaelis-Menten equation. [1]

For efficacy testing in a rat model of acute pancreatitis (AP), male Sprague-Dawley rats were used. AP was induced at laparotomy by a pressure-controlled retrograde biliopancreatic infusion of glycodeoxycholic acid followed by a 6-hour infusion of caerulein. GSK180 was administered therapeutically 1 hour after AP induction as an intravenous bolus of 24 mg/kg followed by a continuous intravenous infusion of 5.5 mg/kg/hour for the remainder of the 6-hour experiment. This regimen delivered stable plasma drug levels of approximately 600 µM. Control animals received vehicle. Tissues and blood were collected at the end of the experiment for analysis. [1]
For pharmacokinetic/pharmacodynamic studies in rats, GSK180 was administered as a single intravenous bolus at 27 mg/kg. Blood samples were taken at various time points to measure plasma drug levels and metabolite (kynurenine, kynurenic acid) concentrations. [1]
For studies in genetically modified mice, Kmo-null and wild-type littermate control mice were used. Experimental AP was induced by retrograde intraductal injection of sodium taurocholate followed by intraperitoneal caerulein. Sham-operated controls underwent the same surgical procedure without toxin infusion. Mice were euthanized at a specified time point for tissue and blood collection. [1]
To assess acute effects of the compound on metabolites, GSK180 was administered to mice as a single bolus injection at 30 mg/kg. Blood was collected one hour post-dose for analysis of drug and metabolite levels. [1]

Following intravenous bolus administration of GSK180 (27 mg/kg) to rats, the volume of distribution (Vss) and clearance (Clp) of the drug were both low (0.45 ml/min/kg), and the half-life (t1/2) was 3 hours. [1] After intravenous bolus administration of 27 mg/kg GSK180 to rats, the initial plasma concentration was close to 600 µM. [1] GSK180 did not bind to rat erythrocytes (blood-to-plasma ratio of 0.46). [1] GSK180 had moderate binding to rat plasma proteins (7.7% free fraction at 1 mM, n=2). [1] GSK180 had high water solubility (24 mg/mL in physiological saline as Tris salt). [1]
GSK180 exhibits high microsomal metabolic stability in various species (clearance in rat, dog, and human tissues is <0.5 ml/min/g). [1]
Treatment with GSK180 results in rapid changes in the levels of kynurenine metabolites in vivo. In rats, intravenous bolus injection leads to a rapid increase in circulating kynurenine and kynuric acid levels, which return to baseline levels as drug concentration decreases. [1]

In Kmo knockout mice, fertility, reproductive capacity, and lifespan (up to 2 years) were unaffected, indicating that the mice tolerated long-term KMO blockade well, with elevated kynurenic acid and kynurenine levels. [1]
GSK180 significantly reduced circulating tryptophan levels in wild-type and Kmo knockout mice, indicating an off-target effect independent of KMO inhibition. This decrease in tryptophan levels was also observed in rats, and this decrease lasted for several hours before recovering as plasma drug concentrations decreased. This effect was attributed to GSK180 displacing tryptophan from plasma proteins in a concentration-dependent manner. [1]
The increase in kynurenic acid levels during KMO blockade may have a sedative effect, as it is an NMDA receptor antagonist. [1]

[1]. Mole DJ, et al. Kynurenine-3-monooxygenase inhibition prevents multiple organ failure in rodent models of acute pancreatitis. Nat Med. 2016 Feb;22(2):202-9.

GSK180 is a potent and specific kynurenine-3-monooxygenase (KMO) inhibitor, a key enzyme in the kynurenine pathway of tryptophan metabolism. [1] KMO inhibition is considered a novel therapeutic strategy for acute pancreatitis-associated multiple organ dysfunction syndrome (AP-MODS) and other potentially serious acute systemic inflammatory diseases. [1] The compound was discovered through a medicinal chemistry strategy based on kynurenine substrate modification, resulting in an oxazolidinone derivative. [1] GSK180 has a low molecular weight (276 Da) and is suitable for intravenous administration. [1] Although GSK180 effectively inhibits KMO and protects organs from damage in disease models, its poor intracellular permeability (low passive permeability) leads to reduced cellular efficacy and, at the high concentrations required for efficacy, can cause off-target effects on tryptophan and kynurenine metabolism. Improving cellular efficacy is considered a key component of future clinical drug candidate development. [1]

C10H7CL2NO4

276.0729

274.975

1799725-26-0

105539874

White to off-white solid powder

2.1

1

4

3

17

338

0

MIGAKMWKMLYGJX-UHFFFAOYSA-N

InChI=1S/C10H7Cl2NO4/c11-5-3-7-8(4-6(5)12)17-10(16)13(7)2-1-9(14)15/h3-4H,1-2H2,(H,14,15)

3-(5,6-dichloro-2-oxo-1,3-benzoxazol-3-yl)propanoic acid

GSK180GSK-180GSK-180

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)

DMSO : ~250 mg/mL (~905.57 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.53 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 20.8 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: ≥ 2.08 mg/mL (7.53 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 20.8 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: ≥ 2.08 mg/mL (7.53 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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