Having an IC50 of 2 nM in Hep3B cells, 9.7 nM in human blood, and 62 nM in mouse plasma, PAT-505 is a strong, selective, noncompetitive, and oral autotaxin inhibitor. Adenosine A3 receptor, MT1 melatonin receptor, prostaglandin E2 EP4 receptor, 5-HT5a serotonin receptor, and GABA-gated Cl- channel binding are all marginally inhibited by PAT-505, which is selective for ATX over other ENPP proteins[1]. The inhibition ranges from 50% to 70% at 10 µM.

With an average IC50 of 62 nM and an average IC90 of 630 nM in mouse plasma and ∼770 nM in rat plasma, PAT-505 inhibits ATX lysoPLD activity. In a mouse model of nonalcoholic steatohepatitis (NASH), PAT-505 (30 mg/kg, po) dramatically lowers the percentage of PSR-positive region, the fibrotic score, and the α-SMA immunoreactivity[1].

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In this study, we describe the preclinical pharmacologic, pharmacokinetic, and pharmacodynamic properties of a novel small-molecule ATX inhibitor, PAT-505 [3-((6-chloro-2-cyclopropyl-1-(1-ethyl-1H-pyrazol-4-yl)-7-fluoro-1H-indol-3-yl) thio)-2-fluorobenzoic acid sodium salt]. PAT-505 is a potent, selective, noncompetitive inhibitor that displays significant inhibition of ATX activity in plasma and liver tissue after oral administration. When dosed therapeutically in a Stelic Mouse Animal Model of nonalcoholic steatohepatitis (NASH), PAT-505 treatment resulted in a small but significant improvement in fibrosis with only minor improvements in hepatocellular ballooning and hepatic inflammation. In a choline-deficient, high-fat diet model of NASH, therapeutic treatment with PAT-505 robustly reduced liver fibrosis with no significant effect on steatosis, hepatocellular ballooning, or inflammation. These data demonstrate that inhibiting autotaxin is antifibrotic and may represent a novel therapeutic approach for the treatment of multiple fibrotic liver diseases, including NASH.[1]

Biochemical Assay with FS-3 Substrate[2]
Starting from 20 μM highest concentration, 10 μL of a dilution series of compound, 1/5 dilution, was added to the wells. Glycosylated human ATX protein (see Supporting Information) was used at a final concentration of 0.4 or 0.64 μg/mL. The enzyme was diluted in 50 mM Tris-HCl (2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride) pH 8.0, 250 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 0.1% fatty acid free BSA in a total volume of 20 μL. The enzyme mixture was added to compounds, and the resulting mixture was incubated for 30 min at room temperature under shaking. The reaction was started by the addition of 20 μL of 0.75 μM FS-3 diluted in the same buffer as described above. Fluorescence was read on an Envision apparatus after 30 min incubation at room temperature (excitation 485 nm, emission 520 nM).[2]

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Biochemical Assay with LPC 16:0 Substrate[2]
Starting from 20 μM highest concentration, 5 μL of a dilution series of compound (1/5 dilution) was added to the wells. Glycosylated human ATX protein (see Supporting Information) was used at a final concentration of 1 or 3 μg/mL. The enzyme was diluted in 50 mM Tris-HCl pH 8.5, 500 mM NaCl, 5 mM KCl, 10 mM CaCl2, and 0.1% fatty acid free BSA in a total volume of 10 μL. The reaction was started by the addition of 10 μL of 150 μM LPC 16:0 diluted in the same buffer as described above, and the mixture was incubated at 37 °C for 30 min. The reaction was terminated and choline quantified by the addition of a 25 μL mixture containing 0.6 U/mL of choline oxidase, 0.6 U/mL of horseradish peroxidase (HRP), 1.8 mM TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium salt dihydrate), 1.2 mM 4-aminoantipyrine, and 20 mM EGTA (ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, stop-developer solution) diluted in the buffer described above. Luminescence was read on an Envision apparatus after 30 min of incubation at room temperature (excitation 555 nm, excitation light = 70%).[2]

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Rat Plasma Assay[2]
Rat plasma was thawed on ice and added into a plate containing a dose range of compound to be tested. After 2 h of incubation at 37 °C, plasma proteins from a 10 μL aliquot were precipitated with an excess of methanol containing LPA 17:0 as internal standard. After centrifugation, the corresponding supernatant was diluted and injected on a C18 column. Analytes were eluted out of the column under isocratic conditions. No calibration curve was prepared for LPA 18:2, and all quantifications were performed based on peak area ratios (LPA 18:2/LPA 17:0). For each concentration of compound, LPA data were expressed as percentage of reduction (% reduction) using the formula: 100 – [((LPA ratio)/(LPA ratio in control sample)) × 100].

NASH is induced in male C57BL/6 mice. Briefly, 5-week-old mice are acclimated for 1 week on normal chow before switching to a choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD) containing 60% kcal% fat and 0.1% methionine. After 4 weeks of CDAHFD feeding, approximately 200 μL of blood is collected from each animal via a submandibular bleed and the serum analyzed for liver enzyme levels. Any animal with a total serum bilirubin level >1 mg/dL is removed from the study prior to compound dosing. Animals are fed CDAHFD for 5 weeks before randomization into treatment groups (n = 7-10 per group). Vehicle or PAT-505 (3-30 mg/kg) is administered by oral gavage in 0.5% methylcellulose (MC) once daily from weeks 5 to 12[1].

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Rat PK/PD with Compound 40 ( an analog of PAT-505)[2]
Male Sprague–Dawley rats were maintained in a controlled environment and dosed at 5 mg/kg with compound 40 formulated in 10% (2-hydroxypropyl)-β-cyclodextrin with pH adjusted to 3 with citric acid (1 mg/mL of 40). Blood samples were collected at the jugular vein via a catheter according to protocols approved by the GALAPAGOS Ethical Committee for animal welfare with the agreement of the Ministère de l’Enseignement Supérieur et de la Recherche and the Direction Départementale de la Protection des Populations, at the following time points: 0.5, 1, and 3 h after dosing and placed into tubes containing Li-heparin as anticoagulant. LPA 18:2 plasma peak areas and compound 40 plasma concentrations were assayed by LC–MS/MS. Plasma concentrations of compound 40 were measured against a calibration curve consisting of eight levels with a 3-log amplitude. Back-calculated values of the QCs (three levels prepared in duplicate) were used for accepting or rejecting the whole batch. The lower limit of quantification was 4 ng/mL for compound 40, using a plasma volume of 25 μL. Plasma proteins were precipitated with an excess of methanol containing the internal standard, and the corresponding supernatant was injected on a C18 column. Analytes were eluted out of the HPLC system by increasing the percentage of the organic mobile phase. Pharmacokinetic parameters were calculated after averaging individual plasma concentrations, by noncompartmental analysis using WinNonlin software (Pharsight, version 5.2). Plasma levels were compiled, and the average of plasma levels of three rats at each sampling time was used. For the analysis of LPA 18:2 plasma peak areas, plasma proteins from a 10 μL aliquot were precipitated with an excess of methanol containing the internal standard, LPA 17:0. After centrifugation, the corresponding supernatant was diluted and injected on a C18 column. Analytes were eluted out of the column under isocratic conditions. No calibration curve was prepared for LPA 18:2, and all quantifications were performed based on peak area ratios (LPA 18:2/LPA 17:0). LPA data were finally expressed as percentage of reduction (% reduction) using the formula: 100 – [((LPA value at time point t)/(mean of LPA value at the same time point t, in vehicle group)) × 100].

[1]. Selective Inhibition of Autotaxin Is Efficacious in Mouse Models of Liver Fibrosis. J Pharmacol Exp Ther. 2017 Jan;360(1):1-13. Epub 2016 Oct 17.

Autotaxin (ATX) is a secreted glycoprotein that converts lysophosphatidylcholine (LPC) to the bioactive phospholipid lysophosphatidic acid (LPA) and is the major enzyme generating circulating LPA. Inhibition of LPA signaling has profound antifibrotic effects in multiple organ systems, including lung, kidney, skin, and peritoneum. However, other LPA-generating pathways exist, and the role of ATX in localized tissue LPA production and fibrosis remains unclear and controversial. In this study, we describe the preclinical pharmacologic, pharmacokinetic, and pharmacodynamic properties of a novel small-molecule ATX inhibitor, PAT-505 [3-((6-chloro-2-cyclopropyl-1-(1-ethyl-1H-pyrazol-4-yl)-7-fluoro-1H-indol-3-yl) thio)-2-fluorobenzoic acid sodium salt]. PAT-505 is a potent, selective, noncompetitive inhibitor that displays significant inhibition of ATX activity in plasma and liver tissue after oral administration. When dosed therapeutically in a Stelic Mouse Animal Model of nonalcoholic steatohepatitis (NASH), PAT-505 treatment resulted in a small but significant improvement in fibrosis with only minor improvements in hepatocellular ballooning and hepatic inflammation. In a choline-deficient, high-fat diet model of NASH, therapeutic treatment with PAT-505 robustly reduced liver fibrosis with no significant effect on steatosis, hepatocellular ballooning, or inflammation. These data demonstrate that inhibiting autotaxin is antifibrotic and may represent a novel therapeutic approach for the treatment of multiple fibrotic liver diseases, including NASH.[1].

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Autotaxin (ATX) is a secreted enzyme playing a major role in the production of lysophosphatidic acid (LPA) in blood through hydrolysis of lysophosphatidyl choline (LPC). The ATX–LPA signaling axis arouses a high interest in the drug discovery industry as it has been implicated in several diseases including cancer, fibrotic diseases, and inflammation, among others. An imidazo[1,2-a]pyridine series of ATX inhibitors was identified out of a high-throughput screening (HTS). A cocrystal structure with one of these compounds and ATX revealed a novel binding mode with occupancy of the hydrophobic pocket and channel of ATX but no interaction with zinc ions of the catalytic site. Exploration of the structure–activity relationship led to compounds displaying high activity in biochemical and plasma assays, exemplified by compound 40. Compound 40 was also able to decrease the plasma LPA levels upon oral administration to rats.[2]

C23H18CLF2N3O2S

473.922729969025

473.077

C, 58.29; H, 3.83; Cl, 7.48; F, 8.02; N, 8.87; O, 6.75; S, 6.76

1782070-22-7

1782070-22-7 (free);1782070-85-2 (sodium);

118094189

Typically exists as Pale purple to off-white solids at room temperature

5.3

1

6

6

32

704

0

BQMMCRXYIIKAOB-UHFFFAOYSA-N

InChI=1S/C23H18ClF2N3O2S/c1-2-28-11-13(10-27-28)29-20(12-6-7-12)22(15-8-9-16(24)19(26)21(15)29)32-17-5-3-4-14(18(17)25)23(30)31/h3-5,8-12H,2,6-7H2,1H3,(H,30,31)

3-[6-chloro-2-cyclopropyl-1-(1-ethylpyrazol-4-yl)-7-fluoroindol-3-yl]sulfanyl-2-fluorobenzoic acid

PAT-505; 1782070-22-7; 53T7TDA5QJ; 3-[6-Chloranyl-2-Cyclopropyl-1-(1-Ethylpyrazol-4-Yl)-7-Fluoranyl-Indol-3-Yl]sulfanyl-2-Fluoranyl-Benzoic Acid; UNII-53T7TDA5QJ; CHEMBL4642052; 3-((6-chloro-2-cyclopropyl-1-(1-ethyl-1H-pyrazol-4-yl)-7-fluoro-1H-indol-3-yl)thio)-2-fluorobenzoic acid; 3-[6-chloro-2-cyclopropyl-1-(1-ethylpyrazol-4-yl)-7-fluoroindol-3-yl]sulfanyl-2-fluorobenzoic acid;

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 : ~48.33 mg/mL (~101.98 mM)

Solubility in Formulation 1: ≥ 4.83 mg/mL (10.19 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 48.3 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: ≥ 4.83 mg/mL (10.19 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 48.3 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.)