IC50: 2 nM (Autotaxin, Hep3B cell), 9.7 nM (Autotaxin, Human blood), 62 nM (Autotaxin, Mouse plasma)[1]
PAT-505 targets autotaxin (ATX), also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), a secreted enzyme that produces lysophosphatidic acid (LPA) from lysophosphatidylcholine. ATX-LPA signaling plays a critical role in cell proliferation, migration, survival, and inflammation. PAT-505 is a noncompetitive inhibitor with an IC50 of 2 nM in Hep3B cells.

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.

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In vitro, PAT-505 potently inhibits autotaxin activity with an IC50 of 2 nM in Hep3B cells, 9.7 nM in human blood, and 62 nM in mouse plasma. It shows high selectivity over other ENPP family members. The compound inhibits ATX-mediated LPA production and downstream signaling pathways involved in cancer progression and fibrosis.

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]

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In vivo, PAT-505 displays significant inhibition of ATX activity in plasma and liver tissue after oral administration. The compound is orally available and shows potent and selective noncompetitive inhibition of ATX. It has been studied in preclinical models of cancer and fibrosis.

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].

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Specific cell-free enzyme/receptor binding assay protocols for PAT-505 involve autotaxin enzyme activity assays using purified recombinant ATX protein. Enzyme activity is measured by detecting LPA production from lysophosphatidylcholine using fluorescent or mass spectrometry-based assays. IC50 values are determined from concentration-response curves.

In vitro cell-based assays for PAT-505 use Hep3B hepatocellular carcinoma cells and other cell lines expressing ATX. Cells are treated with PAT-505, and LPA production in culture supernatants is measured by ELISA or mass spectrometry. The compound's effects on ATX-LPA signaling are assessed by measuring downstream pathway activation (e.g., AKT, ERK phosphorylation).

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].

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In vivo animal studies for PAT-505 have been conducted in mouse models. The compound is administered orally, and ATX activity is measured in plasma and liver tissue. Efficacy studies in cancer and fibrosis models assess tumor growth inhibition or fibrosis reduction. Pharmacodynamic markers such as LPA levels are measured.

PAT-505 has a molecular formula of C23H18ClF2N3O2S and a molecular weight of 473.92 g/mol. The chemical name is 3-{[6-chloro-2-cyclopropyl-1-(1-ethyl-1H-pyrazol-4-yl)-7-fluoro-1H-indol-3-yl]sulfanyl}-2-fluorobenzoic acid. It has a UNII of 53T7TDA5QJ. The compound is orally available.

Specific toxicological data for PAT-505 are not detailed in the available literature. The compound is classified for research use only and is not intended for human therapeutic applications. As an ATX inhibitor that blocks LPA production, potential toxicities may relate to effects on cell proliferation and migration.

[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 secretory glycoprotein that converts lysophosphatidylcholine (LPC) into biologically active phospholipid lysophosphatidic acid (LPA) and is the main enzyme generating circulating LPA. Inhibition of the LPA signaling pathway has significant anti-fibrotic effects in multiple organ systems, including the lungs, kidneys, skin, and peritoneum. However, LPA is generated through multiple pathways, and the role of ATX in local tissue LPA generation and fibrosis remains unclear and controversial. This study describes the preclinical pharmacology, pharmacokinetics, and pharmacodynamics 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-fluorobenzoate sodium salt]. PAT-505 is a potent, selective, non-competitive inhibitor that significantly inhibits ATX activity in plasma and liver tissue after oral administration. In a Stelic mouse model of nonalcoholic steatohepatitis (NASH), PAT-505 treatment significantly improved liver fibrosis, but had little effect on hepatocellular ballooning degeneration and liver inflammation. In a choline-deficient, high-fat diet-induced NASH model, PAT-505 treatment significantly reduced the degree of liver fibrosis, but had no significant effect on steatosis, hepatocellular ballooning degeneration, or inflammation. These data suggest that inhibiting autosecodylin has an antifibrotic effect and may represent a novel treatment for a variety of fibrotic liver diseases, including nonalcoholic steatohepatitis. [1] Autosecodylin (ATX) is a secretase that plays an important role in the production of lysophosphatidylcholine (LPA) in the blood by hydrolyzing lysophosphatidylcholine (LPC). The ATX-LPA signaling pathway has attracted much attention in the field of drug development because it is associated with a variety of diseases, including cancer, fibrotic diseases, and inflammation. A series of imidazo[1,2-a]pyridine ATX inhibitors were discovered through high-throughput screening (HTS). The co-crystal structure of one of the compounds with ATX revealed a novel binding mode: the compound occupied the hydrophobic pockets and channels of ATX but did not interact with the zinc ions at the catalytic site. Exploration of the structure-activity relationship revealed compounds that exhibited high activity in biochemical and plasma assays, such as compound 40. Compound 40 also reduced plasma LPA levels after oral administration to rats. [2]

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PAT-505 (CAS 1782070-22-7) is a potent, selective, noncompetitive, orally available autotaxin inhibitor with IC50s of 2 nM (Hep3B cells), 9.7 nM (human blood), and 62 nM (mouse plasma). It shows high selectivity over other ENPP family members. The compound inhibits ATX activity in plasma and liver tissue after oral administration.

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.)