Autotaxin (IC50 = 2.8 nM)

Additionally, PF-8380 inhibits rat autotaxin, a substrate for FS-3, with an IC50 of 1.16 nM. When fetal fibroblast-produced enzymes were combined with lysophosphatidylcholine (LPC) as a substrate, PF-8380's efficacy remained intact. When human whole blood was treated with PF-8380 for two hours at an IC50 of 101 nM, autocrine motility factors were suppressed [1]. Lysophospholipase D (lysoPLD) activity is exhibited by the enzyme autotaxin (ATX), which catalyzes the conversion of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA). After applying 1 μM PF-8380 as a pretreatment to GL261 and U87-MG cells, they were exposed to 4 Gy of radiation, which led to a decrease in clone survival, reduced migration (33% in GL261; P=0.002 and 17.9% in U87-MG; P=0.012), decreased invasion (35.6% in GL261; P=0.0037; 31.8% in U87-MG; P=0.002), and attenuate radiation-induced Akt phosphorylation [2].

PF-8380's pharmacokinetic properties were assessed over the course of 24 hours at intravenous dosages of 1 mg/kg and oral doses ranging from 1 to 100 mg/kg. PF-8380 has an effective t1/2 of 1.2 hours, a steady-state distribution volume of 3.2 L/kg, and an average clearance rate of 31 mL/min/kg. There is moderate oral bioavailability, with a range of 43% to 83%. As single oral dosages are increased, plasma concentrations rise as well; however, the rate of increase in Cmax is less than proportional to doses of 10 to 100 mg/kg but about proportionate to doses of 1 to 10 mg/kg. Up to 100 mg/kg, exposure to PF-8380 is roughly dose-proportional and linear, as indicated by the area under the curve. The amounts of plasma C16:0, C18:0, and C20:0 LPA were tested right away following collection. With the 3 mg/kg dose, the largest drop in LPA levels was seen at 0.5 hours, and within 24 hours, all LPAs were returned to or exceeded baseline [1]. Tumor-associated vascularity increased by a moderate 20% after treatment with 10 mg/kg PF-8380 (P=0.497). 45 minutes prior to 4 Gy irradiation, PF-8380 treatment decreased vascularity in mice treated relative to controls by approximately 48% (P=0.031) and by 65% (P=0.011) in mice treated with radiation alone[2].

ATX ELISA and ATX activity assay. [3]
BOS and Non-BOS cell lines were cultured in 60-mm dishes until confluent. Cells were washed once with PBS and then serum starved for 24 hours. Serum-free supernatant was collected, and ATX levels were measured with a Human ENPP-2/Autotaxin Quantikine ELISA Kit according to the manufacturer’s protocol. Absorbance at 450 nm was measured using a SpectraMax M3 multi-mode microplate reader. For ATX activity, cell supernatant was collected, centrifuged at 17,000 g for 10 minutes at 4°C to sediment floating cells or debris, and concentrated to one-eighth of the original volume with an Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-3 membrane. After measurement of protein concentration, an equal amount of total protein was subjected to ATX activity assay with the fluorogenic phospholipid ATX substrate FS-3. Briefly, 30 μl supernatant and 40 μl FS-3 solution (containing 5 μM FS-3, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 50 mM Tris-HCl pH 8.0, and 1 mg/ml BSA) were mixed and loaded to a Costar 96-well black-wall, clear-bottom plate. Fluorescence of samples was measured using a SpectraMax M3 multi-mode microplate reader at excitation and emission wavelengths of 485 nm and 528 nm, respectively. For ATX activity assays in lung lysates, 20 μl allograft lysate and 40 μl FS-3 solution were mixed, and ATX activity was measured similarly for placebo- and PF-8380–treated lung allografts.

Co-culture clonogenic survival assay [2]
HUVEC (1.0 × 106) and bEnd.3 cells (1.0 × 106) were plated in 100 mm plates and after 24 h, U87-MG (2 × 106) and GL261 (2 × 106) cells were plated onto transwell inserts. After co-culture for 24 h, cells were treated with 1 μM of PF-8380 or vehicle control DMSO for 45 min prior to irradiation with 0, 2, 4, 6, or 8 Gy. After the treatments as co-culture with either PF-8380 or DMSO calculated numbers of U87-MG and GL261 cells were plated to enable normalization for plating efficiencies. After 7 to 10 day incubation plates were fixed with 70% EtOH and stained with 1% methylene blue. Colonies consisting of>50 cells were counted by viewing the plates under a microscope. The survival fractions were calculated as (number of colonies/number of cells plated)/(number of colonies for corresponding control/number of cells plated). Survival curves were analyzed by curve fitting to the alpha/beta model calculating D0 and n.

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Wound healing/scratch assay for cell migration[2]
GL261 or U87-MG cells were plated in triplicate onto 6 cm plates and allowed to grow to 70% confluence. The semi-confluent cell layer was scratched with a sterile 200 μL pipette tip to create a scratch devoid of cells and plates were washed once with PBS to remove non-adherent cells and debris. For radiosensitization drug studies, cells were treated with 1 μM PF-8380 or DMSO for 45 min prior to irradiation with 4 Gy, and then incubated at 37°C in 5% CO2. Control plates were monitored for cell migration (20–24 h). Cells were fixed with 70% ethanol and stained with 1% methylene blue. To quantify migration, cells in three randomly selected high power fields (HPFs) in the scratched area were counted and normalized for surrounding cell density. Mean and standard error for each treatment group were calculated.

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Tumor transwell-invasion assays[2]
The tumor transwell matrigel invasion assay has previously been used to aid in quantitation of the tumor endothelium interactions and transmigrations. GL261 (1.0 × 106 cells/well) or U87-MG (0.6 × 106 cells/well) were suspended in serum-free media and added onto the upper chamber (inserts) that was matrix-coated polycarbonate membrane filters with 8 μm pores. Five hundred microliters of fresh medium was added to the bottom chamber. For radiosensitization drug studies, both chambers were then treated with vehicle DMSO or 1 μM PF-8380 for 45 min prior to irradiation with 4 Gy. After 36 h, remaining cells in the upper chamber of the membrane inserts were removed using a wet cotton swab. The cells that adhered on the outer surface of the transwell insert membrane which had invaded through the matrigel were fixed with 100% methanol, and stained. Invaded cells in 7–10 HPF from each sample were counted using Image J Software, and the average number of invaded cells per HPF was calculated. Mean and standard error for each treatment group were calculated.

Mice, treatment, and tumor growth delay [2]
All animal procedures used in this study were approved by IACUC. Handling of animals and housing was followed as per DCM guidelines. GL261 cells (1 × 106) were injected into the right hind limb of nude mice. Once tumors were palpable the mice were serpentine sorted into groups of six to seven animals representing similar distributions of tumor sizes (range = 240 mm3). Tumor bearing mice were injected intraperitoneally with vehicle (DMSO) or PF-8380 at 10 mg/kg body weight once daily for five consecutive days. Forty five minutes after drug injection, mice were anesthetized with isoflurane and positioned in the RS2000 irradiator. They were then irradiated with 2 Gy daily for five consecutive days for a total of 10 Gy. Lead blocks (10 mm thick) were used to shield the head, thorax, and abdomen. Tumor size was monitored longitudinally using an external traceable digital caliper.

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Oral gavage was performed in a containment room of the animal facility. PF-8380 and AM095 were dissolved in PEG 400 at a concentration of 6 mg/ml. Body weights of animals were measured daily. Treatment with PF-8380 or AM095 was administered by oral gavage twice daily at a dosage of 30 mg/kg body weight starting from day 14 after lung transplantation. Placebo-treated mice were given vehicle (PEG 400) via oral gavage ingestion. On day 40 after lung transplantation, mice were sacrificed, and lung allografts were harvested for Western blotting, hydroxyproline assay, or immunohistochemistry.

[1]. A novel autotaxin inhibitor reduces lysophosphatidic acid levels in plasma and the site of inflammation. J Pharmacol Exp Ther. 2010 Jul;334(1):310-7.

[2]. Autotaxin Inhibition with PF-8380 Enhances the Radiosensitivity of Human and Murine Glioblastoma Cell Lines. Front Oncol. 2013 Sep 17;3:236.

[3]. Autocrine lysophosphatidic acid signaling activates β-catenin and promotes lung allograft fibrosis. J Clin Invest. 2017 Apr 3;127(4):1517-1530.

Autotaxin is the enzyme responsible for the production of lysophosphatidic acid (LPA) from lysophosphatidyl choline (LPC), and it is up-regulated in many inflammatory conditions, including but not limited to cancer, arthritis, and multiple sclerosis. LPA signaling causes angiogenesis, mitosis, cell proliferation, and cytokine secretion. Inhibition of autotaxin may have anti-inflammatory properties in a variety of diseases; however, this hypothesis has not been tested pharmacologically because of the lack of potent inhibitors. Here, we report the development of a potent autotaxin inhibitor, PF-8380 [6-(3-(piperazin-1-yl)propanoyl)benzo[d]oxazol-2(3H)-one] with an IC(50) of 2.8 nM in isolated enzyme assay and 101 nM in human whole blood. PF-8380 has adequate oral bioavailability and exposures required for in vivo testing of autotaxin inhibition. Autotaxin's role in producing LPA in plasma and at the site of inflammation was tested in a rat air pouch model. The specific inhibitor PF-8380, dosed orally at 30 mg/kg, provided >95% reduction in both plasma and air pouch LPA within 3 h, indicating autotaxin is a major source of LPA during inflammation. At 30 mg/kg PF-8380 reduced inflammatory hyperalgesia with the same efficacy as 30 mg/kg naproxen. Inhibition of plasma autotaxin activity correlated with inhibition of autotaxin at the site of inflammation and in ex vivo whole blood. Furthermore, a close pharmacokinetic/pharmacodynamic relationship was observed, which suggests that LPA is rapidly formed and degraded in vivo. PF-8380 can serve as a tool compound for elucidating LPA's role in inflammation. [1]

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Purpose: Glioblastoma multiforme (GBM) is an aggressive primary brain tumor that is radio-resistant and recurs despite aggressive surgery, chemo, and radiotherapy. Autotaxin (ATX) is over expressed in various cancers including GBM and is implicated in tumor progression, invasion, and angiogenesis. Using the ATX specific inhibitor, PF-8380, we studied ATX as a potential target to enhance radiosensitivity in GBM. Methods and materials: Mouse GL261 and Human U87-MG cells were used as GBM cell models. Clonogenic survival assays and tumor transwell invasion assays were performed using PF-8380 to evaluate role of ATX in survival and invasion. Radiation dependent activation of Akt was analyzed by immunoblotting. Tumor induced angiogenesis was studied using the dorsal skin fold model in GL261. Heterotopic mouse GL261 tumors were used to evaluate the efficacy of PF-8380 as a radiosensitizer. Results: Pre-treatment of GL261 and U87-MG cells with 1 μM PF-8380 followed by 4 Gy irradiation resulted in decreased clonogenic survival, decreased migration (33% in GL261; P = 0.002 and 17.9% in U87-MG; P = 0.012), decreased invasion (35.6% in GL261; P = 0.0037 and 31.8% in U87-MG; P = 0.002), and attenuated radiation-induced Akt phosphorylation. In the tumor window model, inhibition of ATX abrogated radiation induced tumor neovascularization (65%; P = 0.011). In a heterotopic mouse GL261 tumors untreated mice took 11.2 days to reach a tumor volume of 7000 mm(3), however combination of PF-8380 (10 mg/kg) with irradiation (five fractions of 2 Gy) took more than 32 days to reach a tumor volume of 7000 mm(3). Conclusion: Inhibition of ATX by PF-8380 led to decreased invasion and enhanced radiosensitization of GBM cells. Radiation-induced activation of Akt was abrogated by inhibition of ATX. Furthermore, inhibition of ATX led to diminished tumor vascularity and delayed tumor growth. These results suggest that inhibition of ATX may ameliorate GBM response to radiotherapy. [2]

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Tissue fibrosis is the primary cause of long-term graft failure after organ transplantation. In lung allografts, progressive terminal airway fibrosis leads to an irreversible decline in lung function termed bronchiolitis obliterans syndrome (BOS). Here, we have identified an autocrine pathway linking nuclear factor of activated T cells 2 (NFAT1), autotaxin (ATX), lysophosphatidic acid (LPA), and β-catenin that contributes to progression of fibrosis in lung allografts. Mesenchymal cells (MCs) derived from fibrotic lung allografts (BOS MCs) demonstrated constitutive nuclear β-catenin expression that was dependent on autocrine ATX secretion and LPA signaling. We found that NFAT1 upstream of ATX regulated expression of ATX as well as β-catenin. Silencing NFAT1 in BOS MCs suppressed ATX expression, and sustained overexpression of NFAT1 increased ATX expression and activity in non-fibrotic MCs. LPA signaling induced NFAT1 nuclear translocation, suggesting that autocrine LPA synthesis promotes NFAT1 transcriptional activation and ATX secretion in a positive feedback loop. In an in vivo mouse orthotopic lung transplant model of BOS, antagonism of the LPA receptor (LPA1) or ATX inhibition decreased allograft fibrosis and was associated with lower active β-catenin and dephosphorylated NFAT1 expression. Lung allografts from β-catenin reporter mice demonstrated reduced β-catenin transcriptional activation in the presence of LPA1 antagonist, confirming an in vivo role for LPA signaling in β-catenin activation.[3]

C22H21CL2N3O5

478.3252

477.085

C, 55.24; H, 4.43; Cl, 14.82; N, 8.78; O, 16.72

1144035-53-9

PF-8380 hydrochloride;2070015-01-7

25265312

White to light brown solid powder

1.4±0.1 g/cm3

1.616

3.63

1

6

7

32

693

0

ClC1C([H])=C(C([H])=C(C=1[H])C([H])([H])OC(N1C([H])([H])C([H])([H])N(C([H])([H])C([H])([H])C(C2C([H])=C([H])C3=C(C=2[H])OC(N3[H])=O)=O)C([H])([H])C1([H])[H])=O)Cl

JMSUDQYHPSNBSN-UHFFFAOYSA-N

InChI=1S/C22H21Cl2N3O5/c23-16-9-14(10-17(24)12-16)13-31-22(30)27-7-5-26(6-8-27)4-3-19(28)15-1-2-18-20(11-15)32-21(29)25-18/h1-2,9-12H,3-8,13H2,(H,25,29)

3,5-dichlorobenzyl 4-(3-oxo-3-(2-oxo-2,3-dihydrobenzo[d]oxazol-6-yl)propyl)piperazine-1-carboxylate

PF-8380; PF 8380; PF8380; 1144035-53-9; 3,5-Dichlorobenzyl 4-(3-oxo-3-(2-oxo-2,3-dihydrobenzo[d]oxazol-6-yl)propyl)piperazine-1-carboxylate; (3,5-Dichlorophenyl)methyl 4-[3-oxo-3-(2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)propyl]piperazine-1-carboxylate; T582DIM5A4; 1-Piperazinecarboxylic acid, 4-[3-(2,3-dihydro-2-oxo-6-benzoxazolyl)-3-oxopropyl]-, (3,5-dichlorophenyl)methyl ester; UNII-T582DIM5A4;

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 : ~100 mg/mL (~209.06 mM)

Solubility in Formulation 1: ≥ 0.67 mg/mL (1.40 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 6.7 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: ≥ 0.67 mg/mL (1.40 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 6.7 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: ≥ 0.67 mg/mL (1.40 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 6.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10 mg/mL (20.91 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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