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].

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].

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Pretreatment 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 [2]

The pharmacokinetic properties of PF-8380 were examined over 24 hours at dosages of 1 mg/kg intravenously and oral doses of 1 to 100 mg/kg. The average clearance rate of PF-8380 is 31 mL/min/kg, the steady-state distribution volume is 3.2 L/kg, and the effective t1/2 is 1.2 h. Oral bioavailability is moderate, ranging from 43% to 83%. Plasma concentrations increase with increasing single oral doses, however the rate of increase in Cmax is approximately proportionate to doses of 1 to 10 mg/kg but less than proportional to doses of 10 to 100 mg/kg. Exposure to PF-8380, measured by the area under the curve, is generally dose-proportional and linear up to 100 mg/kg. Plasma C16:0, C18:0 and C20:0 LPA levels were tested immediately after collection. The highest reduction in LPA levels was found at 0.5 hours with the 3 mg/kg dosage, with all LPAs recovering to or above baseline by 24 hours [1]. Treatment with 10 mg/kg PF-8380 resulted in a modest 20% increase in tumor-associated vascularity (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].

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In a heterotopic mouse GL261 tumor model, untreated mice took 11.2 days to reach a tumor volume of 7000 mm³, while combination of PF-8380 (10 mg/kg) with irradiation (five fractions of 2 Gy) took more than 32 days to reach the same tumor volume [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.

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The concentration of PF-8380 was determined based on the kinetics of inhibition of LysoPLD activity of purified ATX [2]

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.

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For clonogenic survival assays, U87-MG and GL261 cells were grown in co-culture with endothelial cells, treated with 1 µM PF-8380 or DMSO for 45 min prior to irradiation with 0, 2, 4, 6, or 8 Gy, incubated for 7–10 days, fixed, stained, and colonies >50 cells were counted [2]
For migration assays, GL261 or U87-MG cells were scratched, treated with 1 µM PF-8380 or DMSO for 45 min prior to 4 Gy irradiation, incubated for 20–24 h, fixed, stained, and migrated cells were counted [2]
For invasion assays, GL261 or U87-MG cells were placed in matrix-coated transwell inserts, treated with 1 µM PF-8380 or DMSO for 45 min prior to 4 Gy irradiation, incubated for 36 h, fixed, stained, and invaded cells were counted [2]
For immunoblot analysis, cells were lysed after treatment and proteins were analyzed using antibodies against phospho-Akt and total Akt [2]

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.

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GL261 cells were injected into the right hind limb of nude mice. Once tumors were palpable, mice were treated intraperitoneally with 10 mg/kg PF-8380 or vehicle daily for five consecutive days. Forty-five minutes after drug injection, mice were irradiated with 2 Gy daily for five days. Tumor size was monitored longitudinally [2]

The pharmacokinetics of PF-8380 have been extensively studied and have shown good oral bioavailability. When administered orally at a dose of 30 mg/kg, it can reduce LPA levels in plasma and inflamed tissues by more than 95% [3].

Long-term use of PF-8380 according to the stated dosage regimen has been shown to have no toxic effects in mice [3]

[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.

Autosecretrin is an enzyme responsible for converting lysophosphatidylcholine (LPC) to lysophosphatidyl acid (LPA), and its expression is upregulated in a variety of inflammatory diseases, including but not limited to cancer, arthritis, and multiple sclerosis. The LPA signaling pathway promotes angiogenesis, mitosis, cell proliferation, and cytokine secretion. Inhibition of autosecretrin may have anti-inflammatory effects in various diseases; however, this hypothesis has not been pharmacologically validated due to the lack of effective inhibitors. This article reports the development of a highly effective autosecretrin inhibitor, PF-8380 [6-(3-(piperazin-1-yl)propionyl)benzo[d]oxazol-2(3H)-one], with an IC50 of 2.8 nM in an enzyme activity assay and 101 nM in human whole blood. PF-8380 has sufficient oral bioavailability and in vivo exposure for in vivo studies of autosecretrin inhibition. This study used a rat air sac model to investigate the role of autosecretin in the production of lysophosphatidic acid (LPA) in plasma and at sites of inflammation. Oral administration of the specific inhibitor PF-8380 at a dose of 30 mg/kg reduced LPA levels in plasma and air sacs by more than 95% within 3 hours, indicating that autosecretin is a major source of LPA during inflammation. PF-8380 at 30 mg/kg reduced inflammatory hyperalgesia, with efficacy comparable to naproxen at 30 mg/kg. Inhibition of plasma autosecretin activity was positively correlated with inhibition of autosecretin activity at sites of inflammation and in isolated whole blood. Furthermore, a close pharmacokinetic/pharmacodynamic relationship was observed, suggesting rapid LPA generation and degradation in vivo. PF-8380 can serve as a tool compound for elucidating the role of LPA in inflammation. [1]

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Objective: Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor with radioresistance, prone to recurrence even with aggressive surgery, chemotherapy, and radiotherapy. Autosoretin (ATX) is overexpressed in various cancers, including GBM, and is associated with tumor progression, invasion, and angiogenesis. We investigated ATX as a potential target for enhancing the radiosensitivity of GBM using the ATX-specific inhibitor PF-8380. Methods and Materials: We used mouse GL261 cells and human U87-MG cells as GBM cell models. Colony formation survival assays and tumor Transwell invasion assays were performed using PF-8380 to evaluate the role of ATX in cell survival and invasion. Radiodependent Akt activation was analyzed by immunoblotting. Tumor-induced angiogenesis was studied using a GL261 dorsal skin fold model. This study used an ectopic mouse GL261 tumor model to evaluate the efficacy of PF-8380 as a radiosensitizer. The results showed that pretreatment of GL261 and U87-MG cells with 1 μM PF-8380 followed by 4 Gy irradiation reduced cell colony formation, migration (33% reduction in GL261 cells, P = 0.002; 17.9% reduction in U87-MG cells, P = 0.012), invasion (35.6% reduction in GL261 cells, P = 0.0037; 31.8% reduction in U87-MG cells, P = 0.002), and radiation-induced Akt phosphorylation levels. In the tumor window model, inhibition of ATX eliminated radiation-induced tumor angiogenesis (reduced by 65%, P = 0.011). In the ectopic mouse GL261 tumor model, it took 11.2 days for untreated mice to reach a tumor volume of 7000 mm³, while it took more than 32 days for PF-8380 (10 mg/kg) combined with radiotherapy (5 fractions, 2 Gy each) to reach a tumor volume of 7000 mm³. Conclusion: PF-8380 inhibition of ATX can reduce the invasiveness of GBM cells and enhance their radiosensitivity. ATX inhibitors can block radiation-induced Akt activation. In addition, ATX inhibitors can reduce tumor angiogenesis and delay tumor growth. These results suggest that inhibition of ATX may improve the response of GBM to radiotherapy. [2]

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Tissue fibrosis is a major cause of long-term graft failure after organ transplantation. In lung transplantation, progressive terminal airway fibrosis leads to irreversible decline in lung function, namely bronchiolitis obliterans syndrome (BOS). This study identified an autocrine pathway linking activated T cell nuclear factor 2 (NFAT1), autocrine factor (ATX), lysophosphatidic acid (LPA), and β-catenin to promote fibrosis progression in lung transplantation. Botanical stem cells (BOS MCs) derived from fibrotic lung transplants exhibit constitutive nuclear β-catenin expression, which depends on autocrine ATX and LPA signaling. We found that NFAT1 is upstream of ATX and regulates the expression of both ATX and β-catenin. Silencing NFAT1 in BOS MCs inhibited ATX expression, while 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 through a positive feedback loop. In an in vivo model of bronchoobacterial syndrome (BOS) induced by orthotopic lung transplantation in mice, LPA receptor (LPA1) antagonists or ATX inhibitors reduced graft fibrosis, accompanied by decreased expression of active β-catenin and dephosphorylated NFAT1. Allogeneic lung transplantation from β-catenin reporter mice showed reduced β-catenin transcriptional activation in the presence of LPA1 antagonists, confirming the in vivo role of LPA signaling in β-catenin activation. [3]

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PF-8380 is a non-lipid small molecule autosecodyne (ATX) inhibitor that inhibits its lysophospholipase D activity, thereby reducing the production of lysophosphatidic acid, which in turn weakens Akt signaling, reduces tumor invasion, migration and angiogenesis, and enhances radiosensitivity in a glioblastoma model. [2]

C22H22CL3N3O5

514.786182880402

513.062

2070015-01-7

PF-8380;1144035-53-9

78357775

White to off-white solid powder

2

6

7

33

693

0

ClC1C=C(C=C(C=1)COC(N1CCN(CCC(C2=CC=C3C(=C2)OC(N3)=O)=O)CC1)=O)Cl.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 hydrochloride

PF8380 HCl; PF-8380; PF-8380 (hydrochloride); PF-8380 hydrochloride; 2070015-01-7; (3,5-dichlorophenyl)methyl 4-[3-oxo-3-(2-oxo-3H-1,3-benzoxazol-6-yl)propyl]piperazine-1-carboxylate;hydrochloride; 1-Piperazinecarboxylic acid, 4-[3-(2,3-dihydro-2-oxo-6-benzoxazolyl)-3-oxopropyl]-, (3,5-dichlorophenyl)methyl ester, hydrochloride (1:1); PF 8380

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 5.2 mg/mL (~10.10 mM)
H2O : < 0.1 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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