Lysophosphatidic acid receptor 1 (LPA₁) (antagonist; IC₅₀ for human LPA₁ = 17 nM; IC₅₀ for mouse LPA₁ = 19 nM in calcium flux assay) [1]
LPA₂ (IC₅₀ = 1.7 μM for human; ~25 μM for mouse) [1]
LPA₃ (IC₅₀ = 1.6 μM for human; 170 nM for mouse) [1]
LPA₄ (IC₅₀ = 7.7 μM for human) [1]
LPA₅ (IC₅₀ = 8.6 μM for human; ~23 μM for mouse) [1]

AM966 is an LPA1 receptor antagonist that is accessible, strong, and selective. Human A2058 melanoma cells (IC50=138±43 nM), human lung fibroblasts IMR-90 (IC50=182±86 nM), and CHO mLPA1 cells (IC50=469±54 nM) are all inhibited by AM966 against LPA1-mediated inhibition[1]. The high-frequency anti-coupling LPA1 but not LPA2-5, AM966 (100 nM), effectively prevented LPA-induced ERK1/2 activation (IC50 = 3.8±0.4 nM). ERK1/2 phosphorylation produced by mianserin is totally blocked by AM966 (100 nM) [2].

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In calcium flux assays using CHO cells stably expressing human or mouse LPA₁ receptors, AM966 potently inhibited LPA-stimulated intracellular calcium release with IC₅₀ values of 17 ± 2 nM (human) and 19 ± 2 nM (mouse). [1]
AM966 demonstrated selectivity for LPA₁ over other LPA receptor subtypes. It was approximately 100-fold more selective for human LPA₁ compared to human LPA₂₋₅, and approximately 10-fold more selective for mouse LPA₁ compared to mouse LPA₂₋₃. [1]
In cell chemotaxis assays, AM966 inhibited LPA-induced migration of cells endogenously expressing LPA₁ receptors. It inhibited chemotaxis of human A2058 melanoma cells (IC₅₀ = 138 ± 43 nM), human IMR-90 lung fibroblasts (IC₅₀ = 182 ± 86 nM), and CHO cells stably expressing mouse LPA₁ (IC₅₀ = 469 ± 54 nM). [1]

In a three-day bleomycin model, AM966 (30 mg/kg, BID) decreases lung damage and inflammation, vascular leakage, and inflammation. 14 days following bleomycin lung injury, AM966 prevents pulmonary fibrosis in a paradigm that lowers lung conditioning and modifies body weight. AM966 decreased tissue damage, vascular leakage, and profibrotic cytokine production in a 14-day bleomycin trial. When compared to pirfenidone, AM966 was more effective in the 14-day bleomycin model. Following bleomycin damage, AM966 can lessen fibrosis and late-stage proliferation [1].

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In a mouse bleomycin-induced lung fibrosis model, oral administration of AM966 (10-60 mg·kg⁻¹, BID) dose-dependently reduced multiple markers of lung injury and fibrosis. [1]
At day 3 post-bleomycin, AM966 (30 mg·kg⁻¹, BID) significantly reduced bronchoalveolar lavage fluid (BALF) protein (a marker of vascular leakage) by 43% and decreased lactate dehydrogenase (LDH) activity (a marker of tissue injury). [1]
At day 7 post-bleomycin, AM966 (30 mg·kg⁻¹, BID) significantly reduced BALF protein, soluble collagen, and total TGFβ1. Histological analysis showed decreased tissue fibrosis and maintenance of normal lung architecture. [1]
At day 14 post-bleomycin, AM966 (30 and 60 mg·kg⁻¹, BID) significantly reduced lung fibrosis as assessed by histopathological scoring and BALF collagen concentrations. It also attenuated body weight loss, reduced total and differential inflammatory cell counts in BALF (macrophages, neutrophils, lymphocytes), decreased BALF protein and LDH activity, and significantly reduced pro-fibrotic biomarkers TIMP-1, TGFβ1, hyaluronan, and MMP-7 in BALF. Dexamethasone (1 mg·kg⁻¹, QD) reduced inflammation but did not affect fibrosis. [1]
In a direct comparison at day 14, AM966 (30 mg·kg⁻¹, BID) demonstrated greater efficacy than pirfenidone (20-400 mg·kg⁻¹, BID) in reducing lung fibrosis, BALF collagen, and tissue injury markers. Pirfenidone showed no significant therapeutic benefit at the tested doses and regimen. [1]
In a 21-day survival study with a high bleomycin dose (5 units·kg⁻¹), AM966 (30 mg·kg⁻¹, BID) significantly improved survival (80% survival vs. 40% in controls) and delayed mortality. [1]
At day 28 post-bleomycin (3 units·kg⁻¹), AM966 (30 mg·kg⁻¹, BID) continued to significantly reduce BALF collagen concentrations. [1]

Calcium Flux Assay: Cells (CHO cells stably expressing LPA receptors or B103 cells transiently expressing receptors) were plated in 96-well plates and cultured overnight. Cells were washed, cultured in serum-free media, then loaded with FLIPR Calcium 4 dye for 1 hour at 37°C. Test compounds (including AM966) were added and incubated for 30 minutes at room temperature. After a 15-second baseline measurement, LPA was added, and intracellular calcium mobilization was measured using a FLEXstation III. IC₅₀ values were calculated. [1]
Cell Chemotaxis Assay: Neuroprobe ChemoTx plates (8 μm pore size) were coated with fibronectin. The lower wells were loaded with 100 nM LPA or vehicle in DMEM/0.1% BSA. Cells (A2058, IMR-90, or CHO mLPA₁) were serum-starved, harvested, and resuspended in media with 0.1-0.2% BSA. Cells were pre-incubated with AM966 or vehicle for 15 minutes at 37°C, then 25,000-50,000 cells were applied to the upper portion of the plate. Plates were incubated for 3-18 hours at 37°C. Cells on the upper filter surface were removed, and the filter was stained. Absorbance was read at 590 nm, and cell numbers were calculated from a standard curve. IC₅₀ values were determined. [1]

Pharmacokinetic Study:** Fasted mice received a single oral dose of AM966 (10 mg·kg⁻¹) in water. Blood was collected via cardiac puncture at 1, 2, 4, 8, and 24 hours post-dose (n=2 per time point). Plasma was analyzed for AM966 concentration by LC-MS/MS. [1]
* **Bleomycin-Induced Lung Fibrosis Model:** Female C57BL/6 mice were lightly anesthetized and given a single intratracheal instillation of bleomycin sulfate (1.5, 3.0, or 5.0 units·kg⁻¹) or saline vehicle. AM966 (1, 10, 30, or 60 mg·kg⁻¹), pirfenidone (20, 100, 400 mg·kg⁻¹), dexamethasone (1 mg·kg⁻¹), or vehicle (water) was administered by oral gavage. Dosing regimens were twice daily (BID) for AM966 and pirfenidone, and once daily (QD) for dexamethasone, starting on day 0 and continuing for the duration of each study (3, 7, 14, 21, or 28 days). At study termination, mice were euthanized, and bronchoalveolar lavage fluid (BALF) and lung tissue were collected for analysis of biomarkers, cell counts, and histopathology. [1]

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Pharmacokinetic Study: Fasted mice received a single oral dose of AM966 (10 mg·kg⁻¹) in water. Blood was collected via cardiac puncture at 1, 2, 4, 8, and 24 hours post-dose (n=2 per time point). Plasma was analyzed for AM966 concentration by LC-MS/MS. [1]
Bleomycin-Induced Lung Fibrosis Model: Female C57BL/6 mice were lightly anesthetized and given a single intratracheal instillation of bleomycin sulfate (1.5, 3.0, or 5.0 units·kg⁻¹) or saline vehicle. AM966 (1, 10, 30, or 60 mg·kg⁻¹), pirfenidone (20, 100, 400 mg·kg⁻¹), dexamethasone (1 mg·kg⁻¹), or vehicle (water) was administered by oral gavage. Dosing regimens were twice daily (BID) for AM966 and pirfenidone, and once daily (QD) for dexamethasone, starting on day 0 and continuing for the duration of each study (3, 7, 14, 21, or 28 days). At study termination, mice were euthanized, and bronchoalveolar lavage fluid (BALF) and lung tissue were collected for analysis of biomarkers, cell counts, and histopathology. [1]

Following a single 10 mg·kg⁻¹ oral dose in mice, AM966 reached a peak plasma concentration of approximately 9 μM within 1 hour. Plasma concentrations declined to 10 nM over 24 hours. [1]
Based on the pharmacokinetic profile and the in vitro IC₅₀ for mouse LPA₁-mediated chemotaxis (469 nM), a twice-daily (BID) dosing regimen was used in efficacy studies to maintain drug coverage above the IC₅₀ over 24 hours. [1]

[1]. A novel, orally active LPA1 receptor antagonist inhibits lung fibrosis in the mouse bleomycin model. Br J Pharmacol. 2010 Aug;160(7):1699-713.

[2]. Antidepressants activate the lysophosphatidic acid receptor LPA(1) to induce insulin-like growth factor-I receptor transactivation, stimulation of ERK1/2 signaling and cell proliferation in CHO-K1 fibroblasts. Biochem Pharmacol. 2015 Jun 15;95(4):311-23.

AM966 [ (4'-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ] is a novel, potent, selective, and orally bioavailable small molecule antagonist of the lysophosphatidic acid receptor 1 (LPA₁). [1]
The therapeutic rationale for AM966 in idiopathic pulmonary fibrosis (IPF) is based on findings that LPA levels are elevated in the BALF of IPF patients, and that LPA₁ receptors mediate fibroblast recruitment and vascular leakage following lung injury. LPA₁ knockout mice are protected from bleomycin-induced fibrosis. [1]
In the mouse bleomycin model, AM966 demonstrated broad anti-fibrotic and anti-inflammatory effects across multiple time points, from the acute injury phase (day 3) through the chronic fibrotic phase (days 14-28). It reduced vascular leakage, tissue injury, inflammatory cell infiltration, and the production of key pro-fibrotic mediators (TGFβ1, TIMP-1, hyaluronan, MMP-7), ultimately leading to reduced collagen deposition, preserved lung architecture, and improved survival. [1]
In a head-to-head comparison, AM966 showed greater efficacy than pirfenidone, an approved anti-fibrotic agent, in reducing fibrosis and tissue injury in the 14-day bleomycin model. [1]

490.13

1228690-19-4

46240292

White to off-white solid powder

6.92

2

6

8

35

708

1

ClC1=C([H])C([H])=C([H])C([H])=C1[C@@]([H])(C([H])([H])[H])OC(N([H])C1C(C([H])([H])[H])=NOC=1C1C([H])=C([H])C(=C([H])C=1[H])C1C([H])=C([H])C(C([H])([H])C(=O)O[H])=C([H])C=1[H])=O

WWQTWEWAPUCDDZ-QGZVFWFLSA-N

InChI=1S/C27H23ClN2O5/c1-16-25(29-27(33)34-17(2)22-5-3-4-6-23(22)28)26(35-30-16)21-13-11-20(12-14-21)19-9-7-18(8-10-19)15-24(31)32/h3-14,17H,15H2,1-2H3,(H,29,33)(H,31,32)/t17-/m1/s1

2-[4-[4-[4-[[(1R)-1-(2-chlorophenyl)ethoxy]carbonylamino]-3-methyl-1,2-oxazol-5-yl]phenyl]phenyl]acetic acid

AM-966 AM966 AM 966

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 (~203.70 mM)

Solubility in Formulation 1: 2.5 mg/mL (5.09 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: ≥ 2.5 mg/mL (5.09 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 10 mg/mL (20.37 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.)