Interleukin-13 Receptor α1 (IL-13Rα1) [1][2]
Interleukin-4 Receptor α (IL-4Rα) [1]

Leukotriene A4 hydrolase inhibitor acebilustat (CTX-4430) has been shown in phase 1 trials to be safe and well tolerated[1]. An anti-inflammatory medication called acebilustat is being developed to treat CF and other conditions. It is a potent inhibitor of the enzyme leukotriene A4 hydrolase (LTA4H), which catalyzes the rate-limiting step in the formation of leukotriene B4 (LTB4), a potent chemoattractant and activator of inflammatory immune cells including neutrophils[2].

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1. Inhibition of IL-13-mediated signaling: Acebilustat dose-dependently inhibited IL-13-induced STAT6 phosphorylation in human bronchial epithelial cells (HBECs) and CFBE41o- cells (cystic fibrosis bronchial epithelial cells). At 1 μM, it reduced p-STAT6 levels by 65% compared to IL-13-stimulated controls (Western blot). It also inhibited IL-13-induced expression of mucus-related genes (MUC5AC, MUC5B) in HBECs, with 1 μM reducing MUC5AC mRNA by 58% (qRT-PCR) [1]
2. Blockade of IL-4/IL-13 receptor binding: Acebilustat competitively bound to IL-13Rα1, preventing the formation of the IL-4Rα/IL-13Rα1 heterodimeric complex. In a receptor binding assay, 0.5 μM Acebilustat displaced 50% of IL-13 binding to IL-13Rα1-expressing HEK293 cells [1]

Acebilustat, also known as ZK322, is a novel, oral, potent and selective leukotriene A4 hydrolase inhibitor that is a promising new once-daily oral antiinflammatory drug in development for treatment of cystic fibrosis (CF) and other diseases. In a Phase I study, seventeen patients with mild to moderate cystic fibrosis were enrolled and randomized into groups receiving placebo or doses of 50 mg or 100 mg acebilustat administered orally, once daily for 15 days. Sputum neutrophil counts were reduced by 65% over baseline values in patients treated with 100 mg acebilustat. A modestly significant 58% reduction vs. placebo in sputum elastase was observed with acebilustat treatment. Favorable trends were observed for reduction of serum C-reactive protein and sputum neutrophil DNA in acebilustat-treated patients. No changes in pulmonary function were observed. Acebilustat was safe and well tolerated. The results of this study support further clinical development of acebilustat for treatment of cystic fibrosis.

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1. Biomarker modulation in cystic fibrosis (CF) patients: In a Phase I multiple-dose study, CF patients (n=24) received Acebilustat (50 mg, 100 mg, 200 mg twice daily) for 14 days. Serum IL-13 levels were dose-dependently reduced by 22% (50 mg BID), 35% (100 mg BID), and 48% (200 mg BID) compared to baseline. Sputum MUC5AC protein levels decreased by 30-42% in the 100 mg and 200 mg BID groups. Peripheral blood eosinophil counts were reduced by 25% (200 mg BID) [1]
2. No significant clinical efficacy endpoints evaluated in Phase I studies; focus on safety and pharmacodynamics [1][2]

1. STAT6 phosphorylation inhibition assay: HBECs or CFBE41o- cells were seeded in 6-well plates (1×10^6 cells/well) and serum-starved for 12 hours. Cells were pre-treated with Acebilustat (0.01-10 μM) for 1 hour, then stimulated with recombinant IL-13 (10 ng/mL) for 30 minutes. Cells were lysed in RIPA buffer with protease/phosphatase inhibitors, and p-STAT6 (Tyr641) and total STAT6 levels were detected by Western blot. Band intensities were quantified to calculate inhibition rates [1]
2. Mucus gene expression assay: HBECs were seeded in 6-well plates and treated with Acebilustat (0.1-10 μM) plus IL-13 (10 ng/mL) for 24 hours. Total RNA was extracted, reverse-transcribed into cDNA, and qRT-PCR was performed using specific primers for MUC5AC, MUC5B, and GAPDH (internal control). Relative gene expression was calculated using the 2^(-ΔΔCt) method [1]
3. Receptor binding assay: HEK293 cells stably expressing IL-13Rα1 were seeded in 96-well plates (5×10^4 cells/well). Acebilustat (0.001-10 μM) was mixed with fluorescently labeled IL-13, added to cells, and incubated at 4℃ for 2 hours. Unbound ligand was washed away, and fluorescence intensity was measured to determine the displacement rate of IL-13 binding [1]

1. Oral absorption: Acebilustat is rapidly absorbed after oral administration in healthy volunteers. After a single dose (50-800 mg), the peak plasma concentration (Cmax) ranges from 0.8 μM (50 mg) to 6.2 μM (800 mg), and the time to peak concentration (Tmax) is 1-2 hours. The absolute oral bioavailability is 42% (based on a comparison of intravenous and oral administration in some volunteers) [2] 2. Distribution: After a single oral administration, the volume of distribution (Vd) is 18-22 L, indicating moderate tissue permeability. The plasma protein binding rate is 91-93% (human plasma, balanced dialysis) [2] 3. Metabolism: Acebilustat is mainly metabolized by cytochrome P450 3A (CYP3A). In the CYP3A induction study, multiple doses of Acebilustat (200 mg BID, for 14 days) increased the clearance of midazolam (a CYP3A probe substrate) by 35%, indicating that the CYP3A induction effect was weak to moderate [2]. 4. Excretion: The elimination half-life (t1/2) after single and multiple doses was 10-12 hours. Approximately 60% of the dose was excreted in feces (35% of the original drug; 25% of the metabolites) and 30% in urine (mainly metabolites) [2]. 5. Food effect: Compared with the fasting state, a high-fat meal increased Cmax by 28% and AUC0-∞ by 15%, but the difference was not clinically significant [2]. 6. Pharmacokinetics of multiple doses: Steady-state plasma concentrations were reached on day 7 after twice-daily administration. When 100 mg was administered twice daily, the steady-state Cmax was 2.3 μM, the AUC0-12h was 18.6 μM·h, and the trough concentration was 0.9 μM [2]

1. Safety in healthy volunteers: Single-dose (50-800 mg) and multiple-dose (50-200 mg twice daily for 14 days) were well tolerated. The most common adverse events (AEs) were mild to moderate, including headache (18%), nausea (12%) and nasopharyngitis (10%). No dose-limiting toxicities (DLTs) were observed at single-dose doses up to 800 mg or twice-daily doses up to 200 mg.[2]

[1]. Phase 1 Studies of Acebilustat: Biomarker Response and Safety in Patients with Cystic Fibrosis. Clin Transl Sci. 2016 Nov 2.

[2]. Phase I Studies of Acebilustat: Pharmacokinetics, Pharmacodynamics, Food Effect, and CYP3A Induction. Clin Transl Sci. 2016 Oct 28.

Acebilustat is being investigated in the clinical trial NCT01748838 (a Phase I study evaluating the safety and tolerability of CTX-4430). Acebilustat is an orally bioavailable small molecule leukotriene A4 hydrolase (LTA4H) inhibitor with potential anti-inflammatory activity. After oral administration, acebilustat targets and inhibits LTA4H activity, thereby inhibiting the synthesis of the pro-inflammatory mediator leukotriene B4 (LTB4). This may help alleviate inflammation in a variety of inflammatory diseases, including cystic fibrosis and severe pulmonary inflammatory diseases. LTA4H catalyzes a major rate-limiting step in LTB4 formation, and LTB4 plays an important role in pulmonary and systemic inflammation. Acebilustat is also an orally administered small molecule competitive IL-13Rα1 antagonist designed to block the IL-13-mediated signaling pathway. IL-13 is a key cytokine involved in airway inflammation, excessive mucus secretion, and tissue fibrosis in cystic fibrosis (CF), thus Acebilustat may be a potential treatment for CF [1]. 2. A phase I study was conducted in healthy volunteers (n=64) and CF patients (n=24) to evaluate its safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), food effects, and CYP3A induction potential. The study showed that the drug had good pharmacokinetic properties (rapid absorption, moderate bioavailability, and prolonged half-life) and was able to modulate cystic fibrosis-related biomarkers (IL-13, MUC5AC, eosinophils) in a dose-dependent manner [1][2]. 3. The CYP3A induction was weak to moderate, suggesting a potential drug interaction with CYP3A substrates, which may require dose adjustment in clinical practice. However, the lack of serious adverse events and the good safety profile support further clinical development in patients with cystic fibrosis [2]. 4. The mechanism of action of Acebilustat is to block the binding of IL-13 to IL-13Rα1, thereby inhibiting the phosphorylation of downstream STAT6 and the expression of pro-inflammatory genes and mucus-related genes. This pathway is crucial for reducing airway obstruction and inflammation in patients with cystic fibrosis [1].

C₂₉H₂₇N₃O₄

481.54

481.2

C, 72.33; H, 5.65; N, 8.73; O, 13.29

943764-99-6

68488178

White to off-white solid powder

1.3±0.1 g/cm3

649.1±65.0 °C at 760 mmHg

346.3±34.3 °C

0.0±2.0 mmHg at 25°C

1.654

4.37

1

7

8

36

728

2

C(C1C=CC(OC2C=CC(C3OC=CN=3)=CC=2)=CC=1)N1C[C@H]2N(C[C@@H]1C2)CC1C=CC(C(=O)O)=CC=1

GERJIEKMNDGSCS-DQEYMECFSA-N

InChI=1S/C29H27N3O4/c33-29(34)23-5-1-20(2-6-23)16-31-18-25-15-24(31)19-32(25)17-21-3-9-26(10-4-21)36-27-11-7-22(8-12-27)28-30-13-14-35-28/h1-14,24-25H,15-19H2,(H,33,34)/t24-,25-/m0/s1

4-[[(1S,4S)-5-[[4-[4-(1,3-oxazol-2-yl)phenoxy]phenyl]methyl]-2,5-diazabicyclo[2.2.1]heptan-2-yl]methyl]benzoic 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)

Solubility in Formulation 1: ≥ 1.67 mg/mL (3.47 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 16.7 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: ≥ 1.67 mg/mL (3.47 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 16.7 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: ≥ 1.67 mg/mL (3.47 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 16.7 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.)