IL-6; IL-10; IL-17A

Compared to AX-000 hit, AX-024 hydrochloride has a potency that is >10,000-fold greater in terms of preventing TCR-triggered T cell proliferation. AX-024 hydrochloride has an inhibitory effect at a concentration of 1 pM or less, but its IC50 in this assay is 1 nM. At a concentration of 10 nM, AX-024 hydrochloride significantly outperforms AX-000 as an inhibitor of cytokine release by human peripheral blood mononuclear cells stimulated with anti-CD3 and significantly reduces the production of interleukin-6 (IL-6), tumor necrosis factor-α (TNFα), interferon-γ (IFN-γ), IL-10, and IL-17A. AX-024 hydrochloride strongly inhibits T cell proliferation in OT1Tg T cells that are WT for the PRS mutation in mice bearing wild-type (WT) CD8+ T cells at a concentration of 0.1 nM. In these cells, coimmunoprecipitation experiments demonstrate that Nck recruitment to the TCR is induced upon stimulation in the absence of drug but is inhibited in the presence of AX-024 hydrochloride in a dose-dependent manner at concentrations beginning at 1 nM[1].

In comparison to the vehicle group, the AX-024 hydrochloride-treated group has fewer scales and lessened skin thickening. Significantly lessening the thickness of both skin layers, AX-024 hydrochloride works best on the dermis, which resembles the skin of mice treated with a control cream devoid of imiquimod (IMQ). In both assays, AX-024 hydrochloride significantly reduces the quantity of inflammatory cells in the airways. In contrast to mice receiving the vehicle, which experience persistent ataxia and loss of the righting reflex, mice receiving AX-024 hydrochloride experience a rapid recovery from neurological impairment and weight loss, becoming symptom-free by day 30[1].

Cell Trace Violet is used to mark the spleen B cells from C57BL/6 mice. The cells are then incubated for 72 hours with either anti-IgM (10 mg/mL) or anti-CD40 (5 mg/mL), as well as IL-4 (5 ng/mL) or LPS (2.5 mg/mL), in the presence of various concentrations of AX-024 hydrochloride. According to the total number of cell divisions, proliferation is calculated[1].

Acute Toxicity Protocol:** Eight-week-old CD-1 mice were injected intraperitoneally with different amounts of the hydrochloride salt of AX-024 HCl dissolved in 0.5 ml of saline. Animals were observed clinically for adverse reactions twice daily over 14 days. A necropsy was performed on each animal on day 14 to examine abdominal, thoracic, and cranial cavities and associated organs [1].
* **EAE Model Protocol (Prophylactic):** Chronic EAE was induced in female C57BL/6 mice by subcutaneous injection of MOG_35-55 emulsified in Freund's complete adjuvant. Mice received daily intraperitoneal (or oral, as specified) doses of AX-024 HCl (e.g., 10 mg/kg, prepared in PBS or saline) for 10 days starting on the day of immunization. Clinical scores and body weight were monitored daily by an observer blind to the treatment groups [1].
* **EAE Model Protocol (Therapeutic):** EAE was induced as above. Treatment began when more than 50% of the animals reached a clinical score higher than 2 (around day 13). Mice received daily oral doses of AX-024 HCl (10 mg/kg) or fingolimod (0.6 mg/kg) from day 13 to day 26. Clinical scores and body weight were monitored daily [1].
* **Psoriasis Model Protocol (IMQ-induced):** Psoriasis-like skin inflammation was induced in BALB/c mice by daily application of IMQ cream on the shaved back and right ear for 6 consecutive days. AX-024 HCl (50 mg/kg in saline) was administered intraperitoneally on days 0, 2, 4, and 6. Disease severity was scored daily, and skin samples were collected for histological and cytokine analysis at the end of the study [1].
* **Asthma Model Protocol (OVA-induced):** BALB/c mice were sensitized with intraperitoneal injections of OVA in alum on days 0, 5, and 12. AX-024 HCl (50 mg/kg in saline) was injected intraperitoneally on days 0 and 5, 2 hours before OVA administration. On day 12, mice were challenged with an aerosolized OVA solution. Lung inflammation was assessed 24 hours post-challenge by FMT imaging and 48 hours post-challenge by analysis of bronchoalveolar lavage fluid (BALF) [1].
* **Ectromelia Virus Infection Protocol:** C57BL/6 mice were infected intranasally with various doses (10² to 10⁵ PFU) of ECTV. Mice were treated with daily oral doses of AX-024 HCl (10 or 40 mg/kg) or vehicle for up to 10 days post-infection. Survival was monitored. Mice surviving the initial infection were rechallenged 60 days later with a lethal intranasal dose (10⁵ PFU) of ECTV without further treatment to assess memory response [1].

[1]. First-in-class inhibitor of the T cell receptor for the treatment of autoimmune diseases. Sci Transl Med. 2016 Dec 21;8(370):370ra184.

C21H23CLFNO2

375.864228487015

375.14

C, 67.11; H, 6.17; Cl, 9.43; F, 5.05; N, 3.73; O, 8.51

1704801-24-0

AX-024;1370544-73-2

129909862

White to off-white solid powder

1

4

4

26

480

0

DGBCIMPGYRVTPD-UHFFFAOYSA-N

InChI=1S/C21H22FNO2.ClH/c1-24-18-8-9-20-19(12-18)21(15-4-6-17(22)7-5-15)16(14-25-20)13-23-10-2-3-11-23;/h4-9,12H,2-3,10-11,13-14H2,1H3;1H

1-[[4-(4-fluorophenyl)-6-methoxy-2H-chromen-3-yl]methyl]pyrrolidine;hydrochloride

AX-024 hydrochloride; AX-024; AX 024; AX024 HCl

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: ~75 mg/mL (~199.5 mM)
Water: ~4 mg/mL (~10.6 mM)
Ethanol: ~18 mg/mL (~47.9 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.65 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 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 (6.65 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 25.0 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: ≥ 2.5 mg/mL (6.65 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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 (Please use freshly prepared in vivo formulations for optimal results.)