Even at concentrations as high as 350 μM, JANEX-1 (WHI-P131) exhibits strong inhibitory activity against JAK3 (IC50 is 78 μM), but not against JAK1 and JAK2, ZAP/SYK family tyrosine kinase SYK, TEC family tyrosine kinase BTK, SRC family tyrosine kinase Acid kinase LYN, or receptor family tyrosine kinase insulin receptor kinase. JAK3-expressing human leukemia cell lines NALM-6 and LC1;19 undergo apoptosis when exposed to JANEX-1, but not melanoma (M24-MET) or squamous cell carcinoma (SQ20B) cells. While JAK3-negative BT-20 breast cancer, M24-MET melanoma, or the SQ20B squamous carcinoma cell line are not inhibited by WHI-P131, it does so in a concentration-dependent way for the JAK3-positive leukemia cell lines DAUDI, RAMOS, LC1;19, NALM-6, MOLT-3, and HL-60. WHI-P131 has an EC50 of 24.4 μM in NALM-6 cells and 18.8 μM in DAUDI cells, which indicates that it suppresses colony formation in a concentration-dependent manner. WHI-P131 reduced several leukemia cell lines' in vitro colony formation by >99% at 100 μM. Conversely, in JAK3-negative M24-MET melanoma or SQ20B squamous carcinoma cell lines, JANEX-1 does not decrease clonogenicity [1].

Even at concentrations as high as 350 μM, JANEX-1 (WHI-P131) exhibits strong inhibitory activity against JAK3 (IC50 is 78 μM), but not against JAK1 and JAK2, ZAP/SYK family tyrosine kinase SYK, TEC family tyrosine kinase BTK, SRC family tyrosine kinase Acid kinase LYN, or receptor family tyrosine kinase insulin receptor kinase. JAK3-expressing human leukemia cell lines NALM-6 and LC1;19 undergo apoptosis when exposed to JANEX-1, but not melanoma (M24-MET) or squamous cell carcinoma (SQ20B) cells. While JAK3-negative BT-20 breast cancer, M24-MET melanoma, or the SQ20B squamous carcinoma cell line are not inhibited by WHI-P131, it does so in a concentration-dependent way for the JAK3-positive leukemia cell lines DAUDI, RAMOS, LC1;19, NALM-6, MOLT-3, and HL-60. WHI-P131 has an EC50 of 24.4 μM in NALM-6 cells and 18.8 μM in DAUDI cells, which indicates that it suppresses colony formation in a concentration-dependent manner. WHI-P131 reduced several leukemia cell lines' in vitro colony formation by >99% at 100 μM. Conversely, in JAK3-negative M24-MET melanoma or SQ20B squamous carcinoma cell lines, JANEX-1 does not decrease clonogenicity [1].

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JANEX-1 treatment inhibited the migration of isolated mouse neutrophils towards interleukin-8 (IL-8) in a concentration-dependent manner in a transwell migration assay. [2]
JANEX-1 treatment inhibited the migration of isolated mouse peritoneal macrophages towards monocyte chemoattractant protein-1 (MCP-1) in a concentration-dependent manner in a transwell migration assay. [2]
Neutrophils and macrophages isolated from JAK3 knockout mice exhibited decreased migration potential towards IL-8 and MCP-1, respectively, mirroring the effect of JANEX-1 treatment. [2]
In myocardial tissue from I/R-injured mice treated with JANEX-1, TUNEL staining showed a significant reduction in apoptotic cells compared to I/R controls. [2]
Western blot analysis of heart tissue from JANEX-1-treated I/R mice showed decreased expression of the pro-apoptotic protein Bax and reduced levels of cleaved caspase-3 and caspase-9 compared to untreated I/R controls. [2]
JANEX-1 treatment did not affect the mRNA expression levels of the chemokines IL-8 and MCP-1 in the myocardium after I/R injury. [2]

There are several dosages of JANEX-1 that can be used: 5 to 100 mg/kg. A dose-response curve with an effective dose 50 (ED50) value of 7.44 mg/kg was found by CPK activity evaluation. The levels of CPK and LDH were significantly lowered in mice given JANEX-1. Furthermore, JANEX-1-treated animals had a considerably smaller infarct size (30.16±2.79%) than I/R-operated mice (65.64±3.76%) [2]. The absorption of JANEX-1 (WHI-P131) occurs quickly; the maximum plasma JANEX-1 concentration (tmax) is reached in 24.7±1.7 minutes. With a 45.6±5.5 minute elimination half-life, JANEX-1 is removed quickly. The systemic exposure level (i.e., AUC) was the same as after intravenous injection, despite the fact that the estimated maximum plasma JANEX-1 concentration of 10.5 ± 0.8 μM is only half the Cmax after intraperitoneal injection of the same bolus dose. The bioavailability was 94.6%. The obtained results (17.1±2.2 μM?h vs. 18.1±1.2 μM?h) were quite similar [3].

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In a murine model of myocardial ischemia (45 min) / reperfusion (24 h) injury, intraperitoneal administration of JANEX-1 (20 mg/kg, 1 h pre-ischemia) significantly reduced serum levels of injury markers creatine phosphokinase (CPK) and lactate dehydrogenase (LDH). [2]
JANEX-1 treatment (20 mg/kg, i.p.) significantly reduced myocardial infarct size (30.16 ± 2.79%) compared to untreated I/R controls (65.64 ± 3.76%) measured by triphenyltetrazolium chloride (TTC) staining. [2]
Echocardiographic assessment 7 days after I/R injury showed that JANEX-1 treatment (20 mg/kg, i.p.) improved cardiac function parameters (ejection fraction, fractional shortening) and reversed I/R-induced left ventricular dilation compared to untreated I/R controls. [2]
Histological analysis (H&E staining) and specific staining for macrophages (F4/80) and neutrophils (myeloperoxidase assay, naphthol AS-D chloroacetate esterase stain) revealed that JANEX-1 treatment significantly reduced the infiltration of both cell types into the infarcted myocardium at 24 hours post-reperfusion. [2]
JANEX-1 treatment significantly reduced the levels of tumor necrosis factor-alpha (TNF-α) in both serum and myocardial tissue after I/R injury. [2]
JAK3 knockout mice subjected to myocardial I/R injury exhibited reduced serum CPK/LDH levels and smaller infarct sizes, phenocopying the protective effects of JANEX-1 pharmacological inhibition. [2]

Neutrophil and Macrophage Migration Assay: Neutrophils were isolated from mouse bone marrow using a negative selection kit. Peritoneal macrophages were elicited by intraperitoneal injection of thioglycollate broth and harvested after 3 days. Cells were pre-incubated with vehicle or JANEX-1 for 2 hours at 37°C. Then, cells were placed in the upper chamber of a transwell insert (3 µm pore for neutrophils, 8 µm pore for macrophages). The lower chamber contained medium with or without the chemokine (IL-8 for neutrophils, MCP-1 for macrophages). After a 2-hour migration period, the number of cells that migrated to the lower chamber was counted using a hemocytometer. A chemotaxis index was calculated. [2]

Dissolved in DMSO in PBS; 20, 50 and 100 mg/kg/day; i.p.
Female NOD mice

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Mouse Myocardial Ischemia/Reperfusion (I/R) Model: Eight-week-old male C57BL/6J or JAK3 knockout mice were anesthetized. A midline sternotomy was performed, and the left coronary artery was ligated for 45 minutes to induce ischemia, followed by release for reperfusion (varying periods: 12h, 24h, 7 days). Sham-operated mice underwent the same surgery without artery occlusion. [2]
Drug Administration: JANEX-1 was dissolved in 10% dimethyl sulfoxide and further diluted 1:100 with phosphate-buffered saline. Mice received a single intraperitoneal injection of JANEX-1 at a dose of 20 mg/kg (or doses ranging from 5-100 mg/kg for dose-response) one hour before the induction of myocardial ischemia. Control mice received vehicle. [2]
Infarct Size Measurement: At the end of reperfusion, the coronary artery was re-occluded, and Evans blue dye was perfused to delineate the area at risk (AAR). The heart was excised, sliced, and incubated with 1% triphenyltetrazolium chloride (TTC) to stain viable tissue red. Infarct size (unstained) was quantified by planimetry and expressed as a percentage of the AAR. [2]
Sample Collection: Blood was collected for serum analysis of CPK, LDH, and TNF-α. Heart tissue was collected for histology, RNA/protein extraction, and myeloperoxidase activity assay. [2]

[1]. Structure-based design of specific inhibitors of Janus kinase 3 as apoptosis-inducing antileukemic agents. Clin Cancer Res. 1999 Jun;5(6):1569-82.

[2]. Inhibition of Janus activated kinase-3 protects against myocardial ischemia and reperfusion injury in mice. Exp Mol Med. 2013 May 17;45:e23.

[3]. In vivo toxicity and pharmacokinetic features of the janus kinase 3 inhibitor WHI-P131 [4-(4'hydroxyphenyl)-amino-6,7- dimethoxyquinazoline. Clin Cancer Res. 1999 Oct;5(10):2954-62.

[4]. IL4 (interleukin 4) induces autophagy in B cells leading to exacerbated asthma. Autophagy. 2018;14(3):450-464.

JANEX-1 (WHI-P131) is a selective Janus kinase 3 (JAK3) pharmacological inhibitor. [2] This study suggests that JANEX-1 does not act directly on cardiomyocytes (cardiomyocytes do not express JAK3), but exerts its protective effect through systemic effects, mainly by inhibiting the migration of JAK3-dependent neutrophils and macrophages to the damaged heart, thereby reducing inflammation-mediated apoptosis damage. [2] The protective effect of JANEX-1 was also observed in JAK3 knockout mice, confirming its targeted mechanism of action. [2] The hypothesized mechanism involves inhibiting inflammatory cell infiltration without affecting the expression of chemokines IL-8 and MCP-1 in the myocardium. [2]

C16H15N3O3

297.31

297.111

202475-60-3

3794

White to gray solid powder

1.3±0.1 g/cm3

468.1±40.0 °C at 760 mmHg

236.9±27.3 °C

0.0±1.2 mmHg at 25°C

1.689

2.73

2

6

4

22

350

0

HOZUXBLMYUPGPZ-UHFFFAOYSA-N

InChI=1S/C16H15N3O3/c1-21-14-7-12-13(8-15(14)22-2)17-9-18-16(12)19-10-3-5-11(20)6-4-10/h3-9,20H,1-2H3,(H,17,18,19)

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: ≥ 2.5 mg/mL (8.41 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 (8.41 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 (8.41 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.)