Androgenetic alopecia (AGA), the most common form of hair loss, affects millions globally. Current treatments have limitations in efficacy and side effects. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway plays a crucial role in hair follicle cycling and inflammation. Aberrant JAK signaling is implicated in AGA pathogenesis. Therefore, targeting JAK kinases with selective inhibitors represents a promising therapeutic strategy. This study focuses on the identification and characterization of MJ04, a novel JAK3 inhibitor developed as a potential treatment for AGA. The study aimed to evaluate MJ04's efficacy, safety, and pharmacokinetic properties in preclinical models. The successful development of a safe and effective treatment for AGA would significantly improve the quality of life for affected individuals. This research is vital due to the large unmet clinical need for effective and well-tolerated AGA therapies. The current options often lack significant efficacy or are associated with unwanted side effects, underscoring the urgency for new treatment modalities.

Extensive research has highlighted the role of the JAK-STAT pathway in hair follicle biology and inflammatory processes associated with AGA. Studies have demonstrated that inhibition of JAK kinases, particularly JAK3, can promote hair growth in preclinical models. Several JAK inhibitors, such as tofacitinib and baricitinib, have shown efficacy in treating inflammatory diseases, but their use in AGA is limited by side effects and lack of hair-specific action. This research builds upon previous findings, investigating the potential of a novel, potentially more specific, JAK3 inhibitor to overcome these limitations and provide a more targeted approach for AGA treatment.

The synthesis of MJ04 involved multiple steps, including a Suzuki cross-coupling reaction between 5-bromo-7-azaindole and 4-fluorophenylboronic acid to yield a 5-aryl substituted 7-azaindole intermediate. Subsequent iodination, Boc protection, and another Suzuki coupling reaction, this time using compound 3, led to the final compound MJ04, purified through sonication and filtration. In vitro studies assessed MJ04's inhibitory activity against JAK1, JAK2, and JAK3 kinases using cell-free assays, determining IC50 values at varying ATP concentrations. The binding affinity and hydrogen bonding interactions of MJ04 with the kinase domains of JAK1, JAK2, and JAK3 were analyzed through molecular docking and molecular dynamics simulations (Figs S1, S2). In vivo studies used a DHT-induced AGA mouse model. C57BL/6J mice received daily topical applications of 0.5% testosterone for an hour before topical treatment with various concentrations of MJ04, tofacitinib, and baricitinib for 28 days. Hair regrowth was monitored through digital photography (Fig S3). MTT assays evaluated the toxicity of MJ04 and tofacitinib in splenocytes (Fig S5). Hematological and biochemical parameters in the mice were assessed (Tables 2, 3). Additionally, studies were performed in a nude mouse model (NU/J Foxn1nu) to further assess efficacy and safety profiles (Table S4). Physicochemical and ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties of MJ04 were predicted using computational tools.

MJ04 demonstrated potent and selective inhibition of JAK3 kinase in vitro, with IC50 values significantly lower than those of tofacitinib. Molecular docking and dynamics simulations revealed MJ04's favorable binding interactions within the JAK3 kinase domain. In vivo studies showed that MJ04 significantly promoted hair regrowth in the DHT-induced AGA mouse model, comparable to or exceeding the effects of tofacitinib and baricitinib at similar or lower doses (Fig S3 and Table S3). The compound showed a dose-dependent effect on hair regrowth. The toxicity studies using splenocytes showed acceptable levels of toxicity in vitro. In vivo hematological analysis showed minimal changes in parameters like WBC, RBC, HGB, HCT, MCV, MCHC, and PLT across treatment groups (Table S3). Similarly, biochemical parameters (Table S3) demonstrated mild to moderate differences between the groups, not exceeding clinically concerning levels. The ADMET predictions suggested MJ04 possessed favorable physicochemical properties, including good absorption, permeability, and relatively low toxicity based on several predictive models. Table S1 shows the detailed inhibition activities against various cell lines. The Natural Product-likeness score indicates that MJ04’s properties are similar to naturally occurring compounds, suggesting possible low toxicity and improved bioavailability. The Lipinski rule, Pfizer rule, and GSK rule assessment provide additional insights into the drug-likeness profile and possible limitations.

The findings demonstrate that MJ04 is a potent and selective JAK3 inhibitor with promising therapeutic potential for AGA. Its superior efficacy in the mouse model of AGA compared to tofacitinib and baricitinib, at comparable or lower doses, suggests a potential for improved clinical outcomes. The favorable ADMET properties further support its suitability as a potential drug candidate. The in vivo safety profile and limited toxicity findings indicate MJ04's potential for better tolerability compared to existing JAK inhibitors used for other inflammatory diseases. However, further studies are required to fully elucidate its mechanism of action and confirm its long-term safety and efficacy in larger animal models and ultimately, in human clinical trials.

MJ04 is a novel JAK3 inhibitor with demonstrated efficacy in a preclinical model of AGA and a favorable safety profile. Its potent and selective JAK3 inhibition, coupled with promising preclinical results, justifies further investigation as a potential treatment for androgenetic alopecia. Future studies will focus on evaluating MJ04's efficacy and safety in larger animal models and ultimately, human clinical trials. Further optimization of its pharmacokinetic profile might also be explored to enhance therapeutic efficacy.

The study was conducted using a mouse model of AGA, which may not fully replicate the complexity of human AGA. The sample size in the in vivo studies was relatively small. Long-term safety and efficacy data are not yet available. The ADMET predictions are based on in silico models and require validation through in vitro and in vivo studies.