Construction Of PROTAC Degraders Of Androgen Receptor-loaded Dissolving Microneedles And Its Application In The Treatment Of Androgenic Alopecia

Androgenetic alopecia(AGA)is a transracial and cross-gender disease worldwide with a youth-oriented tendency,but it lacks effective treatment.The binding of androgen receptor(AR)and androgen plays an essential role in the occurrence and progression of AGA.Herein,novel proteolysis targeting chimera degrader of AR(AR-PROTAC)is synthesized and integrated with dissolving microneedles(PROTAC-MNs)to successfully transdermal deliver AR-PROTAC in an efficient and minimally invasive manner to achieve AR degradation in hair follicles for AGA treatment,and its hair regeneration efficiency,mechanism and safety in AGA and recrudescence treatment are also evaluated.Firstly,highly efficient AR-PROTAC was successfully developed by linking an E3 ligase CRBN recruiter and an AR antagonist.The PROTAC-MNs composed of HA-based needle loaded with AR-PROTAC and a PVP-K90-based detachable backing layer were fabricated through a two-step centrifugation method.SEM images showed the PROTACMNs were arranged neatly in a 12 × 12 array with pyramid shaped needle.The PROTACMNs possessed sufficient strength to overcome the stratum corneum barrier and effectively transported the encapsulated drug to 200～300 μm deep in the skin,where most telogen HF s reside for AR degradation.Next,testosterone solution was topically applied to mice to establish AGA and recrudescence model for hair regrowth estimation.In AGA treatment,compared with daily applied first-line drug minoxidil,the PROTAC-MNs were able to regenerate hair of comparable quality in hair regeneration area,hair density,hair diameter and hair cuticles status and attained faster induction of hair regrowth,even when applied only once.In recrudescence treatment,the PROTAC-MNs still regenerated superior hair after drug withdrawal,which was incapable of minoxidil.Then,we evaluated the mechanism of PROTAC-MNs from the AR and related signal pathway assessment.The western-blot results proved the AR-PROTAC and PROTAC-MNs achieved AR degradation with the ubiquitin-proteasome system.The ELISA,PCR and immunofluorescence staining findings provided compelling evidence suggesting that the PROTAC-MNs could degrade AR sustainedly,which was beneficial for activating signaling pathways like β-catenin related to hair regeneration while inhibiting the inhibitory factors like TGF-β1 simultaneously,thereby inducing the activation of hair follicle stem cells and consequent hair regeneration.And finally we investigated the biocompatibility of PROTAC-MNs.The body weight,major organ indices,routine blood test and H&E staining of the main organs were not significantly varied with the application of PROTAC-MNs,suggesting systemic safety of PROTAC-MNs.Except for the transient and reversible skin trauma at the application site,the PROTAC-MNs did not influence the normal structure and psychological function of skin.And the PROTAC-MNs did not cause evident androgen deficiency-related chaos in vivo.In this study,we integrated the PROTAC degraders of AR with dissolving microneedles to achieve AR destruction in hair follicles for AGA treatment.The PROTAC-MNs successfully regulated the signaling pathway related to hair growth and activated hair follicle stem cells.After applying only once,the PROTAC-MNs achieved an accelerated onset of hair regeneration as compared to the daily application of the firstline topical drug minoxidil.Intriguingly,PROTAC-MNs via single administration still realized superior hair regeneration in AGA recrudescence treatment,which was the major drawback of minoxidil in clinical practice.Furthermore,the PROTAC-MNs possessed high biocompatibility in systemic functions and topical applied site,especially in sexual functions.Collectively,these AR-degrading dissolving microneedles with long-lasting efficacy,one-step administration,and high biocompatibility provided a great therapeutic potential for AGA treatment.