Effects of oxytocin receptor agonists on promoting hair growth

OXTR, a 7-transmembrane G protein-coupled receptor, activates multiple signaling cascades such as: B. the mitogen-activated protein kinase, protein kinase C or phosphoinositide-specific phospholipase C pathways¹⁷. These signaling pathways maintain homeostasis in various organs and tissues, and activation of OXTR could be a therapeutic strategy for certain diseases. Previous studies have reported that OXTR agonist improves social interactions in a mouse model of autism¹⁴reduces alcohol consumption in male mice¹⁸and increases social and cognitive functions in a rat model of schizophrenia¹⁹. In the skin, dermal fibroblasts and keratinocytes express OXTR, and oxytocin signaling plays a role in regulating cell proliferation, inflammation, senescence, and oxidative stress responses²⁰. However, to the best of our knowledge, there are no studies on the effects of OXTR on cells in hair follicles. In the present study, we demonstrated that LIT001 and WAY267464, non-peptidergic specific agonists of OXTR, have hair growth promoting effects, which are promising as drug candidates for the treatment of alopecia. Thus, these findings will accelerate the search for novel OXTR-targeting drugs.

To address the stability and skin permeability issues of oxytocin, this study examined OXTR agonists. Molecules with MW >500 reportedly have difficulty penetrating healthy skin²¹. LIT001 and WAY267464, with MWs of 531 and 655, respectively, are smaller than oxytocin (MW 1007), but still exceed this value. However, since the target of OXTR agonists is hair follicles, particularly DP cells, these molecules could reach DP cells through the pores of hair follicles. This is partially supported by a previous report that bioactive peptides with relatively large MW (MW of 509 and 774) were delivered to the DP via follicular pores and promoted hair growth in the skin²². They used nanoliposomes to load and deliver the peptides to DP. The penetration kinetics and effects of LIT001 and WAY267464 on human skin samples will be our next topic. In addition, other OXTR agonists with smaller molecular weights and high penetration kinetics, as well as combinations with a drug delivery system via follicular pores²³ could provide better approaches.

Bioinformatics analysis showed low OXTR expression levels in DP cells from patients with alopecia (Fig. 2a). This result may be due to various factors, including genetic factors, which are common in patients with alopecia; psychological environmental factors such as happiness and stress; and growth factors caused by hair loss. This result can also be viewed as both a cause and a consequence of alopecia. In the present study, OXTR knockdown in DP cells suppressed hair growth-related gene expression (Fig. 2f). This indicates that low levels of OXTR expression are mainly associated with hair loss. However, the sample size for bioinformatics analysis was small. Further studies are needed to elucidate the association between OXTR expression and androgenetic alopecia (AGA) involving different samples of different ages and genders.

OXTR activation can be achieved through the use of oxytocin itself or OXTR agonists. Oxytocin is produced in both men and women in response to skin stimulation (e.g., hugging and massaging), music therapy, and interaction with pets^5,24,25. Lifestyle improvement promotes OXTR activation, and its simplicity is a benefit of using oxytocin. However, due to low receptor binding specificity and activation of other receptors, continuous administration of oxytocin may reduce its effectiveness and cause side effects. OXTR agonists with high receptor binding specificity may reduce these risks.

The top ten KEGG terms associated with WAY267464 or LIT001 treatment included neuroactive ligand-receptor interactions, oxytocin signaling, and cytokine-cytokine receptor interactions. Of note, these signaling pathways were upregulated by oxytocin treatment in our previous study⁸. Thus, WAY267464 and LIT001 are potential hair growth-promoting alternatives to oxytocin, but several signaling pathways were differentially upregulated by LIT001 and WAY267464. These agonists may have different mechanisms and provide additional hair growth benefits when used in combination with LIT001 and WAY267464. Future research should elucidate the mechanisms and examine the effects of the combination. In addition, more than ten oxytocin agonists are available. Future studies will classify them based on the genes activated in DP cells and test their effectiveness in inducing hair growth using hair follicles.

We used OXTR agonists to activate oxytocin signaling, but an approach to increase OXTR using RNA-based therapy is also possible. Using RNA-based therapy, the target genes can be selectively controlled^26.27. In hair research, the expression of androgen receptors, which mediate a series of biomolecular changes leading to hair loss, can be selectively silenced by RNA interference²⁸. Clinical studies on the use of siRNA on the scalp have confirmed the hair growth-promoting effect²⁹. An approach to increasing the expression level of OXTR using mRNA therapy could also be effective in treating hair loss, which we will investigate in future studies.

In our previous study, we developed a hair follicle production technique that efficiently regenerates hair follicles in vitro^16,30,31. The hair follicles are created through epithelial-mesenchymal interaction³²and reconstruction of the interaction between epithelial and mesenchymal cell populations (including DP cells) enabled the production of hair follicles in vitro. In hair follicles, DP cells produce multiple growth factors that stimulate epithelial cell proliferation to elongate the hair shaft. Hair growth promoting drugs increase hair lengthening by activating growth factor secretion by DP cells. Therefore, hair follicles can be used to evaluate the hair growth promoting effect of drugs by using the elongation of the stick-like structure as an indicator after the addition of the drug candidate¹⁶. Treatment with an existing hair growth-promoting drug, such as minoxidil, promotes the lengthening of the rod-like structure¹⁶. In the present study, hair follicles were cultured in culture medium containing WAY267464 or LIT001. These drug candidates elongated the rod-like structure, but the seed length was shorter with WAY267464 or LIT001 treatment than with minoxidil treatment. To enhance the hair growth effect of treatment with WAY267464 or LIT001, it may be necessary to optimize culture conditions, including drug concentration, timing, duration, and drug combinations. Minoxidil was originally developed as an antihypertensive agent and its ability to increase blood flow has been suggested as a mechanism for promoting hair growth; However, several other mechanisms have since been proposed, including opening of potassium channels, activation of β-catenin signaling as well as extracellular signal-regulated kinase and Akt signaling, and increased release of growth factors such as FGF7^33,34,35,36. Combination with minoxidil, which has a different mechanism of action than OXTR agonists, could be an effective treatment for alopecia.

OXTR agonists increased the expression of hair growth-related genes in DP cells and accelerated the elongation of hair follicles sprouting from hair folloids. These results were further supported by results from organ culture of human hair follicles, a standard test for testing hair growth-promoting agents. However, testing many drugs using this approach is difficult due to the limited availability of hair follicles and the variability of results due to individual follicle conditions, such as: B. their in vivo anagen/telogen phase and the isolation process, a challenge. To address this limitation, the present study frequently used an approach to reconstruct hair follicle organoids from cells distributed from multiple hair follicles. A significant problem with the use of hair folloids for drug testing is the lack of a method to expand human follicular keratinocytes without rapidly losing their ability to form hair. In this study, hair folloids were prepared using human follicular keratinocytes without passage of culture to preserve their functions. Consequently, the number of experiments using cells from the same human samples was limited, requiring control experiments using the same vial of cells to normalize all hair extension data. To alleviate this limitation in hair follicle drug testing, a novel method to expand follicular keratinocytes while preserving their functions is required.

Our results represent the first step in demonstrating the hair growth-promoting effects of OXTR agonists. Future research needs to provide more evidence of their effects using organ culture and animal models. Specifically, organ culture studies should examine four categories: (i) prolongation of the anagen phase or inhibition of the catagen phase; (ii) promoting proliferation or inhibiting apoptosis in the hair matrix; (iii) regulation of important hair growth mediators at the protein expression level, particularly in DP cells, the hair matrix and/or the outer root sheath; (iv) observation of the OTXR-specific effects of WAY267464 and LIT001, documenting significant antagonism by OTXR silencing. Animal studies will investigate effective conditions by optimizing the concentrations of WAY267464 and LIT001 as well as the time and frequency of administration. Although the cells used in this study were from healthy donors, the efficacy of WAY267464 and LIT001 should be evaluated in the future using donor cells isolated from patients with alopecia. We will continue these analyzes to develop novel therapeutics to treat hair loss.

In conclusion, OXTR expression levels were low in DP cells from patients with alopecia. OXTR knockdown in DP cells suppressed the expression of hair growth-related genes. WAY267464 and LIT001 increased the expression of hair growth-related genes in DP cells and accelerated the elongation of hair follicles sprouting from hair folloids. Thus, our results demonstrated that WAY267464 and LIT001 are potential hair growth promoting agents targeting OXTR, facilitating their application as treatments. Our findings will accelerate the development of therapies against OXTR.