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The process of hair shedding, termed exogen, is believed to occur independently of the hair cycle  and is thought to be driven by enzymatic mechanisms [2, 3]. Unwanted hair loss is a common problem and is suffered by much of the world population in both women and men. The condition is characterized by premature hair loss and excessive hair shedding caused by an interruption in the normal hair growth cycle, which can lead to the development of baldness with insufficient replacement of hairs in human scalp. This can have negative effects on the individuals' self-esteem and thus affect their interpersonal relationships . Therefore, a better understanding of the factors underlying processes leading to hair shedding may provide insights into how to address the problem of early or excessive hair shedding.
The hair follicle is a regenerating biological system whose primary function is to produce a hair fibre. The hair growth cycle consists of phases of growth (anagen), regression (catagen) and rest (telogen), which occur continuously throughout the follicle lifetime. During catagen, the growing fibre produced in anagen becomes detached from the follicular matrix and is subsequently referred to as the ‘club fibre’. The club fibre eventually sheds from the follicle in a process termed exogen or teloptosis. Although the club fibre is moving up the follicle prior to release, the adjacent follicle continues to cycle and moves back into anagen. In animals, it is not unusual for a new cycle to start producing a growing hair, although the old club fibre is retained alongside in its epithelial silo. In mice, there can be two or three fibres within the same follicle, whereas only one is a growing fibre [5, 6], so they always have a full coat of hair. The final timing of club fibre shedding, or exogen, appears to occur independently from the phases of the hair cycle [1, 7, 8].
The vast majority of hair biology research has focused on the hair growth cycle, and little attention has been devoted to studying the mechanisms involved in exogen. Although a number of mouse and human molecular and genetic studies have uncovered several pathways involved in hair follicle cycling, the molecular signal(s) for exogen remain elusive. The process of hair shedding has been proposed from two viewpoints: one suggesting that the process is a passive event caused by the physical force from the new growing fibre, which dislodges the club fibre , and a second suggesting that exogen is an active process controlled by signals, leading to the release of the club fibre [7, 10]. Club fibres are regarded to be in exogen from the moment of their complete formation (early exogen, with actively retained club fibres) through to their release (late exogen), with club fibre still within the follicle, but ready to be shed . In this study, our hair collection method was devised to try and collect human club fibres in late exogen.
Understanding the factors that control exogen may shed light on routes for intervention to reduce hair loss in humans. The club fibre is anchored within an outer root sheath (ORS), and this ORS can be genetically ablated resulting in loss of the club fibre from its silo . There is evidence showing a changing gradient of proteins and/or RNA expression of proteolytic enzymes and their respective inhibitors in the transition of early exogen to late exogen . In animals, it is thought that an exogen signal results in the release of an enzyme from its inhibitor, which in turn causes the release of the club hair fibre . Additionally, in humans, there is an evidence indicating the presence of specific enzymes in or surrounding the club fibre, for example transglutaminases , and enzyme inhibitors such as plasminogen activator inhibitor type 2  and tissue inhibitor of metalloprotease 3 , which may have roles in retaining the club fibre. It is therefore reasonable to postulate that proteolytic mechanisms may be involved in the detachment of the club fibre by reducing anchorage to the follicle.
The base of the club fibre is comprised of trichilemmal keratin, which is believed to be important for anchoring the club fibre within the follicle . Between the trichilemmal keratin and the surrounding epithelial silo (or ‘trichilemmal sac’), there is an abundance of desmosomal complexes. Milner  observed cytoplasmic breakdown in the cells surrounding shed exogen club fibres and subsequently proposed that club fibre release was mediated through proteolytic degradation, indicating that proteolytic activity within the trichilemmal sac was a major activity contributing towards release of the club fibre. Desmosomes in the skin have been shown to play a role in cell cohesion in the stratum corneum and may also be involved in club fibre anchorage. Desmoglein 3 (Dsg3) is a desmosomal transmembrane glycoprotein [16, 17] expressed and assembled into desmosomes in the basal and suprabasal keratinocytes . However, patients with pemphigus vulgaris (PV) or pemphigus foliaceus develop auto-antibodies against Dsg3 or Dsg1, respectively , which results in the loss of cell adhesion. Interestingly, patients with PV and also Dsg3 knockout mice (DSG3-/-) developed hair loss of club fibres, whereas anagen hairs remained firmly anchored . Inactivation of Dsg1 in DSG3-/- mice results in loss of anchorage of anagen hair follicles . In addition, a heterozygous non-sense mutation of the corneodesmosin gene has been associated with hypertrichosis simplex of the scalp, a form of alopecia . The retention mechanism of the club fibre during exogen could prove invaluable in identifying the therapeutic routes for stabilizing excessive hair shedding and lessen suffering and concern in hair loss.
Investigating disease types resulting in symptoms of hair loss can often provide clues on processes involved. Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a serine protease inhibitor whose defective expression is observed in a skin disease termed ‘Netherton syndrome’. It is encoded by serine protease inhibitor Kazal type 5 (SPINK5)  and is produced as a precursor that is cleaved by furin, generating a number of LEKTI fragments . Some of the secreted LEKTI fragments have shown specific or differential inhibition of human kallikreins (KLKs) 5, 7 and/or 14. Interestingly, hair shaft defects have been observed in patients with Netherton syndrome . In human hair follicles, KLKs are suggested to be important for hair growth and differentiation and trichilemmal keratinization [26, 27].
Although the complexities of exogen and the events leading to exogen are little understood, reducing hair loss through targeting of processes involved in exogen may provide a means for alleviating symptoms of excessive or premature hair shedding. Because serine proteases have been shown to play a role in corneodesmosome degradation, a similar role may be played by proteases in degrading the bonds that anchor the club hair during the telogen or exogen phases of the human hair follicle cycle. In this study, we describe protein expression of specific proteases in the human hair follicle and the presence of protease activity in the material surrounding human exogen club fibres in a range of ethnic groups. Furthermore, as proteases have been implicated in exogen, we searched for a cosmetically acceptable active system that would be capable of inhibiting the protease activity found in the hair follicle with the potential to reduce hair shedding. This study reports the findings that a combination of Trichogen® and Climbazole is able to inhibit hair follicle proteases and that this phenomenon can be observed in a range of ethnic groups. Finally, we demonstrate that this combination is capable of increasing the force required to remove hair in an ex vivo skin model system.
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Evidence from animal studies has indicated a potential role for proteases in hair shaft anchorage. Further studies have demonstrated the presence of proteases and protease inhibitors in human anagen and telogen hair follicles. We postulated that serine proteases may have a role in mediating exogen in humans and therefore investigated the activity and expression of specific serine proteases surrounding human club fibres. Our data indicate that protease activity can be detected in the material surrounding club hairs, and protein expression can be observed around club hairs. Because serine proteases have been shown to play a role in corneodesmosome degradation, a similar role may be played by proteases in degrading the bonds that anchor the club hair during the exogen phase of human hair follicle.
We hypothesized that materials with anti-protease activity may inhibit the proteases, present in the club root of the human hair, that have been implicated in hair shedding (exogen). Trichogen® is a mixture of ingredients including zinc gluconate, and zinc salts are known to inhibit protease activity . Interestingly, we found that Trichogen® could not only inhibit trypsin activity. Climbazole is an imidazole anti-fungal agent commonly used to treat dandruff, but has no known protease inhibition activity . However, we sought to combine a protease inhibition agent with an anti-dandruff agent and surprisingly found that trypsin activity was further reduced with the addition of climbazole compared with Trichogen® alone. In addition, the combination of Trichogen®+climbazole was capable of inhibiting the proteases present in club root extracts from ethnic and geographical groups across the world. This finding indicates that this combination of ingredients may be able to provide a global solution to reduce excessive hair fall.
Furthermore, we developed in ex vivo model (using pig skin) for measuring force required to remove club hairs from skin. Consequently, we were able to demonstrate that a combination of Trichogen®+climbazole can increase the extraction force required to remove club hairs from the pig skin. This advance demonstrates that we were successfully able to translate our in vitro studies to an ex vivo skin model. Utilizing a Trichogen®+climbazole in cosmetic applications may serve to increase the retention strength of club hairs and therefore reduce unwanted club hair shedding to promote a fuller appearance of hair on the scalp.