A NEW STRATEGY TO MODULATE ALOPECIA 51 Study design. The study was designed as a randomized, placebo-controlled study. One half of the subjects (15 people) received the “active” lotion, whereas the other half was given a placebo lotion. The active lotion composed of 5% of the test product formulated in a solution consisting of water (75%) and alcohol (20%) the placebo lotion composed of 80% water and 20% alcohol. The test product composed of a mixture of clover extract (titrated to reach 15 ppm biochanin A) and 300 ppm acetyl tetrapeptide-3. The study lasted for 4 months (T4). Each evening, patients had to apply 20 drops of the test product or placebo lotion on balding areas and gently massage it into the whole scalp. For each week of the study, the patients received one plastic bag to collect all hair found on their pillow, comb, and clothes on a daily basis. The hair thus collected in a bag had to be returned to the laboratory for counts. Evaluation procedures. Effi cacy was objectively evaluated by instrumental measurements (digital trichogram with TrichoScan™). TrichoScan™ is a noninvasive method, combin- ing standard epiluminescence microscopy with automatic digital image analysis, for the measurement of human hair (28,29). For determining total hair density, a mask was po- sitioned on the volunteer head to delimitate a shaving area of 1.8 cm2 on the zone to be studied. Three days later, as hair may not always contrast well enough with the scalp (due to the presence of gray or fair hair), hair was dyed and subsequently cleaned with alcohol. Images were then recorded with a camera for the purpose of hair count. Patients were asked not to wash their hair for 2 days before the TrichoScan™ examination. Following acquisition, the digital images were processed using software capable of analyzing ana- gen, telogen, and total hair density. The software was calibrated for an average anagen growth rate of 0.3 mm/day and no telogen growth. According to the defi nition of the TrichoScan™ procedure, an anagen (A) hair is a hair that is detectable 3 days after complete hair shaving. Within this time, only anagen hair should grow signifi cantly at a rate of approximately 0.3 mm/day. Nongrowing hair is by defi nition a terminal hair in the telogen (T) phase. The anagen/telogen (A/T) ratio is an indication of the percentage of active hair follicles. £ A/T ratio = activation of hair growth ¤ A/T ratio = loss of hair growth activity RESULTS AND DISCUSSION INHIBITION OF 5-α-REDUCTASE ACTIVITY Biochanin A, a phytoestrogen that, like many other polyphenols including EGCG from green tea, has shown 5-α-reductase inhibitory activity in vitro (30). EGCG is also known to stimulate hair growth ex vivo (23,24). In an intact cell assay, biochanin A proved to be a potent inhibitor of 5-α-reductase activ- ity and was superior in that aspect to EGCG (Figure 3). In this assay, biochanin A (100 μM) inhibited both type I and type II isoforms of 5-α-reductase by −64% and −93%, respec- tively, compared to those of −11% (type 1) and −5% (type 2) for EGCG. Both isomers are found in the scalp and there is strong evidence that type II contributes to male pattern

JOURNAL OF COSMETIC SCIENCE 52 alopecia (31). This is supported by the fact that men genetically defi cient in type II 5-α-reductase do not present hair loss (8). On the one hand, Finasteride, a selective in- hibitor of type II 5-α-reductase, slows the progression of baldness in a majority of men (8,32) and also in some women (33). On the other hand, dutasteride, a dual inhibitor of type I and type II 5-α-reductase activities revealed to be even more potent than fi naste- ride in improving hair growth in balding men (although not approved by the FDA for this indication), suggesting there might be an advantage in inhibiting both isozymes for this condition (31). These results suggest that extracts enriched in biochanin A, by inhib- iting both type I and type II 5-α-reductase activity in the scalp, may fi nd application in male pattern alopecia management. STIMULATION OF ECM PROTEIN SYNTHESIS The size of a hair follicle is thought to be determined by the volume of its dermal papilla, which depends on the number of cells and on the volume of the ECM (3). Regulation of the size of dermal papilla is a dynamic process involving hormonal infl uence and complex cell–matrix interactions. ECM proteins expressed in the hair follicle include collagens I, III, and VII, as well as laminins. Collagens I and III. Type I and III collagens are the most abundant types of collagen in the skin. Not surprisingly, they are also found in the hair follicle where they have the par- ticularity of presenting an unusual high ratio of collagen III/I. Collagen III is a fi brillar protein with elastic properties it is tempting to speculate that these attributes might be of particular importance for hair follicle development and maintenance. Collagen I and III are produced in the human dermal papilla throughout the hair cycle (34) and are also major constituents of the connective tissue sheet of the hair follicle (4). Laminins. Laminins are a family of large glycoproteins comprising more than 50 members that are the major constituents of basement membranes. They display a remarkable Figure 3. Inhibition of type I and type II isoforms of 5-α-reductase activity in intact cells by biochanin A and comparison to EGCG.