J. Cosmet. Sci., 64, 45–58 (January/February 2013)
A new strategy to modulate alopecia using a combination
of two speciﬁ c and unique ingredients
ESTELLE LOING, ROGER LACHANCE, VIRGINIE OLLIER,
and MICHEL HOCQUAUX, R&D Department, Lucas Meyer
Cosmetics, place de la cité-Tour de la cité 2600, boulevard Laurier, #900
Québec (QC) G1V 4W2, Canada (E.L., R.L.) and R&D Department, Lucas
Meyer Cosmetics, 18 avenue de l’Europe, 31520 Ramonville Saint Agne,
France (V.O., M.H.).
Accepted for publication June 6, 2012.
Male pattern hair loss is a major cosmetic concern affecting both genders with a preference for men. Major
causes of hair loss in genetically predisposed individuals include hormonal dysfunction, loss of extracellular
matrix (ECM) proteins in the follicular bed, and localized microinﬂ ammation. Few options are yet available
to correct the problem. For this purpose, a cosmetic active ingredient was developed by combining a Trifolium
pratense ﬂ ower extract and a biomimetic peptide and tested clinically for the prevention of hair loss. Thirty
volunteers with recessing hair were recruited for this randomized, placebo-controlled study. Clinical efﬁ cacy,
following daily topical application of the mixture to the scalp, was checked using TrichoScan™ for the mea-
surement of human hair. Within 4 months of application, anagen hair increased at an average by +13%,
telogen hair density decreased by −29%, and the anagen/telogen (A/T) ratio increased by +46% over baseline
in the treated group. Results strongly differed from those of the placebo group (anagen, −2%; telogen, +23%;
A/T ratio, −33%). Investigation of the potential mechanisms involved in the positive effects of the test
product on hair growth pointed at inhibition of 5-α-reductase activity, reduction of inﬂ ammatory reactions,
and stimulation of ECM protein synthesis in the vicinity of the hair follicle.
The presence of hair is a characteristic of mammals. Other species may have some kind of
ﬁ lamentous outgrowth but that certainly do not qualify as hair. Hair is a unique ﬁ lamen-
tous structure that extends from the dermis to the outside of the body, normally covering
some areas entirely, such as the scalp, while sparing other zones, such as the palms and
the soles. Human hair naturally comes in a variety of shapes, sizes, lengths, and colors,
depending mainly on the speciﬁ c genetic makeup of each individual. In mammals in
general, hair serves diverse functions, such as providing protection, thermal regulation,
camouﬂ age, warning and mating signals, and a sense of touch (1). But for humans, it is
Address all correspondence to E. Loing at email@example.com
JOURNAL OF COSMETIC SCIENCE46
even more than that. Hair is linked to our personality, appearance, sex, and social status;
plays a role in seduction; and is instrumental to nonverbal communication. For all these
reasons, the absence of hair, especially on the scalp, has a huge impact on one’s life.
Each hair is composed of two distinct structures: the dynamic hair follicle located in
the dermis and the hair shaft, a hard keratinized part that extends above the skin sur-
face. The hair follicle is made of dermal and epidermal compartments closely interact-
ing in the regulation of hair growth. The central structure of the hair follicle is the
dermal papilla rich in mesenchymal cells (2). The papilla is connected to the capillary
bed in the dermis and is embedded in a hair matrix consisting of epidermal cells capable
of dividing rapidly to give rise to hair. The hair shaft comprises three layers: a cuticle,
a medulla, and a cortex. Thickness of the shaft totally depends on the size of the papilla;
the bigger the papilla, the stronger the shaft (3).
Hair grows in cycles delimitated by three distinct phases: anagen, catagen, and telogen
(Figure 1). Anagen is the growth phase during which new materials are deposited in the
hair shaft by rapidly dividing follicular cells. Anagen scalp hair grows by 1 cm per month
for a period of 2–6 years. The duration of the anagen period dictates the maximal length
of hair and is genetically determined. Catagen is a transition phase, lasting for about 2–3
weeks, marked by a stop of hair growth. During this phase, the hair follicle involutes,
becomes attached to the hair shaft and keratinizes forming a club hair that is pushed
upward toward the scalp, as the dermal papilla breaks away. Telogen is the resting phase.
The hair follicle regresses, becomes fully keratinized, and can easily be pulled out. The
telogen phase lasts around 3 months for scalp hair. Following shedding, the next hair can
start growing as the papilla and the follicle join again. An adult healthy scalp normally
bears 70–85% hair in the anagen phase and 10–15% in the telogen phase, the rest being
in the catagen phase (4). Male pattern alopecia is generally associated with a shortening
of the anagen phase and premature entry into the catagen phase (5).
Figure 1. Normal hair physiology: hair cycles through anagen (growth phase), catagen (transition phase),
and telogen (resting and falling phase) before reentering early anagen to initiate the growth of a new hair.
A NEW STRATEGY TO MODULATE ALOPECIA 47
Up to a hundred hair are shed every day (6). Over that number, pathological hair loss
(alopecia) is most likely to occur. Male pattern hair loss (androgenic alopecia) is the most
common type of baldness, affecting roughly 50% of Caucasian men by the age of 50 years
and 13% of Caucasian women before menopause increasing to 75% by the age of 65 years
in women (5). Asians and African are less affected than Caucasians and the incidence is
lowest in Native Americans and Eskimos (6). Patterns of hair loss may vary among gen-
ders. Indeed in men, the crown and temples are more likely to be ﬁ rst affected, a pattern
that eventually progresses to baldness, whereas in women hair loss is generally rather dif-
fuse (7). In most cases, hair thinning appears to precede hair loss (8).
The causes of male pattern hair loss are still a matter of debate, but genetic predisposi-
tion, hormonal dysfunction, loss of extracellular matrix (ECM) proteins in the follicular
bed, and localized microinﬂ ammation are recognized as major triggers (Figure 2).
From a hormonal point of view, androgens are known to be important regulators of hair
growth and the enzyme 5-α-reductase is pivotal to their effect. In the scalp, testosterone
is metabolized to the stronger androgenic signal 5-α-dihydrotestosterone (DHT) by
5-α-reductase. Besides its action on androgen receptors in the follicle, DHT also stimu-
lates the synthesis of transforming growth factor (TGF)-β in dermal papilla cells. TGF-β
signaling is associated with inhibition of keratinocyte growth and induction of cell
apoptosis (9). Pathological expression of TGF-β is a source of inﬂ ammation and ﬁ brotic
matrix deposition (10). In hair physiology, TGF-β is a catagen inducer that also pre-
vents reentry from telogen to anagen, thus suppressing hair growth (11). Higher
5-α-reductase activity, resulting in high levels of DHT and dysregulated TGF-β sig-
naling, is found in bald scalp (12).
Cell–matrix interactions are also key regulatory steps in hair cycling (13). The ECM is in
constant remodeling during the different phases of hair growth. Maintenance of the ECM
composition is mainly assumed by the dermal papilla ﬁ broblasts, but proper exchange
with hair matrix keratinocytes is mandatory for this function. These exchanges take place
at the basement membrane zone (BMZ) located at the epithelial–mesenchymal interface
of the hair follicle. Matrix proteins found at this interface serve as anchors to maintain
Figure 2. Pathological mechanisms involved in recessing hair: combined effects of hormones, inﬂ ammation,
and ECM dysfunction alter the hair growth cycle and lead to hair loss.
JOURNAL OF COSMETIC SCIENCE48
epithelial–mesenchymal contact and stabilize the BMZ. These include laminins, some
integrins, and collagen VII (14,15). They have a crucial role in the maintenance of the
hair follicle and the control of its volume.
Microinﬂ ammation is suspected to be a precipitating factor in male pattern alopecia (16),
but the concept has yet to be integrated into treatment strategies. Exposure to irritants,
pollution, and UV radiation has the potential to turn keratinocytes into mediators
of inﬂ ammation (17). Under stress conditions, keratinocytes react by increasing their
production of interleukin (IL)-1α, a pro-inﬂ ammatory cytokine. The latter acts on
ﬁ broblasts to stimulate their production of IL-8, a cytokine involved in the recruit-
ment of neutrophils. Both IL-1α and IL-8 are inducible at the dermal papilla and
were found in plucked hair samples of subjects with male pattern alopecia (18) sug-
gesting their participation in the pathology (19). Cytokine-driven persistent inﬂ am-
mation also activates matrix metalloproteinases involved in tissue remodeling and
perifollicular ﬁ brosis (20).
Limited treatments are currently available for male pattern alopecia. The most popular
are the following: minoxidil (Rogain® McNeil-PPC, Johnson & Johnson, New Bruns-
wick, New Jersey, USA.), an over-the-counter vasodilator that is believed to optimize
blood supply to the dermal papilla (5); ﬁ nasteride (Propecia® Merck, Whitehouse Station,
New Jersey), a drug that acts by inhibiting the enzyme that converts testosterone to
DHT (5); and diaminopyrimidine oxide (Aminexil®, l’Oréal, Paris, France), a patented
compound that prevents perifollicular ﬁ brosis. Each of these treatments targets one aspect
of hair loss and offers a certain level of efﬁ cacy for those who respond. However, for improved
results, it may be desirable to simultaneously target several aspects of the problem. The
new cosmetic active ingredient (a mixture of clover extract and acetyl tetrapeptide-3),
described in this article, represents a new, more integrative approach to hair loss.
MATERIAL AND METHODS
The test material consisted of a mixture of Trifolium pratense (clover) ﬂ ower extract (total
isoﬂ avone ≥98% and biochanin A ≥12%, determined by high-performance liquid chro-
matography (HPLC)) and acetyl tetrapeptide-3 (pure peptide obtained by solid phase
peptide synthesis, purity ≥90% determined by HPLC). The clover extract fraction is
standardized using biochanin A, a phytoestrogen ﬂ avonoid with documented health-
promoting activities (20). The tetrapeptide is a biomimetic derived from a signal peptide
found in matrix proteins, such as collagen and ﬁ brin, and also in HGF, which is a growth
factor ﬁ rst isolated from human plasma (21). The peptide is normally liberated by prote-
olysis in the course of tissue damage. Its release and activation stimulates tissue remodel-
ing following the initial phase of wound healing.
The components of the mixture were tested either together or alone, according to their
expected roles in hair care, as could be deduced from the existing literature (20,22). An effect
of the biochanin A component was documented on 5-α-reductase activity, while acetyl tetra-
peptide-3 was investigated for its inﬂ uence on ECM components, including collagens III and
VII, and laminins. The mixture of both components was tested for anti-inﬂ ammatory activity
before being clinically tested in humans to evidence efﬁ cacy in reducing hair loss.
A NEW STRATEGY TO MODULATE ALOPECIA 49
The enzyme 5-α-reductase catalyzes the conversion of testosterone to DHT. The effects
of biochanin A on 5-α-reductase activity were studied in intact cells expressing type 1 or
type 2 isoforms of the enzyme and compared to that of epigallocatechin-3-gallate (EGCG)
from green tea. EGCG is a known in vitro inhibitor of 5-α-reductase (23). In this assay,
radio-labeled testosterone served as substrate.
The activity of biochanin A on 5-α-reductase activity was shown by Hiipakka et al. (24).
Brieﬂ y, 5-α-reductase-expressing cells were plated at 50,000 per well in a 24-well plate
in speciﬁ c medium for 18 h at 37°C. The medium was then changed to 0.5 ml of serum-
free medium and 5 µl of biochanin A (100 µM) or EGCG (100 µM) was added and kept
for 1 h at 37°C before the addition of 14C-testosterone, at a ﬁ nal concentration of 1.5 µM.
Cells were then incubated for an additional 3 h and radioactive steroids were extracted
with ethyl acetate. The amounts of labeled testosterone and DHT in extracts were next
determined by thin layer chromatography, as a measure of 5-α-reductase activity (for
more details, see Ref. 24).
IMMUNOFLUORESCENT LABELING OF COLLAGEN III AND LAMININS
The effect of acetyl tetrapeptide-3 on the expression of different ECM proteins (collagen III
and laminins) was evaluated by selective immunoﬂ uorescence in comparison with un-
treated ﬁ broblasts. For this experiment, 3 × 104 human ﬁ broblasts (MRC5 from ATCC
CCL) were incubated in Dulbecco’s modiﬁed Eagle’s medium (DMEM) (Eurobio Labora-
tories, Courtaboeuf, France) containing 10% fetal calf serum (FCS) and supplemented
with 1% penicillin/streptomycin. Cells were maintained in a humidiﬁ ed incubator at
37°C with 5% CO2 atmosphere to reach conﬂ uence. Cells were then incubated in the
presence or absence of acetyl tetrapeptide-3 (10−7 M, equivalent to 0.05 ppm) for 3 days.
These cells were rinsed with phosphate-buffered saline (PBS) and ﬁ xed on slides using
methanol (for 10 min at −20°C) followed by acetone ﬁ xation (for 10 min, at 4°C). The
slides were then dried at room temperature and rinsed with PBS at a pH of 7.6 for 10 min.
The presence of collagen III and laminins in cells was detected by incubating the slides
with speciﬁ c antibodies diluted at 1/50e overnight at 4°C, that is, type III anti-collagen
(rabbit, Rockland, Gilbertville, PA) and anti-laminin (rabbit, Sigma, St. Louis, MO),
respectively. Detection of type III anti-collagen and anti-laminin antibodies was done
using a goat anti-rabbit IgM + IgG rhodamine (TRITC) conjugate diluted at 1/100e
(Southern Biotech, Birmingham, AL). The corresponding ﬂ uorescent signal was moni-
tored using confocal microscopy (Axioplan and Zeiss LSM510 Oberkochen, Germany),
allowing for semiquantitative evaluation.
IMMUNOHISTOLOGICAL LABELING OF COLLAGEN VII
The effect of acetyl tetrapeptide-3 on the expression of collagen VII, a major constituent
of anchoring ﬁ brils found in the middle part of the follicular BMZ and around the hair
papilla was evaluated using immunohistological techniques. As the junction around the
anagen hair follicle and its adjacent connective tissue is similar in terms of composition
JOURNAL OF COSMETIC SCIENCE50
and structure to the dermal–epidermal junction (DEJ) (25), the effect of acetyl tetrapeptide-3
on type VII collagen synthesis was evaluated on human skin explants.
Four human skin explants were obtained from abdominal part of patients undergoing
plastic surgery (Caucasian women, 35–45 years old) and maintained in culture. On day 1,
a corticoid cream (Diprosone® with 0.05% betamethasone) was applied at the surface of
the skin explants to induce skin atrophy as seen in aging (26). Acetyl tetrapeptide-3 was
then added to the culture media for 2 days at 10−5 M concentration (equivalent to 5 ppm).
On day 3, the skin explants were prepared for speciﬁ c collagen VII immunohistological
labeling using Avidin-Biotin Complex ABC Peroxidase Kit (Vector Laboratories Burlingame,
CA, USA) and revealed by AEC substrate (brown color).
Visual scoring of collagen VII expression (n = 4) was performed, using a scale ranging
from 0 (negative) to 4 (maximum) deﬁ ned as follows: 0, absence of the protein; 1, slight
expression; 2, moderate expression; 3, normal expression; and 4, overexpression.
MODULATION OF IL-8 PRODUCTION
Low-grade chronic inﬂ ammation is increasingly seen as a contributing factor in male
pattern alopecia (27). Under stress conditions, keratinocytes in the vicinity of the hair
follicle may respond by releasing IL-1, a proinﬂ ammatory cytokine that commands the
production of additional inﬂ ammatory agents such as IL-8 acting as chemoattractants for
inﬂ ammatory cells. IL-1-induced IL-8 production in keratinocytes was used as a model to
document the anti-inﬂ ammatory activity of red clover extract alone and in combination
with acetyl tetrapeptide-3. Dexamethasone (DMS), a glucocorticoid with potent anti-
inﬂ ammatory properties, served as a positive control for anti-inﬂ ammatory action.
Monolayers of cells derived from normal human dermal ﬁ broblasts (NHDFs) (Life tech-
nologies, Saint Aubin, France) were cultured to conﬂ uence for 24 h in DMEM (Eurobio
Laboratories) containing 10% FCS and supplemented with 200 mM L-glutamine and 1%
penicillin/streptomycin in a humidiﬁ ed incubator at 37°C with 5% CO2 atmosphere.
Cells were then challenged by adding IL-1α (0.0075 ng/ml from Eurobio Laboratories)
to the culture media (without FCS) in the presence or absence of the test products (red
clover extract alone or red clover extract + acetyl tetrapeptide-3) (0.5–1%) or DMS (1 µM)
for 24 h. At the end of this period, IL-1α-induced IL-8 production was measured using a
highly sensitive and speciﬁ c enzyme immunoassay kit (human CXCL8/IL-8 DuoSet;
R&D system Minneapolis, MN).
Study population. Thirty healthy volunteers with active mild to moderate hair loss enrolled
in the study. Patients were clinically evaluated and individual case histories were recorded
to rule out possible pathologies such as iron deﬁ ciency anemia, thyroid-related condi-
tions, or others that may inﬂ uence hair growth. Patients were asked to use only “basic”
shampoo and to avoid hair care treatment lotion according to the protocol. Hair count
evaluation was done at the preselection step. As an inclusion criterion, less than 70% of
all hair had to be in the anagen phase.
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 SCIENCE52
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.
A NEW STRATEGY TO MODULATE ALOPECIA 53
repertoire of functions. Their interaction with integrin receptors at the surface of cells
anchors skin cells to basement membranes and modulates cellular processes such as pro-
liferation, apoptosis, differentiation, and motility (35). Laminin-511 (laminin-10) is the
predominant laminin at the epithelial–mesenchymal interface of the hair follicle and is
crucial for hair morphogenesis and anagen hair growth (36). Laminin-511 stimulates hair
growth in vitro, and preventing its expression at the dermal papilla leads to hair regres-
sion and alopecia, a defect that can be prevented by application of the puriﬁ ed protein
(37). Chemotherapy-induced alopecia has recently been associated with downregulation
of laminin-511 expression, providing a new target for the prevention of this adverse effect
(37). The effects of the matrix protein on hair follicle are mediated through the binding
and activation of speciﬁ c integrins at the surface of cells (38).
Collagen VII. Collagen VII is a long-chain collagen synthesized and secreted by keratino-
cytes and dermal ﬁ broblasts. This matrix protein is a major component of anchoring
ﬁ brils that are specialized attachment complexes at epithelium–mesenchyme interface, in
a number of tissues. In human skin, anchoring ﬁ brils extend from the lower portion of the
dermal–epidermal basement membrane to the underlying upper papillary dermis, form-
ing U-like structures that entrap interstitial collagen ﬁ bers (39). In the hair follicle, col-
lagen VII is found along the BMZ outside the hair follicle and at the dermal papilla
junction inside the hair bulb. Collagen VII colocalized with integrins and laminins-511
at the epithelial–mes enchymal interface, suggesting a role in hair growth (14,15). The
close interaction of collagen VII with laminins and interstitial collagen provides stability
to the epithelial–mesenchymal interface.
Effects of acetyl tetrapeptide-3 on matrix proteins. The effect of acetyl tetrapeptide-3 (10−7 M)
on the protein expression of type III collagen and total laminins in human ﬁ broblasts
(MRC5) was evaluated by selective immunoﬂ uorescence in comparison with untreated
ﬁ broblasts. Results showed a signiﬁ cant stimulation in the synthesis of both ECM pro-
teins in the presence of the peptide (Figure 4); expression of type III collagen was in-
creased by +65% while expression of laminins raised by +285% over untreated cells.
Figure 4. Effects of acetyl tetrapeptide-3 (10−7 M) on the protein expression levels of type III collagen and
total laminins in human ﬁ broblasts (MRC5); (A) immunoﬂ uorescence staining of type III collagen and lami-
nins in control and acetyl tetrapeptide-3-treated cells; (B) histogram representation of results. Untreated cells
served as control.
JOURNAL OF COSMETIC SCIENCE54
The potential effect of acetyl tetrapeptide-3 on protein expression levels of collagen VII
at a mesenchymal–epithelial junction was documented using human skin explants.
Skin atrophy was ﬁ rst induced through corticoid application in an effort to mimic the
atrophic state of a deﬁ cient hair follicle. The explants were then treated with acetyl
tetrapeptide-3, and collagen VII expression at the DEJ was revealed semiquantitatively
by immunohistological staining. Microscopic observations of normal untreated skin
revealed a strong labeling of collagen VII along the DEJ. Corticoid treatment resulted
in a drastic and signiﬁ cant diminution (−70% on visual scoring) of collagen VII stain-
ing but baseline expression was completely restored in the presence of acetyl tetrapep-
tide-3 (Figure 5).
Given the importance of collagen III, collagen VII, and laminins for hair morphogenesis
and growth, the positive impact that acetyl tetrapeptide-3 had on their expression in
vitro and ex vivo supports the use of the peptide to help maintain an adequate ECM bed
for optimized hair anchorage.
INHIBITION OF INFLAMMATORY SIGNALS
As mentioned in the Introduction section, microinﬂ ammation in the vicinity of the der-
mal papilla appears to precipitate or at least contribute to male pattern alopecia. To
mimic inﬂ ammatory events that may prevail in this context, NHDFs were stimulated
with IL-1α in the presence or absence of red clover extract alone, or a mixture of red
clover extract and acetyl tetrapeptide-3, at two different concentrations (0.5% and 1%).
DMS, a glucocorticoid with anti-inﬂ ammatory properties, served as positive control for
inhibition of IL-8 secretion.
As was expected, within 24 h of IL-1α stimulation of NHDF cells, a massive release of
IL-8 was observed in the culture media, an effect that was partially blocked by DMS
(−17%). The production of IL-8 was also slightly inhibited in the presence of increasing
concentrations (0.5% and 1%) of red clover extract alone (−11% and −20%, respec-
tively). However, the mixture of red clover extract and acetyl tetrapeptide-3 was much
more potent and showed a dose–effect relationship, inhibiting IL-8 release by −33% and
−48%, at the lowest and the highest concentrations, respectively (Figure 6). From this
Figure 5. Effects of acetyl tetrapeptide-3 on protein expression levels of collagen VII at the DEJ, as mea-
sured by immunohistological labeling of human skin explants: (A) normal skin; (B) corticoid-treated (0.05%)
skin; (C) corticoid-treated (0.05%) skin, cultured in the presence of acetyl tetrapeptide-3; and (D) visual
scoring of collagen VII expression.
A NEW STRATEGY TO MODULATE ALOPECIA 55
experiment, it was concluded that the mixture of red clover extract and acetyl tetrapeptide-3
has the potential to modulate cutaneous inﬂ ammation in conditions similar to those
found in the course of male pattern alopecia.
CLINICAL REDUCTION IN HAIR LOSS
Clinical efﬁ cacy of the mixture of red clover extract and acetyl tetrapeptide-3 (TP) in
reducing hair loss was addressed using TrichoScan™. The technology combines epilu-
minescence microscopy with digital image analysis for the measurement of hair growth
in vivo. The method is suitable for the determination of the density of hair (n/cm2) in
the anagen and telogen phases, the hair diameter (µm), the hair growth rate (mm/day),
and the A/T ratio. For a more detailed description of the method, see the Material and
Of the two groups of 15 participants originally enrolled, 15 placebo people and 14 treated
people completed the 4-month study. The product or the placebo lotion was applied to
the scalp at night, as described earlier. Overall tolerability of the topical formulations was
excellent and no adverse events were reported. Results, which are expressed graphically in
Figure 7, show that treatment with a mixture of red clover extract and acetyl tetrapeptide-3
statistically increased anagen hair density after 4 months of treatment (p < 0.1), whereas
there is no statistic difference in the anagen hair density after treatment with the placebo
lotion. Within 4 months, the number of hair in the anagen phase increased by an average
of +13% in the treated group, whereas the number decreased by −2% in the placebo
group. Treatment with the test product also induced a strong reduction in telogen hair
density compared to the placebo group (Figure 7B). Average telogen hair density
decreased by −29% over baseline in the treated group but increased by +23% in the pla-
cebo group, over the 4-month study. In accordance with these results, the A/T ratio in-
creased signiﬁ cantly (p ≤ 0.05) by +46% over baseline in the treated group, whereas it
decreased signiﬁ cantly (p ≤ 0.05) by −33% in the placebo group for the same period. The
Figure 6. Effects of red clover extract (0.5% and 1%) and a mixture (TP) of acetyl tetrapeptide-3 and red
clover extract (0.5% and 1%) on IL-1-induced IL-8 secretion from NHDFs. DMS at 1 µM served as positive
control for inhibition of IL-8 secretion.
JOURNAL OF COSMETIC SCIENCE56
A/T ratio being a comparison of the number of anagen to telogen hair follicles, the observed
increase in A/T ratio attests that the test product is able to promote hair growth in people
suffering from recessing hair.
Taken together, these results support the efﬁ cacy of the test product in improving hair
growth by rebalancing the A/T ratio and promoting healthy hair growth. The mixture
combines the beneﬁ ts of both biochanin A-enriched red clover extract and biomimetic
acetyl tetrapeptide-3 to simultaneously address multiple factors involved in the progres-
sion of male pattern alopecia. The mixture acts on recessing hair by limiting hormonal
inﬂ uence through inhibition of 5-α-reductase activity, preventing aggravation of hair
loss due to microinﬂ ammation and supporting hair growth through stimulation of ECM
protein synthesis. Clinical efﬁ cacy in supporting hair growth was successfully addressed
in a 4-month randomized, placebo-controlled study involving volunteers presenting
signs of male pattern alopecia.
Figure 7. Effect of topical application of 20 drops of a 5% mixture of acetyl tetrapeptide-3 and red clover
extract (TP) on hair cycle: (A) average anagen hair density at 4 months (T = 4) of treatment, compared to
baseline (T = 0); (B) average telogen hair density at 4 months of treatment (T = 4), compared to baseline
(T = 0); and (C) average ratio of hair in the anagen versus telogen phase of the hair cycle, at 4 months of treat-
ment (T = 4), compared to baseline (T = 0).
A NEW STRATEGY TO MODULATE ALOPECIA 57
(1) M. R. Schneide, R. Schmidt-Ullrich, and R. Paus, The hair follicle as a dynamic miniorgan, Curr. Biol.,
19, R132–142 (2009).
(2) B. A. Bernard, La biologie du follicule pileux, J. Soc. Biol., 199, 343–348 (2005).
(3) K. Elliott, T. J. Stephenson, and A. G. Messenger, Differences in hair follicle dermal papilla volume are
due to extracellular matrix volume and cell number: Implications for the control of hair follicle size and
androgen responses, J. Invest. Dermatol., 113, 873–877 (1999).
(4) B. A. Bernard, La vie révélée du follicule de cheveu humain, Medecine/Sciences, 22,138–143 (2006).
(5) R. P. Feinstein, Androgenic alopecia, emedecine.medscape.com, http://emedicine.medscape.com/article/1070167-
overview (2010) accessed March 3 (2011).
(6) J. Thomas, Androgenetic alopecia: Current status, Indian J. Dermatol., 50, 179–190 (2005).
(7) F. M. Camacho-Martínez, Hair loss in women, Semin. Cutan. Med. Surg., 28, 19–32 (2009).
(8) E. S. Schweiger, O. Boychenko, and R. M. Bernstein, Update on the pathogenesis, genetics and medical
treatment of patterned hair loss, J. Drug. Dermatol., 9, 1412–1419 (2010).
(9) T. Hibino and T. Nishiyama, Role of TGF-beta2 in the human hair cycle, J. Dermatol., 35, 9–18 (2004).
(10) A. G. Li, S. L. Lu, G. Han, K. E. Hoot, and X. J. Wang, Role of TGF-beta in skin inﬂ ammation and
carcinogenesis, Mol. Carcinog., 45, 389–396 (2006).
(11) S. Huh, J. Lee, E. Jung, S. C. Kim, J. I. Kang, J. Lee, Y. W. Kim, Y. K. Sung, H. K. Kang, and D. Park,
A cell-based system for screening hair growth-promoting agents, Arch. Dermatol. Res., 301, 381–385
(12) S. Inui, Y. Fukuzato, T. Nakajima, K. Yoshikawa, and S. Itami, Androgen-inducible TGF-beta1 from
balding dermal papilla cells inhibits epithelial cell growth: A clue to understand paradoxical effects of
androgen on human hair growth, FASEB J., 16, 1967–1969 (2002).
(13) M. Yamao, M. Inamatsu, Y. Ogawa, H. Toki, T. Okada, K. E. Toyoshima, and K. Yoshizato, Contact
between dermal papilla cells and dermal sheath cells enhances the ability of DPCs to induce hair growth,
J. Invest. Dermatol., 130, 2707–2718 (2010).
(14) J. Gao, M. C. De Rouen, C. H. Chen, M. Nguyen, N. T. Nguyen, H. Ido, K. Harada, K. Sekiguchi,
B. A. Morgan, J. H. Miner, A. E. Oro, and M. P. Marinkovich, Laminin-511 is an epithelial message
promoting dermal papilla development and function during early hair morphogenesis, Genes Dev., 22,
(15) S. Joubeh, O. Mori, K. Owaribe, and T. Hashimoto, Immunoﬂ uorescence analysis of the basement
membrane zone components in human anagen hair follicles, Exp. Dermatol., 12, 365–370 (2003).
(16) M. El-Domyati, S. Attia, F. Saleh, and H. Abdel-Wahab, Androgenetic alopecia in males: A histo-
pathological and ultrastructural study, J. Cosmet. Dermatol., 8, 83–91 (2009).
(17) R. M. Trüeb, Molecular mechanisms of androgenetic alopecia, Exp. Gerontol., 37, 981–990 (2002).
(18) Y. F. Mahé, B. Buan, N. Billoni, G. Loussouarn, J. F. Michelet, B. Gautier, and B. A. Bernard, Pro-
inﬂ ammatory cytokine cascade in human plucked hair, Skin Pharmacol., 9, 366–375 (1996).
(19) Y. F. Mahé, J. F. Michelet, N. Billoni, F. Jarrousse, B. Buan, S. Commo, D. Saint-Léger, and B. A.
Bernard, Androgenetic alopecia and microinﬂ ammation, Int. J. Dermatol., 39, 576–584 (2000).
(20) N. L. Booth, C. E. Piersen, S. Banuvar, S. E. Geller, L. P. Shulman, and N. R. Farnsworth, Clinical stud-
ies of red clover (Trifolium pratense) dietary supplements in menopause: A literature review, Menopause,
13, 251–264 (2006).
(21) L. Pickart and M. M. Thaler, Tripeptide in human serum which prolongs survival of normal liver cells
and stimulates growth in neoplastic liver, Nature New Biol., 243, 85–87 (1973).
(22) L. Pickart, The human tri-peptide GHK and tissue remodeling, J. Biomater. Sci. Polym. Ed. 19(8),
(23) O. S. Kwon, J. H. Han, H. G. Yoo, J. H. Chung, K. H. Cho, H. C. Eun, and K. H. Kim, Human hair
growth enhancement in vitro by green tea epigallocatechin-3-gallate (EGCG), Phytomedicine, 14,
(24) R. A. Hiipakka, H. Z. Zhang, W. Dai, Q. Dai, and S. Liao, Structure-activity relationships for inhibi-
tion of human 5alpha-reductases by polyphenols, Biochem. Pharmacol. 63(6), 1165–1176 (2002).
(25) Y. H. Chuang, D. Dean, J. Allen, R. Dawber R, and F. Wojnarowska, Comparison between the expres-
sion of basement membrane zone antigens of human interfollicular epidermis and anagen hair follicle
using indirect immunoﬂ uorescence, Br. J. Dermatol., 149, 274–281 (2003).
JOURNAL OF COSMETIC SCIENCE58
(26) L. Kolbe, A. M. Kligman, V. Schreiner, and T. Stoudemayer, Corticosteroid-induced atrophy and
barrier impairment measured by non-invasive methods in human skin, Skin Res. Technol., 7, 73–77
(27) P. Hirsso, U. Rajala, L. Hiltunen, J. Jokelainen, S. Keinanen-Kiukaanniemi, and S. Nayha, Obesity
and low-grade inﬂ ammation among young Finnish men with early-onset alopecia, Dermatology, 214,
(28) R. Hoffmann, TrichoScan: Combining epiluminescence microscopy with digital image analysis for
the measurement of hair growth in vivo, Eur. J. Dermatol., 11, 362–368 (2001).
(29) R. Hoffmann, TrichoScan: A novel tool for the analysis of hair growth in vivo, J. Investig. Dermatol. Symp.
Proc., 8, 109–115 (2003).
(30) R. A. Hiipakka, H. Z. Zhang, W. Dai, Q. Dai, and S. Liao, Structure-activity relationships for inhibi-
tion of human 5alpha-reductases by polyphenols, Biochem. Pharmacol., 63, 1165–1176 (2002).
(31) E. A. Olsen, M. Hordinsky, D. Whiting, D. Stough, S. Hobbs, M. L. Ellis, T. Wilson, and R. S.
Rittmaster (Dutasteride Alopecia Research Team), The importance of dual 5alpha-reductase inhibition
in the treatment of male pattern hair loss: Results of a randomized placebo-controlled study of dutaste-
ride versus ﬁ nasteride, J. Am. Acad. Dermatol., 55, 1014–1023 (2006).
(32) D. Van Neste, V. Fuh, P. Sanchez-Pedreno, E. Lopez-Bran, H. Wolff, D. Whiting, J. Roberts, D. Kopera,
J. J. Stene, S. Calvieri, A. Tosti, E. Prens, M. Guarrera, P. Kanojia, W. He, and K. D. Kaufman, Finasteride
increases anagen hair in men with androgenetic alopecia, Br. J. Dermatol., 143, 804–810 (2000).
(33) S. Keene and A. Goren, Therapeutic hotline: Genetic variations in the androgen receptor gene and
ﬁ nasteride response in women with androgenetic alopecia mediated by epigenetics, Dermatol. Ther., 24,
(34) A. G. Messenger, K. Elliott, A. Temple, and V. A. Randall, Expression of basement membrane proteins
and interstitial collagens in dermal papillae of human hair follicles, J. Invest. Dermatol., 96, 93–97 (1991).
(35) M. Aumailley and P. Rousselle, Laminins of the dermo-epidermal junction, Matrix Biol., 18, 19–28
(36) J. Li, J. Tzu, Y. Chen, Y. P. Zhang, N. T. Nguyen, J. Gao, M. Bradley, D. R. Keene, A. E. Oro, J. H.
Miner, and M. P. Marinkovich, Laminin-10 is crucial for hair morphogenesis, EMBO J., 22, 2400–2410
(37) H. Imanishi, D. Tsuruta, C. Tateishi, K. Sugawara, R. Paus, T. Tsuji, M. Ishii, K. Ikeda, H. Kunimoto,
K. Nakajima, J. C. Jones, and H. Kobayashi, Laminin-511, inducer of hair growth, is down-regulated
and its suppressor in hair growth, laminin-332 up-regulated in chemotherapy-induced alopecia,
J. Dermatol. Sci., 58, 43–54 (2010).
(38) C. Margadant, R. A. Charafeddine, and A. Sonnenberg, Unique and redundant functions of integrins in
the epidermis, FASEB J., 24, 4133–4152 (2010).
(39) H. J. Chung and J. Uitto, Type VII collagen: The anchoring ﬁ bril protein at fault in dystrophic epider-
molysis bullosa, Dermatol. Clin., 28, 93–105 (2010).