Active
ingredients

Technical File
Capixyl™
Anti-Aging
Hair Care Complex
Biomimetic peptide
combined with a
red clover extract
For stronger and thicker
hair and lashes

TABLE OF CONTENT

Capixyl™
SUMMARY2

INTRODUCTION

3

EFFICACY STUDIES

10

Effect of Acetyl Tetrapeptide-3 on Collagen synthesis by Fibroblasts In Vitro
11
Effect of Acetyl Tetrapeptide-3 on the synthesis of ECM proteins by Fibroblasts In Vitro
13
Effect of Acetyl Tetrapeptide-3 on the synthesis of COLLAGEN VII In Human Skin Explants Ex Vivo
15
Effect of Acetyl tetrapeptide-3 on Human Hair follicles Ex Vivo
18
Effect of Acetyl tetrapeptide-3 on Human Hair follicles Ex Vivo – Comparative study
20
Effect of Biochanin A on 5α-Reducatse Activity In Vitro
21
Effect of Capixyl™ and Red Clover on IL-8 Production by Human Fibroblasts
24

CLINICAL STUDY ON HAIR LOSS

26

CLINICAL STUDY ON EYELASHES

32

CONCLUSION40

COSMETIC APPLICATIONS40RECOMMENDATION OF USE

40

FORMULATION EXAMPLE FOR EYELASH TREATMENT

42

REFERENCES

43[Tapez un texte]Technical File
CapixylTM

INCI NAME
CAS

Butylene Glycol (1) Water (and) (2) (and) Dextran (3) (and) Acetyl Tetrapeptide-3(4) (and) Trifolium Pratense (Clover) Flower Extract (5)107-88-0 (1), 7732-18-5 (2), 9004-54-0 (3), 827306-88-7 (4), 85085-25-2 (5)EINECS

203-529-7 (1), 231-791-2 (2), 232-677-5 (3), - (4). 285-356-7 (5)ORIGIN

Biomimetic peptide combined with red clover extract rich in Biochanin A

COSMETIC
PROPERTIES• Improves ECM proteins renewal for optimal hair follicle anchoring• Reduces IL-8 pro-inflammatory cytokine• Reduces 5-α-reductase enzyme activity• Reduces hair loss
• Stimulates / Boosts hair growth

SKIN BENEFITS / • Improves hair follicle anchoring by maintaining proteins matrix support andPOTENTIAL CLAIMS
integrity• Reduces inflammation in the scalp

APPLICATIONSRECOMMENDED
DOSAGE•

Anti-hair loss•

Tonics•Hair regrowth•

Anti-aging hair care product•

Eye lashes•

Treatment for seasonal hair loss•

Leave-on•Eyelash mascara•

Hair treatment for•

Eyelash serum

menopaused women•

Eyelash conditioner•

Scalp treatment•

Eyelash treatment•

Lotions•

Active makeup•

Intensive treatment: 5%•

Preventive care: 0.5 – 2.5%

USAGE PH RANGE

pH: 4 – 8

INCORPORATIONAt the end of the formulation (< 40˚C)

INCOMPATIBILITIESNot known

PATENTSWO 2005/009456 – US7057719

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The importance of hair in our lives cannot be overstated. Whether men or
women lose their hair, they lose much more than their natural, youthfulappearance. People also lose their self-esteem and self-confidence
associated with healthy looking hair. Hair loss also called alopecia is acommon problem affecting both men and women. There are six major
types of hair loss, but the most common one is Androgenetic/Androgenic alopecia which seems to represent
95% of all hair loss.
Androgenetic alopecia is said to affect roughly 50% of men and perhaps as many women older than 40 years.
Androgenetic alopecia (AGA) is also called male pattern baldness (MPB). By the age of 35 two-thirds ofAmerican men will experience some degree of appreciable hair loss. Approximately 25% of men who suffer
with MPB begin the painful process before they reach the age of twenty-one. AGA affects an estimated 35
million men in the United States and about 21 million women. In France 10 millionof people are affected by hair loss, which represents 2 men out of 3 and one
woman out of 5. In Japan, 30% of the male population experiences balding,usually later in life after 45 years of age. The prevalence of AGA in Chinese men
is 21.3% and 6% for women, these rations are lower than in Caucasians but
similar to that in Koreans. Approximately 13% of premenopausal womenreportedly have some evidence of AGA.

However, the incidence of AGA
1

increases greatly in women following menopause , and it may affect 75% of
2

women older than 65 years .
According to The Washington Post, American hair loss sufferers spend more than
3.5 billion dollars a year attempting to treat their hair loss. There are numerous
products on the market addressing this condition. The most popular are Minoxidil®

(Regain ) an OTC vasodilator medication known for its ability to slow or stop hair®

loss and promote hair regrowth, Finasteride (Propecia ) also a drug that acts by
inhibiting the enzyme that converts testosterone to dihydrotestosterone (DHT) in®

3

AGA and Aminexil a patented molecule by l’Oréal .

In today’s image-conscious society, people are looking for an affordable cosmetic hair loss solution thatdelivers on its promises. This presents an opportunity for high performance, clinically proven anti-hair loss
products.

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Hair Science
Hair appears much more complex that they look andthey play a vital role in our appearance.

They arecomposed of strong structural protein called keratin.
Below the surface of the skin is the hair root, which is
enclosed within a hair follicle. At the base of the follicle
is the dermal papilla. The dermal papilla is feed by thebloodstream which carries nourishment to produce new
hair. The dermal papilla plays a crucial role in thedermal-epidermal interactions and is of great importance4for the hair formation and growth cycle .
Each human head carries roughly 100,000 hair follicles.
Each follicle can grow many hairs over a lifetime: on average, each follicle grows a new hair around twenty
times. Hair grows about 3-4 mm/day. This hair growth and loss is completely random. At any given time, arandom number of hairs will be in various stages of growth and shedding. Over the years, the number offollicles capable of growing hair declines naturally. The decline is especially noticeable on the top of the head.

Between starting to grow and falling out years later, each hair passes through three distinct growth stages.
- Anagen: the Growth phase. Hair is actively growing; this phase can last from 2 to 6 years.
- Catagen: the Transition phase.

The dermal papilla breaks away and the hair stops growing andremains in this phase for only two to three weeks.
- Telogen: the Resting phase. The hair stays attached to the follicle and falls out only to be replaced by
the next building hair in the anagen phase. This phase lasts approximately 100 days.

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At the end of the telogen phase the hair follicle re-enters the anagen phase. The dermal papilla and the baseof the follicle join together again and a new hair begins to form. If the old hair has not already been shed the
new hair pushes the old one out and the growth cycle starts all over again.
In an adult scalp, 70-85% of hairs are in anagen phase and 10% in telogen phase. As we age, the percentageof hair in the telogen phase increases for a slower hair growth rate and the replacement hair gets finer andthinner.

WHAT CAUSES HAIR LOSS?
Although causes of hair loss are still not yet fully understood, it can be theresults of several factors.

The causes of hair loss can be genetic, due tohormonal changes or imbalances (childbirth, menopause), linked to nutrition(deficiency in certain vitamins and minerals), stress, diseases like diabetes orlupus or medications, hair treatment (overstyling and excessive brushing, dye,bleaching) and of course aging.
Hair loss can be permanent or temporary.
Most cases of hair loss are due to AGA. Approximately 50% of men by the age of 50 years and 15% of women
before the time they reach menopause have some degree of AGA. In men, it begins at the crown, temples, orboth. In women, the hair becomes thinner all over the head, and the hairline does not recede. AGA in women
rarely leads to total baldness.
A normal hair growth cycle is not affected by any hormonal dysfunction. The hairfollicle is surrounded by good quality of connective tissue and dermal papilla andhas minimal or absence of neither inflammation nor oxidative stress.
Hair loss sufferers will most of the time have the hair growth cycle disrupted by an increase of
dihydrotestosterone (DHT) hormone, loss of connective tissue integrity surrounding the hair follicle, anincrease in the inflammatory process in the scalp and is submitted to higher oxidative stress. Of course withaging, the decrease number of hair follicle and size of follicle in the scalp will also lead to thinner hair shaft andan increase in hair loss.

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EFFECT OF DHT AND 5-ΑLPHA-REDUCTASE
Researchers have determined that AGA is related to hormones called androgens, particularly testosterone
5and its related hormone dihydrotestosterone (DHT) .
DHT is formed by the action of the enzyme 5α-reductase on testosterone.

DHT causes hair loss by

shortening the growth phase of the hair cycle (decreasing the anagen phase (growth) and increasing the
telogen phase (resting)), causing miniaturization (decreased size) of the follicles, and producing progressively
shorter, finer hairs. Eventually these hairs totally disappear. It has been found that in spite of their normal
testosterone levels people suffering from alopecia have higher levels of 5α-reductase, DHT and androgen
receptors. In men, the 5α-reductase activity is higher in the balding area.

5-α reductase
DHT

Testosterone
DHT

DHT (dihydrotestosterone)

DHT
DHT shortens the hair growth cycle(shorter anagen phase & longer telogen
Miniaturization of the follicles
Producing progressively shorter, finer hairs

Hair loss

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EFFECT OF CONNECTIVE TISSUE INTEGRITY AND RENEWAL
The dermal papilla consists of a highly active groupof cells (mainly fibroblasts) shown to be capable ofinducing follicle development from the epidermis andproduction of hair fibers

6,7. Dermal papilla directsand dictates the generation of a hair follicle andretains this ability throughout the hair cycle. A big
and healthy dermal papilla will produces more cells,
and will lead to a production of thicker hair fiber by
the hair follicle.
Furthermore, the size of hair follicles is determined by the volume of its dermal papilla which depends on the
8number of cells it contains and on the volume of the extracellular matrix . Dermal papilla cells revealed thetypical morphology and growth pattern which is similar to cutaneous fibroblasts. Dermal papilla cells are foundto produce considerable amounts of matrix proteins (similar to Extracellular Matrix (ECM)) such as fibronectin,
collagen type I and type III

9,10. A reduction in size of the matrix cells of the hair bulb and its dermal papilla has
11been associated with AGA condition .

It has been demonstrated that molecules which have an activity on the ECM proteins by stimulating the skin
12

remodeling, also increase hair follicle size . Furthermore, skin repair and hair growth enhancement effects
13

are closely linked . Moreover, the quality of the ECM proteins in the dermal papilla is of major importance forthe cyclical growth of hair. Healthy dermal papilla will produce good ECM proteins such as collagen type III
and anchoring fibers such as laminin and collagen VII which will favor a good hair anchoring in the bulb
surrounding tissue. It has also been observed that ECM proteins thickness decreases in alopecia.

If there is improper ECM renewal, hair will eventually lack vigor and will thin. Cycle after cycle, the follicle
14

becomes smaller and finally, miniaturized and fall .

EFFECT OF INFLAMMATION
15Chronic inflammation of the hair follicle is considered a contributing factor in the pathogenesis of AGA . The
16

follicular microinflammation also plays an integral role in the pathogenesis of AGA in early cases . The term
«microinflammation» was proposed by Mahe and colleagues because the process of inflammation in patternbaldness adopts a slow, subtle, painless and lethargic course, in contrast to the inflammatory and destructive
17

process that has been seen in the classical inflammatory scarring alopecia . Among the possible causes citedare heat, frictions, UV radiation and microflora.

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LUCAS MEYER COSMETICS UNIQUE SOLUTION TO HAIR LOSS
Capixyl™ is an innovative and unique active complex designed to prevent and stop the hair loss process andstimulate hair growth.

Capixyl™ is composed of a high tech patented four amino acids biomimetic peptide with a red clover
extract rich in Biochanin A.

Red Clover Extract
Red clover (Trifolium Pretense) flowers have been found throughout central andnorthern Europe and Asia. Red clover was traditionally used to treat asthma, cancer,
gout, and various inflammatory skin disorders like eczema and psoriasis.

Biochanin A is the major isoflavone in red clover. Biochanin A is known to be an effective inhibitor of
18

5α-reductase type I & II activity . Researches have demonstrated that
Biochanin A modulates chronic inflammation and that red clover’s
isoflavones may also function as important antioxidants, limiting free radical
damage to the skin and scalp.

Acetyl Tetrapeptide-3 Biomimetic Peptide
This unique patented peptide is a stimulator of extracellular matrix proteins favoring abetter hair anchoring. It is derived from a signal peptide which has potent tissue
protective properties and stimulates tissue remodeling after the initial phase of wound healing. The
peptide has a direct effect on hair follicle. The remodeling signal will increase the size of hair follicle forbetter hair number and vitality.

Capixyl™ efficacy is based on the combined action of its two ingredients, allowing direction action on DHT
preventing the hair cycle to shorten and hair follicle miniaturization, improving the ECM proteins in dermal
papilla surrounding hair papilla for better anchoring and promoting an increase of follicle size. Capixyl™ also
inflammation which is a contributing factor in hair loss.

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CAPIXYL™ MECHANISM OF ACTION•

Inhibition of 5α-reductase, thereby modulating the conversion of testosterone
to DHTNo miniaturization of follicles•

ECM proteins renewal

Better hair anchoring•

Inflammation reduction

Normal hair cycle

Capixyl™ prevents and stops the hair loss process and stimulates hair growth. Ex vivo experiments havedemonstrated better results in hair growth stimulation than Minoxidil. Clinical data shows that it improves,

alopecia by increasing the Anagen/Telogen ratio. Capixyl™ is a safe alternative to Minoxidil and provides fast
results.

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EFFECT OF ACETYL TETRAPEPTIDE-3 ON COLLAGEN SYNTHESISBY FIBROBLASTS IN VITROBACKGROUND
The anatomy of the hair follicle, by contrast with the surface epidermis, is not static or rigid. Human hair grows
in a continuous cyclic pattern and the bulbar portion of the follicle undergoes almost total disintegration during
the catagen phase. The renewal of the proteins implicated in the formation of new hair follicle through thedermal papilla is thus of importance.
The hair follicle can be recognized as a separate entity within the skin. The dermal papilla consists of a highly
active group of cells, especially fibroblasts, shown to be capable of inducing follicle development from theepidermis and production of hair fiber

19,20

.
21

The major biosynthetic product of dermal fibroblasts is collagen . Collagen as a major protein component of
the dermis plays an important role in the skin cohesion and renewal.

PROTOCOL
Principle
The dermis is constituted of cells like fibroblasts which produce collagen. Collagen is composed of threechains wound together in a tight triple helix. Collagen is characterized by the presence of 2 specific aminoacids: hydroxylysine and overall hydroxyproline (OH-proline). Through the dosage of hydroxyproline, it ispossible to correlate with the quantity of collagen produced by cells.
Thus hydroxyproline estimation may be used to give an indication, unaffected by the presence of otherproteins, of the mature collagen content of biological tissue. It may also be used to give an indication of
the degree of collagen breakdown in physiological or disease processes.

Biological material
It has been established and demonstrated that proteins and collagen synthesis of the dermal-hair papilla cells
22

is very similar to skin fibroblasts . We thus used human fibroblasts (MRC5 cell line) for this assay.

Evaluation of activity-7

Cells are incubated or not with the acetyl-tetrapeptide-3 at 10 M for 7 days. After incubation period,
Hydroxyproline (OH-proline) dosage is assessed with the Chloramine T reaction and measured by optical23

density (OD) at 540 nm compared with a range standard .
The procedure is based on alkaline hydrolysis of the tissue homogenate and subsequent determination of thefree Hydroxyproline in hydrolyzates. Chloramine-T was used to oxidize the free Hydroxyproline for the
production of a pyrrole. The addition of Ehrlich's reagent resulted in the formation of a chromophore that canbe measured at 540 nm.

Results analysis
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The effect of the Acetyl-tetrapeptide-3 and its potential activation on collagen synthesis on fibroblasts wasevaluated by comparing the quantity of Hydroxyproline produced by MRC5 cells incubated with or without
Acetyl-tetrapeptide-3.

RESULTSAs represented in the figure above, the cells treated with Acetyl tetrapeptide-3 show highly elevated level of
Hydroxyproline direclty correlated with mature collagen cotent in cells.

CONCLUSION
By stimulating the collagen synthesis and collagen content in fibroblasts, Acetyl-tetrapeptide-3 can be used torenew dermal papilla dermis collagen.

Acetyl-tetrapeptide-3 stimulates collagen production for better ECM
integrity for a better hair follicle anchoring.

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EFFECT OF ACETYL TETRAPEPTIDE-3 ON THE SYNTHESIS OF
ECM PROTEINS BY FIBROBLASTS IN VITROBACKGROUNDAs already described, dermal part of hair follicle, such as dermal papilla cells and dermal sheath cells are24

thought to play an important role in the induction and maintenance of hair growth . Those cells have the25

ability to induce hair follicle formation by interaction with the epithelial cells of hair follicle .
26

The major biosynthetic product of dermal fibroblasts is collagens . It is present in more than ten geneticallytypes found in different tissues and synthesized by specialized connective tissues cells. Dermal fibroblasts
27

synthesize mostly type I and type III, and in addition small amounts of types V and VI collagen . Collagentypes I and III are not uniformly distributed in the dermal connective tissues showing an accumulation of type28

III collagen in the papillary dermis . Furthermore, the presence of laminin was also detected in the papilla29

dermis cells and seems to be important in the first days of growth for proper follicle anchoring .
The size of hair follicle is thought to be determined by the volume of its dermal papilla which depends on the
30

number of cells and on the volume of the extracellular matrix .

PROTOCOL
Principle
The effect of Acetyl tetrapeptide-3 on the synthesis of different extracellular matrix proteins (collagen III &laminin) was evaluated by selective immunofluorescence in comparison with untreated fibroblasts.
Immunofluorescence is a technique allowing the visualization of a specific protein (collagen III or laminin) incells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as
rhodamine (red color).

Biological material
As previously described, fibroblasts from dermal papilla and dermis fibroblasts are similar. Thus, humanfibroblasts (MRC5 cell line) were used for this assay.

Evaluation of activity-7

Cells are incubated or not with Acetyl tetrapeptide-3 at 10 M for 3 days. Cells were then fixed on the slide andproteins (collagen type III or laminin) were detected with specific antibodies coupled to a fluorochrome which
can be detected and quantified by a confocal microscope (Axioplan and Zeiss LSM510), which allowed asemi-quantitative evaluation.

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Results analysis
The effect of Acetyl tetrapeptide-3 and its potential activation on collagen types III or laminin synthesis on
fibroblasts was evaluated by comparing the fluorescence intensity observed on cells incubated with or without
Acetyl tetrapeptide-3.

RESULTS
Represented here, are the microscopic images of the fibroblasts, untreated (left) or treated (right) with Acetyl
tetrapeptide-3. The graphs represent the semi quantitative evaluation of the extra cellular matrix protein
synthesis (type III collagen and laminin).

These results showed a significant increase of the synthesis of extracellular matrix (type III collagen andlaminin) proteins in fibroblasts treated with Acetyl tetrapeptide-3.

CONCLUSION
Acetyl tetrapeptide-3 powerfully induces the synthesis of ECM proteins by fibroblasts such as type III
collagen and laminin. It has been demonstrated that molecules which have an activity on the ECM proteins by
31

stimulating the skin remodeling, also increase hair follicle size .

Acetyl tetrapeptide-3 stimulates dermal papilla extracellular matrix proteins thus has adirect effect on hair follicle size and better anchoring

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EFFECT OF ACETYL TETRAPEPTIDE-3 ON THE SYNTHESIS OF COLLAGEN VII IN
HUMAN SKIN EXPLANTS EX VIVOBACKGROUND
Little is known regarding the junction that separates the follicular epithelial and its adjacent connective tissue,
the connective tissue-epithelial junction (CEJ). However, this junction presents around the anagen hair follicle
is closely similar to that seen at the DEJ

32

and proteins found in the normal interfollicular epidermis were33

expressed mainly during the anagen phase .
Collagen VII (the major constituent of anchoring fibrils), is located in the middle part of the follicular basement
membrane zone and around the hair papilla. As the composition and structure of the anagen hair follicle
dermal-epidermal junction (DEJ) is similar to the interfollicular skin, the effect of Acetyl-tetrapeptide-3 on type
VII collagen synthesis was evaluated on human skin explants.

PROTOCOL
Principle
This biological model uses human normal skin explants. The skin metabolism is experimentally decreased by
the application of corticoïds, which modified the skin metabolism like the one observed in natural aging.
34

Furthermore, it has been described that corticoïds decrease the hair papilla cells (fibroblasts) growth . The
repair of dermal-epidermal junction with the Acetyl tetrapeptide-3 is then evaluated by the analysis of
immunohistological staining of collagen VII proteins.

Biological material
Four Human skin explants were obtained from patients undergoing plastic surgery (Caucasian women 35-45
years old) and maintained in culture.

Evaluation of activity
35-3

The first day, a surface skin application of corticoïds was done . Acetyl tetrapeptide-3 at 10 M was added on
the cell culture for 2 days. Skin explants were prepared for specific collagen VII immunohistological labellingusing ABC peroxydase Kit and revealed by AEC substrate (brown color).

Results Analysis: Semi-quantitative histological evaluation of collagen VII labeling:
Scores ranging from 0 (negative) to 4

No collagen VII labelling

Score 0(maximum)Slight Collagen VII labelling

Score 1Moderate Collagen VII labelling

Score 2

Normal Collagen VII labelling (normal skin)

Score 3Over-expression of Collagen VII labelling

Score 4

weredefinedfollowing parameters:

as

the

RESULTS
Semi-quantitative evaluation of the Collagen VII staining in the human skin explants
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Microscopic observations of the collagen VII immunohistological labelling
The presence of collagen VII in the dermal-epidermal junction is evidenced by its specific labelling using
coupled antibodies (brown-red color along the DEJ).

The microscopic observations of normal untreated skin show a strong labelling of the collagen VII along thedermal-epidermal junction (Figure A). This labelling is quantified using a score ranging evaluation table andcorresponds to a score equal to 3.
We clearly observed a dramatically decrease of the collagen VII staining after application of dermocorticoïds
on skin (Figure B). The semi-quantification evaluation of this labelling represents a decrease of 70% in
collagen VII (score 0.9) which demonstrates a strong alteration of the dermal-epidermal junction properties.
The application of Acetyl tetrapeptide-3 on the skin (Figure C), in addition to dermocorticoïds restores thenormal amount of collagen VII along the DEJ as the score calculated after the labelling is the same than theone obtained for the untreated skin (score 3) (Figure A).

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CONCLUSION
In this aging human skin experimental model we can observe an alteration of the collagen VII along thedermal-epidermal junction after application of dermocorticoïds.
The addition of Acetyl tetrapeptide-3 on the skin surface restores totally the dermal-epidermal junction by thestimulation and/or the protection of collagen VII.
These results obtained in this skin model can be correlated with the interfollicular skin (in anagen hair follicle)
as the expression and the distribution of the collagen VII in the DEJ is similar in both types.

Acetyl tetrapeptide-3 provides a repairing effect at the dermal-epidermal junction level,
improving hair anchoring

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EFFECT OF ACETYL TETRAPEPTIDE-3 ON HUMAN HAIR FOLLICLES EX VIVOBACKGROUND
In 1990, Philpott and al. reported the first time the successful maintenance and growth of human hair follicles
36

in vitro . The importance of this model to hair follicles biology had been demonstrated. This study wasassessed in order to measure the growth speed of hair shafts on isolated human hair follicles in cultured with
Acetyl tetrapeptide-3.

PROTOCOL
Principle
Human anagen hair follicles were isolated by microdissection from human scalp skin. Isolation of the hairfollicles was achieved by cutting the follicle at the dermo-subcutaneous fat interface using scalpel blad.

Biological Material
Hair follicles recovered from human scalps are immersed in a specific culture medium. Successive washing is
performed and the follicles dissection is made under binocular microscope according to the Philpott techniquein order to specifically select follicles which are in the anagen phase.
-9

The follicles are cultured in a specific medium added or not with Acetyl tetrapeptide-3 at 4x10 M for 8 days.
Untreated follicles but cultured in the same conditions represent our negative control.
The numbers of follicles per treatment are:–n = 6 follicles treated with Acetyl tetrapeptide-3–

n = 6 untreated follicles

Evaluation of activity
Growth of hair follicles in culture was assessed by measuring increases in follicle length over the 8 days
culture period, each 24 hours with a micrometer incorporated in the microscope optical.
The percentage of growth was calculated as follows:

Percentage of growth:

Tx: Time x after treatment

Length at Tx – Length at T0
Length at T0x 100

T0: Time 0, at the beginning of the treatment

Results analysis
The growth percentage was determined after 8 days treatment of follicles with Acetyl tetrapeptide-3 incomparison with untreated follicles.

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RESULTSIn these experimental conditions, we observed a strong stimulation of hair growth by 35% in comparison withuntreated follicles.

CONCLUSION
Acetyl tetrapeptide-3 is able to strongly stimulate the human hair growth after 8 days treatment.

Acetyl tetrapeptide-3 stimulates hair growth

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EFFECT OF ACETYL TETRAPEPTIDE-3 ON HUMAN HAIR FOLLICLES
EX VIVO – COMPARATIVE STUDYBACKGROUND
In 1990, Philpott and al. reported the first time the successful maintenance and growth of human hair follicles
37

in vitro . The importance of this model to hair follicles biology had been demonstrated.
This study was assessed in order to measure the growth speed of hair shafts on isolated human hair follicles
in cultured with Acetyl tetrapeptide-3 in comparison with the Minoxidil.

PROTOCOL
Principle
Human anagen hair follicles were isolated by microdissection from human scalp skin. Isolation of the hairfollicles was achieved by cutting the follicle at the dermo-subcutaneous fat interface using scalpel bald.

Biological Material
Hair follicles recovered from human scalps are immersed in a specific culture medium. Successive washing is
performed and the follicles dissection is made under binocular microscope according to the Philpott techniquein order to specifically select follicles which are in the anagen phase.
The follicles are cultured in a specific medium added or not with the test substances: Acetyl tetrapeptide-3 at-7-7

10 M or the reference Minoxidil at 120x10 M for 7 days. Untreated follicles but cultured in the same
conditions represent our negative control.
The numbers of follicles per treatment are:–n = 15 follicles treated with Acetyl tetrapeptide-3–

n = 27 follicles treated with Minoxidil–

n = 27 untreated follicles (control)

Evaluation of activity
Growth of hair follicles in culture was assessed by measuring increases in follicle length over the 7 days
culture period, each 24 hours with a micrometer incorporated in the microscope optical.
The percentage of growth was calculated as follows:
Percentage of growth:

Acetyl tetrapeptide-3 stimulates

x 100

hair growth
Tx: Time x after treatment

T0: Time 0, at the beginning of the treatment

In parallel, the normalized activity after treatment (“hair growth induced by the treatment”) was calculated
following the equation:
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Activity =(length P D7 – length P D0) – (length C D7 – length C D0)(length C D7 – length C D0)

C = control (untreated follicles) ;X 100

P = Acetyl tetrapeptide-3

Results analysis
The growth percentages and activity were determined after a 7 days treatment of follicles with Acetyl
tetrapeptide-3 or Minoxidil in comparison with untreated follicles.

RESULTS
Hair growth activity compared to untreated+ 156%

160% of activity

120
80

+52%

40
0

Minoxidil

Acetyltetrapeptide-3

We observed a strong stimulation of hair growth in these experimental conditions. The activity is better with
Acetyl tetrapeptide-3 and has demonstrated a 47% growth which is higher than the growth obtained with
Minoxidil.

CONCLUSION
Acetyl tetrapeptide-3 is able to strongly stimulate the human hair growth. Results obtained with Acetyl
tetrapeptide-3 are higher than with Minoxidil, a reference in hair growth treatment.

Acetyl tetrapeptide-3 stimulates hair growth with higher activity than
the reference hair growth product, Minoxidil

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EFFECT OF BIOCHANIN A ON 5α-REDUCATSE ACTIVITY IN VITROBACKGROUND
The enzyme 5α-reductase catalyses the conversion of testosterone to dihydrotestosterone (DHT), a process
thought to amplify the androgenic response.
DHT causes hair loss by shortening the growth phase of the hair cycle (decreasing the anagen phase (growth)and increasing the telogen phase (resting)), causing miniaturization (decreased size) of the follicles, andproducing progressively shorter, finer hairs.

PROTOCOL
Principle
In this experiment, the capacity of Biochanin A to modulate the activity of both type I and type II 5α-reductase
activity was measured in intact cells.

Biological Material
EGCG (epigallocathechin gallate) isolated from green tea served as a positive control for the inhibition of 5α38

reductase activity .
ECGC and Biochanin A were used at 100 µM.

Evaluation of activity
Cells were plated and incubated with test compounds (Biochanin A or positive control) for 1 hour at 37°C
before the addition of

14

C-testosterone, at a final concentration of 1.5 µM. Cells were incubated for an

additional 3 hours and radioactive steroids were extracted. The amount of labelled testosterone and DHT were
determined by TLC (Thin Layer Chromatography) in order to quantify the 5α-reducatse activity.

Results analysis
Results are expressed in percent inhibition of 5α−reductase activity in the presence of 100 µM concentrationof Biochanin A or ECGC.

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RESULTS

These results showed that 5α−reductase (type I and II) activity is significantly decrease in presence ofBiochanin A at 100 µM.
The inhibition observed with Biochanin A is higher than the effect observed with ECGC, a 5α−reductase
activity inhibitors reference.

Biochanin A decreases decrease 5-α-reductase activity and thus
reduce the conversion of testosterone into DHT

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EFFECT OF CAPIXYL™ AND RED CLOVER ON IL-8 PRODUCTIONBY HUMAN FIBROBLASTSBACKGROUND
Androgenetic alopecia is a common cosmetic hair disorder, resulting from interplay of genetic, endocrine, andaging factors leading to a patterned follicular miniaturization. Micro-inflammation seems to be a potential active
player in this process

39.

In AGA we observed clusters of abnormal inflamed streamers or fibrous tracts
40

surrounding the hair follicle .
Interleukine-8 (IL-8) is a cytokine secreted by several cell types and is one of the major mediators of theinflammatory response.
Decreasing the level of inflammation on the scalp and hair bulb area can have a positive effect on hair loss
prevention.

PROTOCOL
Principle
In this experiment, the capacity of red clover extract and Capixyl™ to modulate the production of IL-8 was
measured in normal human fibroblasts, in order to evaluate its anti-inflammatory potential.
Inflammation is chemically induced in keratinocytes using IL-1α a physiological pro-inflammatory agent.

Biological Material
Experiments were done using monolayers of cells derived from normal human fibroblasts (NHDF). IL-1α isused at 0.0075 ng/mL to induce inflammation in fibroblasts.
41

Dexamethasone (DMS) at 1µM served as a positive control and anti-inflammatory agent . Dexamethasone isa potent synthetic member of the glucocorticoïd class of steroid hormones and acts as an anti-inflammatory
with potency about 20-30 times that of hydrocortisone 42.

Evaluation of activity
After seeding, confluent NHDF cells were cultured for 24 hours. At the end of this period, the fibroblasts wereincubated in the presence or absence of Capixyl™, red clover or DMS and IL-1α was added concomitantly tothe medium in order to mimic an inflammatory reaction during 24 hours. IL-8 content was quantified in themedium, using a highly sensitive and specific enzyme immunoassay (EIA) kit.

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Results analysis
Results are expressed as pg/ml of IL-8 produced by fibroblasts.

RESULTS
These results showed that fibroblasts secreted IL-8 after stimulation by IL-1α, acytokine that is triggering the
inflammation process in cells. When cells are treated with DMS, red clover extract or Capixyl™, we observed
a significant decrease in the synthesis of IL-8 attesting a decrease in the inflammation process.

The effect of Capixyl™ has a dose dependant effect and is higher than the effect observed with the DMS, ananti-inflammatory reference. We also observed a better activity of the Capixyl™ in comparison with the red
clover extract alone, which demonstrates a synergistic effect of the peptide and the red clover extract.

Capixyl™ decreases pro-inflammatory cytokines with a synergistic
action compared to red clover extract alone

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OBJECTIVES
The goal of this clinical study was to evaluate the efficacy of Capixyl™ in vivo as a hair care active ingredient
to prevent hair loss and to promote hair growth under normal use conditions. Furthermore, acceptability of the
product upon normal use was also evaluated.
Efficacy was objectively evaluated by using instrumental measurements (digital trichogram with
TrichoScan™). Measurements from a group of 15 Capixyl™-treated volunteers were compared with those of
15 Placebo-treated ones. Every one of the volunteers evaluated acceptability of the product on a daily basis.
The assay was carried out on volunteers suffering AGA, who had been chosen using the following criteria: 200
hairs or less on digital trichogram hair count, and/or less than 70% hairs in the anagen phase.

PRINCIPLE OF TRICHOSCAN
TrichoScan is suitable for the analysis of human scalp hair in AGA. TrichoScan is a non-invasive method,combining standard epiluminescence microscopy with automatic digital image analysis, for the measurementof human hair. The most important advantages are:-Total hair counts can be analyzed within the same day-

The same target site can be used to calculate the number of telogen and anagen hairs.

Determination of total hair density
1.

A shaving mask is positioned on the volunteer head in order to shave a2

1.8 cm area on the zone or zones to be studied.

2.

Three days later, as hairs do not normally contrast well enough with the scalp (due to gray or fairhairs) skin for digital photography, hairs must be dyed and subsequently cleaned with alcohol.

3.

Images were recorded with the equipment camera in order to evaluate theanagen and telogen phases.

Patients were asked not to wash their hair for two days prior to the evaluation with
TrichoScan.

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Recording the images
After acquisition, the digital images are transmitted to specific software for the analysis of the total hair density(anagen + telogen).
1. Original image
2. Detection of hair by the software
3. Specific marked hair
4. Detection of hair in anagen and telogen phases:-

Red: telogen phase;-

Green: anagen phase;-

Yellow: hair is touching the edge of the picture, grouping follows via a special statisticalprocedure.

Original

Detected hairs

1

2

3

4

Marked hairs

Anagen/Telogen hairs

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PROTOCOL
Formulations used in the clinical study•

Capixyl™ lotion: 5% Capixyl™, 20% alcohol, 75% water (pH = 5.4)•

Placebo lotion: 20% alcohol, 80% water

Biological Material•

30 healthy volunteers suffering from AGA, (average age 46).

•

Patients were clinically evaluated and individual case histories were recorded in order to rule out
possible pathologies as iron deficiency anemia, thyroid related conditions or other possible
pathologies.

•

Twenty (20) drops of the tested products were applied in the evening and gently distributed with thefingertips on the experimental area during 4 consecutive months.

•

Digital trichogram was performed using professional TrichoScan. The number of hairs per sample had
to be 200 or less and/or the proportion of anagen hairs had to be less than 70%.

•

Every week, patients were given a plastic bag, where they had to collect all the hairs on their pillows,
combs and clothes on a daily basis; they had to bring the bag to the laboratory for hairs to be counted.

Evaluation of activity
The TrichoScan™ software quantifies the number of hairs in the studied area and the proportion of these hairsin the anagen and the telogen phase. This software is calibrated on the basis of 0.3 mm hair growth per day
during the anagen phase and no hair growth during the telogen phase. Two measurements – one at the
beginning and one at the end of the study – were taken to each volunteer.

Results analysis
2

Anagen hair density (n/cm ): in the definition of the TrichoScan procedure, an anagen hair is a hair which isdetectable three days after complete hair shaving. Within this time only anagen hairs should grow significantly.
2

Telogen hair density (n/cm ): by definition a terminal hair will not grow whereas anagen hairs will. When
images are taken three days after clipping, growing hairs can be differentiated from non-growing hairs basedon different hair length. TrichoScan identifies non-growing hairs as telogen hairs and growing hairs as anagen
hairs.
Ratio A/T: Comparison of the numbers of anagen and telogen hair, which is an indication of the percentage ofactive hair follicles.
 A/T ratio = activation of hair growthefficacy of treatment

 A/T ratio = loss of hair growth activity alopecia continues

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RESULTS
Determination of anagen hair count
The TrichoScan software defines anagen hairs basedon the knowledge that anagen grows at approximately
0.3 mm/day whereas telogen and catagen hairs do not
grow.
During successful treatment, the anagen hair countshould increase and therefore this approach can beused to monitor a treatment response.

Determination of telogen hair countIn the software sense a telogen hair is a non-growing
hair.
The software will measure the length of hair and bystatistical analysis will discriminate between growing
versus non-growing hairs. (catagen and exogen hairs
will be judged as non-growing hairs).

Capixyl™ induces a clear increase in the anagen hair density in comparison with placebo, demonstrating anefficacy of the treatment for hair growth after 4 months application.
Capixyl™ also induces a strong reduction in the telogen hair density in comparison with the placebo, whichclearly indicates a reduction of hair loss with Capixyl™ treatment.

Determination of Anagen/Telogen ratio (A/T)
The A/T ratio is a comparison of the numbers ofanagen and telogen hair follicles, which is anindication of the percentage of active hair follicles.

The graphic shows the averaged beginning-of-study
and end-of-study measurements for the Placebotreated and the Capixyl™-treated groups.

Capixyl™ statistically increases the Anagen/Telogen ratio (which represents an increase in the number of hairin the growing phase with a reduction of the number of hair in the resting phase) of 46% compared to a
reduction of -33% for the placebo.
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Before & After Pictures

Capixyl™ increases the A/T ratio of 46% compared to a reduction of -33% for the placebo
attesting the efficacy for stimulating hair growth and reducing hair loss

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As eyelash characteristics are very similar to those of scalp hair, Capixyl™ represents the ideal and perfectsolution to modulate eyelash growth in order to avoid eyelash loss and stimulate biological mechanisms
behing eyelash growth.
Compared with human hair, little is knowm about eyelashes. In the embry, eyelashes develop between the 7thth

and 8 week. They grow in imperfect rows of five to six in the upper and three to43four in the lower lid .
Their mean number is 90-160 in the upper and 75-80 in the lower lid, while theirlength varies from 8 to 12 mm in the upper and from 6 to 8 mm in the lower
44 45

lid , .
The terminal eyelash fibre is characterized by a regular curve shape, more or less46

marked depending on ethnic origin . Microscopy of eyelash revealed a structure very
close to that of hair fibre, with three compartments from the outside to the inside.
However, the eyelash fibre is much shorter than scalp hair, due to a shorter hair cycle.
The growth rate is approximately 120 µm daily

47

and the duration of anagen and telogen

phases is estimated to be 1-4 and 4-9 months respectively. At any given time, 59-85% of
48eyelash follicles are in the telogen phase, depending on whether upper or lower lid .

Comparison between hair growth cycle and eyelash growth cycle

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OBJECTIVES
The aim of the study was to evaluate the efficacy of Capixyl™ versus placebo under normal conditions of use.
Relevancy
Cosmetic products present effects on ciliary characteristics, which are shown through biometrologicalmeasurements and subjective evaluations. These observations were performed in normal conditions of use.

Study design
Design: The study was open and randomized. The treated eye on which Capixyl™ gel was applied was
randomized.
The average age of the subjects was: 49.4 ± 14.4 years old (extreme values: 25 - 68 years old).
17 subjects were included according to the following criteria:

Conditions of use (application)
Capixyl™ or placebo gel was applied on the upper and lower lashes of the eye designed by the
randomization, in the morning, before the application of the usual mascara and a second time, alone, in theevening, during 8 weeks.
2 steps application of 2.5% Capixyl™ eye gel - twice a day (morning and evening) for 8 weeks:
1. Application of the gel to skin using applicator: «draw the applicator carefully along theskin at the base of the upper and lower eyelashes (where the eyelashes meet the skin)”.

2. Application of the gel on lashes using the brush as close to the roots of your lashes as
possible and wiggle before brushing out to get more product applied to every lash. Lightly brushthe tip back and forth across lower lashes.

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Parametres evaluation
Three biological parameters were evaluated at D0, after 4 and 8 weeks:

1. Lash density:
At D0, 4 8 weeks, photographs of the upper lashes were taken with a camera fixed on abiomicroscope. The number of lashes in the specific area were then analyzed andcounted.

After counting, the surface where the lashes growth wasmeasures and the density was determined.

2. Evaluation of the number of young lashes:
Young lashes have the followed characteristics (in comparison with adult lashes):- less coloring- thinner- shorter
At D0, 4 weeks and 8 weeks, photographs of the upper lashes were taken with acamera fixed on a biomicroscope.
The number of new lashes according the morphology and characteristics of young
lashes were then counted..

3. Length of the lashes:
Assessment of the mean length of the upper ciliary fringe (for each eye)At D0, 4 weeks and 8 weeks, photographs of the upper ciliary fringe were taken with a digital camera
The mean of the length of the lashes (mm) was measured

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Results
Evaluation of lash density
Lash density was measured after 4 and 8 weeks for each volunteers; results showed here after presented theaverage of lash density evolution of all the volunteers.

We observed a clear and significant evolution of lash density when eyelashes were treated with 2.5%
Capixyl™ in comparison with placebo treatment (on the other eye).
The lash density is increased up to 27% after 8 weeks application of Capixyl™ gel.
It is important to note that after 4 weeks, 73% of the subjects improved in lash density and 93% had an effectafter 8 weeks.
The improvement in lash density also indicated that Capixyl™ treatment induced an increase in new lashes
and decrease in falling lashes.

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Evaluation of number of new lashes
New lashes were identified and counted after 8 weeks treatment with gel containing 2.5% Capixyl™ orplacebo gel.

We observed a significant increase in new lashes after application of Capixyl™ gel by 45.5% in comparison
with placebo treatment no new lashes were measured.
Illustration showed here after attest the evolution between 4 and 8 weeks as we can cleary observed newlashes at the upper lid area.

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Evaluation of lashes lenght
The length of lashes were measured at D0 and after 4 and 8 weeks treatment of gel containing or not
Capixyl™ 2.5%.

A significant increase of lash length was observed after 4 and 8 weeks treatment with Capixyl™. We
measured up to 0.70 mm growth after 4 weeks treatement.
The evolution of lash length is 3 and 5 times better after Capixyl™ application (4 and 8 weeks respectively) incomparison with placebo.
After 8 weeks 73% of the subjects saw an improvement in the length of their lashes.

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Consumer perception

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CONCLUSION

Technical File
CapixylTM

Capixyl™ is an innovative and powerful cosmetic ingredient providing fuller, thicker and healthier looking hair.
Capixyl™ promotes thicker, fuller and more luscious looking lashes!
It is a complex combination of red clover extract rich in Biochanin A with a patented signal peptide.
It has outstanding results.
It has higher activity than Minoxidil.
It has clinical results to prove its significant anti-hair loss effect and hair regrowth.
Capixyl™ is an innovative & powerful active for eyelashes growth and for decreasing the lash loss process.
Capixyl™ has clinical results to prove its significant anti-lash loss effect and lash regrowth.

COSMETIC APPLICATIONS

















Hair treatment
Men hair care productLeave-on
Scalp treatment
TonicsAnti-aging hair care productTreatment for seasonal hair loss
Hair treatment for menopaused women
Eyelashes
Eyelash growth mascara
Eyelash serum
Eyelash conditioner
Anti-lash loss
Eyelash treatment
Active makeup

RECOMMENDATION OF USE
Capixyl™ is easy to use and has excellent stability.
Capixyl™ should be incorporated at the end of the manufacturing process, at a temperature below 40°C.

Recommended dosage:- Intensive treatment: 5%- Preventive care: 0.5 – 2.5%

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Mixing Procedure
1. Solubilize Sodium Phosphate in water, after complete solubilization, add citric acid.
2. One phase A is homogenous, introduce Phase B ingredients one at the time (in indicated order) and homogenize after each
ingredient addition.
3. Mixed the solubilisant with the fragarance and then slowly add this pre-mix to the mix (phase A & B)..
(NB : the fragrance pre-mix should be completly transparent before introducing it into the mix).
4. Check pH.
Caractéristiques
Aspect : Clear solution
pH = 5,50 – 5,70
Stabilitiy : 1 month at 45°C / 3 month at 40°C

The information given concerning the application and product technology represents our state of knowledge and no claims are
made as to its completeness. The information given does not free the user of his own responsibility for comprehensive testing
before production. We therefore disclaim any responsibility for the accuracy of the information given.

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Formulation Example for Eyelash Treatment

Formulation (gel) developed in partnership with SECOS SAS (Cheops group)

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1

Raine-Fenning NJ, Brincat MP, Muscat-Bron Y. Skin aging and menopause : implications for treatment. Am
J Clin Dermatol. 2003;4(6):371-8.
2

Androgenetic Alopecia. Robert P Feinstein, MD, Associate Clinical Professor, Department of Dermatology,
Columbia University College of Physicians and Surgeons3

Suzuki M. Hair Loss mechanisms and treatment materials : a review. Cosmetics and toileterries. 2008. 9,123; 51-56.

4

Bernard BA. Hair biology: an update. Int J Cosmet Sci. 2002. 24(1):13-6.

5

Randall VA. Androgens and human hair growth. Clin. Endocrinol. 1994. 40(4):439-57.

6

Oliver RF. Whisker growth after removal of the dermal papilla and lengths of follicle in the hooded rat. J
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7

Oliver RF. Regeneration of dermal papillae in rat vibrissae. J Invest Dermatol. 1966. 47(5): 496-497.

8

Elliott K, Stephenson TJ, Messenger AG. Differences in hair follicle dermal papilla volume are due toextracellular matrix volume and cell number: implications for the control of hair follicle size and androgen
responses. J Invest Dermatol. 1999 Dec;113(6):873-7.
9

Couchman J.R. Rat hair follicle dermal papillae have an extracellular matrix containing basement
membrane components. J Invest Dermatol. 1986; 87(6): 762-767.

10

Katsuoka K., Mauch C., Schell H., Hornstein O.P., Krieg T. Collagen-type synthesis in human-hair papillacells in culture. Arch. Dermatol. Res. 1988; 280(3): 140-144.
11

van Scott EJ, Ekkel TM. Geometric relationships between the matrix of the hair bulb and its dermal
papilla in normal and alopecic scalp. J Invest Dermatol. 1958. 31(5): 281-287.
12

Fuchs E. Beauty is skin deep: the fascinating biology of the epidermis and its appendages. Harvey Lect.
1998-1999; 94: 47-77.

13

Almond-Roesler B, Schön M, Schön MP, Blume-Peytavi U, Sommer C, Löster K, Orfanos CE. Cultured
dermal papilla cells of the rat vibrissa follicle. Proliferative activity, adhesion properties and reorganizationof the extracellular matrix in vitro. Arch Dermatol Res. 1997 Nov;289(12):698-704.

14

El-Domyati M, Attia S, Saleh F, Abdel-Wahab H. Androgenetic alopecia in males: a histopathological andultrastructural study. J Cosmet Dermatol. 2009. 8(2): 83-91.
15

Chittur S, Parr B, Marcovici G. Inhibition of Inflammatory Gene Expression in Keratinocytes Using a
Composition Containing Carnitine, Thioctic Acid and Saw Palmetto Extract. Evid Based Complement Alternat
Med. 2009 Aug 19. [in printing]

16

El-Domyati M, Attia S, Saleh F, Abdel-Wahab H. Androgenetic alopecia in males: a histopathological andultrastructural study. J Cosmet Dermatol. 2009. 8(2): 83-91.
17

Mahé YF, Michelet JF, Billoni N, Jarrousse F, Buan B, Commo S, Saint-Léger D, Bernard BA. Androgenetic
alopecia and microinflammation. Int J Dermatol. 2000. 39(8): 576-584.
18

Evans BA, Griffiths K, Morton MS. Inhibition of 5 alpha-reductase in genital skin fibroblasts and prostate
tissue by dietary lignans and isoflavonoids. J Endocrinol. 1995. 147(2): 295-302.
19

Oliver RF, Jahoda CA. Dermal-epidermal interactions. Clin Dermatol. 1988 Oct-Dec;6(4):74-82.

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