m

Technical Report

Product
ADIFYLINE®

Date
January 2013

Revision
3

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Contents

JUVENILE LOOK AND DESIRED SILHOUETTE 3

AGING AND AIDPOSE TISSUE 4
BREAST IMPORTANCE AND MORPHOLOGY 5
ADIPOCYTE MATURATION 6
PGC-1α AND VOLUME INCREASE 7
ADIFYLINE®, PERFECT CURVES AGAINST GRAVITY 8
IN VITRO EFFICACY
PGC-1α expression in human adipocytes 9
Effect on lipid accumulation 10
IN VIVO EFFICACY
Instrumental evaluation of facial volume increase 11
Instrumental evaluation of breast volume increase 12
COSMETIC PROPERTIES 13
COSMETIC APPLICATIONS 13
TECHNICAL DATA
INCI name of the active ingredient 14
Presentation and Preservative 14
APPLICATION DATA
Processing 14
Incompatibilities 14
Solubility 14
Dosage 14
REFERENCES 15

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Juvenile look and desired silhouette
Nowadays, age related consequences on human body are not considered attractive.
However, time does not obey to canons of beauty and its effects can be seen in all organs,
including skin which is the most important for personal appearance. As we age, skin gets
thinner, loses firmness and gravity also acts pulling the skin downwards, leading to a loose,
lax and saggy skin [1]. These facts, together with a loss of the adipose tissue, which can act
as a supporting tissue of the skin, alter facial morphology and provoke undesired and visible
changes in people when aging.

Considering the whole body, the beauty stereotypes established by society are not always in
accordance to real women and men silhouettes. Women need to have a perfect and feminine
figure for being considered attractive and men are supposed to have an athletic but also well-
developed shape as well. In most cases, people do not have these specific silhouettes.

As a result of these imposed canons of

beauty, most people are not fully satisfied
with their personal image and would like
to change specific areas of their anatomy
to feel themselves more attractive. As the
most extensive organ of the body, the skin
and the fatty tissue beneath can perform
these changes easily and without invasive
treatments.

Without any doubt, the face is the most

exposed and visible body area of a person,
so the aging process produces important
modifications on it. If the adipose tissue
volume lost when aging is regained, the
unpleasant consequences of aging would
be in part diminished. The result would be a
younger and good-looking appearance.
People want to find an efficient
This increase of adipose tissue can be and non aggressive way to
stimulated in certain areas of the whole increase tissue volume where
body (breast, buttocks, lips or hands) in they specifically want, including
order to change silhouette and personal facial and body areas.
image according to personal needs and
desires.

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Aging and adipose tissue
Adipose tissue is mostly localised beneath the skin and it is a specialised connective tissue
with two variants, depending on the main function of the tissue and the kind of adipocytes.
Brown Adipose Tissue (BAT) is present in a really small percentage in humans but it is found to
be necessary in newborns and early stages of human life. The aging process diminishes even
more these already low levels of brown adipose tissue so White Adipose Tissue (WAT) is the
most abundant type in adult life.

The main features of human BAT include Young and old people have a very different
an elevated expression of mitochondrial adipose tissue distribution and quantity.
genes and polygonal adipocytes, which With age, fat tissue decreases and gets
present a high number of mitochondria in redistributed. During this natural process,
the cytoplasm and numerous lipid droplets. fat has a tendency to go from the
The principal purpose of the brown subcutaneous stores to visceral depots,
adipocytes is to generate and dissipate muscle, liver and other ectopic sites [1].
heat from stored energy. Therefore, several body regions see their
volume decreased with age (cheeks, facial
On the contrary, the major human storage oval, hands or breast), depending also on
site for the lipids incorporated by food the personal genetics. As a result,
intake is the WAT. This tissue stores lipids silhouette and facial morphology suffer
until the body requires energy, moment in remarkable changes, which are not
which they are burned. Round adipocytes always expected or happily accepted.
with a unique lipid droplet are the typical
cells of WAT together with its precursor
cells, known as preadipocytes. Apart from
these two cell types, WAT also contains
macrophages, fibroblasts, leukocytes and
many collagen fibres, which act as the
supporting structure for all these cells.

White adipocytes contain a wide lipid

droplet (80% of cell content), forcing the
nucleus and cytoplasm to remain in the
periphery of the cell. This ability to store
triglycerides (and cholesterol esters) is one If the adipose tissue and adipocyte fat
of the most important differences between deposits can be increased, the global
adipocytes and its precursors, as volume of the selected area will also
preadipocytes cannot accumulate lipids. augment. This situation will produce a
development of local volume with the
consequent improvement on facial
appearance and curvy silhouette.

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Breast importance and morphology
The relevance of breast in the silhouette of women is clear in the contemporary cultures. It
seems one of the most important areas to define either a woman is considered attractive or
not. For this reason, since its early development in girls (normally between 9-14 years old) it
becomes an aesthetical concern for all women.

Breast can be described as a modified As a function of some personal

sudoriferous gland situated at both sides circumstances or physiological global
of the sternum and positioned between the changes, breast volume can vary a bit as it
skin and the pectoral muscle. These happens during pregnancy, for example.
semispherical glands are basically formed
by lobules, connective tissue, adipose The adipose tissue represents a high
tissue and Cooper’s ligament, although percentage of the total gland composition
they also have blood vessels, nerves and as it is placed around the lobules and also
lymphatic vessels, which make it sensitive. between the gland and the muscle behind.
For this reason, its size variation has a great
The lobules of the breast are separated by effect on global breast size.
connective tissue but also connected and
joined together in a final channel which
leads to the nipple. The adipose tissue is basic to
determine breast shape and size.

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Adipocyte maturation
The volume of WAT varies as a result of its adipocyte number and size. For this reason, the
increase of volume can be produced by an increment of the number of adipocytes or by an
increase of lipid content.

In adult life, the equilibrium is maintained White adipocyte precursors represent 15-
between adipocyte maturation rate and 50% of the total adipose tissue cells [2], so
death rate, so the total number of changing them would highly modify
adipocytes remains constant in number. adipose tissue.

Having this into consideration, increasing

the differentiation process rate would
modify this equilibrium resulting in a
higher number of mature white
adipocytes, which are the cells capable of
storing lipids in WAT (Fig. 1). When
adipocyte differentiation rate is superior to
adipocyte death rate, a replenishing effect
can be perceived because lipid storage
increases. Fig. 1. Result of increasing adipocyte differentiation.

Preadipocyte differentiation is
the key route to locally
increase lipid storage in WAT.
A local augment of the
adipogenesis process leads
to an extra volume in the
desired areas; face, breast or
buttocks among them.

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PGC-1α and volume increase
Adipogenesis is the process by which preadipocytes convert into mature adipocytes. It is a
complex process where the typical genes of mature adipocytes must be expressed while the
distinctive genes of preadipocytes need to be downregulated or almost inhibited [3]. As a result
of this gene regulation and many interacting factors, the adipocyte phenotype is obtained.
There are some key factors without which the differentiation would not be successful. One of
the key factors is the Peroxisome proliferator-activated receptor-Gamma Coactivator 1 alpha
(PGC-1α) because of its coactivation of a key receptor known as PPARγ.

PPARγ belongs to the Peroxisome

Proliferator-Activated Receptors (PPARs)
family, which is a group of nuclear
receptor proteins. These receptors act as
transcriptional factors and regulate gene
Preadipocyte
expression in cellular differentiation
processes. PPARγ is essential in the
adipose tissue and it forms heterodimers
with Retinoid X Receptors which bind to
specific regions on the DNA of target genes
and regulate their expression. This receptor
is strictly necessary but not sufficient for
Mature adipocyte
preadipocytes to differentiate.

PGC-1α is a transcriptional coactivator Fig. 2. PGC-1α effect in adipogenesis.

that increases the probability of certain
genes of being transcribed by interacting
with a broad range of transcriptional factors Considering this increase, it is confirmed
and nuclear receptors (including PPARγ) [4]. that PGC-1α coactivates PPARγ
In WAT cells, a high induction of PGC-1α potentiating the expression of genes
expression during ex vivo human linked to adipocyte differentiation,
subcutaneous preadipocyte finishing in a stimulation of adipogenesis
differentiation has been seen, rising to the (Fig. 2).
levels found in mature adipocytes [5].

If there was an increase of PGC-1α expression, the adipocyte

maturation rate would raise, resulting in a growth of the
number of adipocytes capable of storing lipids. As a
consequence, fat depots would be more easily formed and the
desired area would see its fat tissue volume augmented.

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ADIFYLINE®, perfect curves against gravity

ADIFYLINE® is a new hexapeptide containing natural amino acids ideal for formulations to
increase fat tissue volume in specific and local areas. It was identified by a combinatorial
chemistry approach from a library of 49,521,980 hexapeptides. The combinatorial peptide
library was screened using the reporter gene assay in a stably transfected cell line where
luciferase expression was controlled by PGC-1α promoter activity.

ADIFYLINE® showed to increase adipocyte differentiation in WAT by raising PGC-1α in

vitro. The increase of mature adipocytes capable of storing lipids in the white adipose tissue
was confirmed in vivo. ADIFYLINE® produced an augment of fat tissue volume in the tested
areas (face and breast), which had an evident increase of volume.

ADIFYLINE® is the perfect ingredient

to diminish the effects that the
aging process causes in the skin.

In facial formulations, it can

increase the supporting tissue
beneath the skin improving facial
appearance in mature individuals as
well as increasing direct volume. In
body products, it can be used to
enhance the volume of certain areas
like breast or buttocks.

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In vitro efficacy
PGC-1α EXPRESSION IN HUMAN ADIPOCYTES

Efficacy of ADIFYLINE® was verified by measuring its effect in human subcutaneous

preadipocytes in culture.

Human subcutaneous preadipocytes were differentiation process, 0.5 or 0.1 mg/mL

incubated during 24 h in the Preadipocyte ADIFYLINE® were added and all samples
Growth Medium (PGMTM-2), which was (including controls) were incubated at 37 ºC
used as the basal control (non-treated non- for 10 days.
differentiated cells). Differentiation was
induced by changing this medium to the Afterwards, cells were lysed, RNA was
Preadipocyte Differentiation Medium (PDM- extracted and reverse transcription was
2), which was also used as a control for carried out. The resulting cDNA was
non-treated differentiated cells. During the analysed by quantitative RT-PCR (Fig. 3).

Fig. 3. PGC-1α mRNA expression relative quantity in human subcutaneous adipocytes after incubation with
ADIFYLINE®.

These values showed that preadipocytes

treated with ADIFYLINE® had a higher ADIFYLINE® increased the
expression of PGC-1α mRNA compared
expression of PGC-1α by 61.1%
to non-treated differentiated cells.
ADIFYLINE® increased the expression of
versus non-treated differentiated
PGC-1α by 25.6% at 0.1 mg/mL and by cells at 0.5 mg/mL.
61.1% at 0.5 mg/mL.

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EFFECT ON LIPID ACCUMULATION

Human subcutaneous preadipocytes were Afterwards, 5 μL of AdipoRedTM reagent

incubated during 24 h in PGMTM-2, which were added to each well and mixed. The
was also used as the basal control (non- AdipoRedTM reagent is a hydrophilic
treated non-differentiated cells). solution that turns into fluorescent in
Differentiation was induced by changing hydrophobic environments, which
this medium to PDM-2 and incubating the facilitates the detection of the levels of
cells for 10 days in the presence of the intracellular lipid droplets accumulated
different treatments. ADIFYLINE® was during preadipocyte differentiation (they
tested at two different concentrations (0.5 become stained).
and 0.1 mg/mL) and PDM-2 was used as a
control for non-treated differentiated cells. Fluorescence values were read at 535 nm
(excitation at 485 nm), normalised with
After 10 days, the supernatants were respect to basal fluorescence and to the
removed and wells were washed. fluorescence of the differentiation medium.

Fig. 4. Lipid accumulation in human adipocytes after different treatments, including ADIFYLINE®.

Results demonstrated that adipocytes

coming from preadipocytes treated with
ADIFYLINE® produced a 32.4%
ADIFYLINE® had an increase in lipid
accumulation compared to non-treated increase of lipid storage versus
differentiated cells. ADIFYLINE® enlarged non-treated differentiated cells
lipid accumulation in white adipocytes by at 0.5 mg/mL.
27.9% at 0.1 mg/mL and by 32.4% at 0.5
mg/mL.

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 In vivo efficacy
INSTRUMENTAL EVALUATION OF FACIAL VOLUME INCREASE

In order to evaluate the efficacy of ADIFYLINE® in increasing skin volume, a group of 22 female
volunteers between 50-60 years old was selected. In this in vivo study, the tested areas were the
cheeks and volunteers were asked to apply the cream containing 2% ADIFYLINE® SOLUTION
twice a day for 14 days. The volume of the cheeks was analysed by fringe projection at the
beginning and at day 14. This technique allows the direct acquisition of the morphology of the
studied area in 3D, being able to obtain the volume in mm3. The volumes obtained at the end of
the treatment were compared to the initial values (Fig. 5).

After 14 days, areas treated with the

cream containing 2% ADIFYLINE®
SOLUTION had a significant 11.9%
volume growth versus initial time.
Results showed as well that with
ADIFYLINE®, 79% of volunteers had a
volume expansion after 14 days.

Fig. 5. Measurement of cheek volume of volunteers.

ADIFYLINE®
produced a visible
cheek volume
increase of almost
12% after 14 days,
versus initial time.

Fig. 6. Real 3D images from a volunteer at the initial time (left) and after 14 days
applying a cream with 2% ADIFYLINE® SOLUTION (right).

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INSTRUMENTAL EVALUATION OF BREAST VOLUME INCREASE

For the evaluation of the in vivo efficacy of ADIFYLINE® in increasing local volume (breast), a
panel of 22 females between 25 and 40 years old was selected. As specific criteria, volunteers
needed to have an 80-90 European bra cup size and a stable weight. They were asked to apply
twice a day the placebo cream in a bust and the cream containing 2% ADIFYLINE® SOLUTION in
the other, for 56 days. Measurements of breast volume were taken at the beginning and at day
14, 28 and 56 using the Fast Optical In vivo Topometry Technique (FOITS), which allows
reconstructing the surface and volume of the breast based on the principle of optical
interferometry. The relative volume of the area and 3D images were obtained. The differences
versus initial time were measured (mm3) as well as the evolution of the breast volume of the
volunteers versus initial time normalised with respect to placebo results.

Fig. 7. Growth of breast volume vs. initial time and evolution of breast volume applying ADIFYLINE® SOLUTION
normalised with respect to placebo results, and versus T0.

Results at the end of the study showed

that ADIFYLINE® generated a clear
positive growing tendency in breast
volume, while placebo did not. Moreover,
the volume increase in the area where the
cream containing 2% ADIFYLINE ®
SOLUTION was applied was 30 times
higher than the placebo at day 56.

ADIFYLINE® improved
breast volume increase by Fig. 8. Real 3D images of a volunteer at the initial time
30-fold compared to (left) and at day 56 (right), after applying a cream with
2% ADIFYLINE® SOLUTION.
placebo results, at day 56.

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Cosmetic properties
ADIFYLINE®:

enhances adipocyte maturation by boosting PGC-1α expression in WAT (closely

linked to adipogenesis). In vitro, it showed to raise PGC-1α levels by 61.1%.

induces a higher lipid accumulation in WAT, which was confirmed in vitro as it

increased lipid storage by 32.4%.

augments local volume by raising subcutaneous adipose tissue, which was

confirmed in vivo when cheek volume grew by almost 12%.

increases body volume by growing adipose tissue volume; in vivo breast volume
raised by 30 times more than placebo.

Cosmetic applications
ADIFYLINE® can be incorporated in many formulations and products to increase local volume.
It includes facial redefining products, breast firming formulations, facial anti-aging products,
make up for mature women, lip care products and make up, specific formulations for the
cleavage area (mature women), and anti-aging products for hands, which lose adipose tissue
with age.

Moreover, it can also be incorporated as a complementary ingredient in hydrating, firming and

sun care products for specific body care areas (buttocks and breast) with a replenishing effect.

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Technical data
INCI NAME OF THE ACTIVE INGREDIENT

Active ingredient INCI name

ADIFYLINE® Acetyl Hexapeptide-38

PRESENTATION AND PRESERVATIVE

Solution containing 0.05% of active ingredient.

Code Product presentation Preservative

®
PD210 ADIFYLINE SOLUTION Preservative free

Application data
PROCESSING
ADIFYLINE® SOLUTION needs to be added in the aqueous phase. In case of emulsions, it should
be added once the emulsion is formed and at temperatures below 40 ºC.

ADIFYLINE® is stable at a pH range between 3.0 and 8.0.

INCOMPATIBILITIES
Not expected.

SOLUBILITY
Soluble in water, ethanol and glycols (glycerin, butylene glycol).

DOSAGE
A dosage of 2% of ADIFYLINE® SOLUTION is recommended in final cosmetic formulations.

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 References

1. Petrofsky J, Prowse M, Lohman E. The influence of ageing and diabetes on skin and
subcutaneous fat thickness in different regions of the body. The Journal of Applied
Research. 8 (1): 55-61, 2008.

2. Tchkonia T, Morbeck DE, Zglinicki T., et al. Fat tissue, aging, and cellular senescence. Aging
Cell. 9: 667-684, 2010.

3. Gerhold DL, Liu F, Jiang G, et al. Gene expression profile of adipocyte differentiation and its
regulation by peroxisome proliferator-activated receptor-gamma agonists. Endocrinology.
143 (6): 2106-18, 2002.

4. Liang H, Ward WF. PGC-1α: a key regulator of energy metabolism. Adv Physiol Educ. 30:
145-151, 2006.

5. Semple RK, Crowley VC, Sewter CP, et al. Expression of the thermogenic nuclear hormone
receptor coactivator PGC-1α is reduced in the adipose tissue of morbidly obese subjects.
International Journal of Obesity. 28: 176-179, 2004.

Note: Graphs and photographs of efficacy tests are available for customer use provided that the final product contains the same
concentration of active as the formulations in our tests. Customers must request written permission for use of the graphic
material and/or ingredient tradenames to Lipotec. Customers are responsible for compliance with local and international
advertising regulations.

The specific situation of the trademark in each country may vary and we recommend that you contact us for updated
information.

Disclaimer:
While the claims and supporting data provided in this publication are believed to be reliable and they are presented free and for
guidance only, there are no warranties of any kind. All expressed and implied warranties are disclaimed. The recipient is solely
responsible for ensuring that products marketed to consumers comply with all relevant laws and regulations. LIPOTEC is the
exclusive holder of the both industrial and intellectual property rights identified herein. Recipient of this publication agrees to
indemnify and hold harmless each entity of the LIPOTEC organization for any and all regulatory action arising from recipient’s
use of any claims or information in this publication, including, but not limited to, use in advertising and finished product label
claims, and not present this publication as evidence of finished product claim substantiation to any regulatory authority.

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