05080081_Pigmentation 8/8/05 2:06 PM Page 1

BACKGROUND

Hyperpigmentation, the heightened expression of melanin in the skin, is a common problem

with many individuals. Some types of this expression are evidenced by freckling, lentigines, and
KERATINOCYTES
broader areas of discoloration such as melasma. The causes for hyperpigmentation in the skin
vary and have been shown to be related to sunlight exposure, hormonal fluctuation, disease,
and even a response to laser therapies such as hair removal, resurfacing, and age spot removal.
Melanosome Transfer
The pigment in skin is called melanin, a polymeric substance generated in a specialized cell
called the melanocyte. This structure in the skin is located in the basal area of the dermis and Tyrosinase
functions through a known set of processes.
Melanocyte Secretory
Function
The most commonly practiced method for hyperpigmentation control has been the use of
hydroqinone and its derivatives arbutin and kojic acid. These chemicals penetrate the dermis
MELANOCYTE
and interfere with the action of the mediating enzyme in the melanocyte tyrosinase, in effect
stopping its function. While these ingredients have been used for a long period of time they
suffer from being single action-oriented to tyrosinase inhibition and melanocyte toxicity.

Unfortunately, the control of the administration of these materials is at the discretion of the
users, and overuse and difficulty in modulation is a common difficulty. The result is patchy
skin color control and over-whitening.
Human Melanocyte
We report here the findings of controlling hyperpigmentation via a more natural and easier to
modulate methodology that works with the progression of melanogenesis in the cell, namely KOJIC ACID
ASCORBIC ACID Tyrosine
by controlling the discrete steps of activation, synthesis, and expression of the melanocyte. ARBUTIN
HYDROQUINONE

Existing skin lighteners function by Tyrosinase is the

direct interaction with tyrosinase to enzyme that converts tyrosine
inhibit its catalytic activity, i.e., the to melanin. It is located within a
production of melanin. vesicle called a melanosome.

ACTIVATION PHASE To be effective, these tyrosinase

inhibitors not only have to penetrate
the stratum corneum to reach the
melanocytes but they also have to
cross both the melanocyte cell and
the melanosome membranes to reach Melanosome Melanin
Activation: melanin production is initiated. the enzyme tyrosinase.

Synthesis: melanin is actually produced by melanocytes.

Expression: melanin is transported to the surface of the skin.

The process of pigmentation consists of three phases: activation, synthesis, and expression.
The first step is the activation of the melanocyte by one of several triggers that begins the
enzymatically-mediated synthesis of the melanin, starting with the amino acid tyrosine. In
other words, the activation phase occurs when the melanocytes receive some sort of stim-
ulus that causes them to go into a defensive mode and begin the melanin production
process. Melanocytes are activated by a variety of stimulants: stress, acne, hormones, and
inflammatory processes such as exposure to UV radiation or other free radical damage. All
these factors trigger the cells in the upper part of the skin to produce a variety of small
chemical messengers, called hormones, and more specifically melanocyte stimulating
hormone (MSH). These hormones then travel to where melanocytes are located and activate
the melanocyte production of melanin.
Activation Receptors

SYNTHESIS PHASE

The synthesis phase is when the melanocyte actually makes the melanin granules called
melanosomes. There are several processes in the synthesis phase that create these packages Tyrosinase
of melanin. When stress or irritation mediators bind to receptors on the cellular membrane of
the melanocyte, the enzyme tyrosinase switches to its active form. Tyrosinase is essential in Pheo-melanin
Tyrosine Dopa Dopaquinone
the synthesis of melanin, acting as a catalyst throughout the synthesis process. Once active,
Eu-melanin
tyrosinase converts the amino acid tyrosine into a molecule called dopa. Tyrosinase then con-
verts dopa into a secondary chemicaldopaquinone. After a series of reactions, dopaquinone
is converted into either dark melanin (eu-melanin) or light melanin (pheo-melanin).

05080081
05080081_Pigmentation 8/8/05 2:06 PM Page 2

SYNTHESIS PHASE CONTINUED

EPIDERMIS
Pheo-melanin production delivers a lighter colored pigment to the skin and eu-melanin pro-
duces a larger, blacker particle. Dark melanin is associated with suntans and dark skin and
light melanin is more predominant in fair skinned people.
KERATINOCYTES
Layers of skin cells.

EXPRESSION PHASE: THE PROCESS

Finally, during the expression phase, the melanosomes are transferred from the
melanocytes to the upper skin cell layers. Once the melanin has been synthesized and filled MELANOCYTE
into the melanosomes, the melanosomes travel out into the arms of the melanocyte. Think Melanin is formed inside
of a melanocyte as a cell that has very long, dendrite-like tentacles. When the melanosomes the melanocyte.

reach the end of these dendrite-like tentacles, they are actually pushed out of the melanocyte
and taken up by keratinocytes, which are the skin cells located above the melanocytes in
the epidermis. The keratinocytes take these melanosomes and carry them all the way up
to the surface of the skin, where they are, in essence, expressed. After this transfer takes
place, the melanin color will eventually become visible on the surface of the skin.

In recognizing that melanin formation is a set of three discrete phases, we designed a series
Melanin becomes visible
of studies to test how to modulate the phases and allow the melanocyte to regulate itself
EPIDERMIS at the suface of the skin.
back to a homeostatic function.

KERATINOCYTES
EXPERIMENTAL PROTOCOL Layers of skin cells.

In-Vitro Studies
Two sets of experiments were designed and carried out. The first was an in-vitro screening
of materials that had geometrical agreement to those entities known to help regulate at least MELANOCYTE
Melanin-filled melanosomes
one of the three phases of melanogenesis. This initial screening was practiced using the move up dendrite-like arms of the
melanocyte and are deposited
Melanoderm technology whereby melanocytes are cultured and their degree of melanin inside the keratinocytes.
production is measured as a colorometric change as a result of controlled dosing of the
aqueous solution with individual ingredients.

Next, each ingredient was re-evaluated for its specific activity on any of the three melanogenesis
steps by observing the degree of particulate definition of color. That is, whether the color
was discrete or diffuse would indicate whether the melanin granule was still in its synthesis
or expression stage. This helped us to differentiate the principal relationship between the Detailed illustration
material under study and its function in the melanogenesis pathway. of melanocyte

Table 1 summarizes the sorting of the 15 most active materials. It shows that there
were materials that could be used to modulate the activity of the melanocyte downward
and thereby promote minimal melanin formation.

In-Vivo Studies
The next set of experiments we conducted were designed to test the materials in Table 1
in a serial fashion on living human skin for controlling hyperpigmentation. Sample com-
positions of a cleanser and a moisturizer base were developed and the ingredients in Table
1 were included at specific concentrations by weight and tested by an independent third
party contract research organization for the ability to decrease hyperpigmentation of areas
on the face.

The cleanser was a simple composition made of: Water (Aqua), Ammonium Lauryl
Sulfate, Lauryl Betaine, Sodium Lauroyl Sarcosinate, Glycerin, PEG-120 Methyl Glucose

05080081
05080081_Pigmentation 8/8/05 2:06 PM Page 3

EXPERIMENTAL PROTOCOL CONTINUED Table 1

Density (>100=25% Melanogenesis
(0=white) cell death) Function
Dioleate, Carbomer, Butylene Glycol, PVP, Sodium Chloride, Fragrance (Parfum),
Melanostatin 3.33 116 AMSH Inhibition
Tetrasodium EDTA, Benzoic Acid, Phenoxyethanol, Chlorphenesin, and Methylparaben.
The tyrosinase inhibitors were then tested in the cleanser for compatability and screened Thiotaine 5.17 99 Tyrosinase Inhibitor,
Antioxidant
for activity using the Melanoderm test methodology described above. Melanosome Transfer
Niacinamide 3.75 78
Inhibitor

Tyrostatase 5.75 88 Tyrosinase Inhibitor,

Similarly, a moisturizing base was constructed from: Water (Aqua), Aloe Vera Gel, Antioxidant

Propylene Glycol, Stearic Acid, Cetearyl Alcohol, Ceteareth-20, Dioctyl Adipate, Octyl Alpha-Arbutin 6.17 88 Tyrosinase Inhibitor

Stearate, Octyl Palmitate, Stearamidopropyl Dimethylamine, Ceteth-2, Glycol Stearate, Licorice Extract 4.75 88 Tyrosinase Inhibitor,
Anti-inflammatory
Glycerin, Tocopheryl Linoleate, Fragrance (Parfum), Caramel, Triethanolamine, Diazolidinyl
Asafetida Extract 4.75 93 Tyrosinase Inhibitor
Urea, Methylparaben, Propylparaben, and Disodium EDTA. Separately, the MSH and
melanosome transfer inhibitors were also tested for efficacy using the Melanoderm test Lemon Peel Extract 4.42 97 Tyrosinase Inhibitor

methodology described above. Wheat Germ Extract 3.92 72 Diverts Melanin Production
to Pheomelanin

*Ethanol 3.25 0 Melanocyte Inhibitor

From these sets of experiments the most potent combinations were selected and a 12-week
Niacinamide Axcorbate 4.33 52 Melanosome Transfer
clinical study against a control and a commercially available skin lightening product Inhibitor, Antioxidant
from Japan based on hydroquinone was conducted as follows: **Kojic Acid 4.33 0 Tyrosinase Inhibitor

Citrus Unshiu Peel Extract 5.5 89 Tyrosinase Inhibitor

12-week clinical study.
Creatine 4.33 82 Melanosome Transfer
29 Japanese women with mild to moderate pigmentation on the face. & Tyrosinase Inhibitor

Skilled clinical graders. No Additions 7.17

Test product applied to whole face twice per day.

We found that the strategy of using a series of materials specifically evaluated for the control
Performance by Clinical Grader
of the three phases of melanogenesis outperformed the control of a hydroquinone product.

120

4 WEEKS
CONCLUSION 100
8 WEEKS
PERCENT OF SUBJECTS

80
WITH IMPROVEMENT

12 WEEKS

60
We have shown that through a screening of materials that could have potential regula-
tory influence on the discrete steps of melanogenesisactivation, synthesis, and 40

expressionit is possible to down-regulate the processes in a manner to provide a gen- 20

tle means of controlling hyperpigmentation.
0
ss

ea

e
ce

s
lo

es

nc
ne

an

Ar
Co

hn

ra
ht

di

ea
ug
en
ig

Ra

pp
Br

Ro
Ev

lA

REFERENCES
al
er
Ov

ATTRIBUTE
1. Cayce KA, McMichael AJ, Feldman SR. Hyperpigmentation: an overview of the common
afflictions. Dermatolo Nurs 2004;16(5):4016, 4136.
2. Stulberg DL, Clark N, Tovey D. Common hyperpigmentation disorders in adults: Part II.
Melanoma, seborrheic keratoses, acanthosis nigricans, melasma, diabetic dermopathy,
tinea versicolor, and postinflammatory hyperpigmentation. Am Fam Physician
2003;68(10);19638.
3. Perez MI. The stepwise approach to the treatment of melasma. Cutis 2005;75(4):21722.
4. Imokawa G. Autocrine and paracrine regulation of melanocytes in human skin and in
pigmentary disorders. Pigment Cell Res 2004;17(2):96110.
5. Hakozaki T, Minwalla L, Zhuang J, Chhoa M, Matsubara A, Miyamoto K, Greatens A,
Hillebrand GG, Bisset DL, Boissy RE. The effect of niacinamide on reducing cutaneous pig-
mentation and suppression of melanosome transfer. Br J Dermatol 2002;147(1):2031.
6. Yokota T, Nishio H, Kubota Y, Mizoguchi M. The inhibitory effect of glabridin from licorice
* 20% Ethanol
extracts on Melanogenesis and inflammation. Pigment Cell Res 1998;11(6):35561. ** 1% Kojic Acid

05080081