Lightening Skin Through a Melanogenesis-interrupting Peptide

Visible skin pigmentation depends primarily on the function of melanocytes in the epidermis, which synthesize melanin through a process termed melanogenesis. Also involved are neighboring keratinocytes that receive and distribute melanin in upper layers of the skin (see Figure 1).¹ Melanocytes reside adjacent to the epidermal basement membrane and synthesize the pigmented polymer, which is then stored in cytosolic organelles known as melanosomes that are transferred to keratinocytes through melanocyte dendrites. Since keratinocytes are continuously desquamated, there is a constant need for the synthesis and transfer of melanosomes from melanocytes to keratinocytes, for cutaneous pigmentation.

Thus, cutaneous pigmentation is determined by: melanogenesis, which induces the formation of melanosomes; the different types of melanin synthesized; the transfer of melanosomes through dendrites; the transfer of melanosomes to surrounding keratinocytes; and finally, the distribution of melanin to suprabasal layers of the skin. To interrupt any of these processes would limit skin pigmentation, consequently achieving a better skin lightening/whitening effect. Here, the authors describe a peptide designed to interrupt specific processes for a skin-whitening effect, the mechanisms of which are first reviewed.

Melanosome Maturation

The maturation of melanosomes can be divided into stages I-IV, shown schematically in Figure 2. In stage I, melanosomes or pre-melanosomes develop from the endoplasmic reticulum (ER). At this stage, melanosomes are spherical vacuoles that lack tyrosinase activity—i.e., the main enzyme involved in melanogenesis. Melanin synthesis begins in stage II, at which point tyrosinase is transported into the melanosome, and melanosomes organize into elongated, fibrillar matrix structures.

The deposition of melanin on the fibrillar matrix is found in stage III melanosomes. This process is mainly determined by the actions of microphthalmia-associated transcription factor (MITF), a basic-helix-loop-helix and leucine zipper transcription factor, which functions as the master gene for melanocyte survival. It also is a key factor in regulating the transcription of the major melanogenic proteins, including tyrosinase and tyrosinase-related proteins (TRP) 1 and 2, which lead to pigmentation. After UV irradiation/exposure, the upregulation of α-melanocyte-stimulating hormone (α-MSH) in the keratinocytes leading to an increase of MITF is observed. Stage IV melanosomes are fully matured, highly dense and dark, and are ready for transport to the keratinocytes.²

Melanosome Transport

The ultimate path for melanosomes in the skin is to be transferred to keratinocytes. Melanosomes are delivered from the perinuclear region of melanocytes to the tips of melanocyte dendrites via the intracellular transport system. Here, they are anchored to the plasma membrane, then transferred to neighboring keratinocytes. Previous research of coat color mutations in mice indicates that melanosome transport within melanocytes is also necessary for skin pigmentation, shown schematically in Figure 3.³

The first key regulator of actin-base melanosome transport is a tripartite protein complex known as Rab27A, which mediates myosin Va binding to melanosomes through another linker protein, melanophilin (Mlph), also referred to as Slac-2. Missing any of these components will cause the perinuclear aggregation of melanosomes and lead to deficient melanosome transfer from microtubules to actin filaments.⁴

The successful transfer of melanosomes to keratinocytes is essential to normal skin pigmentation; thus an effective whitening ingredient could act by impacting Rab27A, myosin Va and/or Mlph regulation to interrupt the binding of melanosomes to actin filaments, preventing them from transferring to neighboring keratinocytes. Through this mechanism, one could better limit skin pigmentation and consequently achieve a better skin lightening effect.⁵

Epidermal Melanin Unit

After melanosomes transfer to keratinocytes, they are arranged in a supranuclear cap to protect DNA against ultraviolet (UV) light irradiation. This substantial interaction has been proposed based on the finding that 36 viable keratinocytes surround each melanocyte to form a specialized cell group called the epidermal melanin unit. The ratio of melanocytes to keratinocytes in the epidermal melanin unit is consistent regardless of human skin color.^{1, 6}

The proliferation and differentiation of melanocytes are believed to be regulated by the keratinocyte-derived factors α-MSH, adrenocorticotropic hormone (ACTH), stem cell factor (SCF) and endothelin-1 (ET-1) (see Figure 4). In human epidermis, α-MSH and ACTH are produced in and released by keratinocytes. They are involved in regulating melanogenesis and forming melanocyte dendrites. After UV exposure, α-MSH and ACTH bind to the melanocyte-specific receptor MC1R, which increases the expression of MITF and triggers the melanin synthesis process.

Regarding ET-1, interleukin-6 (IL-6) and UV exposure are known to induce its expression in keratinocytes, which enhances the expression of MC1R although through its own EDNRB receptor on melanocytes. Other melanogenic factors including SCF also are produced by keratinocytes in response to UV, and all are involved in regulating the proliferation, melanogenesis and formation of melanocytes and dendrites in normal or UV-irradiated skin, which may be related to the tanning response, i.e., pigmentation, as shown schematically in Figure 4.

Melanogenesis

Melanogenesis is regulated by a wide range of mechanisms in melanocytes and keratinocytes, and produces different types of melanin. Eumelanin primarily provides black and dark colors found in human hair and skin. It also imparts grey, black, yellow and brown color in hair. Pheomelanin produces reddish colors. In humans, it is more abundant in women’s skin than in men; so women’s skin overall is slightly redder. It also occurs in hair and is the main agent in red hair color.

Three key enzymes control the melanogenesis process: tyrosinase, TRP-1 and TRP-2.⁷ A critical step in melanin biogenesis is the oxidation of tyrosine by the enzyme tyrosinase. Tyrosinase therefore directly regulates the amount of melanin produced; the others modify the type of melanin synthesized. TRP-1 functions as a 5,6-dihydroxyindole-2-carboxylic acid (DHICA) oxidase in the melanogenic pathway. The catalytic activity of TRP-1 promotes the oxidation and polymerization of DHICA monomers into melanin. However, TRP-2 catalyzes the transformation of dopachrome to DHICA. TRP-2, similar to TRP-1, is considered a eumelanogenic enzyme that also stabilizes tyrosinase activity.⁸ Thus, both TRP-1 and TRP-2 can act as enzymes, modifying eumelanogenesis velocity as regulators and stabilizers of the eumelanogenic apparatus in vivo, and perhaps as regulators of other melanocyte functions. Ultimately, an effective skin lightener would downregulate tyrosinase, TRP-1 and TRP-2 activity, as shown schematically in Figure 5.

Skin Lightening Peptide

Enzymic browning of fruits and vegetables is caused by Polyphenoloxidase (PPO)—the main enzyme catalyzing the biochemical conversion of phenolics to produce quinones, which undergo further polymerization to yield dark, insoluble polymers referred to as melanins.⁹ Glycyl-peptides were investigated for their ability to inhibit the browning process of DOPA-treated apple and potato. The aim was to develop a glycine derivative that is safe, effective, applicable, stable and convenient safety, efficacy, applicability, stability and convenience. The resulting material, acetyl glycyl β-alanine^a, is a white crystalline powder with 99% minimal purity. It is water-soluble, making it easy to use in formulations; it also is stable and safe. As will be shown, this peptide interrupts specific processes for skin melanogenesis for good skin whitening efficacy.

Western Blot: Effects on Tyrosinase, TRP-1 and TRP-2

To determine the skin lightening efficacy of the peptide, a Western blot test was carried out using lysate B16F10/A375 melanoma cells stimulated by α-MSH. The melanoma cells were seeded in a six-well dish and cultured for 24 hr. The media were then replaced with DMEM with 10% fetal bovine solution (FBS) containing the peptide at 0.125, 0.25 or 0.5%, and α-MSH. The melanocytes were then incubated for 72 hr and after incubation, protein was extracted to perform the Western blot. One well included a sample without culture medium, which served as the untreated control; β-actin served as an internal control.

When cells were stimulated by α-MSH, the protein expression of tyrosinase, TRP-1 and TRP-2 increased significantly. Treatment with β-actin showed no change in protein level; however, the addition of the new peptide inhibited the expression of tyrosinase, TRP-1 and TRP-2 in α-MSH-stimulated cells in a dose-dependent manner. In Figure 6, the x axis shows the test samples, and the y axis shows tyrosinase expression. These results show the peptide inhibited tyrosinase activity by 36.5% compared to the control, which was stimulated by α-MSH at 100%.

Moreover, as shown in Figure 7, the peptide presented excellent dose-dependent inhibition of TRP-1 formation. Also, while TRP-2 expression did not significantly change in the controls (see Figure 8), the peptide inhibited TRP-2 levels up to 91.9%. These results indicated the potential for the peptide to downregulate the activities of tyrosinase, TRP-1 and TRP-2—three important enzymes involved in melanogenesis.

PCR: Effects on Melanosome Transport

The polymerase chain reaction (PCR) is a biochemical technology used to amplify a specific region of a DNA target. Real-time quantitative PCR, also known as qPCR, combines PCR amplification and detection in a single step; with qPCR, the amount of PCR product is measured after each amplification while with traditional PCR, the amount of PCR product is measured only at the end point of amplification.

In qPCR reactions, the amplification products are measured using a fluorescent label as they are being produced. The fluorescence signal is directly proportional to DNA concentration over a broad range, and the linear correlation between PCR product and fluorescence intensity is used to calculate the amount of template present at the beginning of the reaction. After the reaction is completed, and the number of cycles to generate the product (expressed in fluorescence intensity) is plotted, the amount generated for complete cycles of PCR product from each reaction can be rendered —i.e., the so-called quantitative real time polymerase chain reaction.

In this experiment, mouse melanoma cells (B16-F10) were treated with the new peptide. MITF, Rab27a, Myosin Va and Mlph were analyzed by qPCR at the mRNA level; if the expression of mRNA was less than the positive control, the peptide was interpreted as having the ability to decrease mRNA levels. According to this assessment, at 0.5%, the peptide effectively inhibited MITF and Mlph up to 38% and 45% respectively, verifying its specific activities (see Figure 9).

ELISA: Epidermal Melanin Unit, ET-1 and SCF

An ELISA^b kit based on a quantitative sandwich enzyme immunoassay technique was used to evaluate the activity of ET-1 and SCF. A monoclonal antibody specific for ET-1/SCF was pre-coated onto a microplate, then control and test samples were pipeted into wells. Any ET-1/SCF present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific for ET-1/SCF is added to the wells. Following another wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of ET-1/SCF bound in the initial step. Finally, color development is stopped and the intensity of the color is measured.

In this experiment, normal human epidermal keratinocytes (NHEK) were pre-treated with the new peptide for 6 hr. The supernatant was discarded and the cell layer was washed twice by phosphate-buffered saline (PBS). To each well, 0.5 mL PBS was added, then the samples were irradiated with 40 mJ/cm² UVB to induce ET-1/SCF secretion. The PBS was discarded, and fresh medium was added for 18 hr. After post-treatment, the supernatant was collected in Eppendorf tubes and stored at -80°C. The secreted ET-1/SCF was then measured by the ELISA kit. The results, shown in Figure 10, indicate the peptide dose-dependently and significantly inhibited the secretion of ET-1 and SCF, which regulate melanocyte migration and melanogenesis.

Ex vivo Depigmenting Effects

Another study was carried out using in vitro tissue models of the human epidermis prepared from cultured human keratinocytes and melanocytes^c, to assess the potential of the peptide to induce changes in tissue pigmentation. The models were stored at 4°C until used. For use, the tissues were removed from the agarose-shipping tray and placed into a 6-well plate containing 1.0 mL of assay medium (37±2°C). All of the agarose was removed from the outside of the tissue culture insert, since any residual agarose could prevent the assay medium from reaching the tissue. A 50-μL sample of test material was then applied to the surface of the tissue. The tissues were incubated at 37±2°C and 5±1% CO₂.

Every 24 hr, the tissues were rinsed with PBS, fresh test material was applied and the media was changed. The treatments were carried out for nine days. Two sets of tissues were prepared for this study; one set was used to determine changes in melanin content while the other was used assess changes in pigmentation via histology. Results are shown in Figure 11. Here, the black areas are melanin from melanocytes and after nine days of treatment, a 2% concentration of the peptide showed excellent skin brightening activity, compared with day one.

Clinical Depigmenting Effects

An independent clinical study of the peptide’s ability to reduce age spots was carried out using a topical formula containing 2% peptide. The formula was applied twice daily for 56 days by 10 healthy women, ages 25-50, exhibiting visible hyperpigmented areas on the face. Skin colorimetric measurements were taken^d to detect subtle changes in color by the three dimensional profile of hue, value and chroma on days 0, 14, 28 and 56. These characteristics were translated into L*, a* and b* color coordinate values, where spacing is considered to correlate with color changes perceivable to the human eye. Any increase in the L* coordinate indicates lightening of the color. As Figure 12 shows, the peptide significantly increased the L* value at each evaluation point.

In addition, detailed, high-resolution digital photographs were taken of the subjects before and after treatment via reverse photo-engineering, and the photographs were processed using image analysis software. This allows age spots to be captured and quantified. Since the area measured varied with each panelist, the change for each individual was calculated as a percentage; then all were averaged together. Figure 13 shows the average effect of age spot reduction according to differences in pixel value. The new peptide demonstrated immediate whitening of age and sun spots.

Figure 14 is the result of one of the 10 total panels used in this study. It compares the treated zone at day 0 with day 56; the mean surface area of spots was reduced significantly by 83.68%. Overall, this clinical study confirmed the ability of the peptides to reduce hyperpigmentation through a cosmetic product.

Stability Study

Historically, the most commonly used whitening ingredients have been vitamin C derivatives or plant extracts, but these often suffer from stability issues, i.e., sensitivities to light or heat, which lead to discoloration and compound degradation. These changes reduce the efficacy in the final product. The new peptide, however, shows a strong stability profile.. The ingredient was stored in a crystalline powder form for three months under four different conditions: room temperature (RT), 4°C, 45°C and in sunlight. Then, the purity and transmittance values were analyzed; if the color and purity remained unchanged, the ingredient was determined to tolerate high temperatures and remain stable.

As shown in Figure 15, the purity did not change, proving the peptide remained stable at RT, 4°C, 45°C and in sunlight for three months. In addition, the 5% aqueous peptide transparent samples remained unchanged at RT, 4°C, 45°C and in sunlight for three months, even at pH values of 4 and 5. Color change was assessed by transmittance (T%), as Figure 16 shows; the higher the T% value, the more transparent the color.

Conclusions

There are three major processes of skin melanogenesis, including melanosome formation, melanin synthesis and melanin transfer. The described peptide was shown to inhibit the formation of melanosomes by reducing the activity of key enzymes: TRP-1 by 42.1%; TRP-2 by 91.9%; and tyrosinase by 36.5%. These effects, in turn, down-regulated melanin synthesis. The peptide also decreased the activity of MITF, a key regulator in the melanogenesis pathway, by 38%. Finally, the peptide inhibited related gene melanosome transport within melanocytes; i.e., Mlph was decreased by 45%; epidermal melanin unit (ET- 1) by 92.7%; and SCF by 61.3%. As stated previously, interrupting these key processes can achieve a better skin lightening effect, and the peptide tested here provides a promising option.

References

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