A Novel Tyrosinase-Inhibiting Oligopeptide Improves the Appearance of Facial Hyperpigmentation

manuscript.pdf

A Novel Tyrosinase-Inhibiting Oligopeptide Improves the Appearance of Facial Hyperpigmentation

Basil M. Hantash, MD, PhD1, Felipe Jimenez, PhD2

1. Elixir Institute of Regenerative Medicine, San Jose, CA

2. Envy Medical, Inc, Westlake Village, CA

CORRESPONDING AUTHOR:

Basil M. Hantash, MD, PhD

Chairman

Elixir Institute of Regenerative Medicine

5941 Optical Court

San Jose, CA 95138

Phone No.: (408) 914-2033

Fax No.: (323) 306-4330

E-mail: basil@elixirinstitute.org

Conflict of Interest:    The research related to decapeptide-12 was initiated, invented, and patented by Dr. Hantash while at Stanford University (Stanford, CA).  The in vitro studies were completed at the Elixir Institute of Regenerative Medicine (San Jose, CA). Dr. Hantash serves as Vice Chairman of the Board of Directors of Envy Medical, Inc.

Financial Support: None

Type of Manuscript: Research Report

Key Words: Melasma, hyperpigmentation, tyrosinase, melanin

INTRODUCTION

Melasma is a common medical condition that presents as patches of skin hyperpigmentation on the cheeks, forehead, upper lip, nose and chin. It presents most frequently in Asian and Hispanic females with Fitzpatrick phototypes IV and higher.1,2 Pregnancy is a common trigger that leads to overproduction of melanin, presumably caused by a fluctuation in hormonal levels, resulting in the appearance of mottled hyperpigmentation commonly know as chloasma or pregnancy mask. 3, 4 Various strategies designed to regulate the overproduction of melanin have previously been described. One such strategy involves inhibiting tyrosinase, a key enzyme involved in initiating the production of melanin in skin, to help improve the appearance of hyperpigmentation over time.5 We present, herein, two previously published studies describing the discovery of a novel tyrosinase-inhibiting peptide (decapeptide-12) and its reduction to clinical practice.6,7 The objective of the in vitro study was to discover short-sequence peptides that exhibit inhibitory activity against the tyrosinase enzyme without toxicity to human melanocyte cells. The goal of the pilot clinical study was to determine if twice-daily topical application of the most potent tyrosinase-inhibiting peptide identified in our in vitro study could improve the appearance of facial melasma after 16-weeks.

MATERIALS & METHODS

In Vitro Study

A mushroom tyrosinase inhibition assay was conducted using L-tyrosine as the substrate and various concentrations of test peptides (P1, P2, P3, P4 (decapeptide-12), P5, P6 & P7). Hydroquinone was used as the positive control inhibitor and 5% DMSO as the negative control. This assay was designed to facilitate the calculation of an IC50 value (amount of test peptide, hydroquinone etc., needed to inhibit the activity of the mushroom tyrosinase enzyme by 50%) and to characterize the dose-dependent inhibition profiles of of the test peptides, hydroquinone, and the negative control. Similar inhibition assays were also conducted testing varying concentrations of mushroom tyrosinase enzyme, substrate (L-tyrosine) and inhibitors (test peptides). These assays were designed to determine the mechanism of action or how each peptide inhibits mushroom tyrosinase (i.e. competitively, non-competitively etc.) in comparison to hydroquinone.

A human tyrosinase inhibition assay was conducted using L-tyrosine (and L-dopa as the co-factor) or L-dopa as the substrate in the presence of varying concentrations of test peptides or hydroquinone. This assay was designed to show dose-dependent inhibition of each of the first two steps in the human melanin synthetic pathway by each of the test peptides versus that of hydroquinone. A cell culture assay was then performed using various concentrations of test peptides or hydroquinone in order to determine their respective effects on melanin production in human melanocyte cells (cells responsible for skin color). Finally, a cytotoxicity assay was performed to measure the potential of varying concentrations of test peptides or hydroquinone to kill human melanocyte cells.

Clinical Study

A split-face, double-blinded, randomized and placebo controlled clinical study was designed to determine if P4 (decapeptide-12, Lumixyl™) could improve the appearance of facial hyperpigmentation. The secondary objective was to evaluate the effect of decapeptide-12 on overall facial appearance. Five females between the ages of 35 and 45 of Asian and Hispanic descent with moderate to severe epidermal melasma were included in the study. All had previously failed a 6-month regimen with twice daily Tri-Luma®. Volunteers washed their face then applied placebo cream on one side of the face and cream containing decapeptide-12 on the other side. Improvement in the appearance of melasma was assessed by all study volunteers and two blinded physicians using digital photography and a ten-point scale, with each point representing a 10% improvement. Overall improvement in facial appearance was also assessed by volunteers and two blinded physicians using digital photography and a four-point global assessment scale, with each point representing a 25% improvement. Volunteers also graded both sides of the face for overall treatment satisfaction using a 5-point nominal scale.

RESULTS

In Vitro Study

Results indicated that two (P3 and P4) of the seven peptides tested were more potent at inhibiting mushroom tyrosinase than hydroquinone. P4 (decapeptide-12) had the lowest IC50 at 40 μM compared to that of hydroquinone at 680 μM suggesting that decapeptide-12 is roughly 17 times more potent at inhibiting mushroom tyrosinase, in vitro, compared to hydroquinone. Moreover, additional enzymatic assays, aimed at discovering the inhibitory mechanism of both P3 and P4, confirmed that both of the tested peptides competitively inhibit mushroom tyrosinase in a similar manner as hydroquinone. Additional testing further demonstrated that both peptides also dose-dependently inhibited human tyrosinase more potently than hydroquinone. Cell culture assays with human melanocytes confirmed that both peptides reduced intracellular melanin content more effectively than hydroquinone (Figure 1). Most importantly, cytotoxicity assays showed that hydroquinone was highly toxic to human melanocyte cells causing 100% cell death, at concentrations greater than or equal to 100 μM, whereas both tested peptides caused only minimal cell death at all tested concentrations.

Clinical Study

Results showed that the test product (base cream plus 0.01% decapeptide-12) was well tolerated and did not elicit observable irritation or allergic reaction. Both volunteers and blinded physician graders observed greater than 40% diminished appearance in melasma after 12 weeks and greater than 50% after 16 weeks on the side of the face that was treated with the test product (Figure 2). Improvement of melasma did not exceed 4% for the side of the face treated with placebo. Moreover, both volunteers and physician graders observed greater than 70% improvement in overall facial appearance on the side of the face treated with the test product after 16 weeks of treatment. The placebo treated side showed less than a 15% improvement in overall facial appearance. Furthermore, all five volunteers said that they were very satisfied or extremely satisfied with their appearance on the test product treated side versus all five volunteers expressing that they were mildly or not satisfied with their appearance on the placebo treated side.

CONCLUSION

Decapeptide-12 is a safe and efficacious tyrosinase inhibitor that improves the appearance of facial melasma. 

REFERENCES

1. Grimes PE. Diseases of hyperpigmentation. In: Sams WM, Lynch PJ, eds. Principles and practice of dermatology. New York: Churchill Livingstone; 1990:807-819.

2. Grimes PE. Melasma. Etiologic and therapeutic considerations. Arch Dermatol. Dec 1995;131(12):1453-1457.

3. Ferrario E. [Observations on cutaneous hyperchromia during pregnancy.]. Riv Ostet Ginecol. Dec 1962;17:793-816.

4. Takase Y, Watanabe K. [Endocrinological study of pigmentation disorders]. Horumon To Rinsho. Sep 1967;15(9):705-710.

5. Parvez S, Kang M, Chung HS, Cho C, Hong MC, Shin MK, Bae H. Survey and mechanism of skin depigmenting and lightening agents. Phytother Res. 2006 Nov;20(11):921-34.

6. ; Abu Ubeid A, Zhao L, Wang Y, Hantash BM. Short-sequence oligopeptides with inhibitory activity against mushroom and human tyrosinase. J Invest Dermatol. 2009 Sep;129(9):2242-9.

7. Hantash BM, Jimenez F. A split-face, double-blind, randomized and placebo-controlled pilot evaluation of a novel oligopeptide for the treatment of recalcitrant melasma. J Drugs Dermatol. 2009 Aug;8(8):732-5.

FIGURE LEGENDS

Figure 1. Effect of P3, P4, and hydroquinone on intracellular levels of melanin.

Figure 2. Level of melasma improvement before and after 16 weeks of twice-daily use of 0.01% decapeptide-12 in an inert cream base. Results represent the average score of two blinded physicians for the series of 5 patients enrolled in the study.