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POTSDAM-REHBRUCKE
, GERMANY; AND NATIONAL CANCER INSTITUTE, NATIONAL INSTITUTES OF HEALTH,
BETHESDA, MD, U.S.A.
ABSTRACT. To discover safe and effective topical skin-lightening agents, we have evaluated alkyl esters of the
natural product gentisic acid (GA), which is related to our lead compound methyl gentisate (MG), and four
putative tyrosinase inhibitors, utilizing mammalian melanocyte cell cultures and cell-free extracts. Desirable
characteristics include the ability to inhibit melanogenesis in cells (IC50 , 100 mg/mL) without cytotoxicity,
preferably due to tyrosinase inhibition. Of the six esters synthesized, the smaller esters (e.g. methyl and ethyl)
were more effective enzyme inhibitors (IC50 ; 11 and 20 mg/mL, respectively). For comparison, hydroquinone
(HQ), a commercial skin bleaching agent, was a less effective enzyme inhibitor (IC50 ; 72 mg/mL), and was
highly cytotoxic to melanocytes in vitro at concentrations substantially lower than the IC50 for enzymatic
inhibition. Kojic acid was a potent inhibitor of the mammalian enzyme (IC50 ; 6 mg/mL), but did not reduce
pigmentation in cells. Both arbutin and magnesium ascorbyl phosphate were ineffective in the cell-free and
cell-based assays. MG at 100 mg/mL exhibited a minimal inhibitory effect on DHICA oxidase (TRP-1) and no
effect on DOPAchrome tautomerase (TRP-2), suggesting that MG inhibits melanogenesis primarily via
tyrosinase inhibition. MG and GA were non-mutagenic at the hprt locus in V79 Chinese hamster cells, whereas
HQ was highly mutagenic and cytotoxic. The properties of MG in vitro, including (1) pigmentation inhibition
in melanocytes, (2) tyrosinase inhibition and selectivity, (3) reduced cytotoxicity relative to HQ, and (4) lack
of mutagenic potential in mammalian cells, establish MG as a superior candidate skin-lightening agent.
BIOCHEM PHARMACOL 57;6:663 672, 1999. 1999 Elsevier Science Inc.
KEY WORDS. melanogenesis; TRP-1; TRP-2; hydroquinone; arbutin; kojic acid; magnesium ascorbylphosphate; cosmeceutical; methyl gentisate; pigmentation
E. V. Curto et al.
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Quantitation of cell number (to determine whether compounds are cytotoxic/cytostatic) was obtained following the
protocol of Dooley et al. [6], except for minor modifications.
After washing the cells with PBS and emptying the wells by
inversion and tapping the inverted plates against paper
towels, 0.5 mL of 0.1% crystal violet in 10% ethanol was
added to each well. After staining the wells for 5 min at
room temperature, the stain was decanted by inversion, and
the plates were rinsed four times in beakers of water. After
rinsing, the plates were emptied once again as described. To
extract crystal violet from the cells, 1 mL of 95% ethanol
was added to each well, and the plates were placed on a
rotating platform for 201 min. For analysis, 100 mL of
solution was transferred from each well into appropriate
wells in 96-well plates. To determine the relative number of
cells in each well, we measured the absorption by crystal
Tyrosinase Inhibitors
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Determinations of
IC50
Values
E. V. Curto et al.
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FIG. 1. Mammalian V79 cell mutagenicity results. The frequencies of mutations (number of colonies 3 1026) at the hprt locus
in Chinese hamster V79 cells are plotted versus the mg/mL
concentrations of the test agents (HQ, ; MG, ; and GA, E).
Concentrations were titrated in cultures of 1 3 106 cells to the
point of cytotoxicity. Frequencies above 20 3 1026 are considered mutagenic. This titration is representative of three experiments.
Tyrosinase Inhibitors
667
We tested four commercially available, putative skinlightening agents and compared them with MG in our
three primary screens. Structures of these compounds are
shown in Fig. 2. The third column of Table 1 presents
tyrosinase enzyme IC50 values for these compounds. Kojic
acid and MG were the most potent free enzyme inhibitors, with IC50 values of ca. 6 and 11 mg/mL, respectively.
HQ and arbutin were relatively poor enzyme inhibitors,
with IC50 values of about 72 and 149 mg/mL, respectively.
Magnesium ascorbylphosphate (MAP) did not inhibit tyrosinase enzyme in these assays, even at 200 mg/mL.
Column 4 of Table 1 presents IC50 values for inhibition of
cultured MelAb melanocyte pigmentation (due to both the
amounts of melanin synthesis and total cell number) for
these same compounds. HQ was a very effective agent with
an IC50 of about 1 mg/mL, albeit due to cytotoxicity. Here
MG was an effective pigmentation inhibitor, with an IC50 of
about 31 mg/mL, falling within the range of numerous prior
similar experiments (range of ca. 15 60 mg/mL). The other
compounds in this series did not inhibit pigmentation in
intact melanocytes at concentrations up to 200 mg/mL.
Column 5 of Table 1 shows MelAb cell cytotoxicity IC50
values for these compounds. MG was relatively non-toxic,
with an IC50 of cytotoxicity/cytostasis of about 119 mg/mL
on MelAb cells. HQ was extremely toxic; it had an IC50
value of about 3 mg/mL in this assay. The other commercial
compounds were non-toxic to MelAb cells up to 200
mg/mL.
Based upon our experience with over 150 compounds in
this assay system [5, 6; this work; and unpublished results],
we arbitrarily define a potentially efficacious skin depigmentation candidate agent as one that inhibits tyrosinase
enzyme with an IC50 of ,25 mg/mL, inhibits melanocyte
cell pigmentation with an IC50 , 100 mg/mL, and is
non-cytotoxic to cells with an IC50 . 100 mg/mL. The
threshold criteria are based upon: (1) our published stan-
Compound
Desired agent
HQ
MG
Kojic acid
Arbutin
MAP
Relative mass
[MG]
Cell-free tyrosinase
IC50 (mg/mL)
Melanocyte pigmentation
IC50 (mg/mL)
0.655
1.000
0.845
1.62
1.68
<25
72 6 25
11.2 6 4
6.2 6 2
149 6 41
.200
<100
1.1 6 0.2
30.9 6 5
.200
.200
.200
Melanocyte cytotoxicity
IC50 (mg/mL)
>100
3.1 6 0.7
118.7 6 12
>200
>200
>200
Assessment
Effective
Toxic
Effective
Ineffective
Ineffective
Ineffective
Experimental in vitro evaluations of putative skin-lightening agents, using tyrosinase enzyme, melanogenesis, and cytotoxicity assays, are displayed along with an overall assessment.
The relative mass of each compound is denoted with respect to MG. Based on the characteristics of MG and our experience with .150 compounds, we designate a desired
candidate as one that inhibits by 50% (IC50) mammalian tyrosinase enzyme at concentrations , 25 mg/mL, inhibits pigmentation in mammalian melanocytes , 100 mg/mL, and
exhibits low cytotoxicity at concentrations . 100 mg/mL. Magnesium ascorbyl phosphate is denoted as MAP.
E. V. Curto et al.
668
dard (i.e. 100 mg/mL) for cell-based assays [6]; (2) a more
stringent standard for free enzyme (i.e. 25 mg/mL), because
the agents must pass through two cellular membranes to
inactivate the enzyme within melanosomes in cultured
cells, and optimal uptake is not necessarily assured; and (3)
our general experience in screening many phenolic compounds in the various assays. Of the compounds tested in
this current study, only MG satisfied all of these requirements. Based on these multiple in vitro endpoints, it is the
best candidate skin-lightening agent that we are aware of
from the literature (and this work), and its behavior in
these assays establishes the standards for further rational
drug design to identify additional comparable or better
active agents. HQ is highly toxic and mutagenic [this work;
6, 10, 11, 22]. Other purportedly active compounds tested
in this series were non-efficacious. They did not act on
MelAb cells or the enzyme, or both. Our assessments of
these compounds are denoted in the last column of Table 1.
R group
Methyl
Ethyl
n-Propyl
i-Propyl
n-Butyl
n-Pentyl
Methoxy-ethyl
Relative
mass
Cell-free tyrosinase
IC50 (mg/mL)
Melanocyte pigmentation
IC50 (mg/mL)
Melanocyte cytotoxicity
IC50 (mg/mL)
1.00
1.08
1.17
1.17
1.25
1.33
1.26
11.2 6 4
19.6 6 7
28.5 6 12
57.5 6 21
128 6 53
.200
100 6 29
30.9 6 5
28.7 6 5
15.6 6 2
27.8 6 8
12.4 6 4
15.6 6 2
43.7 6 8
118.7 6 12
65.9 6 20
56.5 6 10
62.7 6 9
60.1 6 10
56.5 6 10
186.5 6 8
Experimental in vitro evaluations of a series of alkyl esters of gentisic acid as potential skin-lightening agents using tyrosinase enzyme, melanogenesis, and cytotoxicity assays. The
relative mass of each compound is denoted with respect to MG.
Tyrosinase Inhibitors
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References
FIG. 4. Conceptual model of gentisate ester binding to the active
site of tyrosinase: Hypothetical two-dimensional model of initial
docking of gentisate ester in the putative active site of mammalian tyrosinase. The two coppers are assumed in octahedral
coordination with a gentisate ester and oxygen.
It inhibits melanocyte pigmentation at moderate concentrations, with minimal cytotoxicity, and specifically inhibits tyrosinase enzyme. Furthermore, it is non-mutagenic,
unlike HQ. No other compound that we are aware of in the
literature has all of these desired characteristics, thus
establishing MG as the superior candidate.
Once MG-containing topical formulations are developed
and tested in vivo in animal models and/or clinically, what
types of products could be marketed? Pre-market approval
and regulatory compliance issues will have significant
effects on the potential commercial availability of MGcontaining products in many countries. If the products
marketing literature or labeling asserts that it alters the
structure or function of skin or makes a specific therapeutic claim (e.g. treatment of a dermatologic disorder), this
compound would likely be formulated for use as an OTC or
prescription drug product. However, in selected global
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emerging concept in dermatologic agent development and
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and cosmetic products, yet vaguely defined and not formally
recognized by the U.S. FDA [4]. Cosmeceutical products,
unlike cosmetics per se, should contain ingredients that
have been demonstrated in the laboratory to be pharmacologically active. In addition, it is preferable if the pharma-
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672
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