Received: 1 June 2023

| Revised: 3 February 2024 | Accepted: 14 March 2024

DOI: 10.1111/exd.15069

RESEARCH ARTICLE

A novel professional-­use synergistic peel technology to
reduce visible hyperpigmentation on face: Clinical evidence
and mechanistic understanding by computational biology andoptical biopsy
Vinay Bhardwaj1 | Marc Zachary Handler2 | Junhong Mao1 | Chloe Azadegan1,3
Pritam K. Panda4,5 | Hans Georg Breunig6 | Isabell Wenskus6 | Isabel Diaz2 |Karsten König6,71
Department of Global Personal Careand Skin Health R&D, Colgate-­Palmolive
Company, Piscataway, New Jersey, USA
2

Dermal Clinical Research, Colgate-­
Palmolive Company, Piscataway, New
Jersey, USA

3

Drexel University College of Medicine,Philadelphia, Pennsylvania, USA
4
Department of Physics and Astronomy,
Uppsala University, Uppsala, Sweden|

Abstract
Topicals and chemical peels are the standard of care for management of facial hyperpigmentation. However, traditional therapies have come under recent scrutiny,such as topical hydroquinone (HQ) has some regulatory restrictions, and high concentration trichloroacetic acid (TCA) peel pose a risk in patients with skin of colour.
The objective of our research was to identify, investigate and elucidate the mechanism of action of a novel TCA-­ and HQ-­free professional-­use chemical peel to man-5

Nerdalytics, Uppsala, Sweden

6

age common types of facial hyperpigmentation. Using computational modelling

JenLab GmbH, Berlin, Germany

and in vitro assays on tyrosinase, we identified proprietary multi-­acid synergistic

7

Department of Biophotonics andLaser Technology, Saarland University,
Saarbrucken, Germany
Correspondence
Vinay Bhardwaj, Department of Global
Personal Care and Skin Health R&D,
Colgate-­Palmolive Company, Piscataway,
NJ 08854, USA.
Email: bhard.vinay@gmail.com
Funding information
Colgate-­Palmolive Company

technology (MAST). After a single application on human skin explants, MAST peel
was found to be more effective than a commercial HQ peel in inhibiting melanin(histochemical imaging and gene expression). All participants completed the casestudy (N = 9) without any adverse events. After administration of the MAST peel
by a dermatologist, the scoring and VISIA photography reported improvements
in hyperpigmentation, texture and erythema, which could be linked to underlying
pathophysiological changes in skin after peeling, visualized by non-­invasive opticalbiopsy of face. Using reflectance confocal microscopy (VivaScope®) and multiphoton tomography (MPTflex™), we observed reduction in melanin, increase in metabolic activity of keratinocytes, and no signs of inflammatory cells after peeling.
Subsequent swabbing of the cheek skin found no microbiota dysbiosis resulting
from the chemical peel. The strong efficacy with minimum downtime and no adverse events could be linked to the synergistic action of the ingredients in the novel
HQ-­ and TCA-­free professional peel technology.
KEYWORDS

acne, hydroquinone-­free, hyperpigmentation, melasma, microbiome, molecular docking,multiphoton tomography, photodamage, reflectance confocal microscopy
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,provided the original work is properly cited.
© 2024 Colgate-Palmolive Company. Experimental Dermatology published by John Wiley & Sons Ltd.
Experimental Dermatology. 2024;33:e15069.
https://doi.org/10.1111/exd.15069

﻿

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I NTRO D U C TI O N|

BHARDWAJ et al.

monotherapy for melasma and PIH. 2,20 Combination treatment inchemical peeling is an effective and convenient method to manage

Globally, there is a burden on dermatologists and skin care profes-

HP, rather than a series of peelings over months using a single peel-sionals to manage the influx of patients with hyperpigmentation

ing agent. 21–23(HP) disorders and skin lightening (SL) demand for cosmetic rea1

sons. Topicals and chemical peels using hydroquinone (HQ) andThe overarching goal of our well-­structured holistic research was
to use state-­of-­the-­art dermatology research techniques to identify,

trichloroacetic acid (TCA) are the dermatology standard first-­  andinvestigate and clinically test a novel TCA-­ and HQ-­free multi-­acidsecond-­line therapy to manage HP and SL, respectively. 2 They havesynergistic technology (MAST) to manage all common types of facial

proven efficacy in treatment of hyperpigmentation conditions such

hyperpigmentation in all Fitzpatrick skin types.

2

as melasma and post-­inflammatory hyperpigmentation (PIH). HQ
has more than 40 years of safety and efficacy history.3 Although
no safety concern is reported on humans,3 prolonged use of HQ isassociated with skin irritation, erythema and ochronosis or discoloration.1,4,5 Consequently there are research efforts, though limited,2|

M E TH O D S

2.1 | In silico computational biology

to find HQ-­free alternative technologies6,7 to meet demands of peoFollowing the previous reports,19,24 molecular docking (MD) and

ple and professionals to manage HP and SL.
Chemical peeling is among the most popular non-­s urgical8molecular dynamic simulations (MDS) were done using open access

The use of Jessner's solution forsoftware Autodock Vina 1.2.2 and GROMACS v.2019.2, respec-

superficial peeling, and its combination with trichloroacetic

tively. A high performance computer was used to screen and under-acid (TCA) to enhance depth of penetration and efficacy has astand molecular interactions of depigmentation and peeling agentslong history of use by dermatologists and plastic surgeons.9,10

with human tyrosinase enzyme (hTYR).

dermatology procedures.

Jessner's and TCA peels are often enhanced by addition of
depigmentation topicals like HQ, kojic acid (K A), azelaic acid
(AzA), resorcinol, etc.11 When properly administered by pro-

2.2 | In vitro enzyme assay

fessionals, a superficial TCA peel is generally well tolerated
and effective to treat melasma and PIH. 2 However, TCA is a

The activity of leads from in silico screening were tested on ty-

strong caustic and protein coagulation agent, hence, TCA peel-rosinase enzyme following manufacturer's protocol (Sigma kit,

ing has major downtime (cosmetically noticeable reactions likecatalogue#MAK257). Following our previous report, 25 the syn-frosting and exfoliation lasting 1 week or so), and less toler-ergy was investigated by testing mixtures of the leads at specificated in particular by skin of colour.

12

Although chemical peel isratios and confirmed using open-­access software CompuSyn fromexpected to work through controlled chemical injury action,13

ComboSyn, Inc. USA. 26 The synergy was defined as the cumula-

unexpected severe adverse events like permanent injury andtive effect of the mixture being more than the additive effect of theinfections may occur from strong deep peels using phenol ortest agents when tested independently, and the CompuSyn value ofhigh-­s trength TCA, and the risks are particularly high if the

combination index is <1. 25,26

peeling is performed by non physician ancillary staff.

14,15

The

micro lesions or wounds after chemical injury from peel may
serve as a habitat for microflora. Depending on the degree

2.3 | Ex vivo comparative study on skin explantsof the chemical injury and the regrowing microflora, chemical
injury may be followed by normal recovery to restoration or

Human skin biopsies/explants were washed, acclimatized andabnormal healing resulting in infection. To improve outcomes,

treated with the peel solution (commercial HQ-­K A peel or synergy

dermatologists need to better understand histological andpeel ‘MAST’) for 20 min. The skin samples were harvested on Day

molecular changes after treatment; however, it is challenging

7 to quantify melanin (histochemical imaging) and gene markers ofin aesthetic medicine/procedures where taking excision of fa-

melanin synthesis (RT-­qPCR). We followed our previously publishedcial skin tissue (biopsies) is unnecessary. Non-­invasive opticalmethod for histochemical imaging and image processing to visualize

biopsy 16,17 and computational molecular modelling (predictive

and quantify melanin.17 For gene markers, biopsies were mechani-

biology) 18,19 can help us decipher the underlying anatomical

cally disrupted and homogenized, RNA was isolated (RNeasy Plus 96

and molecular changes in skin after treatment, without the

kit from Qiagen, Inc. USA) and 100 ng of extracted RNA was usedneed of invasive procedures.

to synthesize cDNA (cDNA reverse transcription Kit from Applied

Using two or more therapeutics (combination/synergy ther-

Biosystems, USA). RT-­qPCR was done using QuantStudio™ 5 real-­apy), rather than a single agent has proven to be an effective way totime PCR (Applied Biosystems, USA) and the three gene markers

treat skin HP disorders. 2,20 One successful example is Tri-­Luma®,of melanin synthesis (TYR, TYRP1 and DCT) were quantified using

a combination of three drugs (HQ, tretinoin and fluocinolone ace-

TaqMan real-­time qPCR assays (Applied Biosystems, USA). For thetonide), which has emerged as more effective treatment than HQ

gene expression study, TenSkin™ model from Ten Bio, Ltd., United

16000625, 2024, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/exd.15069 by Uppsala University Library, Wiley Online Library on [22/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

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Kingdom was used to grow skin explants under optimum mechanicaltension to mimic the real in vivo environment.

2.6 | Non-­invasive optical biopsy imaging

27

Optical biopsies of the facial skin were taken using VivaScope 1500
and MPTflex, as reported previously.30,31 Medical grade ECG rings

2.4 | Subject recruitment and facial peelingperformed by US board-­certified dermatologist

were placed on regions of interest on the cheeks to monitor thesame spot over the course of the clinical study (longitudinal imaging). Images of skin anatomy (keratinocytes, melanin, collagen, etc)

Nine subjects representing different ethnicities, Fitzpatrick skinwere acquired following manufacturer's protocol, using a tissue cap

types (I–V) with dark or hyperpigmented patches on the face (pho-and conducting gel for VivaScope 1500 imaging (830 nm laser withtodamage, melasma and acne-­induced post-­inflammatory hyperpig-

maximum power 20 mW) and a magnetic ring with coverslip, immer-

mentation) were recruited for the proof-­of-­concept testing (casesion oil and water for MPTflex imaging (tunable laser at 760 andstudy). Table S1 (Supplementary information) captures the demo-

890 nm with maximum power 39 mW). Dermoscope images weregraphic details of the subjects and Appendix S1 (supplementaryalso acquired prior to imaging using vivascope 1500. VivaScope im-

information) lists the inclusion and exclusion criteria for subject re-ages were studied primarily to visualize melanin,30 while MPTflex

cruitment. The study protocol was approved by the US Institutional

images were used to visualize keratinocytes.31 Similar to the previ-

Review Board, Inc. in Miami, FL (U.S.IRB2022/CP01).The signed in-ous report,32 FLIM images and photon counts were acquired at twoformed consent was taken from the subjects before their participa-

different wavelengths (760 and 890 nm) to accurately discriminatetion in the study. MAST Peel was dispensed by a US board-­certified

and quantitate the NADH from other autofluorophores in the skindermatologist, Dr. Handler, following manufacturer's guidelines forlike melanin and FAD. FLIM images were processed offline using

peeling, post-­peel care and for tolerability assessment (Appendix S2,

SPCImage software from Becker and Hickl GmbH, Germany, as re-

supplementary information). There were two peeling sessions,ported previously.33

4 weeks apart. See Table 1 for summary of proof-­of-­concept clinicalstudy design including subject visits for peeling and measurements.

2.5 | VISIA-­CR imaging and dermatologist's scoring

2.7 | Facial swabbing for microbiome analysis
Swabbing of the cheeks was performed following the procedurereported in previous report,34 but with some modifications. The

There were three measurement sessions: before peel (Week 0),

special large contour FLOQSwabs® from Copan Diagnostics, Inc.

4 weeks after first peel (Week 4) and 4 weeks after second peel (Week

USA (Cat#528C) was used to enable faster collection and efficient

8). Visia-­CR 4.3.1 photography with special optics and RBX® image

extraction of the samples for DNA extraction. The entire cheek wasprocessing technology from Canfield Scientific, USA, to separate red

swabbed after impregnating the applicator of the sterile swab infrom brown was used to capture and analyse images of the face from

PBS. The cheek was swabbed for 30 s by dabbing in a rolling manner

three angles (front, left and right).28,29 The study dermatologist as-

and in horizontal directions to ensure that the entire surface area ofsessed changes in facial global appearance (uneven pigmentation/

the cheek was covered. After the sample was collected, the tip of theskin tone, skin texture, fine lines/wrinkles and redness) using a 5-­pointswab was broken from breakpoint and placed in a sterile tube, stored

scale (0 = No, 1 = minimal, 2 = mild, 3 = moderate and 4 = severe).

immediately at −80°C until tested for microbiological information.
16S rRNA sequencing was done using the MiSeq device (Illumina)

TA B L E 1 Summary of case study design (N = 9 participants).
Intervention/study week
Procedure

Screening

Informed consent

X

and the data were processed to obtain alpha and beta diversity indices, as reported previously. 29,35 Further analysis was done on two
most abundant species, P. acne and S. epidermidis to monitor changes

0 wk

4 wks

8 wks

Subject self-­assessment

X

X

X

Facial swabbing

X

X

X

Visual assessment bydermatologist

X

X

X

VISIA-­CR imaging

X

X

X

VivaScope imaging

X

X

X

MPTflex imaging

X

X

X

Subject self-­assessment(post-­toner application)

X

X

Chemical-­peeling

X

X

before and after peel application.

3|

R E S U LT S

3.1 | Computational modelling revealed surprisingfindings on molecular interactions between
depigmentation agents and hTYR
Molecular docking (Figure 1A, i) shows binding of some well-­known
peeling and depigmentation agents on the catalytic and allostericsites of hTYR, enabling us to predict their binding affinity to inhibit
hTYR (Figure 1A, ii). Ellagic acid (commonly found in pomegranate),

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glabridin and glycyrrhizic acid (commonly found in licorice), butyl and(Figure 1A, i); however, binding to the allosteric site is more stable

ethyl derivatives of resorcinol and arbutin (a derivative of HQ) showed(Figure SI, and Videos SI and SII, supplementary information). KA

strongest inhibition with binding affinity < −6 kcal/mol, highlighted byforms two hydrogen bonds at active site, while three hydrogen bonds

Tier I (Figure 1A, ii). KA, AzA, HQ, tranexamic acid (TXA), ascorbic

at the allosteric site, which could be the possible rationale behindacid, mandelic acid and hexyl derivative of resorcinol show moderatehigher binding stability at the allosteric site (Figure SI).

activity (affinity between −4 and −6 kcal/mol, Tier II). Surprisingly, KA

Mandelic acid binds to allosteric site while TXA binds to cat-

was found to bind to catalytic site (active) as well as allosteric site#1

alytic site (Figure 1A, i and 1B, i). Figure 1B shows the reaction

F I G U R E 1 In silico computational biology (A, B), and in vitro assay (C) showing screening results to identify LMP-­T XA synergistic
technology to inhibit tyrosinase enzyme. Binding site (A, i) and binding affinity (A, ii) of different skin lightening agents using moleculardocking. Mechanistic understanding behind synergistic effect between mandelic acid and TXA using molecular dynamic simulations (B).
Synergistic effect between lactic (L), mandelic (M) and pyruvic (P) acids (C, i) and the synergy validation by CompuSyn (C, ii), green line meanssynergistic effect, thicker the line stronger the synergistic effect. TXA, tranexamic acid.

16000625, 2024, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/exd.15069 by Uppsala University Library, Wiley Online Library on [22/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

4 of 13kinetics to elucidate interactions of TXA and mandelic acid withunstable binding of HQ to hTYR could be because HQ is reported

hTYR. That reveal the possible mechanism of synergistic action: ato act as substrate as well as inhibitor. 36

conformational change in mandelic acid at allosteric site (Figure 1B,

The strongest triple combination synergy effect between alphaii) around ~50 ns of simulated reaction lead to the stabilize of the(lactic and mandelic acid) and keto acid (pyruvic acid) was identified

hTYR structure in presence of TXA that has stable binding to cata-(Figure 1C, i) showing ~10 times higher anti-­T YR activity than theirlytic site (Figure 1B, iii). The inhibition of hTYR by TXA is compara-

additive effect (21.9% vs. 2.5%). Among all mixtures, lactic and man-ble to KA but stronger than HQ with binding affinity −5.42 versus

delic mixtures show strongest in vitro synergistic effect (data not−5.36 versus −5.13 kcal/mol, respectively (Table 2). This could beshown), which was consistent with CompuSyn results (Figure 1C, ii),

because TXA and KA form three hydrogen bonds with the enzyme

thickness of the green line denotes strength of the synergy.

(vs HQ that form two bonds), and compared to KA, TXA binds
stably to copper in the catalytic site (Table 2, Figure SII). TXA isstable inhibitor of the enzyme as compared to HQ, which was
not stable (Videos SIII and SIV, supplementary information). The

TA B L E 2 Comparison of TXA versus HQ versus KA binding totyrosinase enzyme.
Activeingredient(ligand)

Bindingaffinity(kcal/mol)

Inhibition
constant(mM)

Number of
H bonds

Bond withcopper

TXA−5.427

0.031

3

Yes

HQ

−5.137

0.119

2

No

KA

−5.36

0.035

3

No

Abbreviations: HQ, hydroquinone; KA, kojic acid; TXA, tranexamic acid.

3.2 | After single application, MAST peel
is superior to commercial HQ-­K A peel ininhibiting melanin
Figure 2 shows the melanin inhibition effect of MAST peel compared
to commercial HQ peel that also contained KA (HQ-­K A peel). There
was no significant reduction in expression of TYRP1 and DCT genes
after treatment with HQ-­K A peel, but MAST peel (Figure 2A).
Histochemical imaging showed similar results (Figure 2B), significant
reduction in melanin intensity after MAST peel (−17%) but nochange after HQ-­K A peel (−1%). Figure 2C–E shows representative
histochemical images used for quantification of melanin. The skinretained its structural integrity after peeling and histochemical

F I G U R E 2 Ex vivo testing on human skin explants to compare melanin inhibition potential of MAST peel versus commercial peelcontaining HQ and KA. (A) Gene markers of melanin synthesis normalized to RPS13 content (N = 4), and (B) melanin quantification from
histochemical images (N = 6). Representative histochemical images of melanin are shown for untreated ‘control’ (C) MAST peel (D) and HQ-­
KA peel (E). *Denotes significant difference versus untreated control (p < 0.05), ** denotes significant difference versus HQ-­K A peel and %change is with respect to untreated (control). DCT, dopachrome tautomerase; Ed, epidermis; M, melanin; RPS13, ribosomal protein S13; SC,
stratum corneum; TYR, tyrosinase, TYRP1, tyrosinase-­related protein 1.

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processing, which is evident through intactness of stratum corneum,

images (Figure 4D) are comparable to what has been reported pre-epidermis and dermis (Figure 2C–E). Melanin, grey/black molecules

viously to image keratinocytes and quantify intracellular NADH

in the histochemical images, was abundantly present at the basal

incvivo on face31 and reconstructed human skin. 32 There seems

layer (epidermis–dermis junction) and showed reduction or fading

to be a noticeable increase in NADH intensity after peeling (blackafter MAST peeling.

and white MPT images); however, it could be the interference
from intracellular melanin, as highlighted by arrows in pseudo-­

3.3 | MAST peel shows clinical implications tomanage hyperpigmentation without any downtime

coloured FLIM images in Figure 4D, i. In pseudo-­coloured FLIM
images, melanin is shown by red and NADH in fluorescent green.
Using FLIM, we were also able to accurately discriminate between
free NADH (~300 picosecond, Figure 4E, ii) and protein-­b oundAll subjects (N = 9) completed the study and there were no adverse

NADH (~2300 picosecond, Figure 4E, iii), values are in agreement

events. No subjects experienced frosting, and only two subjects de-with previous reports using MPTflex. 33,37 There was a significant

veloped minor visible exfoliation, but not enough to avoid attending% decrease in lifetime of NADH before vs after peel (Figure 4E),

office and social gatherings (no downtime). A clear decrease in brownfree NADH, Tau 1 (Figure 4E, ii), protein bound NADH, Tau 2

patches and redness was observed in most cases. Figure 3 shows(Figure 4E, iii) and mean lifetime ‘Tau M’ (Figure 4E, iv).

before and after VISIA photographs to represent cases of photodamage (A, B), melasma (C, D), and acne-­PIH (F) with improvement
after two peeling sessions. A decrease in redness and brown spots iseven more appreciated after processing of VISIA images using RBX®

3.5 | Chemical peeling does not induces
microbial dysbiosis

image processing to separate brown from red (Figure SIII, supplementary information). The acne-­induced PIH case reported a reac-

Owing to the virtue of special FLOQSwab® technology, all swab sam-tion to sunscreen and was corrected after replacing the sunscreen

ples (9 × 3 subjects × time points) produced enough microbial yieldwith a commercial sunscreen specially designed for oily and acne-­

to give more than 10 000 reads, except one sample that produced

prone skin. Figure 3F shows dermoscope and VivaScope images to7342 reads (Figure SIV). Figure 5A shows the abundance of top 30

reveal reduction of acne-­induced PIH spots and the complete closure

species found on cheek, and propionibacterium acne (14%–91% abun-of all three comedones after peeling.

dance) and staphylococcus epidermidis (0.5%–26% abundance) being

Figure 3E shows the average score calculated from the dermatol-

most abundant, findings similar to previous report.35 S. aureus, typi-ogist's objective visual assessment of each subject before and aftercally related to infection and atopic dermatitis,34 was below the de-chemical peeling. There is a global improvement in most, if not all

tection limit, except two subjects with <0.1% abundance. Althoughsubjects, with an average score improvement in uneven pigmenta-

not significantly different, there was a noticeable change in species

tion/skin tone, skin texture, redness (Erythema) and fine lines/wrin-

richness (Shannon diversity) after chemical peeling (Figure 5B), which

kles. Three out of four cases of photodamage showed one to two

is in agreement with increase in richness of operational taxonomic

step improvement in discoloration, all four cases of melasma showed

unit (Figure SIV, Supplementary Information). There was no struc-

improvement, but only by 0.5 points on the global appearance scor-tural difference (beta diversity) before and after peeling (Figure 5C).

ing scale. Decrease in redness was evident in the majority of the

A decrease in P. acne after each peeling session can be observed insubjects, and more pronounced in melasma cases.

Figure 5A, and appreciated after quantification (Figure 5D). As expected the PIH case with acne showed P. acne abundance, but surpris-

3.4 | Optical biopsy revealed underlying
structural and functional changes in facial skinafter peeling
For illustration purposes, Figure 4 shows non-­i nvasive optical

ingly it was >90% of total facial microbiome. VISIA-­CR images of thisacne-­induced PIH case (Figure 5E), shows a decrease in Porphyrin, afluorescent metabolite produced by P. acne.38,39

4|

DISCUSSION

biopsy imaging instruments, VivaScope (Figure 4A) and MPTflex(Figure 4B). The insets shows example of a participant with an

Using in vitro enzyme assays and in silico computational biology,

ECG ring used for longitudinal imaging. After peeling, a decrease

we were able to identify a synergistic mixture of four acids (Lactic,

in visible superficial discoloration was observed under the dermo-

mandelic, pyruvic and tranexamic acids), aka MAST peel technol-

scope, which could be due to the underlying melanin reduction

ogy (Figure 1). Our in silico predictions using molecular docking

in the epidermis layer visualized by VivaScope (Figure 4C, i, ii).

(Figure 1B) were similar to in vitro results (data not shown), except

Melanin (small solid white dots) and melanin-­containing keratino-in the case where ellagic acid and arbutin corresponded to lower

cytes (honeycomb structure) are visible under VivaScope; how-

activity in vitro. This variance could be explained by (1) the purity

ever, keratinocytes are even better appreciated under MPT-­FLIM

of arbutin and ellagic acid was not 100% in vitro experiments, (2)images (Figure 4D, i, ii). The quality of ultra-­high resolution MPT

the source of tyrosinase enzyme was mushroom for in vitro assay.19

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F I G U R E 3 Untouched raw images before and after MAST peel. Visia images to show global improvement in two representative cases ofphotodamage (A, B), and two representative cases of melasma (C, D). Acne-­induced PIH case (F) showing reduction in hyper-­pigmentation(dermoscope) and complete closure of all three comedones (VivaScope) after peeling. Graph (E) shows the average score of all subjects(N = 9) after objective visual assessment scores given by the dermatologist who performed the peeling. Grading scale was 0 = absent,
1 = Minimal, 2 = mild, 3 = moderate and 4 = SEVERE. Arrows highlight open comedones, PIH = post-­inflammatory hyperpigmentation. D: Thissubject did not give consent to use full-­face photographs, but only the cropped part of the face area.

Using MD, KA was found to have affinity to bind to catalytic as welland Videos SI and SII). The strong binding of KA to the allosteric siteas allosteric site (Figure 1A), however, MDS revealed that binding

is in agreement with another contradictory finding,36 that opposesof KA to allosteric site is more stable than catalytic site (Figure S1,classical KA binding theory to the copper in the catalytic site. Similar

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F I G U R E 4 Non-­invasive optical biopsy imaging using VivaScope (A) and MPT-­FLIM (B) to visualize structural and functional changes inskin before and after MAST peeling. The changes in melanin by VivaScope (C) and changes in keratinocytes' metabolic activity by MPT (D)and the quantification of NADH lifetime in the keratinocytes to measure metabolic activity ‘cellular energy’ using FLIM (E). The % decreasein decay time (E,i) and lifetime of Tau1 (E, ii), Tau2 (E, iii) and TauM (E, iv) is shown before and after MAST peel. The inset in Figure A andB, shows the photo of a melasma patient to highlight ECG ring that was used to improve longitudinal imaging. *highlights intracellular
NADH (fluorescent green) inside Kc (keratinocytes) in MPT-­FLIM images, and significant difference (p < 0.05 vs. before peel) in FLIM
graphs. Arrows in (D) and (I) highlights significance of FLIM to avoid interference of melanin (white in MPT image, and red in FLIM image) in
NADH quantification. FLIM, fluorescence lifetime imaging; Kc, keratinocytes; M-­Kc, melanin-­containing keratinocytes; MPT, multiphoton
tomography; NADH, nicotinamide adenine dinucleotide hydrogen.
observations were made for HQ. HQ binds strongly to the catalytic

surgeons to combine peeling acid with depigmentation agent to tar-site but binding was unstable (Video SIII). Similar findings on insta-

get complex pathology of refractory melasma.40 The peeling acids

bility of the HQ binding with tyrosinase enzyme has been reported(lactic, mandelic, and pyruvic acids) induce an effect, thereby facil-

previously and it could be because HQ acts as a substrate as wellitating TXA penetration across the skin barrier. Usually, a 4% TXA

as inhibitor of tyrosinase enzyme.36 We believe the unstable inter-peel/solution is combined with microneedling to enhance delivery ofactions could be the reason why HQ is associated with refractory

TXA and achieve better clinical outcomes.5 However, we got good5

melasma. Compared to HQ, we found that TXA was a strong andresults with low level of TXA (1% in MAST peel), which could bestable inhibitor of tyrosinase (Table 2, and Video SIV), hence it was

because of 1) penetration enhancement due to the peel's keratolytic

identified as potential replacement to HQ. In the last couple years,

effect, and 2) Synergy between TXA and mandelic acid to target ty-

TXA has emerged as a well-­tolerated effective alternative to HQ inrosinase enzyme from two different sites (Figure 1C). Using MDS, we

treating refractory melasma.5

were able to elucidate the mechanism of synergistic action between

The multi-­pronged synergistic action of our MAST peel design

TXA and mandelic acid: TXA binding to catalytic site of the tyros-

is supported by a deceptive simple solution identified by plastic

inase enzyme induced conformational change in the mandelic acid

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FIGURE 4(Continued)(Figure 1C, ii), which lead to a stable binding (Figure 1C, iii). Synergy

exfoliation was minimal (only noticed by two subjects). Participants

could be the rationale behind our surprising superior performance ofwith skin of colour, for example acne-­induced PIH case in Figures 3E

MAST peel over a commercial peel containing HQ and KA (Figure 2).

and 5E (Fitzpatrick V, African-­American) were able to tolerate andSignificant inhibition of melanin after a single peel is a significant

show improvement after MAST peel. Although the sample size ofadvantage of MAST over commercial HQ-­K A peel (Figure 2A,B), be-

our proof-­of-­concept clinical study was small (N = 9 subjects), based

cause prolonged use of HQ and KA is associated with skin irritation,

on dermatologist's experience in peeling and the fair representation

erythema, ochronosis, contact dermatitis and sunburns.5,41of people from different ethnicities and Fitzpatrick skin type I–V

Another evidence of synergy in action may be stipulated fromclinical outcomes after MAST peeling. A decrease in hyperpigmen-(Table S1), it is reasonable to suggest that the MAST peel may bewell tolerated by all skin types.

tation and surprisingly also in erythema was observed in most of the

By using VivaScope® and MPTflex™, we were able to success-cases recruited for the study (Figures 3A–D and SIII). A decrease in

fully visualize and monitor real-­t ime changes in skin anatomy after

erythema may be attributed to (1) TXA, a tyrosinase and plasmino-

peeling (Figure 4). There were no obvious signs of inflammation

gen inhibitor whose multi-­pronged action can target melanogenesis

or inflammatory cells after MAST peeling, unlike our previous

as well as angiogenesis,42–44 (2) synergistic action between our mild

study on TCA peel that induces temporary inflammation.13 After

peeling acids and (3) less acidic, because MAST peel is buffered to

MAST peel, a decrease in melanin content ex vivo (Figure 2D) andpH 3.6 to bring the pH closer to skin's physiological pH 4.5 and hencein vivo (Figure 4C) could be the underlying mechanism behind

expect little to no inflammatory response.45 All subjects were able

visible surface improvement in discoloration imaged by VISIA-­CR

to tolerate maximally applied two layers of the peel and completed(Figure 3A–D), dermoscope (Figure 4C) and dermatologist's vi-

both peeling sessions. Unlike TCA peels, there was no frosting andsual assessment (Figure 3E). Using the VivaScope we were able

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F I G U R E 5 Microbiome analysis of cheek before and after MAST peel showing no microbial dysbiosis in, abundance of top 30 species (A)and their Shannon alpha diversity (B), and beta diversity (C). Quantitative analysis of two abundant species, P. acne (D). Visia-­CR image ofacne/acne-­induced PIH case, before and after peeling (E).
to monitor acne-­induced PIH microenvironment. VivaScope wasdermatologist’ eye and even after using a dermoscope with 15×

able to visualize three invisible comedones/papules at the site

optical zoom (Figure 3F). An improvement in acne/acne-­inducedof acne-­induced PIH spots, which were otherwise not visible to

PIH case after MAST peel (Figure 3F) may be attributed to pyruvic

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acid and mandelic acid, which are clinically reported to be well

5|

CO N C LU S I O N S

tolerated and effective alternatives to azelaic acid and salicylic
acid peels to treat acne.46–48 Due to resolution limitations of

Owing to the synergistic action of the proprietary multi-­acid blend,

the VivaScope, it is difficult to decisively label the surrounding

the MAST peel when administered by an experienced professional

highly reflective organelles around the open comedones (before

showed strong: (1) ex vivo anti-­pigmentation activity on human skinpeel) as inflammatory cells or melanin. Using ultra-­h igh resolution

explants as compared to a commercial professional peel of similar

MPTflex, we were able to visualize keratinocytes as well as mela-strength but containing HQ and KA, and (2) clinical efficacy with mini-

nin containing keratinocytes, which were otherwise poorly visible

mum downtime (no frosting and little visible exfoliation) to effectively

under VivaScope (Figure 4). Although quality of our MPTflex im-

manage common hyperpigmentation conditions like melasma, photo-ages are comparable to previous reports using MPT to visualize

damage and acne-­induced PIH. The MAST peel induced no dysbiosis

and quantify NADH, 31 we did not use their approach to quan-of the facial microbiome and showed good tolerance by all people who

tify NADH because there are other molecules in skin that aut-

participated in the study. However, it was small proof-­of-­concept clini-

ofluoresce when excited at wavelength ~760 nm (NADH, FAD,

cal study (N = 9) and further investigation is required to conclude safety

Melanin). Therefore we used two laser wavelengths (760 andand efficacy on people with skin of colour and sensitive skin.

890 nm) to discriminate between aforementioned fluorophores,

Using computational biology and optical biopsy, we were able toand acquired their fluorescence lifetime information using FLIM

gain new insights into the pathophysiology of skin after chemical peel.

detector to accurately discriminate (Figure 4D) and quantify

The visible improvement in dull, hyperpigmented and acne could be

32,33

The decrease in lifetime of the intracellular

correlated to invisible histological changes in intracellular NADH (en-NADH after peeling (Figure 4E) indicates increase in metabolic ac-

ergy marker in mitochondria of keratinocytes), melanin and comedo-

tivity of the keratinocytes. Similar findings on decrease in NADH

nes visualized real-­time by non-­invasive optical biopsy using MPTflex™

lifetime have been correlated with healing of wounded skin, 37,49

and VivaScope®. Based on our mechanistic findings from computa-

NADH (Figure 4E).

and recovery of UV-­d amaged skin.

32

Skin aging (chronological andtional biology (TXA vs. HQ), performance testing on skin explants

UV-­induced), dyspigmentation and other skin health conditions/(MAST peel with TXA vs. commercial peel with HQ), and a comprehen-diseases have been linked to mitochondria dysfunction, whichsive review of 46 articles on comparing TXA versus HQ for melasma

may be treated by boosting energy of the mitochondria. 50

treatment,5 we can conclude that TXA is a promising alternative to HQ.

After MAST peel, we did not find any significant change in microbiome, but a trend showing increase in species diversity and

AU T H O R C O N T R I B U T I O N S

decrease in P. acne (Figure 5D). However, it was a small proof-­of-­Conceptualization, Vinay Bhardwaj and Junhong Mao; Data curation,

concept study (N = 9), and no conlcusion can be drawn on the disup-Vinay Bhardwaj, Marc Zachary Handler, Chloe Azadegan and Pritam

tive potential of the MAST technology on microbiome. Considering

Panda; Investigation, Vinay Bhardwaj and Marc Zachary Handler;chemical peel is a popular intervention for management of acne,

Methodology, Vinay Bhardwaj, Chloe Azadegan, Marc Zacharymelasma, etc. our findings set foundation for further research in

Handler, Hans Georg Breunig; Software, Vinay Bhardwaj and Isabellpeels and post-­peel regimens to intervene microbiome in patients

Wenskus; Supervision, Vinay Bhardwaj, Junhong Mao and Isabelwith acne (P.acne/C.acne phylotypes), atopic dermatitis (S. aureus),

Diaz; Writing—original draft, Vinay Bhardwaj; Writing—Editing, Marc

melasma (Actinomyces, Collinsella) etc.34,51–53
Although we were able to record real-­time the blood flow in thecapillaries (Video SV, supplementary information), detailed anal-

Zachary Handler, Karsten Koenig, Isabel Diaz, Writing—review, Allco-­authors. All authors have read and agreed to the published version of the manuscript.

ysis and models need to be developed to make any conclusion onchanges in blood volume and dynamics before and after peeling.

AC K N OW L E D G E M E N T S

It will be particularly important for melasma, acne, PIH and other

RTL genomics for 16S rRNA sequencing. TenBio, Ltd UK for genesensitive and inflammatory skin conditions like psoriasis, atopic

expression study using their TenSkin™ model. Jin Namkoong anddermatitis, Rosacea, etc., where redness and inflammation are sa-

Joanna Wu from Colgate-­Palmolive Company to provide consulta-

lient features. Using powerful features of ultrafast in situ VivaScopetion on designing gene expression study.

imaging (turnaround time <1 min) we can get rich anatomical information on skin health. Using this information, a trained dermatol-

F U N D I N G I N FO R M AT I O Nogist/professional can take pathology-­driven real-­time decisions toThis research was fully funded by Colgate-­Palmolive Company.

personalize treatment for better outcomes. For example, by using
VivaScope as a guide to understand melanin distribution in different

C O N FL I C T O F I N T E R E S T S TAT E M E N T

skin layers, dermatologists and surgeons can make a better choice

The authors declare no conflict of interest.

in selecting a peel (ingredients, depth, layers, application pressure,etc.) and/or their synergistic combination with laser, microneedling,

DATA AVA I L A B I L I T Y S TAT E M E N T

etc. to treat different types of HP, such as epidermal melasma versus

The data that support the findings of this study are available fromdermal melasma.

the corresponding author upon reasonable request.

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BHARDWAJ et al.

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BHARDWAJ et al.

I N S T I T U T I O N A L R E V I E W B OA R D S TAT E M E N T
The study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by the US Institutional Review
Board, Inc. in Miami, FL (U.S. IRB2022/CP01).
I N FO R M E D C O N S E N T S TAT E M E N T
Informed consent was obtained from all subjects involved in the
study before participating in any study-­related activities. Written informed consent was obtained from the patients to use their personal
information (including microbiome data and photos) for the manuscript publication.
ORCID
Chloe Azadegan

https://orcid.org/0000-0002-1266-1637

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S U P P O R T I N G I N FO R M AT I O N
Additional supporting information can be found online in the
Supporting Information section at the end of this article.
Video SI: Kojic acid binding to active (catalytic) site of humantyrosinase enzyme.
Video SII: Kojic acid binding to allosteric site of human tyrosinase
enzyme.
Video SIII: MD simulation showing binding of HQ to humantyrosinase enzyme is not stable.
Video SIV: MD simulation showing TXA binding to human tyrosinase
is stable.
Video SV: Real-­time video captured from VivaScope to visualize
blood flow in facial skin.
Appendix S1: Inclusion and exclusion criteria.
Appendix S2: Peeling procedure.

How to cite this article: Bhardwaj V, Handler MZ, Mao J, et al.
A novel professional-­use synergistic peel technology to reduce
visible hyperpigmentation on face: Clinical evidence andmechanistic understanding by computational biology andoptical biopsy. Exp Dermatol. 2024;33:e15069. doi:10.1111/
exd.15069

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