Usage of Synthetic Peptides in Cosmetics for Sensitive Skin

Abstract

Sensitive skin is characterized by symptoms of discomfort when exposed to environmental factors. Peptides are used in cosmetics for sensitive skin and stand out as active ingredients for their ability to interact with skin cells by multiple mechanisms, high potency at low dosage and the ability to penetrate the stratum corneum. This study aimed to analyze the composition of 88 facial cosmetics for sensitive skin from multinational brands regarding usage of peptides, reviewing their synthetic pathways and the scientific evidence that supports their efficacy. Peptides were found in 17% of the products analyzed, namely: acetyl dipeptide-1 cetyl ester, palmitoyl tripeptide-8, acetyl tetrapeptide-15, palmitoyl tripeptide-5, acetyl hexapeptide-49, palmitoyl tetrapeptide-7 and palmitoyl oligopeptide. Three out of seven peptides have a neurotransmitter-inhibiting mechanism of action, while another three are signal peptides. Only five peptides present evidence supporting their use in sensitive skin, with only one clinical study including volunteers having this condition. Noteworthy, the available data is mostly found in patents and supplier brochures, and not in randomized placebo-controlled studies. Peptides are useful active ingredients in cosmetics for sensitive skin. Knowing their efficacy and synthetic pathways provides meaningful insight for the development of new and more effective ingredients.

Full text

pharmaceuticals
Review
Usage of Synthetic Peptides in Cosmetics for Sensitive Skin
Diana I. S. P. Resende 1, 2, † , Marta Salvador Ferreira 3, 4, † , JoséManuel Sousa-Lobo 3,4 , Emília Sousa 1, 2, *
and Isabel Filipa Almeida 3, 4, *


Citation: Resende, D.I.S.P.; Ferreira,
M.S.; Sousa-Lobo, J.M.; Sousa, E.;
Almeida, I.F. Usage of Synthetic
Peptides in Cosmetics for Sensitive
Skin. Pharmaceuticals 2021,14, 702.
https://doi.org/10.3390/ph14080702
Academic Editors: Giovanni
N. Roviello and Rosanna Palumbo
Received: 18 June 2021
Accepted: 16 July 2021
Published: 21 July 2021
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Copyright: © 2021 by the authors.
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Attribution (CC BY) license (https://
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4.0/).
1CIIMAR–Centro Interdisciplinar de Investigação Marinha e Ambiental, Avenida General Norton de Matos,
S/N, 4450-208 Matosinhos, Portugal; dresende@ff.up.pt
2Laboratory of Organic and Pharmaceutical Chemistry, Department of Chemical Sciences,
Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal
3Associate Laboratory i4HB-Institute for Health and Bioeconomy, Faculty of Pharmacy, University of Porto,
4050-313 Porto, Portugal; msbferreira@ff.up.pt (M.S.F.); slobo@ff.up.pt (J.M.S.-L.)
4UCIBIO–Applied Molecular Biosciences Unit, MedTech, Laboratory of Pharmaceutical Technology,
Department of Drug Sciences, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal
*Correspondence: esousa@ff.up.pt (E.S.); ifalmeida@ff.up.pt (I.F.A.); Tel.: +351-220-428-621 (I.F.A.)
† These authors equally contributed to this work.
Abstract:
Sensitive skin is characterized by symptoms of discomfort when exposed to environmental
factors. Peptides are used in cosmetics for sensitive skin and stand out as active ingredients for
their ability to interact with skin cells by multiple mechanisms, high potency at low dosage and the
ability to penetrate the stratum corneum. This study aimed to analyze the composition of 88 facial
cosmetics for sensitive skin from multinational brands regarding usage of peptides, reviewing their
synthetic pathways and the scientific evidence that supports their efficacy. Peptides were found in
17% of the products analyzed, namely: acetyl dipeptide-1 cetyl ester, palmitoyl tripeptide-8, acetyl
tetrapeptide-15, palmitoyl tripeptide-5, acetyl hexapeptide-49, palmitoyl tetrapeptide-7 and palmitoyl
oligopeptide. Three out of seven peptides have a neurotransmitter-inhibiting mechanism of action,
while another three are signal peptides. Only five peptides present evidence supporting their use in
sensitive skin, with only one clinical study including volunteers having this condition. Noteworthy,
the available data is mostly found in patents and supplier brochures, and not in randomized placebo-
controlled studies. Peptides are useful active ingredients in cosmetics for sensitive skin. Knowing
their efficacy and synthetic pathways provides meaningful insight for the development of new and
more effective ingredients.
Keywords: peptides; cosmetics; sensitive skin; chemical synthesis
1. Introduction
Sensitive skin is a condition characterized by the occurrence of symptoms such as
tightness, stinging, burning or pruritus, which are triggered by stimuli that do not normally
produce unpleasant sensations, such as cold, heat, sun, pollution, cosmetics or moisture [
1
].
The skin may also present erythema, dryness and desquamation, but these signs are typ-
ically absent [
2
]. Sensitive skin is thought to affect 71% of the general adult population,
and the epidemiological studies have also shown that the symptoms are more frequent
on the face [
3
,
4
]. The causes for this condition are unknown, but genetics, poor mental
health, and microbiome imbalance have been proposed as contributing factors [
5
–
7
]. The
pathophysiological mechanisms involved in sensitive skin remain unknown, but three
hypotheses have been pointed in scientific literature: increased stratum corneum perme-
ability, an exacerbated immune response and a hyperactivity from the somatosensory
and vascular systems [
8
]. While the two former hypotheses have been questioned and
remain poorly understood, there is growing evidence linking sensitive skin to abnormal
responses from the somatosensory system. The lower sensitivity threshold in individuals
with sensitive skin may be due to a dysfunction in the communication with central nervous
Pharmaceuticals 2021,14, 702. https://doi.org/10.3390/ph14080702 https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021,14, 702 2 of 22
system, leading to pain sensations and neurosensory defects, namely the hyperactivation
of endothelin receptors and transient receptor potential channels (TRP), which are present
in cutaneous nerve fibers such as unmyelinated C fibers and keratinocytes [
4
,
9
]. The
activation of cutaneous nerve fibers by physical and chemical stimuli, such as heat, low pH
solutions, or known irritants such as capsaicin, results in the release of neuropeptides, such
as substance P or calcitonin gene related peptide (CGRP), which activate keratinocytes,
mast cells, and antigen-presenting cells and T cells nearby, causing a burning pain sen-
sation [
10
]. A lower density of unmyelinated C-fibers was detected in individuals with
sensitive skin, which may be due to degeneration following the contact with the environ-
mental factors, which are thought to be responsible for the occurrence of skin sensitivity.
Paradoxically, the lower density of unmyelinated C-fibers may generate hyperreactivity
of the existing ones [
11
]. On the other hand, the inflammatory responses associated to
itching sensations are initiated by the activation of transient receptor potential vanilloid
type 1 (TRPV1), which is stimulated by heat, capsaicin, and cations, therefore promoting
the release of IL-23 by dendritic cells [
10
]. Individuals with sensitive skin are thought to
present an overexpression TRPV1, thus increasing neuronal excitability [
12
,
13
]. Overall,
these mechanisms may be exacerbated by an impairment in the skin barrier, which fails to
protect nerve endings adequately [10].
The synthesis of glutathione in the 1930
0
s and the isolation of oxytocin in the 1950
0
s pro-
moted an increase in the research on peptide synthesis, isolation, as well as their chemical,
biochemical, and biological characterization [
14
,
15
]. After the surge of conformational/top-
ographic-biological activity relationships, which allowed to determine the affinity and
specificity for target receptors, peptide leads emerged, offering several advantages over
small molecules (increased specificity) and antibodies (small size) [
15
,
16
]. Peptide ligands
may act as agonists or antagonists at cell receptors and acceptors modulating cell function
and animal behavior. This area encompasses approximately 50% of current drugs, and
it is likely to keep evolving in the future. In the cosmetic industry, peptides have been
used since the late 1980s, with growing notoriety during the first decade of the XXI cen-
tury
[17–19]
. Peptides used in cosmetic products present a molecular weight lower than
500 Da and hydrophilic properties, thus achieving a moderate penetration through the stra-
tum corneum [
20
]. Focusing on this challenge, chemical modifications such as esterification
with alkyl chains, are usually required. Peptide leads typically derived from three sources:
isolated from nature (also known as bioactive peptides); from chemical libraries, or by
genetic/recombinant libraries [
16
]. According to Gorouhi and Maibach, peptides used in
cosmetics may be classified as enzyme inhibitory, carrier, neurotransmitter-inhibitory, and
signal peptides [
17
]. Neurotransmitter-inhibitory peptides are able to mimic amino acid
sequences involved in neuron excitability, thus modulating the nervous response, while
signal peptides stimulate cells’ activity and growth [21]. Accordingly, these peptides may
be useful for modulating the neurogenic symptoms associated with sensitive skin, as well
as the synthesis of pro-inflammatory cytokines.
We have previously characterized the trends in the use of peptides in anti-aging
cosmetics [
22
]. As the usage of these ingredients in the sensitive skin care segment remains
unknown, the present study aims to fill this gap.
2. Materials and Methods
2.1. Data Collection
The composition of a pool of skin care facial cosmetic products from multinational
manufacturers, marketed in Portuguese parapharmacies and pharmacies was collected
in 2019, in order to access the most used active ingredients for sensitive skin. Skin care
products were included in the study if they exhibited in the label one of the following
expressions: “sensitive skin” OR “reactive skin” OR “intolerant skin”. All the information
available in the product’s label was collected, along with the information available on the
manufacturers’ websites.

Pharmaceuticals 2021,14, 702 3 of 22
2.2. Data Analysis
The products ingredient lists were analyzed by visual inspection in order to find
peptides, and they were listed according to the International Nomenclature of Cosmetic
Ingredients (INCI). Data were analyzed with respect to the following parameters:
2.2.1. Peptides Usage Frequency
The relative amount of cosmetic products for sensitive skin containing peptides were
evaluated and expressed in percentage.
2.2.2. Top Peptides for Sensitive Skin
The peptides were identified from INCI lists and ranked in descending order of
occurrence to disclose the top.
2.2.3. Scientific Evidence Supporting the Efficacy in Sensitive Skin Care
The efficacy data of each peptide were searched on the on-line databases PubMed,
Scopus, Cochrane, KOSMET, and SciFinder. Due to the lack of studies regarding the
applicability of active ingredients in cosmetics for sensitive skin, a broader search was
performed, using the keywords (“INCI name” OR “synonyms”, when applicable).
3. Results and Discussion
Following these criteria, 88 skin care facial products were selected from 19 multina-
tional brands. Fifteen cosmetic products contained one or more peptides in their com-
position, making up about 17% of products analyzed. Noteworthy, only two products
contained more than one peptide in their composition.
3.1. Top Ingredients for Sensitive Skin
The peptides were identified (Figure 1) and ranked in descending order according to
their relative usage (Table 1).
Table 1.
Peptides found at INCI lists of cosmetic products for sensitive skin and their relative
usage (%).
INCI Classification Relative Usage (%)
Acetyl Dipeptide-1 Cetyl Ester Neurotransmitter-inhibiting 5.7
Palmitoyl Tripeptide-8 Neurotransmitter-inhibiting 4.5
Acetyl Tetrapeptide-15 Neurotransmitter-inhibiting 2.3
Palmitoyl Tripeptide-5 Signal 2.3
Acetyl Hexapeptide-49 Unknown 1.1
Palmitoyl Tetrapeptide-7 Signal 1.1
Palmitoyl Oligopeptide Signal 1.1
Overall, acetyl dipeptide-1 cetyl ester was the most used ingredient in cosmetic
products for sensitive skin, being present in more than 5% of all products. Palmitoyl
tripeptide-8 achieved the second place, followed by acetyl tetrapeptide-15 and palmitoyl
tripeptide-5. Acetyl hexapeptide-49, palmitoyl tetrapeptide-7, and palmitoyl oligopeptide
were only found in the composition of one cosmetic product.

Pharmaceuticals 2021,14, 702 4 of 22
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 4 of 22
N
H
OH
NN
H
O
O
NH2
O
NH
NNH
NH2
HN
Palmitoyl tripeptide-8
N
H
OH
NN
H
O
O
OH
O
NH2
Palmitoyl oligopeptide
HN
N
Acetyl dipeptide-1 cetyl ester
O
ON
H
O
NH
O
OH
H
N
H2N
NH
H
NN
H
NH2
O
O
O
NO
H
N
O
HO
Acetyl tetrapeptide-15
N
H
OH
NN
H
O
O
OH
O
NH2
Palmitoyl tripeptide-5
NH2
H
NN
H
NH2
Palmitoyl tetrapeptide-7
O
O
O
O N
N
H
O
OOH
H
N
H2N
HN
Palmitoyl tripeptide-1
O
N
HO
H
N
O
N
HO
H
N
O
N
ONH
O
OH
(CH2)14
H3C
(CH2)14
H3C
Palmitoyl hexapeptide-12
(CH2)14
H3C
(CH2)14
H3C
(CH2)14
H3C
OR
(CH2)15
H3C
(retired INCI)
Figure 1. Structures of peptides found at INCI lists from cosmetic products for sensitive skin.
Figure 1. Structures of peptides found at INCI lists from cosmetic products for sensitive skin.

Pharmaceuticals 2021,14, 702 5 of 22
3.2. Scientific Evidence Supporting the Efficacy in Sensitive Skin Care
The search results are summarized below (Figure 2):
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 5 of 22
3.2. Scientific Evidence Supporting the Efficacy in Sensitive Skin Care
The search results are summarized below (Figure 2):
Figure 2. Flow chart of selected articles according to four different parts of the search process: iden-
tification, screening, eligibility, and inclusion.
3.2.1. Acetyl Dipeptide-1 Cetyl Ester
Acetyl dipeptide-1 cetyl ester is the INCI name for the peptide N-acetyl-L-tyrosyl-L-
arginine hexadecyl ester (Figure 1). This compound was based on the bioactive dipeptide
Tyr-Arg, for its alleviating and decontracting properties of muscle fibers, and is obtained
by chemical synthesis, through initial esterification of L-arginine×HCl (1) with palmitol (2)
[23] to give hexadecyl ether of L-arginine (3) (Scheme 1). Activation of N-acetyl-L-tyrosine
(4) with N-hydroxysuccinimide (NHS) and coupling with L-arginine (3) allows to obtain
the acetyl dipeptide-1 cetyl ester [23].
Scheme 1. Synthesis of acetyl dipeptide-1 cetyl ester. PTSA: p-toluenesulfonic acid; TEA: triethyla-
mine; NHS: N-hydroxysuccinimide; THF: tetrahydrofuran; DCC: N,N’-dicyclohexylcarbodiimide;
rt: room temperature; h: hours. Adapted from [23].
Figure 2.
Flow chart of selected articles according to four different parts of the search process:
identification, screening, eligibility, and inclusion.
3.2.1. Acetyl Dipeptide-1 Cetyl Ester
Acetyl dipeptide-1 cetyl ester is the INCI name for the peptide N-acetyl-L-tyrosyl-L-
arginine hexadecyl ester (Figure 1). This compound was based on the bioactive dipeptide
Tyr-Arg, for its alleviating and decontracting properties of muscle fibers, and is obtained
by chemical synthesis, through initial esterification of L-arginine
×
HCl (
1
) with palmitol
(
2
) [
23
] to give hexadecyl ether of L-arginine (
3
) (Scheme 1). Activation of N-acetyl-L-
tyrosine (
4
) with N-hydroxysuccinimide (NHS) and coupling with L-arginine (
3
) allows to
obtain the acetyl dipeptide-1 cetyl ester [23].
Acetyl dipeptide-1 cetyl ester promotes the pro-opiomelanocortin (POMC) gene ex-
pression. POMC incurs post-translational processing and originates the biologically active
peptides melanocyte-stimulating hormones (MSHs) and adrenocorticotropin (ACTH), in-
volved in melanin synthesis, as well as
β
-endorphin, which contains met-enkephalin’s pep-
tide sequence, providing an opiate activity [
24
,
25
].
α
-MSH is able to bind to melanocytes
melanocortin receptors, thus inducing melanin synthesis, but it also intervenes in the
reduction of the inflammatory response by modulating the nuclear factor
κ
-
β
activity (NF-
κβ
) [
26
]. Noteworthy, there are
α
-MSH peptide fragments which do not elicit significant
melanogenic activity, such those that are used in palmitoyl tripeptide-8. Furthermore the
opioid
β
-endorphinreduces CGRP release. CGRP is able to activate TRPV1 in multiple
cells thus initiating an inflammatory response [
27
]. Consequently, acetyl dipeptide-1 cetyl
ester reduces the stinging sensation and inflammation resulting from the skin exposure to
heat, contact with specific substances, such as capsaicin, and mechanical stress.

Pharmaceuticals 2021,14, 702 6 of 22
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 5 of 22
3.2. Scientific Evidence Supporting the Efficacy in Sensitive Skin Care
The search results are summarized below (Figure 2):
Figure 2. Flow chart of selected articles according to four different parts of the search process: iden-
tification, screening, eligibility, and inclusion.
3.2.1. Acetyl Dipeptide-1 Cetyl Ester
Acetyl dipeptide-1 cetyl ester is the INCI name for the peptide N-acetyl-L-tyrosyl-L-
arginine hexadecyl ester (Figure 1). This compound was based on the bioactive dipeptide
Tyr-Arg, for its alleviating and decontracting properties of muscle fibers, and is obtained
by chemical synthesis, through initial esterification of L-arginine×HCl (1) with palmitol (2)
[23] to give hexadecyl ether of L-arginine (3) (Scheme 1). Activation of N-acetyl-L-tyrosine
(4) with N-hydroxysuccinimide (NHS) and coupling with L-arginine (3) allows to obtain
the acetyl dipeptide-1 cetyl ester [23].
Scheme 1. Synthesis of acetyl dipeptide-1 cetyl ester. PTSA: p-toluenesulfonic acid; TEA: triethyla-
mine; NHS: N-hydroxysuccinimide; THF: tetrahydrofuran; DCC: N,N’-dicyclohexylcarbodiimide;
rt: room temperature; h: hours. Adapted from [23].
Scheme 1.
Synthesis of acetyl dipeptide-1 cetyl ester. PTSA: p-toluenesulfonic acid; TEA: triethy-
lamine; NHS: N-hydroxysuccinimide; THF: tetrahydrofuran; DCC: N,N’-dicyclohexylcarbodiimide;
rt: room temperature; h: hours. Adapted from [23].
Furthermore, Khmaladze et al. demonstrated that acetyl dipeptide-1 cetyl ester
significantly upregulates the expression of Aquaporin 3 (AQP3), Filaggrin (FLG), caspase
14, and keratin 10 genes, thus contributing to the improvement of the epidermal barrier [
28
].
Another study showed that acetyl dipeptide-1 cetyl ester is able to significantly reduce
PGE
2
secretion and decrease NF
κ
B signaling
in vitro
[
29
]. PGE
2
has been proposed to be
associated with neurogenic inflammation in sensitive skin [2].
The ingredient supplier reports a reduced ability to perceive heating sensations [
30
].
The efficacy of a cream containing 3% acetyl dipeptide-1 cetyl ester was assessed regarding
the interference in the ability of 21 volunteers to discriminate between four distinct levels of
heat: warm, hot, very hot, and painful. The heat perception was reduced very significantly
for temperatures which provide hot, very hot, and painful sensations. Additionally, the
efficacy of the same cream for reducing the unpleasant sensations provoked by sandpaper
aggression on one hand, using the other hand as control, was evaluated in a double-blind
study including 18 volunteers. The subliminal response to discomfort was measured by a
lie detector. The hand in which the cream was applied revealed greater comfort after the
sandpaper aggression. Due to the above-mentioned effects, the supplier concludes acetyl
dipeptide-1 cetyl ester is expected to reduce some of the unpleasant sensations of sensitive
skin associated with hyperactivity of the somatosensory system.
Schoelermann et al. compared the ability of a cosmetic product containing acetyl
dipeptide-1 cetyl ester with another product with 4-t-butylcyclohexanol for inhibiting
capsaicin-induced stinging in a clinical study including 31 volunteers with sensitive to very
sensitive skin. Volunteers’ self-perception stinging/burning sensations and photographs
of signs of skin inflammation were used for performing the evaluations. The authors
concluded that the product containing 4-t-butylcyclohexanol presented a greater efficacy
by significantly reducing neuronal activation, compared to the one with acetyl dipeptide-1
cetyl ester, which had no significant effect [
31
]. However, this study alone does not allow to
conclude that 4-t-butylcyclohexanol is more efficacious than acetyl dipeptide-1 cetyl ester
due to differences in the cosmetic bases containing each active ingredient, which could also
interfere with study results.
Moreover, there are several manufacturers who invested in the registration of patents
of cosmetic products for sensitive skin that include acetyl dipeptide-1 cetyl ester, demon-
strating that researchers and cosmetic manufacturers recognize the value and usefulness of
these ingredients for future applications [32–36].
3.2.2. Palmitoyl Tripeptide-8
Palmitoyl tripeptide-8 is a synthetic peptide ester based on a
α
-melanocyte stimulating
hormone (
α
-MSH), originating from POMC, and it is composed by the sequence N-(1-

Pharmaceuticals 2021,14, 702 7 of 22
oxohexadecyl)-L-histidyl-D-phenylalanyl-L-argininamide (Figure 1). This peptide can
be obtained via a solid-phase peptide synthesis using the fluorenylmethyloxycarbonyl
(Fmoc) strategy on an ACT496S2 automated synthesizer with PS-Rink amide (RAM) resin
(
Scheme 2)
[
37
]. The deprotection and coupling steps are carried out until the desired
sequences are synthesized. Final side-chain deprotection and cleavage from the resin
with a cleavage cocktail (trifluoroacetic acid/water/triisopropylsilane), affords palmitoyl
tripeptide-8 [37].
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 7 of 22
(Scheme 2) [37]. The deprotection and coupling steps are carried out until the desired se-
quences are synthesized. Final side-chain deprotection and cleavage from the resin with
a cleavage cocktail (trifluoroacetic acid/water/triisopropylsilane), affords palmitoyl
tripeptide-8 [37].
Scheme 2. Solid-phase synthesis of palmitoyl tripeptide-8. HBTU: N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-
yl)uronium hexafluorophosphate; DMF: N,N-dimethylformamide; NMP: pyrrolidone-2-methyl-N; TIS: triiso-
propylsilane; TFA: trifluoroacetic acid. Adapted from [37].
The ingredient supplier performed several efficacy tests. In in vitro models, palmitoyl
tripeptide-8 showed the ability to significantly inhibit IL-8 production up to 32% in UVB-
irradiated keratinocytes, which was comparable to α-MSH, and also in IL-1 stimulated
fibroblasts, reaching 64% inhibition which is greater that the achieved by α-MSH [38]. In
skin explants exposed to substance P, palmitoyl tripeptide-8 significantly reduced the
number of dilated capillaries and the size of dilated vessels up to 30% and 51%, respec-
tively. Edema was also reduced by 60% due to palmitoyl tripeptide-8 [26]. The supplier
also performed two clinical studies. In one study, eight individuals with no reported skin
conditions applied both the control and the test formula containing palmitoyl tripeptide-
8, three times a day, on separate areas of the volar side of the forearm, for eight days. After
this period, single patches containing 250 µL of an aqueous 0.5% sodium dodecyl sulfate
(SDS) solution were applied for 24 h, following their removal and a 24 h resting period.
Then, the test areas were photographed using a video microscope and their temperature
was measured using Thermovision, a temperature measurement device through infrared
camera. The supplier reported that photographs demonstrated a redness reduction when
palmitoyl tripeptide-8 was applied to the skin, although no quantitative measure was pre-
sented. In areas where palmitoyl tripeptide-8 was applied, a significant reduction in the
skin temperature after an increase caused by SDS was found. Control results were not
statistically significantly different. In another study, which included 13 individuals with
no reported skin condition, the same patches containing 250 µL 0.5% SDS solution were
applied for 24 h on separate areas of the volar side of the forearm. After this period, the
patches were removed and both the test and control formulas were applied three times
daily for two days. Again, the test areas were photographed and their temperature was
measured using the same equipment. Skin redness decreased in the areas, which the test
formula was applied, with no quantitative measurement, and skin temperature increase
induced by SDS presented an average 78% reduction, with statistical significance, con-
trary to control results. Together, these results indicate that palmitoyl tripeptide-8 is able
to prevent and soothe an irritative response [26].
There is only one study in the scientific literature, which addresses in vivo the effi-
cacy of a formulation containing palmitoyl tripeptide-8 for the treatment of persistent red-
ness in patients with rosacea who had been successfully treated with topical or oral ther-
apy [39]. Twenty-five patients (23 women and 2 men) were asked to continue using their
prior medication, while applying a lotion containing caffeine, zinc gluconate, bisabolol,
Eperua falcata bark extract, and palmitoyl tripeptide-8 for 8 weeks. Clinical and patients’
assessments for efficacy and tolerability were performed at weeks 4 and 8 using Visia CR
device photographs. The evaluation of the product’s efficacy showed a statistically signif-
icant improvement in redness, flushing, skin tone, and overall rosacea severity. Skin
Scheme 2.
Solid-phase synthesis of palmitoyl tripeptide-8. HBTU: N,N,N
0
,N
0
-tetramethyl-O-(1H-benzotriazol-1-yl)uronium
hexafluorophosphate; DMF: N,N-dimethylformamide; NMP: N-methyl-2-pyrrolidone; TIS: triisopropylsilane; TFA: trifluo-
roacetic acid. Adapted from [37].
The ingredient supplier performed several efficacy tests. In
in vitro
models, palmitoyl
tripeptide-8 showed the ability to significantly inhibit IL-8 production up to 32% in UVB-
irradiated keratinocytes, which was comparable to
α
-MSH, and also in IL-1 stimulated
fibroblasts, reaching 64% inhibition which is greater that the achieved by
α
-MSH [
38
].
In skin explants exposed to substance P, palmitoyl tripeptide-8 significantly reduced the
number of dilated capillaries and the size of dilated vessels up to 30% and 51%, respectively.
Edema was also reduced by 60% due to palmitoyl tripeptide-8 [
26
]. The supplier also
performed two clinical studies. In one study, eight individuals with no reported skin
conditions applied both the control and the test formula containing palmitoyl tripeptide-8,
three times a day, on separate areas of the volar side of the forearm, for eight days. After
this period, single patches containing 250
µ
L of an aqueous 0.5% sodium dodecyl sulfate
(SDS) solution were applied for 24 h, following their removal and a 24 h resting period.
Then, the test areas were photographed using a video microscope and their temperature
was measured using Thermovision, a temperature measurement device through infrared
camera. The supplier reported that photographs demonstrated a redness reduction when
palmitoyl tripeptide-8 was applied to the skin, although no quantitative measure was
presented. In areas where palmitoyl tripeptide-8 was applied, a significant reduction in
the skin temperature after an increase caused by SDS was found. Control results were not
statistically significantly different. In another study, which included 13 individuals with
no reported skin condition, the same patches containing 250
µ
L 0.5% SDS solution were
applied for 24 h on separate areas of the volar side of the forearm. After this period, the
patches were removed and both the test and control formulas were applied three times
daily for two days. Again, the test areas were photographed and their temperature was
measured using the same equipment. Skin redness decreased in the areas, which the test
formula was applied, with no quantitative measurement, and skin temperature increase
induced by SDS presented an average 78% reduction, with statistical significance, contrary
to control results. Together, these results indicate that palmitoyl tripeptide-8 is able to
prevent and soothe an irritative response [26].
There is only one study in the scientific literature, which addresses
in vivo
the efficacy
of a formulation containing palmitoyl tripeptide-8 for the treatment of persistent redness
in patients with rosacea who had been successfully treated with topical or oral therapy [
39
].
Twenty-five patients (23 women and 2 men) were asked to continue using their prior
medication, while applying a lotion containing caffeine, zinc gluconate, bisabolol, Eperua
falcata bark extract, and palmitoyl tripeptide-8 for 8 weeks. Clinical and patients’ assess-

Pharmaceuticals 2021,14, 702 8 of 22
ments for efficacy and tolerability were performed at weeks 4 and 8 using Visia CR device
photographs. The evaluation of the product’s efficacy showed a statistically significant
improvement in redness, flushing, skin tone, and overall rosacea severity. Skin radiance,
texture, and overall appearance also improved. Regarding patient’s tolerance, there was a
significant improvement in skin erythema, dryness, edema, and stinging. This finding may
be particularly relevant for patients with sensitive skin, who also present this symptom.
However, the presence of other active ingredients in the product composition and the lack
of control do not allow to draw conclusions regarding palmitoyl tripeptide-8
0
s efficacy. In
addition to the cosmetic products found in our investigation, here are also several patented
cosmetic formulations for sensitive skin containing palmitoyl tripeptide-8 [40–43].
3.2.3. Acetyl Tetrapeptide-15
Deriving from endomorphin-2 (Tyr-Pro-Phe-Phe-NH
2
), a human
µ
-opioid agonist
with selective anti-nociceptive effect, acetyl tetrapeptide-15 is a synthetic peptide consti-
tuted by the sequence N-acetyl-L-tyrosyl-L-prolyl-L-phenylalanyl-L-phenylalaninamide
(Figure 1) [
44
,
45
]. Although this peptide is widely used in skincare formulations for sensi-
tive skin, its synthesis is not fully described. However, the synthesis of a novel biologically
active compound, the conjugate of jasmonic acid and of acetyl tetrapeptide-15, discloses
that the synthesis of this tetrapeptide proceeds via a solid-phase method using AM RAM
resin and the Fmoc/But procedure (Scheme 3) [
46
]. After initial treatment of the resin,
the synthesis of the tetrapeptide proceeds with the addition of the resin to a mixture of
hydroxybenzotriazole (HOBt) and Fmoc-L-Phe-OH (
9
). The reaction proceeds with anchor-
ing of the Fmoc-L-Phe-OH (
9
) to the Rink amide resin (RAM) followed by protection of
unreacted hydroxyl groups of the resin by capping, and deprotection of the Fmoc group.
Further addition of protected amino acids Fmoc-L-Phe-OH (
9
), Fmoc-L-Pro-OH (
10
), Fmoc-
L-Tyr(tBu)-OH (
11
) to the obtained amide, capping, N-Fmoc deprotection, and cleavage
from the resin allows to obtain acetyl tetrapeptide-15 [46].
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 8 of 22
radiance, texture, and overall appearance also improved. Regarding patient’s tolerance,
there was a significant improvement in skin erythema, dryness, edema, and stinging. This
finding may be particularly relevant for patients with sensitive skin, who also present this
symptom. However, the presence of other active ingredients in the product composition
and the lack of control do not allow to draw conclusions regarding palmitoyl tripeptide-
8′s efficacy. In addition to the cosmetic products found in our investigation, here are also
several patented cosmetic formulations for sensitive skin containing palmitoyl tripeptide-
8 [40–43].
3.2.3. Acetyl Tetrapeptide-15
Deriving from endomorphin-2 (Tyr-Pro-Phe-Phe-NH2), a human µ-opioid agonist
with selective anti-nociceptive effect, acetyl tetrapeptide-15 is a synthetic peptide consti-
tuted by the sequence N-acetyl-L-tyrosyl-L-prolyl-L-phenylalanyl-L-phenylalaninamide
(Figure 1) [44,45]. Although this peptide is widely used in skincare formulations for sen-
sitive skin, its synthesis is not fully described. However, the synthesis of a novel biologi-
cally active compound, the conjugate of jasmonic acid and of acetyl tetrapeptide-15, dis-
closes that the synthesis of this tetrapeptide proceeds via a solid-phase method using AM
RAM resin and the Fmoc/But procedure (Scheme 3) [46]. After initial treatment of the
resin, the synthesis of the tetrapeptide proceeds with the addition of the resin to a mixture
of hydroxybenzotriazole (HOBt) and Fmoc-L-Phe-OH (9). The reaction proceeds with an-
choring of the Fmoc-L-Phe-OH (9) to the Rink amide resin (RAM) followed by protection
of unreacted hydroxyl groups of the resin by capping, and deprotection of the Fmoc
group. Further addition of protected amino acids Fmoc-L-Phe-OH (9), Fmoc-L-Pro-OH
(10), Fmoc-L-Tyr(tBu)-OH (11) to the obtained amide, capping, N-Fmoc deprotection, and
cleavage from the resin allows to obtain acetyl tetrapeptide-15 [46].
Scheme 3. Solid-phase synthesis of acetyl tetrapeptide-15. HOBt: hydroxybenzotriazole; DIC: N,N′-
diisopropylcarbodiimide; DMAP: 4-(dimethylamino)pyridine; DIPEA: N,N-diisopropylethylamine.
Reproduced from reference with permission from the Centre National de la Recherche Scientifique
(CNRS) and the Royal Society of Chemistry [46].
Acetyl tetrapeptide-15 was developed with the aim to reduce skin hyperreactivity
producing inflammatory, chronic and neuropathic pain, by increasing the threshold of
neuronal excitability in µ-opioid receptor via an endorphin-like pathway [47,48]. The ef-
ficacy of this peptide was demonstrated both in vitro and in vivo by the supplier [44].
Firstly, acetyl tetrapeptide-15 was tested regarding its ability to modulate the release of
Scheme 3.
Solid-phase synthesis of acetyl tetrapeptide-15. HOBt: hydroxybenzotriazole; DIC: N,N
0
-
diisopropylcarbodiimide; DMAP: 4-(dimethylamino)pyridine; DIPEA: N,N-diisopropylethylamine.
Reproduced from reference with permission from the Centre National de la Recherche Scientifique
(CNRS) and the Royal Society of Chemistry [46].
Acetyl tetrapeptide-15 was developed with the aim to reduce skin hyperreactivity
producing inflammatory, chronic and neuropathic pain, by increasing the threshold of

Pharmaceuticals 2021,14, 702 9 of 22
neuronal excitability in
µ
-opioid receptor via an endorphin-like pathway [
47
,
48
]. The
efficacy of this peptide was demonstrated both
in vitro
and
in vivo
by the supplier [
44
].
Firstly, acetyl tetrapeptide-15 was tested regarding its ability to modulate the release of
CGRP. CGRP is released after the activation of TRVP1 by capsaicin, heat, or depolarizing
agents such cations [
45
]. The test was performed by incubating sensory neurons with
acetyl tetrapeptide-15 (0.0003% and 0.001%), capsazepine (10
µ
M, a TRPV1 antagonist), or
verapamil (100
µ
M, a calcium channel blocker) for 6h, which were then exposed to KCl
and capsaicin. Acetyl tetrapeptide-15 0.001% reduced CGRP release very significantly, both
when neurons were exposed to capsaicin and KCl, performing better than capsazepine
10
µ
M and similarly to verapamil 100
µ
M. The ability of acetyl tetrapeptide-15 to activate
µ
-opioid receptors from cultured sensory neurons in a capsaicin media was evaluated in
competition with naloxone, a receptor antagonist. Capsaicin binds to TRVP1 receptors,
thus eliciting a calcium influx through the cell membrane that produces CGRP, as well as
a nervous influx signaling pain and discomfort. The presence of acetyl tetrapeptide-15
significantly reduced the CGRP release by capsaicin-stimulated neurons, but this effect was
compromised in the presence of naloxone, reinforcing that acetyl tetrapeptide-15 binds to
µ
-opioid receptors. The activation from
µ
-opioid receptors inhibits the TRVP1 response by
reducing the phosphorylation of adenylate cyclase (ADC) to protein kinase A (PKA). Lastly,
a split-faced single-blind clinical study elucidated the ability of acetyl tetrapeptide-15 to
reduce skin sensitivity after the exposure to capsaicin in 20 individuals. The protocol
started with the application of increasing concentrations of a capsaicin solution in the
nasolabial folds, to determine the concentration, which induced discomfort. A vehicle
solution was applied to the other side of the face. Then, a 0.0015% solution with acetyl
tetrapeptide-15 was applied twice daily, for four days, and the application of increasing
capsaicin concentrations was repeated. Overall, there was a significantly increase in the
capsaicin threshold which provoked discomfort in volunteers.
There is a patent referring to the use of acetyl tetrapeptide-15 in a cosmetic product
for sensitive skin [
45
], but no studies were found in scientific literature for this compound.
3.2.4. Palmitoyl Tripeptide-5
Palmitoyl tripeptide-5 is a fragment of Thrombospondin I (TSP-1) presenting the se-
quence, N-(1-oxohexadecyl)-L-lysyl-L-valyl-L-lysine (Figure 1) [
21
,
49
]. Two different liquid-
phase methodologies were described for the synthesis of this tripeptide [
50
,
51
], designed
to surpass some of the disadvantages associated with the solid-phase synthetic method-
ologies (high costs and pollution to the environment) [
52
]. One methodology (
Scheme 4
)
involves a convergent synthesis with the initial formation of a N-carboxyanhydride
14
by reaction of L-valine (
12
) with phosgene (
13
) [
50
]. Boc-L-lysine (
15
) is then coupled to
the N-carboxyanhydride
14
, forming Boc-protected dipeptide
16
. In a convergent route,
N-acylated aminoacid
18
is prepared from Boc-L-lysine (
15
) and palmitoyl chloride (
17
).
EDC/NHS Activation of the carboxyl group of Pal-Lys(Boc)-OH (
18
) produces Pal-Lys(Boc)-
OSu (
19
). Coupling of intermediates
16
and
19
and further Boc deprotection furnishes
palmitoyl tripeptide-5 [50].
The other synthetic methodology reported for the preparation of palmitoyl tripeptide-
5 [
51
], although via a linear strategy (Scheme 5), is quite similar to the depicted in Scheme 4.
Initial formation of palmitoyl chloride (
17
) from palmitic acid
8
, followed by coupling with
benzyloxycarbonyl (Cbz)-L-lysine
21
forms Pal-Lys(Cbz)-OH (
22
) [
51
]. Further activation of
the carboxyl group of Pal-Lys(Cbz)-OH (
22
) with NHS (
23
) and coupling with L-valine (
12
),
followed by a second activation of the carboxyl group of
25
with NHS (
23
) and coupling
with Cbz-L-lysine (
27
) furnishes Pal-Lys (Cbz)-Val-Lys(Cbz)-OH (
28
). Final deprotection of
the Cbz groups allows to obtain palmitoyl tripeptide-5 [51].

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Scheme 4. Liquid-phase synthesis of palmitoyl tripeptide-5. EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
Adapted from [50].
The other synthetic methodology reported for the preparation of palmitoyl tripep-
tide-5 [51], although via a linear strategy (Scheme 5), is quite similar to the depicted in
Scheme 4. Initial formation of palmitoyl chloride (17) from palmitic acid 8, followed by
coupling with benzyloxycarbonyl (Cbz)-L-lysine 21 forms Pal-Lys(Cbz)-OH (22) [51]. Fur-
ther activation of the carboxyl group of Pal-Lys(Cbz)-OH (22) with NHS (23) and coupling
with L-valine (12), followed by a second activation of the carboxyl group of 25 with NHS
(23) and coupling with Cbz-L-lysine (27) furnishes Pal-Lys (Cbz)-Val-Lys(Cbz)-OH (28).
Final deprotection of the Cbz groups allows to obtain palmitoyl tripeptide-5 [51].
Scheme 4.
Liquid-phase synthesis of palmitoyl tripeptide-5. EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
Adapted from [50].
Palmitoyl tripeptide-5 was used in a cosmetic ingredient mix from a raw material
supplier, also containing spent grain wax and conjugated linoleic acid (CLA), for reduc-
ing skin redness in type I rosacea [
53
]. This ingredient mix is currently unavailable.
Palmitoyl tripeptide-5 is proposed to reduce metalloproteases (MMP’s) expression and
pro-inflammatory cytokine syntheses, causing vasodilation and capillary permeability [
54
].
However, neither efficacy studies for the use of palmitoyl tripeptide-5 alone or in this mix
in rosacea of sensitive skin are available. This peptide has also been used in patented
cosmetic formulations for sensitive skin [
55
–
57
]. Palmitoyl tripeptide-5 is also used in
anti-aging cosmetic products, due to its ability to reduce MMP’S and promote the synthesis
of type I and type II collagen from extracellular matrix, as well as for inhibiting melanin
production by reducing tyrosinase activity [51,58].

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Scheme 5. Liquid-phase synthesis of palmitoyl tripeptide-5. Adapted from [51].
Palmitoyl tripeptide-5 was used in a cosmetic ingredient mix from a raw material
supplier, also containing spent grain wax and conjugated linoleic acid (CLA), for reducing
skin redness in type I rosacea [53]. This ingredient mix is currently unavailable. Palmitoyl
tripeptide-5 is proposed to reduce metalloproteases (MMP’s) expression and pro-inflam-
matory cytokine syntheses, causing vasodilation and capillary permeability [54]. How-
ever, neither efficacy studies for the use of palmitoyl tripeptide-5 alone or in this mix in
rosacea of sensitive skin are available. This peptide has also been used in patented cos-
metic formulations for sensitive skin [55–57]. Palmitoyl tripeptide-5 is also used in anti-
aging cosmetic products, due to its ability to reduce MMP’S and promote the synthesis of
type I and type II collagen from extracellular matrix, as well as for inhibiting melanin
production by reducing tyrosinase activity [51,58].
3.2.5. Acetyl Hexapeptide-49
Although acetyl hexapeptide-49 is widely used in the cosmetic industry, neither the
structure nor the synthesis was reported to date in the literature.
Scheme 5. Liquid-phase synthesis of palmitoyl tripeptide-5. Adapted from [51].
3.2.5. Acetyl Hexapeptide-49
Although acetyl hexapeptide-49 is widely used in the cosmetic industry, neither the
structure nor the synthesis was reported to date in the literature.
This compound aims to regulate proteinase activated receptor 2 (PAR-2) from mast
cells by trypsin-like serine proteases, thus reducing the inflammatory response which
leads to IL-6 and IL-8 production, as well as TRVP-1 activation and subsequent CGRP
release [
59
,
60
]. The supplier presents three studies for elucidating acetyl hexapeptide-
49 efficacy. Primary human epidermal keratinocytes were incubated with vehicle or
increasing concentrations of an acetyl hexapeptide-49 solution, and then exposed to 50
µ
M
PAR-2 agonist. Cytokine production was determined by an ELISA test. At 0.5 mg/mL
acetyl hexapeptide-49, there was a 69.6% and 71.5% decrease in IL-6 and IL-8 production,
respectively. Moreover, cicatrization and barrier function recovery assays were performed
using the same
in vitro
model. In this regard, keratinocytes treated with acetyl hexapeptide-
49 solution were subject to an injury (cell-free area) induced by scraping a monolayer with
a pipette tip, and then a cell proliferation assay was performed through the enzymatic

Pharmaceuticals 2021,14, 702 12 of 22
conversion of the non-fluorescent calcein. The barrier function was recovered in both essays
(concentrations are not disclosed). Another study in a reconstructed epidermis model was
performed for evaluating the ability of a 4% acetyl hexapeptide-49 solution for reducing
response to cosmetic allergens. The skin model was exposed both to hexyl cinnamal and
farnesol, allergens, for 24 h, and the IL-8 expression was determined by an ELISA test. The
4% acetyl hexapeptide-49 allowed to reduce IL-8 expression in 58.2% comparing to positive
control. Noteworthy, the skin model used in this study is not revealed, which would be
important to evaluate its susceptibility to these allergens. Additionally, a clinical study was
performed using 25 volunteers (24 to 67 years) who were selected based on their lactic acid
stinging susceptibility. At the beginning of the study, volunteers applied a 10% lactic acid
solution on the nasolabial fold, followed by a cream containing 2% acetyl hexapeptide-49.
The soothing effect was evaluated after one hour, and volunteers reported an improvement
in the stinging sensation. Then, the cream was applied twice a day for 7 days, and
once again, the stinging sensation was assessed. After this period, a 32% reduction in
volunteers experiencing stinging was found. Lastly, the supplier reported another clinical
study including 20 volunteers (18 to 55 years) who applied a cream containing 2% acetyl
hexapeptide-49 on the left leg, and a vehicle formulation on right leg twice a day for four
weeks. Skin moisturization was evaluated by corneometry, and a clinical assessment of skin
dryness, scaling, smoothness, softness, and suppleness was performed by a dermatologist.
After four weeks, the supplier reported a significant increase in skin hydration comparing
to vehicle, and the skin appeared less dry and scaly, smoother, softer, and more supple.
Two patents for cosmetics with acetyl hexapeptide-49 have also been found [60,61].
3.2.6. Palmitoyl Tetrapeptide-7
Palmitoyl tetrapeptide-7 is a fragment of immunoglobulin G presenting the sequence
N-(1-oxohexadecyl)glycyl-L-glutaminyl-L-prolyl-L-arginine (Figure 1) [
62
]. Two different
solid-phase methodologies were described for the synthesis of this tetrapeptide [
63
,
64
]. In
the first methodology (Scheme 6), a preloaded H-Arg(Boc)-HMPB-ChemMatrix resin (
29
)
(functionalized support acylated with Riniker’s super-acid-sensitive (4-hydroxymethyl-
3-methoxyphenoxy)butanoic acid handle) is used and the first amino acid is attached by
coupling with 2,7-disulfo-9-fluorenylmethoxycarbonyl (Smoc)-proline sodium salt (
30
).
After resin wash and Smoc deprotection, coupling of the next amino acid is performed
(Smoc-glutamine (
31
), and a solution of Smoc-glycine (
32
)), which was used without
side-chain protecting group) until the desired peptide is completed. Oxyma [ethyl 2-cyano-
2-(hydroxyimino)acetate] is an additive in the coupling medium safer than benzotriazole-
based additives such as HOBt. Palmitoylation and cleavage of the peptide from the resin
followed by precipitation and lyophilization gives the desired palmitoyl tetrapeptide-7 [
63
].
The second methodology involves the presence of a soluble fragment to improve the
water solubility of the palmitoyl tetrapeptide-7, so that it is easier to purify [
64
]. Hence,
five hydrophilic lysines are continuously coupled on the amino resin, then a connecting
arm of p-hydroxybenzoic acid is introduced, and finally the remaining amino acids are
coupled according to the peptide sequence of the palmitoyl tetrapeptide-7 (Pal-Gly-Gln-
Pro-Arg-OH) [
64
]. HOBt/DIC methodology is adopted as a coupling approach when
other amino acid residues (except the first lysine) are coupled [
64
]. Removal of the Fmoc
protecting groups, resin cleavage, and further purification gives the refined peptide which
is hydrolyzed to the target peptide palmitoyl tetrapeptide-7 [64].

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Scheme 6. Solid-phase synthesis of palmitoyl tetrapeptide-7. Adapted from [63].
The second methodology involves the presence of a soluble fragment to improve the
water solubility of the palmitoyl tetrapeptide-7, so that it is easier to purify [64]. Hence,
five hydrophilic lysines are continuously coupled on the amino resin, then a connecting
arm of p-hydroxybenzoic acid is introduced, and finally the remaining amino acids are
coupled according to the peptide sequence of the palmitoyl tetrapeptide-7 (Pal-Gly-Gln-
Pro-Arg-OH) [64]. HOBt/DIC methodology is adopted as a coupling approach when other
amino acid residues (except the first lysine) are coupled [64]. Removal of the Fmoc pro-
tecting groups, resin cleavage, and further purification gives the refined peptide which is
hydrolyzed to the target peptide palmitoyl tetrapeptide-7 [64].
Palmitoyl tetrapeptide-7 decreases IL-6 secretion, reduces inflammation after UVB
exposure and stimulates laminin IV and V as well as collagen VII production [62]. In this
regard, palmitoyl tetrapeptide-7 has been used in anti-aging cosmetics [65,66]. Although
this mechanism of action is promising in the regulation of skin inflammation, namely for
wound healing, no studies were found revealing palmitoyl tetrapeptide-7′s efficacy in this
regard, nor for controlling the symptoms of sensitive skin [67]. Two patents however de-
scribe its use in cosmetic products for sensitive skin [68,69].
3.2.7. Palmitoyl Oligopeptide
The name “palmitoyl oligopeptide” was “removed” in 2013, since was used to des-
ignate two distinct molecules from the time of its development in 1994. The two com-
pounds were renamed as palmitoyl tripeptide-1 (Pal-GHK) and palmitoyl hexapeptide-
12 (Pal-KTTKS) in order to clarify the composition of cosmetic products [70].
Palmitoyl tripeptide-1 is a collagen fragment presenting the sequence N-(1-oxohexa-
decyl)glycyl-L-histidyl-L-lysine (Figure 1) [71]. To date, three different methodologies
were reported for the synthesis of this tripeptide [63,72,73]. The first methodology
(Scheme 7) [73] consists in an initial EDC-mediated coupling of H-Lys(Z)-OBzl×HCl (33)
and Boc-His trityl(Trt)-OH (34) followed by removal of the trityl and Boc protecting
groups affords H-His-Lys (Z)-OBzl (36). Coupling of this dipeptide 36 with Pal-Gly-ONb
(39), previously synthesized from Pal-Gly-OSu (37) gives Pal-Gly-His-Lys (Z)-OBzl (40)
which, after removal of Cbz and Bzl protecting groups affords palmitoyl tripeptide-1 [73].
Scheme 6. Solid-phase synthesis of palmitoyl tetrapeptide-7. Adapted from [63].
Palmitoyl tetrapeptide-7 decreases IL-6 secretion, reduces inflammation after UVB
exposure and stimulates laminin IV and V as well as collagen VII production [
62
]. In this
regard, palmitoyl tetrapeptide-7 has been used in anti-aging cosmetics [
65
,
66
]. Although
this mechanism of action is promising in the regulation of skin inflammation, namely for
wound healing, no studies were found revealing palmitoyl tetrapeptide-7
0
s efficacy in
this regard, nor for controlling the symptoms of sensitive skin [
67
]. Two patents however
describe its use in cosmetic products for sensitive skin [68,69].
3.2.7. Palmitoyl Oligopeptide
The name “palmitoyl oligopeptide” was “removed” in 2013, since was used to desig-
nate two distinct molecules from the time of its development in 1994. The two compounds
were renamed as palmitoyl tripeptide-1 (Pal-GHK) and palmitoyl hexapeptide-12 (Pal-
KTTKS) in order to clarify the composition of cosmetic products [70].
Palmitoyl tripeptide-1 is a collagen fragment presenting the sequence N-(1-oxohexade-
cyl)glycyl-L-histidyl-L-lysine (Figure 1) [
71
]. To date, three different methodologies were re-
ported for the synthesis of this tripeptide [
63
,
72
,
73
]. The first methodology (
Scheme 7
) [
73
]
consists in an initial EDC-mediated coupling of H-Lys(Z)-OBzl
×
HCl (
33
) and Boc-His
trityl(Trt)-OH (
34
) followed by removal of the trityl and Boc protecting groups affords
H-His-Lys (Z)-OBzl (
36
). Coupling of this dipeptide
36
with Pal-Gly-ONb (
39
), previ-
ously synthesized from Pal-Gly-OSu (
37
) gives Pal-Gly-His-Lys (Z)-OBzl (
40
) which, after
removal of Cbz and Bzl protecting groups affords palmitoyl tripeptide-1 [73].
Another reported methodology for the synthesis of this tripeptide is performed by us-
ing the same methodology as used for the synthesis of palmitoyl tetrapeptide-7 (
Scheme 6
).
A preloaded H-Lys(Boc)-HMPB-ChemMatrix resin to which were coupled Smoc-L-Hys and
Smoc-Gly is used in a solid-phase approach. Deprotection of the Smoc protecting group,
palmitoylation, cleavage of the peptide from the solid support, and further lyophilization
affords palmitoyl tripeptide-1 [63].
The third methodology is initiated through the protection of the carboxylic moiety of
Boc-Lys-(Z)-OH (
41
) to obtain Boc-Lys(Z)-OBzl (
43
) (Scheme 8) [
72
]. Boc removal in acidic
conditions, followed by coupling with Boc-His-OH (
44
), under usual coupling conditions
(HOBt, NMM, DIPEA) gives Boc-His-Lys(Z)-OBzl (
45
) which, after deprotection/coupling
with Boc-Gly-OH (
47
) and deprotection/palmitoylation with palmitic acid (
8
), and final
deprotection of the carboxylic moiety with Pd/C furnishes the desired palmitoyl tripeptide-1.

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Scheme 7. Liquid-phase synthesis of palmitoyl tripeptide-1. NHS: N-Hydroxysuccinimide. Adapted from [73].
Another reported methodology for the synthesis of this tripeptide is performed by
using the same methodology as used for the synthesis of palmitoyl tetrapeptide-7 (Scheme
6). A preloaded H-Lys(Boc)-HMPB-ChemMatrix resin to which were coupled Smoc-L-
Hys and Smoc-Gly is used in a solid-phase approach. Deprotection of the Smoc protecting
group, palmitoylation, cleavage of the peptide from the solid support, and further lyoph-
ilization affords palmitoyl tripeptide-1 [63].
The third methodology is initiated through the protection of the carboxylic moiety of
Boc-Lys-(Z)-OH (41) to obtain Boc-Lys(Z)-OBzl (43) (Scheme 8) [72]. Boc removal in acidic
conditions, followed by coupling with Boc-His-OH (44), under usual coupling conditions
(HOBt, NMM, DIPEA) gives Boc-His-Lys(Z)-OBzl (45) which, after deprotection/coupling
with Boc-Gly-OH (47) and deprotection/palmitoylation with palmitic acid (8), and final
deprotection of the carboxylic moiety with Pd/C furnishes the desired palmitoyl tripep-
tide-1.
Scheme 7. Liquid-phase synthesis of palmitoyl tripeptide-1. NHS: N-Hydroxysuccinimide. Adapted from [73].
Palmitoyl hexapeptide-12 is an elastin fragment presenting the sequence N-(1-oxohex-
adecyl)-L-valyl-glycyl-L-valyl-L-alanyl-L-prolyl-glycine (Figure 1). The methodology for
the preparation of this peptide is similar to the previously reported for other liquid-
phase peptide syntheses, consisting in a series of deprotection/coupling reactions starting
with the initial coupling of Boc-L-proline (
50
) and benzyl glycinate (
51
), followed by the
coupling of Boc-L-alanine (
53
), Boc-L-valine (
54
), Boc-glycine (
55
), and Boc-L-valine (
54
)
(Scheme 9) [74].
Although both peptides have been used in anti-aging cosmetics [
22
], neither studies
or patents revealing the use of palmitoyl oligopeptide, palmitoyl tripeptide-1, or palmitoyl
hexapeptide-12 for reducing sensitive skin symptoms were found.

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Scheme 8. Liquid-phase synthesis of palmitoyl tripeptide-1. HOBt: hydroxybenzotriazole; DIPEA: N,N-diisopropylethyl-
amine; N-methylmorpholine; DIC: N,N′-diisopropylcarbodiimide; EDC: 1-ethyl-3-carbodiimide hydrochloride. Adapted
from [72].
Palmitoyl hexapeptide-12 is an elastin fragment presenting the sequence N-(1-
oxohexadecyl)-L-valyl-glycyl-L-valyl-L-alanyl-L-prolyl-glycine (Figure 1). The methodol-
ogy for the preparation of this peptide is similar to the previously reported for other liq-
uid-phase peptide syntheses, consisting in a series of deprotection/coupling reactions
starting with the initial coupling of Boc-L-proline (50) and benzyl glycinate (51), followed
by the coupling of Boc-L-alanine (53), Boc-L-valine (54), Boc-glycine (55), and Boc-L-valine
(54) (Scheme 9) [74].
Scheme 8.
Liquid-phase synthesis of palmitoyl tripeptide-1. HOBt: hydroxybenzotriazole; DIPEA: N,N-diisopropylethylamine;
N-methylmorpholine; DIC: N,N0-diisopropylcarbodiimide; EDC: 1-ethyl-3-carbodiimide hydrochloride. Adapted from [72].

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Scheme 9. Synthesis of palmitoyl hexapeptide-12. Adapted from [74].
Although both peptides have been used in anti-aging cosmetics [22], neither studies
or patents revealing the use of palmitoyl oligopeptide, palmitoyl tripeptide-1, or pal-
mitoyl hexapeptide-12 for reducing sensitive skin symptoms were found.
3.2.8. Highlights in the Usage of Synthetic Peptides in Cosmetics for Sensitive Skin
Peptides disclosed in the composition of the facial cosmetics for sensitive skin inves-
tigated are all from synthetic origin. In contrast to natural peptides that have variable di-
mensions, reaching high molecular weights, and may contain allergenic moieties and their
extraction can be costly [22], the synthesis of simpler peptides has the advantage of reach-
ing both the pharmacophoric portion and improved bioavailability. Most peptides found
in cosmetic products for sensitive skin are based on the pharmacologically active portions
of endogenous molecules, whose low molecular weight and subsequent hydrophobi-
zation provide a better penetration through the stratum corneum. Noteworthy, acetyl di-
peptide-1 cetyl ester, palmitoyl tripeptide-8, and acetyl tetrapeptide-15 are neurotransmit-
ter-inhibiting peptides acting as agonists from cutaneous opioid system, such as µ recep-
tor, which interacts with TRVP1 receptors through intracellular signaling. Therefore, these
peptides reduce the activation of cutaneous nerve fibers, especially through TRVP1, thus
preventing the release from CGRP. Conversely, acetyl hexapeptide-49 also reduces CGRP
release after TRVP1 activation as well as the pro-inflammatory cytokine production by a
signaling pathway involving proteinase activated receptor 2 (PAR-2). Interestingly, no
peptide acting directly on TRVP1 receptors has been found. There are several TRVP1 an-
tagonists reported in the scientific literature, but no peptides have been described, possi-
bly due to the specificity of the receptor’s binding site [75].
Concerning synthetic methodologies, these usually proceed via a linear approach,
although a few convergent approaches were also described. Additionally, the fine-tuning
of the chemistry associated with the synthetic methodology, ranging from standard cou-
pling procedures (EDC/NHS) to new and improved methodologies, such as greener meth-
ods (EDC/Oxyma) can also contribute for the marked increase of peptides in this industry.
The main bottleneck of these procedures is related to the activation of one of the carboxylic
groups before the occurrence of the coupling reaction. This activation step, along with the
next coupling reaction can lead to a potential loss of chiral integrity at the carboxyl residue
Scheme 9. Synthesis of palmitoyl hexapeptide-12. Adapted from [74].
3.2.8. Highlights in the Usage of Synthetic Peptides in Cosmetics for Sensitive Skin
Peptides disclosed in the composition of the facial cosmetics for sensitive skin inves-
tigated are all from synthetic origin. In contrast to natural peptides that have variable
dimensions, reaching high molecular weights, and may contain allergenic moieties and
their extraction can be costly [
22
], the synthesis of simpler peptides has the advantage
of reaching both the pharmacophoric portion and improved bioavailability. Most pep-
tides found in cosmetic products for sensitive skin are based on the pharmacologically
active portions of endogenous molecules, whose low molecular weight and subsequent
hydrophobization provide a better penetration through the stratum corneum. Notewor-
thy, acetyl dipeptide-1 cetyl ester, palmitoyl tripeptide-8, and acetyl tetrapeptide-15 are
neurotransmitter-inhibiting peptides acting as agonists from cutaneous opioid system, such
as
µ
receptor, which interacts with TRVP1 receptors through intracellular signaling. There-
fore, these peptides reduce the activation of cutaneous nerve fibers, especially through
TRVP1, thus preventing the release from CGRP. Conversely, acetyl hexapeptide-49 also
reduces CGRP release after TRVP1 activation as well as the pro-inflammatory cytokine
production by a signaling pathway involving proteinase activated receptor 2 (PAR-2).
Interestingly, no peptide acting directly on TRVP1 receptors has been found. There are
several TRVP1 antagonists reported in the scientific literature, but no peptides have been
described, possibly due to the specificity of the receptor’s binding site [75].
Concerning synthetic methodologies, these usually proceed via a linear approach,
although a few convergent approaches were also described. Additionally, the fine-tuning of
the chemistry associated with the synthetic methodology, ranging from standard coupling
procedures (EDC/NHS) to new and improved methodologies, such as greener methods
(EDC/Oxyma) can also contribute for the marked increase of peptides in this industry. The
main bottleneck of these procedures is related to the activation of one of the carboxylic
groups before the occurrence of the coupling reaction. This activation step, along with the
next coupling reaction can lead to a potential loss of chiral integrity at the carboxyl residue
undergoing activation. Although the above-described methodologies for the preparation
of these peptides are fully optimized, the development of new procedures might need
to take this challenge into account. New stand-alone coupling reagents, such as HOAt,
containing better leaving groups can be used to enhance coupling rates and reduce the

Pharmaceuticals 2021,14, 702 17 of 22
risk of racemization. Oxyma, a highly efficient leaving group, is safer and less hazardous
than HOAt. Oxyma exhibited the same efficiency as HOAt and greater performance than
HOBt. Of similar importance, is also the use of protecting groups, which can be used
to maximize the yield of the desired products, as well as to minimize undesirable side
reactions such as polymerization of the amino acids, usual in the synthesis of complex
peptide-based structures.
3.3. Applicability of the Described Synthetic Peptides in Pharmeceuticals
Synthetic peptides whose mechanism of action has been previously act indirectly on
TRPs. These receptors are associated with several skin diseases [
76
]. Pruritus, also known
as itch, can be idiopathic or secondary to different pathologies, such as atopic dermatitis,
psoriasis, urticaria, chronic renal failure, or liver diseases [
77
]. TRP channels, namely
transient receptor potential cation channel subfamily A member 1 (TRPA1) and TRPV1
have shown to be greatly involved in itch development both under both physiological
and pathological conditions. Rosacea is also aggravated by neuroinflammation, and the
overexpression of TRPV1, TRPV2, TRPV4, and TRPV4 receptors has been proved in distinct
subtypes from the disease [
78
]. Palmitoyl tripeptide-8, which is present in the formulation
of a cosmetic product with proven efficacy for the treatment of rosacea patients, may be
a prime candidate for the development of pharmaceuticals aimed at alleviating the signs
and symptoms of this condition [
39
]. Moreover, the overexpression of TRPV1, TRPV4,
and TRPV6 has been associated to nonmelanoma skin cancer, but their carcinogenic effect
remains unknown [76].
Therefore, the recognition of the effectiveness from these synthetic and the further
improvement of their molecular structures, may be useful for modulating TRP associated
pathways, providing alternative treatments and/or symptom management for multiple
diseases. These compounds are known to be safe for topical application, and their toxicity
has been assessed both by the manufacturer, through material safety data sheets, and
by the independent committee Cosmetic Ingredient Review supporting the Federal drug
Administration in the US [79].
4. Conclusions
This study characterizes the usage of peptides in cosmetic products for sensitive skin
for the first time. These ingredients were present in about 17% of the facial cosmetics for sen-
sitive skin analyzed in 2019. Seven distinct peptides were found, namely acetyl dipeptide-1
cetyl ester, palmitoyl tripeptide-8, acetyl tetrapeptide-15, palmitoyl tripeptide-5, and acetyl
hexapeptide-49, for which experimental data is reported to support use in cosmetics for
sensitive skin, along with palmitoyl terrapeptide-7 and palmitoyl oligopeptide (the old
name for the peptides palmitoyl tripeptide-1 and palmitoyl hexapeptide-12), whose ef-
ficacy is only documented for anti-aging cosmetics. Most of the available information
regarding these ingredients is not reported in peer-reviewed scientific journals, but rather
in patents and supplier brochures. Additionally, the small number of randomized clinical
studies, and especially the fact that only one study included volunteers with sensitive skin
(acetyl dipeptide-1 cetyl ester), hinders a robust evidence of the
in vivo
efficacy of these
peptides. More clinical studies with good methodological quality are needed to provide
sound evidence of the peptide’s efficacy in sensitive skin care.
From a chemical perspective, the increasingly use of these peptides as ingredients
in the cosmetic industry can be explained as a result of the development of solid-phase
syntheses and automated methodologies. Finally, the implementation of reverse-phase
high-performance liquid chromatography for peptide purification, in combination with
the previously mentioned factors, has allowed the production of complex peptides in
multi-kilogram amounts that was impossible to envisage only a few decades ago and that
contributed to a boost in the use of peptides as ingredients in cosmetic formulations.
Peptides are useful ingredients in cosmetics for sensitive skin that may also be relevant
for medical devices or medicines intended to treat or prevent the symptoms of diseases

Pharmaceuticals 2021,14, 702 18 of 22
in which neurogenic inflammation plays an important role, such as rosacea and atopic or
seborrheic dermatitis. Given the worldwide prevalence of sensitive skin and the growing
interest in peptides by the cosmetic industry, it is foreseeable that the market for these
products may increase in the coming years, fostering the design of new and more effective
compounds. In the future, it is possible to see a further exploration of signaling pathways
involving cutaneous opioid receptors, through the development of peptides acting up-
stream in this pathway, or as agonists of opioid receptors. Moreover, the development of
peptides acting directly on TRVP1 receptors, either extra or intracellularly, could provide
promising results.
Author Contributions:
Conceptualization: I.F.A. and E.S.; Data collection and analysis: M.S.F.;
Writing-Original draft preparation, and final manuscript: M.S.F. and D.I.S.P.R.; Supervision: J.M.S.-L.;
Writing-Reviewing and Editing: I.F.A. and E.S., Funding acquisition J.M.S.-L. and E.S. All authors
have read and agreed to the published version of the manuscript.
Funding:
This work was financially supported by the Applied Molecular Biosciences Unit-UCIBIO
which is financed by national funds from FCT/MCTES (UID/Multi/04378/2020), UIDB/04423/2020,
UIDP/04423/2020, and under the project PTDC/SAUPUB/28736/2017 (reference POCI-01-0145-
FEDER-028736), co-financed by COMPETE 2020, Portugal 2020 and the European Union through the
ERDF and by FCT through national funds, as well as CHIRALBIOACTIVE-PI-3RL-IINFACTS-2019.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing not applicable.
Acknowledgments:
This work received support and help from FCT regarding M.S.F. doctoral grant
(ref. SFRH/BD/144864/2019).
Conflicts of Interest:
The authors declare no conflict of interest the funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
Limitations:
This study was performed for the Portuguese cosmetic market, which is dominated
by multinational cosmetic brands. Therefore, this may result in discrepancies when comparing
with other markets. Many ingredients found in cosmetic products from the market lack scientific
literature regarding their efficacy. Therefore, some of the information used in this study was collected
in technical documents and patents from suppliers.
Abbreviations and Acronyms
α-MSH α-melanocyte stimulating hormone
ACTH adrenocorticotropin
ADC adenylate cyclase
AQP3 aquaporin 3
Boc tert-Butyloxycarbonyl
Bu Butyl
Bz benzoyl
Cbz benzyloxycarbonyl
CLA conjugated linoleic acid
DCC N,N’-dicyclohexylcarbodiimide
DIC N,N0-diisopropylcarbodiimide
DIPEA N,N-diisopropylethylamineDMAP
DMAP 4-(dimethylamino)pyridine
DMF N,N-dimethylformamide
EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
FLG filaggrin
Fmoc fluorenylmethyloxycarbonyl
h hours
HBTU N,N,N0,N0-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate

Pharmaceuticals 2021,14, 702 19 of 22
HOBt hydroxybenzotriazole
INCI international nomenclature of cosmetic ingredients
MMP’s metalloproteases
MSHs melanocyte-stimulating hormones
NF-κβ nuclear factor κ-β
NHS N-hydroxysuccinimide
NMP N-methyl-2-pyrrolidone
Pal Palmitic acid
PAR-2 proteinase activated receptor 2
PKA protein kinase A
POMC pro-opiomelanocortin
PTSA p-toluenesulfonic acid
RAM Rink amide
rt room temperature
SDS sodium dodecyl sulfate
Smoc 2,7-disulfo-9-fluorenylmethoxycarbonyl
Su succinimide
TEA triethylamine
TFA trifluoroacetic acid
THF tetrahydrofuran
TIS triisopropylsilane
Trt Trityl
TRPV Transient Receptor Potential Cation Channel Subfamily V
TPPA Transient Receptor Potential Cation Channel Subfamily A
TSP-1 thrombospondin I
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