ORIGINAL ARTICLE

Treatment with Synthetic Pseudoceramide

Improves Atopic Skin, Switching the Ceramide
Profile to a Healthy Skin Phenotype
Koichi Ishida1, Akihiko Takahashi2, Kotatsu Bito3, Zoe Draelos4 and Genji Imokawa5,6

Little is known about the pathophysiological linkages between altered ceramide profiles in the stratum cor-
neum (SC) of patients with atopic dermatitis and their impaired skin barrier and water-holding functions. We
studied those characteristics following topical treatment with a designed synthetic pseudoceramide (pCer) and
analyzed that pathophysiological linkage by microanalyzing ceramides using normal phase liquid
chromatographyeelectrospray ionization mass spectrometry. Four weeks of treatment with pCer significantly
reduced skin symptoms, accompanied by significant decreases in transepidermal water loss and increases in
water content. In the SC ceramide profiles, ceramides containing nonhydroxy fatty acids and 6-
hydroxysphingosines (Cer[NH]) and ceramides containing nonhydroxy fatty acids and phytosphingosines
(Cer[NP]) increased, whereas ceramides containing nonhydroxy fatty acids and sphingosines (Cer[NS]) and
ceramides containing a-hydroxy fatty acids and sphingosines (Cer[AS]) decreased, with larger alkyl chain
lengths in Cer[NS], distinctly representing a switch from an atopic dermatitis to a healthy skin phenotype. The
level of pCer that penetrated into the SC was significantly correlated with the SC water content but not with
transepidermal water loss. The level and the average carbon chain length of Cer[NS] were closely correlated
with the pCer level in the SC. These findings indicate that the penetrated pCer contributes to shift the ceramide
profile from an atopic dermatitis to a healthy skin phenotype. Taken together, the observed clinical efficacy of
treatment with pCer provides a deep insight into the pathogenesis of atopic dermatitis as a ceramide-deficient
disease.
Journal of Investigative Dermatology (2020) 140, 1762e1770; doi:10.1016/j.jid.2020.01.014

INTRODUCTION dry skin and functionally by cutaneous barrier disruption and

Atopic dermatitis (AD) is a recurrent dermatitis with a high
susceptibility to itching, irritants, and allergens even in
nonlesional skin, which is characterized clinically by severe
1
R&D Strategy, Kao Corporation, Tokyo, Japan; 2Skin Care Products
Research Laboratories, Kao Corporation, Kanagawa, Japan; 3Analytical
Science Laboratories, Kao Corporation, Tochigi, Japan; 4Dermatology
Consulting Services, High Point, North Carolina, USA; 5Center for
Bioscience Research & Education, Utsunomiya University, Tochigi, Japan;
and 6Research Institute for Biological Functions, Chubu University, Aichi,
Japan
Correspondence: Genji Imokawa, Center for Bioscience Research &
Education, Utsunomiya University, 350 Mine Utsunomiya, Tochigi, 321-
8505 Japan. E-mail: imokawag@dream.ocn.ne.jp
Abbreviations: AD, atopic dermatitis; Cer[ADS], ceramides containing
a-hydroxy fatty acids and dihydrosphingosines; Cer[AH], ceramides con-
taining a-hydroxy fatty acids and 6-hydroxysphingosines; Cer[AP], ceramides
containing a-hydroxy fatty acids and phytosphingosines; Cer[AS], ceramides
containing a-hydroxy fatty acids and sphingosines; Cer[EOH], ceramides
containing ester-linked fatty acids, o-hydroxy fatty acids, and
6-hydroxysphingosines; Cer[EOP], ceramides containing ester-linked fatty
acids, o-hydroxy fatty acids, and phytosphingosines; Cer[EOS], ceramides
containing ester-linked fatty acids, o-hydroxy fatty acids, and sphingosines;
Cer[NDS], ceramides containing nonhydroxy fatty acids and dihy-
drosphingosines; Cer[NH], ceramides containing nonhydroxy fatty acids and
6-hydroxysphingosines; Cer[NP], ceramides containing nonhydroxy fatty
acids and phytosphingosines; Cer[NS], ceramides containing nonhydroxy
fatty acids and sphingosines; pCer, pseudoceramide; SC, stratum corneum;
TEWL, transepidermal water loss
Received 20 September 2019; revised 13 January 2020; accepted 21 January
2020; accepted manuscript published online 1 February 2020; corrected
proof published online 4 March 2020
impaired water-holding function. Many studies have shown
that the barrier-disrupted dry skin of patients with AD is
mainly attributable to significantly decreased levels of
ceramides in the stratum corneum (SC) (Arikawa et al., 2002;
Choi and Maibach, 2005; Di Nardo et al., 1998; Imokawa
et al., 1991a; Ishibashi et al., 2003; Ishikawa et al., 2010;
Jungersted et al., 2010; Kim et al., 2017; Okamoto et al.,
2003; Sugiura et al., 2014). That mechanism is based on
evidence that ceramide can function as a water reservoir
(Imokawa and Hattori, 1985; Imokawa et al., 1991b) and also
as a permeability barrier because of the formation of multi-
layered lamellar structures with other lipids, such as choles-
terol ester and fatty acids, between the SC layers (Holleran
et al., 1994a, 1994b, 1993, 1991a, 1991b). Further, the
integrity of lipid lamellae in the SC of AD skin is distinctly
diminished as a result of alterations in the ceramide profile,
including the total ceramide level, its composite species, and
its alkyl chain properties (Ishikawa et al., 2010; Janssens
et al., 2011). Further, the disrupted barrier function and wa-
ter deficiency that occurs in both the nonlesional and lesional
skin of patients with AD (Matsuki et al., 2004a) and in
essential fatty acidedeficient mice or is elicited by surfactant
or solvent treatment of normal skin can be repaired by the
topical application of natural ceramides (Imokawa, 2004;
Imokawa et al., 1989c, 1986) or by synthetic pseudocer-
amides (pCer) (Funasaka et al., 2004; Hata et al., 2002b;
Imokawa, 2004, 2001, 1999, 1994; Matsuki et al., 2004a;
Mizutani et al., 2001; Nakamura et al., 2000; Takagi et al.,

1762 Journal of Investigative Dermatology (2020), Volume 140 ª 2020 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.

2005; Takashima et al., 2002; Umeda et al., 1997; Yamanaka
et al., 2001; ). Thus, pCer was designed and screened based
on its ability to form multilamellar structures in vitro and its
water-retaining abilities, as well as its in vivo water-holding
capacity and ameliorating effects on acetone-ether or
surfactant-induced roughened normal skin (Imokawa, 2001,
1999, 1994; Imokawa et al., 1989a, 1989b). The sum of
these findings indicated that decreased ceramide levels and
alterations of the ceramide profile in the nonlesional SC of
patients with AD are, at least in part, attributable to defects in
barrier and water-folding functions. Those conditions
frequently result in itching sensations because of the exces-
sive dry skin, and many irritating and allergenic substances
easily penetrate through the SC, resulting in eliciting in-
flammatory reactions. Such a high susceptibility to irritants
and allergens is also supported by our clinical study using
photoacoustic spectrometry (Hata et al., 2002a), in which the
in vivo penetration rates of lipophilic and hydrophilic
chemicals were significantly higher in the nonlesional skin of
patients with AD than in healthy controls and were increased
in proportion to the severity of AD.
Recently, microanalysis of ceramides by normal phase
liquid chromatographyeelectrospray ionization mass spec-
trometry demonstrated that in addition to the ceramide
deficiency, there are alterations in the balance of ceramide
classes and an increased level of short alkyl chain bearing
ceramides in the SC of patients with AD (Ishikawa et al.,
2010; Jungersted et al., 2010). Thus, compared with healthy
skin, AD skin exhibits lower levels of the classes of ceramides
containing nonhydroxy fatty acids and 6-
hydroxysphingosines (Cer[NH]) and ceramides containing
nonhydroxy fatty acids and phytosphingosines (Cer[NP]) and
higher levels of the classes of ceramides containing non-
hydroxy fatty acids and sphingosines (Cer[NS]) and ceram-
ides containing a-hydroxy fatty acids and sphingosines (Cer
[AS]).In contrast, ceramides with a shorter alkyl chain length
exist in the Cer[NS] class. On the basis of the influence of
sphingoid base composition on lamellar membrane archi-
tecture in the SC, Loiseau et al. (2018) hypothesized that
altered sphingoid base profiles contribute to the barrier ab-
normality in AD skin. Further, in pediatric patients with AD,
Shen et al. (2018) reported that two ceramide subclasses, Cer
[AS] and Cer[NS], were elevated with a close correlation with
the severity scoring of AD. However, it remains unclear as to
whether these ceramide alterations are specific for the atopic
diathesis, because these studies on AD were conducted in
comparison with healthy controls and did not include con-
trols for cutaneous inflammatory disorders such as contact
dermatitis or chronic eczema.
To provide deeper insights into the biological and physio-
logical mechanisms by which phenotypic changes in the
altered ceramide profile are associated with the impaired
barrier and water functions in the nonlesional SC of patients
with AD, it is important to determine what phenotypic
changes in the altered ceramide profile in the nonlesional SC
of patients with AD appear to be expressed and to identify
which changes are associated with the improvement of bar-
rier and water-holding functions when compensating
ceramides replenish the barrier and water functions
concomitant with the amelioration of atopic dry skin. This
K Ishida et al.
Role of Ceramide in Atopic Dermatitis
approach also seems to be key for preventing the refractory
nature of the dermatitis (Imokawa and Ishida, 2014) and,
further, to give a mechanistic insight into the pathogenesis of
AD.
The goal of this study was to characterize the pathophysi-
ological linkages between altered ceramide profiles in the SC
of patients with AD and their impaired skin barrier and water-
holding functions. We first determined the clinical and SC
functional efficacy following topical treatment with a
designed synthetic pCer and characterized that pathophysi-
ological linkage by microanalysis of ceramides using normal-
phase liquid chromatographyeelectrospray ionization mass
spectrometry. The results of this study indicated that topical
treatment with pCer significantly reduced skin symptoms,
accompanied by significant decreases in transepidermal
water loss (TEWL) and increases in water content. Those
changes were associated with a distinct switch of the cer-
amide profile from an AD to a healthy skin phenotype
without any increased level of endogenous natural ceram-
ides. Taken together, the observed clinical efficacy of treat-
ment with pCer provides a deep insight into the pathogenesis
of AD as a ceramide-deficient disease.
RESULTS
Clinical efficacy of the pCer test lotion
The mean clinical symptom severity scores through the 4
weeks of the study are shown in Figure 1. All clinical scores
tested, including erythema, scaling, lichenification, and
excoriation, were significantly improved across all time
points from 1 week through 4 weeks relative to the baseline
at the onset of the clinical study using the pCer lotion
(Figure 1a). Clinical evaluation of skin sensations revealed
that sensation factors, such as stinging, burning, and itchi-
ness, were significantly improved after 1 week of treatment
with the pCer lotion and continued to improve significantly
through the 4 weeks of treatment compared with the baseline
(0 week) (Figure 1b and Supplementary Figure S1).
Changes in the water content and the water evaporation
Water content in the SC of lesional and nonlesional AD skin
was measured by skin conductance and was markedly
increased 1 day after treatment with the pCer lotion and
remained at a significantly higher level during the 4 weeks of
treatment with the pCer lotion and the 7 days of the regres-
sion phase (Figure 2a). TEWL values decreased significantly
at week 2 and 4 during the 4 weeks of treatment with the
pCer lotion and remained at a lower level at day 3 in the
regression phase in the AD lesional skin (Figure 2b). This
indicates that the water evaporation (inside-out barrier) was
markedly improved by treatment with the pCer lotion in the
AD lesional skin.
Changes in the levels of endogenous ceramide and
penetrated pCer in the SC
Analysis of the ceramide content in the SC revealed that there
was no increase in endogenous ceramide levels in the SC
even after 4 weeks of treatment with the pCer lotion
(Figure 2c). Quantitative analysis of the applied pCer
revealed that the same average level of pCer as endogenous
ceramides accumulated in the SC at week 4 of treatment
(Figure 2c).
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 K Ishida et al.
Role of Ceramide in Atopic Dermatitis

a b

Severe (4) Erythema Severe (4) Stinging

Desquamation Burning
Lichenification Itching
Severity Score

Severity Score
Moderate (3) Moderate (3)
Excoriation

Mild (2) Mild (2)

**** ** ** **
Slight (1) **** ** Slight (1)
**** ** **
** ** **
** ** ** ** ****
None (0) None (0)
Baseline Week 1 Week 2 Week 4 Baseline Week 1 Week 2 Week 4

Figure 1. Improvement of clinical symptoms of patients with AD during 4 weeks of treatment with the pCer lotin. (a) Improvement of clinical skin
appearance. n ¼ 38, data represent mean  SD. (b) Improvement of skin sensations. n ¼ 38, data represent mean  SD. **P < 0.01 versus baseline by Wilcoxon
signed-rank test. AD, atopic dermatitis; pCer, pseudoceramide.

Changes in ceramide distributions before and after 4 weeks containing a-hydroxy fatty acids and dihydrosphingosines
of treatment with the pCer lotion (Cer[ADS]), ceramides containing a-hydroxy fatty acids and
The distribution profiles of ceramide species in the SC of 6-hydroxysphingosines (Cer[AH]), and ceramides contain-
nonlesional AD skin revealed that, whereas levels (by ng/mg ing a-hydroxy fatty acids and phytosphingosines (Cer[AP])
protein) (Figure 3) or class ratios (by weight %) significantly increased after 4 weeks of treatment, those of
(Supplementary Figure S2) of Cer[NH], Cer[NP], ceramides Cer[NS]; Cer[AS]; ceramides containing ester-linked fatty

a b
500 12
Lesion Lesion
10
400 Non-lesion Non-lesion
Conductance (µS)

TEWL (g/m2/hr)

8 ††
300 ** **
**
6
** * ††
** ††
200 †† *
** ** **
** 4

100
** †† ††
2

0 0
Baseline Day 1 Week 1 Week 2 Week 4 Day 3 Day 7 Baseline Week 1 Week 2 Week 4 Day 3 Day 7

Treatment Regression Treatment Regression

c
120
Levels of Total Ceramides

100
and pCer (ng/µg protein)

N. S.
80

60

40

20

0
Ceramides pCer Ceramides pCer
Baseline Week 4

Figure 2. Changes in skin conditioneassociated factors and endogenous ceramide levels of patients with AD during 4 weeks of treatment with the pCer
lotion. (a) Changes in water content (conductance) of the SC of lesional and nonlesional AD skin during 4 weeks of treatment with the pCer lotion and in the 7 days of
regression phase. n ¼ 38, data represent mean  SD. (b) Changes in the barrier function (TEWL) of the SC of lesional and nonlesional AD skin during 4 weeks of
treatment with the pCer lotion and in the 7 days of regression phase. n ¼ 38, data represents mean  SD. (c) Endogenous ceramide levels in the SC and pCer that
penetrated into the SC in the nonlesional AD skin on the volar forearm before and after 4 weeks of treatment with the pCer lotion. n ¼ 38, data represent individual
values and mean  SD. *P < 0.05 and **P < 0.01 versus baseline; †P < 0.05 and ††P < 0.01 versus week 4 by repeated measures ANOVA followed by Bonferroni’s post
hoc analysis. AD, atopic dermatitis; N.S., not significant by paired t-test; pCer, pseudoceramide; SC, stratum corneum; TEWL, transepidermal water loss.

1764 Journal of Investigative Dermatology (2020), Volume 140

 K Ishida et al.
Role of Ceramide in Atopic Dermatitis

Cer[NDS] Cer[NS] Cer[NH] Cer[NP] Cer[ADS] Cer[AS]

7.0 6.0 20.0 20.0 1.2 3.0
N. S. N. S. N. S. N. S. p<0.001 N. S.
6.0 5.0 1.0 2.5
15.0 15.0
Level of Ceramide

5.0
(ng/µg protein)

4.0 0.8 2.0

4.0
3.0 10.0 10.0 0.6 1.5
3.0
2.0 0.4 1.0
2.0
5.0 5.0
1.0 1.0 0.2 0.5

0.0 0.0 0.0 0.0 0.0 0.0

BL W4 BL W4 BL W4 BL W4 BL W4 BL W4

Cer[AH] Cer[AP] Cer[EOS] Cer[EOH] Cer[EOP] Total

12.0 20.0 5.0 4.0 1.2 80.0
p<0.01 p<0.05 p<0.001 N. S. p<0.001 N. S.
10.0 1.0
4.0
15.0 3.0 60.0
Level of Ceramide
(ng/µg protein)

8.0 0.8
3.0
6.0 10.0 2.0 0.6 40.0
2.0
4.0 0.4
5.0 1.0 20.0
2.0 1.0
0.2

0.0 0.0 0.0 0.0 0.0 0.0

BL W4 BL W4 BL W4 BL W4 BL W4 BL W4

Figure 3. Changes in the levels (ng/mg protein) of endogenous ceramide species in the SC of nonlesional AD skin on the forearm before and after the 4 weeks
of treatment with the pCer lotion. n ¼ 38, data represent individual values and mean  SD. P-values were analyzed by paired t-test. AD, atopic dermatitis;
Cer[ADS], ceramides containing a-hydroxy fatty acids and dihydrosphingosines; Cer[AH], ceramides containing a-hydroxy fatty acids and
6-hydroxysphingosines; Cer[AP], ceramides containing a-hydroxy fatty acids and phytosphingosines; Cer[AS], ceramides containing a-hydroxy fatty acids and
sphingosines; Cer[EOH], ceramides containing ester-linked fatty acids, o-hydroxy fatty acids, and 6-hydroxysphingosines; Cer[EOP], ceramides containing
ester-linked fatty acids, o-hydroxy fatty acids, and phytosphingosines; Cer[EOS], ceramides containing ester-linked fatty acids, o-hydroxy fatty acids, and
sphingosines; Cer[NDS], ceramides containing nonhydroxy fatty acids and dihydrosphingosines; Cer[NH], ceramides containing nonhydroxy fatty acids and
6-hydroxysphingosines; Cer[NP], ceramides containing nonhydroxy fatty acids and phytosphingosines; Cer[NS], ceramides containing nonhydroxy fatty acids
and sphingosines; N.S., not significant; SC, stratum corneum; pCer, pseudoceramide.

acids, o-hydroxy fatty acids, and sphingosines (Cer[EOS]);
ceramides containing ester-linked fatty acids, o-hydroxy
fatty acids, and 6-hydroxysphingsines (Cer[EOH]); and
ceramides containing ester-linked fatty acids, o-hydroxy
fatty acids, and phytosphingosines (Cer[EOP]) significantly
decreased, which indicates that the ceramide profile of AD
skin acquires a feature characteristic of healthy skin
(Ishikawa et al., 2010).
Changes in ceramide carbon numbers before and after 4
weeks of treatment with the pCer lotion
Comparisons of ceramide carbon numbers in each ceramide
species before and after 4 weeks of treatment with the pCer
lotion revealed that the average alkyl chain lengths of Cer
[NS] and Cer[ADS] significantly increased (Figure 4). In
contrast, the average alkyl chain lengths of Cer[NP], Cer[AS],
Cer[AH], Cer[AP], Cer[EOS], Cer[EOH], and Cer[EOP]
became significantly shorter, whereas those of ceramides
containing nonhydroxy fatty acids and dihydrosphingosines
(Cer[NDS]) and Cer[NH] remained unchanged. The distri-
bution of total carbon numbers of endogenous ceramides in
the SC of nonlesional AD skin before and after treatment with
the pCer lotion for 4 weeks demonstrated that there was a
distinct distribution change, with decreased levels of
endogenous ceramide species with shorter alkyl chain
lengths and increased levels of endogenous ceramide species
with longer alkyl chain lengths in the Cer[NS] and Cer[ADS]
classes, respectively (Supplementary Figure S3). In contrast,
there were no distinct changes in the distribution of total
carbon numbers of endogenous ceramide species with
shorter or longer alkyl chain lengths in the other ceramide
species (Cer[NDS], Cer[NH], Cer[NP], Cer[AS], Cer[AH], Cer
[AP], Cer[EOH], Cer[EOS], and Cer[EOP]). However, there
were increased or decreased levels of ceramide species
without distinct distribution changes of alkyl chain lengths in
the Cer[NH], Cer[NP], Cer[AH], and Cer[AP] classes and the
Cer[EOS], Cer[EOH] and Cer[EOP] classes, respectively.
Thus, a comparison between average alkyl chain length and
its distribution indicates that the increased average alkyl
chain lengths in Cer[NS] and Cer[ADS] after treatment with
the pCer lotion are mainly attributable to the decreased levels
of ceramide species with shorter alkyl chain lengths and the
increased levels of ceramide species with longer alkyl chain
lengths, respectively.
Relationships between levels of ceramide classes and
penetrated pCer and skin conditioneassociated factors
Although there were no significant correlations between all
endogenous ceramide species and skin conditioneassociated
values, barrier and water-holding functions, comparisons of
the penetrated and accumulated levels of pCer in the SC with
skin conditioneassociated factors revealed that there is a

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 K Ishida et al.
Role of Ceramide in Atopic Dermatitis

Cer[NDS] Cer[NS] Cer[NH] Cer[NP] Cer[ADS] Cer[AS]

47.0 47.5 45.5 47.0 45.0 44.0
N. S. p<0.001 N. S. p<0.05 p<0.001 p<0.01

47.0
Average Total Carbon
Number of Ceramide

46.5 43.0
45.0 46.0 44.0
46.5
46.0 42.0
46.0
44.5 45.0 43.0
45.5 41.0
45.5

45.0 45.0 44.0 44.0 42.0 40.0

BL W4 BL W4 BL W4 BL W4 BL W4 BL W4

Cer[AH] Cer[AP] Cer[EOS] Cer[EOH] Cer[EOP]

45.0 45.5 69.5 68.2 70.0
p<0.001 p<0.001 p<0.001 p<0.05 p<0.001
Average Total Carbon
Number of Ceramide

44.5 45.0 69.5

68.0

44.0 44.5 69.0 69.0

67.8
43.5 44.0 68.5

43.0 43.5 68.5 67.6 68.0

BL W4 BL W4 BL W4 BL W4 BL W4

Figure 4. Changes in the average of total carbon numbers of endogenous ceramide species in the SC of nonlesional AD skin on the forearm before and after
the 4 weeks of treatment with the pCer lotion. n ¼ 38, data represent individual values and mean  SD. P-values were analyzed by paired t-test. AD, atopic
dermatitis; Cer[ADS], ceramides containing a-hydroxy fatty acids and dihydrosphingosines; Cer[AH], ceramides containing a-hydroxy fatty acids and
6-hydroxysphingosines; Cer[AP], ceramides containing a-hydroxy fatty acids and phytosphingosines; Cer[AS], ceramides containing a-hydroxy fatty acids and
sphingosines; Cer[EOH], ceramides containing ester-linked fatty acids, o-hydroxy fatty acids, and 6-hydroxysphingosines; Cer[EOP], ceramides containing
ester-linked fatty acids, o-hydroxy fatty acids, and phytosphingosines; Cer[EOS], ceramides containing ester-linked fatty acids, o-hydroxy fatty acids, and
sphingosines; Cer[NDS], ceramides containing nonhydroxy fatty acids and dihydrosphingosines; Cer[NH], ceramides containing nonhydroxy fatty acids and
6-hydroxysphingosines; Cer[NP], ceramides containing nonhydroxy fatty acids and phytosphingosines; Cer[NS], ceramides containing nonhydroxy fatty acids
and sphingosines; N.S., not significant; SC, stratum corneum; pCer, pseudoceramide.

significant correlation (r ¼ 0.447, P ¼ 0.005) with the SC
water content but not with TEWL (Supplementary Table S1)
(Figure 5). That suggests that pCer that penetrated into the SC
contributes mainly to the increased water-holding function in
the SC.
Relationships between levels of pCer that penetrated into
the SC and endogenous ceramide profiles
We next determined the relationships between the levels of
pCer that penetrated into the SC and endogenous ceramide
profiles. Comparison of levels of pCer that penetrated into
the SC with endogenous ceramide species (expressed as
weight %) indicated significant correlations only between
the levels of Cer[NS] (r ¼ 0.325, P ¼ 0.047) and average
carbon chain of Cer[NS] (r ¼ 0.393, P ¼ 0.015) and the
penetrated levels of pCer (Supplementary Table S2)
(Figure 5).
DISCUSSION
In this clinical study, daily continued use of the pCer-
containing lotion on mild to severe AD skin improved
the clinical symptoms of both lesional and nonlesional
skin of patients with AD. These benefits increased pro-
gressively during the four weeks of treatment and
continued even through the regression phase (7 days).
Improvements of the skin symptoms were confirmed by the
investigator’s clinical scoring and by instrumental
measures for barrier (TEWL) and water-holding functions
(conductance). Significant benefits from the pCer lotion
were noted specifically in the clinical scoring of erythema.
Taken together, this study demonstrates the value of the
pCer lotion to reduce the clinical symptoms associated
with mild to severe AD as well as improving skin barrier
and water-holding functions.
To elucidate the cutaneous mechanisms involved in the
improvements of skin symptoms as well as the SC barrier
and water-holding functions by treatment with the pCer
lotion, we found that, whereas the decreased levels of
endogenous ceramides in the SC of treated nonlesional AD
skin remained unchanged after 4 weeks of treatment, the
ceramide profiles of endogenous ceramides, even though
present at still decreasing levels, were significantly altered
in terms of their species and carbon chain number. In
contrast, the pCer applied was additionally accumulated at
a similar average level to existing endogenous ceramides in
the treated nonlesional SC of AD skin. These findings sug-
gest the following possibilities as the biological mechanisms
involved in the improvement of skin symptoms and SC
functions:
(1) The changes in ceramide profiles of endogenous ceram-
ides in the SC of AD skin are in part responsible for the
improvements.

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 K Ishida et al.
Role of Ceramide in Atopic Dermatitis

a b
500 15.0
y = 0.006x + 4.997
400 12.5 r = -0.118, p = 0.480
Conductance (µS)

TEWL (g/m2/hr)
10.0
300
7.5
200
5.0
100 y = 1.187x + 239.4 2.5
r = 0.500, p = 0.001
0 0.0
0 20 40 60 80 100 120 0 25 50 75 100 125
pCer (ng/µg protein) pCer (ng/µg protein)

c d
5.0 47.5
y = 0.013x + 5.920 y = 0.004x + 46.116
r = -0.325, p = 0.047 r = 0.393, p = 0.015
Class Ratio of Cer[NS]

Average Total Carbon

4.0

Number of Cer[NS]
47.0
(weight %)

3.0
46.5
2.0

46.0
1.0

0.0 45.5
0 20 40 60 80 100 120 0 25 50 75 100 125
pCer (ng/µg protein) pCer (ng/µg protein)

Figure 5. Relationships between pCer level penetrated into the SC and skin conditioneassociated factors and endogenous Cer[NS] level after 4 weeks of
treatment with the pCer lotion. (a) pCer level versus skin conductance. n ¼ 38, data represent individual values and linear regression. Correlation coefficient
r ¼ 0.447 and P ¼ 0.005 were analyzed by Pearson’s correlation coefficient. (b) pCer level versus TEWL. n ¼ 38, data represent individual values and linear
regression. Correlation coefficient r ¼ 0.101 and P ¼ 0.545 were analyzed by Pearson’s correlation coefficient. (c) pCer level versus class ratio (weight %) of Cer
[NS]. n ¼ 38, data represent individual values and linear regression. Correlation coefficient r ¼ -0.325 and P ¼ 0.047 were analyzed by Pearson’s correlation
coefficient. (d) pCer level vs average of total carbon numbers of Cer[NS]. n ¼ 38, data represent individual values and linear regression. Correlation coefficient
r ¼ 0.393 and P ¼ 0.015 were analyzed by Pearson’s correlation coefficient. Cer[NS], ceramides containing nonhydroxy fatty acids and sphingosines; pCer,
pseudoceramide; SC, stratum corneum; TEWL, transepidermal water loss.

(2) The penetrated and accumulated pCer plays an essential
role in ameliorating the SC barrier and water-holding
functions, in turn leading to the improved skin symptoms.
Ceramides in human SC have been assigned to 12 distinct
groups based upon their fatty acid and sphingoid base
structures (Masukawa et al., 2008), as shown in
Supplementary Figure S4. In the skin of patients with AD,
Ishikawa et al. (2010) used normal-phase liquid
chromatographyeelectrospray ionization mass spectrometry
to demonstrate that the levels of endogenous ceramides are
significantly downregulated in lesional SC, which is consis-
tent with previous studies using other analytical methods
(Arikawa et al., 2002; Choi and Maibach, 2005; Di Nardo
et al., 1998; Imokawa et al., 1991a; Ishibashi et al., 2003;
Ishikawa et al., 2010; Jungersted et al., 2010; Kim et al.,
2017; Okamoto et al., 2003; Sugiura et al., 2014). In
contrast, whereas the levels of Cer[NH], Cer[NP], Cer[EOS],
Cer[EOH], and Cer[EOP] classes are significantly down-
regulated compared with healthy skin, Cer[NS] and Cer[AS]
classes occur at rather higher levels. Further, although Cer
[NS], Cer[NDS], Cer[NH], Cer[AS], and Cer[AH] with larger
alkyl chain lengths (>50 total carbons) occur at lower levels,
Cer[NS], Cer[NDS], and Cer[AS] with smaller alkyl chain
lengths (<40 total carbons) are expressed at higher levels
than in healthy skin. Janssens et al. (2012) also reported that
the relative ratio (but not quantitative levels) of ceramides
with smaller chain lengths is distinctly decreased in the SC of
AD skin, which is mainly associated with aberrant lipid or-
ganization and an impaired skin barrier function.
In this study, the improvement of skin symptoms and bar-
rier and water-holding functions by 4 weeks of treatment with
the pCer lotion was accompanied by distinct changes in
ceramide profiles, including carbon chains. Whereas the
levels of Cer[NH], Cer[NP], Cer[ADS], Cer[AH], and Cer[AP]
significantly increased in the SC of AD skin after 4 weeks of
treatment with the pCer lotion, levels of Cer[NS], Cer[AS],
Cer[EOS], Cer[EOH], and Cer[EOP] significantly decreased.
Although the average alkyl chain lengths of Cer[NS] and Cer
[ADS] significantly increased, the average alkyl chain lengths
of Cer[NP], Cer[AS], Cer[AH], and Cer[AP] tended to
decrease, and those of Cer[NDS] and Cer[NH] remained
unchanged. Therefore, based upon the report by Ishikawa
et al. (2010), it is likely that the increased levels in the Cer
[NH] and Cer[NP] classes as well as the increase of average
carbon number in Cer[NS] classes observed in this study
could be considered as a possible shift from an AD pheno-
type to a healthy phenotype of ceramide profiles, acquiring a

www.jidonline.org 1767
 K Ishida et al.
Role of Ceramide in Atopic Dermatitis
feature specific for healthy skin. In contrast, the levels of the
Cer[EOS], Cer[EOH], and Cer[EOP] classes, which were
significantly diminished in the lesional SC of AD skin
(Ishikawa et al., 2010) were not ameliorated by 4 weeks of
treatment with pCer lotion. The failure to increase the levels
of Cer[EOS], Cer[EOH], and Cer[EOP] classes, an acylcer-
amide type that plays a role in maintaining the permeability
barrier function (Imokawa et al., 1994; Matsuki et al., 2004b),
in the pCer-treated skin of patients with AD seems to be
attributable in part to the much lower impaired levels of
barrier function (TEWL) in the AD skin tested in this study
than in those in the study of Ishikawa et al. (2010). Thus, the
ameliorating effect of pCer on the water-holding function
(conductance) is much greater than on the barrier function
(TEWL), which seems to reflect the increased levels of the Cer
[NH] and Cer[NP] classes, which play a role in the water-
holding function in the pCer lotionetreated skin of patients
with AD (Matsuki et al., 2004b).
In contrast, it should be noted that because similar patterns
of ceramide profiles including carbon chain lengths were
observed in both the lesional skin of patients with AD and of
patients with psoriasis (Koyano et al., 2010), these alterations
in ceramide profiles seem to be not AD-specific or psoriasis-
specific but are inflammation-specific. Similar inflammation-
associated alterations in ceramide profiles were reported by
Kim et al. (2017), who demonstrated that the SC of nonle-
sional skin in allergic contact dermatitis exhibits abnormal-
ities in barrier function and ceramide profiles similar to those
in AD skin. Thus, it is plausible that the disrupted keratini-
zation process that results from cutaneous inflammation is
mainly responsible for the ceramide profile changes, thereby
exacerbating the pathological changes.
As for the association of structural ceramide profile
changes with distinct improvements of the SC barrier and
water-holding functions by treatment with the pCer lotion, in
this study, there was no significant correlation between any
ceramide class and skin conditioneassociated values and
barrier and water-holding functions. It is likely that the failure
of endogenous ceramide levels to increase by treatment with
the pCer lotion results in no substantial contribution to the
improvement of SC functions. In contrast, the relationship
between the penetrated levels of pCer and skin
conditioneassociated factors revealed that there is a signifi-
cant correlation of the penetrated pCer levels with the SC
water content but not with the barrier function (TEWL). Based
on the much lower impaired levels of barrier function (TEWL)
in the skin of patients with AD enrolled in this study
compared with those in the study by Ishikawa et al. (2010),
the stated findings strongly suggest that the penetrated and
accumulated pCer in the SC contributes mainly to the
increased water-holding function in the SC, resulting in the
amelioration of inflammatory conditions, which is associated
with the improvement of skin symptoms. The penetrated
levels of pCer are only significantly correlated with the
decreased levels of the Cer[NS] class as well as the increased
levels of average carbon chain lengths of the Cer[NS] class
among all Cer classes. Thus, the levels and the average car-
bon numbers of Cer[NS], which are characteristically
increased or shortened as a result of the increased levels of
shorter carbon numberebearing ceramides, respectively, in
1768 Journal of Investigative Dermatology (2020), Volume 140
the SC of AD skin (Ishikawa et al., 2010) tended to decrease
and become longer with the decreased levels of shorter car-
bon numberebearing ceramides, respectively, by treatment
with the pCer lotion in a proportional fashion with the
penetrated levels of the pCer. These results strongly suggest
that the penetration and accumulation of pCer distinctly
contributes to the preferable shift of the ceramide profile from
an AD phenotype to a healthy phenotype, accompanied by
the amelioration of itchy or dry skin mainly because of the
improvement of downregulated levels of water content in the
SC of AD skin.
In the mechanistic linkage between the altered ceramide
profiles, especially for the reduced levels of Cer[NS] with
long-chain fatty acids and causative inflammatory factors
such as cytokines, Tawada et al. (2014) speculated that a
potent proinflammatory T helper type 1etype cytokine, IFN-
g, whose mRNA and protein levels are distinctly upregulated
in lesional AD skin during the cutaneous inflammatory pro-
cess (Bieber, 2010; Grewe et al., 1998, 1994), is mainly
associated with the decreased levels of ceramides with long-
chain fatty acids resulting from the downregulated expression
levels of ELOVL1 (Ohno et al., 2010) and ceramide synthase
3 (Mizutani et al., 2008). In this study, because inflammatory
reactions were remarkably diminished by the topical treat-
ment with the pCer lotion, it is likely that the levels of IFN-g
may be downregulated in the pCer-treated AD skin, thus
supporting the possibility that treatment with pCer amelio-
rates the dermatitis by repairing the impaired barrier and
water-holding functions. That may result in the down-
regulation of IFN-g expression, leading to the recovery of
levels of Cer[NS] with long-chain fatty acids.
In conclusion, an intriguing point for the observed clinical
improvements by the pCer lotion is that these clinical effi-
cacies can be achieved without any recovery of the decreased
levels of endogenous ceramides but with compensated pCer
levels similar to existing endogenous ceramides, despite the
fact that the ceramide profiles shifted from an AD phenotype
to a healthy phenotype in the pCer lotionetreated SC of AD
skin. This suggests that ceramide levels in the SC are more
essential to maintaining the barrier and water-holding func-
tions than are the differential ceramide profiles. This hypoth-
esis is supported by the fact that altered ceramide profiles in
AD skin are not atopy-specific but are inflammation-specific
and can be impaired by upregulating the barrier and water-
holding functions. Thus, the observed clinical efficacy using
the pCer lotion provides a deep insight into the pathogenesis of
AD as a ceramide-deficient disease.
MATERIALS AND METHODS
Materials
The pCer test lotion was an oil-in-wateretype lotion that contains
3% of Cetyl-PG hydroxyethyl palmitamide as a pseudoceramide (see
Supplementary Materials for more information about the pCer and
lotion formula).
Study design overview
This study was conducted in an open-label, single-center method.
Subjects with mild to severe AD were enrolled in this 6-week study.
Each subject completed a washout of all topical medications and
lotions for 1 week, then applied the pCer test lotion (whose

formulation is shown in Supplementary Table S3) twice daily
(morning and evening) for 4 weeks. At the completion of the four
weeks of treatment, subjects discontinued the use of the pCer test
lotion for a 1-week regression period. During the study, subjects did
not use any medications on their skin and all skin care products
remained unchanged and no new products were added. All subjects
underwent skin assessments at the onset of the treatment phase
(baseline, i.e., week 0); 24 hours after the first application of the test
lotion; and after 1, 2, and 4 weeks of treatment. Investigator as-
sessments were performed at baseline and at the completion of 1, 2,
and 4 weeks of treatment. Assessments of TEWL were performed at
baseline and at weeks 1, 2, and 4. Corneometry was performed at
baseline, 24 hours, and weeks 1, 2 and 4. Tape-stripping for cer-
amide sampling was performed at baseline and at week 4. A subset
of 10 subjects participated in surface imaging at baseline, 24 hours,
week 2, and week 4, as well as a methyl nicotinate insult for
assessing barrier function at baseline and at week 4. All assessments
were made at least 12 hours following test sample application.
During the regression phase, subjects returned to the test facility for
evaluation at day 3 and day 7, and all subjects participated in TEWL
and corneometry assessments on both days. A subset of 10 subjects
also underwent surface imaging and barrier function testing at day 7.
Study population
This study enrolled 38 subjects. The demographics of the study group
were 36 females and 2 males, ranging from 29 to 71 years old, and
included 21 African Americans and 17 Caucasians. AD severity,
according to the diagnostic criteria of Hanifin and Rajka (1980),
were 14 mild, 15 moderate, and 9 severe.
Study approval
This test was registered under the number UMIN000037554. Ethics
approval was obtained on the basis of the study protocol (study
number: DCS-79-09) by the institutional review board of Concordia
Clinical Research (Cedar Knolls, NJ, USA, IRB number: 175V).
Written informed consent was obtained from each subject before
performing any study procedures.
Clinical assessment
A dermatologist (ZD) performed all clinical assessments. The eval-
uation parameters consisted of stinging, burning, itching (sensory
attributes assessed by querying the subject), erythema, desquama-
tion, lichenification and excoriation. The following rating scale was
used for each evaluation: 0 ¼ none, 1 ¼ minimal, 2 ¼ mild, 3 ¼
moderate, and 4 ¼ severe.
Skin conductance and TEWL measurements
Skin conductance and TEWL values were measured using a Der-
maLab Moisture Meter with a pin probe and a DermaLab TEWL
module, respectively. These measurements were taken at both
lesional and nonlesional skin sites according to the methods previ-
ously reported (Draelos and Raymond, 2018).
Statistical analyses
Statistical analyses were performed using SPSS for Windows, version
25 (SPSS Inc., Chicago, IL). For the changes over time, Wilcoxon
signed-rank test, repeated measures ANOVA followed by Bonferro-
ni’s post hoc analysis, or paired t-test was used. For correlation
studies, Pearson’s correlation coefficient was used. All data are
expressed as mean  SD, and a P-value of 0.05 or less was
considered statistically significant.
K Ishida et al.
Role of Ceramide in Atopic Dermatitis
Other details of the Materials and the Methods used (sources of
materials, barrier function test, and ceramide assessment method)
can be found in Supplementary Materials and Methods.
Data availability statement
No datasets were generated or analyzed during this study.
ORCIDs
Koichi Ishida: https://orcid.org/0000-0001-6293-507X
Akihiko Takahashi: https://orcid.org/0000-0001-5081-2590
Kotatsu Bito: https://orcid.org/0000-0002-3197-7880
Zoe Draelos: https://orcid.org/0000-0001-9803-7415
Genji Imokawa: https://orcid.org/0000-0001-7312-4475
CONFLICT OF INTEREST
KI, AT, and KB have been employed by Kao Corporation. ZD received an
educational grant from Kao to conduct the research detailed in this manu-
script. GI states no conflict of interest.
ACKNOWLEDGMENTS
This study was financially supported by Kao Corporation, Tokyo, Japan and
was conducted in Dermatology Consulting Services, High Point, NC. The
authors thank Anthony Simion, Lisa Adams, Andrew DiMuzio, Ward
Billhimer, and Desiree Butcher (Kao USA) for excellent technical and
administrative assistance; Yoshinori Masukawa (Kao Corporation, Japan) for
help with the microanalysis of ceramides using normal-phase liquid
chromatographyeelectrospray ionization mass spectrometry; and Tsujimura
Hisashi and Ayano Naoe (Kao Corporation, Japan) for help with the ceramide
analysis.
AUTHOR CONTRIBUTIONS
Conceptualization: KI, AT, KB; Formal Analysis: GI, KI; Investigation: KI, AT,
KB, ZD; Writing - Original Draft Preparation: GI, KI
SUPPLEMENTARY MATERIAL
Supplementary material is linked to the online version of the paper at www.
jidonline.org and at https://doi.org/10.1016/j.jid.2020.01.014.
REFERENCES
Arikawa J, Ishibashi M, Kawashima M, Takagi Y, Ichikawa Y, Imokawa G.
Decreased levels of sphingosine, a natural anti-microbial agent, may be
associated with vulnerability of the stratum corneum from patients with
atopic dermatitis to colonization by Staphylococcus aureus. J Invest Der-
matol 2002;119:433e9.
Choi MJ, Maibach HI. Role of ceramides in barrier function of healthy and
diseased skin. Am J Clin Dermatol 2005;6:215e23.
Di Nardo A, Wertz P, Giannetti A, Seidenari S. Ceramide and cholesterol
composition of the skin of patients with atopic dermatitis. Acta Derm
Venereol 1998;78:27e30.
Draelos ZD, Raymond I. The efficacy of a ceramide based cream in mild-to-
moderate atopic dermatitis. J Clin Aesthet Dermatol 2018;11:30e2.
Funasaka Y, Bito T, Yamamoto M, Nishigori C, Ishihashi M, Nakamura T, et al.
Clinical evaluation of “Curel UV Milk” and “Curel UV Cream” in subjects
with low barrier function’s skin. Skin Res 2004;31:62e74 [in Japanese].
Hanifin JM, Rajka G. Diagnostic features of atopic dermatitis. Acta Derm
Venereol (Stockh) 1980;92(Suppl):44e7.
Hata M, Tokura Y, Takigawa M, Sato M, Shioya Y, Fujikura Y, et al. Assessment
of epidermal barrier function by photoacoustic spectrometry in relation to
its importance in the pathogenesis of atopic dermatitis. Lab Invest
2002a;82:1451e61.
Hata M, Tokura Y, Takigawa M, Tamura Y, Imokawa G. Efficacy of using
pseudoceramide-containing cream for the treatment of atopic dry skin in
comparison with urea cream. Nishinihon J Dermatol 2002b;64:606e11 [in
Japanese].
Holleran WM, Feingold KR, Man MQ, Gao WN, Lee JM, Elias PM. Regulation
of epidermal sphingolipid synthesis by permeability barrier function. J Lipid
Res 1991a;32:1151e8.
Holleran WM, Ginns EI, Menon GK, Grundmann JU, Fartasch M,
McKinney CE, et al. Consequences of beta-glucocerebrosidase deficiency
in epidermis. Ultrastructure and permeability barrier alterations in Gaucher
disease. J Clin Invest 1994a;93:1756e64.
www.jidonline.org 1769
 K Ishida et al.
Role of Ceramide in Atopic Dermatitis
Holleran WM, Man MQ, Gao WN, Menon GK, Elias PM, Feingold KR.
Sphingolipids are required for mammalian epidermal barrier function. In-
hibition of sphingolipid synthesis delays barrier recovery after acute
perturbation. J Clin Invest 1991b;88:1338e45.
Holleran WM, Takagi Y, Menon GK, Jackson SM, Lee JM, Feingold KR, et al.
Permeability barrier requirements regulate epidermal beta-glucocere-
brosidase. J Lipid Res 1994b;35:905e12.
Holleran WM, Takagi Y, Menon GK, Legler G, Feingold KR, Elias PM. Pro-
cessing of epidermal glucosylceramides is required for optimal mammalian
cutaneous permeability barrier function. J Clin Invest 1993;91:1656e64.
Imokawa G. In vitro and in vivo models Elsner P. In: Berardesca E,
Maibach HI, editors. Bioengineering of Skin: Water and the stratum cor-
neum. Boca Raton, FL: CRC Press; 1994. p. 23e47.
Imokawa G. Skin lotions: development and clinical use ceramides. In:
Loden M, editor. Dry skin and lotions. Boca Raton, FL: CRC Press; 1999. p.
269e99.
Imokawa G. Ceramides as natural moisturizing factors and their efficacy in
dry skin. In: Leyden JJ, Rawlings AV, editors. Skin moisturization. Boca
Raton, FL: CRC Press; 2001. p. 267e302.
Imokawa G. Surfactant-induced depletion of ceramides and other intercel-
lular lipids: implication for the mechanism leading to dehydration of the
stratum corneum. Exog Dermatol 2004;3:81e98.
Imokawa G, Abe A, Jin K, Higaki Y, Kawashima M, Hidano A. Decreased
level of ceramides in stratum corneum of atopic dermatitis: an etiologic
factor in atopic dry skin? J Invest Dermatol 1991a;96:523e6.
Imokawa G, Akasaki S, Hattori M, Yoshizuka N. Selective recovery of
deranged water-holding properties by stratum corneum lipids. J Invest
Dermatol 1986;87:758e61.
Imokawa G, Akasaki S, Kawamata A, Yano S, Takaishi N. Water-
retaining function in the stratum corneum and its recovery properties
by synthetic pseudo-ceramides. J Soc Cosmet Chem 1989a;40:
273e85.
Imokawa G, Akasaki S, Kuno O, Zama M, Kawai M, Minematsu Y, et al.
Function of lipids on human skin. J Disp Sci Tech 1989b;10:617e41.
Imokawa G, Akasaki S, Minematsu Y, Kawai M. Importance of intercellular
lipids in water-retention properties of the stratum corneum: induction and
recovery study of surfactant dry skin. Arch Dermatol Res 1989c;281:
45e51.
Imokawa G, Hattori M. A possible function of structural lipid in the water-
holding properties of the stratum corneum. J Invest Dermatol 1985;84:
282e4.
Imokawa G, Ishida K. Role of ceramide in the barrier function of the stratum
corneum, Implications for the pathogenesis of atopic dermatitis. J Clin Exp
Dermatol Res 2014;5:1000206.
Imokawa G, Kuno H, Kawai M. Stratum corneum lipids serve as a bound-
water modulator. J Invest Dermatol 1991b;96:845e51.
Imokawa G, Yada Y, Higuchi K, Okuda M, Ohashi Y, Kawamata A. Pseu-
doacylceramide with linoleic acid produces selective recovery of
diminished cutaneous barrier function in essential fatty acid deficient rats
and has an inhibitory effect on epidermal hyperplasia. J Clin Invest
1994;94:89e96.
Ishibashi M, Arikawa J, Okamoto R, Kawashima M, Takagi Y, Ohguchi K,
et al. Abnormal expression of the novel epidermal enzyme, glucosylcer-
amide deacylase and the accumulation of its enzymatic reaction product,
glucosylsphingosine in the skin of patients with atopic dermatitis. Lab
Invest 2003;88:397e408.
Ishikawa J, Narita H, Kondo N, Hotta M, Takagi Y, Masukawa Y, et al.
Changes in the ceramide profile of atopic dermatitis patients. J Invest
Dermatol 2010;130:2511e4.
1770 Journal of Investigative Dermatology (2020), Volume 140
Janssens M, van Smeden J, Gooris GS, Bras W, Portale G, Caspers PJ, et al.
Lamellar lipid organization and ceramide composition in the stratum
corneum of patients with atopic eczema. J Invest Dermatol 2011;131:
2136e8.
Janssens M, van Smeden J, Gooris GS, Bras W, Portale G, Caspers PJ, et al.
Increase in short-chain ceramides correlates with an altered lipid organi-
zation and decreased barrier function in atopic eczema patients. J Lipid Res
2012;53:2755e66.
Jungersted JM, Scheer H, Mempel M, Baurecht H, Cifuentes L,
Høgh JK, et al. Stratum corneum lipids, skin barrier function and
filaggrin mutations in patients with atopic eczema. Allergy 2010;65:
911e8.
Kim D, Lee NR, Park SY, Jun M, Lee K, Kim S, et al. As in atopic dermatitis,
nonlesional skin in allergic contact dermatitis displays abnormalities in
barrier function and ceramide content. J Invest Dermatol 2017;137:
748e50.
Koyano S, Hatamochi A, Yamazaki S, Ishikawa J, Kitahara T, Narita H, et al.
Psoriasis patients have abnormal ceramide profile in stratum corneum.
Nishinihon J Dermatol 2010;72:494e9 [in Japanese].
Loiseau N, Obata Y, Moradian S, Sano H, Yoshino S, Aburai K, et al. Altered
sphingoid base profiles predict compromised membrane structure and
permeability in atopic dermatitis. J Dermatol Sci 2013;72:296e303.
Masukawa Y, Narita H, Shimizu E, Kondo N, Sugai Y, Oba T, et al. Charac-
terization of overall ceramide species in human stratum corneum. J Lipid
Res 2008;49:1466e76.
Matsuki H, Kiyokane K, Matsuki T, Sato S, Imokawa G. Re-characterization of
the non-lesional dry skin in atopic dermatitis through disrupted barrier
function. Exog Dermatol 2004a;3:282e92.
Matsuki H, Kiyokane K, Matsuki T, Sato S, Imokawa G. Re-evaluation of the
importance of barrier dysfunction in the non-lesional dry skin of atopic
dermatitis through the use of two barrier creams. Exog Dermatol 2004b;3:
293e302.
Mizutani H, Takahashi M, Shimizu M, Kariya K, Sato H, Imokowa G. Usage of
pseudoceramide cream in atopic dry skin in comparison with 10% urea
cream. Nishinihon J Dermatol 2001;63:457e61 [in Japanese].
Nakamura T, Satoh H, Imokawa G, Miyachi Y. Clinical test of skin care
products on atopic dry skin. Skin Res 2000;42:264e9 [in Japanese].
Okamoto R, Arikawa J, Ishibashi M, Kawashima M, Takagi Y, Imokawa G.
Sphingosylphosphorylcholine is upregulated in the stratum corneum of
patients with atopic dermatitis. J Lipid Res 2003;44:93e102.
Shen CP, Zhao MT, Jia ZX, Zhang JL, Jiao L, Ma L. Skin ceramide profile in
children with atopic dermatitis. Dermatitis 2018;29:219e22.
Sugiura A, Nomura T, Mizuno A, Imokawa G. Reevaluation of the non-
lesional dry skin in atopic dermatitis by acute barrier disruption: an
abnormal permeability barrier homeostasis with defective processing to
generate ceramide. Arch Dermatol Res 2014;306:427e40.
Takagi Y, Nakagawa H, Higuchi K, Imokawa G. Characterization of
surfactant-induced skin damage through barrier recovery induced by
pseudoacylceramides. Dermatology 2005;211:128e34.
Takashima I, Hasebe K, Okuda M, Imokawa G. Efficacy of pseudoceramide-
containing detergent formulation for treatment of atopic dermatitis and
asteatotic eczema. Nishinihon J Dermatol 2002;64:612e20 [in Japanese].
Umeda Y, Mizutani H, Imokawa G, Shimizu M. Topical ceramide corrected
epidermal cell hyperproliferation and stratum corneum dysmaturation in
atopic eczema. In: Johannes R, Heidrun B, Dieter V, editors. New trends in
allergy IV. Berlin, Heidelberg: Springer; 1997. p. 237e9.
Yamanaka M, Ishikawa O, Takahashi A, Sato H, Imokawa G. Clinical eval-
uation of Curel medicated cream in patients with atopic dermatitis. Skin
Res 2001;43:278e85 [in Japanese].

SUPPLEMENTARY MATERIALS AND METHODS
Materials
Cetyl-PG hydroxyethyl palmitamide (Supplementary Figure S5)
(Imokawa, 2004, 2001, 1999, 1994; Imokawa et al., 1989a,
1989b; Matsuki et al., 2004; Takagi et al., 2005), developed
by Kao Corporation (Tokyo, Japan), was used as the synthetic
pseudoceramide (pCer). An oil-in-wateretype lotion containing
3% pCer and 0.9% eucalyptus extract that is known to increase
ceramide content in the stratum corneum (Ishikawa et al., 2012)
was used as the test lotion for this study. The formula of the pCer
test lotion is shown in Supplementary Table S3.
Barrier function test
The methyl nicotinate insult method was used to evaluate the
barrier function of the volar forearm skin. A 1-cm diameter
Whatman filter paper #1 disc was placed on the test site, and
10 ml of a 10 mM aqueous solution of methyl nicotinate was
pipetted onto the filter paper disc. After drying for 2 minutes,
the filter paper disc was removed from the skin (blotted dry if
necessary), and the erythema index was measured using a
Cortex Technology DSM II Color meter. Transient sensations
by subject assessment were evaluated immediately and at 5,
10, and 15 minutes after removal of the disc.
Ceramide assessment method
The level and total carbon number of endogenous ceramides
and pCer in the stratum corneum were determined by
HPLCemass spectrometry analysis of tape strips taken from
the volar surface of the forearms of all subjects. At the speci-
fied sampling intervals (baseline and week 4), each subject
had a nonlesional volar surface of the forearm, approximately
2.5 cm  2.5 cm, stripped 10 times with a polyphenylene
sulfide film tape (Nichiban, Tokyo, Japan). All tape strips were
divided in half so that one portion was designated for analysis
of total soluble protein and the other half was designated for
the determination of ceramides and pCer contents. For each
subject, tapes from strippings 2 through 10 were combined for
extraction and analysis. Ceramides were extracted from half of
the tape-stripped specimens according to a previously
described method (Sugiura et al., 2014). The concentration of
soluble proteins was determined using a bicinchoninic acid
protein assay kit (Pierce, Rockford, IL) with a calibration
curve established using BSA as a standard (Smith et al.,
1985). Analysis of the ceramides and pCer in the tape
strips was performed according to normal-phase liquid
chromatographyeelectrospray ionization mass spectrometry,
as described previously (Masukawa et al., 2009, 2008), which
can separate ceramides in the extracted lipid mixture with
normal-phase gradient liquid chromatography, and ceramides
were detected using their m/z of molecular-related ions in the
selected ion monitoring mass spectrometry, as shown in part
by Supplementary Figure S6. An Agilent 1100 Series LC/MSD
single-quadrupole system equipped with an electrospray
ionization source (Agilent Technologies, Santa Clara, CA) and
an Inertsil SIL 100A-3, 1.5 mm inner diameter  150 mm
column (GL Science, Tokyo, Japan) were used in the analysis.
K Ishida et al.
Role of Ceramide in Atopic Dermatitis
The levels of each ceramide were quantified by using N-
stearoyl-D-erythro-sphingosine (C18 Ceramide [d17:1/18:0]
Avanti Polar Lipids, Alabaster, AL) as an internal standard,
according to specific equations for quantification of all cer-
amide species that were established by using synthetic natural
ceramide standards (Masukawa et al., 2009).
SUPPLEMENTARY RESULTS
Change in the permeability barrier
Evaluation of the permeability barrier by the methyl nicotinate
test indicated that in the nontreated atopic dermatitis skin (at
baseline), erythema induced by methyl nicotinate was signif-
icantly accentuated at 10 and 15 minutes after application
(Supplementary Figure S7). In the pCer lotionetreated nonle-
sional atopic dermatitis skin at week 4 and in the regression
phase, erythema did not significantly appear at 5, 10, and 15
minutes after application. These findings suggest that the
penetration (outside-in) barrier in the pCer-treated skin was
markedly improved compared with the baseline.
SUPPLEMENTARY REFERENCES
Imokawa G. In vitro and in vivo models Elsner P. In: Berardesca E,
Maibach HI, editors. Bioengineering of Skin: Water and the stratum cor-
neum. Boca Raton, FL: CRC Press; 1994. p. 23e47.
Imokawa G. Skin lotions: development and clinical use ceramides. In:
Loden M, editor. Dry skin and lotions. Boca Raton, FL: CRC Press; 1999. p.
269e99.
Imokawa G. Ceramides as natural moisturizing factors and their efficacy in
dry skin. In: Leyden JJ, Rawlings AV, editors. Skin moisturization. Boca
Raton, FL: CRC Press; 2001. p. 267e302.
Imokawa G. Surfactant-induced depletion of ceramides and other intercel-
lular lipids: implication for the mechanism leading to dehydration of the
stratum corneum. Exog Dermatol 2004;3:81e98.
Imokawa G, Akasaki S, Kawamata A, Yano S, Takaishi N. Water-retaining
function in the stratum corneum and its recovery properties by synthetic
pseudo-ceramides. J Soc Cosmet Chem 1989a;40:273e85.
Imokawa G, Akasaki S, Kuno O, Zama M, Kawai M, Minematsu Y, et al.
Function of lipids on human skin. J Disp Sci Tech 1989b;10:617e41.
Ishikawa J, Shimotoyodome Y, Chen S, Ohkubo K, Takagi Y, Fujimura T, et al.
Eucalyptus increases ceramide levels in keratinocytes and improves stratum
corneum function. Int J Cosmet Sci 2012;34:17e22.
Masukawa Y, Narita H, Sato H, Naoe A, Kondo N, Sugai Y, et al. Compre-
hensive quantification of ceramide species in human stratum corneum.
J Lipid Res 2009;50:1708e19.
Masukawa Y, Narita H, Shimizu E, Kondo N, Sugai Y, Oba T, et al. Charac-
terization of overall ceramide species in human stratum corneum. J Lipid
Res 2008;49:1466e76.
Matsuki H, Kiyokane K, Matsuki T, Sato S, Imokawa G. Re-evaluation of the
importance of barrier dysfunction in the non-lesional dry skin of atopic
dermatitis through the use of two barrier creams. Exog Dermatol 2004;3:
293e302.
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD,
et al. Measurement of protein using bicinchoninic acid. Anal Biochem
1985;150:76e85.
Sugiura A, Nomura T, Mizuno A, Imokawa G. Reevaluation of the non-
lesional dry skin in atopic dermatitis by acute barrier disruption: an
abnormal permeability barrier homeostasis with defective processing to
generate ceramide. Arch Dermatol Res 2014;306:427e40.
Takagi Y, Nakagawa H, Higuchi K, Imokawa G. Characterization of surfac-
tant-induced skin damage through barrier recovery induced by pseudoa-
cylceramides. Dermatology 2005;211:128e34.
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 K Ishida et al.
Role of Ceramide in Atopic Dermatitis

Erythema Stinging
Baseline Baseline
Week 1 Week 1
Week 2 Week 2
Week 4 Week 4

0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100%
Severe Moderate Mild Minimal None Severe Moderate Mild Minimal None

Desquamation Burning
Baseline Baseline
Week 1 Week 1
Week 2 Week 2
Week 4 Week 4

0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100%
Severe Moderate Mild Minimal None Severe Moderate Mild Minimal None

Lichenification Itching
Baseline Baseline
Week 1 Week 1
Week 2 Week 2
Week 4 Week 4

0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100%
Severe Moderate Mild Minimal None Severe Moderate Mild Minimal None

Excoriation Overall
Baseline Baseline
Week 1 Week 1
Week 2 Week 2

Week 4 Week 4

0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100%
Severe Moderate Mild Minimal None Severe Moderate Mild Minimal None

Supplementary Figure S1. Assessments of skin symptoms during 4 weeks of treatment with the pCer lotion. n ¼ 38, data represent the percentage of severity
score for each skin symptom. pCer, pseudoceramide.

1770.e2 Journal of Investigative Dermatology (2020), Volume 140

 K Ishida et al.
Role of Ceramide in Atopic Dermatitis

Cer[NDS] Cer[NS] Cer[NH] Cer[NP] Cer[ADS] Cer[AS]

10.0 12.0 35.0 30.0 2.0 8.0
N. S. p<0.001 p<0.001 p<0.05 p<0.001 p<0.001
10.0
Class Ratio of Ceramide

8.0
30.0 25.0 6.0
8.0 1.5
(weight %)

6.0
6.0 25.0 20.0 4.0
4.0
4.0 1.0
20.0 15.0 2.0
2.0
2.0

0.0 0.0 15.0 10.0 0.5 0.0

BL W4 BL W4 BL W4 BL W4 BL W4 BL W4

Cer[AH] Cer[AP] Cer[EOS] Cer[EOH] Cer[EOP]

25.0 30.0 10.0 8.0 2.0
p<0.001 p<0.01 p<0.001 p<0.001 p<0.001
Class Ratio of Ceramide

25.0 8.0
6.0 1.5
20.0
(weight %)

20.0 6.0
4.0 1.0
15.0 4.0
15.0
2.0 0.5
10.0 2.0

10.0 5.0 0.0 0.0 0.0

BL W4 BL W4 BL W4 BL W4 BL W4

Supplementary Figure S2. Changes in the class ratio (weight %) of endogenous ceramide species in the SC of nonlesional AD skin on the forearm. Before and
after 4 weeks of treatment with the pCer lotion. n ¼ 38, data represent individual values and mean  SD. P-values were analyzed by paired t-test. AD, atopic
dermatitis; Cer[ADS], ceramides containing a-hydroxy fatty acids and dihydrosphingosines; Cer[AH], ceramides containing a-hydroxy fatty acids and
6-hydroxysphingosines; Cer[AP], ceramides containing a-hydroxy fatty acids and phytosphingosines; Cer[AS], ceramides containing a-hydroxy fatty acids and
sphingosines; Cer[EOH], ceramides containing ester-linked fatty acids, o-hydroxy fatty acids, and 6-hydroxysphingosines; Cer[EOP], ceramides containing ester-
linked fatty acids, o-hydroxy fatty acids, and phytosphingosines; Cer[EOS], ceramides containing ester-linked fatty acids, o-hydroxy fatty acids, and
sphingosines; Cer[NDS], ceramides containing nonhydroxy fatty acids and dihydrosphingosines; Cer[NH], ceramides containing nonhydroxy fatty acids and
6-hydroxysphingosines; Cer[NP], ceramides containing nonhydroxy fatty acids and phytosphingosines; Cer[NS], ceramides containing nonhydroxy fatty acids
and sphingosines; N.S., not significant; SC, stratum corneum.

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 K Ishida et al.
Role of Ceramide in Atopic Dermatitis

a Cer[NDS] b Cer[NS] c Cer[NH]

0.6 0.5 3.0
Baseline Baseline Baseline
Cer[NDS] (ng/µg protein)

Cer[NH] (ng/µg protein)

Cer[NS] (ng/µg protein)
0.5 0.4 2.5
Week 4 Week 4 Week 4
0.4 2.0
0.3
0.3 1.5
0.2
0.2 1.0

0.1 0.1 0.5

0.0 0.0 0.0

C32
C33
C34
C35
C36
C37
C38
C39
C40
C41
C42
C43
C44
C45
C46
C47
C48
C49
C50
C51
C52
C53
C54

C32
C33
C34
C35
C36
C37
C38
C39
C40
C41
C42
C43
C44
C45
C46
C47
C48
C49
C50
C51
C52
C53
C54

C32
C33
C34
C35
C36
C37
C38
C39
C40
C41
C42
C43
C44
C45
C46
C47
C48
C49
C50
C51
C52
C53
C54
Total Carbon Number of Cer[NDS] Total Carbon Number of Cer[NS] Total Carbon Number of Cer[NH]

d Cer[NP] e Cer[ADS] f Cer[AS]

2.0 0.2 0.4
Baseline Baseline Baseline

Cer[ADS] (ng/µg protein)

Cer[NP] (ng/µg protein)

Cer[AS] (ng/µg protein)

1.5 Week 4 Week 4 0.3 Week 4
0.1

1.0 0.2

0.1
0.5 0.1

0.0 0.0 0.0

C32
C33
C34
C35
C36
C37
C38
C39
C40
C41
C42
C43
C44
C45
C46
C47
C48
C49
C50
C51
C52
C53
C54

C32
C33
C34
C35
C36
C37
C38
C39
C40
C41
C42
C43
C44
C45
C46
C47
C48
C49
C50
C51
C52
C53
C54

C32
C33
C34
C35
C36
C37
C38
C39
C40
C41
C42
C43
C44
C45
C46
C47
C48
C49
C50
C51
C52
C53
C54
Total Carbon Number of Cer[NP] Total Carbon Number of Cer[ADS] Total Carbon Number of Cer[AS]

g Cer[AH]
h Cer[AP] i Cer[EOS]
1.5
2.5 2.0
Baseline

Cer[EOS] (ng/µg protein)

Baseline Baseline
Cer[AH] (ng/µg protein)

Cer[AP] (ng/µg protein)

2.0 Week 4
Week 4 1.5 Week 4
1.0
1.5
1.0
1.0
0.5
0.5
0.5

0.0 0.0 0.0

C66:2

C67:2

C68:2

C69:2

C70:2

C71:2

C72:2
C32
C33
C34
C35
C36
C37
C38
C39
C40
C41
C42
C43
C44
C45
C46
C47
C48
C49
C50
C51
C52
C53
C54

C32
C33
C34
C35
C36
C37
C38
C39
C40
C41
C42
C43
C44
C45
C46
C47
C48
C49
C50
C51
C52
C53
C54

Total Carbon Number of Cer[AH] Total Carbon Number of Cer[AP]

Total Carbon Number of Cer[EOS]

j Cer[EOH] k Cer[EOP]
1.0 0.20
Baseline Baseline
Cer[EOH] (ng/µg protein)

Cer[EOP] (ng/µg protein)

0.8 Week 4 Week 4

0.15

0.6
0.10
0.4

0.05
0.2

0.0 0.00
C66:2

C67:2

C68:2

C69:2

C70:2

C71:2

C72:2

C66:2

C67:2

C68:2

C69:2

C70:2

C71:2

C72:2

Total Carbon Number of Cer[EOH] Total Carbon Number of Cer[EOP]

Supplementary Figure S3. Changes in the total carbon number distributions of endogenous ceramide species in the SC of nonlesional AD skin on the forearm. Before
and after treatment with the pCer lotion for 4 weeks. n ¼ 38, data represent mean  SD and moving averages. (a) Cer[NDS]. (b) Cer[NS]. (c) Cer[NH]. (d) Cer
[NP]. (e) Cer[ADS]. (f) Cer[AS]. (g) Cer[AH]. (h) Cer[AP]. (i) Cer[EOS]. (j) Cer[EOH]. (k) Cer[EOP]. AD, atopic dermatitis; Cer[ADS], ceramides containing a-
hydroxy fatty acids and dihydrosphingosines; Cer[AH], ceramides containing a-hydroxy fatty acids and 6-hydroxysphingosines; Cer[AP], ceramides containing
a-hydroxy fatty acids and phytosphingosines; Cer[AS], ceramides containing a-hydroxy fatty acids and sphingosines; Cer[EOH], ceramides containing ester-
linked fatty acids, o-hydroxy fatty acids, and 6-hydroxysphingosines; Cer[EOP], ceramides containing ester-linked fatty acids, o-hydroxy fatty acids, and
phytosphingosines; Cer[EOS], ceramides containing ester-linked fatty acids, o-hydroxy fatty acids, and sphingosines; Cer[NDS], ceramides containing
nonhydroxy fatty acids and dihydrosphingosines; Cer[NH], ceramides containing nonhydroxy fatty acids and 6-hydroxysphingosines; Cer[NP], ceramides
containing nonhydroxy fatty acids and phytosphingosines; Cer[NS], ceramides containing nonhydroxy fatty acids and sphingosines; pCer, pseudoceramide; SC,
stratum corneum.

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 K Ishida et al.
Role of Ceramide in Atopic Dermatitis

Supplementary Figure S4. Chemical

structures of endogenous ceramide
species in the SC of human skin.
Ceramides in human SC can be
divided into 11 groups according to
their fatty acid and sphingoid base
structures. CER[NDS] contains
nonhydroxy fatty acids [N] and
dihydrosphingosines [DS]; CER[NS]
contains [N] and sphingosines [S];
CER[NH] contains [N] and 6-
hydroxysphingosines [H]; CER[NP]
contains [N] and phytosphingosines
[P]; CER[ADS] contains a-hydroxy
fatty acids [A] and [DS]; CER[AS]
contains [A] and [S]; CER[AH]
contains [A] and [H]; CER[AP]
contains [A] and [P]; CER[EOS]
contains ester-linked fatty acids and
o-hydroxy fatty acids [EO] and [S];
CER[EOH] contains [EO] and [H]; and
CER[EOP] contains [EO] and [P].
These classes can be further
subdivided into many species based
on their chain length. To date, more
than 350 species of ceramides have
been identified in human SC using
NPLC-ESI-MS. NPLC-ESI-MS, normal-
phase liquid chromatographye
electrospray ionization mass
spectrometry; SC, stratum corneum.

Supplementary Figure S5. Chemical

structure of natural ceramide and
synthetic pCer. (a) Natural ceramide:
Cer[NS]. (b) Product name of pCer:
SOFCARE CERAMIDE SL-E (Kao
Corporation), INCI Name: Cetyl-PG
hydroxyethyl palmitamide, Molecular
weight: 598 (C37H75O4N), Melting
point: 69.077.0 C, Purity >97%. Cer
[NS], ceramides containing
nonhydroxy fatty acids and
sphingosines; pCer, pseudoceramide.

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 K Ishida et al.
Role of Ceramide in Atopic Dermatitis

Supplementary Figure S6. Multi-SIM chromatograms of ceramide species in the SC using NPLC-ESI-MS. (a) At the baseline and (b) after 4 weeks of use of the
pCer test lotion. Internal standard: N-stearoyl-D-erythro-sphingosine (Avanti Polar Lipids, Alabaster, AL). NPLC-ESI-MS, normal-phase liquid
chromatographyeelectrospray ionization mass spectrometry; SC, stratum corneum; SIM, selected ion monitoring.
Mean Erythema Change from Baseline

Baseline
Week 4
5.0 Regression Day 7
(⌬Color Meter Index)

4.0
**
3.0 **
2.0

1.0

0.0

0 min 5 min 10 min 15 min

Time after Methyl Nicotinate Insult

Supplementary Figure S7. Change of the skin barrier function evaluated by

the erythema index. Function was evaluated by color meter after using the
pCer lotion for 4 weeks and after the 7 days regression phase, following
application of methyl nicotinate on the nonlesional AD skin on the volar
forearm. n ¼ 9, data represent mean  SD. **P < 0.01 versus baseline
analyzed by repeated measures ANOVA and post hoc Bonferroni correction.
AD, atopic dermatitis; pCer, pseudoceramide.

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 K Ishida et al.
Role of Ceramide in Atopic Dermatitis

Supplementary Table S1. Relationships between Endogenous Ceramide Species and Penetrated Levels of pCer and
Skin ConditioneAssociated Factors
Conductance (mS) TEWL (gm/m2/hr)

pCer and Endogenous Ceramide Species r-value P-value r-value P-value

1
pCer 0.447 0.005 0.101 0.545
Total ceramides 0.224 0.177 0.078 0.640
Cer[NDS] 0.213 0.199 0.135 0.419
Cer[NS] 0.198 0.232 0.174 0.296
Cer[NH] 0.194 0.243 0.015 0.929
Cer[NP] 0.135 0.419 0.161 0.335
Cer[ADS] 0.178 0.286 0.239 0.149
Cer[AS] 0.224 0.177 0.086 0.606
Cer[AH] 0.207 0.213 0.002 0.992
Cer[AP] 0.244 0.141 0.274 0.096
Cer[EOS] 0.198 0.233 0.133 0.427
Cer[EOH] 0.204 0.219 0.030 0.859
Cer[EOP] 0.184 0.270 0.135 0.420
Abbreviations: AD, atopic dermatitis; Cer[ADS], ceramides containing a-hydroxy fatty acids and dihydrosphingosines; Cer[AH], ceramides containing a-
hydroxy fatty acids and 6-hydroxysphingosines; Cer[AP], ceramides containing a-hydroxy fatty acids and phytosphingosines; Cer[AS], ceramides containing
a-hydroxy fatty acids and sphingosines; Cer[EOH], ceramides containing ester-linked fatty acids, o-hydroxy fatty acids, and 6-hydroxysphingosines; Cer
[EOP], ceramides containing ester-linked fatty acids, o-hydroxy fatty acids, and phytosphingosines; Cer[EOS], ceramides containing ester-linked fatty acids,
o-hydroxy fatty acids, and sphingosines; Cer[NDS], ceramides containing nonhydroxy fatty acids and dihydrosphingosines; Cer[NH], ceramides containing
nonhydroxy fatty acids and 6-hydroxysphingosines; Cer[NP], ceramides containing nonhydroxy fatty acids and phytosphingosines; Cer[NS], ceramides
containing nonhydroxy fatty acids and sphingosines; pCer, pseudoceramide; SC, stratum corneum; TEWL, transepidermal water loss.
Ceramide levels were measured in ng/mg protein in the SC of nonlesional AD skin on the forearm after 4 weeks of use of the pCer lotion. n ¼ 38, correlation
coefficients and P-values were analyzed by Pearson’s correlation coefficient.
1
P < 0.01.

Supplementary Table S2. Relationships between pCer Levels and Class Ratio of Ceramide Species and Average of
Total Carbon Number of Endogenous Ceramides
Class ratio of Ceramides Average of total carbon number of
(weight %) Ceramides

Endogenous Ceramide Species r-value P-value r-value P-value

Cer[NDS] 0.185 0.265 0.177 0.287

Cer[NS] 0.325 0.0471 0.393 0.0151
Cer[NH] 0.090 0.590 0.167 0.318
Cer[NP] 0.126 0.450 0.215 0.194
Cer[ADS] 0.027 0.871 0.295 0.072
Cer[AS] 0.117 0.484 0.092 0.584
Cer[AH] 0.021 0.902 0.107 0.524
Cer[AP] 0.105 0.529 0.289 0.079
Cer[EOS] 0.091 0.586 0.315 0.054
Cer[EOH] 0.085 0.613 0.012 0.942
Cer[EOP] 0.193 0.245 0.255 0.123
Abbreviations: AD, atopic dermatitis; Cer[ADS], ceramides containing a-hydroxy fatty acids and dihydrosphingosines; Cer[AH], ceramides containing a-
hydroxy fatty acids and 6-hydroxysphingosines; Cer[AP], ceramides containing a-hydroxy fatty acids and phytosphingosines; Cer[AS], ceramides containing
a-hydroxy fatty acids and sphingosines; Cer[EOH], ceramides containing ester-linked fatty acids, o-hydroxy fatty acids, and 6-hydroxysphingosines; Cer
[EOP], ceramides containing ester-linked fatty acids, o-hydroxy fatty acids, and phytosphingosines; Cer[EOS], ceramides containing ester-linked fatty acids,
o-hydroxy fatty acids, and sphingosines; Cer[NDS], ceramides containing nonhydroxy fatty acids and dihydrosphingosines; Cer[NH], ceramides containing
nonhydroxy fatty acids and 6-hydroxysphingosines; Cer[NP], ceramides containing nonhydroxy fatty acids and phytosphingosines; Cer[NS], ceramides
containing nonhydroxy fatty acids and sphingosines; pCer, pseudoceramide; SC, stratum corneum.
pCer levels were measured in ng/mg protein in the SC of the nonlesional AD skin on the forearm after 4 weeks of use of the pCer lotion. Class ratio was
measured in weight %. n ¼ 38, correlation coefficients and P-values were analyzed by Pearson’s correlation coefficient.
1
P < 0.05.

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 K Ishida et al.
Role of Ceramide in Atopic Dermatitis

Supplementary Table S3. pCer Test Lotion Formula

Phase Ingredient

Phase A Cetyl-PG Hydroxyethyl Palmitamide (pCer); 3.0%

Bis-Methoxypropylamido Isodocosane
Cholesterol
Stearyl Alcohol / Cetyl Alcohol
Isopropyl Palmitate
Polyglyceryl-2 Diisostearate
Sorbitan Stearate
Polyoxyethylene (20) Sorbitan Monostearate
Phase B Allantoin
Glycerin
Sodium Methyl Stearoyl Taurate
Citric Acid
Paraben
Deionized Water
Phase C Eucalyptus Globulus Leaf Extract (BG/Water)
0.9% Dimethylpolysiloxane,
Abbreviations: O/W, oil-in-water; pCer, pseudoceramide.
Preparation Method: O/W lotion was prepared by mixing phase A and phase B at 80e85 C, adding phase C, and cooling to room temperature.
pH of formulation (by 10-fold dilution): 4.5e5.0.

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