Life Sciences 168 (2017) 24–27

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Life Sciences

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Clinical study of a retinoic acid-loaded microneedle patch for seborrheic

keratosis or senile lentigo
Sachiko Hirobe a, Risa Otsuka a, Hiroshi Iioka b, Ying-Shu Quan c, Fumio Kamiyama c, Hideo Asada b,
Naoki Okada a,⁎, Shinsaku Nakagawa a,⁎
a
Laboratory of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
b
Department of Dermatology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan
c
CosMED Pharmaceutical Co. Ltd., 32 Higashikujokawanishi-cho, Minami-ku, Kyoto 601-8014, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Aims: Pigmented lesions such as of seborrheic keratosis and senile lentigo, which are commonly seen on skin of
Received 7 October 2015 people N 50 years of age, are considered unattractive and disfiguring because of their negative psychological im-
Received in revised form 5 December 2015 pact. Drug therapy using all-trans retinoic acid (ATRA) is an attractive option for self-treatment at home. We have
Accepted 30 December 2015 developed an ATRA-loaded microneedle patch (ATRA-MN) and confirmed the pharmacological effects of ATRA-
Available online 3 January 2016
MN application in mice. Here, we describe a clinical study to evaluate the safety and efficacy of ATRA-MN in sub-
jects with seborrheic keratosis or senile lentigo.
Keywords:
Microneedle
Main methods: ATRA-MN was applied to the lesion site of each subject for 6 h once per week for 4 weeks. The skin
Clinical study irritation reaction was scored to assess adverse reactions and blood tests were performed to evaluate the pres-
All-trans retinoic acid ence of systemic adverse reactions. To assess the treatment effect using ATRA-MN, the desquamation and whit-
Seborrheic keratosis ening ability of the investigational skin was observed.
Senile lentigo Key findings: Desquamation of the stratum corneum was observed following four ATRA-MN applications at 1-
week intervals, but ATRA-MN applications did not induce severe local or systemic adverse effects.
Significance: These results showed that ATRA-MN treatment is promising as a safe and effective therapy for seb-
orrheic keratosis and senile lentigo.
© 2016 Elsevier Inc. All rights reserved.

1. Introduction specialized equipments, patients must undergo regular outpatient

Seborrheic keratosis and senile lentigo are characterized
by pigmented lesions that are commonly seen on skin of
people N 50 years of age [1–3]. Seborrheic keratosis is characterized as
sharply demarcated brownish plaques with verrucous surfaces. Origi-
nating from keratinocytes, they can develop anywhere on the skin, ex-
cept the palms and soles, and may become irritated and itchy. Senile
lentigo appears on sun-exposed areas of the skin such as the face and
back of the hands. These lesions are considered unattractive and
disfiguring and may have negative psychological impacts. Thus, they
are often removed for cosmetic reasons.
Cryotherapy and laser surgery are common treatments for seborrhe-
ic keratosis and senile lentigo. Cryosurgery using liquid nitrogen is the
standard and most widely practiced treatment for seborrheic keratosis.
Another surgical option is laser ablation using erbium YAG or CO2 lasers
[4,5]. However, these treatments incur complications such as scarring,
hyperpigmentation, and recurrence. Additionally, because they require
⁎ Corresponding authors.
E-mail addresses: okada@phs.osaka-u.ac.jp (N. Okada), nakagawa@phs.osaka-u.ac.jp
(S. Nakagawa).
treatments for complete removal of the lesions, incurring high costs.
Drug therapy for seborrheic keratosis and senile lentigo is an attrac-
tive treatment option for self-treatment at home but is not well
established. Retinoids are natural and synthetic metabolites and analogs
of vitamin A and are important regulators of epidermal proliferation and
differentiation [6,7]. All-trans retinoic acid (ATRA), a natural retinoid, is
the major biologically active form of retinoids. ATRA induces heparin-
binding epidermal growth factor-like growth factor (HB-EGF) expres-
sion in keratinocytes. HB-EGF can bind to the epidermal growth factor
receptor (EGFR) to play an important role in re-epithelialization by in-
creasing keratinocyte proliferation [8]. ATRA treatment leads to epider-
mal hyperplasia via keratinocyte-derived HB-EGF [6]. Thus, ATRA
increases basal keratinocyte proliferation, inducing the accelerated
turnover of epidermal cells, leading indirectly to epidermal thickening
[9]. These effects could be beneficial in seborrheic keratosis and senile
lentigo treatments.
ATRA has some drawbacks such as its poor water solubility and
photostability, and skin irritation reactions limit its topical use [10,11].
Furthermore, its skin permeability is relatively low [12]. To overcome
these disadvantages, we have developed an ATRA-loaded microneedle
patch (ATRA-MN) [13]. Microneedles [14,15] made of dissolving

http://dx.doi.org/10.1016/j.lfs.2015.12.051
0024-3205/© 2016 Elsevier Inc. All rights reserved.
 S. Hirobe et al. / Life Sciences 168 (2017) 24–27 25

polymers can puncture the stratum corneum (the physical barrier to Table 2
material permeation), dissolve in water in the skin, and directly deliver Schedule of clinical study for assessing the safety and efficacy of the ATRA-MN.

ATRA to the epidermis. Previously, we reported that ATRA-MN applica-
tion induced HB-EGF expression in the skin and epidermal hyperplasia
and shortened the stratum corneum turnover time in mice [13]. We
also confirmed that an ATRA-MN application potentially will not cause
serious adverse events in humans [13].
We conducted a clinical study to evaluate the safety and efficacy of a
novel treatment method using ATRA-MN for seborrheic keratosis and
senile lentigo.
2. Materials and methods
2.1. ATRA-MN preparation
As described previously [14–19], ATRA-MN was prepared at CosMED
Pharmaceutical Co. Ltd. (Kyoto, Japan) using micromolding technolo-
gies with sodium hyaluronate as the base material. To form the transcu-
taneous patch system, ATRA-MN with an area of 0.8 cm2 (containing
200 microneedles) was affixed to an adhesive film with 2.3 cm2 area.
The length of the microneedles on ATRA-MN was 800 μm. The amount
of ATRA loaded into the microneedles was 1.6 μg, using a high-
performance liquid chromatography method as reported previously
[15,20].
2.2. Clinical study protocol
Eight patients volunteered for and were enrolled in the study
(Table 1). Written informed consent was received before enrollment.
All procedures in humans were performed at Nara Medical University
in accordance with a protocol (Table 2) approved by the ethics commit-
tee of the Nara Medical University. ATRA-MN was applied to the lesion
site of each subject for 6 h once per week for 4 weeks. The investigated
skin was observed every week, and the skin irritation reaction was
scored according to the classification of the International Contact Der-
matitis Research Group (ICDRG) [21] system (Table 3) to assess adverse
reactions. A general blood test and biochemical tests of the liver and
renal function were performed before the first and after the last applica-
tion to evaluate the presence of systemic adverse reactions. To assess
the treatment effect using ATRA-MN, the desquamation and whitening
ability of the investigational skin was observed.
3. Results
3.1. Safety assessment of ATRA-MN applications
The primary aim of this study was to demonstrate that multiple
ATRA-MN applications are safe in humans. Faint erythema, classified
as ?+ by the ICDRG scale, was observed as the application number in-
creased (Fig. 1), whereas positive reactions were not induced by
ATRA-MN applications. This faint erythema was a temporary reaction
that disappeared completely after 3 months. Administration of ATRA-
MN did not induce detectable adverse effects, as determined by a gener-
al peripheral blood test and biochemical tests of liver and renal function.
Day 0 7 14 21 28 // 111
ATRA-MN application • • • •
Skin observation • • • • •
Blood test • •
Thus, multiple ATRA-MN applications did not induce local or systemic
adverse effects, indicating that ATRA-MN could be safely administered
to humans.
3.2. Efficacy assessment of ATRA-MN applications
Having confirmed the safety of multiple ATRA-MN applications in
humans, we next evaluated the efficacy of the novel treatment method
for seborrheic keratosis and senile lentigo. On the investigational skin,
desquamations of the stratum corneum were observed in three subjects
after three ATRA-MN applications (Fig. 2). After four applications, the
stratum corneum of an additional subject also desquamated. These des-
quamations were inferred to be caused by acceleration of stratum
corneum turnover, resulting from the pharmacological effect of ATRA
delivered into the skin. However, a clear skin-whitening effect and des-
quamation of the seborrheic keratosis lesion skin were not observed
during ATRA-MN applications or 3 months after four applications.
4. Discussion
In this study, we applied ATRA-MN four times at 1-week intervals.
The pharmacological effect of ATRA was confirmed in humans, but
these results showed that this novel treatment method using ATRA-
MN needed improvement for the complete cure of seborrheic keratosis
and senile lentigo.
The first point is the ATRA dose. In a study, when 0.025% ATRA gel
was applied at 5 mg gel/cm2 (1.25 μg ATRA/cm2), the dose of ATRA de-
livered to the epidermis was 20% (0.25 μg ATRA/cm2) after 40 h [22]. It is
estimated that the dose of ATRA delivered to the epidermis is
1–4 μg/cm2 in 0.1%–0.4% ATRA used in Japan [23]. Therefore, we esti-
mated that a safe delivery dose to the epidermis would be a few micro-
grams, and in fact, we used 1.6 μg/0.8 cm2/patch (2 μg/cm2). In a study
of intradermal ATRA injection, a maximum of 5 mL of 0.2 mg/mL ATRA
solution was administered to a subject [24]. Although the number of ad-
ministration sites was unclear, we assumed that 1 mg ATRA was the
maximum dose administered to a subject. We would be able to increase
the ATRA dose to at least 25 μg/patch while ensuring safe ATRA
application.
Moreover, we propose that the negative result is because of the
small number of applications and short-term treatment. Although we
have no data about ATRA clearance in the skin, we have shown that an-
tigens delivered by MN into mouse skin remained over 48 h [15]. We
consider the possibility that the clearance of ATRA with hyaluronic
acid, which is a high molecular polymer and a component of MN, is
slow. Because the primary endpoint in this study was the safety of the
approach, we performed ATRA-MN application once per week, after

Table 1 Table 3
Patients' information in clinical study. Scoring of skin local reactions according to ICDRG.

Factor Group Number of subjects Score Reactions

Race Asian 8 − Negative reaction

Gender Male 5 ?+ Doubtful reaction; faint erythema only
Female 3 + Weak (non-vesicular) positive reaction; erythema, infiltration and
Age (years) 51–60 1 possibly papules
61–70 2 ++ Strong (vesicular) positive reaction; erythema, infiltration, papules,
71–80 5 vesicles
Lesion Senile lentigo 6 +++ Extreme positive reaction; bullous reaction
Seborrheic keratosis 2 IR Irritant reaction
26 S. Hirobe et al. / Life Sciences 168 (2017) 24–27

In addition, we believe that the coadministration of several active

Fig. 1. Local adverse effects after ATRA-MN applications. ATRA-MN was applied to the
lesion site of each subject for 6 h once per week for 4 weeks. The investigational skin
was observed every week, and the skin irritation reaction was scored according to the
ICDRG classification system.
which most ATRA disappeared and skin status recovered. In another
study, twice-daily topical application of 0.1% tazarotene (a retinoid)
cream resulted in clinical and histological improvement of seborrheic
keratosis in 7/15 patients [25]. Because our study showed that the stra-
tum corneum was desquamated by only three applications at 1-week
intervals, we expect that numerous applications (once every few days
or daily) and long-term treatment would improve the efficacy of the
novel treatment method using ATRA-MN.
ingredients is important. Pigmentation was found in several sub-
jects, but it was expected by a dermatologist to disappear in several
weeks from a level of the pigmentation. Given that in this study we
confirmed the disappearance of the pigmentation 3 months later,
we expected that these reactions are temporary and, if they occur,
they will disappear after the end of the treatment. However, for
safer and more effective treatment, we need to develop a
microneedle patch containing both active ingredients, which has
the potential to increase the therapeutic effect, because it has been
reported that the coadministration of retinoic acid and hydroqui-
none is effective against pigmented lesions [26].
Improvement of the microneedles may also be necessary.
Microneedles must be able to puncture the lesion skin with swollen
stratum corneum and increased hardness.
We further plan to optimize the ATRA dose, schedule of ATRA-MN
applications, and coadministration of other active ingredients to estab-
lish our novel treatment method for seborrheic keratosis and senile
lentigo.
5. Conclusions
We showed that ATRA-MN treatment is promising as a safe and ef-
fective therapy for seborrheic keratosis and senile lentigo.

Fig. 2. The investigational skin of four subjects in which the stratum corneum desquamated. ATRA-MN was applied to the lesion site of each subject for 6 h once per week for 4 weeks. The
investigational skin was observed every week. Each image shows the ICDRG score and the presence of desquamation.
 S. Hirobe et al. / Life Sciences 168 (2017) 24–27
Acknowledgments
Our work was supported by JSPS KAKENHI Grant Number 25293038
and 26670076 from the Ministry of Education, Culture, Sports, Science,
and Technology of Japan.
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