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ORIGINAL RESEARCH
A Single-Arm, Open-Label, Phase IV Study to Evaluate
the Efficacy of a Topical Formulation
for Hyperkeratotic Actinic Keratosis Lesions
Maria Pia De Padova .Barbara Marzani .Daniela Pinto .
Fabio Rinaldi
Received: June 20, 2018
ÓThe Author(s) 2018
ABSTRACT
Introduction: Actinic keratosis (AKs) are epi-
dermal lesions that commonly occur in skin
exposed to chronic cumulative UV irradiation.
Untreated AK lesions can advance to squamous
cell carcinoma. Current treatments of AK have
many shortcomings; for instance, not all treat-
ments can be used for the hyperkeratotic form
of AK. The aim of this study was to test the
efficacy and tolerability of a topical product
containing 2,4,6-octatrienoic acid and urea for
the treatment of hyperkeratotic AK lesions.
Methods: Forty male and female subjects with
at least two hyperkeratotic AK lesions were
enrolled in this single-arm, open-label phase IV
study. The product was applied twice daily for
two consecutive months. The efficacy end-
points were the reductions in the mean number
of AK lesions per subject from baseline (T0) to
the end of the trial (T1) and to three months
after the end of the treatment period (T2).
Results: At T0, the mean (SD) number of
lesions per subject was 3.65 (1.25). At the end of
the treatment period (T1), this number had
dropped (significantly, p\0.0001) by 83.56%.
The mean number of lesions per subject then
decreased by 41.47% (p\0.0001) between T1
and the three-month follow-up visit (T2).
Complete elimination of lesions had occurred
in 57.5% of the subjects at T1, and 82.5% (55%
who had remained completely clear of lesions
since T1, and 27.5% who had fully eliminated
their lesions during the period from T1 to T2) at
T2. No side effects were reported.
Conclusion: The application of a topical com-
bination of 2,4,6-octatrienoic acid and urea
twice daily for 60 consecutive days is a safe and
effective treatment for hyperkeratotic AK
lesions.
Funding: Giuliani SpA.
Keywords: Hyperkeratotic actinic keratosis;
Keratolytic agent; 2,4,6-octatrienoic acid;
Topical treatment; Urea
INTRODUCTION
The chronic skin condition actinic keratosis
(AK) is characterized by intraepithelial atypical
proliferation of keratinocytes in sun-exposed
skin regions, especially on the face, as well as
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M. P. De Padova
Dermatology, Nigrisoli Private Hospital, Bologna,
Italy
B. Marzani D. Pinto
R&D, Giuliani SpA, Milan, Italy
F. Rinaldi (&)
International Hair Research Foundation (IHRF),
Milan, Italy
e-mail: fabio.rinaldi@studiorinaldi.com
Dermatol Ther (Heidelb)
https://doi.org/10.1007/s13555-018-0252-3
the scalp, ears, forearms, and backs of the
hands, although it can also appear on the trunk
and legs [1]. AK lesions are considered a clinical
sign of the photoaging of photodamaged skin
that has undergone subclinical genetic changes:
field cancerization [2,3]. AK is a worldwide
problem with a continuously increasing inci-
dence. In theory, anyone can develop AK, but
AK lesions typically arise in immune-compro-
mised patients and in fair-skinned (phototypes
I–III) individuals who have undergone excessive
and chronic sun exposure and frequent sunburn
[4]. AK risk is associated with UV radiation and
increases with chronological aging.
AK lesions are classified using clinical and
histological grading systems. Olsen et al. pro-
posed a clinical evaluation of AK lesions based
on their thickness: either slightly palpable
(grade 1), moderately thick (grade 2), or very
thick, hyperkeratotic, and/or obvious (grade 3)
lesions [5]. Later, Roewert-Huber and collabo-
rators introduced a histological classification
based on atypical keratinocytes in the epider-
mis: in grade AK I (mild), atypical keratinocytes
are restricted to basal and suprabasal layers and
limited to the lower third of the epidermis; in
grade AK II (moderate), atypical keratinocytes
extend to the lower two-thirds of the epidermis;
and in grade AK III (severe), full-thickness aty-
pia of the epidermis is present [6]. Unfortu-
nately, these two classification schemes do not
correlate well [7]. Although the histological
scheme is better at describing and characteriz-
ing lesions, the clinical grading scheme is
commonly used in clinical practice and in
clinical trials (i.e., to determine patient
eligibility).
Guidelines recommend that AK should be
treated, even if spontaneous lesion regression is
possible [8,9], in order to eliminate visible
lesions and subclinical AK and thus minimize
the risk of progression to squamous cell carci-
noma (SCC) [10]. There are a number of differ-
ent treatment options for AK, which can be
categorized into two main types: lesion and
field-directed therapies. The treatment selection
process should consider various factors, includ-
ing the number of AK lesions (solitary or mul-
tiple), their localization and duration, as well as
patient age, comorbidities, and treatment
compliance. In particular, for some categories of
patients (i.e., immunosuppressed patients) and
when AK lesions are present in particular body
areas (the lips, nose, eyelids, or ears), a more
intense therapeutic approach is required [1].
The available therapies are highly effective but,
unfortunately, not all of the approved treat-
ments (i.e., ingenol mebutate and imiquimod)
are indicated for hyperkeratotic lesions due to
the penetration barrier.
For years, the terms ‘‘hypertrophic’’ and
‘‘hyperkeratotic’’ in the treatment guidelines
were considered to be synonymous with thicker
AK lesions, with no clear distinction between
clinical entities [11,12]. Recently, however, in
an editorial, the expert clinicians of the
AKTeam
Ò
suggested that the term ‘‘hyperkera-
totic’’ should be used for clinically nonsuspi-
cious AK with thick scale [10]. Because the
thickness of the lesion is a limiting factor for
many treatments, it would be useful to better
understand the dimensions of this problem. An
Australian cohort study from 2014 reported that
up to 25% of AK lesions are hyperkeratotic,
although this proportion depended on the body
area considered: it was 10.3% of the AK lesions
on a bald scalp, 11.5% of those on the face and
ears, 13.7% of the lesions on the forearms, 16%
of those on the dorsal hands, and 25% of the AK
lesions on the legs [13].
There is no standard therapeutic strategy for
hyperkeratotic AK, and the treatment applied
can vary with the experience of the physician,
the pathology of the lesion, and various patient
factors. Multiple methods of enhancing the
efficacy of keratosis reduction have been
described. Pretreatment with curettage,
debulking, tape stripping, as well as microder-
mabrasion or laser ablation and excisional sur-
gery can be effective approaches for
hyperkeratotic lesions [14]. In the case of mul-
tiple hyperkeratotic AK lesions, the guidelines
propose pretreatment with topical keratolytics
and subsequent topical or physical treatment
modalities. Salicylic acid and urea are the main
topical keratolytic compounds that are used to
induce exfoliation of the superficial epidermal
layers through the cleavage of corneodesmo-
somes [15]. This is a simple, atraumatic,
Dermatol Ther (Heidelb)
inexpensive, and painless method of reducing
keratosis on photodamaged skin.
Another compound with demonstrated effi-
cacy for photodamaged skin and mild to mod-
erate AK is 2,4,6-octatrienoic acid. Preclinical
data published on this parrodiene derivative
demonstrate its activity in skin cell lines and a
commercial skin model as an endogenous skin-
protective agent. Its biological mechanism of
action involves PPARcactivation, which helps
to reduce oxidative damage and induce DNA
repair mechanisms. In UVB-irradiated NHKs
post-treated with 2,4,6-octatrienoic acid, the
authors observed not only a decrease in the
total expression of c-H2AX with respect to UVB-
irradiated cells, but also a lower percentage of
super-bright-positive cells, i.e., cells in which
the cell cycle to repair DNA damage has been
arrested [18,19].
Because data on and treatment options for
hyperkeratotic AK lesions are currently lacking,
the aim of the study reported in the present
paper was to evaluate the safety and efficacy of a
topical product containing keratolytic urea and
2,4,6-octatrienoic acid when applied to hyper-
keratotic AK lesions.
METHODS
Study Design
This study was a spontaneous single-center,
single-arm, open-label, phase IV study.
Subjects and Study Assessment
In the study, 40 subjects with at least two or
more clinically hyperkeratotic AK lesions on the
face (including the periorbital region, eyelids,
ears, and lips) were enrolled. Subjects were
asked to avoid using any topical product con-
taining retinoic acid, retinol, vitamin C, sali-
cylic acid, or alpha-hydroxy acids in the treated
area one week before and during the entire
study period. Subjects were not enrolled if they
had actinic keratosis treated with ablative laser
within six months of the study, with other
topical therapies (including cryotherapy)
within three months of the study, or with
intense pulsed light treatment within
one month of the study. Patients with skin
conditions who underwent interventions that
could interfere with the diagnosis and/or eval-
uation of AK were also excluded. Subjects were
asked to apply the topical product (2,4,6-octa-
trienoic acid 0.3% and urea 15%) twice daily for
two consecutive months. A sunscreen (Licht-
ena
Ò
Dermosolari 50 ?) was also provided.
Photographs were taken at baseline and at
the end of the treatment period. A dermato-
logical evaluation and epiluminescence micro-
scopy analysis (Molemax HD, Derma Medical)
were carried out before the treatment (T0), at
the end of the treatment period (T1), and after
three months of follow-up (T2) to check that
the AK had visibly completely cleared. Because
defining treatment success as the complete
clearance of all AK lesions could underestimate
the clinical benefits of treatment in patients
with multiple lesions (e.g., the resolution of
nine out of ten lesions would be classed as
treatment failure, and patients frequently
experienced an increase in the lesion count
during the study due to the appearance of sub-
clinical lesions), an endpoint of partial clear-
ance (C75%) was introduced. Partial clearance
is a more clinically powerful endpoint than
complete clearance, and has been used in other
clinical trials investigating AK treatments
[16,17]. Treatment efficacy was therefore mea-
sured based on the following clearance per-
centage scale: partial clearance (C75%); mild
clearance (C10% and \75%); minimal to no
clearance (\10), i.e., worsened (observable
increase in AK lesions), and unable to be
assessed.
Statistical Analysis
Descriptive statistics (i.e., mean and standard
deviation, SD) were calculated for all continu-
ous variables, while frequencies were deter-
mined for all categorical variables. Comparisons
between visits were performed by means of the
two-sample ttest. Pvalues of less than 0.05 were
considered clinically significant.
Dermatol Ther (Heidelb)
Compliance with Ethics Guidelines
All procedures followed were in accordance
with the Helsinki Declaration of 1964, as revised
in 2013. Informed consent was obtained from
all patients before they were included in the
study.
RESULTS
A total of 40 subjects (26 men and 14 women)
with a mean age of 71.075 ±7.426 years and a
mean number of AK lesions of 3.650 ±1.252
were enrolled and received treatment (Table 1).
AK lesion clearance was assessed after
two months of treatment with 2,4,6-octa-
trienoic acid and urea (Table 2). Compared to
baseline, the treatment reduced the mean
number of AK lesions by 83.56%, from 3.650 at
baseline to 0.600 at the end of the treatment
period (P\0.0001) (Fig. 1). The AK count after
three months of follow-up showed a reduction
in the number of AK lesions since the end of the
treatment period of 41.47% (p\0.0001)
(Fig. 1).
Table 2shows that 23 subjects (57.5% of all
subjects) presented complete clearance of all AK
lesions at T1. Partial clearance (C75% of all
lesions cleared; including patients with com-
plete clearance) was observed in 32 patients
(80%), while mild clearance was seen in six
subjects (15%). No clearance was reported in
two patients.
During the three-month follow-up visit,
82.5% of the subjects showed total clearance, as
the AK lesions had completely cleared during
the period from T1 to T2 in 11 patients
(Table 3). New AK lesions were observed in four
subjects at T2.
Some representative images before treatment
and at the end of treatment are shown in Fig. 2.
In general, no side effects were reported,
even during treatment of the periorbital region
(Fig. 2).
Fig. 1 Mean number of AK lesions per subject before
treatment (T0, baseline), at the end of the treatment
period (T1), and at the three-month follow-up visit (T2
FU). ***p\0.0001 vs T0
Table 3 AK treatment efficiency at T2
AK lesion status Subjects
(N= 40), n(%)
Complete clearance (100%) 33 (82.5%)
Complete clearance maintained from
T1
22 (55%)
Complete clearance achieved during
period from T1 to T2
11 (27.5%)
New AK lesions observed at T2 4 (10%)
Table 1 Subject characteristics
Demographic Subjects (N= 40),
n(%)
Men (n, %) 26 (65%)
Women (n, %) 14 (35%)
Age (years, mean ±DS) 71.075 ±7.426
No. of AK lesions (n, mean ±DS) 3.650 ±1.252
Table 2 AK treatment efficacy at T1
Proportion of AK lesions cleared Subjects
(N= 40), n(%)
Complete clearance (100%) 23 (57.5%)
Partial clearance (C75%, including
subjects with complete clearance)
32 (80%)
Mild clearance (C10 and \75%) 6 (15%)
No clearance 2 (5%)
Dermatol Ther (Heidelb)
Fig. 2a–b Representative images of two subjects (aand b) at baseline (A) and at the end of the treatment period (B)
Dermatol Ther (Heidelb)
DISCUSSION
AK is very common, and there is a risk that
untreated lesions will progress to SCC. Many
therapies for actinic keratosis are tolerated well
and produce excellent outcomes, but only a few
of those can be used for hyperkeratotic AK.
Treatment efficacy depends strongly on the
penetration of the topical drug into the epi-
dermis. As actinic keratosis lesions are com-
monly hyperkeratotic, they can be difficult to
penetrate, so the use of a keratolytic agent is
highly recommended.
Recently, some studies have reported the
efficacy of a product containing 2,4,6-octa-
trienoic acid for the prevention and treatment
of nonhyperkeratotic actinic keratosis lesions
[20]. The presence of a hyperkeratotic lesion has
usually been an exclusion criterion in relevant
clinical trials, so few studies have addressed this
type of lesion. Available methods of treating
hyperkeratotic AK lesions show some disad-
vantages, such as pain, discomfort, possible
infections, and hypo- or hyper-pigmentation in
the case of curettage or excision. Chemical
keratolytic agents present a better safety profile.
The combination of 2,4,6-octatrienoic acid
with urea should be a treatment option for hyper-
keratotic AK lesions. Subjects with two or more AK
lesions treated twice daily with 2,4,6-octatrienoic
acid and urea for 60 consecutive days showed a
significant reduction in the number of lesions,
with complete clearance of lesions observed by the
end of the treatment period in 55% of the subjects
and complete clearance achieved in 27.5% of
subjects during the first three months after the
treatment period (i.e., the follow-up period). The
present study showed that hyperkeratotic AK
lesions can be cleared through the topical appli-
cation of 2,4,6-octatrienoic acid and urea. This
topical formulation was tolerated well during the
two-month treatment period, including when it
wasusedintheperiorbitalarea,andnoadverse
effects of this treatment were reported.
Limitations
A major limitation/shortcoming of the current
study is that our evaluation includes the results of
applying a no-drug treatment in a small number
of subjects. Moreover, in an attempt to perform
the investigation under real-life conditions, sub-
jects from the same region of Italy were chosen for
the study. A future study with a shorter treatment
period, a longer follow-up period, and larger
clinical trials should more accurately establish the
efficacy of this alternative treatment.
CONCLUSIONS
In conclusion, 2,4,6-octatrienoic acid and urea
twice daily for 60 consecutive days was found to
be a safe and effective alternative treatment for
hyperkeratotic AK lesions.
ACKNOWLEDGEMENTS
We thank the participants of the study.
Funding. The study and article processing
charges were funded by Giuliani SpA. All authors
had full access to all of the data in this study and
take complete responsibility for the integrity of
the data and the accuracy of the data analysis.
Authorship. All named authors meet the
International Committee of Medical Journal
Editors (ICMJE) criteria for authorship for this
manuscript, take responsibility for the integrity
of the work as a whole, and have given final
approval for the version to be published.
Disclosures. B. Marzani is a full-time
employee of Giulini SpA. D. Pinto is a full-time
employee of Giuliani SpA. M.P. De Padova and
F. Rinaldi have nothing to disclose.
Compliance with Ethics Guidelines. All
procedures followed were in accordance with
the Helsinki Declaration of 1964, as revised in
2013. Informed consent was obtained from all
patients before they were included in the study.
Data Availability. The datasets used and/or
analyzed during the current study are available
from the corresponding author on reasonable
request.
Dermatol Ther (Heidelb)
Open Access. This article is distributed
under the terms of the Creative Commons
Attribution-NonCommercial 4.0 International
License (http://creativecommons.org/licenses/
by-nc/4.0/), which permits any noncommer-
cial use, distribution, and reproduction in any
medium, provided you give appropriate credit
to the original author(s) and the source, provide
a link to the Creative Commons license, and
indicate if changes were made.
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