ARTICLE
Auteur(s) : Audrey Guéniche1,
Anca Hennino2, Catherine Goujon2, Karima
Dahel2, Philippe Bastien3, Richard
Martin4, Roland Jourdain1, Lionel
Breton1
1L’OREAL Recherche, Clichy, France
2Clinical Immunology and Allergology, Lyon-Sud Hospital,
Pierre Bénite and INSERM U503, IFR 128, Lyon, France
3L’Oréal Recherche, Aulnay-sous-bois, France
4C/O CRN, L’OREAL, Notre Dame D’OE, France
accepté le 9 Février 2006
Atopic dermatitis (AD) is a chronically relapsing inflammatory skin
disease. It is characterized by phases of intense pruritus,
numerous lesions with erythema, excoriation, erosion accompanied by
a serous exudate, lichenification, papules, dry skin and a
proneness to cutaneous infections [1]. One of the main
abnormalities of AD skin is a defect in the epidermal barrier
function leading to increased penetration of environmental
compounds which activate and perpetuate the AD skin symptoms [2].
The daily use of emollients is the first line therapy of AD [3, 4].
Emollients can improve AD skin symptoms so sufficiently as to be
the only treatment in some patients with mild and moderate AD. In
others, the use of topical corticosteroids and/or immunomodulators
together with emollients is necessary to control the symptoms [5].
However, very few studies have addressed the question of the best
composition of emollients specifically formulated for AD skin.
Emollients restore to some extent the skin barrier defect and may
improve AD skin symptoms by preventing the penetration of
environmental compounds. Furthermore, emollients may contain
ingredients endowed with properties which balance cutaneous immune
homeostasis and therefore may be more efficient for the improvement
of eczema and the prevention of relapses.Vitreoscilla filiformis
(V.f.) is a non photosynthetic non fruiting gliding bacterium. This
filamentous micro-organism is found in sodic sulphureous thermal
springs recognized for several years in phytomedicine for their
local moisturising properties. Furthermore, V.f. extracts
demonstrated anti-irritant properties and promoted wound healing in
preliminary studies. These observations suggested that a V.f.
extract could be used in skin care preparations to improve the AD
skin symptoms. In the present randomised, double-blind,
vehicle-controlled clinical study, we confirm this hypothesis and
report that a 5% V.f. extract-containing ointment improves the
inflammatory skin symptoms of mild to moderate AD.
Material and methods
Preparation of V.f. bacterium extract
V. f. is a non photosynthetic, non fruiting gliding bacterium
belonging to the Beggiatoales order as defined in the Bergey’s
classification [6]. Culture conditions may be briefly described as
follows.
The culture medium is prepared with an autolytic yeast
extract (2 g/L), a soya papainic peptone (2 g/L), glucose (3 g/L),
Heller microelements (1 mL/L) and CaCl2, 2
H2O (60 mg/L).Then the pH is adjusted at 7.20 before
sterilization. After sterilization (121 °C/30’) the bioreactor
medium is inoculated with V. f. (20 mL/L) issued of an
Erlenmeyer batch culture. The batch step of the process is
monitored on the following parameters: dissolved oxygen
(> 10%), T° (28 °C) and pH (pH stat = 7.00). Due to
its sheer stress sensitivity, we used a draft tube agitation system
to cultivate this strain. After 24~36 hours, the continuous culture
step is engaged. According to the μmax (0.2 H–1) the
culture is controlled by the injection of fresh medium with a μ =
0.12 H–1. In the same time we harvested equivalent
volume of the whole culture in order to maintain the same culture
volume in the bioreactor. The cells were separated from this
continuous flow of the whole culture by centrifugation
(10,000 g for 10 min). Finally, the biomass was
stabilized by heat treatment (121 °C for 30 min). The
reproducibility of the biomass is improved by this continuous
production.
Verum and vehicle ointments
In order to demonstrate the efficacy of V.f. on AD skin
inflammation we chose a standard base cream which contains a
mixture of glyceryl mono/distearate and polyethylene glycol
stearate, isoparaffin and cyclopentadimethylsiloxane. This base
corresponds to an ointment not specifically designed for atopic
skin. The vehicle was the base cream and the verum was prepared by
mixing 5% of the V.f. biomass with the base cream.
Study design
The monocenter study was performed during autumn and early winter
in order to avoid seasonal variations of the disease. The study
involved a screening visit for patient selection and 3 visits for
clinical examination at the start of the treatment (Day 0) then
after 2 and 4 weeks treatment respectively. The ethics committee
reviewed the protocol and granted approval of the study before its
implementation.
Patient selection
Thirteen male or female AD patients aged 14 years and older (mean
35.7 ± 16.5) with a slight to moderate AD according to Rajka &
Langeland criteria [7] and disease involvement of 5% to 60% of the
total body surface area (BSA) were enrolled in the study. The main
exclusion criterion was a serious skin disorder other than AD
requiring treatment. Each patient (or parents for younger patients)
gave written informed consent.
Randomisation and blinding
Each patient was assigned a number at baseline visit. For all
patients, treatment was randomised according to body side (ointment
containing 5% V.f. extract versus vehicle ointment). Both ointments
(verum and placebo) were provided in tubes alike in all respects to
safeguard blinding. Treatment boxes for each body side bore no
information that might suggest they contained the study ointment or
vehicle.
Treatment
At baseline, investigators defined symmetrical areas to be treated,
gave the first ointment supply box, and explained the dosing
procedure to patient. Treatment procedures consisted of applying a
thin layer of ointment containing 5% V.f. extract twice daily to
affected areas on one side of the body and applying the vehicle on
the corresponding lesions on the contralateral side of the body.
The following treatments were prohibited during the study: topical,
inhaled, intranasal or systemic corticosteroids, antimicrobials,
histamines, coal tar, topical non steroidal anti-inflammatory
drugs, non steroidal immunosuppressants, UV light treatments,
hypnotics, sedatives, other investigational drugs, bath oil and
nonmedicated emollients.
Assessment
At baseline (Day 0), and after 2 and 4 week treatment, the
investigator scored the following symptoms on each side of the body
(right and left): erythema, edema-induration-papulation,
excoriations, and lichenification using a scale 0 to 3 and
estimated the percentage of total BSA affected by AD (0%-100%) in 4
body regions (head and neck, trunk, upper limbs, and lower limbs).
Patients self-assessed the intensity of itching experienced during
the 24 hours preceding clinical examination using a 10-cm visual
analog scale, with 0 cm indicating “no itch” and 10-cm
indicating “worst imaginable itch” at each side of the body (right
and left). This assessment was used to calculate the modified
eczema area and severity index (mEASI) at each side of the body
(right and left). The mEASI is a variant of the eczema area
severity index (EASI) developed by Hanifin et al. [8]. The mEASI is
almost identical to the EASI but the former also includes an
assessment of itching, considered as a primary symptom of AD [16].
Both EASI and mEASI have the advantage of including severity scores
for individual symptoms of AD weighted according to the extent of
affected BSA.
For each body region (head and neck, upper limbs, trunk and
lower limbs) and each side of the body (right or left), the
following steps were carried out: (a) an affected area score of 0
to 6 was assigned for the percentage of affected BSA (0%-100%); (b)
the individual ratings for erythema, edema-induration-papulation,
excoriations and lichenification were totaled (0 to 3 for each of
the 4 symptoms); (c) the sum of individual symptoms (maximum = 12)
was multiplied by affected area score (maximum = 6), with a maximum
of 72; (d) since the patients were older than 7 years, the head and
neck subtotal was multiplied by 0.1, the upper limb subtotal by
0.2, the trunk subtotal by 0.3, and the lower limb subtotal by 0.4
(e). All components were summed (maximum EASI = 72); and (f) the
patient’s assessment of itching intensity was converted to an
ordinal scale of 0 to 3 and then multiplied by the investigator’s
total affected area score (0-6), with a maximum itching score of
18. The EASI was summed with the itching score, with a maximum
mEASI of 90 (the sum of 72 and 18). This system of scoring AD is
similar to the SCORAD index developed by the European Task Force on
AD [9].
Investigators also assessed overall clinical improvement in the
physician’s global evaluation of clinical response. “Cleared”
indicated improvement by 100%, “excellent” by 90% to 99%, “marked”
by 75% to 89%, “moderate” by 50% to 74%, “slight” by 30% to 49% and
“no appreciable improvement” by 0% to 29% respectively. “Worse”
indicated a worsening of the condition.
Adverse events were monitored on an ongoing basis. An adverse
event was defined as any undesirable experience that occurred in a
patient during the clinical trial, regardless of whether it was
considered related to the product applied or not. Causally related
adverse events were those assessed by the investigators as having a
highly probable, possible, or not assessable relationship to the
treatment product applied or where noted, adverse event data were
presented irrespective of causal assessment.
Statistical analyses
The primary population was an intent-to-treat population, which
comprised all patients who were randomised and received at least
one application of study ointment. The primary endpoint was mEASI.
The evolution of mEASI, pruritus and affected body surface area
from baseline to D14 and to D28 (end of the treatment) were
analysed with paired non parametric tests (Wilcoxon signed ranks
sum test) [10]. Due to the limited number of patients, asymptomatic
results from inferential analyses could be misleading. Therefore,
exact significance testing procedures based on two-sided
non-parametric tests were performed. Statistical analyses were
carried out using SPSS (SPSS Inc. Chicago, Illinois, release 11.0).
The significance level was set at 5%.
Results
Efficacy
The study was conducted in 13 Caucasian AD patients (5 males and 8
females) aged 14 and older (mean age 35.7 ± 16.5). All patients had
an active and extensive disease at baseline (in average one third
of total body area affected by AD). Randomization resulted in a
good matching between treatment sides. No statistically significant
difference in the severity of AD between patient’s sides was
observed at baseline. Patients applied the verum and vehicle on all
symmetrical AD skin lesions.
AD elementary clinical symptoms were documented for each patient
by mEASI. Comparison was done between the 5% V.f. ointment treated
side and vehicle treated side (figures 1A and 2). Improvement
in mEASI was observed in V.f. treated side at day 28 with a 42.9%
decrease in mEASI, whereas vehicle treated side showed a decrease
by 24.8% only (p = 0.008; Wilcoxon signed ranks test). Pictures of
skin symptoms were obtained at selected sites during the course of
the study. Representative patients are showed in ( figure 3 ).
Statistical analyses were also performed as secondary endpoints
on the different parameters involved in the mEASI index including
EASI, the pruritus severity index and the affected body surface
area. There was a significant decrease in EASI index in favour of
the treated side at day 28 (p = 0.012; Wilcoxon signed ranks test)
( (figure 1B) ).
The severity of pruritus was evaluated as a subjective measurement
of clinical efficacy. At baseline, no significant differences were
noted between body sides (right or left). After 28 days of
treatment, the level of pruritus was significantly decreased in the
V.f. treated side compared to the vehicle treated side (p = 0.046)
( (figure 1C) ).
No significant difference in pruritus was observed at Day 14, as
the affected body surface area at any assement time ( (figure 1D) ).
Tolerance
The most common adverse events were pricking and burning
sensations, with the same intensity in both sides and thus they
were likely to be related to the vehicle. Three patients reported
these events in the context of local discomfort. The incidence and
intensity of these side effects shortly decreased and never lasted
more than 15 minutes after topical application. Only a few
sensations of dryness appeared (7%) on both treatment sides.
Discussion
In this study, the ointment containing 5% V.f. extract was found to
significantly alleviate the signs and symptoms of atopic skin in
the study population when applied twice daily on all AD skin
lesions compared to symmetrical AD lesions which received the
placebo. It showed a relatively rapid onset of action, with
beneficial effects observed after only 15 days of treatment and
tending to increase thereafter.
V.f. topical ointment was well tolerated, with most adverse
events being localized, transient in nature and of moderate
severity. These reactions of skin discomfort expressing as burning
and stinging were observed with the vehicle and are to be related
to the base formulation chosen to formulate the V.f. extract.
Indeed, since emollients are part of the treatment of AD and could
improve skin lesions when used alone we decided to test the
efficacy of V.f. in a topical preparation devoid of strong
emollient properties. Since the V.f. extract was able to improve
atopic skin lesions despite the non optimal formulation of the
vehicle, we postulate that its effect would be better when
formulated in an emollient specifically designed for atopic skin.
Future studies will test this hypothesis.
Although V.f. extract was efficient in reducing the inflammatory
signs of AD skin lesions, we do not know which components in the
V.f. are responsible for the positive effects and by which
mechanisms they improved the AD skin symptoms. The bacterial
extract could contain some molecules able to restore the AD skin
barrier impairment and therefore participate to the limitation of
the penetration of pro-inflammatory environmental factors.
Alternatively, some components of the V.f. bacterial extract could
bear activities comparable to those found in cell walls of
saprophyte bacteria from the gut [11]. Some unpublished studies
have revealed that topical application of V.f. extract could
decrease the intensity of arachidonic acid-induced skin
inflammation and could down-regulate oxazolone-induced contact
hypersensitivity. Moreover, unpublished studies have shown that
V.f. extract promotes healing of epidermis and dermis in wound
healing studies following suction blisters or skin incisions.
Finally, a pilot clinical study has shown that the extract of V.f.
includes moisturizing properties for the skin and protects skin
against ultraviolet alterations.
While further investigations need to be done to more precisely
define the mode of action of the V.f. extract, our results show
that its incorporation into the formulation of emollients may help
to reduce the AD skin symptoms.
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