ARTICLE
Auteur(s) :, D.D. VERMA1,*, S.
VERMA1, K.J. McELWEE2, P.
FREYSCHMIDT-PAUL2, R. HOFFMAN2, A.
FAHR3
1Institute for Pharmaceutical Technology and
Biopharmacy, Department of Pharmaceutical Sciences, Bouve College
of Health Sciences, 312 Mugar Building, Northeastern
University, 360 Huntington Avenue, Boston, MA 02115, USA
2Department of Dermatology, Philipp University, Marburg,
Germany
3Institute of Pharmaceutical Technology, FSU Jena,
Germany
*D.D. Verma, Fax: (+1)-617-373-8886. E-mail:
ddverma@yahoo.com
accepté le 26 Avril 2004
Alopecia areata (AA) is a chronic cutaneous disease with a
suspected autoimmune origin [1]. The severity of AA ranges from
localized patchy areas of hair loss to a total loss of scalp hair
(Alopecia totalis) or a loss of all body hair (Alopecia
universalis). Histopathologic features of AA include a
perifollicular lymphocytic infiltrate involving the anagen hair
follicles with subsequent miniaturization of these follicles [2].AA
has been described in various animals, including cats, dogs, horses
and non-human primates [3, 4]. In the larger species, AA is poorly
characterized. The animals are outbred and are not readily
available for study, which makes them of limited practical use as
research models. A type of reversible hair loss, that closely
resembles human AA, both clinically and histopathologically, has
been described in C3H/HeJ mice [5] and the Dundee Experimental Bald
Rat (DEBR) model. In the DEBR, AA arises spontaneously with a low
frequency in rats beginning at about 6 months of age. In
females the expression frequency can approach 70% by 18 months
of age [6]. Affected animals first develop patchy hair loss on the
head with hair loss on both the ventral and dorsal surfaces
developing shortly thereafter. Histopathologic examination shows
that these rats develop nonscarring alopecia with dystrophic anagen
hair follicles surrounded by mononuclear cell infiltrates with a
predominance of CD4+ lymphocytes and small numbers of
CD8+ cells [7].CyA, a cyclic endecapeptide, is a T
cell-specific immunosuppressant and, due to its lack of bone marrow
toxicity, has assumed a leading role in therapy after organ
transplant surgery. CyA has provided new approaches in the
treatment of several diseases. It has shown remarkable efficacy in
psoriasis and has shown potential usefulness in other
dermatological diseases that are thought to have an inflammatory
T-cell-mediated pathogenesis [8]. One of the most common
dermatological side effects of oral CyA is hypertrichosis [9]. This
stimulating effect on hair growth has encouraged a number of
investigators to consider CyA not only for the treatment of AA, but
also for androgenetic alopecia (AGA). However, while oral
application has proved successful, the beneficial effect of topical
application is very limited in both AA and AGA [10-11].Liposomes
for topical drug delivery were first introduced in 1980 and since
then have attracted considerable interest and generated many
speculative claims concerning their potential utility both as a
drug carrier to the hair follicle [12] and reservoir for the
controlled release of drugs within various layers of the skin [13].
Liposomes are microscopic vesicles consisting of amphipathic lipids
arranged in one or more concentric bilayers. These
thermodynamically stable, lamellar structures form spontaneously
when the lipid is brought into contact with an aqueous phase.
Unlike micelles, emulsions, and microemulsions, liposomes have an
entrapped, discontinuous aqueous phase separated by 4-nm thick,
bilayered lamellae from the continuous aqueous phase [14]. Besides
reducing the undesirable high systemic absorption of drugs, the
major advantage of applying liposomes topically, in comparison to
other formulations such as ointments or creams, is based on their
ability to create a depot from which the drug is slowly released.
In addition, they mediate the permeation into the skin of
therapeutic amounts of hydrophilic or lipophilic drugs with
otherwise poor penetration properties [14].First results on topical
liposomal delivery were reported by Mezei and co-workers. These
in vivo studies documented comparisons between liposomal and
conventional formulations of triamcinolone acetonide [15, 16]. In
both studies, the application of the liposomal preparations was
associated with greater steroid concentrations in the epidermis and
dermis as well as with less systemic absorption as compared to the
regular formulations. Further permeation studies in animals have
demonstrated that liposomal encapsulation can improve the
penetration of various molecules. Enhanced delivery into the skin
has been reported for lidocaine [17] and caffeine in hairless rats
[18, 19]. The penetration kinetics of molecules from liposomes has
also been assessed in in vitro skin studies. Incorporation
into liposomes, resulted in the increased uptake of hydrocortisone
[20], insulin [21] and cyclosporin [22] into the cornified layer of
hairless mice and/or guinea pigs. In another study, localized and
prolonged activity of a local anaesthetic contained in liposomes
was reported, when compared with an equivalent conventional topical
application [23].In a growing number of topical studies, vesicles
have been shown to target drug delivery to the pilosebaceous unit
[24]. Experiments with the Syrian hamster ear model have
demonstrated that carboxyfluorescein-loaded liposomes delivered
much higher drug concentrations into the sebaceous glands than
conventional carboxyfluorescein formulations [25]. Li and
co-workers found that liposomal entrapment of calcein [26], melanin
[27] and DNA [28] resulted in specific delivery into the hair
follicles of histocultured mouse skin, while aqueous control
solutions of these molecules showed no preferential drug
localization within the follicle. Liposome-entrapped melanins,
proteins, genes, and small-molecules have also been selectively
targeted to the hair follicle and hair shafts of mice in
vivo. Liposomal delivery of these molecules is time dependent
and negligible amounts of molecules enter the dermis, epidermis, or
bloodstream demonstrating selective follicle targeting [12].While
AA in humans is known to respond to systemically administered CyA
[29], systemic CyA administration is associated with serious side
effects, especially nephrotoxicity. A topical CyA formulation is
one approach to limit the risk of side effects. Here we describe a
preliminary investigation into the potential of specially designed
vesicles that contain CyA, for topical immunotherapy of AA using
the DEBR model.
Materials and Methods
Vesicle formulation
NAT 8539 was purchased from Nattermann Phospholipid GmbH,
Cologne, Germany, and contained phosphatidylcholine and
lysophosphatidylcholine as ≥ 75 and 5% of the dry
residue respectively, natural oils, sterol 5%, and 25% w/w ethanol.
The terpenes, d-limonene, citral and 1,8 cineole, were
purchased from Sigma-Aldrich, Seelze, Germany. CyA was purchased
from Galena A.S., Opava, Czech Republic. All other chemicals were
of analytical grade and the water used was double distilled,
deionized and filtered with a Milli-Q system (Darmstadt, Germany).
The invasomal systems investigated here were composed of 10% wt.
NAT 8539, 0 or 2% wt. of a mixture of terpenes [d-limonene:
citral: cineole - 10:45:45 (PE)], CyA 5 mg/ml and phosphate
buffered saline (PBS) pH 7.4 to 100%. Vesicles were prepared
by the ethanol dissolution method [30]. To produce vesicles
containing PE (CyA vesicles PE), the drug and PE were dissolved in
an ethanolic solution of lipids. In the case of vesicles without PE
(CyA vesicles), the drug alone was dissolved in an ethanolic
solution of lipids. The mixture was vortexed for 5 min
followed by sonication (Branson ultrasonic cleaner, Connecticut,
USA) to form a clear, transparent solution. PBS was added to this
mixture with the help of a syringe attached to a needle (21G x 2”,
0.8 x 50) under constant vortex mixing. The vortexing was
continued for an additional 5 min. This mixture was sonicated
for 5 min. This lipid coarse suspension was kept at room
temperature for 5 h and then was extruded through
polycarbonate membranes of different pore sizes (400 to
50 nm) (Nuclepore GmbH, Tubingen, Germany) using a
high-pressure homogenizer, Emulsiflex C5 (Avestin, Ottawa,
Canada).
> 0.3) indicates a high degree of heterogeneity. The
polydispersity index was in the range of 0.25 to 0.44.
Drug application
Fifteen male and female DEBR were used at a mean age of
19 months (range 14-26). The rats were individually caged with
acidified water and food ad libitum (Altromin diet 1324, Altromin
International, Lage, Germany) for the duration of the study. Hair
loss ranged from large bald areas on the flanks and hairless
patches on the head to almost complete loss of the head and body
hair.
Rats were matched for hair loss, age, and sex allocated into
three groups of five animals per group (group I, II and III). The
study was conducted with vesicles containing 0.5% of CyA in
2 liposomal formulations, namely with and without PE and with
0.5% CyA in ethanol. Vehicle control formulations were prepared in
the same manner but without CyA. Liposomal formulations were
applied non-occlusively to the rats. For dose application, a
specific area of 4 cm2 was marked with tattoos on
the left and right flanks. The dose application started with
20 μl/cm2 in the first week,
40 μl/cm2 in the second week,
50 μl/cm2 in the third week and
80 μl/cm2 from four to six week for all
3 groups. Each rat received treatment two times a day everyday
for 6 weeks within the marked area on one bald flank, while
the contralateral flank was treated with the control formulation.
Group I was used to study the potential of CyA vesicles PE on hair
growth. Group II was used to study the potential of CyA vesicles
without PE and Group III rats received an ethanolic solution of
CyA.
Morphological changes were examined and documented weekly.
Photographs were taken before, during, and at the end of the study.
Hair growth density within the tattooed, treated areas was recorded
through visual evaluation by two investigators (DDV, KJM) on an
arbitrary scale of 7 intervals from no visible hair (category
0) to a pelage coat of normal hair density (category 6). The same
scale was used in previous topical treatment studies employing the
DEBR model [31]. After 6 weeks of treatment the rats were
necopsied. The drug treated and vehicle treated skin was removed
and fixed in Fekete’s acid-alcohol-formalin solution, processed
routinely, sectioned at 5 μm and stained with hematoxylin and
eosin [32]. Histological examination was conducted on frozen
sections from biopsies taken from the CyA vesicles treated and
control formulation treated sites. Cellular inflammation was
recorded on an arbitrary scale of: No visible infiltrate present
(category 0) < sparse infiltrate present (category
2) < moderate infiltrate present (category
4) < dense infiltrate present (category 6).
Analysis of raw hair density and histological data was conducted
with the Mann-Whitney-Wilcoxon test. Statistically significant
differences between the drug treated site and control formulation
treated site were determined with
P < 0.05 regarded as a minimal level of
significance. From raw hair density categorization, a hair growth
index was calculated by counting the number of category changes
between baseline and the hair growth status as recorded in the
first, second and sixth weeks for each rat on drug treated and
control treated sites. For inflammation intensity categorization,
the recorded category values for drug treated and control treated
skin in each rat were added and averaged for each group.
Results
Macroscopic observations
Before treatment, rats had large, bald flanks on the abdominal,
dorsal, head and shoulders areas (( Fig. 1a and 1b )). The
formulation was applied on the left side of dorsal surface to an
area of 4 cm2. The control formulation was applied
on the right side of the dorsal surface of each rat. All
5 rats in group I treated with CyA vesicles PE first exhibited
hair growth on the drug treated site in the first week of drug
application (P value from 0.5 on week 0 to 0.06 on
week 1). In the second week, one rat was dropped from the study due
to an allergic response to the tattoo marks. An allergic reaction
to the tattoo ink was confirmed in subsequent studies of tattooed,
but untreated rats (data not shown). However, the rat showed good
hair growth on the drug treated site and no hair growth was
observed on the control treated area in the first week of drug
application. This pharmacodynamic effect was supported by
histological examination that suggested a decrease in the
inflammatory infiltrate in drug treated skin after the first week
of treatment. After three weeks of drug application, three rats of
four in group I exhibited sparse to moderate hair growth from drug
treated skin and one rat showed sparse hair growth over the whole
body, but more pronounced on the drug treated site. This effect was
probably due to either spontaneous remission or licking of the
applied formulation by the rat. Further hair growth was rapid on
all 4 rats and by the end of week five, the pelage coat had
reached its maximum density where the formulation was applied and
persisted until necropsy (( Fig. 1a-1e )). As hair
growth continued at the application site, there was progressive
hair loss elsewhere on some of the rats.
The first sign of hair regrowth was seen in all rats in group II
on the drug treated site after two weeks. No hair growth was
observed on the control-treated area. One rat from this group was
removed from the study due to illness. Hair continued to grow
within, and immediately adjacent to the drug application sites of
the remaining 4 rats by the end of the third week of drug
application (( Fig. 2 )). There were
regions of sparse to moderate growth of hair in all the rats. After
five weeks of the drug application, three out of four rats
exhibited drug treated skin with moderate hair growth and one rat
showed sparse to moderate growth of hair. The hair growth was
progressive and reached a maximum density at the site of drug
application by the end of week six. A rise in P value from week
three to week six (Table I( Table I )) could be explained on the basis
that one rat showed extensive hair regrowth over the whole body.
Group III rats, which received an ethanolic solution of CyA, showed
no visible signs of hair growth within the drug application area
and there was a progressive hair loss on all rats.
Table I The P values calculated using the
Mann-Whitney-Wilcoxon test showing the statistical difference in
hair growth between the drug treated and control treated site for
rats within each treatment group
|
P values
|
|
Group
|
Wk0
|
Wk1
|
Wk2
|
Wk6
|
|
I
|
0.5
|
0.06
|
0.01
|
0.01
|
|
II
|
0.5
|
0.23
|
0.08
|
0.23
|
|
III
|
0.42
|
0.3
|
0.1
|
0.32
|
Histological investigation
The drug treated and the control treated sites in all the rats in
groups I, II and III were investigated for the degree of
inflammatory infiltrate in and around the hair follicles (( Fig. 3 )).
Histological examination of tissue from group I and II rats
consistently revealed that the control treated skin site (( Fig. 4a, 4b ))
contained a higher number of inflammatory infiltrate cells
associated with extensive hair follicle dystrophy, as compared to
the drug treated site (( Fig. 4c, 4d )). In
group I treated with CyA vesicles PE, all rats showed some degree
of reduction in the inflammatory infiltrate in treated skin versus
control vehicle treated skin. Two rats exhibited the presence of a
moderate infiltrate while two rats had a sparse to moderate
infiltrate on the drug treated site. Overall the rats in group I
showed a statistically significant reduction in inflammation
intensity on the drug treated site as compared to control treated
site (P value 0.02). In group II treated with CyA vesicles alone,
two of five rats demonstrated a significant reduction in the
infiltrate in drug treated skin, as compared to the control treated
skin. The statistical analysis showed a P value of 0.07 after
the drug treatment for this group. In group III treated with an
ethanolic solution of CyA, two rats did not show any difference in
inflammatory intensity between drug treated and control treated
sites and for the remaining rats the difference was minimal (P
value 0.33).
Discussion
An effective topical preparation without systemic absorption or
toxicity is the ultimate goal of CyA topical therapy. CyA inhibits
T cell activation by interfering with the production of
interleukin-2 (IL-2) and by inhibiting IL-2 gene expression,
probably through the inhibition of calcineurin [9]. CyA is a
specific inhibitor of lymphocyte activation and, therefore, may be
useful in treating patients with AA [29]. This drug has been
reported to not only clear immune cells from the hair follicles,
but also to alter the balance of regulatory lymphocytes. CyA has
been reported to restore hair growth, when administered orally in
the DEBR model for AA [33] and in humans [29, 34]. On the other
hand, AA has been described in renal or liver transplant recipients
on high doses of CyA [35-37]. Large studies on AA treated with CyA
alone are lacking. However, as the activity of AA is localized to
epidermal appendages, it is neither necessary nor desirable to
suppress the entire immune system by systemic application. Systemic
immune suppression may release AA- affected hair follicles from
their dystrophic state, but it can potentially leave the patient
partially exposed to infection and side effects, including
neurotoxicity and impairment of renal function [9]. Topical CyA
application for treatment of AA has thus far met with little
success [10, 38].
Recent approaches in modulating drug delivery through the skin
have involved various classes of novel vesicular carriers based on
phospholipids and single, or mixtures of several membrane modifying
agents. The size of these vesicles can be modulated from tens of
nanometers to microns. These vesicular systems have been found to
be very efficient for the enhanced delivery of molecules with
different physico-chemical characteristics to and through the skin.
They can be modulated to permit enhanced delivery into the skin
strata as far as the deep dermis [39] or to facilitate transdermal
delivery of lipophilic and hydrophilic molecules [39-41]. The skin
permeation of minoxidil was reportedly enhanced by specially
designed vesicles in vitro as compared with either
ethanolic, hydroethanolic solutions or phospholipid ethanolic
micellar solution of minoxidil. In addition, the transdermal
delivery of testosterone from lipid vesicles patches was greater
both in vitro and in vivo than from commercially
available patches [40].
Ultradeformable carrier vesicles have been shown to be versatile
carriers for the local and systemic delivery of various steroids,
proteins and hydrophilic macromolecules [42]. The highly deformable
property facilitates their rapid penetration through the
intercellular lipid of the stratum corneum [43]. The osmotic
gradient, caused by the difference in water concentrations between
the skin surface and skin interior, has been proposed as the major
driving force for penetration of these vesicles [44]. Gap junction
proteins (GJP) incorporated into ultradeformable carriers and
applied to the intact skin surface gave rise to specific antibody
titers marginally higher than those elicited by subcutaneous
injections of GJP in vesicles, mixed lipid micelles or liposomes
[45]. Diclofenac association with ultradeformable carriers has been
shown to have a longer effect and to produce 10-times higher
concentrations in the tissues under the skin in comparison to the
drug from a commercial hydrogel [46]. Enhanced skin absorption has
been reported for the delivery of macromolecules, such as insulin,
when associated with ultradeformable carriers [43]. Drawing on the
published data described above, the approach to develop our
vesicles started with the fact the phospholipids and ethanol act as
penetration enhancers for substances into the skin. We further
tried to improve the penetration enhancement of substances by
making vesicles of acceptable size (~ 100 nm) and high
deformation ability by using the penetration enhancement potential
of phospholipids plus ethanol and a mixture of terpenes.
In this study, hair growth was visible at the CyA in liposome
application site in all rats in groups I and II within two weeks of
starting therapy. The hair regeneration was uniform rather than a
wave-like band of hair growth moving over the region as would be
seen in a normal rat pelage coat. This suggests that the hair
follicles were arrested in a dystrophic anagen state and were then
released into normal anagen activity by CyA, as has been described
following oral CyA treatment [33]. As hair fiber length in the CyA
vesicles PE treated regions seemed to be longer than would normally
be expected by completion of the study, it is possible that CyA may
directly act on dystrophic follicles. However, the primary
mechanism is likely to be through a localised reduction in the
mononuclear cell infiltrate activity.
The terpenes used in this formulation probably played an
important role in delivering the CyA into and around the hair
follicles. Terpenes act by increasing the partition coefficient of
the drug into the skin thereby increasing the thermodynamic
activity and solubility of the drugs in the vehicle [47]. A
synergistic effect of ethanol and terpenes can not be ruled out for
the enhanced delivery of CyA. There is some evidence in the
literature to support this notion. Cornwell and Barry reported the
additive effects of sesquiterpene and ethanol on the rate of
absorption of the model hydrophilic permeant, 5-fluorouracil [48].
The addition of 3% terpene in combination with 47% ethanol
increased the penetration of thyrotropin-releasing hormone and
pGlu-3-methyl-His2-Pro amide [49]. Kikuchi et al. determined
the amounts of d-limonene, ethanol and indomethacin which were
transferred from aqueous gel ointments to the rat skin. The
increase of d-limonene concentration in the gel ointments was
directly proportional to the accumulation of ethanol in the skin
and the amount of ethanol in the skin was closely associated with
the percutaneous absorption of indomethacin [50]. These and other
studies [51-53] show the enhancing activity of terpenes is
significantly affected by the concentration of ethanol in the
formulation. The presence of ethanol makes the bilayer of the lipid
more deformable [40], which may allow the vesicles to pass along
the side of the hair shaft to the hair follicle bulb region.
Therefore, the synergistic effect of ethanol and phospholipids
might be an explanation for the enhanced delivery of CyA and any
accumulation of the vesicles in the hair follicle might have
resulted in controlled delivery of CyA over a prolonged time
period.
The observations in this proof of concept preliminary study
indicated that CyA in liposomes had a localized hair growth
promoting effect, but may have been unable to penetrate through, or
diffuse laterally within the dermis at least not in a sufficient
amount for a pharmacological hair growth inducing response, as hair
elsewhere on some treated rats continued to be lost. CyA in
vesicles may have promising potential for use in humans. However, a
larger scale, detailed study is required to determine the dose
related efficacy of CyA vesicular formulations for human skin
application. Liposomal formulations of other drugs may also have an
application in the treatment of AA.
Acknowledgements
We are thankful to the Deutscher Akademischer Austausch Dienst
(DAAD) for their financial support of DDV. This work was supported
in part by the Deutsche Forschungsgemeinschaft (Ho 1598/8-1, PF-P,
RH). KJM is a recipient of the Alfred Blaschko Memorial Fellowship,
Marburg. We also would like to express our gratitude to Ms.
Kissling and Mr. Schaefer, Dept. of Dermatology, Philipp University
Marburg, for their technical assistance.
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