In vivo evaluation of piperine and synthetic analogues as potential treatments for vitiligo using a sparsely pigmented mouse model

Abstract

Piperine and its analogues have been reported to stimulate melanocyte replication in vitro and may be useful in treating the depigmenting disease, vitiligo.
To investigate the ability of piperine (PIP) and three analogues to stimulate pigmentation in a strain of sparsely pigmented mice.
The test compounds were PIP [5-(3,4-methylenedioxyphenyl)-2,4-pentadienoylpiperidine], tetrahydropiperine [THP, 5-(3,4-methylenedioxyphenyl)-pentanoylpiperidine], a cyclohexyl analogue of piperine [CHP, 5-(3,4-methylenedioxyphenyl)-2,4-pentadienoylcyclohexylamine], and reduced CHP [rCHP, 5-(3,4-methylenedioxyphenyl)-2,4-pentanoylcyclohexylamine]. Sparsely pigmented, HRA/Skh-II mice were randomized to receive topical treatment with test compounds or vehicle twice a day for five days a week, with or without ultraviolet (UV) irradiation on 3 days a week. Treatment was either continuous or interrupted to evaluate fading and repigmentation. Skin inflammation and pigmentation were evaluated regularly during treatment. DOPA+ melanocytes were determined histologically at the termination of treatment.
Four weeks of treatment with one of the compounds PIP, THP or rCHP, but not CHP, induced greater pigmentation than vehicle with low levels of inflammation. Additional exposure to UVR led to darker pigmentation than did the compound or UVR alone, and greater numbers of DOPA+ melanocytes were found. The combination produced an even pigmentation pattern, contrasting with the speckled, perifollicular pattern produced by UVR alone. Treatment interruption led to a decrease in pigmentation but not its loss. Repigmentation was achieved by administering one of the compounds, UVR or both, and occurred faster than in naïve mice.
Treatment with PIP, THP or rCHP and UVR induced a marked pigmentation response in HRA/Skh-II mice, with clinically better results than UVR alone. This result supports the potential use of these compounds in treating vitiligo.

Full text

CLINICAL AND LABORATORY INVESTIGATIONS DOI 10.1111/j.1365-2133.2008.08464.x
In vivo evaluation of piperine and synthetic analogues
as potential treatments for vitiligo using a sparsely
pigmented mouse model
L. Faas,*,–R. Venkatasamy,* R.C. Hider,* A.R. Young,# and A. Soumyanath*,,#
*Department of Pharmacy King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NN, U.K.
St John’s Institute of Dermatology, Division of Genetics and Molecular Medicine, King’s College School of Medicine, King’s College London, Guy’s Hospital,
London SE1 9RT, U.K.
#Equal last authors.
–Present address: Department of Biology, University of York, Heslington, York YO10 5DD, U.K.
Present address: Department of Neurology, Oregon Health & Science University, Portland, Oregon 97239, U.S.A.
Correspondence
A. Soumyanath.
E-mail: soumyana@ohsu.edu
Accepted for publication
4 September 2007
Key words
HRA ⁄Skh-II, melanocyte, pigmentation, piperine,
tetrahydropiperine, vitiligo
Conflicts of interest
None declared.
Summary
Background Piperine and its analogues have been reported to stimulate melano-
cyte replication in vitro and may be useful in treating the depigmenting disease,
vitiligo.
Objective To investigate the ability of piperine (PIP) and three analogues to stimu-
late pigmentation in a strain of sparsely pigmented mice.
Methods The test compounds were PIP [5-(3,4-methylenedioxyphenyl)-2,4-penta-
dienoylpiperidine], tetrahydropiperine [THP, 5-(3,4-methylenedioxyphenyl)-
pentanoylpiperidine], a cyclohexyl analogue of piperine [CHP, 5-(3,4-methyl-
enedioxyphenyl)-2,4-pentadienoylcyclohexylamine], and reduced CHP [rCHP,
5-(3,4-methylenedioxyphenyl)-2,4-pentanoylcyclohexylamine]. Sparsely pigmen-
ted, HRA ⁄Skh-II mice were randomized to receive topical treatment with test
compounds or vehicle twice a day for five days a week, with or without ultra-
violet (UV) irradiation on 3 days a week. Treatment was either continuous
or interrupted to evaluate fading and repigmentation. Skin inflammation and
pigmentation were evaluated regularly during treatment. DOPA+ melanocytes
were determined histologically at the termination of treatment.
Results Four weeks of treatment with one of the compounds PIP, THP or rCHP,
but not CHP, induced greater pigmentation than vehicle with low levels of
inflammation. Additional exposure to UVR led to darker pigmentation than did
the compound or UVR alone, and greater numbers of DOPA+ melanocytes were
found. The combination produced an even pigmentation pattern, contrasting
with the speckled, perifollicular pattern produced by UVR alone. Treatment inter-
ruption led to a decrease in pigmentation but not its loss. Repigmentation was
achieved by administering one of the compounds, UVR or both, and occurred
faster than in naı
¨ve mice.
Conclusions Treatment with PIP, THP or rCHP and UVR induced a marked pigmenta-
tion response in HRA ⁄Skh-II mice, with clinically better results than UVR alone.
This result supports the potential use of these compounds in treating vitiligo.
The skin disorder vitiligo is the most common acquired hypo-
melanosis, affecting approximately 1% of the world’s popula-
tion, with serious cosmetic and psychological effects.
1
The
characteristic depigmentation can be restricted to a limited
skin area (segmental vitiligo) or generalized in symmetrical
patches (nonsegmental vitiligo). In most cases, loss of skin
colour corresponds with melanocyte loss, first in the epider-
mal compartment, and later in the follicular reservoir where
most melanocytic stem cells are probably situated.
2
Treatment of vitiligo is often difficult and disappointing.
This is most probably because the aetiopathogenesis is
unknown, and a treatment directed to the cause has not
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists •British Journal of Dermatology 2008 158, pp941–950 941

been established. Several treatment modalities, such as PUVA
[psoralen + UVA (320–400 nm) radiation], broad-band (280–
320 nm) and narrow-band (311 nm) UVB, and local corticos-
teroids are currently used. However, it has been reported that
these standard treatments result in limited success; less than
25% of patients responded successfully to topical corticoster-
oids.
3
Moreover, corticosteroids applied either systemically or
topically carry the risk of significant side effects in long-term
therapy.
4
Alternatively, PUVA therapy seldom achieves extensive
repigmentation that is cosmetically acceptable, and treatment
response is often followed by relapse.
5
A recent Cochrane
review
6
highlights the lack of research on current treatments
as well as the need to identify novel clinical approaches for
vitiligo.
Several clinical studies
7–10
strongly suggest that reservoirs in
hair follicles are the source of melanocytes in skin repigmented
by standard therapies. Small circular areas of repigmentation
centred around hair follicles enlarge and eventually coalesce.
Consequently, the identification of stimuli that activate outer
root sheath melanocytes is a prospective means of developing
new treatments for vitiligo.
Recent evidence from our laboratory indicates that piperine
[5-(3,4-methylenedioxyphenyl)-2,4-pentadienoylpiperidine; PIP]
has a potent stimulatory effect on mouse melanocytes in vitro.
Culture media supplemented with Piper nigrum (black pepper)
fruit extract or its main alkaloid, PIP, induced nearly 300%
stimulation of melan-a mouse melanocyte proliferation after
8 days of treatment in vitro.
11
The increase of growth was
effectively inhibited by RO-31-8220, a broad-spectrum pro-
tein-kinase C (PKC) inhibitor, suggesting that PKC signalling
is involved in its activity. Both Piper nigrum extract and PIP also
induced an increase in the number and length of cell den-
drites.
11
Melanin synthesis, however, was not stimulated. We
have also shown that several synthetic derivatives of piperine
share these in vitro effects.
12
The aim of the present study was to evaluate the melano-
cyte-stimulatory activity of PIP and three of its synthetic deriv-
atives (Fig. 1) in vivo as a putative new chemical group for the
treatment of vitiligo, alone or in association with UVR. Studies
were performed in HRA.HRII-c ⁄+⁄Skh mice, a hairless, spar-
sely-pigmented mouse model
13
that has white skin except for
the ears and tails. This line, congenic with albino inbred
HRA ⁄Skh mice, segregates into albino and pigmented pheno-
types and was developed by Dr P. Forbes, Temple University
Centre for Photobiology, Philadelphia, PA, U.S.A. These mice
have melanocytes in the epidermal layer (as in human skin),
whereas in many other pigmented mouse strains, melanocytes
are found only in the dermis. The numbers of epidermal mel-
anocytes in this model are small – two or three DOPA+ mela-
nocytes mm
)2
.
14
However, unlike albino mice, pigmentation
in HRA.HRII-c ⁄+⁄Skh mice is inducible
13,14
with melanocyte
numbers reaching close to 600 mm
)2
. As with vitiligo, peri-
follicular pigmentation is evident after exposure to UVR with
and without photosensitizers.
13–15
We therefore advocate the
induction of pigmentation in this strain as an in vivo model for
repigmentation in vitiligo.
Materials and methods
Animals
Male and female inbred HRA.HRII-c ⁄+⁄Skh hairless pigmented
mice, age-matched (8–16 weeks old), were used. Animals
were bred by the Biological Services Division, KCL, University
of London, U.K. and the Rayne Institute, St Thomas’s Hospital,
London, U.K. Animals were killed by cervical dislocation and
skin samples removed surgically when required.
Chemicals
PIP [5-(3,4-methylenedioxyphenyl)-2,4-pentadienoylpiperi-
dine] was purchased from Sigma-Aldrich Ltd (Dorset, U.K.).
PIP derivatives, i.e. tetrahydropiperine [5-(3,4-methylenedi-
oxyphenyl)-pentanoylpiperidine; THP], a cyclohexyl analogue
of piperine [5-(3,4-methylenedioxyphenyl)-2,4-pentadienoyl-
cyclohexylamine; CHP] and reduced CHP [5-(3,4-methyl-
enedioxyphenyl)-2,4-pentanoylcyclohexylamine; rCHP] were
synthesised in our laboratory.
12
Selection of vehicles by ex vivo skin assays
To determine the optimum vehicle for delivery of test agents,
ex vivo permeation studies were conducted with vertical Franz
diffusion cells using a modification of reported methods.
16
O
N
O
O
1'
2'
3'
4'
5'
1
2
3
4
5
6
7
8
9
10
11
5-(3,4-methylenediox y phe ny l)-2,4-pentadieno yl pi peridine (PIP)
O
N
O
O
1'
2'
3'
4'
5'
1
2
3
4
5
6
7
8
9
10
11
5- (3,4-methylenedioxypheny l)-pentano yl piperidine (THP)
1'
2'
3'
4'
5'
1
2
3
4
5
6
7
8
9
10
11
O
NH
O
O
6'
5- (3,4-methylenedioxypheny l)-2,4-pentadienoylc yc lohexy lamine (CHP)
1'
2'
3'
4'
5'
1
2
3
4
5
6
7
8
9
10
11
O
NH
O
O
6'
5- (3,4-methy le nedioxy pheny l)-2,4-pentano yl cy clohexylamine (rCHP)
Fig 1. Piperine (PIP) and structural analogues tetrahydropiperine
(THP), a cyclohexyl analogue of piperine (CHP) and reduced CHP
(rCHP).
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists •British Journal of Dermatology 2008 158, pp941–950
942 Evaluation of piperine and analogues for vitiligo, L. Faas et al.

Each cell consists of a chamber with upper (donor) and lower
(receiver) compartments divided by the mounted skin sample.
The skin acts as a seal between the two half-cells when they
are clamped together. The upper, stratum corneum side is
filled with the drug formulation and the lower one (dermal
side) with receiving fluid. Samples of the receiving fluid are
taken at intervals to quantify the amount of the drug passing
through the skin. The cells used in this study had a 10 mL
capacity receptor compartment and a 1Æ75 cm
2
diffusion area.
A circular piece of full-thickness dorsal skin from HRA.HRII-
c+ ⁄Skh mice was carefully mounted onto the receiver com-
partment of the diffusion cells with the stratum corneum
facing the donor compartment. The receptor compartment
was filled with phosphate buffered saline; PBS, pH 7Æ4) which
was continuously stirred with a magnetic bar. Test solutions
[175 mmol L
)1
PIP in ethanol, diethylene glycol monoethyl
ether (Transcutol

, Gattefosse
´, Saint-Priest Cedex, France),
dimethyl sulfoxide (DMSO), polyethylene glycol (PEG) or 5%
oleic acid (OA) in PEG] were added into the donor compart-
ment of each cell (n= 4 for each formulation). Samples of
fluid from the receiver cell were taken at 3, 19 and 22 h and
the concentration of PIP was determined by high performance
liquid chromatography (HPLC) using a model 3100 pump
(LDC Analytical, Riviera Beach, FL, U.S.A.) with a Spectro-
monitor 3100 UV detector (LDC Analytical) and Hewlett Pack-
ard 3390 A integrator. A 4Æ6·25 cm, 10 lm, C18 Econosil
reverse phased column (Alltech U.K., Stamford, U.K.) was
used, eluting with methanol : water (60 : 40; HPLC grade,
1 mL min
)1
). The detector wavelength was set at 348 nm.
Under these conditions, PIP eluted at 10Æ59 min. Results were
expressed as mg mL
)1
according to a previously determined
calibration curve (0Æ003–0Æ1mgmL
)1
PIP in PBS).
Topical application of test compounds in vivo
Test agents were dissolved in vehicle (either OA ⁄PEG or in
DMSO) to a final concentration of 175 mmol L
)1
and 100 lL
(17Æ5lmoles) applied with a micropipette on the central area
of mouse dorsal skin (2–3 cm
2
), twice a day (weekdays only)
with an interval of 5–6 h between applications. In protocols
with UVR exposure, the irradiations were carried out every
Monday, Wednesday and Friday immediately prior to the first
daily application, to avoid a possible photosensitizing effect
and ⁄or photodamage
17
to the test compound.
UV irradiation and dosimetry
The UVR source was a bank of eight Bellarium SA-1-12-100W
fluorescent tubes (Wolff, Erlangen, Germany), the emission
spectrum of which has been published.
13
This UVR source emits
4Æ1% UVB (280–320 nm) and 95Æ8% UVA (320–400 nm), but
the UVB accounts for the 71Æ5% erythemally effective energy
when biologically weighted with the human erythema spec-
trum.
18
Given that tanning and erythema action spectra are very
similar
19
it is probable that the small UVB component accounts
for most of the tanning effect. Irradiations were carried out in a
purpose-built unit with ventilation, temperature and humidity
controls. The irradiance was monitored daily immediately
before irradiation with an International Light radiometer (IL
422A; Newburyport, MA, U.S.A.) equipped with UVR sensors.
The radiometer was calibrated for the source, as described
before.
13
Irradiance measured at mouse level was typically about
0Æ16 mW cm
)2
. Animals were irradiated unrestrained in metal
cages with a dose of 354 mJ cm
)2
,
13
confirmed to be sub-
inflammatory from a single exposure (increase in skin fold
thickness (SFT) < 10%; data not shown). Irradiations lasted for
a maximum of 1 h. The position of cages was systematically
rotated to ensure even UVR exposure.
Experimental groups
In initial experiments, animals were treated topically with PIP,
THP and CHP dissolved in either OA ⁄PEG or in DMSO or with
vehicle alone for 9 weeks with concomitant exposure to UVR
during weeks 5–9. Further experiments (summarized in
Fig. 2) were conducted using compounds dissolved in DMSO,
with DMSO as control. For continuous treatment, animals
(n‡4) were treated topically with PIP, THP, rCHP or DMSO
for up to 13 weeks (Fig. 2, Group A). A second group
(Fig. 2, Group B) received the same treatment, but was add-
itionally exposed to UVR from week 5 to 13. For studies on
discontinuous treatment, animals were treated as in Group B
up to week 7. All treatment was then suspended for 3 weeks
(weeks 8–10) and re-started as topical application only
(Fig. 2, Group C), UVR only (Fig. 2, Group D), or topical
application plus UVR (Fig. 2, Group E) for weeks 11–13.
Mice exposed only to UVR (i.e. no vehicle treatment) from
week 5 onwards (Fig. 2, Group F) were used as controls for
all groups treated with UVR.
Group Week
12345678910111213
A
B
C
D
E
F
Untr
No treatment Compound UVR
3
Fig 2. Treatments. Mice were treated for 13 weeks with topical
compounds alone (A) or with additional ultraviolet radiation (UVR)
exposure (B). For other groups, treatment was interrupted and
restarted as compounds only (C), UVR only (D), or compounds +
UVR (E). Group F received UVR alone.
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists •British Journal of Dermatology 2008 158, pp941–950
Evaluation of piperine and analogues for vitiligo, L. Faas et al. 943

Assessment of inflammation and pigmentation
Dorsal SFT was recorded to evaluate potential inflammatory
effects of treatments, Measurements were taken every day dur-
ing the first week of treatment and twice a week thereafter
with a spring-loaded micrometer (Mitutoyo, Kawasaki, Japan).
Pigmentation was assessed independently by two investigators
and the average score calculated. The first type of assessment
was conducted visually, every day, and pigmentation scored
from 0 to 5 according to the following scheme: 0 = no
pigmentation; 1 = first signs of pigmentation (freckles);
2 = light brown; 3 = medium brown; 4 = dark brown;
5 = black. Pigmentation was also assessed histologically by
DOPA staining at the end of the experiment. Animals were
killed and skin samples from representative dorsal areas
(1 cm
2
) were removed surgically and incubated in 2 mol L
)1
NaBr in PBS for 2 h at 37 C. The epidermis was carefully
removed with tweezers and further incubated in 0Æ1% L-DOPA
in PBS (pH 7Æ2) for 4 h at 37 C. The DOPA solution was
changed periodically to prevent auto-oxidation. Finally, epi-
dermal sheets were fixed in 4% paraformaldehyde in PBS (pH
7Æ4) for 15 min, dehydrated through a graded series of alco-
hol concentrations and mounted on glass microscope slides
for examination. The number of DOPA+ cells per mm
2
was
calculated from at least 30 fields per sample (n= 4 animals).
DOPA+ cells were also classified as highly or poorly melanized
according to their melanin granule content. The percentage of
cells in each category per mm
2
was calculated.
Results
Selection of vehicles based on skin penetration
measured with Franz cells
Skin penetration of PIP when dissolved in five vehicles was
compared using Franz cells. At 22 h after application, the con-
centration of PIP in the receiver compartment was highest
with DMSO followed by OA ⁄PEG in both male and female
skin (Fig. 3). PIP was undetectable when delivered in other
vehicles (ethanol, diethylene glycol monoethyl ether and
PEG). No PIP was detected at shorter time periods (3 h and
19 h) with any vehicle. DMSO and OA ⁄PEG were therefore
chosen as vehicles for the in vivo studies.
Inflammatory and irritant effects
Differences in inflammatory response were seen depending on
the vehicle, test compound and sex of animal. OA ⁄PEG based
formulations induced stronger adverse effects than those with
DMSO in both male and female mice (Figs 4 and 5). However,
in males, PIP and THP solutions in OA ⁄PEG induced a stronger
inflammatory response (more than 30% increase in SFT;
Fig. 4a), than in females where THP had only a mild
inflammatory effect (20% increase in SFT, Fig. 5a). The
inflammatory effect of CHP was comparable to vehicle alone in
both males and females (around a 20% increase in SFT, Figs 4a
and 5a). The inflammatory response induced by formulations
0
0·001
0·002
0·003
0·004
0·005
0·006
0·007
0·008
DMSO EtOH Transc OA/PEG PEG
PIP Concentration (mg mL–1)
Males
Females
Fig 3. DMSO is the best of five skin penetration enhancers for
piperine (PIP). Penetrance of PIP dissolved in ethanol (EtOH), DMSO,
diethylene glycol monoethyl ether (Transc), polyethylene glycol (PEG)
and 5% oleic acid in PEG (OA ⁄PEG) through HRA.HRII-c+ ⁄Skh
mouse dorsal skin was tested using Franz cells.
–10
10
30
50
70
Increase in SFT (%)Increase in SFT (%)
123456789
Weeks
123456789
Weeks
PIP
THP
CHP
(a)
(b)
V
UV
.
–10
10
30
50
70
Fig 4. Inflammation in male mice treated with piperine (PIP),
tetrahydropiperine (THP) or cyclohexyl analogue of piperine (CHP) in
5% oleic acid in polyethylene glycol (OA ⁄PEG) (a) or DMSO (b).
Inflammation was assessed as percentage increase in skin fold
thickness (SFT). Only mean values (n= 5) are given for clarity (% CV
not more than 30%). V, vehicle; UV, ultraviolet radiation.
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists •British Journal of Dermatology 2008 158, pp941–950
944 Evaluation of piperine and analogues for vitiligo, L. Faas et al.

in DMSO was observed to be milder than with OA ⁄PEG. DMSO
alone had a mild inflammatory effect (up to 20% increase in
SFT; Figs 4b and 5b). Both vehicles and test solutions had a
transient irritant effect, producing redness and desquamation
of treated areas during the first 10 days of treatment (data not
shown). Irritancy induced by DMSO (and DMSO-based solu-
tions) was milder than with OA ⁄PEG. Redness decreased after
an hour of topical application, and desquamation was less
severe than with OA ⁄PEG. PIP and THP in OA ⁄PEG showed the
most powerful irritant effects, in agreement with the strong in-
flammatory response observed. The irritant effects were over-
come by week 2 of topical treatment. UVR alone had lower
inflammatory effects than any of the topical treatments.
Pigmentation
Four weeks of topical treatment with PIP or THP, in either
DMSO (Fig. 6a) or OA ⁄PEG (not shown), induced a light,
even pigmentation of the treated area compared with vehicle
control whereas CHP had virtually no effect. The vehicles used
also showed some effect, as previously reported for DMSO
20,21
and OA.
22–24
For PIP (Fig. 6b) and THP, subsequent subery-
themal exposure to two UVR exposures alone significantly
enhanced pigmentation induced by the test compounds com-
pared with controls treated with vehicle and UVR, or UVR
alone. Pigmentation was observed as a dark, even pattern after
6–8 exposures (Fig. 6c,d). The pigmentation induced by topi-
cal treatment with vehicle was lighter and uneven (DMSO,
Fig. 6a–d). The pigmentation induced by UVR alone (Fig. 6c)
was observed to be perifollicular and therefore speckled, in
contrast to the even pigmentation of PIP and THP alone
(Fig. 6a), or in combination with UVR (Fig. 6b–d).
Different pigmentation responses observed in vivo corre-
sponded with changes in the number of DOPA+ cells mm
)2
in the skin (Fig. 7). Pigmentation responses were slower and
less evident in females (scores not shown) than in males. In
male mice, treatment with PIP and THP in either DMSO or
OA ⁄PEG significantly (P<0Æ05) increased the number of
DOPA+ cells compared with vehicles. The lower pigmenta-
tion responses in female mice corresponded with a smaller
mean number of DOPA+ cells mm
)2
under all treatment
conditions compared with males receiving equivalent treat-
ments (Fig. 7). In females the stimulatory effects of THP and
PIP on pigmentation reached statistical significance only with
PIP in OA ⁄PEG and THP in DMSO (Fig. 7) although a trend
towards an increase was apparent with both compounds in
either vehicle. CHP, in contrast, did not show any effect on
the number of DOPA+ cells compared with vehicles in either
males and females, in agreement with the low pigmentation
levels observed on visual examination of the animals
(Fig. 6).
Further experiments were carried out in order to determine
the persistence of the pigmentation effect after the cessation of
treatment and the stimuli needed to restore pigmentation if
lost, according to protocols summarized in Fig. 2. PIP, THP
and a novel compound, the reduced form of CHP (rCHP)
were tested. rCHP was chosen because of its high stimulatory
activity on melanocyte proliferation in vitro.
12
All solutions
were prepared in DMSO because of its better performance as a
penetration enhancer (Fig. 3) and milder inflammatory effects
than OA ⁄PEG (Figs 4 and 5). The results of this experiment
are shown in Fig. 8. The application of the compounds alone
for 13 weeks (Group A) stimulated pigmentation up to a
maximum of level 2 (light brown). The first change was
observed at 4 weeks and maximum pigmentation was reached
by week 6. The application of concomitant UVR (Group B)
significantly enhanced pigmentation reaching up to level 5
(black) by week 7. This was greater than the highest mean
scores obtained with either compound alone (Group A; score
2, light brown) or UVR alone (Group F; score 3, medium
brown). In mice treated topically with a compound, four
exposures of suberythemal doses of UVR were sufficient to
induce a pigmentation score of 3 (Group B), in contrast with
the greater number of UVR exposures (more than 10) needed
–10
10
30
50
70
Increase in SFT (%)
123456789
Weeks
–10
10
30
50
70
Increase in SFT (%)
PIP
THP
CHP
V
UV
123456789
Weeks
(a)
(b)
Fig 5. Inflammation in female mice treated with piperine (PIP),
tetrahydropiperine (THP) and cyclohexyl analogue of piperine (CHP)
in 5% oleic acid in polyethylene glycol (OA ⁄PEG) (a) or DMSO (b).
Inflammation was assessed as percentage increase in skin fold
thickness (SFT). Only mean values (n= 5) are given for clarity (% CV
not more than 20%). V, vehicle; UV, ultraviolet radiation.
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists •British Journal of Dermatology 2008 158, pp941–950
Evaluation of piperine and analogues for vitiligo, L. Faas et al. 945

to obtain a similar, but less even, response in naı
¨ve mice
(Group F). This data clearly shows a combined pigmentation
enhancing effect of PIP, THP and rCHP with UVR. Pigmenta-
tion in Group B was maintained up to week 13 with contin-
ued treatment with the compounds plus UVR.
After three weeks without treatment (Week 8–10), the
degree of pigmentation decreased in animals treated with a
compound and UVR (Fig. 8, Groups C, D and E) compared
with week 7 pigmentation levels, but did not disappear
completely. By contrast, there was no remaining detectable
pigmentation after week 9 in animals treated with only
DMSO and UVR in both male (Fig. 8, Group C, D and E)
and female (data not shown) mice. Retreatment with topical
solutions, UVR, or a combination of both, all resulted in re-
pigmentation after 3 weeks (Week 11–13; Fig. 8, Group C,
D and E). The rate of increase in pigmentation was faster than
the initial pigmentary response (weeks 1–4), reaching scores
of 2 or more within 2 weeks of retreatment (Week 12;
Group C, D and E). Retreatment with the compounds alone
(Fig. 8, Group C) increased pigmentation to levels comparable
with those obtained by continuous topical treatment alone
(Fig. 8, Group A). Retreatment with UVR alone (Fig. 8,
Group D) or combined topical applications plus UVR (Fig. 8,
Group E) resulted in higher pigmentation levels (score 3)
than after retreatment with the compounds alone (score about
2), but comparable with UVR alone (Fig. 8, Group F). The
pigmentation patterns resulting from a compound alone
(Group C) or a compound plus UVR (Group E) were both
even (Fig 6c and data not shown). In contrast, pigmentation
induced by retreatment with UVR (Fig. 8, Group D) resem-
bled the spotted pattern obtained with continuous UVR
exposure (Fig. 6c).
Histological analysis of skin melanocyte numbers (Fig. 9)
again showed a good correlation with visually observable dif-
ferences in pigmentation for both male and female mice.
Group B animals (compound plus UVR) showed significantly
more melanocytes mm
)2
than those receiving compounds
(Group A) or UVR (Group F) alone. Based on the fading of
pigmentation, treatment withdrawal (Groups C–E, weeks
8–10) is assumed to have caused a decrease in the activity of
melanocytes. In males (Fig. 9a), retreatment with UVR alone
(Group D) or with topical compounds plus UVR (Group E)
increased the number of DOPA+ cells mm
)2
at the end of
Week 13 to levels comparable with those in Group B which
(a)
(c)
(b)
(d)
DMSO
PIP
CHP
THP
Fig 6. Pigmentation induced in male mice by
piperine (PIP) and derivatives applied in
DMSO. (a) PIP and tetrahydropiperine (THP)
[but not cyclohexyl analogue of piperine
(CHP)] applied for 4 weeks induce greater
pigmentation than DMSO. After two
ultraviolet radiation (UVR) exposures (b) or
eight UVR exposures (c,d), pigmentation is
darker in mice treated with PIP or THP (but
not CHP) than in mice treated with DMSO or
previously untreated (UVR) mice.
Compounds produce an even pigmentation
compared with the speckles (c, arrows)
caused by UVR alone.
0
100
200
300
400
500
600
700
Cells/mm2
M F M F M F M F M F M F M F M F M F
PIP+ M F
PIP+ THP+ THP+ CHP+ CHP+ UVR Untr
DMSO DMSO
DMSO
DMSOOA OA OA
OA
*
*
*
*
**
Fig 7. Increase of DOPA+ cell number after continuous treatment
with piperine (PIP), tetrahydropiperine (THP) or cyclohexyl analogue
of piperine (CHP) in DMSO or 5% oleic acid in polyethylene glycol
(OA ⁄PEG) for 9 weeks, with exposure to ultraviolet radiation (UVR)
from week 5 to 9 (mean ± SD; n= 4). (M), male; (F), female.
*P<0Æ05 compared with vehicle (Student’s t-test). Untr, untreated
(naı
¨ve).
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists •British Journal of Dermatology 2008 158, pp941–950
946 Evaluation of piperine and analogues for vitiligo, L. Faas et al.

had received continuous treatment with UVR and a com-
pound. The number of cells mm
)2
in Group D animals was
significantly higher than in the group that received continuous
UVR (Group F) or continuous topical treatment (Group A).
Retreatment with a compound alone significantly increased
the number of DOPA+ cells mm
)2
compared with vehicle
control (Group C), reaching comparable levels with those in
animals treated continuously with a compound alone (Group
A). However, the number of cells mm
)2
was lower than for
groups that were retreated with UVR alone (Group D) or with
a compound and UVR (Group E). The results obtained in
female mice (Fig. 9b) showed the same trends as in males,
except that in animals retreated with UVR alone (Group D) or
UVR with a compound (Group E) no significant differences
were observed compared with vehicle controls.
To investigate whether the differences in pigmentation
observed were due to an increase in melanocyte number or in
melanin production, DOPA+ cells were classified as highly or
poorly melanized according to the content of pigment gran-
ules, and the percentage of DOPA+ cells in each category per
mm
2
of skin was calculated for each experimental group. As
expected, UVR exposure considerably increased the degree of
melanization of DOPA+ cells (Fig. 10c and Groups B, D, E
and F in Fig. 11a,b) compared with mice treated with a
compound alone (Fig. 10b or Groups A and C in Fig. 11a,b),
where poorly melanized cells were predominant.
4 5 6 7 8 9 10 11 12 13
Weeks
0
1
2
3
4
5
6
Pigmentation
DMSO
PIP
THP
rCHP
78910111213
Weeks
0
1
2
3
4
5
6
Pigmentation
****
7 8 9 10111213
Weeks
0
1
2
3
4
5
6
Pigmentation
*
*
**
Group A: Compound only 13 weeks Group B: As group A, plus UV on weeks 5–13
Group C: Interrupt treatment,
then compound only Group D: Interrupt treatment, then UV only
Group E: Interrupt treatment,
then compound + UV
0
1
2
3
4
5
6
Pigmentation
0
1
2
3
4
5
6
Pigmentation
4 5 6 7 8 9 10 11 12 13
Weeks
*****
78910111213
Weeks
*
*
0
1
2
3
4
5
6
Pigmentation
567891011
Weeks
** ** ** ** ** **
Group F: Treat with UV only
Fig 8. Pigmentation response of male mouse skin (n= 4) to continuous (Groups A, B, F) or discontinuous (Groups C–E) treatment as
summarized in Figure 2. Mice were treated for 13 weeks with topical compounds alone (A) or with additional ultraviolet radiation (UVR)
exposure (B). For other groups, treatment was interrupted and restarted as compounds only (C), UVR only (D), or compounds + UVR (E).
Group F received UVR alone. Pigmentation scores range from 1 (freckles) to 5 (black). *P<0Æ05 compared with vehicle; **P<0Æ05 compared
with Group B (Mann–Whitney U-test).
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists •British Journal of Dermatology 2008 158, pp941–950
Evaluation of piperine and analogues for vitiligo, L. Faas et al. 947

Discussion
Topical treatment of HRA ⁄Skh-II mice with PIP, or two of its
synthetic derivatives, THP and rCHP, stimulates the develop-
ment of even skin pigmentation in vivo after four or more
weeks of continuous topical application. The darkening of skin
in treated areas corresponds with an increase in the number
of DOPA+ melanocytes. This in vivo finding correlates well
with our previous studies showing the stimulation of in vitro
melanocyte proliferation by PIP and chemically related com-
pounds.
11,12
Animals treated with PIP or analogues before
UVR exposure showed more rapid and darker pigmentation
than those treated with UVR exposure or a compound alone
(Fig. 8). These findings highlight the potential of these com-
pounds as novel treatments for vitiligo. Notably, supplement-
ing UVR with these compounds may offer a means of
reducing UV exposure in vitiligo therapy, thereby reducing
the risk of developing skin cancer.
The degree of skin pigmentation is a consequence of both
number of melanocytes and their degree of melanization.
UVR, for example, stimulates both melanocyte proliferation
0
100
200
300
400
500
600
700
800
900
DOPA+ cells/mm2
DOPA+ cells/mm2
A B C D E F Untr
A B C D E F Untr
DMSO
PIP
THP
rCHP
**
*
*
***
*
*
*
0
100
200
300
400
500
600
700
800
900
*
*
*
*
*
**
*
*
**
(a)
(b)
Fig 9. DOPA+ cell numbers (mean ± SD) in male (a) and female (b)
mouse skin (n= 4) after continuous and discontinuous treatment as
in Fig. 2, Groups A–F). Cell numbers correlate well with visually
determined pigmentation scores. *Significant increase compared with
vehicle; **Significant decrease compared with groups B and D
(P<0Æ05; Student’s t-test).
(a) (b) (c)
Fig 10. Histology of DOPA+ melanocytes in skin from animals treated with (a) vehicle, (b) piperine (PIP) and (c) PIP + ultraviolet radiation
(UVR). The ratio of highly (arrowheads) to poorly (arrows) pigmented melanocytes increases in the order a < b < c. Original magnification
·200.
(a)
(b)
Fig 11. Percentage of highly (black bars) vs. poorly (grey bars)
melanized melanocytes in male (a) and female (b) mice (mean ± SD;
n= 4). Groups receiving ultraviolet radiation (UVR) (B, D, E and F)
show greater melanization than those receiving a compound alone
(A, C) prior to histology. Treatment groups as shown in Figure 2.
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists •British Journal of Dermatology 2008 158, pp941–950
948 Evaluation of piperine and analogues for vitiligo, L. Faas et al.

and melanin synthesis.
21,25
The relatively low pigmentation
scores in the absence of UVR (Fig. 8) and the low degree of
melanization of DOPA+ cells observed in skin treated with a
compound alone (Fig. 11) suggests that these compounds
stimulate melanocyte proliferation rather than melanin synthe-
sis. This is in good agreement with in vitro data showing that
PIP derivatives do not stimulate melanin production although
they stimulate melanocyte proliferation.
11,12
Retreatment with
a compound alone induced a higher difference in DOPA+ cell
numbers between compound and vehicle (Fig. 9a,b, Group C)
than did retreatment with UVR alone (Fig. 9a,b, Group D),
This again suggests that the primary effect of piperine is to
stimulate rapid melanocyte proliferation and population of
epidermal areas. This phenomenon, as well as effects on mela-
nocyte differentiation by PIP analogues could be further exam-
ined through bromodeoxyuridine incorporation experiments
and immunohistochemical determination of specific markers
such as Kit, Mitf, TRP-1 and TRP-2, indicative of different
developmental stages of melanocytes.
26
A gender difference in induced pigmentation was observed
in these studies, with males showing a greater response than
female mice. However, skin penetration of PIP was the same
in both sexes using a Franz cell model (Fig. 3), suggesting
equal bioavailability in both sexes. However, the mild inflam-
matory and irritant effects seen with PIP and its analogues
(Figs 4 and 5) may be significant, in explaining the activity of
the compounds per se, as well as the differences in pigmentary
response of male and female animals. Females showed a lower
inflammatory response than males. Gender differences in sen-
sitivity to UVR have also been observed in humans with males
showing a greater sensitivity and lower MED.
27
An important feature of treatment with PIP and its analogues
is the even pigmentation pattern that is obtained with or with-
out additional UVR (Fig. 6). This correlates well with the find-
ing that DOPA+ melanocytes in treated skin (Fig. 10) were
distributed in interfollicular areas rather than associated with
hair follicles, and suggests an active epidermal distribution of
melanocytes after treatment with PIP or its analogues. An exam-
ination of the in vivo cutaneous absorption and distribution of
PIP and its analogues, particularly the relative roles of the
stratum corneum and hair follicles, would be of interest in
determining their site of action and understanding the repig-
mentation patterns seen. Hair follicles are known to play a sig-
nificant role in the percutaneous absorption of many drugs.
28
The use of PIP and its analogues in vitiligo clearly offers po-
tential cosmetic advantages over the use of PUVA or UVR alone
(common current treatments for vitiligo) if an even pattern can
be obtained in humans. PUVA repigmentation, when successful,
progresses from a perifollicular pattern in early stages of ther-
apy, with the circular patches of pigment coalescing after fur-
ther treatment to a more even pattern in humans
5
and in
mice.
15
A similar progression has been observed using therapies
based on UVR.
9,29
Mice treated with UVR alone in the present
study also showed this speckled pattern (Fig. 6c).
Continuous treatment appears to be needed to maintain
pigmentation as shown by the gradual, though not complete,
loss of pigmentation when treatment is suspended. Retreat-
ment with either UVR alone, topical compounds alone or the
combination of both, restored pigmentation over a shorter
period of time than in naı
¨ve mice. This indicates the possible
presence of poorly melanized melanocytes but in greater num-
bers than in naı
¨ve skin. Consistent with our previous observa-
tions, the resulting pigmentation after retreatment with UVR
often showed darker perifollicular areas, in contrast to the
even pattern produced by retreatment with a compound alone
or a compound plus UVR.
Although our results suggest that the melanocyte is the main
target for these compounds, no known melanocytic receptor
for PIP or its derivatives has been identified to date. Interest-
ingly, the presence of one of the subtypes of vanilloid receptor,
the receptor for PIP and PIP-related molecules, has recently
been shown in keratinocytes.
30
In this respect, it is well known
that melanocytes and keratinocytes exhibit a close functional
relationship. Keratinocytes are known to produce several fac-
tors that regulate melanocyte activity and survival, such as
nerve growth factor, granulocyte-monocyte colony stimulating
factor, basic fibroblast growth factor, endothelin-1, stem cell
factor and other cytokines.
31–37
It has recently been shown
that some of these molecules are imbalanced in vitiligo skin,
38
suggesting that the deregulation of the melanocyte microenvi-
ronment could be involved in the selective destruction of mel-
anocytes in vitiligo. Indeed, an impairment of keratinocyte
function is observed in perilesional skin.
39
It is reasonable to
speculate that PIP and PIP analogues could have an effect on
modulating cytokine production by keratinocytes in vivo, con-
sequently stimulating melanocyte replication or activity, which
could result in an increase in pigmentation. Nevertheless, we
have observed an effect on PKC activation by PIP in vitro that is
suggestive of a direct effect on melanocytes.
11
In summary, we have shown that topical treatment with
PIP, and two of its synthetic analogues, THP and rCHP, stimu-
lates even pigmentation in mice. Topical treatment in combin-
ation with low dose UVR significantly enhances the
pigmentation response with results that are cosmetically better
compared with conventional vitiligo therapies when applied
to mice. Although fading may occur when the treatment is
interrupted, a good pigmentation response is readily achieved
again after short periods of retreatment. Side effects, such as
irritation and inflammation, were transient and tolerable.
These data provide strong support for the future clinical evalu-
ation of PIP and its derivatives as novel treatments for vitiligo.
Acknowledgments
This work was funded by BTG International Ltd and by an
Overseas Research Student Award to RV. We thank Dr Marc
Brown and Richard Harper of the Pharmacy Department,
King’s College London for, respectively, guidance on the Franz
cell assay and photography of the mice. At St John’s Institute
of Dermatology, we acknowledge the technical support in
histology provided by Guy Orchard and thank Dr Susan
Walker for helpful discussions and critical reading of the
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists •British Journal of Dermatology 2008 158, pp941–950
Evaluation of piperine and analogues for vitiligo, L. Faas et al. 949

manuscript. Dr A Soumyanath and Professor AR Young held
joint responsibility for the supervision of this work.
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2008 The Authors
Journal Compilation 2008 British Association of Dermatologists •British Journal of Dermatology 2008 158, pp941–950
950 Evaluation of piperine and analogues for vitiligo, L. Faas et al.