Investigation of the absorption of hypericin into the skin of hairless mice

Article abstract of DOI:10.1211/0022357001774264

The skin absorption of hypericin was evaluated in hairless mice to develop an optimised hypericin topical formulation that could be used in the clinical study of psoriasis. Hypericin (0.01-1.0%) in Beeler basis, polyethylene glycol ointment, carbopol gel, cetomacrogol cream, petrolatum or emulsifying ointment, with and without skin-absorption enhancers (isopropylidene glycerol and diethylene glycol monoethyl ether), was tested in-vivo on hairless mice skin. Using a skin-stripping technique and the intrinsic fluorescence of hypericin under standardised UV₃₆₅ irradiation, it was demonstrated that the absorption of hypericin very much depended on the vehicle used. The concentrations of hypericin in the skin were then estimated by HPLC analysis. For this purpose, two vehicles were employed, with which hypericin penetrated the skin of hairless mice well (emulsifying ointment with isopropylidene glycerol) or very poorly (polyethylene glycol ointment). In the case of emulsifying ointment with isopropylidene glycerol (0.05% hypericin), a substantial concentration of hypericin (8.6±3.2 μg g^-1) (mean±s.d., n=5) was found in the skin. With polyethylene glycol ointment, however, only a limited hypericin skin concentration (0.38±0.34 μg g^-1, n= 5) was achieved. These results show that emulsifying ointment with polyethylene glycol holds promise as an effective topical vehicle for the treatment of skin diseases, such as psoriasis, with hypericin.

Full text of DOI:10.1211/0022357001774264

J. Pharm. Pharmacol. 2000, 52: 487±494
Received November 17, 1999
Accepted January 11, 2000
# 2000 J. Pharm. Pharmacol.
Investigation of the Absorption of Hypericin into the
Skin of Hairless Mice
APPOLINARY R. KAMUHABWA, KAREL R. GEBOES* AND PETER A. DE WITTE
Laboratorium voor Farmaceutische Biologie en Fytofarmacologie,
Faculteit Farmaceutische Wetenschappen and *Laboratorium voor Histochemie en Cytochemie,
Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Abstract
The skin absorption of hypericin was evaluated in hairless mice to develop an optimised
hypericin topical formulation that could be used in the clinical study of psoriasis.
Hypericin (0Á01±1Á0%) in Beeler basis, polyethylene glycol ointment, carbopol gel,
cetomacrogol cream, petrolatum or emulsifying ointment, with and without skin-absorp-
tion enhancers (isopropylidene glycerol and diethylene glycol monoethyl ether), was tested
in-vivo on hairless mice skin. Using a skin-stripping technique and the intrinsic ¯uores-
cence of hypericin under standardised UV365 irradiation, it was demonstrated that the
absorption of hypericin very much depended on the vehicle used. The concentrations of
hypericin in the skin were then estimated by HPLC analysis. For this purpose, two vehicles
were employed, with which hypericin penetrated the skin of hairless mice well (emulsi-
fying ointment with isopropylidene glycerol) or very poorly (polyethylene glycol oint-
ment). In the case of emulsifying ointment with isopropylidene glycerol (0Á05% hypericin),
1
a substantial concentration of hypericin (8Á6Æ 3Á2 mg g ) (meanÆ s.d., n ˆ 5) was found
in the skin. With polyethylene glycol ointment, however, only a limited hypericin
1
skin concentration (0Á38Æ 0Á34 mg g , n ˆ 5) was achieved.
These results show that emulsifying ointment with polyethylene glycol holds promise as
an effective topical vehicle for the treatment of skin diseases, such as psoriasis, with
hypericin.
Hypericin (Figure 1), a hydroxylated phenanthro-
perylenequinone derivative, is a constituent of a
number of plants of the genus Hypericum.
Hypericin is a potent photosensitizer and its
photosensitising properties exhibit interesting bio-
logical activity both in-vitro and in-vivo. The
compound is under consideration as a potential tool
in photodynamic therapy (Diwu & Lown 1994).
Hypericin also has a high ¯uorescence yield, so the
presence of hypericin in body tissues can be easily
visualised. In vehicles or solvents, the intensity of
¯uorescence indicates the degree of solubility of
the compound (Yamazaki et al 1993; Lavie et al
1995).
compound exhibits a powerful in-vitro inhibition of
the enzymatic activity of casein kinase 2 (CK-2; a
Ser=Thr kinase) (Agostinis et al 1995) and epi-
dermal growth factor (EGF; a tyrosine kinase)
receptor (de Witte et al 1993; Agostinis et al 1996).
It has been suggested that both of these enzymes
are involved in the aetiology of psoriasis (Nanney
et al 1986; Mitev et al 1992). Moreover, epidermal
hyperproliferation is a prominent feature of psor-
iasis, and photoactivated hypericin has been
reported to exhibit antiproliferative effects on dif-
ferent human cell lines (Ritcher & Davies 1995;
Ugwu et al 1998).
Thus far, only a few photosensitizers such as 5-
aminolevulinic acid (Szeimies et al 1994) have
been employed topically for the photodynamic
therapy of skin diseases, probably because of the
limited skin penetration of the compounds. The
possibility of using hypericin, a natural photo-
sensitizer, topically for dermatological purposes
has not been explored. Consequently, as a ®rst step
Some data suggest that photoactivated hypericin
might be a potential antipsoriatic agent. For
instance, it has been shown that the photoactivated
Correspondence: P. A. de Witte, Laboratorium voor Farm-
aceutische Biologie en Fytofarmacologie, Faculteit Farmaceu-
tische Wetenschappen, Van Evenstraat 4, B-3000 Leuven,
Belgium. E-Mail: peter.dewitte@farm.kuleuven.ac.be

488
APPOLINARY R. KAMUHABWA ET AL
Sil G=UV254 plates (Macherey-Nagel, Duren,
Germany)) with the solvent toluene=ethylacetate=
formic acid (5 : 4 : 1, v=v). The latter was further
puri®ed by Sephadex LH20 (Pharmacia, Uppsala,
Sweden) column chromatography with dichloro-
methane=methanol=acetone (11 : 6 : 3, v=v) as elu-
ent. The yield was 28% and its purity was > 99%
as assessed by HPLC.
Hypericin was characterised with 1H NMR
(Gemini 200 MHz, Varian), LSI mass spectrometry
(Kratos Concept IH), and UV=Vis spectro-
photometry (Hewlett-Packard, CA). The data
obtained were comparable with literature data (Falk
& Schoppel 1992; Falk et al 1993).
Figure 1. The chemical structure of hypericin.
Topical vehicles
The vehicles used were petrolatum, polyethylene
glycol ointment (polyethylene glycol 400, 60% and
polyethylene glycol 4000, 40%), emulsifying oint-
ment (30 g emulgent cetyl-stearyl alcohol, 50 g
petrolatum and 20 g liquid paraf®n), Beeler basis
(15 g cetyl alcohol, 1 g cera alba, 10 g propylene
glycol, 2 g sodium lauryl sulphate and 72 g water),
carbopol gel (2 g carboxypolymethylene, 15 g pro-
pylene glycol, 0Á05 g disodium ethylenediamine
tetra-acetate, 2Á5 g triethanolamine and 80Á45 g
water) and cetomacrogol cream (7Á2 g lanette O,
1Á8 g cetomacrogol 1000, 15 g white petrolatum, 6 g
liquid paraf®n and 70 g water). These vehicles were
supplemented (or not) with 10% of the skin
absorption enhancers diethylene glycol monoethyl
ether or isopropylidene glycerol. Compositions
of the vehicles were taken from the Belgian
National Formulary (NF VI) or Pharmacopoeia
(Ph. Belg. V).
this study was undertaken to evaluate and optimise
the in-vivo absorption of hypericin in hairless mice
skin by using topical formulations with different
compositions. By utilising the intrinsic ¯uorescence
of hypericin, a skin-stripping technique was
employed as a straightforward way of screening
hypericin topical vehicles. Some results obtained
with the skin-stripping technique were quantita-
tively con®rmed using HPLC. The results show that
hypericin incorporated in a suitable vehicle is
absorbed by the skin of hairless mice.
Materials and Methods
Hypericin
Hypericin was synthesised as described by Falk
et al (1993). Brie¯y, 2Á5 g of emodin (isolated from
cortex frangulae) was dissolved in 125 mL acetic
acid and re¯uxed. SnCl2.2H2O (30Á0 g) dissolved in
75 mL conc. HCl was added. After 2 h, the ¯ask
was cooled at room temperature and the emodin
anthrone that was formed was ®ltered off and dried
in vacuum (yield 2Á0 g; 80%). Emodin anthrone
(2Á0 g) was heated in a mixture of pyridine=
piperidine (10 : 1, v=v), 4Á0 g pyridine 1-oxide, and
0Á1 g FeSO4.7H2O at 110ꢀC under nitrogen and in
darkness. Crude protohypericin was obtained after
precipitation (by adjusting the solution to pH 4 with
HCl) and centrifugation. The crude mixture was
then further puri®ed by silica chromatography
(Merck, 0Á063±0Á200 mm) with ethylacetate=
water (100 : 2Á5, v=v), followed by ethylaceta-
te=acetone=water (80 : 20 : 2Á5, v=v) as eluent.
Protohypericin was dissolved in acetone and
converted to hypericin by light irradiation, as
monitored by thin-layer chromatography (Alugram
Preparation of hypericin formulations
When skin-absorption enhancers were not used, the
appropriate amount of hypericin was ®rst dissolved
in acetone. Then acetone solutions of hypericin
(200±500 mL) were pipetted onto a ¯at glass plate.
After acetone evaporation, given weights of vehi-
cles were added and thoroughly mixed to give the
desired hypericin concentrations (0Á01±1Á00%).
When skin-absorption enhancers were used,
hypericin was ®rst dissolved in the enhancer fol-
lowed by addition of the vehicle.
Application of hypericin formulations onto the
hairless mice skin
Male and female hairless mice, aged 8±12 weeks
and weighing 24±26 g (NMRI-HR-HR, Charles
River, Germany), were used. Control and test

ABSORPTION OF HYPERICIN INTO HAIRLESS MOUSE SKIN
489
vehicles containing varying amounts of hypericin
with or without skin-absorption enhancers were
uniformly and gently applied with a spatula
dermis, as con®rmed by histological microscopy)
was weighed and homogenised (Ultra-Turrax) in
2 mL of water. The samples were then further
homogenised with 8 mL acetone and centrifuged
(11 000 6 g, 10 min). The supernatants were
removed and the samples were re-extracted using
the same procedure. The pooled supernatant frac-
tions were dried under reduced pressure, and the
residue dissolved in dimethylsulphoxide. Finally,
the dimethylsulphoxide samples were centrifuged
(7000 6 g, 5 min) to remove debris, and the
supernatants analysed by HPLC.
2
(20 mg cm ) onto the dorsal side of the mice
anaesthetised with sodium pentobarbital (i.p.,
1
90 mg kg ). Four different vehicles were applied
to each mouse. After predetermined application
times (1±6 h), the excess ointment was washed off,
and the mice were killed. Treated skin samples of
uniform surface were excised and mounted on a ¯at
glass plate for stripping.
To determine the yield of the extraction proce-
dure described above, the following experiment
was carried out. Skin samples (1 cm2) were excised
from the dorsal side of the hairless mice and were
heated in water for 1 h at 70ꢀC to remove the fatty
layer. The rest was then homogenised in 2 mL
water and incubated with known amounts of
hypericin at 37ꢀC for 1 h. Acetone (8 mL) was then
added, after which the samples were processed as
described above.
Skin stripping
Skin samples were stripped by repetitively applying
and removing a piece of transparent adhesive
polyethylene tape. For each strip, a fresh piece of
tape was used. In order to standardise the skin-
stripping technique, a 1-kg weight was placed on
the tape in contact with the skin for 30 s before the
peeling. Immediately after every 5, 10, 15, 20, or
25 strippings, the skin samples were visualised
under standardised UV365 irradiation and pictures
taken to document the presence and extent of
¯uorescence of hypericin in the skin. The skin
could be stripped up to 20±25 times, after which
it glistened and could no longer be ef®ciently
stripped.
In evaluating tissue removal with skin stripping,
the skin was treated with the vehicles described
including different concentrations of hypericin.
After 4 h, the treated skin was excised and stripped
as described. Skin samples were immediately ®xed
with formalin and routinely processed after paraf®n
embedding. Eight semi-serial sections of each
sample were stained with haematoxylin and eosin
(H&E) and examined by light microscopy.
Hypericin analysis
An HPLC (L-6200 intelligent pump; Merck Hita-
chi, Tokyo, Japan) with a Lichrosphere 100 RP-18
(5 mm, 4 6 100 mm) column (Merck) and ¯uores-
cence detection (F-1050, Merck Hitachi, Tokyo,
Japan) (excitation wavelength: 470 nm, emission
wavelength: 600 nm) was used to analyse the
samples. As eluent, an isocratic mixture (20 : 80) of
30 mM citrate buffer (pH 4) and methanol=acetone
1
(2 : 1, v=v), with the ¯ow rate set at 1 mL min ,
was used. The retention time of hypericin was
6 min. The method was linear over the range 0Á05±
50 ng of hypericin (r2: 0Á9998). The reproducibility
of the method was determined by replicate analysis
(n ˆ 5) of 3Á1 ng hypericin. The relative standard
deviation obtained was 2Á4%.
Determination of hypericin concentration
in hairless mice skin
Hypericin in emulsifying ointment with iso-
propylidene glycerol (0Á05% hypericin) or poly-
ethylene glycol ointment (0Á5% hypericin) was
applied onto the skin as described. After 4 h, the
unabsorbed ointment was washed off, the mice
were killed, and the treated skin samples excised.
Freshly excised skin samples were heated in water
for 1 h at 70ꢀC to dislodge the fatty layer from the
rest of the skin. This treatment did not extract
hypericin from the epidermis or dermis since
hypericin is virtually insoluble in water. Conse-
quently, hypericin was not found in the water used
to heat the tissues. The fatty layer was then care-
fully peeled off from the skin with the help of
tweezers. The remaining skin tissue (epidermis and
Statistical analysis
Results are presented as meansÆ s.d., n ˆ 5. Stan-
dard statistical evaluation of the data was per-
formed with Student's t-test. P < 0Á05 was
considered signi®cant.
Results
Tissue removal associated with skin stripping
Microscopic analysis of the stripped skin tissues
revealed gradual removal of parts of the stratum
corneum with successive stripping. No effect was

490
APPOLINARY R. KAMUHABWA ET AL
Table 1. The extent of hypericin absorption into the skin of hairless mice, after 4 h of application of different hypericin
concentrations in different vehicles with or without skin-absorption enhancers.
Vehicle
Absorption enhancer
Hypericin concn (%)
Fluorescence intensity
Petrolatum
Polyethylene glycol ointment
None
None
0Á01±1Á0
0Á01±1Á0
0Á001±1Á0
0Á01±1Á0
0Á01±1Á0
0Á01±1Á0
0Á01±1Á0
0Á01
Isopropylidene glycerol
Diethylene glycol monoethyl ether
None
Isopropylidene glycerol
Diethylene glycol monoethyl ether
None
Carbopol gel
Beeler basis
‡
None
0Á05±1Á0
0Á01
‡ ‡
Isopropylidene glycerol
Isopropylidene glycerol
Diethylene glycol monoethyl ether
Diethylene glycol monoethyl ether
None
Isopropylidene glycerol
Diethylene glycol monoethyl ether
None
‡ ‡
0Á05±1Á0
0Á01
‡ ‡ ‡
‡ ‡
0Á05±1Á0
0Á05±1Á0
0Á05±1Á0
0Á05±1Á0
0Á05±1Á0
0Á05±1Á0
0Á05±1Á0
‡ ‡ ‡
‡
Emulsifying ointment
Cetomacrogol cream
‡ ‡ ‡
‡ ‡
‡ *
Isopropylidene glycerol
Diethylene glycol monoethyl ether
‡ ‡ *
‡ ‡ *
, no ¯uorescence of hypericin detected in the skin; ‡ , ‡ ‡ and ‡ ‡ ‡ , intensity of the ¯uorescence of hypericin in the skin
( ‡ < ‡ ‡ < ‡ ‡ ‡ ). *Lateral spreading of hypericin in the skin.
observed on the stripping ef®ciency after applica-
tion of the different vehicles used. Micro-
photographs of the stripped mouse skin indicated
almost complete removal of the stratum corneum
over large areas of skin after 20 strippings (results
not shown).
ing ointment, with which the hypericin was con-
centrated on the applied spot.
There was virtually no hypericin absorption into
the skin when the compound was incorporated in
polyethylene glycol ointment, petrolatum or car-
bopol gel. With petrolatum and carbopol gel, there
was no ¯uorescence of hypericin detected in the
skin after the ®rst stripping, which indicates that
there was no hypericin absorption into the skin
from these vehicles. Hypericin delivered from
polyethylene glycol ointment could no longer be
detected in the skin after the ®rst ®ve strippings, so
the compound was only absorbed into the very
super®cial areas of the epidermis. Comparison of
the different concentrations of hypericin in the
vehicle showed no signi®cant differences in terms
of absorption, except for the lowest hypericin
concentration (0Á01%), which produced less
absorption of hypericin than the other concentra-
tions (Table 1).
Addition of the skin-absorption enhancers (iso-
propylidene glycerol and diethylene glycol mono-
ethyl ether) markedly increased hypericin
absorption from Beeler basis and emulsifying
ointment at all hypericin concentrations (Table 1).
However, in the emulsifying ointment, iso-
propylidene glycerol appeared to be much more
effective than diethylene glycol monoethyl ether,
while in Beeler basis the two enhancers were
equally effective. With skin-absorption enhancers,
there were also no signi®cant differences in the
amount of hypericin absorbed in the skin for the
different hypericin concentrations used, except for
Absorption of hypericin into the skin
The extent of hypericin absorption into the skin was
assessed by visualisation of its ¯uorescence under
standardised UV365 irradiation. The results of dif-
ferent hypericin concentrations incorporated in
different vehicles following 4 h of application on
the hairless mice skin are listed in Table 1. Figure
2 shows some typical ¯uorescence present in the
hairless mice skin, as visualised under standardised
UV365 irradiation after 4 h of topical application
with some vehicles containing hypericin with and
without skin-absorption enhancers, before and after
5, 15, and 20 strippings of the treated skin samples.
Hypericin was found to penetrate the skin when
incorporated in Beeler basis, emulsifying ointment
and cetomacrogol cream. However, the absorption
into the skin from cetomacrogol cream was dif-
ferent from that of Beeler basis or emulsifying
ointment. Hypericin with cetomacrogol cream
appeared to be absorbed in a lateral spread, as
indicated by scattering of the hypericin ¯uores-
cence over a much larger surface area in the skin.
Thus, hypericin ¯uorescence intensity in the skin
from cetomacrogol cream could not be compared
very well with that from Beeler basis or emulsify-

ABSORPTION OF HYPERICIN INTO HAIRLESS MOUSE SKIN
491
Figure 2. Photographs showing the ¯uorescence of hypericin in hairless mice skin (4-h application). The following vehicles were
used: (I) (A) hypericin (0Á05%) incorporated in (a) Beeler basis, (b) polyethylene glycol ointment, (c) emulsifying ointment and (d)
hypericin (0Á5%) in Beeler basis, before washing off the unabsorbed ointments and after (B) 5, (C) 15 and (D) 20 strippings of the
skin, and (II) hypericin (0Á05%) in Beeler basis with (a) diethylene glycol monoethyl ether or (d) isopropylidene glycerol and in
emulsifying ointment with (b) diethylene glycol monoethyl ether or (c) isopropylidene glycerol, (A) before washing off the
unabsorbed ointments and after (B) 5, (C) 15, and (D) 20 strippings of the skin.

492
APPOLINARY R. KAMUHABWA ET AL
the lowest concentration (0Á01%), which showed
less absorption of the compound. There was no
hypericin absorption with carbopol gel, petrolatum
or polyethylene glycol ointment, even after the
addition of the skin-absorption enhancers.
For those vehicles which allowed better absorp-
tion of hypericin into the skin (Beeler basis,
emulsifying ointment, cetomacrogol cream), the
amount of hypericin appeared to increase with
increasing application time. After 1 h of applica-
tion, hypericin could be detected only in the ®rst 5±
10 strippings, while the compound could still be
detected even after 25 strippings after being applied
to the skin for 4 and 6 h (results not shown).
skin of hairless mice. As a control, an experiment
was conducted to determine the amount of unab-
sorbed hypericin that could not be washed away
before the samples were analysed. In this case, the
ointments were applied to the skin for only 1 min.
The results of the control experiment and the 4-h
application periods are presented in Table 2. With
polyethylene glycol ointment, the average hyper-
icin concentration present was limited after the
skin
1
4-h application time (0Á38Æ 0Á34 mg g
(epidermis and dermis), n ˆ 5) and was not
signi®cantly different from that of the control
experiment (P > 0Á05). Conversely, with emulsify-
ing ointment plus isopropylidene glycerol, the
concentration of hypericin in the skin for the con-
trol experiment was very small compared with that
measured for the 4-h application time (P < 0Á01).
After a 4-h application, the average hypericin
Hypericin concentration in hairless mice skin
1
Extraction yield. The extraction yield experiment
was carried out to determine the ef®ciency of the
extraction procedure employed in extracting hyper-
icin from the skin. The extraction yield was deter-
mined with three doses of hypericin (20 ng, 100 ng,
and 300 ng) added to a homogenate of the epider-
mis and dermis present in 1 cm2 skin. The choice of
these three doses was based on the results of the
amounts of hypericin (20 and 200 ng) that were
extracted from the skin following the topical appli-
cation of the compound in polyethylene glycol
ointment or emulsifying ointment with isopropyli-
dene glycerol, respectively. About 65±86% of the
original amount of hypericin incubated with skin
homogenate could be recovered.
concentration was 8Á61Æ 3Á18 mg g , n ˆ 5. The
average hypericin concentration was approximately
23 times higher in the skin when emulsifying
ointment with isopropylidene glycerol was used
than when the hypericin was delivered in poly-
ethylene glycol ointment (P < 0Á01).
Discussion
After being topically applied in a vehicle, hypericin
present in the skin could be visualised well under
strong UV365 irradiation because of its bright
¯uorescence. This allowed the evaluation of the
progressive fading of ¯uorescence that accom-
panied the stripping of the skin, which gradually
removed layers of the stratum corneum. Conse-
quently, the effectiveness of different vehicles in
delivering hypericin into the skin could be inves-
tigated. The decrease in ¯uorescence of the tissue
was due to removal of cell layers containing
hypericin and not due to photodegradation of
hypericin under UV365 irradiation, since prolonged
irradiation of tissue containing hypericin without
stripping did not reduce the ¯uorescence.
Amount of hypericin extracted from skin. Hypericin
penetrated into the skin much better with some
vehicles than with others. To corroborate quantita-
tively these visual observations, the concentrations
of hypericin in the skin were estimated by HPLC
analysis. For this purpose, two vehicles were used
with which hypericin penetrated well (emulsifying
ointment with isopropylidene glycerol) or very
poorly (polyethylene glycol ointment) into the
Table 2. Average concentration of hypericin present in hairless mouse skin following topical application either in polyethylene
glycol ointment or in emulsifying ointment with isopropylidene glycerol.
1
)
Vehicles
Application time
Hypericin concn in skin (mg g
Polyethylene glycol ointment
1 min (control)
4 h
1 min (control)
4 h
0Á21Æ 0Á14
0Á38Æ 0Á34
0Á40Æ 0Á028
8Á61Æ 3Á18**
Emulsifying ointment plus isopropylidene glycerol
Values are expressed as meansÆ s.d., n ˆ 5. **P < 0Á01 compared with application of hypericin in polyethylene glycol ointment
for 4 h.

ABSORPTION OF HYPERICIN INTO HAIRLESS MOUSE SKIN
493
The ¯uorescence yield of hypericin in the vehi-
cles also appeared to be higher in some vehicles
than in others. For instance, hypericin showed high
¯uorescence in polyethylene glycol ointment and
carbopol gel while moderate to low ¯uorescence
was observed in the other vehicles. Fluorescence of
hypericin in the vehicle or solvent indicates the
extent of its solubility, and an increase in ¯uores-
cence indicates an increase of solubility (Yamazaki
et al 1993; Lavie et al 1995). It is well known that
hypericin readily dissolves in polar organic sol-
vents (e.g. polyethylene glycol) to develop red
¯uorescent solutions, but forms non-¯uorescent
high-molecular-weight aggregates in aqueous
media (Lavie et al 1995). In addition, hypericin
does not dissolve, and consequently is not ¯uor-
escent, in vegetable oils and non-polar organic
solvents (data not shown). The higher ¯uorescence
of hypericin in carbopol gel is likely to be due to
the dissolution of hypericin caused by the presence
of propylene glycol. Moreover, hypericin is known
to form ion pairs with organic bases such as tri-
ethanolamine, which is present in carbopol gel. The
various ion pairs of hypericin have varying solu-
bility rates in water depending on the cation (Falk
& Schmitzberger 1992) and it is likely that, in
combination with propylene glycol, triethanol-
amine enhanced the solubility of hypericin in the
aqueous medium.
It is interesting to note that there was an inverse
correlation between observed ¯uorescence and the
skin absorption of hypericin: the compound was
poorly absorbed in the skin from the vehicles in
which it was highly ¯uorescent but readily absor-
bed from those vehicles in which it showed mod-
erate to low ¯uorescence. It is not obvious from this
study why hypericin tends to penetrate the skin
from those vehicles in which it is only slightly
soluble but not from those vehicles in which it is
well solubilized. It can, however, be speculated that
in the former vehicles there is high af®nity between
the drug and the vehicle that might prevent the
release of the compound from the vehicle to the
skin. Alternatively, it can also be assumed that in
the vehicles in which hypericin is insoluble, the
compound is also not able to leave the vehicle and
penetrate the skin due to the bigger sizes (e.g.
aggregates).
might facilitate its interaction with the stratum
corneum constituents, may explain why hypericin
forms a depot in the stratum corneum, as docu-
mented for other lipophilic drugs (Michaels et al
1975).
This study also shows that hypericin skin
absorption is enhanced by the addition of skin-
absorption enhancers (isopropylidene glycerol and
diethylene glycol monoethyl ether) to the vehicles.
The two enhancers, at 10% concentration, have
been found by other investigators to be non-toxic to
mice and other animal skins (Lashmar et al 1989)
and effective in promoting percutaneous absorption
of different drugs (Harrison et al 1996). In this
study it is interesting to note that this enhancement
was only observed with those vehicles from which
the compound could already ef®ciently absorb into
the skin. This might imply that the action of these
enhancers was on the skin rather than on the
vehicles. However, there was a small increase in
hypericin ¯uorescence in the vehicles after the
addition of the skin-absorption enhancers, which
indicates an effect on the solubility of hypericin by
the enhancers themselves.
The quantitative results obtained using an extrac-
tion procedure and HPLC further demonstrate the
validity of the skin-stripping technique. Sub-
stantially higher concentrations of hypericin were
extracted from the skin when it was applied in
emulsifying ointment with isopropylidene glycerol
than when it was applied in polyethylene glycol
ointment. This large ( ꢁ 23-fold) difference is con-
sistent with the visual results obtained with the skin-
stripping technique, indicating that absorption of
hypericin into the skin indeed depends on the vehicle
that is used to deliver the compound to the skin.
The extraction procedure employed to extract
hypericin from the skin indicates that 65±86% of
the applied dose could be extracted. These data are
in agreement with the recovery of hypericin
reported from human urine (63±75%) but some-
what lower than the recovery of hypericin from
plasma of mice, monkeys and humans
(96Á6Æ 5Á8%) (Liebes et al 1991). The relatively
lower recovery of hypericin in this study could be
explained by the fact that hypericin interacts with
body ¯uid components such as human serum
albumin and lipoproteins (Lavie et al 1995). In
addition, mice that received hypericin in their diets
remain sensitive to light for more than a week after
transfer to hypericin-free food, which indicates that
the pigment has a high af®nity for tissue matter
(Giese 1980). In this regard, it is likely that
hypericin also strongly interacts with skin con-
stituents, leading to a lower recovery of the com-
pound from the skin homogenate.
The results of this study further demonstrate the
in¯uence of the application time on the absorption
of hypericin into the skin. Hypericin diffuses
slowly through the stratum corneum layers. Thus,
after the hypericin diffused from the vehicle, its
absorption into the layers of the skin is determined
by the stratum corneum. The lipophilicity as well as
the hydrogen-bonding potential of hypericin, which

494
APPOLINARY R. KAMUHABWA ET AL
Falk, H., Meyer, J., Oberreiter, M. (1993) A convenience
We conclude that the absorption of hypericin into
semisynthetic route to hypericin. Monatshefte fuÈr Chemie.
124: 339±341
Giese, A. C. (1980) Hypericin. Photochem. Photobiol. Rev. 5:
229±255
the skin very much depends on the composition of
the incorporating vehicle. We have proved that the
solubility of hypericin in the vehicle, as assessed by
the ¯uorescence of the compound, could be used as
a good indication for its delivery to the skin.
Hypericin absorption into the inner layers of the
skin is controlled by the stratum corneum. Emul-
sifying ointment with isopropylidene glycerol pro-
mises to be an effective topical vehicle in the
treatment of skin diseases (such as psoriasis) with
hypericin.
Harrison, J. E., Watkinson, A. C., Green, D. M., Hadgraft, J.,
Brain, K. (1996) The relative effect of Azone1 and Trans-
cutol1 on permeant diffusivity and solubility in human
stratum corneum. Pharm. Res. 13: 542±546
Lashmar, U. T., Hadraft, J., Thomas, N. (1989) Topical
application of penetration enhancers to the skin of nude
mice: a histopathological study. J. Pharmacol. 41: 118±121
Lavie, G., Mazur, Y., Lavie, D., Meruelo, D. (1995) The
chemical and biological properties of hypericin ± a com-
pound with a broad spectrum of biological activities. Med.
Res. Rev. 15: 111±119
Liebes, L., Mazur, Y., Freeman, D., Lavie, D., Lavie, G.,
Kudler, N., Mendoza, S., Levin, B., Hochster, H., Meruelo,
D. (1991) A method of quanti®cation of hypericin, an
antiviral agent, in biological ¯uids by high-performance
liquid chromatography. Anal. Biochem. 195: 77±85
Michaels, A. S., Chandrasekran, S. K., Shaw, J. E. (1975) Drug
permeation through human skin: theory and in-vitro experi-
mental measurement. AIChE. J. 21: 985±996
Mitev, V. I., Miteva, L., Dourmishev, A. (1992) Casein kinase
II activity in psoriatic epidermis. Arch. Dermatol. Res. 284:
111±113
Nanney, L. B., Stoscheck, C. M., Magid, M., King, L. E.
(1986) Altered [125I]epidermal growth binding and receptor
distribution in psoriasis. J. Invest. Dermatol. 86: 260±265
Ritcher, A., Davies, D. E. (1995) Effects of anthralin and
hypericin on growth factor signalling and cell proliferation
in-vitro. Biochem. Pharmacol. 50: 2039±2045
Szeimies, R. M., Sassy, T., Landthaler, M. (1994) Penetration
of topical applied delta-aminolevulinic acid for photo-
dynamic therapy of basal cell carcinoma. Photochem. Photo-
biol. B. 6: 143±148
Ugwu, F., Vandenbogaerde, A. L., Merlevede, W. J., de Witte,
P. A. (1998) A comparative study on phototoxicity of
hypericin on A431 cells using three different assays. Anti-
cancer Res. 18: 1181±1184
References
Agostinis, P., Vandenbogaerde, A., Donella-Diana, A., Pinna,
L. A., Lee, K. T., Goris, J., Merlevede, W., Vandenheede, J.
R., de Witte, P. (1995) Photosensitised inhibition of growth
factor-receptor protein kinases by hypericin. Biochem. Phar-
macol. 49: 1615±1622
Agostinis, P., Donella-Diana, A., Cuveele, J., Vandenbo-
gaerde, A., Sarno, S., Merlevede, W., de Witte, P. (1996)
A comparative analysis of the photosensitised inhibition
growth factor regulated protein kinases by hypericin-deri-
vatives. Biochem. Biophys. Res. Commun. 220: 613±617
de Witte, P., Agostinis, P., Van Lint, J., Merlevede, W.,
Vandenheede, J. R. (1993) Inhibition of epidermal growth
factor receptor tyrosine kinase activity by hypericin. Bio-
chem. Pharmacol. 16: 1929±1936
Diwu, Z., Lown, W. (1994) Phototherapeutic potential of
alternative photosensitizers to porphyrins. Pharmacol.
Ther. 63: 1±35
Falk, H., Schmitzberger, W. (1992) On the nature of ``soluble''
hypericin in Hypericum species. Monatshefte fuÈr Chemie.
123: 731±739
Falk, H., Schoppel, G. (1992) On the synthesis of hypericin by
oxidative trimethylemodin anthrone and emodin anthrone
dimerization: isohypericin. Monatshefte fuÈr Chemie. 123:
931±938
Yamazaki, T., Ohta, N., Yamazaki, I., Song, P.-S. (1993)
Excited-state properties of hypericin: electronic spectra
and ¯uorescence decay kinetics. J. Phys. Chem. 97: 7870±
7875