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Skin penetration properties of cosmetic formulations using a perfused bovine udder model

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Abstract

Bioactives are a major focus of the recent developments in cosmetics. Most of these ingredients will work only if they penetrate the stratum corneum and reach the living epidermis. The extent and rate of penetration of bioactive ingredients from cosmetic products is of paramount interest. A systematic in vitro study of penetration into stratum corneum and viable skin was performed using the isolated perfused bovine udder skin (BUS) model. As bioactive ingredients, oil-soluble vitamin E, vitamin E acetate, and water-soluble D-panthenol were incorporated into different cream formulations and a shower gel. Under leave-on conditions (exposure periods of one hour and five hours), the changes in phase behavior and viscosity have a strong impact on the penetration pattern. Generally, the water-soluble D-panthenol penetrates faster and in a higher amount into the stratum corneum than the oil-soluble vitamin E. Regarding the choice of the galenic vehicles, the penetration of both the oil-soluble and water-soluble vitamin is faster and much higher from w/o-cream than from lamellar o/w-cream due to changes in phase behavior. In contrast to the lamellar o/w-emulsion, for the w/o-emulsion there is no difference in the penetration of vitamin E between one hour and five hours (e.g., a steady state is already reached after an exposure period of 1 h). The top ten skin layers (200 μm depth) again reflect the influence of the emulsion formulation on the vitamin E absorption. From the w/o-cream the penetration is faster and more efficient than from the lamellar o/w-cream. Under rinse-off conditions (3*2 minutes), the water-soluble D-panthenol penetrates in a higher amount than the oil-soluble vitamin. However, the penetration effect is detectable even after the extremely short period of exposure. Due to the fact that the shower gel will not change the phase behavior, different routes of penetration may be considered for water-and oil-soluble vitamins.
j. Cosmet. Sci., 50, 147-157 (May/June 1999)
Skin penetration properties of cosmetic formulations using
a perfused bovine udder model
TH. F(SRSTER, W. PITTERMANN, M. SCHMITT, and
M. KIETZMANN, Henkel KGaA, D-40191 Diisseldorf and Institute
of Pharmacology, Toxicology and Pharmac•y, School of Veterinary
Medicine, D-30559 Hannover, Germany
Accepted for publication April 30, 1999. Presented at the XXth
Congress of the International Federation of the Societies of Cosmetic
Chemists, Cannes, France, September 14-18, 1998. Presented in part at
Penetration, Leiden, The Netherlands, September 22-26, 1998.
Synopsis
Bioactives are a major focus of the recent developments in cosmetics. Most of these ingredients will work
only if they penetrate the stratum corneum and reach the living epidermis. The extent and rate of pen-
etration of bioactive ingredients from cosmetic products is of paramount interest. A systematic in vitro study
of penetration into stratum corneum and viable skin was performed using the isolated perfused bovine udder
skin (BUS) model.
As bioactive ingredients, oil-soluble vitamin E, vitamin E acetate, and water-soluble D-panthenol were
incorporated into different cream formulations and a shower gel. Under leave-on conditions (exposure
periods of one hour and five hours), the changes in phase behavior and viscosity have a strong impact on the
penetration pattern. Generally, the water-soluble D-panthenol penetrates faster and in a higher amount into
the stratum corneum than the oil-soluble vitamin E. Regarding the choice of the galenic vehicles, the
penetration of both the oil-soluble and water-soluble vitamin is faster and much higher from w/o-cream than
from lameIlar o/w-cream due to changes in phase behavior. In contrast to the lamellar o/w-emulsion, for the
w/o-emulsion there is no difference in the penetration of vitamin E between one hour and five hours (e.g.,
a steady state is already reached after an exposure period of 1 h). The top ten skin layers (200 pm depth)
again reflect the influence of the emulsion formulation on the vitamin E absorption. From the w/o-cream
the penetration is faster and more efficient than from the lameliar o/w-cream.
Under rinse-off conditions (3*2 minutes), the water-soluble D-panthenol penetrates in a higher amount
than the oil-soluble vitamin. However, the penetration effect is detectable even after the extremely short
period of exposure. Due to the fact that the shower gel will not change the phase behavior, different routes
of penetration may be considered for water- and oil-soluble vitamins.
INTRODUCTION
Today's cosmetic emulsions are not simply mixtures of oil, water, and emulsifier, but
they also contain a lot of active ingredients whose function is to ameliorate the condition
147
148 JOURNAL OF COSMETIC SCIENCE
of the skin, e.g., use in skin hydration or on the barrier properties of the stratum
corneum. Most of these ingredients will work only if they penetrate into the stratum
corneum or the deeper layers of the epidermis. The extent and rate of penetration of an
active ingredient from a topical formulation depends on its passive diffusion into the
skin, which in principle obeys Fick's law of diffusion. This takes account of the con-
centration of the active ingredient in the galenic vehicle, the mobility (diffusion con-
stant) of the active ingredient molecule, and--in the form of the distribution coeffi-
cient-the interaction of the active ingredient with the vehicle and the skin (overview
in references 1 and 2). In practice, the application of this simple law is frequently
difficult, because the composition of a topical formulation only rarely remains constant
during the course of application; volatile components vaporize, water evaporates (3-5),
and the mobility and the distribution coefficient of the active ingredient change as a
function of its concentration and of the viscosity and phase behavior of the galenic
vehicle.
Moreover, the penetration properties of a formulation are also dependent on the inter-
action of the vehicle with the skin (2,6). The application of the emulsion can cause an
occlusive effect, under which the hydration of the horny layer increases, resulting in
increased penetration. Certain components of the formulation may accumulate in the
outermost skin layer, the actual diffusion barrier, thus accelerating penetration (2,7).
Simple penetration models cannot take these different, mutually interacting effects into
account, so that an optimal galenical can only be developed on the basis of experimental
penetration studies.
On ethical grounds, in vivo studies on humans and animals are only carried out on a
limited scale, mainly in connection with dermatics (8). The isolated perfused bovine
udder skin (BUS) model is a good substitute for in vivo tests on humans (9). As a living
skin model it takes also into account metabolic processes in the skin, so that it can be
used to study both skin penetration and skin irritation by cosmetic formulations (10,11).
Due to the different rheology of the emulsions under living conditions (physiological
surface temperature, physiological TEWL) and the expected identical substantivity
(same components as far as possible), it was intended to compare the emulsions under the
conditions of infinite dose, i.e., more than 10 or 20 mg/cm 2. The high dosage prevents
any depletion of vitamin concentration in the vehicle during the penetration experiment.
Furthermore, the study design allows a comparison with the results of the testing of
certain types of ointments and dermatics using this in vitro model (9).
Based on an extensive investigation of the influence of emulsion type and structure on
vitamin penetration into the skin (10), the objective of this work was to study the
kinetics of the penetration of oil- and water-soluble vitamins such as vitamin E, vitamin
E acetate, and D-panthenol. An emulsion of the water-in-oil (w/o) type and a lamellar
oil-in-water (o/w) emulsion were chosen as leave-on products since they showed big
differences in phase behavior and penetration properties (10). At an extreme, the short-
term penetration from a surfactant-based shower gel is investigated under rinse-off
conditions.
MATERIALS AND METHODS
CHEMICALS
The galenic formulations selected for the penetration studies (Table I) exhibit basic
SKIN PENETRATION PROPERTIES 149
physicochemical differences. Two creams (w/o and lameliar o/w emulsions) were studied
as typical leave-on products. As a rinse-off product, a shower gel formulation based on
an aqueous surfactant solution was chosen.
All surfactants, emulsifiers and, consistency-imparting factors were Henkel products.
The surfactants used were sodium lauryl sulfate (Texapon © N70), lauryl glucoside
(Plantacare © 1200), potassium cocoyl hydrolyzed collagen (Lamepon © S), PEG-7 glyc-
eryl cocoate (Cetiol © HE), and cocoamidopropyl betaine (Dehyton © K). The emulsifiers
used were PEG-7 hydrogenated castor oil (Dehymuls © HRE 7) and sodium cetearyl
sulfate (Lanette © E); the consistency-imparting products and iipids were glyceryl oleate
(Monomuls © 90-O-18), cetearyl alcohol (Lanette © O), and behenyl alcohol (Lanette ©
22).
The oils used were dicaprylyl ether (Cetiol © OE, Henkel KGaA), decyl oleate (Cetiol ©
V, Henkel KGaA), and dimethicone (Bayslion © 350, Bayer AG). The vitamins were
natural vitamin E (Copherol © F1300, Henkel KGaA), vitamin E acetate (Hoffmann-La
Roche), and provitamin B5 (D-panthenol, Hoffmann-La Roche). Glycerol and
MgSO4*7H20 (p.a. quality, J.T. Baker).
METHODS
Sample preparation. Lameliar oil-in-water (o/w) emulsions were prepared by mixing the
Table I
Galenic Formulations (in wt.% active substance)
O/W cream
W/O cream (lameliar) Shower gel
Ingredients
Dicaprylyl ether 7.0 7.5
Decyl oleate 7.0 7.5
Behenyl alcohol 7.0
Glyceryl oleate 1.2
Sodium lauryl sulfate 9.5
Lauryl glucoside 5.4
Cocoamidopropyl betaine 1.0
Potassium cocoyl hydrolyzed collagen 1.0
PEG-7 glyceryl cocoate 1.0
Laureth-2 0.5
Sodium cetearyl sulfate 0.2
PEG-7 hydrogenated castor oil 3.5
Dimethicone 0.5
Vitamin E 0.7 0.7
Vitamin E acetate 0.1
D-Panthenol 0.4 0.4 0.2
Glycerol 5.0 5.0
MgSO4*H20 0.7
NaC1 0.5
Preservative 0.3 0.3 1.4
Water 75.4 71.9 78.2
Physical parameters
Droplet size/pm 0.7 _+ 0.2 2.4 _+ 2.2
Viscosity/mPas at 30/s 1000 1000 1160
150 JOURNAL OF COSMETIC SCIENCE
components with an Ikavisc MR-D1 mixer (Janke & Kunkel) for 30 minutes during a
heating and cooling cycle between 25 øC and 95 øC. The emulsions were homogenized for
five minutes at 40øC.
The w/o cream was made by heating the oil/emulsifier mixture to 80øC and stirring in
the hot water phase. The mixture was stirred for five minutes at 80øC and then cooled
to room temperature while being stirred.
The shower gel was prepared by mixing the components for 30 minutes at room
temperature.
Phase behavior of the emu/siom. Liquid crystalline phases were identified by polarization
microscopy (Zeiss, K61n, Germany). The emulsion type (o/w or w/o) was determined by
conductivity measurements (Radiometer, Copenhagen, Denmark).
Viscosity measureme, t. Flow and viscosity curves in the shear rate range from 0 to 100/s
were studied with a thermosrated, shear-rate-controlled rotation rheometer RFS 2
(Rheometrics, Piscataway, NJ) with a plate-plate measuring system (2 mm gap) at 25 øC.
Partide sizes. The particle size distribution in the undiluted emulsions was determined
with an optical microscope with the help of the Optimetrix digital image analyser
(Stemmer, Meerbusch, Germany).
Bovine udder skin (B US) model. The in vitro "isolated perfused bovine udder skin" model
makes use of material from slaughterhouses. Immediately after an animal has been
slaughtered, the udder skin is perfused with heated and oxygen-enriched Tyrode's
solution (80-100 mm Hg pressure; approx. 120 ml/minute) under laboratory condi-
tions. The viability of the skin is monitored biochemically in the perfusate by deter-
mining the pH, the lactate-dehydrogenase activity, and the lactate and glucose concen-
trations. The skin surface temperature (approx. 32øC) and skin fold thickness 3mm are
measured physically (9). The skin of the udder is thin and exhibits all the morphological
characteristics of mammalian skin, including the cutaneous appendages such as seba-
ceous glands and hair follicles. The skin of the udder functionally resembles human skin
(9). The experimental procedures such as preparation, perfusion, viability checks, the
topical application of the coded test substances, and sampling were performed at
SIMRED GmbH (Gro[3burgwedel, Germany).
Skin penetration. The penetration of oil- and water-soluble vitamins from the different
formulations into perfused bovine udder skin was studied in two ways.
Leave-on application. Fifteen minutes after perfusion started, 3 g of each different vita-
min-containing cream formulation were applied topically to skin areas measuring 75
cm 2. This high dosage was intended to prevent any depletion of the vitamin concen-
tration in the vehicle during the penetration experiment (infinite dose). After one hour
and five hours, respectively, the residual cream was carefully removed with a paper towel,
and then adhesive tape strips and samples for preparing dermatome sections were taken.
The adhesive strips and skin samples were kept deep-frozen at -20øC.
Rinse-off application. Fifteen minutes after perfusion started, 3 g of each different vi-
tamin-containing shower gel formulation were applied topically to skin areas measuring
75 cm 2. After two minutes the shower gel was rinsed off with a surplus of warm water
(40øC). The skin was carefully dried with a paper towel. This washing-rinsing procedure
was repeated three times, and then adhesive tape strips were taken.
SKIN PENETRATION PROPERTIES 151
Adhesive tape stripping. Stripping (Tesa © 4204, BDF, Hamburg) was used to remove the
outermost layers of the stratum corneum in sequence; about ten layers of horny-layer
cells (roughly 10 l•m) were removed. The mass of the removed horny-layer cells was
constant for all layers, being approx. 1.10 mg per adhesive strip (1.9'10 cm), with a
standard deviation of +0.36 mg (n = 20). The dermatome sections parallel to the skin
surface were cut into 20-pm-thin sections and went down 200 pm through the epider-
mis into the upper layer of the dermis.
Detection of the vitamins. The detection of vitamin E, vitamin E acetate, and D-panthenol
in the adhesive strips from the outermost skin layer and in the dermatome sections of the
dermis and epidermis was based on extraction of the analytes and subsequent liquid
chromatographic analysis of the obtained extracts. The selective determination of the
vitamins in the complex matrix required substance-specific detection to be carried out
after the chromatographic separation. The quantification was based on external standard
calibrations and comparison of the treated and untreated skin.
Detection of vitamin E and vitamin E acetate. Vitamin E/vitamin E acetate were extracted
from the relevant skin sections with ethanol, and then the solvent was quantitatively
removed and the residue was taken up in hexane. Extraction from adhesive strips was
carried out directly with hexane. Vitamin E was separated from unwanted accompanying
substances in a diol phase (Lichrospher 100 Diol) by means of liquid chromatography.
The chromatography was carried out under isocratic conditions with n-hexane/te•-t.
butyl methyl ether as eluent. Selective detection was performed with a fluorescence
detector. The chromophoric system in the vitamin E molecule enabled the work to be
carried out at an excitation wavelength of 295 nm.
Detection ofpanthenol. In contrast to vitamin E and vitamin E acetate, panthenol has no
molecular properties that would facilitate sensitive and selective detection in difficult
matrices. After extraction from the skin or adhesive strips, therefore, the obtained extract
was first hydrolyzed alcoholically and the panthenol quantitatively converted to ami-
nopropanol. This hydrolysis product was separated from unwanted accompanying sub-
stances in an ion-exchange column with diluted sodium hydroxide solution as eluent,
and was converted to a strongly fiuorescing isoindole derivative in a postcolumn reaction
with orthophthaldialdehyde. The fluorescence emission was measured with an HPLC
fluorescence detector at a wavelength of 455 nm.
RESULTS AND DISCUSSION
PHASE BEHAVIOR OF THE COSMETIC FORMULATIONS
For the leave-on products, consideration also has to be given to their behavior after open
topical application. When they are applied, the emulsions are spread over the skin,
forming a film (approx. 400 l•m). As the temperature of the emulsions increases to skin
temperature, components with a high vapor pressure, in our case water, start to evaporate
(3,4). Drying experiments on thin emulsion films show that•clepending on atmo-
spheric humidity and the thickness of the layer--almost all the water escapes from the
emulsions within five to ten minutes. During this period the viscosity and sometimes
also the structure of the emulsion change (10).
152 JOURNAL OF COSMETIC SCIENCE
Directly after application, the lameliar cream contains 70% water and has a moderate
viscosity of about 1000 mPas (Table I). When water evaporates, the lameliar layers are
packed together more densely, leading to an appreciable viscosity increase (about 7,000
mPas). When the water content becomes less than about 15%, the amount of water is
insufficient to allow o/w-emulsions to be formed any longer, and w/o-emulsions are
formed (10).
The situation is different with the w/o-cream. Here the oil is the outer phase and the
viscosity rises exponentially by increasing the proportion of the inner water phase (10).
During drying, therefore, the viscosity of the w/o-cream decreases below 10 mPas, in
contrast to the o/w-emulsion.
For the shower gel, the phase behavior will not change during the short application
period. The surfactant product is washed away with a surplus of water after only two
minutes. Therefore, phase changes caused by water loss will not take place under
rinse-off conditions.
KINETICS OF VITAMIN PENETRATION INTO THE SKIN UNDER LEAVE-ON CONDITIONS
After application on the skin, an active ingredient begins to diffuse out of the galenic
vehicle into the top layers of the skin. If, for purposes of simplification, it is assumed that
the horny layer is a homogeneous diffusion barrier, then, according to Fick's law (Equa-
tion 1), the penetration rate after a steady state has been reached depends only on the
mobility of the active-ingredient molecule, given by the diffusion constant D, the
coefficient of distribution K of the active ingredient between the vehicle and the stratum
corneum layer, and the concentration c of the active ingredient (1,2).
Penetration rate J = K/d ß D * c (Eq. 1)
where d is the thickness of the horny layer, D is the diffusion constant, c is the
concentration, and K is the distribution coefficient.
Figures 1 to 4 show the penetration profiles of vitamin E and D-panthenol into the
stratum corneum from w/o and o/w creams. The amount of naturally occurring vitamin
E in the skin is several orders of magnitude smaller than the amount absorbed after
topical application of the creams. For the oil-soluble vitamin E, a steep concentration
gradient over the stratum corneum is observed (Figures 1 and 2), which indicates that
the stratum corneum is the main penetration barrier. In contrast to the lameliar o/w
emulsion, for the w/o emulsion there is no difference in penetration between 1 hour and
5 hours. This means that a steady state is already reached for the vitamin E penetration
from the w/o emulsion after 1 hour. In general, the penetration is faster and much higher
from the w/o cream than from the lameliar o/w cream.
The water soluble D-panthenol penetrates faster and in a higher amount into the stratum
corneum compared to the oil-soluble vitamin E (Figure 3, 4). The concentration gradient
is smaller than with vitamin E, indicating a low skin barrier capability against pen-
etration. Again, absorbed amounts are much higher for the w/o than for the o/w emul-
sion.
The reason for the poor vitamin penetration from the o/w emulsion relative to the w/o
cream is the lameliar gel network that is responsible for the viscosity buildup in this o/w
SKIN PENETRATION PROPERTIES ! 5 3
1,6
1,2
0,4
0,0
1st 2rid 3rd 4th 5th 6th 7th 8th 9th loth
Tape strip
-•- untreated --•-w/o cream (1 h) --•-w/o cream ($ h)
Figure I. Kinetics of vitamin E penetration (pg/cm 2 tape strip) from a w/o cream into the stratum corneum
after exposure periods of one hour and five hours (SEM for n = 4).
cream (12--14). These lameliar layers impede the free diffusion of vitamin E from the oil
phase into the skin (10,15). There are no differences in the oil components and vitamin
contents between both emulsion types and only small differences in emulsifiers. There-
fore, the differences observed in the penetration properties are attributed mainly to the
1,2
0,8
0,4
0,0
1st 2nd 3rd 4th 5th 6th 7th 8th 9th loth
Tape strip
-e-untreated -x- o/w cream (1 h) -•- o/w cream ($ h)l
Figure 2. Kinetics of vitamin E penetration (pg/cm 2 tape strip) from a lameilar o/w cream into the stratum
corneum after exposure periods of one hour and five hours (SEM for n = 4).
154 JOURNAL OF COSMETIC SCIENCE
5,0
•4,0
3,0
1,0
0,0 1st 2nd 3rd 4th 5th 6th 7th 8th 9th loth
Tape strip
--•-w/o cream (1 h) --*-w/o cream (5 h)]
Figure 3. Kinetics of D-panthenol penetration (tig/cm 2 tape strip) from a w/o cream into the stratum
corneum after exposure periods of one hour and five hours (SEM for n = 4).
different phase and viscosity behavior of the emulsions in the course of drying on the
skin.
After passing the stratum corneum, which represents the actual penetration barrier, the
vitamins enter into deeper skin layers. Therefore, for vitamin E not only adhesive tape
1,0 .......... ,
0,0 1st 2nd 3rd 4th 5th 6th 7th 8th 9th loth
Tape strip
-•-o/w cream (1 h) -*-o/w cream (5 h)l
Figure 4. Kinetics of D-panthenol penetration (pg/cm 2 tape strip) from a lameliar o/w cream into the
stratum corneurn after exposure periods of one hour and five hours (SEM for n = 4).
SKIN PENETRATION PROPERTIES 155
Table II
Kinetics of Vitamin E Penetration From W/O and Lameliar O/W Emulsions
O/W cream
W/O cream (lameliar)
Amount of vitamin E in 200 lain skin in pg/cm 2
% Absorbed vitamin E in 200 pm skin
After 1 h 24.9 + 4.6 14.5 + 2.7
After 5 h 30.0 + 5.1 21.8 + 2.7
After 1 h 8.9 5.1
After 5 h 10.7 7.8
Total applied amount of vitamin E = 280 pg/cm 2 skin; SEM for n = 4.
Table III
Penetration Properties of Oil- and Water-Soluble Vitamins From O/W Emulsions and a Shower Gel
Leave-on Rinse-off
(5 h) (3* 2 min)
O/W cream
W/O cream (lameliar) Shower gel
Applied total Vitamin E 280 280
amount of vitamins Vitamin E acetate 3'40
in pg/cm 2 D-panthenol 160 160 3*80
Amount of vitamins Vitamin E 4.3 + 1.4 2.9 + 1.0
in 10 stratum Vitamin E acetate 0.1 + 0.02
corneum strips in ].lg/cm 2 D-panthenol 25.3 + 8.5 6.6 _+ 1.2 1.2 + 0.09
% Absorbed vitamin Vitamin E 1.5 1.0
in 10 stratum Vitamin E acetate 0.1
corneum strips D-panthenol 15.8 4.1 0.5
SEMforn = 4.
strips of the outermost stratum corneum layers but also skin sections were examined to
get information about vitamin absorption in the living epidermis and dermis.
In general, the cumulative absorption of vitamin E in the top ten skin layers (200 pm
skin depth) is determined by the penetration of vitamin E into the stratum corneum. As
a consequence, the influence of the emulsion formulation on vitamin E absorption is the
same for the stratum corneum as for the epidermis. From the w/o cream the penetration
is faster and higher than from the lameliar o/w cream (Table II).
The naturally occuring vitamin E content in the skin (200 pm) is about 0.312 (1 hour)
and 0.191 (5 hours) i•g/mg. Thus the results in Table II show that, by typical leave-on
applications, high amounts of vitamin E are transported from the cream vehicle into the
outer skin.
VITAMIN PENETRATION FROM SHOWER GEL UNDER RINSE-OFF CONDITIONS
The rinse-off conditions chosen in the experimental setup should resemble the short
exposure periods found for body cleansing products on one hand and the frequent
156 JOURNAL OF COSMETIC SCIENCE
Vitamin E acetate and D-panthenol (rinse-off)
BUS-model (rinse off): Penetration of Vitamin E Acetate and
D-panthenol into the horny layer (n=4)
0,16
0,14
0,10
0,08
0,06 ........................................................................................
0,04 I ......................
0,02
0,001 ,
1st 2rid 3rd 4th 5th 6th 7th 8th 9th 10th
tape strip
•Vitamin E acetate •D-panthenol 1
Figure 5. Kinetics of D-panthenol and vitamin E penetration (tlg/cm 2 tape strip) from a surfactant-based
shower gel (rinse-off condition) into the stratum corneum after an exposure period of 3 * 2 minutes (SEM
for n = 4).
applications on the other hand. Considering the very short exposure periods (two min-
utes compared to one or five hours for topical leave-on application), the low vitamin
concentrations in the surfactant formulation, and the missing of any vitamin enrichment
caused by water evaporation, it is astonishing that penetration of vitamins into the skin
is achievable at all (Table III, Figure 5).
The vitamin penetration from a surfactant-based shower gel formulation after three
rinse-off applications is much lower than from the cream formulations under leave-on
conditions, but still detectable. Again, as found for the leave-on products, the water-
soluble D-panthenol penetrates to a higher extent into the stratum corneum than the
oil-soluble vitamin E acetate. It can be assumed that the difference is also influenced by
different routes of penetration into the stratum corneum, e.g., the lipid-soluble vitamin
is following the intercellular spaces whereas the water-soluble vitamin penetrates more
through the corneocytes.
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Structure activity relationships, Progr. Colloid Polymer Sci., 72, 37-42 (1986).
... Mit der Verwendung des BUS-Modells zur Untersuchung der Penetration eines rinse-off Produktes (zeitlich begrenzt appliziertes Produkt, z. B. Shampoo) wurde nachgewiesen, dass die Penetration sehr schnell und differenziert einsetzt [11] . Weitere Untersuchungen für leave-on Produkte (zeitlich unbegrenzt applizierte Produkte , z. B. Händedesinfektionsmittel) bestätigten diesen Sachverhalt. ...
... Die Entwicklung einer Zellschädigung und Zellreizung ist abhängig von der aktuellen Penetration durch die Hornschicht. Daher wurde der BUS-Verträglichkeitstest standardisiert über drei träglichkeit, Penetration) sowie für den betrieblichen Hautschutz adaptiert8910111213. Im BUS-Modell werden Rindereuter (unmittelbar nach der Schlachtung) isoliert und anschließend kontinuierlich über die linke und rechte Arteria pudenda ext. ...
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Hintergrund: Für die Untersuchung der Hautverträglichkeit von Händedesinfektionsmitteln existiert keine objektive In-vivo-Methode, mit der die beiden wichtigen Komponenten einer möglichen Hautirritation, die Zellreizung und die Zellschädigung, wirklichkeitsnah, vergleichend, objektiv und praxisnah abgebildet werden können. Daher wurde die Haut des isoliert perfundierten Rindereutermodells (BUS-Modell) als In-vitro-Methode verwendet, das eine reife, lebensfähige Haut mit intakter Reservoir- und Barrierefunktion aufweist. Methode: Im standardisierten BUS-Hautverträglichkeitstest wurden parallel fünf verschiedene als Arzneimittel zugelassene Händedesinfektionsmittel (kodiert als 01-01, 01-03, 01-05, 01-06 und 01-07) offen appliziert. Als Negativkontrolle diente Aqua dest., als Positivkontrolle 10 % SLS. Nach einer Expositionsdauer von 30 min, 1 h und 5h wurden Ganzhautbiopsien der behandelten Haut (sowie unbehandelter Haut als Kontrolle) entnommen und biochemisch auf deren Zellschädigung (MTT-Test) und Zellreizung (Prostaglandin E2-Konzentration) hin untersucht. Zur Bestimmung der Hautverträglichkeit wurde der Gesamtscore aus der Summe der Werte für die Zellschädigung und Zellreizung berechnet. Ergebnisse: Hinsichtlich der Zellreizung waren zwischen den Händedesinfektionsmitteln keine wesentlichen Unterschiede über alle drei Expositionszeiten zu erkennen. Nach 30 min lagen auch keine wesentlichen Unterschiede in der Zellschädigung vor. Erst nach 1 h und 5 h wurden formulierungsabhängige Profile sichtbar. Dabei zeigte sich, dass die Positivkontrolle sowie drei Händedesinfektionsmittel nach einer (1 h: 01-07; 5 h: 01-06) oder nach zwei Expositionszeiten (1 h und 5 h: 01-05) die hautkritische Marke von Gesamtscore 3.0 erreichten, nicht jedoch die beiden Formulierungen 01-01 und 01-03 oder die Negativkontrolle. Schlussfolgerung: Die signifikant bessere Hautverträglichkeit der Händedesinfektionsmittel 01-01 und 01-03 könnte zum einen durch die im Vergleich zu den anderen Formulierungen geringere Alkoholkonzentration, vor allem aber auf den Einsatz von wirksamen Rückfettersystemen zurückgeführt werden.
... These variations constitute an important category of factors affecting the penetration of various pharmaceutical and cosmeceutical compounds [16]. Few in vitro studies have reported the effect of these variations of skin on the penetration of various actives [17,18] and some studies tried to explain the varying skin responses towards external agents using these intra-and inter-subject variations [19][20][21]. ...
Article
The present research work deals with the in vivo determination of intra- and inter-individual human stratum corneum biochemical variability using confocal Raman spectroscopy. This study was carried out at three different spots on the volar forearm region of healthy human participants in the extended fingerprint and high wavenumber spectral regions. All the preprocessed spectra were compared by correlation matrices, which were calculated using Pearson's correlation coefficient (r). The results indicate that the participated volunteers in this study are homogenous with respect to the stratum corneum in the forearm region in both extended fingerprint and high wavenumber regions as there is high correlation between their Raman spectra at both intra- and inter-individual levels. This study confirms the application of confocal Raman spectroscopy as an accurate and precise technique for in vivo studies involving human skin.
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Background: No objective in vivo method has yet been established for evaluating the skin compatibility of hand disinfectants, in particular with respect to a realistic, comparative, objective and practically relevant test of the two important constituents of potential skin irritation, cell irritation and cytotoxicity. Therefore the standardized in vitro BUS (bovine udder skin) test system was implemented, enabling experiments with a mature, viable skin with intact skin barrier and reservoir functions. Method: In the BUS skin compatibility test five approved pharmaceutical formulations of hand disinfectants (coded 01-01, 01-03, 01-05, 01-06 und 01-07) were openly applied to the skin. Distilled water was used as negative control and SLS 10% as positive control. Full-thickness skin biopsies were taken from treated as well as from untreated skin after three exposure times (30 min, 1 h and 5 h). These biopsies were then biochemically analysed with regard to their inflammatory (prostaglandin E2 concentration) and their cytotoxic (MTTassay) potentials. In order to determine skin compatibility, the total score was computed from the sum of cytotoxicity and cell irritation values. Results: Regarding the irritation potential of the hand disinfectants no significant differences between the formulations were observed at any exposure time. Also, there were no differences in the cytotoxic potential after 30 minutes of exposure. However, after 1 h and 5 hours of exposure, significant formulation-specific differences were found. The positive control SLS 10% and also three of the hand disinfectants reached a skin-critical score of 3.0 at one exposure time (1 h: 01-07, 5 h: 01-06) or at two exposure times (1 h and 5 h: 01-05). In contrast, neither the formulations 01-03 and 01-01 nor the negative control reached a critical cytotoxic level of a total score of 3.0. Conclusion: The significantly better skin compatibility of the formulations 01-03 and 01-01 could possibly be explained by medizinithe lower total alcohol concentration but is primarily achieved through the use of an effective moisturizing and lipogeneous agent systems.
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We have studied the emulsion particle size change, the stability, and the possibility for applications of cosmetics depending on the concentration and variety of phosphatidyl choline (PC). The relationship between the emulsion particle size and the concentration of PC was checked using lecithin that included either 39% or 70% PC. The greater PC concentration gave the smaller particle size because of the critical packing parameter (CPP) of each lecithin. The cell toxicity of lecithin is similar to that of nonionic surfactants, and so it could be used as a skin-compatible material in cosmetics that use its benefits. PC, the main ingredient of lecithin, forms stable vesicles because its CPP is 1/2 ~ 1. Single-chain surfactants resulted in smaller particle when used with lecithin; Tween 60 made the smallest particles, so we believe its CPP is similar to that of PC. The particle size of a sample over the phase inversion temperature was smaller than one below it. In a study of emulsions using oils of different densities, silicone oil (the highest density) was worked positively in emulsion particles. The moisturizing effect of an emulsion using lecithin was better than that of an emulsion using nonionic surfactant. Finally, we prepared emulsions using unsaturated lecithin and saturated lecithin at the same concentration and found that unsaturated lecithin creates smaller particles.
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Aim of the study: There is a widely accepted need for systemic investigations regarding the efficacy of skin protection products used in occupational health care. The efficacy of such products has been established for water-soluble irritants, such as SLS, but the use in studies of lipid-soluble irritants, such as toluene, poses experimental and ethical problems. The aim of the in-vitro study using toluene was to demonstrate the skin protecting properties of eight commercially available products and two positive controls (lanolin alcohol, petrolatum) and one market product (W/O emulsion) used as negative control. Four of the eight products were emulsions of the O/W type, three products were suspensions and one product was described as a hydrogel formulation. All of them claimed to be protective either against water-insoluble or water-soluble/water-insoluble irritants. Method: The viable, natural skin of the isolated, perfused, bovine udder (BUS model) was used as the in-vitro model. Continuously oxygenised perfusion maintains skin metabolism and the performance of the barrier within the horny layer for more than 8 hours. Using whole skin biopsies the degree of irritation is measured by evaluating the irritancy (PGE2 concentration) and cytotoxicity (MTT assay). The BUS study design allows the simultaneous evaluation of the status of the untreated skin, the skin-compatibility of the products/reference substances, the irritation capability of toluene itself, and finally the irritation capability of toluene applied to the skin pre-treated with the products 15 minutes previously. The first samples of whole skin biopsies were taken 15 minutes after the toluene was applied. The following exposure periods were 1 hour and a prolonged exposure period of 5 hours. In addition to the main study using the hairy skin (follicular) of the lateral udder (n = 4) in a 2nd study (n = 2) the hairless skin of the teats was used for the assay. Results: The local effects of toluene are based on progressive cytotoxicity and a slight increase in the PGE2 concentration in the skin tissue. Both reference substances (lanolin alcohol, petrolatum) reduced significantly the toluene-related alterations by approx. 70 % within 15 minutes after application of the toluene. In contrast, the negative reference (W/O emulsion) did not influence the tissue reactions induced by toluene at all. Two (suspension, O/W emulsion) of the eight market products were comparable to the reference substances regarding their protective potential. Three market products (suspension, O/W emulsion) had a moderate degree of protective potential, while three other market products (suspension, O/W emulsion, and hydrogel) had a low degree of protective potential. After exposure for 1 hour and 5 hours, only the negative control and the products with the low performance demonstrated a distinct increase in protective potential. No distinct difference was observed between the results after exposure for 1 hour and 5 hours. Similar results were obtained after application on the hairless skin of the teats. Conclusions: None of the eight market products was comparable with the negative reference (market product) formulated as a W/O emulsion. This product claimed to protect against water-soluble irritants only. In this assay it proved to be ineffective against lipid-soluble irritants, such as toluene. All skin protection products tested in the BUS model showed a certain degree of protective potential against the lipophilic model toxicant, toluene.
Article
Dermatology studies focussing on the stratum corneum were carried out by infrared attenuated total reflection (ATR) spectroscopy using flexible fibre-optic probes made from square cross-section polycrystalline silver halide material. We compared the upper horny layer of human and bovine udder skin (BUS). As a substitute for human in vivo tests, the in vitro model of the isolated perfused bovine udder skin (BUS-model) has been used. For depth profiling of endogenous and topically applied compounds, subsequent stripping of corneocyte layers by adhesive tape application was carried out in combination with in situ fibre-probe ATR-measurements. The non-invasive measurement technique was also applied to patients with various skin abnormalities. Owing to the remote sensing capability, the technique presented is promising, since it opens the field for new medical and cosmetic applications, which are otherwise not possible with conventional sampling compartment based ATR-accessories.
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Synopsis The skin activation and penetration capability of vitamin E acetate as an ingredient in a basic o/w cream (lameliar type), in liposomes (Rovisome ©) and microparticles (Roviparts©), was investigated under in vitro conditions (BUS model) by the adhesive stripping method. The aim of the study was to compare the analytical results obtained by UV spectroscopy (transmission) and the conventional HPLC method. For the quantitative spectrometric assay, a classical least-squares evaluation of the spectra between 265 and 350 nm, based on the constituent spectra, was used. UV spectroscopy is an economic analytical method for evaluating a large population of samples of the horny layer taken by the adhesive tape stripping method, which is an established tool for depth profiling of substances within the stratum corneum. With regard to the irritation test, no cytotoxicity was recorded for all formulations tested. However, the Roviparts © and Rovisome © cream formulations induced a considerable activation of the epidermal cells that may contribute to the penetration efficiency of Rovisome©-formulated vitamin E acetate. The Rovisome ©-formulated cream delivered a maximum amount of vitamin E acetate into the horny layer compared to the other formulations tested. The difference can be explained by an alteration of the plasticity of the horny layer inducing a strong reservoir capacity and an activation of upper epidermal cells. Moreover, the opening of the potential pathway for a follicular penetration may be part of the increased reservoir capacity.
Article
Mid-infrared fiber probes allow an extended use of attenuated total reflection (ATR) measurements for topical in vivo skin analysis, which were otherwise not possible with conventional sample compartment accessories. Evanescent wave spectroscopy using a flexible fiber-optic probe from silver halide fibers of square cross-section was employed for stratum corneum characterization and keratinocyte quantification on adhesive tapes. Such a method of quantifying the amount of keratin, which can be repetitively removed from the skin surface by adhesive tape application, is essential for the study of substances topically applied and penetrating into the horny layer. For calibration, the weight of keratinocytes was determined using an ultramicro-balance. Best results were obtained with difference spectroscopy and the evaluation of the amide I absorption band intensity (correlation coefficient r ¼ 0:983). Lowest amounts per cm 2 were reached for the range down to 5 mg/cm 2 . The heterogeneity in the surface density of keratinocytes clinging to the tape was investigated by microscopy, and the thickness of some individual keratinocytes was tested by ATR-microspectroscopy and atomic force microscopy. q 2003 Elsevier B.V. All rights reserved.
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Infrared-spectroscopy has proved to be a powerful method for the study of various biomedical samples, in particular for in-vitro analysis in the clinical laboratory and for non-invasive diagnostics. In general, the analysis of biofluids such as whole blood, urine, microdialysates and bioreactor broth media takes advantage of the fact that a multitude of analytes can be quantified simultaneously and rapidly without the need for reagents. Progress in the quality of infrared silver halide fibers enabled us to construct several flexible fiber-optic probes of different geometries, which are particularly suitable for the measurement of small biosamples. Recent trends show that dry film measurements by mid-infrared spectroscopy could revolutionize analytical tools in the clinical chemistry laboratory, and an example is given. Infrared diagnostic tools show a promising potential for patients, and minimal-invasive blood glucose assays or skin tissue pathology in particular cannot be left out using mid-infrared fiber-based probes. Other applications include the measurement of skin samples including penetration studies of vitamins and constituents of cosmetic cream formulations. A further field is the micro-domain analysis of biopsy samples from bog mummified corpses, and recent results on the chemistry of dermis and hair samples are reported. Another field of application, for which results are reported, is food analysis and bio-reactor monitoring.
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Atopic dermatitis (AD) is a common chronic relapsing disease particularly affecting children. The emollient used for protection of skin barrier function is the standard treatment for patients with AD. Currently, there is a growing interest in the use of nonsteroidal anti-inflammatory agents such as dexpanthenol (vitamin B5) as an alternative treatment. To compare the effectiveness of 5% dexpanthenol (DT) ointment with 1% hydrocortisone (HC) ointment in childhood AD therapy. Patients were treated topically with 5% DT ointment on the right side of the body and 1% HC ointment on the other side twice daily for 4 weeks. The clinical responses were evaluated by SCORAD (Scoring Atopic Dermatitis index) with statistical analysis using paired t-test. Of the 30 children enrolled, 26 completed the protocol; mean age was 7.19 years. The average baseline SCORAD score of the DT-treated side and the HC-treated side was 30.95 and 30.54, respectively. There was no statistically significant difference in SCORAD score reduction between the 2 agents. The edematous score of the HC-treated side exhibited faster resolution than that of the DT-treated side, with a statistically significant difference at week 1 and without a statistically significant difference at weeks 2 to 4. The lichenification response rate of HC treatment was more rapid than that of DT treatment; however, there was no statistical group difference. No adverse events were observed with either agent. The effectiveness of 5% DT ointment is equal to that of 1% HC ointment. DT ointment may be used as alternative treatment in mild to moderate childhood AD therapy.
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Synopsis Cosmetic oil-in-water emulsions such as lotions and creams are complex multiple-phase systems. In their preparation, combinations of fatty amphiphiles (glyceryl monoesters or fatty alcohols) and ionic or nonionic surfactants are widely used. The mixed emulsifier combinations interact in the aqueous continuous phases to form lameliar or crystalline structures. These both stabilise and sometimes control the consistencies of emulsions between wide limits. There is, however, confusion as to the type of lameliar phase that forms in a specific emulsion. The majority of the literature fails to distinguish between the lamellar liquid crystalline state and the equally important lamellar gel state. Although liquid crystalline phases form in many emulsions at the high temperatures of manufacture, these often convert to gel phases when the emulsion cools so that the properties of this phase dominate the emulsion. In this article the structures and swelling properties of the different lameliar phases that occur in emulsions are discussed, as well as the formation of other crystalline phases. Attention is given to the conditions over which each type of phase forms and, in particular, the relevance of the gel-liquid crystalline transition temperature to emulsions. It will be shown how the behaviour of many complex emulsions during manufacture , storage, and use can be related to the component phases.
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The action of skin lotions is discussed against time dependent changes in their composition and structure and the specific influence on the skin by the different final structures is reviewed.
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The evaporation from thin layers of water/hydrocarbon emulsions was followed gravimetrically and the state of the emulsion was observed optically during the process.For a system with a hydrocarbon of high vapor pressure (cyclohexane) the evaporation rate was changed suddenly at the depletion of oil for the W/O emulsion while the O/W emulsions gave a gradual reduction. The system water/hexadecane gave the opposite behavior, water evaporated faster leaving hexadecane.In the water/decane system evaporation was similar and the rate dependence show no special feature, except, the emulsion with O/W ratio equal to 70/30. It was initially a double emulsion of the O/W/O type. Because the decane being the continuous phase evaporated slightly faster from a thin layer, coalescence was observed of the water droplets with the smaller oil droplets inside the water droplets remaining intact.
Chapter
This study deals with crystalline nonionic surfactant-water mixtures having lamellar gel structures. The release of an extremely hydrophilic drug (nicotinamide) from mixtures with various water contents was measured and the results compared with Differential Scanning Calorimetry data. It is concluded that the lamellar gel structure fits a simple pore model in which the interlamellar channels behave as pores, the porosity being determined by the free (i. e. unbound) water content, the tortuosity by the orientation of the lamellae.
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Synopsis Oil-in-water emulsions stabilized with nonionic emulsifiers change to water-in-oil emulsions as the temperature rises when the hydrophilic and lipophilic properties of the mixed emulsifier are just balanced. Preparation above the phase inversion temperature followed by rapid cooling yields emulsions that exhibit very fine droplet size and extreme long-term stability. Cosmetic emulsions were prepared by this phase inversion temperature (PIT) method using typical raw materials such as polar oils, e.g. decyl oleate, 2-octyl dodecanol or isopropyl myristate, and nonionic emulsifiers, e.g. ceteareth-12 or polyoxyethylene eicosyl/docosyl ether combined with cetostearyl alcohol as a co-emulsifier. The phase inversion temperature was measured as a function of the oil polarity and the concentration of mixed emulsifier. The relationship between phase inversion temperature, droplet size and emulsion stability was investigated. In addition, self-bodying agents such as cetostearyl alcohol or monoglycerides were added to these thin, fine disperse emulsions to adjust the consistency. The influence of these ingredients on phase inversion temperature, droplet size, yield value and emulsion stability was studied.
Using udders from slaughtered cows as a new in vitro model of percutaneous drug absorption, the tissue viability and the percutaneous absorption of dexamethasone, benzoyl peroxide, and etofenamate were studied. The organ was perfused with gassed tyrode solution for up to 6 hr. As shown by measurement of glucose consumption, lactate production, lactate dehydrogenase activity, and pH in the perfusate, the tissue was viable over a 6-hr period. This was confirmed by a histological examination. Determination of the udder skin-fold thickness demonstrated that no edema developed within the perfusion period. A maximum skin penetration of dexamethasone was found after administration of dexamethasone dissolved in acetone with dimethyl sulfoxide, followed by ointment with salicylic acid, ointment without salicylic acid, and acetone solution. Experiments with benzoyl peroxide and etofenamate demonstrated that the perfused udder skin was capable of metabolizing drugs in vitro. In conclusion, the isolated perfused bovine udder is a new in vitro model, which maintains bovine udder skin with an isolated vasculature in a viable state. Using this in vitro model, we note it is possible to compare the dermal penetration, metabolism, and absorption of substances after topical administration of different drug formulations.