permeation of topical caffeine_trauer 2009

8/11/2019 Permeation of Topical Caffeine_Trauer 2009

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Permeation of topicallyapplied caffeine through

human skin a comparisonof in vivo and in vitro dataSindy Trauer,1,2 Alexa Patzelt,1 Nina Otberg,1 Fanny Knorr,1

Christel Rozycki,3 Gabor Balizs,3 Rolf Bttemeyer,4

Michael Linscheid,5 Manfred Liebsch2 & Jrgen Lademann1

1Department of Dermatology, Charit-Universittsmedizin Berlin, 2Zebet at The Federal Institute of Risk

Assessment (BfR), 3Federal Institute of Risk Assessment (BfR), 4Department of Surgery,

Charit-Universittsmedizin and5Institute of Chemistry of the Humboldt University,Berlin, Germany

CorrespondenceAlexa Patzelt, MD,Department

of Dermatology, Charit

Universittsmedizin Berlin,Charitplatz 1,

10117 Berlin

Tel.:+49 30 450 518 106

Fax: +49 30 450 518 918

E-mail: alexa.patzelt@charite.de----------------------------------------------------------------------

Keywordscaffeine, follicular penetration,hair follicle

----------------------------------------------------------------------

Received18 December 2008

Accepted5 May 2009

WHAT IS ALREADY KNOWN ABOUT

THIS SUBJECT

The hair follicles represent important shunt

routes into the skin for a multiplicity of drugs and

chemicals. Recently, it has been shown that the

hair follicles are responsible for a fast delivery of

topically applied substances. After topical

application, caffeine was already detected in the

blood of the volunteers after 5 min, whereas,

when the hair follicles were selectively blocked

utilizing the newly developed Follicular ClosingTechnique (FCT), caffeine was detectable only

after 20 min. Because of ethical reasons, in vivo

investigations are not always applicable.

Therefore, appropriatein vitromethods have to

be developed and compared with the available in

vivodata, in order to identify their transferability.

WHAT THIS STUDY ADDS

In the present study, the FCT was adapted forin

vitrouse in the Franz diffusion cell and the

penetration of caffeine was investigated and

compared with the previously obtainedin vivo

data. It was shown that the combination of FCT

and Franz diffusion cell represents a valuable

method to estimate the follicular penetration

processin vitro, which revealed comparable

resultsin vivo, whereas the kinetics of caffeine

penetration were significantly different. These

findings are of importance and need to be kept

in mind when evaluating the results obtained in

in vitrostudies.

AIMS

Due to ethical reasons, in vivopenetration studies are not applicableat all stages of development of new substances.Therefore, thedevelopment of appropriatein vitromethods is essential, as well as the

comparison of the obtainedin vivoandin vitrodata, in order to identifytheir transferability. The aim of the present study was to investigate thefollicular penetration of caffeinein vitroand to compare the data with

thein vivoresults determined previously under similar conditions.

METHODS

The Follicular Closing Technique (FCT) represents a method to

investigate the follicular penetration selectively. In the present study,FCT was combined with the Franz diffusion cell in order to differentiatebetween follicular and intercellular penetration of caffeine into thereceptor mediumin vitro. Subsequently, the results were compared

with the data obtained in an earlier study investigating follicular andintercellular penetration of caffeinein vivo.

RESULTS

The comparison of the data revealed that thein vitroexperiments werevaluable for the investigation of the follicular penetration pathway,

which contributedin vivoas well asin vitroto approximately 50% ofthe total penetration, whereas the kinetics of caffeine penetration wereshown to be significantly different.

CONCLUSIONS

The combination of FCT with the Franz diffusion cell represents avaluable method to investigate follicular penetrationin vitro.

Nevertheless, in vivoexperiments should not be abandoned as in vitro,structural changes of skin occur and blood flow and metabolism areabsent, probably accounting for reduced penetration ratesin vitro.

British Journal of ClinicalPharmacology

DOI:10.1111/j.1365-2125.2009.03463.x

Br J Clin Pharmacol / 68:2 / 181186 / 181 2009 The AuthorsJournal compilation 2009 The British Pharmacological Society


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Introduction

For most topically applied pharmaceuticals and cosmetics,

penetration through the skin barrier is essential for devel-

oping their effects. However, regarding optimization and

the development of new substances, it is consequently of

the highest relevance to be familiar with the correspond-

ing penetration pathways. In principle, four differentpenetration pathways are available for topically applied

substances. On the one hand, penetration can occur inter-

cellularly along the lipid layers or intracellularly. Addition-

ally, penetration via the sweat glands as well as via the hair

follicles is feasible. In the past, the intercellular penetration

pathway was supposed to represent the most important

pathway and took scientific centre stage [1-6].On the con-

trary, the shunt routes (i.e. hair follicles and sweat glands)

have been somewhat neglected. The hair follicles were

assumed to cover only 0.1% of the skin surface and there-

fore were considered to be irrelevant for skin penetration

processes [7]. During the past years, this opinion has dras-tically changed.Scientists recognized that the hair follicles

represent weak spots in the skin barrier. Moreover, hair

follicles represent invaginations of the epidermis extend-

ing deep into the dermis, thus providing a greater actual

area for potential absorption [8]. In the meantime, a multi-

plicity of studies has shown that the hair follicles represent

important penetration pathways, as well as a long-term

reservoir for topically applied substances [915]. However,

the development of a method to investigate the follicular

penetration selectively represents a particular challenge.

Available skin absorption tests, such as the tape stripping

procedure or the Franz diffusion cell do not allow a clear

differentiation between the different penetration path-ways. Recently, the Follicle Closing Technique (FCT), an

in vivo method to investigate the follicular penetration

pathway, was introduced by Teichmann et al. [16]. After

closing the hair follicles with a varnish wax mixture, the

penetration of topically applied caffeine was investigated

and compared with caffeine penetration through a skin

area with open hair follicles. In the case of the open hair

follicles, the caffeine was detectable in the blood signifi-

cantly earlier (detection after 5 min),whereas in the case of

closed hair follicles, caffeine was not detectable in the

blood until 20 min after administration [14]. Recently,

the FCT was also successfully established for in vitro usein the Franz diffusion cell (FD-C) [17].

The aim of the present study was to investigate follicu-

lar penetration of caffeinein vitroutilizing the FCT in com-

bination with the FD-C and to compare these data with the

available data on in vivofollicular penetration of caffeine,

derived from the study of Otberg et al. [14].

Due to ethical reasons,in vivo studies are not applicable

at all stages of development of new substances; therefore,

the development of equivalent in vitro models seems

highly reasonable. However, as in vitro data cannot com-

pletely reflect thein vivosituation, a comparison ofin vivo

and in vitro results is essential in order to identify the trans-

ferability.

Methods

Preparation of test formulation

Caffeine 2.5 g (Sigma Aldrich, Steinhagen, Germany) wasadded to 30 g of ethanol 70% (ethanol p.a. analytical

grade, Merck, Darmstadt, Germany). Subsequently, 67.5 g

of propylene glycol (Henry Lamotte GmbH, Bremen,

Germany) was added and the composition was homog-

enized in an ultrasonic bath for 15 min corresponding to

the formulation utilized by Otberg et al. [14].

Skin absorption testThein vitroexperiments were performed according to the

OECD Test Guideline 428 [18].

Pre-calibrated static Franz diffusion cells with an area of

1.76 cm2

available for diffusion and receptor compartmentvolume of approximately 12 ml were used for the skin

absorption tests. The receptor compartment was carefully

filled with Dulbeccos phosphate buffered saline (DPBS)

with Ca++ and Mg++ from PANBiotech GmbH (Aidenbach,

Germany) and stirred with a small magnetic stir bar to

ensure adequate mixing.

Skin samplesHuman full thickness skin was obtained during plastic

surgery from the breast region and from four different

subjects (female, aged 3562 years). The study had been

approved by the Ethics Committee of the Charit.The hair

follicle density was 22 follicles cm-2 on average. The hairfollicle density in thein vivostudy by Otberget al.[14] was

20 to 32 follicles cm-2.

Follicular closing techniqueThe follicular closing technique (FCT) was performed on

the skin test samples, which had been placed beforehand

between the donor and receptor chamber of the static

Franz diffusion cell.

The follicular orifices of the test samples were closed by

small drops of a varnish wax mixture in accordance with

Otberget al. [14].The method has been described in detail

elsewhere [14, 16, 17]. The varnish wax mixture was alsoapplied to the control samples, but only in the vicinity of

the follicles, so that the shunts were not blocked. In both

cases, the penetration area was reduced on the same

surface.

Application protocol and samplingFor the in vitro study, 17.6 ml of the caffeine formulation

was applied to a skin area of 1.76 cm2.The test formulation

contained 25 mg caffeine ml-1.Thus,250 mg cm-2 of caffeine

was applied, which corresponded to a five-fold increase in

the amount of caffeine having been applied in vivo by

S. Trauer et al.

182 / 68:2 / Br J Clin Pharmacol


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Otberg et al. [14] (50 mg cm-2). This was inevitable as the

detection limit of caffeine for the analysis of the in vitro

samples with HPLC was significantly lower (25 ng ml-1) in

comparison with the detection limit of the in vivoblood

samples (1 ng ml-1) determined by SI/MS.

After application of the caffeine formulation, samples

from the sampling port (400 ml receptor fluid) of the static

Franz diffusion cell were taken at the time points 0, 1, 2, 5,8 and 24 h, and immediately replaced by fresh receptor

medium of equal volume and temperature.

The recovery rate was determined after 24 h in all Franz

diffusion cell experiments for four different components

(donor, epidermis, dermis and receptor fluid). Samples

were extracted using an ultrasonic bath for 1 h in isopro-

panol (Isopropanol SupraSolv analytical grade, Merck

Darmstadt, Germany) or DPBS, respectively.

High performance liquid chromatographyA WATERS liquid chromatography equipped with a

WATERS 510 high-pressure pump,as well as a 712WISP anda WATERS photo diode array detector were employed in

combination with a Reversed Phase column TYPE WATERS

RESOLVE C18. 5 mm, 3.9 mm 150 mm.

To prepare the samples for calibration, the donor solu-

tion was used. For every run, these calibration samples

were analyzed and a calibration curve was calculated.

The HPLC detection limit for caffeine was 25 ng ml-1 at a

wavelength of 262 nm.

For analysis, 50 ml of each test sample was used. The

elution mixture for caffeine was 40 : 60 methanol : ammo-

nium acetate buffer (pH 5.35) (both analytical grade,

Merck, Darmstadt, Germany).

The in vitro investigations were performed according tothe experimental conditions of the in vivo study conducted

by Otberg et al. [14]. In both studies, the investigations

were performed on breast skin providing a follicular

density of 2032 follicles cm-2 in vivoand 22 follicles cm-2

on average in vitro. The artificial blocking of the hair fol-

licles was carried out in accordance with the FCT devel-

oped by Teichmann et al. [16]. In both cases, the same

varnish wax mixture was utilized. Due to the closing of the

hair follicles and the corresponding application of the

varnish wax mixture to the control area, the penetration

surface was reduced by 10% 0.76%. In vivo, the penetra-

tion surface was reduced to 8% [14]. In all experiments,thesame caffeine formulation was applied, although different

amounts had to be employed due to different detection

limits of the analytical methods.

Results and discussion

Although the first studies on follicular penetration had

already been performed 40 years ago [20, 21], in 2006,

Akomeah [19] criticized the consistent lack of an adequate

in vitro technique to investigate shunt route penetration

and to differentiate between different penetration path-

ways. Since then, follicular penetration has become more

and more important.Moreover, it has been recognized that

the hair follicles offer interesting therapeutic target sites,as

they represent complex and dynamic three-dimensional

structures [22].In particular particulate substances,such as

nanoparticles or liposomes have been shown to penetrate

preferentially into the hair follicles. These findings allow aselective targeting of specific structures within the hair

follicles and offer new possibilities, for example, for selec-

tive gene therapy or topical vaccination [22, 23]. Never-

theless, the development of an adequate method to

investigate the follicular penetration selectively still repre-

sents a particular challenge.

The Follicle Closing Technique, established by Teich-

mann et al. [16], permitted thein vivoinvestigation of the

follicular penetration pathway selectively. Recently, Trauer

et al. [17] implemented a combination of FCT with FD-C,

enabling the quantification of the follicular penetration

pathwayin vitro,for the first time.The aim of the study was the comparison of thein vivo

data,obtained by Otberg et al.[14] in a previous study,with

thein vitrodata generated in the present experiments, in

order to assess the transferability of the in vitrodata to the

in vivosituation.Therefore, the experimental conditions of

thein vitroexperiments were adapted as far as possible to

thein vivoconditions.

The comparison of the in vivo and in vitro data both

revealed a number of similarities, as well as significant dif-

ferences.

The in vivo results obtained by Otberg et al.[14] showed

a penetration of caffeine into the blood already after a few

minutes, following topical application (see Figure 1). Themaximum of caffeine penetration was reached after 1 h

(control samples) or 2 h (test samples). In the case of

0

2

4

6

8

10

12

14

16

5 10 20 30 60 120 300 480 1440%o

ftopica

llyappliedcaffeine

Time after caffeine application (min)

Figure 1Kinetics of caffeine penetration forcontrol andtest skinsitesin relation to

the topically applied amount of caffeine, determined as 100%.Thein vivo

values were determined in the blood,the in vitrovalues were determined

in the receptor medium at different time points. in vivo test ();in vivo

control ( ); in vitro test ( ); in vitro control ( )

Follicular pathwayin vivo vs in vitro

Br J Clin Pharmacol / 68:2 / 183


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the open hair follicles (control skin), more caffeine was

detected in the blood of the volunteers than in the case of

the closed hair follicles (test skin). During the test period of

24 h, the caffeine concentration found in the blood in the

case of the closed hair follicles and the open hair follicles

decreased continuously.

In comparison, the in vitro investigations revealed

detectable concentrations of caffeine in the receptormedium, initially, 2 h after topical application. In the recep-

tor medium of the test skin (closed hair follicles), only

0.09% caffeine was found. However, in the receptor

medium of the control skin (open hair follicles), 0.39%

of the topical caffeine was detected, which implies a

significantly increased penetration rate of caffeine in the

case of the open hair follicles (U-test after Wilcoxon/

MannWhitney, P< 0.05).

A possible explanation for the faster occurrence of

caffeine in the blood in comparison with the receptor

medium might represent the still existent blood flow in

vivo. Around the infundibulum region of the hair follicles,the blood vessels form a relatively dense capillary network

[22] being responsible for a fast evacuation of the perme-

ated substances. In vitro, this mechanism is absent and this

might explain the longer period of time needed for the

caffeine to be detectable in the receptor medium. After

permeating the hair follicle, the caffeine reaches the living

tissue.The evacuation via the blood system in vivo is absent

in vitro; therefore,the caffeine has to penetrate through all

skin layers to reach the receptor medium. In 1979, Zesch

et al. [24] found that due to the absence of blood and

lymph flow, a 450-fold higher caffeine concentration could

be detected in the corium after 1000 min.They found com-

parable concentrations of caffeine after a 5 h penetration

time, as in the present study.

Up until the end of the in vitroexperiments (after 24 h),

the concentration of the caffeine in the receptor medium

increased continuously in the control as well as in the

test skin. After 24 h, 11.82% of the applied caffeine weredetected in the receptor medium of the control skin,

whereas a significantly lower concentration of caffeine

(5.45%) was found in the receptor medium of the test skin

(P


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the caffeine penetration values of the control skin areas.

This calculation revealed comparable follicular penetration

rates in vivoand in vitro.In vitro, 58.6% of the permeated

caffeine penetrated via the follicular pathway, whereas in

vivo, 50.2% of the penetrated caffeine utilized the follicular

pathway.In vitro, the follicular penetration rate was addi-

tionally determined for the different skin compartments.

It was calculated that 51.0% of the caffeine, which pen-etrated into the epidermis and 63.9% of the caffeine that

penetrated into the dermis utilized the follicular penetra-

tion pathway.

In the case of closed hair follicles, the penetration of

caffeine can be considered as significantly lowered in vitro,

in comparison within vivo,although the 24 h values were

higher. On the one hand, this might be due to the accumu-

lation of the caffeine in the receptor medium, on the other

hand it is known that the permeability of the skin in the

FD-C increases as time goes by. Significant differences

between in vivo and in vitro penetration rates were also

found for the control areas with open hair follicles (P 0.05, F-test,

t-test) at this time point, whereas in vitro, a significant

difference (5.45%vs 11.82%) for test and control samples

was established (P


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2 Choi EH, Lee SH, Ahn SK, Hwang SM. The pretreatment effect

of chemical skin penetration enhancers in transdermal drugdelivery using iontophoresis. Skin Pharmacol Appl SkinPhysiol 1999; 12: 32635.

3 Elias PM. Epidermal lipids, barrier function, and desquama-tion. J Invest Dermatol 80 (Suppl): 1983; 44s49s.

4 Elias PM. Stratum corneum defensive functions: an

integrated view. J Invest Dermatol 2005; 125: 183200.

5 Hadgraft J. Modulation of the barrier function of the skin.Skin Pharmacol Appl Skin Physiol 2001; 14 (Suppl. 1): 7281.

6 Moser K, Kriwet K, Naik A, Kalia YN, Guy RH. Passive skinpenetration enhancement and its quantificationin vitro.Eur

J Pharm Biopharm 2001; 52: 10312.

7 Schaefer H,Redelmeier T. Skin barrier: Principles of percuta-

neous absorption. Basel: Karger, 1996;56.

8 Agarwal R, Katare OP, Vyas SP. The pilosebaceous unit: a

pivotal route for topical drug delivery. Methods Find ExpClin Pharmacol 2000; 22: 12933.

9 Barry BW. Drug delivery routes in skin: a novel approach.

Adv Drug Deliv Rev 2002; 54 (Suppl. 1): S3140.

10Genina EA, Bashkatov AN, Sinichkin YP, Kochubey VI,

Lakodina NA, Altshuler GB, Tuchin VV. In vitroandin vivostudy of dye diffusion into the human skin and hair follicles.J Biomed Opt 7: 2002; 4717.

11Lademann J, Knorr F, Richter H, Blume-Peytavi U, Vogt A,Antoniou C, Sterry W, Patzelt A. Hair follicles an efficientstorage and penetration pathway for topically applied

substances. Skin Pharmacol Physiol 2008; 21: 1505.

12Lauer AC, Ramachandran C, Lieb LM, Niemiec S, Weiner ND.

Targeted delivery to the pilosebaceous unit via liposomes.Adv Drug Deliv Rev 1996; 18: 31124.

13Ogiso T, Shiraki T, Okajima K, Tanino T, Iwaki M, Wada T.Transfollicular drug delivery: Penetration of drugs throughhuman scalp skin and comparison of penetration betweenscalp and abdominal skinsin vitro. J Drug Target 2002; 10:

36978.

14Otberg N, Patzelt A, Rasulev U, Hagemeister T, Linscheid M,

Sinkgraven R, Sterry W, Lademann J. The role of hair folliclesin the percutaneous absorption of caffeine. Br J ClinPharmacol 2008; 65: 48892.

15Teichmann A, Jacobi U, Ossadnik M, Richter H, Koch S,Sterry W, Lademann J. Differential stripping: determination

of the amount of topically applied substances penetratedinto the hair follicles. J Invest Dermatol 2005; 125: 2649.

16Teichmann A, Otberg N, Jacobi U, Sterry W, Lademann J.Follicular penetration: development of a method to block

the follicles selectively against the penetration of topically

applied substances. Skin Pharmacol Physiol 2006; 19:21623.

17Trauer S, Patzelt A, Knorr F, Richter H, Liebsch M, Rozycki C,

Balizs G, Buettemeyer R, Linscheid M, Franz TJ, Lademann J.Development of an in vitro modified skin absorption test forthe investigation of the follicular penetration pathway ofcaffeine. Skin Pharm Physiol 2009 (in press).

18OECD. OECD (Organisation for Economic Co-operation andDevelopment) Guidelines for Testing Chemicals-428 Skinabsorption: In vitromethod. OECD TG 428 2004.

19Akomeah FK, Martin GP, Brown MB. Variability in human skinpermeability in vitro: comparing penetrants with different

physicochemical properties. J Pharm Sci 2007; 96: 82434.

20Maibach HI, Feldman RJ, Milby TH, Serat WF. Regionalvariation in percutaneous penetration in man. PesticidesArch Environ Health 1971; 23: 20811.

21Scheuplein RJ. Mechanism of percutaneous absorption. II.Transient diffusion and the relative importance of variousroutes of skin penetration. J Invest Dermatol 1967; 48: 7988.

22Patzelt A, Knorr F, Blume-Peytavi U, Sterry W, Lademann J.

Hair follicles, their disorders and their opportunities. DrugDiscovery Today 2008; 5: 17381.

23Li L, Hoffman RM. The feasibility of targeted selective gene

therapy of the hair follicle. Nature Med 1995; 1: 7056.24Zesch A, Schaefer H, Stuttgen G. The quantitative distribution

of percutaneously applied caffeine in the human skin. ArchDermatol Res 1979; 266: 27783.

25Patzelt A, Richter H, Buettemeyer R, Huber HJ,Blume-Peytavi U, Sterry W, Lademann J. Differential stripping

demonstrates a significant reduction of the hair folliclereservoir in vitro compared to in vivo. Eur J Pharm Biopharm2008; 70: 2348.

S. Trauer et al.

186 / 68:2 / Br J Clin Pharmacol

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