Abstract
AIMS
Due to ethical reasons, in vivo penetration studies are not applicable at all stages of development of new substances. Therefore, the development of appropriate in vitro methods is essential, as well as the comparison of the obtained in vivo and in vitro data, in order to identify their transferability. The aim of the present study was to investigate the follicular penetration of caffeine in vitro and to compare the data with the in vivo results 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 differentiate between follicular and intercellular penetration of caffeine into the receptor medium in vitro. Subsequently, the results were compared with the data obtained in an earlier study investigating follicular and intercellular penetration of caffeine in vivo.
RESULTS
The comparison of the data revealed that the in vitro experiments were valuable for the investigation of the follicular penetration pathway, which contributed in vivo as well as in vitro to approximately 50% of the total penetration, whereas the kinetics of caffeine penetration were shown to be significantly different.
CONCLUSIONS
The combination of FCT with the Franz diffusion cell represents a valuable method to investigate follicular penetration in vitro. Nevertheless, in vivo experiments should not be abandoned as in vitro, structural changes of skin occur and blood flow and metabolism are absent, probably accounting for reduced penetration rates in vitro.
Keywords: caffeine, follicular penetration, hair follicle
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 Closing Technique (FCT), caffeine was detectable only after 20 min. Because of ethical reasons, in vivo investigations are not always applicable. Therefore, appropriate in vitro methods have to be developed and compared with the available in vivo data, in order to identify their transferability.
WHAT THIS STUDY ADDS
In the present study, the FCT was adapted for in vitro use in the Franz diffusion cell and the penetration of caffeine was investigated and compared with the previously obtained in vivo data. It was shown that the combination of FCT and Franz diffusion cell represents a valuable method to estimate the follicular penetration process in vitro, which revealed comparable results in 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 vitro studies.
Introduction
For most topically applied pharmaceuticals and cosmetics, penetration through the skin barrier is essential for developing their effects. However, regarding optimization and the development of new substances, it is consequently of the highest relevance to be familiar with the corresponding penetration pathways. In principle, four different penetration pathways are available for topically applied substances. On the one hand, penetration can occur intercellularly along the lipid layers or intracellularly. Additionally, 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 contrary, 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 therefore were considered to be irrelevant for skin penetration processes [7]. During the past years, this opinion has drastically changed. Scientists recognized that the hair follicles represent weak spots in the skin barrier. Moreover, hair follicles represent invaginations of the epidermis extending deep into the dermis, thus providing a greater actual area for potential absorption [8]. In the meantime, a multiplicity of studies has shown that the hair follicles represent important penetration pathways, as well as a long-term reservoir for topically applied substances [9–15]. 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 pathways. 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 significantly 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 use in the Franz diffusion cell (FD-C) [17].
The aim of the present study was to investigate follicular penetration of caffeine in vitro utilizing the FCT in combination with the FD-C and to compare these data with the available data on in vivo follicular 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 completely reflect the in vivo situation, a comparison of in vivo and in vitro results is essential in order to identify the transferability.
Methods
Preparation of test formulation
Caffeine 2.5 g (Sigma Aldrich, Steinhagen, Germany) was added 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 homogenized in an ultrasonic bath for 15 min corresponding to the formulation utilized by Otberg et al. [14].
Skin absorption test
The in vitro experiments 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 compartment volume of approximately 12 ml were used for the skin absorption tests. The receptor compartment was carefully filled with Dulbecco's 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 samples
Human full thickness skin was obtained during plastic surgery from the breast region and from four different subjects (female, aged 35–62 years). The study had been approved by the Ethics Committee of the Charité. The hair follicle density was 22 follicles cm−2 on average. The hair follicle density in the in vivo study by Otberg et al. [14] was 20 to 32 follicles cm−2.
Follicular closing technique
The 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 Otberg et al. [14]. The method has been described in detail elsewhere [14, 16, 17]. The varnish wax mixture was also applied 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 sampling
For the in vitro study, 17.6 µl of the caffeine formulation was applied to a skin area of 1.76 cm2. The test formulation contained 25 µg caffeine µl−1. Thus, 250 µg cm−2 of caffeine was applied, which corresponded to a five-fold increase in the amount of caffeine having been applied in vivo by Otberg et al. [14] (50 µg 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 vivo blood samples (1 ng ml−1) determined by SI/MS.
After application of the caffeine formulation, samples from the sampling port (400 µl 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 isopropanol (Isopropanol SupraSolv® analytical grade, Merck Darmstadt, Germany) or DPBS, respectively.
High performance liquid chromatography
A WATERS liquid chromatography equipped with a WATERS 510 high-pressure pump, as well as a 712 WISP and a WATERS photo diode array detector were employed in combination with a Reversed Phase column TYPE WATERS RESOLVE™ C18. 5 µm, 3.9 mm × 150 mm.
To prepare the samples for calibration, the donor solution 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 µl of each test sample was used. The elution mixture for caffeine was 40 : 60 methanol : ammonium acetate buffer (pH 5.35) (both analytical grade, Merck, Darmstadt, Germany).
The in vitro investigations were performed according to the 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 20–32 follicles cm−2in vivo and 22 follicles cm−2 on average in vitro. The artificial blocking of the hair follicles was carried out in accordance with the FCT developed 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 penetration surface was reduced to 8% [14]. In all experiments, the same 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 pathways. 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 a selective targeting of specific structures within the hair follicles and offer new possibilities, for example, for selective gene therapy or topical vaccination [22, 23]. Nevertheless, the development of an adequate method to investigate the follicular penetration selectively still represents a particular challenge.
The Follicle Closing Technique, established by Teichmann et al. [16], permitted the in vivo investigation 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 pathway in vitro, for the first time.
The aim of the study was the comparison of the in vivo data, obtained by Otberg et al. [14] in a previous study, with the in vitro data generated in the present experiments, in order to assess the transferability of the in vitro data to the in vivo situation. Therefore, the experimental conditions of the in vitro experiments were adapted as far as possible to the in vivo conditions.
The comparison of the in vivo and in vitro data both revealed a number of similarities, as well as significant differences.
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). The maximum of caffeine penetration was reached after 1 h (control samples) or 2 h (test samples). In the case of 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.
Figure 1.
Kinetics of caffeine penetration for control and test skin sites in relation to the topically applied amount of caffeine, determined as 100%. The in vivo values were determined in the blood, the in vitro values were determined in the receptor medium at different time points. in vivo test (□); in vivo control (
); in vitro test (
); in vitro control (
)
In comparison, the in vitro investigations revealed detectable concentrations of caffeine in the receptor medium, initially, 2 h after topical application. In the receptor 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/Mann–Whitney, 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 permeated 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 comparable concentrations of caffeine after a 5 h penetration time, as in the present study.
Up until the end of the in vitro experiments (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 were detected 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 < 0.05, F-test, t-test). In comparison, in vivo, the concentration decreased continuously until the 24 h end point.
A possible explanation might again be the absence of blood flow and metabolism in vitro. Due to a continuous evacuation and degradation of the caffeine in vivo, the concentration gradient is kept up, whereas, in vitro, a static Franz diffusion cell is applied. Here, the receptor medium does not change, as only after sampling, a small amount of fresh medium is replaced. Thus, the in vitro data represent cumulative values of caffeine permeation.
Additionally, the in vitro experiments allowed the balancing of the control and test skins. The total recovery rate of caffeine in the test skin was 89.8% ± 3.36; and in the control skin 88.8% ± 6.15%. In Table 1, the mean values and SD of the cumulative caffeine recovery rates over 24 h are given for the different skin compartments.
Table 1.
Cumulative caffeine recovery rates of different skin compartments (donor, epidermis, receptor and total) after a 24 h penetration time. Values are given as percentage of applied concentration. Data are given as mean values and SD for control and test skins
| Donor (%) | Epidermis (%) | Dermis (%) | Receptor (%) | Total (%) | |
|---|---|---|---|---|---|
| Test skin | 75.7 ± 3.38 | 4.7 ± 0.57 | 2.6 ± 1.02 | 7.0 ± 1.79 | 89.8 ± 3.36 |
| Control skin | 55.3 ± 3.68 | 9.6 ± 1.09 | 7.2 ± 1.34 | 16.9 ± 3.90 | 88.8 ± 6.15 |
In Table 2, the follicular penetration rates were calculated for the in vivo and in vitro situation. The caffeine penetration values of the test skin areas were subtracted from the caffeine penetration values of the control skin areas. This calculation revealed comparable follicular penetration rates in vivo and 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 additionally determined for the different skin compartments. It was calculated that 51.0% of the caffeine, which penetrated into the epidermis and 63.9% of the caffeine that penetrated into the dermis utilized the follicular penetration pathway.
Table 2.
Mean cumulative penetration of caffeine as % of applied dose in different compartments (in vitro: epidermis, dermis, receptor; in vivo: blood) for control and test skin sites. The follicular penetration rate was calculated by subtracting the values of the test samples from the control samples. The relative value is given in relation to the total penetration rate
| In vitro experiments | In vivo experiments | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Control skin – test skin = follicular penetration (%) | Control skin – test skin = follicular penetration (%) | ||||||||
| Compartment | Control skin (%) | Test skin (%) | abs. | rel. | Compartment | Control skin (%) | Test skin (%) | abs. | rel. |
| Epidermis | 9.6 ± 1.09 | 4.7 ± 0.57 | 4.9 | 51.0 | N.d. | – | – | – | – |
| Dermis | 7.2 ± 1.34 | 2.6 ± 1.02 | 4.6 | 63.9 | N.d. | – | – | – | – |
| Receptor | 16.9 ± 3.90 | 7.0 ± 1.79 | 9.9 | 58.6 | Blood | 24.9 ± 1.05 | 12.4 ± 0.90 | 12.5 | 50.2 |
N.d., not determined; abs., absolute value in relation to applied amount; rel., relative value in relation to permeated amount.
In the case of closed hair follicles, the penetration of caffeine can be considered as significantly lowered in vitro, in comparison with in vivo, although the 24 h values were higher. On the one hand, this might be due to the accumulation 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, U-test after Wilcoxon/Mann–Whitney). Moreover, some further key data were investigated and significant differences were found when comparing the in vivo and in vitro test skin data and also for the control skin data (U-Test after Wilcoxon/Mann–Whitney, P < 0.05). a) For the first detection of caffeine in blood (in vivo) and the receptor medium (in vitro), the detected concentration of caffeine was significantly higher in vivo than in vitro for both test and control samples. b) The overall highest caffeine concentration was found in vitro after 24 h. c) At the endpoint, after 24 h, the caffeine concentration was significantly higher in vitro in the receptor medium than in vivo in the blood. In vivo, no significant differences between test and control samples (1.19% vs 1.44%) were detectable (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 < 0.05, F-test, t-test).
Additional explanations for the differences in the in vivo and in vitro results might be due to differences in the investigated skin. For the in vivo study, male volunteers were observed, whereas the breast skin, utilized for the in vitro experiments, was derived from female patients.
Masculine breast hair is mainly of terminal origin, which means that the hair follicles are large and even reach into the subcutaneous fat tissue. According to OECD TG 428 [18], full thickness skin utilized for in vitro experiments has to be disburdened from the subcutaneous tissue, which leads inevitably to a destruction of the pilosebaceous unit and could possibly influence penetration experiments. Therefore, in the present study, skin derived from female patients providing exclusively vellus hair follicles was utilized, as these hair follicles are significantly smaller and do not reach the subcutaneous fat tissue. Moreover, it is known that skin contracts after excision. By mounting the skin sample onto the Franz diffusion cell, the skin is again extended, but the multiple elastic fibres around the hair follicle remain contracted, which reduces the follicular reservoir by up to 90% [25]. Also, this aspect might represent a feasible explanation for the reduced and slower permeation of caffeine in vitro. Additionally, the lower detection limit of caffeine in vitro should also be taken into consideration, which was probably only partially compensated by the increased amount applied.
In conclusion, the combination of the Franz diffusion cell and the Follicle Closing Technique represents a suitable method for the investigation of the follicular penetration of topically applied substances in vitro. This combined method contributes helpful information in terms of risk assessment of active agents and formulations and presents a new opportunity to evaluate the efficacy of newly developed substances. The comparison of the in vivo and in vitro data revealed that the in vitro experiments were valuable for the investigation of follicular penetration processes. In summary, the FCT represents a suitable method to investigate follicular penetration in vivo and in vitro. It was shown that in the case of caffeine, approximately half of the permeated concentration utilized the follicular pathway.
Nevertheless, in vitro experiments can be applied in order to estimate approximately the penetration pathway preferably utilized by a test substance. However, in vivo experiments should not be abandoned, as in vitro, structural changes of the skin occur and blood flow and metabolism are absent. In static Franz diffusion cells, no continuous evacuation of the test substance is available. Therefore, the diffusion gradient between the different compartments cannot be investigated. Thus, in vitro experiments are less feasible to investigate the penetration kinetics of a test substance.
Competing interests
None declared.
We would like to thank the Foundation ‘Skin Physiology’ of the Donor Association for German Science and Humanities for financial support.
We would also like to thank the ‘Centre for Alternative Methods to Animal Experiments – ZEBET’ at the Federal Institute of Risk Assessment for laboratory equipment and the unit ‘Residues of Medicinal Products’ at the Federal Institute of Risk Assessment, for analytical support.
Finally, we would like to thank Ms Elisabeth Schmidt for valuable advice whilst creating the study design, as well as for the many hours of helpful scientific discussions.
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