Through Human Stratum Corneum. A. Winkler, C. C. Müller-Goymann. Institut für Pharmazeutische Technologie, Technische Universität Braunschweig, ...
Comparison Of The Diffusion Properties Of δ-Aminolevulinic Acid And Its N-Butyl Ester Through Human Stratum Corneum A. Winkler, C. C. Müller-Goymann Institut für Pharmazeutische Technologie, Technische Universität Braunschweig, Mendelssohnstr. 1, D-38106 Braunschweig INTRODUCTION
EXPERIMENTAL METHODS • Materials ALA was provided by Medac GmbH, Wedel, Germany. ABE was synthesized according to Kloek et al. [1]. They used n-butanol and thionyl chloride to esterify ALA. n-Butanol was purchased from Heraeus GmbH, Karlsruhe, Germany, thionylchloride from Merck KGaA, Darmstadt, Germany. The product ABE was purified by recrystallisation in diethylether (all chemicals were reagent grade). Diethylether was purchased from Fluka, Neu-Ulm, Germany. Acetonitrile, methanol and acetic acid (all chemicals were HPLC quality) were purchased from J.T. Baker, VA Deventer, Netherlands. o-Phthalaldehyde (OPA) (HPLC quality) was purchased from Fluka, Neu-Ulm, Germany, mercaptoethanol (pro analysi) and all buffer substances (pro analysi) from Merck KGaA, Darmstadt, Germany. Boric acid (HPLC quality) was purchased from Carl Roth GmbH, Karlsruhe, Germany, absolute ethanol (HPLC quality) from Riedel de Haën, Seelze, Germany. Water was used in bidistilled quality. •
In vitro permeation studies
The in vitro permeation studies were performed with a modified Franz cell which is represented in figure 1. For the preparation of stratum corneum trypsination was used (Kligman and Christophers [2]). The stratum corneum was placed on a polycarbonate filter (Isopore membrane filters, type TMTP, 5.0 µm, Millipore, Ireland) for a higher mechanical stability. The donor was Excipial Fettcreme (Hans Karrer GmbH, Königsbrunn, Germany) enriched with ALA and ABE, respectively. The concentration of the drugs were in both cases 10 % (w/w). The receiver was a phosphate buffer of pH 5.0 (Ph. Helv. 8) to guarantee the stability of ALA and ABE. The acceptor was stirred and heated to 37 °C. Samples were taken over 35 hours as shown in figure 2. The permeated amounts of both substances were measured by HPLC with fluorometric detection using OPA as a derivatization reagent [3]. •
HPLC analysis
The HPLC system consisted of a fluorescence HPLC monitor RF-353 (Shimadzu, Kyoto, Japan) and a Spectroflow 400 solvent delivery system (Kratos Analytical, New Jersey, USA) equipped with a Rheodyne 7125 syringe loading sample injector (Rheodyne, Cotati, USA). The injector was fitted with a 20 µl loop.
polycarbonate filter
buffer pH 5.0 stirring bar
Figure 1: Schematic representation of the modified Franz cell
40 permeated amounts [µg/cm ²]
δ-Aminolevulinic acid (ALA) is applied as a prodrug of protoporphyrin IX (Pp IX). The fluorescense of Pp IX induced by ALA is used for the treatment of skin diseases as well as of cancer of the lung, oesophagus, and bladder, respectively. To improve the limited permeation through biomembranes Kloek et al. [1] synthesized δ-aminolevulinic acid-n-butyl ester (ABE) besides other derivatives of ALA. They detected an increase of the fluorescence of Pp IX after application of ABE. Therefore ABE had a higher bioavailability because of its higher lipophilicity than ALA. To investigate, if ABE is able to penetrate better into skin than ALA, permeability studies through stratum corneum, the lipophilic barrier, are necessary. For this purpose a modified Franz cell was used. This report shows the results of the in vitro permeation studies of ALA and ABE through excised human stratum corneum.
RESULTS AND DISCUSSION
donor stratum corneum
35 30
ABE, n=6 ALA, n=9
25 20 15
ALA and ABE had significantly different permeation profiles as shown in figure 2. The permeated amounts of ABE were higher than those of ALA. Permeation data are summarized in figure 3. For each substance the lag-time was determined from the intercept of the linear part of the graph with the time axis. The calculated lag-time of ABE was 825 minutes. Permeated ABE could be proven from 6 hours onwards. For ABE the ascent becomes linear between 14 and 24 hours. The detection of permeated ALA also succeeded after 6 hours. The permeated amounts of ALA increase linearly after a lag-time of 215 minutes. For each substance the linear ascent of the curve was used to determine the flux J. The permeation coefficient P was calculated as quotient of flux and concentration for each substance. The mean values of flux and permeation coefficient of ALA through excised human stratum corneum were 2.85⋅10-11 g/cm²s and 3.01⋅10-10 cm/s, respectively. Flux and permeation coefficient of ABE were nearly ten times higher than those of ALA with mean values of 3.19⋅10-10 g/cm²s and 3.00⋅10-9 cm/s, respectively.
10 5
CONCLUSION
0 0
500
1000 time [min]
1500
2000
Figure 2: Comparison of the permeation profiles of ALA and ABE through excised human stratum corneum
donor
ALA in Excipial Fettcreme, 10 % (m/m) ABE in Excipial Fettcreme, 10 % (m/m)
flux J [g/cm²s]
permeation coefficient P [cm/s]
2.85⋅10-11
3.01⋅10-10
(+/- 7.04⋅10-12)
(+/- 7.44⋅10-11)
3.19⋅10-10
3.00⋅10-9
(+/- 8.54⋅10-11)
(+/- 8.04⋅10-10)
lag-time [min]
This study shows that stratum corneum is more permeable for ABE than for ALA. The increase in lipophilicity of the esterified molecule results in an improvement of diffusivity through stratum corneum. Therefore the present investigation agrees with the results from Kloek et al. [1] who observed an increase of the Pp IX concentration after application of ABE. ABE shows a progressive permeation behaviour. Further studies have to show, if the increase in lipophilicity is the only reason for this behaviour or if an enhancement effect exists for ABE.
215
ACKNOWLEDGEMENTS 825
Figure 3: Permeation data
The analytical column (250 mm x 4.6 mm) was connected in line with a guard column (10 mm x 4.6 mm). Both columns were packed with Hypersil ODS (particle size 5 µm, Grom, Herrenberg, Germany). For determining ABE the mobile phase was prepared according to Graser et al. [4]. An aqueous monobasic potassium phosphate buffer (12.5 mM, pH 7.2) was mixed with acetonitrile in the ratio of 5.5 to 5.0. ABE was isolated by isocratic elution at a flow rate of 1.5 ml/min at ambient temperature. ALA was eluted at ambient temperature with a mixture of an aqueous sodium acetate buffer (22mM) and methanol in the ratio of 7.0 to 5.0, adjusted to pH 3.38 with acetic acid. The flow rate was 1.3 ml/min. Prior to the measurement derivatization of ALA and ABE were performed according to Ho et al. [3], too. 100 µl of the sample were mixed with 100 µl of a derivatization reagent enriched with OPA. Immediately after a reaction time of exactly 2 minutes 100 µl monobasic potassium phosphate buffer (0.1 M) were added to the mixture to stop the reaction. The fluorescence of the reaction products of both substances were monitored with an excitation wavelength of 330 nm and an emission wavelength of 418 nm.
We would like to thank Medac GmbH, Wedel, Germany for their generous donation of ALA and Hans Karrer GmbH, Königsbrunn, Germany for support. Dr. Flory (Hollwede Hospital, D-Braunschweig) is thanked for the donation of skin samples.
REFERENCES [1]
J. KLOEK, G. M. J. BEIJERSBERGEN VAN HENEGOUWEN: Prodrugs of 5-Aminolevulinic Acid for Photodynamic Therapy, Photochem. and Photobiol., 64(6), 994-1000 (1996)
[2]
A.M. KLIGMAN, E. CHRISTOPHERS: Preparation of Isolated Sheets of Human Stratum Corneum, Arch. Dermatol., 88, 702-705 (1964)
[3]
J. HO, R. GUTHRIE, H. TIECKELMANN: Detection of δ-Aminolevulinic Acid, Porphobilinogen and Porphyrins related to Heme biosynthesis by High-Performance Liquid Chromatography, J.Chromatogr., 375, 57-63 (1986)
[4]
T.A GRASER, H. G. GODEL, S. ALBERS, P. FÖLDI, P. FÜRST: An Ultra Rapid and Sensitive High Performance Liquid Chromatographic Method for Determination of Tissue and Plasma Free Amino Acids, Analytical Biochemistry, 151, 142-152 (1985)
