Posters / European Journal of Pharmaceutical Sciences 2 (1994) 117-194 131

P49 PARACELLULAR ABSORPTION ENHANCEMENT IN INTESTINAL CACO-2 MONOLAYERS BY SODIUM CAPRATE T. Lindmsrkl, N. Schipperl, L. Lazorova 1, A.G. de Boer2, P. Arlurssonl I Dept. of Pharmacy, Uppsale Univendty, 75123 Uppsala, Sweden 2 Dept. of Pharmacology, Leiden University, Leiden, Netherlands

Sodium caprate (C10) increases paracellular permeability of small

molecules, e.g. mannitol and penicillin V, in the intestinal epithelium (1).

The aim of this study was to investigate the effects of CIO on the transport

of molecules of different molecular weight across Caco-2 monolayers

grown on permeable supports. The transport of radioactively labetled

monodisperse polyethylene glycols (MW=238-546 g/mole) and peptides

and proteins (MW=1.208-69.000 g/mole) was studied in the presence of

10-13 mM C10. Transmission electron microscopy with ruthenium red, an

electron dense marker, (MW=860 g/mole), and confocal fluorescence

microscopy (using FITC, MW=390 g/mole, and HTC labelled dextrans,

MW--4.000 g/mole and 20.000 g/mole) were used to visualise passage of

macromolecules across the epithelium. The permeability of all PEGs was

increased by 10 mM C10. C10 (13 raM) also enhanced the transport of

proteins smaller than 6000 g/mole, while the transport of larger proteins,

was unaffected. Transmission electron microscopy and confocal

microscopy showed that C10 increased transport across the epithelium

through the paraceUular mute. We conclude that C10 is an effective

absorption enhancer for macromolecules and the increased transport is

mainly due to an increased paracellular permeability.

1) E.K. Anderberg, T. Lindmark and P. Artursson,

Pharm. Res. 10 (1993) 857-864

PS0 SUBZERO THERMAL ANALYSIS OF HUMAN STRATUM CORNEUM H. Tanojo, J.A. Bouwstra, H.E. Junginger, H.E. Bodd6 Die. Pharmaceutical Technology, Leiden/Amsterdam Center for Drug Research, Leiden University, 2300 RA Leiden, Nethedands

The major problem in administering drugs transdermaUy arises from the barrier capacity of the stratum comeum, the outermost layer of the skin. This problem may be partially overcome by including in the formulation certain substances, known as penetration enhancers, which are able to reversibly alter the barrier properties of stratum comeum. In order to understand the mode of action of enhancers on this skin layer, several techniques have been used, including thermal analysis. As a lot of enhancers exhibit thermal transitions at temperatures lower than 0°C (subzem), it becomes the aim of this study to investigate the thermal behaviour of stratum corneum, with or without enhancers, at the subzero temperature range.

Isolated human stratum comeum sheets were studied using differ- ential thermal analysis within the temperature range -130 ° to 120°C. In the analysis curves of dehydrated as well as hydrated stratum corneum sheets a particular subzero transition, at approximately -9°C (264 K), was noticed. This transition could be distinguished from the water peak found only in hydrated stratum corneum samples.

To further characterise this transition, thermal analysis was performed on stratum comeum sheets: (i) after undergoing lipid extraction, (ii) after the treatment of propylene glycol and (iii) after the treatment of oleic acid/ propylene glycol solution. The last two treatments were done on the hydrated as well as the dehydrated sheets. From the results, it was concluded that the subzero transition (-9°C) belongs to low melting lipid components of stratum comeum. This finding can lead to a new method in studying the nature of the interaction between the enhancers and the stratum corneum.

PSl ltq"r.RACllONS BE1WEEN UPOSOMES AND HUMAN SIGN: STUDIED BY CONFOCAL LASER SCANNING MICROSCOPY M.E.M.J. Meuwissent, C. Cullander 1,2, H.E. Jungingerl, J,A, Bouwstrat 1 Division of Pharmecatnica] Technology, Leiden/Amsterdam Center for Drug Research, 2300 RA leiden, Netherlands 2 Departments of Pharmacy and Pherrnaoeutical Chemistw, School of Pharmecy, University of California, San Franctseo, USA

The main barrier of the skin is the upper layer of the skin, the stratum corneum, which consists of comeocytes embedded in lipid lamellar regions. Liposomes am phospholipid vesicles that have been introduced as drug delivery systems for dermal or transdermal application. Applying a drug to the skin in a liposomal form, may enhance the penetration rate of the drug. Several studies have dealt with the penetration effects of vesicles', like liposomes, hut the interactions of liposomes and skin are still not established. In this study DLPC liposomes labelled with fluorescent bilayer markers were used, A concentration of 0.1 tool % of fluorescein=DPPE or texas red-DPPE or both labels was added during the vesicle preparation. The stratum comeum side of dermatomized (250 pro) human skin was incubated with these labelled liposomes at 32"C. After 17 hours of incubation the liposomes were washed off the skin and the skin was carefully cut into tiny pieces and examined by the confocal laser scanning microscope (CLSM). The CLSM could optically section a piece of skin and obtained focal plane images parallel to the surface from deep within the skin. The CLSM was a Bio-Rad MRC 600 with a krypton-argon laser. Serial XY-sections of the incubated skin revealed that fluorescence of the labelled liposomes was present in deeper layers of the skin, until an apparent depth of approximately I0 to 20 pro. The fluorescence of the labels was mainly present between the corneocytes and was probably associated with the intercellular lipid regions. From these results it may be concluded that material originally present in the liposomal bilayer can appear in the lipohilic matrix of the skin barrier, although the mechanism of liposome - skin interactions is still not clear. Further it may concluded that CLSM is a useful technique for studying these interactions.

" Schreier, H. and Bouwstra, J.A.; accepted in J. Contr. Rcl. (1994)

P52 TRANSPORT OF BIPHOSPHONATES IN CULTURED CACO-2 CELLS AS AN IN VITRO MODEL OF GASTROINTESTINAL EPITHELIUM I.M. Twiss Department of Clinical pharmacy, University Hospital Leiden, 2300 RC Leiden, Netherlands

8iphosphonatas are new drugs in the treatment of various diseases of bone and

calcium-metabolism of which pamidronate (ADP) and dimethylaminehydroxypropyl-

idenediphosphonic acid (dimethylADP) belong to the second generation. Despite the

use of orally administered ADP and dirnethylADP the cytotoxioity and the transport

(and -mechanism) for intestinal epithelium has never been investigated. The aim of

our study was to investigate the transport and the cytotoxicity of ADP and

dimethylADP in an in vitro model using cultured Caco-2 cells. Cultured Caco-2 cells

form a well established human cell line and are regarded to represent a good model

for gastro-intastinel epithelium.

The effect of ADP and dimethylADP on lactate dehydrogenase (LDH) release

(biochemical cytotoxicity assay), on the morphology and on the transepithelial

electrical resistance (a measure for the permeability and thus for tightness of the

tight junctions of the cells) of the Caco-2 cells was investigated. The morphology was

studied by electron microscopy, The transport of ADP was measured by HPLC.

The results indicate that after treatment of the Caco-2 cells with increasing

concentrations of ADP LDH loss increased more than after treatment with

dimethylADP. The morphology, revealed by electron microscopy, showed alterations

due to the treatment in accordance with the biochemical assay. Transport

percentage of ADP was 1% after treatment of the cells with non-toxic doses and 13%

with a toxic dose. After treatment of the cells with increasing doses of ADP the TEER

decreased more than after treatment with dimethylADP.

In conclusion ADP is more cytotoxic for Caco-2 cells than dimethylADP. As the

transport percentage of non-toxio doses of ADP is low and ADP reduces the TEER, it

might be concluded that ADP is transported paracellulariy. Transport of high doses is

probably due to diffusion through necrotized areas.