Development and evaluation of an in vitro method for prediction of human drug absorption: I. Assessment of artificial membrane composition
Introduction
In recent years the development of parallel synthesis and combinatorial chemistry in the drug discovery field have generated an ever increasing number of hits and leads for potential therapeutic use. Therefore, the availability of reliable high throughput screening methods for rapid evaluation and prediction of the biopharmaceutical properties of such molecules, and prompt individuation of potential bioavailability problems is needed. Dissolution rate in gastro-intestinal fluids and permeability through the lipid bilayer of epithelial cells lining the intestinal wall are considered the most important factors in determining oral drug absorption properties (Lobenberg and Amidon, 2000, FDA, 2001). Various official apparatus for in vitro drug dissolution testing are available in the different Compendia (e.g. USPXXV, 2002 and European Pharmacopoeia, 2002), and formal guidelines exist which provide useful recommendations for their correct use (FDA, 1997).
However, although several methods have been proposed and investigated for in vitro evaluation and prediction of gastrointestinal permeability of drugs, no official methods are available at present. The in silico techniques, based on pure computational calculations of some physico-chemical properties of drug molecules (such as solubility, o/w partition coefficient, lipophilicity, molecular polar surface area) to estimate their oral absorption potential, are very attractive, but their predictive power is at present unsatisfactory (Norris et al., 2000, Ekins et al., 2001, Bergstrom et al., 2003). The proposed in vitro experimental methodologies include systems based on the use of animal or human excised tissues or cells and systems based on the use of artificial membranes. Among the first series of methods, Caco-2 cell monolayer (a colon rectal adenocarcinoma cell line of human origin) is the most advanced and widely used in vitro model because it mimics most transport pathways in the gastrointestinal tract and it has demonstrated to be a particularly valuable tool for estimating human drug absorption potential (Artursson, 1990, Yamashita et al., 2000, Artursson et al., 2001). However, its use as a high throughput screening tool is limited by long cell growth cycles, possible risks of microbial contamination, high implementation costs and rather elevated inter-experimental and inter-laboratory variability. In vitro methods using artificial membranes represent an interesting alternative to the use of animal tissues or cells owing to their greater simplicity, rapidity, cost-effectiveness and reproducibility of results (Avdeef, 2003). Since the majority of drugs are mainly absorbed through passive transport, the use of artificial membranes which mimic passive diffusion offers a potentially effective high throughput approach for the assessment of drug absorption potential. Among these methods, the Parallel Artificial Membrane Permeability Assay (PAMPA) system is based on a filter-supported lipidic membrane initially introduced for the study of passive absorption processes (Kansy et al., 1998). This method has been currently gaining acceptance by several pharmaceutical industries and it is continuously subjected to new studies for further improvement and optimization (Sugano et al., 2001a, Sugano et al., 2003, Sugano et al., 2004, Kansy et al., 2004, Ruell and Avdeef, 2004). However, it has some disadvantages such as the impossibility of maintaining sink conditions during permeation experiments, due to the very small volume of the donor and receptor compartments; moreover, being a static method, the presence of the unstirred water layer (UWL) in both the acceptor and the donor wells, could reduce drug permeation rate and lead to its underestimation.
Therefore, the aim of the present study was to realize an in vitro method based on the use of artificial membranes that could assure dynamic conditions of the permeability assay and overcome the problems and limitations demonstrated by the above described techniques and enable a rapid and reliable evaluation and prediction of the absorption properties of drugs. Naproxen was selected as reference model drug in this preliminary study, since its apparent permeability was recently determined by using a dynamic Caco-2 cell based system (Maestrelli et al., 2004) and a strong link has been demonstrated between permeability measured in Caco-2 cells and the fraction of dose absorbed in vivo in humans (Pade and Stavchansky, 1998).
The development of a suitable artificial membrane simulating the behaviour of the natural gastrointestinal one constitutes the critical step for the fulfilment of this research. The artificial membrane is a multi-component system in which the interactions among the constituents have to be considered. Therefore an in-depth investigation has been undertaken, in order to select the best kind of artificial support and the most suitable composition of the lipidic mixture to be used for support impregnation. This first series of studies aims to find the most effective artificial gastrointestinal model membrane able to give the highest reproducibility in the results and the naproxen apparent permeability value closest to the in vitro reference data obtained using the Caco-2 cell dynamic methodology (Maestrelli et al., 2004). The effect on drug permeability of the composition of the lipid mixture used for support impregnation was investigated by using mixture design strategy. In fact, this strategy is suitable for multiple components for which the proportions in the mixture are dependent on each other and for which the sum is 100% (Cornell, 1990).
The next step of this research will be to evaluate the general applicability of the proposed method for estimating drug absorption potential, by testing it on a large set of drugs with a wide range of solubility and permeability properties.
Section snippets
Materials
Porous supports (see Table 1) were kindly supplied by Sartorius Italia S.p.A. (Florence, Italy), Whatman International Ltd (Milan, Italy) and Millipore Italia S.p.A. (Milan, Italy). The lipid phase used for the impregnation of the porous supports consisted of a mixture of phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, lysophosphatidylethanolamine) (Lipoid® E 80) kindly donated by Lipoid (Ludwigshafen, Germany). Cholesterol, n-octanol and
Results and discussion
The Sartorius apparatus used in this study was selected to perform diffusion experiments on the basis of the following characteristics which were particularly suitable for our study purposes: it is endowed with donor and receptor compartments whose volume can be varied as needed from 30 to 100 mL and adequately thermostated in a temperature range between 35 and 40 °C; a peristaltic pump allows the continuous circulation of both donor and receptor phases in the related compartments and sides of
Conclusion
The availability of effective, highly reproducible, economic and rapid in vitro permeation assays for a prediction of the drug absorption properties in humans is highly desirable for the preliminary screening of new drug compounds in the early stages of the drug discovery process, thus improving efficiency and the probability of success in the development of efficacious pharmaceutical formulations.
Therefore, a research aimed at the development of a valid and powerful in vitro permeation
Acknowledgment
Financial support from MIUR is gratefully acknowledged.
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