Computational Models for the Prediction of Intestinal Membrane Permeability

Transcription

1 Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 247 Computational Models for the Prediction of Intestinal Membrane Permeability BY PATRIC STENBERG ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2001

2 Dissertation for the Degree of Doctor of Philosophy (Faculty of Pharmacy) in Pharmaceutics presented at Uppsala University in 2001 ABSTRACT Stenberg, P., Computational Models for the Prediction of Intestinal Membrane Permeability. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy pp. Uppsala. ISBN Lead compounds generated in high-throughput drug discovery programs often have unfavorable biopharmaceutical properties, resulting in a low success rate for such drug candidates in clinical development. Efficient and reliable methods that predict biopharmaceutical properties, such as intestinal permeability and solubility are therefore required in order to reduce the attrition rate during development of these compounds. One aim of this thesis was to identify molecular properties that are important for intestinal drug permeability using a wide range of drugs and model compounds. A second aim was to develop computational models for predicting intestinal drug permeability based on these properties. The calculated molecular descriptors ranged from the simple counting of atoms and fragments to more complex descriptors derived from molecular mechanics and quantum mechanics calculations. Particular attention was given to descriptors associated with molecular surface areas. Descriptors calculated by the various methods were used to establish structure-permeability relationships for conventional drugs, peptide derivatives and large, lipophilic compounds generated by high-throughput pharmacological screening. Caco-2 cell monolayer permeabilities were determined for a structurally diverse set of compounds and were used to predict human intestinal membrane permeability and to develop computational models. From these investigations, several new models for the computational prediction of intestinal membrane permeability were developed. Models were developed that are suitable for the prediction of membrane permeability to specific types of drugs, as well as models that are more generally applicable. One of these general models is based on partitioned total molecular surface areas, and this model can be used to predict intestinal membrane permeability to structurally diverse compounds. It was also demonstrated how these models can be applied in a manner that increases both the accuracy of the prediction and the throughput. In addition, a simplified protocol based on Caco-2 cells for the experimental prediction of intestinal permeability was developed. These improvements can be used to construct highly effective experimental and computational filters for use in drug discovery and development. Patric Stenberg, Department of Pharmacy, Uppsala Biomedical Centre, Box 580, SE Uppsala, Sweden Patric Stenberg 2001 ISSN ISBN Printed in Sweden by Uppsala University, Tryck & Medier, Uppsala 2001

3 CONTENTS 1. PAPERS DISCUSSED 5 2. ABBREVIATIONS 6 3. INTRODUCTION Mechanisms of intestinal membrane permeation Passive transcellular transport Paracellular transport Carrier-mediated transport and efflux 3.2 In vitro models for predicting intestinal membrane transport Partitioning in isotropic systems Interaction with and transport across artificial membranes Transport across cultured epithelial cell monolayers 3.3 Computational models for predicting intestinal membrane transport Solute transport across lipid bilayers Generation of calculated descriptors Model development 4. AIMS OF THE THESIS MATERIALS AND METHODS Drugs and radiolabeled markers Experimental Cell culture Transport studies Sample analyses 5.3 Calculation of molecular descriptors Hydrogen bond strength Lipophilicity and molar refractivity Electrotopological state indexes Molecular surface properties Descriptors derived by quantum mechanics 5.4 Statistical methods Linear and multiple linear regression analyses Principal component analyses Partial least squares projections to latent structures Training set selection 5.5 Datasets RESULTS AND DISCUSSION In vitro drug permeability (IV) Passive drug permeability Prediction of intestinal permeability in vivo 3

4 6.2 Polar molecular surface areas for the prediction of intestinal drug transport (I, II, III, IV) Conventional drugs Peptide derivatives Compounds generated by pharmacological screening Structurally diverse compounds 6.3 Influence of non-polar molecular surface areas on intestinal drug transport (II, III, IV) Enabling fast calculations of molecular surface areas (II, III, IV) A physico-chemical description of molecular surface properties (II, IV) Comparison of computational models for the prediction of intestinal drug transport (III, IV) Compounds generated by pharmacological screening Structurally diverse compounds 7. CONCLUSIONS PERSPECTIVES ACKNOWLEDGEMENTS REFERENCES AND NOTES APPENDIX 66 4

5 1. PAPERS DISCUSSED This thesis includes the following papers, which will be referred to by Roman numerals in the text: I. Palm, K.; Stenberg, P.; Luthman, K.; Artursson, P. Polar molecular surface properties predict the intestinal absorption of drugs in humans. Pharm. Res. 1997, 14, II. Stenberg, P.; Luthman, K.; Artursson, P. Prediction of membrane permeability to peptides from calculated dynamic molecular surface properties. Pharm. Res. 1999, 16, Published erratum appears in Pharm. Res. 1999, 16, III. IV. Stenberg, P.; Luthman, K.; Ellens, H.; Lee, C.-P.; Smith, P. L.; Lago, A.; Elliott, J. D.; Artursson, P. Prediction of the intestinal absorption of endothelin receptor antagonists using three theoretical methods of increasing complexity. Pharm. Res. 1999, 16, Stenberg, P.; Norinder, U.; Luthman, K.; Artursson, P. Experimental and computational screening models for the prediction of intestinal drug absorption. Submitted to J. Med. Chem. Reprints were made with permission from the journal. 5

6 2. ABBREVIATIONS log P a-b b-a FA HBSS HTS log P h/eg log P o/w MTS NPSA NPSA d P app P c PCA P-gp PLS PSA PSA d PTSA Q 2 QSAR QSPR R 2 RMSE VIP VP difference between the logarithm of the octanol/water partition coefficient and the logarithm of an alkane-water partition coefficient apical-to-basolateral basolateral-to-apical fraction of drug absorbed after oral administration to humans Hank s balanced salt solution high-throughput screening logarithm of the heptane/ethylene glycol partition coefficient logarithm of the octanol/water partition coefficient medium-throughput screening non-polar molecular surface area dynamic non-polar molecular surface area apparent permeability coefficient cellular permeability coefficient principal component analysis P-glycoprotein partial least squares projections to latent structures polar molecular surface area dynamic polar molecular surface area partitioned total surface area leave-one-out cross-validated coefficient of determination quantitative structure-activity relationship quantitative structure-property relationship coefficient of determination root mean square error variable importance in the prediction verapamil 6

7 3. INTRODUCTION It is often desired that new drug compounds are given orally because of the convenience of this administration route. However, not all compounds possess properties that are compatible with oral administration. In fact, the clinical development of new drugs is often terminated due to their poor bioavailability after oral administration [1-3], leading to significant costs for pharmaceutical companies [4]. The research costs for a compound increase dramatically as it enters clinical development, which means that there is an economic incentive to identify and discontinue the development of poor drug candidates as soon as possible. Ideally, poor biopharmaceutical properties such as low oral bioavailability, should be identified even before the compound is synthesized. The introduction of modern technologies, such as combinatorial chemistry and highthroughput pharmacological screening in drug discovery, has resulted in a vast increase in the number of lead compounds identified. The compounds generated in highthroughput drug discovery programs are generally more lipophilic, less soluble and of higher molecular weight than conventional drugs [5]. These characteristics often entail unfavorable biopharmaceutical properties which keep the success rate of such drug candidates in clinical development at a low level [6]. Therefore, there is a growing research effort aimed at developing simple experimental and theoretical methods that predict these biopharmaceutical properties earlier. In order for a drug to be effective after oral administration, a significant part of the administered dose must reach the systemic blood circulation unchanged. The oral bioavailability is defined as the percentage of the dose that reaches the systemic blood circulation intact after oral administration, and it is the result of a rather complex series of events (fig. 1). Besides stability issues introduced by chemical and enzymatic degradation, the solubility, and hence the dissolution in the gastrointestinal fluids and the permeation of the intestinal wall have to keep pace with the intestinal transit in order for the ingested drug to reach the systemic circulation and subsequently its site of action [7]. The solubility and permeability of a drug are considered to be two of the most important properties that determine absorption and the influence of these two properties on the extent of absorption from the intestinal tract has received considerable attention [e.g. 8-10]. While the solubility of a drug compound can often be modified to some extent by appropriate formulation, the possibilities of improving intestinal membrane permeability are more limited. It is therefore crucial to produce a compound whose structure ensures a sufficiently high permeability, or at least to be able to efficiently identify at an early stage compounds whose permeabilities are too low. This thesis describes and evaluates models that predict the process of passive drug permeation across the intestinal mucosa. Exploratory studies were performed to identify molecular properties which are important for intestinal permeability and to validate the generality of these properties using a wide range of drugs and model compounds. By applying this fundamental knowledge, computational models aimed at providing an accurate and yet fast estimation of intestinal drug permeability were constructed. In addition, an existing experimental model was simplified without loss of accuracy. 7

8 Solid dosage form Dissolution Drug in solution Intestinal membrane permeation Enzymatic and chemical degradation Enzymatic degradation, efflux Drug in portal vein Liver passage Enzymatic degradation Drug in systemic circulation Figure 1. Schematic representation of drug transport from a solid dosage form into the systemic blood circulation. The fraction of drug that reaches the systemic circulation unchanged is termed the bioavailability of the drug. The horizontal arrows indicate events which decrease the bioavailability. The dissolution and membrane permeation steps both influence the absorption of a drug into the systemic circulation. This thesis deals with one of these steps, the intestinal membrane permeation. To be applicable in a drug discovery setting, the models have to be accurate in their predictions as a high level of false negative predictions would sort out compounds with the potential of becoming good drugs, whereas a high level of false positive predictions would render the models less useful. The number of compounds under consideration in drug discovery and development will rapidly decrease as these processes proceed. Therefore, experimental and computational models of different complexity can be applied at different stages of the discovery and development processes. At an early stage in drug discovery when many compounds remain under consideration, the model applied must have a high-throughput. One possible scheme of permeability screening is presented in fig. 2. As a reference, the corresponding pharmacological screening activities are also shown. Quantitative structure-property relationships (QSPR) based on fast computational methods may be used to guide the early stages of the design of a library that contains the highest possible number of high permeability compounds [see, for example, 5, 11]. The compounds are synthesized, and then rapid in vitro screening gives a rough estimate of the permeability and provides information to help in the lead selection. Although there are no in vitro methods of determining permeability available today that can match their pharmacological counterparts in terms of throughput, significant progress towards the development of fast methods have been made [12, 13]. During lead optimization, the experimental data can be used to construct improved QSPR models. At this stage, the number of compounds is lower, and more sophisticated and computationally demanding 8

9 QSPR models can be applied. Finally, thorough in vitro permeability studies can be performed to obtain more reliable measurements and information about transport mechanisms. It should be noted that, while separated in fig. 2 for clarity, design and optimization of pharmacological properties and permeability are ideally performed simultaneously. This thesis deals with computational models of a wide range of complexity. These models can therefore be applied at the different stages of the discovery process outlined above. Identification and validation of target QSAR Compound library design QSPR Library optimized with regard to both pharmacological activity and permeability Synthesis Pharmacological HTS PermeabilityHTS/MTS QSAR Lead identification Compound redesign QSPR Lead optimization Training of QSAR model Synthesis Lead optimization Training of QSPR model Thorough pharmacological/permeability characterization Candidate selection Drug development Figure 2. Flow chart illustrating a possible application of computational and experimental permeability screening in drug discovery. The width of the shaded areas symbolizes the number of compounds dealt with during the discovery process. The right side illustrates application of permeability models. For comparison, the left side shows efforts aimed at optimization of the pharmacological properties. In the early discovery phase (top of figure), simple and fast QSPR models are used as guides to optimize the library and obtain a high proportion of well absorbed compounds for synthesis. Although not available as of today, a high-throughput (HTS) in vitro permeability screen would theoretically increase the ratio of high permeability compounds among the identified leads. Instead, a medium-throughput (MTS) permeability screen can be used for selected compounds. During the lead optimization phase, results from the in vitro permeability characterization are used to aid the candidate selection and also to construct more complex (and hopefully more accurate) QSPR models that can be used for compound redesign. By updating the QSPR models with data from the in vitro permeability characterization, these models will be tailored for the particular class of compounds investigated in the discovery phase. 9

10 3.1 Mechanisms of intestinal membrane permeation A solute encounters several sequential barriers (such as the mucus layer, the unstirred water layer, the epithelial cell layer and its underlying tissues) during the transport from the intestinal lumen into the blood. It is generally assumed that the permeation of the epithelium lining the intestine is the rate-limiting step [14, 15]. There are two pathways by which a solute can cross the epithelial membrane: the transcellular pathway, which requires the drug actually penetrating the intestinal cell membranes, and the paracellular pathway, in which diffusion occurs through water-filled pores of the tight junctions between the cells. Both passive and active processes may contribute to the permeability to drugs transported by the transcellular pathway. All of these pathways and processes are distinctly different, and the molecular properties that influence drug transport by these routes are also different, as will be discussed in the following sections. Before the onset of this thesis, it was crucial to investigate which of these pathways and processes that is most important and therefore should be considered first when developing experimental and theoretical models of drug permeability Passive transcellular transport A solute must penetrate the membrane surrounding the epithelial cell in order to transverse the cell. The cell membrane consists of an interrupted double layer of phospholipids [16] and has traditionally been described by the fluid mosaic model [17], and more recently, by the more complex superlattice model [18]. The presence of different lipids and different embedded proteins provides unique properties to cell membranes throughout the body. The epithelial cells are polarized, and there are appreciable differences in membrane properties even within one epithelial cell. For instance, the part of the cell membrane facing the intestinal lumen (the apical side) and the part of the membrane facing the sub-epithelial tissues (the basolateral side) have different protein and lipid compositions, and thus different permeability properties. The lipoid nature of the cell membranes restricts the transport of ions and hydrophilic molecules (see also section 3.3.1) [19]. The ordered structure of the membrane also imposes detrimental effects on the diffusion rate in the membrane as the solute size is increased [20]. The first step of passive transcellular transport is the penetration by the solute of the apical membrane, which is followed by diffusion through the cytoplasm of the cell interior (if the solute is very lipophilic, the transport across the cell interior may also involve lateral diffusion in the lipid bilayer of the cell membrane). Finally, the solute exits through the basolateral membrane. The diffusion of small solutes in the cytoplasm is normally a rapid process, and thus the rate of passive transcellular permeability is mainly determined by the rate of transport across the apical cell membrane [21]. Even though transport by this pathway requires a reasonably lipophilic solute of moderate size, numerous studies indicate that the vast majority of well absorbed drugs are transported passively across the cell membranes. Some of these studies are reviewed in ref 22. Thus, the rather complex process of intestinal drug permeation can often be satisfactorily described by considering passive transport across the apical cell membranes only. This is why experimental and theoretical models describing transport by this mechanism have received particular attention [23, 24]. However, an increasing 10

11 number of active transporters and efflux mechanisms that may influence drug permeability are being discovered, although the contributions of these to the in vivo absorption of drugs remain to be determined Paracellular transport Transport across the epithelial cell layer can also occur via water-filled pores between the cells, a process known as paracellular transport. Some investigators have reported that a saturable and substrate specific mechanism operates during transport by this pathway [25, 26], but paracellular transport is generally considered to be a passive process that follows Fick s law (i.e. the transport rate is proportional to the drug concentration at the surface of the apical membrane). The paracellular pathway offers an opportunity for hydrophilic compounds, which do not penetrate the cell membranes of the epithelial cells, to be transported, but the surface area presented by the pores constitutes only a small fraction (0.01%-0.1%) of the total intestinal membrane surface area [27, 28]. In addition, tight junctions provide a seal between adjacent epithelial cells, which restricts solute transport via this route [29, 30]. The tight junctions further limit transport by the paracellular pathway in the distal parts of the intestine [31, 32]. It is therefore unlikely that this pathway contributes significantly to the overall transport of most drugs in vivo, although small molecules (<200 Da) may be exceptions to this statement [33, 34] Carrier-mediated transport and efflux Active and facilitated transport. Solutes can also be transported in the apical to basolateral direction by proteins embedded in the cell membrane. These proteins extract nutrients and other compounds essential for the organism from the luminal contents by various carrier-mediated mechanisms. The most notable features of carrier-mediated transport are its substrate specificity, saturability and regional variability [35]. Substrate specificity is crucial to prevent the entry of unwanted compounds into the body, but the substrate specificity is not absolute and this transport route is available to a limited number of drugs. These include β-lactam antibiotics, ACE inhibitors and phosphate analogs, all of which are structurally similar to the native substrates of transport proteins [36, 37]. Saturability will become apparent when the carrier protein is faced with a high substrate concentration. From a drug delivery perspective, saturation will manifest itself as non-linearity in the dose-response relationship. Similarly, carrier proteins are expressed to different degrees in different regions of the intestinal tract, and the substrate will be absorbed better in areas of the intestines where expression of the carrier protein is high (usually upper intestine). In summary, carrier-mediated mechanisms enhance the transcellular permeability to a limited number of drugs. The extent of this enhancement depends not only on the structural similarity between the drug and the natural substrate of the transporter, but also on drug concentration and physiological factors. Receptor-mediated transcytosis. Transcellular transport can also occur by a process known as receptor-mediated transcytosis, in which the solute binds to a receptor on the cell surface and is then internalized by endocytosis. The endocytotic vesicle that is formed then proceeds to the opposite membrane surface. This route has a very low 11

12 capacity and is only significant for highly potent macromolecular drugs that are effective at low concentrations [38]. Efflux mechanisms. In contrast to the carrier-mediated mechanisms that promote drug permeability, efflux proteins have the potential to limit the overall permeability by pumping drugs in the basolateral to apical direction. Drug efflux into the intestine is often attributed to proteins, such as P-glycoprotein (P-gp) and multidrug resistanceassociated protein 2 (MRP2), in the apical cell membrane [39-41]. Several researches have attempted to define the substrate specificity of these efflux proteins, but the exact structural requirements remain uncertain [40, 42-44]. The function of the efflux system may be to prevent uptake of toxic substrates or to facilitate the excretion of such substrates across the mucosa of the intestinal tract [45]. This would explain the broad and sometimes overlapping substrate specificity of the efflux proteins. For example, the chemotherapeutic agent etoposide is effluxed across the intestinal wall not only by P-gp, but also by other efflux systems [46]. Although an increasing number of investigations report the involvement of efflux in drug permeation, the in vivo relevance of this mechanism in the therapeutic environment is not clear [47 and further discussed in ref 22]. In summary, a number of transport mechanisms are available to orally administered drugs. A high passive transcellular permeability is important for the satisfactory absorption of most drugs. Other processes contribute significantly to the transport in certain cases, but the contribution of these processes vary widely between different regions of the intestinal tract, making knowledge of passive transcellular permeability important also in these cases. In conclusion, the development of models that predict passive transcellular permeability is highly motivated, and such models are the focus of this thesis. 3.2 In vitro models for predicting intestinal membrane transport In vivo studies of intestinal membrane permeability to lead compounds in humans are costly and, if performed with hazardous compounds, potentially harmful for the volunteers. Therefore, permeability can only be determined in this manner for a small number of well characterized drugs. In the early stages of the drug discovery or development processes, there are many compounds to be considered and these compounds are poorly characterized. Such studies can be performed in experimental animals instead of humans, but the cost and throughput still severely restrict the capacity of these experiments. Therefore, a number of in vitro models that have a much higher capacity than in vivo models have been developed to enable predictions of intestinal membrane permeability of larger compound libraries. An additional advantage of using in vitro methods is that these methods may provide a more fundamental understanding of the different steps in the absorption process [48, 49]. The in vitro methods range from simple determination of drug partitioning between an aqueous solvent and an organic solvent to more complex methods based on animal tissues. In this thesis, in vitro methods of various complexity have been used. The methods range from the determination of partition coefficients and drug transport across monolayers of 12

13 cultured intestinal epithelial cells to experiments using whole tissue. These methods have been employed to develop computational models and to predict intestinal permeability. A brief overview of some of the less complex models is given in this section Partitioning in isotropic systems The structure of octanol resembles that of the major lipids of the cell membrane, which can be summarized as a polar group at the end of an alkyl chain. This suggests that solute transport from water into octanol models the process of solute transport into a cell membrane (fig. 3A). In fact, it has been speculated that micro-domains within the hydrated octanol phase can accommodate solute molecules by providing a range of localized environments that are similar to lipid bilayers [50]. Indeed, the logarithm of the octanol/water partition coefficient (log P o/w ) for solutes correlates roughly with cell membrane permeability and has therefore been widely used to predict the rate of membrane transport [e.g. 14, 51-57]. The widespread use of log P o/w has also led to this descriptor becoming the benchmark of the lipophilicity of a solute. A potential limitation of log P o/w as a predictor of membrane transport is that it does not describe hydrogen bonding properties well. Octanol can act as a hydrogen bond acceptor and, to a lesser extent, as a hydrogen bond donor [58]. Therefore, a solute that is transported from the water phase into the octanol phase does not experience much change in the hydrogen bond accepting properties of the solvent. A B C D Figure 3. Schematic representation of in vitro experimental methods for the assessment of intestinal membrane permeability. (A) Drug partitioning into isotropic solvents gives information about physico-chemical properties. Immobilized lipids allow drug-membrane interaction studies (B), while passive drug transport can be studied across artificial membranes (C). Cell monolayers (such as Caco-2) allow studies of passive transcellular transport and passive paracellular transport as well as active transcellular transport and efflux mechanisms (D). The arrows symbolize drug transport across and interaction with the membranes. However, complete desolvation is probably also required before solutes can enter the interior of biological membranes. This means that the ability of a molecule to permeate a cell membrane also depends on the number and the strength of the hydrogen bonds that the molecule forms with water [59, 60]. Since log P o/w is a poor descriptor of hydrogen bonding, partition coefficients determined in a system consisting of two 13

14 solvents, only one of which can form hydrogen bonds, have been introduced. This partitioning is often expressed as log P, the difference between the octanol/water partition coefficient and an alkane/water partition coefficient [61, 62]. log P has been shown in several studies to correlate well with membrane permeability [see, for example, 63-65]. The main disadvantage of this descriptor is that it requires two experimental determinations for each compound (octanol and alkanes are miscible, and it is not possible to measure log P directly). A single log P determination between two immiscible solvents, such as heptane and ethylene glycol (log P h/eg ), has been suggested as a less experimentally demanding method of assessing hydrogen bonding capacity [64, 66]. Log P h/eg was strongly correlated with membrane permeability to a series of peptide derivatives [67]. However, as discussed in Paper II, log P h/eg does not always provide a pure measure of hydrogen bonding. In contrast to the experimental simplification introduced by log P h/eg, it has been suggested that a critical quartet of partition coefficients in four different solvent systems is required to describe membrane partitioning accurately [68]. Such a multiple partitioning approach may be appealing for mechanistic studies of the interactions between drugs and membranes, but the large number of experiments required is probably more time-consuming than the determination of the actual membrane transport in a cell culture or tissue model. Although several successful attempts have been made to adopt partition coefficients such as log P o/w and log P to predict membrane transport, these descriptors frequently fail to account for differences in permeability in datasets containing structurally diverse compounds [14, 51, 52, 56, 57]. Partition coefficients can also be readily obtained by computational methods [69, 70] and the rationale for using these descriptors to predict membrane transport is further discussed in Section Interaction with and transport across artificial membranes Phospholipids resemble biological membranes more closely than simple isotropic solvents, and useful information may be obtained by determining the partition coefficients into phospholipid phases. Experiments using ph-metric titrations can be performed in liposome suspensions [71]. This method successfully described human intestinal absorption in a structurally diverse series of compounds for which log P o/w failed [72]. While this method is limited to protolytic compounds (since it is based on ph-metric titrations), it does not require the development of an analytical method that is specific for each compound. The development of compound-specific analytical assays was also circumvented in a system in which liposomes were coupled to a biosensor surface. It was subsequently demonstrated that 27 drug compounds could be roughly classified as having high, moderate or low intestinal absorption in humans based on results from the biosensor system [12]. Chromatographic methods that model the intestinal permeability to drugs have also been developed. The permeability is related to the retention time of the drug on stationary phases that mimic the lipid bilayer [see, for example, 73, 74]. The stationary 14

15 phase may be immobilized liposomes or immobilized phospholipids. These chromatographic methods are attractive for screening purposes, since they require only small amounts of solute, the processes can be automated and the retention times can often be modified by the use of organic solvents [75] or by altering the phospholipid content [76]. The retention times of solutes on these columns are determined by a combination of two effects: electrostatic interactions between the drug and the lipid surface, and the partitioning into and across the lipid phase (fig. 3B) [77]. This means that drug retention on such columns will not always reflect the transport across a cell membrane. Thus, these methods have sometimes resulted in correlations with drug permeability that were not stronger than those obtained with conventional measurements of octanol/water partition coefficients [76, 78]. The development of a membrane system that allows transport across rather than interaction with the membrane lipids is therefore desired. Recently, a small scale method based on filter chambers inserted into a microtiter plate has been described [13]. The filter chambers are equipped with filters that have been impregnated with a mixture of phospholipids and an inert organic solvent to form an artificial lipid membrane. This allows high-throughput studies of the passive transmembrane transport of drugs (fig. 3C). A potential limitation in setting up this method is the difficulty in forming a homogenous bilayer that is representative of the cell membrane. This is probably one of the reasons why it is hard to find publications where the promising initial results reported in ref 13 are followed up [79]. However, at least one commercial system using such artificial membranes is now available [80] Transport across cultured epithelial cell monolayers Drug transport across membranes can also be studied using cultured epithelial cells grown on semi-permeable filter supports. The human colon adenocarcinoma cell line Caco-2 is the most widely used cell line for the determination of drug permeability [81, 82]. Caco-2 cells spontaneously differentiate into enterocyte-like cells with a paracellular permeability comparable to that of human colon. In spite of its colonic origin, a number of active transport mechanisms normally found in the absorptive enterocytes of the small intestine are present in this cell line [83]. A number of other intestinal epithelial cell lines can be induced to differentiate and form confluent monolayers of polarized cells, and these cell lines have been used for studies of drug transport under controlled conditions [see, for example, 84-86]. Such cell lines include HT29, T84 and 2/4/A1. One disadvantage of cell culture models is that the cells must be grown on filters for up to several weeks before they can be used for drug transport experiments. Caco-2, for example, normally requires culture on filters for three weeks before it can be used for drug transport experiments [87]. The problem can be circumvented to some extent by using cell lines that grow and differentiate faster [86, 88], or by optimizing culturing conditions for Caco-2 to allow the transport studies to be performed only a few days after seeding to filters [89]. Furthermore, studies of permeability in cell monolayers are currently being automated [90, 91], and several compounds have been successfully tested at the same time using cassette dosing [92-94]. During transport studies, the experiments can be further optimized by selecting the number and the timing of 15

16 sampling points to obtain the maximum amount of information in the shortest possible time [95]. One advantage of cell culture models is that they measure the transport of the drug across a cell membrane, rather than an interaction of the drug with the lipid bilayer. Another advantage is that parallel transport routes, both passive and active, can be studied (fig. 3D). In fact, much of our recent knowledge on active drug transport mechanisms in the intestine has been derived from cell culture studies, as has new information on passive transcellular and paracellular transport mechanisms [49]. However, quantitative results obtained in cell culture experiments are generally poorly correlated with the levels of active drug transport in vivo [96, 97]. In summary, the in vitro experimental models that have been developed for the screening of intestinal permeability describe different mechanisms, fig. 3. It is important to understand these differences in order to select the specific model that most efficiently provides information about the oral absorption of the compounds under study. All experimental models require the synthesis of sufficient amounts of the compounds, which may be a serious drawback. 3.3 Computational models for predicting intestinal membrane transport The previous section has shown that in vitro models may be useful for the estimation of intestinal drug permeability. However, a thorough understanding of the molecular properties that govern intestinal drug permeability is essential for the design of compound libraries. This understanding can be reached by applying proper computational methods to establish structure-permeability relationships. Furthermore, computational determinations of intestinal membrane permeability do not require synthesis of the compounds and can therefore be applied at any stage of the drug discovery and development processes. In silico models of different levels of complexity are available, and different models will be suitable at different stages of the drug discovery process (fig. 2). The interpretation of results obtained using these models requires a fundamental understanding of the molecular properties that determine passive membrane transport. In the following section, therefore, these properties will be briefly discussed Solute transport across lipid bilayers Passive transport across the cell membrane was initially thought to occur according to the solubility-diffusion model [98]. The cell membrane is treated in this model as a homogenous barrier, and the transport proceeds by distribution into and subsequent diffusion across the membrane. The ph-partition theory predicts that only the uncharged species of protolytic compounds will be partitioned into the membrane [99]. Thus, according to the solubility-diffusion model and the ph-partition theory, membrane-water partitioning, charge and solute size (assuming that diffusion in the membrane is reflected by the solute size) describe the transport process. While it is indisputable that the transport of a molecule across a cell membrane is impaired when the molecule is ionized, transport does not completely cease [100]. The solubility- 16

17 diffusion model also implies that membrane partitioning is a one step process. This is why drug partitioning in isotropic solvent systems, such as the octanol/water system, has frequently been employed to predict passive membrane transport with some success (see Section 3.2.1). However, this simplistic model, in which membrane partitioning is considered to be a one-step process, fails to account for the anisotropic nature of the cell membrane [101]. Two observations that result from this anisotropic nature are that the diffusion rate of a solute is different in various regions of the membrane [102] and, perhaps more importantly, the forces that govern partitioning into the membrane are different in various regions of the membrane [103]. The anisotropic nature of the membrane has been included in a model in which membrane partitioning is considered to be a two-step process, and this model has been successfully applied to describe drug transport [64]. The two-step partitioning process can be rationalized by considering the insertion of a polar, but lipophilic, solute into a phospholipid membrane. Lipophilicity constitute the major driving force of solute accommodation into the region of the phospholipid head groups. In contrast, transfer of the solute into the interior of the phospholipid bilayer depends mainly on energetically unfavorable interactions between the bilayer and the polar parts of the solute. This process depends to a lesser extent on lipophilicity, and can be largely accounted for by hydrogen bonding and polarity [103]. Evidence that this mechanism is indeed correct comes from molecular dynamics simulations where four separate regions in the membrane was identified (fig. 4) [101]. These simulations suggest that partitioning of polar solutes into the dense apolar region of the membrane interior is often the main barrier in the transport process [101, 103, 104]. It should be emphasized that the interactions of drugs with real membranes containing multiple components can be considerably more complex than simulated drug-membrane interactions, [see, for example, 105] Resistance to water permeation Figure 4. Schematic representation of the four-region membrane model. In the perturbed water layer (1), an approaching solute starts to experience the polar head groups. The membrane reaches its highest density in the interphase (2) and the charges presented by the polar head groups restricts the movement of water molecules. In the soft polymer region (3), a high tail density restricts solute diffusion. Due to its apolar nature, this region will not allow the incorporation of water molecules. The tail density decreases in the middle of the bilayer. In the absence of water and with a high percentage of free volume, the decane region (4) allows for the incorporation of hydrophobic solutes [101]. The curve represents the resistance that a water molecule experiences during the passage through the membrane. 17

18 In conclusion, regardless of whether the solubility-diffusion model or the four region membrane model is adopted to describe passive membrane permeation, the transport process is largely accounted for by the hydrogen bonding capacity, lipophilicity, size and charge of the solute. A large number of descriptors related to hydrogen bonding capacity, lipophilicity, size and charge can be obtained using various computational methods. Several of these are discussed in the next section Generation of calculated descriptors The first step in the development of a model for the prediction of membrane permeability is the construction of a description of the solute molecule. In its simplest form, this description may be the number of atoms in the solute molecule (the general trend would show that the lower the atom count, the higher the permeability). Such a simple descriptor, however, would generate a scattered relationship with membrane permeability, and more finely tuned descriptions are often used. Descriptors based on a two-dimensional representation. Molecules can be represented by their two-dimensional (2D) structure or by their SMILES code [106]. Such representations identify atom types and functional groups, and this information can be used to rapidly calculate physico-chemical properties such as hydrogen bonding capacity [107], lipophilicity [108, 109] and charge. In addition, a number of topological descriptors can be derived from the 2D structure [see, for example, 110]. Descriptors based on a three-dimensional representation. Two-dimensional representations provide incomplete information about a molecule, and three dimensional (3D) structures may be required. Further, using the 3D structure of the molecule allows several different spatial arrangements that are not accounted for in the 2D representation to be distinguished. These 3D structures depend on the environment of the molecule and can be obtained by performing molecular mechanics calculations. Descriptors generated from 3D structures are therefore unique for a particular molecular conformation and are considered to better reflect the intramolecular interactions. A single 3D conformation can be generated in fractions of a second, but a full conformational search based on molecular mechanics using the Monte Carlo algorithm or a molecular dynamics simulation may require computations for up to several weeks in order to cover the entire conformational space. Descriptors such as molecular surface areas [111], volume and conformationally dependent lipophilicity [MLP, see 112] can then be derived from the 3D structures that are generated. Descriptors based on wave functions. The 2D and 3D structures do not generally provide an accurate description of the electron distribution of the molecule. In order to obtain information about molecular valence properties, the molecules must be represented by wave functions, which are generated by quantum mechanics calculations. These representations contain a massive amount of information and allow virtually every known computational descriptor to be calculated. A disadvantage of quantum mechanics calculations is that they are very time consuming even if only one conformation of each compound is considered and these calculations are therefore not practical for application in larger compound libraries. 18

19 Representation Descriptors 2D structure I Atom counts, fragment counts, topological indexes 3D structure IIa Molecular surface properties and volume Conformational space IIb Dynamic molecular surface properties and volume Wave function III Molecular valence properties Figure 5. The three levels of complexity for molecular structure representation discussed in the text are shown in the left panel. Some of the descriptors that can be derived from these representations are shown in the right panel. The 2D structure is the basis for the molecular mechanics or molecular dynamics calculations, and an appropriate 3D structure is used as input for the quantum mechanics calculations. These methods thus constitute different levels of complexity of the representation of a molecule (fig. 5). Descriptors of the corresponding (or lower) complexity can be derived from each of these levels. However, complex descriptors have been successfully predicted from less complex ones [ ]. Whether simple or complex, the descriptors outlined in fig. 5 may be related to membrane permeability by appropriate statistical methods and in this way provide predictive models of intestinal membrane permeability Model development During the past few years, several computational models have been developed for the prediction of passive intestinal membrane permeability [see, for example, 22, 24]. These models can be quantitative or qualitative. These two types of model are often constructed using different approaches and they allow different types of predictions to be made. The models are therefore treated separately in the following sections. Qualitative models of drug-likeness. It is assumed that a drug or a compound that has reached phase II in the development process is sufficiently well absorbed to meet clinical demands (since oral administration is preferred for most drugs) and that it possesses other properties that are associated with drugs. Such a compound may be defined as drug-like [116]. Qualitative models have been developed by examining groups of drug-like compounds and looking for systematic patterns in the structures of 19

20 these compounds. Several investigators have used this method to compare a set of druglike compounds with a set of compounds that are not drug-like [ ], or to analyze molecular descriptors in a single set of drug-like compounds [5, ]. One advantage of these qualitative models is that they do not require the conduct of experiments, and this allows the study of a large number of compounds (provided that the databases have been validated to actually consist of drugs and, when applicable, non-drugs, respectively). However, the model treats the difference between drugs and non-drugs in a manner which may be too simple since a drug may differ from a nondrug in many respects. The reason that a compound is not a drug may be poor permeability, but it may also be the result of a lack of efficacy, lack of pharmacological activity, toxicity, extensive metabolism or poor solubility. These factors need to be considered when such models are interpreted. The qualitative model that is probably the best known is the rule of five [5]. This model was developed by Lipinski and co-workers, who analyzed 2245 phase II compounds, and identified four easily calculated descriptors that did not exceed certain limits for most of the compounds. The descriptors (limits) were: the number of hydrogen bond donors (more than five), the number of hydrogen bond acceptors (more than ten), molecular weight (greater than 500) and log P o/w (greater than five). The rule of five states that if two or more of these limits are exceeded, the compound in question is not likely to be a drug [5]. The merit of this model is the ease with which the descriptors can be determined and interpreted. This is probably also the major reason for its widespread use. However, the relevance of the rule of five has recently been questioned [126]. Quantitative models of intestinal membrane permeability. These models have been developed by relating experimental permeability to one or more calculated molecular descriptors. The relationships can be derived using various statistical methods, ranging from simple linear regressions [e.g. 55] to complex methods involving neural networks [e.g. 127]. In general, simple models containing few descriptors are easier to interpret than those that contain many descriptors that are related to permeability in a complex way. Calculated lipophilicity, hydrogen bonding capacity and molecular size derived from fragment and atom counts (fig. 5, level I) were among the first descriptors to be used for the description of passive membrane permeability [59, 128]. These descriptors have subsequently been correlated to passive membrane permeability with some success [see, for example, 55, ]. Other descriptors based on fragment and atom counts that have been employed for the description of membrane transport include solubility parameters [133], electrotopological state indexes [134] and a composite ensemble of physico-chemical properties [135]. The results obtained using these simple methods were generally comparable to the results obtained using more computationally demanding methods [136, 137], which suggest that the computational procedures can be simplified with little effect on the correlations with membrane permeability. The use of molecular surface properties derived from molecular mechanics calculations (fig. 5, level II) have been explored as a means to describe various physico-chemical properties, such as lipophilicity [112, 138], solvation energy [139] and solubility [140]. 20