In Vitro Cell Culture Models And Its Implications In Drug Discovery
By - 12/22/2004
Hemant P. Joshi and Sarasija Suresh*
Department of Pharmaceutics, Al-Ameen College of Pharmacy, Hosur Road, Bangalore- 560027,Karnataka, INDIA. Ph: 91-80-22234619 Fax: 91-80-22225834 *e-mail: sarasija_s@hotmail.com
Recent advances in drug discovery have led the way towards more efficient techniques to synthesize and screen new chemical entities (NMEs) and have increased subsequently the number of leads identified. Compounds with good biological activity may fail to become drugs due to insufficient oral absorption. Selection of drug development candidates with adequate absorption characteristics should increase the probability of success in the developmental phase. To assess the absorption potential of new chemical entities numerous in vitro and in vivo model systems have been tried.
Many laboratories rely on cell culture models for intestinal permeability such as, Caco-2, HT-29 and MDCK etc. and also to attempt an increase in the rapid screening of permeability measurements, several physicochemical methods such as, immobilized artificial membrane (IAM) columns and parallel artificial membrane permeation assay (PAMPA) etc. have been used. More recently, much attention has been given to the development of in silico (computational methods) to predict drug absorption. However, it is clear that no single method will prove sufficient for studying drug absorption, but most likely a combination of systems will be needed. Higher throughput, less reliable methods could be used to discover loser compounds, whereas lower throughput, more accurate methods could be used to optimize the absorption properties of lead compounds. Finally, accurate methods are needed to understand absorption mechanisms (efflux –limited absorption, carrier-mediated, intestinal metabolism) that may limit intestinal drug absorption. This information could be extremely valuable for medicinal chemists in the selection of favorable chemical entities. The present review looks into the process and mechanisms of absorption, one of the most important requirements in drug discovery, its evaluation techniques and the recent cell culture systems developed as in vitro models to hasten drug discovery.
Keywords: in vitro, in vivo, IAM artificial membrane, PAMPA, in silico, drug absorption, Caco-2.
INTRODUCTION
The drug discovery process is rapidly evolving due to technological development in target identification along with automation of combinatorial synthesis. Drug discovery requires an early appraisal of all factors impacting on the likely success of a drug candidate in the subsequent pre-clinical, clinical and commercial phases of drug development thus for every new drug that reaches the market, pharmaceutical companies must screen approximately 5,000 compounds 1. Combinatorial chemistry and automation has made possible to synthesize thousands of compounds in a day. Through the use of receptor cloning and cell-based assays it is possible to evaluate the biological effect of these compounds at an unpredictable speed. In some cases, the implementation of high throughput screening (HTS) and ultra-high throughput screening (UHTS) has presented pharmaceutical scientists with an opportunity to test the biological activity of 100,000 compounds in a day 2 leading to the rapid identification of large number of biologically active compounds (i.e. hits). However, despite of this dramatic increase in the speed at which new chemical entities are produced and tested, the number of compounds that survives through numerous hurdles associated with the drug development process is extremely low. Approximately 40% of the compounds that enters the development phase fail to reach the market 3. The main reason for failure is poor biopharmaceutical properties like, low aqueous solubility, chemical instability, insufficient intestinal absorption, intestinal and or hepatic metabolism, biliary excretion, and high systemic clearance. Though compound is found to be highly potent it will not be developable unless it exhibits adequate pharmaceutical profile. For example, to be an effective, orally administered compound, must be absorbed across the intestinal mucosa. Thus, a major challenge for medicinal chemists is the design of chemical compounds that exhibit the right pharmaceutical characteristics, including intestinal absorption. In other words, to increase the probability of success of drug development candidates intended for the oral administration, the drug design process must be approached with absorption considerations in mind. Failure to do this will lead to optimization of potency without sufficient knowledge of absorption potential. Once the potency has been optimized, any change aimed at increasing absorption will most likely have a detrimental effect on the biological activity. For medicinal chemists to be able to incorporate absorption considerations in the design phase they must have prompt access to absorption data on their current leads. Intestinal drug absorption depends on two major parameters:
1) permeability across the epithelial mucosa and 2) gastrointestinal transit time. Because gastrointestinal transit time can only be evaluated in vivo, all in vitro systems can determine only mucosal permeability. Under the assumption of a similar gastrointestinal transit time for different compounds, a difference in mucosal permeability should suggest a difference in potential intestinal absorption in vivo.
MORPHOLOGY OF THE SMALL INTESTINE
Small intestine in an average adult human is about 280 cm long and 4 cm in diameter playing a major role as a selective permeability barrier in case of oral absorption. It permits the absorption of nutrients such as, sugars, amino acids, peptides, lipids and vitamins 4, 5 it is also accomplished with varied functions, structures and cells with unique characteristics thus limiting the absorption of xenobiotics, digestive enzymes, and bacteria. Drug and nutrient absorption takes place in the intestinal mucosa. The intestinal mucosa consists of three layers: the muscularis mucosa, the lamina propria, and the epithelial cell layer (Fig. 1). The muscularis mucosa is located at the boundary between the mucosa and the submucosa. Intestinal epithelial cells are joined at intercellular junctional complexes. These junctional complexes, which are approximately 0.5-2 nm wide consists of three components; tight junctions, intermediate junctions and spot desmosomes 6. The tight junctions, located at the apical end of the lateral membrane of adjacent cells, eliminate the intercellular space over a variable distance 6. The tight junctions of ileal absorptive cells are deeper and denser than tight junctions of the jejunum. The depth and density of the tight junctions between absorptive cells are greater than those of undifferentiated crypt cells and goblet cells. Compared to the intermediate junctions and spot desmosomes, the tight junctions are the most significant diffusion barrier component of the junctional complexes.
Fig. 1. The intestinal mucosa consists of three layers: the muscularis mucosa, the lamina propria, and the epithelial cell layer. (Ref. 17)
BARRIER PROPERTIES OF THE INTESTINE
The physical barrier is the result of the tight junctions and the lipid character of the cell membrane. The permeability resistance of the intestinal mucosa consists of both physical and biochemical barriers. The biochemical component is the result of the actions of membrane enzymes and transporters; however the driving forces for intestinal transport are the concentration gradient, electrical potential difference, and hydrostatic pressure gradients.
Physical Barrier
Tight junctions constitute the main barrier to paracellular diffusion. The diameters of the tight junction pores are approximately 4-8 Å and 10-15 Å in humans and animals, respectively 7. Because in humans the paracellular route will not allow the passage of molecules with diameters greater than ~8 Å, this route is unlikely to play an important role in the absorption of most compounds of pharmaceutical interest. In addition to the narrow diameter of the tight junctions, this pathway is of little importance for most drugs because of the small surface area of the tight junctions, which accounts for 0.01% of the total surface area i.e. cell membrane plus tight junctions 8,9.However, evidence have shown that the diameter of tight junctions can be increased by cellular regulatory processes10, thus efforts to increase the paracellular permeability of poorly absorbed compounds through co-administration of agents that open up tight junctions 11, 12. At present, the potential application of this approach is limited by safety concerns with the few possible exceptions.
Biochemical Barrier
The biochemical component of the mucosal barrier consists of drug metabolizing enzymes. The enzymes expressed by enterocytes include aminopeptidases N/P/W, dipeptidyl peptidase IV, isozymes of the cytochrome P450 (CYP) superfamily such as, 1A1, 1A2, 2D6, 3A4, 2C9, and 2C19, esterases, phenol sulfotransferase (PST), and UDP glucuronyltransferase (UDPGT) 6, 13,14. CYP3A4, which accounts for approximately 60% of the total amount of CYP enzymes found in the intestine, metabolizes a large number of compounds and limits absorption 15, 16.
Absorption Pathways
The intestinal mucosa is a selective permeability barrier. Drugs administered into intestinal lumen can cross the mucosal epithelium by the paracellular and transcellular routes (Fig. 2) 17. In a general sense, the transport pathways can be divided into a) passive paracellular transport, b) passive transcellular transport, c) carrier-mediated transcellular transport, and d) vesicular transport.
Fig. 2. Diagrams of pathways and mechanisms mediating transepithelial transport
- Passive paracellular transport.
- Carrier mediated transport across apical membrane.
- Carrier mediated transport across apical and baslolateral membrane.
- Carrier mediated efflux transport across the membrane.
a)Passive Paracellular Transport
The paracellular pathway is an aqueous, extracellular route across the epithelium. The driving forces for passive paracellular diffusion are the electrochemical potential gradients derived from differences in concentration, electrical potential and hydrostatic pressure between the two sides of the epithelium. The main barrier to passive Para cellular diffusion is the tight junction. In general, the transepithelial permeation of hydrophilic compounds occurs mainly through the paracellular route.
b)Passive Transcellular Transport
The passive transcellular pathway involves the movement of solute molecules across the apical membrane, through the cell cytoplasm, and across the basolateral membrane. This is the main route of permeation for hydrophobic compounds. The surface area of the transcellular route i.e. cell membrane is much larger (i.e. 99.9% versus 0.01%) than the surface area of the paracellular route (tight junctions) 8, 9. Generally, compounds whose permeability is limited to the paracellular pathway have low absorption and compounds that readily traverse the cell membrane have high absorption 18. However, the involvement of uptake or efflux transporters can distort this pattern. In case of some hydrophilic molecules such as, peptides and proteins undergo carrier-mediated transport, and thus, exhibit absorption values higher than expected from their intrinsic membrane permeability characteristics 19.
c)Carrier-Mediated Transport
The small intestinal mucosa expresses large numbers of absorption transporters, which are responsible for the absorption of nutrients and vitamins. In addition to transporting vitamins and nutrients, these transporters seem to have shown to mediate the absorption of some drugs. Transporters for di-/tripeptides (PEPT1), large neutral amino acids, bile acids, nucleosides, and monocarboxylic acids have received a great deal of attention for their perceived potential to deliver drugs across the intestinal mucosa 20- 28. Recent studies have reported the presence of two additional peptide transporters, PTR3 and PHT1, in human and rat intestine 24. The full potential impact in drug absorption of these transporters, and others remains to be established. Influx transporters can increase intestinal drug absorption whereas efflux transporters can have the opposite effect. Influx transporters can bind compounds that are dissolved in the intestinal fluid and translocate them across the apical membrane of enterocytes, thus facilitating the drug absorption process. of these, PEPT1 seem to mediate the transport of peptide drugs such as, angiotensin converting enzyme (ACE) inhibitors, b- lactam antibiotics, and renin inhibitors 21-24, 25. Compounds that are substrates for these transporters exhibit intestinal absorption higher than expected from their diffusion across cell membranes.
Carrier-Limited Transport (Apical Efflux)
In contrast to the role of absorption transporters, which can enhance intestinal drug absorption, other transporters may have the opposite effect. Efflux transporters mediate the extrusion of compounds from the cell cytoplasm to the intestinal lumen through a process known as apical efflux. Two families of transporters that belong to the ATP binding cassette (ABC) superfamily of transporters mediate apical drug efflux. They are the multidrug resistance (MDR) and multidrug resistance-associated protein (MRP) families 26. 27. P-glycoprotein (Pgp), the most studied member of the apical efflux transporters, is the product of the MDR1 gene. Pgp has 2 subunits with 6 trans-membrane domains and 2 ATP binding sites 28, 29. Located on the apical membrane of normal enterocytes, it has been shown to limit intestinal absorption of a large number of drugs 30-33 of these, three (MRP1, MRP2/cMOAT, and MRP3) have been found in human duodenum whereas MRP1, MRP3, and MRP5 are found in human colon 34. Their location on the apical membrane of enterocytes and colonocytes, together with their efflux function, makes these transport a potentially formidable barrier to gastrointestinal drug absorption. So far, this family of transporters has been shown to export glutathione S conjugates and organic anions 35, 36. The extent to which they are involved in intestinal drug transport will yet be identified. The MRP family of transporters is emerging as potentially important in determining drug absorption and excretion.
Vesicular Transport
While the action of intestinal enzymes limits the extent to which a compound can permeate the intestinal mucosa, enterocytes also possess vesicular transport processes that can facilitate drug absorption. These include fluid-phase endocytosis (pinocytosis), receptor-mediated endocytosis (RME), and transcytosis. Fluid-phase endocytosis (FPE) is a process by which solute molecules dissolved in the luminal fluid are incorporated by bulk transport into the fluid-phase of endocytic vesicles. This process starts when the plasma membrane forms invaginations that pinch off to form vesicles or pinosomes, which migrate inwardly. Molecules dissolved in the vesicle are transported to endosomes (prelysosomal vesicles), which subsequently fuse with lysosomes. There is evidence that the mucosal uptake of some peptides and proteins involves fluid-phase endocytosis 37. Receptor-mediated endocytosis is generally of importance only for the mucosal permeation of macromolecules, but not for small molecules. It involves the binding of the macromolecule to a binding site (receptor) on the membrane followed by clustering of the receptor-ligand complex into clathrin-coated pits. After endocytosis, the fate of the elements of the receptor-ligand complex is determined through a process called sorting. Usually, the sorting process results in the destruction of the ligand in the lysosomes while the receptor can either undergo lysosomal destruction or recycling back to the cell membrane 38-40. The ligand dissolved in the endocytic vesicle, following FPE or RME, can by-pass the lysosomes and undergo release across the basolateral membrane 41. This process, known as transcytosis, may result in the intestinal absorption of molecules unable to permeate the cell membrane by simple diffusion.
MODELS OF INTESTINAL DRUG ABSORPTION
The ability of an orally administered compound to permeate the intestinal mucosa may be limited by the physical or the biochemical component of the intestinal mucosal barrier. Thus, in vitro intestinal permeability models should not only predict intestinal drug absorption potential but also provide some understanding of the absorption mechanism(s). This can be accomplished to the extent in which the permeability model incorporates the functionality of the physical and biochemical barrier components.
In Vivo Drug Absorption
In general, drug absorption in animals is believed to be a good predictor of absorption in humans. Animals integrate all the biological factors that may affect drug absorption. Unlike in vitro systems, in which a correlation to in vivo data must be established, this step is unnecessary when animals are used. An important advantage of whole animals is that the species used in absorption studies could be the same one used in pharmacology /or toxicology evaluations. They also can be used to evaluate complex formulations, which would be very difficult to test in vitro. Some of the disadvantages of studies with whole animals include the need for relatively large amounts of material, the complexity of the analytical methods needed for plasma analysis, the time-consuming and labor-intensive nature of experiments, and the fact that they provide little mechanistic information on drug absorption.
In Vitro Models of Intestinal Drug Absorption
The successful application of in vitro models of intestinal drug absorption depends on the extent to which the model comprises the relevant characteristics of the in vivo biological barrier. Despite the obvious difficulties associated with trying to reproduce in vitro all the characteristics of the intestinal mucosa, various systems have been developed which mimic, to varying degrees, the relevant barrier properties of the intestinal mucosa. These systems include excised tissue e.g. isolated intestinal segments, everted sacs, intestinal rings, stripped and unstripped mucosal sheets, cultured cells such as Caco-2, HT-29, T84, and MDCK, physicochemical methods like Log D/P, immobilized artificial membrane, parallel artificial membrane permeation assay, mucosal cell membrane vesicles, isolated mucosal cells, and computational (in silico) methods.
Excised tissue
Excised intestinal tissues have been used to study intestinal drug and nutrient absorption. The solution containing the drug is applied to one side (mucosa or serosa) of the mucosa and the rate of drug absorption is determined by measuring either the disappearance of drug from the dosing solution or appearance of drug in the serosal side. Although they vary in complexity and versatility, excised tissue preparations share the two important advantages: a) preservation of the architectural integrity and b) ability to determine absorption across different gastrointestinal segments. A common disadvantage is the limited viability of this type of preparations.
Perfused Intestinal Segments
Isolated intestinal segments comprise the absorptive cells and the underlying muscle layers. As it is commonly used, this technique only allows sampling from the mucosal side; drug disappearance is assumed to be equal to drug absorption. This assumption is valid when apical uptake is the rate-limiting step in drug absorption. But, considering that drug absorption is not the only factor responsible for luminal drug disappearance, this assumption could be misleading. For example, mucosal metabolism and mucosal accumulation could lead to an overestimation of true drug absorption. Studies found that, for several b- lactam antibiotics, absorption based on luminal disappearance was roughly twice the true amount transported across the tissue 42.
Perfused intestinal loops offer a few advantages over other drug absorption models. Unlike whole animals, perfused intestinal loops can be used to study segmental differences in drug absorption and metabolism without the interference from physiological factors such as, gastric emptying, surface area of the segment and/or small intestinal transit time. With respect to throughput and complexity, this technique offers a few advantages over whole animals, but not over other in vitro techniques. It also has numerous disadvantages. First, the determination of absorption based on luminal disappearance is potentially misleading. Second, it requires large amounts of compound, relative to other in vitro systems. Third, the number of intestinal segments that can be obtained from one animal is limited. Fourth, as is the case with other excised tissue preparations, the viability of perfused intestinal segments is limited. As a result of these limitations, this technique is not likely to be useful as a screening tool. It has greater value in the elucidation of transport mechanisms. It may also be useful to evaluate the absorption of drugs whose poor solubility requires the use of complex dosing vehicles, which could not be presented to other in vitro systems such as, culture cells.
Everted Sacs
The everted sac was one of the first in vitro techniques used to study intestinal drug absorption. It is prepared by inverting a piece of intestine using a glass rod. The two ends of the intestinal segment are tied and the inside of the resulting sac is filled with an oxygenated buffer. The sac is then placed in a container that has the test compound. Drug absorption is measured by sampling the solution inside and outside the sac. It includes the mucosa plus underlying muscle layers. This differs from the in vivo situation and could lead to biased values for transport. The presence of this muscle layer could accumulate drugs, and thus lead to poor recovery. This technique was popular a few decades ago, but its utilization in recent years has been greatly reduced. It is unlikely that it will constitute an important absorption-screening tool in the future.
Intestinal Mucosa (Stripped or Unstripped)
Mounted in Ussing Chambers
To prepare intestinal mucosal sheets suitable for mounting in Ussing chambers a longitudinal cut of an intestinal segment is made to produce a long mucosal sheet. This sheet is cut as needed to produce mucosal strips of adequate size to fit in the opening of the diffusion (Ussing) chamber. Although mucosal sheets are used with or without the underlying muscle layer, in vivo, the muscle layer is not a barrier to absorption. Thus, the removal of this muscle layer, a process known as stripping, is advantageous for two reasons. First, it removes an artificial permeability barrier, and second, stripped tissues can be oxygenated more efficiently. Usually, Ussing chambers are connected to voltage clamps, which are used to make electrical measurements during the course of experiments. These measurements can be used to monitor tissue integrity and viability. This technique is very valuable. It allows the determination of transport polarity, which is indicative of the involvement of carrier or efflux mechanisms. This technique also allows determination of segmental-dependence of transport. It has been used to evaluate the in vitro permeability with varying degrees of success. For instance, one study found that rat intestinal mucosa mounted in Ussing chambers were useful in describing the regional variability in gastrointestinal absorption of mixed series of compounds 42. Also, a comparison between the Ussing chamber technique and human jejunal permeability found a good correlation for a discrete small series of 12 compounds 43.
In general, the in vitro technique generally underestimated the transport of compounds that undergo carrier-mediated absorption 43. While this small discrepancy may be reflect the limitation of the in vitro-in vivo correlation between these systems, it is also possible that the intrinsic level of expression of the carriers involved, may differ between rat and human. It has also been argued that the presence of the muscularis mucosa in the Ussing technique constitutes an artificial barrier not present in vivo, which can bias the permeability data. And because in vivo the rate limiting barrier is the epithelial cell layer, in vitro cell culture models may better reflect the absorption potential of a compound 44. However, the complexity of this technique and the amount of compound needed in studies, are likely to limit the application of this model system to the study of transport mechanisms.
Isolated Enterocytes
Isolated intestinal cells have been used to study intestinal drug absorption. To prepare these cells, first, an intestinal segment is cut and then the mucosal surface is treated with mechanical forces, enzymes or chelating agents to dissociate the cells from the underlying tissues. This technique is not commonly used because of its limited utility. The process of cell isolation destroys many cells and greatly diminishes cell viability. Because cells must be used as a suspension, they lack the polarity that characterizes intestinal mucosal cells in vivo. Thus, isolated enterocytes can be used to study drug uptake, but not transepithelial transport or transport polarity.
Membrane Vesicles (BBMV, BLMV)
Cell membrane preparations include brush-border membrane vesicles and basolateral membrane vesicles. The membrane vesicles are suspended in a physiologic buffer that contains the test compound. Uptake into these vesicles is supposed to mimic transport across cell membranes. The vesicles are useful to study drug uptake or metabolism by plasma membrane-bound enzymes. These preparations constitute simplified models to study mucosal absorption. They have been proved useful to study specific membrane processes such as, binding of organic cations and uptake of PEPT1 substrates by small intestine 45, 46. Advantages of this preparation include, the short duration of experiments, the small amount of material needed, and the ease with which vesicles can be made. However, they also possess many disadvantages. Their lack of cellular metabolism makes it difficult to study important aspects of ATPdependent transport. In addition, uptake into membrane vesicles does not provide any insight into paracellular transport.
Caco-2 Cells
Numerous cultured cells have been used to study intestinal permeability. Caco-2, HT-29, and MDCK cells have been used with varying degrees of success 47-50. Caco-2 cells, first characterized as an intestinal permeability model in 198949 have been used to study various aspects of intestinal permeability. They exhibit morphological features of small intestinal cells (e.g., tight intercellular junctions and microvilli) and express intestinal enzymes (e.g., aminopeptidases, esterases, sulfatases, and cytochrome P450 enzymes) and transporters (e.g., bile acid carrier, large neutral amino acid carrier, biotin carrier, monocarboxylic acid carrier, PEPT1, and p-glycoprotein) 51-56. Results from many laboratories suggest a correlation between in vitro permeability coefficient values in Caco-2 monolayer and indicators of in vivo absorption (e.g., bioavailability, absorption, pharmacological effect) Encouraging in vitro -in vivo correlations have led to the widespread use of this model. The ease with which Caco-2 cells can be cultured has permitted their utilization in many laboratories. In many instances, little, if any, effort has been made to implement control measurements for monitoring the performance of the cells. Thus, the heterogeneity of wild type Caco-2 cells and the different culturing conditions used in various laboratories can give rise to the selection of different cell populations. This gradual selection, referred to as phenotypic drift, could contribute to the large inter-laboratory variability in Caco-2 permeability measurements. For example, transepithelial electrical resistance (TEER) and the transepithelial permeability of a paracellular flux marker such as, mannitol are commonly used to monitor monolayer integrity or cell damage. And it has been argued that mannitol permeability can be used to normalize the permeability values of test compounds 57. The interlaboratory variability in Caco-2 permeability is not limited to the mannitol. The difference in permeability values may be due to biologic factors associated with cell culture and/or study conditions. Although numerous correlations between in vitro permeability and in vivo absorption have been demonstrated, comparison of data between laboratories will be hindered by the variability in permeability values. This is particularly problematic when data from multiple laboratories are combined to increase the sized of databases used to develop models for in silico prediction of permeability or absorption potential. In the absence of serious efforts to normalize the permeability values from different laboratories, the utility of in silico permeability or absorption models is likely to remain limited to small series of carefully chosen compounds. The combination of data obtained under different conditions in various laboratories or resulting from high throughput permeability screening most likely will exhibit too much variability to be useful in trying to develop mathematical models truly predictive of intestinal absorption. To produce this type of data, permeability experiments must be conducted with this objective in mind.
Madin-Darby Canine Kidney (MDCK)
MDCK cells have received attention as an alternative to Caco-2 cells for permeability measurements. When grown under standard culture conditions, MDCK develop tight junctions and form monolayers of polarized cells. Cultured on filters they can be used to study not only cellular uptake but also vectorial fluxes. The main advantage over Caco-2 cells is the shorter culture times, which in some laboratories equals 24 hours. The transepithelial electrical resistance of MDCK cells is lower than that of Caco-2 cells and thus closer to the TEER of the small intestine. Several studies have found a good correlation between permeability coefficient values in Caco-2 and MDCK cells 58, 59. The permeability coefficients of hydrophilic compounds are usually lower in Caco-2 cells than in MDCK cells 58, 59. This is consistent with the higher transepithelial electrical resistance of Caco-2 cells. The expression of intestinal transporters in MDCK cells has not been well characterized. The true utility of MDCK as a model system of intestinal permeability is yet to be established
HT29
Several clones of HT29 cells have been used to study different aspects of intestinal drug absorption. Wild type HT29 cells form multilayer of undifferentiated cells, which are not useful for intestinal permeability studies. Culturing wild-type HT29 cells in media containing galactose instead of glucose leads to the selection of a subclone of HT29 that form monolayers of polarized cells. Several HT29 clones differentiate into enterocytic cells or goblet cells, which secrete mucus. An enterocytic HT29 clone, HT29-18-C1, was proposed as a model of intestinal permeability 71; however, these cells grow very slowly and a large number of cultures failed to develop acceptable barrier characteristics (e.g. transepithelial electrical resistance and mannitol permeability). Two HT29 clones that secrete mucus are HT29-H and HT29- MTX 60. The HT29-H clone was used to demonstrate the role of mucus on the permeability of testosterone. HT29-H monolayers had TEER values equal to 159 Wcm2, whereas the corresponding TEER value for Caco- 2 monolayers was 401 Wcm2 60. The presence of a mucus layer in HT29-H monolayers resulted in lower permeability coefficients in HT29-H (6.8 x 10-6 cm/s) than Caco-2 monolayers (31.5 x 10-6 cm/s). This indicates that the mucus layer accounts for most of the permeability resistance to testosterone. With the exception of a few studies the role of mucus on drug transport has been largely ignored as most pharmaceutical scientist rely only on Caco-2 monolayers to predict intestinal permeability. In an attempt to incorporate into Caco-2 cell monolayers characteristics more representative of the intestinal mucosa, Caco-2 cells were cocultured with HT29-MTX or HT29-H cells 61, 62.
Immobilized Artificial Membrane (IAM) Columns
IAM columns are essentially reverse-phase liquid chromatographic columns where the usual hydrocarbon phase that coats the solid support is replaced with lipids 63, 64. These lipids are supposed to mimic the lipid environment of the cell membrane. IAM columns have been developed under the premise that permeation of solute across the cell membrane is limited by its ability to partition into the lipid domain. Compounds that interact with the lipid phase of IAM have longer retention in the column. Presumably, a compound that has a long retention in the column (i.e. large capacity factor, k’) should have a good permeability across lipid bilayers. This assumption may not be accurate because for a compound to cross the epithelial cell layer it must also diffuse across the outer and inner leaflets of the lipid bilayer and back into the aqueous environment of the cytoplasm. One of the perceived advantages of this technique is that it can be conducted in a higher throughput compared with cell-based permeability assays. Because it eliminates the need to use a biological system, it can be faster; however, in most cases, the bottleneck in the evaluation of permeability is drug analysis, which is not eliminated with this technique. Unlike usual LC/MS and LC/MS/MS which require limited chromatographic separation, this technique requires greater separation because the absorption potential is based on k’, the capacity factor, which requires separation of a hydrophilic, which has poor retention. The system has been validated by showing correlation between k’ and permeability across Caco-2 cell monolayers 63, 64. Closer examination shows potential problems with this type of validation. This validation method has a problem. Because k’ is a physicochemical measurement, it is reasonable to expect reproducible results from different laboratories. However, considering the dramatic interlaboratory variability in Caco-2 permeability measurements, it is unrealistic to expect good correlations between k’ and Caco-2 permeability in different laboratories. The conclusions drawn from this study were that, as is the case for log P or log D, IAM columns may provide a reasonable permeability ranking for homologous, but not for diverse series of compounds. Some of the general disadvantages of this technique are that it does not consider the potential role of paracellular transport, carrier-mediated transport, drug metabolism, and efflux transporters on permeability. Overlooking the possible influence of all these factors on intestinal permeability seems dangerous and may provide a very limited view of the true intestinal absorption potential of a compound.
Parallel Artificial Membrane Permeation Assay (PAMPA)
PAMPA is run in a 96-well plate that consists of two parts. The bottom is a standard 96-well plate. All the wells at the bottom are filled with buffer. The top part contains a series of filters, which match with the wells in the bottom part. One half of the filters on the top part are impregnated with an organic solvent, which supposedly mimics the cell membrane, and the other half are wetted with methanol/buffer. The drug solution is applied to the top filters and the rate of appearance in the bottom wells should reflect the diffusion across the lipid layer. A recent study found a good correlation between flux in the PAMPA system and % absorption in humans for a selected series of compounds 64. PAMPA was portrayed as a high throughput assay because, unlike the IAM, it does not require HPLC analysis. However, it has important disadvantages. For example, it requires UV absorbance, which many compounds do not exhibit. The limited sensitivity of UV detection and the small diffusional surface area may require long experimental time. The incubation time a recent study was 15 hours 64. In a screeningprogram, such a long incubation time may present problems with unstable compounds. Like IAM columns, it ignores the role of enzymes, influx and efflux transporters, and the paracellular pathway in intestinal drug absorption.
IN SILICO METHODS
Assessment of absorption potential using computational methods instead of experimental data has promoted in recent years. The attractiveness of this strategy is undisputable. The suitability of chemical structures could be determined even before the compound is synthesized. This could make it possible to save money, time and effort spent in bad compounds. It also has an ethical component in that it would avoid the need to use either animals or animal tissues to test absorption potential. Within the in silico screening area there have been a number of approaches. For instance, Lipinski’s “rule of five” predicts poor absorption for compounds exhibiting the following characteristics: 1) molecular weight > 500, 2) H-bond donors >5, 3) H-bond acceptors > 5, 4Mlog P> 4.15, and 5) c LogP > 5. This technique appears to have some success for closely related analog series. Other groups have used more traditional quantitative structure-transport relationship (QSTR) models to predict permeability. A recent QSTR study, found a reasonable correlation between observed and predicted Caco-2 permeability for small series of compounds. The fact that different equations had to be used is consistent with physicochemical methods such as, Log P, in that it may be useful only for small series of compounds. QSTR models tend to be deterministic, but usually provide very little understanding of the molecular determinants of permeability. A different approach has consisted in generating numerous molecular descriptors from structural information on the compounds and to assess the importance of these descriptors in predicting permeability. These studies have shown that absorption is influenced by H-bonding capacity and molecular size, but that the dynamic polar surface area (PSAd) was the stronger predictor of absorption. While work in this area continues and several software packages claim to be able to predict absorption 65 and/or Caco- 2 permeability, the common theme voiced by several researchers 65 is the lack of reliable data on which to base the development of the models. Unless the models are derived from diverse and reliable databases, their utility will probable remain limited to compounds closely related to the training sets used to develop the models.
CONCLUSIONS
As the cost and time associated with the discovery and development of new drug products continues to increase, so does the drive to find ways to accelerate this process. Insufficient intestinal absorption represents a formidable obstacle to the successful development of an oral drug product. Thus, knowledge of the absorption potential of new chemical entities while the compounds are still in the discovery phase will increase the probability of success in the development phase. This realization by pharmaceutical scientists will likely continue to push for the development and refinement of intestinal absorption methods. In the near future, it is likely that companies will become more familiar with the cost-effectiveness of killing compounds with low absorption potential as soon as possible. To achieve this goal, the ongoing tendency to shift the use of absorption methods to earlier phases of the drug discovery process, is likely to continue. As a result, decisions regarding the selection of lead compounds will include its absorption potential. Shifting permeability assays upstream in drug discovery may make it possible to screen larger numbers of compounds. This may help medicinal chemist select the most promising chemical leads. This potential advantage explains the widespread interest in so-called high throughput permeability screening. While high speed assays are needed if a real attempt to screen large numbers of compounds is made, it is important to keep in mind that speed should not be pursued at the expense of quality. Failure to recognize this will lead to the generation of data that is too variable to be used in making any important decisions on compound selection. This will ultimately undermine the credibility in and utility of in vitro permeability assays. One characteristic of compounds that greatly influences our ability to test their permeability coefficients is aqueous solubility. Aqueous solubility, like membrane permeability, is a critical determinant of drug absorption. To be absorbed, a compound must have a minimum degree of solubility and membrane permeability. The minimum aqueous solubility depends on factors such as, therapeutic indication, dose, biological half-life, and gastrointestinal site of absorption. Poorly soluble compounds probably affect biological activity tests as much as they affect drug permeability results; however, solubility receives much less attention during biological activity tests than during permeability measurements. The reason for this situation is that new chemical entities are screened first for biological activity, and permeability is conducted later, usually only on compounds found to be biologically active. During activity screening, insoluble compounds are probably considered to be inactive (false negatives). But, once a compound is found to be active, its value increases drastically and any result that could hinder the ability to move it forward is carefully scrutinized. Evaluation of the permeability of poorly soluble compounds is extremely complicated various reasons. Because of the low solubility the dosing concentration must be low. Poorly soluble compounds tend to undergo extensive nonspecific binding (adsorption) to the surfaces of the device(s) used to run the in vitro permeability tests. Due to the small amounts of the compound that undergo transport to the receiver side, the analytical method must be sensitive. Low dosing concentrations, nonspecific binding and cell/tissue uptake result in poor recovery of the compound. To test the permeability of biologically active compounds known to have low aqueous solubility several approaches are taken. Commonly, DMSO or organic solvents are added as co-solvents. Despite the popularity in the use of co-solvents, very little data has been published in which the effect of the co-solvents on the viability and integrity of the biological systems is adequately evaluated. Co-solvents that cause a detrimental effect on the barrier functions (integrity) or the viability of the biological systems could result in artificially high permeability coefficient values. The use of internal or post-experiment controls of system integrity or viability could help reduce this type of false positives. Another approach used to evaluate the permeability of poorly soluble compounds is the use of protein (e.g. bovine serum albumin, BSA)119. While BSA does not have the potentially damaging effect of co-solvents, it increases the difficulty of the analytical procedure used to measure drug concentrations. One approach that is receiving a great deal of attention is in silico permeability screening. Although conceptually attractive, the real utility of in silico methods remains to be proven. Considering the variability in the most commonly used permeability assay (Caco-2 cells), it is very unlikely that truly predictive models can be developed without a concerted effort to generate the appropriate data for this task.
REFERENCES:
1)Brennan, M. B. Drug Discovery. Filtering out failures early in the game. Chem. & Eng. News, June 5, 2000, p. 63.
2)Upham, L. Contract services turbocharge screening. Drug Disc. Today, Jan 1999, 42-44.
3)Lipper, R. A. E Pluribus product. 55-60. Modern Drug Disc., Jan/Feb 1999.
4)Said, H. M.; Redha, R.; Nylander, W. A carrier-mediated Na+gradient-dependent transport for biotin in human intestinal brush-border membrane vesicles. Am. J. Physiol., 1987, 253, G631-G636.
5)Strum, W.B. Characteristics of the transport of pteroylglutamate and amethopterin in rat jejunum. J. Pharmacol. Exp. Ther., 1981, 216, 329-333.
6)Trier, J. S.; Madara, J. L. Functional Morphology of the Mucosa of the Small Intestine. In Physiology of the Gastrointestinal Tract (Johnson, L.R., Ed.) Raven Press, New York, 1981, p. 926.
7)Smith, P.L.; Wall, D. A.; Wilson, G. Drug Carriers for the Oral Administration and Transport of Peptide Drugs Across the Gastrointestinal Epithelium. In Multiparticulate Oral Drug Delivery (Ghebre-Sellassie, I., Ed) Marcel-Dekker, N. Y., 1994, p111.
8) Pappenheimer, J. R.; Reiss, K. Z. Contribution of solvent drag through intercellular junctions to absorption of nutrients by the small intestine of the rat. J. Membr. Biol., 1987, 100, 123-136.
9) Madara, J. L.; Pappenheimer, J. R. Structural basis for physiological regulation of paracellular pathways in intestinal epithelia. J. Membr. Biol., 1987, 100, 149-164.
10)Levitt, D. G.; Hakim, A. A.; Lifson, N. Evaluation of components of transport of sugars by dog jejunum in vivo. Am. J. Physiol., 1969, 217, 777-783.
11) Fix, J A. Strategies for delivery of peptides utilizing absorption-enhancing agents. J. Pharm. Sci., 1996, 85, 1282-1285.
12)Brayden, D. J.; Creed, E.; Meehan, E.; O’Malley, K. E. Passive transepithelial diltiazem absorption across intestinal tissue leading to tight junction openings. J. Control. Rel., 1996, 38, 193-203.
13) McDonnell, W. M.; Scheimani, J. M.; Traber, P. G. Induction o f cytochrome 450IA genes (CYP1A) by omeprazole in the human alimentary tract. Gastroenterology, 1992, 103, 1509-1516.
14) Peters, W. H. M.; Kock, L.; Nangestat, F. M.; Kremers, P. G. Biotransformation enzymes in human intestine: critical low levels in the colon? Gut, 1991, 32, 408-412.
15) Watkins, P. B.; Wrighton, S. A.; Schuetz, E. G.; Molowa, D. T.; Guzelian, P. S. Identification of glucocorticoidinducible cytochromes P-450 in the intestinal mucosa of rats and man. J. Clin. Invest., 1987, 80, 1029-1036.
16) Hebert, M. F.; Roberts, J. P.; Prueksaritanont, T.; Benet, L. Z. Bioavailability of cyclosporine with concomitant rifampin administration is markedly less than predicted by hepatic enzyme induction. Clin. Pharmacol. Ther., 1992, 52, 453-457.
17) Smith, P.L.; Wall, D. A.; Gochoco, C. H.; Wilson, G. (D) Routes of Delivery: Case Studies. (5) Oral absorption of peptides and proteins. Adv. Drug Deliv. Rev., 1992, 8, 253- 290.
18) Pade, V.; Stavchansky, S. Estimation of the relative contribution of the transcellular and paracellular pathway to the transport of passively absorbed drugs in the Caco-2 cell culture model. Pharm. Res., 1997, 14, 1210-1215.
19)Tsuji, A.; Tamai, I. Carrier-mediated intestinal transport of drugs. Pharm. Res., 1996, 13, 963-977.
20) Bai, J. P. F.; Hu, M.; Subramanian, P.; Mosberg, H. I.; Amidon, G. L. Utilization of peptide carrier system to improve intestinal absorption: Targeting prolidase as a prodrug-converting enzyme. J. Pharm. Sci., 1992, 81, 113- 116.
21) Hu, M.; Amidon, G. L. Passive and carrier-mediated intestinal absorption components of captopril. J. Pharm. Sci., 1988, 77, 1007-1011.
22)Kramer, W.; Girbig, F.; Gutjahr, U.; Kleemann, H.-W.; Leipe, I.; Urbach, H.; Wagner, A. Interaction of rennin inhibitors with the intestinal uptake system for oligopeptides and beta-lactam antibiotics. Biochim. Biophys. Acta, 1990, 1027, 25-30.
23)Dyer, J.; Beechey, R. B.; Gorvel, J.-P.; Smith, R. T.; Wooton, R.; Shirazi-Beechey, S. P. Glycyl-L-Proline transport in rabbit enterocytes basolateral-membrane vesicles. Bichem. J., 1990, 269, 565-571.
24)Herrera-Ruiz, Dea,; Wang, Q.; Gudmundsson, O. S.; Cook, T.; Smith, R. L.; Faria, T. N.; Knipp, G. T. Spatial expression patterns of peptide transporters in the human and rat gastrointestinal tracts, Caco-2 in vitro cell culture model, and multiple human tissues. AAPS Pharm. Sci., 2001, 3, (Issue 1, art. 9), 1-10.
25)Dyer, J.; Beechey, R. B.; Gorvel, J.-P.; Smith, R. T.; Wooton, R.; Shirazi-Beechey, S. P. Glycyl-L-Proline transport in rabbit enterocytes basolateral-membrane vesicles. Bichem. J., 1990, 269, 565-571.
26)Lorico, A.; Rappa, G.; Finch, R. A.; Yang, D.; Flavell, R. A.; Sartorelli, A. C. Disruption of the murine MRP (multidrug resistance protein) gene leads to increased sensitivity to etoposide (VP-16) and increased levels of glutathione. Cancer Res., 1997, 57, 5238-5242.
27)Keppler, D.; Leier, I.; Jedlitschky, G. Transport of glutathione conjugates and glucuronides by the multidrug resistance proteins MRP1 and MRP2. Biol. Chem., 1997, 378, 787-791
28)Ling, V. Multidrug resistance: molecular mechanisms and clinical relevance. Cancer Chemother.Pharmacol., 1997, 40, (Suppl.), S3-S8.
29)Patel, N.; Rothenberg, M. L. Multidrug resistance in cancer chemotherapy. Invest. New Drugs, 1994, 12, 1-13.
30)Stephens, R. H.; O’Neill. C.A.; Warhurst, A.; Carlson, G. L.; Rowland, M.; Warhurst, G. Kinetic profiling of p-glycoprotein- mediated drug efflux in rat and human intestinal epithelia. J. Pharmacol. Exp. Ther., 2001, 296, 584-591.
31)Huisman, M. T.; Smit, J. W.; Wiltshire, H. R.; Hoetelmans, M. W.; Beijnen, J. H.; Schinkel, A. H. P-glycoprotein limits oral availability, brain, and fetal penetration of saquinavir even with high doses of ritonavir. J. Pharmacol. Exp. Ther., 2001, 59, 806-813.
32)Hochman, J. H.; Chiba, M.; Yamazaki, M.; Tang, C.; Lin, J.H. P-glycoprotein-mediated efflux of indinavir metabolites in Caco-2 cells expressing cytochrome P450 3A4. J. Pharmacol. Exp. Ther., 2001, 298, 323-330.
33)Dautrey, S.; Felice, K.; Petiet, A.; Lacour, B.; Carbon, C.; Farinotti, R. Active intestinal elimination of ciprofloxacin in rats: modulation by different substrates. Br. J. Pharmacol., 1999, 127, 1728-1734.
34)Kool, M.; de Haas, M.; Scheffer, G. L.; Scheper, R. J.; van Eijk, M. J. T.; Juijn, J. A.; Baas, F.; Borst, P. Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines. Cancer Res.,1997, 57, 3537-3547.
35)Hirohashi, T.; Suzuki, H.; Takikawa, H.; Sugiyama, Y. ATP-dependent transport of bile salts by rat multidrug resistance-associated protein 3 (Mrp3). J. Biol. Chem., 2000, 275, 2905-2910.
36)Keppler, D.; Leier, I.; Jedlitschky, G; Konig, J. ATPdependent transport of glutathione S conjugates by the multidrug resistance protein MRP1 and its apical isoform MRP2. Chem.-Biol. Interact., 1998, 111-112, 153-161.
37)Clark, S. L., Jr. The ingestion of proteins and colloidal materials by columnar absorptive cells of the small intestine in suckling rats and mice. J. Biophys. Biochem. Cytol., 1959, 5, 41-50.
38)Kraehenbuhl, J. P.; Campiche, M. A. Early stages of intestinal absorption of specific antibodies in the newborn. An ultra structural, cytochemical and immunological study in the pig, rat, and rabbit. J. Cell Biol., 1969, 42, 345-365.
39)Rodewald, R. Selective antibody transport in the proximal small intestine of the neonatal rat. J. Cell Biol., 1970, 45, 635-640.
40)Rodewald, R.; Kraehenbuhl, J. P. Receptor-mediated endocytosis of IgG. J. Cell Biol., 1984, 99, 159S-164S.
41)Sugawara, M.; Saitoh, H.; Iseki, K.; Miyazaki, K.; Arita, T. Contribution of passive transport mechanisms to the intestinal absorption of beta-lactam antibiotics. J. Pharm. Pharmacol., 1990, 42, 314-318.
42)Ungell, A.-L.; Nylander, S; Bergstrand, S; Sjoberg, A.; Lennernas, H. Membrane transport of drugs in different regions of the intestinal tract of the rat. J. Pharm. Sci., 1998, 87, 360-366.
43)Lennernas, H.; Nylander, S.; Ungell, A.-L. Jejunal permeability: A comparison between the Ussing chamber technique and the single-pass perfusion in humans. Pharm. Res., 1997, 14, 667-671.
44)Yamashita, S.; Tanaka, Y.; Endoh, Y.; Taki, Y.; Sakane, T.; Nadai, T.; Sezaki, H. Analysis of drug permeation across Caco-2 monolayer: Implication for predicting in vivo drug absorption. Pharm. Res., 1997, 14, 486-491.