Human Intestinal Permeability

Human Intestinal Permeability HANS LENNERNA¨ S* Contribution from the Department of Pharmacy, Group of Biopharmaceutics, Box 580, Uppsala University, S-751 23 Uppsala, Sweden. Received August 21, 1997. Final revised manuscript received November 18, 1997. Accepted for publication January 12, 1998. Abstract 0 This review focuses on permeability measurements in humans, briefly discussing different perfusion techniques, the relevance of human Peff values, and various aspects of in vivo transport mechanisms. In addition, human Peff values are compared with corresponding data from three preclinical transport models. The regional human jejunal perfusion technique has been validated in several important ways. One of the most important findings is that there is a good correlation between the measured human effective permeability values and the extent of absorption of drugs in humans determined by pharmacokinetic studies. Estimations of the absorption half-lives from the measured Peff agree very well with the time to maximal amount of the dose absorbed achieved after an oral dose in humans. We have also shown that it is possible to determine the Peff for carrier-mediated transported compounds and to classify them according to the proposed biopharmaceutical classification system (BCS). Furthermore, human in vivo permeabilities can be predicted using preclincal permeability models, such as in situ perfusion of rat jejunum, the Caco-2 model, and excised intestinal segments in the Ussing chamber. The permeability of passively transported compounds can be predicted with a particularly high degree of accuracy. However, special care must be taken for drugs with a carrier-mediated transport mechanism, and a scaling factor has to be used. Finally, the data obtained in vivo in humans emphasize the need for more clinical studies investigating the effect of physiological in vivo factors and molecular mechanisms influencing the transport of drugs across the intestinal and as well as other membrane barriers. It will also be important to study the effect of antitransport mechanisms (multidrug resistance, MDR), such as efflux by P-glycoprotein(s) and gut wall metabolism, for example CYP 3A4, on bioavailability.

Introduction Various regions of the gastrointestinal tract are normally exposed to a large range of different molecules and microorganisms. Consequently, the intestinesapart from the major role for digestion and uptake of nutrientsshas to protect the organism from systemic exposure by minimizing the absorption of various toxins, antigens, and microorganisms. The oral route is also the most convenient way to administer drug products, with absorption across the intestinal barrier being prerequisite to clinical effect for most drugs. Therefore it has been of great interest to study intestinal permeability in humans even if the possibilities are limited for obvious reasons. Measurement of differential urinary excretion following oral administration of test substances, such as lactulose, poly(ethylene glycol)s, 51Cr-EDTA, provides a specific index of intestinal permeability.1-3 However, such an approach to assessing intestinal permeability is affected by intestinal transit, metabolism (if any), and the renal elimination rate, which * Telephone: + 46 18 471 43 17. Fax: +46 18 471 42 23. E-mail: [email protected].

© 1998, American Chemical Society and American Pharmaceutical Association

means that it does not reflect true intestinal mucosal permeability. A more specific way to study these issues is to perfuse specific regions along the intestinal tract. Different approaches have been developed such as open, semiopen, or closed perfusion of an intestinal segment, perfusion of a tied loop during surgery, colorectal perfusion, and colonic load via endoscope.4-8 In general, it is viewed that intestinal perfusion, based on the difference between solute concentration entering and leaving the region of interest, offers the most direct way to measure the human intestinal permeability. Physiological and biochemical factors controlling transport mechanisms of various molecules across the human intestinal barrier in vivo are still controversial and require further research.9-13 For instance, the validity of PEGs as permeability markers for the paracellular route, and the contribution of the paracellular route to quantitative intestinal absorption of small hydrophilic molecules (e.g. atenolol) have been discussed in the literature.12,13 Another basic issue that remains unresolved is to what extent water is transported by trans- and paracellular routes. It is wellestablished that the small intestine is lacking the aquaphorines that facilitate the water reabsorption in the kidney tubuli.14 An alternative hypothesis regarding water transport has been advanced by Loo et al.15 They calculated that 260 water molecules are absorbed simultaneously with each molecule of D-glucose transported across the intestinal cell membrane via active transcellular transport. It indicates that water movement across the intestinal barrier most likely occurs transcellularly instead of paracellularly when approximately isotonic conditions are present.10,11,15 In oral biopharmaceutics, the impact of metabolism in and multidrug-resistant (MDR)-mediated efflux from the enterocyte of both peptides and nonpeptide drugs needs to be better understood.16-20 There is also a need for an improved mechanistic understanding of the relationship between molecular structure and the in vivo factors determining drug transport (both passive and carrier-mediated) across intestinal epithelial cells in humans.21-24 The overall aim of perfusion studies is to develop a quantitative understanding of the above-mentioned steps involved in the transport of drugs into the systemic circulation. We are currently trying to better understand the interplay between these processes in various preclinical models and then validate and further study them in our in vivo models in humans. This paper will focus on permeability measurements in humans, emphasizing the different in vivo transport mechanisms.

Intestinal Perfusion Techniques Accurate determinations of effective intestinal permeability (Peff) for drugs and nutrients are difficult to study in vivo in humans, but different single-pass perfusion techniques have been developed and used during the last 40 years. The basic principle of perfusion experiments is

S0022-3549(97)00332-8 CCC: $15.00 Published on Web 03/12/1998

Journal of Pharmaceutical Sciences / 403 Vol. 87, No. 4, April 1998

Figure 1sSchematic diagram of three different perfusion methodologies for human use: A, open; B, semiopen; C, double balloon. For the open and semiopen, the hydrodynamics are best described by the parallel tube model (see dotted line for the concentration profile over the intestinal length). The well-stirred model is the best hydrodynamic model for the double balloon perfusion technique.

that the absorption is calculated from the disappearance rate of the drug from the perfused segment. The absorption rate can be calculated in different ways, but we have found that the intestinal Peff provides the best description of the transport process across the intestinal barrier.7-8,11,25-27 Calculation of Peff is dependent on the hydrodynamics within the segment, which in turn is determined by the perfusion technique, perfusion rate, and the degree of intestinal motility. For the open and semiopen perfusion methods, the parallel tube model is applied, as the concentration will decrease in an exponential fashion along the perfused segment (Figure 1).28 This model is a consequence of the fact that the drug solution enters the segment proximally and leaves it distally and that the absorption occurs along the perfused segment. The wellstirred model, however, is the most applicable for regional perfusion techniques using the double-balloon approach.26 This hydrodynamic model predicts that the measured outlet concentration is the best approximation of the concentration throughout the intestinal segment between the two balloons.29 This is in accordance with the experimental setup, in which the perfusion solution enters via a central port and leaves the intestine through two holes at each end of the segment (Figure 1). This means that the perfusion solution goes in two directions, which is similar to movement of the fluid back and forth over a short region during physiological intestinal contractions. The intestinal Peff represents a direct measurement of the local absorption rate in man and reflects the transport velocity across the epithelial barrier, expressed in centimeters per second. The enterocyte is the most common cell type in the epithelial barrier, which also contains a significant number of lymphocytes, mast cells, and macrophages. The Peff for passively transcellularly transported drugs most likely reflects the diffusion across the complex apical membrane into the cytosol lying close to the cytoplasmic leaflet of the apical enterocyte membrane.11,30-33 Therefore, intestinal perfusion models, which measure the disappearance of the drug from the perfusion solution, directly describe uptake into the epithelial cell. The apical membrane is very complex and apparently represents the major resistance barrier under in vivo conditions. Fur404 / Journal of Pharmaceutical Sciences Vol. 87, No. 4, April 1998

thermore, it has been discussed that the exofacial leaflet is responsible for the lower permeability of the apical membrane.30-32 However, as concluded by Lande et al. in 1995, more studies are required in order to determine the role of bilayer asymmetry and membrane proteins in determining the unique permeability properties of the barrier epithelial apical membrane.31,32 Intracellular metabolism in the enterocytesfor instance CYP 3A4 and diand tripeptidasessdoes not occur in the vicinity of the outer leaflet and is therefore considered less likely to influence the disappearance rate (Peff). Intracellular metabolism is, however, a part of the first-pass effect and can therefore be a further limitation to the bioavailability of the drug. In contrast to intracellular metabolism, metabolism in the lumen and/or at the brush-border will interfere directly with the determination of the Peff.34,35 Three main perfusion methods have been employed in the small intestine: (i) a triple lumen tube including a mixing segment, (ii) a multilumen tube with a proximal occluding balloon, (iii) and a multilumen tube with two balloons occluding a 10 cm long intestinal segment (Figure 1).4-8,36-37 In the triple lumen tube method, entering perfusion solution and gastrointestinal fluids are mixed in a “mixing segment”, and at the distal end of the mixing segment a sample is taken which is considered to be the inlet concentration of the test segment. The absorption is then calculated from a second outlet sample taken at the end of the test segment, which usually is 20-30 cm distal to the mixing segment.4,36,37 A major disadvantage of this method is that the composition of the perfusate will change along both mixing and test segments, which makes it difficult to define the absorption conditions and therefore to determine reference drug permeability at well-defined luminal conditions.4,7-8,38-39 Perfusate can flow in either direction and it is difficult to estimate the actual segment length in an open system.4,40,41 In 1965 Ewe and Summerskill introduced a multilumen tube which overcame the problem of proximal contamination by using an occluding balloon proximal to the test segment.42 There is also a separate tube that terminates aboral to the balloon, to have continuous drainage and prevent proximal leakage into the test segment. This method decreases proximal leakage and therefore the luminal composition will be kept at equilibrium and drug permeability can de determined under welldefined conditions. However, both of these methods have the disadvantage that they have a low recovery of the nonabsorbable volume marker, usually PEG 4000, and they also use rather high perfusion flow rates, typically between 5 and 20 mL/min.4 These flow rates are significantly higher than physiological flow rates of about 1-3 mL/min.43 One of the main advantages of these techniques, on the other hand, is the ability to perfuse distal parts of the small intestine, as shown in several open-perfusion studies by Gramatte´ et al.36,37 Recently, new experimental techniques have been introduced for perfusions in both the proximal small intestine (Loc-I-Gut) and colorectal regions (Loc-ICol).5-8 When using Loc-I-Gut, a 10 cm segment is created between two balloons, enabling single-pass perfusion of a well-defined region of jejunum. One of the main advantages with these techniques is that the occlusion of the segment between two intraluminal balloons minimizes contamination with luminal fluids both proximally and distally into the perfused segment. In addition, the leakage out from the segment over the balloons is small, so the recovery of the nonabsorbable marker is almost complete.5-8 These qualities enable control of the absorption conditions in the intestinal segment and thus facilitate the study of mechanisms of transport and metabolism of xenobiotics and nutrients in the human intestine. Another important advantage is the ability to assess the degree of first-pass

effect in the gut and liver by simultaneously determining the extent of absorption and bioavailability.7-8,10,27,46 This cannot be done with the same accuracy using other perfusion techniques, since in those cases the degree of absorption cannot be estimated unless radiolabeled compound is used. One criticism that has often been leveled on perfusion techniques in general is that they may perturb the physiology, especially the motility patterns. In a study reported by Read et al. in 1984, the effect of a tube that reached the terminal ileum on gastrointestinal physiology was investigated by monitoring the passage of a solid meal through the stomach and small intestine with γ-scintigraphy.44 They found that the presence of the tube slowed the gastric emptying half-life (mean ( SD) from 1.2 ( 1.3 to 1.5 ( 0.7 h, and the small intestinal transit time decreased from 6.5 ( 2.0 to 5.3 ( 1.7 h.44 Thus, the effects of the tube are minimal and do not throw into question the pharmaceutical relevance of drug absorption data collected using these perfusion methods.45 The mass-balance issue regarding transport across the intestinal barrier cannot be directly assessed, since it is not possible to measure the concentration of a drug in the cytosol of the enterocyte or in the vena porta in humans. Instead, the peripheral venous blood is usually used as the in vivo reference on the receiver side. For example, we have shown that the peripheral plasma concentration is about 100 times less than the luminal concentration for antipyrine.7 These data suggest that sink conditions apply: antipyrine is a drug with a small volume of distributionsapproximately 40 L in a 70 kg personsand is assumed to be evenly distributed in the body.7,47 In addition, we have reported that the liver extraction of fluvastatinsa CYP 2C9 substrateswas identical following jejunal perfusion and intravenous infusion, which further supports the hypothesis that the mass balance between disappearance and appearance rates of the investigated drug is obtained by Loc-I-Gut.27 We have also shown a good correlation between the measured Peff and extent of absorption for drugs representing a wide range of Peff.26,48-51 Loc-I-Gut and Loc-I-Col are two methods with which one can assess accurate and direct in vivo estimates of the local absorption rate in humans, i.e., Peff (cm/s), across the intestinal barrier.5-8,25-27,49-51 At a perfusion rate of 2.0 or 3.0 mL/min the recovery of PEG 4000 is more than 95%.5-8,10,26-27,46,48 This indicates that the barrier is intact: it is generally viewed that large hydrophilic molecules are good probes for diagnosis of decreased tissue viability and integrity.52 Whether the motility is altered by the presence of one or two balloons and whether the blood supply to the perfused segment is affected have also been discussed.53-58 Previous comparison between the triple lumen technique and the single balloon technique has clearly demonstrated that no difference was found in electrolyte and water transport.41 In addition, studies in which perfusion with a balloon inflated versus deflated are compared tends to show no difference.40,41,53,56,57 Furthermore, distension of the canine intestine at a pressure of 20 mmHg does not reduce the capillary area open for tissue perfusion.58 However, an increase in the intraluminal pressure to 100 mmHg significantly affected capillary blood perfusion in these animals.58 As a comparison, the regional perfusion jejunal segment with two balloons in humanss each with a pressure of about 20 mmHgsshows a very rapid absorption of both passively and actively transported high permeability compounds (absorption half-life of about 4-5 min for D-glucose).7,46 A rough comparison of the absorption rate of antipyrine from perfusion experiments and an oral pharmacokinetic study also indicated that the absorption rate constants were very similar.7 That obser-

Figure 2sThe effective permeability (mean) of five compounds with different physicochemical properties in the human jejunum and ileum. Peff is recalculated using the parallel tube model (see ref 28) and the data is obtained from an open perfusion study in humans (see ref 60). Table 1sAbsorption Half-lives Estimated from Human Jejunal Peff (Loc-I-Gut), and Their Relation to Maximal Dose Absorbed over a Certain Time (for refs see text) Peff compound (10-4, cm/s) D-glucose

atenolol terbutaline antipyrine metoprolol

10 0.15 0.3 5 1.5

ka (min-1)

t1/2 abs fraction abs time to max abs (min) (%) (min)

0.2 4.5 2.4 × 10-3 287 4.7 × 10-3 147 0.08 8.5 0.02 30

100 45 65 100 100

20−30 300 300 40−50 150

vation can be interpreted as demonstrating that no major change in blood flow exists, since it has been reported that the absorption of antipyrine is sensitive to changes in blood flow.59 In addition, maintenance of the functional viability of the mucosa during perfusion was demonstrated by the rapid transmucosal transport of D-glucose and L-leucine from the regional jejunal segment, and complete recovery (>95%) of PEG 4000 (a nonabsorbable volume marker) in the perfusate leaving the jejunal segment.7,10,26,27,46,48 Some human permeability data have been reported from studies in which a triple lumen tube was used to perfuse 80 cm segments in jejunum and ileum at 5 mL/min.60 The approximated permeability data are shown in Figure 2. These data were obtained using an isotonic electrolyte solution with all drugs perfused concurrently.60 From these data it can be speculated that the Peff values of these low permeability compounds are somewhat higher than measured by the regional perfusion method. This apparent discrepancy may be explained by pH differences (our Peff data were usually generated at a specific pH) and/or by the open nature of the triple lumen perfusion technique, which may have allowed absorption from a much longer segment than it actually was designed for (due to uncontrolled flow of perfusate in both directions).4 A direct validation of the in vivo relevance of the measured jejunal Peff (with Loc-I-Gut) is shown in Table 1. The absorption half-lives (t1/2) are calculated from eq 1:

t1/2 )

V ln 2 Peff × 2πRl

(1)

where 2πRl is the cylinder surface area of the jejunal Journal of Pharmaceutical Sciences / 405 Vol. 87, No. 4, April 1998

segment (radius (R) ) 1.75 cm; length of the segment (l) ) 10 cm), and V is the estimated volume of the fluid inside the perfused intestinal segment (V ≈ 40 mL).7,26 All five compounds in Table 1 are metabolically stable both in the lumen and at the brush-border, highly soluble and are not involved in any luminal complex reaction in the intestine. The absorption half-life of D-glucose at a concentration of 5-10 mM is about 4-5 min, which means that the absorption will be complete (>90%) within 20 min (four half-lives).7 This agrees with intubation studies in humans by Borgstro¨m et al., which indicate that absorption of D-glucose should be complete in the mid-jejunum (small intestinal transit time to midjejunum is about 20-30 min).61 The estimated absorption half-lives of antipyrine and metoprolol agrees with the time for complete absorption from the small intestine when these drugs are given in solution or immediate release products.7,47,62-65 The time for complete absorption was estimated to 40-50 min and 2.5 h for antipyrine and metoprolol, respectively, which agrees very well with their absorption half-lives (Table 1)7,47,62-65 For atenolol, the absorption half-life was 4-5 h, which agrees very well with the extent of absorption of 40-50%.66 Atenolol is a hydrophilic drug which has a limited absorption from colon, with most absorption occurring in the small intestine.66,67 In an open perfusion study, it was shown that atenolol was absorbed in human ileum (Figure 2).60 Terbutaline, another hydrophilic compound with limited colonic uptake, has an absorption-half-life of 140 min, which is consistent with a somewhat higher extent of absorption (60%) than atenolol.68 All together, these data in Table 1 support our earlier conclusions that the measured jejunal Peff values with the human perfusion technique are physiologically relevant.7,10,27,46,48 It is obvious that these human intestinal perfusion experiments are useful for generating knowledge about the direct in vivo membrane transport. This jejunal perfusion system (Loc-I-Gut) has also been used extensively to investigate secretion of endogenous substances. In addition, these techniques might be used to study (a) the firstpass effect of drugs in the liver, (b) drug metabolism in intestinal tissue by measuring the metabolite(s) in the outlet perfusate, (c) in vivo dissolution of drugs, (d) local pharmacological studies of drugs, (e) nutrient absorption, (f) biological mechanisms of different gastrointestinal diseases, (g) food-drug interactions, and (h) intestinal secretion of drugs and endogenous compounds.7,10,18,27,46,69-71

Is the Unstirred Water Layer an Important Barrier in Vivo in Humans? The unstirred water layer (UWL) is a more or less stagnant layer of water, mucus, and glycocalyx adjacent to the intestinal wall that is created by incomplete mixing of the luminal contents near the intestinal mucosal surface.72 Whether the UWL has any effect on the absorption of drugs from the lumen is suggested to depend on the intestinal Peff value.72 The rate-limiting step in the transmucosal uptake of a low permeability compound is the transport across the apical membrane, rather than the diffusion through the UWL. Hence, the UWL can be considered as a negligible barrier to the uptake of slowly absorbed drugs.72 For a rapidly permeating solute (effective intestinal permeability value, Peff ) 2 × 10-4 cm s-1),7,26-27,48 the UWL is suggested to contribute to the major resistance to intestinal absorption.72-74 Since absorbed drug is only slowly replaced by new molecules from the bulk solution due to slower diffusion across the UWL, a concentration gradient is created between the exterior side of the UWL and the intestinal wall. The effective thickness of the UWL (∂) can be defined by this concentra406 / Journal of Pharmaceutical Sciences Vol. 87, No. 4, April 1998

tion difference.75 The ∂ is actually not a true value of the layer thickness, rather it is an operational value of the distance to the apical membrane from the point where the linear extrapolation of the concentration gradient reaches the homogeneous concentration in the well-stirred bulk phase. Thus, compounds that have high permeabilities should have the same Peff values if their diffusion coefficients across the UWL are the same. Previously reported values of thickness of UWL were approximately 300-800 µm, based on transport data for highly permeable compounds using various techniques.73-78 However, reanalyzed absorption data in humans as well as in vivo studies in unanesthetized animals resulted in estimations of the UWL thickness of about 30-100 µm. Similar values were found in humans with the regional perfusion technique (Loc-I-Gut). We could not detect any significant changes in the Peff of two high permeability compounds, D-glucose (∼10 × 10-4 cm/s) and antipyrine (∼4 × 10-4 cm/s), or aqueous film thickness over a 4-fold range of flow rates (1.5-6.0 mL min-1).79 This conclusion has also been shown to be valid in cell culture models. Efficient stirring in the Caco-2 model led to a permeability of antipyrine which was similar to human in vivo jejunal Peff.50 The lower UWL value in concious animals and in humans can be attributed to a more effective intestinal motility and stirring in vivo.80-83 In conclusion, the resistance of the UWL to intestinal absorption of highly permeable drugs has previously been overestimated, and instead, the intestinal absorption in humans appears to be membrane-controlled for both low and high permeability compounds, irrespective of the transport mechanism. Furthermore, the high correlation between the individual Peff values of D-glucose (carrier-mediated) and antipyrine (passive) (r2 ) 0.79; p ) 0.0001; n ) 53) suggests that the available surface of the apical membrane of the intestinal mucosa is the main barrier for both actively and passively absorbed solutes.7,79

Paracellular versus Transcellular Quantitative Absorption in Humans in Vivo A hypoosmolar solution at the luminal side of the intestinal epithelium stimulates water absorption which might, in parallel, increase the uptake of compounds that are freely or partly transported with water (solvent drag).12,13,87-91 It has been suggested that the transport route is paracellular for both intestinal water and hydrophilic compounds under conditions of so-called convective water transport, but conclusive evidence is lacking.12,13,91-95 In a number of in vivo studies in humans, we have stimulated water absorption using hypotonic solutions (170-180 mosm/L), but the jejunal Peff was not increased for hydrophilic drugs with molecular weights between 225 and 348 Da (terbutaline, atenolol, and enalaprilat).10,11,48,96,97 These results suggested that quantitative paracellular absorption does not occur for any compound greater than 200 Da in the proximal human small intestine in vivo.10,11,48,96,97 This is consistent with the small area of the paracellular route, which is estimated to be about 0.01% of the total available surface area, and suggests molecular/pore size ratios greater than unity.10,11,48,98,99 However, water transport might affect drug absorption in more distal regions of the small intestine, as suggested for talinolol in an open perfusion study of the human ileum.36 With the regional perfusion model in humans (Loc-I-Gut) we have failed to induce absorption of water and other hydrophilic compounds by adding high concentrations of nutrients, such as D-glucose and L-leucine, at isotonic conditions.10,46,48,96-97 Thus, although this nutrient-induced phenomenom has frequently been shown in vitro, the effect

seems to be negligible in vivo. Further support for passive transcellular transport of small and hydrophilic compounds is the observation that terbutaline (MW 225, log D6.5 ) -1.3) is undergoing extensive first-pass metabolism in the gut.22,121 This sulfate conjugation occurs intracellularly in the enterocyte which requires an entrance across the cell membrane. The main absorption route for water has not yet been clarified, even though several authors claim that at least 50% of the absorption occurs through the paracellular route during convective transport.92,100-102 Across other membranes, for example, in the proximal tubuli and distal collecting duct in the kidney, special proteinssaquaphorinessenable water transport.14 However, there is no evidence that this mechanism exists in the small intestine.14 The lack of such a transport protein in the intestinal membrane is also in accordance with the finding that the intestinal Peff for water is 10 times lower in jejunum than in the kidney.97 Instead, water transport might occur by passive transcellular diffusion as a consequence of its small size.103,104 It has long been known that water movement across the intestine is linked to solute transport, such as salt and sugar. A new hypothesis for glucose and water absorption was presented by Loo et al. in 1996.15 They suggested that water is cotransported with water, sodium, and sugar by sodium/glucose cotransporter (SGLT1) across the brush-border membrane. This transport occurs via conformational changes of the protein and they demonstrated that in Xenopus oocytes 260 water molecules are transported for every sugar molecule.15 However, when we used D2O as a marker for water transport, we found that human jejunal Peff (in vivo) was approximately 4-5 times lower than for D-glucose at 5-10 mM in the lumen.97 This discussion clearly demonstrates that the physiological mechanisms controlling intestinal absorption rate and transport routes of small drugs (<500 Da), endogenous compounds, and water in humans in vivo may vary from those in commonly used absorption models.9-13,91-95,105-107 More studies of these mechanisms in vivo and validation of in situ/in vitro models are therefore necessary.11,105-107 On the basis of previous discussion, we assume that membrane transport is the dominating absorption route for drugs irrespective of their physicochemical properties.10,11,48,96,97 The partitioning-diffusion model which originated from a report by Overton at the end of the previous century is, however, a great simplification of the very complex interactions that occur during transcellular transport of a drug by passive diffusion through a membrane barrier.30,108 This simplification is well-recognized and further theoretical discussion can be found elsewhere.30 Numerous attempts have been made to predict the passive diffusion of drugs across the intestinal mucosasas well other membranessfrom their physicochemical properties. Recently, drug transport across the membranes in the Caco-2 model and the blood-brain barrier have been predicted using conformational analysis followed by an estimation of the polar surface area.109,110 Palm et al. reported that the extent of oral drug absorption in humans for structurally diverse drugs could be well predicted from the polar surface area of the most plausible conformation.21 However, correlation between physicochemical properties (both experimentally and theoretically) and direct in vivo measurements of intestinal membrane transport are rare in the literature. Recently, we reported data where we found that log D (octanol/water; pH 6.5) correlated better to the human jejunal Peff values than molecular size and the number of hydrogen bond donors for four drugs (antipyrine, atenolol, metoprolol, and fluvastatin).27 To improve our understanding of the in vivo performance, we are investigating a larger number of structurally diverse

Figure 3sThe effective permeability (Peff; mean ± SD) of two compounds with different transport mechanisms in the human jejunum and rectum.

compounds (>20). We have found that hydrogen bond number, polar surface area, and log D (octanol/water; pH 6.5) together predicted the human jejunal Peff for passively transported drug with high accuracy.22 These recent reports reflect the ongoing effort to integrate aspects of biopharmaceutics and pharmacokinetics earlier within the discovery and design process for new drugs.11,21-24,27,30,111,112

In Vivo Transport Data of Nutrients and Drugs Regional perfusion experiments in human jejunum and rectum with the double balloon techniques have provided interesting permeability data for compounds transported by various mechanisms. This human perfusion methodology is currently being used to establish a human permeability database, which will be used to further develop the Biopharmaceutics Classification Scheme (BCS).25,27,113,114 The jejunal Peff for L-leucine is high, about 6.2 × 10-4 cm/ s, despite a luminal concentration of the amino acid above the Km value for the amino acid carrier.46 The jejunal Peff for phenylalanine is 7 × 10-4 cm/s at 0.06 mM.115 The transport of D-glucose is also very rapid in the human jejunum (∼10 × 10-4 cm/s) at 10 mM, but no absorption was observed in human rectum despite similar study conditions (Figure 3).7,8 This clearly shows that nutrients are very rapidly transported in the human jejunum and that transport can easily be detected using this single-pass perfusion technique in humans. These transport rates for nutrients represent very efficient absorption with absorption half-lives between 4 and 10 min (Table 1). Interestingly the jejunal Peff for water, during luminal hypotonic condition, was about 2.5 × 10-4 cm/s.97 This suggests that water is more slowly absorbed than nutrients transported by the carriers for hexoses and amino acids. A tight regulation of water transport across the intestinal epithelium would circumvent rapid changes in the fluid volume in the intestinal lumen. Jejunal Peff of drugs such as fluvastatin, antipyrine, metoprolol, (R/S)-verapamil and naproxen are reported to be within approximately 1-10 × 10-4 cm/s.7,18,27 L-Dopa is a drug that is efficiently absorbed from the human jejunum (3.5 × 10-4 cm/s at 2.5 mM) via the carrier for large neutral amino acids.46 All these drugs are considered to be high-permeability drugs according to the BCS, which should be interpreted as meaning that they are rapidly transported across the apical cell membrane of the human enterocyte.25 A plot of jejunal Peff versus extent of intestinal drug absorption (defined as the extent of the given oral dose that is transported into the enterocyte) shows a very steep relationship in the region 0.3-0.8 × 10-4 cm/ s.26,49-51 We have also studied a number of low permeJournal of Pharmaceutical Sciences / 407 Vol. 87, No. 4, April 1998

Table 2sComparison of the Effective Permeabilities (Peff, Papp) Determined in Four Different Absorption Models (data from refs 49−51) Peff compound

Papp (Caco-2) (10-4, cm/s)

rat, Ussing (10-4, cm/s)

rat, perfusion (10-4, cm/s)

D-glucose L-leucine L-dopa

0.25 ± 0.001 0.005 ± 8 × 10-4 0.01 ± 2 × 10-4

0.57 ± 0.21 0.71 ± 0.27 0.36 ± 0.20

1.3 ± 0.4

atenolol terbutaline creatinine enalaprilat inogatran

0.006 ± 5 × 0.004 ± 3 × 10-4 0.008 ± 3 × 10-4

0.06 ± 0.003 0.07 ± 0.08 0.08 ± 0.01 0.06 ± 0.06 0.03 ± 0.01

0.06 ± 0.07 0.05 ± 0.08 0.02 ± 0.05

metoprolol antipyrine naproxen

0.92 ± 0.04 2.7 ± 0.07 2.1 ± 0.07

0.35 ± 0.07 0.40 ± 0.07 0.45 ± 0.45

0.33 ± 0.20 1.6 ± 0.40 2.1 ± 0.41

10-4

ability compounds such as terbutaline, atenolol, enalaprilat, and inogatran which have Peff values ranging from 0.1 to 0.3 × 10-4 cm/s.10,27,48-51 The variability, expressed as coefficient of variation (CV), was about 50-100% for these low permeability compounds (Table 2). These drugs also have a low bioavailibilty, due to their poor Peff, and the CVs of their oral bioavailability estimates (in vivo) are very similar to those for Peff.116

Comparison of Human Jejunal Peff with Preclinical Permeability Models Direct correlations of permeability from three different preclinical models with human Peff data obtained in the regional jejunal perfusion technique are summarized in Table 2.49-51 The comparison between human and Caco-2 data was not influenced by the effect of an unstirred water layer, and therefore the Peff of the rapidly and completely absorbed drugs differed only 2-4 times between the two models.50 This quantitative agreement is in accordance with the hypothesis that such drugs are absorbed at the tip of the villus, due to the fact that there is no absorptive area difference for rapidly and passively transported drugs between the flat monolayer and the folded human jejunum. The permeability values of the low permeability compounds, such as creatinine, atenolol, and terbutaline, were on average 50 times lower in the Caco-2 monolayers than in the human jejunum (Table 2).50,117 The lower mean permeability in the Caco-2 monolayers might be due to a lower paracellular and/or a larger area available in vivo in humans, as it is assumed that the absorption of hydrophilic compounds is so slow that a larger surface area of the intervillous space is exposed.50,95,118 Thus, the permeability values of hydrophilic compounds in the Caco-2 monolayers are closer to those seen in the human colon.50 The permeabilities of various polyethylene glycols were almost 100-fold lower in the cell monolayer.119 The effective permeability values of carrier-mediated transported compounds such as L-dopa, D-glucose, and L-leucine were lower in Caco-2 cells than in human jejunum in vivo at the same concentrations (Table 2). This is consistent with a variable and generally lower expression of carriermediated transport than that seen in vivo.120 Previously, it has been concluded that the passive diffusion of drugs across the human jejunal mucosa in vivo can be predicted and classified in the Caco-2 model (Table 2).50 However, for the BCS, it is necessary to use a set of reference compounds in order to account for the interlaboratory variation as well as the relative differences in permeability between Caco-2 cell monolayers and human permeability data. 408 / Journal of Pharmaceutical Sciences Vol. 87, No. 4, April 1998

2.0 ± 0.6

human perfusion (10-4, cm/s) 10 ± 8.2 6.2 ± 2.9 3.4+1.7 0.12 ± 0.2 0.3 ± 0.3 0.3 ± 0.2 0.2 ± 0.3 0.03 ± 0.03 1.2 ± 0.9 5.6 ± 1.6 10 ± 4.7

extent abs (human) (%) 100 100 100 50 60 <10 >95 100 100

The Peff values for compounds transported by both passive diffusion and carrier-mediated mechanisms across the rat jejunal segment mounted in the Ussing Chamber have been compared with corresponding human data.51 The rank order for Peff values was the same for passively transported compounds in the excised rat jejunal segment (in vitro) and in the human jejunum (in vivo). There was a high correlation between the two models for passively transported compounds, and the human Peff estimates were in general about 4-5 times higher than those in the rat, irrespective of permeability class. The carrier-mediated transport for D-glucose, L-dopa, and L-leucine was approximately 5-15 times higher in the in vivo human model (Table 2).51 The lower in vitro Peff is probably due to the lack of blood flow and/or a less pronounced concentration gradient across the jejunal barrier. It might also be affected by a lower supply of cofactors in vitro, which is crucial for an optimal function of transport protein(s).51 We have determined Peff in a single-pass perfusion model in anesthetized rats in situ (thiobutabarbital Na+) at a perfusion flow rate of 0.2 mL/min. The human Peff estimates for drugs transported by passive diffusion were on average 3.6 times higher in humans in vivo than in the rats in situ, irrespective of the permeability classification of the drug.49 The rank order for passively absorbed drugs was the same in perfused proximal jejunal segments of both humans and rats. Carrier-mediated transport of D-glucose was about 10 times lower in the anesthetized rat, which is in accordance with results of other groups demonstrating that D-glucose absorption is very sensitive to anesthesia (Table 2).49,107 The transport of L-dopa was, however, only 2 times lower. This suggests that each drug where carriermediated transport is involved needs to be carefully investigated in order to elucidate the transport mechanism(s) and to arrive at a suitable scaling factor. However, both human and rat Peff values predict the quantitative amount of drug absorbed in vivo in human very well.49 In an overall comparison of all four models the trend is that they can be used to investigate and classify passive transport with high accuracy, even if there are some quantitative differences between the models (Table 2). The major difference among them is the lower transport rate of carrier-mediated transported compounds in the Caco-2 model. However, successful expression of the carrier of interest for drug transport could lead to improvements in the predictiviness of cell cultures. It is also a trend that the in vitro model, the Ussing chamber, had the lowest permeabilities for carrier-mediated transport among the three remaining models. This is most likely due to the lack of blood supply and its consequences (see the discussion above).

Conclusions The regional human jejunal perfusion technique has been validated in several important ways. One of the most important findings is that there is a good correlation between the measured human effective permeability values and the extent of absorption of drugs in humans determined by pharmacokinetic studies. We have also shown that it is possible to determine the Peff for carrier-mediated transported compounds. Absorption half-lives estimated from the measured human Peff values predict the time to maximal absorption with high accuracy. Human in vivo permeabilities can also be predicted using preclinical permeability models, such as in situ perfusion of rat jejunum, the Caco-2 model, and excised intestinal segments in the Ussing chamber. In particular, passive transport was more precisely estimated than carriermediated transport, which requires introduction of a scaling factor. In the use of preclinical permeability values for predictions of human permeabilities according to BCS, a set a reference compounds is necessary to account for the interlaboratory variation regarding both passively and actively absorbed drugs. We have also obtained in vivo data in humans suggesting that the membrane and the transcellular route is the major transport barrier for drugs with a MW larger than 200 Da, regardless of the physicochemical properties. The data obtained in vivo in humans emphasize the need for more clinical studies investigating the effect of physiological factors and molecular mechanisms influencing the transport of drugs across the intestinal barriers. In particular, the influence of antitransport mechanisms, such as multidrug resistant (P-glycoprotein) and gut wall metabolism (e.g. CYP 3A4), on the bioavailability of drugs warrants further investigation.

References and Notes 1. Tagesson, C.; Sjo¨dahl, R. Scand. J. Gastoenterol. 1984, 19, 315-320. 2. Menzies, I. S. Biochem. Soc. Trans. 1974, 2, 1040-1046. 3. Bjarnason, I.; Peters, T. J.; Levi, A. J. Dig. Dis. Sci. 1986, 4, 83-92. 4. Ewe, K.; Wanitscke, R.; Staritz, M. Csa´ky, T. Z. (Eds.) In Pharmacology of intestinal permeation II; Springer-Verlag: New York, 1984; pp 535-571. 5. Knutson, L.; Odlind, B.; Ha¨llgren, R. Am. J. Gastroenterol. 1989, 84, 1278-1284. 6. Raab, Y.; Ha¨llgren, R.; Knutson, L.; Krog, M.; Gerdin, B. Am. J. Gastroenterol. 1995, 10, 1453-1459. 7. Lennerna¨s, H.; Ahrenstedt, O ¨ .; Ha¨llgren, R.; Knutson, L.; Ryde, M.; Paalzow, L. Pharm. Res. 1992, 9, 1243-1251. 8. Lennerna¨s, H.; Fagerholm, U.; Raab, Y.; Gerdin, B.; Ha¨llgren, R. Pharm. Res. 1995, 12, 426-432. 9. Bjarnason, I.; Macpherson, A.; Hollander, D. Gastroenterology 1995, 108, 1566-1581. 10. Lennerna¨s, H.; Ahrenstedt, O ¨ .; Ungell, A.-L. Br. J. Clin. Pharmacol. 1994, 37, 589-596. 11. Lennerna¨s, H. Pharm. Res. 1995, 12, 1573-1582. 12. Ma, T. Y.; Krugliak, P. Gastroenterology 1996, 110, 967968. 13. Cox, M. A.; Iqbal, T. H.; Lewis, K. O.; Cooper, B. T. Gastroenterology 1997, 112, 669-670. 14. van Os, C. H.; Deen, P. M. T.; Dempster, J. A. Biochim. Biophys. Acta 1994, 1197, 291-309. 15. Loo, D. D. S. F.; Zeuthien, T.; Chandy G.; Wright E. M. Proc. Natl. Acad. Sci. 1996, 93, 13367-13370. 16. Benet, L. Z.; Wu, C.-Y.; Hebert, M. F.; Wacher, V. J. J. Controlled Release 1996, 39, 139-143. 17. Hsing, S.; Gatmaitan, Z.; arias, I. M. Gastroenterology 1992, 102, 879-886. 18. Sandstro¨m, R.; Knutson, L.; Karlsson, A.; Lennerna¨s, H. Pharm. Res. submitted 1997. 19. Burton, P. S.; Conradi, R. A.; Ho, N. F. H.; Hilgers, A. R.; Borchardt, R. T. J. Pharm. Sci. 1996, 85, 1336-1340.

20. Karlsson, J.; Kuo, S.-M.; Ziemniak, J. A.; Artursson, P. Br. J. Pharmacol. 1993, 110, 1009-1016. 21. Palm, K.; Stenberg, P.; Luthman, K.; Artursson, P. Pharm. Res. 1997, 14, 568-571. 22. Winiwarter, S.; Bonham, N.; Hallberg, A.; Lennerna¨s, H.; Karle´n, A. Eur. J. Pharm. Sci. 1997, 5 (suppl. 2) S50. 23. Testa, B.; Carrupt, P.-A.; Gallard, P.; Billois, F.; Weber, P. Pharm. Res. 1996, 13, 335-343. 24. Waterbeemd, H., Camenisch, G. P., Folkers, G.; Raevsky, O. A. Quant. Struct.-Act. Relat. 1996, 15, 480-490. 25. Amidon, G. L. A.; Lennerna¨s, H.; Shah, V.; Crison J. Pharm. Res. 1995, 12, 413-420. 26. Lennerna¨s, H.; Lee, I.-D.; Fagerholm, U.; Amidon, G. L. A. J. Pharm. Pharmacol. 1997, 49, 682-686. 27. Lindahl, A.; Sandstro¨m, R.; Ungell, A.-L.; Abrahmsson, B.; Knutson, L.; Knutson T.; Lennerna¨s H. Clin. Pharm. Ther. 1996, 60, 493-503. 28. Ho, N. F. H.; Park, J. Y.; Ni, P. F.; Higuchi, W. I. In Animal models for oral drug delivery in man; in situ and in vivo approaches; Crouthamel, W., Sarapu, A. C., Eds.; American Pharmaceutical Association: Washington D.C., 1983; pp 27106. 29. Amidon, G. L.; Kou, J.; Elliott, R. L.; Lightfoot, E. N. J. Pharm. Sci. 1980, 69, 1369-1373. 30. Conradi, R. A.; Burton, P. S.; Borchardt, R. T. In Pliska V.; Testa B.; Lipophilicity in Drug Action; Waterbeemd, H., Eds.; VCH: Basel, Switzerland. 31. Lande, M. B.; Priver, N. A.; Zeidel, M. L Am. J. Physiol. 1994, 267, C367-C374. 32. Lande, M. B.; Donovan, J. M.; Zeidel, M. L. J. Gen. Physiol. 1995, 106, 67-84. 33. Lennerna¨s, H.; Crison, J.; Amidon, G. L. J. Pharmacokinet. Biopharm. 1995, 23, 333-337. 34. Langguth, P.; Merkle, H. P.; Amidon, G. L. Pharm. Res. 1994, 11, 528-535. 35. Krondahl, E.; Orzechowski, A.; Ekstro¨m, G.; Lennerna¨s, H. Pharm. Res. 1997, 14, 1780-1785. 36. Gramatte´, T.; Desoky, E. E.; Klotz, U. Eur. J. Pharmacol. 1994, 46, 253-259. 37. Gramatte´, T.; Oertel, R.; Terhaag, B.; Kirch, W. Clin. Pharmacol. Ther. 1996, 59, 541-549. 38. Mekhjian, H.; Phillips, S. F.; Hofmann A. F. J. Clin. Invest. 1971, 50, 1569-1577. 39. Wingate, D. L.; Phillips, S. F.; Hofmann, A. J. Clin. Invest. 1973, 52, 1230-1236. 40. Modigliani, R.; Bernier J. J. Gut 1971, 12, 184-193. 41. Modigliani, R.; Ramboud, J. C.; Bernier, J. J. Dig. Dis. Sci. 1978, 23, 720-722. 42. Ewe, K.; Summerskill, W. H. J. J. Lab. Clin. Med. 1965, 65, 839-847. 43. Kerlin, P.; Zinsmeister; Phillips, S. Gastroenterology 1982, 82, 701-706. 44. Read, N. W.; Janabi, M. L.; Bates, T. E.; Barber, D. C. Gastroeneterology 1983, 84, 1568-1572. 45. Wilding, I. Eur. J. Pharm. Sci. 1997, 5 (suppl. 2) S18-S19. 46. Lennerna¨s, H.; Nilsson, D.; Aquilonius, S.-M.; Ahrenstedt, O ¨ .; Knutson, L.; Paalzow, L. K. Br. J. Clin. Pharmacol. 1993, 35, 243-250. 47. Danhof, M.; Zuilen, A.; Boeijinga, J. K.; Breimer, D. D. Eur. J. Clin. Pharmacol. 1982, 21, 433-441. 48. Fagerholm, U.; Borgstro¨m, L.; Ahrenstedt, O ¨ .; Lennerna¨s, H. J. Drug. Targeting 1995, 3, 191-200. 49. Fagerholm, U.; Johansson, M.; Lennerna¨s, H. Pharm. Res. 1996, 13, 1335-1341. 50. Lennerna¨s, H.; Palm, K., Fagerholm, U.; Artursson, P. Int. J. Pharm. 1996, 127, 103-107. 51. Lennerna¨s, H., Nylander, S.; Ungell, A.-L. Pharm. Res. 1997, 14, 667-671. 52. Lundin, P. D. P.; Westro¨m, B. R.; Pantzar, N.; Karlsson, B. W. Dig. Dis. Sci. 1997, 42, 677-683. 53. Phillips, S. F.; Summerskill, W. H. J. Mayo Clin. Proc. 1966, 41, 224-231. 54. Modigliani, R.; Ramboud, J. C.; Bernier, J. J. Digestion 1973, 9, 176-192. 55. Whalen, G. E.; Harris, J. A.; Geenen, J. E.; Soergel, K. H. Gastroenterology 1966, 51, 975-984. 56. Slades, G. E.; Dawson, A. M. Gut 1970, 11, 947-954. 57. Slades, G. E.; Dawson, A. M. Clin. Sci. 1969, 36, 133-145. 58. O ¨ hman, U. A. P.; Shepherd, Granger, D. N. (Eds.) in Physiology of the intestinal circulation; Raven Press: New York, 1984; pp 321-334. 59. Winne D. Role of blood flow in intestinal permeation. In Pharmacology of intestinal permeation II, Csa´ky, T. Z., Ed.; Springer-Verlag: Berlin, 1984; pp 301-347. 60. Sutcliffe, F. A.; Riley, S. A.; Kaser-Liard, B.; Turnberg, L. A.; Rowland, M. Br. J. Clin. Pharmacol. 1988, 206P-207P. 61. Borgstro¨m, B, Dahlqvist, A.; Lundh, G.; Sjo¨vall, J. J. Clin. Invest. 1957, 36, 1521-1536.

Journal of Pharmaceutical Sciences / 409 Vol. 87, No. 4, April 1998

62. Eichelbaum, M.; Ochs, H. R.; Roberts, G.; Somogyi, A. Arzheim.-Forsch./Drug Res. 1982, 32, 575-578. 63. Oberle, R. L.; Chen, T.-S.; Lloyd, C.; Barnett, J. L.; Owyang, C.; Meyer J.; Amidon G. L. Gastroeneterology 1990, 99, 1275-1282. 64. Sandberg, A.; Abrahamsson, B.; Sjo¨gren, J. Int. J. Pharm. 1991, 68, 167-177. 65. Abrahamsson, B.; Alpsten, M.; Jonsson, U. E.; Lundberg, P. J.; Sandberg, A.; Sundgren, M.; Svenheden, A.; To¨lli, J. Int. J. Pharm. 1996, 140, 229-233. 66. Johnsson, G.; Regårdh, C.-G. Clin. Pharmacokin et. 1976, 1, 233-263. 67. Fagerholm, U.; Lindahl, A.; Lennerna¨s, H. J. Pharm. Pharmacol. 1997, 687-690. 68. Borgstro¨m, L.; Nyberg, L.; Jo¨nsson, S.; Lindberg, C.; Paulson, J. Br. J. Clin. Pharmacol. 1989, 27, 49-56. 69. Knutson, L.; Ahrenstedt, O ¨ .; B. Odlind, B.; Ha¨llgren, R. Gastroenterology 1990, 98, 849-854. 70. Ahrenstedt, O ¨ .; Knutson, L.; Nilsson, B.; Nilsson-Ekdahl, K.; Odlind, B.; Ha¨llgren, R. N. Eng. J. Med. 1990, 322, 13451349. 71. Bo¨nlo¨kke, L.; Christensen, F. N.; Knutson, L.; Kristensen, H. G.; Lennerna¨s, H. Pharm. Res. 1997, 14, 1490-1492. 72. Thomson, A. B. R.; Dietchy, J. M. Csaky, T. Z. Eds. International Pharmacology of Intestinal Permeation II; Springer-Verlag: Berlin, 1984. 73. Levitt, M. D.; Aufderheide, T.; Fetzer, C. A.; Bond, J. H.; Levitt, D. G. J. Clin. Invest. 1984, 74, 2056-64. 74. Winne, D. Naun-Schm. Arch. Pharmacol. 1987, 335, 20415. 75. Winne, D.; Go¨rig, H.; Mu¨ller, U. Naunyn-Schm. Arch. Pharmacol. 1987, 335, 204-15. 76. Read, N. W.; Barber, D. C.; Levin, R. J.; Holdsworth, C. D. Gut 1977, 18, 865-76. 77. Ho¨gerle, M. L.; Wimme, D. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1983, 322, 249-355. 78. Wetsrgard, H.; Holtemu¨ller, K. H.; Dietschy, J. M. Am. J. Physiol. 1986, 250, G727-735. 79. Fagerholm, U.; Lennerna¨s, H. Eur. J. Pharm. Sci. 1995, 3, 247-253. 80. Andersson, B. W.; Levine, A. S.; Levitt, D. G.; Kneip, J. M.; Levitt, M. D. Am. J. Physiol. 1988, 254, G843-848. 81. Levitt, M. D.; Furne, J. K.; Strocchi, A.; Anderson, B. W.; Levitt, D. G. J. Clin. Invest. 1990, 86, 1540-1547. 82. Levitt, M. D.; Strocchi, A.; Levitt, D. G. Am. J. Physiol. 1992, 262, G593-596. 83. Levitt, M. D.; Furne, J. K.; Levitt, D. G. Gastroenterology 1992, 103, 1460-1466. 84. Karasov, W. H.; Diamond, J. M. Am J. Physiol. 1983, 245, G443-462. 85. Diamaond, J. M. News Physiol. 1991, 6, 92-96. 86. Strocchi, A.; Levitt, M. D. Dig. Dis. Sci. 1993, 38, 385-387. 87. Ochsenfahrt, H.; Winne, D. Naunyn-Schm. Arch. Pharmacol. 1974, 281, 175-196. 88. Karino, A.; Hayashi, M.; Horie, T.; Awazu, S.; Minami, H.; Hanano, M. J. Pharm. Dyn, 1982, 5, 410-417. 89. Powell, D. W. Intestinal water and electrolyte transport. In Physiology of the Gastro-Intestinal Tract, 2nd ed.; Johnson, L. R., Ed.; Raven Press: New York, 1987. 90. Hunt, J. B.; Elliot, E. J.; Fairclough, P. D.; Clark, M. L.; Farthing, M. J. G. Gut 1992, 33, 479-83. 91. Krugliak, P.; Hollander, D.; Schlaepfer, C. C.; Nguyen, H.; Ma, T. Y. Dig. Dis. Sci. 1994, 39, 796-801. 92. Pappenheimer, J. R.; Reiss, K. Z. J. Membr. Biol. 1987, 100, 123-136. 93. Fine, K. D.; Santa Ana, C. A.; Porter, J. L.; Fordtran, J. S. Gastroenterology 1993, 105, 1117-1125.

410 / Journal of Pharmaceutical Sciences Vol. 87, No. 4, April 1998

94. Fine, K. D.; Santa Ana, C. A.; Porter, J. L.; Fordtran, J. S. Gastroenterology 1994, 109, 1391-1396. 95. Schwartz, R. M.; Furne, J. K.; Levitt, M. D. Gastroenterology 1995, 109, 1206-1213. 96. Nilsson D.; Fagerholm U.; Lennerna¨s H. Pharm. Res. 1994, 11, 1540-1544. 97. Fagerholm U.; Nilsson D.; Knutson L.; Lennerna¨s H. Acta Physiol. Scand. In press. 98. Madara, J. L.; Pappenheimer, J. R. J. Membr. Biol. 1987, 100, 149-164. 99. Nellans, N. H. Adv. Drug Del. Rev. 1991, 7, 339-364. 100. Armstrong, W. M. Cellular mechanisms of ion transport in the small intestine. In Physiology of the gastrointestinal tract, 2nd ed.; Johnson, L. R., Ed.; Raven, New York, 1987; pp 1251-65. 101. Madara, J. L., Trier, J. S. The functional morphology of the mucosa of the small intestine. In Physiology of the gastrointestinal tract. Johnson, L. R., ed.; 3rd ed.; Raven: New York, 1994; pp 1577-622. 102. Shi, X.; Gisolfi, C. V. Am. J. Med. Sci. 1996, 311, 107-112. 103. Nagle, J. F. J. Bioenerg. Biomembr. 1987, 19, 413-26. 104. Deamer, D. W.; Nichols, J. W. J. Membr. Biol. 1989, 107, 91-103. 105. Diamond, J. M. Nature 1995, 376, 117-118. 106. Turner, J. R.; Madara, J. L. Gastroenterol. 1995, 109, 13911396. 107. Uhing, M. R.; Kimura, R. E. J. Clin. Invest. 1995, 95, 2799805. 108. Overton, E. Vierteljahrsschr. Naturforsch. Ges. Zuerich. 1899, 44, 88-135. 109. Palm, K.; Luthman, K.; Ungell, A.-L.; Strandlund, G.; Artursson, P. J. Pharm. Sci. 1996, 85, 32-39. 110. Waterbeemd, H.; Kansy, M. Chimia 1992, 299-303. 111. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Del. Rev. 1997, 23, 3-25. 112. Smith, D. A.; Jones, B. C.; Walker, D. K. Med. Res. Rew. 1996, 16, 243-266. 113. Lennerna¨s, H.; Knutson, L.; Knutson, T.; Lesko, L.; Salmonson, T.; Amidon, G. L. Pharm. Res. 1995, 12, 396. 114. Lennerna¨s, H.; Knutson, L.; Knutson, T.; Lesko, L., Salmonson, T.; Amidon, G. L. Pharm. Res. 1995, 12, 295. 115. Takamatsu, N.; Welage, L. S.; Idkaidek, N. M.; Liu, D.-Y.; Lee, P.-I.; Hayashi, Y.; Rhie, J. K.; Lennerna¨s, H.; Barnett, J. L.; Shah, V. P.; Lesko, L.; Amidon, G. L. Pharm. Res. 1997, 14, 1127-1132. 116. Hellriegel, E. T.; Bjornsson, T. D.; Hauck, W. W. Clin. Pharm. Ther. 1996, 60, 601-606. 117. Karlsson, J., Ph.D. Thesis, University of Uppsala, Sweden, 1995. 118. Artursson, P.; Palm, K.; Luthman, K. Adv. Drug Del. Rev. 1996, 22, 67-84. 119. Artursson, P.; Ungell, A.-L.; Lo¨frorth, J.-E. Pharm. Res. 1993, 10, 1123-1129. 120. Hu, M.; Borchardt, R. T. Pharm. Res. 1990, 7, 1313-1319. 121. Pacifici, G. M.; Eligi, M.; Giuliani, L. Eur. J. Clin. Pharmacol. 1993, 45, 483-487. 122. Stein, W. D. Transport and diffusion across cell membranes; Academic Press: New York, 1986.

Acknowledgments This work was in part financed by grants the Swedish Medical Research Council K97-14X-11584-02B. The interest of this article from the reviewers is also acknowledge.

JS970332A