ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2018

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 260

Intestinal absorption of drugs

The impact of regional permeability, nanoparticles,and absorption-modifying excipients

CARL ROOS

ISSN 1651-6192ISBN 978-91-513-0484-7urn:nbn:se:uu:diva-363908

Dissertation presented at Uppsala University to be publicly examined in A1:107A, UppsalaBiomedical Centre, Husargatan 3, Uppsala, Friday, 7 December 2018 at 09:15 for thedegree of Doctor of Philosophy (Faculty of Pharmacy). The examination will be conductedin English. Faculty examiner: Professor Martin Brandl (University of Southern Denmark).

AbstractRoos, C. 2018. Intestinal absorption of drugs. The impact of regional permeability,nanoparticles, and absorption-modifying excipients. Digital Comprehensive Summaries ofUppsala Dissertations from the Faculty of Pharmacy 260. 73 pp. Uppsala: Acta UniversitatisUpsaliensis. ISBN 978-91-513-0484-7.

For successful delivery of orally given drug products, the drug compounds must have adequatesolubility and permeability in the human gastrointestinal tract. The permeability of a compoundis determined by its size and lipophilicity, and is usually evaluated in various pre-clinical models,including rat models.

This thesis had three major aims: 1) investigate regional permeability in human andrat intestines and evaluate two different rat models, 2) investigate the mechanisms behindabsorption in nanosuspensions, and 3) investigate the effect of food on the absorption ofdrug molecules in solutions and suspensions, and also food’s effect on absorption modifyingexcipients (AMEs).

Effective human permeability values obtained using regional intra-intestinal dosing anda deconvolution method agreed with values established by perfusion from the jejunum,demonstrating the accuracy and validity of the intra-intestinal bolus-dosing approach. Single-pass intestinal perfusion (SPIP) in rats showed better correlation with human effectivepermeability than the Ussing chamber, and was therefore deemed the better model for predictingdrug permeability in humans.

Absorption of microsuspensions and nanosuspension was investigated using rat SPIP,which showed that microsuspensions are subject to pronounced food effects, probably bypartitioning of drug into the colloidal structures formed by bile acids, lecithin, and fattyacids. Nanosuspensions were less affected by food, which was attributed to fewer availablenanoparticles in the fed state due to partitioning into colloidal structures, and becausenanoparticles are able to cross the aqueous boundary layer on their own, increasing theconcentration of drug adjacent to the epithelial membrane.

AMEs had less effect in the fed state than the fasted state when investigated using SPIP. Thisdifference may be caused by AMEs partitioning into luminal colloidal structures, decreasingthe AMEs’ effects on the intestinal membrane. It thus seems that AMEs as well as drugcompounds are subject to food-drug interactions, which may either increase or decrease theeffect or absorption, something that needs to be considered during development of new drugproducts.

In summary, this thesis has improved the knowledge of pre-clinical absorption models andthe understanding of several biopharmaceutical mechanisms important for drug absorption.

Keywords: Permeability, intestinal absorption, regional permeability, nanoparticles,nanosuspensions, absorption-modifying excipients, AMEs

Carl Roos, Department of Pharmacy, Box 580, Uppsala University, SE-75123 Uppsala,Sweden.

© Carl Roos 2018

ISSN 1651-6192ISBN 978-91-513-0484-7urn:nbn:se:uu:diva-363908 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-363908)

“I have not failed. I've just found10,000 ways that won't work.”

Thomas A. Edison

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Dahlgren, D., Roos, C., Lundqvist, A., Abrahamsson, B., Tan-

nergren, C., Hellström, P. M., Sjögren, E., Lennernäs, H. (2016) Regional intestinal permeability of three model drugs in human. Mol. Pharm., 13(9):3013–3021.

II Roos. C., Dahlgren, D., Sjögren, E., Tannergren, C., Abra-hamsson, B., Lennernäs, H. (2017) Regional intestinal permea-bility in rats: a comparison of methods. Mol. Pharm., 14(12):4252-4261.

III Roos, C., Dahlgren, D., Berg, S., Westergren, J., Abrahamsson, B., Tannergren, C., Sjögren, E., Lennernäs, H. (2017). In vivo mechanisms of intestinal drug absorption from aprepitant nanoformulations. Mol. Pharm., 14(12): 4233-4242.

IV Roos. C., Dahlgren, D., Sjögren, E., Sjöblom, M., Hedeland, M., Lennernäs, H. (2018). Jejunal absorption of aprepitant from nanosuspensions: role of particle size, prandial state and mucus layer. Eur. J. Pharm. Biopharm., 132:222-230.

V Roos. C., Dahlgren, D., Sjögren, E., Sjöblom, M., Hedeland, M., Lennernäs, H. (2018). Effect of three permeation enhancers on the jejunal absorption of four compounds in fasted and fed con-ditions. Eur. J. Pharm. Biopharm – submitted.

Reprints were made with permission from the respective publishers.

Additional papers not included in this thesis

i. Dahlgren, D., Roos, C., Sjögren, E., Lennernäs, H. (2014) Direct In Vivo Human Intestinal Permeability (Peff) Determined with Dif-ferent Clinical Perfusion and Intubation Methods. Journal of Phar-maceutical Sciences, 104(9):2702–2726.

ii. Sjögren, E., Dahlgren, D., Roos, C., Lennernäs, H. (2015) Human in vivo regional intestinal permeability: quantitation using site-spe-cific drug absorption data. Molecular Pharmaceutics, 12(6): 2026-2039.

iii. Dahlgren, D., Roos, C., Johansson, P., Lundqvist, A., Tannergren, C., Abrahamsson, B., Sjögren, E., Lennernäs, H. (2016) Regional intestinal permeability in dogs: biopharmaceutical aspects for de-velopment of oral modified-release dosage forms. Mol. Pharm. 13(9):3022−3033.

iv. Dahlgren, D., Roos, C., Lundqvist, A., Tannergren, C., Langguth, P., Sjöblom, M., Sjögren, E., Lennernäs, H. (2017) Preclinical Ef-fect of Absorption Modifying Excipients on Rat Intestinal Transport of Model Compounds and the Mucosal Barrier Marker 51Cr-EDTA. Mol. Pharm. 14(12):4243–4251.

v. Forner, K., Roos, C., Dahlgren, D., Kesisoglou, F., Konerding, MA., Mazur, J., Lennernäs, H., Langguth, P. (2017) Optimization of the Ussing chamber setup with excised rat intestinal segments for dissolution/permeation experiments of poorly soluble drugs. Drug development and industrial pharmacy, 43(2):338−346.

vi. Dahlgren, D., Roos, C., Johansson, P., Tannergren, C., Langguth, P., Lundqvist, A., Sjöblom, M., Sjögren, E., Lennernäs, H. (2018) The effects of three absorption-modifying critical excipients on the in vivo intestinal absorption of six model compounds in rats and dogs. Int. J. Pharm. 547(1–2): 158−168

vii. Dahlgren, D., Roos, C., Lundqvist, A., Tannergren, C., Sjöblom, M., Sjögren, E., Lennernäs, H. (2018) Effect of absorption-modify-ing excipients, hypotonicity, and enteric neural activity in an in vivo model for small intestinal transport. Int. J. Pharm, 459(1-2):239-248.

viii. Dahlgren, D., Roos, C., Lundqvist, A., Tannergren, C., Sjöblom, M., Sjögren, E., Lennernäs, H. (2018) Time dependent effects of absorption-modifying excipients on small intestinal transport. Eur. J. Pharm. Biopharm, 132:19-28.

ix. Roos, C., Westergren, J., Dahlgren, D., Lennernäs, H., Sjögren, E., (2018). Mechanistic modelling of intestinal drug absorption – the in vivo effects of nanoparticles, hydrodynamics, and colloidal structures. Eur. J. Pharm. Biopharm, 133:70−76.

Contents

Introduction ................................................................................................... 11 Gastrointestinal anatomy and physiology ................................................ 13 

The human gastrointestinal tract .......................................................... 13 Functions of different regions of the gastrointestinal tract .................. 14 Rat gastrointestinal anatomy ................................................................ 16 Predicting human permeability from rat permeability ......................... 17 

The concepts of intestinal drug permeation processes ............................. 17 Transcellular permeation ..................................................................... 18 Paracellular permeation ....................................................................... 19 Intestinal metabolism and efflux transport .......................................... 20 Apparent and effective permeability.................................................... 20 

In vitro and in vivo methods for determining drug permeability .............. 22 Artificial membranes and cell-based methods ..................................... 22 Ussing chamber ................................................................................... 23 Single-pass intestinal perfusion (SPIP) in rat ...................................... 24 Human intestinal perfusion .................................................................. 25 Intra-intestinal bolus dosing ................................................................ 26 

Overcoming biopharmaceutical hurdles in intestinal drug absorption ..... 27 Increasing solubility and/or dissolution rate ........................................ 27 Formulation of nanosuspensions ......................................................... 29 Increasing permeation .......................................................................... 29 

Aims of the thesis.......................................................................................... 31 

Methods ........................................................................................................ 32 Model compounds .................................................................................... 32 Study subjects ........................................................................................... 33 

Healthy volunteers in the clinical study ............................................... 33 Rats used in the pre-clinical studies ..................................................... 33 

Investigation of regional intestinal permeability ...................................... 33 Human intra-intestinal bolus dosing .................................................... 34 Regional intestinal permeability in the Ussing chamber ..................... 35 Regional permeability in single-pass intestinal perfusion ................... 35 Jejunal absorption of nanosuspensions in single-pass intestinal perfusion .............................................................................................. 36 

Jejunal absorption of nano- and microsuspensions in single-pass intestinal perfusion............................................................................... 37 Effect of AMEs in simulated intestinal fluids in SPIP ........................ 38 

Bioanalytical methods .............................................................................. 38 Data analysis ............................................................................................ 39 

Human intra-intestinal bolus dosing .................................................... 39 Regional permeability in rats ............................................................... 39 Absorption of nanosuspensions in single-pass intestinal perfusion ..... 40 Jejunal absorption of nano- and microsuspensions in single-pass intestinal perfusion............................................................................... 40 Effect of absorption-modifying excipients in simulated intestinal fluids .................................................................................................... 41 

Statistical analyses .................................................................................... 41 

Results and Discussion ................................................................................. 42 I. Human regional intestinal permeability ................................................ 42 II. Pre-clinical assessment of regional intestinal permeability ................. 43 

Comparing pre-clinical to clinical permeability .................................. 45 III. In vivo absorption mechanisms of aprepitant nanosuspensions ......... 46 IV. Absorption of nanosuspensions compared to microsuspensions ....... 48 V. Effects of AMEs in simulated intestinal fluids .................................... 51 

Conclusions ................................................................................................... 55 

Populärvetenskaplig sammanfattning ........................................................... 57 

Acknowledgements ....................................................................................... 59 

References ..................................................................................................... 61 

Abbreviations

ABL aqueous boundary layer AME absorption-modifying excipient API active pharmaceutical ingredient AUC area under the plasma concentration-time curve BCS biopharmaceutics classification system C concentration CL clearance CLCr-EDTA blood-to-lumen clearance of 51Cr-EDTA CM carrier-mediated EG gut-wall extraction EH hepatic extraction fabs fraction absorbed GI gastrointestinal HBA/HBD hydrogen bond acceptor/donor i.v. intravenous Japp lumen-to-blood absorptive flux Jdisapp luminal disappearance flux Log P n-octanol−water coefficient Log D7.4/6.5 n-octanol−water partition coefficient at pH 7.4/6.5 MW molecular weight NAC N-acetyl-cysteine Papp apparent permeability Peff effective permeability PK pharmacokinetic(s) pKa dissociation constant PSA polar surface area Q flow rate SDS sodium dodecyl sulfate SPIP single-pass intestinal perfusion

11

Introduction

The use of oral drug products to treat diseases have long been the favored route of administration 1. Benefits for health care systems and patients include convenience and ease of administration. For pharmaceutical companies, these patient-related benefits mean that oral formulations are the most desirable, not only from the compliance and manufacturing perspective, but also from the sales and marketing perspective. Indeed, three of the five top-selling drug products in the US in 2013 were orally administered formulations (IMS Health 2013). The most basic type of oral formulation is the immediate release (IR) tablet. This tablet can be fairly simple, consisting of an active pharmaceutical ingredient (API), excipients to achieve an adequately sized tablet with desired properties, and in some cases a coating to facilitate swallowing or mask un-pleasant tastes. The fate of an IR tablet when it enters the gastrointestinal (GI) tract is as follows (Figure 1): 1, it disintegrates from a solid tablet to smaller fragments; 2, it dissolves into drug monomers (drugs dissolve directly from the large solid tablet also, but at a lower rate because of less total surface area); 3, the drug monomers permeate the enterocytes lining the intestinal wall 2.

Figure 1. An overview of three key processes involved in the gastrointestinal ab-sorption of solid dosage forms. 1, Disintegration; 2, dissolution; 3, permeation.

12

In addition to the processes described in Figure 1, the API can be subject to processes in the intestinal lumen that may decrease the bioavailability (F) or decrease the rate of absorption across the enterocytes. These luminal processes include precipitation, enzymatic degradation, and complexation 3. It is im-portant to consider these processes when formulating a new drug product, as they may affect the rate and extent of absorption. The fraction of the dose given that is not affected by these processes and is absorbed into the entero-cytes is called the fraction absorbed (fabs). The fabs together with the extraction in enterocytes (EG) and the extraction in the liver (EH) constitute the three fac-tors describing F (Equation 1)4:

1 1 Eq. 1

Once the API has crossed the apical membrane of the enterocyte, it enters the cytosol, and the API (or drug compound as it is usually called after entering the cells) may be metabolized by a variety of enzymes, including the cyto-chrome P450-enzymes (abbreviated CYP or CYP450). The drug compound subsequently permeates across the basolateral membrane of the cell, after which it enters the mesenteric blood. Smaller capillary vessels are joined to form the vena porta, which transports the drug to the liver (the final step before entering the systemic circulation). The liver is the body’s main metabolic or-gan, responsible for metabolizing, inactivating, and detoxifying both endoge-nous and exogenous molecules in the blood. Similarly to the metabolism in the gut, there are an abundance of CYP-enzymes present, which are important for metabolizing drug compounds 5, 6. The hepatocytes in the liver express a number of uptake transport proteins, the most important for drug compounds belonging to the solute carrier family (SLC) 7, 8. In addition to metabolism in the liver, the drug compound may also be subject to biliary excretion via efflux proteins like P-glycoprotein (P-gp) and the breast cancer resistance protein (BCRP) 9. A drug compound that escapes the liver reaches the systemic circu-lation and may now exert its desired pharmacological actions.

Drug candidates can be classified based on their biopharmaceutical prop-erties (solubility and permeability) according to the biopharmaceutics classi-fication system (BCS, Figure 2) 4. The ideal drug candidate has high solubility in the gastric and intestinal fluids and high permeability across the enterocytes. Such compounds are classified as BCS class I. Class II compounds have low solubility but high permeability, while class III compounds have a high solu-bility but a low permeability. Finally, class IV compounds have both low sol-ubility and low permeability. Class IV is obviously the least desirable from an oral formulation perspective, as the challenges of getting a sufficient amount of API into the systemic circulation may prove insurmountable. An in-depth description of intestinal permeability and different methods for evaluating per-meability is presented starting on page 17.

13

Figure 2. The biopharmaceutics classification system (BCS).

The biopharmaceutical properties of a drug compound are determined by its physicochemical characteristics. According to the well-known “Lipinski’s rule-of-5”, the ideal oral drug candidate should be less than 500 Da large, have fewer than five hydrogen-bond donors, fewer than 10 hydrogen-bond accep-tors, and have a calculated log P under 5 10. Historically, adhering to this rule has proven a successful strategy in the development of drugs. However, most drugs developed over the last decades do not adhere to this rule, as many are larger and/or more lipophilic, extending beyond the rule-of-5 11, 12. Clearly there should be a balance between hydrophilicity and lipophilicity; a very hy-drophilic API may have high solubility but a limited permeability, with the opposite being true for a very lipophilic API 10, 13. There is also a strong cor-relation between the polar surface area (PSA) of the molecule and the perme-ability or fraction absorbed, where PSA is defined as the contributions of ni-trogen and hydrogen atoms to the total surface area 14, 15. In general, it has been shown that molecules that have a PSA above 140 Å2 do not have an adequate potential for permeation 14, 16. If a drug candidate has sub-optimal biopharma-ceutical properties, there may still be opportunities for achieving sufficient plasma exposure by using different oral formulation strategies. Some of these strategies, which include particle size reduction and the use of absorption modifying excipients (AMEs), will be discussed starting on page 29.

Gastrointestinal anatomy and physiology The human gastrointestinal tract The human gastrointestinal (GI) tract is an open-ended canal lined with epi-thelium, starting at the mouth and ending at the anus 17. The definition usually comprises (at least) the stomach, duodenum, jejunum, ileum, colon, rectum, and anus, however the definition sometimes also includes parts in front of the stomach, namely the buccal cavity, pharynx, and esophagus 17. For the sake of

14

relevance to this thesis, only the stomach, duodenum, jejunum, ileum, and co-lon will be included here when the acronym GI tract is used, as these are the primary sites of interest for conventional oral dosages 17. A summary of ana-tomical and physiological variables of the GI tract are presented in Table 1.

Table 1. A summary of key anatomical and physiological variables for different gastrointestinal regions in human in vivo 17-24.

Region pH fasted pH fed Length Water volume

Stomach 1.0-3.0 3.0-6.0 n/a Fasted: <50 mL, Fed: depending on food intake, up to approx. 1 L

Duodenum 6.0-7.0 5.0-5.5 25 cm Small intestine: Water present in pock-ets, total volume around 50-100 mL

Jejunum 6.0-7.7 5.0-6.5 150 cm Ileum 6.5-8.0 6.5-8.0 150 cm Colon 5.5-8.0 5.5-8.0 150 cm Colon: negligible

The hollow inside of the GI tract is usually called the intestinal lumen. The inner surface of the entire GI tract is lined with a mucous membrane, or mu-cosa, which is composed of a single layer of epithelial cells covering connec-tive tissue containing nerve fibers and small blood vessels (the lamina propria) 25. The epithelial cells contain small membrane protrusions called microvilli, which increase the surface area of these epithelial cells 26. The mucosa extends into the intestinal lumen as finger-like protrusions known as villi. Underlying the mucous membrane, there is a thin muscular layer called the muscularis mucosae 25. Contractions of the muscularis mucosae allow the villi to move back and forth, possibly increasing the flow of molecules across the epithelial cells by reducing the stagnant layer (the aqueous boundary layer, ABL, or the unstirred water layer) adjacent to the membrane 17, 27. Under the muscularis mucosae is the submucosa, which is a second region of connective tissue con-taining larger blood vessels than the lamina propria 25. The submucosa also contains the submucosal plexus, one of two major enteric nervous system net-works 28. The final muscular layer of the GI tract is the muscularis externa, which consists of two layers of muscle fibers, with the inner layer encircling the GI tract and the outer layer oriented longitudinally 25. Together, these mus-cle layers produce propulsive and mixing motions of the luminal contents, the action of which is controlled by the myenteric plexus, the other major enteric nervous system networks 28, 29. Finally, another layer of connective tissue (the serosa) lines the outer wall of the tube of the GI tract 25.

Functions of different regions of the gastrointestinal tract The stomach can be divided into three functional parts 30. The upper part of the stomach is the fundus, which acts as a reservoir for any ingested food. Below the fundus is the corpus or body, which is the main part of the stomach. The third part is the antrum, which sieves and mills particles, and enables

15

emptying through the pyloric sphincter into the duodenum 30. Once ingested, food (or an oral formulation) enters the stomach and is immediately subject to digestion. The pH in the stomach in the fasted state is around 1–3 in healthy humans 19, 31. After ingestion of food, the stomach’s pH in the subsequent fed state rises to around 5, and gradually returns to the fasted state pH 30, 32, 33. Peristaltic motions know as mixing waves combined with the low pH in the stomach begins digesting macronutrients (larger protein, lipids, and carbohy-drates), and the low pH also activates pro-enzymes secreted by cells in the stomach mucosa 34. Digestion is a necessary step for breaking down macronu-trients into their respective smaller constituents. For drug products the peri-staltic motion of the stomach combined with the low pH may may facilitate dissolution, especially for basic compounds as they are more soluble at acidic pH 35, 36. However, for drug molecules sensitive to low pH or to peptidases (which might be found in e.g. oral peptide formulations), the stomach is a hostile environment. These potentially detrimental conditions can be amelio-rated by using gastro-resistant coatings on tablets (entero-coatings) 37.

The stomach also serves another important (and possibly rate-limiting) function, which is the pyloric sphincter’s regulation of the flow of the stomach contents (now called chyme) into the duodenum. The rate of flow is normally limited to 2–4 mL/min, although up to 40 mL/min have been observed after ingestion of particularly large volumes 35, 38, 39. Depending on the composition of the meal, gastric emptying time can vary substantially. For instance, non-caloric liquids are emptied within a few minutes, whereas a solid meal high in fat content may not have fully emptied after as much as 6 hours 40, 41.

After leaving the stomach through the pyloric sphincter, the chyme enters the 25 cm long duodenum, where the low pH is quickly neutralized by bicar-bonate secretions from Brunner’s glands, bile, and pancreatic secretions 42. The chyme is then propelled along the small intestine by peristaltic contrac-tions. Throughout the small intestine, nutrients are continuously absorbed, and the pH gradually increases from around 6.0–6.5 in the duodenum to 7.4–7.8 in the distal ileum 23, 42, 43. The chyme, which has changed in composition and consistency, enters the first part of the colon (the caecum) approximately 4 hours after first entering the duodenum 44-46. In the caecum, the pH is rapidly lowered to around 5.0–6.0 by bacterial fermentation of undigested carbohy-drates 47. Transit time through the colon is highly variable, but is generally assumed to be 6–48 hours 44, 46, 48. In the colon, the majority of the remaining water and nutrients is absorbed, further altering the nature of the chyme (form-ing feces) compared to the contents of the stomach 22. This difference in com-position and texture may have implications for the solubility, dissolution, and diffusion of drug molecules 49.

In addition to the differences in pH and composition of the chyme along the GI tract, several additional factors may influence regional differences in rate and extent of absorption. First, the different regions have quite different

16

epithelial surface areas. In the small intestine, intestinal folds, villi, and mi-crovilli all help to multiply the epithelial surface area by around 300–750 (alt-hough it has recently been suggested that the size enlargement in fact is more modest, based on macroscopic data from non-invasive techniques) 26, 50, 51. The stomach and large intestine lack these permanent folds, although both have folds induced by muscular contractions 26. The large intestine has microvilli, but even so, the result is a much lower surface area enlargement than in the small intestine, reflecting the large intestine’s smaller role in nutrient absorp-tion 26. Second, there is a big difference in transporter protein expression and abundance. The small intestine has a wider and greater expression of trans-porter proteins than the large intestine, allowing for a higher active uptake of different molecules (including drug molecules) 52. Third, the space between the epithelial cells in the colon (the colonocytes) is narrower than the corre-sponding space between the cells in the small intestine (the enterocytes) 53. This means that the potential for paracellular transport is greater in the small intestine, which may be of importance for small and hydrophilic molecules (discussed starting on page 19).

Rat gastrointestinal anatomy The morphology of the rat intestine is similar to that of humans at the epithelial level, and drug absorption in rat has shown many similarities to human 17, 54. There are however important differences that need to be accounted for, if drug absorption is to be predicted in humans based on drug absorption observed in rats. For one thing, the surface area enlargement is less pronounced in the rat due to a lack of intestinal folds and lesser enlargement from villi 55, 56. The length of the human GI tract is only five times longer than the rats, even though the standard human weighs over 200 times more than the standard lab rat 57. However, total available surface area available is around 200 times higher in humans, demonstrating the importance of the size enlargement 17. The radii of the different intestinal regions for humans are 1.75, 1.5, and 2.0 cm, for the jejunum, ileum, and colon, respectively 58. The corresponding radii for the rat are 0.2, 0.2, and 0.35 cm 17, 54. The relative lengths of the GI tract also differ. In rat, the small intestine accounts for 83% of the total length of the GI tract, and within that, 90% is the jejunum 17, 59. In human, the small intestine is 81% of the total GI tract length and the jejunum comprises 38% of the small intestine. Furthermore, the rat has a large caecum comprising 26% of the total length of the colon, compared to 5% in humans 17, 59. Even though there is such a large difference in the dimensions of the intestines, the transit time through the small intestine is similar in both species (3-4 h) 17, 54.

There are also pronounced differences in the nature of the luminal contents throughout the intestinal tract. Chyme in rats has a paste-like consistency when entering the duodenum, while the human chyme is more watery 17, 54. This difference is even more pronounced in the colon, where rats feces is

17

formed as semi-solid pellets. This difference in consistency may have impli-cations for the mucosa, and consequently affect the possibilities for permea-tion or absorption.

Predicting human permeability from rat permeability Using rat as a model species for predicting drug permeability in humans has been proven successful, primarily for passively transported compounds, but also for those transported via carrier proteins 4, 60-66. It has been shown that the rat and human intestinal tract share similar expression profiles in terms of many absorptive transporters, however there are significant differences in the expression of various metabolizing enzymes in the enterocytes. For instance, expression of CYP3A4 (or the rat isoform CYP3A9) and glucuronosyltrans-ferase, which are both involved in the metabolism of many drug molecules, showed 12- to 193-fold differences when comparing rat to human models, while also exhibiting pronounced regional differences within the GI tract 67-69. Despite this, rat models are generally well-suited for investigating drug per-meation, as long as the metabolic differences are understood and accounted for when using rat plasma data for predicting human absorption.

The concepts of intestinal drug permeation processes For a drug molecule to have a systemic effect, it first needs to enter the sys-temic circulation. Only dissolved monomers of a drug can cross the apical and basolateral membranes of enterocytes and reach the circulation. The flux (J) of a drug compound from the intestinal lumen can be described by Equation 2:

Eq. 2

where P is the permeability, A is the surface area of the intestine, and ΔC is the concentration gradient across the membrane 4, 70. There are two different paths by which a drug compound can enter the systemic circulation, namely transcellular and paracellular (Figure 3).

18

Figure 3. Schematic representation of the different processes of the enterocytes (beige boxes) associated with permeation. The intestinal lumen is at the top of the figure, while the circulatory system is at bottom. 1, Paracellular transport through the spaces between cells; 2, active carrier-mediated transport via transport proteins requiring ATP or co-transport of another solute; 3, facilitated diffusion via transport proteins; 4, passive lipoidal diffusion directly through the cell membrane ; 5; intra-cellular metabolism of API; 6, efflux transport of API (or subsequent) metabolite via efflux proteins; 7, basolateral transport of the API or its metabolites to the circula-tion through either passive or carrier-mediated transport.

Transcellular permeation Most drug molecules enter the circulation by crossing the apical and basolat-eral membranes of the epithelial cells 71. This can either be done by passive lipoidal diffusion or by carrier-mediated (CM) transport, which can be further divided into active (primary or secondary) and facilitated CM transport . There is (and has been for a very long time) a consensus that passive lipoidal diffu-sion is the most important mechanism for absorption 72, 73. This consensus has been challenged in the last few years by the suggestion that CM transport is the major mechanism, and that yet-to-be discovered transporter proteins are responsible for the uptake that has previously been accredited to passive dif-fusion 74-76. Although an intriguing thought, there are no data yet to support this claim, meaning that the extensive data supporting passive diffusion as the key route of intestinal absorption is more compelling at this point in time. Lipophilic molecules (logP > 0) are predominantly absorbed through passive lipoidal diffusion, and it has been estimated that over 80% of drug molecules are lipophilic; therefore most drugs are absorbed via this route 71.

The rate-limiting step for passively transported drugs is the crossing of the apical membrane 77, 78. The exact mechanism of lipoidal diffusion has not been shown, but it includes partitioning from the aqueous intestinal lumen to the

19

lipid membrane, diffusion within the membrane, and partitioning from the membrane to the cytosol of the enterocyte 78, 79. Passive diffusion can be de-scribed by Fick’s law of diffusion, which states that the driving force for ab-sorption is a concentration difference, and diffusion increases the total entropy in the system (Eq. 2) 80. Hence, the process does not require any other source of energy, while in contrast additional energy is needed for active CM transport 80. Properties such as low molecular weight, high lipophilicity, and low PSA have all been shown to be associated with a high passive lipoidal diffusion 14, 71, 78, 81.

There are two main forms of CM transport, namely facilitated diffusion and active transport 80. In facilitated diffusion, molecules move via membrane pro-teins down the concentration gradient in a process that does not consume en-ergy. Active CM transport can be either primary or secondary 80. Primary transport means that molecules are moved against their concentration gradi-ents at the cost of hydrolyzing ATP, whereas secondary transport means that the transporters simultaneously transport ions (like sodium ions) down the concentration gradient (which can be in either direction), thus enabling transport of another molecule against its concentration gradient 80.

CM transport may become the main mechanism for absorption if the drug molecule has a high affinity for one or more transport proteins. For nutrients like glucose, vitamins, and amino acids, CM transport is dominant 82. Transport can be categorized as either influx or efflux depending on the direc-tion 80. The efflux transporters which are of most clinical importance are P-gp, the multidrug resistance proteins (MRPs) 1-5, and BCRP 83. Among the potentially important influx transporters that can be used for drug compounds are the oligonucleotide transporter PEPT1 and the organic anion transporters (OATPs) 84, 85.

Paracellular permeation Paracellular transport of molecules occurs in the intercellular spaces between enterocytes. Because there is no membrane barrier, the process is energy-in-dependent, and the driving force for transport is therefore only the concentra-tion gradient 86. The spaces between cells are sealed by negatively charged tight junctions, making the pore size too narrow for molecules larger than around 4-8 Å 53, 61. The primary proteins responsible for sealing the tight junc-tions are the claudins, with important contributions also from the transmem-brane protein occludin 86, 87. For the majority of drug molecules, the contribu-tion of paracellular transport is limited, but for small (<200-300 Da) and hy-drophilic compounds, this pathway may contribute substantially 61, 88, 89.

20

Intestinal metabolism and efflux transport Once drug molecules have entered the cytosol of the enterocytes, they may be subject to metabolism. The most important metabolizing enzymes are the CYP-enzymes 5, 6. CYP3A4 is the most abundant isoform the intestine, ac-counting for approximately 70% of the total CYP content 90. It has been esti-mated that around 50–60% of all orally administered drugs will be subject to oxidative metabolism by CYP3A4, which demonstrates the clinical im-portance of this enzyme 91. Despite the relatively low expression of CYPs in the intestine compared to the liver, metabolism in the intestine may have an impact on the bioavailability of drug compounds. This is thought to be in part attributed to a substrate overlap between CYPs and efflux transporters in gen-eral, and specifically between CYP3A4 and the efflux transporter P-glycopro-tein (P-gp) 92. Such a synergistic interplay would effectively prolong the resi-dence time for the drug compounds in the enterocytes, and thus increase the fraction of the drug compound which may undergo metabolism 92. The mag-nitude of this apical recycling has not been shown to be clinically relevant in vivo, but it remains clear that P-gp and other efflux transporters may have a role in limiting the bioavailability of xenobiotics, which includes drug com-pounds 90, 93. Consequently, inhibiting the activity of either the metabolizing enzymes or the efflux transporters may increase the bioavailability of a drug compound.

Apparent and effective permeability Permeability is defined as the speed at which a molecule is transported across a membrane (cm/s) regardless of which type of mechanism is responsible for the transport 58. Permeability is the most common way of comparing the po-tential of different compounds for transport across the intestinal membrane. The permeability of a drug compound depends both on the membrane perme-ability and the permeability through the ABL 70, 94. Two different variants of permeability are commonly described in literature, namely effective permea-bility (Peff) and apparent permeability (Papp). Peff is determined by measuring the disappearance of an API from the intestinal lumen by perfusion, according to Equation 3:

⁄ Eq. 3

where Qin is the perfusate flow rate, Cout and Cin are the concentrations of API leaving and entering the intestinal segment, and A is the surface area of the perfused intestinal segment, which is assumed to be a smooth cylinder 95. De-pending on whether the tube is open, semi-open, or the closed Loc-I-Gut

21

(which influences the hydrodynamics of the perfused segment), the Peff can be calculated by either Eq. 3 (open and semi-open) or Equation 4 (Loc-I-Gut):

/ Eq. 4

where Qin is the perfusate flow rate, Cout and Cin are the concentrations of drug compound leaving and entering the intestinal segment, and A is the surface area of the intestinal segment, assumed to be a smooth cylinder. Eq. 3 is based on a parallel tube model and Eq. 4 is based on a well-mixed model, which has been shown to best describe the hydrodynamics within the segment during a perfusion using the respective perfusion tubes 70, 95, 96. Because Peff only measures disappearance from the intestinal lumen, this method gives no infor-mation about cytosolic processes within the intestinal cells or basolateral transport. Peff is hence a direct measurement of the permeability of a drug com-pound across the apical membrane of the epithelial cells. Peff is exclusively determined in vivo in humans or rats, which has several advantages, the major one being that permeability is determined in a living organism (and therefore respiration, blood flow, and other physiological characteristics that could af-fect permeability are held intact) 60, 64, 97.

Papp is the second common measure of permeability, differing from Peff in that it measures the amount of drug compound that crosses the membrane. This means that Papp takes into account not only transport across the apical membrane, but also transport across the basolateral membrane, and potential metabolism and/or intracellular binding 98, 99. Papp is determined by measuring the appearance of an drug compound on the basolateral side (which represents the blood) of a membrane or a cell monolayer, and is usually determined using either excised intestinal tissue from rat or human in Ussing chamber, or by using a cell monolayer or an artificial membrane in multi-well systems 100-103. The rate of drug compound appearance in the basolateral chamber gives Papp according to Equation 5:

Eq. 5

where dQ/dt is the appearance over time in the basolateral chamber, A is the area of the membrane, and C0 is the concentration in the apical chamber 102.

22

In vitro and in vivo methods for determining drug permeability Determining solubility and permeability for a drug candidate is important for assessing whether or not that candidate has the appropriate biopharmaceutical properties for successful oral drug delivery. Depending on the stage in the development process, there are a variety of different methods for determining permeability. In general, the more complex and time-consuming the experi-ment is, the more information can be gained. It is important to note that in some stages of development, the need to generate data quickly for many can-didate drugs is more important than generating in-depth mechanistic infor-mation for a single drug candidate.

Artificial membranes and cell-based methods Artificial membrane emulating cell membranes were first used experimentally in 1962 104. By placing phospholipid or lipid solutions on a microfilter, an artificial membrane with a bilayer structure similar to that in intestinal epithe-lia can be formed. After adding the drug candidate in buffer to the apical side of the membrane, its appearance over time is monitored in a buffer on the basolateral side. Papp can then be calculated according to Eq. 5. These experi-ments are referred to as parallel artificial membrane permeability assays, or PAMPA. Because the membrane used in these assays is artificial, there is no CM transport, paracellular transport, or metabolism. The Papp determined is thus only based on passive lipoidal diffusion. In a high-throughput setting, where limited information on many compounds is desired, PAMPA assays can be very useful, because they are cheap, easy to set up in a multi-well plate, and have shown a reasonable correlation to fraction dose absorbed in human 100.

Figure 4. Illustration of a Caco-2 monolayer set-up. An apical (representing the in-testinal lumen) and a basolateral chamber (representic the circulation), each filled with buffer, are separated by a monolayer of Caco-2 cells that have been grown on a permeable polycarbonate membrane (PCM).

23

Once initial permeability screening has been conducted using PAMPA, more in-depth knowledge is needed to assess whether the candidates selected are suitable for continued development. Rather than going directly to animal stud-ies at this stage, it is common to use a cell-based method to investigate not only passive lipoidal diffusion, but also CM transport 105, 106. The most com-mon cell-line used in these studies is the Caco-2 cell line, which is derived from human colon adenocarcinoma 107. Compared to other cells lines, these cells are robust, show decent reproducibility, and most importantly, they have spontaneous differentiation, which allows them to express morphological and biochemical properties similar to in vivo 101. The Caco-2 cells have tight junc-tions and many of the transporters and enzymes found in the human intestine, although in many cases at different levels of expression108-110. Using Caco-2 cell lines have shown a good correlation towards human permeability for com-pounds that are absorbed via passive lipoidal diffusion 111. There are however significant inter-laboratory differences in protein expression, which may re-sult in very different permeability estimates from different laboratories 112. Furthermore, the tight junctions in Caco-2 cells are narrower than those in the human small intestine, meaning that the paracellular transport derived from Caco-2 data may be underestimated 113. Other cell-lines, like the kidney-de-rived MDCK and the colon-derived HT29, may also be used to investigate intestinal absorption 114, 115.

Ussing chamber The Ussing chamber, an in vitro method for examining excised animal tissue, dates back to the 1950s, when Hans Ussing investigated electrical currents in frog skin 116. In an Ussing chamber, small tissue specimens are mounted be-tween two chambers filled with buffer and nutrients, where the temperature is maintained at 37○C by water jackets. The compound(s) under investigation is added to the donor compartment, which can be the apical or the basolateral side, depending on which direction of transport is being investigated. The ap-pearance or accumulation of drug in the receiver chamber is monitored over time, and apparent permeability (Papp) can be calculated according to Eq. 5.

In permeability assessments, the most common type of tissue is excised rat intestine, or preferably human intestine from gastric bypass or colon cancer patients. Human intestinal tissue is expected to generate more predictive data, although permeability values based on rat tissue have been shown to be well correlated to human Peff and fabs 65. The integrity of the tissue is usually as-sessed by monitoring the electrical parameters epithelial potential difference (PD) and short-circuit current (Isc). Using electrical parameters instead of ref-erence marker compounds (such as mannitol) to monitor integrity has the ad-vantage of removing potential effects of the markers on the membrane and/or the investigated compounds, and this method is routinely used, either alone or in combination with marker compound 117, 118.

24

Because the tissue used in the Ussing chamber is excised from living or-ganisms, all parts of the intestinal wall are present, not only the epithelial cells, which may improve predictions based of this model 117, 118. The expression of transporters and metabolizing enzymes is also similar to that of the in vivo situation, especially if human tissue is used 97. The drawback of the method is that since the tissue is removed from the animal, it loses viability over time 117. Certain measures can be taken to improve viability, such as bubbling the tissue with carbogen gas and adding nutrients to the buffer 102. Despite these improvements, the experiments generally cannot last more than 2–3 hours. In addition, using excipients or compounds that affect the membrane may in-crease the tissue’s stress response compared to in vivo 64. The activity of the transport proteins and metabolizing enzymes may also decrease as the viabil-ity decreases, so careful consideration needs to be taken when designing and analyzing experiments.

Single-pass intestinal perfusion (SPIP) in rat Determination of the effective permeability (Peff) can be done with intestine in situ using a variety of methods based on the same general procedure: a solu-tion containing one or several drug compounds are perfused (either in a single pass or recirculated) through an intestinal segment, and this solution is quan-titatively collected after a given distance. The Peff is calculated by relating the concentration in the perfusate entering the intestine to the concentration in the perfusate leaving the intestine, according to Eq. 3. Typically, a non-permeable marker (like PEG-4000) is present to account for potential differences in water content due to absorption or secretion in the segment. The water flux can also be quantified by the gravimetric method, that is by weighing the collected samples, and relating those weights to the flow rate multiplied by the time span during which the sample was collected 119.

The most commonly used method is the single-pass intestinal perfusion (SPIP, Figure 5). The basic method is as described above: a ~10 cm segment of intestine is cannulated using e.g. polypropylene tubing; a solution or sus-pension is perfused through the segment in a single pass; the perfusate is quan-titatively collected into fractions and the drug concentration is determined us-ing a method like LC-MS. After an equilibration period, the disappearance rate reaches a steady state, and Peff can be calculated using Eq. 3. This method has been used to evaluate permeability for several decades and has shown a high correlation to both human permeability and fraction dose absorbed 61, 64,

97. To further improve the method, blood can be simultaneously sampled from either systemic or mesenteric veins or arteries 120. Doing so allows the appear-ance rate of a drug compound in the blood to be calculated, which can give information complementary to the disappearance rate.

25

It is possible to place the cannulated intestinal segment within the abdomen and use sutures to close the abdomen, or to place the cannulated segment out-side the animal and cover the segment and the opening with transparent plastic wrap. Placing the segment within the abdomen makes temperature control eas-ier, and may also reduce stress caused by the environment. It does however increase the risk of uneven flows and removes the possibility of optical mon-itoring.

Figure 5. Graphical illustration of a single-pass intestinal perfusion (SPIP) set-up in rat with the intestinal segment placed on the outside of the abdomen. 1, The perfu-sion solution/suspension on heating plate with constant stirring, perfused using a peristaltic pump; 2, a syringe pump allows co-administration of additions like en-zymes; 3, the perfusate is collected after passing through an intestinal segment; 4, in-travenous infusion of a blood to lumen marker; 5, blood sampling from the femoral artery; 6, body temperature is monitored using a rectal probe, connected to a heating pad; 7, blood pressure and heart rate are monitored in the femoral artery.

Using a living animal has several advantages compared to excised tissue or cell-based assays. The intact blood supply and functioning respiratory system provide continuous oxygenation and combined with normal neural and endo-crine regulations allow for adequate responses to stress or harmful stimuli 97. Monitoring the animal’s body temperature, heart rate, and blood pressure gives detailed information of the status of the animal, which in turn allow for a better understanding of deviating data points. Because the animal is alive throughout the entire experiment, experiment durations can be at least twice as long as Ussing chamber experiments, provided that a suitable anaesthetiz-ing agent is used and that breathing is facilitated through a tracheotomy.

Human intestinal perfusion To intubate and perfuse drug solution in situ in healthy human volunteers has long been considered the gold standard of permeability determinations. This approach has been used for over 50 years, and Peff values determined by this method give the best indication as to whether a drug compound has a sufficient potential for absorption 58, 95, 121-125. To perfuse a drug solution in a human, a

26

radiopaque perfusion tube containing multiple small channels is inserted through the mouth, and positioned into the desired intestinal segment, usually confirmed by fluoroscopy. Typically only the proximal to mid-small intestine is available for such experiments due to the relative bulkiness of the perfusion tube. Once the position of the tube has been verified, a perfusion solution is continuously administered through one of the channels in the tube, and subse-quently collected from other channels in the tube. Like with the rat perfusion experiments, a non-permeable marker is often used to account for water flux.

The obvious key benefit to using human perfusion is that values determined in the human intestine give the most accurate information about the absorption behavior of the candidate drug in vivo. Furthermore, determining permeability in situ in a healthy volunteer, as opposed to excised tissue from gastric bypass patients in Ussing chambers, comes with all the benefits previously mentioned of using living models. One drawback to this technique is that it is difficult to investigate food effects, since undigested food may physically prevent the tube from reaching the desired intestinal site. Obviously, the largest drawback to human perfusions are that they are demanding of both time and resources.

Intra-intestinal bolus dosing A more convenient method of regional administration in humans is intra-in-testinal bolus-dosing, using capsules that can be remotely triggered to release their contents once in the correct position. There are several different versions of this technique, but they can generally be divided into standalone capsules and capsules attached to a thin tube 126-128. In the standalone capsule, the drug compound(s) is contained within the capsule and released by for instance radio frequency triggering, whereas for the capsule attached to the tube, the drug compound in solution is contained in a syringe at the other end of the tube and is injected through the tube out through the capsule. Both devices typically contain a contrast agent that allows for verifying intestinal position before ad-ministration. The primary advantage of using the standalone capsule is the limited discomfort associated with swallowing a capsule (typically 1 x 3 cm). The dimensions of the capsule does however limit the amount of drug solution that can be administered, making it more suitable for use with powder. The capsule attached to tubing allows for much larger doses to be administered, as they are injected using a syringe. This process is however restricted to the administration of solutions or suspensions, and the long length of the small diameter tubing means that highly viscous fluids may not work well. Using a tube gives an additional opportunity for verifying intestinal position by meas-uring the length of tube that has entered through the nose, and calculating the distance to different intestinal segments 127.

In contrast to intestinal perfusion, intra-intestinal bolus dosing does not al-low for sampling of the contents in the intestinal lumen. Thus, in order to de-

27

termine drug permeation, blood sampling is required. However, simply deter-mining the drug concentration in the plasma can not distinguish absorption from distribution and elimination, and in certain cases it is imperative to know the differences in permeation or flux across the enterocytes in order to deter-mine the mechanisms underlying any observed changes in absorption. Quan-tifying flux is typically done using the deconvolution method, which briefly means that an intravenous reference injection of the drug compound is given from which distribution and elimination rates can be calculated. These rates are then subtracted from the plasma concentration-time curves following the intra-intestinal bolus dosing, and when accounting for first-pass metabolism an absorption-time profile is generated. From this profile, Peff or flux can be calculated by relating the rate of appearance of the drug in the plasma to the concentration in the intestinal lumen and the area of the exposed intestinal segment. The Peff determined using this method has shown very good correla-tion with values determined using human intestinal perfusion, making this an excellent alternative for human studies when regional permeability is of inter-est 127, 129.

Overcoming biopharmaceutical hurdles in intestinal drug absorption In recent decades, a large number of new drugs and drug candidates in devlop-ment have less than optimal biopharmaceutical properties; that is, they are compounds in BCS class II, III, or IV 13, 130. Problems with limited solubility and/or permeability may decrease the fraction dose absorbed, and reduce the absorption rate to such an extent that plasma exposure may be subpar. Fur-thermore, drugs with low solubility/permeability may exhibit highly variable plasma exposure in different individuals, and may have pronounced food-drug interactions. To increase the absorption and the bioavailable dose, and to re-duce the inter- and intra-individual variability, more extensive formulation strategies may be required.

Increasing solubility and/or dissolution rate The number of lipophilic drug candidates in early development are increasing as a result of techniques like in silico screening of receptor-ligand interactions 32, 131. While in silico screening proposes candidates with high potentials for interacting with designated target receptors, these candidates are usually quite lipophilic 130. Lipophilic compounds tend to have adequate permeability across enterocytes, but at the same time have limited solubility and dissolution 13. Several strategies attempt for addressing this issue, the most common of which is probably to form the salt of an acidic or basic drug compound with a

28

counter ion such as HCl, which increases the solubility and dissolution rate in the diffusion layer adjacent to the particles 132. Two other commonly used strategies are (i) to alter the solid state properties of the compounds from crys-talline stable structures to metastable polymorphs with lower intra-molecular cohesive forces, or (ii) to reduce the particle size, which increases the surface area of the drug compound and consequently the dissolution rate according to the Noyes-Whitney equation:

Eq. 6

where dC/dt is the dissolution rate, D is the diffusion coefficient, A is the total surface area of all particles of the drug compound, Cs is the concentration of drug compound in the diffusion layer, C is the concentration of drug com-pound in the bulk, and h is the thickness of the diffusion layer adjacent to the particle 132, 133.

Figure 6. Illustration of the factors involved in the dissolution of a particle in an un-restricted volume.

Reducing the size of the particles increases the dissolution rate and conse-quently increases the bioavailability of drug compounds with dissolution-lim-ited absorption. However, as particle sizes decrease to the scale of nanometers, increases are seen in absorption that cannot be explained solely by increased dissolution rate and other mechanisms must be present 134.

29

Formulation of nanosuspensions As a logical consequence of size reduction, and due to improvements in pro-cessing and manufacturing, particle sizes on the order of nanometers are be-coming increasingly popular. Nanosuspensions have the benefit of greatly in-creased dissolution rates compared to suspensions at the higher micrometer scale, and have been shown to increase the bioavailability of several poorly soluble compounds 135-139. Theoretically, size reduction will reach a point at which dissolution is no longer the rate-limiting step in absorption, meaning that further reducing size will not result in increased absorption 32. However, at present, absorption has continued to increase with decreasing particle size, which has raised the possibility that other absorption-promoting processes are occurring simultaneously 2, 94, 134. The most prevalent theory is that the nano-particles may be crossing the lumen’s aqueous boundary layer (ABL) to a greater extent than larger particles like microparticles 140. Commercially avail-able nanoparticles are typically around 100–300 nm, which would potentially allow them to traverse the mucin-based mesh structure in the mucous layer to a larger extent 141. Since the diffusivity for a spherical particle increases with decreasing radius, smaller particles cross the ABL adjacent to the enterocytes more easily than larger particles 142. The primary mechanism of transport through this layer is diffusion, compared to the bulk of the intestinal lumen, where the primary mechanism of transport is convection 27, 143. It seems likely that mucous penetration and increased diffusivity working together are suffi-cient to increase absorption for nanoparticles compared to larger particles.

Increasing permeation There are not as many viable strategies for dealing with poorly permeable drug compounds as there are for poorly soluble compounds, and the strategies that are available often come with potentially big drawbacks or risks. Two ap-proaches for managing poorly permeable compounds are (i) altering the mol-ecule or (ii) manipulating the intestinal membrane. By altering the chemical structure of the molecule, it is possible to mask regions like polar groups that limit passive lipoidal diffusion. This masking can be either permanent or re-versible upon entry into the cells or the systemic circulation (such temporary masking is called making a pro-drug). However, when the chemical structure of a molecule is changed, there is always a risk of changing the pharmacoki-netics and the pharmacological/toxicological potency of the molecule. Thus, an increase in absorption may come at the cost of more undesirable side-ef-fects or reduced clinical effects. By using the pro-drug approach, it is possible to work around these drawbacks, as the original molecules are recreated in vivo by enzymatic cleaving. This approach is not readily available for all mol-ecules, because it requires the molecules to have specific structural elements for metabolizing enzymes to recognize and cleave.

30

The other approach is to manipulate the intestinal barrier, to increase the transport of drug molecule across the enterocytes and into the systemic circu-lation. Typically this manipulation is done by using absorption-modifying ex-cipients (AMEs), which are chemicals or compounds that interact with some component of the membrane and reduce membrane integrity 144, 145. Generally, either the fluidity/integrity of the membrane or the paracellular space between the enterocytes is affected, depending on the mechanism of action of a given AME 146. While this approach may be very useful for increasing the flux of drug compound into the blood stream, it also increases the possibility that other xenobiotics, even bacteria and virus, could enter the systemic circula-tion. It is therefore important that the effect is transient, allowing the drug compound to enter the circulation in sufficient quantity but without producing any lingering or persistent changes to the membrane’s barrier function and integrity.

31

Aims of the thesis

The overall aim of this thesis is to increase the understanding of physiological and biopharmaceutical factors influencing intestinal drug absorption. The in-vestigations focus specifically on increasing the understanding of regional in-testinal drug permeability, absorption processes of nanoparticles, and the ef-fect of absorption-modifying excipients (AMEs). The individual aims of the papers included in the thesis are

Paper I: for the first time, to investigate the regional intestinal permea-bility in humans for three model compounds, to establish reference human values, and to validate the method of determining permeability using de-convolution.

Paper II: to investigate regional intestinal permeability in two different rat models, and to determine which model is the most robust and predic-tive of the human values determined in Paper I, and thus most suitable for use in complex biopharmaceutical mechanistic investigations

Paper III: to investigate the influence and interplay of nanoparticles and colloidal structures and how that interplay affects intestinal absorption of a poorly soluble, highly permeable compound.

Paper IV: to investigate the differences in intestinal absorption between microsuspensions and nanosuspensions of a poorly soluble, highly perme-able model compound, and to investigate different luminal conditions (i.e. different prandial states) and the effect of the mucous layer on intestinal absorption.

Paper V: to investigate the effect of three different AMEs with different mechanisms of action on the intestinal absorption of four drug compounds with different physicochemical properties in different luminal conditions (i.e. different prandial states).

32

Methods

The following sections summarize the methods used in the research papers that form the basis for this thesis. For more detailed descriptions, the reader is referred to the respective papers.

Model compounds Two sets of model compounds were used for the different studies. The key physicochemical and biopharmaceutical properties of these model compounds are summarized in Table 2. In Paper I, atenolol, metoprolol, and ketoprofen were investigated. Papers II and V investigated atenolol, metoprolol, and ke-toprofen as well, with the addition of enalaprilat. In Papers III and IV, the primary compound was aprepitant, but ketoprofen was also investigated as a high-permeability marker compound. The primary mechanism of intestinal absorption for all compounds is passive lipoidal diffusion, but the selected compounds represent different classes of the biopharmaceutics classification system (BCS).

Table 2. A summary of key physicochemical and biopharmaceutical properties of the investigated drug compounds 147.

Atenolol Metoprolol Enalaprilat Ketoprofen Aprepitant

Papers I, II, V I, II, V II, V I, II, III, IV, V III, IV

BCS class III I III II II

MW (Da) 266 267 348 254 535

pKa(s) 9.6b 9.7b 3.2b/7.8a 3.9a 2.4b/9.2a

PSA 88 58 102 54 88

fabs 0.4-0.6 0.9-1 0.2 1 -

Human Peff 0.2 1.3 0.2 8.7 -

Log P 0.18 2.1 -0.13 3.4 4.7

Log D6.5 <-2 -0.5 -1.0 0.8 -

Log D7.4 2.0 0.0 -1.0 0.1 -

Footnotes: aacidic pKa, bbasic pKa. Abbreviations: MW – molecular weight, PSA – polar surface area, fabs – fraction absorbed, Peff – effective permeability, Log P – uncharged oc-tanol-water partitioning coefficient, Log D6.5 – octanol-water partitioning coefficient at pH 6.5, Log D7.4 –octanol-water partitioning coefficient at pH 7.4.

33

Study subjects In Paper I, healthy volunteers were used as study subjects. In the remaining four papers, rats were used. The experiments for Papers II and III were con-ducted at AstraZeneca in Gothenburg, and the experiments for Papers IV and V were conducted at the Biomedical Centre in Uppsala. In both labs, the same strain of rats, weighing approximately the same and obtained from the same breeder, were used to minimize inter-laboratory differences.

Healthy volunteers in the clinical study Paper I included 14 volunteers (11 male and 3 female) between 18 and 55 years of age with body mass indices (BMI) of 19–30 kg/m2. The study was conducted in association with Clinical Trial Consultants AB at the University Hospital in Uppsala. The study was approved by the Swedish Medical Prod-ucts Agency (No. 5.1-2015-49712), the regional ethics committee for human research (No. 2015/243), and the radiation protection committee (No. D15/27). The study was conducted in accordance with the Declaration of Hel-sinki, and all participations signed their informed consent and were subject to a health screening before entering the trial.

Rats used in the pre-clinical studies In Papers II–V, male Wistar Han rats (strain 273) from the same breeder were used. Papers II and III were conducted at animal facility at AstraZeneca in Gothenburg, Sweden, and were approved by the local ethics committee for animal research (No. 48-2013). Papers IV and V were conducted at the De-partment of Physiology, Uppsala University, Sweden, and were approved by the local ethics committee for animal research (No. 64/16). All animals arrived at the respective animal facility least one week prior to the experiments to allow for acclimatization.

Investigation of regional intestinal permeability The regional intestinal permeability of atenolol, metoprolol, and ketoprofen were evaluated in three models: a clinical human intra-intestinal bolus dosing in Paper I and excised rat intestinal tissue in Ussing chamber and single-pass intestinal perfusion in rats in Paper II. In the two rat models, the BCS class III compound enalaprilat was also included.

34

Human intra-intestinal bolus dosing To establish reference human Peff values from all parts of the intestine, a clin-ical study was conducted in Paper I. A total of 14 healthy volunteers were randomly assigned to one of three study groups, each of which had a different order of the intestinal regions dosed (see Table 3). All participants received an intravenous (i.v.) reference dose of the study compounds at least one week prior to the first intra-intestinal dose to acquire individual disposition values, which were then to be used in the deconvolution of the plasma concentration-time curves following the intra-intestinal dosing. Between each administra-tion, there was at least one week of washout-period. The study participants were fasted for at least 8 hours prior to the start of the i.v. and the intra-intes-tinal dosing, to reduce food-effects and to ensure unrestricted capsule transit. Approximately 4 hours after the intravenous dose and 1 hour after intra-intes-tinal administration, the participants were given a standardized meal.

Table 3. Order of visits to the clinic for three study groups.

Visit number

1 2 3 4

Study group 1 IV Jejunum Ileum Colon Study group 2 IV Colon Jejunum Ileum Study group 3 IV Ileum Colon Jejunum

Preparation of study solutions The study solutions were all manufactured from commercially available GMP products for injection. The ketoprofen solution for intravenous dosing was made by diluting the commercial product with saline to a final concentration 5 mg/mL. The concentration of the the commercially available atenolol and metoprolol tartrate solutions were 0.5, and 0.78 mg/mL, respectively.

Administration of study solutions On the first study occasion, the intravenous reference dose was given as serial injections in a peripheral vein in the forearm. The doses were 1 mg atenolol (2 mL volume), 0.78 mg metoprolol tartrate (1 mL), and 5 mg ketoprofen (1 mL). After the final injection, the catheter was rinsed with 3 mL saline solu-tion to ensure complete administration of the drug compounds. Venous blood samples were collected immediately prior to administration, and after 5, 10, 20, 30, 40, 50, 60, 120, 240, 360 min, and after 8 and 24 h.

On the second, third, and fourth study occasions, intra-intestinal admin-istration in different regions were conducted. 10 mL of solution containing 10 mg atenolol, 25 mg metoprolol, and 5 mg ketoprofen were administered using the Bioperm capsule at each of the occasions (jejunum, ileum, colon). The Bioperm capsule is a five meter long tube (outer and inner diameter of 0.6 and 0.3 mm, respectively) attached to a radiopaque capsule serving as a position

35

verifier (by fluoroscopy) and to allow the tube to descend through the GI tract. After the capsule reached its intended position, the solution was injected through the tube and rinsed with saline to ensure a complete administration. Venous blood samples were collected immediately prior to administration, and after 5, 10, 20, 30, 40, 50, 60, 120, 240, 360 min.

Regional intestinal permeability in the Ussing chamber Preparation of study buffers Two phosphate buffers were prepared, one at pH 6.5 and one at pH 7.4. Amino acids and glucose were added to both buffers to increase the viability of the intestinal tissue. Atenolol, metoprolol, ketoprofen, and enalaprilat were added to the respective buffers from DMSO stocks. The final concentration of each of the drug compounds were 25 µM.

Experimental procedures Both regional differences as well as pH effects on permeation were investi-gated in the Ussing chamber in Paper II. Tissue from rat jejunum, ileum, or colon was excised from anesthetized male Wistar Han rats (strain 273). Im-mediately after excision, the intestinal tissue was put in ice-cold Krebs-Ringer buffer (KRB) bubbled by carbogen gas (95% O2, 5% CO2). A tissue specimen was mounted in the Ussing chamber, and ice-cold KRB was added to both the serosal and mucosal side. The tissue was allowed a 30 min equilibration pe-riod, over which the temperature was increased to 37○C by water jackets. After 30 minutes, the KRB was replaced by KRB containing drug compounds and with a pH of 6.5 or 7.4 in the mucosal chamber, and KRB without drug com-pounds and a pH of 7.4 in the serosal chamber. Samples were taken from the mucosal chamber at times 0 and 150 min, and from the serosal chamber every 30 minutes for 150 minutes total. Equivalent volumes of KRB were added to the chambers after sampling. The samples were frozen immediately and kept at –20○C pending analysis. For a tissue segment to be deemed viable, cut-off values for the potential difference (PD) and the electrical resistance (R) over the tissue segment were applied. For the jejunum and the ileum, these values were –4 mV (PD) and 30 Ohm • cm2 (R), and for the colon they were –6 mV (PD) and 70 Ohm • cm2 (R). Tissue segments with values below these were replaced with a new tissue segment.

Regional permeability in single-pass intestinal perfusion The regional differences and the pH effects on permeation were also investi-gated using single-pass intestinal perfusion (SPIP) in Paper II. In total, three different buffers containing atenolol, metoprolol, ketoprofen, and enalaprilat were investigated in situ in jejunum, ileum, and colon.

36

Preparation of the study buffers Three phosphate buffers were prepared: 1, a pH 6.5 buffer with low buffer strength (6.7 mM, hereafter called LBS); 2, a pH 6.5 buffer with high buffer strength (67 mM); 3, a pH 7.4 buffer with high buffer strength (100 mM). Atenolol, metoprolol, ketoprofen, and enalaprilat were all added from DMSO stock, yielding a final concentration of 100 µM for each of the drug com-pounds.

Surgical and experimental procedure Male Wistar Han rats weighing 270–370 g were anesthetized with Isoflurane and placed on a heating table (37○C). A midline incision in the abdomen was made, and an intestinal segment of 6–12 cm was cannulated using polypro-pylene tubing. After rinsing the segment with saline solution, the segment was placed inside the abdomen, and the abdomen was sutured closed to prevent heat and moisture loss. The respective intestinal segment was single-pass per-fused at 0.2 mL/min with the respective buffers containing 100 µM atenolol, metoprolol, ketoprofen, and enalaprilat. Outlet samples were collected at 45, 60, 75, 90, and 105 min and immediately stored at –20 ○C for later analysis.

Jejunal absorption of nanosuspensions in single-pass intestinal perfusion The intestinal absorption of aprepitant from nanosuspensions in buffer and in fasted state simulated intestinal fluid (FaSSIF) was investigated using rat SPIP in Paper III. Two different concentrations of aprepitant (20 and 200 µM) were investigated in both phosphate buffer and in FaSSIF. Nanoparticles were present in all compositions except for 20 µM in FaSSIF, as the apparent solu-bility of aprepitant in FaSSIF is 39 µM, meaning all aprepitant was either dis-solved or partitioned into colloidal structures (such as micelles or vesicles), consisting of bile acids and lecithin present in FaSSIF.

Preparation of study formulations A phosphate buffer of pH 6.5 was prepared. From this buffer, FaSSIF was prepared by addition of lecithin and sodium taurocholate. The pH of FaSSIF was also 6.5. To produce the aprepitant nanosuspensions, a stock suspension was made each day by suspending a commercially available aprepitant cap-sule (Emend®) in water, sonicating the mixture to disrupt potential aggre-gates, and aspirating the resulting suspension. Depending on the concentration of the final nanosuspension, different volumes of the stock were added to pro-duce 20 or 200 µM nanosuspensions in buffer, a 20 µM solution in FaSSIF, or a 200 µM nanosuspension in FaSSIF. Ketoprofen and phenol red were added from DMSO stocks to give a final concentration of 139 and 25 µM.

37

Surgical and experimental procedures The surgical and experimental procedures followed that described above for Paper II, with the addition of a catheter being surgically inserted in the jugular vein of the anesthetized rat to allow for blood sampling.

Jejunal absorption of nano- and microsuspensions in single-pass intestinal perfusion The jejunal absorption of nano- and microsuspensions in buffer, FaSSIF, and fed state simulated intestinal fluid (FeSSIF) was investigated in Paper IV. The single-pass intestinal perfusion used in Paper IV was modified from the method used in Papers II and III.

Preparation of the study formulations A phosphate buffer was prepared using the same procedure as for Paper III. To produce FaSSIF and FeSSIF, freeze-dried instant FaSSIF or FeSSIF pow-der was added to the phosphate buffer, and the mixture was allowed to equil-ibrate for 1 hour before start of the experiment.

The nanosuspensions were produced using a commercial aprepitant capsule as described for Paper III. The microsuspensions were produced by suspend-ing microsized aprepitant in buffer, FaSSIF, or FeSSIF. The final concentra-tion of apreptiant was 200 µM in all the suspensions. Ketoprofen was added from a stock solution (in phosphate buffer) to give a final concentration of 100 µM dissolved ketoprofen in both the aprepitant nano- and microsuspensions.

Surgical and experimental procedure The surgical procedure for Paper IV was modified from that used in Paper II and III. Male Wistar Han rats weighing 250-350 g were anesthetized by an intraperitoneal injection of pentobarbital. A tracheotomy was performed to ensure unobstructed breathing. The body temperature was kept at 37.5○C and important physiological functions were monitored by a catheter in the right femoral artery, which was also used for blood sampling. The abdomen was opened with a 3–5 cm incision and an approximately 10 cm segment of the jejunum was cannulated using polypropylene tubing. The common bile duct was cannulated adjacent to the intestine to prevent build up of secretions in front of the segment. An injection of the blood-to-lumen permeability marker 51Cr-EDTA was given through the right femoral vein, followed by a constant infusion over the course of the experiment.

After an equilibration period of 30 min, a single-pass perfusion of one of the study formulations was commenced. After the first 75 min (control pe-riod), the mucolytic agent N-acetyl-cysteine (NAC) was added to the formu-lation and perfused for an additional 75 min (study period). Blood was sam-

38

pled at time 0 and every 15 minutes for 150 minutes. The blood was immedi-ately centrifuged and the plasma stored at –80○C to await analysis. Equivalent volumes of 7% saline bovine serum albumin solution were given to mitigate any blood loss effects.

Effect of AMEs in simulated intestinal fluids in SPIP Preparation of study formulations Two maleic acid buffers were prepared, one for FaSSIF with pH 6.5, and one for FeSSIF with pH 5.8. On the day of each experiment, freeze-dried instant FaSSIF or FeSSIF powder was added to the respective buffer, and atenolol, metoprolol, ketoprofen, and enalaprilat were added from a stock solution (in maleic acid buffer). The final concentration of all drug compounds were 200 µM.

Surgical and experimental procedures The surgical procedure for Paper V followed that described for Paper IV. The experimental procedure was similar with a 75 minute control period of perfusing only the study drugs in FaSSIF or FeSSIF; however, in addition to NAC, the effects of sodium dodecyl sulphate (SDS) and sodium caprate were also investigated. All excipients were investigated at two different concentra-tions, 0.1 and 0.5%, and in both FaSSIF and FeSSIF. As 0.5% NAC changed the pH substantially, an additional group of 0.5% NAC where the pH was adjusted to 5.8 or 6.5 was included, labelled 0.5% NAC pH adj.

Bioanalytical methods The plasma samples from Papers I, III, IV, and V were analyzed using liquid chromatography and tandem mass spectrometry (LC-MS/MS). In addition, the perfusate samples in Paper III were analyzed using by dissolving all par-ticles present in the perfusate and then quantifiying the samples using LC-UV. In Paper II, the concentrations of the samples from the Ussing chamber and SPIP were analyzed using LC-MS (single quadropole). In all studies, quanti-fications were performed in a single run, and only the parent compounds were quantified (no metabolites were investigated). The specific details for each analytical method can be found in the research papers this thesis is based on, but on a general level all methods adhered to the European Medicines Agen-cys’s guidelines for method validation, except for the perfusate analysis in Paper III, which used an in-house designed and validated method at Astra-Zeneca, Gothenburg.

39

Data analysis Human intra-intestinal bolus dosing In Paper I, a deconvoluted effective permeability (Peff) was calculated. An input rate of the different drug compounds was acquired by deconvolution of the plasma concentration-time profiles following intra-intestinal bolus dosing, using the disposition parameters established by individual intravenous admin-istration. The input rate was compensated for first pass metabolism which was based on known PK values of blood flow, fraction metabolized, and assuming a blood-to-plasma ratio of 1. A deconvoluted Peff was calculated from the com-pensated input rate by relating it to the radius of the intestinal segment and the amount of drug compound in the lumen according to Equation 6:

Eq. 6

The mean Peff during the first 30 minutes for each compound was used in fur-ther calculations and comparison in order to reduce the effect of dispersion in the intestine, thereby ensuring that the absorption occurred in the pre-deter-mined intestinal segment.

Regional permeability in rats In Paper II, the apparent permeability (Papp) in the Ussing chamber was cal-culated according to Eq. 5 during the time when the increase in the donor chamber was linear, typically from 30–150 min. The exposed tissue surface area was 1.14 cm2.

The effective permeability (Peff) in the SPIP was calculated according to Equation 3 using a flow rate of 0.2 mL/min and radius of 0.2, 0.2, and 0.35 cm for jejunum, ileum, and colon, respectively. The concentration of the per-fusate leaving the segment was corrected for water flux according the change in concentration of the absorption marker phenol red:

1.15 ,

, Eq. 7

where Ccorr is the corrected concentration, Csample is the concentration deter-mined using LC-MS, 1.15 is an additional correction factor to account for ab-sorption of phenol red, and Cin,PhRed and Cout,PhRed are the concentrations of phe-nol red entering and leaving the segment 119.

40

Absorption of nanosuspensions in single-pass intestinal perfusion

In Paper III, the Peff for both ketoprofen and aprepitant was calculated as de-scribed previously (Eq. 3), including the corrections for water flux (Eq. 7). For most preparations of aprepitant, the total concentration of drug compound was above the solubility (except for 20 µM in FaSSIF), meaning the total concen-tration of drug compound was used (dissolved and particles).

In addition to standard Peff, deconvoluted absorption flux (Japp) and disap-pearance flux (Jdisapp) were also calculated. An intravenous injection of aprep-itant allowed for acquiring disposition PK parameters, in analogy with that described for Paper I. The Jdisapp was calculated according to Equation 8 with the concentration corrected for water flux as previously described:

Eq. 8

In analogy, Japp was calculated based on the deconvolution approach described for Paper I (Eq. 9), but without correcting for the luminal amount:

Eq. 9

Jejunal absorption of nano- and microsuspensions in single-pass intestinal perfusion In Paper IV, Japp was calculated according to the method described for Paper III using the disposition parameters acquired in Paper III as well.

A blood-to-lumen clearance of 51Cr-EDTA (CLCr-EDTA) (infused intrave-nously during the entire experiment) was also calculated based on the 51Cr-EDTA activity in all luminal samples, and relating them to the activity in the blood. The CLCr-EDTA, expressed in mL/min/100g wet tissue, was calculated using Equation 10:

100 Eq. 10

where Cperfusate is the radioactivity in the perfusate sample (cpm), Cplasma is the radioactivity at the corresponding time in the plasma, Qin is the flow rate en-tering the intestinal segment (0.2 ml/min), and the tissue weight is the ex vivo weight of the perfused intestinal segment (g). Mean CLCr-EDTA from 15–75 min was used to represent the control period and from 90–150 min for the study period.

41

Effect of absorption-modifying excipients in simulated intestinal fluids In Paper V, Japp for atenolol, metoprolol, ketoprofen, and enalaprilat, and the CLCr-EDTA were calculated according to the method described previously for Paper IV. The disposition PK parameters used were based on previous studies from the same laboratory using the same drug compounds and the same type of rats from the same breeder 148.

Statistical analyses Values (Peff, Papp, Japp, Jdisapp, CLCr-EDTA) from all papers are expressed as mean ± standard deviation (SD) unless otherwise specifically stated. The number of experiments conducted were based on power calculations and assumptions based on previous experiences from the respective laboratories. Statistical dif-ferences were evaluated using one-way ANOVA, two-way ANOVA, or mul-tiple t-tests, with post hoc corrections as appropriate. Log transformations of values were performed when the data were not normally distributed. A p-value less than 0.05 was considered statistically significant for all comparisons.

42

Results and Discussion

I. Human regional intestinal permeability The traditional way of determining the human intestinal permeability in vivo has been by using different perfusion techniques. Because these methods have limitations in reaching the distal parts of the intestine, other methods are com-monly used, such as regional bolus dosing with different capsule techniques. The goal of the study in Paper I was to use one such technique, the Bioperm capsule, to establish reference permeability values from all three parts of the intestine using a deconvolution technique, and to compare this approach to previously published human jejunal Peff values. Even though the small intes-tine is the primary site for absorption of nutrients and drug molecules, the co-lon may play an important role in the delivery of modified release formula-tions or when the drug molecule is not fully absorbed in the small intestinal. The regional intestinal permeabilities determined in Paper I are shown in Ta-ble 4.

Table 4. Human effective permeability (Peff) of atenolol, metoprolol, and ketoprofen in the jejunum, ileum, and colon, determined in vivo using the Bioperm capsule and a deconvolution approach. The values were determined in 14 healthy volun-teers and are expressed as median values (range).

Peff (• 10-4 cm/s)

Intestinal site Atenolol Metoprolol Ketoprofen

Jejunum 0.45 (0.07-1.46)a 1.72 (0.016-3.38) 8.85 (0.49-16.14)a

Ileum 0.15 (0.06-0.42)a 0.72 (0.09-7.53) 6.53 (1.49-10.44)

Colon 0.013 (0.01-0.10) 1.30 (0.54-7.74) 3.37 (0.98-9.07)

Footnotes: a significantly higher than the corresponding value in colon for that drug com-pound.

For the BCS class III compound atenolol, the permeability was significantly lower in the colon compared to both jejunum and ileum (>10 fold difference). For the BCS class I compound metoprolol, there were no significant differ-ences, while for the BCS class II compound ketoprofen, there was a significant difference between the jejunum and the colon, however the permeability was still high in both regions. This observation is in line with the common theory that poorly permeable compounds use the entire villi for absorption, while highly permeable compounds only use the villi tips 149. The smaller surface

43

area of the colon compared to the small intestine would thus have a higher impact on the poorly permeable compounds. The results also confirm that modified release formulations are more suitable for highly permeable com-pounds, as the permeability in colon is sufficient for a high absorption from this region.

Figure 7 shows a comparison between the jejunal values determined in Pa-per I and values determined using the Loc-I-Gut perfusion method (based on disappearance) 71, 95. There was a good agreement between the results, sug-gesting that intra-intestinal bolus dosing coupled with deconvolution is a valid and valuable tool in the determination of regional intestinal permeability.

Figure 7. Comparison between the deconvoluted jejunal Peff determined in Paper I and jejunal Peff that had been determined using Loc-I-Gut intestinal perfusion.

II. Pre-clinical assessment of regional intestinal permeability Even though determining intestinal permeability in human volunteers is the “gold standard”, it is not possible to routinely evaluate permeability this way. Instead, several pre-clinical models have been established, of which the Ussing chamber and single-pass perfusion in rats (SPIP) are two common models. The aim of Paper II was to investigate regional permeability in these pre-clinical models, for the compounds investigated in Paper I, with the ad-dition of one poorly permeable compound (enalaprilat). The effect of luminal pH on absorption was also evaluated to allow a better understanding of how the pH in the different intestinal regions may influence absorption.

44

The apparent permeabilities (Papp) and effective permeabilities (Peff) for the different compounds in the different intestinal regions, with the two different buffers, are shown in Table 5 and Table 6, respectively.

Table 5. Regional intestinal apparent permeability (Papp) determined using excised intestinal rat tissue in the Ussing chamber.

Atenolol Metoprolol Ketoprofen Enalaprialt

pH 6.5

Jejunum 11.5 ± 4.09a,b 4.22 ± 1.42b 26.0 ± 13.4a 4.74 ± 3.25a,b Ileum 7.81 ± 4.76 16.8 ± 7.67a 24.6 ± 8.38a 2.87 ± 1.69 Colon 12.2 ± 3.88a,b 7.66 ± 3.12b 17.9 ± 10.6a 4.21 ± 1.24a pH 7.4 Jejunum 3.41 ± 1.76 3.39 ± 1.91b 15.8 ± 9.24 1.42 ± 0.74 Ileum 4.58 ± 2.29 23.9 ± 11.8 13.8 ± 6.66 1.56 ± 0.64

Colon 3.69 ± 2.65 10.1 ± 6.55b 8.22 ± 3.92 1.62 ± 1.14

Footnotes: a significantly different from the same segment at pH 7.4, b significantly different from ileum at the same pH.

Table 6. Regional intestinal effective permeability (Peff) determined using single- pass intestinal perfusion in rats.

Atenolol Metoprolol Ketoprofen Enalaprilat

pH 6.5

Jejunum 0.21 ± 0.13 0.63 ± 0.16 2.92 ± 0.70 0.32 ± 0.19 Ileum 0.28 ± 0.10a 0.92 ± 0.26a 2.10 ± 0.44 0.34 ± 0.05 Colon 0.24 ± 0.10a 0.64 ± 0.10 1.53 ± 0.48 0.29 ± 0.13 pH 7.4 Jejunum 0.30 ± 0.04 1.04 ± 0.19 2.35 ± 0.70 0.45 ± 0.06 Ileum 0.34 ± 0.14a 1.10 ± 0.49a 2.88 ± 0.67 0.36 ± 0.16 Colon 0.20 ± 0.10a 0.72 ± 0.19 1.33 ± 0.46 b 0.29 ± 0.04 pH 6.5 LBS Jejunum 0.50 ± 0.19b 0.99 ± 0.22b 3.63 ± 1.70b,c 0.98 ± 1.30 Ileum 0.79 ± 0.30 1.60 ± 0.41 2.01 ± 0.29 0.77 ± 0.30 Colon 0.51 ± 0.26 0.80 ± 0.20b 1.41 ± 0.29 0.45 ± 0.13

Footnotes: a significantly different from the same segment at pH 6.5 LBS, b significantly dif-ferent from ileum with the same buffer, c significantly different from colon with the same buffer.

In both models, ketoprofen and metoprolol showed high permeability in all segments and in all buffers, with one exception: the Papp for metoprolol in the jejunum was substantially lower than in the other intestinal segments. This trend is not seen for the Peff, indicating that the lower permeability observed in the Ussing chamber might be related to processes within the enterocytes, such as metabolism within the cytosol 150. The values determined for both ke-toprofen and metoprolol were in line with historical data 151-153.

45

Atenolol had an unexpectedly high Papp for all segments at pH 6.5. The reason for this remains unclear, but it is evident that there were no issues with barrier integrity, because the electrical parameters showed no adverse signals and the permeability of the other three drug compounds remained stable. Apart from this, the two poorly permeable compounds atenolol and enalaprilat ex-hibited low permeability across all conditions and in both models, which was in agreement with previously published values 65, 154.

When perfusing with the pH 6.5 LBS buffer in SPIP, the pH changed dra-matically in the ileum, from 6.5 in the inlet perfusate to 8.3 in the outlet per-fusate. This difference shows the very high buffering capacity of the intestine, and consequently demonstrates the importance of using buffers with sufficient strength if investigating potential pH effects. Furthermore, there were signifi-cant increases for both atenolol and metoprolol when perfused with the LBS buffer, which is in line with the pH partitioning theory, as both compound are weak bases with pKa’s of 9.6 and 9.7.

Comparing pre-clinical to clinical permeability To assess the correlation between the values determined using the pre-clinical models in Paper II, a correlation was established between these values and the deconvoluted values for human intestinal permeability from Paper I.

Figure 8. Deconvoluted human intestinal effective permeability (Peff) values for atenolol, metoprolol, and ketoprofen administered using a Bioperm capsule, plotted against the corresponding (upper panels) rat intestinal effective permeability (Peff) and (lower panels) rat apparent permeability (Papp) values determined with the Ussing chamber. In the upper panel, circles (●) represent values for the three drugs determined with the pH 6.5 buffer, while squares (■) represent values determined with the pH 7.4 buffer, and triangles (▲) represent values determined with the pH 6.5 buffer at low buffer strength (LBS). In the lower panel, the three circles (●) rep-resent values for the three drugs determined at pH 6.5 and the squares (■) represent values determined at pH 7.4. Note that the Peff scales are different for the different regions.

46

For the Peff determinations in rat, there was a good linear correlation with the deconvoluted human Peff in all intestinal regions (Figure 8, upper panels). The highest correlations were found when applying a buffer with a pH similar to that found in the respective region (jejunum 6.5, R2 = 0.99; ileum 7.4, R2 = 0.99; colon 6.5, R2 = 1.0). In contrast, no significant correlations were present between the Papp values determined in the Ussing chamber and the human in-testinal Peff (Figure 8, lower panels). This pattern may, to some extent, be be-cause disappearance and appearance does not always follow the same pattern due to factors like intracellular metabolism or binding. However, the results clearly demonstrate that SPIP is the more useful method because it has the highest potential for accurately predicting intestinal permeability in humans.

III. In vivo absorption mechanisms of aprepitant nanosuspensions Having established that the single-pass intestinal perfusion was better suited for pre-clinical evaluation of drug absorption than the Ussing chamber, SPIP was used to investigate the absorption of nanosuspensions under different lu-minal conditions in Paper III.

Table 7. Effective permeability (Peff), appearance flux (Japp), and disappearance flux (Jdisapp) for aprepitant in the four investigated formulations. All values are shown as means ± SD.

Peff

(10-4 cm/s) Japp

(nmol/(h × cm2)) Jdisapp

(nmol/(h × cm2))

20 µM in buffer 1.49 ± 0.20 1.26 ± 0.17 2.38 ± 0.32 20 µM in FaSSIF 1.68 ± 0.16a 2.38 ± 0.43 2.23 ± 0.24

200 µM in buffer 0.54 ± 0.07a,b 6.31 ±1.26a 9.36 ± 1.11a,b

200 µM in FaSSIF 0.93 ± 0.41b 14.2 ± 3.96a,b,c 14.8 ± 5.78a.b

Footnotes: a significantly different from 20 µM in buffer, b significantly different from 20 µM in FaSSIF, c significantly different from 200 µM in buffer.

The Peff (calculated by Eq. 3) for aprepitant from the four different investigated formulation are shown in Table 7. The highest permeability was seen for 20 µM aprepitant solution in FaSSIF, however that 20 µM aprepitant in FaSSIF should have the highest Peff was not consistent with the plasma-concentration time profiles (Figure 9), but rather was due to the fact that Eq. 3 uses the total concentration of API, not only the dissolved concentration. Instead, an appear-ance flux (Japp) was calculated from Eq. 9 (see Table 7). The rank order of the Japp values were 20 µM in buffer < 20 µM in FaSSIF < 200 µM in buffer < 200 µM in FaSSIF, which is in line with the plasma concentration-time pro-files. Evidently, increasing the number of nanoparticles as well as increasing

47

the apparent solubility (by including colloidal structures which the drug mol-ecules can partition into) increases the intestinal absorption. The increase seen when increasing the concentration ten-fold was consistent between both buffer and FaSSIF, indicating a similar behavior of nanoparticles in both cases. Be-cause the dissolution rate of aprepitant achieved in systems containing nano-suspensions is sufficient to keep the lumen constantly saturated, we propose that the increase in absorption is due to diffusion/deposition of nanoparticles across the ABL 32. It has previously been suggested that nanoparticle may in fact cross the ABL to provide a higher total concentration of API closer to the enterocytes apical membrane 134. Furthermore, an increased deposition of na-noparticle compared to microparticles has been found in the mucus, further supporting this theory 155.

Figure 9. Plasma-concentration time profiles for aprepitant after single-pass perfu-sion of the four different formulations in the rat jejunum. Values are shown as means ± SD.

In addition to Japp, disappearance flux (Jdisapp) was calculated based on the con-centrations in the perfusate using Eq. 8 (Table 7). Jdisapp values were similar to Japp values for the formulations in FaSSIF, while Jdisapp values were signifi-cantly lower than Japp values for the formulations in buffer, indicating that there is a luminal process present in buffer that is not present in FaSSIF. This luminal process could possibly result from particles drifting into the mucus, which seems to be more pronounced in buffer potentially due to a higher num-ber of particles compared to FaSSIF and/or an effect of partitioning into col-loidal structures present in FaSSIF.

The effect seen in the presence of nanoparticles and colloidal structures can be explained as an increased ability of the API to cross the ABL. In quantita-tive terms, this effect can be explained as an increase in the effective diffusion

48

coefficient (Deff) across the ABL, or as a reduction in the effective thickness of the ABL (Figure 10)134. Deff values increased 6-fold for the composition with 20 µM aprepitant in FaSSIF compared to 20 µM in buffer. The possibility of partitioning into the additional colloidal structures increases the ability to cross the ABL compared to unpartitioned particles at this concentration. The same level of increase is seen comparing the 200 µM in buffer and the 20 µM in buffer, which means that an increase in the number of particles also has a positive effect on absorption. This increase is further evidence that the disso-lution rate is not the only factor responsible for nanosuspensions increase in absorption, because the bulk is constantly saturated even at 20 µM. Finally, the increase is 17-fold comparing the formulation with 200 µM in FaSSIF to 20 µM in buffer, which demonstrates the very powerful absorption-promoting effect of combining colloidal structures and nanoparticles. These findings mean that in addition to the API monomers diffusing across the ABL, nano-particles and API partitioned into the colloidal structures also cross the ABL94. Even though the diffusion of nanoparticles and colloidal structures probably occurs at a slower speed due to their much larger size, their contributions may still be substantial because the particles consist of a high number of molecules.

Figure 10. A visualization of how the effective diffusion coefficient (Deff) affects the concentration gradient across the aqueous boundary layer (ABL). Cb = concentration of API in the bulk.

IV. Absorption of nanosuspensions compared to microsuspensions Having established that both the drifting/deposition of nanoparticles and col-loidal structures may contribute to an increase in the absorption of nanosus-pensions, the next objective was to further investigate the mechanisms for the increased absorption. We also wanted to further develop and validate the rat

49

SPIP model as a tool for investigating complex biopharmaceutical issues. In Paper IV, absorption of microsuspensions was compared to absorption of nanosuspensions against a background of buffer and fasted and fed state sim-ulated intestinal fluids (FaSSIF and FeSSIF). To evaluate the influence of the mucus layer on absorption, a mucolytic agent (N-acetyl-cysteine) was also in-cluded during the study period.

The plasma concentration time-curves following perfusion of the two dif-ferent suspensions in buffer, FaSSIF, and FeSSIF are shown in Figure 11. The luminal fed condition had a pronounced effect on microsuspensions (Figure 11A), while the effect of food was largely eliminated with nanosuspensions (Figure 11B). The plasma AUC0-150min for microsuspensions in FeSSIF were 9- and 38-fold higher than microsuspensions in FaSSIF and buffer, respec-tively. For the nanosuspensions, no significant differences were observed in plasma AUC0-150min. These differences are in line with previously published data from both dog and humans, where it was seen that nanosuspensions seemed to prevent variation in plasma exposure between different prandial states, while microsuspensions show significant food-effects 156-158. The dif-ferences between microsuspensions and nanosuspensions in the different prandial states (that is, differences in buffer and FaSSIF but no difference in FeSSIF) are also in line with previously published data 156, 159.

Figure 11. Mean (± SEM) plasma concentration-time profiles for aprepitant follow-ing jejunal single-pass perfusion. A, Microsuspension in buffer (●), FaSSIF (■), or FeSSIF(▲). B, Nanosuspension in buffer (●), FaSSIF (■), or FeSSIF(▲). The mu-colytic agent N-acetyl-cysteine (NAC) was added after 75 minutes (highlighted ar-eas).

To quantify the flux of aprepitant across the enterocytes, Japp was calculated according to Eq. 9 (Table 8). There were no significant differences between any of the formulations, which was rather surprising considering the differ-ences in plasma exposure. There were, however, large non-significant differ-ences for the microsuspensions; for instance, the differences in Japp for mi-crosuspensions in FeSSIF were 64 and 4.8 times higher than for the microsus-pensions in buffer and FaSSIF, respectively, during the control period. For the

50

nanosuspensions the Japp were all within a 1.6-fold range, again reiterating the effective elimination of a luminal food effect by the nanoformulation.

Table 8. In vivo absorption flux (Japp) for aprepitant nano- and microsuspensions determined in the jejunum of the single-pass perfused rat. During the control pe-riod, buffer, FaSSIF, and FeSSIF were perfused without excipient, and after 75 minutes the mucolytic NAC was added to the perfusate for duration of the study period.

Flux (nmol / (hr x cm2)

Control period Study period

Formulation Media Average SD Average SD Study:control

ratio

Nanosuspension Buffer 2.91 1.02 1.21 0.639 0.42

Nanosuspension FaSSIF 3.57 1.97 3.73 1.40 1.04

Nanosuspension FeSSIF 2.25 1.69 3.44 4.62 1.52

Microsuspension Buffer 0.104 0.0810 0.113 0.0780 1.09

Microsuspension FaSSIF 1.58 2.10 1.36 2.31 0.86

Microsuspension FeSSIF 6.63 7.32 7.23 7.67 1.09

To investigate the influence of mucus on absorption, a study period was in-cluded during which NAC was co-administered in the perfusate. There were no differences between the study period and the control period for any of the investigated compounds (Figure 12). There was a 58% non-significant de-crease in flux for nanosuspension in buffer during the study period. This result could be interpreted as a reduction in the amount or the integrity of the mucus, which would reduce the possibility for particles get caught in the mucus, with the consequence that a larger number of particles would leave the intestine unabsorbed. The opposite trend was seen for nanosuspensions in FeSSIF, where there is a non-significant increase in flux of 52%.

Figure 12. Mean (± SEM) absorption flux ratios (study period after addition of NAC/control period without NAC) for aprepitant for all six investigated composi-tions. Nanosized aprepitant suspensions in buffer, FaSSIF, and FeSSIF are shown as filled circles (●), squares (■), and triangles (▲), respectively. Microsized aprepitant suspensions in buffer, FaSSIF, and FeSSIF are shown as hollow circles (○), squares (□), and triangles (∆), respectively.

51

One explanation for the increase in flux in fed-state nanosuspensions is varia-bility (as the relative standard deviation is above 100%). Mechanistic studies on the role of mucus in absorption of nanosuspensions are needed to be able to fully elucidate whether the mucus is a barrier to or enhancer of absorption of nanosuspensions.

V. Effects of AMEs in simulated intestinal fluids While nanosuspensions clearly have great potential for solubility limited drugs, this approach is generally not considered for highly soluble drug com-pounds with limited intestinal permeability, hence other pharmaceutical ap-proaches are needed to increase the permeation. As previously discussed, one such approach is the use of absorption-modifying excipients (AMEs). In pre-vious publications, the effect of three AMEs were evaluated with the SPIP model using buffer as the perfusion media and also with the rat intra-intestinal bolus model 148, 160. The magnitude of absorption increase was different in the two models, and the SPIP tended to overestimate the effect. In Paper V, we sought to investigate the effect of these AMEs on absorption of the four model compounds used in Paper II, and to see whether using simulated intestinal fluids could predict the AME effect observed in vivo.

The plasma concentration-time profiles for each of the AMEs in FaSSIF is shown for atenolol (Figure 13) as an example of these profiles (all of which are presented in Paper V). During the control period, the plasma concentra-tion increased evenly in all formulations. However, after addition of AME, the profiles differ significantly depending on the type and concentration of AME.

Figure 13. Mean (± SEM) plasma concentration-time profiles for atenolol in FaSSIF with different AEMs. The white area is the control period (75 min) and the shaded area is the study period (75 min) in which the respective AEMs were added.

52

Japp values were calculated for control and study periods according to Eq. 9. The fluxes of the investigated compounds from the control periods in FaSSIF and FeSSIF are shown in Figure 14. For all investigated compounds, the flux was higher in FaSSIF than in FeSSIF, which suggests a greater extent of par-titioning of drugs into colloidal structures in FeSSIF, which reduces the free monomer concentration available for permeation. The flux ratios between fasted and fed luminal conditions were 1.9 for atenolol, 1.5 for enalaprilat, 1.4 for ketoprofen, and 13 for metoprolol. These results are corroborated by pre-vious reports using the same or similar techniques 161-163. Metoprolol was shown to have an increased bioavailability in a human in vivo study when given with food 164. However, this increase can not be attributed to increases in absorption, because metoprolol has complete absorption in all prandial states 71. The increase seen in the in vivo study must therefore be due to changes in the first pass metabolic extraction, possibly because of increases in splanchnic blood flow while ingesting and digesting food 165, 166.

Figure 14. Jejunal absorption flux (Japp) values (mean ± SEM) from the control pe-riod in all experiments. Asterisks * indicate statistical significance (p-value < 0.05).

The Japp ratios for the study and control periods for both FaSSIF and FeSSIF are shown in Figure 15. Unsurprisingly, the two poorly permeable com-pounds, enalaprilat and atenolol, are the most affected by addition of AMEs 148, 160. For enalaprilat (human fraction absorbed <10%), SDS had a very strong effect on absorption in both media, with significant increases in both media for 0.5% SDS and for 0.1% SDS in FeSSIF 71, 167, 168. The 26-fold increase seen in for 0.1% SDS in FaSSIF was not statistically significant, but it is clear that SDS does indeed greatly increase absorption of enalaprilat. Caprate shows a weak non-significant effect on the absorption of enalaprilat, which is con-sistent with previous reports, indicating that caprate’s effect on transcellular permeation is very weak at best 148, 160, 169. A significant increase in the absorp-tion of enalaprilat was seen with 0.5% NAC, but no such increase was seen with 0.5% NAC pH adj. It can therefore be assumed that the increase was due

53

to an effect of low luminal pH rather than NAC itself. The effect of acidic pH on the jejunum has previously been shown to increase leakage from the blood to the lumen of the barrier integrity marker 51Cr-EDTA, which agrees with our findings 170. Atenolol (human fraction absorbed 50–60%) exhibits a similar pattern to enalaprilat, which is in line with previous results 71, 148, 160, 167. In general, the changes are smaller in magnitude, reflecting the lower potential for increasing absorption using AMEs.

Figure 15. Ratios between study and control periods jejunal absorption flux (Japp) for atenolol, enalaprilat, ketoprofen, and metoprolol in FaSSIF (●) and FeSSIF (■). Val-ues are shown as mean ± SEM. Asterisks * indicate statistical significance (p-value < 0.05).

For the two highly permeable compounds metoprolol and ketoprofen, none of the AMEs had positive effects on Japp. The lack of effect on compounds with a fraction absorbed of 0.95–1 is not surprising, and in line with previous find-ings 148, 160, 169. Ketoprofen showed several significant reductions in Japp during the study period, which is most likely not due to the AMEs. Rather, one ex-planation may be that sink conditions were not maintained due to the high

54

concentration in the perfusate and the long duration of the experiments 171. Regardless of the mechanism behind this decrease, it is evident that no addi-tional benefit is gained from combining AMEs with BCS class I and II com-pounds.

The blood to lumen clearance of 51Cr-EDTA agreed with the results seen for the poorly permeable drug compounds in this study, and also with previous reports 148, 169. In summary, the increases seen in CLCr-EDTA were higher in FaS-SIF than in FeSSIF.

As observed both for investigated drug compounds and CLCr-EDTA, the ef-fects of AMEs were greater in FaSSIF than in FeSSIF. Both these simulated intestinal media contain bile acids and lecithin, but they differ in relative amounts. In FeSSIF, the concentrations of bile acids and lecithin are 10 and 3 mM respectively, while in FaSSIF the concentrations are 3 and 0.2 mM re-spectively 172. In addition, FeSSIF also contains 5 mM glyceryl monooleate. In solution, in FaSSIF the colloidal structures formed are fewer in number but larger in size (40–100 nm), while in FeSSIF the colloidal structures are more abundant but smaller in size (10–20 nm) 173, 174. These differences result in a generally higher capacity for drug partitioning into colloidal structures in FeS-SIF, as evident by the differences in apparent solubility in FeSSIF and FaSSIF for a vast number of compounds 175, 176. Both drug compounds and AMEs can partition into these colloidal structures to a higher degree in FeSSIF, meaning that the free fractions of drug compound and AME decrease. Since only free drug monomers can cross the apical membrane of the enterocytes, the driving force for absorption is thus decreased in FeSSIF compared to FaSSIF. By anal-ogy, only the free AMEs can interact with the membrane (or another site of action), meaning the AME effect is decreased in FeSSIF. This theory has pre-viously been shown valid for maltosides, both in Caco-2 cells and in rat per-fusion 177. Finally, when comparing the results from our study to a previous perfusion study using plain phosphate buffer (without colloidal structures) and an intra-intestinal bolus study in rat and dog, the results using simulated intes-tinal fluid could better predict the in vivo values from the bolus study 148, 160. Using simulated intestinal fluids instead of phosphate buffer when conducting AME evaluations could therefore be beneficial.

Comparing historical data from Caco-2 cell models to our results, it is ap-parent that Caco-2 cells have tended to over predict the magnitude of AMEs’ effects. For instance, 0.005–0.025% SDS increased the flux of mannitol 20–142-fold in two different studies 178, 179. In contrast, in in vivo methods (such as intestinal instillations and perfusion), the addition of 0.1–2% SDS gave in-creases in flux of zero to 6-fold, which is in line with our findings using SPIP 180-183. The same pattern was seen for caprate as well, further validating this conclusion about Caco-2 cells 108, 184, 185. The differences in results can proba-bly be attributed to the lower biorelevance of the Caco-2 model, meaning that results based on the cell lines usually used in laboratories should be interpreted with caution 101, 116, 146.

55

Conclusions

Papers I and II investigated the absorption in different intestinal regions of four drug compounds in humans and in two pre-clinical rat models. In Paper II, the impact of luminal pH was also assessed in two pre-clinical models. The main findings from these investigations were: The human permeability of the poorly permeable atenolol was signifi-

cantly lower in the colon than in the jejunum or ileum. The highly perme-able compounds metoprolol and ketoprofen showed no such pronounced differences.

The deconvoluted Peff values from Paper I showed a good agreement with historical human Peff values from in vivo intestinal perfusion. The decon-volution approach is therefore deemed suitable for determining region-ally-based Peff in human intestine.

Single-pass intestinal perfusion determination of Peff showed a higher cor-relation to human Peff than did Papp determined with the Ussing chamber, indicating that SPIP is the more predictive of the two pre-clinical models.

Neither of the two pre-clinical models in Paper II were able to capture the intestinal region differences observed in Paper I. However, if appro-priate correction factors are applied, these models may still be useful for regional determinations of permeability.

Papers III and IV investigated the mechanisms behind the greater absorption seen in nanosuspensions than in microsuspensions. The main findings from these investigations were: Increases in the number of nanoparticles increases the absorption, even

after reaching a dissolution rate which maintains the dissolved concentra-tion at saturation.

Colloidal structures facilitate transport across the aqueous boundary layer (ABL) by drug partitioning into them, followed by diffusion across the ABL. This process results in an increased concentration of free drug mon-omer adjacent to the apical membrane of the enterocytes, thereby increas-ing the driving force for absorption.

Nanoparticles are more likely than microparticles to drift into the mucus, which increases the concentration closer to the apical membrane of the enterocytes, similar to the effect seen for colloidal structures.

56

The role of the mucus layer in absorption of nanoparticles is not fully un-derstood. It seems that a reduction in the mucus layer decreases absorp-tion, possibly by reducing the number of drifting particles that are caught by the mucus, thus decreasing the transport of drug through the ABL.

Paper V investigated the effect of absorption-modifying excipients on the rat intestinal mucosa when single-pass perfused in simulated intestinal fluids. The main findings from this investigation were: No positive effects on absorption could be seen for the highly permeable

compounds metoprolol and ketoprofen, showing the limited benefits of combining AMEs with BCS class I and II compounds.

The poorly permeable (BCS class III) compounds atenolol and enalaprilat both showed concentration-dependent increases in absorption for sodium dodecyl sulphate, an agent that interferes with the membrane of the enter-ocytes and increases the transcellular flux.

The effect of the AMEs were more potent in fasted state simulated intes-tinal fluid (FaSSIF) than in fed state simulated intestinal fluid (FeSSIF). The magnitude of the effects with FeSSIF closely resembled that seen the in vivo in rat under fed state, highlighting the benefits of using simulated intestinal fluids in permeability assessments.

In summary, these studies using the single-pass intestinal perfusion in rats, and investigating simulated intestinal fluid, absorption modifying excipients, and nanoparticles, have increased the understanding of several physiological and biopharmaceutical aspects that affect gastrointestinal absorption.

57

Populärvetenskaplig sammanfattning

De flesta av läkemedlen som används ges via munnen och då det för de flesta är enkelt att svälja en tablett, förväntas detta sätt att ta läkemedel fortsätta vara dominerande. För att ett läkemedel som tas på detta sätt ska kunna ha effekt måste det aktiva ämnet i tabletten absorberas, dvs. tas upp över tarmslemhin-nan in i blodet. Hur väl ämnet absorberas avgörs dels av molekylens storlek och dess fettlöslighet, men också av sammansättningen av tarmslemhinnan. En liten och fettlöslig läkemedelsmolekyl har bättre förutsättningar att absor-beras snabbt än en stor och vattenlöslig molekyl. Eftersom tarmslemhinnan ser olika ut beroende på vilken del av tarmen som undersöks, innebär det också att läkemedelsabsorptionen kan vara olika snabb i olika delar. Absorptionen av läkemedel, men även näringsämnen sker framförallt i den första delen av tunntarmen. Tarmarnas rörelser gör både så att innehållet blandas om, men också så att innehållet transporteras framåt. Om ett läkemedel inte hinner ab-sorberas i den första delen av tunntarmen, kan det fortfarande absorberas i den senare delen av tunntarmen eller i tjocktarmen. Detta kan vara viktigt för lä-kemedelstabletter som har en långsam absorption och således inte hinner ab-sorberas i den första delen av tunntarmen, eller för tabletter som av olika an-ledningar löses upp väldigt sent.

För att undersöka om ett läkemedel absorberas snabbt eller långsamt, är det vanligt i forskningsvärlden att använda sig av olika råttmodeller för att studera transporten över tarmslemhinnan. Kunskapen om hur väl dessa djurmodeller kan förutsäga absorptionen i människa är dock inte fullständig. Första delen av denna avhandling hade därför som mål att utvärdera två råttmodeller. Detta gjorde dels genom en studie i människa för att studera absorption i alla delar av tarmen och dels genom en studie där absorptionen från alla delar av tarmen i råtta studerades i två olika modeller: en modell där sövda råttor användes och en modell där utskuren tarm användes. I människa visade det sig att läkemedel som har en snabb absorption i första delen av tunntarmen, också har en snabb absorption i den senare delen av tunntarmen och i tjocktarmen. För läkemedel med långsam absorptionshastighet i tunntarmen, var absorptionshastigheten i tjocktarmen ännu långsammare. I båda råttmodellerna kunde man se att läke-medel som absorberades snabbt i människans tunntarm också absorberades snabbt i råttans tunntarm. Däremot såg man inte den stora skillnaden mellan absorptionen i tunntarm för läkemedel som absorberades långsamt som man såg i människa. Dessutom visades det att den av råttmodellerna som använde

58

sövda råttor var bättre på att förutsäga absorptionen i människa, jämfört med den modell som använde enbart utskuren tarm från råttan.

För läkemedel som kroppen inte kan ta upp fullständigt på grund av att de har svårt att lösa sig i tarmvätskan, är det vanligt att man försöker förbättra lösligheten eller göra så att läkemedlet löser upp sig snabbare. Ett sätt att göra detta är att göra läkemedelspartiklarna mindre, för att större yta ska komma i kontakt med vätskan i tarmen. När partiklarna blir mindre än en tusendels mil-limeter, brukar man kalla dessa partiklar för nanopartiklar. I avhandlingens andra del undersöktes vilka anledningar det finns till att läkemedel som består av nanopartiklar kan absorberas så mycket bättre än läkemedel som består av större partiklar. Genom att studera absorptionen i tarmen på sövda råttor kunde det konstateras att dessa nanopartiklar kan ta sig in mycket närmare tarmslem-hinnan än vad större partiklar kan. När de kommit nära tarmslemhinnan kan de släppa ifrån sig många enskilda molekyler som sedan kan ta sig över celler i tarmslemhinnan och in i blodet. Det visade sig även att det finns olika struk-turer inuti tarmen som bildats av nedbrytningsprodukter från mat och från äm-nen från kroppen. Dessa strukturer har förmågan att fånga upp läkemedelsmo-lekyler och transportera dem till tarmslemhinnan för att sedan släppa ifrån sig läkemedelsmolekyler där. Detta gör att man kan få skillnader i hur mycket läkemedel som kroppen kan ta upp om man tar en tablett med eller utan mat. Om tabletten däremot innehöll nanopartiklar såg man att det inte var någon skillnad i hur mycket som togs upp, vare sig det var med mat eller utan.

För läkemedel som har låg absorptionshastighet över tarmslemhinnan kan man ibland använda så kallade absorptionsförbättrande tillsatser för att för-bättra absorptionen över tarmslemhinnan. Den sista delen av avhandlingen hade som syfte att försöka förstå hur intag av dessa tillsatser, med eller utan mat, kan påverka transporten av läkemedel över tarmslemhinnan. Effekten på läkemedelsabsorptionen av tre olika tillsatser som gavs med och utan mat stu-derades i sövda råttor. Det kunde fastställas att effekten av tillsatserna var större utan mat än med mat. Detta tros bero på att de strukturer som består av nedbrytningsprodukter av mat blandat med ämnen som utsöndras från krop-pen kan fånga upp tillsatser och på så vis hindra dem från att utöva en effekt på tarmslemhinnan. Man kunde även se att de absorptionsförbättrande tillsat-serna endast förbättrade transporten över tarmslemhinnan för läkemedel med en långsam absorption. För läkemedel med en snabb absorption ger således denna typ av tillsatser ingen effekt och bör därför inte användas.

Sammantaget har denna avhandling bidragit till att förbättra kunskapen om försöksdjurs nytta vid forskning om läkemedelsabsorption, förbättrat förståel-sen för hur läkemedel med nanopartiklar kan tas upp av kroppen och slutligen ökat förståelsen för hur födointag kan påverka effekten av absorptionsförbätt-rande tillsatser.

59

Acknowledgements

The work presented in this thesis was conducted at the Department of Phar-macy, Uppsala University, at AstraZeneca, Gothenburg, and at Uppsala Uni-versity Hospital. I would like to thank the Oral Biopharmaceutics Tools pro-ject (Orbito) within the Innovative Medicines Initiative (EU) for funding my PhD-studies and giving me the possibility to collaborate with great researchers all over Europe. I would also like to thank Apotekare CD Carlsson stiftelse (Apotekarsocieteten) for the grants which allowed me to travel to Edinburgh, Scotland on a conference.

Hans Lennernäs – main supervisor. Thank you for taking me under your wings and allowing me the liberty to influence the direction of my PhD and the studies conducted. I have greatly appreciated your scientific and entrepre-neurial knowledge, but also your many, many, many anecdotes and stories!

Erik Sjögren – co-supervisor. It has been great going a nano-adventure with you! I have really appreciated all our discussions and the assistance that you have given me. Your enthusiasm and knowledge, coupled with a handful of nerdiness and rebel attitude makes you a formidable supervisor and re-searcher. I will never stop hoping for that Surf-n-Turf!

I would like to thank Staffan Berg, Charlotta Vedin, Pernilla Johansson, Jan Westergren, Anna Thorén, Anders Lundqvist, Christer Tannergren, and Bertil Abrahamsson at AstraZeneca in Gothenburg for teaching me so much and al-ways having a positive attitude whenever I would visit.

Thank you to Markus Sjöblom, Tobias Olander, and Olof Nylander at the De-partment of Neuroscience, for a very productive and interesting collaboration.

Thank you to Mikael Hedeland and Margareta Sprycha at SVA for assisting with the quantification of plasma samples and for allowing me to gain deeper insights into the world of analytical chemistry.

Thank you to all the present and former PhD students in the Biopharmacy group Elsa, Ilse, Emelie, Johanna, Oliver, and Fredrik. Emelie, it has been fantastic to discuss everything from failed science and sourdough bread to the latest trends within the hipster society with you.

60

To my closest co-worker David. Embarking on this “quest” with you has given me the speed to complete distances quickly but also the time to sometimes relax and enjoy the ride. I admire your productiveness and ability to always get things done, while at the same time always having a positive attitude, jok-ing or quoting “Sagan”. As Sam once said: “There’s some good in this world, Mr. Frodo… and it’s worth fighting for”.

To all the present and past PhD student at the Department of Pharmacy who in some way contributed to making my time at the department better: thank you! A special recognition to Magnus for handing me the key to sourdough-heaven, for our many DG-rounds, TB and Oslipat, and for our amazing “es-pacci”-sessions!

All my friends, old and new. Thank you for always making me happier and remembering the most important things in life.

Finally, to my wife Helena, our children Emil and Gustav, my parents Claes and Mariann, and my brothers Olof and Erik. Thank you for always support-ing and encouraging me. Without you, this thesis could never have been made!

61

References

1. Lennernäs, H.; Abrahamsson, B. The use of biopharmaceutic classification of drugs in drug discovery and development: current status and future extension. Journal of pharmacy and pharmacology 2005, 57, (3), 273-285. 2. Sugano, K. Aqueous boundary layers related to oral absorption of a drug: from dissolution of a drug to carrier mediated transport and intestinal wall metabolism. Molecular pharmaceutics 2010, 7, (5), 1362-1373. 3. Lennernäs, H. Does fluid flow across the intestinal mucosa affect quantitative oral drug absorption? Is it time for a reevaluation? Pharmaceutical research 1995, 12, (11), 1573-1582. 4. Amidon, G. L.; Lennernas, H.; Shah, V. P.; Crison, J. R. A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharmaceutical research 1995, 12, (3), 413-20. 5. Rendic, S. Summary of information on human CYP enzymes: human P450

metabolism data. Drug metabolism reviews 2002, 34, (1-2), 83-448. 6. Huang, S. M.; Strong, J. M.; Zhang, L.; Reynolds, K. S.; Nallani, S.; Temple,

R.; Abraham, S.; Habet, S. A.; Baweja, R. K.; Burckart, G. J. New era in drug interaction evaluation: US Food and Drug Administration update on CYP enzymes, transporters, and the guidance process. The Journal of clinical pharmacology 2008, 48, (6), 662-670.

7. Funk, C. The role of hepatic transporters in drug elimination. Expert opinion on drug metabolism & toxicology 2008, 4, (4), 363-379.

8. Giacomini, K. M.; Huang, S.-M.; Tweedie, D. J.; Benet, L. Z.; Brouwer, K. L.; Chu, X.; Dahlin, A.; Evers, R.; Fischer, V.; Hillgren, K. M. Membrane transporters in drug development. Nature reviews Drug discovery 2010, 9, (3), 215-236.

9. Klaassen, C. D.; Watkins, J. B. Mechanisms of bile formation, hepatic uptake, and biliary excretion. Pharmacological Reviews 1984, 36, (1), 1-67.

10. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced drug delivery reviews 1997, 23, (1), 3-25.

11. Doak, B. C.; Over, B.; Giordanetto, F.; Kihlberg, J. Oral druggable space beyond the rule of 5: insights from drugs and clinical candidates. Chemistry & biology 2014, 21, (9), 1115-1142.

12. Matsson, P.; Doak, B. C.; Over, B.; Kihlberg, J. Cell permeability beyond the rule of 5. Advanced drug delivery reviews 2016, 101, 42-61.

13. Lipinski, C. Poor aqueous solubility—an industry wide problem in drug discovery. Am Pharm Rev 2002, 5, (3), 82-85.

14. Palm, K.; Stenberg, P.; Luthman, K.; Artursson, P. Polar molecular surface properties predict the intestinal absorption of drugs in humans. Pharmaceutical research 1997, 14, (5), 568-571.

62

15. van de Waterbeemd, H.; Kansy, M. Hydrogen-bonding capacity and brain penetration. CHIMIA International Journal for Chemistry 1992, 46, (7-8), 299-303.

16. Clark, D. E. Rapid calculation of polar molecular surface area and its application to the prediction of transport phenomena. 1. Prediction of intestinal absorption. Journal of pharmaceutical sciences 1999, 88, (8), 807-814.

17. DeSesso, J.; Jacobson, C. Anatomical and physiological parameters affecting gastrointestinal absorption in humans and rats. Food and Chemical Toxicology 2001, 39, (3), 209-228.

18. Schiller, C.; FRÖHLICH, C. P.; Giessmann, T.; Siegmund, W.; Mönnikes, H.; Hosten, N.; Weitschies, W. Intestinal fluid volumes and transit of dosage forms as assessed by magnetic resonance imaging. Alimentary pharmacology & therapeutics 2005, 22, (10), 971-979.

19. Fallingborg, J.; Christensen, L.; INGEMAN⁷ NIELSEN, M.; Jacobsen, B.; Abildgaard, K.; Rasmussen, H. pH⁷ Profile and regional transit times of the normal gut measured by a radiotelemetry device. Alimentary pharmacology & therapeutics 1989, 3, (6), 605-614.

20. Chial, H.; Camilleri, C.; Delgado⁷ Aros, S.; Burton, D.; Thomforde, G.; Ferber, I.; Camilleri, M. A nutrient drink test to assess maximum tolerated volume and postprandial symptoms: effects of gender, body mass index and age in health. Neurogastroenterology & Motility 2002, 14, (3), 249-253.

21. Dressman, J. B.; Berardi, R. R.; Dermentzoglou, L. C.; Russell, T. L.; Schmaltz, S. P.; Barnett, J. L.; Jarvenpaa, K. M. Upper gastrointestinal (GI) pH in young, healthy men and women. Pharmaceutical research 1990, 7, (7), 756-761.

22. Murray, K.; Hoad, C. L.; Mudie, D. M.; Wright, J.; Heissam, K.; Abrehart, N.; Pritchard, S. E.; Al Atwah, S.; Gowland, P. A.; Garnett, M. C. Magnetic resonance imaging quantification of fasted state colonic liquid pockets in healthy humans. Molecular pharmaceutics 2017, 14, (8), 2629-2638.

23. Koziolek, M.; Grimm, M.; Becker, D.; Iordanov, V.; Zou, H.; Shimizu, J.; Wanke, C.; Garbacz, G.; Weitschies, W. Investigation of pH and temperature profiles in the GI tract of fasted human subjects using the Intellicap® system. Journal of pharmaceutical sciences 2015, 104, (9), 2855-2863.

24. Camilleri, M. Integrated upper gastrointestinal response to food intake. Gastroenterology 2006, 131, (2), 640-658.

25. Silverthorn, D. U.; Ober, W. C.; Garrison, C. W.; Silverthorn, A. C.; Johnson, B. R., Human physiology: an integrated approach. Pearson/Benjamin Cummings San Francisco, CA:: 2004.

26. Helander, H. F.; Fändriks, L. Surface area of the digestive tract–revisited. Scandinavian journal of gastroenterology 2014, 49, (6), 681-689.

27. Wang, Y.; Brasseur, J. G. Three-dimensional mechanisms of macro-to-micro-scale transport and absorption enhancement by gut villi motions. Physical Review E 2017, 95, (6), 062412.

28. Lundgren, O.; Svanvik, J.; Jivegård, L. Enteric nervous system. Digestive diseases and sciences 1989, 34, (2), 264-283.

29. Huizinga, J. D. Gastrointestinal peristalsis: joint action of enteric nerves, smooth muscle, and interstitial cells of Cajal. Microscopy research and technique 1999, 47, (4), 239-247.

30. Koziolek, M.; Garbacz, G.; Neumann, M.; Weitschies, W. Simulating the postprandial stomach: physiological considerations for dissolution and release testing. Molecular pharmaceutics 2013, 10, (5), 1610-1622.

63

31. Sjögren, E.; Abrahamsson, B.; Augustijns, P.; Becker, D.; Bolger, M. B.; Brewster, M.; Brouwers, J.; Flanagan, T.; Harwood, M.; Heinen, C. In vivo methods for drug absorption–comparative physiologies, model selection, correlations with in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. European Journal of Pharmaceutical Sciences 2014, 57, 99-151.

32. Sjögren, E.; Westergren, J.; Grant, I.; Hanisch, G.; Lindfors, L.; Lennernäs, H.; Abrahamsson, B.; Tannergren, C. In silico predictions of gastrointestinal drug absorption in pharmaceutical product development: application of the mechanistic absorption model GI-Sim. European Journal of Pharmaceutical Sciences 2013, 49, (4), 679-698.

33. Minekus, M.; Alminger, M.; Alvito, P.; Ballance, S.; Bohn, T.; Bourlieu, C.; Carriere, F.; Boutrou, R.; Corredig, M.; Dupont, D. A standardised static in vitro digestion method suitable for food–an international consensus. Food & function 2014, 5, (6), 1113-1124.

34. Hinsberger, A.; Sandhu, B. Digestion and absorption. Current Paediatrics 2004, 14, (7), 605-611.

35. Kong, F.; Singh, R. Disintegration of solid foods in human stomach. Journal of food science 2008, 73, (5), R67-R80.

36. Kong, F.; Singh, R. P. A human gastric simulator (HGS) to study food digestion in human stomach. Journal of food science 2010, 75, (9), E627-E635.

37. Nakamura, T.; Takeuchi, T.; Tando, Y. Pancreatic dysfunction and treatment options. Pancreas 1998, 16, (3), 329-336.

38. Kwiatek, M. A.; Menne, D.; Steingoetter, A.; Goetze, O.; Forras-Kaufman, Z.; Kaufman, E.; Fruehauf, H.; Boesiger, P.; Fried, M.; Schwizer, W. Effect of meal volume and calorie load on postprandial gastric function and emptying: studies under physiological conditions by combined fiber-optic pressure measurement and MRI. American Journal of Physiology-Gastrointestinal and Liver Physiology 2009, 297, (5), G894-G901.

39. Indireshkumar, K.; Brasseur, J. G.; Faas, H.; Hebbard, G. S.; Kunz, P.; Dent, J.; Feinle, C.; Li, M.; Boesiger, P.; Fried, M. Relative contributions of “pressure pump” and “peristaltic pump” to gastric emptying. American Journal of Physiology-Gastrointestinal and Liver Physiology 2000, 278, (4), G604-G616.

40. Hellström, P. M.; Grybäck, P.; Jacobsson, H. The physiology of gastric emptying. Best Practice & Research Clinical Anaesthesiology 2006, 20, (3), 397-407.

41. Camilleri, M.; Malagelada, J.; Brown, M.; Becker, G.; Zinsmeister, A. Relation between antral motility and gastric emptying of solids and liquids in humans. American Journal of Physiology-Gastrointestinal and Liver Physiology 1985, 249, (5), G580-G585.

42. Flemstrom, G.; Garner, A.; Nylander, O.; Hurst, B.; Heylings, J. Surface epithelial HCO3 (-) transport by mammalian duodenum in vivo. American Journal of Physiology-Gastrointestinal and Liver Physiology 1982, 243, (5), G348-G358.

43. Turnberg, L. A.; Bieberdorf, F. A.; Morawski, S. G.; Fordtran, J. S. Interrelationships of chloride, bicarbonate, sodium, and hydrogen transport in the human ileum. The Journal of clinical investigation 1970, 49, (3), 557-567.

44. Sarosiek, I.; Selover, K.; Katz, L.; Semler, J.; Wilding, G.; Lackner, J.; Sitrin, M.; Kuo, B.; Chey, W.; Hasler, W. The assessment of regional gut transit times in healthy controls and patients with gastroparesis using wireless motility technology. Alimentary pharmacology & therapeutics 2010, 31, (2), 313-322.

64

45. Davis, S.; Hardy, J.; Fara, J. Transit of pharmaceutical dosage forms through the small intestine. Gut 1986, 27, (8), 886-892.

46. HARDY, J. G.; WILSON, C. G.; WOOD, E. Drug delivery to the proximal colon. Journal of Pharmacy and Pharmacology 1985, 37, (12), 874-877.

47. Bown, R.; Gibson, J.; Sladen, G.; Hicks, B.; Dawson, A. Effects of lactulose and other laxatives on ileal and colonic pH as measured by a radiotelemetry device. Gut 1974, 15, (12), 999-1004.

48. Khosla, R.; Davis, S. Gastric emptying and small and large bowel transit of non-disintegrating tablets in fasted subjects. International journal of pharmaceutics 1989, 52, (1), 1-10.

49. Tannergren, C.; Bergendal, A.; Lennernäs, H.; Abrahamsson, B. Toward an increased understanding of the barriers to colonic drug absorption in humans: implications for early controlled release candidate assessment. Molecular pharmaceutics 2009, 6, (1), 60-73.

50. Niess, J. H.; Reinecker, H. C. Dendritic cells in the recognition of intestinal microbiota. Cellular microbiology 2006, 8, (4), 558-564.

51. MacDonald, T. T.; Monteleone, G. Immunity, inflammation, and allergy in the gut. Science 2005, 307, (5717), 1920-1925.

52. Englund, G.; Rorsman, F.; Rönnblom, A.; Karlbom, U.; Lazorova, L.; Gråsjö, J.; Kindmark, A.; Artursson, P. Regional levels of drug transporters along the human intestinal tract: co-expression of ABC and SLC transporters and comparison with Caco-2 cells. European Journal of Pharmaceutical Sciences 2006, 29, (3), 269-277.

53. Van Itallie, C. M.; Holmes, J.; Bridges, A.; Gookin, J. L.; Coccaro, M. R.; Proctor, W.; Colegio, O. R.; Anderson, J. M. The density of small tight junction pores varies among cell types and is increased by expression of claudin-2. Journal of cell science 2008, 121, (3), 298-305.

54. DeSesso, J. M.; Williams, A. L. Contrasting the gastrointestinal tracts of mammals: factors that influence absorption. Annual reports in medicinal chemistry 2008, 43, 353-371.

55. Hebel, R.; Stromberg, M. W., Anatomy and embryology of the laboratory rat. BioMed Verlag: 1986.

56. Collett, A.; Walker, D.; Sims, E.; He, Y.-L.; Speers, P.; Ayrton, J.; Rowland, M.; Warhurst, G. Influence of morphometric factors on quantitation of paracellular permeability of intestinal epithelia in vitro. Pharmaceutical research 1997, 14, (6), 767-773.

57. Houpt, T.; Swenson, M.; Reece, W. Dukes Physiology of domestic animals. 1993.

58. Dahlgren, D.; Roos, C.; Sjögren, E.; Lennernäs, H. Direct in vivo human intestinal permeability (P eff) determined with different clinical perfusion and intubation methods. Journal of pharmaceutical sciences 2015, 104, (9), 2702-2726.

59. Kararli, T. T. Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals. Biopharmaceutics & drug disposition 1995, 16, (5), 351-380.

60. Lennernas, H. Human intestinal permeability. Journal of pharmaceutical sciences 1998, 87, (4), 403-10.

61. Fagerholm, U.; Nilsson, D.; Knutson, L.; Lennernäs, H. Jejunal permeability in humans in vivo and rats in situ: investigation of molecular size selectivity and solvent drag. Acta physiologica Scandinavica 1999, 165, (3), 315-324.

65

62. Watanabe, E.; Takahashi, M.; Hayashi, M. A possibility to predict the absorbability of poorly water-soluble drugs in humans based on rat intestinal permeability assessed by an in vitro chamber method. European Journal of Pharmaceutics and Biopharmaceutics 2004, 58, (3), 659-665.

63. Fagerholm, U.; Johansson, M.; Lennernäs, H. Comparison between permeability coefficients in rat and human jejunum. Pharmaceutical research 1996, 13, (9), 1336-1342.

64. Lennernas, H. Human jejunal effective permeability and its correlation with preclinical drug absorption models. The Journal of pharmacy and pharmacology 1997, 49, (7), 627-38.

65. Lennernäs, H.; Nylander, S.; Ungell, A.-L. Jejunal permeability: a comparison between the ussing chamber technique and the single-pass perfusion in humans. Pharmaceutical research 1997, 14, (5), 667-671.

66. Chiou, W. L. The validation of the intestinal permeability approach to predict oral fraction of dose absorbed in humans and rats. Biopharmaceutics & drug disposition 1995, 16, (1), 71-75.

67. Cao, X.; Gibbs, S. T.; Fang, L.; Miller, H. A.; Landowski, C. P.; Shin, H.-C.; Lennernas, H.; Zhong, Y.; Amidon, G. L.; Lawrence, X. Y. Why is it challenging to predict intestinal drug absorption and oral bioavailability in human using rat model. Pharmaceutical research 2006, 23, (8), 1675-1686.

68. Zhang, Q.-Y.; Dunbar, D.; Ostrowska, A.; Zeisloft, S.; Yang, J.; Kaminsky, L. S. Characterization of human small intestinal cytochromes P-450. Drug Metabolism and Disposition 1999, 27, (7), 804-809.

69. Paine, M. F.; Khalighi, M.; Fisher, J. M.; Shen, D. D.; Kunze, K. L.; Marsh, C. L.; Perkins, J. D.; Thummel, K. E. Characterization of interintestinal and intraintestinal variations in human CYP3A-dependent metabolism. Journal of Pharmacology and Experimental Therapeutics 1997, 283, (3), 1552-1562.

70. Amidon, G. L.; Kou, J.; Elliott, R. L.; Lightfoot, E. N. Analysis of models for determining intestinal wall permeabilities. Journal of pharmaceutical sciences 1980, 69, (12), 1369-73.

71. Lennernas, H. Intestinal permeability and its relevance for absorption and elimination. Xenobiotica; the fate of foreign compounds in biological systems 2007, 37, (10-11), 1015-51.

72. Sugano, K.; Kansy, M.; Artursson, P.; Avdeef, A.; Bendels, S.; Di, L.; Ecker, G. F.; Faller, B.; Fischer, H.; Gerebtzoff, G. Coexistence of passive and carrier-mediated processes in drug transport. Nature reviews Drug discovery 2010, 9, (8), 597-614.

73. Di, L.; Artursson, P.; Avdeef, A.; Ecker, G. F.; Faller, B.; Fischer, H.; Houston, J. B.; Kansy, M.; Kerns, E. H.; Krämer, S. D. Evidence-based approach to assess passive diffusion and carrier-mediated drug transport. Drug discovery today 2012, 17, (15), 905-912.

74. Dobson, P. D.; Kell, D. B. Carrier-mediated cellular uptake of pharmaceutical drugs: an exception or the rule? Nature Reviews Drug Discovery 2008, 7, (3), 205-220.

75. Kell, D. B.; Dobson, P. D.; Oliver, S. G. Pharmaceutical drug transport: the issues and the implications that it is essentially carrier-mediated only. Drug discovery today 2011, 16, (15), 704-714.

76. Kell, D. B.; Dobson, P. D.; Bilsland, E.; Oliver, S. G. The promiscuous binding of pharmaceutical drugs and their transporter-mediated uptake into cells: what we (need to) know and how we can do so. Drug discovery today 2012.

66

77. Lande, M. B.; Donovan, J. M.; Zeidel, M. L. The relationship between membrane fluidity and permeabilities to water, solutes, ammonia, and protons. The Journal of general physiology 1995, 106, (1), 67-84.

78. Xiang, T.-X.; Anderson, B. The relationship between permeant size and permeability in lipid bilayer membranes. The Journal of membrane biology 1994, 140, (2), 111-122.

79. Kraemer, S. D.; Lombardi, D.; Primorac, A.; Thomae, A. V.; Wunderli⁷Allenspach, H. Lipid⁷ bilayer permeation of drug⁷ like compounds. Chemistry & biodiversity 2009, 6, (11), 1900-1916.

80. MORRIS, M. E. Overview of drug transporter families. Drug Transporters: molecular characterization and role in drug disposition 2007, 4, 1.

81. Winiwarter, S.; Bonham, N. M.; Ax, F.; Hallberg, A.; Lennernäs, H.; Karlén, A. Correlation of human jejunal permeability (in vivo) of drugs with experimentally and theoretically derived parameters. A multivariate data analysis approach. Journal of medicinal chemistry 1998, 41, (25), 4939-4949.

82. Mueckler, M. Facilitative glucose transporters. European journal of biochemistry 1994, 219, (3), 713-725.

83. Schinkel, A. H.; Jonker, J. W. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Advanced drug delivery reviews 2003, 55, (1), 3-29.

84. Steffansen, B.; Nielsen, C. U.; Brodin, B.; Eriksson, A. H.; Andersen, R.; Frokjaer, S. Intestinal solute carriers: an overview of trends and strategies for improving oral drug absorption. European journal of pharmaceutical sciences 2004, 21, (1), 3-16.

85. Tamai, I. Oral drug delivery utilizing intestinal OATP transporters. Advanced drug delivery reviews 2012, 64, (6), 508-514.

86. Anderson, J.; Van Itallie, C. Tight junctions and the molecular basis for regulation of paracellular permeability. American Journal of Physiology-Gastrointestinal and Liver Physiology 1995, 269, (4), G467-G475.

87. Van Itallie, C. M.; Anderson, J. M. The molecular physiology of tight junction pores. Physiology 2004, 19, (6), 331-338.

88. Lennernas, H.; Ahrenstedt, Ö.; Ungell, A. Intestinal drug absorption during induced net water absorption in man; a mechanistic study using antipyrine, atenolol and enalaprilat. British journal of clinical pharmacology 1994, 37, (6), 589-596.

89. Nilsson, D.; Fagerholm, U.; Lennernäs, H. The influence of net water absorption on the permeability of antipyrine and levodopa in the human jejunum. Pharmaceutical research 1994, 11, (11), 1540-1544.

90. Lennernäs, H. Intestinal drug absorption and bioavailability: beyond involvement of single transport function. Journal of pharmacy and pharmacology 2003, 55, (4), 429-433.

91. Willson, T. M.; Kliewer, S. A. PXR, CAR and drug metabolism. Nature reviews Drug discovery 2002, 1, (4), 259.

92. Benet, L. Z.; Cummins, C. L. The drug efflux–metabolism alliance: biochemical aspects. Advanced drug delivery reviews 2001, 50, S3-S11.

93. Sandström, R.; Karlsson, A.; Knutson, L.; Lennernäs, H. Jejunal absorption and metabolism of R/S-verapamil in humans. Pharmaceutical research 1998, 15, (6), 856-862.

94. Amidon, G. E.; Higuchi, W. I.; Ho, N. F. Theoretical and experimental studies of transport of micelle⁷ solubilized solutes. Journal of pharmaceutical sciences 1982, 71, (1), 77-84.

67

95. Lennernäs, H.; Ahrenstedt, Ö.; Hällgren, R.; Knutson, L.; Ryde, M.; Paalzow, L. K. Regional jejunal perfusion, a new in vivo approach to study oral drug absorption in man. Pharmaceutical research 1992, 9, (10), 1243-1251.

96. LENNERNÄS, H.; LEE, I. D.; FAGERHOLM, U.; AMIDON, G. L. A residence⁷ time distribution analysis of the hydrodynamics within the intestine in man during a regional single⁷ pass perfusion with Loc⁷ I⁷ Gut: in⁷ vivo permeability estimation. Journal of pharmacy and pharmacology 1997, 49, (7), 682-686.

97. Lennernäs, H. Animal data: the contributions of the Ussing Chamber and perfusion systems to predicting human oral drug delivery in vivo. Advanced drug delivery reviews 2007, 59, (11), 1103-1120.

98. Söderholm, J.; Hedman, L.; Artursson, P.; Franzen, L.; Larsson, J.; Pantzar, N.; Permert, J.; Olaison, G. Integrity and metabolism of human ileal mucosa in vitro in the Ussing chamber. Acta physiologica Scandinavica 1998, 162, (1), 47-56.

99. Lampen, A.; Christians, U.; Gonschior, A. K.; Bader, A.; Hackbarth, I.; von Engelhardt, W.; Sewing, K. F. Metabolism of the macrolide immunosuppressant, tacrolimus, by the pig gut mucosa in the Ussing chamber. British journal of pharmacology 1996, 117, (8), 1730-1734.

100. Avdeef, A. The rise of PAMPA. Expert opinion on drug metabolism & toxicology 2005, 1, (2), 325-342.

101. Hidalgo, I. J.; Raub, T. J.; Borchardt, R. T. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 1989, 96, (3), 736-749.

102. Sjöberg, Å.; Lutz, M.; Tannergren, C.; Wingolf, C.; Borde, A.; Ungell, A.-L. Comprehensive study on regional human intestinal permeability and prediction of fraction absorbed of drugs using the Ussing chamber technique. European Journal of Pharmaceutical Sciences 2012.

103. Ungell, A.-L. In vitro absorption studies and their relevance to absorption from the GI tract. Drug development and industrial pharmacy 1997, 23, (9), 879-892.

104. Mueller, P.; Rudin, D. O.; Tien, H. T.; Wescott, W. C. Reconstitution of excitable cell membrane structure in vitro. Circulation 1962, 26, (5), 1167-1171.

105. Yee, S. In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man—fact or myth. Pharmaceutical research 1997, 14, (6), 763-766.

106. Artursson, P.; Borchardt, R. T. Intestinal drug absorption and metabolism in cell cultures: Caco-2 and beyond. Pharmaceutical research 1997, 14, (12), 1655-1658.

107. Pinto, M. Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol. cell 1983, 47, 323-330.

108. Lindmark, T.; Nikkilä, T.; Artursson, P. Mechanisms of absorption enhancement by medium chain fatty acids in intestinal epithelial Caco-2 cell monolayers. Journal of Pharmacology and Experimental Therapeutics 1995, 275, (2), 958-964.

109. Lindmark, T.; Kimura, Y.; Artursson, P. Absorption enhancement through intracellular regulation of tight junction permeability by medium chain fatty acids in Caco-2 cells. Journal of Pharmacology and Experimental Therapeutics 1998, 284, (1), 362-369.

68

110. Ölander, M.; Wiśniewski, J. R.; Matsson, P.; Lundquist, P.; Artursson, P. The proteome of filter-grown Caco-2 cells with a focus on proteins involved in drug disposition. Journal of pharmaceutical sciences 2016, 105, (2), 817-827.

111. Artursson, P.; Palm, K.; Luthman, K. Caco-2 monolayers in experimental and theoretical predictions of drug transport1. Advanced drug delivery reviews 2001, 46, (1-3), 27-43.

112. Hayeshi, R.; Hilgendorf, C.; Artursson, P.; Augustijns, P.; Brodin, B.; Dehertogh, P.; Fisher, K.; Fossati, L.; Hovenkamp, E.; Korjamo, T. Comparison of drug transporter gene expression and functionality in Caco-2 cells from 10 different laboratories. European Journal of Pharmaceutical Sciences 2008, 35, (5), 383-396.

113. Linnankoski, J.; Mäkelä, J.; Palmgren, J.; Mauriala, T.; Vedin, C.; Ungell, A. L.; Lazorova, L.; Artursson, P.; Urtti, A.; Yliperttula, M. Paracellular porosity and pore size of the human intestinal epithelium in tissue and cell culture models. Journal of pharmaceutical sciences 2010, 99, (4), 2166-2175.

114. Irvine, J. D.; Takahashi, L.; Lockhart, K.; Cheong, J.; Tolan, J. W.; Selick, H.; Grove, J. R. MDCK (Madin-Darby canine kidney) cells: A tool for membrane permeability screening. Journal of pharmaceutical sciences 1999, 88, (1), 28-33.

115. Collett, A.; Sims, E.; Walker, D.; He, Y.-L.; Ayrton, J.; Rowland, M.; Warhurst, G. Comparison of HT29-18-C1 and Caco-2 cell lines as models for studying intestinal paracellular drug absorption. Pharmaceutical research 1996, 13, (2), 216-221.

116. Ussing, H. H.; Zerahn, K. Active Transport of Sodium as the Source of Electric Current in the Short⁷ circuited Isolated Frog Skin. Acta physiologica Scandinavica 1951, 23, (2⁷ 3), 110-127.

117. Polentarutti, B. I.; Peterson, A. L.; Sjöberg, Å. K.; Anderberg, E. K. I.; Utter, L. M.; Ungell, A.-L. B. Evaluation of viability of excised rat intestinal segments in the Ussing chamber: investigation of morphology, electrical parameters, and permeability characteristics. Pharmaceutical research 1999, 16, (3), 446-454.

118. Ungell, A. L.; Nylander, S.; Bergstrand, S.; Sjöberg, Å.; Lennernäs, H. Membrane transport of drugs in different regions of the intestinal tract of the rat. Journal of pharmaceutical sciences 1998, 87, (3), 360-366.

119. Sutton, S. C.; Rinaldi, M.; Vukovinsky, K. Comparison of the gravimetric, phenol red, and 14C-PEG-3350 methods to determine water absorption in the rat single-pass intestinal perfusion model. AAPS pharmSci 2001, 3, (3), 93.

120. Brouwers, J.; Mols, R.; Annaert, P.; Augustijns, P. Validation of a differential in situ perfusion method with mesenteric blood sampling in rats for intestinal drug interaction profiling. Biopharmaceutics & drug disposition 2010, 31, (5⁷ 6), 278-285.

121. Modigliani, R.; Bernier, J. Absorption of glucose, sodium, and water by the human jejunum studied by intestinal perfusion with a proximal occluding balloon and at variable flow rates. Gut 1971, 12, (3), 184-193.

122. Modigliani, R.; Rambaud, J. C.; Bernier, J. J. The method of intraluminal perfusion of the human small intestine. I. Principle and technique. Digestion 1973, 9, (2), 176-92.

123. Modigliani, R.; Rambaud, J.; Bernier, J. Validation of the use of a tube with a proximal occlusive balloon for measurement of intestinal absorption in man. The American journal of digestive diseases 1978, 23, 720-722.

69

124. Lennernas, H.; Nilsson, D.; Aquilonius, S.; Ahrenstedt, O.; Knutson, L.; Paalzow, L. The effect of L⁷ leucine on the absorption of levodopa, studied by regional jejunal perfusion in man. British journal of clinical pharmacology 1993, 35, (3), 243-250.

125. Chen, M.-L.; Amidon, G. L.; Benet, L. Z.; Lennernas, H.; Lawrence, X. Y. The BCS, BDDCS, and regulatory guidances. Pharmaceutical research 2011, 28, (7), 1774-1778.

126. Stevens, H. N.; Wilson, C. G.; Welling, P. G.; Bakhshaee, M.; Binns, J. S.; Perkins, A. C.; Frier, M.; Blackshaw, E. P.; Frame, M. W.; Nichols, D. J. Evaluation of Pulsincap™ to provide regional delivery of dofetilide to the human GI tract. International journal of pharmaceutics 2002, 236, (1-2), 27-34.

127. Nyberg, L.; Månsson, W.; Abrahamsson, B.; Seidegård, J.; Borgå, O. A convenient method for local drug administration at predefined sites in the entire gastrointestinal tract: experiences from 13 phase I studies. european journal of pharmaceutical sciences 2007, 30, (5), 432-440.

128. Wilding, I.; Hirst, P.; Connor, A. Development of a new engineering-based capsule for human drug absorption studies. Pharmaceutical science & technology today 2000, 3, (11), 385-392.

129. Sjögren, E.; Dahlgren, D.; Roos, C.; Lennernäs, H. Human in vivo regional intestinal permeability: quantitation using site-specific drug absorption data. Molecular pharmaceutics 2015, 12, (6), 2026-2039.

130. Lipinski, C. A. Drug-like properties and the causes of poor solubility and poor permeability. Journal of pharmacological and toxicological methods 2000, 44, (1), 235-249.

131. Refsgaard, H. H.; Jensen, B. F.; Brockhoff, P. B.; Padkjær, S. B.; Guldbrandt, M.; Christensen, M. S. In silico prediction of membrane permeability from calculated molecular parameters. Journal of medicinal chemistry 2005, 48, (3), 805-811.

132. Serajuddin, A. T. Salt formation to improve drug solubility. Advanced drug delivery reviews 2007, 59, (7), 603-616.

133. Noyes, A. A.; Whitney, W. R. The rate of solution of solid substances in their own solutions. Journal of the American Chemical Society 1897, 19, (12), 930-934.

134. Sugano, K. Possible reduction of effective thickness of intestinal unstirred water layer by particle drifting effect. International journal of pharmaceutics 2010, 387, (1-2), 103-109.

135. Jinno, J.-i.; Kamada, N.; Miyake, M.; Yamada, K.; Mukai, T.; Odomi, M.; Toguchi, H.; Liversidge, G. G.; Higaki, K.; Kimura, T. Effect of particle size reduction on dissolution and oral absorption of a poorly water-soluble drug, cilostazol, in beagle dogs. Journal of controlled release 2006, 111, (1-2), 56-64.

136. Merisko-Liversidge, E.; Liversidge, G. G.; Cooper, E. R. Nanosizing: a formulation approach for poorly-water-soluble compounds. European Journal of Pharmaceutical Sciences 2003, 18, (2), 113-120.

137. Müller, R.; Jacobs, C.; Kayser, O. Nanosuspensions as particulate drug formulations in therapy: rationale for development and what we can expect for the future. Advanced drug delivery reviews 2001, 47, (1), 3-19.

138. Hens, B.; Brouwers, J.; Corsetti, M.; Augustijns, P. Gastrointestinal behavior of nano-and microsized fenofibrate: in vivo evaluation in man and in vitro simulation by assessment of the permeation potential. European Journal of Pharmaceutical Sciences 2015, 77, 40-47.

70

139. Sigfridsson, K.; Lundqvist, A. J.; Strimfors, M. Particle size reduction for improvement of oral absorption of the poorly soluble drug UG558 in rats during early development. Drug development and industrial pharmacy 2009, 35, (12), 1479-1486.

140. Lai, S. K.; O'Hanlon, D. E.; Harrold, S.; Man, S. T.; Wang, Y.-Y.; Cone, R.; Hanes, J. Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus. Proceedings of the National Academy of Sciences 2007, 104, (5), 1482-1487.

141. Khanvilkar, K.; Donovan, M. D.; Flanagan, D. R. Drug transfer through mucus. Advanced drug delivery reviews 2001, 48, (2-3), 173-193.

142. Einstein, A. On the motion of small particles suspended in liquids at rest required by the molecular-kinetic theory of heat. Annalen der physik 1905, 17, 549-560.

143. Roos, C.; Westergren, J.; Dahlgren, D.; Lennernäs, H.; Sjögren, E. Mechanistic modelling of intestinal drug absorption–the in vivo effects of nanoparticles, hydrodynamics, and colloidal structures. European Journal of Pharmaceutics and Biopharmaceutics 2018.

144. Aungst, B. J. Intestinal permeation enhancers. Journal of pharmaceutical sciences 2000, 89, (4), 429-442.

145. Aungst, B. J. Optimizing oral bioavailability in drug discovery: an overview of design and testing strategies and formulation options. Journal of pharmaceutical sciences 2017, 106, (4), 921-929.

146. Maher, S.; Mrsny, R. J.; Brayden, D. J. Intestinal permeation enhancers for oral peptide delivery. Advanced drug delivery reviews 2016, 106, 277-319.

147. Branchu, S.; Rogueda, P. G.; Plumb, A. P.; Cook, W. G. A decision-support tool for the formulation of orally active, poorly soluble compounds. european journal of pharmaceutical sciences 2007, 32, (2), 128-139.

148. Dahlgren, D.; Roos, C.; Lundqvist, A.; Tannergren, C.; Langguth, P.; Sjöblom, M.; Sjögren, E.; Lennernäs, H. Preclinical Effect of Absorption Modifying Excipients on Rat Intestinal Transport of Model Compounds and the Mucosal Barrier Marker 51Cr-EDTA. Molecular pharmaceutics 2017, 14, (12), 4243-4251.

149. Nilsson, A.; Peric, A.; Strimfors, M.; Goodwin, R. J.; Hayes, M. A.; Andrén, P. E.; Hilgendorf, C. Mass spectrometry imaging proves differential absorption profiles of well-characterised permeability markers along the crypt-villus axis. Scientific reports 2017, 7, (1), 6352.

150. Yoon, I.-S.; Choi, M.-K.; Kim, J. S.; Shim, C.-K.; Chung, S.-J.; Kim, D.-D. Pharmacokinetics and first-pass elimination of metoprolol in rats: contribution of intestinal first-pass extraction to low bioavailability of metoprolol. Xenobiotica; the fate of foreign compounds in biological systems 2011, 41, (3), 243-251.

151. Zur, M.; Gasparini, M.; Wolk, O.; Amidon, G. L.; Dahan, A. The low/high BCS permeability class boundary: physicochemical comparison of metoprolol and labetalol. Molecular pharmaceutics 2014, 11, (5), 1707-1714.

152. Lozoya-Agullo, I.; Zur, M.; Beig, A.; Fine, N.; Cohen, Y.; González-Álvarez, M.; Merino-Sanjuán, M.; González-Álvarez, I.; Bermejo, M.; Dahan, A. Segmental-dependent permeability throughout the small intestine following oral drug administration: Single-pass vs. Doluisio approach to in-situ rat perfusion. International journal of pharmaceutics 2016, 515, (1-2), 201-208.

71

153. Zakeri-Milani, P.; Barzegar-Jalali, M.; Tajerzadeh, H.; Azarmi, Y.; Valizadeh, H. Simultaneous determination of naproxen, ketoprofen and phenol red in samples from rat intestinal permeability studies: HPLC method development and validation. Journal of pharmaceutical and biomedical analysis 2005, 39, (3-4), 624-630.

154. Salphati, L.; Childers, K.; Pan, L.; Tsutsui, K.; Takahashi, L. Evaluation of a single⁷ pass intestinal⁷ perfusion method in rat for the prediction of absorption in man. Journal of pharmacy and pharmacology 2001, 53, (7), 1007-1013.

155. Lamprecht, A.; Schäfer, U.; Lehr, C.-M. Size-dependent bioadhesion of micro-and nanoparticulate carriers to the inflamed colonic mucosa. Pharmaceutical research 2001, 18, (6), 788-793.

156. Shono, Y.; Jantratid, E.; Kesisoglou, F.; Reppas, C.; Dressman, J. B. Forecasting in vivo oral absorption and food effect of micronized and nanosized aprepitant formulations in humans. European Journal of Pharmaceutics and Biopharmaceutics 2010, 76, (1), 95-104.

157. Wu, Y.; Loper, A.; Landis, E.; Hettrick, L.; Novak, L.; Lynn, K.; Chen, C.; Thompson, K.; Higgins, R.; Batra, U. The role of biopharmaceutics in the development of a clinical nanoparticle formulation of MK-0869: a Beagle dog model predicts improved bioavailability and diminished food effect on absorption in human. International journal of pharmaceutics 2004, 285, (1-2), 135-146.

158. Majumdar, A. K.; Howard, L.; Goldberg, M. R.; Hickey, L.; Constanzer, M.; Rothenberg, P. L.; Crumley, T. M.; Panebianco, D.; Bradstreet, T. E.; Bergman, A. J. Pharmacokinetics of aprepitant after single and multiple oral doses in healthy volunteers. The Journal of Clinical Pharmacology 2006, 46, (3), 291-300.

159. Kesisoglou, F.; Panmai, S.; Wu, Y. Nanosizing—oral formulation development and biopharmaceutical evaluation. Advanced drug delivery reviews 2007, 59, (7), 631-644.

160. David, D.; Carl, R.; Pernilla, J.; Christer, T.; Anders, L.; Peter, L.; Markus, S.; Erik, S.; Hans, L. The effects of three absorption-modifying critical excipients on the in vivo intestinal absorption of six model compounds in rats and dogs. International journal of pharmaceutics 2018.

161. Stappaerts, J.; Wuyts, B.; Tack, J.; Annaert, P.; Augustijns, P. Human and simulated intestinal fluids as solvent systems to explore food effects on intestinal solubility and permeability. European Journal of Pharmaceutical Sciences 2014, 63, 178-186.

162. Bannwarth, B.; Lapicque, F.; Netter, P.; Monot, C.; Tamisier, J.; Thomas, P.; Royer, R. The effect of food on the systemic availability of ketoprofen. European journal of clinical pharmacology 1988, 33, (6), 643-645.

163. Melander, A.; Stenberg, P.; Liedholm, H.; Schersten, B.; Wåhlin-Boll, E. Food-induced reduction in bioavailability of atenolol. European journal of clinical pharmacology 1979, 16, (5), 327-330.

164. Melander, A.; Danielson, K.; Schersten, B.; Wåhlin, E. Enhancement of the bioavailability of propranolol and metoprolol by food. Clinical Pharmacology & Therapeutics 1977, 22, (1), 108-112.

165. Regårdh, C. G.; Borg, K. O.; Johansson, R.; Johnsson, G.; Palmer, L. Pharmacokinetic studies on the selectiveβ 1-receptor antagonist metoprolol in man. Journal of pharmacokinetics and biopharmaceutics 1974, 2, (4), 347-364.

72

166. McLean, A. J.; McNamara, P. J.; Gibaldi, M.; Lalka, D. Food, splanchnic blood flow, and bioavailability of drugs subject to first⁷ pass metabolism. Clinical Pharmacology & Therapeutics 1978, 24, (1), 5-10.

167. Lawrence, X. Y.; Amidon, G. L. A compartmental absorption and transit model for estimating oral drug absorption. International journal of pharmaceutics 1999, 186, (2), 119-125.

168. Ulm, E. H. Enalapril maleate (MK-421), a potent, nonsulfhydryl angiotensin-converting enzyme inhibitor: absorption, disposition, and metabolism in man. Drug metabolism reviews 1983, 14, (1), 99-110.

169. Dahlgren, D.; Roos, C.; Lundqvist, A.; Tannergren, C.; Sjöblom, M.; Sjögren, E.; Lennernäs, H. Effect of absorption-modifying excipients, hypotonicity, and enteric neural activity in an in vivo model for small intestinal transport. International journal of pharmaceutics 2018.

170. Nylander, O.; Kvietys, P.; Granger, D. N. Effects of hydrochloric acid on duodenal and jejunal mucosal permeability in the rat. American Journal of Physiology-Gastrointestinal and Liver Physiology 1989, 257, (4), G653-G660.

171. Borgå, O.; Borgå, B. Serum protein binding of nonsteroidal antiinflammatory drugs: a comparative study. Journal of pharmacokinetics and biopharmaceutics 1997, 25, (1), 63-77.

172. Jantratid, E.; Janssen, N.; Reppas, C.; Dressman, J. B. Dissolution media simulating conditions in the proximal human gastrointestinal tract: an update. Pharmaceutical research 2008, 25, (7), 1663.

173. Riethorst, D.; Baatsen, P.; Remijn, C.; Mitra, A.; Tack, J.; Brouwers, J.; Augustijns, P. An in-depth view into human intestinal fluid colloids: intersubject variability in relation to composition. Molecular pharmaceutics 2016, 13, (10), 3484-3493.

174. Fatouros, D. G.; Walrand, I.; Bergenstahl, B.; Müllertz, A. Colloidal structures in media simulating intestinal fed state conditions with and without lipolysis products. Pharmaceutical research 2009, 26, (2), 361.

175. Augustijns, P.; Wuyts, B.; Hens, B.; Annaert, P.; Butler, J.; Brouwers, J. A review of drug solubility in human intestinal fluids: implications for the prediction of oral absorption. European Journal of Pharmaceutical Sciences 2014, 57, 322-332.

176. Dressman, J. B.; Reppas, C. In vitro–in vivo correlations for lipophilic, poorly water-soluble drugs. European journal of pharmaceutical sciences 2000, 11, S73-S80.

177. Gradauer, K.; Nishiumi, A.; Unrinin, K.; Higashino, H.; Kataoka, M.; Pedersen, B. L.; Buckley, S. T.; Yamashita, S. Interaction with mixed micelles in the intestine attenuates the permeation enhancing potential of alkyl-maltosides. Molecular pharmaceutics 2015, 12, (7), 2245-2253.

178. Anderberg, E. K.; Nyström, C.; Artursson, P. Epithelial transport of drugs in cell culture. VII: Effects of pharmaceutical surfactant excipients and bile acids on transepithelial permeability in monolayers of human intestinal epithelial (Caco⁷ 2) cells. Journal of pharmaceutical sciences 1992, 81, (9), 879-887.

179. Anderberg, E. K.; Artursson, P. Epithelial transport of drugs in cell culture. VIII: Effects of sodium dodecyl sulfate on cell membrane and tight junction permeability in human intestinal epithelial (Caco⁷ 2) cells. Journal of pharmaceutical sciences 1993, 82, (4), 392-398.

180. Aungst, B. J.; Saitoh, H.; Burcham, D. L.; Huang, S.-M.; Mousa, S. A.; Hussain, M. A. Enhancement of the intestinal absorption of peptides and nonpeptides. Journal of controlled release 1996, 41, (1-2), 19-31.

73

181. Lecluyse, E. L.; Sutton, S. C.; Fix, J. A. In vitro effects of long-chain acylcarnitines on the permeability, transepithelial electrical resistance and morphology of rat colonic mucosa. Journal of Pharmacology and Experimental Therapeutics 1993, 265, (2), 955-962.

182. NAKANISHI, K.; MASADA, M.; NADAI, T. Effect of pharmaceutical adjuvants on the rectal permeability of drugs. III. Effect of repeated administration and recovery of the permeability. Chemical and pharmaceutical bulletin 1983, 31, (11), 4161-4166.

183. KIMURA, T.; NAKAMURA, J.; SEZAKI, H. Effect of synthetic surfactants on drug absorption in the presence of a physiologic surfactant, sodium taurocholate, in rats. Journal of pharmacobio-dynamics 1985, 8, (8), 661-668.

184. Brayden, D. J.; Walsh, E. Efficacious intestinal permeation enhancement induced by the sodium salt of 10-undecylenic acid, a medium chain fatty acid derivative. The AAPS journal 2014, 16, (5), 1064-1076.

185. Tomita, M.; Hayashi, M.; Horie, T.; Ishizawa, T.; Awazu, S. Enhancement of colonic drug absorption by the transcellular permeation route. Pharmaceutical research 1988, 5, (12), 786-789.

 

Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 260

Editor: The Dean of the Faculty of Pharmacy

A doctoral dissertation from the Faculty of Pharmacy, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofPharmacy. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Pharmacy”.)

Distribution: publications.uu.seurn:nbn:se:uu:diva-363908

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2018