Caco-2 cell cultures in the assessment of intestinal ...ethesis.helsinki.fi/julkaisut/far/farma/vk/laitinen/caco2cel.pdf · Caco-2 cell cultures in the assessment of intestinal absorption:

Drug Discovery and Development Technology Center

Division of Biopharmaceutics and Pharmacokinetics

Faculty of Pharmacy

University of Helsinki

Finland

Caco-2 cell cultures in the assessment of intestinal absorption:

Effects of some co-administered drugs and natural compounds in biological

matrices

by

Leena Laitinen

Academic Dissertation

To be presented, with the permission of the Faculty of Pharmacy of the University of Helsinki,

for public criticism in Lecture hall 2 at Viikki Infocentre (Viikinkaari 11)

on June 3rd, 2006, at 12 noon

Helsinki 2006

Supervisors: Docent Ann Marie Kaukonen Drug Discovery and Development Technology Center Division of Pharmaceutical Technology Faculty of Pharmacy University of Helsinki Finland Professor Jouni Hirvonen Division of Pharmaceutical Technology Faculty of Pharmacy University of Helsinki Finland Professor Martti Marvola Division of Biopharmaceutics and Pharmacokinetics Faculty of Pharmacy University of Helsinki Finland Professor, Docent Pia Vuorela Division of Pharmaceutical Biology Faculty of Pharmacy University of Helsinki Finland Reviewers: Dr. Marjukka Suhonen Department of Pharmaceutics Faculty of Pharmacy University of Kuopio Finland Dr. Staffan Tavelin Department of Chemistry Faculty of Science and Technology University of Umeå Sweden Opponent: Professor Albin Kristl Department of Biopharmacy and Pharmacokinetics Faculty of Pharmacy University of Ljubljana Slovenia © Leena Laitinen 2006 ISBN 952-10-3124-7 (print) ISBN 952-10-3125-5 (pdf, http://ethesis.helsinki.fi/) ISSN 1795-7079 Helsinki University Printing House Helsinki 2006 Finland

To my family, Mira, Mikko and Eino

ABSTRACT Several different in vitro absorption models are used in the screening of new drug candidates. One of them is the Caco-2 model, a widely used in vitro model for small intestinal absorption. Caco-2 cells, which originate from a colon carcinoma, differentiate spontaneously to cells that resemble mature small intestinal enterocytes and express carrier proteins similar to the small intestine, and can therefore be used for the assessment of active transport processes during intestinal absorption. Due to variation in cell line differentiation and selection of sub-populations, permeability data obtained from different laboratories is seldom directly comparable. Hence, the use of several drugs or compounds with known permeability characteristics are recommended for model standardisation and the use of internal standards in every experiment could offer a solution to the problem.

In this study, the simultaneous use of several drugs as internal standards was evaluated. Drugs with different permeability characteristics (high and low permeability, passive and active transport and active efflux) were used to detect their possible effects on cell viability, monolayer integrity and possible interactions during active transport processes. Five to ten drugs in one experiment at individual 50 µM concentrations did not cause problems in cell viability (MTT test) or monolayer integrity (transepithelial electrical resistance (TEER) measurements, and mannitol diffusion test). Drugs with passive permeability can be included in cocktails; no interactions between them are expected. Drugs, which occupy the same binding sites of a transport protein, cannot be used simultaneously.

After validation of the use of several drugs simultaneously as internal standards, the usefulness of the method was further probed by testing the possible interactions during absorption between drugs and plant extracts that are used as food supplements, functional foods, or natural laxatives. These extracts contain several active compounds, such as flavonoids, alkyl gallates, or anthraquinones, which are able to partition into the cell membranes and thus affect e.g. the fluidity of the membranes, or diffuse across the cell monolayers, either by using active transport mechanisms or passively. The effects of different concentrated plant extracts, which may contain high concentrations of several different active compounds, are difficult to predict, because their interactions may be mediated via several active transport proteins, such as OCT, MDR1 and different MRP´s, or additionally via the effects on the rigidity of cell membranes.

Whereas several food-drug interactions have often been attributed to the inhibition of drug metabolizing enzymes, information regarding the effects of food components on transporters during absorption, distribution and excretion of drugs is still limited. Caco-2 cell monolayers, when expressing active transport and efflux proteins, are therefore suitable as in vitro model for this type of studies.

_____________________________________________________________________________

TABLE OF CONTENTS TABLE OF CONTENTS ............................................................................................................................. I

LIST OF ABBREVIATIONS AND ACRONYMS ...................................................................................II

LIST OF ORIGINAL PUBLICATIONS .................................................................................................III

1. INTRODUCTION ....................................................................................................................................1

2. THEORY AND REVIEW OF THE LITERATURE ............................................................................3 2.1. BARRIERS TO DRUG ABSORPTION ..............................................................................................................3 2.1.1. Unstirred water layer ..................................................................................................................................3 2.1.2. Intestinal epithelium ....................................................................................................................................4

2.2. ABSORPTION OF DRUGS .................................................................................................................................5

2.3. METHODS FOR PREDICTING INTESTINAL ABSORPTION .......................................................................7 2.3.1. In vitro biophysical (cell free) methods .......................................................................................................8 2.3.2. In vitro biological (cell based) methods ......................................................................................................9

2.4 INTERACTIONS DURING ABSORPTION .....................................................................................................16 2.4.1. Interactions between drugs ........................................................................................................................16 2.4.2. Interactions between drugs and plant extracts ..........................................................................................18

3. AIMS OF THE STUDY ........................................................................................................................20

4. EXPERIMENTAL ................................................................................................................................21 4.1. MATERIALS .....................................................................................................................................................21

4.1.1. Permeation markers (drugs and compounds used) (I-V)...........................................................................21 4.1.2. Natural compounds and derivatives (IV)...................................................................................................21 4.1.3. Intestinal epithelial cells (I-V)....................................................................................................................21

4.2. METHODS ........................................................................................................................................................22 4.2.1. Plant material and extraction (III) ............................................................................................................22 4.2.2. Preparation of the permeation marker solutions and extracts (I-V)..........................................................24 4.2.3. Evaluation of cytotoxicity (I-V)..................................................................................................................24 4.2.4. Evaluation of monolayer integrity (I-V).....................................................................................................25 4.2.5. Transepithelial permeability (I-V)..............................................................................................................26 4.2.6. Analytical methods (I-V).............................................................................................................................27 4.2.7. Determination of permeability coefficients (I-V)........................................................................................27

5. RESULTS AND DISCUSSION ............................................................................................................28

5.1. EFFECTS ON MONOLAYER VIABILITY AND INTEGRITY ......................................................................28 5.1.1. Cytotoxicity(I-V).........................................................................................................................................28 5.1.2. Integrity of Caco-2 monolayers (I-V).........................................................................................................30

5.2. PERMEABILITY EXPERIMENTS ...................................................................................................................33 5.2.1. The effects of cocktail dosing on individual drug permeability (I, II)........................................................33 5.2.2. Food supplements and food fractions (III, IV)...........................................................................................40 5.2.3. Anthranoid laxatives (V).............................................................................................................................47

6. CONCLUSIONS ....................................................................................................................................49

ACKNOWLEDGEMENTS ........................................................................................................................51

REFERENCES.............................................................................................................................................53

Original publications I-V

I

_____________________________________________________________________________

LIST OF ABBREVIATIONS AND ACRONYMS

AP-BL apical-to-basolateral

API atmospheric pressure ionisation

APPI atmospheric pressure photoionisation

BBMV brush border membrane vesicles

BCRP Breast cancer resistance protein (=MXR)

BL-AP basolateral-to-apical

CHO chinese harmster ovary cells

DMEM Dulbecco´s modified Eagle´s medium

DMSO dimethyl sulphoxide

DOPC dioleylphosphatidylcholine

DPPC dipalmitoylphosphatidylcholine

ESI electropray ionisation

GI tract gastrointestinal tract

HBSS Hank´s balanced salt solution

HEPES N-[2-hydroxyethyl]piperazine-N´-[-2-ethanesufonic acid]

hPepT1 human intestinal small peptide carrier

HPLC high performance liquid chromatography

IAM immobilized artificial membrane

Kd partition coefficient

LC/MS/MS liquid chromatography coupled with tandem mass spectrometry

LDH lactate dehydrogenase

MDCK Madin Darby canine kidney cells

MDR1 multidrug resistance protein 1 (=P-glycoprotein)

MRP multidrug resistance accociated protein

MTT 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide

OATP organic anion transporting polypeptides

PAMPA parallel artificial membrane permeability assay

Papp apparent permeability coefficient

Pgp P-glycoprotein (= MDR1)

POPC 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine

Rho123 rhoadamine123

TEER transepithelial electrical resistance

UGT uridine-5′-diphospho-glucuronosyltransferase enzymes UWL unstirred water layer

II

_____________________________________________________________________________

LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following original publications referred to the text by the Roman

numerals I - V.

I Laitinen, L., Kangas, H., Kaukonen, AM., Hakala, K., Kotiaho, T., Kostiainen, R.,

and Hirvonen J., 2003. N-in-one permeability studies of heterogeneous sets of

compounds across Caco-2 cell monolayers. Pharmaceutical Research 20, 187 - 197.

II Hakala, K., Laitinen, L., Kaukonen, A.M., Hirvonen, J., Kostiainen, R., and Kotiaho,

T., 2003. Development of LC/MS/MS methods for cocktail dosed Caco-2 samples

using atmospheric pressure photoionization and electrospray ionization. Analytical

Chemistry 75, 5969-5977.

III Laitinen, L., Tammela, P., Galkin, A., Vuorela, H., Marvola, M., and Vuorela, P.,

2004. Effects of extracts of commonly consumed food supplements and food

fractions on the permeability of drugs across Caco-2 cell monolayers.

Pharmaceutical Research 21, 1904 - 1916.

IV Tammela, P., Laitinen, L., Galkin, A., Wennberg, T., Heczko, R., Vuorela, H., Slotte,

P., and Vuorela, P., 2004. Permeability characteristics and membrane affinity of

flavonoids and alkyl gallates in Caco-2 cells and in phospholipid vesicles. Archives

of Biochemistry and Biophysics 425, 193 - 199.

V Laitinen, L., Takala, E., Vuorela, H., Vuorela, P., Kaukonen, AM., and Marvola, M.,

2005. Anthranoid laxatives influence the absorption of poorly permeable drugs in

human intestinal cell culture model (Caco-2), submitted.

Reprinted with the permission of the publishers.

III

INTRODUCTION _____________________________________________________________________________

1. INTRODUCTION

The epithelium of the small intestine is a highly dynamic system, being spatially separated into

proliferative, differentiating, and functional cells in the lower and upper crypt regions and on the

villi, respectively. Partly because of that, normal intestinal epithelial cell lines are not available.

Therefore, most of our knowledge about processes during absorption has been derived from

experimental animals (Kédinger et al. 1987, Evans et al. 1994) or human colon cancer cell lines

(Rousset 1986, Whitehead and Watson, 1997). Animal models offer limited information about

absorption in humans; for example, major differences in intestinal cell differentiation in humans

and rodents have been detected (Simon-Assman et al. 1994). Additionally, major differences in

animal and human intestinal function, such as luminal pH conditions and leakiness of the small

intestine, are observed. Therefore, information obtained from experimental animal models

cannot always be transposed to human situation. Many efforts of producing longliving cultures

of fully differentiated or proliferative human intestinal epithelial cells have been undertaken, but

the major problems, i.e. low viability and short life-time, are still not overcome.

Because in vivo studies performed with humans and laboratory animals are expensive,

time consuming and often even unethical, in vitro methods, as accurate as possible, are needed

in screening of new drug candidates. Immortalised, often of cancer origin, animal and human

cell cultures have been used for estimation and prediction of human drug absorption. Several

possible in vitro human cell models are available for this purpose, one of which is the Caco-2

cell model, a well characterised cell line. According to Biopharmaceutics Classification System

(BCS) and FDA approval, Caco-2 cells can be used as a screening method for new drug

candidates during drug discovery and development (Guidance for Industry, FDA 2000,

Artursson and Borchardt 1997, Rubas et al. 1996). For the suitability and reliability of the

method, permeability of several model compounds with known intestinal absorption in humans

has to be demonstrated. FDA recommends the use of compounds with high, low, and zero

permeability, passive and active transport, and use of efflux markers for this purpose. The

simultanous use of model compounds requires that they do not express cytotoxicity, do not

interact with each other during permeation, and that they are easily detected. Therefore, the use

of different sets of model compounds has to be validated before the actual experiments with

drug candidates can be performed.

Because of an increased interest in preventive health care, the food industry produces

food supplements and food products, fortified with different fractions of herb extracts, berries

1

INTRODUCTION _____________________________________________________________________________

and other plant materials known to possess beneficial effects. These might contain several active

compounds, such as flavonoids, that are able to interact with co-administered drugs by affecting

cell membranes, thus altering the barrier function of the lipid bilayer, or active transport

mechanisms (Saija et al. 1995, Spahn-Langguth and Langguth 2001, Vaidyanathan and Walle

2001, Walle 2004). Additionally, several potent drugs of natural origin, such as anthranoid

laxatives, are widely used. Senna leaves contain several active compounds, not only dianthrones,

but also anthraquinones, flavonoids and phenolic acids. Anthrones and anthraquinones, that are

able to interact with intestinal epithelium leading to accelerated intestinal transit and changes in

water absorption and excretion across the intestinal wall, may affect absorption of drugs when

ingested simultaneously. Because people often use food supplements and natural drugs intended

for preventive health care simultaneously with other medication, it is important to study the

possible effects of the active compounds in these plant extracts on absorption of drugs.

In this thesis, intestinal epithelial cell monolayers, Caco-2, are evaluated as an in vitro

model for simultaneous use of several drugs during one experiment. Seven different sets of drug

cocktails are probed and their effects on individual drug permeabilities across cell monolayers

are evaluated. Their effects on monolayer integrity and viability are also assessed. The method is

then further evaluated by testing the effects of different plant extracts and natural products

containing several active compounds on permeability of some commonly used drugs and marker

molecules. The absorption behaviour of several compounds present in plants is also evaluated.

2

THEORY AND REVIEW OF THE LITERATURE _____________________________________________________________________________

2. THEORY AND REVIEW OF THE LITERATURE 2.1. BARRIERS TO DRUG ABSORPTION 2.1.1. Unstirred water layer (UWL)

Permeability of the drugs may be affected by several physiological features at the site of

absorption. Before the compound is able to diffuse across the absorptive cells, it has to diffuse

across a mucus layer adjacent to the enterocytes (Figure 1). This mucus layer possesses a

hydrogel character and is composed mainly of mucin molecules (large glycoproteins, net

negative charge that forms the gel structure of mucus) secreted from goblet cells, and water.

These mucin molecules form large polymeric complexes by disulphide bonds. Additionally, the

mucus layer contains lipids and cellular proteins, luminal and cellular debris and secreted

immunoglobulins and sloughed cells (Specian and Oliver, 1991, Wikman Larhed et al. 1998).

Figure 1. The structure of intestinal epithelial cells. Above the epithelial cells, in the

intestinal lumen, an acidic microclimate is formed by the mucus layer and the glycocalyx. Modified from Avdeef (2001).

Characteristic for the apical surfaces of the absorptive cells are the microvilli.

Glycoproteins and oligosaccharide side branches are protruding into the luminal fluid. This

environment close to the epithelial surface is called the glycocalyx. The glycocalyx and mucus

layer together form a major part of unstirred water layer (UWL) in the vicinity of the absorbing

cells in the intestinal lumen and thus form a pre-epithelial diffusion barrier for rapidly

permeating compounds (Figure 1) (Lennernäs 1998). The pH of the UWL is between 5.2 and

6.2 and it is regulated independently of the variable luminal pH. According to Shiau et al.

3


(1985), the mucus layer plays the main role in regulating the pH of the epithelial cell surface. It

has been proposed that the amphiphilic character of the mucus is necessary to the maintenance

of an acidic microclimate at the mucosal surface.

2.1.2. Intestinal epithelium

The main barrier for drug absorption is the epithelium of the intestine (Figure 2). This

epithelium is a one cell thick layer and consists of several different types of cells, of which the

absorptive epithelial cells (enterocytes) are most abundant. These cells are formed in the crypts

and differentiate during migration to the tips of the villi over a period of 3-5 days. During the

differentiation, the cells adopt a columnar appearance and develop microvilli on their apical

surfaces and tight junctions between the neighbouring cells (Figure 2a,b). When the

differentiated enterocytes have reached the tips of the villi, they are sloughed off into the lumen

of the intestine (Madara and Trier 1994). In addition, a number of other cell types are present in

the villi: goblet cells (production of mucin) and M-cells in the Peyer´s patches (Madara and

Trier 1994).

a b Figure 2 Schematic structure of an intestinal villus covered by a monolayer of absorptive epithelial cells (enterocytes) and mucus producing cells (goblet cells) (a), and the epithelial cell junctional complex with a tight junction, an adherent junction and a desmosome (b).

These cells together form a tight protective barrier through several different intercellular

junctions (Figure 2b). The junctional complex is comprised of several structures (Denker and

4


Nigam 1998): desmosomes that attach cells together by intermediate filaments; adherent

junctions, which keep adjacent cells together via calcium-dependent cell-cell adhesion

molecules that are linked to actin and myosin filaments; and the tight junctions (Figure 2b).

Additionally, gap junctions, which mediate communication between the adjacent cells and allow

small molecules to move between neighbouring cells, are present in the intestinal epithelium

(Kumar and Gilula 1996). The tight junctions, that are the most apical components of the

junctional complex, are responsible for the tightness of the epithelium.

2.2. ABSORPTION OF DRUGS

Drug permeability across the intestinal epithelium is divided into passive and active

mechanisms. Passive diffusion can occur either through the epithelial cells (transcellularly) or

via the intercellular spaces (paracellularly) (Figure 3A). A paracellularly-permeating drug is

usually polar or highly water soluble and small (Hidalgo, 1996). The transcellular pathway is the

most common route for drug permeability, since the cellular absorbing surface is over 1000

times greater than the area of the paracellular spaces (Pappenheimer and Reiss 1987).

Figure 3. Schematic figure of intestinal epithelium as a selective barrier against the entry of compounds to circulation. A: passive trans- and paracellular diffusion; B: carrier mediated absorption at apical and basolateral membranes; C: active efflux transporter on apical membrane, acting during absorption; D: active efflux transporter on apical membrane, offering an additional route for drug clearance from the circulation; E: intracellular metabolising enzymes localized inside the enterocytes, possibly combined with an active efflux transporter on apical and basolateral membranes. Modified from Chan et al. 2004.

Transcellular permeability requires that the drug molecule is able to partition between the

aqueous contents of intestinal lumen, the phospholipid bilayer of the apical cell membranes, the

aqueous contents of the enterocytes, and again, the phospholipid bilayer of the basolateral cell

membranes. Finally, the drug molecule has to be hydrophilic enough to be able to leave the cell

membranes when entering the circulation. Very lipophilic molecules may remain solubilized in

the cell membrane (Karlson and Artursson 1992, Madara and Trier 1994). Most drugs permeate

5


mainly transcellularly, but a variable contribution of the paracellular route is possible (Pade and

Stavchansky 1997, Flanagan et al. 2002a,b). As mentioned before, the capacity of the

paracellular route is limited because the spaces between absorbing enterocytes are small

compared to the total absorptive area (Pappenheimer and Reiss, 1987).

Concentration (µM)

0 500 1000 1500 2000 2500 3000

Flux

(nm

ol/h

cm

2 )

0

5

10

15

20total flux

active transport

passive flux

Figure 4. The concentration dependent permeability of an actively transported compound. Total permeability (•-• ); passive flux (....) and the active part (--) of the total permeability. The concentration dependent saturation of active transport is characteristic.

Active transport of drugs and nutrients is mediated by several membrane transporter

proteins located in the cell membranes (Figure 3B, C, D, E). The membrane transporter carriers

can be classified based on their energy requirements to facilitated diffusion, primary active and

secondary active transport. Facilitated diffusion does not require energy for its function. Primary

active transport is an energy demanding process. The needed energy is gained from hydrolysis

of ATP to ADP, where energy is liberated from the high-energy phosphate bond. Secondary

active transport uses energy from ion gradients (mostly Ca2+, Na2+, and H+ -gradients) across the

cell membranes generated by primary active ion pumps (for example Na+/K+-ATPase), and are

also called symporters or antiporters. Characteristic for active transport is the saturability of the

carrier protein (Figure 4). Interaction between the drug and a specific carrier-protein leads to

higher permeation than with passive diffusion. Several absorptive transport systems with the

primary function of transporting nutrients (amino acids, oligopeptides, monocarboxylic acids,

monosaccharides, organic anions and cations, bile acids and severeal water soluble vitamins) are

present in the small intestine (Steffansen et al. 2004). These saturable carriers show sometimes

pH dependency due to proton co-transport, and their transport capacity varies considerably in

different parts of the intestine (Hidalgo and Li 1996, Putnam et al. 2002b).

6


According to the barrier function of the intestinal mucosa, the access of more or less

cytotoxic substances into the animal body is limited via the GI tract. For that reason, the

intestinal epithelium contains a number of secretory systems (Figure 3C,D,E) such as multidrug

resistance protein 1 (MDR1, P-glycoprotein, P-gp) and multidrug resistance associated proteins

(MRP), which modulate the absorption of a variety of substances. Both systems belong to the

superfamily of ATP-binding cassette (ABC) membrane transport proteins (Gottesman and

Pastan 1993, Fujita et al. 1997, Lorico et al. 1997).

2.3. METHODS FOR PREDICTING INTESTINAL ABSORPTION

The most accurate method for determination of intestinal drug absorption is to measure the

disappearance of the drug from the GI tract, and later, the appearance of the drug in the portal

blood. These methods offer information about the fraction of drug absorbed across the intestinal

wall and possibly about the amount of metabolism during absorption. Animal in vitro, in situ

and in vivo techniques are available for these kinds of studies (Amidon et al. 1988, Stewart et al.

1995). In addition, human “in situ” techniques (Loc-I-Gut® and Loc-I-Col) are available for

studies where drug absorption in humans has to be evaluated (Lennernäs et al. 1992, Lennernäs

et al. 1995). In contrast to earlier open and semi-open small intestinal and colonic human

perfusion techniques, these methods provide more controlled conditions for the experiments; for

example standardised length of the intestinal segment, and complete recovery of a non-absorbed

marker molecule.

screen order

- methods based on physico-chemical propterties of the drug

- cell assays

- tissue assays

- whole animal assays

INCREASED COMPLEXITY

Figure 5. Methods for assessment of per oral drug absorption.

Even though above mentioned in vivo absorption experiments give the most

comprehensive results, simpler in vitro and in situ predictive methods are important when

intestinal absorption of a new drug candidate has to be clarified, especially during drug

7


development and screening of large molecule libraries (Figure 5). The use of these simpler

methods requires that these applications mimic closely enough the in vivo conditions of humans

and their predictive capacity is validated.

2.3.1. In vitro biophysical (cell free) methods

Since most drugs are absorbed by passive diffusion, valuable information about the permeability

across the GI epithelium can be gained from experiments performed with non-living material.

Based on physicochemical parameters of a drug, such as aqueous solubility, molecular weight,

surface charge and conformational flexibility, as well as partition coefficient log P or

distribution coefficient log D, that reflect partition into the lipid phase, and polar surface area,

passive permeability characteristics can often be predicted (Burton et al. 1996, Wils et al. 1994,

Palm et al. 1997, Camenisch et al. 1998, Avdeef 2001). A good correlation between log D and

drug permeability across epithelial cell monolayers and absorption in mice have been

determined for some drugs (Ungell 1997). To predict the behaviour of a disperse group of

compounds by using a single above mentioned parameter is yet not possible.

The simplest in vitro method for mimicking the absorption is the use of phospholipid

vesicles. These vesicles are prepared to mimic the outer leaflet of a lipid bilayer of intestinal

enterocytes. The main phospholipid components of the outer leaflet are phosphatidylcholine and

phosphatidylethanolamine (Dudeja et al. 1991). By using these vesicles, the uptake of

compounds to the cellular lipid bilayer can be studied (Saija et al. 1995, Taillardat-Bertschinger

et al. 2002). For example, multilamellar dipalmitoylphosphatidylcholine (DPPC) liposomes are

prepared in the presence and absence of the studied compounds. The drug content can be

analysed by differential scanning calorimetry. This technique allows convenient and sensitive

determination of drug interactions with the lipid bilayer: the presence of drug molecules

(amphipathic of lipophilic in nature) in the ordered lipid bilayer structure might modify the lipid

chain packing, causing variations in the transition temperatures of the pure lipid and/or changes

in the enthalpy of chain melting (Cater et al. 1974). Drug-membrane interactions can be studied

as a function of time by performing several scans of the same sample. Also, drug transfer from

lipid vesicles to aqueous medium can be followed by conventional HPLC analyses of the

aqueous medium (Saija et al. 1995).

Immobilized artificial membrane (IAM) columns have been used for measuring drug-

membrane partitioning for predicting membrane permeability (Pidgeon et al. 1995, Yen et al.

8


2005). The method is based on different retention of drugs on covalently immobilised cell

membrane phospholipids on inert (propylamine-)silica surfaces. Also unilamellar liposomes can

be immobilised to the columns (Liu et al. 2002). The surface is thus mimicking one half of the

membrane bilayer in vivo. Drug molecules are retained on the column depending on their

lipophilicity. The environment resembles in vivo cell membranes by the fact that the molecule

meets the polar head groups of the phospholipids first before entering the lipid bilayer.

Phosphatidylcholine, which is the major phospholipid found in cell membranes, and its analogs

are the basic constituents of IAM surfaces (Ollila et al. 2002, Yen et al. 2005).

Figure 6. Cross section of a 96-well microtitre plate Pampa sandwich assembly

The use of a parallel artificial membrane permeability assay (PAMPA), first introduced

by Kansy et al. (1998), has been adapted to many laboratories around the world (Figure 6). The

PAMPA assay is based on a “sandwich” system with a 96-well microtiter plate and a 96-well

microfilter plate (125 µm thick with 0.45 µm pores), which is impregrated with e.g.

phospholipid - dodecane solutions (Avdeef, 2001). In this system, two separated compartments,

donor and acceptor, are formed, thus mimicking the in vivo situation with intestinal lumen and

circulation. The microfilter disc is impregnated with a 2-5% dodecane solution of

dioleoylphosphatidylcholine (DOPC) under conditions where multilamellar bilayers are assumed

to form inside the filter channels. This method is very suitable to assess passive transcellular

permeability during early stages of drug discovery.

2.3.2. In vitro biological (cell based) methods

Absorption of drugs can be investigated by using several different biological in vitro methods.

Compared to the biophysical methods, these offer more physiological conditions, where

absorption mechanisms (passive diffusion and/or active transport) at cellular or even sub-cellular

level can be studied. Additionally, these in vitro biological methods can be used to determine

drug transport rate and uptake to cells. In vitro cell based methods are also suitable as screening

9


methods since they require small quantities of compounds tested and are less laborious than in

situ animal experiments.

Brush border membrane vesicles

The simplest biological method is the use of subcellular fractions, for example brush border

membrane vesicles (BBMV). Several techniques are employed in the isolation procedure of

small intestinal BBMV, including cell disruption followed by fractionation usually by density

gradient centrifugation (Maenz et al. 1991), and selective precipitation of other membranes

using divalent cations Mg2+ or Ca2+ (Kessler et al. 1978, Hauser et al. 1980). The use of divalent

cations has been observed to activate membrane-associated enzymes (different lipases).

Precipitation with polyethylene glycole is one of the most recent methods in preparing brush-

border membrane vesicles (Prabhu and Balasubramanian 2001). The used tissue sources are

mostly human, rabbit, guinea pig and rat.

BBMV are used for mechanistic studies of enzyme interactions or ion transport - coupled

transport processes (Ungell 1997). This method offers information, whether the compound of

interest is partitioning into the lipid membrane or being transported into the intravesicular space.

Much effort has been used to elucidate active and facilitated transport of nutrients and

endogenous compounds, such as D-glucose and amino acids (Dudeja et al. 1991), bile acids

(Kramer et al. 1994, Nowicki et al. 1997), biotin (Said et al. 1990), monocarboxylic acids (Tsuji

et al. 1990), peptides (Inui et al. 1988, Yasa et al. 1993). Brush border membrane vesicles,

which contain several hydrolytic enzymes, give valuable information about drug stability during

absorption. This method has therefore been especially used in studying prodrug reconversion at

the intestinal wall (Hashimoto et al. 1994).

Freshly isolated cells

Fully differentiated human intestinal cells are probably not possible to be cultured for

several passages in vitro, most likely because their sensitivity to environmental factors (Young

and Das 1990). Several research groups have tried to culture freshly isolated intestinal epithelial

cells to develop a transport model of human origin, so far without total success. Because

enterocytes are already differentiated in vivo, they are not able to form a uniform cell monolayer

with characteristic intestinal morphology. Therefore, because of their nonproliferative status,

normal freshly isolated enterocytes can only be maintained in vitro as primary cultures. Several

10


techniques for isolating the intestinal cells have been developed. One example is the sequential

vibrational technique, by which crypt cells are separated from villus-tip cells (Harrison and

Webster, 1969). Weiser (1973) developed an isolation technique that utilized dithiothreitol and

EDTA for cell dissociation. Hartmann et al. (1982) introduced a method in which collagenase

was perfused through the vascular bed of an intestinal segment. During this procedure, intestinal

cells were sloughed into the lumen and collected by the following perfusions in a sequential

manner from villus tip to crypt. In this manner, several viable populations of mature enterocytes

were obtained. The first attempts to prepare an in vitro model for drug absorption were based on

comparisons between the cell isolation techniques of Weiser (1973) and Hartmann et al. (1982)

by Osiecka et al. (1985) and Porter et al. (1985). The obtained intestinal cells can be used for

uptake studies, as opposed to transport across the monolayer.

Undifferentiated primary epithelial cells from a human fetus grown in their mesenchymal

environment are able to develop to normal differentiated enterocytes (Sanderson et al. 1996).

Additionally, considerations in cell isolation include the harvesting of a homogeneous cell

population, as well as sufficient viability of the cell preparation, which must be maintained over

the course of the uptake experiments. As the lifetime of the mature enterocytes is short, this can

be particularly challenging.

Two more recent attempts to generate human normal intestinal cell lines might lead to

better and successful isolation and culturing of these cells. A human intestinal cell line with

normal proliferative crypt cell characteristics has been developed (Perreault and Beaulieu 1996,

1998). This cell line shows characteristic intestinal physiological functions, i.e. cell-cell and cell-

matrix interactions, growth factor effects and metabolism. Secondly, production of conditionally

immortalized human intestinal primary cultures with a temperature sensitive SV40 large T

antigen has been described (Quaroni and Beaulieou 1997). At lower temperatures (32ºC), these

cells proliferate and display crypt cell morphology, but a shift to higher temperature (39ºC)

results in an irreversible growth arrest, leading to enterocyte-like phenotype differentiation.

However, in vitro cell models of fully differentiated normal intestinal epithelial cells are still a

question for the future.

Intestinal rings, everted sacs

Intestinal slices or rings have been used for kinetic analyses of carrier mediated transport of

nutrients and drugs (Osiecka et al. 1985, Kim et al. 1994, Leppert and Fix 1994). In this method,

11


the intestine of an animal is cut into slices or rings, the dissolved drug(s) are added to the

incubation medium, and after a desired period of time, drug concentration samples of the

incubation medium are analysed.

The everted sacs are prepared by everting a short part of an intestine, which is tied at one

end, on a glass rod. By this everting, the serosa becomes the inside of the sac and the mucosa the

outside facing the incubation medium. The whole sac is put into a flask, which contains the

dissolved drug(s), and samples are taken from the incubation medium and analysed.

By using the above-mentioned methods, the disappearance of the drug studied from the

incubation medium and tissue uptake can be followed. The advantages of these two methods are

that the preparation procedure is easy and rapid and that several drugs can be studied

simultaneously (n-in-one). The disadvantages are that the viability of the intestinal tissue is

maintained only for a short time period (a few hours at best), although efficient stirring and

oxygenation of the incubation medium prolongs the viability time.

Cell cultures

As the use of isolated human intestinal cells in continuous culture as a working model for

intestinal absorption poses many technical difficulties, the use of immortalized other cell lines is

ubiquitous for this purpose. Intestinal adenocarcinoma cell lines, Caco-2 (Fogh et al. 1977,

Hidalgo et al. 1989, Artursson 1990, Hilgers et al. 1990, Cogburn et al. 1991), HT29, HT29-18

and HT29-H (Huet et al. 1987, Wikman et al. 1993), and T84 (Dias and Yatscoff 1994), have

been used for permeability experiments by several laboratories around the world because of their

capability to differentiate in culture (Neutra and Louvard 1989). Cell lines from animals, such as

MDCK (Madin Darby canine kidney), CHO (Chinese hamster ovary), and 2/4/A1 (rat), are also

intensively used as in vitro model for intestinal absorption (Cho et al. 1989, Covitz et al. 1996,

Tavelin et al. 1999).

MDCK cells

The Madin Darby canine kidney (MDCK) cell line is one of the most common epithelial cell

lines for studying cell growth regulation and metabolism. From the late 1990, these cells have

been used for studying transport mechanisms in renal epithelia (Hunter et al. 1993, Irvine et al.

1999). MDCK cells have been shown to differentiate into columnar epithelial cells with well

formed tight junctions close to the apical surface of the cells when cultured on semi permeable

12


supports (Irvine et al. 1999). In addition to investigations for renal transport processes, MDCK

cells are used as a cell culture model for intestinal absorption (Cho et al. 1989). This cell line has

been well characterised (morphology, electrical resistance, polarisation of the cells in culture),

and provides a good in vitro model for drug transport and interaction studies with drugs

(Rothen-Rutishauser et al. 1998).

Polarisation of the cell monolayer and formation of tight junctions can be followed by

measuring the transepithelial electrical resistance (TEER). There exist several subclones of

MDCK cells, of which some show characteristic transepithelial electrical resistance (TEER): one

subclone with TEER about 4000 Ωcm2 and another with lower resistance; 200-300 Ωcm2. For

drug transport studies, the later mentioned subclone is normally used because of the leakiness

which resembles small intestine (Rothen-Rutishauser et al. 1998). Compared with original “wild

type” MDCK cells, this cell line exhibits similar growth curves, but reaches the stationary phase

later (7-10 days after seeding) (Braun et al. 2000). These MDCK cells form tight junctions

between cells during the first day in culture (Rothen-Rutishauser et al. 1998). During the

following days, confluent monolayers are formed, and cytoskeleton rearrangement happens. The

cells can be used for permeability experiments after one week in culture. After 15 days, the cell

monolayers start to detach spontaneously from the permeable supports.

The permeability of several highly and passively diffused drugs correlates well with

experiments performed using other cell cultures as in vitro intestinal absorption model (Irvine et

al. 1999). Weaker correlation was found for compounds of low permeability, partially due to

loose tight junctions and partially to different active transport properties. In the parental MDCK

cell line, no evidence for active transport of large neutral amino acids and bile acids was

detected, whereas transport of monocarboxylic acids and small peptides was active (Putnam et

al. 2002a, b). The big advantage in using MDCK cells is the minimum culturing time of three

(parent line), 7-10 (subclone 2) days, whereas for example Caco-2 cells need 21 days for the

formation of fully differentiated polarised cell monolayer.

The subclone 2 MDCK cells have been used for transfection with the MDR1 gene, which

codes for P-glycoprotein. These cells form irregular multilayers, tight junctions are found also

between the cell layers, not only close to the apical surfaces. TEER values are much higher

(>1000 Ωcm2) than characteristic for subclone 2 cells (Braun et al. 2000). This cell line has been

tested for using as in vitro model for blood-brain barrier (Wang et al. 2005). Additionally,

13


MDCK cell lines that overexpress human MRP´s (1, 2, 3, and 5) have been transfected

(Flanagan et al. 2002a, Evers et al. 1998, Tang et al. 2002, König et al. 1999).

CHO cells

Chinese hamster ovary cell cultures are widely used in cell biology for several decades. Their

use in drug transport experiments has been increased when CHO/hPepT1 cell line was

constructed (Covitz et al. 1996). By using this cell line, the membrane transport characteristics

of several D- and L-amino acid esters of acyclovir and zidovudine, several di- and tripeptides,

amino acids and ß-lactam antibiotics has been characterised (Covitz et al. 1996, Han et al. 1998,

Surendran et al. 1999 etc). This cell line is used mainly for characterizing substrates and

inhibitors of hPepT1.

2/4/A1 cells

A conditionally immortalized rat intestinal cell line 2/4/A1 forms polarized monolayers 4-6 days

after seeding onto permeable supports (Tavelin et al. 1999). The cells formed continuous

multilayers at 33ºC, but at 37ºC monolayers are formed. These cells express low TEER values

(20-25 Ωcm2) 2-12 days after seeding onto semi-permeable supports. The pore radius of 2/4/A1

cells is similar to that in the human small intestine, about 0.9 nm (9.0 Å), whereas in Caco-2

cells it is about 0.37 nm (3.7 Å) (Tavelin et al. 2003). The paracellular permeability of

hydrophilic model drugs is very well comparable to that of small intestine, as well as the

diffusion of low permeable model drugs (Tavelin et al. 1999, Tavelin et al. 2003). According to

the authors, this cell line is a promising alternative to Caco-2 monolayers for studies of passive

drug transport.

The advantages of this cell culture method are many. The experiments are rapid to

perform; the prediction of drug absorption in humans is more precise than in vivo animal studies.

One of the disadvantages is the origin of the cells; as they are of rodent origin, the regulation of

gene expression during differentiation is probably not similar to that in humans (Perreault and

Beaulieu 1998).

HT29 cells

When colon carcinoma HT29 cells are cultured in the presence of glucose, they grow as a

multilayer of unpolarised, undifferentiated cells without expression of any characteristics of

enterocytes (Zweibaum et al. 1985). However, if the cells are grown in the presence of

14


galactose, the cells express moderate enterocytic differentiation (highly polarized cells, which

express micovillar hydrolases and resemble enterocytes and goblet cells) (Zweibaum et al. 1985,

Zweibaum 1991).

Several mucus producing goblet cell sublines have been established from human HT29

cells (Huet et al. 1987). Upon differentiation, the cloned subpopulation HT29-18, was shown to

be heterogeneous: 90% of the cells exhibited morphologically absorptive characteristics, and

10% goblet cell characteristics. The HT29-18 clone was proposed to be multipotent, being able

to give rise to distinct differentiated cells with properties resembling intestinal enterocytes,

goblet cells, and possibly also paneth cells (Huet et al. 1987). In HT29-H, another subpopulation

from parent HT29 line, cell monolayers has been shown to contain over 80% cells with

morphology of mature goblet cells (Wikman et al. 1993, Karlsson et al. 1993). The HT29-MTX

cell line, which has been developed by adapting the cells to a medium containing methotrexate,

is also a subpopulation of parental HT29 cells, and express the morphological and mucin

producing characteristics of goblet cells (Lesuffleur et al. 1990, Pontier et al. 2001). The HT29

cells have been used for drug permeation studies as such or co-cultured with Caco-2 cells

(Wikman et al. 1993, Wikman Larhed and Artursson, 1995, Walter et al. 1996, Hilgendorf et al.

2000).

Caco-2 cells

Caco-2 cell line was established from a moderately well differentiated colon adenocarcinoma

obtained from a 72-year-old patient (Fogh et al. 1977). Caco-2 cells differentiate spontaneously

in culture and exhibit structural and functional differentiation patterns characteristic of mature

enterocytes (Pinto et al. 1983). Caco-2 cells reach confluency within 3-6 days and reach the

stationary growth phase after 10 days in culture (Braun et al. 2000). The differentiation is

completed within 20 days (Pinto et al. 1983). The differentiated cells exhibit high levels of

alkaline phosphatase, sucrase isomaltase and aminopeptidase activity characteristic to enterocyte

brush border microvilli. The structural and functional differentiation of the microvilli is

associated with the polarization of the monolayer after confluency. The structural polarity is also

apparent from the presence of tight junctions, which are formed during the differentiation. The

monolayers exhibit a barrier function as judged by high TEER values (200-600 Ωcm2, grown on

polycarbonate filters) and a minimal permeability of mannitol (m.w. 182 g/mol), Lucifer yellow

(453 g/mol), polyethylene glycol (4000 g/mol), inulin (5000 g/mol), and dextran (70000 g/mol)

(Hidalgo et al. 1989). Fully differentiated Caco-2 cells form an epithelial membrane with a

15


barrier function similar to the human colon (Artursson et al., 1993) but express carrier proteins

similar to the small intestine (Baker and Baker 1992, Hidalgo et al. 1989, Wilson et al. 1990).

Caco-2 cell monolayers have been used for studying mechanisms of passive paracellular

(Artursson et al., 1993, Artursson et al. 1996, and many others) and passive transcellular

permeability (Artursson 1990 and others), carrier mediated absorptive transport of amino acids

(Thwaites et al. 1995b), amino acid analogues (Hu and Borchardt 1990, Thwaites et al 1995a),

oligopeptides (Thwaites et al. 1993), ß-lactam antibiotics and ACE-inhibitors (Inui et al. 1992),

and peptidomimetic thrombin inhibitors (Walter et al. 1995). Carrier mediated efflux (combined

with metabolism inside the enterocytes) of several drugs has been intensively studied over the

last years (Benet et al. 1999, Walgren et al. 1999, Polli et al. 2001, Eneroth et al. 2001, Tran et

al. 2002, Troutmann and Thakker 2003, Collett et al. 2004 and many others), as well as cocktail

dosing of several different drugs (Bu et al. 2000, Markowska et al. 2001, Tannergren et al. 2001,

Larger et al. 2002, Augustijns and Mols 2004, Palmgren et al. 2004).

Although Caco-2 cells express many enzymes and transporters present in the small

intestine, the high TEER and poor paracellular permeability properties resemble colonic cells

(Grasset et al. 1984, Artursson et al. 1993). This fact leads to very low permeability for

hydrophilic, mostly paracellularly permeating compounds. The best correlation to the in vivo

situation is obtained for transcellularly passively permeating drugs. Many active transport

proteins are expressed in the Caco-2 cells (Table 1), but at different quantities than in small

intestine. The lack of the crypt-villus axis, which is important for fluid and ion transport in vivo,

is one of the major disadvantages of all cell based in vitro models, as well as the lack of the

mucus producing goblet cells, which leads to the lack of a prominent mucus layer (Wikman

Larhed and Artursson 1995, Hilgendorf et al. 2000).

2.4. INTERACTIONS DURING ABSORPTION

2.4.1. Interactions between drugs Clinically significant drug interactions occur when the toxicity or efficacy of a drug is altered by

another drug or substance. The effects of several, simultaneously ingested drugs might cause

problems during absorption, distribution, metabolism, and excretion processes. Drug-drug

interactions are mostly caused by interactions during liver metabolism, intestinal metabolism

and drug transport. The occurrence of interactions during liver metabolism is commonly known,

but problems during absorption are generally not. One potential source for interactions is that

16


drugs are sharing the same active transport mechanisms and/or metabolic pathway inside the

enterocytes during absorption (Table 1).

Table 1. Some membrane transporters involved in intestinal drug absorption

Membrane transporter

Location

Expression in Caco-2

Substrate

Inhibitor

Effect measured

Reference

hPepT1

apical

+

Cefixime Valacyclovir Gly-Sar

Nifedipine cephalosporines

biovailability ↓

Duverne et al. 1992, Behrens et al. 2004

MCT1

apical

+

Salicylic acid Ketoprofen

Fluorescein L-lactic acid

absorption ↓

Konishi et al. 2002 Choi et al. 2005

OATP-B

apical

?

Estrone-3-sulphate Pravastatin

Pravastatin

absorption ↓

Ito et al. 2005 Kobayashi et al. 2003

BCRP

apical

+

Topotecan

GF120918

bioavailability ↑

Jonker et al. 2000

MDR1

apical

+

Talinolol Digoxin

Verapamil Guinidine Verapamil

absorption ↑ efflux ↓ plasma conc. ↑

Augustijns and Mols, 2004 Greiner et al. 1999 Collett et al. 2005

MRP1

basolateral

+

Methotrexate Estradiolglucor.

Probenecid MK571

absorption ↓

Endres et al. 2006

MRP2

apical

+

Grepafloxacin Vinblastine

MK571, Furosemide Indomethacin

absorption ↑ bioavailability ↑

Lowes and Simmons, 2002 Endres et al. 2006

MRP3

basolateral

+

Methotrexate Estradiolglucor.

Hydroxycotinine O-glucuronide

absorption ↓

Endres et al. 2006 Létourneau 2005

MRP4

apical

+

Estradiolglucor.

Probenecid

absorption ↑

Endres et al. 2006

MRP5

basolateral

+

Methotrexate

Probenecid, MK571

plasma conc. ↓

Pratt et al. 2006

MRP6

basolateral

+

Mercaptopurine

Probenecid, Indomethacin

absorption ↓

Endres et al. 2006 Boraldi et al. 2004

Several active uptake transporters are located in the epithelium of the small intestine

(Table 1). On the apical membrane of the enterocytes, a peptide transporter (hPepT1) takes care

of the transport of di- and tripeptides. Competitive inhibition of this carrier has been studied with

several drugs, such as ACE inhibitors, valacyclovir and β-lactam antibiotics (Han et al. 1998,

Ganapathy et al. 1995, Zhang et al. 2002). The monocarboxylic acid transporter (MCT1)

enhances the absorption of several acidic compounds, among which are also ketoprofen and

naproxen (Tamai et al. 1995, Choi et al. 2005). Benzoic acid and L-lactic acid are able to

decrease cellular uptake of 0.5 mM ketoprofen in Caco-2 cells. In addition, ketoprofen (0.44

mM) and naproxen (0.25 mM) inhibited the cellular uptake of benzoic acid (Choi et al. 2005).

However, ketoprofen transport across rat jejunal segment has not been detected to be saturable

over a concentration range from 0.125 to 5 mM, nor inhibited by benzoic acid or L-lactic acid

17


(Legen and Kristl 2003). This may be due to higher Km values for MCT1 mediated transport in

rat jejunum than in Caco-2 cells.

Transporters may also play a crucial role in limiting drug absorption through drug

secretion into the intestinal lumen. Primary active efflux transporters (ABC transporters), such

as multidrug resistance protein 1 (MDR1), limits the uptake of substrates such as digoxin and

cyclosporin (Gottesman et al. 2002). Additional secretory pathways are mediated by multidrug

resistance-associated proteins (MRP) and breast cancer resistance protein (BCRP), which are

also expressed in the membranes of intestinal enterocytes and Caco-2 cells. BCRP, MRP 2 and 4

are expressed in the apical membranes and MRP1, 3, 5 and 6 in the basolateral membranes

(Chan et al. 2004). The expression of the efflux transporters in the Caco-2 cells correlates well

with expression in normal human jejunum (Taipalensuu et al. 2001).

Because transport proteins play an important role in the absorption of a wide variety of

drugs, transporter-based drug interactions are possible during intestinal absorption, although

interactions at the transporter level alone have been reported only in a few cases. The

metabolism by CYP3A and by UGTs in human intestine, accompanied with efflux by MDR1,

MRP2 and/or BCRP, is a major factor limiting oral bioavailability (Zhang and Benet, 2001). The

function of efflux proteins without metabolism plays a minor role in drug-drug interactions

during absorption. Yet, for example co-administration of erythromycin or verapamil enhances

plasma concentration of orally administered talinolol. Talinolol is a MDR1 substrate and is not

metabolised during absorption. The renal clearance is not affected by erythromycin, but

increased intestinal absorption has been observed (Schwarz et al. 2000).

2.4.2 Interactions between drugs and plant extracts

The human diet contains many different nutrients, several compounds that are not nutrients, and

additives. According to studies where effects of carbohydrates, fat and proteins (macronutrients)

were determined on drug absorption and metabolism, changes in micro- and macronutrient

composition of the diet can affect absorption and/or elimination of drugs (for example Welling,

1996). The effects of non-nutrients and additives in food may also exert an effect on

bioavailability of drugs. Some drugs, which normally are not interacting with food components,

might interact during absorption with the components of plant material, causing unexpected drug

effects, such as several times higher or lower drug concentrations in the circulation (Wallace and

Amsden 2002). Food supplements manufactured from for example St. John´s Wort (Hypericum

perforatum), Ginkgo Biloba, and licorice (Glycyrrhiza glabra) are frequently used by patients,

18


believing that plant extracts possess only beneficial effects. Additionally, the patients do not

normally report the use of food supplements to the physicians responsible for their health care.

Polyphenols (e.g. anthocyanins, coumarins, flavonoids, lignans and tannins), present in

herbs, vegetables, fruits, flowers, and leaves in many plants, are believed to be beneficial to

human health by exerting biological effects such as free radical scavenging (Heim et al. 2002).

Bioavailability of polyphenols has been studied in vivo (Hollman et al. 1995, Miyazawa 2000)

and in vitro (Kuo et al. 1998, Murota et al. 2000, Murota et al. 2002). Most dietary flavonoids

present in food exist as O-glycosides (with glucose, glucorhamnose, galactose, arabinose, or

rhamnose) (Heim et al. 2002). The β-linkage of these glycosides resist hydrolysis caused by

acidity in the stomach and the attack by pancreatic enzymes. However, β-endoglucosidases

present in small intestine are able to hydrolyse flavonoid glycosides (Spencer et al. 1999).

Additionally, colonic microflora hydrolyses the sugar moiety from the flavonoid aglycone, thus

increasing the absorption of flavonoids. During absorption, the flavonoids may interact with

metabolising enzymes and transport proteins, and thus affect the uptake of co-administered

drugs. Indeed, polyphenols are potent inhibitors or inducers of CYPs, UGTs and transport

proteins, if consumed in large amounts (Zhai et al. 1998, Conseil et al. 1998, Ohnishi et al.

2000). Dietary supplements of plant origin are not only possible candidates for drug interactions,

but rather, when used as concentrated products for prolonged time, they really pose a great risk

for interactions with conventional drugs.

Pharmacokinetic herb-drug interactions might be due to phytochemical mediated

alternations in the activity of xenobiotic metabolising enzymes (CYPs and UGTs) or/and

transport proteins (Wilkinson 1997, Ioannides 1999). For example, furanocoumarins present in

grapefruit juice inhibit intestinal CYP3A4 and have shown to increase the peroral bioavailability

of drugs that are CYP3A4 substrates, e.g. felodipine, midazolam, cyclosporine (Ioannides,

1999). Furanocoumarins and bioflavonoids present in fruit juices are also inhibitors of intestinal

OATP, leading in reduction of bioavailability, when co-administered with fexofenadine (Dresser

et al. 2002). The inhibitory effect of grapefruit juice on MDR1 has recently been determined to

be modest, but the effect is more pronounced in OATP inhibition (Mizuno et al. 2003).

Additionally, broccoli, cabbage, and watercress contain appreciable amounts of glucosinolates

that are capable of inhibiting CYP1A2 and CYP2E1 while inducing various phase II enzymes

(Smith and Yang 2000, Leclercq et al. 1998).

19

AIMS OF THE STUDY _____________________________________________________________________________

3. AIMS OF THE STUDY The use of Caco-2 cell monolayers as an in vitro model for intestinal absorption is accepted by

many laboratories around the world. Because the permeability data obtained from different

laboratories is not directly comparable, it is important to standardise the experimental

conditions. One possibility is to include several standard drugs or compounds with known

permeability characteristics in every permeability experiment. The ultimate aim of this study

was to evalueate the usefulness simultaneous use of several model drugs as internal standards,

and to detect possible interactions during their permeation across Caco-2 cell monolayers. Drugs

utilizing different permeability characteristics: high and low permeability, passive and active

transport and active efflux were used.

To achieve the overall aims the following investigations were conducted:

1) To compare the effects of several actively or passively permeating drugs and

compounds on the permeability of the individual compounds in the cocktails

and on their permeability without prescence of other compounds [I, II].

2) To determine the effects of different studied sets of drugs on the integrity (14C-

mannitol permeability, TEER measurements) and viability of Caco-2 cell

monolayers [I, II]

After introducing several drugs and compounds, which can be used as internal standards without

interactions or toxic effects on cell monolayers, the usefulness of the method was further probed

by conducting the following experiments.

3) To probe if food supplements and food fractions (all potential functional foods)

[III] or laxatives of natural origin [V] affect the permeability of simultaneously

administered permeation markers with different transport mechanisms.

Additionally, their effects on monolayer integrity and toxicity were also

assessed.

4) Because the food supplements, food fractions and anthranoid laxatives contain

many active compounds capable to interact with co-administered drugs, the

permeation behaviour of some flavonoids and alkyl gallates was studied [IV].

20

EXPERIMENTAL _____________________________________________________________________________

4. EXPERIMENTAL

Detailed descriptions of the materials and methods can be found in the respective papers (I - V).

All used methods are presented in Table 2.

4.1. MATERIALS

4.1.1. Permeation markers (drugs and compounds used) (I-V) Antipyrine (I, II) was purchased from Aldrich Chemical Company Inc. (Milwaukee, WI, USA),

buspirone (I), hydroxyzine (I), and metoprolol bitartrate (II, III) from Sigma Aldrich Chemie

(St. Louis, MO, USA), ketoprofen (I, II, III, IV, V), naproxen (I), propranolol hydrochloride (I,

II, V), and verapamil hydrochloride (I, II, III, V) from ICN Biomedicals Inc. (Aurora, Ohio,

USA), midazolam (I, II) from Hoffman-La Roche (Basle, Switzerland). Procaine hydrochloride

(I) was donated by the Helsinki University Pharmacy, Finland, furosemide (III, V), paracetamol

(III, IV, V), and timolol maleate (I) by Orion Pharma (Espoo Finland). D-[1-14C]-mannitol (I,

II, III, IV, V) was bought from DuPont NEN (Boston, MA, USA) and Amersham Pharmacia,

ranitidine (II), fluorescein natrium (II), hydrochlorothiazide (II), cephalexin hydrate (II) from

Sigma-Aldrich Chemie (Steinheim, Germany), and rhodamine 123 (Rho123) (III, V) from Fluka

Chemie GmbH (Buchs, Switzerland).

4.1.2. Natural compounds and derivatives (IV) Since the plant extracts (III) contain several active flavonoids and alkyl gallates, the

permeability of these pure compounds (test compounds in IV) was assessed across Caco-2 cell

monolayers. Luteolin and naringin were purchased from Extrasynthese S.A. (Genay, France), (-

)-epicatechin, (+)-catechin and propyl gallate from Sigma (St. Louis, MO, USA), flavone, morin

and naringenin from Carl Roth GmbH & Co (Karlsruhe, Germany), methyl gallate and octyl

gallate from Fluka Chemie (Buchs, Swizerland), quercetin from Merck & Co (Darmstadt,

Germany).

4.1.3. Intestinal epithelial cells (I-V) Caco-2 cells, originating from a human colorectal carcinoma, were obtained from the American

Type Culture Collection (ATTC # HTB-37, Rockville, MD, USA). The cells were cultivated as

described in articles I-V. Briefly, Dulbecco´s Modified Eagle´s Medium (DMEM) supplemented

with 10% heat inactivated foetal bovine serum and 1% non-essential amino acids, 1% L-

glutamine and 100 IU/ml penicillin + 100 µg/ml streptomycin was used as culture medium.

21

EXPERIMENTAL _____________________________________________________________________________

Cells were seeded at 6.8 x 104 cells per cm2 onto Transwell polycarbonate filters (Corning

Costar Corp., Cambridge, MA, USA) with 0.4 µm mean pore size and growth area of 1.1 cm2

(12-well format, I, II, III, V) or 0.33 cm2 (24-well format, IV) and used for experiments 21 - 28

days after seeding. Cells of passages 31 - 42 were used. All cell culture media were obtained

from Gibco Invitrogen Corp. (Life Technologies Ltd. Paisley, Scotland) or BioWhittaker

(Cambrex Bio Science, Verviers, Belgium).

4.2. METHODS

4.2.1. Plant material and extraction (III) Extraction of flax seed (Linum usitatissimum L.) groats (Neomed Ltd., Somero, Finland) and dry

powdered purple loosestrife (Lythrum salicaria L.), of Finnish origins, was performed in 80%

aqueous methanol, sonicated, centrifuged and concentrated by rotary evaporation, followed by

freeze drying. The Scots pine (Pinus sylvestris L.) bark extract was prepared by freeze drying

commercial Scots-pine-bark aqueous extract (Ravintorengas Oy, Siikainen, Finland).

Extracts of bilberries (Vaccinum myrtillus L.), cowberries (Vaccinum vitis-idaea L.) and

raspberries (Rubus idaeus L.), of Finnish origins, were prepared in aqueous ethanol containing

L-ascobic acid and concentrated by rotary evaporation (glycosidic fractions). The aglycone

fractions were prepared by boiling the homogenates in 6M HCl followed by filtration and rotary

evaporation.

The herbs oregano (Origanum vulgare L.), rosemary (Rosmarinum officinalis L.), and

sage (Salvia officinalis L.), all purchased from Paulig Group Ltd (Helsinki, Finland) were

extracted with deionized water using a European Pharmacopoeia hydrodistillation apparatus.

The water-extraction fractions were filtered and freeze-dried.

Determination of total phenolics in the extracts (III)

To determine the quantities of total phenolics present in the extracts, the Folin-Ciocalteau

procedure was used (Singleton et al. 1999). The Folin-Ciocalteau reagent was added to the

freeze-dried extract - HBSS (pH 7.4) samples, and the resultant absorbances were measured at

760 nm. Total phenolic content was expressed as gallic-acid equivalents (GAE) in mg/g of dry

material.

22

EXPERIMENTAL _____________________________________________________________________________ Table 2. Methods used in the publications I-V.

Permeation marker(s)

Drugs or compounds

Permeation time, min

pH apical/ basolateral

Experiments

Paper discussed

antipyrine1,2

buspirone1

cephalexin2

fluorescein2

hydroxyzine1

hydrochloro- thiazide2

ketoprofen1,2

metoprolol2

midazolam1,2

naproxen1

procaine1

propranolol1,2

ranitidine2

timolol1

verapamil1,2

Cocktail 1*

Cocktail 2*

Cocktail 3*

Cocktail 4*

Cocktail 5* Cocktail 6*

Cocktail 72

120

5.5 / 7.4

-Cytotoxicity MTT, LDH -Monolayer integrity before and after exp. 14C-mannitol, TEER -Permeation of markers as single markers in cocktails -Analysis LC-ESI/MS/MS Scintillation counting

I, II

furosemide3

ketoprofen mannitol metoprolol paracetamol Rho1233

verapamil

Extracts of flax seed purple loosestrife Scots pine bark oregano rosemary sage bilberry cowberry raspberry

90

1203

7.4 / 7.4 -Total phenolics Folin Ciocalteau -Cytotoxicity: MTT -Monolayer integrity during the experiment 14C-mannitol TEER before and after -Permeation of markers alone and with extracts -Analysis: fluorescence plate reader, HPLC, Scintillation counting

III

ketoprofen paracetamol mannitol

flavone luteolin morin quercetin naringenin naringin methyl gallate octyl gallate propyl gallate (+)-cathecin (-)-epicathecin

90 7.4/7.4 -Cytotoxicity: MTT -Monolayer integrity during the experiment: 14C-mannitol TEER before and after -Permeation of markers and compounds -Partitioning into POPC -Analysis: HPLC, Scintillation counting

IV

furosemide3

ketoprofen paracetamol propranolol Rho1233

verapamil

rhein danthron sennidin A+B sennoside A+B senna leaf infusion

90

1203

5.8 / 7.4 -Cytotoxicity: MTT -Monolayer integrity before and after exp. 14C-mannitol, TEER long-term effects (8h) 14C-mannitol -Permeation of markers ± laxatives laxatives -Analysis: fluorescence plate reader, HPLC, Scintillation counting

V

1 Permeation markers presented in Paper I 2 Permeation markers presented in Paper II 3 Permeability of furosemide and Rho123 assessed over 120 minutes *The contents of different cocktails are presented in Table 4

23

EXPERIMENTAL _____________________________________________________________________________

4.2.2. Preparation of the permeation marker solutions and extracts (I-V) All the permeation marker solutions were prepared in Hank´s balanced salt solution (HBSS)

containing 10 mM HEPES (with the pH adjusted to 7.4) or 10 mM MES (with the pH adjusted

to 5.5, or 5.8).

The freeze-dried extracts of food supplements, herbs and glycosidic fractions of berries

(III) were prepared in HBSS. The aglycone fractions of berry extracts were first dissolved in

DMSO or ethanol and added further into HBSS. The final DMSO and ethanol concentrations

were 5% (w/v and v/v, respectively). The flavonoids and alkyl gallates (IV) were firstly

dissolved in DMSO and these solutions were then added into HBSS. The final DMSO

concentration was 5% (w/v).

The anthranoid laxatives (rhein, danthron, sennidin and sennoside) (V) were first

dissolved in DMSO and the solution was further added into prewarmed HBSS. The final DMSO

concentration was 2% (w/v). The senna leaf infusion was prepared by infusing senna leaves in

boiling deionised water (90% of the total water volume). After the infusion, HBSS concentrate

(10x) was added with 1M MES buffer solution. Finally, pH was adjusted with 0.1M NaOH and

deionised water was added to volume.

4.2.3. Evaluation of cytotoxicity (I-V) The reduction of viability of Caco-2 cells by all used compounds and solvents was evaluated

prior to the permeability experiments to eliminate their effects on possible active transport

(Table 2). Firstly, the cytotoxicity was assessed by using the MTT test (I-V), which determines

the effects of the compounds on intracellular dehydrogenase activity (Anderberg et al. 1992).

MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (Sigma Chemical Co, St.

Louis, MO, USA) is a water soluble yellow tetrazolium salt that is cleaved to insoluble purple

formazan by active mitochondrial succinate dehydrogenases in living cells (Mosman 1983).

Secondly, the LDH test (CytoTox 96® kit, Promega, Madison, WI, USA) was used (I). Lactate

dehydrogenase is a stable cytosolic enzyme that is released during cell lysis into the extracellular

fluid and can be detected with an enzymatic assay. The reactions with LDH ends with the

conversion of a tetrazolium salt into a red formazan product (Decker and Lohmann-Matthes

1988). The combined permeation marker and extract solutions (III-V) were dark coloured, thus

causing interference with the LDH assay. Hence, LDH assay could not be used as a viability test

with plant extracts and natural compounds.

24

EXPERIMENTAL _____________________________________________________________________________

4.2.4. Evaluation of monolayer integrity (I-V) [14C]Mannitol transport

The integrity of the cell monolayer (= barrier function of the cell culture) can be studied by

determining the paracellular permeability of hydrophilic marker molecules like mannitol.

Mannitol is a water soluble, cell membrane impermeable and non-ionisable small molecule with

mw of 182 g/mol and its molecular radius is 4.1 nm (Hidalgo 1996) Mannitol permeability is

very low (<0.5% per hour) when monolayer integrity is good (Hidalgo 1996).

The integrity of the monolayers was assessed with 14C-mannitol (Table 2): 1) after the

actual permeability experiments (I, II, V), 2) during the permeability experiments without and

with the extracts and permeation markers (III, IV), or 3) during an 8 h long assay, where the

recovery of cell monolayers was monitored after anthranoid laxative pre-treatment (V).

The monolayers were equilibrated with HBSS at experimental pH conditions at 37°C

before starting the mannitol permeability experiments. The experiments were initiated by

addition of 14C-mannitol solutions to the apical (or basolateral) side of the monolayer. Samples

from the opposite side (apical or basolateral, respectively) were withdrawn at appropriate time

points. The amounts of 14C-mannitol in the samples were determined using a liquid scintillation

counter WinSpectral 1414 Liquid Scintillation Counter (Wallac, Turku, Finland).

TransEpithelial Electrical Resistance (TEER) (I-V)

Another method for determining the monolayer integrity is to measure the electrical resistance

produced by a tight cell monolayer (Table 2). TEER determines the ion permeability through

paracellular spaces in a cell monolayer (Madara 1989, Hidalgo 1996). This provides a quick and

reliable method for assessing monolayer integrity. The TEER of Caco-2 cell monolayers was

measured after equilibrating the monolayers in transport buffers with a Millicell® ERS

Voltohmmeter (Millipore Corp. Bedford, MA, USA). Additionally, the TEER was measured

after each permeability experiment to confirm monolayer integrity.

In experiments where long-term effects of laxatives on the monolayer integrity were

assessed, TEER of the monolayers was followed over an 8 hour period (V). During this assay

the recovery of tight junctions in cell monolayers was followed by mannitol permeability and

TEER measurements.

25

EXPERIMENTAL _____________________________________________________________________________

4.2.5. Transepithelial permeability (I-V) Drug permeability

The experiments were performed in the apical to basolateral (AP-BL) (I, II, III, IV, V) and

basolateral to apical (BL-AP) (I, V) directions under iso-pH conditions (pH 7.4 on both sides,

iso-pH 7.4) (III, IV) or pH-gradient conditions (apical pH 5.5 [I, II] or 5.8 [V], basolateral pH

7.4) (Table 2). All permeability experiments were performed on an orbital shaker in an incubator

at +37ºC at constant stirring rate of 50-75 rpm.

Prior to the permeability experiments, all cell monolayers were washed with HBSS

solution buffered with 10 mM HEPES to pH 7.4. After washing, the apical and basolateral

transport buffer solutions were added (same pH conditions as during the permeability

experiment). After 30 min of equilibration at 37ºC, the TEER of the monolayers was measured.

Values lower than 220 - 250 Ωcm2 would indicate poor integrity (< 5%) and monolayers were

discarded.

The AP-BL permeability experiment was started by adding pre-warmed HBSS containing

the drug(s) of interest on the apical side. The basolateral side contained fresh HBSS. The

samples were taken at appropriate time points by transferring the cell inserts into new wells

containing fresh HBSS. The BL-AP permeability experiment was started by adding HBSS (pH

7.4) containing the compound(s) on the basolateral side. The samples were taken from the apical

side by emptying the whole apical volume and replacing the volume with fresh HBSS. After the

experiment, the monolayers were washed once with fresh HBSS (pH 7.4), the TEER was

measured and mannitol permeability experiment was performed (I, IV). Samples were kept at

-22ºC (for no longer than 35 days) (III, V) or -70ºC (up to 75 days) (I, II, IV) prior to analysis

by HPLC or LC/MS/MS.

Permeability of natural compounds and derivatives (IV)

The experiments were performed in the AP-BL direction under iso-pH conditions (Table 2). The

permeability of each compound was studied in 24-well plates with the 0.33 cm2 growth area per

insert. The permeability experiments were performed as described in Section “Drug

Permeability”.

After the 30-min equilibrium time at +37ºC, TEER of the monolayers was measured, and

pre-warmed HBSS containing the active compounds and 14C-mannitol was added to the apical

side, and the permeability experiment was started under constant stirring rate of 75 rpm.

26

EXPERIMENTAL _____________________________________________________________________________

Samples were collected from the basolateral side after 15, 30, 45, 60 and 90 min. After the

permeability experiment, the TEER of the monolayers was measured to ensure the integrity of

the monolayers. The samples were kept at -70ºC until analysed.

4.2.6. Analytical methods (I-V) Samples from the cocktail dosed Caco-2 permeability experiments were analysed with an LC-

MS/MS system using atmospheric pressure ionisation (API) (I), atmospheric pressure

photoionisation (APPI) or electrospray ionisation (ESI) (II). All the compounds were detected

by positive ion multiple reaction monitoring with API (I). Antipyrine, ketoprofen, propranolol,

metoprolol, fluorescein, midazolam and verapamil were detected using positive and ranitidine

and hydrochlorothiazide using negative ion multiple reaction monitoring with APPI (II).

Samples from the drug permeability experiments (III-V) were analysed by reversed-

phase HPLC. An isocratic system with a UV-detector was used, except for furosemide, where a

scanning fluorescence detector was used. Analyses of flavonoid and alkyl gallate samples (IV)

and senna laxative samples (V) were carried out with reversed-phase HPLC with a diode array

detector and performed by gradient elution. Samples containing radio-labelled mannitol were

analysed using liquid scintillation counting. Rho123 samples were analysed by fluorometry.

4.2.7. Determination of permeability coefficients (I-V) The apparent permeability coefficients (Papp, cm/s) were calculated based on the equation:

Papp = (dQ/dt) / (A . C0 . 60),

where dQ/dt is the cumulative transport rate (µmol/min, nmol/min or µg/min) defined as the

slope obtained by linear regression of cumulative transported amount as a function of time

(min), A is the surface area (1.1 or 0.33 cm2 in 12- and 24-wells, respectively) of the

monolayers, C0 is the initial concentration of the compounds on the donor side (µmol/ml,

nmol/ml or µg/ml), and 60 is the coefficient when minutes are converted to seconds.

27

RESULTS AND DISCUSSION _____________________________________________________________________________

5. RESULTS AND DISCUSSION

5.1. EFFECTS ON MONOLAYER VIABILITY AND INTEGRITY The viability of the Caco-2 cell monolayers during the experiments is prerequisite for reliable

permeability values. Although non-living cells would not fall off from the inserts, the transport

proteins would not function properly, if the mitochondria were not producing energy for active

transport. Paracellular permeability is dependent on the integrity of the monolayers. A majority

of ionisable drugs and compounds are able to permeate to some extent via the paracellular

spaces, and therefore, their permeability might be affected by decreased monolayer integrity.

The administration of several drugs and/or compounds simultaneously might affect the viability

and integrity of the cell monolayers, if the individual concentrations were too high. Therefore, it

is important to examine the effects of these cocktail solutions on both viability and integrity of

cell monolayers.

5.1.1. Cytotoxicity (I-V) DMSO, the solubility enhancing excipient, showed low toxicity on Caco-2 cells at

concentrations below 5.0% during a 90 minutes long experiment (Figure 7). At higher

concentrations (5.0-15.0%), slightly increased dehydrogenase activity was measured. More

clearly toxic effects were noticed at DMSO concentrations above 15.0%. Ethanol, which also

was used at 5.0% concentrations as solubility enhancing excipient, showed more clear toxicity at

concentrations over 10.0%, which already was slightly toxic to Caco-2 cells (Figure 7).

Concentration (%)

0 10 20 30 4

Enzy

me

activ

ity (%

)

00

20

40

60

80

100

120

140

160DMSO Ethanol

Figure 7. Effects of DMSO and ethanol on the intracellular dehydrogenase activity of Caco-2 cells (mean ± SD, n = 4).The cells were exposed to DMSO for 90 min and to ethanol for 60 min.

28

RESULTS AND DISCUSSION _____________________________________________________________________________ Table 3. The MTT toxicity tests. Caco-2 cells were exposed to the compounds studied for 60 min. Results are expressed as percentage of the control value (100%) obtained after exposure to HBSS only (mean ± SD, n = 4-8).

Compound

Concentration, mg/ml

Enzyme activity, % of the control

Paper

Extracts of

Flax seed1

0.02 20.0

115 ± 26 106 ± 7

III

Purple loosestrife1

0.02

20.0 110 ± 16 106 ± 17

III

Scots pine bark1 0.02

20.0 129 ± 15 120 ± 5

III

Bilberry glycosidic aglycone2

20.0

0.20

103 ± 8 86 ± 5

III

Cowberry glycosidic aglycone2

20.0

0.20

126 ± 7 83 ± 8

III

Raspberry glycocidic aglycone2

20.0

0.20

112 ± 16 87 ± 11

III

Sage1 0.02

20.0 101 ± 31 79 ± 7

III

Oregano1 0.02

20.0 88 ± 28 77 ± 16

III

Rosemary1 0.02

20.0 105 ± 19 94 ± 15

III

Rhein3 0.015

0.15 91 ± 6 45 ± 10

V

Danthron3 0.012 0.12

95 ± 8 28 ± 11

V

Sennidin A/B3 0.025 0.125

97 ± 19 54 ± 27

V

Sennoside A/B3 0.645 0.32

102 ± 13 45 ± 11

V

Senna infusion 2.5 10.0

106 ± 23 92 ± 12

V

1 Contains 5% (w/v) DMSO 2 Contains 5% (v/v) ethanol 3 Contains 2.5% (w/v) DMSO

DMSO concentrations in the experiments (5.0% in solutions of the aglycone fractions of

the berry extracts [III] and solutions of the flavonoids and alkyl gallates [IV], and 2.0% in all of

the anthranoid laxative solutions [except the senna leaf infusion] [V]), were low enough not to

affect the viability of cell monolayers during permeability experiments. Ethanol concentration

(5.0%) was also low enough not to alter viability of the Caco-2 cells.

29


The studied drugs and compounds (antipyrine, buspirone, cephalexin hydrate, fluorescein

natrium, furosemide, hydrochlorothiazide, hydroxizine, ketoprofen, metoprolol bitartrate,

midazolam, naproxen, paracetamol, procaine hydrochloride, propranolol hydrochloride,

ranitidine, timolol maleate, verapamil hydrochloride, Rho123, catechin, epicatechin, flavone,

luteolin, methyl gallate, morin, naringenin, naringin, octyl and propyl gallate, quercetin) as

single or in cocktails induced no toxic effects on the Caco-2 cells at the used concentrations

measured either by the LDH (I) or by the MTT test (I-V).

No toxic effects in relation to mitochondrial enzyme activity (MTT test) were observed

with extracts made from flax seed, purple loosestrife, Scots pine bark, bilberries, cowberries,

raspberries, sage, oregano or rosemary at 0.02 mg/ml concentrations (III). However, slightly

toxic effects with decreased enzyme activities were observed with 20 mg/ml sage and oregano

extracts (Table 3). The laxatives (rhein, danthron, sennidin A/B, and sennoside A/B) caused a

clear reduction in mitochondrial enzyme activity at higher concentrations (Table 3) (V). Because

the solubility of the laxatives in HBSS was insufficient, 2.5% DMSO was included in the test

solutions for toxicity experiment to enhance the solubility. However, as stated earlier in this

Section, this has been shown not to affect the viability of the cells during the 90 min experiment

time (Figure 7). The senna leaf infusion exhibited no toxicity at used concentrations (2.50 -

10.00 mg/ml).

5.1.2. Integrity of Caco-2 monolayers (I - V) Mannitol permeability and transepithelial electrical resistance (TEER) across Caco-2 cell

monolayers were assessed after the actual permeability experiment in Papers I, II, and V.

Co-administration of several drugs in the same experiment had no effect on the tightness

of cell monolayers; only 0.19 - 0.43% (control monolayers, treated with HBSS only, 0.16 ±

0.02%) of the total mannitol amount diffused paracellularly to the basolateral compartment

during the 60 min experiment time (Figure 8A) (I, II). According to Hidalgo (1996), values

below 0.50%/h offer evidence for tight monolayers. Higher mannitol permeability in BL-AP

than AP-BL direction (Figure 8A) might be due to diffences in morphology of the apical and

basolateral membranes. The observed TEER values before and after experiments were 470 - 540

and 470 - 660 Ω.cm2, respectively, which indicate lack of irreversible damage caused by the

cocktails.

30


Mannitol diffusion across cell monolayers after laxative-permeation marker experiment

was not significantly enhanced (Figure 8B) (V). Compared to the control (HBSS without

laxatives), treatment with rhein and danthron enhanced mannitol permeability 2.1 and 1.7 fold,

respectively, but the values were still under the 0.50%/h adequate integrity limit (Figure 8B).

However, treatment with laxatives rhein and danthron resulted in a slight reduction of resistance,

which is consistent with the slight enhancement in mannitol diffusion (Figure 8B).

Perm

eate

d 14

C-m

anni

tol,

% o

f the

don

or

A

*********

*****

1.0

0.8

0.6

0.4

0.2

0.0

AP-BL BL-AP

II VIVIVIIIIcontrol

600700

500

400

TEER

, ohm

. cm

2

BKetoprofenParacetamol Verapamil Furosemide Rho123

500400300

200

TEER

, ohm

. cm2

* * * *

**

0.2

0.4

0.6

0.0

0.8

1.0

control rhein danthron sennidinA+B

sennosideA+B

sennainfusion

Figure 8. The effects of co-administered drugs as different cocktails (A) or laxatives (B) on Caco-2 cell monolayer integrity. On the left axis, 60-min mannitol diffusion measured after the actual permeability experiment as % of the total mannitol amount in the donor compartment (mean ± SD, n=3-4). The dashed line at 0.5% permeated mannitol indicates the highest value for tight monolayer and adequate monolayer integrity. On the right axis, transepithelial electrical resistance (TEER) measured before the mannitol diffusion test after the actual permeability experiment.Control bars represent results from mannitol permeability test after inbubation with HBSS only, without different cocktails (A) or laxatives (B).

Monolayer integrity was assessed during the actual permeability experiments, i.e. 14C-

mannitol was added in some of the permeation marker solutions when food supplement, herb

and berry extracts (III), and flavonoids and alkyl gallates (IV) were present. Three cell

monolayers were studied using only 14C-mannitol, without other permeation markers (controls).

Flax seed, purple loosestrife and Scots pine bark extract did not cause major effects on

mannitol diffusion. Minor changes in mannitol permeability compared to the controls and high

TEER values indicated tightly closed paracellular spaces (Figure 9). Herbs oregano, rosemary

and sage, on the contrary, exhibited stronger effects on cell monolayer integrity (Figure 9).

Rosemary extract had the highest effect on the paracellular spaces, leading to mannitol

permeability, which was 165% higher than the control value, when Caco-2 cell monolayers were

exposed to 1.0 mg/ml extract. Additionally, reduced but reversible TEER values were observed.

31


The effects of sage were similar, yet not as pronounced. Interestingly, the effects caused by

oregano were opposite to those of rosemary and sage: high concentrations inhibited but low

concentration enhanced mannitol diffusion. Although the herb extracts are able to open

paracellular spaces between the Caco-2 cells, the opening is reversible. Berry extracts,

glycosidic and aglycone fractions of bilberries, cowberries and raspberries, did not cause

significant differences in either mannitol diffusion or TEER values compared to the controls

(data not shown).

165 73

Differs from the Papp value of the control(no extract = 0), %

-40 -20 0 20 40 60

0.01 mg/ml0.1 mg/ml1.0 mg/ml

Scots pine bark

Flax seed

Sage

Rosemary

Oregano

Purple loosestrife

******

*** ****

******

*

Figure 9. The effects of plant extracts purple loosestrife, Scots pine bark and flax seed, and herbs (sage, rosemary and oregano) on the integrity of the cell monolayers measured as mannitol diffusion during the extract treatment. Values are mean percentage differences (n = 3) ± SD from the mannitol permeability in the absence of extracts (value of 0%, control). *p < 0.05, **p < 0.01, ***p < 0.001.

Mannitol diffusion during permeability experiments with flavonoids and alkyl gallates

(IV) varied between 0.19 and 0.48%/h (control monolayers 0.21 ± 0.02%) (data not shown).

In order to study long term effects of anthranoid laxatives on the cell monolayer integrity,

an eight hour experiment with 14C-mannitol was performed (V). Pre-treatment (60 min) with 100

µM rhein (0.03 mg/ml) and danthron (0.025 mg/ml) enhanced mannitol permeability. This

enhancement was observed not before than 60-100 minutes after the start of mannitol diffusion

experiment (Figure 4 in Paper V). Same effect was noticed in both AP-BL and BL-AP

permeability, the latter being more prominent. Characteristic for this was that the enhanced

mannitol permeability evened out again at time 240 minutes, and the slope of the cumulative

permeability curve was similar to that of the control. As expected, decreased TEER values were

32


observed during elevated mannitol permeability (Figure 4 in Paper V). However, all cell

monolayers recovered during the eight-hour experiment, leading to almost control like values at

the end of the experiment.

The integrity of Caco-2 cell monolayers, when measured as 14C-mannitol permeability

and TEER, was not affected by co-administration of several drugs (I, II), most of the food

fractions (III), natural compounds (IV), and anthranoid laxatives (V). In every case, when

elevated mannitol flux and decreased TEER values were observed, the effect was reversible

during or shortly after the experiment. Therefore, it can be stated that the results of the

experiments described in the following Section 5.2., are not affected by impairment of the Caco-

2 cell monolayers.

5.2. PERMEABILITY EXPERIMENTS

5.2.1. The effects of cocktail dosing on individual drug permeability (I, II) Modern combinatorial synthesis increase the number of drug candidates in drug discovery. For

that reason, pressure towards higher throughput also in permeability screening and more

effective use of expensive and time consuming in vitro absorption models is substantial.

Additionally, because of the non-comparable permeability data between different laboratories, standardisation of the use of these in vitro models is needed. For this purpose, the simultaneous

use of several model compounds as internal standards is indispensable. To standardise the use of

several model compounds simultaneously, the possibility for drug-drug interactions has to be

clarified. Because drug-drug interactions may differ significantly from cocktail to cocktail,

several different sets of drugs with high and low permeability, passive and active transport and

active efflux, were probed.

Correlation between single compounds and compounds in cocktails

First, the effects of five to ten drugs dosed simultaneously in different cocktails (1-6) (Table 4)

were studied on permeabilities of individual compounds (I). By this comparison, possible drug-

drug interactions in a cocktail could be detected. The Papp values obtained from the experiments

using single compounds were used as reference when compared with those obtained from the

different sets of cocktails containing 5-10 compounds. All six cocktails contained basic drugs

buspirone, hydroxizine, procaine, and timolol, which might act as substrates of the MDR1 efflux

protein (Kan et al. 2001, Terao et al. 1996). The acidic compounds ketoprofen (cocktails 4-6)

33


and naproxen (cocktails 5 and 6), possible substrates for an active H+ co-transporter (Ogihara et

al. 1996, Choi et al. 2005) were also included to elucidate the potential effects in active AP-BL

transport. All the cocktails contained also midazolam, a substrate for CYP 3A isoenzymes, but a

non-substrate for MDR1, expected to exhibit passive permeability (Paine et al. 1996, Kirn et al.

1999). Antipyrine, another passively permeating drug, was included in cocktails 5 and 6.

Verapamil, a known inhibitor of MDR1 (Saitoh and Aungst 1995), was added to cocktails 1 and

6 to probe, if inhibition of MDR1 would affect permeability of other potential MDR1 substrates.

Propranolol, which was also considered a substrate for MDR1 (Hunter and Hirst 1997, Yang et

al. 2000), was included in coctails 1 and 6 (contained verapamil), and in cocktail 2 (no added

verapamil), to further investigate the possible interactions between verapamil and MDR1. The

individual permeability data of the single compounds and the cocktails are presented in Table I

in Paper I.

Table 4. Summary of all drugs present in the cocktails 1-6 (I). The apical pH was 5.5 and basolateral pH 7.4.

Drug

Cocktail 1

Cocktail 2

Cocktail 3

Cocktail 4

Cocktail 5

Cocktail 6

Single conc. µM

Buspirone

x

x

x

x

x

x

50

Hydroxyzine

x

x

x

x

x

x

50

Midazolam

x

x

x

x

x

x

50

Procaine

x

x

x

x

x

x

50

Timolol

x

x

x

x

x

x

50

Propranolol

x

x

x

50

Verapamil

x

x

50

Antipyrine

x

x

50

Naproxen

x

x

50

Ketoprofen

x

x

x

50

total conc.*

350

300

250

300

400

500

50 * total cumulative concentration of all the compounds in the cocktail, µM

The permeability of midazolam or antipyrine was not affected by other drugs in the

cocktails. The AP-BL as well as BL-AP permeability was only slightly affected by other

compounds in cocktails, confirming absence of active efflux (Table I in Paper I). The AP-BL

permeabilities of basic drugs in almost all cocktails were very consistent with the results

obtained using single compounds. In cocktail 1, where both verapamil and propranolol were

present, the permeability of verapamil was 45% lower than when assessed as single compound,

indicating drug interaction between the two drugs. The Pappvalues of all compounds, including

ketoprofen, in cocktail 4, correlated well with the Papp values of the single compounds (Figure

34


2d in Paper I), but when naproxen was added (cocktails 5 and 6), the correlation was poorer,

indicating for a competitive utilization of a shared active transport pathway. It has been reported

that ketoprofen and naproxen are able to inhibit cellular uptake of benzoic acid (IC50 values 440

and 250 µM, respectively) in Caco-2 cells (Choi et al. 2005). According to the authors, kinetic

analysis using Lineweaver-Burk plots gave inhibition constant values 220 and 380 µM for

naproxen and ketoprofen, respectively. Further, cellular uptake of ketoprofen was inhibited by

the presence of benzoic acid and L-lactic acid, supporting the theory of ketoprofen being a

substrate of MCT1. Drug concentrations in experiments discussed here were much lower (50

µM), and yet permeability of both acidic drugs was significantly decreased, naproxen more than

ketoprofen.

In cocktails 1 to 4, the BL-AP permeability of all other compounds except procaine

correlated well with the permeabilities of individual single compounds. In cocktails 5 and 6, all

eight and ten compounds correlated strongly with the permeabilities of single compounds

(Figure 2 in Paper I).

In the second study, the effects of ten compounds in one cocktail (“cocktail 7” in Table 2

in Page 23) (II) were probed on their individual permeability. The effects of using different cell

batches were also probed (Table 4 in Paper II). The total concentration of the compounds in the

cocktail was 500 µM, giving individual concentrations of 50 µM. The permeation markers

verapamil, propranolol, ketoprofen, antipyrine and midazolam (also present in cocktails studied

in Paper I), and fluorescein (active transport by monocarboxylic acid H+ cotransport system,

Kuwayama et al. 2002), cephalexin (substrate of PepT1 transporter, Bretschneider et al. 1999),

ranitidine (absorptive permeability paracellular, probably substrate of MDR1 efflux (Takano et

al. 2006), and passively diffusing hydrochlorothiazide and metoprolol were included in the

cocktail.

The individual Papp values of the compounds in the cocktail correlated closely with those

obtained from single-compound experiments (Figure 10), although most single results were

from earlier data and thus obtained using another ESI method and different cultivation batch of

Caco-2 cells (Table 4 in Paper II). The Papp values of fluorescein, hydrochlorothiazide,

ketoprofen, and midazolam were very similar, when single and cocktail results were compared,

showing differences as low as ± 9-16%, indicating absence of interactions with other compounds

in the cocktail.

35


Most of the compounds with low permeability at the used pH gradient conditions showed

somewhat poorer correlation (Figure 10). The permeability of ranitidine, which is able to cause

concentration dependent decrease of its own permeability at high drug concentrations by

tightening the paracellular spaces between Caco-2 cells (Gan et al. 1998), showed a slightly

lower permeation in cocktail. According to Lee and Thakker (1999), active absorptive transport

is not involved with ranitidine permeability, because ATPase inhibitor ouabain and metabolic

inhibitors were not able to affect the transport.

ketoprofen

antipyrine

fluorescein

midazolam

y = 0.857x + 1.1646R2 = 0.9839

0

20

40

60

80

100

0 20 40 60 80 100

Papp (10-6) cm/s single compounds

Papp

(10-6

) cm

/s in

coc

ktai

l

52 1

6

4

3

y = 1.9551x - 0.6875R 2 = 0.9447

0

2,5

5

7,5

0 2,5 5

Figure 10. Linear regression of the Papp values between the compounds as single and in a cocktail of ten compounds (II). The permeability was conducted at individual 50 µM donor concentration. The apical pH was 5.5 and basolateral pH 7.4. Results are expressed as means ± SD (n = 3-4). The regression equation and correlation value are calculated with all detected ten compounds. Linear regression of the insert graph is calculated without midazolam, fluorescein, antipyrine and ketoprofen. 1= cephalexin; 2=hydrochlorothiazide; 3=metoprolol; 4=propranolol; 5=ranitidine; 6=verapamil

Simultaneous analysis of nine highly heterogeneous compounds was possible within 5-7

minutes (20 min in method presented in Paper I), giving remarkable saving in time and cost of

analysis. Cocktail dosing is also an effective technique to increase throughput, especially during

drug development and pre-clinic experiments. Standard testing of Caco-2 functionality requires

the use of at least 3-4 reference compounds (very low, low, moderate and high permeability),

depending on the characteristics of the studied drug candidates. Which compounds to choose for

this purpose is dependent on characteristics of drug candidates.

Palmgren et al. (2004) studied the possible interactions during AP-BL and BL-AP

permeability between seven heterogeneous drugs in a cocktail. They used different drug

concentrations, depending on transport mechanism: 20 µM verapamil, 50 µM propranolol, 20

36


µM antipyrine, 20 µM ibuprofen, 200 µM atenolol, 300 µM cephalexin and 500 µM baclofen.

Both AP-BL and BL-AP permeabilities correlated well as single and in cocktail, indicating that

interactions between drugs did not occur. The analysis was performed with a typical

UV/fluorescence HPLC, which was accurate enough for this set of experiments. Yet, drug

concentrations in the experiments have to be modified according to the analytical method used.

Low concentrations of slowly permeating compounds may not be detectable when using an

HPLC method. LC/MS/MS method is therefore more suitable for determining low

concentrations of slowly permeating drug candidates. One advantage in using LC/MS/MS

method is the possibility for detection of low drug concentrations and time saving, but the use of

HPLC offers often lower costs.

Effect of cell batches and age of monolayers (I)

Results of permeability experiments with Caco-2 cells show not only interlaboratory differences,

but also differences between the cell batches, experiment days and persons performing the

experiments. Therefore, regression between results of different experiment days was calculated.

When permeability data between same cell batches and same age of cell monolayers were

compared, the variability was further minimized. Experiments with cocktails 1 and 4, 2 and 3, 5

and 6 were conducted during same working day, using same cell passages and same age of

monolayers. When these results are compared by linear regression, they exhibit a strong

correlation in both AP-BL and BL-AP directions (Figure 3 in Paper I). Additionally, the good

correlation between cocktails 1 and 4 shows negligible effects of verapamil and propranolol in

cocktail 1 on the permeability of other MDR1 substrates in cocktail 4, where these substrates

were not present. Further, the only difference between cocktails 2 and 3 is the absence of

propranolol in cocktail 3, indicating that propranolol did not affect the permeability of other

possible substrates of MDR1 (Figure 3b in Paper I). And finally, the permeability values of all

basic compounds in cocktail 5, where neither verapamil nor propranolol were present, correlated

strongly with those in cocktail 6 (verapamil, propranolol and all basic and acidic compounds

present) (Figure 3c in Paper I).

The use of Caco-2 cells with different passage numbers and different age of monolayers

has been documented to affect permeability of drugs. For example when atenolol and

propranolol permeability was assessed during two different days, different Papp values were

obtained according to Markowska et al. (2001). During co-administration of four drugs in a

cocktail, propranolol permeability was significantly enhanced in cocktail. The authors explain

37


these results by the use of a heterogeneous cell line. This is indeed true. If such different results

after repeating the experiments are obtained, the results can be compared only when control

experiments are performed during same time and using same cell passages. On the other hand,

equal results can be obtained (Paper II).

Involvement of active transport (I)

The ratio between BL-AP and AP-BL permeability values is often used as a marker for a

compound to be substrate of efflux proteins. This kind of permeability studies are usually

performed with cell monolayers expressing MDR1 or other transport proteins (Polli et al. 2001).

Characteristic for the function of MDR1 is that the permeability of substrates is asymmetrical,

BL-AP being higher (≥2) than AP-BL at iso-pH conditions. Hence, if pH on both sides is the

same, high BL-AP values may indicate for active efflux. If the ratio between BL-AP and AP-BL

permeabilities is very low, the absorptive permeability is probably active.

According to the results in Paper I, all of the basic compounds would be substrates of

efflux proteins, showing high values for the ratio between BL-AP and AP-BL, when apical pH is

lower than the basolateral one. In this situation, the ionisation of weak bases and acids is

dramatically different compared to iso-pH conditions. For bases, BL-AP permeability exceeds

the AP-BL permeability, and the opposite happens for acids. On the other hand, according to

results in Paper I, none of the basic compounds would act as substrates of MDR1, if iso-pH

conditions are involved (Table III in Paper I). Under iso-pH conditions (7.4), a possibility for

over estimation of efflux in permeability of weak bases is possible, because their ability to

partition into lipid bilayers may be over estimated (Neuhoff et al. 2003).

The expression of functional efflux proteins in Caco-2 cells varies significantly not only

between different laboratories but also between different cell passages. Therefore it is important

to characterise the expression of the efflux proteins of the used Caco-2 passages. By using

reverse transcriptase polymerase chain reaction (RT-PCR), the mRNA levels of different efflux

proteins can be determined. According to experimental data from our laboratory, Caco-2 cells

express efflux proteins MDR1 and MRP1-6 (Siissalo et al. 2005). The expression of functional

MDR1 has been observed to be relatively high at low cell passage numbers (P29-32), but during

further cultivation of Caco-2 cells, the expression of functional MDR1 seems to decrease (P36-)

(Siissalo et al. 2004). According to these results, the expression of functional MDR1 was

medium to low in the Caco-2 cells used for Paper I (passage numbers 34-37).

38


It has been recognised that fast transmembrane permeability might hide efflux transport,

resulting in evening of the ratio between BL-AP and AP-BL transport. This is a phenomenon

where a drug might appear to be uninfluenced by MDR1, but in reality the drug is able to

traverse the cell membrane faster than MDR1 is able to efflux the drug (Eytan et al. 1996, Lenz

et al. 2000). In addition, low permeability may also mask a MDR effect. In the case of a MDR1-

substrate drug, which is absorbed via paracellular spaces, MDR1 may be less effective in

limiting transport, because the intracellular drug concentration is low. With a low intracellular

concentration, a drug may not manifest the ratio between BL-AP and AP-BL above 1, because

only small drug amounts are effluxed (Lenz et al. 2000).

The lack of MDR1 effects of verapamil and propranolol on the permeability of other

compounds in cocktails might be due to several factors. Although both compounds interact with

MDR1, they have not exhibited asymmetric permeability in in vitro models (Polli et al. 2001).

Verapamil is considered to be one of the most effective stimulators of MDR1-associated ATPase

activity. Additionally, verapamil has been shown to inhibit the MDR1 mediated efflux of several

drugs (Pauli-Magnus et al. 2000, Ayrton and Morgan 2001, Fromm 2002, Adachi et al. 2003,

among others). Passive diffusion of these compounds is perhaps rapid enough to traverse the cell

membranes faster than MDR1 is able to efflux (Litman et al. 2001). Like verapamil, propranolol

is also able to activate MDR1-associated ATPase, and interact with MDR1 (Collett et al. 2004),

but some studies do not confirm propranolol as a substrate for MDR1 (Wang et al. 2000, Polli et

al. 2001). Both drugs, verapamil (50 µM) and propranolol (200 µM) were able to induce a

marked increase (four to six-fold) in MDR1 mRNA levels compared to control cells, and were

both able to up-regulate expression of MDR1 in LS180 cells after relatively short periods of

exposure (3-6h) (Störmer et al. 2002). It has been shown that some drugs that do not show up as

being MDR1 substrates in a Caco-2 transport assay (probably because of high permeability), are

yet able to up-regulate the expression of MDR1 in LS180 cells if exposed over a very long

period.

Verapamil absorption from the intestine is half-maximal at concentrations between 4 and

31 µM (reported Km values: jejunum 31, ileum 29, colon 4.4) (Lin 2003). For propranolol and

verapamil, 50-300 µM concentrations are consistent with those likely to be present in the gut

lumen during oral dosing (Collett 2004). Thus, the used concentrations (50 µM and 250 µM) are

high enough to saturate active efflux, and are high enough for predicting their in vivo behaviour.

These concentrations are yet high enough to induce rapid increase of MDR1 in vivo, and thus

likely to affect permeability of other efflux substrates (Collett et al. 2004).

39


According to results in Papers I and II, several drugs and compounds can be included in

one permeability experiment, if the drug-drug interactions are not expected during the assay.

Several other reports regarding similar results are available. Markowska et al. (2001) studied

effects of a mixture of four (atenolol, nadolol, metoprolol and propranolol) and eight (RWJ-

53308, atenolol, terbutaline, propranolol, naproxen, piroxicam, topiramate and furosemide)

drugs in a cocktail with 10 µM individual drug concentrations. The use of several β-blockers in

same experiment is possible when active transport is not involved or the drugs are not sharing

same binding sites of the transport proteins. According to Larger et al. (2002), 100 µM

propranolol, co-administered with atenolol and phenylalanine, showed similar permeabilities as

single and in cocktail, as did also 50 µM propranolol in a cocktail with other drugs

(diphenhydramine, fluconazole, haloperidol, orphenadrine, propaphenone) (Bu et al. 2000).

The use of several permeation markers as internal standards in every in vitro permeability

assay is possible, but according to our results, standard testing of Caco-2 functionality does not

require the use of more than 3-4 compounds with very low, low, moderate and high permeability

(including substrates for transporters), depending on the characteristics of the studied drug

candidates.

5.2.2. Food supplements and food fractions (III, IV)

After validation of the use of several drugs as internal standards, the usefulness of the

method was further probed by experiments with food supplements and food fractions. These

contain several active compounds, such as flavonoids, alkyl gallates, which may interact with

simultaneously administered drugs. The permeability behaviour of some flavonoids and alkyl

gallates, which could offer valuable information about possible interactions, was also assessed.

The permeabilities of MDR1 substrates verapamil and Rho123 (Tsuruo et al. 1981,

Orlowski et al. 1996; Lee et al. 1994, Troutman and Thakker 2003), passively permeating

metoprolol and paracetamol, actively absorbing ketoprofen (Ogihara et al. 1996, Choi et al.

2005), and furosemide (passive trans- and paracellular absorption combined with possible active

transport by OAT transporters, MDR1 and/or MRPs efflux transporters, or another, yet not

characterised transporter [Flanagan and Benet 1999, Flanagan et al. 2002a,b, Chen et al.

2003a]), were studied with or without extracts from flax seed, purple loosestrife, Scots pine

bark; bilberries, cowberries and raspberries; oregano, rosemary and sage under iso-pH (7.4)

conditions.

40


The permeabilities of all the compounds, except verapamil and Rho123, were decreased

when flax seed extract was present during the permeability assay (Figure 1 in Paper III). Flax

seeds contains phytoestrogen and lignan precursors, flax seed oil (contains α-linolenic acid),

proteins, mucilage, and phenolic acids (Table II in Paper III). The effects on drug permeabilities

were significant, although flax seeds were not used as such, which is the normal way to use

them. When used as laxative, fluids with dissolved drugs etc. are adsorbed on the fibers of flax

seed, leading to swelling of intestinal contents. This could result in decreased absorption of

drugs from the intestine in vivo.

Scots pine bark and purple loosestrife extract contain phenolic acids, and permeability of

basic compounds verapamil and metoprolol was decreased. The effects on acidic compounds

were not as significant. Finnish berry extracts, which contain many phenolic acids and tannins,

did not affect drug permeaibility.

Neither oregano nor rosemary extracts were able to affect the transport of highly

permeable drugs studied. All herb extracts enhanced furosemide permeability: oregano (1mg/ml)

by 35%, rosemary (1mg/ml) by 365%, and sage (1mg/ml) by 13% (Table III in Paper III). The

strong enhancement of furosemide when co-administered with 1 mg/ml rosemary extract might

be partially due to widened paracellular spaces, as strongly enhanced mannitol permeability also

was observed (Figure 9 in Page 32). This is possible, because a considerable part of furosemide

absorption occurs via the paracellular spaces at pH 7.4 (Pade and Stavchansky 1997).

Methanolic extracts of rosemary have been observed to inhibit MDR1 efflux in cells over

expressing MDR1 (Plouzek et al. 1999), leading to enhancement of MDR1 substrates.

Additionally, rosemary has been described to induce CYP (2B) and UGT (1A6, 2B1) enzymes

in rat liver (Debersac et al. 2001), but effects on intestinal enzymes has not been determined.

The effects of herb extracts on Rho123 permeability were detectable, but not as significant.

Rosmarinic acid is the main phenolic component of all herb extracts used in our

experiments (Table 5). Other phenolic compounds are flavonoids (apigenin, naringin, luteolin).

The various phenolic compounds in plant extracts may affect the active transport of co-

administered drugs. Their effects on metabolising enzymes inside the enterocytes is more poorly

understood, but usually the efflux proteins MDR1 and MRP2 co-operate with CYP and and

UGT enzymes, respectively (Ofer et al. 2005).

Flavonoids are polyphenolic compounds, plant secondary metabolites, widely occurring

in plants. They consist of over four thousand of naturally occurring substances. Flavonoids are

41


defined chemically as substances composed of a common phenylchromanone structure (C6-C3-

C6), with one or more hydroxyl substituents, including derivatives (Figure 11).

Table 5. Phenolic compounds present in studied plant extracts (III). The permeability of these individual compounds were studied in Paper IV

Species

Active chemical components in the extracts

Total phenolics mg of GAE/g dw*

Flax seed Linum usitatissimum L.

phenolic acids: gallic acid

26.2

Purple loosestrife Lythrum salicaria L.

flavonoids phenolics: methyl gallate

42.1

Scots pine bark Pinus sylvestris L.

flavonoids: catechin phenolic acids

111.9

Bilberry Vaccinum myrtillus L.

flavonoids: catechin, quercetin, quercetin glycosides phenolic acids

37.8 G

279.0 A

Cowberry Vaccinum vitis-idaea L.

flavonoids: quercetin, quercetin glycosides phenolic acids

25.7 G

317.5 A

Raspberry Rubus idaeus L.

flavonoids: catechin, quercetin, quercetin glycosides phenolic acids

44.7 G

335.0 A

Oregano Origanum vulgares L.

flavonoids: apigenin, luteolin glycosides phenolic acids: rosmarinic acid

149.0

Rosemary Rosmarinus officinalis L.

flavonoids: apigenin, naringin phenolic acids: rosmarinic acid

185.0

Sage Salvia officinalis L.

flavonoids: apigenin, luteolin, luteolin glycosides phenolic acids: rosmarinic acid

166.0

*Determined as gallic acid equivalents (GAE) according to Folin-Ciocalteu procedure. Values are means of three analyses. 1 Glycosidic fraction (G) 2 Aglycone fraction (A)

O

O

OH

OH

OHOH

O

OH

O

OH

OH

OH

Figure 11 Structures of quercetin, a flavonoid, and genistein, an isoflavone

42


In contrast to flavonoids, isoflavonoids possess a 3-phenylchroman skeleton,

biogenetically derived from the 2-phenylchroman of flavonoids (Figure 11). Flavonoids and

isoflavonoids occur commonly as ester, ether, or glycoside derivatives of mixtures thereof, and

embrace over 4000 compounds (Harborne 1989). Flavonoids exist in foods as β-glycosides,

although it is the aglycone which is responsible for the pharmacologic activity.

Apparent permeability coefficients for flavone, naringenin, methyl- and propyl gallate

were calculatable. The highest Papp value was obtained for flavone, 380 (10-6) cm/s, indicating

very rapid transcellular passive permeation (Table 6) (Kuo et al. 1998). Naringenin, a flavonone

aglycone, with three hydroxyl groups, is able to permeate across Caco-2 cell monolayers, but

naringin, the correspondent glycosidic flavonoid did not permeate (Table 6). Addition of a sugar

moiety to a flavonoid molecule increases the number of hydroxyl groups and also the size of the

molecule, leading to a situation where the molecule is probably unable to partition into the cell

membranes.

Luteolin, morin and quercetin, which contain four, five and five hydroxyl groups,

respectively, were not absorbed across Caco-2 cell monolayers, but were able to partition into

apical cell membranes (measured as decrement of donor concentration) (Table 6). Molecular

configuration, i.e. the planarity of the flavonoids seems to contribute to the poor permeability.

According to results of van Dijk et al. (2000), planar configuration of flavonols might strongly

favour their intercalation into the cell membranes. Flavanones (for example naringenin) possess

a more tilted configuration, which favours less intercalation into the membranes and leads to

enhanced permeability compared to flavonols.

Catechin isomers (+)-catechin and (-)-epicatechin were not able to permeate across Caco-

2 cells (Table 6). Additionally, no accumulation in Caco-2 cells were observed (amount of

isomers in the apical solution after 90 minutes was practically unchanged) (Figure 2 in Paper

IV). According to other experiments, no absorptive permeability across Caco-2 cells has been

observed, but excretive permeability has been determined to be about as high as for mannitol

(Vaidyanathan and Walle 2001). According to these results, the minimal absorptive permeability

might be due to efflux mechanisms (MDR1 or MRPs). Indeed, when MK-571, an inhibitor of

MRP, was co-administered, the absorptive permeability of epicatechin was enhanced and

excretive decreased, indicating the role of MRPs in the minimal absorption. When verapamil, a

substrate of MDR1 was added, no effects on either absorptive or excretive permeability were

observed (Vaidyanathan and Walle 2001).

43


Table 6. Apparent permeability coefficients of flavonoids, alkyl gallates and model drugs. Values are means ± SD (n = 3-4).

Compound

Papp (10-6) cm/s

Compound

Papp (10-6) cm/s

O

O flavone

n(-OH) = 0

380.0 ± 32.0

OH

OOH

OH

OHOH

(+)-catechin

n(-OH)= 5

0

naringenin n(-OH) = 3

29.4 ± 3.1

OH

OOH

OHOH

OH

(-)-epichatechin

n(-OH)=5

0

naringin

n(-OH) = 2

0

methyl gallate -CH3 n(-OH)=3

2.9 ± 0.2

luteolin n(-OH) = 4

0

propyl gallate -(CH2)2- n(-OH)=3

7.3 ± 0.5

morin n(-OH) = 5

0

octyl gallate -(CH2)7- n(-OH)=3

0

O

OH

OH

O

OH

OH

OH

O

O

OH

OH

Glu-Rha-O

O OH

OH

OH

O

O

O

OH

OH

OH

OH

O OH

OH

OH

O

O

OH

O

OH

OH

OH

OH

OH

O

OOH

OH O

OH

OH

OH O quercetin

n(-OH) = 5

0

OH NH

CH3O

paracetamol

22.0 ± 5.0

O CH3

O

OH

ketoprofen

24.6 ± 4.8

44


Alkyl gallates, with three hydroxyl groups, were all taken up by Caco-2 cells (measured

by disappearance from donor concentration) (Table 6). Octyl gallate with the longest alkyl chain

distributed extremely rapidly from the apical solution into the cell membranes within 60 min,

whereas methyl and propyl gallate disappeared more slowly from the apical solution (Figure 2

in Paper IV). The length of the alkyl chain seemed to affect the transepithelial permeability;

methyl and propyl gallate were able to traverse the monolayer, while octyl gallate did not. The

slight lengthening of the alkyl chain seemed not to affect the permeability characteristics of alkyl

gallates. When the alkyl chain was significantly longer, interactions with cell membranes might

be stronger, and the molecules remain in the membrane, as happened with octyl gallate.

Flavonoid and isoflavone aglycones are small enough (about 250 g/mol) to diffuse passively

across absorptive epithelia. Apigenin, which is present in all herbs, is a flavonoid analogue of

genistein (Figure 11, Page 42). It is rapidly absorbed (Liu and Hu 2002), but extensively

metabolised by UGT1A1 in Caco-2 cells (Chen et al. 2003b).

Absorption of aglycones apigenin and genistein is rapid and temperature-dependent

followed by extensive phase II metabolism via glucuronidation and sulfation in the upper small

intestine and Caco-2 cells (Figure 12) (Liu and Hu 2002, Oitate et al. 2001). The apical-to-

basolateral transport of genistein was significantly decreased by flavones rutin and quercetin,

and flavanones (+)-catechin and (-)-epicatechin. Rutin and quercetin are potent inhibitors of

MRP´s; the permeability was decreased to about 50% (Oitate et al. 2001). Additionally,

genistein was able to inhibit Rho123 efflux in a rapidly reversible manner, indicating possible

interaction also with MDR1 (Castro and Altenberg, 1996).

Figure 12. Transport and metabolism pathways of flavonoid aglycones in the intestine.

FG: flavonoid glycoside F: flavonoid aglycone BSβG: broad-specific β-glucosidase UGT: UDP-glucuronosyltransferase SULT: sulphotransferase Fg: flavonoid glucuronide Fs: flavonoid sulphate MRP2, MRP3: multidrug resistance-associated proteins Modified from Walle (2004)

The absorption of flavonoids has been studied across Caco-2 monolayers by Murota et al.

(2000) and Walle (2004), among others. The amount of flavonoid aglycones in the apical side

45


was significantly decreased during two hours of experiment, resulting in the appearance of free

and conjugated forms in the cellular extracts. As compared with flavonoid aglycones, none of

the glucosides were absorbed efficiently from apical side. On the other hand, some flavonoid

glucosides are absorbed actively by a glucose transporter according to Walle (2004), but

quercetin probably not (Murota et al. 2000). The ingested flavonoids are supposed to be taken up

passively by enterocytes. Inside the cells, the flavonoid is metabolised, and the metabolites are

actively effluxed out of the enterocytes by MRP2 (to the lumen of intestine) and MRP3 (to the

circulation), because they are more water soluble than unmetabolised flavonoids (Figure 12)

(Petri et al. 2003).

Daily consumption of 100g of berries (black currants, lingonberries and bilberries) has

been observed to significantly increase serum quercetin concentration by almost 50% (Erlund et

al. 2003). According to the researchers, their preliminary studies have shown, that quercetin is

bioavailable from all of the berries mentioned. In general, berries are an important source of

quercetin in Finnish diet.

Several flavonoids are substrates of efflux proteins. Nguyen et al. (2003) investigated the

effects of 22 dietary flavonoids on accumulation of daunomycin and vinblastine in human

pancreatic adenocarcinoma cell line Panc-1, which over express MRP1, but does not express

MDR1. Flavonoids, such as morin, genistein, quercetin are capable of modulating MRP1-

mediated drug transport of daunomycin and vinblastine (Nguyen et al. 2003). Several of the 22

flavonoids could inhibit MRP1-mediated efflux after 2-hour exposure. The mechanism

underlying this inhibition is not known, but direct binding interactions are possible (Nguyen et

al. 2003). According to other researchers, quercetin, naringenin, and naringin are able to interact

with MDR1 efflux protein present in Caco-2 cells (Ofer et al. 2005).

As a summary, the effects of different food extracts, which contain high concentrations of

several flavonoids, are difficult to predict on permeability of simultaneously ingested drugs.

Flavonoid concentrations present in normal food are low enough not to excert any influence on

drug permeability (Nguyen et al. 2003). The flavonoids interact with several active transport

proteins, such as OCT, MDR1, and MRPs. The exact mechanisms behind the interactions with

these transporters are difficult to clarify, because absorptive cells (enterocytes) and Caco-2 cells

express several efflux proteins, and specific inhibitors for all of these are not available.

Additionally, flavonoids and other active compounds in the extracts might interact with each

other during absorption, thus affecting their own permeability and binding to transporter

proteins.

46


5.2.3. Anthranoid laxatives (V) Senna leaves are a widely used “natural” laxative. Senna leaf infusion contains several active

compounds, such as sennosides A and B, aloe emodin, rhein glucosides and several flavonoids,

but also salicylic acid and oxalate (Franz 1993, Bruneton et al. 1995). These active compounds

are able to influence absorption of simultaneously dosed drugs by affecting transport proteins

and membrane fluidity. Therefore, the effects of senna leaves were assessed on drug

permeability. The effects of active compounds in senna leaves, i.e. rhein, sennosides and

sennidins were also probed on permeability of several drugs. Danthron, which is a synthetic

anthranoid laxative, has been withdrawn from the market because of carcinogenic effects.

The effects of anthranoid laxatives on permeability of paracetamol, verapamil and

propranolol were minor (Figure 3 in Paper V). These drugs possess high transmembrane passive

permeability, although verapamil and propranolol are able to interact with the efflux protein

MDR1. If the studied laxatives interact with efflux proteins, the effects are not detectable,

because of high passive permeability of the marker drugs.

OH O OH

Ketoprofen, also a highly permeable drug in the experiment, was more sensitive to

laxative treatment; rhein and danthron were able to decrease ketoprofen permeability slightly

(Figure 3 in Paper V). The absorption of ketoprofen is in part active, mediated by the

monocarboxylic acid transporter, located on the apical membrane of enterocytes and Caco-2

cells (Ogihara et al. 1996, Choi et al. 2005). Rhein and danthron, which are relatively small

(m.w. < 300 g/mol) planar molecules (Figure 13), are possibly able to intercalate with cell

membranes, leading to changes in membrane fluidity (increased rigidity) and perhaps affect the

transport proteins. They enhanced furosemide permeability 3.6 and 3.0 fold, respectively. The

ability of these compounds to participate with the cell membranes could explain the behaviour of

furosemide. Additionally, the effects of rhein and danthron could partly be explained by

reversibly opened paracellular spaces, indicated as elevated mannitol permeability and decreased

TEER values during the experiment (Figure 8 in Page 31). Furosemide is only partially

absorbed paracellularly, and therefore, a partial opening of the paracellular spaces would not

R2R1O

Figure 13. The chemical structure of anthraquinones rhein (R1 = H, R2 = COOH) and danthron (R1, R2 = H).

47


lead to very strong enhancement of permeability. The absorptive permeability of Rho123, a

substrate for MDR1 efflux, was not affected by rhein and danthron, because rhodamine transport

occurs through paracellular spaces in this direction (Troutman and Thakker, 2003). The

excretive permeability, on the other hand, was significantly affected by rhein (130%) and

danthron (75%).

Senna infusion, which contains mainly sennosides A and B, small amounts of other

sennosides, aloe-emodin (anthraquinone, R1=H, R2=CH2OH, Figure 13), rhein glucosides and

flavonoids (kaempferol, kaempferin and isorhamnetin) and naphthalene precursors, but also

salicylic acid and oxalate (Franz 1993, Bruneton et al. 1995), significantly enhanced ketoprofen

permeability (Figure 3 in Paper V). This infusion contains stronger acids than ketoprofen, but

also many active compounds; therefore it is not easy to explain what could happen between

drugs and these compounds. Oxalic acid and tannins have been observed to react with iron (II)

salts, Ca2+, Mg2+ ions (Geisser 1990). Interestingly, tannins (tannic, ellagic and gallic acids) have

been noticed to interact with proteins on the plasma membranes (Simon et al. 2003). Because

these acidic compounds are able to adhere to the cell membranes, regional changes in the pH

near to the site of absorption could affect the permeability of ionisable compounds according to

the pH partition theory.

The mechanisms behind the inteactions during absorption between drugs and “natural”

drugs, such as senna infusion, are not easily detected. The knowledge about how these mixtures

of several active components interact with drugs during absorption is not sufficient. Not only the

active substances of senna infusion, but also the metabolites of different sennosides, sennidines,

rhein and danthron may influence absorption of co-administered drugs. On the other hand, the

concentration of active components in senna infusion is not as high as in enriched herb extracts,

and therefore safer to use. Highly permeable drugs, such as verapamil, propranolol, paracetamol

and ketoprofen, are most probably not affected but more slowly permeating drugs, especially if

they are substrates of active absorptive or excretive transport and metabolism might interact with

flavonoids and other active compounds in the senna infusion.

48

CONCLUSIONS _____________________________________________________________________________

6. CONCLUSIONS

Caco-2 cell cultures were assessed as an in vitro model for intestinal absorption during

simultaneous administration of several drugs and natural compounds. The most significant

information to emerge from this study was that Caco-2 cell monolayers can be used as an

absorption model during drug development when several drugs are probed simultaneously and

higher throughput has to be gained, although possible interactions between drugs and natural

compounds in biological matrices are not easy to detect.

The following conclusions can be drawn from the results of this study:

1. When determining permeability of drug candidates, the use of several standard

compounds is possible, if drug-drug interactions between the standards are

not expected. These standards, if they are substrates for different transport

proteins, should not interact with each other. This is important, because drugs

may use several active transport mechanisms, both absorptive and excretive,

simultaneously. The choice of standards is mainly dependent of the characteristics

of the studied drug candidates and analytical methods used.

2. When choosing standards, their effects on cell monolayer viability and integrity have

to be assessed. The concentration of standards should be low enough not to affect

viability of the cell monolayers but high enough to be detectable by the analytical

methods available. For the monolayer integrity testing, the use of paracellular marker

molecules, for example 14C-mannitol can be used, if radioactive labelling does not

disturb nalytical methods used.

3. The variation between results from different experiment days or using different cell

batches can be minimised by using appropriate standards and careful working

routines.

49

CONCLUSIONS _____________________________________________________________________________

4. To probe effects of enriched food fractions or “natural” drugs, standard drugs with

known absorption behaviour can be used. A proper set of several standard drugs in a

cocktail is a more demanding task for the future, because possible interactions

between drugs and active compounds in the food fractions and natural drugs are

complicated and might be severe for the consumer. The behaviour of several different

active compounds in biological matrices during absorption is not yet clarified.

5. The possibility to increase throughput by using several compounds simultaneously

was reached, both during permeability experiments and analyses by LC/MS/MS.

The permeability experiments with 24-well inserts were successfully performed,

and compound amounts in the samples were high enough for HPLC analysis. If

several drugs are probed simultaneously on cell cultures grown on 24-well inserts,

HPLC methods can most probably not be used because of very low drug

concentrations and small sample volumes.

Whereas several food-drug interactions have been attributed to the inhibition of drug

metabolising enzymes, the effect of food components on transporters during absorption,

distribution and excretion of drugs is still limited. Caco-2 cell monolayers, if expressing active

transport and efflux proteins, are therefore suitable as in vitro model for these types of studies.

Caco-2 cells used for the experiments described in this thesis, express efflux transporters MDR1,

BCRP, MRP1-6, determined by RT-PCR.

50

ACKOWLEDGEMENTS _____________________________________________________________________________

ACKNOWLEDGEMENTS

The study was carried out at Viikki Drug Discovery and Development Technology Center

(DDTC) and Division of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy,

University of Helsinki, during the years 2000-2005.

I wish to express my appreciation to Professor Martti Marvola, Head of the Division of

Biopharmaceutics and Pharmacokinetics, for his support during this study and for providing

excellent facilities for my work.

My sincere gratitude is due to my supervisor docent Ann Marie Kaukonen for valuable ideas and

excellent advices as well as for encouraging guidance and positive way of thinking during the

course of this work. She has always been ready for both scientific and also non-scientific

discussions during this work. Additionally, she has always been ready for sharing the

accommodation during scientific meetings and providing me a bed in her home, when needed.

I want to thank all my co-authors. Professor Pia Vuorela has provided me an opportunity to dive

into a world of plant extracts and to present our results in scientific and professional meetings.

Professor Jouni Hirvonen has offered me enormous support in writing my first scientific paper

and an opportunity to join his working group, Professor Heikki Vuorela is acknowledged for his

skills in statistics and contacts around the world. Other co-workers professors Risto Kostiainen

and Tapio Kotiaho, docent Päivi Tammela, Heli Kangas, Kati Hakala, Anna Galkin and Elina

Takala are thanked for their contribution in this work.

I thank most sincerely the reviewers Dr. Marjukka Suhonen and Dr. Staffan Tavelin, for their

constructive criticism and for giving me valuable suggestions for improvement of my thesis.

I offer my thanks to senior laboratory technician Erja Piitulainen for her skills in sample

determinations by HPLC.

I express my sincere thanks to all my colleagues at the Division of Biopharmaceutcs and

Pharmacokinetics, for creating a supportive and pleasant atmosphere, and to Professor Arto Urtti

and my new colleagues at DDTC for giving me the time I needed for completing this thesis.

51

ACKOWLEDGEMENTS _____________________________________________________________________________

Finally, my warmest thanks belong to my dear husband Eino for his neverending and

understanding support, and my children Mira and Mikko who have grown up during this work.

Mira will be thanked for helping with some of the figures in this thesis. And finally, Sarah is

thanked for taken care of my outdoor recreation.

Helsinki, May 2006.

Leena Laitinen

52

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