caco-2 cell cultures in the assessment of intestinal ...anthranoid laxatives influence the...
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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.
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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
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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
THEORY AND REVIEW OF THE LITERATURE _____________________________________________________________________________
(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
THEORY AND REVIEW OF THE LITERATURE _____________________________________________________________________________
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
THEORY AND REVIEW OF THE LITERATURE _____________________________________________________________________________
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
THEORY AND REVIEW OF THE LITERATURE _____________________________________________________________________________
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
THEORY AND REVIEW OF THE LITERATURE _____________________________________________________________________________
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
THEORY AND REVIEW OF THE LITERATURE _____________________________________________________________________________
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
THEORY AND REVIEW OF THE LITERATURE _____________________________________________________________________________
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
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THEORY AND REVIEW OF THE LITERATURE _____________________________________________________________________________
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
THEORY AND REVIEW OF THE LITERATURE _____________________________________________________________________________
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
THEORY AND REVIEW OF THE LITERATURE _____________________________________________________________________________
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
THEORY AND REVIEW OF THE LITERATURE _____________________________________________________________________________
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
THEORY AND REVIEW OF THE LITERATURE _____________________________________________________________________________
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
THEORY AND REVIEW OF THE LITERATURE _____________________________________________________________________________
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
THEORY AND REVIEW OF THE LITERATURE _____________________________________________________________________________
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
THEORY AND REVIEW OF THE LITERATURE _____________________________________________________________________________
(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
THEORY AND REVIEW OF THE LITERATURE _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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
RESULTS AND DISCUSSION _____________________________________________________________________________
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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