THE EFFECT OF SELECTED HYDROXY FLAVONOIDS 
ON THE IN VlTRO EFFLUX TRANSPORT OF 
RHODAMINE 123 USING RAT JEJUNUM 
S. VAN HUYSSTEEN B. Pharm 
Dissertation submitted in the partial fulfilment of the requirements for the degree 
Magister Scientiae in Pharmaceutics at the North West University 
Supervisor: Dr. M.M. Malan 
Go-Sugervisor: M.-K.Sv\rart 
2005 
POTCHEFSTROOM 

CHAPTER 1 
INTRODUCTION AND STATEMENT OF THE PROBLEM 
1 .I Introduction 
Oral bioavailability requires the absorption of drugs across the intestinal epithelium. This 
may be mediated by either the paracellular andlor transcellular routes. Passive 
transcellular absorption requires the appropriate physicochemical properties to allow 
permeation across the apical and basolateral membrane domains. Compounds 
demonstrating these properties are more likely to be recognised as substrates for 
intracellular metabolism, such as by cytochrome P450 isozymes andlor secretory drug 
efflux systems, including Pgp, such that oral bioavailability will be limited. Pgp, which 
leads to MDR in tumor cells, is an ATP-dependent secretory drug efflux pump, encoded 
by the MDRI gene in humans. One of the working mechanisms of Pgp is to clear the 
membrane lipid bilayer of lipophilic drugs in the manner of a flippase. In the intestine, as 
well as at specific other epithelial and endothelial sites, Pgp expression is localised at 
the apical membrane, consistent with secretory detoxifying and absorption limitation 
functions. Other secretory efflux systems, such as multidrug-resistance associated 
protein (a glutathione S-conjugate transporter), fluorochrome efflux systems and the 
methotrexate efflux systems, together with drug ionic charge and the intestinal pH 
microclimate, may mediate intestinal secretion of a wide variety of drugs. Direct 
evidence for Pgp limiting drug absorption comes from studies in vitro with human Caco-2 
cells and includes non-linear dependence of absorption on substrate concentration, 
increased absorption upon saturation of secretion and increased absorption upon 
inhibition of Pgp function, with modulators such as verapamil. Pgp function may be 
integrated with drug metabolism, with several drugs being common substrates for Pgp 
and cytochrome P450 (CYP3A4) (Hunter & Hirst, l997:lZ9). 
Other potential problems concerning Pgp involve specificity. If the inhibitor is not 
specific enough, it blocks multiple ATP-binding cassette proteins (ABC) members, or it 
binds with such high affinity that it does not dissociate, then normal 
physiologiclprotective functions of these transporters may be compromised, leading to 
unwanted effects (Chan et a/., 2004:43). Many Pgp inhibitors are compounds which 
occur in foods such as soy beans and grapefruit juice and inhibition as well as induction 
of this protein could lead to enhanced or diminished absorption of drugs that are 
substrates for Pgp (Wagner et a/., 2001 :S14). 
Different compounds have been shown to reverse the Pgp-mediated MDR. MDR cells 
can be sensitised to anticancer drugs when treated with a Pgp inhibitor, which is known 
as a chemosensitiser (Choi et a/., 2004:672). The search for chemosensitisers which 
have the advantages of being a non-transportable inhibitor without side effects, has led 
to a great deal of research in flavonoids derived from plants (Conseil et a/., 1998:9831). 
During this study a few selected flavonoids (morin, galangin, kaempferol and quercetin) 
were used to test whether inhibition of active transport can be facilitated in this model. 
To assess the absorption potential of chemical entities numerous in vitro and in vivo 
model systems have been used. Many laboratories rely on cell culture models of 
intestinal permeability such as Caco-2 cell monolayers. The successful application of in 
vitro models of intestinal drug absorption depends on the extent to which the model 
comprises the relevant characteristics of the in vivo biological barrier (Hidalgo, 
2001:389). For the purposes of this study, an in vitro method using intestinal mucosal 
sheets suitable for mounting in Ussing chambers (Sweetana-Grass diffusion cells) was 
chosen. Using this method, the rate of transport of selected compounds can be 
determined in the presence of Pgp inhibitors. 
The aims of this study are to: 
m study the effects of selected flavonoids (morin, galangin, kaempferol and 
quercetin) on the transport of the Pgp substrate Rhodamine 123 (Rho 123) 
across rat jejunum using Sweetana-Grass diffusion cells; 
m evaluate the structure activity relationships (SAR) of the selected flavonoids with 
reference to the inhibition of Pgp, and 
a compare the effect of the selected flavonoids at two different concentrations. 
1.2 Structure of this dissertation 
In this dissertation, the introductory chapter is followed by a review of the relevant 
literature (Chapter 2). In Chapter 3, flavonoids as modulators will be discussed with 
reference to the inhibition of Pgp. In Chapter 4 the experimental procedure as well as 
the validation of this method will be given. In Chapter 5 the results of the study will be 
reported and discussed. In Chapter 6 final conclusions are drawn and recommendations 
are made. 
CHAPTER 2 
DRUG DELIVERY AND ORAL BlOAVAlLABlLlTY 
2.1 Introduction 
The oral route is the most convenient and widely used means of drug administration. 
Estimates of effective clinical dose are based on a combination of estimates of oral 
absorption, bioavailability, clearance and volume of distribution (Pelkonen et al., 
2001:622). Bioavailability indicates a measurement of the rate and amount of 
therapeutically active drug that reaches the systemic circulation and is available at the 
site of action (Shargel & Yu, 1999:247). Bioavailability relates to more than just 
absorption. It is determined by the extent of absorption and presystemic metabolism. It 
is also the result of several competing processes, some of which favor the entry into the 
systemic circulation and others which impede it. The most important processes are the 
following: 
Extent of absorption which is determined by passive diffusion, active and 
facilitated transport, paracellular transport, endocytosis and gut flora metabolism; 
Efflux proteins located at the apical membrane of the small intestine, which 
include P-glycoprotein (Pgp; MDRI) and MRP2 (multidrug resistance-associated 
protein), may drive compounds from inside the cell back into the intestinal lumen, 
preventing their absorption into the blood, thus although lipophilic compounds 
may readily diffuse across the apical plasma membrane, their subsequent 
passage across the basolateral membrane and into the blood is by no means 
guaranteed and 
Presystemic metabolism which describes the rapid metabolism of orally 
administered drugs prior to reaching the general circulation. It is determined by 
cytochrome P450 (CYP)3A4, other CYPs and phase4 enzymes in the gut and 
by contribution of the liver. 
(Chan et a/., 2004:25; Hunter & Hirst, 1997:132; Pelkonen et a/., 2001:622; Shargel & 
Yu, 1999:378). 
Efforts to formulate oral products to optimise their bioavailability are continually being 
researched. These efforts also include changes in the physico-chemical and dissolution 
properties of the drugs to be able to formulate such products. It is however widely 
recognised that intestinal metabolism and active drug efflux must also be addressed in 
order to maximise oral bioavailability and decrease variability that exist in drug delivery 
(Wacher et a/. , 1 998: 1 322). 
The principal site of absorption of most nutrients is the small intestine which has a more 
elaborate system that determines its permeability (Csaky, 1 984:sl). In order to 
understand the absorption of drugs as well as the efflux of drugs that may take place, 
the anatomy of the small intestine as well as the different absorption processes and 
mechanisms of the efflux of drugs that may take place will be discussed. 
2.2 Anatomy of the small intestine 
The small intestine represents the principal site of absorption for any ingested 
compound, whether dietary, therapeutic, or toxic (Chan et a/., 2004:26). The three 
anatomic divisions of the small intestine are the duodenum, jejunum and ileum with a 
total length of approximately 6 m in humans (Carr & Toner, 1984:l). 
The intestinal mucosa is divided into three distinct layers (Figure 2.1). The deepest layer 
is the muscularis mucosa, a continuous thin sheet of smooth muscle separating the 
mucosa from the submucosa. Contraction of the muscularis mucosa facilitates emptying 
of crypt luminal contents by causing luminal compression. The second layer, the lamina 
propria is the connective tissue inside the villus and surrounding crypt and carries out 
several immunologic functions as well as providing structural support to the epithelial 
layer (Trier & Madara, 1981:926). The epithelial layer, which is the third layer, is a 
continuous sheet of epithelial cells that is only one cell thick. It lines the villi and their 
surrounding crypts. It consists of a monolayer of heterogenous cells and includes 
absorptive cells, crypt cells, endocrine cells and goblet cells which secrete mucin. This 
layer is in direct contact with the luminal content. The epithelium is separated from the 
underlying lamina propria by a thin, continuous basement membrane. In certain parts of 
the intestine (Peyer's patches) the epithelial cell layer contains M (microfold) cells, which 
sample antigens from the intestinal lumen to the lymph (Trier & Madara, 1981:928). The
anatomy of the villi of the small intestine is shown in Figure 2.1 (Trier & Madara,
1981 :926).
C~11
extru&ion zene
tQ"'
t
" I
Absorp Ive(
crlls ....
..."
"Blood vuse l$
Lymph vesuls
N@rvi!S
Smooth muscle l Lamina
Connective tlnue l' proprio
Lymp~ytes
Plasma cells
Eos.inophi les
/
,
,
,
,
/
,
VilIous
epithelium
Muscularis
mucoso ----
Figure 2.1: Schematic presentation of the intestinal mucosa (Trier & Madara,
1981:926).
The surface facing the lumen has a fuzzy appearance called the brush border or
microvilli (Rogers, 1983:201). The microvillous border increases the surface area
available for absorptionby a factor of 25 (Carr & Toner, 1984:13).
Although the primary function of the villus epithelium is absorption, other functions can
also be performed. Known functions of the crypt epithelium include epithelial cell
renewal by means of undifferentiated and young goblet cells and exocrine secretion into
the crypt lumen by means of goblet, Paneth and undifferentiated cells and endocrine
secretion by means of the endocrine epithelial cells (Trier & Madara, 1981 :929).
6
--
- ---
--- --:.- - -
2.3 Passage of drugs across biologic membranes
The absorption, distribution, biotransformation and excretion of a drug involve its
passage across cell membranes (Benet et al., 1996:139). The extent to which a
compound is absorbed by the intestinal epithelium is therefore a critical factor in
determining its overall bioavailability (Chan et al., 2004:26). In general, biological
membranesdisplay the property of a porous lipid membrane through which water and a
few small polar particles can easily pass, but the passage of the vast majority of
hydrophilic molecules is markedly restricted. Yet, many essential polar nutrients pass
across the intestinal barrier (Csaky, 1984:53). There are two principal routes by which
compounds may cross the intestinal epithelium namely paracellular or transcellular
(Chan et al., 2004:26). The different pathways and mechanisms that initiate
transepithelialtransport are shown in Figure 2.2 (Chan et al.,2004:26).
LUMEN
.-
.-
,8
.
. . o.
BLOOD
Figure 2.2: Diagram of pathways and mechanisms that mediate transepithelial
transport (Chan et al., 2004:26).
In Figure 2.2 (A) passive paracellular transport is illustrated on the left side followed by
(B) carrier-mediated transport, (C) efflux transporters, (D) apical efflux, (E) intracellular
metabolising enzymes (F) apical efflux and intracellular metabolising enzymes (G)
passive transcellular diffusion and (H) vesicular transport which is illustrated on the right
side (Chan et al., 2004:26).
7
- -
-- ----
In general, the transport pathways can thus be divided into the following:
A. Passive paracellular transport (passive diffusion). This is an aqueous,
extracellular route across the epithelium and the driving forces for this route are
the electrochemical potential gradients derived from differences in concentration,
electrical potential and hydrostatic pressure between the two sides of the
epithelium. The transepithelial permeation of hydrophilic compounds occurs
mainly through the paracellular route (Hidalgo, 2001 :388).
B. Carrier-mediated transporters (active transport). Transcellular absorption
from the lumen to the blood requires uptake across the apical membrane,
followed by transport across the cytosol, then exit across the basolateral
membrane and into the blood. Transcellular absorption of hydrophilicdrugs may
be facilitated via specific carrier-mediated pathways by means of utilising the
same route of absorptionfollowed by nutrients and micronutrients. Many orally
administered drugs are lipophilic and undergo passive transcellular absorption
(Hunter & Hirst, 1997:131).
C. Effluxtransporters. Drugs that cross the apical membrane may be substrates
for apical efflux transporters, which extrude compounds back into the lumen
(Evers et al., 1998:1318).
D. Apical efflux. These apical efflux transporters are principallyABC proteinssuch
as Pgp and MRP2, and are ideally situated to act as the first line of defence by
limiting the absorption of potentially toxic compounds. Compounds that are
already present in the blood may undergo active blood-to-Iumen secretion
facilitated by these transporters(Watkins, 1992:520).
E. Intracellular metabolising enzymes. The transcellular route of absorption
exposes drugs to intracellular metabolic systems, small intestinal enterocytes
(which provides the first site for cytochrome P450 (CYP)-mediated metabolism of
orally ingested drugs) and xenobiotics (Watkins, 1992:520).
8
F. Apical efflux transporters and intracellular metabolising enzymes. The
CYP-system (phase I metabolism), as well as other intracellular metabolic
systems, such as phase II conjugatingenzymes, may yield metabolites that are
themselves substrates for effluxpumps, thus providingadditionalpossibilitiesfor
interactions(Keppleret a/., 1999:237).
It is however only passive diffusion,active transport and facilitateddiffusionthat are of
importance in drug transport (Shargel &Yu, 1993:112).
2.3.1 Passive diffusion
The most common method of drug absorption is passive diffusion,the process where
molecules diffuse spontaneously from a region of higher concentration to a region of
lower concentration. This process is passive because no external energy is needed
(Shargel & Yu, 1993:112). A prerequisite for passive diffusion is that the compound
should be lipophilicand of low molecular weight. The rate of transport across a
membrane by means of passive diffusionis derivedfrom the first law of diffusionby Fick
which states that the rate of transport is dependant on the surface area of the diffusion
coefficientand the concentrationgradient across the membrane (Lund,1994:220).
Accordingto Fick (as quoted by Csaky, 1984:51) diffusion is the unrestricted flow of
substances from one compartmentto the other. It is caused by thermal agitation of the
molecules. Fick's law quantitativelyexpresses the rate of diffusion by means of the
followingequation:
dn =DA de
dt dx
Where dn is the number of molecules (ions)crossing an area A intime dt in proportionto
the concentrationdifferencede over a distance of dx. D is the diffusioncoefficientand is
expressed by the amount of substance diffusingacross unit area per unit time where
dc/dx=1.
9
Permeation is essentially a two-way process, namely the flow of substancesfrom the
lumen into the bloodstream (absorption) and the flow from the bloodstream into the
lumen (exorption),which occurs simultaneously. The primary physiologicfunction ofthe
intestine is absorption. Therefore, the net result of permeation is usually absorption.
Nonetheless, exorption cannot be completely neglected (Csaky, 1984:52). Lipids or
lipid-soluble substances, including the majority of drugs and other xenobiotics, are
transported by passive diffusion (Csaky, 1984:52).
2.3.2 Active transport
Active transport is characterised by the transport of drug against a concentration
gradient which is, from regions of low drug concentrations to regions of high drug
concentrations. Therefore, this is an energy-consuming system. In addition, active
transport is a specialised process requiringa carrierthat binds the drugto form a carrier-
drug complexthat shuttles the drug across the membrane and then dissociatesthe drug
on the other side of the membrane. The carrier molecule may be highlyselectivefor the
drug molecule. If the drug structurally resembles a natural substrate that is actively
transported, then it is likely to be actively transported by the same carrier mechanism.
Therefore, drugs of similar structure may compete for sites of adsorptionon the carrier.
Furthermore,because only a fixed number of carriers are available, all the bindingsites
on the carrier may become saturated with an increased drug concentration (Shargel &
Yu, 1999:105).
2.3.3 Facilitated diffusion
Facilitated diffusion is also a carrier-mediated transport system, differing from active
transport in that the drug moves along a concentration gradient. Therefore, this system
does not require an energy input. However, because this system is carrier mediated, it
is saturable and structurally selective for the drug and shows competition kinetics for
drugs of similar structure. In terms of drug absorption, facilitated diffusion seems to play
a very minor role (Shargel & Yu, 1999:106).
10
Absorption mechanisms such as efflux may limit intestinal drug absorption. Therefore,
drug concentrations may not be satisfactory to deliver the desirable therapeutically
effects. It is of great importanceto study processes like efflux more extensivelyin order
to find ways to circumvent these mechanisms and to improve a drug's absorption and
ultimately,its bioavailability.
2.4 Effluxmechanisms
Pgp is the first member of what is now a large and diverse superfamily comprising
aroundfifty humanATP-binding cassette (ABC) proteins that perform many and varied
functions (Chan et al., 2004:25). Overexpression of Pgp is found to prevent the
accumulation of cytotoxic drugs in resistant cells. MDR (multidrug resistance) is of
clinical importance because many human tumors have inherent or acquired Pgp-
mediated drug resistanceand do not respondto chemotherapy. Pgp acts as an energy-
dependent pump to translocate a wide variety of structurally and functionally diverse
substrates. These compounds tend to be moderately hydrophobic, planar and natural
productswhich are often substrates for/or metabolitesof detoxification enzymessuch as
cytochromes P450 (CYPs) (Bard, 2000:357). CYP represents a superfamily of
microsomal heme-thiolate enzymes that catalyse the biotransformation (oxidation) of
numerous endogenous and foreign compounds (McKinnon et al., 1995:259). CYPs
identified in the human intestine include CYP1A1, CYP2C8-10, CYP 2D6, CYP2E1
(trace amounts), CYP3A4 and, more recently, CYP3A5 (Wacher et al., 2001:90). The
major congener of the CYP3A superfamily is CYP3A4, the predominantform in the liver
and intestine. The enzyme is considered to be the most important enzyme in drug
metabolism (Wacher et al., 2001 :90). Pgp also prevent the cellular accumulation of
endogenous metabolites, phospholipids and xenobiotics in exposed animals and cell
cultures (Bard,2000:357).
Drugs may also be modified by biotransformation processes which include phase I and
phase II reactions. The process biotransformation or metabolism is the enzymatic
conversion of a drug to a metabolite. Phase I, or asynthetic reactions, include oxidation,
reduction and hydrolysis. Phase II reactions, or synthetic reactions include conjugations
(Shargel & Yu, 1999:367). These processes may not only render the drug ineffective,
11
but it may also produce metabolites that are themselves substrates for Pgp and/or
MRP2 (Chan et al., 2004:25).
Drugs that eventually reach the blood are transported to the liver, where they are subject
to further metabolic processes and biliary excretion, often by a system of ABC
transporters and enzymes similar to those present in the intestine. Thus a synergistic
relationship exists between intestinal drug metabolising enzymes and apical efflux
transporters, a partnership that proves to be a critical determinant of oral bioavailability.
The effectiveness of this system is optimised through dynamic regulation of transporter
and enzyme expression. Tissue has a remarkable capacity to regulate the amounts of
protein in order to maintain homeostasis (Chan et al., 2004:25).
Members of the ABC family of transporter proteins and knowledge on the mechanism of
action of Pgp as a representative of this fascinating, rapidly growing group of membrane
transport proteins, is of great interest and are involved in diverse physiological
processes which include antigen presentation, drug efflux from cancer cells, bacterial
nutrient uptake and cystic fibrosis. In order to understand the role of these transporters,
an integrated approach combining structural, pharmacological and biochemical methods
are being studied extensively (Germann, 1996:928; Kerr, 2002:47).
2.4.1 ABC transporters
Over the years, tremendous progress has been made to understanding the
pharmacologicaland toxicological impact of ABC drug efflux transporters. Once thought
to be perhapsjust of relevance in making cancer cells resistant to anticancer drugs, it is
now clear that they can have a pronounced role in the oral bioavailability and
hepatobiliary, direct intestinal and most likely renal excretion of an extensive range of
drugs and toxins. In addition, they contribute to important pharmacological sanctuary
sites such as the brain, cerebrospinal fluid, testis and fetus and they can protect
individual cells from drug and toxin penetration (Schinkel & Jonker, 2003:22). Many
other xenotoxins, pre-carcinogensand endogenous compounds are also influenced by
the ABC transporters(Schinkel& Jonker, 2003:3).
12
The typical structure of an ABC protein consists of membrane-embedded
transmembrane domains (TMD) and ATP binding domains (ABC) (Figure 2.3).
Typically, the transmembrane regions anchor the protein to the membrane and form a
pore through which the transport of a surprisinglylarge variety of substrates occurs.The
cytoplasmic nucleotide binding domains provide the molecular compartment where the
energy of ATP is released(SigmaAldrich, 2005).
MDR1
InU'aCcllulollt
ExU'4«!Uuinr
Figure 2.3: Schematic presentation of membrane topology of an ABC transporter
(Sigma Aldrich, 2005).
2.4.1.1 P-glycoprotein
With the realisation of the importance of drug efflux transporters in the treatment of
diseases, inhibitors of these transporters have been sought to use as adjuncts during
treatment with these specific drugs. To date, inhibitors that have best been studied are
modulators of Pgp, a transporter that is important in the removal of anticancer agents
from cells and over expression of this transporter will result in multidrug resistance
(Dantzig et al., 2003:133). In vitro and in vivo studies have demonstrated that Pgp plays
a significant role in drug absorption and disposition. Like cytochrome P450 enzymes,
inhibition and induction of Pgp has been reported as the cause of drug-drug interactions
(Lin, 2003:53).
13
--- - -----
--
2.4.1.2 Structure of Pgp
Pgp is a transmembrane protein which is 1280 amino acids long and consists of two
homologous halves of 610 amino acids joined by a flexible linker region of 60 amino
acids. Each half has an N-terminal hydrophobic domain containing six transmembrane
domains followed by a hydrophilic domain containing a nucleotide binding site. The
nucleotide binding sites can bind ATP and its analogues and both sites are essential
since inactivation of either site inhibits substrate-stimulatedATPase activity. However,
the two sites are likelyto be functionally independentand cleavage probablyonly occurs
at one site at a time (Loo & Clarke, 1999:24759). The membranetopology of the human
multidrug resistance protein (MDR1) is illustrated in Figure 2.4. The ATP-bindingsites
are circled (Sarkadiet a/., 1996:216).
Figure 2.4: Schematic model of the human multidrug resistance gene product,
Pgp and its functional domains (Sarkadi et al., 1996:216).
The nucleotide binding domain must interact with the membrane spanning domains in
order to transmit conformational changes resulting from the hydrolysis of ATP. In
eukaryotic systems fusion of the domains into one polypeptide chain assures their
physical proximity (Schneider& Hunke, 1998:12).
14
2.4.1.3 Location and function of Pgp
Pgp expression is detected in three distinct subsets of normal human tissue namely
those involved in secretionand absorptionor those that have a barrier function. It is also
present in some tumors derived from these tissues which include a subset of columnar
epithelial cells, endothelial cells of capillary beds in specific anatomic locations and
placental trophoblasts. The trophoblast is a thin layer of ectoderm that constitutes the
wall of a mammalian blastula and is important for the nutrition and implantation of an
embryo (van der Heydenet a/., 1995:223).
It is suggested that Pgp helps to maintain the integrity of the blood-brain and blood-testis
barrier in addition to preventing accumulation of xenobiotics in multiple organs (Bard,
2000:368). Studies by Borst and Schinkel (1996:985) support the positive expectation
that clinical chemosensitiser treatment in cancer patients to block functional Pgp, may
not have major effects on human physiology, beyond altered drug metabolism. The
location and functions of Pgp is represented in Table 2.1 (Delph, 2000).
15
--
Table 2.1: Location and function of Pgp (Delph, 2000).
Gut
Apical(luminal)surfaces of
superficialcolumnar
epithelialcells
Hepatocytes on biliary
canalicularcells of small
biliaryductules
Apicalsurfaces of epithelial
cells of small biliary
ductules
Hepatocytes
. Colon
. Jejunum
Liverand biliarysystem
Pancreas
Apicalsurfaces of epithelial
cells of small ductules
Apicalsurfaces of epithelial
cells of proximaltubules
Luminalsurfaces of
endothelialcells of cerebral
capillaries
Choroidplexus
Endothelialcells of nerve
capillaries
Kidney
Brain
Peripheral nerves
Uterus
Fetally-devided epithelial
cells of placenta
Testis and ovary
Steroid-producingcells of
endometrialglands
Capillaryendothelialcells
16
Intestinalexcretionand
reduced absorption of
drugs and toxins
Hepatobiliaryexcretion of
drugs and toxins
Regulation of cytochrome
expression
Unknown
Urinaryexcretionof drugs
and toxins
Contributes to the blood-
brain barrier, keeping drugs
and toxins out of the brain
Unknown
Contributesto the blood-
nerve barrier, keeping
drugs and toxins out of the
nerves
Contributesto the matemo-
fetal barrier, keeping drugs
and toxins out of the fetus
Unknown- possible role in
steroid production
Contributesto the blood-
testis/ovary barrier,keeping
-- - --
2.4.1.4 Mechanism of efflux
For many years, the model for drug resistance conferred by Pgp has been a relatively
simple one. According to that model, cytotoxic drugs are actively transported out of cells
that express Pgp against a concentration gradient thereby reducing intracellular drug
accumulation and inhibit drug-mediated cell death. Three models have been described
by Bard (2000:361-362) to explain Pgp-mediated transport and include the following:
. Hydrophobic vacuum cleaner model (Higgins & Gottesman,1992:18);
. Flippase (Higgins & Gottesman, 1992:18) and
. Extrusion from the inner leaflet (Eytan & Kuchel, 1999:217).
17
-- - - - -
drugs and toxins out of the
gonads
Immune system
Skin dendritic cells
Migration of dendritic cells
to Iymph nodes
Activated lymphocytes Transport of some
cytokines (especially
interleukins-1 ,2,4 and
interferon-y) out of cell
Natural killer and CD8+
Reduced cytolytic activity
cytotoxic T cells
Bone marrow
Hematopoietic stem cells May remove drugs and
toxins from the bone
marrow
Adrenal gland
Medulla and cortex
Unknown - possible role in
steroid production
Large arteries
Endothelium
Unknown - possiblerole in
accumulation of intracellular
cholesterol ester in
atherosclerotic lesions
In the hydrophobic vacuum cleaner model, drugs were transported from the plasma
membraneto the extracellular medium (Higgins & Gottesman, 1992:18). This modelwas
not satisfactory because Pgp has been shown to specifically bind to substrates with
different specifities(Liu & Sharom, 1996:11873).
The second model proposed that Pgp flips drugs from the inner to the outer leaflet of the
plasma membrane against an intramembrane concentration gradient and is shown in
Figure 2.5 (Higgins & Gottesman, 1992:19; Borst & Schinkel, 1997:220).
Membrane A Membrane
7 ~. T ~Ion
Medium
P;JYCDprotean
ftipping drug
Ftip-ftop
( (stow}
~\P4.
Oytosol
Figure 2.5: Flippase model for Pgp action (Borst & Schinkel, 1997:220).
This model is based on the analogy between amphipathic drugs and the normal
phospholipids constituents of membranes. The term fIippase was originally used to
describe membrane enzymes that translocate phospholipids. Although the lateral
mobility of phospholipids is high, movement between the leaflets is low because the
polar head group of the phospholipids has difficulty passing through the hydrophobic
interior of the membrane. Enzymes that can speed up this flipping reaction have been
described and these enzymes are called flippases or phospholipids translocators (Borst
& Schinkel, 1997:220).
A third model has been proposed by Eytan and Kuchel (1999:218), which combines
elements of the two previous models and is shown in Figure 2.6.
18
--- -
--
- - - ---
--
Figure 2.6: Schematic diagram describing movement of MDR-type drugs in Pgp-
overexpressing cells (Eytan & Kuchel, 1999:180).
In this model, MDRdrugs added to the extracellularmedium, bindto and are in practical
equilibration(K1)with the drug pool in the outer leaflet of the plasma membrane. The
drugs flip-flopacross the plasma membrane (f1and fo). The drug pool inthe inner leaflet
of the membrane is in "effective"equilibriumwith the drug pool in the cytoplasm (K2).
Drugs in the cytoplasm are largely bound and form equilibrium(K3)with intracellular
molecular sinks represented here, for simplicity,as DNA. Pgp seems to extract its
substrates fromthe inner leaflet ofthe plasma membrane and flipthose outwards (Eytan
&Kuchel,1999:180).
2.4.1.5 The ATPaseo activity of P-glycoprotein
It has been previously stated that ABC-transporters have two NBDs. Both of these
domains were found to be able to hydrolyse ATP, but substrate-stimulated ATPase
19
--
-- -
activity requires interaction between the two halves of the molecule (Borges-Walmsley &
Walmsley, 2001:76; Schneider & Hunke, 1998:11; Sharom et a/., 1999:329). Both
domains are sensitive to the same set of inhibitors and seem to be functionally identical
(Schneider & Hunke, 1998:15). Inactivation by chemical modification of either of the
NBDs causes a loss of activity, suggesting Cooper-activity between the sites. In addition
to the site for the nucleotide, the NBDs of Pgp have a site for ligands, such as
flavonoids, which modulate drug transport, probably by inhibiting ATPase activity
(Borges Walmsley & Walmsley, 2001 :76).
It has also been suggested that mammalian sites function by an alternating-site
mechanism. This implies that one site hydrolyses ATP and inactivates the other site for
that period. After hydrolysation, the site becomes inactive and the previously inhibited
site becomes able to hydrolyse ATP (Borges-Walmsley & Walmsley, 2001 :76; Schneider
& Hunke, 1998:7). This alternating-site drug transport process and its relationship to the
hydrolysis of ATP is shown in Figure 2.7 (Senior et a/., 1995:288).
A1P
~
Figure 2.7: Postulated alternating catalytic sites cycle of ATP hydrolysis by p.
glycoprotein (Senior et a/., 1995:288).
In Figure 2.7 the rectangles represent the two Pgp trans-membrane domains (TMD).
The circles, squares and the hexagon, represent different conformationsof the N- and
20
C-catalytic sites (NBS1 and NBS2, respectively). The mechanism can be explained in
the followingfour steps:
. The N-catalytic site is empty where the C-catalytic site has bound ATP and the
drug is bound at the inside-facingtransport site (top left).
. In the illustration on the top right side it is suggested The ATP binding at the N-
site allows ATP hydrolysis at the C-site, inducing a conformation at the C-site
which prohibits hydrolysisat the N-site(top right). The conformationat the C-site
immediately after bound-cleavage is a high chemical potential state with bound
ADP.Pj(shownas a hexagon).
. Relaxationof the C-site conformationoccurs and coupled to the drug movement
from the inside-facing, higher-affinity to the outside-facing, lower-affinity, and Pi is
released (bottom right).
. The drug and ADP dissociate (bottom left). The drug binds at the inner side and
the N- and C-sites have now reversed their relationship (top left). In the next
cycle, ATP hydrolysiswill occur in the N-site (Senior et al., 1995:288).
2.4.1.6 Multidrug resistance-associated protein - MRP
Several of the substrates and inhibitors of MRP are anionic. In contrast Pgp has
typically been associated with cationic substrates. MRP is inhibited by anionic
compounds, such as probenecid and indomethacin (Aungst, 1999:110). Since the
discovery of MRP in 1992, understanding of its biological properties has progressed
rapidly. It is now firmly established that MRP can confer a multidrug resistance
phenotypethat in vitro is similar in many respects to that conferred by Pgp. Insufficient
data is available to assess whether these two proteins playa similar role in vivo in drug
resistant malignancies, but the normal physiological functions of these two proteins
clearly differ (Loe et al., 1996:950).
Human MRP is a 190 kDa protein and its primary structure shares approximately15%
amino acid identity with Pgp (Quan et al., 2000:197). The expression of MRP1 in the
body is widespread, with high levels in the intestine, brain, kidney, lung, testis and lower
expression in the liver (Cherringtonet al., 2002:97). MRP1 is localisedto the basolateral
membranesof polarised epithelial cells includingthose of the intestinal crypt, renaldistal
21
and collecting tubules (Peng et a/., 1999:757) and liver (Mayer et a/., 1995:138). The
basolateral localisation of MRP1 in normal tissues suggests that it protects cells by
extruding its substrates into blood. Indeed, confocal microscopic studies show that
MRP1-positive staining on the basolateral membranes of rat hepatocytes is markedly
increased following bile duct ligation (Pei et a/., 2002:59), indicating an adaptive
response to maintain cellular detoxification when elimination into bile is restricted. The
increased levels of toxins present in the blood presumably undergo subsequent
elimination by the intestine and kidneys. The barrier formed by MRP1 against the
cellular accumulation of toxins is yet more effective when coupled with apical Pgp
expression (Tadaet a/.,2002:634). '
2.5 Synergism between Pgp and Cyp3A4
Many of the same drugs that are transported by Pgp are metabolised by some of the
cytochromes (CYPs), especially CYP450 3A which constitutes 30% and 70% of the total
CYP450s in most human livers and intestines, respectively. CYP3A substrates include
HIV protease inhibitors, the antibiotic erythromycin, the lipid-lowering agent lovastatin;
the calcium channel blocker nifedipine; rifampicin; and the sedative midazolam. CYP3A
can be induced by many agents, including glucorticoids, barbiturates and rifampicin.
Human variation in CYP3A expression is believed to influence drug response for up to
one-third of all drugs (Delph, 2000). There are several mechanisms according to which
the functions of Pgp and CYP3A4 could be complimentary and are shown in Figure 2.8
(Watkins, 1997:166).
22
-----
Figure 2.8: Potential functional metabolism between enterocyte Pgp and CYP3A4
(Watkins, 1997:166).
Firstly, Pgp may prevent parent drug diffusion across the apical brush border membrane
and therefore prevent absorption. Secondly, Pgp may function to prolong the duration of
absorption. This might increase the duration of exposure of drugs to CYP3A4 and,
hence, the extent of metabolism by enterocyte CYP3A4. Finally, Pgp may preferentially
remove primary drug metabolites that are themselves substrates for CYP3A4. This
would prevent or limit product inhibition by these metabolites and facilitate primary
metabolism catalysed by CYP3A4 (Watkins, 1997:161).
Tissue are equipped with two lines of defense namely phase I (e.g. hydroxylationvia
cytochromes P450) and phase II (e.g. conjugation with glutathione) detoxification
enzymes which metabolise xenobiotics (as well as endogenous substrates) to more
hydrophilic compounds, which can be more easily excreted from the body. An
examination of the structure-activity relationships and molecular characteristics for
xenobiotic transport substrates and inhibitory ligands of Pgp indicate that Pgp may
function in the elimination of hydroxylated metabolites of xenobiotics after modification
by phase I enzymes (Bain et aI, 1997:812). Many phase II conjugates are also
transported by MRP. A schematic presentation of the roles of ABC transporters and
phase I and II enzymes in xenobiotic resistance are shown in Figure 2.9 (Bard,
2000:370).
23
- -
DRUG I . DRUG
1
CYP3A4
metabolite'" c:: 'mlJ I .. metabolite
x
x X..oH G-S-X
A~ I A~P ATP ~p
Phase:I I Phasen
1
X ... X-OH(OS ... G..X
cyp GST
OSH
intracellular
membrane
Diffusion extracellular
c:::::> Aclive trnUl>pOrt
Figure 2.9: A schematic presentation of the roles of ABC transporters and phase I
and IIenzymes in xenobiotic resistance (Bard, 2000:370).
This is a speculative model of xenobiotic resistance provided by transmembrane active
transporters, Pgp and MRP, and phase I and II detoxification enzymes, cytochromes
P450 (CYP) and glutathione-S-transferase (GST) which gives an indication of the
different pathways by which each exerts its action. A moderately hydrophobic natural
product (X) diffuses in and out of the cell and at low concentrations little accumulates in
the cell due to the active efflux of parent compound by Pgp. At high concentrations, X
accumulates and is metabolised by one or more of the CYP enzymes to form a
hydroxylated metabolite (X-OH). The hydroxylated metabolite can either be removed by
Pgp mediated transport or may further be modified by conjugation to glutathione (GSH)
catalysed by GST. The glutathione conjugate (G-S-X) is then expelled from the cell by
MRP active transport as illustrated in Figure 2.9 (Bard, 2000:370).
24
- - -
-- --
2.6 Drug substrates and inhibitors that interact with Pgp
The most conspicuous feature of the transport activity of Pgp is the diversity of its
substrates. An enormous number of unrelated neutral or cationic lipophilic organic
compounds are transported by Pgp. A similarly diverse array of compounds, known as
"chemosensitisers", "resistance modulators" or "reversing agents", inhibit transport by
Pgp. Many, but not all of these latter compounds are themselves transported by Pgp
(Shapiro & Ling, 1998:228). Evidence suggests that there are different categories of
substrates due to multiple binding sites or to a complex bindingsite with different regions
of interaction(Scala et al., 1997:1024).
Studies done by Scala et al. (1997:1024) defined compounds as Pgp substrates if
cytotoxicity was increased more than four times by the addition of cyclosporin, and as
Pgp antagonists if inhibitionof efflux increased Rhodamine 123 (Rho 123) accumulation
more than four times. Their studies also imply that antagonistsbind to Pgp but fail to be
transported, therefore, prevent the transport of other agents. Pgp substrates are
transported but do not block the transport of other substrates. Pgp substrates and
inhibitors are shown in Tables 2.2 and 2.3 respectively(Delph,2000).
25
--
Table 2.2: Pgp substrates (Delph, 2000).
Cancer drugs
. Doxoubicin
. Sequinavir
Cardiac drugs
. Digoxin
. Quinidine
Antiemetic
. Ondansetron
Anti-diarrhoeal agent
. Loperamide
Anti-gout agent
. Colchicine
Antibiotic
. Erythromycin
Anti-helminthic agent
. Ivermectin
. Teniposide
. Etoposide
Immunosuppressivedrugs
. Cyclosporin A
. FK506
Lipid-loweringagent
. Lovastatin
Antihistamine
. Terfenadine
Steroids
Dopamine antagonist
. Domperidone
26
- -- - -----
.
Daunorubicin
.
Vinblastine
.
Vincristine
.
Actinomycin D
.
Paclitaxel
.
Amprenavir
.
Indinavir
.
Nelfinavir
.
Ritonavir
.
Aldosterone
.
Hydrocortisone
.
Cortisol
.
Corticosterone
.
Dexamethasone
Table 2.3: Pgp inhibitors (Delph, 2000).
Cyclopropyldibenzosuberane
. LY335979
Anti-estrogen cancer agent
. Tamoxifen
Antiarrhythmic agent
. Quinidine
Antifungal agent
. Ketoconazole
Sedative
. Midazolam
Acridonecarboxamide derivative
Immunosuppressant
. Cyclosporin A
· Valspodar (PSC833)
HIVprotease inhibitors
Calcium channel blocker
. Verapamil
Progesterone antagonist
· Mefipristone (RU486)
. GG918 (GF120918)
Peptide chemosensitisers
. Reversin 121
· Reversin205
It has also been known for some time that flavonoids present in the diet are capable of
interfering with drug metabolism in vitro, and it was thought that a flavonoid present in
grapefruit juice might be responsible for the interaction with CYP3A4 substrates (Evans,
2000:133). One of the most abundant flavonoids in grapefruit juice is naringen. The
compound is converted in vivo to the aglycone, naringenin (Ho et al., 2000:379).
Naringenin, like many other flavonoids, is a potent inhibitor of CYP3A4 (Fuhr, 1998:265).
It was found by Mitsunaga et al. (2000:199) that naringenin increased the uptake of
vincristine into MBEC4 cells, indicating inhibition of Pgp activity.
Non-cytotoxic compounds such as fluorescent Rho 123 dye and the calcium channel
blocker verapamil, a cardiovascular medication, have been used respectively as a model
substrate as well as a model competitive inhibitor (Neyfakh, 1998:168). The competitive
inhibition of Rho 123 efflux in a model vesicle or cellular system by the addition of
verapamil is considered evidence of Pgp-activity. Furthermore, the ability of a chemical
to inhibit Rho 123 transport in an assay system is considered evidence of a potential
Pgp-substrate or inhibitor (Bard 2000:363).
27
--- -
.
Ritonavir
.
Sequinavir
.
Nelfinavir
.
Indinavir
Verapamil which can competitively inhibit the transport of Pgp substrates functions as a
chemosensitiser or modulator of multidrug resistance. MDR chemosensitisers tend to
be more lipophilic than other substrates and once extruded by Pgp, diffuse back into the
cell before being transported again (Sharom, 1997:162). Drugs referred to as substrates
are found to diffuse relatively slowly across membranes during experimental studies (in
the order of minutes to hours) compared to chemosensitisers which diffuse bilayers at a
speed unable to measure (Eytan et al., 1996:12901). Clinicians have co-administered
verapamil with chemotherapeutic drugs in the hope to eradicate MDR tumors in cancer
patients. Unfortunately, the chemosensitiser dose required to inhibit Pgp provokes
cardiovascular side effects (Lehnert et al., 1998:1155).
2.7 Substrate binding of Pgp
Pgp has wide substrate specificity and is an intriguing problem and much work has been
directed toward characterising the binding site(s). Recently Shapiro and Ling (1998:227)
presented evidence that Pgp contains at least two distinct substrate binding and
transport sites and that these sites interact in a positively cooperative fashion. They
propose that one site marked R (for the substrate Rho 123 and anthracyclenes) binds
one set of compounds and the other site H (for the substrate Hoechst 33342 and
colchicine) binds a second set as shown in Figure 2.10 (Shapiro & Ling, 1998:227).
28
-- - ----
Figure 2.10: Two transport-competent drug binding sites of Pgp with distinct but
overlapping specificities (Shapiro &Ling, 1998:227).
Each R substrate stimulates the Pgp-mediated transport of an H substrate and vice
versa. Two R substrateswould competefor the R site and inhibit binding and transport
of the other. Competitiveinhibitionwould also be observed betweentwo H substrates or
between substrates which can bind to both Rand H sites. A third drug-bindingsite (for
the substrates prazosin and progesterone)on the Pgp exerts a positive allosteric effect
on drug transport by the Hand R sites but is not thought to be capable of drug transport
itself (Shapiro et a/., 1999:848). The three-site model described by Shapiro and Ling is
consistent with previous observations of both competitive and non-competitive
interactionsbetween Pgp substrates(Bard, 2000:362).
2.8 Structure-activity relationships of Pgp substrates
Little information is available regarding the structure-activity relationships of Pgp
substrates. It is, however, evident that the substrates are of large molecular weight,
amphipathic and contain at least one aromatic ring (Wacher et a/., 1998:1323). Other
important structure-activity relationship properties that influence the complexation
interaction (between a drug and a drug-binding site on Pgp) include large molecular
surface, high polarisability and hydrogen binding properties (Osterberg & Norinder,
2000:295). The only physical similarities substrates share are that they are moderately
hydrophobic, cationic or neutral but never anionic and that they are natural products
(Gottesman & Pastan, 1988:55).
29
2.9 Conclusion
According to Steyn (1999:579) the properties of Pgp that has to be taken into account in
attempting to test for possible modulators or inhibitors of this protein are summarised as
follows:
· Pgp is an intrinsic membraneprotein with a polypeptide sequence that suggests
it consists of two similar halves. Each half has six sectors that cross the
membraneas well as an ATP-bindingcytoplasmic region;
· Pgp is a transport ATPase and can transport its substrates out of the cell in
which it is present. The substrates are pumped from the membrane by itself.
Substrates can be pumpedfrom the outer bilayer of the membrane,Le., before it
crossesthe membraneand entersthe cell, as well as from the inner half bilayer;
· Pgp displays a very broad specificityto its substrates. Most are weak bases, but
non-protonable molecules can also be good substrates, as well as molecules
bearing a permanent positive charge. Although all the substrates should be
lipophilicit is not clear whetherextremelylipophilic molecules can be substrates;
· Transport out of the cell can be blocked by reversers or modulatorsof Pgp. The
latter consist of a very wide range of molecular types with the same general
characteristicsas the substratesthemselves;
· Pgp is an ATPase of moderateATP-splitting capacity. The rate of hydrolysisof
ATP is enhanced up to 5- to 10-fold by many substrates and modulatorsof Pgp's
function, but reduced by others;
· Transport of substrates by Pgp is not influenced by the membrane potential, is
independent of the pH gradient and is not associated with any co- or
countertransportof protons, cations or anions. Thus Pgp seems to be a primary
ATPase;
.. No evidence has been found for an intermediate phosphorylated state of Pgp
when it hydrolysesATP, nor for a tightly bound ATP molecule and
· The two ATP-binding sites in Pgp appear to co-operate in the transport of
substrate but can independently hydrolyse ATP. The two halves of Pgp thus
seem to interact during substratetransport. Consistent with this is the evidence
30
-
that pairs of substrate molecules and of some modulators and inhibitors, co-
operate in the action of Pgp.
(Stein, 1997:579)
At present, available therapy cure a little more than half cancer patients. Surgery is
considered the most efficient treatment, possibly because it is used to cure very small
tumours, which are rarely accompanied by metastasis. Chemotherapy appears to be
less efficient, possibly because it is used when metastases are already present, the
tumour is large, spreading and moreover, anticancer drug resistance is frequent. A
successful solution to this resistance would lead to a great increase in therapeutic
success. As novel anti-cancer drugs often offer only a small improvementover older
agents, a change in the target of chemotherapymust be considered. Insteadof looking
for a new cytotoxic drug, it may be an option to look for methods of circumventingdrug
resistance. In order to address this problem most efficiently, it is essential to establish
the dominant mechanism of resistance to chemotherapy in human tumours. This is
often consideredto be the so~calledMDRmechanism (Desoize& Jardillier, 1999:194).
Nevertheless, although the first drug able to reverse MDR was discovered more than 20
years ago, a potent, specific and safe drug, able to reverse multidrug resistance and to
assist in the chemotherapy of cancer is still lacking. However, several drug candidates
presently under development look very promising and some of them, such as valspodar,
biricodar and LY 335979 are in a good position to gain approval in the near future
(Teodori et a/., 2002:405).
Present data are consistent with the hypothesis that certain flavonoids affect MRP-
mediated transport of anticancer drugs by a direct interaction with MRP (Hooijberg et a/.,
1997:344). The benefits of flavonoids as chemopreventive dietary or dietary
supplemental agents are still only potential. Much has been learned about possible
mechanisms of action of these agents, but whether they can reach their multiple
intended sites of action, particularly in humans, is largely unknown (Walle, 2004:829).
In the following study, with reference to the inhibition of Pgp. Certain flavonoids will be
extensively studied to provide an inspiration for a rational drug and/or chemopreventive
31
---- --- ---
agent design of future pharmaceuticals. The potential inhibition of selected flavonoids
will be evaluatedusing an in vitrotransport model with Rho 123 as a Pgp substrate.
32
-- - --
CHAPTER 3
THE POTENTIAL STRUCTURE ACTIVITY RELATIONSHIP
OF FLAVONOIDS WITH REFERENCE TO THE
INHIBITION OF P-GLYCOPROTEIN
3.1 Introduction
Diverse compounds have been shown to reverse the Pgp-mediated MDR, including
calcium channel antagonists such as verapamil (Tsuruo et a/., 1981:1967) and
immunosuppressants such as cyclosporin A (Twentyman et a/., 1991:24). MDR cells
can be sensitisedto anticancer drugs when treated with a Pgp inhibitor, which is known
as a chemosensitiser. ThE!search for chemosensitiserswhich have the advantagesof
being a non-transportable inhibitor without side effects, has led to a great deal of
research inflavonoids derivedfrom plants (Conseilet a/., 1998:9831).
The diverse nature of the cDmpounds which interact with Pgp has lead to the realisation
that there is a greater possibility for drug-drug interaction than was initially thought.
Many of the Pgp inhibitors are compounds which occur in foods such as soya beans and
grapefruit juice, and inhibition as well as induction of this protein could lead to enhanced
or diminished absorption of drugs that are substrates for Pgp (Wagner et a/., 2001:S14).
3.1.1 Flavonoids
Flavonoids represent a group of phytochemicalsexhibiting a wide range of biological
activities arising mainly from their antioxidant properties and ability to modulate several
enzymes or cell receptors Flavonoids have been recognised to exert anti-bacterial,
anti-viral, anti-inflammatory, anti-angionic, analgesic, anti-allergic, hepato-protective,
cytostatic, apototic, estro!~enic and anti-estrogenic properties. However, not all
flavonoids and their actions are necessarilybeneficial. Someflavonoids have mutagenic
33
and/or pro-oxidant effects and can also interfere with essential biochemical pathways.
Cytochromes P450, mono-oxygenases, metabolising xenobiotics (e.g. drugs and
carcinogens) and endogenous substrates (e.g. steroids) also playa prominent role
among the proteins that interact with flavonoids. Flavonoids in the human diet may
reduce the risk of various types of cancers, especially hormone-dependentbreast and
prostate cancer, as well as preventing menopausal symptoms. For these reasonsthe
structure-function relationship of flavonoids is extensively studied to provide an
inspiration for a rational drug and/or chemopreventive agent design of future
pharmaceuticals(Hodek,2002:1).
3.1.2 Basic flavonoid structure
The basic flavonoid structure is the flavan nucleus, which consists of 15 carbon atoms
arranged in three rings (CS-C3-CS),which are labeled A, B, and C (Figure 3.1a). The
various classes of flavonoids differ in the level of oxidation and pattern of substitution of
the C ring, while individual compounds within a class differ in the pattern of substitution
of the A and Brings (Pietta, 2000: 1035-1036).
The benzene ring A is condensed with a six-member ring (C), which in the 2-position
carries a phenyl pyran, which yields flavanols (catechins)and anthocyanidins,or pyrone
which yields flavonols, flavones and flavonones. The term 4-oxo-fiavonoids is often
used to describe flavonoids, such as flavanols (catechins), flavanones, flavonols and
flavones, which carry a carbonyl group on C-4 of ring C (Figure 3.1b) (Aherne & O'Brien,
2002:75).
The chemical nature of the flavonoids depends on structural
hydroxylation, other substitutions and conjugations and degree
(Harborne, 1986:15).
class, degree of
of polymerisation
34
3'
(a)
5 4
3'
(b)
Figure 3.1: (a) Flavan nucleus and (b) 4-oxo-flavonoid nucleus (Aherne & O'Brien,
2002:75).
The structures of the basic flavonoids are given in Figure 3.2 (Aherne & O'Brien,
2002:75).
35
--
- -
Anthocyanidins
Catechins
Flavones Flavonols
Flavonones
Isoflavones
Figure 3.2: Structures of the basic flavonoids (Aherne & O'Brien, 2002:76).
3.2 Flavonoids and MDR
It has previously been demonstrated that flavonoids are efficient Pgp inhibitors, by
binding to cytosolic sites which partly overlap the ATP-binding site and the modulators
interacting region (Conseil et a/., 1998:9831). It has been reported that quercetin
affected the expression of MDR1 in the human hepatocarcinomacell line HepG2. The
increase of Pgp synthesis (the gene product) and MDR1 mRNA accumulation in these
36
- --
cells caused by exposure to arsenite were inhibited by quercetin (Kioka et al., 1992:308).
This appeared to be the first report to describe the inhibition of MDR1 expression by any
chemical. Not only did certain flavonoids inhibit the expression of the multidrug
resistance gene but, in addition, could act as potent stimulators of the Pgp-mediated
efflux of the carcinogen 7,12-dimethylbenz[a]anthracene, resulting in a decreased
intracellular burden of this polycyclic compound. The active flavonoids were kaempferol,
quercetin and galangin (Phang et al., 1993:5979).
In a study conducted by Chieli and co-workers (1995:1748), it has been shown that
flavonols are able to modulate liver Pgp activity in vitro and that the interference with the
transporter is related not only to the flavonol concentration and to the Pgp amount, but,
more interestingly, to the kind of substrate used. In particular, the resulting effect may
also be of opposite sign. Thus the overall pattern would be consistent with the possibility
that the flavonols are substrates of Pgp. In fact, this hypothesis could explain the dose-
dependent flavonol-depressed Rho 123 efflux from hepatocytes as a competitive
inhibition on the same transporter.
It has also been shown that (iso) flavonoids genistein, kaempferol and flavopiridol
stimulate the membrane ATPase activity of the MRP-overexpressing cell line
CLC4/ADR. This data is consistent with the hypothesis that certain (iso)flavonoids affect
MRP-mediated transport of anticancer drugs by a direct interaction with MRP (Hooijberg,
1997:344).
However, contradictory effects were reported with different multidrug resitant cell lines.
Quercetin, kaempferol and galangin were found to increase adriamycin efflux from HCT-
15 colon cells whereas quercetin and a methoxylated derivative of quercetin inhibited
Rho 123 efflux and reverted MDR in MCF-7 breast cells (Conseil et al., 1998:9831).
3.3 Structure-activity relationship between Pgp and flavonoids
To date, no elements of molecular recognition have been found to interact with Pgp.
However, the analysis of three-dimensional structures of a much larger (n>100) and
chemically more heterogeneousset of compounds than used in previous investigations
revealed specific recognitionpatterns for Pgp. They consist of hydrogen bond acceptor
37
- - -- - -
(or electron donor) groups (e.g. carbonyl, ether, hydroxyl, or tertiary amino groups) with
a specific spatial separation. Recognition patterns formed by two electron donor groups
with a spatial separation of 2.5:f:0.3 A are called type I and recognition elements formed
either by two electron donor groups with a spatial separation of 4.6:f:0.6 A, or three
electron donor groups with a spatial separation of the two outer groups of 4.6:f:0.6 A are
called type II patterns. For binding with Pgp at least one of the type lor, one of the type
II pattern are required. Binding was shown to increase with the strength and the number
of electron donor groups involved in type I and type II patterns. For transport at least
two of the type I patterns and one of the type II patterns are required. The same
substrate recognition principle was also observed for MRP. Type II patterns are also
responsible for Pgp induction and thus the development of drug resistance (Seelig &
Landwojtowicz, 2000:32).
Studies of structure-activity relationships have concluded that flavones are more efficient
than isoflavones, while flavonols and chalcones are even more active (Boumendjel,
2001 :75). Hydroxyl groups at positions 3 and 5 are essential for high-affinity binding to
Pgp as in quercetin, galangin, kaempferol and morin shown in Figure 3.4. In a recent
study conducted with chalcones, Boumendjel and co-workers (2001 :76) showed that the
presence of a hydrophobic substituent on the B-ring, considerably enhanced the binding
affinity.
In a study conducted by Conseil et al., (1998:9831) the binding of flavonols to the
purified C-terminal nucleotide-binding domain (NBD2) of Pgp was directly measured by
the quenching of protein intrinsic fluorescence due to a single tryptophan residue.
Flavones (like quercetin or apigenin) bound more strongly than flavanones (naringenin),
isoflavones (genistein), or glycosylated derivatives (rutin). Kaempferide, a 4' -methoxy
3,5,7-trihydroxy flavone, was even more reactive and induced a complete quenching of
H6 -NBD2 intrinsic fluorescence. Kaempferide binding was partly prevented by
preincubation with ATP, or partly displaced upon ATP addition. Interestingly,
kaempferide was also able to partly prevent the binding of the anti progestin RU 486 to a
hydrophobic region similar to that recently found, close to the ATP site, in the N-terminal
cytosolic domain. Conversely, RU 486 partly prevented kaempferide binding, the effect
being additive to the partial prevention by ATP. Furthermore, MANT-ATP binding, that
occurred at the ATP site and extended to the vicinal steroid interacting hydrophobic
38
- -- --
region, was completely prevented or displaced by kaempferide. Flavonoids behave as
bifunctional modulators able to bind partially to the ATP-binding site and partially to a
vicinal hydrophobic steroid-binding site. Despite several studies on the structural
features of flavonoids that lead to inhibition of MDR transport, nothing is known about
the orientation of the flavonoid molecules within the binding pocket. It has been
indicated that the flavonoid, luteolin and it's 7-O-glucosidebind to the purified NBD2
fragment (Conseil et aI, 1998:9831). This ability qualifies them as suitable ligands for
investigating the alignment of both flavonoids inside the ATP binding pocket (Nissler et
a/., 2004:1045). The assumed bindingof luteolin (1) and luteolin 7-0-glucoside (2) in the
binding pocket of the NBD2is presentedin Figure 3.3 (Nissler et a/.,2004:1048).
The flavonoid molecule is bound to the protein by its polar groups on C-5, C-4, and C-3'
(solid lines in Figure 3.3). The latter requires a conformation of the side chain phenyl ring
as drawn. This molecular attachment causes the protein to transmit the magnetisation
mainly to the protons H-6, H-3, and H-2' (dashed lines in Figure. 3.3) and this holds for
both the luteolin 1 and its glucoside derivative 2. Since the side-chain phenyl ring is
attached by a single bond, it is more flexible and thus the protons H-6' and H-5' also
obtain some magnetisation transfer. It could also be that the protein pocket reaches
around the side-chain phenyl ring. These structural requirements predict that
modification of the polar group at C-5 should result in less pronounced binding and/or
functional activity of the flavonid derivative. Indeed, by using quenching of intrinsic
tryptophan fluorescence (see above) it was found that by introducing a methoxy group at
C-5 of luteolin increases the ECsovalue from 22.9 to 33.4 M, and substitution with hexyl
groups at C-5 and C-3' further enhanced it to 46.5 M (Nissler et a/., 2004:1048).
39
--
- --
Figure 3.3: Assumed binding of luteolin (1) and luteolin-7-0-glucoside (2) in the 
binding pocket of the NBD2 (Nissler et a/., 2004:1048). 
The structures of the flavones that will be investigated in this study are given in 
Figure 3.4. 
OH
HO
HO
OH 0
OH 0
Quercetin (3,5,7,3' ,4'-
pentahydroxyflavone)
Galangin (3,5,7-trihydroxyflavonone)
HO
HO
OH
Kaempferol (3, 4',5, 7-tetrahydroxyflavone)
Morin (2', 3, 4', 5, 7-
pentahydroxyflavone)
Figure 3.4: Flavones thatwill be evaluated duringthis study.
3.4 Rhodamine 123
Rho 123 is the model compound that will be used in this study. Rho 123 is a fluorescent
lipophilic cation and is classified as a typical Pgp substrate and subject to Pgp-
dependent extrusion through the plasma membrane. Rho 123 shows selectivity for
mitochondrial compartments. Due to its fluorescence, dye levels can easily be
measured in cell extracts and accumulation can be observed in intact cells. Thus, Rho
123 accumulation in (or efflux from) cells is often used as a measure of Pgp-dependent
41
- - - - - --
--
transport activity. Initially used to study mitochondria, Rho 123 has been introduced in
the study of multidrug resistance by Neyfakh (1988:168), who showed that this dye was
a definite substrate for Pgp. Since then, various investigators have used the dye as a
probe for the investigation of Pgp functional activity (Hirsch-Ernst et a/., 4001 :48;
Marques-Santoset a/., 1999:103).
3.5 Conclusion
The effectivenessof Pgp modifiersin chemosensitisingmultidrug resistancecancer cells
has stimulated a serious effort to define a common pharmacophore necessary to
circumvent Pgp mediated transport (Seelig & Landwojtowicz, 2000:31). Many
successful anticancer drugs are themselves either natural products or have been
developed from naturally occurring lead compounds. Great interest is currently being
given to flavonoids. Flavonoids are one of the major classes of natural products with
widespread distribution in fruits, vegetables,spices, tea and soy-basedfoodstuff(Pouget
et a/.,2001:3095).
Flavonoids constitute a class of bifunctional modulators which are able to partly overlap
the ATP binding site and a vicinal hydrophobic region interacting with steroids, within a
cytosolic domain of Pgp. This important finding opens exciting perspectives for studying
the molecular mechanism of interaction with flavonoid modulators through structural and
functional approaches and for further design of a new generation of bifunctional and
specific inhibitors of Pgp activity (Conseil et a/., 1998:1836).
The aim of this study is to determine the potential inhibition of Pgp by selected
flavonoids using an in vitro transport model and to explore the potential structure-activity
relationship and requirements of the selected flavonoids in order to identify an optimal
candidateto circumvent MDR.
42
CHAPTER 4
EXPERIMENTAL PROCEDURE
4.1 Introduction
The ability of an orally administered compoundto permeate the intestinal mucosa may
be limited by the physical andlor the biochemicalcomponents of the intestinal mucosal
barrier. Thus, in vitro intestinal permeability models should not only predict intestinal
drug absorption potential but also provide some understanding of the absorption
mechanism(s). This can be accomplishedto the extent in which the permeabilitymodel
incorporates the functionality of the physical and biochemical barrier components
(Hidalgo, 2001:389).
4.1.1 In vitro models of intestinal drug absorption
The successful application of in vitro models for intestinal drug absorption depends on
the extent to which the model comprises the relevant characteristics of the in vivo
biological barrier. Despite the obvious difficulties associated with trying to reproduceall
the characteristics of the intestinal mucosa in vitro, various systems have been
developed which mimic, to varying degrees, the relevant barrier properties of the
intestinal mucosa. These systems include excised tissue (e.g. rat and rabbit), cell
cultures (e.g. Caco-2, HT-29, T84 and MOCK),physicochemicalmethods (e.g. Log DIP,
immobilised artificial membrane and parallel artificial membrane permeation assay),
mucosal cell membrane vesicles, isolated mucosal cells and computational (in silico)
methods (Hidalgo,2001 :389).
- - ---
---
--
The mobile phase was mixed using HPLC grade reagents and Milli Q50 water for HPLC.
The mobile phase was filtered through a MN 85/90 glass fibre filter (Macheney-Nagel,
Germany) prior to use.
4.2.1 Method validation
Whether the methods used to predict the behavior of drugs in vivo in humans are in
vitro, ex vivo or in experimental animals, some validation is necessary. Validation is a
process by which the reliability and relevance of a procedure are established for a
particular purpose. With the respect to the importance of in vitro methods in the
assessmentof chemicals,there is an urgent need to perform this exercise (Pelkonenet
al., 2001:627).
For the validation of the HPLC method, the following parameters for Rho 123 were used:
t!J linearity
t!J precision (intra-day and inter-day)
t!J sensitivity
t!J selectivity
t!J system repeatability
4.2.1.1 Linearity
The linearity for Rho 123 was determined by performing linear regression analysis on
the plot of the peak AUC versus concentration. Six standard solutionswere preparedas
describedin Appendix 8, to obtain concentrationsranging from 34.38to 68.751Jg/ml.The
regressionvalue (~) was greater than 0.9940 and the V-intercept was 31.129.
- -- - -
- --
--
4.1.2 In vitro measurement of gastrointestinal tissue
permeability using a diffusion cell
A diffusion cell (Sweetana-Grassdiffusion chambers),derived from the Ussingchamber,
was developed for the measurement of tissue permeability. This cell incorporatesthe
attributes of using a single material and laminar flow across the tissue surface. In
addition, the design allows the cell to be manufacturedin a wide range of sizes to allow
optimisation of surface area to velume for a variety of tissue. The apparatus is
applicable for the evaluation of transport of compounds through mucosal/epithelial
barriers such as the gastrointestinal tissue. Active transport, permeability enhancers,
enzymatic degradationand absorption in various tissue sections can be explored(Grass
& Sweetana, 1988:373).
4.2 HPLC Analysis and validation
A high pressure liquid chromatographic method was validated to analyse the samples,
using the apparatusand conditionsgiven below:
Apparatus: Pump:
Autosampler:
Detector:
Integrator:
Spectra Physics SP 8810
Spectra Physics AS 3000
Spectra Physics FL 2000
Computerised integration system, with
Chromquest chromatographic database for
Windows@ NT as software
Column:
Luna 5J.1C18(2)reverse phase
Conditions:
Mobile phase:
Injection volume: 200 J.1L
Flow rate: 1.5 mUmin
32% Acetonitrile: 68% K2HP04 0.1M
(pH = 3.2)
510 nm
546 nm
Excitation wavelength:
Emission wavelength
4.2.1.2 Precision
Intra-day precision:
Precision (repeatability) was determined by, performing HPLC analyses (n=5) of five
different concentrationsranging from 34.38 to 68.75 ug/ml of Rho 123 on the same day.
Data for the intra-dayprecision is given in Table 4.1.
Table 4.1: Data obtained for intra-day precision
Inter-day precision:
The inter-day precision was determined by performing HPLC analyses (n=3) on a low,
medium and a high concentration (34.38 ~g/ml, 44.20~g/ml and 68. 75 ~g/ml) of Rho 123
on three consecutive days. The inter-day precision complied with pharmaceutical
standards and is presented in Table 4.2.
Table 4.2: Data obtained for inter-day precision
46
!t Rllo<1.;28.CQQc;eQnti_si!
l"'CQ¥d ,1
S1!@g' gjclti9
R5m;
i(pgJriil) i{J)) .' i() (o/q);
34.38 106.46 12.16 7.93
39.29 106.57 7.51 4.27
44.20 107.45 6.55 3.27
54.02 100.55 9.75 4.25
68.75 97.05 8.11 2.87
I
Rho23nC$1tratJon llean;trecoyereg.
Standalid\de¥iation RSD
(R9friil) (o/q) ()
..
Co/ci>
34.38 104.09 5.56 3.71
44.20 106.14 5.79
2.23
68.75 97.61 8.11 2.77
4.2.1.3 Sensitivity
The sensitivity of an analytical method can be measured by determining the limit of
quantification and limit of detection. The limit of quantification is defined as the lowest
concentration of an analyte in a sample that can be quantitatively determined with
acceptable precision and accuracy (RSD < 15%). The limit of detection is defined as
the lowest concentration of an analyte in a sample that can be detected, but not
necessarily quantified as an exact value. The limit of quantificationfor Rho 123 studied
was 7.79 ~g/mland the limit of detection was 23.43 ~g/ml.
4.2.1.4 Selectivity
Selectivity is the ability of an analytical method to determinethe analyte selectivelyand
accurately in the presence of other compoundsthat may interfere. A test for selectivity
is imperativewhen the drug is unstable and degradationproducts are formed which may
interfere with the analysis or when any other compound in the sample may cause
interference. The Krebs-ringer bicarbonate buffer (KR) (pH 7.4) and the mobile phase
were separately analysed by HPLC. This method was selective since there were no
interferingpeakswith the same retentiontime as Rho 123.
4.2.1.5 System repeatability
In order to evaluate the repeatability of the peak area and the retention time, a sample of
Rho 123 with a known concentration (44.2 ~g/ml) was injected six times. The variation
in the response (RSD) of the detection system when six determinations of Rho 123 were
made on the same day, and under the same conditions, was found to be 3.00% for the
peak area and 1.20% for retention time. The system repeatability for Rho 123 was
within acceptable limits.
47
4.3 Instrument used for in vitro transport studies
To prepare intestinal mucosal sheets suitable for mounting in diffusion chambers a
longitudinal cut of an intestinal segment of the jejunum is made to produce a long
mucosal sheet. This sheet is cut as needed to produce mucosal strips of adequatesize
to fit in the openingof the diffusion chamber. Although mucosal sheets are usedwith or
without the underlyingmuscle layer, the removal of this muscle layer, a processknown
as stripping, is advantageous for two reasons. Firstly, it removes an artificial
permeability barrier, and secondly, stripped tissues can be oxygenated more efficiently
(Ungell, 1998:360). In a trial study conducted with Rho 123 transport across isolated
jejunum segments mounted in Sweetana-Grass diffusion cells, it was found that the
serosa had to be removed in order for the Rho 123 to be transported (Hattingh,
2002:48).
The rate of Rho 123 transport under the influence of selected flavonoids was determined
using a vertical diffusion chamber system, comprising four Sweetana-Grass diffusion
chambers, one heating block and one gas manifold (Coming Costar Corporation,
Cambridge, USA) (Slide 1).
Slide1
48
---
- -- --
4.3.1 Materials
KR, Rho 123, galangin, kaempferol, morin and quercetin (Sigma Chemical Company
Ltd., S1. Louis, Missouri, USA) were obtained from Sigma-Aldrich (Pty) Ltd,
Johannesburg and absolute ethanol, acetonitrile for HPLC, K2HP04were qbtainedfrom
Merck (Pty) Ltd, Germiston. Certificates of analysis for Rho 123, galangin, kaempferol,
morin, quercetin and Krebs-ringerbicarbonate buffer are presentedin Appendix C.
4.3.2 Tissue preparation
The use of unfasted adult male Sprague-Dawley rats (350-370g) obtained from the
Animal Research Centre (North-West University) were approved by the Ethical
Committee of the North-West University under protocol number 03D03 (Appendix C).
The rats were anaesthetised by inhalation of halothane. An abdominal incision was
made and starting 10 cm from the stomach a 20-30 em strip of the jejunum was excised,
rinsed with ice cold Krebs-Ringerbicarbonate buffer (KR) through which 95% O2/ 5%
C02 had been bubbled for 10 minutes (Slide 2) and then pulled onto a glass rod
(Slide 3).
49
--
-- - -- --
Slide 2 Slide 3
One hand was used to lift and hold the one end of the rod. The jejunum was then gently
scoured long the mesenteric border with the back of a scalpel (Slide 4). Using the index
finger along the length of the jejunum, the serosa and muscle layer were then gently
pushed back and peeled off (Slide 5). The jejunum was kept moist with cold KR at all
times which was kept in an ice bath. The jejunum was cut above and along the
mesenteric border with a scalpel blade (Slide 6). The jejunum was then washed off the
glass rod with a buffer-filled syringe onto a strip of filter paper (Slide 7).
50
Slide 4
+
Slide 6
Slide 5
Slide 7
The jejunum and filter paper were then cut simultaneously into lengths approximately 3
cm (Slide 8). The segments were kept moist with ice cold KR and were kept on ice
during the procedure. Segments containing Peyer's patches were identified visually and
avoided (Slide 9), as these lymph like tissues would cause greater variation in the rates
of transport because of altered morphology and thickness of the epithelial layer.
51
Slide 8
Slide 9
The segments were then carefully mounted onto the half cells (preheated to 37°C) with
the side containing the basolateral side of the jejunum face-down onto the pins (Slide 10
& 11). The filter paper was then removed and the matching half-cells were then carefully
clamped together (Slide 12 & 13). Thereafter the assembled chamber was placed in the
heating block (37°C) and 5 ml KR preheated to 37°C was added. Circulation of the
buffer was maintained by a gas-lift using 95% O2 I 5% C02 at a flow rate of 15-20
ml/min.
Slide 10
--
- - -- --
Slide 11
52
Slide 12 Slide 13
After all four cells were mounted with jejunum segments and filled with KR, the cells
were left for 15 min to reach a state of equilibrium before a transport study was started
by adding the various compounds under investigation to the donor cell. The temperature
of the cells was maintained at 37°C throughout the experiment.
4.3.3 Procedures used during transport studies
The transport of Rho 123 was determined in the apical to basolateral (AP-BL) direction
for the first two cells and basolateral to apical (BL-AP) direction for the last two cells.
Galangin, kaempferol, morin and quercetin were screened as potential Pgp modulators.
Each experiment was done in quadruplet for each modulator. Galangin, kaempferol,
morin and quercetin were dissolved in 700 IJI absolute ethanol as these flavonoids are
poorly soluble in water. These solutions were then made up to volume with KR
containing Rho 123. The final ethanol concentration in the various sample solutions was
kept ~1%, a concentration proven to not alter cell viability or permeability (Soldner et a/.,
1999: 479).
The effects of these compounds on the transport of Rho 123 were assessed by the
concomitant addition of the individual compounds with Rho 123 in the first two
chambers. Sufficient Rho 123, to give a final Rho 123 concentrationof 10.1 IJMin the
donor cell, was added to the apical side and 2 ml KR was added to the basolateralside.
For the last two chambers, Rho 123 (sufficient to give 10.1 IJM) was added to the
---
53
--- ---
basolateral side and the 2 ml KR was added to the apical side. The total volume in each
half chamber after the final additions was 7 ml. The total exposed tissue surface area
was 1.78 cm2. 250 IJI aliquots were .taken from the receiver cell of the chambers at 30,
60, 90, and 120 min after the addition of Rho 123, and replaced with an equivalent
amount of fresh KR. It was previously shown (Hattingh, 2002:83) that the maximum
period to perform transport studies was approximately 120 min before structural damage
to the epithelium occurred. The aliquots were analysed by HPLC.
4.3.4 Apparent permeability coefficient
The average apparent permeability coefficient (Papp)was calculated according to the
following equation:
dQ/dt
Papp=60xAxCo
Where dQ/dTis the transport rate, Co is the initial concentration of Rho 123 (100%) and
A is the area of exposed tissue (1.78 cm2). An example of the calculations performed is
presented in Appendix B.
4.4 Statistical analysis
Statsoft@ Statistica for Windows (Statsoft Inc., Tulsa, Oklahoma, USA) was used to
perform the statistical analyses on the data obtained. For the comparison of all the
values with the control a non-parametric one-tailed Kruskal-Wallis test was done (Steyn
et al., 1998:604). This test was also used to test for statistical differences between
different modulators at the same concentration. Statistically, this was the preferred test
to perform the statistical analysis on the data obtained because each experiment was
done in quadruplet and normality could not be assumed. Effect sizes were performed to
determine whether a practical significance exist between each modulator at two different
concentrations.
54
--- -
--
CHAPTER 5
RESULTS AND DISCUSSION
5.1 Transport studies
The effect of different flavonoid modulators on the transport of Rho 123 was investigated
using the Sweetana-Grass diffusion method comprising four cells. Each experiment was
done in quadruplet. The transport of Rho 123 was calculated in the apical to basolateral
(AP-BL) direction for the first two cells and basolateral to apical (BL-AP) direction for the
last two cells. The net transport of Rho 123 was determined and the ratio of the Papp
value in the BL-AP to the Pappvalue in the AP-BL direction was calculated. The
individual values for the cumulative transport of Rho 123 in the absence and in the
presence of selected flavonoids are presented in Appendix A. Statistical analyses were
used to compare the results of the experiments with the control.
5.1.1 Transport of Rho 123
An example of the cumulative transport of Rho 123 with no modulators added, is
presented in Figure 5.1 and the Pappvalues in Table 5.1. The ratio's obtained served as
the control values to which the ratio's obtained after addition of the modulators, could be
compared.
55
--
- --- ---
---
-.------.--.--.-.-...---..--...---.---..-........-.--.----.--.......-
Table 5.1: Individual and mean Pappvalues of Rho 123 transported (AP-BLand BL-
AP) with no modulators present.
*Each value represents the mean f: standard deviation
-
~4
t:
o
g. 3
c:
~ 2
Q)
.~
12 1
:]
E
:]
()
. BUAP
· AA'BL
o
o 20 40 60
Tlrre(rrin)
80 100 120
Figure 5.1: Example of the cumulative transport of Rho 123 with no modulators
added.
Conclusion
Accordingto Neyfakh(1998:168), Rho 123 is classifiedas a typical Pgp substrateand is
subject to Pgp-dependent extrusion through the plasma membrane. Thus, Rho 123
accumulation in (or efflux from) cells is used as a measure of MDR1-dependent
transport activity. The results obtained during these experiments also indicatedthat Rho
123 is a substrate for active transporters. The mean ratio calculated was 3.29. The
mean ratio's observed in the absence of a modulatorwere comparedto the mean ratio's
obtained after the addition of the modulators. Table 5.2 presentsthe p-values obtained
56
- - -
, A' '_>
Pappoc1f01*rcMls iPappxiit)'t;mls'
JMean!Ratio"
EXpenmenti
Ratio
No. AP':BL BL--AP BL-AP/AP-BL BL"!APJAP..;BL
1 1.67 5.60 3.35 3.29f:0.220*
2 1.15 3.63 3.15
3 0.67 2.40 3.57
4 3.59 11.05 3.07
by the statistical analysis (Kruskal-Wallis test) to show which mean ratio's showed a
statistical significant difference from the control. p<0.05 was taken as statistical
significant.
Table 5.2: Multiple comparisons p-values (1-tailed) obtained by the non-parametric
Kruskal-Wallistest comparing the Papp ratio's in the presence of modulators with
the Pappratio obtained in the control experiment.
Control
0.024864* 0.042755*
0.500000
0.2108245
0.500000
0.500000
0.182349
0.500000
0.500000
0.500000
Morin 0.024864*
Galangin 0.042755* I 0.500000
Kaempferol 0.210825 0.500000 I 0.500000
Quercetin 0.182349 0.500000 I0.500000
* Value representinga statisticalsignificantdifference
0.500000
In order to determine whether a practical significance exists between the two
concentrations of each modulator, effect sizes were determined. Tables for the
descriptive statistics of each modulatorare given in Table 5.3.
57
--- -
Control
0.006252* I 0.050887 I 0.242980 I 0.500000
Morin
0.006252* 10.500000
I 0.500000 I0.098850
Galangin
0.050890 I 0.500000 I I 0.500000 I 0.471320
Kaempferol
0.242980 I 0.500000 10.500000
I I 0.500000
Quercetin
0.500000 I 0.098850 I 0.471322 I 0.500000
Table 5.3 Descriptive statistics of modulators at concentrations 10 JIMand 20 JIM.
Results of the effect size were determined by the following equation:
d=1
Where d is the effect size, XF;o-XF;ois the difference between the means of the two
concentrations at 10 IJMand 20 IJMand Smax is the ratio at the maximum standard
deviation (Steyn, 1999:28). The results of the effect sizes between the two
concentrationsof each modulatorare given in Table 5.4.
58
Control 3.245 0.277
Morin 1.880
0.091
Galangin
2.184 0.375
Kaempferol
2.514 0.554
Quercetin 2.976
0.200
Control 3.245 0.277
Morin 1.490 0.611
Galangin
1.604 0.544
Kaempferol
1.816 0.557
Quercetin 1.952 0.272
Table 5.4: Effect size results.
*Valuerepresenting practicalsignificance
Guidelines for the interpretation of the effect sizes are the following:
(a) Small effect: d S 0.2
(b) Mediumeffect: 0.2 S d S 0.8
(c) Large effect: d ~ 0.8
Data where d ~ 0.8 is considered as practically significant as it is the result of a
differencehavinga large effect (Cohen, 1988).
5.1.2 Transport of Rho 123 in the presence of selected
flavonoids
The modulating effect of four selected flavonoids on the transport of Rho 123 was
investigated.Duringscreeningforflavonoid chemosensitisers in a study done by Choi
and co-workers (2004:672), it was found that a certain flavonoid 5,7,3',4',5'-
pentamethoxyflavone(PMF) was equipotent to verapamil in vitro with respect to the
chemosensitising effect. However, the effect of PMF on the level of Rho 123
accumulationwas lower than that of the Pgp substrate, verapamil. In this study, morin,
galangin, kaempferol and quercetin (all flavonoids) were investigated as modulators.
The effect of these components on the transport of Rho 123 across rat jejunum was
evaluated using two concentrations (10 IJM and 20 IJM). The concentrations were
chosen according to findings by Scambia and co-workers (1994:459). They have
demonstrated that concentrations of quercetin ranging between 1 and 10 IJM had a
dose-dependent effect on reducing Pgp in MCF-7 adriamycin-resistant human breast
cancer cells. Van Zanden and co-workers (2005:699) found that quercetinand
59
- ---
Morin 0.64
Galangin
1.06*
Kaempferol
1.30*
Quercetin
3.80*
kaempferol were potent MRP inhibitors at concentrations 25 IJM and 19.4 IJM
respectively. They also found that the highest flavonoid concentration tested was 50
IJM, because some flavonoids are either cytotoxic or poorly soluble at concentrations
above 50 IJM.
Structure activity relationships (SAR) of the selectedflavonoids were also investigatedin
this study with reference to inhibition of Pgp. The effect of the different hydroxyl group
positioning on the B-ring was investigated. SAR studies for the inhibition of Pgp ATP-
ase activity by flavonoids showed that the presence of a 5-hydroxyl group, the 3-
hydroxyl group and the C2-C3 double bond are requiredfor high potency bindingto the
NBD of Pgp (Boumendjel et a/., 2002:512). In a study by van Zanden and co-workers
(2005:704),multiple regressionanalysiswas used for the identificationand quantification
of the effects of different structural characteristics regarding potent inhibition of MRP1
and MRP2 activity. For MRP1, an optimal multiple parameter aSAR model was
obtained which revealedthat three structural characteristicsare of importancefor MRP1
inhibition:
'(!J The total number of methoxylated moieties;
'(!J The total number of hydroxyl groups and
'(!J The dihedral angle between the B- and C-ring which quantifies the planarity of the
flavonoid modulators.
The measured lipophilicity and calculated dihedral angle between the B- and C-ring for
the flavonoids tested are given in Table 5.5. The lipophilicity of the flavonoids was
calculated using the capacityfactor (K') calculatedby:
K' = tr-to
to
in which K' is the capacity factor, tr is the retention time (min) and to is the retention time
of unretained substances (min) (van Zanden et a/., 2004:1613).
60
-- - --- -----
-- -
Table 5.5: Measured lipophilicity (K') and calculated dihedral angle between the B-
and C-ring for the following flavonoids tested (van Zanden et al., 2004:705).
As seen from the results in Table 5.5, galangin is the most lipophilic compound. It is
also interesting to take note that galangin is the only flavonoid without hydroxyl groups at
the B-ring. Therefore it can be expected that galangin has a higher net transfer across
the brush border and therefore probably will have a profound effect on inhibiting Rho 123
transport. Morin is shown to have the lowest lipophilicity. It may be due to the presence
of the two hydroxyl groups at the B-ring. Therefore it can be expected for morin to have
a less inhibitory effect on the transport of Rho 123. Kaempferol has only one hydroxyl
group at the B-ring and also has a have relative high lipophilicity. Therefore kaempferol
also may have a relative effect on the inhibition of Rho 123 efflux. There exists a definite
correlation between the amount of hydroxyl groups at the B-ring and the lipophilicity of
the flavonoids. According to the dihedral angles given in Table 5.5, there does not seem
to be major differences between the angles of the compounds. Morin however is shown
to have the largest dihedral angle (19.3°).
5.1.2.1 Transport of Rho 123 in the presence of morin
The effect of morin on the transport of Rho 123 across rat jejunum was investigatedin
this study using two concentrations. An example of the cumulative transport of Rho 123
in the presence of morin (10 IJM and 20 IJM) is presented in Figures 5.2 and 5.3
respectivelyand the Pappvalues in Table 5.6.
61
---
Morin
2.8 19.3
Galangin
18.3 14.5
Kaempferol
8.8 I 14.4
Quercetin
4.4 I 14.7
Table 5.6: .Individual and mean Pappvalues of Rho 123 transported (AP-BL and BL-
AP) in the presence of morin (10 JIMand 20 JIM).
-
~4
-
8. 3
UJ
c:
~ 2
Q)
~ 1
"3
E
:J
U
o
o
. BUAP
. AP/BL
20 40 60
lime (ITin)
80 100
120
Figure 5.2: Example of cumulative transport of Rho 123 in the presence of marin
(10 JIM).
62
-- - -- - -
Morin (10 JIM)
1 2.24 4.25 1.90
I 1.88:i:0.091*
2 4.49 8.98 2.00
3 4.52 8.21 1.82
4 1.59 2.86 1.80
Morin (20 JIM)
1 6.65 12.07 1.82
I 1.49:tO.611*
2 5.15 11.20 2.18
3 9.62 8.24
0.86
4 22.65 25.20
1.11
*Each value represents the mean :i:standard deviation
Figure 5.3: Example of cumulative transport of Rho 123 in the presence of morin
(20 pM).
Conclusion
At concentrations of 10 ~M and 20 ~M the mean ratio's calculated were 1.88 and 1.49
respectively, indicating that the transport of Rho 123 in the basolateral to apical direction
was decreased. The p-values at the two concentrations were 0.006252 and 0.024864
respectively. Both p-values were < 0.05 and therefore indicated that there was a
statistically significant difference between the mean ratio of morin and the mean ratio of
the control. Morin however, did not show a practically significant effect between the two
different concentrations according to the effect size. It can be concluded that morin at
concentrations of 10 ~M and 20 ~M inhibited the ration of transport of Rho 123. It acts
therefore as an inhibitor of the transporter proteins responsible for the efflux of Rho 123.
The chemical structure of morin is given in Figure 5.4.
63
---- -----
4
-
...
3
CIJ
c:
CtJ
2
CD
>
1 . BUAP
"3
E
. APlBL
::J 0
()
0 20 40
60 80
100 120
lime (rrin)
HO
Figure 5.4: Chemical structure of morin (2',3,4',5,7-pentahydroxyflavone).
Morinis the onlyflavonoidinvestigated in this study possessinga hydroxyl group at the
C2' atom. In a study done by Nguyen and co-workers(2003:250) it was found that the
most potent inhibitors on MRP1-mediated transport in Panc-1 cells were morin >
kaempferol > quercetin > genistein. Therefore, it can be assumed for the purposes of
this study that the hydroxylgroups at the C2' atom plays a definite role in the inhibitionof
Rho 123 efflux. Contradictoryresults were found regardingthe lipophilicity. InTable 5.5
morin had the lowest lipophilicity, but regardless of that, still showed inhibition of Rho
123 transport more effectively than the other flavonoids. Morin also has the largest
dihedral angle which probably played a definite role in the Rho 123 transport inhibition
and this may be due to the hydroxylgroup on the C2' atom of the 8-ring.
5.1.2.2 Transport of Rho 123 in the presence of galangin
The effect of galangin on the transport of Rho 123 across rat jejunum was investigated
using two concentrations. An example of the cumulative transport of Rho 123 in the
presence of galangin (10 IJM and 20 IJM) is presented in Figures 5.5 and 5.6
respectively and the Pappvalues in Table 5.7.
64
-
Table 5.7: Individual mean Papp values of Rho 123 transported (AP-BL and BL-AP)
in the presence of galangin (10 J.lMand 20 J.lM).
Figure 5.5: Example of the cumulative transport of Rho 123 in the presence of
galangin (10 J.lM).
65
----
Galangin (10 J.lM)
1 4.20 11.38 2.71
I 2.18:1:0.375*
2 16.76 36.58 2.18
3 19.36 36.21 1.87
4 13.25 26.11 1.97
Galangin (20 J.lM)
1 22.54 23.58 1.05
I
1.60:1:0.544*
2 9.91 21.27 2.15
3 4.33 8.60 1.99
4 17.76 21.93 1.23
*Each value represents the mean :I: standard deviation
?;:4
---
I I
-
...
g, 3
!J)
c::
ro
.;: 2
Q)
>
1 J
-------- I. BUAP
:J
E
I -------- I. APfBl
<3 0
0 20 40 60 80 100
120
Time (rrin)
-
o
~4
1::
8. 3
C/)
c:
~ 2
Q)
>
~ 1
"3
E
:J
()
o
o
. BUAP
. AA'BL
20 40 60
lirre (rrin)
80 100 120
Figure 5.6: Example of the cumulative transport of Rho 123 in the presence of
galangin (20 IJM).
Conclusion
At a concentrationof 10 J.lMthe mean ratio calculated was 2.18 indicatinga decrease in
transport of Rho 123 but this was not statistical significant. At the concentration of 20
J.lMthe mean ratio calculated was 1.60. The p-value at the 20 J.lMconcentration was
0.042755 which is <0.05 and therefore indicated that there was a statistical significant
difference between the mean ratio of galangin and the mean ratio of the control. It can
be concluded that galangin at concentration 20 J.lMinhibitedthe rate of transport of Rho
123 in the basolateral to apical direction. It acts therefore as an inhibitorof the
transporter proteins responsible for the efflux of Rho 123. This corresponds to the
findings of Conseil and co-workers (1998:3835) who reported that galangin was able to
modulate drug effluxin MDRcancer cells. Galangin did have a practicallysignificant
difference at the two differentconcentrations according to the effect size. The chemical
structure of galangin is given in Figure 5.7.
66
-- -- - ----
Figure 5.7: Chemical structure of galangin (3,5,7-trihydroxyflavonone).
Galangin is the onlyflavonoidused in this study possessing no hydroxylgroups at the B-
ring. Van Zanden and co-workers (2004:1607) investigated the structural requirements
for inhibitionof glutathione-S-transferase P1-1 (GSTP1-1)and MRP1and MRP2activity
by structurallyrelated flavonoids. They found that especiallygalangin was able to inhibit
almost all cellular GSTP1-1 activityupon exposure of the cells to a concentration of 25
IJM. They found that the absence of B-ringhydroxylgroups in galangin might result in
relativelyhigher GSTP1-1 inhibitionpotency. The results obtained in this study may be
correlated to the above findings where galangin with no B-ring hydroxyl groups,
successfully inhibitedthe transport of Rho 123 at a concentration of 20 IJM.Although
galangin was shown to be the most lipophiliccompound inTable 5.5, its inhibitionof Rho
123 transport was smaller than that of morin. The dihedral angle of galangin does not
significantlydifferfromthat of kaempferoland quercetin.
5.1.2.3 Transport of Rho 1,23in the presence of kaempferol
The effect of kaempferol on the transport of Rho 123 across rat jejunum was
investigated using two concentrations. An example of the cumulativetransport of Rho
123 in the presence of kaempferol (10 IJMand 20 IJM)is presented in Figures 5.8 and
5.9 respectively and the Pappvalues inTable 5.8.
67
- - - -- -- - - - -
3'
;-.......
4'
HO _O
l B) 5'
6'
Table 5.8: Individual mean Pappvalues of Rho 123 transported (AP-BL and BL-AP)
in the presence of kaempferol (10 JIMand 20 JIM).
Kaempferol (10 JIM)
1 14.37
2 7.14
3 2.74
4 13.90
Kaempferol (20 JIM)
1 5.28
2 11.20
3 5.15
4 3.82
40.83
17.10
4.90
42.14
2.84
2.39
1.79
3.03
2.51:1:0.533*
9.43
24.40
5.35
8.63
1.79
2.18
1.04
2.26
1.82:1:0.577*
*Each value representsthe mean :I:standard deviation
~4
t
& 3
CJ)
c
~ 2
CD
>
~ 1
:;
§ 0
u
o
. BUAP
. APlBL
20 40 60
Time (rrin)
80 100 120
Figure 5.8: Example of the cumulative transport of Rho 123 in the presence of
kaempferol (10 JIM).
68
- -- -
Figure 5.9: Example of the cumulative transport of Rho 123 in the presence of
kaempferol (20 JIM).
Conclusion
At concentrations 10 ~M and 20 ~M the mean ratio's calculated were 2.51 and 1.82
respectively but this was not statistical significant. According to the effect size,
kaempferol seemed to have a practical significant difference between the two different
concentrations.
In a study done by Phang and co-workers (1993:5979) it was found that kaempferol,
together with galangin and quercetin was one of the most active compounds on the
activity of Pgp. Hooijbergand co-workers (1997:347)found that kaempferol also had a
stimulating effect on the ATPase activity on the MRP overexpressing cell line
GLC4/ADR. The chemicalstructureof kaempferol is given in Figure5.10.
69
-
0
4
-
....
0
g. 3
c
2
Q)
>
. . BLJAP
1
"3
T
§ 0
..
. AP/BL
()
0 20 40 60 80 100 120
Time(mn)
3'
OH
HO
6'
OH 0
Figure 5.10: Chemical structure of kaempferol (3, 4', 5, 7-tetrahydroxyflavone).
Kaempferolhas four hydroxylgroups. The hydroxylgroup at C4' on ring B is of interest.
In a study done by Conseil and co-workers(1998:9831)itwas found that the additionof
a hydroxyl group at C4' of ring B may have a negative effect on the inhibitionof
modulators (kaempferol compared to galangin). Results obtained in this study also
indicated that, although kaempferol was able to inhibit Rho 123 efflux at both
concentrations, ithad a slightlesser inhibitoryeffect compared to galangin.
5.1.2.4 Transport of Rho 123 in the presence of quercetin
The effect of quercetin on the transport of Rho 123 across rat jejunum was investigated
using two concentrations. An example of the cumulative transport of Rho 123 in the
presence of quercetin (10 11Mand 20 11M)is presented in Figures 5.11 and 5.12
respectivelyand the Pappvalues inTable 5.9.
70
--- - - - --- - -
Table 5.9: Individual mean Pappvalues of Rho 123 transported (AP-BLand BL-AP)
in the presence of quercetin (10 IJMand 20 IJM).
~4
1::
& 3
IJJ
C
~ 2
Q)
>
~ 1
:;
~ 0
() 0
. BUAP
. APfBl
20 40 60
Time(rrin)
80 100 120
Figure 5.11: An example of the cumulative transport of Rho 123 in the presence of
quercetin (10 IJM).
71
-- ---
---
Quercetin (10 IJM)
1 15.45 50.53 3.27
I
2.98:1:0.200*
2 5.90 16.70 2.83
3 14.97 43.36 2.90
4 15.15 44.06 2.91
Quercetin (20 IJM)
1 3.74 8.14 2.17
I
1.95:f:0.335*
2 3.49 5.45 1.56
3 8.80 18.56 2.11
4 8.65 16.93 1.96
*Eachvalue represents the mean :f:standard deviation
. BUAP
. APfBl
20 40 60
Time(rrin)
80 100 120
Figure 5.12: An example of the cumulative transport of Rho 123 in the presence of
quercetin (20 JIM).
Conclusion
At concentrations 10 ~M and 20 ~M the mean ratio's calculated were 2.98 and 1.95
respectively indicating a decrease in transport of Rho 123 but this was not statistical
significant. The results however, indicated that quercetin is an inhibitor of Pgp. These
results con-elatewith those found by van Zanden and co-workers (2005:703), who
demonstratedthat quercetin was able to inhibit more than 50% of MRP1 activity at 25
~M. There was also a practical significant difference between the two different
concentrations of quercetin according to the effect size. The chemical structure of
quercetin is given in Figure 5.13.
72
---
----
-
4
t
3
UJ
c:
CtI
2
J::;
(J)
>
1CtI
"3
E
0:!
()
0
OH
HO
Figure 5.13: Chemical structure of quercetin (3,5,7,3',4'-pentahydroxyflavone).
Quercetin, shown to be the most important MRP1 and MRP2 inhibitor,contains five
hydroxyl groups including a B-ring 3',4'-catechol moiety. The presence of a 3',4'-
dihydroxymoietyon the B-ringresults in strong inhibitionas is shown by comparison of
quercetin to morinand kaempferol (van Zanden et a/., 2005:1615). However,this study
indicatedthat morinshowed better Rho 123 effluxinhibition. This may be due to the low
concentrations used. Regarding the lipophilicityand dihedral angle, quercetin also did
not seem to deviate specificallyfrom the other flavonoids tested. Quercetin was the
weakest inhibitorof Rho 123.
5.2 Summary
Figure 5.14 gives a comparative overviewof results obtained in the differentstudies (as
summarised in Table 5.10). As can be seen from this chart, the most pronounced
inhibitoryeffectwas due to morinat 20 ~M(M2). As previouslystated, itwas onlymorin
(10 ~M and 20 ~M)and galangin (20 ~M)that showed statistical significantresults on
the transport of Rho 123, although the other flavonoidconcentrations did show inhibition
of Rho 123.
73
-- -
----
Table 5.10: Mean ratio's obtained for all the modulators examined.
Figure 5.14: Bar chart showing the relative mean ratio's of the various flavonoids
* Values representing statistical significant differences
compared to the mean ratio of the control.
74
- -
Modulator BarChart Ratio :Standard
Idenft"fier
¥(Nlean) ;
Deviation
Control Cont 3.29
0.22
Morin 10 IJM
M1 1.88 0.09
Morin 20 IJM
M2 1.49 0.61
Galangin 10 IJM
G1 2.18 0.38
Galangin 20 IJM
G2 1.60 0.54
Kaempferol10 IJM
K1 2.51 0.55
Kaempferol 20 IJM
K2 1.82 0.56
Quercetin 10 IJM
Q1 2.98 0.27
Quercetin 20 IJM
Q2 1.95 0.27
4.00
3.50
3.00
.2 2.50
.....
c:: 2.00
co
Q)
1.50
1.00
0.50
0.00
Cont M1* M2* G1 G2* K1 K2 01
02
CHAPTER 6 
CONCLUSION AND RECOMMENDATIONS 
6.1 Introduction 
Because of the deleterious effect of Pgp on chemotherapeutic efficacy, compounds that 
modify its function are of potential clinical value. Numerous modulators or 
chemosensitisers are known that inhibit the ability of Pgp, maintaining subtoxic 
intracellular drug concentrations (Gottesman & Pastan, 1993:385). Examples include 
calcium channel blockers like verapamil and trifluoperazine, detergents like Triton X-100 
and immunosuppressants like cyclosporine A (Shapiro & Ling, 1997587). In contrast to 
chemosensitisers, three flavonoids, kaempferol, galangin and quercetin have been found 
to stimulate efflux of two Pgp substrates, 7,12-dimethylbenz[a]anthracene and 
Adriamycin, from multidrug-resistant cells (Phang et al., 1 993:5977). This remarkable 
observation could be of importance for chemotherapy because flavonoids are very 
common constituents of substances and an increase in the activity of Pgp could 
potentially reduce the efficacy of chemotherapy (Gottesman & Pastan, 1993:385). 
6.2 Conclusion 
The final conclusions of this study are as follows: 
a Of all the modulators used, morin was the strongest modulator of Rho 123 
transport. At a concentration of 10 pM it decreased the mean Pa,, ratio from 3.29 
in the control to 1.88 and at a concentration of 20 pM it decreased the mean Pam 
ratio from 3.29 to 1.49. Both concentrations caused a statistical significant 
decrease of the transport ratio- of _R_ho 123. Slrudure actirJi#yrelationhship {SAR) 
---------------- 
with reference to inhibition of Pgp indicated that the hydroxyl groups at the C2' 
atom at the B-ring probably played an important role in the inhibition Rho 123 
transport. 
rn Galangin decreased the mean Pap, ratio from 3.29 in the control to 2.18 at a 
concentration of 10 pM, indicating decreased efflux activity. This decrease was, 
however, not statistically significant probably due to the low concentration used. 
At a concentration of 20 pM it decreased the mean Pap, ratio from 3.29 to 1.60, 
also indicating decreased efflux activity and showed a significant difference 
according to the Kruskal-Wallis test. This decrease in mean Pap, ratio showed 
reduced efflux of Rho 123. 
u Kaempferol decreased the mean Pap, ratio from 3.29 in the control to 2.51 at 
concentration 10 pM and from 3.29 to 1.82 at a concentration of 20 pM indicating 
inhibition of active transport of Rho 123. No statistical significant differences 
were observed. SAR with reference to inhibition of Pgp may be due to the 
hydroxyl group present at C4' at the B-ring. 
u Quercetin decreased the mean Pap, ratio from 3.29 in the control to 2.98 at 
concentration 10 pM and from 3.29 to 1.95 at a concentration of 20 pM. The 
decrease at both concentrations however, were not statistically significant. SAR 
with reference to inhibition of Pgp may be due to the 3',4'-dihydroxy moiety on 
the B-ring. 
u Regarding the effect of the two different concentrations used, effect sizes 
showed that galangin, kaempferol and quercetin showed practical significant 
differences. Morin however did not show any practical significant differences at 
the two different concentrations. 
u The lipophilicity and dihedral angle did not seem to have a pronounced effect on 
the efflux of Rho 123 as the flavonoids tested did not deviate from each other. 
u The order of the inhibitory potency on the transport of Rho 123 was as follow: 
morin > galangin > kaempferol > quercetin. 
rn In summary, this study described the inhibitory interaction of selected flavonoids 
with Pgp. Structure activity relationships were identified describing the inhibitory 
potency of the flavonoids based on hydroxyl groups positioning. Moreover, this 
study proved that the Sweetana-Grass diffusion model seems to be efficient and 
sensitive enough to be used to determine differences in rates of transport caused 
by various modulators. 
6.3 Recommendations 
In continuation of this study the following should be investigated: 
P An extensive study on structure activity relationships of other selected flavonoid 
inhibitory activities on Pgp will provide a valuable inspiration for a rational drug 
and/or chemopreventive agent design of future pharmaceuticals in the prevention 
and/or treatment of several life-threatening diseases such as cancer. 
P Further in vivo studies should be pursued by comparing concentrations of in vitro 
studies and whether it will have therapeutic plasma concentrations as a result in 
vivo . 
a The potential therapeutic application of flavonoids in cancer therapy, either alone 
or in combination with other conventional cytotoxic drugs. 
a As the potency of Pgp inhibition is largely dependent on the hydrophobic 
substituent on the B-ring at C4' (Conseil et a/., 1998:9836), further studies on 
different substitutions at this position should be pursued as well as their 
therapeutic applications. 
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Table A.1: Cumulative transport amounts of Rho 123 (10.1 pM) in the AP-BL and
BL-AP direction with and without (control) modulators.
90
- -
i Component
' _ "A ,
MeanfStandarddeyjation
l1me
Cumulative traportOfRhodamine 123
i
L-AP
. .
AP';BL
. BL-AP
,
AP4SL
Ci;)aniberf
,)Chamber:
D1aniber'
Clamber', '"
'1. <,
.2 .,' .!
It '
.2 .
, "
,vw c
Control 3 30 0.03 0.06 0.07 0.02 0.045
:!: 0.021 0.045 :t 0.035
60 0.04 0.08 0.15 0.13 0.060 :!: 0.028 0.140 :t 0.014
90 0.11 0.10 0.32 0.33 0.105
:!: 0.007 0.325 :t 0.007
........
120 0.18 0.24 0.62 0.53 0.210
:!: 0.042 0.575 :t 0.064
.eontrol2 30 0.02 0.02 0.08 0.06 0.020 :!: 0.000 0.070
:t
0.014
60 0.04 0.07 0.13 0.16 0.055 :!: 0.021 0.145 :t 0.021
90 0.07 0.09 0.23 0.23 0.080 :!: 0.014 0.230 :!: 0.000
...... 120 0.09 0.17 0.40 0.46 0.130 :!: 0.057 0.430 :t 0.042
1Control.3: 30 0.09 0.08 0.08 0.08 0.085 :!: 0.007 0.080
:t
0.000
60 0.09 0.09 0.10 0.12 0.090 :!: 0.000 0.110
:!: 0.014
90 0.11 0.10 0.23 0.20 0.105
:!: 0.007 0.215 :t 0.021
120 0.15 0.15 0.33 0.27 0.150 :!: 0.000 0.300 :t 0.042
CoRftol4 30 0.05 0.07 0.07 0.07 0.060 :!: 0.014 0.070
:!:
0.000
60 0.08 0.10 0.13 0.13 0.090 :!: 0.014 0.130 :t 0.000
90 0.17 0.24 0.48 0.55 0.205 :!: 0.049 0.515 :t 0.049
120 0.32 0.49 1.05 1.07 0.405 :!: 0.120 1.060 :t 0.014
30 0.09 0.11 0.09 0.04 0.100 :!: 0.014 0.065 :!: 0.035
60 0.14 0.11 0.23 0.17 0.125 :!: 0.021 0.200 :t 0.042
90 0.16 0.19 0.26 0.27 0.175 :!: 0.021 0.265 :t 0.007
120 0.33 0.32 0.56 0.44 0.325 :!: 0.007 0.500 :t 0.085
30 0.04 0.04 0.13 0.12 0.040 :!: 0.000 0.125 :t 0.007
60 0.10 0.09 0.23 0.26 0.095 :!: 0.007 0.245 :t 0.021
90 0.20 0.14 0.57 0.53 0.170 :!: 0.042 0.550 :t 0.028
120 0.57 0.42 0.97 1.00 0.495 :!: 0.106 0.985 :t
0.021
30 0.04 0.04 0.10 0.11 0.040 :!: 0.000 0.105 :t 0.007
60 0.04 0.04 0.20 0.23 0.040 :!: 0.000 0.215 :t 0.021
90 0.14 0.15 0.51 0.49 0.145 :!:
0.007 0.500 :!: 0.014
120 0.49 0.49 0.84 0.93 0.490 :!: 0.000 0.885 :t 0.064
30 0.04 0.04 0.24 0.23 0.040 :!: 0.000
0.235
:t
0.007
60 0.60 0.64 0.54 0.55 0.620 :!: 0.028 0.545 :t 0.007
90 0.92 1.02 2.03 1.37 0.970 :!:
0.071 1.700 :t 0.467
120 1.53 1.71 3.50 2.30 1.620 :!:
0.127 2.900 :t 0.849
30 0.11 0.25 0.37 0.14 0.180 :!: 0.099 0.255 :t
0.163
60 0.45 0.53 0.66 0.62 0.490 :!:
0.057 0.640 :t 0.028
120 0.76 0.96
1.52 1.34 0.860 :!: 0.141 1.430 :!: 0.127
30 0.23 0.15 0.17 0.26 0.190
:!: 0.057 0.215 :!: 0.064
60 0.33 0.25 0.31 0.47 0.290
+
0.057 0.390 :t 0.113
90 0.39 0.59 0.80 0.91
0.490 :!: 0.141 0.855 :!: 0.078
91
- - - - -- - ----
-- -
120 0.64
0.71
1.12 1.40 0.675
0.049 1.260 :!: 0.198
30 0.03
0.04 0.11
0.10 0.035
0.007 0.105 :!: 0.007
60 0.11
0.15 0.22
0.12
0.130
+
0.028 0.170 :!: 0.071
90 0.28
0.50 0.46
0.37 0.390 I
0.156 0.415 :!: 0.064
120 0.81
1.14
0.89 0.91 0.975
0.233 0.900 :I: 0.014
30 0.20
0.20 0.08
0.08 0.200
0.000 0.080 :!: 0.000
160
0.62 0.63
0.44 0.29
0.625 0.007
0.365 :!: 0.106
90 1.25
1.28 1.24
0.92 1.265
0.021 1.080 :I: 0.226
120 2.25
2.56 2.57
2.50 2.405 0.219
2.535 :!: 0.049
30 0.03
0.10 0.00
0.00 0.065 0.049
0.000 :I: 0.000
60 0.12 0.19
0.24 0.26
0.155 0.049 0.250 :I: 0.014
90 0.20 0.32
0.60 0.66
0.260 0.085 0.630 :!:
0.042
120
0.51 0.45 1.13
1.06 0.480
0.042 1.095 :I: 0.049
30 0.20 0.06
0.23 0.13
0.130 :!: 0.099 0.180 :!: 0.071
60 0.25
0.39 0.94
1.04 0.320 :I: 0.099
0.990 :!: 0.071
90 0.68
0.67 1.77 2.33
0.675 0.007 2.050 :!:
0.396
120 2.03 1.57 3.44
4.03 1.800 0.325 3.735 :!:
0.417
30 0.01 0.01
0.10
0.19 0.010 0.000 0.145 :!: 0.064
60 0.26 0.39
0.68 0.77
0.325 0.092 0.725 :!: 0.064
90 0.78
1.18 1.84 1.98 0.980
0.283 1.910 :I: 0.099
120 1.56 2.17
3.49 3.74 1.865
0.431 3.615 :!: 0.177
30
0.05 0.03 0.11
0.13 0.040 0.014
0.120 :!: 0.014
60 0.45 0.30
0.46 0.68 0.375 0.106
0.570 :!: 0.156
90 0.91
0.67 1.23 1.73 0.790
:I: 0.170 1.480
:I: 0.354
120 1.57
1.07 2.13 3.08 1.320
0.354 2.605 :!: 0.672
30 0.20 0.23
0.12 0.12
0.215 0.021 0.120 :!: 0.000
60 0.70 0.64 0.53
0.45 0.670 0.042
0.490 :!: 0.057
90 1.41 1.22 0.99
1.15 1.315 0.134 1.070
:!: 0.113
, 120 2.64 2.17 2.44
2.45 2.405 0.332 2.445 :!: 0.007
30 0.18
0.18 0.15 0.14 0.180
0.000 0.145 :I: 0.007
60 0.24 0.32
0.29 0.35 0.280
0.057 0.320 :I: 0.042
90 0.46 0.81
1.07 1.01 0.635 0.247
1.040 :!: 0.042
120 0.92 1.32
2.45 1.89 1.120 0.283 2.170 :I: 0.396
30 0.06 0.06
0.21 0.16 0.060 0.000 0.185 :!: 0.035
60 0.20 0.10
0.43 0.53 0.150 0.071
0.480 :!: 0.071
90 0.37 0.16
0.81 0.75 0.265 0.148
0.780 :I: 0.042
120 0.69 0.28 1.07 0.95
0.485 0.290 1.010 :I:
0.085
30 0.30 0.27 0.38
0.34 0.285 0.021 0.360 :!: 0.028
60 0.83 0.94
1.12 0.85 0.885 0.078 0.985 :I: 0.191
!!; 90 1.36 1.61 1.75
1.69 1.485 0.177
1.720 :I: 0.042
120 1.85 2.11 2.46
2.45 1.980 0.184 2.455 :I: 0.007
30 0.06 0.36 0.17 0.14
0.210 0.212 0.155 :!: 0.021
60 0.25
0.79 1.53 1.21 0.520
0.382 1.370 :!: 0.226
90
0.71 1.25 2.89 2.49 0.980
0.382 2.690 :!: 0.283
92
- - --
- --- -- -
--
120 1.07 2.10
4.14 4.01 1.585
:i: 0.728 4.075
:I: 0.092
30 0.15 0.21 0.25
0.16 0.180
:i: 0.042 0.205 :!: 0.064
60 0.34
0.52
0.55 0.47 0.430 :i:
0.127 0.510 :!: 0.057
90 0.46
0.71 1.11
1.41 0.585 :i: 0.177
1.260 :!: 0.212
120 0.70 1.08
1.78 1.78
0.890 :I: 0.269 1.780
:!: 0.000
30 0.23 0.14
0.23 0.27
0.185 :i: 0.064 0.250
:!: 0.028
60
0.29 0.25 0.38
0.43 0.270
:i: 0.028 0.405 :!: 0.035
90 0.35
0.36 0.52
0.62 0.355 :i: 0.007
0.570 :!: 0.071
120 0.42
0.47 0.65
0.79 0.445
+
0.035 0.720 :!:
0.099
30 0.08 0.25 0.20
0.10 0.165 :i:
0.120 0.150
:!: 0.071
60 0.32 0.31 1.12
0.79 0.315
+
0.007 0.955 :!: 0.233
90 0.98 0.91
2.36 1.94
0.945 :i: 0.049 2.150
:I:
0.297
120 0.53
1.35 4.87 3.63 0.940
:i: 0.580 4.250 :!: 0.877
30 0.07 0.07
0.19 0.15 0.070 :i: 0.000
0.170 :!: 0.028
60 0.16 0.16
0.28 0.32 0.160
:i: 0.000 0.300 :!: 0.028
90 0.25
0.18 0.59 0.62
0.215 :i: 0.049 0.605 :!: 0.021
120 0.62 0.60
1.22 0.93 0.610 :i: 0.014 1.075 :I: 0.205
- -
30 -0.20 -0.11
-0.20 -0.09 0.155 :i: 0.064 0.145 :!:
0.078
60 0.02
0.01 0.18 0.38 0.015
+
0.007 0.280 :I: 0.141
90 0.20 0.28 0.78 0.85
0.240 :t 0.057 0.815 :I: 0.049
120 0.96 0.96
2.25 2.32 0.960 :i: 0.000 2.285
:!: 0.049
30 0.12 0.18
0.17 0.11 0.150 :i: 0.042 0.140
:I: 0.042
60 0.27 0.24 0.30
0.25 0.255
+
0.021 0.275 :!: 0.035
90 0.38 0.43
0.45 0.44 0.405 :i: 0.035 0.445 :I:
0.007
120 0.60 0.70 0.60 0.71 0.650
:i: 0.071 0.655 :!: 0.078
30 0.12 0.14 0.20
0.11 0.130 :i: 0.014 0.155
:!: 0.064
60 0.18 0.21
0.35 0.26 0.195 :i: 0.021 0.305 :I: 0.064
90 0.28 0.36
0.57 0.53 0.320 :i: 0.057 0.550
:!: 0.028
120 0.36 0.63 1.01
0.98 0.495 :i: 0.191 0.995
:!: 0.021
rQuercetin':1 30
0.05 0.08 0.51 0.46
0.065
+
0.021 0.485 :!: 0.035
(.'1iQIiM)
60 0.24 0.37 1.55
1.65 0.305 :i: 0.092 1.600
:!: 0.071
;
, 90 0.70
1.04 3.14 3.60 0.870 z 0.240 3.370
:!: 0.325
120 1.16 1.89 5.04 5.55 1.525
:i: 0.516 5.295 :!: 0.361
..Quercetin:2!
30 0.08 0.11 -0.01
0.21 0.095
+
0.021 0.100 :!: 0.156
(C1Q.....M)
. 60 0.33 0.28 0.54
0.73 0.305 :i: 0.035 0.635 :!: 0.134
90 0.50 0.45 1.05
1.54 0.475 :i: 0.035 1.295 :!: 0.346
120 0.70 0.64 1.34
1.99 0.670 :i: 0.042 1.665 :I: 0.460
, Quercetin 3 30
0.05 0.07 0.40 0.42 0.060 :i: 0.014 0.410
:!: 0.014
(1;p
60 0.26 0.33 1.34
1.51 0.295
+
0.049 1.425 :!: 0.120
90 0.67 0.97 2.91 3.45 0.820 :i: 0.212 3.180
:!: 0.382
93
--
----
---
120 1.16
1.80 3.99
4.92 1.480 :!: 0.453
4.455
:!: 0.658
Qurcefin:4 30
0.06 0.08 0.37
0.46 0.070
:!: 0.014 0.415
:!: 0.064
'('Q,p)......
60 0.24
0.37 1.19
1.61 0.305 :!: 0.092
1.400
:!:
0.297
'<
r
90 0.69 1.03
2.30
3.48 0.860 :!: 0.240 2.890
:!: 0.834
<
,< 120
1.14 1.86 3.75
5.48 1.500 :!:
0.509 4.615
:!: 1.223
. Quercefin ..
30 0.09 0.07
0.16 0.09 0.080
:!: 0.014 0.125
:!:
0.049
. (2QipM)
60 0.13
0.16 0.40 0.46
0.145 :!: 0.021 0.430
:!: 0.042
90 0.28 0.29
0.57 0.60 0.285
:!: 0.007 0.585
:!: 0.021
120 0.45 0.41 0.91
0.97 0.430 :!: 0.028
0.940
:!: 0.042
Queroetin2 30
0.18 0.20
0.09 0.09 0.190
:!: 0.014 0.090
:!:
0.000
;(!OJ1'l '..';=
60 0.20 0.21 0.09
0.18 0.205
:!: 0.007 0.135
:!:
0.064
90 0.35
0.27 0.29 0.21 0.310
:!: 0.057 0.250
:!: 0.057
120 0.54
0.51 0.69 0.58
0.525 :!: 0.021 0.635
:!: 0.078
.Ql.leroefin;a ..
30 0.14 0.23 0.20
0.10 0.185
:!: 0.064 0.150 :t 0.071
(2OsPM)
60 0.28 0.29 0.63
0.31 0.285 :!: 0.007 0.470
:!: 0.226
i::= :::
90 0.53
0.52 1.24 0.63 0.525 :!:
0.007 0.935 :!: 0.431
120 1.08
1.01 2.62 1.34
1.045 :!: 0.049 1.980
:!:
0.905
Queroefin4 . 30 0.04
0.07 0.17
0.12 0.055 :!: 0.021 0.145
:!: 0.035
i (2Qp1Vn
60 0.32
0.48 0.52 0.16 0.400
:!: 0.113 0.340 :t 0.255
90 0.56 0.77
0.75 0.87 0.665
+
0.148 0.810
:!: 0.085
120 0.78 1.00 1.76
1.83 0.890
:!: 0.156 1.795 :t 0.049
APPENDIX B
94
--- --- - -
Table B.1: Example of the values and calculations done to determine the apparent
permeability coefficient (P8PP)'
Table B.2: Values used to obtain standard curve.
4.58
9.17
13.75
18.33
11
36
55
72
10
48
60
81
4.7855 -8.8333 0.999216
24
57
82
12
36
57
78
a)
Reference concentration =Concentration of Rh0123 in 7mL
=275~g x 1000 120ml x 2ml/7ml
=3928.57ng/mL
b) It is the transport corrected for dilution at one time interval divided by 28.
This value is then added to the value at the next time interval
Example: 38/28 + 56 = 57.35
The value of 28 is obtained by dividing the volume of the cell (7000 ~L) by the
volume of the replaced buffer (250 ~L) (7000 ~U250 ~L = 28)
95
3928.57 I
Cell 1
0 38 38 9.79 0.25
J 0.01830
1.61 1.71
30 93 94 21.56 0.55
90 197 200 43.71 1.11
120 319 326 69.98 1.78
Cell 2
30 21 21 6.23 0.16
J 0.01174
1.81
60 79 79 18.35 0.47
90 257 257 55.51 1.41
120 325 325 69.77 1.78
By using the standard curve generated for each experiment, the peak area is 
converted to a concentration by using the standard equation for a straight line 
(y = mx + c). 
Slope (m): 4.7855 
y-intercept (c): -8.8333 nglmL 
Thus x = (y-c)/m 
x = (38 - (-8.8333 nglmL)/4.7855 
x = 9.786 nglmL 
d) 
Value calculated by dividing the concentration at each time by the 100% 
concentration and expressing it as a percentage 
Example: 9.78613928.57 x I00 = 0.249 
e) 
Slope of line obtained by plotting relative transport against time. 
f) 
Calculated by the equation on page 54 given in the experimental procedure 
chapter 
Papp= 0.0183 
60x 1.78~ 100 
= 1.71 x lo-' cmls 
APPENDIX C 
Product Name 
Product Number 
Product Brand 
CAS Number 
Molecular Formula 
Molecular Weight 
TEST 
APPEARANCE 
SOLUBILITY 
IR SPECTRUM 
WATER BY KARL 
FISCHER 
CARBON * 
NITROGEN * 
PURIM BY HPLC 
SHELF LIFE SOP QC- 
12-006 
QC ACCEPTANCE 
DATE 
Rhodarnine 123, 
R8004 
SIGMA 
62669-70-9 
C~IHI~N~O~. HCI 
380.82 
SPECIFICATION 
RED TO BROWN POWDER 
LOT 013K37 
RESULTS 
RED-BROWN 
POWDER 
CLEAR TO SLIGHTLY HAZY YELLOW-ORANGE TO RED CLEAR YELLOW- 
SOLUTION AT lMG/ML IN ETHANOL ORANGE 
CONFORMS TO STRUCTURE CONFORMS 
NMT 10% 7.2% 
4 YEARS DECEMBER 2003 
JANUARY 2003 
Lori Schult, Manager 
Analytical Setvices 
St. Louis, Missouri USA 
Product Name 
Product Number 
Product Brand 
CAS Number 
Molecular Formula 
Molecular Weight 
TEST 
APPEARANCE 
SOLUBILITY 
WATER BY KARL FISCHER 
PH TEST 
PH TEST WITH NAHC03 
OSMOLALITY 
OSMOLALITY WITH NAHC03 
GLUCOSE 
ENDOTOXIN ASSAY 
KEY ELEMENTS BY ICP 
CELL CULTURE TEST 
CELL LINE 
EXPIRATION DATE 
QC ACCEPTANCE DATE 
Krebs-Ringer Bicarbonate Buffer, 
powder cell culture tested 
K4002 
SIGMA 
SPECIFICATION 
WHITE TO OFF-WHITE POWDER 
CLEAR SOLUTION AT 9.5 G/L IN WATER 
NMT 2.0% 
6.1 - 6.7 
7.0 - 7.6 
235 - 260 MOSM/KG H20 
253 - 280 MOSM/KG H20 
17.0 - 20.8% 
NMT 1.0 EU/ML AT IX 
CONSISTENT WITH FORM UMTION 
PASS 
24 MONTHS 
LOT 034K8309 RESULTS 
PASS 
PASS 
0.8% 
6.4 
7.4 
237 MOSM/KG H20 
265 MOSM/KG H20 
18.6% 
c0.05 EU/ML 
PASS 
PASS 
M2E6 
MARCH 2006 
APRIL 2004 
Lori Schulz, Manager 
Analytical Serbices 
St. Louis, tvlissouri USA 
Product Name 
Product Number 
Product Brand 
CAS Number 
Molecular Formula 
Molecular Weight 
Morin, 
powder 
M4008 
SIGMA 
480-16-0 
CisH1007 
302.24 
TEST 
APPEARANCE 
SOLUBILITY 
WATER BY KARL FISCHER 
UV-VIS SPECTRUM 
PURfTY BY HPLC 
QC ACCEPTANCE DATE 
PRODUCT CROSS REFERENCE INFORMATION 
LOT062K2504RESULTS 
BROWN POWDER 
RED SOLUTION AT 0.5 G PLUS 10 ML OF METHANOL 
7.1% 
El% = 417 AT LAMBDA MAX 360 NM IN METHANOL 
88.g0/o 
JUNE 2002 
REPLACEMENT FOR ALDRICH #PI87630 
Lori Schuls tvlanager 
Analytical S enice s 
St. Louis, Missouri USA 
GMA-ALDRICH 
Product Name 
Product Number 
Product Brand 
CAS Number 
Molecular Formula 
Molecular Weight 
Galangin, 
28,220-0 
ALDRICH 
548-83-4 
C15H1005 
270.24 
TEST 
QC Acceptance date 
APPEARANCE - COLOUR 
APPEARANCE - STATE 
HPLC - PURITY 
FT-IR SPECTROSCOPY - FTIR SPECTRUM 
MELTING POINT 
LOT SO8982 RESULTS 
23-NOV-200 1 
YELLOW . 
POWDER 
96.10°/0 
CONFORMS TO STRUCTURE 
220.5-223.6 DEG C 
Claudia P34ayer, Manager 
Quality Control 
Steinheim Germany 
Product Name 
Product Number 
Product Brand 
CAS Number 
Molecular Formula 
Molecular Weight 
TEST 
ASSAY (HPLC) 
APPEARANCE 
WATER 
INFRAREDSPECTRUM 
DATE OF QC-RELEASE 
Kaempferol, 
BioChemika 296% (HPLC) 
60010 
FLUKA 
520-18-3 
CisHio06 
286.24 
LOT 437695/1 RESULTS 
97.8 % REL 
LIGHT YELLOW POWDER 
6.1 % 
CORRESPONDS 
02/MAY/02 
Fluka guarantees the 'Sales-Specification' values only, non-specified tests may be included as additional 
information. The current 'Sales-Specifications' sheet is available on request. For further inquiries, please 
contact our Technical Service. Fluka warrants, that its products conform to the information contained in 
this and other Fluka publications. Purchaser must determine the suitability of the product for its particular 
use. See reverse side of invoice for additional terms and conditions of sale. The values given on the 
'Certificate of Analysis' are the results determined at the time of analysis. 
Dr. Gert van Look, Manager 
Quality Control 
Buchs Switzerland 
Product Name 
Product Number 
Product Brand 
CAS Number 
Molecular Formula 
Molecular Weight 
TEST 
APPEARANCE 
SOLUBILITY 
PURITY BY HPLC 
QC ACCEPTANCE 
DATE 
Quercetin dihydrate, 
r98O/0 (HPLC) powder 
Q0125 
SIGMA 
6151-25-3 
CI~HIOOT 2H20 
338.27 
SPECIFICATION 
LOT 039H0598 
RESULTS 
YELLOW TO YELLOW WITH A GREEN TO BROWN CAST ' 
POWDER 
POWDER 
DARK RED SOLUTION AT 200 MG PLUS 4 ML OF 1 M 
SODIUM HYDROXIDE 
CONFORMS 
NOT LESS THAN 98% 99.7% 
APRIL 1999 
Lnri Schulz, Manager 
Analytical S ewi ce s 
St. Luuis. t;dissouri USA 
............................... 
2.6 Drug substrates and inhibitors that interact with Pgp 
25 
................................................................... 
2.7 Substrate binding of Pgp 
28 
.................................... 
2.8 Structure-activity relationships of Pgp substrates 
29 
.................................................................................... 
2.9 Conclusion -30 
CHAPTER 3: THE POTENTIAL STRUCTURE ACTIVITY 
RELATIONSHIP OF FLAVONOIDS WITH REFERENCE TO THE 
INHIBITION OF P-GLYCOPROTEIN ........................................................ 33 
................................................................................... 3.1 Introduction -33 
........................................................................................... . 3.1 1 Flavonoids 33 
3.1.2 Basic flavonoid structure ........................................................................ 34 
........................................................................ 3.2 Flavonoids and MDR 36 
3.3 Structure-activity relationship between Pgp and flavonoids ....................... 37 
............................................................................. 3.4 Rhodamine 123 -41 
3.5 Conclusion ................................................................................... -42 
.......................................... CHAPTER 4: EXPERIMENTAL PROCEDURE 43 
................................................................................... 4 . I Introduction -43 
4.1 . 1 in vitro models of intestinal drug absorption .............................................. 43 
4.1.2 in vitro measurement of gastrointestinal tissue permeability using a 
....................................................................................... diffusion cell 44 
............................................................ 4.2 HPLC Analysis and validation 44 
............................................................................... 4.2.1 Method validation -45 
...................................................................................... 4.2.1.1 Linearity -45 
4.2.1.2 Precision ...................................................................................... 46 
..................................................................................... 4.2.1.3 Sensitivity 47 
.................................................................................... 4.2.1.4 Selectivity -47 
....................................................................... 4.2.1.5 System repeatability -47 
4.3 Instrument .................................................................................... -48 
........................................................................................... 4.3.1 Materials -49 
.............................................................................. 
4.3.2 Tissue preparation -49 
................................................. 
4.3.3 Procedures used during transport studies 
53 
............................................................. 
4.3.4 Apparent permeability coefficient 54 
.......................................................................... 
4.4 Statistical analysis -54 
CHAPTER 5: RESULTS AND DISCUSSION ............................................. 55 
5.1 Transport studies ........................................................................... -55 
........................................................................... 5.1 . 1 Transport of Rho 123 55 
5.1.2 Transport of Rho 123 in the presence of selected flavonoids ........................ 59 
5.1 .2.1 Transport of Rho 123 in the presence of morin ..................................... 61 
5.1.2.2 Transport of Rho 123 in the presence of galangin ................................. 64 
5.1 . 2.3 Transport of Rho 123 in the presence of kaempferol .............................. 67 
5.1 . 2.4 Transport of Rho 123 in the presence of quercetin ................................ 70 
5.2 Summary ...................................................................................... -73 
.......................... CHAPTER 6: CONCLUSION AND RECOMMENDATIONS 75 
6.1 Introduction .................................................................................. -75 
6.2 Conclusion ................................................................................... -75 
......................................................................... 6.3 Recommendations -77 
................................................................................................ BIBLIOGRAPHY 78 
...................................................................................................... APPENDIX A 89 
...................................................................................................... APPENDIX B 94 
...................................................................................................... APPENDIX C 97 
iii 
My gratitude to the following people: 
Unto my heavenly Father; Your everlasting love and mercy makes everything in my life 
possible and I praise You with all my heart and soul. 
My dear parents Stefan and Estelle van Huyssteen. Thank you for all your unconditional 
love, advice and support. I will always look up to you. You are everything that I am and 
I love you dearly. 
My sisters Andri and Este van Huyssteen. You were always a beam of sunshine to 
come home to after work. Thank you for all your love and support. 
To my future husband Stanley Dodd. Without you, my days would have been dark and 
difficult. Thank you for all your help, patience and love. I treasure it deeply. 
My dearest grandpartents Pine and Rensa Pienaar and Lettie van Huyssteen. Thank 
you for all your love and support. 
Mr. Kobus Swart, you were always someone to rely on and thank you for helping me 
with every aspect of this study. 
Dr. Maides Malan, I treasure all your support, leadership and help deeply within my 
heart. Thank you for taking over the project and in helping me to get onto my feet again. 
Prof. D.G. Muller, you will always be in my thoughts and I am privileged to know 
someone like you. 
Prof. A.W. Kotze, thank you for the use of the diffusion cells. 
Mrs. Antoinette Fick and Mr. Cor Bester. Thank you for always providing me with the 
rats for the experiments. 
Mrs. J.W. Breytenbach, thank you for your help with the statistical analysis of the data. 
To everyone at Kosmos, Secunda and President Pharmacies, thank you for all your 
caring and support. 
To all my friends and the friendly faces at the Department of Pharmaceutics, thank your 
for your friendship and support. 
LIST OF TABLES 
............................................. 
Table 2.1 : Location and function of Pgp (Delph. 2000) 
16 
.......................................................... 
Table 2.2. Pgp substrates (Delph. 2000) 
26 
Table 2.3. Pgp inhibitors (Delph. 2000) .................................................................... 27 
......................................................... Table 4.1 : Data obained for intra-day precision 46 
........................................................ Table 4.2. Data obtained for inter-day precision 46 
Table 5.1 : lndividual and mean Papp values of Rho 123 transported 
................................................ (AP-BLand BL-AP) with no modulators present 56 
Table 5.2: Multiple comparisons p-values (1-tailed) obtained by the 
non-parametric Kruskal-Wallis test by comparing the Pap ratio's 
in the presence of modulators with the Papp ratio obtained in the control 
experiment ........................................................................................................... 57 
Table 5.3: Descriptive statistics of modulators at concentrations 10 pM and 
20 pM .................................................................................................... 58 
.......................................................................... Table 5.4. Effect size results 59 
Table 5.5: Measured lipophilicity (Kg) and calculated dihedral angle between 
the 6- and C-ring for the following flavonoids tested 
(van Zanden et a/., 2004.705) .................................................................. 61 
Table 5.6: lndividual and mean Pap values of Rho 123 transported 
(AP-BL and BL- AP) in the presence of morin (10 pM and 20 pM) ................. 62 
Table 5.7: lndividual and mean Pap values of Rho 123 transported 
(AP-BL and BL-AP) in the presence of galangin (10 pM and 20 pM) ............... 65 
Table 5.8: lndividual and mean Pap, values of Rho 123 transported 
(AP-BL and BL-AP) in the presence of kaempferol (1 0 pM and 20 pM) ........... 68 
Table 5.9: lndividual and mean Pap values of Rho 123 transported 
(AP-BL and BL- AP) in the presence of quercetin (10 pM and 20 pM) ............. 71 
Table 5.1 0: Mean ratio's obtained for all the modulators examined ..................... 74 
LIST OF FIGURES 
Figure 2.1 : Schematic presentation of the intestinal mucosa 
....................................................................... 
(Trier & Madara. 1981 .926) 
6 
Figure 2.2: Diagram of pathways and mechanisms that mediate 
.......................................... transepithelial transport (Chan et a/.. 2004.26) -7 
Figure 2.3: Schematic representation of membrane topology of an ABC 
transporter (Sigma Aldrich. 2005) ........................................................... 13 
Figure 2.4: Schematic model of the human multidrug resistance gene 
product, Pgp and its functional domains (Sarkadi et al., 1996:216) ............. 14 
Figure 2.5. Flippase model for Pgp action (Borst & Schinkel. 1997:220) .............. 18 
Figure 2.6: Schematic diagram describing movement of MDR-type 
drugs in Pgp-overexpressing cells (Eytan & Kuchel. 1999: 180) .................. 19 
Figure 2.7: Postulated alternating catalytic sites cycle of ATP hydrolysis 
by P-glycoprotein (Senior et a/.. 1995.288) ............................................... 20 
Figure 2.8: Potential functional metabolism between enterocyte Pgp 
and CYP3A4 (Watkins. 1997: 166) ............................................................ 23 
Figure 2.9: A schematic presentation of the roles of ABC transporters 
and phase I and II enzymes in xenobiotic resistance (Bard. 2000:370) ........ 24 
Figure 2.10: The transport-competent drug binding sites of Pgp 
with distinct but overlapping specifities (Shapiro & Ling. 1998.227) ........... 29 
Figure 3.1: (a) Flavan nucleus and (b) 4-0x0-flavonoid nucleus 
(Aherne & O'Brien. 2002.75) .................................................................. 35 
Figure 3.2. Structure of the basic flavonoids (Aherne & O'Brien. 2002:76) ........... 36 
Figure 3.3: Assumed binding of luteolin (I) and luteolin-7-0-glucoside (2) 
..................... in the binding pocket of the NBD2 (Nissler eta/.. 2004:1048) 40 
Figure 3.4. Flavones that will be evaluated during this study ................................... 41 
Figure 5.1: Example of the cumulative transport of Rho 123 with no 
modulators added ............................................................................... -56 
Figure 5.2: Example of the cumulative transport of Rho 123 in the 
..................................................................... presence of morin (10 pM) 62 
Figure 5.3: Example of the cumulative transport of Rho 123 in the 
..................................................................... presence of morin (20 pM) 63 
........... Figure 5.4. Chemical structure of morin (2'.3.4'.5. 7-pentahydroxyflavone) 64 
Figure 5.5: Example of the cumulative transport of Rho 123 in the 
.................................................................. presence of galangin (1 0 pM) 65 
Figure 5.6: Example of the cumulative transport of Rho 123 in the 
presence of galangin (20 pM) .................................................................. 66 
Figure 5.7. Chemical structure of galangin (3.5.7.trihydroxyflavone) .................. 67 
Figure 5.8: Example of the cumulative transport of Rho 123 in the 
presence of kaempferol (1 0 pM) .............................................................. 68 
Figure 5.9: Example of the cumulative transport of Rho 123 in the 
presence of kaempferol (20 pM) .............................................................. 69 
Figure 5.10. Chemical structure of kaempferol (3.4.5.7-tetrahydroxyflavone) ...... 70 
Figure 5.11: Example of the cumulative transport of Rho 123 in the 
presence of quercetin (1 0 pM) ............................................................... -71 
Figure 5.12: Example of the cumulative transport of Rho 123 in the 
presence of quercetin (20 pM) ................................................................ 72 
Figure 5.1 3: Chemical structure of quercetin (3.5.7.3'.4'-pentahydroxyflavone) .... 73 
Figure 5.14: Bar chart showing the relative mean ratio's of the 
various compounds compared to the mean ratio of the control .................. 74 
Background: 
Multidrug resistance (MDR) is resistance of cancer cells to multiple 
classes of chemotherapeutic drugs that can be structurally unrelated. MDR involves 
altered membrane transport that results in a lower cell concentration of cytotoxic drugs 
which plays an important role during cancer treatment. P-glycoprotein (Pgp) is localised 
at the apical surface of epithelial cell in the intestine and it functions as a biological 
barrier by extruding toxic substances and xenobiotics out of cells (Lin, 2003:54). The 
ATP-binding-cassette superfamily is a rapidly growing group of membrane transport 
proteins and are involved in diverse physiological processes which include antigen 
presentation, drug efflux from cancer cells, bacterial nutrient uptake and cystic fibrosis 
(Germann, 1996:928; Kerr, 2002:47). A number of drugs have been identified which are 
able to reverse the effects of Pgp, multidrug resistance protein (MRPI) and their 
associated proteins on multidrug resistance. The first MDR modulators discovered and 
studied during clinical trials were associated with definite pharmacological actions, but 
the doses required to overcome MDR were associated with the occurrence of 
unacceptable side effects. As a consequence, more attention has been given to the 
development of modulators with proper potency, selectivity and pharmacokinetic 
characteristics that it can be used at a lower dose. Several novel MDR reversing agents 
(also known as chemosensitisers) are currently undergoing clinical evaluation for the 
treatment of resistant tumors (Teodori et a/., 2002:385). Aim: The aim of this study was 
to investigate the effect of selected flavonoids (morin, galangi n, kaem pferol and 
quercetin) at two different concentrations (10 pM and 20 pM) on the transport of a known 
Pgp substrate, Rhodamine 123 (Rho 123) across rat intestine (jejunum) and to 
investigate structure activity relationships (SAR) of the selected flavonoids with 
reference to the inhibition of Pgp. Methods: Morin, galangin, kaempferol and quercetin 
were evaluated as potential modulators of Rho 123 transport, each at a concentration of 
10 pM and 20 pM across rat jejunum using Sweetana-Grass diffusion cells. This study 
was done bidirectionally, with two cells measuring transport in the apical to basolateral 
direction (AP-BL) and two cells measuring transport in the basolateral to apical direction 
(BL-AP). The rate of transport was expressed as the apparent permeability coefficient 
(Pap,) and the extent of active transport was expressed by calculating the ratio of BL-AP 
to AP-BL. Results: The BL-AP to AP-BL ratio calculated for Rho 123 with no 
modulators added was 3.29. Morin decreased the BL-AP to AP-BL ratio to I .88 at a 
concentration of 10 pM and to 1.49 at a concentration of 20 pM. Galangin decreased 
the BL-AP to AP-BL ratio to 1.60 at a concentration of 20 pM. These two flavonoids 
showed statistically significant results and inhibition of active transport were clearly 
demonstrated. However, the other flavonoids inhibited active transport of Rho 123 but 
according to statistical analysis, the results were not significantly different. The two 
different concentrations (10 pM and 20 pM) indicated that galangin, kaempferol and 
quercetin showed practically significant differences according to the effect sizes. Morin, 
however, did not show any practically significant differences at the different 
concentrations. Regarding .the SAR, it was shown by Boumendjel and co-workers 
(2002:512) that the presence of a 5-hydroxyl group and a 3-hydroxyl group as well as 
the C2-C3 double bond are required for high potency binding to the nucleotide binding 
domain (NBD) of Pgp. All the flavonoids tested had the above-mentioned 
characteristics. Conclusion: All the selected flavonoids showed inhibition of active 
transport of Rho 123 and should have an effect on the bioavailability of the substrates of 
Pgp and other active transporters. This study described the inhibitory interaction of 
selected flavonoids on Pgp activity. Practical significant differences between the same 
modulator at different concentrations were also observed. Structure activity 
relationships were identified describing the inhibitory potency of the flavonoids based on 
hydroxyl group positioning. 
Xeywords: Pgty~~otetni Rhodaminel2& rnerin, galangin, kaempteml, querctin, 
structure activity relationship, Sweetana-Grass diffusion cells 
UITTREKSEL 
Agtergrond: Multi-geneesrniddel resistensie (MGR) is die weerstandigheid van 
kankerselle teen verskeie klasse struktureel onvetwante chemoterapeutiese 
geneesrniddels. MGR behels die veranderde rnembraan transport wat lei tot 'n laer 
sellul6re konsentrasie sitotoksiese geneesmiddels en speel dus 'n belangrike rot tydens 
kankerbehandeling. P-glikoprotei'en (Pgp) is gelokaliseerd in die apikale oppervlakte 
van die epiteelselle in die dunderm en funksioneer as 'n biologiese skans deur toksiese 
bestandele en xenobiotika uit selle te vewyder (Lin, 2003:54). Pgp is 'n groep transport 
protei'ene wat vinnig uitbrei en is betrokke in diverse fisiologiese prosesse wat insluit 
antigeen uitdrukking , geneesrniddel effluks vanuit kankerselle, bakteriele nutrient 
opnarne en sistiese fibrose (Gemann, l996:928; Kerr, 2002:47). 'n Aantal 
geneesmiddels is reeds gei'dentifiseer wat die verrnoe het om die effekte van Pgp, 
rnultigeneesrniddel resistente proteiien (MRPI) en hul verwante prote'iene tydens MGR 
om te keer. Die eerste MGR rnoduleerders wat tydens kliniese toetse ontdek en 
bestudeer is het definitiewe farrnakologiese eienskappe besit, in so 'n mate dat die 
dosisse wat nodig was om MGR om te keer, geassosieer was met 'n onaanvaarbare hoe 
voorkoms van newe-effekte. Dit het daartoe aanleiding gegee dat rneer aandag 
geskenk was aan die ontwikkeling van moduleerders, met 'n aanvaarbare sterkte, 
selektiwiteit en farmakokinetiese eienskappe, wat gebruik kan word by laer dosisse om 
MGR te verhoed. Verskeie bestaande MGR rnoduleerders (ook bekend as 
chemosensitiseerders), word tans klinies geevalueer vir die behandeling van resistente 
turnore (Teodori ef a/., 2002:385). Doel: Die doel van die studie was om die effek van 
geselekteerde flavenoi'ede (morien, galagnien, kaernpferol en kwersetien) op die 
---- ---- 
----- ---- 
- - - - 
transport van 'n bekende Pgp substraat, ~hodamien 123 (Rho1 23J oorPr6t Jejenum te 
ondersoek en om die struktuuraktiwiteitsvetwantskappe (SAR) van die geselekteerde 
flavenoi'ede vas te stel met verwysing na die inhibisie van Pgp. Metode: Morien, 
galagnien, kaernpferol en kwersetien was bestudeer as potensiele rnoduleerders van 
Rho 123 transport, elk by 'n konsentrasie van 10 pM en 20 pM oor rot jejenurn deur 
middel van Sweetana-Grass diffusie karners. Hierdie studie was bidireksioneel 
uitgevoer, met twee selle wat die transport in die apikaal na basolaterale (AP-BL) rigting 
en Wee selle wat die transport in die basolateraal na apikale (BL-AP) rigting gerneet het. 
Die tempo van transport was uitgedruk as die waarneembare penneabiliteits koeffisient 
(Pa,) en die mate van die aktiewe transport was uitgedruk deur die verhouding van BL- 
AP tot AP-BL te bereken. Resultate: Die BL-AP tot AP-BL verhouding wat bereken 
was vir Rho 123 met geen moduleerden teenwoordig nie, was 3.29. Morien het die BL- 
AP tot AP-BL verhouding venninder na 1.88 by 'n konsentrasie van 10 pM en na 1.49 
veninder by 'n konsentrasie van 20 pM. Galagnien het die BL-AP tot AP-BL 
verhouding na 1.60 verminder by 'n konsentrasie van 20 pM. Hierdie twee flavenoi'ede 
se verlaging in die verhouding was statisties betekenisvol en inhibisie van aktiewe 
transport was aangetoon. Alhoewel die ander flavenoi'ede aktiewe transport van 
Rho 123 gei'nhibeer het, was dit volgens die statistiese analise nie statisties betekenisvol 
nie. Die effek tussen die verskillende konsentrasies (10 pM en 20 pM) het aangetoon 
dat galagnien, kaempferol en kwersetien prakties betekenisvolle verskille volgens die 
berekende effekgroottes getoon het. Morien het egter geen prakties betekenisvolle 
verskille by die verskillende konsentrasies getoon nie. Wat SAR betref, was dit 
voorheen aangedui deur Boumendjel en mede-werkers (2002:512) dat die aanwesigheid 
van 'n 5-hidroksielgroep en 'n 3-hidroksielgroep sowel as 'n C2-C3 dubbel binding nodig 
is vir sterk binding van die flavenoied aan die nukleotied-bindings area (NBD) van Pgp. 
Al die flavenoi'ede wat ondersoek was het hierdie karakteristieke getoon. 
Gevolgtrekking: Die geselekteerde flavenoi'ede het almal inhibisie van aktiewe 
transport van Rho 123 getoon en behoort 'n effek te he op die biobeskikbaarheid van 
Pgp substrate en ander aktiewe transporteerders. Hierdie studie beskryf die inhibisie 
van geselekteerde flavenoi'ede op Pgp aktiwiteit. Praktiese betekenisvolle verskille 
tussen dieselfde moduleerder by verskillende konsentrasies is ook waargeneem. SAR 
is ook geidentifiseer wat die sterkte van inhibisie van flavenoi'ede aandui, gebasseer op 
die posisievandk hidroksietgmqx- - - - - - - - - - - - - - - - - - - - - - - - - 
Sleutelwoorde: P-glikoproteh, Rhodamien 123, morien, galagnien, kaempferol, 
kwersetien, stnrktuuraktiwiteitsvenvantskappe, Sweetana-Grass diffusie kamers 
xii