9

Chapter 1

________________________________

Introduction

Abstract

This chapter presents an introduction to the issues of platinum antitumor chemistry and

biochemistry, which are discussed in this thesis. The thesis deals with investigation of

antitumor-active dinuclear platinum complexes. It includes the design of new dinuclear

platinum antitumor drugs, study of intracellular distribution of dinuclear platinum anticancer

complexes, investigation of their mechanism of action on cellular level, and evaluation of the

toxicity of dinuclear platinum drugs. Besides, application of dinuclear platinum complexes in

drug targeting is described.

This chapter starts with the history of cisplatin, the first platinum drug, which is widely

used for therapeutic purposes nowadays. Subsequently, the advantages and disadvantages of

cisplatin are discussed. An overview of other clinically approved platinum drugs and

potential platinum anticancer agents is presented, with special attention given to polynuclear

platinum complexes. Mechanism of action of platinum drugs on molecular and cellular level

is described in detail. Nature of cisplatin resistance is discussed. Mechanisms of cisplatin

nephrotoxicity are reviewed. The concept of drug targeting to the therapeutic site is

introduced, and an overview of the strategies used for targeting platinum drugs is presented.

Finally, the content of this thesis is briefly described.

10

1.1. General introduction

Cis-diamminedichloroplatinum(II) (1), usually referred to as cisplatin, was first

described in the chemistry literature in 1845.1 Accidental discovery of a cisplatin effect on

cell growth and division by Rosenberg in the 1960s,2 gave this complex a new important

meaning.

Pt

H3N

H3N

Cl

Cl

1

Figure 1.1. Structural formula of cisplatin (1).

Rosenberg investigated the influence of an electric field on growth and division of

E.coli. He observed filamentous growth of the cells, which normally occurs when DNA

replication is blocked. This striking effect appeared not to result from a direct action of the

electric field on bacterial cells but rather to occur due to the products of electrolysis of the

platinum electrodes used in the experiment. Rosenberg’s co-workers clearly identified

cisplatin as a compound responsible for the anti-proliferative effect.3 Subsequently, a series

of successful experiments with cancer cells were performed, and cisplatin developed into one

of the most widely used antitumor drugs.4 Nowadays it is a standard treatment of testicular,

ovarian, head and neck, esophageal, and small-cell lung cancer.5-8

Cure rates are especially

high for testicular and ovarian cancer9,10

and exceed 90% if tumors are diagnosed in an early

state.7,11

Cisplatin is also used in combination with other antitumor drugs, 5-fluorouracil12

and arabinofuranosylcytosine.13

These drugs reduce repair of cisplatin-DNA lesions, thereby

increasing the activity of cisplatin.

Nevertheless, cisplatin has some serious disadvantages. The toxic side effects, e.g.

nausea, vomiting, nephro-, neuro- and ototoxicity, limit the dose that can be given to

patients.14,15

A recent study with high-dose cisplatin treatment in solid tumors showed that

42% of the treated patients suffered from nephrotoxic injury.16

To reduce nephrotoxicity,

intravenous hydration and diuresis have been applied.6,17

The use of serotonin receptor

antagonists has helped to overcome nausea and vomiting.18,19

A number of so-called

‘protective agents’ (given before cisplatin treatment) and ‘rescue agents’ (administered after

treatment), such as mesna, amifostine (W R-2721), diethyldithiocarbamate, and thiosulfate

have been used to control cisplatin toxicity.20-22

Besides, cisplatin has limited solubility in

aqueous solution and is administered intravenously generating another inconvenience for

clinical use. In fact, its solubility (~ 1mg/mL) approaches the practical limit of solubility for

a cytotoxic agent that is administered parenterally.23

The major problem encountered in the

clinic is resistance of tumor cells to cisplatin treatment.24-27

Some tumors have intrinsic

resistance to cisplatin, and other tumors develop resistance during the chemotherapy. The

11

issues of cisplatin toxicity and cisplatin resistance will be further addressed in Sections 1.4

and 1.5, respectively.

The disadvantages of cisplatin described above have stimulated an intensive

investigation of its mechanism of action and an intensive search for new potential anticancer

agents.

1.2. Cisplatin: mechanism of action

In the clinic, cisplatin is administered intravenously, and blood transports the complex

throughout the body. In the blood stream, cisplatin is maintained in a neutral state by a

relatively high concentration of chloride ions (≈100 mM). Evidence of cisplatin binding to

cysteine and methionine residues of albumin during transport has been reported.28

Several

studies also suggested that albumin-bound cisplatin has antitumor activity.29

Cisplatin is

believed to enter the cells mainly by passive diffusion.8,30

However, there is accumulating

evidence of involvement of active transport in cisplatin uptake.31-33

The mechanism by which

cisplatin is taken up by the cells is not well elucidated. This issue is discussed in some more

detail in Section 1.3.

Within the cell, where the chloride concentration is much lower (between 2 and

30 mM), cisplatin readily hydrolyses yielding monoaqua ([Pt(NH3)2Cl(H2O)]+) and diaqua

species ([Pt(NH3)2(H2O)2]2+

).34

These aqua species are very reactive towards cellular

nucleophiles because a water molecule is a much better leaving group than Cl-.35

Many potential targets are available for reactive platinum species inside the cells.

Cisplatin may bind to phospholipids36

and form a coordination complex with

phosphatidylserine,37,38

located in the cell membrane. In the cytosol and in the nucleus, DNA,

RNA, proteins, peptides, etc. are available for cisplatin binding. Small ions and molecules

(e. g. Cl-, HPO4

2-) can also react with the drug. The first clue for identifying the ultimate

cellular target of cisplatin was the filamentous growth observed in the early experiments by

Rosenberg.2 This phenomenon is characteristic of DNA-damaging agents like, for instance,

UV radiation.39

Subsequent experiments more clearly indicated that cisplatin binding to

genomic DNA is largely responsible for its antitumor acivity.30,40,41

However, a high degree

of mitochondrial DNA platination has been reported,30,42-44

and damage of mitochondrial

DNA might also be involved in activation of cytotoxic pathways. Furthermore, only 5 to

10% of intracellular cisplatin is bound to DNA, while 75-85% of the drug reacts with

proteins abundantly present in the cytoplasm.40,45

Cisplatin binding may affect the activity of

receptors, enzymes, and other proteins, and the resulting protein damage may contribute to

cytotoxicity. One of the most important targets of cisplatin in cytoplasm is the tripeptide

glutathione (GSH). Glutathione is a tripeptide of glutamate (Glu), cysteine (Cys) and glycine

(Gly) that contains an unusual γ-peptide bond between glutamate and cysteine (γ-GluCysGly,

2). This bond prevents GSH from being hydrolyzed by most peptidases. Intracellularly,

12

glutathione is kept in its thiol form by glutathione disulfide reductase. Ready interaction of

the drug with GSH is a result of a relatively high intracellular concentration of the tripeptide

(0.5 – 10 mM)24

and high affinity of platinum for sulfur. Binding of cisplatin to glutathione

and other thiol-containing peptides and proteins (e.g. metallothionein) is thought to be

disadvantageous for the anticancer activity of the drug and is associated with toxicity and

resistance (see also Sections 1.4 and 1.5).26

On the other hand, it has been suggested that

cisplatin bound to glutathione may act as a drug reservoir, thereby modulating the kinetics of

DNA platination.21

Figure 1.2. Structural formula of glutathione (2).

Thus, cisplatin ultimately reaches the DNA of the genome. Although all DNA

nucleobases, adenine (A), guanine (G), thymine (T) and cytosine (C) (Figure 1.3), have

potential sites for platinum coordination, the N7 nitrogen atoms of guanine and adenine are

the most preferred under physiological conditions.46

Other potential binding positions are

either protonated, or involved in DNA base pairing at physiological pH (7.2 – 7.4).

HH

OH

H HO

PO

O-

HHH

H HO

O

O

PO

O-

HHH

H HO

O

O

PO

O-

HH

OH H

H HO

O

HO

N

HN

O

O

N

N

N

N

NH2

N

N

N

NH

O

NH2

N

N

NH2

O

1'2'3'

4'

5'

1

1

1

1

3

3

3

3

5

6

6

7

7

9

9

= possible platinum binding site

= possible platinum binding site

after loss of H+

C

G

A

T

Figure 1.3. Potential binding sites of cisplatin on DNA nucleobases.

-O

HN

O

NH3+

OSH

NH

OH

O

O

2

13

Initially, monofunctional adducts are formed, but most of them react further to form

intra- and interstrand crosslinks, which block replication and prevent transcription of

DNA.47,48

The major products are 1,2-intrastrand d(GG) crosslinks comprising 60-65% of all

the adducts formed and 1,2-intrastrand d(AG) crosslinks comprising another 20-25% (Figure

1.4). Minor adducts include 1,3-intrastrand and interstrand crosslinks.30,46

DNA-protein

crosslink formation has also been reported.49,50

Figure 1.4. Schematic representation of cisplatin-DNA adducts.

Thus, 1,2-intrastrand d(GG) crosslinks predominate among the products of cisplatin

interaction with genomic DNA. Therefore, they are believed to be responsible for the

cytotoxic effect of the drug. This hypothesis has not been absolutely proven but is supported

by various findings. For example, the inactive isomer of cisplatin, transplatin is unable to

form 1,2-d(GG) crosslinks, for steric reasons. Transplatin mainly produces 1,3-intrastrand

and interstrand crosslinks.51

Furthermore, 1,2-intrastrand adducts are less effectively repaired

by nucleotide excision repair (NER), an important DNA repair system.52,53

High-mobility

group (HMG) proteins have been found to specifically recognize this type of crosslinks, and

it has been suggested that this event is a first step toward the induction of apoptosis.30,42

However, the importance of other adducts in cisplatin cytotoxicity should not be excluded.

The formation of cisplatin-DNA crosslinks structurally distorts the double helix. In

1,2-d(GG) intrastrand adducts, cisplatin induces a kink on DNA of 40-70º towards the major

groove (Figure 1.5).54-56

DNA platination also causes partial unwinding of the double helix.

Other types of adducts are also structurally distorted. In the 1,3-d(GTG) adduct, DNA is bent

27-33º towards the major groove,57

and interstrand crosslinks result in bending of the double

helix towards the minor groove (20-40°) and a high degree of DNA unwinding (≈80°).58

The DNA damage induced by cisplatin affects essential cellular processes. Several

proteins are known to recognize cisplatin-DNA adducts. As mentioned above, HMG-box

14

proteins (e.g. HMG1 and HMG2) selectively bind to the DNA lesions structurally distorted

by 1,2-d(GG) crosslinks (Figure 1.6).30,59

Figure 1.5. A “ribbon” representation of the NMR solution structure of

d(CCTG*G*TCC)⋅d(GGACCAGG) containing 1,2-intrastrand d(GG) cross-link.54

Figure 1.6. Structure of domain A of HMG1 protein bound to a cisplatin 1,2-d(GG)

intrastrand adduct.60

Two mechanisms have been proposed to explain how HMG proteins might regulate

cisplatin cytotoxicity. One of them, so-called “repair shielding model”, suggests that HMG

proteins protect cisplatin-DNA crosslinks from recognition by the DNA repair system of the

cell.61

The other one, “hijacking model”, postulates that HMG binding changes cell cycle

events, which eventually results in triggering apoptosis.62

It is generally accepted that futile attempts to repair DNA damage finally result in

apoptosis.42,63

Apoptosis, or programmed cell death, is a normal component of the

development and health of multicellular organisms. Cells die in response to a variety of

stimuli and during apoptosis they do so in a controlled, regulated fashion. Apoptosis is a

15

genetically regulated mechanism that takes place during embryonic development, normal

cellular homeostasis, and spontaneous or drug-induced cell death.64

It requires energy from

ATP hydrolysis and de novo protein synthesis. There is evidence that protein damage might

play an important role in triggering apoptotic pathways.65

The apoptotic process is divided in

three stages.42

In the initiation phase, the stimulus is received and one of several possible

responses is initiated. In the effector phase, all signals are integrated, and the decision to live

or die is made. In the last stage, a common irreversible execution phase, proteins autodigest,

and DNA is cleaved by endonuclease. A distinct feature of the execution phase is specific

degradation of a series of proteins by the cysteine aspartate-specific proteinases (caspases).42

Cisplatin-induced damage induces release of reactive oxygen species and Ca2+

, which are

known to disrupt mitochondria.66

These processes subsequently result in release of

cytochrome c from mitochondria, which activates the caspases,67

finally leading to the

formation of so-called apoptotic bodies.

Besides, necrosis, cell death due to general cell machinery failure through undefined

pathways, has also been reported in several cell lines.26,68

Moreover, apoptosis and necrosis

might take place together in the same cell population.69

In fact, both mechanisms of cell

death are linked, and intracellular ATP levels appear to determine whether the cell will die by

apoptosis or necrosis.70,71

1.3. Cellular uptake and distribution of platinum antitumor drugs

As mentioned in Section 1.2, passive diffusion is thought to be the main mechanism of

cisplatin uptake. Indeed, cisplatin uptake is proportional to its concentration, is not saturable

and is not inhibited by structural analogs.31,72

However, several recent findings strongly

suggest that a certain degree of cisplatin uptake is energy dependent.31-33,73,74

A number of

pharmacological agents that do not alter membrane permeability, like Na+/K

+-ATPase

inhibitors and membrane-interactive drugs, inhibit cisplatin uptake.31,33,75

Given that, the

involvement of a gated ion channel has been proposed by Andrews and Albright.76

They

have also shown that cisplatin accumulation is mediated by Na+/K

+-ATPase.

77 Inhibition of

cisplatin uptake by copper has recently been reported and led to intensive investigations of

the role of copper transport proteins in accumulation of the drug.78

The copper transporter

CTR1 has been found to regulate cisplatin uptake in some models.32,79

Accumulation of

cisplatin in non-proliferating (healthy) cells has also been investigated in order to elucidate

the mechanism of toxicity. Several studies have shown that the organic cation transporter

(OCT) mediates cisplatin uptake73

and cisplatin toxicity80,81

in kidney epithelial cells (see

also Section 1.4).

Cellular distribution of cisplatin and other platinum-based anticancer drugs has not

been thoroughly investigated. Various methods, such as fluorescence microscopy,82-84

electron microscopy,44,85,86

synchrotron radiation-induced X-ray emission87

and X-ray

16

microanalysis,88,89

have been applied to study subcellular localization of platinum antitumor

drugs. As fluorescence microscopy has been widely used in the studies presented in this

thesis, it deserves a more detailed description. Fluorescence is defined as the emission of

light by molecules, so-called fluorophores, which results from the absorption of energy by

these molecules. Absorption of a photon leads to the transition of an electron to a state with

higher energy (excited state). The electron transition back to the ground state is accompanied

by release of energy in the form of light. Thus, fluorescent molecules (fluorophores) can be

detected inside cells by fluorescence microscopy. This enables investigation of static and

dynamic subcellular localization of compounds in cancer cells. A great advantage of this

method is that the cells can be observed in living state. Unfortunately, most of the platinum-

based drugs are not intrinsically fluorescent. Development of new antitumor platinum

complexes with fluorescent ligands has proved to be a successful approach to combine drug

design and investigation of cellular distribution of platinum-based anticancer compounds.84

Otherwise, established platinum antitumor drugs can be modified with a fluorophore tag, and

the labeled molecules can be introduced in living cells, so that they could mimic the behavior

of the parent label-free compounds. This approach has also been successfully applied.83

Cellular distribution and processing of platinum-based anticancer drugs, as described in

the literature, vary significantly between different tumor types. Most of the studies report

accumulation of platinum complexes in the nucleus.83,85-88

Digital fluorescence microscopy

investigations have shown that platinum anticancer drugs localize in the nucleus within an

hour.83,84

In the nucleus, the drugs mainly target nucleoli, which contain highly concentrated

double-stranded DNA.84

Platinum-DNA adducts are recognized by certain proteins and

subsequently processed by the cellular machinery.30,52,59

Some damage-recognition proteins

(e.g. NER proteins) perform DNA repair by excision of cisplatin-DNA lesions.52,53

The

excised DNA lesions may be excreted from the nucleus to the cytoplasm for being

exocytosed. This hypothesis is supported by evidence that platinum concentration in the

nucleus decreases with time.83

However, other studies have shown irreversible accumulation

of platinum drugs in the nucleus, indicating that a fraction of the drug remains bound to

nuclear DNA.85,87

Platination of mitochondrial DNA has also been reported.43,44,90,91

Some in vitro

experiments on ovarian and melanoma cells as well as in vivo studies revealed higher

incorporation of cisplatin into mitochondrial DNA than into genomic DNA.43,90,91

However,

a higher degree of mitochondrial DNA platination may partly result from the following

features of this type of DNA. Mitochondria contain double-stranded circular DNA, which

has no histones, and as a result lacks nucleosomal organization.92

Therefore, mitochondrial

DNA is more easily accessible for DNA-damaging agents than genomic DNA. Moreover,

mitochondria are not so efficient in DNA repair, as they do not perform nucleotide excision

repair.93

The impact of mitochondrial DNA platination on cell viability is not well elucidated.

17

Mitochondria are known to be involved in apoptotic processes.66,94

Thus, binding of

platinum-based anticancer drugs to mitochondrial DNA may play a role in induction of

cytotoxic effects.

As mentioned in Section 1.2, a major fraction of cisplatin binds to peptides and proteins

and is excreted by the cell efflux system. The Golgi complex83

and lysosomes84,89,95,96

were

found to be involved in transport of platinum drugs out of the cell. As will be suggested in

Chapter 4, in some cell lines cisplatin-protein conjugates and excised cisplatin-DNA lesions

may be excreted as a part of a general mechanism of protein recycling, by which secretory

proteins are transported via the Golgi complex to the outer cell surface. The role of

lysosomes in exocytosis of platinum drugs is not well elucidated. It is not clear, for example,

whether the drugs are degraded in lysosomes or only stored.

1.4. Mechanisms of cisplatin nephrotoxicity

As mentioned in Section 1.1, clinical use of cisplatin is limited due to acute and chronic

renal toxicity. Cisplatin and other platinum complexes exert their nephrotoxicity mainly in

the proximal tubule of the kidney.97,98

Kidney tubules are formed by epithelial cells.

Epithelium is one of the four basic tissue types present in the body. It separates the interior of

the body and various body organs from the external world, and it is responsible for the

transport of water and mineral salts. Epithelium forms secretory cavities of the body: glands,

intestine, urine vessels and kidney tubules.99

Epithelial cells possess distinct structural

features, which enable them to perform their protective and transport function. These cells

have a polar structure: the cell membrane exposed to the lumen (apical membrane) is

distinguished from the cell membrane oriented towards the blood side (basolateral

membrane). The solutes are absorbed through the epithelial layer from the apical to the

basolateral side, and their secretion occurs in the opposite direction. Epithelial cells are

connected to each other by means of so-called tight junctions, narrow bands close to the

apical surface. Tight junctions ensure that materials pass the epithelial layer by actually

entering the cells, and not through the space between cells. This pathway provides control

over the transport of substances. Tight junctions also prevent the movement of membrane

proteins between the apical and basolateral surfaces of the cell. Thus, the special functions of

each surface, for example, receptor-mediated endocytosis at the apical side and exocytosis at

the basolateral side, can be preserved.

As mentioned above, the major site of renal injury is the proximal tubule of the

kidney. Cisplatin concentrations in proximal tubular epithelial cells are approximately five

times higher than plasma concentrations. This is at least in part due to active uptake via an

energy-dependent process, which involves organic cation transporters (OCT).73,100

These

proteins are located in the basolateral, but not in the apical membrane of renal epithelial

cells.101 In vitro toxicity studies have shown that the nephrotoxic potential of platinum

18

complexes is higher when the compounds are added to the basolateral medium.80,81

Thus,

membrane transport proteins are very likely to partly mediate the nephrotoxicity of platinum-

based drugs.

Inside renal epithelial cells, cisplatin has been reported to localize in cytosol,

mitochondria, nuclei and lysosomes.102

The low concentration of chloride ions in the

cytoplasm facilitates cisplatin hydrolysis, which yields much more reactive and, therefore,

more toxic, aquated species. The toxic effects of cisplatin are usually divided into primary

effects, which promote cellular damage, and secondary effects, which are considered the

consequences of established cell damage. The former include inhibition of protein synthesis,

DNA damage, glutathione depletion and disruption of lysosomal function, the latter ones are

lipid peroxidation, mitochondrial damage and subsequent activation of apoptotic or necrotic

pathways.102

Inhibition of protein synthesis is the first cellular reaction to the toxic platinum species.

The detailed mechanism is not clearly understood. It has been suggested that disruption of

the nucleolus, where ribosome biosynthesis takes place, results in decrease in ribosome

number and ultimately in inhibition of protein synthesis.103

Another study has shown that

cisplatin interferes with joining of two ribosomal subunits, thereby preventing translation.104

Platinum aqua species readily react with nuclear DNA of epithelial cells, as well as they

interact with the DNA in cancer cells.105

Binding of platinum complexes to the nucleobases

blocks DNA transcription and may subsequently lead to the cell death.

Inhibition and gradual loss of lysosomal function has been observed in renal epithelial

cells following cisplatin exposure.103

Platinum complexes have been reported to inhibit

protein degradation in lysosomes. It has been proposed that platinum-induced toxicity may

result in failure of lysosomal acidification, which subsequently affects lysosomal activity.103

The tripeptide glutathione functions as a cellular oxidant defense system, and it is

responsible for control of lipid peroxidation in cells. Inside the cell, glutathione is

concentrated in cytosol (30%) and in mitochondria (70%).102

Mitochondrial GSH appears to

be important for the regulation of inner membrane permeability. High affinity of platinum for

sulfur facilitates rapid interaction of cisplatin with the SH group of glutathione. Although

cisplatin binding to GSH initially appears as a detoxification process, it finally results in a

series of biochemical events, which lead to the cell damage. Important consequences of

glutathione interaction with cisplatin are dysfunction of membrane associated and

cytoplasmic proteins (e.g. Na+/phosphate and Na

+/glucose cotransporters)

106 and inactivation

of important enzyme systems (e.g. glutathione-S transferase and peroxidase).107

Furthermore,

platinum binding to glutathione decreases the amount of GSH available for scavenging free

oxygen radicals.108,109

Abundance of the latter inside the cell may lead to lipid peroxidation

and subsequently induce oxidative stress in mitochondria. Reactive oxygen species, such as

hydrogen peroxide, superoxide anion and hydroxyl radicals, play an important role in

19

cisplatin toxicity. They are normally generated in kidney cells and immediately scavenged by

intracellular antioxidants (glutathione, catalase and superoxide dismutase).102

GSH depletion

by cisplatin allows intracellular accumulation of reactive oxygen species, which may lead to

membrane lipid peroxidation and DNA damage.102

Less reactive drugs, such as carboplatin

and oxaliplatin, decrease the GSH concentration significantly slower and therefore reduce the

negative effect of glutathione depletion on cell viability. Various radical scavengers and

antioxidants can effectively protect against platinum-induced toxicity most likely by

preventing or delaying membrane lipid peroxidation, and not by repletion of cellular

glutathione content.110

Mitochondrial injury is a central event in cell damage caused by nephrotoxicity of

platinum complexes. Among all cellular organelles, mitochondria accumulate the highest

concentration of cisplatin in vivo. Functional changes in mitochondria include inhibition of

mitochondrial Ca2+

uptake, inhibition of Na+/K

+-ATPase and the resulting loss of the normal

intracellular-to-extracellular Na+ gradient, lipid peroxidation and collapse of the

mitochondrial membrane potential.102,111

Mitochondrial injury is thought to be a consequence

of mitochondrial GSH depletion.112

The latter is a major cause of oxidative stress, which

leads to lipid peroxidation and collapse of membrane potential.

Inhibition and loss of vital cellular functions, which result from nephrotoxic injury,

eventually lead to cell death through apoptosis or necrosis. High concentrations of cisplatin

(800 µM) were found to result in necrotic cell death, whereas lower concentrations (up to

300 µM) induce apoptosis.108

In non-proliferating (healthy) cells, as well as in the case of

cancer cells, apoptotic mechanisms involve activation of caspases.113

Caspases receive an

apoptotic signal when cytochrome c is released from collapsed mitochondria into the

cytosol.114

1.5. Cisplatin resistance

Resistance of tumor cells to drug treatment is a common problem in cancer

chemotherapy, and cisplatin is no exception.24

Some tumors, such as colorectal cancer and

non-small cell lung cancer, are inherently insensitive to cisplatin (intrinsic resistance).115,116

Other tumors, for example, testicular and ovarian cancer, develop resistance after repeated

administrations of the drug (acquired resistance).25,26

The factors determining both intrinsic

and acquired resistance vary significantly between different tumors. Besides, several

resistance mechanisms usually act simultaneously in a given cell type. Thus, versatility of

cisplatin resistance makes it a major problem of the chemotherapy and presents a major

challenge for scientists.

The mechanisms of resistance to cisplatin are generally divided into two groups: pre-

binding and post-binding mechanisms26

(Figure 1.7). Pre-binding mechanisms hamper

cisplatin reaching its intracellular target, nuclear DNA, and post-binding mechanisms prevent

20

induction of cell death (by apoptosis or necrosis) after cisplatin interaction with DNA. The

former include decreased platinum accumulation (decreased uptake and increased efflux),

sequestration of the drug in cellular organelles and deactivation of reactive cisplatin species

by intracellular thiols, like glutathione and metallothionein. The latter mechanisms are

increased repair of cisplatin-DNA lesions, increased DNA damage tolerance and failure of

apoptotic pathways.

Figure 1.7. Schematic representation of the mechanisms of cisplatin resistance.

Decreased uptake and increased efflux lower intracellular concentrations of the drug.117

It is generally believed that decreased uptake is much more important in cisplatin resistance

than increased efflux, as the main multidrug resistance efflux pump, P-glycoprotein, is

normally not overexpressed in cisplatin-resistant cells.31,33

However, other multidrug

resistance transporters, such as multidrug resistance proteins (MRP), have been reported to

be involved in cisplatin transport out of the cell. MRP proteins normally transport drugs by

conjugation with sulfate, glucoronate or glutathione. Evidence suggests that MRP1 and

MRP2 confer resistance to cisplatin by pumping out the drug coupled to GSH.118,119

The only known example of sequestration of platinum complexes away from the

nucleus as a mechanism of resistance is presented in Chapter 3. Sequestration of various

organic anticancer drugs, such as doxorubicin and daunorubicin, in cellular organelles

(lysosomes and Golgi complex) is a common mechanism of multidrug resistance.120-122

In

this case, altered intracellular distribution is usually associated with overexpression of drug

efflux pumps, such as P-glycoprotein and MRP proteins.121-123

Changes in intracellular pH

drug

drug-DNAdrug-DNA

apoptosis

sequestration in cellular

organelles

deactivation by cellular proteins

decreased

accum ulation

increased

DNA repair

increased tolerance

failure of apoptotic

pathways

post-binding m echanism s

pre-binding m echanism s

21

gradients also appear to be important.121,124,125

In the case of cisplatin-resistant cells, the

factors responsible for altered drug distribution inside the cell, e.g. sequestration in cellular

organelles, are not well elucidated. It is proposed in Chapter 4 that alkalinization of

lysosomes, which results in disruption of normal endocytosis, might be involved.

Increased deactivation of reactive platinum species by coordination to intracellular

thiols, glutathione and metallothionein, is a clearly established mechanism of resistance.25,26

Glutathione, which is present in the cytosol at high concentrations (0.5 – 10 mM),24

reacts

with the aquated form of cisplatin, spontaneously, or with the help of glutathione

S-transferase enzyme,126

and resulting conjugates are readily excreted by the ATP-dependent

S-conjugate export pump.127,128

GSH can also inhibit conversion of Pt-DNA monoadducts to

potentially cytotoxic crosslinks.47,129

Many cisplatin-resistant cell lines exhibit elevated GSH

levels compared to their sensitive counterparts.130-132

The agents, which deplete intracellular

GSH in resistant cell lines, like L-buthionine sulfoximine, are able to partially reverse

resistance.133-135

Increased deactivation of cisplatin by glutathione is generally accepted as a

mechanism of resistance, but other findings concerning the effect of GSH are also

interesting. These include the role of elevated GSH levels in increased DNA repair,136

or

increasing the inhibitory effect on apoptosis by buffering drug-induced oxidative stress.137

Besides, increase in intracellular metallothionein concentration has been found in some

cell lines with acquired resistance to cisplatin.24,25

Mammalian metallothionein (MT) is a

small protein of 62 aminoacids, which contains 20 cysteine residues. MT is involved in

intracellular detoxification of heavy metal ions such as Cd2+

and Pb2+

, and it also readily

binds cisplatin.26,138

However, while metallothionein overexpression has been associated with

cisplatin resistance in some models, no relation was found in the other models.139-141

A major mechanism of resistance to cisplatin is increased repair of DNA damage by

nucleotide excision repair (NER) proteins.26

Increased NER in resistant cell lines as

compared to their sensitive counterparts has been reported for both intra- and interstrand

crosslinks.42,142

In some cell lines, interstrand crosslink repair is favored over intrastrand

adduct repair.143

However, in most tumor types removal of 1,2-intrastrand crosslinks by NER

is of particular importance.144

NER is an ATP-dependent multiprotein complex that

recognizes the DNA kink, induced by 1,2-intrastrand crosslinks and excises the damaged

segment, as a 27-29 base-pair oligonucleotide. The remaining gap is subsequently filled in by

DNA polymerase.145

NER can be inhibited by HMG1 protein, which is also able to recognize

1,2-intrastrand crosslinks and can shield cisplatin-DNA adducts from repair proteins.61

Thus,

increased nucleotide excision repair of platinum-DNA crosslinks contributes to cisplatin

resistance, and conversely, defective NER confers hypersensitivity of some cell lines to the

drug.145

The post-binding mechanism, so-called “increased tolerance”, appears to be one of the

most general mechanisms of resistance to DNA-binding drugs.146

In this case, cisplatin

22

resistance is mediated through increased ability to tolerate drug-induced damage without

NER taking place. Post-replication repair is defined as the replication of damaged DNA

without introduction of the gaps and/or the immediate repair of the gaps following

replication.146

As the presence of discontinuities in the DNA can be lethal for the cell, post-

replication repair is a major mechanism of DNA-damage tolerance. Since post-replication

repair in human cells mainly occurs during replication, it is often considered a replicative

bypass. Enhanced post-replicative bypass, the ability of the replication complex to synthesize

DNA past cisplatin-induced adduct, has been found in some resistant cell lines.145,147,148

The

biochemical mechanisms conferring increased tolerance of DNA damage are not clear.

However, defects in a second DNA-repair process, mismatch repair (MMR), are believed to

be involved.148-150

The mismatch repair system includes at least 5 proteins (MLH1, MSH2,

MSH3, MSH6 and PMS2) and is responsible for correction unpaired or mispaired

nucleotides.151,152

DNA replication along the cisplatin 1,2-d(GpG) intrastrand crosslink

results in imperfect base pairing. In cisplatin-sensitive MMR-proficient cells, MSH2

recognizes (but does not remove) 1,2-d(GpG) intrastrand adducts of cisplatin with DNA,153

initiating futile cycles of excision of the lesions containing mispaired nucleotides and DNA

resynthesis, which eventually lead to cell death.145,154

Thus, MMR attempts to repair the

lesion, but in failing to do so activates the apoptotic signal. In some cisplatin-resistant cells,

the loss of MMR activity results in increase in ability to replicate DNA past cisplatin-induced

adduct.145,149,150

Thus, MMR-deficient tumors acquire resistance by blocking futile cycles of

mismatch correction.

It is believed that apoptosis can be triggered if cellular damage has passed a certain

threshold level.42

This threshold level varies between different types of cancer cells.155

However, damaged genes are quite common in cancer cells, and apoptotic pathways might

malfunction. In some tumors, this appears to be the main mechanism conferring intrinsic

resistance to cisplatin chemotherapy.156

Activation of the p53 protein is known to be critical

for cisplatin-induced apoptosis. Therefore, tumor cells that have defects in the apoptotic

function of p53 fail to activate the cell death program and, thus, they tolerate DNA

damage.24,27,157

A major factor affecting the loss of apoptotic function is p53 gene

mutation.158

Cell lines with mutant p53 sequence have been reported to have a much higher

degree of cisplatin resistance that those containing a wild-type p53 protein.159

p53 also

directly affects the expression of the genes that regulate pro- and antiapoptotic responses to

cisplatin-induced DNA-damage.24,155

The bcl2 family of genes encodes several proapoptotic

(e.g. Bax, Bak) and antiapoptotic (e.g. Bcl-2) proteins that regulate the effector phase of

apoptosis.160,161

It has been suggested that the ratio of apoptotic to antiapoptotic proteins (for

example, Bax to Bcl-2) ultimately determines whether or not the cell would undergo

apoptosis.155

Thus, activation of antiapoptotic pathways (e.g. through overexpression of Bcl-

23

2) and/or disruption of proapoptotic pathways (e.g. through downregulation of Bax and Bak

proteins) may prevent induction of apoptosis and thereby confer resistance to cisplatin.162,163

1.6. New platinum anticancer compounds

As mentioned above, the disadvantages of cisplatin have triggered intensive search for

new platinum-based anticancer drugs. However, from over 3000 compounds tested in vitro

only 28 complexes have entered clinical trials.6,164

Some of them were approved for clinical

application after a series of successful experiments.

Carboplatin (Paraplatin, 3) is widely used in cancer chemotherapy since 1986. Its

pharmacological profile is similar to that of cisplatin, however, it appears to be slightly less

cytotoxic.15

On the other hand, carboplatin is significantly less toxic for kidneys and the

nervous system and causes less nausea and vomiting.165

Nowadays, it is mainly used for

treatment of ovarian cancer in patients suffering from severe side effects.166

Nedaplatin (4) is

approved for clinical use in Japan since 1995. It exhibits good antitumor activity in

combination with a reduced toxicity.165,167

Decreased toxicity of carboplatin and nedaplatin,

as compared to cisplatin, is associated with their relatively low reactivity, e.g. towards

glutathione, due to the more inert leaving group, a didentate dicarboxylate (the mechanism of

nephrotoxicity of platinum anticancer drugs is described in more detail in Section 1.4).168

However, both complexes show cross-resistance with cisplatin.23,169

Beside lower toxicity,

oxaliplatin (Eloxatin, 5) exhibits a different cytotoxic profile and overcomes cisplatin

resistance in some cell lines.170

This drug is increasingly used in the clinic.164,171

In Germany

and France, it is one of a few drugs approved for treatment of colorectal cancer.164,165

Oxaliplatin contains the 1,2-cyclohexane carrier ligand, which increases the lipophilicity of

the drug and therefore improves its uptake. Better cellular uptake and DNA adducts that are

more effective in inhibiting DNA chain elongation, confer the ability of oxaliplatin to

circumvent cisplatin resistance.172

O

O

Pt

H3N

H3N

O

O

O

Pt

O

O

Pt

ONH3

NH3

O O

ONH2

H2

N

3 4 5

Figure 1.8. New platinum drugs used in the clinic.

Platinum complexes with carrier ligands seem promising anticancer drugs, as a carrier

ligand may target a platinum drug to tumor cells, may facilitate the uptake and transport, or

may increase the affinity for DNA. This group of anticancer compounds will be briefly

reviewed in Section 1.8.

Compounds with decreased reactivity have also been intensively investigated. They are

expected to be less toxic and less susceptible to deactivation by glutathione prior to DNA

24

binding.21,173

One of the most prominent complexes in this group is cis-[PtCl2(NH3)(2-

methylpyridine)] (ZD0473, formerly known as AMD0473 and JM473) (6), which is now in

Phase II clinical trials.165,174,175

Another example is the first orally administered platinum

drug JM216 (satraplatin) (7) that has entered Phase III clinical trials.23,165

This complex

shows no cross-resistance with cisplatin and carboplatin and has low nephro- and

neurotoxicity.176,177

PtH3N

NH2

Cl

Cl

OCOCH3

OCOCH3

PtN

H3N

Cl

Cl

6 7

Figure 1.9. Sterically hindered platinum anticancer complexes.

Since increased repair of cisplatin-DNA adducts is one of the most important

mechanisms of cisplatin resistance (see Section 1.5), platinum complexes able to form

structurally different DNA adducts are expected to escape from the DNA repair system in the

cell, and therefore to be less susceptible to the resistance mechanisms. A number of platinum

complexes with a different mode of DNA binding have been designed to circumvent cisplatin

resistance. Examples of successful application of this approach are platinum complexes with

intercalators and DNA groove binders that are discussed in more detail in Section 1.8.

Another group of compounds with a different mode of interaction with DNA are trans

platinum complexes.178

Although transplatin, the trans isomer of cisplatin, is not antitumor

active, various analogues, which possess other ligands than ammonia, exhibit high

cytotoxicity.179-181

The ability of trans complexes to overcome cisplatin resistance is believed

to result from a high proportion of interstrand crosslinks formed upon interaction of these

complexes with nuclear DNA.182

Dinuclear (and polynuclear) platinum complexes can form various types of DNA

adducts including interstrand crosslinks and long-range inter- and intrastrand crosslinks,

which are not recognized by DNA repair proteins.178,183-186

This feature helps these

complexes to escape from DNA repair system of the cell, and to overcome resistance. This

class of promising anticancer drugs is separately reviewed in Section 1.7.

1.7. Polynuclear platinum complexes as potential anticancer drugs

As mentioned earlier in this chapter, intrinsic and acquired resistance of tumors to drug

treatment is a major problem of cancer chemotherapy. It is generally accepted that the

cytotoxic effect of platinum complexes results from the crosslinks formed by these

compounds on genomic DNA.40,41

Resistance to cisplatin is often conferred by increased

repair of cisplatin-DNA lesions and increased tolerance to DNA damage. Therefore,

platinum complexes, which are able to form structurally different DNA adducts compared to

25

cisplatin and its analogues, are supposed to have improved cytotoxic profiles and to

overcome cisplatin resistance.185,187

Polynuclear platinum complexes contain two or more linked platinum centers, which

can each bind to DNA, and thus form a very different array of DNA adducts compared to

cisplatin.187

Structurally different platinum-DNA lesions are supposed to escape DNA

damage recognition and repair proteins, and therefore, to be much less susceptible to the

mechanisms of cisplatin resistance.188,189

The first polynuclear complexes have been designed linking cisplatin-,190,191

carboplatin-192

and transplatin193

moieties by aliphatic diamines (8, 9). These compounds can

form 1,2-d(GG) intrastrand crosslinks similarly to cisplatin, but they are also able to form

long-range adducts.194

Mechanistic studies have indeed shown that these dinuclear platinum

complexes produce a high percentage of interstrand crosslinks.194

Many of the complexes

exhibit good activity in cisplatin resistant cell lines.194

H2N(CH2)nNH2

Pt

Cl

H3N Cl

Pt

Cl

Cl NH3

H2N(CH2)nNH2

Pt

Cl

Cl NH3

Pt

Cl

H3N Cl

8 9

Figure 1.10. First polynuclear platinum complexes designed as potential antitumor

drugs.

A number of other linking ligands have been used to develop similar dinuclear

platinum-based drugs. A series of double-bridged complexes with 4,4’-dipyrazolylmethane,

such as 10, have been prepared and tested for antitumor activity.195

Subsequently, the series

has been extended to single-bridged complexes, like 11, which showed even higher

cytotoxicity.196

Although the neutral complexes with bifunctional platinum centers exhibit

relatively high antitumor activity, their poor water solubility due to the large lipophilic

bridging ligands makes them inappropriate for clinical use.

10 11

N

HN

N

NH

N

NH

N

HN

PtPt

Cl

Cl

Cl

Cl

N

NH

N

HN

PtPt

Cl

Cl

Cl

Cl

NH3 H3N

Figure 1.11. Dinuclear platinum complexes with 4,4’-dipyrazolylmethane.

Cationic dinuclear platinum complexes present another class of promising polynuclear

platinum anticancer drugs. Due to their positive charge, they are well soluble in water and

have a high affinity for the negatively charged DNA. On the other hand, positive charge and

26

larger molecular weight of these complexes may suggest poor uptake by cancer cells, as

compared to cisplatin. Passive diffusion, the main mechanism of cisplatin uptake, is unlikely

in the case of charged dinuclear species.197

However, a number of studies in various cancer

cell lines have shown that accumulation of cationic dinuclear complexes is comparable to or

higher than that of cisplatin.197,198

Most of these compounds can be divided into two groups: complexes with flexible

linkers and complexes with rigid linkers. The former type, which utilizes flexible aliphatic

di- and polyamines as linking ligands, has been designed to achieve long-range inter- and

intrastrand crosslinking on DNA.187

Besides, in contrast to their neutral counterparts, these

charged complexes have been found to induce transition of the normal right-handed B-DNA

helix into the left-handed Z-DNA helix, which is believed to help to escape from DNA

damage recognition and repair systems, and therefore, to overcome cisplatin resistance.199-201

The first reported dinuclear complexes with flexible linkers consist of two cis- or transplatin

units linked by diamines with varied length of the aliphatic chain (12, 13).199,202

These compounds were tested for antitumor activity in vitro and in vivo, and a

structure-activity relationship has been developed.186,188

Hexanediamine (n = 6) appeared to

be the ideal bridging ligand: analogous complexes with a shorter (n = 2,3) or longer (n = 7)

aliphatic chain are less cytotoxic.186

Geometrical isomerism is also of importance.203,204

Complexes with the chloride ligand in either cis- or trans-position with respect to the

diamine show good antitumor activity with cytotoxic profiles different from that of cisplatin.

However, trans-complexes are usually much more cytotoxic in resistant cell lines in contrast

to their cis-isomers, which are often cross-resistant with cisplatin.183,187

Such difference in

activity of geometric isomers has been associated with the ability of trans-complexes to form

various long-range intra- and interstrand crosslinks whilst cis-complexes only produce

interstrand crosslinks.189,204,205

Indeed, steric hindrance prevents formation of intrastrand

adducts by dinuclear complexes of cis-geometry. Besides, kinetics of DNA binding might

also play a role: some trans-complexes have been reported to react with guanine faster than

their cis-counterparts.204

H2N(CH2)nNH2

Pt

NH3

Cl NH3

Pt

H3N

H3N Cl

H2N(CH2)nNH2

Pt

NH3

H3N Cl

Pt

H3N

Cl NH3

12 13

2+ 2+

n = 2-7 n = 2-7

Figure 1.12. Cationic dinuclear platinum complexes 1,1/c,c (12) and 1,1/t,t (13).

This class of dinuclear complexes has been further extended using other bridging

diamines. Linking transplatin moieties with naturally occurring polyamines spermine and

spermidine were reported to yield highly cytotoxic dinuclear complexes 14 and 15, which

overcome cisplatin resistance in several resistant cell lines.206,207

These complexes are

27

expected to enter Phase I clinical trials.185

Their improved antitumor activity in sensitive and

resistant cells, compared to 1,1/t,t complexes 13, is attributed to the hydrogen-bond donor

capacity of the linking polyamines.186

14

15

3+

4+

H2N

H2

NNH2

Pt

H3N

ClNH3

Pt

H3N

NH3

Cl

H2N

H2

NNH2

NH2Pt

H3N

NH3

Cl

Pt

H3N

NH3

Cl

H2NNH2

Pt

H2

N

Pt

H3N

NH3

Cl

Cl

Cl

NH2

Pt

H3N

NH3

Cl

4+

16 (BBR3464)

Figure 1.13. Promising polynuclear platinum antitumor drugs.

Hydrogen bonding provides stronger pre-association with genomic DNA, and this

significantly affects the rate and site of platination, as increased local concentration will

increase the probability of binding at these sites. Moreover, hydrogen bonding stabilizes

flexible long-range crosslinks formed by 14 and 15. Another approach was to introduce an

additional platinum atom in the linker. The central platinum atom brings additional positive

charge, and therefore, stronger interaction with a polyanion DNA. This approach has led to

the development of highly antitumor active complex BBR3464 (16),183

which has entered

Phase II clinical trials.185

This trinuclear complex has a cytotoxic profile very different from

that of cisplatin and is very active against cisplatin-resistant tumors. It forms various types of

long-range crosslinks on genomic DNA, which confers its ability to overcome cisplatin

resistance.208

Thus, cationic dinuclear platinum complexes with flexible linking ligands present a

promising class of new antitumor drugs. Their different cytotoxic profiles and their ability to

circumvent cisplatin resistance is attributed to the formation of long-range intra- and

interstrand crosslinks on nuclear DNA and induction of B Z DNA transition.

The dinuclear and polynuclear complexes with rigid linkers have been less intensively

investigated. Farrell and colleagues186,209

have claimed that linking two platinum moieties by

a sterically rigid ligand (e.g. 1,4-diaminocyclohexane or dipyrazolylmethane) results in

reduced cytotoxicity compared to the complexes with flexible bridging ligands. However, the

28

dinuclear complexes with isomeric azines (17, 18, 19) have been found to exhibit good

anticancer activity and largely or partly circumvent cisplatin resistance.210

N N

N NPtH3N

NH3

Pt

Cl

NH3

NH3Cl

N N

2+

=N NN

N,

or

17 18 19

Figure 1.14. Dinuclear platinum complexes with isomeric azines.

An interesting type of cationic dinuclear platinum complexes with rigid linkers, such as

hydrazine211-213

and azoles (20, 21)184

features a bridging leaving group, either chloride or

hydroxide. These complexes have been designed to induce minimal distortion in the DNA

double helix upon binding. The resulting 1,2-d(GG) intrastrand crosslink is expected to be

not recognized by DNA repair proteins, which is beneficial for antitumor activity, especially

in resistant cell lines. The dinuclear complexes with hydrazine and azoles are indeed highly

cytotoxic and overcome cisplatin resistance.184,212

X-Ray structural investigation of the

adduct of 20 with 9-ethylguanine revealed that the two guanine rings are almost parallel, so,

their orientation is strikingly similar to the orientation of nucleobases in the undistorted

DNA.214

N N

Pt

OH

Pt

H3N

H3N

NH3

NH3

N N

N

Pt

OH

Pt

H3N

H3N

NH3

NH3

2+2+

20 21

Figure 1.15. Dinuclear platinum complexes with azole ligands designed to induce

minimal distortion on DNA.

The NMR and molecular mechanics studies of the adduct of this complex with a DNA

oligomer have indicated that the distortion of the helix is limited to an unwinding of 10-15º at

the site of lesion.215,216

Thus, the structure of 1,2-d(GG) intrastrand adduct of 20 is different

from that of cisplatin-DNA adduct and has only minor deviations from unplatinated genomic

DNA, which probably accounts for the high activity of the complex in resistant cell lines. In

contrast to 20, the triazole-bridged complex 21 undergoes isomerization on the triazole ring

upon binding to 9-ethylguanine.217

The same isomerization also occurs during interaction of

21 with a double-stranded oligonucleotide.218

Besides, the azole-bridged complexes 20 and

21 possess bridging hydroxide as a leaving group. This makes the compounds less reactive

than cisplatin, which may result in their lower toxicity.

29

Thus, dinuclear and polynuclear platinum complexes present a new approach in

platinum-based anticancer drug design and offer a big potential as new antitumor agents.

1.8. Drug targeting to the therapeutic sites

As mentioned above, cancer chemotherapy is often accompanied with side effects

caused by the toxicity of antitumor agents. Development of new anticancer drugs with lower

toxicity proved to be a good solution of this problem. Another, very elegant, strategy is

specific delivery of a drug to the tumor tissue and/or its specific uptake by cancer cells.

Targeting a drug to the site of therapeutic action will enhance its pharmacological effect

minimizing at the same time undesirable side effects.

Targeted drugs usually consist of a drug moiety bound to a carrier, which directs the

drug to the desired tissue or cell type. After delivery to the site of action, release of the drug

from the carrier must be achieved, so that the drug would perform its therapeutic function. In

the case of platinum antitumor agents, a carrier can be introduced into the leaving group or

can be attached to the amine ligands. The former approach is more favorable, as release of

the drug from the carrier occurs automatically upon hydrolysis of the platinum complex

inside the cell. In the latter case, it is more difficult to attain drug release. If the carrier

remains bound to a platinum moiety, it will probably change the chemical and biological

properties of the platinum drug, and may even impair its biological activity.

A carrier may not only deliver a drug to specific cells but may also facilitate its uptake.

The latter can be achieved using carriers with a high affinity for a certain membrane

transporter. Since most resistant tumors exhibit decreased drug accumulation, development

of drugs that target active uptake mechanisms are of special interest. Increasing affinity of

anticancer agents for their ultimate cellular target, genomic DNA (DNA targeting), may

improve cytotoxic activity of the drugs in sensitive and resistant tumors.

Drug targeting strategies described in the literature can be roughly divided into four

types: passive targeting, receptor-mediated targeting, enzymatically activated prodrug

delivery and DNA targeting.219

Passive tumor targeting is based on so-called enhanced permeability and retention

effect (EPR effect), namely, the hyperpermeability of tumors towards macromolecules.220

Tumors can easily accumulate macromolecules because of their compromised vasculature

combined with a lack of effective lymphatic drainage. Thus, the EPR effect would direct a

drug bound to a macromolecule or encapsulated in a nano-sized particle, to the tumor tissue.

Passive targeting has proven to be a successful approach. Encapsulation of a well-known

antitumor drug doxorubicin in liposomes provided a new promising chemotherapeutic agent,

Doxil®

.221

Various liposomal formulations of cisplatin have been prepared despite the

difficulties associated with its low lipophilicity.178

These species have a high affinity for

tumor tissue. High specificity of their delivery to cancer cells results in very low toxicity.

30

Some cisplatin-containing liposomes, such as lipoplatin165

and a stealth liposome-

encapsulated cisplatin (SPI-77),222,223

are now in clinical trials. Another example of EPR-

based drug targeting is a complex containing a diammineplatinum unit linked to the N-(2-

hydroxypropyl)methacrylamide (HPMA) copolymer (AP5280, 22). Inside the cell, the

platinum moiety is thought to be released from the polymer either via hydrolysis of the

platinum-peptide bond or via proteolytic cleavage of the short peptide bridge between the

platinum-based therapeutic moiety and the polymer carrier.224,225

AP5280 has been reported

to be more cytotoxic and at the same time less toxic than the structurally related antitumor

drug carboplatin.219

This promising polymer-tethered drug has entered Phase I clinical

trials.226

C

OHN

HO

CH2 C

OHN

NH

O

OHN

O

NH

ON

OH

O

CH2

Pt

O

O

H3NNH3

x y

22

Figure 1.16. Promising polymer-tethered drug AP5280.

A drug can be specifically delivered to cancer cells if it is bound to a carrier with a high

affinity for a certain receptor on the cell surface. Receptors regulating cell growth and

division are overexpressed in tumor cells and can, therefore, be easily targeted. Some breast

and prostate tumors overexpress an estrogen receptor. Platinum complexes with polyaromatic

ligands227,228

and estradiol derivatives229

have been found to be taken up by this receptor.

However, these compounds exhibit little improved activity in the cells overexpressing an

estrogen receptor as compared to the cells that lack it. Another widely investigated target is a

folate receptor abundant in human cancer cells. Several platinum complexes have been

shown to be internalized via the folate receptor.230

Unfortunately, these compounds did not

show the desired increase in cytotoxicity. Nevertheless, further exploration of receptor-

mediated drug targeting may lead to the development of new promising antitumor agents.

Antibody-directed enzyme prodrug therapy (ADEPT) is based on the administration of

the enzyme conjugated to the tumor-specific antibody followed by a prodrug, which is

activated by this enzyme.231

Some enzymes are concentrated around the tumor. In this case,

31

an enzymatically activated prodrug can be administered directly (prodrug monotherapy).232

These approaches have been successfully applied, for example, for development of platinum-

based anticancer prodrugs, which can be activated by -lactamase233

or -glucoronidase.234

As the cytotoxic effect of antitumor drugs is believed to result from their interaction

with genomic DNA, increasing drug affinity for DNA is likely to improve anticancer

activity. A moiety with high DNA affinity incorporated in the drug structure will not only

target the drug to DNA, but may also provide structurally different DNA adducts able to

escape from the DNA repair system. Thus, DNA targeting may also help to overcome drug

resistance. An additional positive charge in platinum complexes, as in the case of the

dinuclear platinum complexes with polyamines and the trinuclear platinum complex

BBR3464, discussed in Section 1.7, results in higher affinity for the negatively charged

DNA, which improves the cytotoxic properties.183,206

Platinum complexes coupled to DNA-

groove binders (23) present an interesting class of promising anticancer compounds. These

compounds may interact with DNA in a way different from the platinum complex and the

groove binder alone resulting in different cytotoxic profiles.235

NH

PtNH2

Cl Cl

HN

O

N

HN

O

N

HN

ON

H2N

23

Figure 1.17. Platinum complex with a DNA-groove binder.

Intercalators have also been used as targeting ligands for platinum complexes.

Intercalators are polyaromatic molecules, which can form non-covalent adducts with DNA

based on stacking interaction of their aromatic system with the aromatic rings of two

neighboring nucleobases. Thus, intercalating ligands in platinum complexes can provide

additional interaction with DNA next to coordination of platinum to guanine bases. Platinum

complexes with various intercalators (24, 25) have been prepared.236-242

Many of these

complexes exhibit high antitumor activity, show less toxicity and overcome cisplatin

resistance.240,243-245

Thus, drug targeting to the site of therapeutic action includes specific delivery of a drug

to a certain organ, to tumor tissue, to cancer cells or to its intracellular target. This approach

aims for increasing the therapeutic effect minimizing at the same time undesirable side

effects.

32

NHPt

NH2

Cl Cl

NH

N

O

NH2

(CH2)n

O

O

PtNH3

NH3

O

O

NHO

O

(CH2)n

n = 2 - 6 n = 3, 6

24 25

Figure 1.18. Platinum complexes with intercalators.

1.9. Aims and scope of this thesis

This thesis deals with the chemical design and biological investigation of dinuclear

platinum complexes as potential antitumor agents. Investigation of the cellular processing of

dinuclear platinum drugs in cancer cells takes a central place. Insights into drug behavior in

different types of tumors are of great importance for pharmacogenomics, a new field of

science, which studies how genes affect a person’s response to drugs. Pharmacogenomics

holds the promise that in the future drugs might be designed for individuals and adapted to

each person’s own genetic inheritance. This thesis also describes the development of new

dinuclear platinum anticancer compounds, a study of nephrotoxicity of promising dinuclear

platinum antitumor agents and an application of dinuclear platinum complexes for targeting

small organic drugs to the therapeutic sites.

Chapter 1 explains the basic concepts, which are used or referred to throughout the

thesis, and presents an overview of the relevant literature. Chapter 2 describes synthesis,

binding to guanine model bases and antitumor activity of the new dinuclear platinum

complexes with azine ligands. Chapters 3 and 4 deal with cellular processing of dinuclear

platinum complexes with fluorescent anthraquinones. The intracellular distribution of these

platinum complexes and the respective free ligands has been studied in different cell lines

using digital fluorescence microscopy. Cellular processing of these compounds in A2780

human ovarian carcinoma cells is described in Chapter 3, and cellular processing in U2-OS

human osteosarcoma cells is discussed in Chapter 4. Chapter 5 presents new fluorescent

labeled dinuclear platinum complexes developed as model compounds for investigation of

the intracellular distribution of promising dinuclear platinum anticancer drugs. Design of a

conjugate for targeting small organic drugs to a therapeutic site using a dinuclear platinum

linking unit between a drug and a carrier protein is described in Chapter 6. The relationship

between the structure of dinuclear platinum complexes and their nephrotoxicity is discussed

in Chapter 7. Chapter 8 includes a summary of the results presented in this thesis, main

conclusions and an outlook.

Some parts of this thesis have been published246-249

or submitted for publication.250

33

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