Part 1:

Tumor Biology and KineticsIntroduction of Cytotoxic Agents

Pharmacologic Anti-Cancer Treatments Seminars 2007:

Carlos Linn, M.D. 林錦洲 醫師Clinical Research Physician, Oncology

Lilly Oncology

Board Certified Gynecologic Oncologist

2

Cellular Kinetics

• Human body contains 5x1013 cells

• Cells can either be - non dividing and terminally

differentiated - continually proliferating- rest but may be

recruited into cell cycle

• Tumour becomes clinically detectable when there is a mass of 109 cells (1g)

3

DEATHDEATH

DIFFERENTIATIONDIFFERENTIATION

DNA content = 2nDNA content = 2n

MitosisMitosis

MM

SSDNA synthesisDNA synthesis

GG22

GG11

GG00

DNA content = 4nDNA content = 4n

The Cell Cycle

4

The Cell Cycle

5

CANCER CELLS NORMAL CELLS

Loss of contact inhibition

Increase in growth factor secretion

Increase in oncogene expression

Loss of tumor suppressor genes

Oncogene expression is rare

Intermittent or coordinatedgrowth factor secretion

Presence of tumor suppressorgenes

FrequentFrequentmitosesmitoses

NucleusNucleus

Blood vesselBlood vessel

AbnormalAbnormalheterogeneous cellsheterogeneous cells

NormalNormalcellcell

FewFewmitosesmitoses

Cancer Cells and Normal Cells

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GrowthFactor

Growth FactorReceptor

Paracrine (Adjacent cells)

Growth Factor and Receptor Synthesis

PostPostreceptor signalreceptor signaltransductiontransductionpathwayspathways

Gene Activation

Oncogenes

Autocrine stimulation

Growth Factors and Oncogenes

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NORMAL GROWTH ANDNORMAL GROWTH ANDDEVELOPMENTDEVELOPMENT

NORMAL EXPRESSION & NORMAL EXPRESSION & RESPONSIVE ONCO SUPPRESSION GENERESPONSIVE ONCO SUPPRESSION GENE

MUTAGENIC orMUTAGENIC orCARCINOGENIC AGENTSCARCINOGENIC AGENTS

VIRAL ONCOGENEVIRAL ONCOGENE

Oncogenesis

CELLULARCELLULARONCOGENEONCOGENE

INCREASED OR ABNORMALEXPRESSION

CANCERGROWTH

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Example: Oncogenesis Integrated by HPV

Beutner, KR et al, "Human Papillomavirus and Human Disease." Am J Med 1997; 102(5A):9-15.

Complete viral Complete viral life cycle with life cycle with KoliocytosisKoliocytosis Virus Virus duplicationduplication

Integration of Integration of HPV DNA HPV DNA genome E6, E7 genome E6, E7 into Host-cell into Host-cell Immortally Immortally malignantmalignantNO more NO more KoliocytosisKoliocytosis Virus stops Virus stops duplicationduplication

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E6, E7 Protein involvement in cell cycle

regulation

Cell cycle proteins, influenced by E6, E7 proteins

E6 Bind and Degrade p53:

Loss of p53-induced apoptosis/G1 arrest of the cell cycle; reduces p53 protein via degradation.

E7 releases the E2F transcription factor by binding Rb (retinoblastoma protein), promoting cell cycle progression

transcriptional deregulation of cell cycle control, uncontrolled cell proliferation

intracellular control - cyclin-intracellular control - cyclin-dependent kinase inhibitors (CKI) dependent kinase inhibitors (CKI)

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CYCLIN DEPENDENT KINASES

tyr15-P

P-thr161

thr14-P

e.g. cdk1 (= cdc2)

- protein kinase- binds to cyclin- kinase domain- regulatory domain- present throughout cell cycle

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CYCLINS

- No intrinsic enzymatic activity- Binds cdk- Synthesized and degraded each cycle- Essential component for cdk activity

e.g. Cyclin Be.g. Cyclin B

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CYCLIN / CDK

tyr15-P

P-thr161

thr14-P

cdk1cdk1(cdc2)(cdc2)

cyclin Bcyclin B

• Regulated by:• - tyr15 phosphorylation

• inhibitory kinases• activating phosphatases

• - Direct interaction• inhibitory proteins

• p21, p27, p57• p16, p15, p18,p19

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CELL CYCLE CHECKPOINTS

G2

G1

S

M

CYCLIN E / cdk 2CYCLIN E / cdk 2

CYCLIN D / cdk 4,5,6CYCLIN D / cdk 4,5,6

CYCLIN A / cdk 2

CYCLIN B / cdk 1CYCLIN B / cdk 1

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Variation in Cell Cycle Cyclins

M G1 G2S M G1

Start

Cell cycle phases

Cyclin-dependent kinases (CDK)

D E A B(A)CyclinsCyclins

CDK 4 CDK 2 CDK 1

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G1

Cell Cycle

G0

6-8 h

DNA, RNA, Protein

3-4 h

RNA, Protein

1 h

Mitosis, Cytokinesis

S

G2 Cyclin D’s

CDK4,6Cyclin B/A

CDK1

Cyclin A

CDK2

M

Cyclin E

CDK2

6-12 h RNA, Protein

p53pRb

LaminH1Abl

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E6, E7 involvement in cell cycle regulation DNA damageDNA damage

Phosphorylation

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DNA Damage - Cell Cycle ArrestDamage Dependent Checkpoints

CELL No.

DNA content DNA content

AsynchronousX-ray treatedX-ray treatedG1/S blockG1/S blockG2/M blockG2/M block(6-9 hours)(6-9 hours)

G1 - S - G2

wild-type

Loss of G1/S in p53 deficientcells

G1 - S - G2

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G1/S CHECKPOINTIN RESPONSE TO DAMAGE

strandbreak

p53 p21

P-tyr15

cdk2

cyclin Ecyclin E

p21 = CKI class (cyclin dependent kinase inhibitors)N-terminal of p21 forms complex with cyclin / cdk - inhibit kinase

X-rays

ATM

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pRbpRb

Cell Cycle Regulation

CDK2CDK2

CECE

E2FE2FEnzymes for DNA synthesis

Passage from G1 to S

DNA damage1. CDK phosphorylation

2. C degradation

3. C & CDK synthesis

4. CDK inhibition

pRbpRb

P

Active p53

p21p21

pRbpRb

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Gene Transcription

G0G1

Priming

S

G2

M

Cell CycleCell Cycle

Growth Factors

++

Growth Factors & Cell Cycle

Receptors

21

Retinoblastoma protein (pRb) & CDK inhibitors: p21, p27, p16

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The Normal Cell Cycle &“Cyclins” of the cell cycle

Normal cell cycle (with tumor suppression and apoptosis)

Neoplastic cells (immortal)

E5 protein

E6, E7: immortalize E6, E7: immortalize human keratinocytehuman keratinocyte

G1 arrest

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Common Chemotherapeutic Agents

• Alkylating agents

• Antimetabolites

• Antitumor Antibiotics

• Alkaloids

• Taxanes

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Classes of antineoplastic drugs

• Alkylating agents• Interact directly with cellular DNA

• Antimetabolites• Resemble cellular metabolites (folic acid, purine, pyrimidine)• Interfere with DNA precursors & cellular metabolism

• Antitumor antibiotics• Derived from soil fungus, some antiinfective activity• Interfere with DNA activity

• Mitotic Inhibitors• Derived from plant extracts• Interfere with formation of mitotic spindle, arresting mitosis

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Alkylating agentsAlkylating agents Carboplatin, cyclophosmamide, melphalan, thiotepa

AntimetabolitesAntimetabolites Methotrexate, fluorouracil, gemcitabine

Natural ProductsNatural Products doxorubicin, docetaxel, vinolbine, topotecan

Endocrine agentsEndocrine agents Anastrozole, tamoxifen, prednisolone, goserelin

Molecularly targeted agentsMolecularly targeted agents Retinoids, trastuzumab, gefitinib

Biologic response modifiersBiologic response modifiers Interferon, thalidomide, filgrastim

(Form bonds with nucelic acids and proteins)

(similar to metabolites involved in nucelic acid synthesis)

(anti tumour antibiotics,mictotubule stabilizer, mitotic inhibitor, topoisomerase inhibiotor)

(Aromatase inhibitors, oestrogen antagonist, corticosteroids, LHRH agonist)

(gene expression, monoclonal antibody, tyrosine kinase inhibitor)

Antineoplastic Agents

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Alkylating Agents

• Interact with DNA causing substitution reactions, cross-linking reactions or strand breaks

• Example: cisplatin

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Antimetabolites

• Cytotoxic effects via similarity in structure or function to naturally occurring metabolites involved in nucleic acid synthesis—either inhibit enzymes involved in nucleic acid synthesis or produce incorrect codes

• Example: methotrexate, pemetrexed, gemcitabine, 5-FU

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Antitumor Antibiotics

• Group of related antimicrobial compounds produced by Streptomyces species in culture

• Affect structure and function of nucleic acids by:– Intercalation between base pairs (doxorubicin),

– DNA strand fragmentation (bleomycin),

– Cross-linking DNA (mitomycin)

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Alkaloids

• Bind free tubulin dimers

• Disrupting balance between microtubule polymerization and depolymerization

• Arrest of cells in metaphase

• Examples: vincristine, vinblastine, vinorelbine

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Taxanes

• Disrupt equilibrium between free tubulin and microtubules

• Stabilization of cytoplasmic microtubules

• Formation of abnormal bundles of microtubules

• Examples: paclitaxel and docetaxel

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Paclitaxel & Docetaxel

1971

1986

OH

European Yew: Taxus baccata

Pacific Yew: Taxus brevifolia

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BUSULFAN CYTOSINE ETOPOSIDE BLEOMYCIN L-ASPARAGINASE

CARMUSTINE ARABINOSIDE TENIPOSIDE DACTINOMYCIN HYDROXYUREA

CHLORAMBUCIL FLOXURIDINE VINBLASTINE DAUNORUBICIN PROCARBAZINE

CISPLATIN FLUOROURACIL VINCRISTINE DOXORUBICIN

CYCLOPHOSPHAMIDE MERCAPTOPURINE VINDESINE MITOMYCIN-C

IFOSFAMIDE METHOTREXATE TAXOIDS MITOXANTRONE

MELPHALAN GEMCITABINE TAXANES PLICAMYCIN

PEMETREXED ANTHRACYCLINES

EPOTHILONES

ALKYLATING

AGENTS

ANTI-

METABOLITES

MITOTIC

INHIBITORSANTIBIOTICS OTHERS

Classification of Cytotoxic Agents

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Antibiotics

Antimetabolites

S(2-6h)

G2

(2-32h)

M(0.5-2h)

Alkylating agents

G1

(2-h)

G0

Vinca alkaloids

Mitotic inhibitors

Taxoids

Sites of Action of Cytotoxic Agents – Cell Cycle Level

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Types of chemotherapy

• Cell cycle dependent – Cell cycle phase specific

• Cell cycle independent – Cell cycle phase non-specific

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Cycle-Specific Agents

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DNA synthesis

AntimetabolitesAntimetabolites

DNA

DNA transcription DNA duplication

Mitosis

Alkylating agentsAlkylating agents

Spindle poisons & Spindle poisons &

Microtuble StablizersMicrotuble Stablizers

Intercalating agentsIntercalating agents

Sites of Action of Cytotoxic Agents – Cellular Level

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6-MERCAPTOPURINE6-THIOGUANINE

METHOTREXATE5-FLUOROURACIL

HYDROXYUREAPEMETREXED

CYTARABINEGEMCITABINE

PURINE SYNTHESIS PYRIMIDINE SYNTHESIS

RIBONUCLEOTIDES

DEOXYRIBONUCLEOTIDES

DNA

RNA

PROTEINS

MICROTUBULESENZYMES

L-ASPARAGINASE

VINCA ALKALOIDS

TAXOIDS

ALKYLATING AGENTS

AKYLATING LIKE

(INTERCALATING)

ANTIBIOTICS

ETOPOSIDE

Sites of Action of Cytotoxic Agents

TOPOISOMER

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EXTRACELLULAR INTRACELLULAR

ATPATP

PGPPGP170170 ATPATP

DrugDrug

DrugDrug

PlasmaPlasmaMembraneMembrane

Drug Resistance

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Effect of tumorgrowth kinetics

Tubulin binding sitemutations

P-gp = P-glycoprotein.Dumontet and Sikic. J Clin Oncol. 1999;17:1061.

P-gp mediated drug efflux

Taxanes

Mechanisms of Taxane Resistance

Inhibition of apoptotic signaling

Altered metabolism by host

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Taxane Resistance Mediated through

Multidrug Resistance (MDR)

• MDR is mediated by mdr1 gene amplification encoding P-gp

• P-gp is a cell membrane protein

• Overexpressed in some chemoresistant tumors

• In chemosensitive tumours, can be upregulated after therapy

• Anthracyclines, taxanes, vinca alkaloids are P-gp substrates

NBF = nucleotide binding factor

2 3 4 5 6 7 8 9 10 11 12

Extracellular

Intracellular

NH2

NBF1 NBF2 COOH

1

me

mbra

ne

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Anti-Folate Transporters

Reduced Folate Carrier (RFC) THFs

Methotrexate, 5-FU,

Raltitrexed (Tomudex)

Pemetrexed (ALIMTA®)

Folate Receptor (FR-α)

Rothberg KG et al., J Cell Biol. 110: 637-649, 1990.Folic Acid, THFs

CB 3717l

Pemetrexed (ALIMTA®)

Efflux by MRP

Westerhof GR et al., Mol. Pharmacol 48: 459-471, 1995Zhao R et al., Clin Cancer Res 6: 3687-3695, 2000Pratt SE et al., Proc. Am. Assoc. Cancer Res 43: 782, 2002

MethotrexatePemetrexed (ALIMTA®)

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ADP

ATP

ALIMTA

MRPs

Multiple Drug Resistance Proteins &

Anti-Folate Drug Resistance

Reduced Folate CarrierReduced Folate Carrier

Low affinity for folic acid

High affinity for antifolates

High activity in malignant tissue

Membrane Folate ReceptorMembrane Folate Receptor

High affinity for folic acid

Low affinity for antifolates

High expression in certain malignancies (mesothelioma, ovary)

MDRs: Multiple Drug MDRs: Multiple Drug Resistance Resistance ProteinsProteins

RFC

Folate receptor

(cell membrane)

MFR

Anti-folate

Anti-folateAnti-folate

Anti-folate

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Tumour kinetic

Growth rate depends on:

growth fraction

-percent of proliferating cells within a given system

-human malignacy ranges from 20-70%

-bone marrow 30 %

cell cycle time

-time required for tumour to double in size

rate of cell loss

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Doubling times of some human tumours

Burkitt’s lymphoma 1.0

Choriocarcinoma 1.5

Hodgkin’s disease 3-4

Testicular embryonal carcinoma 5-6

Colon 80

Lung 90

Tumour Doubling times (days)

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Tumor Kinetics – Original Hypothesis

• Conventional views in the field of oncology support the notion that:

– tumor growth is exponential

– chemotherapy treatment is designed to kill in log intervals (kills constant fractions of tumor)

• Currently, chemotherapy for ovarian cancer is administered in 3-week intervals.

• Combination therapy and increased drug dose levels aim at improving ovarian cancer chemotherapy.

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Gompertzian Growth

• Growth rates are exponential at early stages of development and slower at later stages of development.

- Biological growth follows this characteristic curve.- Biological growth follows this characteristic curve.

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Gompertzian growth model

Initial tumour growth is first order, with later growth

being much slower

Smaller tumour grows slowly but large % of cell dividing

Medium size tumour grows more quickly but with

smaller growth fraction

Large tumour has small growth rate and growth fraction

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number of number of cancer cellscancer cells

diagnosticdiagnosticthresholdthreshold

(1cm)(1cm)

timetime

undetectable undetectable cancercancer

detectable detectable cancercancer

limit ofclinical

detection

hostdeath

10 10 1212

10 10 99

Tumor Growth

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Rationales in Human Cancers

• Small tumors grow faster than larger tumors

• Human cancers grow by non-exponential Gompertzian kinetics

50

Principle of chemotherapy

First order cell kill theory

- a given dose of drug kills a constant percentage of tumour cells rather than an absolute number

Maximum kill

Broad coverage of cell resistance

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Theoretical Tumor Kinetics

Tumour Surviving cells Viable mass Recovery of tumour kill (%) (doubling time)

untreated 109 1g -90 (1-log) 108 100mg 3.33 days99 (2-log) 107 10mg 6.66 days99.9 (3-log) 106 1mg 9.99 days99.99 (4-log) 105 100μg 13.3 days

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3 LOG KILL, 1 LOG REGROWTH

Time

TU

MO

R C

EL

L N

UM

BE

R

Chemotherapy

53

The rate of tumor volume regression is proportional to the rate of growth.

Tumor cell regrowth can be prevented if tumor cells

are eradicated using a denser dose rate of cytotoxic therapy.

Tumors given less time to grow in between treatments are more likely to be destroyed.

Hypothesis of Alternative Intervals

54

Principle of chemotherapy

Rationale for combination chemotherapy

Different drugs exert their effect through different mechanisms and at different stages of the cell cycle, thus maximize cell kill

Decease the chance of drug resistance

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Thanks for Your Attention

To Be Continued…..

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CYCLOPHOSPHAMIDE

4-OH CYCLOPHOSPHAMIDE

ALDOPHOSPHAMIDE

PHOSPHORAMIDE

MUSTARD

4-KETOCYCLOPHOSPHAMIDE

CARBOXYPHOSPHAMIDE

ACROLEIN

HEPATICCYTOCHROMES

P 450ACTIVATION

CYTOTOXICITYTOXICITY

INACTIVATION

ALDEHYDE

DEHYDROGENASE

Example: Metabolism of Cyclophosphamide