Part 1: Tumor Biology and Kinetics Introduction of Cytotoxic Agents

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Part 1 Tumor Biology and KineticsIntroduction
of Cytotoxic Agents

• Pharmacologic Anti-Cancer Treatments Seminars
  2007

Carlos Linn, M.D. ??? ?? Clinical Research
Physician, Oncology Lilly Oncology Board
Certified Gynecologic Oncologist
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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)

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The Cell Cycle
DEATH
G0
DIFFERENTIATION
Mitosis M S DNA synthesis
DNA content 2n
DNA content 4n
G2 G1
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The Cell Cycle
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Cancer Cells and Normal Cells
CANCER CELLS NORMAL CELLS
Frequent mitoses
Normal cell Few mitoses
Nucleus
Blood vessel
Abnormal heterogeneous cells
Oncogene expression is rare Intermittent or
coordinated growth factor secretion Presence of
tumor suppressor genes
Loss of contact inhibition Increase in growth
factor secretion Increase in oncogene
expression Loss of tumor suppressor genes
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Growth Factors and Oncogenes
Paracrine (Adjacent cells)
Autocrine stimulation
Growth Factor and Receptor Synthesis
Growth Factor
Growth Factor Receptor
Post receptor signal transduction pathways
Gene Activation
Oncogenes
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Oncogenesis
NORMAL GROWTH AND DEVELOPMENT
NORMAL EXPRESSION RESPONSIVE ONCO SUPPRESSION
GENE
MUTAGENIC or CARCINOGENIC AGENTS
CELLULAR ONCOGENE
VIRAL ONCOGENE
INCREASED OR ABNORMAL EXPRESSION CANCER GROWTH
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Example Oncogenesis Integrated by HPV

• Integration of HPV DNA genome E6, E7 into
  Host-cell
• Immortally malignant
• NO more Koliocytosis
• Virus stops duplication

Complete viral life cycle with Koliocytosis ?
Virus duplication
Beutner, KR et al, "Human Papillomavirus and
Human Disease." Am J Med 1997 102(5A)9-15.
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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-dependent kinase
inhibitors (CKI)
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CYCLIN DEPENDENT KINASES
tyr15-P
thr14-P
P-thr161
- protein kinase - binds to cyclin - kinase
domain - regulatory domain - present throughout
cell cycle
e.g. cdk1 ( cdc2)
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CYCLINS
- No intrinsic enzymatic activity - Binds cdk -
Synthesized and degraded each cycle - Essential
component for cdk activity
e.g. Cyclin B
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CYCLIN / CDK
tyr15-P
thr14-P

• Regulated by
• - tyr15 phosphorylation
• inhibitory kinases
• activating phosphatases
• - Direct interaction
• inhibitory proteins
• p21, p27, p57
• p16, p15, p18,p19

cdk1 (cdc2)
P-thr161
cyclin B
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CELL CYCLE CHECKPOINTS
CYCLIN A / cdk 2
CYCLIN B / cdk 1
CYCLIN D / cdk 4,5,6
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Variation in Cell Cycle Cyclins
Cyclin-dependent kinases (CDK)
Cyclins
Cell cycle phases
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Cell Cycle
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E6, E7 involvement in cell cycle regulation
DNA damage
Phosphorylation
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DNA Damage - Cell Cycle ArrestDamage Dependent
Checkpoints
G1 - S - G2
G1 - S - G2
CELL No.
wild-type
DNA content
DNA content
Asynchronous
X-ray treated G1/S block G2/M block (6-9 hours)
Loss of G1/S in p53 deficient cells
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G1/S CHECKPOINTIN RESPONSE TO DAMAGE
X-rays
P-tyr15
cdk2
strand break
p53
p21
ATM
cyclin E
p21 CKI class (cyclin dependent kinase
inhibitors) N-terminal of p21 forms complex with
cyclin / cdk - inhibit kinase
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Cell Cycle Regulation
DNA damage
1. CDK phosphorylation
2. C degradation
3. C CDK synthesis
CDK2
4. CDK inhibition
CE
pRb
pRb
pRb
E2F
Enzymes for DNA synthesis
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Growth Factors Cell Cycle
Receptors
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Retinoblastoma protein (pRb) CDK inhibitors
p21, p27, p16
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The Normal Cell Cycle Cyclins of the cell
cycle
E6, E7 immortalize human keratinocyte
E5 protein
G1 arrest
Neoplastic cells (immortal)
Normal cell cycle (with tumor suppression and
apoptosis)
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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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Antineoplastic Agents
(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)
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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 synthesiseither 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
Pacific Yew Taxus brevifolia
OH
1986
European Yew Taxus baccata
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Classification of Cytotoxic Agents
ALKYLATING AGENTS
ANTI- METABOLITES
MITOTIC INHIBITORS
ANTIBIOTICS
OTHERS

• BUSULFAN CYTOSINE ETOPOSIDE BLEOMYCIN L-ASPARAGINA
  SE
• CARMUSTINE ARABINOSIDE TENIPOSIDE DACTINOMYCIN HYD
  ROXYUREA
• CHLORAMBUCIL FLOXURIDINE VINBLASTINE DAUNORUBICIN
  PROCARBAZINE
• CISPLATIN FLUOROURACIL VINCRISTINE DOXORUBICIN
• CYCLOPHOSPHAMIDE MERCAPTOPURINE VINDESINE MITOMYCI
  N-C
• IFOSFAMIDE METHOTREXATE TAXOIDS MITOXANTRONE
• MELPHALAN GEMCITABINE TAXANES PLICAMYCIN
• PEMETREXED ANTHRACYCLINES
• EPOTHILONES

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Sites of Action of Cytotoxic Agents
Cell Cycle Level

• Antibiotics
• Antimetabolites

S (2-6h)
G2 (2-32h)
Vinca alkaloids
M (0.5-2h)
Mitotic inhibitors
Taxoids
Alkylating agents
G1 (2-h)
G0
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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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Sites of Action of Cytotoxic Agents
Cellular Level

• DNA synthesis

Antimetabolites
Alkylating agents
DNA
DNA transcription
DNA duplication
Mitosis
Intercalating agents
Spindle poisons Microtuble Stablizers
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Sites of Action of Cytotoxic Agents
PURINE SYNTHESIS
PYRIMIDINE SYNTHESIS

• 6-MERCAPTOPURINE
• 6-THIOGUANINE
• METHOTREXATE
• 5-FLUOROURACIL
• HYDROXYUREA
• PEMETREXED
• CYTARABINE
• GEMCITABINE

RIBONUCLEOTIDES
ALKYLATING AGENTS AKYLATING LIKE
(INTERCALATING) ANTIBIOTICS
DEOXYRIBONUCLEOTIDES
DNA
ETOPOSIDE
RNA
TOPOISOMER
L-ASPARAGINASE VINCA ALKALOIDS TAXOIDS
PROTEINS
MICROTUBULES
ENZYMES
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Drug Resistance

• EXTRACELLULAR INTRACELLULAR

PGP170 ATP
Drug
ATP
Drug
Plasma Membrane
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Mechanisms of Taxane Resistance
Effect of tumor growth kinetics
Altered metabolism by host
Taxanes
P-gp mediated drug efflux
Tubulin binding sitemutations
Inhibition of apoptotic signaling
P-gp P-glycoprotein. Dumontet and Sikic. J Clin
Oncol. 1999171061.
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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

Extracellular
membrane
1
2
3
4
5
6
7
8
9
10
11
12
COOH
NBF1
NBF2
NH2
Intracellular
NBF nucleotide binding factor
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Anti-Folate Transporters
Reduced Folate Carrier (RFC)
THFs Methotrexate, 5-FU, Raltitrexed
(Tomudex?) Pemetrexed (ALIMTA)
Folate Receptor (FR-a) Rothberg KG et al., J
Cell Biol. 110 637-649, 1990.
Folic Acid, THFs CB 3717l Pemetrexed (ALIMTA)
Methotrexate Pemetrexed (ALIMTA)
Efflux by MRP
Westerhof GR et al., Mol. Pharmacol 48 459-471,
1995 Zhao R et al., Clin Cancer Res 6 3687-3695,
2000 Pratt SE et al., Proc. Am. Assoc. Cancer Res
43 782, 2002
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Multiple Drug Resistance Proteins Anti-Folate
Drug Resistance
Reduced Folate Carrier
Anti-folate
Anti-folate
RFC
Low affinity for folic acid
High affinity for antifolates
High activity in malignant tissue
ALIMTA
Folate receptor
Membrane Folate Receptor
Anti-folate
Anti-folate
MFR
ADP ATP
High affinity for folic acid
Low affinity for antifolates
MRPs
High expression in certain malignancies
(mesothelioma, ovary)
(cell membrane)
MDRs Multiple Drug Resistance Proteins
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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
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.

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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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Tumor Growth

• number of
• cancer cells

10 12 10 9
diagnostic threshold (1cm)
time
detectable cancer
undetectable cancer
host death
limit of clinical detection
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Rationales in Human Cancers

• Small tumors grow faster than larger tumors
• Human cancers grow by non-exponential Gompertzian
  kinetics

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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 days 99
(2-log) 107 10mg 6.66 days 99.9
(3-log) 106 1mg 9.99 days 99.99
(4-log) 105 100µg 13.3 days
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3 LOG KILL, 1 LOG REGROWTH
TUMOR CELL NUMBER
Time
Chemotherapy
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Hypothesis of Alternative Intervals

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

Tumors given less time to grow in between
treatments are more likely to be destroyed.
Tumor cell regrowth can be prevented if tumor
cells are eradicated using a denser dose rate of
cytotoxic therapy.
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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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Example Metabolism of Cyclophosphamide

• CYCLOPHOSPHAMIDE

HEPATIC CYTOCHROMES P 450
ACTIVATION
INACTIVATION
4-OH CYCLOPHOSPHAMIDE ALDOPHOSPHAMIDE
4-KETOCYCLOPHOSPHAMIDE CARBOXYPHOSPHAMIDE
ALDEHYDE DEHYDROGENASE
PHOSPHORAMIDE MUSTARD
ACROLEIN
TOXICITY
CYTOTOXICITY