Part 1: Tumor Biology and Kinetics Introduction of Cytotoxic Agents Pharmacologic Anti-Cancer Treatments Seminars 2007: Part 1: Tumor Biology and Kinetics Introduction of Cytotoxic Agents Carlos Linn, M.D. 林錦洲 醫師 Clinical Research Physician, Oncology Lilly Oncology Board Certified Gynecologic Oncologist

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)

The Cell Cycle G0 M S G2 G1 DEATH DIFFERENTIATION Mitosis DNA synthesis DNA content = 4n G2 G1 DNA content = 2n

The Cell Cycle Cells go through a cycle that consists of four stages: G1, S, G2, and M. During G1, growth occurs as organelles double. During the S stage, DNA replication occurs as chromosomes duplicate. During the G2 stage, growth occurs as the cell prepares to divide. During the M stage, mitosis and cytokinesis occur.

Cancer Cells and Normal Cells CANCER CELLS NORMAL CELLS Frequent mitoses Normal cell Few mitoses Nucleus Blood vessel Abnormal heterogeneous 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 coordinated growth factor secretion Presence of tumor suppressor genes

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

RESPONSIVE ONCO SUPPRESSION GENE 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

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.

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)

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)

CYCLINS - No intrinsic enzymatic activity - Binds cdk - Synthesized and degraded each cycle - Essential component for cdk activity e.g. Cyclin B

CYCLIN / CDK Regulated by: cyclin B - tyr15 phosphorylation cdk1 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

CELL CYCLE CHECKPOINTS CYCLIN A / cdk 2 CYCLIN B / cdk 1 CYCLIN D / cdk 4,5,6

Variation in Cell Cycle Cyclins Cyclin-dependent kinases (CDK) CDK 4 CDK 2 CDK 1 Cyclins D E A B(A) M G1 G2 S Start G1: gap phase 1 S: DNA synthesis phase G2: gap phase 2 M: mitosis Cdk: cyclin-dependent kinase Cell cycle phases

Cell Cycle RNA, Protein Mitosis, Cytokinesis G2 M G0 CDK1 CDK4,6 1 h Cyclin B/A CDK1 3-4 h RNA, Protein 1 h Mitosis, Cytokinesis Lamin H1 Abl M G0 G2 Cyclin D’s CDK4,6 G1 6-12 h RNA, Protein S 6-8 h DNA, RNA, Protein Cyc: cyclin CDK: cyclin dependent kinase RNA: ribonucleic acid p53: cell cycle control protein, MW 53kDa, also involved in other phases of cell cycle pRb: retinoblastoma protein DNA: deoxyribonucleic acid H1: histone H1 Abl: Abelson interacting protein Cyclin A CDK2 p53 pRb Cyclin E CDK2

E6, E7 involvement in cell cycle regulation DNA damage Phosphorylation

DNA Damage - Cell Cycle Arrest Damage 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

G1/S CHECKPOINT IN RESPONSE TO DAMAGE X-rays P-tyr15 cdk2 strand break ATM p53 p21 cyclin E p21 = CKI class (cyclin dependent kinase inhibitors) N-terminal of p21 forms complex with cyclin / cdk - inhibit kinase

Cell Cycle Regulation 1. CDK phosphorylation DNA damage Active p53 2. C degradation 3. C & CDK synthesis p21 CDK2 4. CDK inhibition CE pRb P CDK2: cyclin dependent kinase 2 CE: cyclin E pRb: retinoblastoma protein E2F: transcription factor E2F DNA: deoxyribonucleic acid p53: cell cycle control protein, MW 53 kDa p21: protein, MW 21 kDa pRb pRb pRb E2F Enzymes for DNA synthesis Passage from G1 to S

Growth Factors & Cell Cycle Gene Transcription Receptors + S Priming G1 G0 G2 Cell Cycle M

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

The Normal Cell Cycle & “Cyclins” of the cell cycle E6, E7: immortalize human keratinocyte E5 protein G1 arrest Normal cell cycle (with tumor suppression and apoptosis) Neoplastic cells (immortal)

Common Chemotherapeutic Agents Alkylating agents Antimetabolites Antitumor Antibiotics Alkaloids Taxanes

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

Antineoplastic Agents Alkylating agents Carboplatin, cyclophosmamide, melphalan, thiotepa (Form bonds with nucelic acids and proteins) Antimetabolites Methotrexate, fluorouracil, gemcitabine (similar to metabolites involved in nucelic acid synthesis) Natural Products doxorubicin, docetaxel, vinolbine, topotecan (anti tumour antibiotics,mictotubule stabilizer, mitotic inhibitor, topoisomerase inhibiotor) Endocrine agents Anastrozole, tamoxifen, prednisolone, goserelin (Aromatase inhibitors, oestrogen antagonist, corticosteroids, LHRH agonist) Molecularly targeted agents Retinoids, trastuzumab, gefitinib (gene expression, monoclonal antibody, tyrosine kinase inhibitor) Biologic response modifiers Interferon, thalidomide, filgrastim

Alkylating Agents Interact with DNA causing substitution reactions, cross-linking reactions or strand breaks Example: cisplatin

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

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)

Alkaloids Bind free tubulin dimers Disrupting balance between microtubule polymerization and depolymerization Arrest of cells in metaphase Examples: vincristine, vinblastine, vinorelbine

Taxanes Disrupt equilibrium between free tubulin and microtubules Stabilization of cytoplasmic microtubules Formation of abnormal bundles of microtubules Examples: paclitaxel and docetaxel

Paclitaxel & Docetaxel 1971 Pacific Yew: Taxus brevifolia OH 1986 European Yew: Taxus baccata

Classification of Cytotoxic Agents ALKYLATING AGENTS ANTI- METABOLITES MITOTIC INHIBITORS ANTIBIOTICS OTHERS 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

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

Types of chemotherapy Cell cycle dependent Cell cycle independent Cell cycle phase specific Cell cycle independent Cell cycle phase non-specific

Cycle-Specific Agents

Sites of Action of Cytotoxic Agents – Cellular Level DNA synthesis Antimetabolites DNA Alkylating agents DNA transcription DNA duplication Intercalating agents Mitosis Spindle poisons & Microtuble Stablizers

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 ENZYMES MICROTUBULES

Drug Resistance EXTRACELLULAR INTRACELLULAR PGP170 ATP Drug ATP Drug Plasma Membrane

Mechanisms of Taxane Resistance Altered metabolism by host Effect of tumor growth kinetics Taxanes P-gp mediated drug efflux Tubulin binding site mutations Inhibition of apoptotic signaling P-gp = P-glycoprotein. Dumontet and Sikic. J Clin Oncol. 1999;17:1061.

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 NBF1 NBF2 COOH NH2 Intracellular NBF = nucleotide binding factor

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®) 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

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 MFR Anti-folate 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

Tumour kinetic Growth rate depends on: growth fraction cell cycle time 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

Doubling times of some human tumours Tumour Doubling times (days)

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.

Gompertzian Growth Growth rates are exponential at early stages of development and slower at later stages of development. - Biological growth follows this characteristic curve.

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

Tumor Growth 10 12 10 9 diagnostic threshold (1cm) time detectable number of cancer cells 10 12 10 9 diagnostic threshold (1cm) time undetectable cancer detectable cancer limit of clinical detection host death

Rationales in Human Cancers Small tumors grow faster than larger tumors Human cancers grow by non-exponential Gompertzian kinetics

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

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

3 LOG KILL, 1 LOG REGROWTH TUMOR CELL NUMBER Time Chemotherapy

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.

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

Thanks for Your Attention To Be Continued…..

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