Pharmacology of Chemotherapy David W. Hedley MD Dept Medical Oncology and Hematology Division of Applied Molecular Oncology

Chemotherapy Cytotoxic agents - generally given by intravenous injection or orally Most chemotherapy drugs act by damaging DNA or inhibiting DNA synthesis Important exceptions are drugs that target microtubules

Evolution of Chemotherapy 1940’s – first use of successful use alkylating agent nitrogen mustard to treat human cancer 1950-1960’s – major alkylating agents and anti-metabolites currently in use synthesized. Effective against wide range of cancer types, particularly rapidly growing leukemias and lymphomas. Scientific principles of cancer chemotherapy developed.

The Science of Chemotherapy Many early concepts derived from radiation biology: - clonogenic survival - fractional cell kill - need to eliminate all clonogenic cells to achieve cure - normal host effects

The Science of Chemotherapy L1210 Mouse Lymphoma: Howard Skipper’s work (1960’s) Rapidly growing tumours Initially used animal survival to infer cancer cell killing in vivo Based on relation between number of cells inoculated and survival

The Science of Chemotherapy Fractional cell kill of L1210 lymphoma inoculated at 1x106 in vivo Single treatment with chemotherapy drug Effect on lifespan with increasing log cell kill Even though you get a lot of cancer cell, difficult to achieve cure!

Clinical Implications of Fractional Cell Kill

The Science of Chemotherapy - Bob Bruce Data Showing Cell Cycle Dependence of Killing by g-Radiation, Nitrogen Mustard, and Tritiated Thymidine in Lymphoma vs. Normal Bone Marrow in vivo (JNCI 1966;37:233-245)

The Science of Chemotherapy Ian Tannock’s PhD Project Using tritiated thymidine autoradiography, showed solid tumours proliferate more slowly further away from blood vessels. Cause of drug resistance

Evolution of Chemotherapy 1970’s - “Golden Age” of medical oncology. Development of effective combination chemotherapy regimens. New classes of drug developed - anthracyclines, platinum compounds Cures achieved in some forms of cancer (lymphomas, leukemias, testis cancer). Significant responses in some common types of cancer (breast, stomach, small cell lung cancer) Effective use of chemotherapy to prevent recurrence in high risk breast cancer patients.

Evolution of Chemotherapy 1980’s – disillusion sets in. Development of increasingly complex, toxic (and expensive) treatment protocols Some improvement in response rates, but hope fades for curing common forms of cancer Intensive search for analogues of existing drugs, hoping for greater anticancer effect or less toxicity Introduction of remaining major types of chemotherapy (taxanes, topoisomerase I inhibitors)

Evolution of Chemotherapy 1990’s –still hard going in the clinic, but ….. Post-operative adjuvant chemotherapy established to reduce mortality in some major causes of cancer death (breast, colon cancer) Biochemical basis of drug resistance established Idea that development of cancers involves suppression of cell death pathways, and that drug resistance results from failure of damaged cells to undergo apoptosis - e.g. bcl2, p53 stories

Evolution of Chemotherapy 2000’s – rapid development of molecular targeted agents as alternatives to classical chemotherapy Evolution of molecular oncology and rational cancer therapeutics - integrating basic science, pharmacology, pathology, and clinical oncology

Classification of Chemotherapy (difficult to do; many drugs first identified by empirical screening for anticancer effect, rather than rational synthesis) DNA damaging agents - alkylating agents - platinum compounds Antimetabolites Topoisomerase inhibitors Anti-mitotic agents

Nitrogen Mustard First chemotherapy agent used in man Prototype alkylating agent Main toxicity comes from DNA cross linkage

DNA Cross Linkage Arrests DNA replication Can result in DNA damage and chromosome breaks Also mutagenic!

Chlorambucil Derivative of nitrogen mustard Much less reactive Well absorbed by mouth Remains major drug for treating low grade lymphomas

Cyclophosphamide More complex activation than nitrogen mustards; requires cytochrome p450 in liver Can be given orally or intravenously Main side effects are bone marrow suppression

Other DNA Damaging Agents Platinum compounds - prototype cisplatin - main effect interstrand cross links - many analogues produced with broader spectrum Nitrosoureas - transfer chloroethyl group to guanine at O6 position

Antimetabolite Drugs Designed to block DNA synthesis Based on idea that cancer cells divide more rapidly than normal cells, so more vulnerable Originally considered to be cytostatic rather than cytotoxic, but now recognized that many produce cell death by triggering apoptosis Unlike most conventional chemotherapy drugs, development by rational synthesis rather than empirical screening for anticancer effects Most are either nucleoside analogues that interfere with DNA synthesis, or block methylation of uracil to thymidylate

Antimetabolite Basics

Nucleoside Analogues - biochemical pharmacology mirrors the uptake and metabolism of normal nucleosides

Cytosine arabinoside (cytarabine; Ara-C) - major chemotherapy drug to treat acute leukemias

Ara-CTP competes with dCTP inhibits DNA polymerases incorporation into DNA produces strand breaks; triggers apoptosis

Ara-C in treatment of acute leukemia High doses to overcome transport resistance Because ara-C targets cells in S-phase, given over 5-7 days to account for slow cycling populations of leukemia cells Major toxicity is suppression of blood counts

Inhibition of Thymidylate Synthesis Pyrimidine base 5-Fluorouracil (5FU) inhibits thymidylate synthase Methotrexate inhibits dihydrofolate reductase, reducing flow of methyl group carried by reduced folate

Effects of TS Inhibition Decrease in dTTP associated with build up of dUTP Mis-incorporation of dUTP into DNA This is removed by DNA repair pathways However, DNA repair synthesis also mis-incorporates dUTP: “futile repair” cycle results in extensive DNA damage 5-fluorouracil and methotrexate both clinically important drugs

Natural Products Discovered by empirically screening compounds for anticancer effects in vitro (similar to antibiotic discovery) Mechanisms of action subsequently identified Important compounds showing effect in lymphoma (and other cancers) are: - topoisomerase inhibitors - microtubule inhibitors

Topoisomerase II Inhibitors Topoisomerase II allows replicated DNA strands to separate by making breaks, then re-ligating Main class of topo II inhibitors are the anthracyclines, originally from Streptomyces Intercalate into DNA and prevent re-ligation step Daunorubicin is the classical anthracycline used to treat acute leukemia

Daunorubicin toxicity Bone marrow suppression is most important early toxicity Also causes gastro-intestinal toxicity - nausea, vomiting, diarrhea Hepatobiliary excretion is major route for drug elimination – toxicity greater in presence of jaundice Cardiac toxicity is most important late effect - risk increases with accumulated dose - can result in fatal cardiac failure

Mechanism of Anthracycline Cardiac Toxicity As well as intercalating into DNA, daunorubicin avidly binds mitochondrial inner membrane of cardiac muscle Daunorubicin chelates iron, which catalyzes formation of the free radical semiquinone Redox cycling transfers high energy electron to oxygen, generating oxygen free radicals Produce lipid peroxidation damage to mitochondrial membranes

Microtubule Inhibitors Vinca alkaloids (from periwinkle plant) - destabilize microtubules - vincristine commonly used to treat acute lymphoblastic leukemias Taxanes (from Pacific yew tree bark) - stabilize microtubules - taxotere most active in current use Main effect of these drugs is to cause metaphase arrest and chromosomal damage - probably have additional effects due to microtubule disruption in interphase cells

Effects of Vincristine on T-cell Leukemia Cell Line

Chemical Structure of Taxol Many natural products are very complex organic molecules. Complexity can make them useful starting points for drug development

Why Don’t We Cure Cancer With Chemotherapy? Toxic side effects limit dose Cancer cells show drug resistance - innate drug resistance, or acquired resistance during treatment

Toxic Effects of Chemotherapy Generic side effects are damage to rapidly dividing normal cells - bone marrow, gut mucosa, hair follicles Nausea due to triggering CNS vomiting centres Drug-specific side effects: - myocardium (anthracylines) - kidney (platinum) - nervous system (microtubule agents) Toxicity increases with dose of drug used

Combination Chemotherapy Drugs selected for combinations based on: - differences in side effects; allows each to be used at full dose - different mechanisms of action; cancer less likely to be cross-resistant Prototype curative combination is MOPP (nitrogen mustard, vincristine (Oncovin), procarbazine and prednisone (glucocorticoid steroid) - 1970 Typically given as outpatient every 3-4 weeks, to allow recovery of normal tissue side effects

How Far Can You Push Chemotherapy? Generally, the more you give the greater the anticancer effect Bone marrow suppression is main lethal side effect of chemotherapy Bone marrow transplantation (either patient’s or normal donor) bypasses this dose-limiting toxicity, allows more chemo. Can be curative in some situations, including acute leukemia Although higher response rates, other side effects may prevent curative doses from being achieved

Drug Resistance Main factor determining if a cancer will be cured with chemotherapy Complex and multifactorial, but main causes of drug resistance are probably now understood

Development of Drug Resistance in a Leukemia Patient Antique chart (from my residency days!) of newly diagnosed AML patient treated with ara-C plus daunorubicin, followed by ara-C plus 6-thioguanine maintenance chemotherapy represents circulating leukemic blasts Shows initial clearance of leukemia with treatment, and reappearance of normal granulocytes and platelets

Development of Drug Resistance in a Leukemia Patient Blood counts remaining fairly normal during maintenance chemotherapy

Development of Drug Resistance in a Leukemia Patient Eventually leukemic blasts reappear in circulation Initially respond to intensified chemotherapy Then rapid accumulation of drug resistant population

High dose Ara-C can overcome transport resistance because transporter is non-saturable Activity of deoxycytidine kinase can be increased by inhibiting endogenous deoxycytidine using ribonucleotide reductase inhibitors - but these can also enhance normal tissue toxicity

Overcoming Drug Resistance Some cellular mechanisms of multidrug resistance (P-glycoprotein-mediated drug efflux, glutathione conjugation) can be reversed pharmacologically Able to enhance anticancer effects in model systems Results in clinical trials disappointing - probably because of multifactorial nature of drug resistance

P-glycoprotein First multidrug resistance mechanism to be characterized (Vic Ling, OCI, 1975) P-glycoprotein is transmembrane ATP-dependent efflux pump Actively transports many types of chemotherapy from cells (anthracyclines, vinca alkaloids, taxanes) Overexpression in cancers causes drug resistance P-glycoprotein inhibitors tested in clinical trials

P-glycoprotein in Acute Leukemia P-glycoprotein overexpressed in some AML patients Higher levels associated with drug resistance and worse prognosis But clinical trials of P-glycoprotein inhibitors fail to show significant improvement in chemotherapy response - other resistance mechanisms also operating - high Pgp levels might be linked to aggressive biology, rather than directly to drug resistance

Targeting Cell Survival Pathways Recent evidence that failure of DNA damaged cells to undergo apoptosis is major cause of multidrug resistance Suppression of apoptosis often occurs due to oncogenic mutations – i.e. common feature of cancers Potential to reverse this mechanism by molecular therapies – e.g. p53 gene therapy or small molecule inhibitors of PI3-kinase pathway

Where is Chemotherapy Going? Incremental improvements in patient outcome continue, using newer drugs and combinations Unlikely that this will result in major improvements in cure rates for common forms of cancer Over past 2-3 years drug development programs refocused on molecular targeted therapeutics Potential for major advances based on new biology Molecular oncology revolution will need close interactions between clinical oncology, pathology, pharmacology, and basic science