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

2. 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

3. Evolution of Chemotherapy
1940’s – first use of successful use alkylating agent nitrogen mustard to treat human cancer
’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.

4. 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

5. 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

6. 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!

7. Clinical Implications of Fractional Cell Kill

8. 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: )

9. 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

10. 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.

11. 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)

12. 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

13. 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

14. 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

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

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

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

18. 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

19. 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

20. 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

21. Antimetabolite Basics

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

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

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

25. 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

26. 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

27. 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

28. 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

29. 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

30. 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

31. 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

32. 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

33. Effects of Vincristine on T-cell Leukemia Cell Line

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

35. 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

36. 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

37. 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)
Typically given as outpatient every 3-4 weeks, to allow recovery of normal tissue side effects

38. 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

39. 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

40. 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

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

42. 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

44. 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

45. 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

46. 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

48. 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

49. 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

50. 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