1. Cancer therapy IJsbrand Kramer i.kramer@iecb.u-bordeaux.fr

2. In this lecture we will treat - The principles of cancer chemotherapy - The principles of clinical trials - The development of novel anti-cancer drugs - the Abl tyrosine kinase inhibitor Gleevec - the HER2 receptor-inhibitor Herceptin Fab

3. Cancer can be treated in four ways: 1) surgical excision, 2) irradiation, 3) chemotherapy and 4) biological response modification. Cancer chemotherapy takes central stage in the cure of cancer and for certain tumours, high survival rates have been achieved.

4. The first anticancer drugs, the nitrogen mustards, were initially developed as toxic gases during the first World War. Victims of gas attacks were found to have very low white blood cell counts. It was then reasoned that these compounds could be developed as anticancer drugs for the treatment of leukaemia’s. These compounds are now known as the alkylating agents and are still an essential ingredient of chemotherapy regimes.

5. Anticancer drugs inhibit cell proliferation and although effective in combating cancer, they cause proliferation arrest in both disordered- and ordered-cells! Their mode of action is not based on a clear exploitable difference.

6. The exploitable difference may reside in the recovery phase after the treatment We may define the exploitable difference for cancer therapy as follows: a reduced capacity of the cancer cell to repair the damage induced by chemotherapy agents. Because of this, cancer cells pass on to their progenitors an increasing number of defects (mutations) that in the end prove to be lethal.

8. Anti-metabolites

13. Alkylating agents Mustard gas (yperite)

14. Alkylating agents

16. Platinum analogues Cisplatin (red) linking two nucleotides (blue)

17. Platinum analogues

18. Cytotoxic antibiotics

19. Inhibitors of microtubule polymerization or depolymerization

25. Development of new anticancer drugs New targets and how to test their clinical efficacy? -Principles of clinical trials -The development of Gleevec -The development of Herceptin Fab

26. 1 dose response test 2 randomized 3 double blind 4 inclusion of control Control: - application of placebo (no drug but this may not always be possible for ethical reasons (disabling or lethal disease)) - application of existing treatment (the disadvantage of such an approach is that you have no direct evidence of the therapeutic action of the drugs) Principals of Clinical trials

27. A Phase I Study: Phase I studies are primarily concerned with assessing the drug's safety. This initial phase of testing in humans is done in a small number of healthy volunteers (20 to 100), who are usually paid for participating in the study. The study is designed to determine what happens to the drug in the human body--how it is absorbed, metabolized, and excreted. A phase I study will investigate adverse effects that occur as dosage levels are increased. This initial phase of testing typically takes several months. About 70 percent of experimental drugs pass this initial phase of testing. NB: For cancer drugs this phase already contains selected patients that respond badly to the standard treatment and are in an advanced state of the disease. Principals of Clinical trials

28. A Phase Two Study: Once a drug has been shown to be safe and a dose response curve has been established, it must be tested for efficacy in a larger number of people. This second phase of testing may last from several months to two years, and involve up to several hundred patients. Randomized, double blind and with control. NB: For cancer therapy a different approach applies; the trial is not random, the patients are selected by criteria such as severity of disease and unresponsiveness to standard treatment. This study will provide the pharmaceutical company and the FDA comparative information about the relative safety of the new drug, and its effectiveness (therapeutic index). Only about one- third of experimental drugs successfully complete both phase I and phase II studies. Principals of Clinical trials

29. A Phase Three Study: In a phase III study, a drug is tested in several hundred to several thousand patients. This large-scale testing provides the pharmaceutical company and the FDA with a more thorough understanding of the drug's effectiveness, benefits, and the range of possible adverse reactions. Most phase III studies are randomized and blinded trials. Phase III studies typically last several years. Seventy to 90 percent of drugs that enter phase III studies successfully complete this phase of testing. Once a phase III study is successfully completed, a pharmaceutical company can request FDA approval for marketing the drug. Principals of Clinical trials

30. 1. Gleevec and the treatment of Chronic myelogenous lymphoma (CML* ) Chronic myelogenous leukemia (leukemia meaning « white blood ») results from an acquired (not inherited) chromosomal translocation between chromosome 9 and 22, resulting in the formation of a large and very small chromosome, called the Philadelphia chromosome. What produces this translocation is not yet understood. The translocation confers a growth and survival advantage on the stem cell and this leads to a massive increase of white blood cells. Unlike acute myelogenous leukemia, chronic myelogenous leukemia permits the development of mature white blood cells and platelets that generally can function normally. This important distinction from acute leukemia accounts for the less severe early course of the disease. * Also known as chronic granulocytic, chronic myelocytic or chronic myeloid leukemia

31. Chronic myelogenous lymphoma (CML) The disease typically begins with a chronic phase and progresses to blast crisis over a period of years. At the time of blast crisis, CML cells often have acquired secondary genetic abnormalities in addition to Brc-Abl. Because all patients express the Bcr-Abl protein, in both chronic and acute phase, it represents an ideal target for drug therapy

32. The incidence of CML increases with age: a good illustration of cancer being an age-related disease 40608020 age (years)

33. Chronic myelogenous lymphoma (CML): symptoms Carriers of the disease tire more easily and may feel short of breath when physically active. They may have a pale complexion from anemia. Discomfort on the left side of the abdomen from an enlarged spleen may be present. They may experience excessive sweating at night, weight loss, and inability to tolerate warm temperatures. Increasingly, the disease is discovered during the course of a periodic medical examination.

34. Abl Bcr Abl So named because this chromosomal translocation (Ph1) was discovered in Philadelphia in 1960. Bcr stands for breakpoint cluster region, a site in chromosome 22 which is frequently involved in translocations Abl stands for Abelson (gene product homologous to oncogenic virus carried by the Abelson leukemia virus v-Abl) Philadelphia chromosome

35. Abl Bcr Abl Bcr Abl-Bcr Fluorescence in situ hybridization, often referred to as FISH, is a method to identify cells with the 9;22 translocation characteristic of CML When Abl and Bcr are fused, the red and green fluorescent probes superimpose and give a yellow signal (so one set of chromosomes is still intact)

36. The translocation produces a fusion protein Abl-Bcr which is associated with Chronic Myelogenous Leukemia The Philadelphia chromosome harbours a fusion gene called Bcr-Abl

37. Abl has different activation states, from inactive (a) to intermediate states (c-f) and fully active (b). Bcr-Abl cannot return to an inactive state (g) and is said to be constitutive active.

38. Treatment of Chronic Myelogenous Leukemia Stem Cell Transplantation is applied when all fails (with matching HLA donor)

39. Interferon-  has been an important addition to the treatment of CML. In 2001, the Food and Drug Administration approved imatinib mesylate (Gleevec™ in the USA, and Glivec™ elsewhere) for patients resistant or unable to tolerate interferon- . The drug has been very effective in this setting and may become the most common first choice for treatment in the future. Treatment of Chronic Myelogenous Leukemia

40. Signal Transduction Inhibitor STI-571 4-(4-METHYL- PIPERAZIN-1-YLMETHYL)-N-[4-METHYL- 3-(4-PYRIDIN-3-YL-PYRIMIDIN-2-YLAMINO)-PHENYL]- BENZAMIDE (Novartis) Also known as Gleevec, Glivec or imatinib In search for an inhibitor of Abl

41. Specificity of inhibition STI-571 inhibits (until today) - Bcr-Abl - PDGF-R - c-kit (receptor for the Stem Cell Factor)

42. FDA approval (in 2002) for treatment of newly diagnosed patients with Gleevec The food and drug administration (FDA) approved Gleevec as a first-line therapy for CML based on the data obtained from a 12 month international randomized trial of Interferon-a versus Gleevec (STI-571). This phase III trial involved 553 patients that were given Gleevec and 553 that were given a standard therapy of Interferon-a and cytarabine (anti- metabolite). The patients treated with Gleevec after one year had significantly fewer cancerous cells in their blood and bone marrow. The rate of progression was also decreased. The lenght of the follow up is still too short to measure long term clinical benefits (generally presented 5 years survival rates)

43. Adverse effects of Gleevec treatment Most of the adverse affects can be managed without stopping therapy. Among others they are: - Fluid retention - Nausea - Vomiting - Muscle cramps - Diarrhoea -Rashes and (very rarely) -heart failure (late onset adverse effect)

44. Timeline of major CML discoveries and drug development A-MuLV : Abelson murine leukemia virus GAG-Abl : fusion product of viral glycosylated antigen with the Abl tyrosine kinase

45. 2. Herceptin Fab and the treatment of Her2-driven metastatic breast cancer - The human epidermal growht factor receptor-2 (ERBB2, also known as neu or Her2) is amplified in 30% of breast cancers. - Amplification of ERBB2 is associated with an aggressive form of the disease (metastaticwith shortened overall survival time. - In vitro experiments have demonstrated a direct role of ERBB2 in the pathogenesis of this cancer

46. ERBB2 is a member of the family of EGF receptors but, until today, lacks a high affinity ligand. Amplification of ERBB2 causes cell transformation without the apparent involvement of a ligand. This may be explained by its « open » conformation (unlike ERBB1, 3 or 4) ERBB2 extracellular domain ERBB2 extracellular domain + antibody dimerization arm

48. treatmentcontroltreatmentcontrol Trastuzumab 4mg/kg (0.22 gr/person), anti-HER2 Fab fragment Anthracycline is doxorubucin, inhibitor of DNA polymerase Cyclophosphamide is a alkylating agent that binds DNA and inhibits DNA polymerase Paclitaxel is taxol, inhibitor of microtubule depolymerization Example of a clinical trial protocol in which Herceptin (trastuzumab) has been tested

49. Survival rates of metastatic ERBB2-positive breast cancer The use of trastuzumab (mAb) increases the percentage of survival by 10% after two years (red lines).