DEVELOPMENT OF NEW TREATMENTS IN CANCER

1. DEVELOPMENT OF NEW TREATMENTS IN CANCER C. Erlichman M.D. Good morning. Thank you Professors Rosso and Colucci for the kind invitation to participate in this exciting conference on colorectal cancer in the beautiful city of Bologna.Good morning. Thank you Professors Rosso and Colucci for the kind invitation to participate in this exciting conference on colorectal cancer in the beautiful city of Bologna.

2. OUTLINE Preclinical Development Trials: Phase 1 Phase 2 Phase 3 Biomarkers in Cancer Treatment Trials Informed Consent and Regulations

3. PRECLINICAL STUDIES

5. NCI Drug Development Stages

6. NCI Drug Development Stages

7. SCREENING FOR NEW DRUGS Testing in tissue culture (in vitro) 60 human cancer cell lines patterns identified that suggest a new way of killing cancer cells interesting agents tested in animal models

9. Advantages and Disadvantages of Cancer Cell Line Drug Screens The outline of my presentation will be to give you an overview of some of the new targets and agents of available to act on these targets. Subsequently, I will describe ongoing studies of four compounds, CI-1033, a HER family tyrosine inhibitor, CI-1040, a MEK inhibitor, PS 341, a proteasome inhibitor, and 17AAG, an HSP-90 inhibitor.The outline of my presentation will be to give you an overview of some of the new targets and agents of available to act on these targets. Subsequently, I will describe ongoing studies of four compounds, CI-1033, a HER family tyrosine inhibitor, CI-1040, a MEK inhibitor, PS 341, a proteasome inhibitor, and 17AAG, an HSP-90 inhibitor.

10. Preclinical Pharmacology Development of Drug Plasma Method Determine the extent of protein binding Plasma elimination kinetics of i.v. administration Other routes of administration such as oral, may be evaluated as well The in vitro and in vivo metabolism is characterized using liver extracts

11. Preclinical Pharmacology Preliminary (Range-Finding) Toxicity: For each drug, it is necessary to establish a maximum tolerated dose (MTD) and dose limiting toxicities (DLT) in both rodent and non-rodent species. Testing in human tumor models growing in mice to determine whether the drug has activity For each drug, the effect of treating animals with different tumors is evaluated looking at stopping tumors from growing and prolonging of the animals life tumor types tested depends in part of the mechanism of action and where there is activity in tissue culture

12. IND Toxicology Single or multiple daily dose schedules such as Dx1, q3hr x 3, q8hr x 15, q4D x 3, etc. Up to twenty-eight days or more of repeated administration of drug to rodents and non-rodents.

13. IND Toxicology Multiple cycle studies may be required if delayed or cumulative toxicity is anticipated. Special studies such as cardiotoxicity, neurotoxicity evaluations, and immunotoxicity studies may be required as part of an existing study or in a separate study.

14. CANCER CLINICAL TRIALS

15. ELIGIBILITY CRITERIA Primarily to ensure safety To ensure some level of uniformity of the study population To select a population of interest

16. COMMON ELIGIBILITY CRITERIA Evidence of cancer Age Performance status Adequate blood counts Adequate liver function blood tests Adequate kidney function Reasonable life expectancy ? 3 months Capable of giving informed consent

17. COMMON INELIGIBILITY CRITERIA Has had therapy (XRT, Chemo) within 4 weeks Pregnancy Breast feeding Prior treatments that may be similar Recovery from surgery Infections Brain metastases Cannot take oral medications

18. Phase I Trial Goals 1. To determine MTD and phase II recommended dose 2. To describe all toxicities 3. To determine the clinical pharmacokinetics (PK) 4. To report any responses 5. To relate PK to toxicities and/or biologic effect 6. To evaluate potential markers of biologic endpoints (pharmacodynamics) 7. To relate PK to genetic polymorphisms

19. Pharmacokinetics Pharmacodynamics Pharmacogenetics Pharmacokinetics What the body does to the drug AUC, clearance, t1/2, volume of distribution, Cmax, CSS, Tmax, renal clearance

20. Pharmacokinetics Pharmacodynamics Pharmacogenetics Pharmacodynamics What the drug does to the body Toxicity Grade Effect on ANC, platelets Tumor response Normal Tissue Surrogates Effect on Imaging characteristics

21. Pharmacokinetics Pharmacodynamics Pharmacogenetics Pharmacogenetics Genetic variation in enzymes that metabolize drugs Single nucleotide polymorphisms (SNPs) Functional SNPs Differences between ethnic groups Frequency could be common or relatively rare Not analyzed for cancer risk, screening, or prevention

22. Phase I Trial Design Considerations Starting dose 1/10 LD10 in mice provided the dose < TLD (toxic low dose) in dog If toxic in dog, then start with1/3 TLD in dog

23. Phase I Trial Design Considerations Standard cohort-escalation design 3 patients per dose level If 2 or 3 DLTs stop escalation If 1 DLT add 3 patients and if no further DLT continue dose escalation, i.e. 1/6 DLT MTD is 1 dose level lower

24. ASSESSMENT OF DRUG TOXICITY (SIDE EFFECTS)

25. TOXICITY GRADING & ATTRIBUTION NCI common toxicity criteria (CTC v3.0) Not related Possible related Probably related Definitely related

27. PHASE 2 AND 3 STUDY GOALS Response Rate: What percent of patients have significant tumor shrinkage Time to Progression: The time it takes for the tumor to grow by a certain amount after starting the study Disease Free Survival: The time it takes for the tumor to come back after starting the study Overall Survival: The time patients live after starting the study

28. PHASE 2 CLINICAL TRIALS Initiated when a safe dose and schedule define in phase I Performed in specific tumor types Tumor types selected for testing depending on a variety of criteria Overall purpose is to determine whether there is any clinically meaningful activity

29. PHASE 2 CLINICAL TRIALS Criteria for tumor types to study hint of effect in phase I specific target is present in that cancer a clinical need is identified competitors in the market place potential market size

30. PHASE 2 CLINICAL TRIALS GOALS To determine the response rate, time to progression and survival To determine the frequency and spectrum of all toxicities To relate potential markers of biologic endpoints to clinical effect To relate pharmacogenetics to toxicity and efficacy

31. Phase I vs PHASE 2 CLINICAL TRIALS Phase I trials include ~20-25 patients with many tumor types and patients are treated at different doses Phase 2 trials can include from 14 to ~100 patients with one tumor type treated with one dose

32. PHASE 2 CLINICAL TRIALS If a phase 2 trial meets its objective then phase 3 trials are needed However, phase 2 trial results can be used for accelerated approval by FDA This needs to be established in consultation with FDA before the plan for the trial is finalized and will still require a phase III trial

33. PHASE 3 CLINICAL TRIALS Definitive comparative trials Requires a randomization Rarely blinded in Cancer trials Primary endpoints can include response rate, time to progression, overall survival and disease-free survival

34. PHASE 3 CLINICAL TRIALS As follow-up on patients may be long results can take 5 and 10 years to be realized Size of the study can vary from a few hundred patients to thousands

35. Molecularly Targeted Therapies A new paradigm for cancer treatment Cancer as a chronic disease like diabetes, hypertension Although cure is still the ultimate goal, control can achieve valuable benefit for the patient Treatment are less toxic and chronic

36. Natural History of Cancer Initial development of cancer is to a large extent the consequence of the accumulated loss of growth constraints that govern the growth and behaviorof normal cells1: Loss of differentiated function (such as milk production by breastepithelial cells), regulated growth, and a finite number of cell divisionscoordinated with programmed cell death (apoptosis) Through activation of oncogenes and inactivation of tumor suppressorgenes, cells lose these constraints, becoming progressively less dependent on exogenous growth factors and less responsive to cell cycle control and apoptotic signals Rapid and expansive growth of cancer requires stimulation of angiogenesis.2 Generation of tumor vasculature derived from, and contiguous with, host vasculature ensures nourishment and provides a conduit for dissemination Progression to malignancy involves tumor cell variants acquiring the ability to actively invade surrounding tissues and to enter vascular channels to effect metastasis.2 Capabilities such as production of enzymes that degrade tissuematrices and ability to localize to, and survive in, distant sites Initial development of cancer is to a large extent the consequence of the accumulated loss of growth constraints that govern the growth and behaviorof normal cells1: Loss of differentiated function (such as milk production by breastepithelial cells), regulated growth, and a finite number of cell divisionscoordinated with programmed cell death (apoptosis) Through activation of oncogenes and inactivation of tumor suppressorgenes, cells lose these constraints, becoming progressively less dependent on exogenous growth factors and less responsive to cell cycle control and apoptotic signals Rapid and expansive growth of cancer requires stimulation of angiogenesis.2 Generation of tumor vasculature derived from, and contiguous with, host vasculature ensures nourishment and provides a conduit for dissemination Progression to malignancy involves tumor cell variants acquiring the ability to actively invade surrounding tissues and to enter vascular channels to effect metastasis.2 Capabilities such as production of enzymes that degrade tissuematrices and ability to localize to, and survive in, distant sites

37. Molecular Events in Cancer Development of cancer and malignant progression require many coordinated molecular events that lead to dedifferentiation and loss of normal regulation of growth, cell division, and apoptotic death, and eventually to growth autonomy.1 Altered gene activity (including oncogene activation and suppressor gene inactivation) perturbs growth factor (GF) signaling pathways.1,2 Constitutive activation of GF receptors independent of GF Acquired responsiveness to exogenous GF (paracrine stimulation) and to self-generated GF (autocrine stimulation), leading to unregulated orautonomous cell growth and proliferation Novel or enhanced production of angiogenic factors Enhanced production and secretion of matrix metalloproteinases (MMP)and other enzymes that degrade extracellular matrix (ECM), to promotelocal invasion of tissues and metastasis Loss of tumor suppressor genes, like p53, reduces cell cycle control and susceptibility to apoptosis.1 Repair of DNA damage may not occur, leading to more genetic alterations that drive malignant progressionDevelopment of cancer and malignant progression require many coordinated molecular events that lead to dedifferentiation and loss of normal regulation of growth, cell division, and apoptotic death, and eventually to growth autonomy.1 Altered gene activity (including oncogene activation and suppressor gene inactivation) perturbs growth factor (GF) signaling pathways.1,2 Constitutive activation of GF receptors independent of GF Acquired responsiveness to exogenous GF (paracrine stimulation) and to self-generated GF (autocrine stimulation), leading to unregulated orautonomous cell growth and proliferation Novel or enhanced production of angiogenic factors Enhanced production and secretion of matrix metalloproteinases (MMP)and other enzymes that degrade extracellular matrix (ECM), to promotelocal invasion of tissues and metastasis Loss of tumor suppressor genes, like p53, reduces cell cycle control and susceptibility to apoptosis.1 Repair of DNA damage may not occur, leading to more genetic alterations that drive malignant progression

39. Molecularly Targeted Therapies Drugs that target receptor tyrosine kinases � Gleevec, Tarceva Antibodies that target receptors � Herceptin, Erbitux Antibodies that target receptor ligands - Avastin

40. Anti-EGF Agents (Antibodies) MoAb Erbitux ICR62 mAb425 Ior egf/r3 Pantuzamab h-R3 EMD72000 Bispecific Antibodies MDX-447 Toxin-Linked Conjugates ScFV (14E1)-ETA ScFV (225)-ETA MRI (Fv)-PE38 TP40 DAB389-EGF

41. Anti-EGF Agents (Drugs) IRESSA TARVECA *LAPATINIB PKI-166 PD158780 AG1478 CGP59326 *CI1033 *EKB569

42. Tumor Angiogenesis:Angiogenic Balance or Switch Tumor cells possessing the angiogenic phenotype recruit their own blood supply by secreting angiogenic growth factors. Effects of angiogenic growth factors on endothelial cells include promotion of cell survival, stimulation of cell growth, and cytoskeletal remodeling. Endothelial cells proliferate, detach from their surrounding extracellular matrix, and migrate through the capillary basement membrane to form a new capillary. New capillaries invade and surround the tumor, feeding its rapid growth. Tumor cells may invade through capillary walls, resulting in tumor cells entering the circulation. Tumor vessels are thought to provide a route for vascular metastasis.3,12 Tumor cells possessing the angiogenic phenotype recruit their own blood supply by secreting angiogenic growth factors. Effects of angiogenic growth factors on endothelial cells include promotion of cell survival, stimulation of cell growth, and cytoskeletal remodeling. Endothelial cells proliferate, detach from their surrounding extracellular matrix, and migrate through the capillary basement membrane to form a new capillary. New capillaries invade and surround the tumor, feeding its rapid growth. Tumor cells may invade through capillary walls, resulting in tumor cells entering the circulation. Tumor vessels are thought to provide a route for vascular metastasis.3,12

43. VEGF Ligands and Receptors Ferrara. J Mol Med. 1999;77:527. Ferrara. J Mol Med. 1999;77:527.

44. Anti-Angiogenesis Agents Tyrosine Kinase Inhibitors Sorefinib Sunitinib CHIR-258 AEE788 BIBF1120 AZD 2171

45. Biomarkers in Cancer Clinical Trials Should we treat only those patients that express the target? Do we need to treat at doses that cause some side effects like with chemotherapy? Should we measure the benefit in the same way e.g. response rate?

46. Biomarkers in Cancer Clinical Trials Should the agent be highly specific or affect several target? If the latter then how many? Should these agents be used in combination with chemotherapy? Should these agents be combined with each other?

49. Biomarkers for Target Effect Presence of the target eg ER, PR, HER2 Effect of treatment on plasma tumor marker eg CA125, PSA, aFP. Effect of treatment on a downstream molecule eg phospho-p70S6K with CCI-779 or RAD001 Depletion of circulating tumor cells after treatment Decrease in metabolic imaging after treatment

50. Types of Biomarkers Biologic Fluids �plasma, serum, urine, CSF, effusions Tissue �Normal or tumor Proteomic, Genomic, DNA Imaging PET scanning 18FDG, 18FLT, H2O15 MRI CT perfusion

54. Therapy of Patients with Metastatic Colon Cancer How do we know if the treatment is working? Wait 2 months and expose the patient to the side effects Does a test that predicts the effectiveness Blood test Imaging

57. Circulating tumor cells Peripheral blood epithelial cells shed from tumor surface of cancer patients Defined by phenotypic characteristics Expression of cytokeratin or mucin markers such as CK18, CK19, and MUC-1 Phenotypically similar to primary and/or metastatic tumor cells

58. Detection Methods Direct methods (enrichment/detection) Immunomagnetic Immunohistochemistry (IHC) PCR analysis Automated fluorescent methods

59. Prognostic and Predictive Value of CTCs in MBC Hypothesis Measurement of CTCs in metastatic breast cancer may: Identify aggressive disease (prognostic value) Provide early determination of treatment efficacy/benefit (predictive value)

61. Change in CTC Count During Therapy Predicts Overall Survival

68. Tamoxifen Pharmacogenetics(Goetz, Ingle, Weishilboum, Flockhart) Does CYP2D6 genetic variation affect the clinical outcomes of women receiving tamoxifen? Do women who are co-prescribed medications which inhibit CYP2D6 have a higher risk of breast cancer relapse?

69. CYP2D6 Gene Encompasses nine exons with an open reading frame of 1383�bp coding for 461 amino acids. Highly polymorphic At least 46 major polymorphic alleles with 4 well-defined phenotypes: Poor metabolizers (PM) Intermediate metabolizers (IM) Extensive metabolizers (EM) Ultra-rapid metabolizers (UM)

70. CYP2D6 Gene CYP2D6 PM: 7-10% of Caucasians lack functional CYP2D6 CYP2D6 *4: Most common variant leading to null CYP2D6 enzyme in Caucasians (accounts for 75% of the Caucasian PM)

71. NCCTG 89-30-52 Follow-up procedures included recording symptoms, side effects, clinical examination results and medication compliance at specific time points after randomization: 3-month intervals in year 1 6-month intervals in years 2 and 3 Annually thereafterFollow-up procedures included recording symptoms, side effects, clinical examination results and medication compliance at specific time points after randomization: 3-month intervals in year 1 6-month intervals in years 2 and 3 Annually thereafter

72. NCCTG 89-30-52 Median follow-up of 12 years Accrual completed in April 1995 No difference between the arms in terms of primary endpoint (breast cancer relapse) Ingle J, Suman V, Mailliard J, et al: Breast Cancer Res Treat (In Press)

73. Tamoxifen only arm 230/257 eligible patients--Formalin fixed paraffin-embedded tumor blocks Three 10 micron sections DNA extracted Genotyping: CYP2D6 (*4) (n=190) CYP3A5 (*3) SULT1A1 (*2)

76. Informed Consent and Regulations

77. Ethics of Phase I Trials No known benefit Risk of toxicity is unknown Patients expectations are high Patients discount risk of toxicity

78. Ethics of Phase I Trials Patient assessment of benefit Phase I 59.8% Standard 36.8% Patient assessment of toxicity Phase I 29.8% Standard 45.6% Based on analyses of multiple phase I trials Benefit (Response) ~5% Toxicity (Death) 0.5%

80. Consent Form Excerpts Why is this study being done? This study is being done to: Learn the highest safe dose of the investigational drug 17-AAG when it is given over 1 hour once each week for 3 out of every 4 weeks to patients with advanced cancer. Learn the side effects of 17-AAG and how the body responds to and removes 17-AAG.

83. Consent Form Excerpts Are there benefits to taking part in this study? No help can be promised by taking part in this study, and the chance of help from this study cannot be accurately predicted.

85. Consent Form Excerpts During the first month of this study, the following additional tests and procedures will be done to test the amount of 17-AAG in your body and to learn what effect it has on your body. These will be done at no additional cost to you and/or your health plan and include: Nineteen (19) blood samples (totaling about 1� cup) will be collected. Before and at one day after your first and third weekly 17-AAG treatments, samples of tissue will be taken by gently scraping the inside of your cheeks or mouth with a tongue depressor. You will need to save your urine for 24 hours before and after your first 17-AAG treatment. If you have a tumor that can be safely and easily biopsied, you may be asked to have two additional biopsies. One before you start and the other one day after the start of your treatment.

86. Consent Form Excerpts (HIPAA) Authorization To Use And Disclose Protected Health Information By signing this form, you authorize Mayo Clinic Rochester and the investigators to use and disclose any information created or collected in the course of your participation in this research protocol. This information may include information relating to sexually transmitted disease, acquired immunodeficiency syndrome (AIDS), or human immunodeficiency virus (HIV). It may also include information relating to behavioral or mental health services or treatment and treatment for substance abuse.

87. Consent Forms in Phase I Oncology Trials S. Horng et al NEJM 2002; 347(26):2134-2140 never promise benefit rarely mention cure communicate risk and its unpredictability do not contribute to patients� expectation of benefit

88. ADHERENCE TO STUDY PROTOCOL IS CRITICAL

89. COMPLIANCE WITH REGULATIONS NCI regulations Cancer Center Cooperative groups FDA regulation OHRP regulations as interpreted by IRB

90. INVESTIGATOR OBLIGATIONS Ensure informed consent is obtained Report serious adverse events Report adverse events Data monitoring safety plan Data safety monitoring committee Protocol compliance Regular auditing of cases on study

91. QUESTIONS ARE WELCOME