Requirements for the Cure of Cancer: Formulating a Plan of Action

1. Requirements for the Cure of Cancer: Formulating a Plan of Action Workshop sponsored by the Van Andel Institute Jan. 10-11, 2007

2. FROM PRINCIPLES TO PRACTICE SESSION VI(B) The development of technologies for targeting cells that express target patterns Arnold Glazier MD

3. General Design Considerations

4. The challenges of multi-drug therapy

5. Ideal Drug Targeting • The tumor would act like a black hole for drug • All drug in the blood flow to the tumor would be irreversibly retained • No drug accumulation in non-target sites

6. Ideal Drug Targeting • Based on a typical blood flow of 0.15 –0.6 ml/min/gm and 24 hours, maximum average tumor levels would be about 200-800 times the average blood level • The biological effects can be even orders of magnitude higher

7. Multiplicative Increases in Concentration can give Exponential Increases in Effect Surviving Cell Fraction versus Drug Concentration Tirapazamine Brown JM, Wouters BG.; . Cancer Res. 1999 Apr 1;59(7):1391

8. Examples of Almost Perfect Targeting Exist • Hormone/ receptor binding • Peanut allergy / anaphylactic shock • Nerve gas

9. Approaches Towards Ideal Drug Targeting • Specific, high affinity or irreversible binding • Slow “off rates” of drug from receptors • Administering the drugs at the lowest concentration needed to saturate “drug accessible” receptors • Decreasing nonspecific binding • Increasing the quantity of drug receptors (exponential PRTT) • Prolonging treatment time Principles that can be applied towards achieving these goals are well known. (Multi-site binding, slow binding, covalent binding, etc..)

10. Major Issues • Chaotic and uneven blood flow • Limited drug penetration into tumors • Slow rates of drug diffusion • Episodic target pattern expression • On a given day only parts of a tumor will be drug accessible The drugs need to be given continuously for prolonged periods of time. (6 months?)

11. The Aim Should be to Deliver Drug to “Drug Accessible” Target Patterns • The important pathology that sustains cancer occurs within a limited zone around blood vessels. • Areas close to blood vessels will be drug accessible. • Drug accessible cells will be killed, new layers of cancer cells will be exposed and killed over time in an “onion peeling effect” • Therapy needs to be sufficiently intense so that the rate of cell loss exceeds the rate of cell production

12. A Minor, Sustained Decrease in the Probability of Cancer Cell Survival can have Profound Effects Data: Berman JJ, Moore GW; Anal Cell Pathol. 1992 Sep;4(5):359-68

13. Drugs Targeted to a Comprehensive Set of Target Patterns will Inhibit • Angiogenesis • Vasculogenic mimicry • Vascular co-option This will achieve Dr. Folkman’s vision by effectively depriving tumor cells of new blood supply, constraining growth and allowing time for the “onion peeling” killing effect to work.

14. Non-synchronous Expression of Target Pattern Elements Targeting specificity should be for • Invasiveness alone, or • Invasiveness and the potential for proliferation Elements of these classes of target patterns are expressed concurrently.

15. Effector Agents Should be Cell Cycle Independent G2/mitotic-specific cyclin-B1 in colon cancer http://www.proteinatlas.org/

16. The Microenvironmental Nature of Invasiveness There is a requirement for approaches that generate a zone of anticancer activity in the local volume that surrounds target patterns

17. Major Requirements The need for: • Pattern specificity • Signal amplification • Multiple, redundant mechanisms of cell killing or inactivation • Prolonged therapy • The ability to simultaneously give multiple drugs • Chemical stability • Lack of antigenicity • Modularity in design

18. The Logic Function of PRTT Drugs A B C Are all the elements of the pattern present ? Yes No Kill Cell Spare Cell Specificity is for the pattern, not the individual elements.

19. 1. Binding 2. Chemical bond formation 3. Breakage of chemical bonds 4. Catalysis of a reaction 5. Dissolution or precipitation Medicinal Chemistry Boils Down To:

20. Modular Building Blocks • Targeting ligands • Triggers • Triggering agents • Effector agents • Linkers and scaffolds • Male and female adaptors • Masking groups • Molecular clocks • Intracellular transport ligand • Solubility modifiers These components exist and are within the scope of current technology.

21. Targeting Ligands Ligand Receptor Complex Ligands are chemical groups that bind together like a lock and key to target receptors.

22. A Urokinase Selective Ligand Kd is in the low nanomolar range. Tamura S Y., et al., Bioorganic Med Chem Lett, 10:983-987 (2000)

23. Triggers and Triggering Agents Trigger Drug molecule Triggering Agent Chemically altered drug Triggers are chemical groups then when acted upon by a triggering agent undergo a chemical change. Enzymes and non-enzymes can serve as triggering agents.

24. Applications of Triggers • To turn on or off a chemical process • To activate a toxin • To inactivate a toxin • To unmask a ligand • To release a toxin

25. Effector Agents • Toxic agents that kill cells • Agents that irreversibly block the potential for cell proliferation • Agents that trigger an immune response • Agents that amplify a response

26. Linkers and Scaffolds Toxin Trigger Linkers Scaffold Targeting Ligands Structural elements that provide the backbone of the drug

27. Cyclodextrins as Scaffold Rigidity, multiple sites for linker attachment, solubility, spatial separation of components, low toxicity

28. Male and Female Adaptors The male and female parts bind specifically and tightly. In the ideal case the binding is irreversible.

29. Triggering Agent Masked Receptor Unmasked Receptor Masking Groups A masking group blocks a receptor. A triggering agent can unmask the receptor.

30. Molecular Clocks Trigger Triggering Agent Chemical change Molecular clocks provide an adjustable time delay between a triggering event and a chemical change.

31. Intracellular Transport Ligands Drug Drug Cell Receptor Transport into Cell Intracellular Transport Ligand Drug Drug Intracellular transporter groups can also work by physical, non-receptor mediated mechanisms. Tumor Cell Tumor Cell

32. A wide range of pattern targeting technologies can be developed by combining these modular building blocks in logical ways.

33. PRTT Approaches • Targeted delivery of a targeted agent • Targeted delivery of a trigger activated drug • Independently targeted synergistically toxic drugs • Multi-site binding • Exponential Pattern Recognition Targeting • Combinations of the above • Other

34. Targeted Delivery of a Targeted Cytotoxic Agent

35. This method is the simplest and requires no new drug technology

36. Targeted Delivery of a Targeted Cytotoxic Agent Drug

37. The Pattern is a Surface Receptor and Intracellular Target For cell killing both must be present

38. Targeted Delivery of a Targeted Cytotoxic Agent The cytotoxic agent is toxic only if its target is present

39. in The Targeting Receptor Can Also be in the Tumor Cell Microenvironment

40. Targeted Delivery of a Trigger Activated Drug

41. Targeted Delivery of a Trigger Activated Drug Only cells that have both the target receptor and the triggering enzyme will be killed.

42. A Urokinase-Activated GMCSF Receptor Targeted Diphtheria Toxin Trigger Urokinase activates Diphtheria toxin Binds to GMCSF Receptor on cells The drug targets the pattern of urokinase and GMCSF receptor. Ralph J. Abi-Habib, Shihui Liu, Thomas H. Bugge, Stephen H. Leppla, and Arthur E. Frankel; Blood, 1 October 2004, Vol. 104, No. 7, pp. 2143

43. Targeting the Microenvironment The drug is targeted to the microenvironment, released by the triggering enzyme, diffuses to the tumor cell and kills it.

44. Advantages of Releasing a Toxin into the Tumor Microenvironment • Invasiveness is a property of both the cancer cell and its microenvironment • A zone of toxicity is created making it easier to kill all the cancer cells Approaches that produce a zone of toxicity are strongly preferred.

45. Paired, Independently Targeted, Synergistically Toxic Drugs

46. Agent 1 Agent 2 Paired, Independently Targeted Synergistically Toxic Drugs

47. Individually, Agent 1 and Agent 2 are Nontoxic, But Toxic in Combination: Agent 1 Agent 1 Agent 2 Agent 2 Tumor cell Normal cell Type B Normal cell Type A No Toxicity Toxicity No Toxicity

48. Multi-Site Binding

49. Multi-Site Binding and Pattern Recognition Multi-site binding can give an enormous increase in the tightness of binding compared to single site binding

50. A Ten Billion Times Increase in Affinity due to Three Site Binding Vancomycin Tri-Vancomycin Ala-Ala Tri- Ala-Ala Kd = 10 – 6 Kd = 10 –17 • Rao J, Lahiri J, Isaacs L, Weis RM, Whitesides GM; Science 280:708-11 (1998)