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Requirements for the Cure of Cancer: Formulating a Plan of Action Workshop sponsored by the Van Andel Institute Jan. 10-11, 2007 FROM PRINCIPLES TO PRACTICE SESSION VI(B) The development of technologies for targeting cells that express target patterns Arnold Glazier MD General Design Considerations The challenges of multi-drug therapy 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 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 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 Examples of Almost Perfect Targeting Exist • Hormone/ receptor binding • Peanut allergy / anaphylactic shock • Nerve gas 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..) 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?) 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 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 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. 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. Effector Agents Should be Cell Cycle Independent G2/mitotic-specific cyclin-B1 in colon cancer http://www.proteinatlas.org/ 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 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 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. Medicinal Chemistry Boils Down To: 1. Binding 2. Chemical bond formation 3. Breakage of chemical bonds 4. Catalysis of a reaction 5. Dissolution or precipitation 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. Targeting Ligands Ligand Receptor Complex Ligands are chemical groups that bind together like a lock and key to target receptors. A Urokinase Selective Ligand HO H N HN O O N H N NH2 OH NH Kd is in the low nanomolar range. Tamura S Y., et al., Bioorganic Med Chem Lett, 10:983-987 (2000) Triggers and Triggering Agents Trigger Drug molecule Triggering Chemically altered drug Agent 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. 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 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 Linkers and Scaffolds Toxin Trigger Linkers Scaffold Targeting Ligands Structural elements that provide the backbone of the drug Cyclodextrins as Scaffold Rigidity, multiple sites for linker attachment, solubility, spatial separation of components, low toxicity Male and Female Adaptors The male and female parts bind specifically and tightly. In the ideal case the binding is irreversible. Masking Groups Masked Receptor Triggering Agent Unmasked Receptor A masking group blocks a receptor. A triggering agent can unmask the receptor. Molecular Clocks Trigger Triggering Agent Chemical change Molecular clocks provide an adjustable time delay between a triggering event and a chemical change. 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 A wide range of pattern targeting technologies can be developed by combining these modular building blocks in logical ways. 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 Targeted Delivery of a Targeted Cytotoxic Agent This method is the simplest and requires no new drug technology Targeted Delivery of a Targeted Targeted Delivery of a Targeted Cytotoxic Agent Cytotoxic Agent Drug Targeted cytotoxic agent Cleavable linker Targeting ligand Target of cytotoxic agent Cell death The Pattern is a Surface Receptor and Intracellular Target For cell killing both must be present Targeted Delivery of a Targeted Cytotoxic Agent The cytotoxic agent is toxic only if its target is present The Targeting Can Also be in The TargetReceptor Pattern of a Receptor in the Microenvironment an Intracellular Target the Tumor Cell and Microenvironment Intracellular target y Cell Receptor xinin microenvironment Linker cleavage Drug internalization Cell With pattern Cell Toxicity Targeted Delivery of a Trigger Activated Drug Targeted Delivery of a Trigger Activated Drug The Pattern is a Triggering Enzyme and a Receptor Toxin Trigger Triggering Enzyme Triggering Enzyme Cell Cell No Toxicity Toxin Trigger Receptor Tumor Cell Toxicity Cell No Toxicity Only cells that have both the target receptor and the triggering enzyme will be killed. A Urokinase-Activated GMCSF Receptor Targeted Diphtheria Toxin Urokinase activates Trigger 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 Targeting the Microenvironment The drug is targeted to the microenvironment, released by the triggering enzyme, diffuses to the tumor cell and kills it. 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. Paired, Independently Targeted, Synergistically Toxic Drugs Paired, Independently Targeted Synergistically Toxic Drugs Agent 1 Agent 2 Individually, Agent 1 and Agent 2 are Nontoxic, But Toxic in Combination: Agent 1 Normal cell Type A No Toxicity Agent 1 Agent 2 Agent 2 Tumor cell Normal cell Type B Toxicity No Toxicity Multi-Site Binding Multi-Site Binding and Pattern Recognition Multi-site binding can give an enormous increase in the tightness of binding compared to single site binding A Ten Billion Times Increase in Affinity due to Three Site Binding Vancomycin Ala-Ala Kd = 10 – 6 Tri-Vancomycin Tri- Ala-Ala Kd = 10 –17 Rao J, Lahiri J, Isaacs L, Weis RM, Whitesides GM; Science 280:708-11 (1998) Multi-Site Binding Toxin Toxin Tumor cell Normal cell Tight Binding No Binding At low concentrations the drug can bind tightly to cells with the target pattern without binding to cells that express only one element of the pattern Advantages of Multi-Site Binding • • • • Specificity for the pattern Potency Slow off rate Immense reductions in the dose of drug required • Reductions in side effects Exponential Pattern Recognition Targeting From one receptor create two, from two create four …. Exponential Pattern Recognition Targeting Instead of consuming receptors, the targeted drug will in effect increase the target receptor density. The more drug that is delivered, the more drug that can be delivered. In this method specificity is for the pattern of a receptor and a triggering enzyme. Components of Exponential Pattern Recognition Targeting Masked Female Adapter Toxin Targeting Ligand Male Ligand 1 2 The male and female parts bind with very high affinity. The Mechanism of Exponential PRTT Triggering Enzymes Unmask the Female Adaptor • Many enzymes that are overexpressed by tumors can be utilized • The triggering enzyme can also be independently targeted to tumor cells Exponential Pattern Recognition Targeting Triggering enzyme Tumor cell Tumor cell 1.) Component 1 binds to cell receptors. 2.) Triggering enzyme(s) unmask female adapter. Toxin Toxin Triggering enzyme Tumor cell Tumor cell 3) Component 2 binds to the unmasked female adaptor. 4) The triggering enzyme unmasks twice as many new female adaptors. Toxin Tumor cell Repetition of the cycle can deposit a large quantity of drug in a tree like structure Massive Amounts of Drug can be Delivered to a Tumor Cell Amplification 1 10 6 1 10 5 1 10 4 1 10 3 100 10 1 5 10 15 Number of Cycles The quantity can increase exponentially 20 Self-Amplifying Exponential Pattern Recognition Targeting Self-Amplifying Exponential PRTT Masked Female Adaptors Unmasked Female Adaptors Toxin Toxin Male Ligand Female Adaptor Tumor cell Spontaneous Tumor cell The very binding of a male ligand and female adaptor creates two new female adaptors without the need for a triggering enzyme. Unmasked Female Adaptor Masked Female Adaptor Male Adaptor Male and Female Covalently Bound Masked Female Adaptor Unmasked Female Adaptor Unmasked Female Adaptor Masked Female Adaptor Masked Female Adaptor Bulky Group Masked Male Adaptor Unmasked Female Adaptor Female Adaptor from a second molecule Bulky Group Unmasked Female Adaptor Female Adaptors can Transform Different Patterns into a Common Target Different Target Patterns Pattern 1 Pattern 2 Pattern 3 A Common Target Pattern 4 Pattern 5 Female Adaptors Pattern 6 Pattern 7 This can enable the efficient delivery of multiple drugs to each target pattern and prevent the development of drug resistance. Multiple Toxins Can be Delivered to a Single Target Pattern Toxin Toxin Toxin A wide range of possibilities and emergent properties can arise with drugs that interact with each other. Amplification and positive feedback can be achieved by delivering enzymes to adaptors which in turn unmask additional adaptors. An other approach is to deliver a marker to the target patterns that make it look to the immune system like a bacterial infection. Massive signal amplification is possible along with a change in scale. To attract and activate one neutrophil requires only a small number of chemotactic molecules. Each neutrophil can deliver billions of molecules of: • • • • • • Hydrogen peroxide Myeloperoxidase MMP-9 Urokinase Elastase Catepsins The system exhibits positive feedback: • • • • • Myeloperoxidase activates neutrophils ROS inactivate protease inhibitors Ros activate MMP’s Ros stimulate MMP production Cathepsins The protease released can also activate MMP-2, MMP-9, and plasminogen. The net result could be a massive signal amplification in and around the target pattern and… a change in scale.
