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Requirements for the Cure of Cancer: Formulating a
Plan of ActionWorkshop 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
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
TirapazamineBrown 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 RequirementsThe 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
Are all the elements of the pattern present ?
Yes No
Kill Cell Spare Cell
BA C
Specificity is for the pattern, not the individual elements.
4. Catalysis of a reaction
5. Dissolution or precipitation
1. Binding
3. Breakage of chemical bonds
2. Chemical bond formation
Medicinal Chemistry Boils Down To:
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
HNHN N
HN NH2
NHOH
O
O
HO
Tamura S Y., et al., Bioorganic Med Chem Lett, 10:983-987 (2000)
Kd is in the low nanomolar range.
Triggers and Triggering Agents
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.
Trigger
Drug molecule
Triggering Agent Chemically altered drug
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 ScaffoldsToxin
Trigger
Targeting Ligands
Linkers
Scaffold
Structural elements that provide the backbone of the drug
Rigidity, multiple sites for linker attachment,solubility, spatial separation of components, low toxicity
Cyclodextrins as Scaffold
Male and Female Adaptors
The male and female parts bind specifically and tightly.
In the ideal case the binding is irreversible.
Masking Groups
A masking group blocks a receptor.A triggering agent can unmask the receptor.
MaskedReceptor
UnmaskedReceptor
Triggering Agent
Molecular Clocks
Molecular clocks provide an adjustable time delay between a triggering event and a chemical change.
Chemical change
Trigger
Triggering Agent
Intracellular Transport Ligands
Tumor CellTumor Cell
Cell Receptor
Transport into Cell
Drug
IntracellularTransport Ligand
Intracellular transporter groups can also work by physical, non-receptor mediated mechanisms.
DrugDrug
Drug
A wide range of pattern targeting technologies can be developed by combining these modular building blocks in logical ways.
PRTT ApproachesPRTT Approaches• Targeted delivery of a targeted agentTargeted delivery of a targeted agent• Targeted delivery of a trigger activated drug• Independently targeted synergistically toxic drugsIndependently targeted synergistically toxic drugs• Multi-site bindingMulti-site binding• Exponential Pattern Recognition TargetingExponential Pattern Recognition Targeting• Combinations of the aboveCombinations of the above• OtherOther
Targeted Delivery of a Targeted Targeted Delivery of a Targeted Cytotoxic AgentCytotoxic Agent
Internalizationlinker cleavage
No Toxicity
Toxicity
Without pattern
With patternInternalizationlinker cleavage
Cell
Cell
Cell
Cell
The cytotoxic agent is toxic only if its target is present
The Targeting Receptor Can Also be in the Tumor Cell Microenvironment
The Target Pattern of a Receptor in the Microenvironment and an Intracellular Target
CellCell
Intracellular target y
Receptor x inmicroenvironment
Linker cleavage
ToxicityWith pattern
Cell Cell
Drug internalization
in
Targeted Delivery of a Trigger Activated Drug
No Toxicity Toxicity No Toxicity
Cell
The Pattern is a Triggering Enzyme and a Receptor
Trigger
Toxin
TriggeringEnzyme
TriggeringEnzyme
Toxin
Trigger
Cell Tumor Cell Cell
Receptor
Only cells that have both the target receptor and the triggering enzyme will be killed.
A Urokinase-Activated GMCSF Receptor Targeted Diphtheria Toxin
Diphtheria toxin
TriggerUrokinase activates
Binds to GMCSFReceptor on cells
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
The drug targets the pattern of urokinase and GMCSF receptor.
Targeting the Microenvironment
The drug is targeted to the microenvironment, released by the triggering enzyme, diffuses to the tumor cell and kills it.
The Pattern can be a Receptor and Triggering Enzyme in the Tumor Cell Microenvironment
Toxin
Trigger TriggeringEnzyme
Tumor Cell
Receptor
Trigger
Toxin
Tumor Cell Toxin
Cell Death
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.
No Toxicity Toxicity No Toxicity No Toxicity Toxicity No Toxicity
Agent 1 Agent 2Agent 1
Tumor cellNormal cellType A
Normal cellType B
Agent 2
Individually, Agent 1 and Agent 2 are Individually, Agent 1 and Agent 2 are Nontoxic, But Toxic in Combination:Nontoxic, But Toxic in Combination:
Multi-Site Binding and Pattern Multi-Site Binding and Pattern RecognitionRecognition
Multi-site binding can give an enormous Multi-site binding can give an enormous increase in the tightness of binding increase in the tightness of binding compared to single site bindingcompared to single site binding
Vancomycin
Ala-Ala
Tri-Vancomycin
Tri- Ala-Ala
A Ten Billion Times Increase in Affinity due to Three Site Binding
Rao J, Lahiri J, Isaacs L, Weis RM, Whitesides GM; Science 280:708-11 (1998)
Kd = 10 –17Kd = 10 – 6
Multi-Site Binding
At low concentrations the drug can bind tightly to cells with the target 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 patternpattern without binding to cells that express only one element of the pattern
Toxin
Tumor cell
Toxin
Normal cell
Tight BindingTight Binding No Binding
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
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.
Exponential Pattern Recognition Targeting
Components of Exponential Pattern Recognition Targeting
Toxin
Targeting Ligand
Masked Female Adapter
Male Ligand
The male and female parts bind with very high affinity.
1 2
Tumor cell
Toxin
3
Tumor cell
Toxin
Toxin +
Tumor cell
Triggering-Enzyme
Tumor cell21
Toxin
Tumor cell
Triggering-Enzyme
4
Unmasked Female Adaptor
Compound 2
Compound 2
Compound 1
Two Unmasked Female Adaptors
5
Toxin
Toxi
n
Toxin
Tumor cell
After Multiple Cycles:
The Mechanism of Exponential PRTT
Triggering Enzymes Unmask the Female Adaptor
• Many enzymes that are over-expressed by tumors can be utilized
• The triggering enzyme can also be independently targeted to tumor cells
Exponential Pattern Recognition Targeting
1.) Component 1 binds to cell receptors. 2.) Triggering enzyme(s) unmask female adapter.
Tumor cell
Triggering enzyme
Tumor cell
Toxin
3) Component 2 binds to the unmasked female adaptor.4) The triggering enzyme unmasks twice as many new female adaptors.
Triggering enzyme
Tumor cell
Toxin
Tumor cell
Toxin
Toxin
Toxin
Toxi
nToxin
Tumor cellRepetition 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
5 10 15 201
10
100
1 103
1 104
1 105
1 106
Amplification
Number of Cycles
The quantity can increase exponentially
Self-Amplifying Exponential PRTT
The very binding of a male ligand and female adaptor creates two new female adaptors without the need for a triggering enzyme.
ToxinToxin
Tumor cellTumor cell
Masked Female Adaptors Unmasked Female Adaptors
Female Adaptor
Male Ligand
Spontaneous
UnmaskedFemale Adaptor
Unmasked Female Adaptor
UnmaskedFemale Adaptor
Male and FemaleCovalently Bound
MaskedFemale Adaptor
MaleAdaptor
MaskedFemale Adaptor
Female Adaptors can Transform Different Patterns into a Common Target
Pattern 2
Pattern 3
Pattern 4
Pattern 5
Pattern 6
Pattern 1
Pattern 7
Different Target Patterns
A Common Target
Female Adaptors
This can enable the efficient delivery of multiple drugs to each target pattern and prevent the development of drug resistance.
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.