Definitions

• Monoclonal antibodies have been employed as targeting agents for the delivery of cytotoxic drugs to tumors.
• monoclonal antibodies are large molecules, and often do not penetrate well into tumors.
• Proteins and oligopeptides have also been used as targeting agents.
• Small molecules described as targeting agents include: folate, sigma receptor binding agents and agmatine.
• a variety of approaches have also been described to target cells by prodrugs, which are activated by enzymes that are increased in tumor cells.
• the present invention relates to the compositions, methods, and applications of a novel approach to selective cellular targeting.
• the purpose of this invention is to enable the selective delivery and/or selective activation of effector molecules to target cells for diagnostic or therapeutic purposes.
• the present invention relates to multi-functional prodrugs or targeting vehicles wherein each functionality is capable of enhancing targeting selectivity, affinity, intracellular transport, activation or detoxification.
• the present invention also relates to ultra- low dose, multiple target, multiple drug chemotherapy and targeted immunotherapy for cancer treatment.
• Binding Affinity - Tightness of binding between a ligand receptor Binding Affinity - Tightness of binding between a ligand receptor.
• Bioreversibly Masked Group - A chemical group that is derivatized in a
• an ester group can be a bioreversibly masked group for a hydroxy group.
• a bioreversible masking group is a chemical group that when bonded with a second group produces a bioreversibly masked group for said second group.
• Bioreversible Protecting Group - A chemical group or trigger that can be
• various connectivity between groups A, B, C include structures such as A-B-C, B-A-C, or A-C-B.
• Connectivity can be direct such as by a covalent bond between an atom of A and B or indirect such as through a covalently bonded linker.
• Effector - An agent that exerts an activity and evokes a physical, chemical or
• biological response such as a pharmacologically beneficial response such as cytotoxicity, or a diagnostic effect.
• two or more components of a drug acting jointly or together is greater than the effect produced by the components acting individually or independently the components "functionally cooperate".
• Linker - A chemical group that serves to attach targeting ligands, triggers and
• Masked Group - A chemical group that is hidden or blocked.or derivatized
• Microenvironment of the target The volume of space around a target cell
• Multifactohal - A function of multiple factors or variables.
• Multivalent Binding- Binding at multiple targeting ligand- target receptor sites Multivalent Binding- Binding at multiple targeting ligand- target receptor sites.
• Non-selective Targeting Ligand- A chemical structure that binds to a
• effector activity such as normal cells, bone marrow stem cells, or normal liver.
• a drug or effector agent such as a cytotoxicity or stimulation of the immune system or a diagnostic effect.
• Targeting Agent- A chemical structure or group of chemical structures
• Targeting Ligand - A chemical structure, which binds with a degree of
• a triggering agent e.g., a triggering agent, an enzyme, or a chemical or biochemical factor that can be used to distinguish between target and non-target.
• Targeting Receptor- A chemical structure at the target that binds with a
• Target Molecules- Biomolecules that are either target receptors or triggering
• agents such as a protein that binds a targeting ligand or an enzyme at the target cell which can activate a trigger and which are increased at a target compared to a non-target but not necessarily all non-targets.
• Trigger- A chemical group which can undergo in vivo chemical modification
• a trigger can be considered as a chemical switch that upon activation gives a consistent and predictable output such as unmasking a chemical group, or detoxifying the drug, or toxifying the drug, or liberating an effector agent.
• Trigger Activation- The process of chemical modification that causes a trigger
• Triggering Factor- An enzyme, biomolecule or other agent which is able to
• Tumor Component - is a biomolecule which is present in tumor cells, on
• tumor cells in the microenvironment of tumor cells, on tumor stromal cells or present in tumor bulk.
• tumor cells or in the microenvironment of tumor cells compared to that of normal cells but not necessarily all types of normal cells.
• Tumor-selective Triggering Agent A triggering agent or triggering factor
• tumor cells that is present in increased amounts on tumor cells, in tumor cells, or in the microenvironment of tumor cells compared to that of normal cells but not necessarilly all types of normal cells.
• Vital Normal Cells- Cells that if destroyed would produce unacceptable clinical toxicity to a patient such as bone marrow stem cells, liver cells and cardiac cells.
• DNA mutations that disrupt critical regulatory pathways that control cell growth, can cause cancer and reinforce the malignant state in cancerous cells.
• the DNA mutations are specific to the cancer cells, targeting the mutations or the defective proteins that result from the DNA mutations may not be practical. It is likely that out of the estimated 140,000 genes in the human genome hundreds or perhaps thousands are capable of causing cancer. It is infeasible to prepare drugs that target each of these primary causes of cancer.
• many DNA mutations are known that induce malignant transformation by the loss of key regulatory proteins. In these cases the only way to distinguish the normal from malignant cells is by the secondary consequences that result from the absence of the regulatory protein. These consequences are the abnormal patterns of normal protein expression that define the malignant state.
• Anti-cancer therapies should be multifactorial unless directed against a causative lesion of cancer.
• malignancy is uncontrolled cell proliferation and tissue invasion.
• the biochemical manifestation of these processes provides the basis for understanding and defining optimal tumor targeting. Neither the processes of cell replication nor the enzymology of tissue invasion (remodeling) are by themselves uniquely diagnostic of malignancy. But jointly, these processes likely provide highly selective criteria to define effective targeting for the treatment of malignancy.
• the current class of multifunctional anti-cancer drugs provides the opportunity to have anti-cancer agents that are targeted simultaneously and jointly to both the proliferative and the invasive character of malignant cells.
• the present invention relates to technologies that can enable multifactorially targeted toxicity that is a consequence of multifactorial target recognition, effector action, or both.
• the present invention relates to a class of multifunctional, multifactorial drugs with pattern recognition capabilities.
• the present technology also relates to compositions and methods by which selective multifactorial toxicity can be achieved by delivering multiple monofactorially targeted effector molecules.
• the invention also relates to key patterns of protein expression useful for selectively targeting cancer.
• the present invention is a technology, which can allow the selective targeting of tumors with ultra-low doses of multiple drugs directed against multiple tumor targets.
• the high selectivity and high affinity of the drugs for tumor cells can enable the total dose of chemotherapy to be reduced thousands of times below current levels.
• the severe side effects currently associated with chemotherapy are not expected with ultra-low dose multiple drug therapy.
• the simultaneous use of multiple drugs directed against multiple tumor targets can potentially eliminate the problem of tumor resistance.
• the probability that a tumor could simultaneously develop resistance to ten independent drugs each capable of giving a 2 log reduction in tumor burden is essentially zero.
• a second major application of the technology described in this patent is targeted immunotherapy in which an intense immune response directed against non- tumor antigens is specifically targeted to tumors to elicit tumor rejection.
• technology is described that can allow the targeted formation of neotumor antigens.
• the present invention relates to the compositions, methods, and applications of a novel approach to selective cellular targeting.
• the purpose of this invention is to enable the selective delivery and/or selective activation of effector molecules to target cells for diagnostic or therapeutic purposes.
• the present invention relates to multi-functional prodrugs or targeting vehicles wherein each functionality is capable of enhancing targeting selectivity, affinity, intracellular transport or activation.
• the present invention can be used to selectively target cells for diagnostic or therapeutic purposes.
• the principle applications are in the field of anti-cancer therapy. However, the applications are not limited to the delivery of antineoplastic drugs and can be employed in other applications where selective drug targeting is beneficial such as in the delivery of immunosuppressants.
• Most current anti-cancer drugs are nonspecific or have low selectivity for tumor cells versus normal cells.
• the present invention seeks to address this problem by exploiting more than one property of tumor cells to define drug selectivity through the use of multi-functional delivery vehicles or prodrugs. Multifunctionality is also exploited to prevent the emergence of tumor drug resistance, and to selectively detoxify the drug in vital normal cells and to selectively toxify the drug in tumor cells.
• the present invention also encompasses (embodiment ET1 ) A compound ET wherein E is comprised of one or more effector agents having pharmacological activity designated as "PA" and T is comprised of a targeting agent comprised of two or more groups each of which functions to specifically enhance the targeting selectivity by either increasing the pharmacological activity PA at targeted cells and/or decreasing the pharmacological activity PA at non-target cells;
• PA effector agents having pharmacological activity designated as "PA”
• T is comprised of a targeting agent comprised of two or more groups each of which functions to specifically enhance the targeting selectivity by either increasing the pharmacological activity PA at targeted cells and/or decreasing the pharmacological activity PA at non-target cells;
• T is comprised of a group designated as a "selective targeting ligand" that binds specifically to a site designated as a “selective targeting receptor” on the target;
• T is not an antibody, or an analog or component of an antibody, or a complex of antibodies, or a bispecific antibody, or an analog of a bispecific antibody, or a natural protein, or a complex of natural proteins, or a protein, or a naturally occurring polymer, or a radiolabelled dimer, or a polymer to which is attached at multiple sites one or more pharmacologically active compounds that mediate the same pharmacological activity PA.
• the present invention also relates to the method of selectively targeting cells by the administration of said compound.
• the present invention addresses the following critical aspects of antitumor drug function:
• Mechanism of Action The mechanisms of actions and scientific basis of the present invention are described beginning on page 122.
• the present invention encompasses a method (embodiment M1 ) to evoke a greater effector activity referred to as the pharmacological activity "PA"; at target cells compared to non-target cells; wherein at the target cells there are present "m” different types of target molecules designated as (p1...pm); at least one of which is present at increased amounts compared to at a non-target cell, and wherein the type of the targeting molecule which is increased on the target cells compared to a non-target cell can be different for a different non-target cell;
• target molecules are biomolecules that are either target receptors or triggering agents
• a target receptor is a chemical structure at the target cells that binds with a useful degree of specificity to a targeting ligand wherein said target receptor is present in increased amounts at the target cells compared to at some non-target cells;
• a "triggering agent” is an enzyme, or biomolecule or other agent which is able to activate a trigger and which is increased at a target compared to at some non-target cells;
• the method is comprised of contacting the cell or cell populations with one or more compounds designated as (C1...Cn), wherein at least one of the compounds has the structure E-iT-i; wherein Ei is comprised of x effector agents that evoke the pharmacological activity PA, and T-i is comprised of the y different targeting ligands, and z different triggers, which increase the pharmacological activity PA at targeted cells and/or decrease the pharmacological activity PA at non-target cells;
• a targeting ligand comprises a chemical structure, which binds with a degree of specificity to a targeting receptor that is enriched at a target cell compared to at a non-target cell;
• a trigger is a chemical group which can undergo in vivo chemical modification either spontaneously or by a triggering agent with the modification leading to trigger activation that modulates the pharmacological activity of the drug;
• the number x is 1 , 2, 3, 4, 5 or about 5; and wherein the number y is 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or about 10; and wherein the number z is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or about 10; and n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or about 10;
• n if n equals one then the sum of y and z is equal to or greater than m; and wherein if n>1 the selectivity of the evoked response in targeted cells is not due solely to internalization and functional cooperation of the different effector groups inside the cells.
• a targeting ligand is a group that binds selectively to a structure associated with the target referred to as a "targeting receptor";
• a trigger is a group that upon in vivo modification by biomolecules referred to as "triggering agents" becomes activated and modulates the activity of ET;
• target cells there are present "m" different types of target molecules designated as (p1...pm); at least one of which is present at increased amounts compared to at a non-target cell, and wherein the type of the targeting molecule which is increased on the target cells compared to a non-target cell can be different for a different non-target cell;
• the number m is 2, 3, 4, 5, or about 6.
• the present invention also encompasses (embodiment ET3) a compound ET in which E is comprised of one or more effector agents having pharmacological activity designated as "PA” and wherein T comprises: a) A group referred to as a "targeting ligand” which selectively binds to a target receptor on the surface of the target cell or in the microenvironment of the target cell; and b) One or more of the following:
• a targeting ligand which selectively binds to a target receptor on the surface of the target cell or in the microenvironment of the target cell;
• a group referred to as a "masked intracellular transport ligand” which can be modified in vivo to give a group referred to as an "intracellular transport ligand” which binds to a target cell receptor that actively transports bound ligands into the target cell;
• a group referred to as a "trigger” that can be modified in vivo, wherein in vivo modification activates the trigger and modulates the pharmacological activity PA;
• IV A group referred to as an "intracellular trapping ligand", which binds to one or more intracellular receptors or a group referred to as a
• T is comprised solely of a targeting ligand a trigger and in vivo modification of the trigger increases the pharmacological activity PA then the in vivo modification which activates the trigger is caused by an enzyme or enzymatic activity that is increased at target cells or decreased at non-target cells;
• T is comprised solely of a targeting ligand a trigger and in vivo modification of the trigger decreases the pharmacological activity PA then the in vivo modification which activates the trigger is caused by an enzyme or enzymatic activity that is decreased at target cells or increased at non-target cells; and provided that T is not an antibody, or an analog or component of an antibody, or a complex of antibodies, or a bispecific antibody, or an analog of a bispecific antibody, or a natural protein, or a complex of natural proteins, or a protein, or a naturally occurring polymer, or a radiolabelled dimer, or a polymer to which is attached at multiple sites one or more pharmacologically active compounds that evoke the same pharmacological activity PA.
• a preferred embodiment, of all the prior embodiments of ET comprises ET wherein ET evokes a greater pharmacological activity PA at the target cell compared to a non-target cell and wherein this target cell selectivity is due to functional cooperation between the components of ET and not due to any single component of ET acting alone.
• a preferred embodiment comprises ET wherein ET is comprised of a compound in which the targeting ligand selectively binds to a target receptor on the surface of the target cell or in the microenvironment of the target cell wherein the concentration of the target receptor is greater on the surface of the target cell or in the microenvironment of the target cell than on the surface or in the microenvironment of non-target cells.
• a preferred embodiment of the present invention comprises ET wherein ET is comprised of a compound with two or more targeting ligands wherein at least one of the targeting ligands binds to a target receptor on the surface of the target cell or in the microenvironment of the target cell wherein the target has an increased amount of that target receptor compared to a non-target cell that binds to a second targeting ligand of the compound. Generally, the increased amount is greater than about two times or greater than about 5 times, or greater than about 10 times.
• a preferred embodiment is comprised of ET in which at least one of the targeting ligands binds to a receptor that is absent or essentially absent from a non-targeted cell.
• Methods for detecting increased amounts of receptors are well known to one skilled in the arts and include immunohistochemistry, radioimmunoassays, enzymatic assays, and a variety of nucleic acid hybridization techniques.
• a preferred embodiment comprises ET wherein ET is comprised of a compound with two or more targeting ligands that binds to a target cell with an affinity that is greater than a non-target cell presenting a target receptor(s) that bind to the targeting ligands of said compound.
• the above mentioned binding affinity to the target cell is at least about 2-5 times greater, or at least about 5-10 times greater, or at least about10-50 times greater, or at least about 50-500 times greater, or at least about 500-5000 times greater, or at least about 5000-50,000 times greater, or at least about 50,000-1 ,000,000 times greater or more then 1 million times greater than to the non-target cell.
• a preferred embodiment comprises ET wherein ET is comprised of a drug with binding affinity to target cells that is approximately the same as to a population of non-target cells however said population of non- target cells have decreased sensitivity to the effects of the effector agent because said normal cells have decreased levels of an intracellular trapping receptor, or decreased sensitivity to the effector agent, or decreased levels of a specific protein necessary for neoantigen formation, or decreased levels of an enzyme that activates a trigger that increases the toxicity of ET, or increased levels of an enzyme that activates a trigger that decreases the toxicity of ET, or by virtue of said normal cells being located in the body at a site such as the brain where the drug ET cannot penetrate to a significant degree.
• a preferred embodiment of ET is comprised of a compound in which the intracellular trapping ligand selectively binds to one or more intracellular receptors wherein the concentration of the intracellular receptors is greater in target cells than in non-target cells.
• a preferred embodiment of ET is comprised of a compound with a trigger that increases the pharmacological activity PA upon in vivo modification and wherein the in vivo modification that activates the trigger is caused by an enzyme or enzymatic activity that is increased at target cells or decreased at non-target cells.
• a preferred embodiment of ET is comprised of a compound with a trigger that decreases the pharmacological activity PA upon in vivo modification and wherein the in vivo modification that activates the trigger is caused by an enzyme or enzymatic activity that is decreased at target cells or increased at non-target cells.
• a preferred embodiment of ET is comprised of a compound in which the intracellular transport ligand binds to a molecule referred to as a "transporter molecule" to form a complex and wherein this complex binds to a target cell receptor that actively transports bound ligands into the target cell.
• a preferred embodiment of ET is comprised of a compound in which the concentration of transporter molecules is increased at the surface of target cells compared to non-target cells.
• a preferred embodiment of ET is comprised of a compound with two targeting ligands that selectively bind to target receptors on the surface of the target cell or in the microenvironment of the target cell wherein the concentration of the target receptors is greater on the surface of the target cell or in the microenvironment of the target cell than on the surface or in the microenvironment of non-target cells.
• these targeting ligands are the same.
• these targeting ligands are different and bind to different types of targeting receptors.
• a preferred embodiment of ET is comprised of a compound with three targeting ligands that selectively bind to target receptors on the surface of the target cell or in the microenvironment of the target cell wherein the concentration of the target receptors is greater on the surface of the target cell or in the microenvironment of the target cell than on the surface or in the microenvironment of non-target cells.
• these targeting ligands are the same.
• these targeting ligands are different and bind to different types of targeting receptors.
• a preferred embodiment of ET is comprised of a compound with four targeting ligands that selectively bind to target receptors on the surface of the target cell or in the microenvironment of the target cell wherein the concentration of the target receptors is greater on the surface of the target cell or in the microenvironment of the target cell than on the surface or in the microenvironment of non-target cells.
• these targeting ligands are the same.
• these targeting ligands are different and bind to different types of targeting receptors.
• ET is comprised of a compound with two or more targeting ligands wherein at least one of the targeting ligands binds to a target receptor on the surface of the target cell or in the microenvironment of the target cell wherein the target has an increased amount of that target receptor compared to a non-target cell that binds to a second targeting ligand of the compound.
• a preferred embodiment of this embodiment comprises a compound with two different targeting ligands that bind to two different targeting receptors.
• Another preferred embodiment of this embodiment comprises a compound with three different targeting ligands that bind to three different targeting receptors.
• Another preferred embodiment of this.embodiment comprises a compound with four different targeting ligands that bind to four different targeting receptors.
• a preferred embodiment (embodiment ET7) of ET is comprised of the following groups:
• N1 targeting ligands which can differ;
• N2 masked intracellular transport ligands which can differ;
• N3 triggers which can differ, designated “detoxification triggers” wherein activation of the trigger decreases the pharmacological activity PA;
• N1 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or about 10
• N2 0, 1 , 2, 3, 4, or about 4
• N3 0, 1 , 2, 3, 4, 5, or about 5
• N4 1 , 2, 3, 4, 5, or about 5;
• the components are covalently coupled directly or by one or more linkers, and wherein the connectivity between groups can vary provided that the functionality of the different components remains intact and wherein the function of ligands is to bind to their respective receptors; the function of the triggers is to be activated and modulate drug activity, and the function of the effector agent is to evoke the pharmacological activity PA; and wherein the linker lengths can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14,
• the connectivity is not critical because the target molecules that the groups interact with are not rigidly fixed in space.
• N4 1
• N4 1
• N6 0
• N6 0
• ETS 1.X 1 , 2, 3, 4, 5, 6...295 and is the number of the structure below:
• A1 ,A2, and A3 designate targeting ligands, which may be the same or different; and B, B1 , and B2, designate triggers that increase the effector activity PA and may be the same or different, and C designates a masked intracellular transport ligand; and D designates an intracellular trapping ligand; or a masked intracellular trapping ligand; and E, E1 , and E2 designate effector agents which may be the same or different, and F designates a trigger that when activated decreases the effector activity PA; and L designates a linker; which may be the same or different from other linkers; and the lines represent the connectivity of the above components:
• a preferred embodiment comprises ET with one selective targeting ligand at
• a preferred embodiment comprises ET with one selective targeting ligand one non-selective targeting ligand.
• a preferred embodiment comprises ET with one selective targeting ligand one non-selective targeting ligand.
• a preferred embodiment comprises ET with two targeting ligands and at least one intracellular trapping ligand or masked intracellular trapping ligand or where
• a preferred embodiment comprises ET with one selective targeting ligand one non-selective targeting ligand.
• a preferred embodiment comprises ET with one selective targeting ligand one non-selective targeting ligand.
• a preferred embodiment comprises ET with three targeting ligands and at least
• Another preferred embodiment of the present invention is comprised of at least one molecule ET that has been covalently linked to a second molecule that binds to a receptor present in increased amounts at a target cell compared to at a non-target cell; and wherein said second molecule is comprised of a monoclonal antibody, or targeting receptor binding fragment of a monoclonal antibody, or an analog or derivative thereof which bears amino acid sequence similarity to portions of a monoclonal antibody.
• the second molecule coupled to ET can be comprised of a natural protein, or a complex of natural proteins, or a protein, or a naturally occurring polymer that binds to the targeting receptor.
• a trigger is a group that upon in vivo modification by biomolecules referred to as "triggering agents" becomes activated and modulates the activity of ET;
• target cells there are present "m" different types of target molecules designated as (p1...pm); at least one of which is present at increased amounts compared to at a non-target cell, and wherein the type of the targeting molecule which is increased on the target cells compared to a non-target cell can be different for a different non-target cell;
• the number m is about 2 to 5.
• the target is comprised of a tumor, or tumor cell, or components of a tumor, or biomolecules present in the microenvironment of the tumor, or stromal cells present in a tumor, and the effector agent or the pharmacalogical activity PA can evoke or can contribute to tumor cell killing and/or comprises a diagnostic agent.
• the target is comprised of a tumor, or tumor cell, or components of a tumor, or biomolecules present in the microenvironment of the tumor, or stromal cells present in a tumor, and the effector agent can evoke or can contribute to tumor cell killing and/or comprises a diagnostic agent.
• a preferred embodiment is an anti-cancer drug ET comprised of effector agents that are cytotoxic drugs, and/or radionuclides, and/or immunostimulatory drugs.
• a preferred embodiment is an anti-cancer drug ET comprised of effector agents that are cytotoxic drugs.
• a preferred embodiment is an anti-cancer drug ET comprised of effector agents that are radionuclides.
• a preferred embodiment is an anti-cancer drug ET comprised of effector agents that are cytotoxic drugs that produce synergistic cytotoxicity.
• a preferred embodiment is an anti-cancer drug ET comprised of effector agents that stimulate the immune system.
• a preferred embodiment is an anti-cancer drug ET comprised of effector agents that stimulate the innate immune system.
• a preferred embodiment is an anti-cancer drug ET comprised of effector agents that irreversibly chemically modify one or more tumor components.
• a preferred embodiment is an anti-cancer drug ET comprised of effector agents that irreversibly chemically modify one or more tumor components that are present in increased amounts in tumor cells or in the microenvironment of tumors compared to vital normal cells.
• a preferred embodiment is an anti-cancer drug ET comprised of effector agents that potentiates the cytotoxic activity of a second effector agent.
• a preferred embodiment is an anti-cancer drug ET with an effector agent that comprises an inhibitor to multi-drug transporter proteins.
• a preferred embodiment is an anti- cancer drug ET with an effector agent that comprises an inhibitor to a membrane protein transporter that faciltates uptake of a nutrient or biomolecule into tumor cells.
• ET is an anti-cancer drug with an effector agent that comprises an inhibitor to nucleoside transporter proteins.
• ET is an anti-cancer drug with targeting ligands that selectively bind to target receptors on the surface of the tumor cell or in the microenvironment of the tumor cell wherein the concentration of the target receptor is greater on the surface of the tumor cell or in the microenvironment of the tumor cell than on the surface or in the microenvironment of normal cells especially vital normal cells.
• ET is an anti-cancer drug with an intracellular trapping ligand that selectively binds to one or more intracellular receptors wherein the concentration of the intracellular receptors is greater in tumor cells then in vital normal cells.
• ET is an anti-cancer drug with a trigger that increases cytotoxicity of the drug upon in vivo modification and wherein the in vivo modification that activates the trigger is caused by an enzyme or enzymatic activity that is increased at tumor cells or decreased at vital normal cells.
• ET is an anti-cancer drug with a trigger that decreases the cytotoxicity of the drug upon in vivo modification and wherein the in vivo modification that activates the trigger is caused by an enzyme or enzymatic activity that is decreased at tumor cells or increased at vital normal cells.
• ET is an anti-cancer drug in which the intracellular transport ligand binds to a molecule referred to as a "transporter molecule" to form a complex and wherein this complex binds to a target cell receptor that actively transports bound ligands into the tumor cell.
• ET is an anti-cancer drug for which the concentration of transporter molecules is increased at the surface of tumor cells compared to vital normal cells
• ET is comprised of an anti- cancer drug with two targeting ligands that selectively bind to target receptors on the surface of the tumor cell or in the microenvironment of the tumor cell wherein the concentration of the target receptors is greater on the surface of the tumor cell or in the microenvironment of the tumor cell than on the surface or in the microenvironment of vital normal cells or normal cells.
• the targeting ligands are the same.
• the targeting ligands are different and bind to different types of targeting receptors.
• ET is an anti-cancer drug with three targeting ligands that selectively bind to target receptors on the surface of the tumor cell or in the microenvironment of the tumor cell wherein the concentration of the target receptors is greater on the surface of the tumor cell or in the microenvironment of the tumor cell than on the surface or in the microenvironment of normal cells or vital normal cells.
• the targeting ligands are the same.
• the targeting ligands are different and bind to different types of targeting receptors.
• ET is an anti-cancer drug with four targeting ligands that selectively bind to target receptors on the surface of the tumor cell or in the microenvironment of the tumor cell wherein the concentration of the target receptors is greater on the surface of the tumor cell or in the microenvironment of the tumor cell than on the surface or in the microenvironment of normal cells or vital normal cells.
• the targeting ligands are the same.
• the targeting ligands are different and bind to different types of targeting receptors.
• ET is an anti-cancer drug comprised of a compound with two or more targeting ligands wherein at least one of the targeting ligands binds to a target receptor on the surface of the target cell or in the microenvironment of the target cell wherein the target has an increased amount of that target receptor compared to a non-target cell that binds to a second targeting ligand of the compound.
• a preferred embodiment of this embodiment comprises a compound with two different targeting ligands that bind to two different targeting receptors.
• Another preferred embodiment of this embodiment comprises a compound with three different targeting ligands that bind to three different targeting receptors.
• Another preferred embodiment of this embodiment comprises a compound with four different targeting ligands that bind to four different targeting receptors.
• the drug binds to at most one type of receptor present on normal cells.
• the anti-cancer compound ET is comprised of one tumor-selective targeting ligand at least one detoxification trigger or where
• compound ET is comprised of one selective targeting ligand one non-selective targeting ligand. In another embodiment of this both targeting ligands are tumor- selective.
• the anti-cancer compound ET is comprised of one selective targeting ligand one non-selective targeting ligand. In another embodiment of this, both targeting ligands are tumor-selective. In another preferred embodiment the anti-cancer compound ET is comprised of two targeting ligands and at least one intracellular trapping ligand or masked
• the anti-cancer compound ET is comprised of one selective targeting ligand one non-selective targeting ligand. In another embodiment of this, both targeting ligands are tumor-selective.
• the anti-cancer compound ET is comprised of one selective targeting ligand one non-selective targeting ligand. In another embodiment of this, both targeting ligands are tumor-selective.
• the anti-cancer compound ET is comprised of three targeting ligands and at least one intracellular trapping ligand or masked
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins. In another embodiment of this, the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is comprised of a drug that stimulates the immune system. In another embodiment of this, the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components. In another embodiment of this, the effector agent is comprised of an inhibitor to multi-drug transporter proteins. In another embodiment of this, the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system. In another embodiment of this, the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components. In another embodiment of this, the effector agent is comprised of an inhibitor to multi-drug transporter proteins. In another embodiment of this, the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins. In another embodiment of this, the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is comprised of a drug that stimulates the immune system. In another embodiment of this, the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components. In another embodiment of this, the effector agent is comprised of an inhibitor to multi-drug transporter proteins. In another embodiment of this, the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system. In another embodiment of this, the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components. In another embodiment of this, the effector agent is comprised of an inhibitor to multi-drug transporter proteins. In another embodiment of this, the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins. In another embodiment of this, the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide. In another embodiment of this, the effector agent is comprised of a drug that stimulates the immune system. In another embodiment of this, the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components. In another embodiment of this, the effector agent is comprised of an inhibitor to multi-drug transporter proteins. In another embodiment of this, the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system. In another embodiment of this, the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components. In another embodiment of this, the effector agent is comprised of an inhibitor to multi-drug transporter proteins. In another embodiment of this, the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the anti-cancer compound ET is comprised of a compound with two targeting ligands wherein at least one of the targeting ligands binds to a target receptor on the surface of the tumor cell or in the microenvironment of the tumor cell wherein the tumor has an increased amount of that target receptor compared to a normal cell or a vital normal cell that binds to a second targeting ligand of the compound.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide. In another embodiment of this, the effector agent is comprised of a drug that stimulates the immune system. In another embodiment of this, the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components. In another embodiment of this, the effector agent is comprised of an inhibitor to multi-drug transporter proteins. In another embodiment of this, the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the anti-cancer compound ET is comprised of a compound with three different targeting ligands that bind to three different target receptors and wherein at least one of the targeting ligands binds to a target receptor on the surface of the tumor cell or in the microenvironment of the tumor cell wherein the tumor has an increased amount of that target receptor compared to a normal cell or a vital normal cell that binds to a second targeting ligand of the compound.
• the anti-cancer compound ET is comprised of a compound with three different targeting ligands that bind to three different target receptors and wherein at least one of the targeting ligands binds to a target receptor on the surface of the tumor cell or in the microenvironment of the tumor cell wherein the tumor has an increased amount of that target receptor compared to a normal cell or a vital normal cell that binds to a second targeting ligand of the compound.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide. In another embodiment of this, the effector agent is comprised of a drug that stimulates the immune system. In another embodiment of this, the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components. In another embodiment of this, the effector agent is comprised of an inhibitor to multi-drug transporter proteins. In another embodiment of this, the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide. In another embodiment of this, the effector agent is comprised of a drug that stimulates the immune system. In another embodiment of this, the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components. In another embodiment of this, the effector agent is comprised of an inhibitor to multi-drug transporter proteins. In another embodiment of this, the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide. In another embodiment of this, the effector agent is comprised of a drug that stimulates the immune system. In another embodiment of this, the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components. In another embodiment of this, the effector agent is comprised of an inhibitor to multi-drug transporter proteins. In another embodiment of this, the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the anti-cancer compound ET is comprised of a compound with four different targeting ligands that bind to four different target receptors and wherein at least one of the targeting ligands binds to a target receptor on the surface of the tumor cell or in the microenvironment of the tumor cell wherein the tumor has an increased amount of that target receptor compared to a normal cell or a vital normal cell that binds to a second targeting ligand of the compound.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• the effector agent is a cytotoxic drug.
• the effector agent is comprised of a radionuclide.
• the effector agent is comprised of a drug that stimulates the immune system.
• the effector agent is comprised of an effector agent that irreversibly chemically modifies one or more tumor components.
• the effector agent is comprised of an inhibitor to multi-drug transporter proteins.
• the effector agent is comprised of an inhibitor to nucleoside transporter proteins.
• ET is an anti-cancer drug comprised of a compound with two or more targeting ligands that binds to a tumor cell with an affinity that is greater than a normal cell presenting a target receptor(s) that bind to the targeting ligands of said compound.
• the above mentioned binding affinity to the tumor cell is at least about 2-5 times greater, or at least about 5-10 times greater, or at least about 10-50 times greater, or at least about 50-500 times greater, or at least about 500-5,000 times greater, or at least about 5,000-50,000 times greater, or at least about 50,000-1 ,000,000 times greater or more than 1 million times greater than to a normal cell or to a vital normal cell.
• the compound has three different targeting ligands.
• the compound has 4 different targeting ligands.
• ET is an anti-cancer drug with binding affinity to tumor cells that is approximately the same as to populations of normal cells.
• said population of normal cells have decreased sensitivity to the toxic effects of the effector agent because said normal cells have decreased levels of an intracellular trapping receptor, or decreased sensitivity to the effector agent, or decreased levels of a specific protein necessary for neoantigen formation, or by virtue of said normal cells being located in the body at a site, such as the brain, where the drug ET cannot penetrate.
• the anti-cancer drug ET is comprised of: I. N1 targeting ligands, which can differ;
• N2 masked intracellular transport ligands which can differ;
• N3 triggers which can differ, designated “detoxification triggers” wherein activation of the trigger decreases the toxicity of the drug;
• N4 effector agents which can differ
• V. N5 triggers which can differ, wherein activation of the trigger increases the toxicity of the drug
• ET evokes a greater toxicity to a tumor cell compared to a non- tumor cell or a vital normal cell and wherein this increased antitumor selectivity is due to functional cooperation between the components of ET and not due to any single component of ET.
• the compound ET is comprised of an anti-cancer drug with at least one targeting ligand that binds to a target receptor selected from the following list:
• cathepsin D 10. cathepsin K 11. cathepsin L 12. cathepsin O 13. fibroblast activation protein
• peripheral benzodiazepam binding receptors 25.
• ET is comprised of an anti- cancer drug with two targeting ligands for receptors that are increased on a tumor cell compared to a normal cell wherein at least one of the targeting ligands binds to a receptor selected from the list given above.
• ET is comprised of an anti-cancer drug with 2 targeting ligands that bind to receptors selected from the above list. In a preferred embodiment these receptors are the same. In a preferred embodiment these receptors are different and bind to different receptors.
• ET is comprised of an anti-cancer drug with 3 targeting ligands that bind to receptors selected from the above list. In a preferred embodiment these receptors are the same. In a preferred embodiment these receptors are different and bind to different receptors.
• ET is comprised of an anti-cancer drug with 4 targeting ligands that bind to receptors selected from the above list. In a preferred embodiment these receptors are the same. In a preferred embodiment these receptors are different and bind to different receptors. In a preferred embodiment of the anti-cancer drug ET the targeting ligands are selected to bind to targeting receptors that are enriched on tumor cells or in the microenvironment of tumor cells.
• a preferred embodiment of the invention comprises an anti-cancer drug comprised of 2 to n targeting ligands designated as “A1”, and “A2" “An” that are connected by a linker designated as “L”, and wherein “An” refers to a targeting ligand that can bind to a targeting receptor designated “an” that is enriched on the surface of or in the microenvironment of the target and to which is also attached aone or more cytotoxic cytotoxic agents.
• the targeting ligand-target receptor complex a1-A1-L-A2-a2 can be stabilized by the binding energy of both the A1-a1 and A2-a2 interactions which can result in extraordinary affinity of E-T to the target cell.
• super high affinity (essentially irreversible) targeting is possible provided that both A1-a1 and A2-a2 are high affinity bindings.
• Doubling the decrease in standard free energy for a reaction squares the equilibrium constant. Although entropic factors can intervene to preclude the addition of a second receptor site from actually doubling the standard free energy change, the impact on the equilibrium constant (binding affinity) can be enormous. Targeting affinity exceeding that seen with monoclonal antibodies can be achieved with low molecular weight compounds.
• A1 and A2 are identical and the target site a1 is present at sufficient density on the target cell then drugs incorporating this structure can induce crosslinking of the cell receptors.
• Many membrane associated proteins are highly mobile within the surface of the cell membrane.
• the binding energy of the drug to the cell can also be substantially increased which can translate into a markedly increased affinity and potency of targeting. If the affinity of A1 to its target site is high then the crosslinked form can be essentially irreversible. Crosslinking of the receptors can also enhance cellular uptake by triggering endocytosis.
• the following reference relates to this subject matter: York S.J.
• the rate of crosslinking can be a function of the square of the receptor concentration. For example, if a tumor cell has 10 times more target sites a1 than normal cells then the tumor cell can form crosslinked receptors at a rate 100 times faster (to a first approximation) than the normal cells. A prerequisite for the successful application of this class of compounds is that the receptor density on the target cell be sufficiently high to allow crosslinking to occur at a meaningful rate.
• the linker length can be selected to optimize crosslinking capacity.
• the rate of crosslinking and essentially irreversible binding of the prodrug to the cell can be a function of the product of the concentration of the receptor target sites a1 and a2. For example, if a tumor cell has 10 times more a1 and 30 times more a2 than normal cells then the tumor cell can form crosslinked receptors at a rate approximately 300 times faster than the normal cells. If the product of the concentration is too low then the magnitude of the avidity enhancement can be minimal. Accordingly, if a1 is a target receptor, which is present only at very low concentrations, then a2 can be selected to be a target receptor, which is present at high concentrations.
• a1 is enriched on the target cell and a2 is present on target and normal cells at equal concentrations
• a1 is a cell membrane protein, which is poorly internalized then a drug complex coupled to a1 can fail to enter the target cell.
• a2 is a cell membrane protein that undergoes facile endocytosis then crosslinked complex can be transported into the cell with increased efficiency.
• A2 can also serve to localize the drug to the cell membrane. For example, if A2 is a simple fatty acid it can partition into the cell membrane in a nonspecific fashion. Nonetheless, this can contribute significantly to the binding energy of the drug to the cell and markedly increase overall target cell affinity.
• the transfer of a fatty acid chain from solution to the lipid phase of the membrane is expected to be a much slower process then the binding of typical high affinty ligand- receptors that are often under diffusion control. Since the equilbrium constant is the ratio of the forward and backward reaction rates (rate of solvation / rate of desolvation), the rate at which the fatty acid group desolvates from the cell membrane can be even slower which can contribute to the retention of the targeted drug to the target cell. Accordingly, the use of a nonspecific group which binds with relatively low energy, and has minimal entropic requirements, in conjunction with a target selective high affinity ligand can markedly enhance targeting effectiveness. It can be noted that the drugs are designed for use in the nanomolar to picomolar range orders of magnitude below the critical micelle concentration.
• the drug has three target selective ligands A1 , A2, and A3.
• Drugs of this type can bind with high affinity to target cells that express all three or any combination of two of the receptors (a1 , a2, a3 or a1 , a2, or a2, a3 or, a1 , a3).
• the advantage of having three receptors is that loss of one receptor is unlikely to confer the tumor resistance to the drug.
• A1 and A2 are selected so as to bind to target sites that are enriched on tumor cells compared to normal cells.
• A1 or A2 can bind to a receptor, structural component, or enzyme located on the tumor cell surface or to an enzyme that binds to the cell surface.
• a preferred embodiment of the invention and of embodiment ET8 has the structure shown below:
• A1 and A2 are tumor-selective targeting ligands, and the L are linkers and B is a trigger that when activated frees the effector agent E from the remainder of the drug ; and wherein E is a cytotoxin.
• Drugs of this class feature two tumor specific high affinity binding ligands covalently coupled via a linker designed to allow both A1 and A2 to interact with receptors a1 and a2 on the tumor cell surface.
• a toxic moiety is coupled covalently to the linker via a functionality which has a trigger mechanism that when activated releases the toxin.
• the requirements for the trigger functionality differ depending upon the nature of the toxin to be delivered and the rate of cellular uptake of E-T. If the free toxin is readily internalized by the target cells then a trigger can be activated by extracellular or ultracellular enzymes or chemical processes. In a preferred embodiment, the trigger can be activated by an enzyme that is enriched in the tumor microenvironment.
• a trigger that is preferentially activated inside cells can be used to free the drug intracellularly. This can be achieved by employing a trigger that is activated by intracellular enzymes. Alternatively, the trigger can be activated by extracelluar enzymes or by spontaneous chemical processes provided that a time delay mechanism is incorporated which allows sufficient time between trigger activation and toxin release for the drug ligand complex E-T to be internalized. Finally, in circumstances where the toxin is effective extracellullary (or intracellularly when still attached to the targeting ligands), the trigger can be omitted entirely.
• the trigger can be activated by an enzyme that is delivered to the target cell via independently selective mechanisms.
• an enzyme that is delivered to the target cell via independently selective mechanisms.
• a significant limitation with Antibody Directed Enzyme Prodrug Therapy (ADEPT), and related approaches is the requirement that for the targeted enzyme to efficiently activate the prodrug, the prodrug can be given at a concentration near the Michaelis Menton constant (Km) for the enzyme substrate interaction which is generally micromolar. Since all drugs are expected to have multiple pathways of metabolism, prodrug activation by non-targeted enzyme mechanisms can result in dose limiting toxicity.
• Km Michaelis Menton constant
• Drugs embodied by the present invention can preferably be used at extremely low concentrations in vivo, generally in the nanomolar to picomolar range or lower. At these concentrations the fate of the drug can be defined by high affinity targeting interactions under perhaps nonequilibrium conditions.
• metabolic enzymes function by forming an enzyme substrate complex that is transformed into the products.
• the Km for enzymes is in the micromolar range. Accordingly, drug metabolism can predominantly occur at the sites where the drug is trapped by the high affinity binding, provided that the drug has a sufficiently long half-life to allow distribution to the target site. If the drug E-T is selectively localized to the tumor surface and the triggering enzyme is also selectively localized to the tumor surface then greatly enhanced antitumor selectivity can result.
• the trigger can be activated by an enzyme which is enriched in non-tumor cells where dose limiting toxicity takes place.
• the (detoxifying) trigger can be activated by an enzyme that is selectively delivered to non-tumor cells.
• the detoxifying trigger can be activated by an enzyme that is coupled to an antibody selective for bone marrow stem cells. This can allow for the selective detoxification of the drug by bone marrow stem cells.
• the scope of the present invention includes a method of sparing vital normal cells of drug toxicity by targeting, to the normal cells, an enzyme that activates a detoxification trigger on the administered targeted drug that detoxifies the drug.
• the scope of the present invention includes the set of a targeted drug with a detoxfication trigger and a targeted enzyme that can activate the detoxification trigger and detoxify or markedly lower the toxicity of the drug.
• the scope of the present invention includes a drug that has a detoxification trigger that when activated functionally detoxifies or lowers the toxicity of the drug by interfering with cellular uptake.
• E-T comprises the following structure:
• A1 and A2 are targeting ligands; B is a trigger that upon activation liberates the effector agent portion of the molecule from the targeting ligands; C is a masked intracellular transport ligand; D is an intracellular trapping ligand or masked intracellular trapping ligand; E is an effector agent; and F is a detoxification trigger that when activated decreases the toxicity or effector activity by interfering with cellular uptake of the effector agent into the cell.
• the drug can bind with very high affinity to targeted tumor cells via receptors a1 and a2.
• either spontaneous chemical processes or enyzymatic processes can trigger the unmasking of the intracellular transporter ligand that is comprised of a ligand that binds to a cellular receptor that then actively transports the complex into the cell.
• Trigger B can either be activated by intracellular enzymes or be activated extracellularly with a delay mechanism that allows sufficient time for the complex to be transported into the cell prior to the release of the toxin.
• the trigger, which unmasks the intracellular transporter ligand can be activated by enzymes that are enriched in the tumor microenvironment, or by ubiquitous enzymes, or by spontaneous chemical processes.
• the unmasking trigger can be activated by a ubiquitous enzyme such as esterase, then it is desirable to incorporate a time delay mechanism.
• the time delay mechanism can serve to allow time for the targeting receptors rather then the intracellular transport functionality to define the specificity of drug distribution.
• a time delay mechanism can be made having a triggering event such as the enzymatic cleavage of an ester that initiates a second chemical reaction that proceeds at a rate with the desired half-life. Triggers are described in detail in a latter section.
• the detoxifying trigger can be activated by an enzyme that is selectively delivered to non-tumor cells.
• the trigger that unmasks the transport ligand can be activated by an enzyme that is selectively and independently targeted to the tumor cells.
• Multifunctional drug delivery vehicles with both toxifying and detoxifying triggers can have the ability to be either toxic or nontoxic to cells depending upon relative rates of activation of the respective trigger functionalities.
• the drugs have a logic circuit with decision-making ability.
• the input corresponds to the levels of enzyme activity available to activate the toxifying and detoxifying triggers respectively.
• the output is increased or decreased drug toxicity for the potential target cell. Glazier previously disclosed a class of anti-cancer drugs that have toxification and detoxification functionalities.
• the following reference relates to this subject matter: 5,274,162, 12/28/93, Glazier, "Antineoplastic Drugs with Bipolar Toxification/Detoxification Functionalities.”; 5,659,061 , 8/19/97, Glazier, "Tumor Protease Activated Prodrugs of Phosphoramide Mustard Analogs with Toxification and Detoxification Functionalities", the contents of which are incorporated herein by reference in their entirety.
• the previously disclosed Antineoplastic Drugs with Bipolar Toxification/Detoxification Functionalities lacked targeting ligands, would need to be used at relatively high doses, and could potentially undergo substantial non-target site metabolism.
• the present invention can allow for very high affinty multifactorial drug targeting.
• the present drugs can be employed at ultra-low doses under conditions in which drug metabolism (activation of triggers) can be defined by the tumor microenvironment.
• the scope of the present invention includes, the class of drugs E-T, wherein the drug binds to the target cell and exerts the biological effector activity of E depending upon the input received by triggers that turn on (or increase) or turn off (or decrease) the biological effector activity of E.
• This class of drugs enables multifactorial targeting in which the factors or properties that define targeting selectivity and biological activity include both the targeting receptors and triggering factors tr1...trn.
• the designation "trn” is used to refer to enzymes or biomolecules or other factors that activate a particular trigger referred to as "trigger N" or "TRN.”
• the scope of the present invention also encompasses the method comprising the following steps:
• Txn-trn The administration of one or more targeted drugs E-T that has one or more triggers TR1...TRN that when activated by the triggering factors tr1...trn undergoes either an increase or decrease in drug effector activity.
• Txn-trn The administration of one or more compounds (Txn-trn) comprised of targeting groups (Txn) linked to a triggering factor (trn), such that the targeting group delivers the triggering factors to selected population of cells (Pxn); and thereby modulates the biological activity of the drug(s) ET at the population of cells Pxn.
• This technology can allow an enhancement of tumor selectivity.
• Vital normal cell populations can be targeted with triggering factors trn that activate the detoxification trigger and decrease the toxicity of the drug E-T.
• tumor cells can be targeted with triggering factors that activate toxifying triggers and thereby enhance the toxicity of the drug E-T.
• the triggering factor trn can be a wide range of enzymes that utilize a component of the trigger as a substrate and thereby activate the trigger functionality.
• the targeting group Txn can be any group or set of groups linked together that bind to the desired population of cells Pxn. Depending upon the context, the targeting group Txn can be selective for tumor cells or for normal cells. A large number of targeting groups selective for tumor cells are described in other sections. Suitable targeting groups include ligands that bind to receptors that are enriched on tumor cells, monoclonal antibodies, monoclonal antibody analogs, Fab portions or an antibody or monoclonal antibody, growth factor or any other structure which binds selectively to the target cell.
• Txn-trn When targeting Txn-trn to normal cells the Txn can be selected to bind to receptors that are enriched on vital normal cells relative to tumor cells.
• Txn can be a monoclonal antibody specific for the CD34 antigen, which is present on the surface of vital bone marrow stem cells but absent from most tumors. The complex Txn-trn could then be used to selectively detoxify the drug E-T on CD34 + bone marrow stem cells.
• Txn-trn is selected such that Txn binds to a protein or factor that is lost or under-expressed on the surface of tumor cells and trn is comprised of an enzyme that activates a detoxification trigger on the drug E-T.
• Txn is a monoclonal antibody or monoclonal antibody analog which binds to one of the following membrane associated proteins which is under-expressed in various human cancers: E-cadherin; Transforming growth factor beta receptors; Syndecan-1; Galectin -3; Deleted in colorectal cancer (DCC); Epil or Epitheal Protein Lost in Neoplasm; KAI1 protein; Connexin 43; H- cadherin; CD38; VLA-2 collagen receptor; P-cadherin; Luminal epithelial antigen (LEA135); Maspin; Mel-Cam; Billiary glycoprotein; Epithelial cell adhesion molecule C-CAM; Beta 4 integrin subunit; and Hemidesmosomal proteins.
• Intracellular delivery is essential for the activity of many drugs.
• a general method to deliver drugs into cells is to couple the drugs to a ligand such as folic acid, which is taken up by cells via receptor mediated endocytosis.
• a ligand such as folic acid
• the following reference relates to this subject matter: 5,688,488, 11/18/97, Low, et al., "Composition and Method for Tumor Imaging.”; 5,416,016, 5/16/95, Low, et al., “Method for Enhancing Transmembrane Transport of Exogenous Molecules.”, the contents of which are incorporated herein by reference in their entirety.
• an intracellular transport ligand such as folic acid
• intracellular transport ligand such as folic acid
• the spectrum of drug distribution and targeting would be significantly defined by the distribution of folate receptors in the body.
• Folate targeted moieties end up largely in the kidney, which is often undesirable.
• Wang S., et al. "Design and Synthesis of [1111nJDTPA-Folate for use as a Tumor-Targeted Radiopharmaceutical," Bioconjug Chem, 8(5):673-9 (1997), the contents of which is incorporated herein by reference in its entirety.
• the properties of a complex of the protein pro-urokinase and saporin serves to illustrate how targeting and internalization can be mechanistically distinct.
• This complex binds to the urokinase receptor of tumor cells and is internalized following binding of the saporin to the low-density lipoprotein transport receptor.
• This example does not involve a masked intracellular transport ligand.
• the following reference relates to this subject matter: Ippoliti R., et al., "Endocytosis of a Chimera between Human Pro-Urokinase and the Plant Toxin Saporin: An Unusual Internalization Mechanism," FASEB, 14(10):1335-1344 (2000), the contents of which is incorporated herein by reference in its entirety.
• a compound ET further comprising a masked intracellular transporter ligand provides a general solution to the problem of efficient intracellular drug transport while retaining targeting selectivity due to the targeting ligands.
• a masked intracellular transporter ligand is comprised of a group which when unmasked is able to bind to cellular receptors that transport bound ligands into the cell.
• the current invention allows targeting to be defined by the targeting ligands.
• a second major advantage is that the cell associated target receptors that provide targeting specificity need not possess the property of being able to transport the targeted drug into the cells.
• the masked intracellular transported ligand provides a means by which to provide a simultaneous plurality of intracellular transport mechanisms that can decrease the development of drug resistance.
• a variety of masked transporter ligands can be employed. Preferably the following factors are considered individually or in combination in selecting the masked ligand: 1.) When unmasked the group can bind with sufficient affinity to a structure on the target cell, which can activate transport into the cell; 2.) The group has a chemical moiety which can be modified in a reversible manner such that the modification impairs the ability of the group to bind productively to the cellular transport mechanism (ie., a group that allows for masking); 3.) The masked transporter group can be capable of being unmasked by interaction with an enzyme, metabolite, or by a spontaneous chemical process; and
• the unmasked intracellular transporter group can bind to a protein or other factor that also binds to a cell membrane receptor and activates intracellular transport of bound ligands.
• the masked intracellular transporter ligand is a folic acid derivative coupled via one of its carboxylate groups, preferably the gamma carboxylate group, through a linker to the rest of the drug, wherein the folic acid is substituted in a bioreversible manner such that binding of the derivative to the folate receptors is impaired in a bioreversible manner.
• Preferred sites of derivatization are nitrogen 10 or at the alpha carboxy group.
• a preferred embodiment comprises substitution at the N10 position of the folic acid by a bioreversible amino protecting group referred to as a "trigger" that can be modified in vivo and which, upon this modification referred to as "trigger activation", unmasks the amino group.
• Another preferred embodiment comprises folic acid substituted at the alpha carboxy group to yield an ester or amide.
• R is a trigger group and R1 is a bioreversible protecting group for -X-H, and wherein X is O, NH, or S.
• Preferred triggers and preferred embodiments of R1 are described in the trigger section of this document. Cleavage of the trigger can unmask the folate and initiate the process of active cell uptake.
• a wide variety of triggers can be employed including esters, phosphoesters, phosphodiesters, amides, substituted disulfides, oligopeptides, and glycosides. In principle, any functionality suited for use in the ADEPT approach as a trigger could be employed along with an appropriately selected target enzyme that cleaves that trigger.
• the trigger can be activated by tumor-selective proteases.
• a clock-like time delay trigger is employed to unmask the intracellular transport ligand. Triggers of this type can allow the drug to have time to bind to the tumor prior to unmasking of the intracellular transport ligand. A variety of clock-like time delay triggers are described in the trigger section of the present invention.
• the masked intracellular transporter ligand comprises biotin that is chemically modified in such a manner as to interfere with receptor binding in a bioreversible manner.
• Biotin can be linked to the remainder of the drug via its carboxylate group and can retain binding affinity to biotin receptors.
• a preferred embodiment comprises biotin with bioreversible substitution of one or more of the ureido amidic protons as illustrated below:
• X and R can be groups as described previously for the masked folate trigger.
• Drugs of this class can be administered in conjunction with one or more transporter moieties to which is coupled a biotin binding factor such as avidin or streptavidin.
• the transporter moieties are selected such, they bind to receptors on the tumor cell surface and are internalized. Binding of the unmasked biotin to the administered avidin- transporter moiety can transport the drug complex into the tumor cell.
• the avidin-transporter moiety can be tumor-selective or non-selective without specificity for tumor cells. Its role is to efficiently deliver the drug already targeted and located on the tumor cell surface into the cell.
• the drug it is preferred to administer the drug first, allow time for the tumor localization to occur and then to administer the avidin-transporter moiety. Although it can be pointed out that high affinity between the drug and the avidin-transporter can only occur after the biotin is unmasked.
• the avidin-transporter can be given intravenously at a sufficiently high dose to allow contact with the tumor cells. It is preferable to use simultaneously at least two different types of avidin-transporters to avoid the selection of tumor drug resistance based on lack of binding or impaired internalization of one particular type of avidin-transporter.
• Avidin can be coupled to the transporter function in a fashion that does not impede high affinity biotin binding; 2.) The transporter function can bind to the target cells and be internalized; and 3.) The avidin-transporter can be of low toxicity.
• biotin-binding moiety such as avidin
• Preferred transporter moieties include: transferrin, alpha 2 macroglobulin, insulin, folic acid, and epidermal growth factor.
• Monoclonal antibodies against receptors or occupied receptor complexes known to undergo endocytosis can also be used. Techniques for coupling biotin- binding factors such as avidin to other moieties are well known.
• the scope of the present invention includes compounds comprised of one or more masked intracellular transport ligands and the method of delivering drugs or other effector molecules into cells by contacting the cells with a compound that has one or more masked intracellular transport ligands.
• the scope of the present invention also includes the method of delivering drugs and effector molecules into cells that comprises contacting the cells with a targeted drug ET and also contacting the cells with one or more targeted transport moiety that facilitates drug transport into the cell.
• the drug ET and targeted transport moiety are each targeted to different targets present on the target cells.
• the target is a tumor cell.
• Preferred tumor-selective targets are described throughout this document.
• the scope of the present invention includes a preferred embodiment that comprises a method of delivering a targeted drug or effector molecule into cells by multiple independent non-target endocytotic receptors. This method can be useful to circumvent drug resistance due to the loss of a single intracellular transport or endocytotic receptor.
• A1 and A2 are targeting ligands; B1 and B2 are triggers that upon activation liberate the effector agent portion of the molecule from the targeting ligands and E is an effector agent.
• This embodiment incorporates, in addition to the features discussed previously, an effector mechanism comprised of two different cytotoxic agents, which can be released by two different triggering mechanisms. This feature can markedly decrease the rate at which tumor resistance develops to the drugs without significantly increasing overall drug toxicity. In addition, this can allow the joint delivery of two drugs that exhibit synergistic toxicity.
• the toxins are selected such that resistance to each is mediated by independent mechanisms.
• a preferred embodiment of the present invention is the anti-cancer compound ET comprised both of a cytotoxic moiety(s) and an inhibitor to multi-drug resistance mechanisms such as MDR1 P- glycoprotein.
• This can allow major mechanisms of tumor drug resistance to be overcome at a target specific level without increasing total systemic toxicity.
• the emergence of tumor resistance to a broad range of unrelated antineoplastic drugs by increased expression of the multi-drug transporter P-glycoprotein, which actively transports the drugs out of the tumor cells, is a major and fundamental limitation in cancer treatment.
• Clinical trials to date have been unsuccessful and complicated by systemic toxicity.
• the present invention can allow for the selective delivery of the multi-drug resistance inhibitors to tumor cells concurrently with the selective delivery of the anti-cancer drugs.
• the present invention can be used to target drugs to essentially any type of cell, cell population, tissue, or tissue type.
• the targeting specificity or targeting domain of multifunctional drug delivery vehicles can be defined as the populations of cells that are subjected to the effector action of the drug.
• the targeting domain of multifunctional drug delivery vehicles is a multifactorial or multivariable function in which the variables are targeting ligands specificity, specificity of triggers, and nature of the effector agent ultimately delivered. It is the interaction between these variables that ultimately defines the targeting domain and can allow reasonably specific tumor targeting despite the fact that no single factor is unique to tumors.
• the initial targeting specificity of the drugs can be defined by the combined high affinity interactions of the drug targeting ligands A1 ,...An with the target cell associated receptors a1 ,...an.
• the drugs are to be administered at a dose sufficient to bind an effective quantity to the targeted cell population or at a dose sufficient to evoke the desired therapeutic activity.
• the concentration range can generally be in the nanamolar to picomolar range or lower.
• the use of excessive concentrations can allow secondary non-targeting factors to dominate the pattern of drug distribution and metabolism with a potential reduction in the targeting selectivity and therapeutic index.
• the present class of drugs can be orders of magnitude more potent for targeted cells than the non-target toxin due to the high receptor mediated affinity of the drug to the targeted cells.
• the targeting ligands A1 ,...An can be selected to bind a large variety of receptors a1 ,...an which are present at increased amounts on the surface of tumor cells compared to vital normal cells.
• target selective and tumor-selective are used in a functional sense in this patent application. Absolute selectivity is elusive. Drugs always have some form of dose limiting toxicity that restricts the therapeutic index.
• a target can be considered tumor- selective if it is enriched on tumor cells compared to vital normal cells in the tissue that ordinarily suffers dose limiting toxicity.
• a receptor or enzyme enriched on tumor cells compared to bone marrow stem cells would be a suitable "tumor-selective" target even if this target is not unique to tumor cells.
• Normal enzymes or receptors in abnormal locations can also function as tumor-selective targets and are a biochemical manifestation of metastasis.
• an enzyme is ordinarily confined to the luminal surface of the gastrointestinal tract the presence of that enzyme on malignant cells metastatic to the liver can be used for selective targeting. This can be accomplished by employing a drug that is given intravenously and fails to penetrate to the luminal surface of the Gl tract.
• the target sites on the normal Gl cells can be blocked by an orally nonabsorbable inhibitor to the receptor or enzyme.
• Useful tumor-selective targets can also be receptors or enzymes that are present on both malignant cells and normal cells provided that the targeted normal cells are not vital for life. Normal enzymes that are present intracellularly in normal cells but released or activated extracellularly in the tumor microenvironment can also be used for selective targeting provided that the drug is designed to remain in the extracellular space.
• the targeted cell receptors can be any chemical moiety that is enriched on the target cells relative to the cell populations which one desires not to target.
• combinatorial chemistry and high throughput automated screening it is now possible to select high affinity ligands that can bind to essentially any biological receptor.
• the following reference relates to this subject matter: Wilson, Stephen R.; Czarnik, Anthony W.(eds.), "Combinatorial Chemistry; Synthesis and Application.” John Wiley & Sons, Inc., the contents of which is incorporated herein by reference in its entirety.
• the steps in this process are well known to one skilled in the arts and include: 1.) Coupling a large library of potential receptor binding ligands to a linker and reporter functionality such as a fluorescent group, an enzyme, or a group such as biotin which can be readily detected; 2.) Coupling the receptor moiety to a solid phase; 3.) Incubating the receptor ligand-detector molecules with the receptor; 4.) Washing to remove unbound ligand; and
• malignancy is uncontrolled cell proliferation and tissue invasion. Neither the processes of cell replication nor the enzymology of tissue invasion (remodeling) are by themselves uniquely diagnostic of malignancy. But jointly, these processes likely can provide highly selective criteria to define effective targeting for the treatment of malignancy.
• the current class of multifunctional anti-cancer drugs provides the opportunity to have anti-cancer agents that are targeted simultaneously and jointly to both the proliferative and the invasive character of malignant cells.
• Antineoplastic agents directed against cell replication are well-known and typified by anti-cancer drugs such as alkylating agents, topoisomerase inhibitors, DNA antimetabolites, DNA polymerase inhibitors, and antimitotic agents.
• tissue invasiveness Targeting such drugs to cells that express the property of tissue invasiveness can significantly increase antitumor selectivity. Since the biochemical expression of tissue invasiveness is an essential component of malignancy, the development of tumor resistance by loss of these properties can be incompatable with persistence of the malignant phenotype. It is precisely for this reason that cytotoxic targeting towards the coupled expression of invasiveness and proliferation is so compelling. It is also important to recognize that tumors are composed of a heterogenous population with the most invasive and malignant cells defining the ultimate clinical outcome.
• receptor- selective targets There are five general classes of receptors which can be employed as "tumor- selective targets": 1.) Enzymes and factors that are related to the biochemical manifestations of uncontrolled cell growth. Some examples include: autocrine growth factors, and abnormal receptor tyrosine kinases;
• Enzymes and molecules that are expressed by the tumor cells or in the microenvironment of tumor cells that are involved in the mechanism of tissue invasion include collagenases, plasmin, urokinases, metalloproteinases, cathepsins, heparanase;
• Normal enzymes and molecules associated with both tumor cells and normal tissue provided that the normal tissue is not vital to life or not sensitive to the delivered anti-cancer drug. Examples include: prostatic membrane surface antigen, prostatic specific antigen, hepsin in ovarian cancer, and neutral endopeptidase in leukemia; and
• Receptors unique to tumor cells such as tumor specific antigens.
• Suitable receptor targets include enzymes that are membrane associated with the target cell or which bind to receptors on the target cell, structural components of the target cell, or hormone receptors on the target cell. It is important to emphasize the point that targets, which individually cannot provide sufficient specificity in combination with the multifunctionality of the present invention, can provide useful targeting selectivity and in preferred cases can provide excellent target specificity. Targets also can be localized to the microenvironment of tumors. This is discussed in more detail in the section on targeted immunotherapy.
• Targeting ligands can also bind to intracellular receptors that are enriched in target cells.
• the biological activity is dependent upon intracellular concentration that is a function of the relative rates of drug influx and drug efflux.
• Many anti-cancer drugs are actively pumped out of cells by p- glycoprotein and related proteins. This is a major mechanism of tumor resistance to antineoplastic drugs.
• Intracellular targeting ligands that bind to intracellular receptors that are enriched in target cells can contribute to drug selectivity by trapping drug selectively in target cells. A variety of specific and non-specific intracellular trapping ligands are described elsewhere in this patent.
• Preferred embodiments include the anti-cancer compounds ET comprised of targeting ligands, triggers, and effector agents that are selective for combinations of the following factors or targeting properties: 1 ) 5'nucleotidase 2) 5-aminoimidazole-4-carboxamide ribonucleotide transferase
• dipeptidyl peptidase IV 32
• emmprin 33 epidermal growth factor receptors and related proteins
• melanocyte stimulating hormone receptor j 62
• tissue factor 1078 tissue plasminogen activator
• transferrin receptors 111 transforming growth factors and their receptors 112 transporter (PEPT1 ) 113 trypsin 114 tumor necrosis factor receptor 115 type IV collagenase 116 uridine/cytidine kinase 117 urokinase 118 vacuolar type proton pump (V- ATPase) 119 xanthine-guanine phosphoribosyltransferase 120 any tumor-selective antigen 121 any tissue specific antigen which is present on tumor cells, but absent from vital normal cells
• targeting ligands described below are preferred embodiments of targeting ligands for anti-cancer drugs ET of the present invention and all targeted anti- cancer drugs that are embodiments of the present invention: Laminin Receptors
• the laminin receptor is a membrane associated protein which binds laminin, elastin and, type IV collagen.
• the receptor facilitates the cell adhesion and migration key components of invasiveness characteristic of malignancy.
• the laminin receptor is over-expressed in a large number of malignancies including: breast, colon, prostate, ovarian, renal, pancreatic, melanoma, thyroid, lung, lymphomas, leukemias, gastric, and hepatocellular cancer. It is strongly associated with metastatic ability and is an independent adverse prognostic in breast, prostate, lung, thyroid and gastric cancer.
• the laminin receptor although highly over-expressed in many malignancies, is a normal cellular component of many tissues especially endothelial cells.
• the very low levels of laminin receptor in normal bone marrow cells is of significance as bone marrow toxicity is dose limiting for most anti-cancer drugs.
• the following references relate to this subject matter: Hand P.H., et al., "Expression of Laminin Receptor in Normal and Carcinomatous Human Tissues as Defined by a Monoclonal Antibody," Cancer Res, 45(6):2713-9 (1985) ;Hilario E., et al., "Presence of Laminin and 67kDa Laminin-Receptor on Endothelial Surface of Lung Capillaries.
• the laminin receptor binds with high affinity to a number of oligopeptides that are related to laminin or elastin.
• Laminin receptor antagonists have been shown to inhibit metastasis in animals.
• Radiolabelled laminin binding analogs and monoclonal antibodies specific for the laminin receptor have been explored as potential diagnostic and or therapeutic agents.
• the following references relate to this subject matter: Maeda M., et al., "Amino Acids and Peptides. XXXIII.
• the targeting ligand comprises the following structures:
• the wavy line is H, OH, NH 2 , or the site of linker attachment to the remainder of the drug complex; and wherein the amino acid residues have the L-configu ration, or the D configuration, or are a racemic mixture.
• Integrin alpha V beta 3 Integrin alpha V beta 3 ( ⁇ v ⁇ 3 ) are cell adhesion molecules which bind to RGD
• tumor cells in a number of important malignancies including: melanoma, breast cancer metastatic to bone, ovarian cancer, and
• ⁇ v ⁇ 3 expression is a strong adverse prognostic indicator in
• ⁇ v ⁇ 3 is not unique to tumors or tumor
• neovasculature and is also expressed by platlets, osteoclasts, endothelial cells during wound repair, and by vascular smooth muscle cells. Antagonists and
• monoclonal antibodies to v ⁇ 3 inhibit angiogenesis and tumor growth.
• Radiolabelled ligands for ⁇ v ⁇ 3 have been described as potential tumor imaging
• Doxorubicin conjugates of integrin ligands have been described as
• Keenan R.M., et al. "Benzimidazole Derivatives as Arginine Mimetics in 1 ,4- Benzodiazepine Nonpeptide Vitronectin Receptor (Alpha V Beta 3) Antagonists," Bioorg Med Chem Lett, 8(22):3165-70 (1998); Hart S.L., et al., "Cell Binding and Internalization by Filamentous Phage Displaying a Cyclic Arg-Gly-Asp- Containing Peptide," J Biol Chem, 269(17): 12468-74 (1994); Keenan R.M., et al., "Conformational Preferences in a Benzodiazepine Series of Potent Nonpeptide Fibrinogen Receptor Antagonists," J Med Chem, 42(4):545-59 (1999); Nicolaou K.C., et al.
• targeting ligand comprised of a structure that binds to ⁇ v ⁇ 3 .
• the targeting ligand is comprised of one of the following structures:
• wavy line is the site of linker attachment to the remainder of the drug complex and Ri is H, or methyl, and amino acids in the cyclopeptide are the L-configu ration except for the tyrosine which is the D-configuration.
• the targeting ligand for ⁇ v ⁇ 3 is used in
• tumor neovasculature such as urokinase, plasmin, MMP-1-, MMP-3.
• target receptors such as urokinase, plasmin, MMP-1-, MMP-3.
• Matrix metalloproteases are enzymes, which degrade connective tissue and which are over-expressed by a large number of tumors and stroma of tumors.
• MMP matrix metalloproteases
• inhibition of MMP activity does not typically produce cytotoxicity and several clinical trials to date have failed to show efficacy of MMP inhibitors as antimetastatic drugs.
• Membrane type metalloproteinases are associated with the cell surface by a hydrophobic transmembrane domains or glycosylphosphatidylinositol anchors.
• Other MMP's become associated with the surface of tumor cells by a variety of mechanisms which include binding to: 1.) MT-1-MMP and TIMP2 (tissue inhibitor of metalloproteinase); 2.) Heparin sulfate proteoglycans; 3.) Hyaluronan receptor CD44; 4.) Integrin alpha V beta 3; and
• EMMPRIN Extracellular matrix metalloproteinase inducer
• ligands which bind to MMP's, can be employed in targeting tumors.
• the following references relate to this subject matter: Sato H., et al., "Cell Surface Binding and Activation of Gelatinase a Induced by Expression of Membrane-Type-1 -Matrix Metalloproteinase (MT1-MMP),” FEBS Lett, 385(3):238-40 (1996); Monsky W.L., et al., “Binding and Localization of M(r) 72,000 Matrix Metalloproteinase at Cell Surface Invadopodia," Cancer Res, 53(13):3159-64 (1993); Yu Q; Stamenkovic I., “Cell Surface-Localized Matrix Metalloproteinase-9 Proteolytically Activates TGF-Beta and Promotes Tumor Invasion and Angiogenesis," Genes Dev, 14(2):163-76 (2000); Menashi S.,
• a preferred embodiment of the present invention is a compound ET with a targeting ligand comprised of a structure that binds to a matrix metalloproteinase.
• Matrix Metalloproteinase 7 is a protease, which is constitutively produced by exocrine epithelial cells. MMP-7 is over-expressed by tumor cells in wide range of malignancies including: ovarian, gastric, prostate, colorectal, endometrial, gliomas, and breast cancer. MMP-7 contrasts with many other metalloproteases, which are over-expressed by tumor stromal elements rather than the tumor cells. At the present time there are no known methods to convert the over-expression of MMP-7 into selective tumor toxicity.

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