Claims for Patent: 8,168,568

Summary for Patent: 8,168,568

Title:	Combinatorial therapy for protein signaling diseases
Abstract:	A method for selecting combinations of drugs for treatment of diseases that arise from deranged signaling pathways is disclosed. The method involves measuring the activity states for signaling proteins in a diseased cell and determining whether the activity states are different from the activity states observed for a reference cell such as a normal cell. Based on the observed differences, combinations of two or more drugs are selected to reduce these differences. Treatment of a subject with the combinations restores the activity states of the signaling proteins of the deranged disease-associated signaling pathways toward the activity states observed in the reference cell. Since the diseased cell and the reference cell can both be obtained from the same subject, combinations of drugs that specifically target patient-specific signaling derangements is possible.
Inventor(s):	Mehta; Arpita I. (Hoffman Estates, IL), Liotta; Lance A. (Bethesda, MD), Petricoin; Emanuel F. (Gainesville, VA)
Assignee:	The United States of America, as represented by the Secretary of the Department of Health and Human Services (Washington, DC) N/A (N/A)
Application Number:	12/581,019
Patent Claims:	1. A method for selecting a combination of therapeutic agents for individualized treatment of a disease in a patient caused by an abnormal network of interconnected cell signaling proteins that leads to an aberrant cellular response in the patient, comprising: measuring activity states for a plurality of different signaling proteins extracted from diseased cells obtained from a tissue of the patient; determining whether the activity states measured for the plurality of signaling proteins extracted from the diseased cells are different from activity states for corresponding signaling proteins from reference cells to detect differences between the activity states of individual signaling proteins from the diseased cells and the activity states of the corresponding individual signaling proteins from the reference cells, thereby providing a snapshot of the signaling proteins in the diseased cells that form an abnormal network of interconnected cell signaling proteins that leads to the aberrant cellular response in the patient; and selecting a combination of at least two different therapeutic agents for the patient to reduce the difference that was detected in the activity states of two or more of the signaling proteins in the network of interconnected cell signaling proteins from the activity states of the corresponding signaling proteins in the reference cells. 2. The method of claim 1, wherein the combination of therapeutic agents provides a synergistic improvement in efficacy of treatment of the aberrant cellular response when compared to the combined efficacies of the agents administered alone at the same dose. 3. The method of claim 1 further comprising isolating the diseased cells from the tissue of the patient. 4. The method of claim 3, wherein isolating the diseased cells comprises microdissection of the diseased cells from the tissue. 5. The method of claim 4, wherein microdissection comprises laser capture microdissection. 6. The method of claim 3, wherein isolating the diseased cells comprises isolating the diseased cells by fluorescence activated cell sorting. 7. The method of claim 1, further comprising extracting the plurality of different signaling proteins from a cell sample comprising the diseased cells. 8. The method of claim 7, wherein the cell sample is a sample of cells obtained by microdissection. 9. The method of claim 7, wherein the cell sample is a sample of cells obtained by laser capture microdissection. 10. The method of claim 1, wherein measuring the activity states of the plurality of signaling proteins comprises measuring the activity states using protein microarray analysis, immunohistochemistry, antibody microarray analysis, or bead capture. 11. The method of claim 1, wherein measuring the activity states of the plurality of signaling proteins extracted from the diseased cells comprises using reverse phase protein microarray analysis. 12. The method of claim 11, wherein the reverse phase protein microarray analysis comprises microarray analysis of phosphorylated signaling proteins using antibodies that specifically bind to a particular phosphorylated signaling protein. 13. The method of claim 12, wherein the microarray analysis comprises microarray analysis of total amounts of signaling proteins using antibodies that specifically bind to particular signaling proteins regardless of their phosphorylation state, and the activity state of the signaling protein is determined as a ratio of the phosphorylated signaling protein to the total amount of the signaling protein. 14. The method of claim 1, wherein the reference cells are normal cells, cells before or after a treatment, or cells before or after a disease or a stage of disease. 15. The method of claim 14, wherein the reference cells are normal cells. 16. The method of claim 14, wherein the reference cells comprise cells that have not been treated with a therapeutic agent. 17. The method of claim 1, wherein the diseased cells and the reference cells are obtained from the same patient. 18. The method of claim 1, wherein the diseased cells are obtained from the patient and the reference cells are obtained from another patient. 19. The method of claim 1, wherein the reference cells are cultured cells. 20. The method of claim 1, further comprising administering the combination to the patient. 21. The method of claim 1, wherein the aberrant cellular response comprises abnormal growth, apoptosis, cytoskeletal remodeling, survival, receptor localization and distribution, gene transcription, motility, differentiation, proliferation, inflammation or angiogenesis. 22. The method of claim 1, wherein measuring the activity states of the signaling proteins comprises determining one or more of a protein-protein interaction, a post-translational modification, a protein cleavage, a translocation to an organelle or compartment, an ion channel activation, a concentration of a soluble mediator that is a product or a substrate of the proteins, a protein-nucleic acid interaction, a protein-lipid interaction, or a protein-carbohydrate interaction. 23. The method of claim 22, wherein the post-translational modification comprises phosphorylation, farnesylation, myristylation, acetylation or ubiquitinization. 24. The method of claim 1, wherein determining differences between the activity states of the plurality of signaling proteins between the diseased cells and the reference cells comprises pattern recognition. 25. The method of claim 1, wherein the combination of therapeutic agents comprises two or more of drugs that separately target a combination of EGFr dimerization, EGFr phosphorylation, AKT phosphorylation, non-voltage gated calcium ion channels, cyclooxygenase-1, cyclooxygenase-2, MEK-1, NFKB/IKB, and P38. 26. The method of claim 1, wherein the combination prevents shunting to or around a signaling pathway. 27. The method of claim 26, wherein the combination includes a drug that inhibits MEK phosphorylation of ERK kinase, and shunting occurs via activation and phosphorylation of CREB. 28. The method of claim 1, wherein the combination comprises a prostaglandin pathway effector and a non-voltage gated calcium influx channel effector. 29. The method of claim 1, wherein the combination comprises CaI and a specific COX-2 inhibitor. 30. The method of claim 29, wherein the specific COX-2 inhibitor comprises Rofecoxib, Celecoxib or LM-1685. 31. The method of claim 1, wherein the combination comprises an AKT kinase inhibitor and either an EGFR dimerization inhibitor or an EGF kinase inhibitor. 32. The method of claim 31, wherein the EGF dimerization inhibitor comprises Trastuzumab and the EGF kinase inhibitor comprises IRESSA. 33. The method of claim 1, wherein the combination comprises a PKCalpha agonist resulting in phosphorylation and activation of PKCalpha. 34. The method of claim 31, wherein the combination comprises an AKT kinase inhibitor and Trastuzumab. 35. The method of claim 1, wherein the disease comprises a neurodegenerative disease, memory loss or cancer. 36. The method of claim 35, wherein the disease is selected from the group consisting of breast cancer, lung cancer, and colon cancer. 37. The method of claim 1, wherein one or more of the signaling proteins in the plurality of different signaling proteins are members of an integrin pathway, a focal adhesion signaling pathway, an Akt signaling pathway, an IL-6R pathway, a growth factor pathway, a chemokine receptor signal pathway, a cell-cycle signaling pathway, a stress signal pathway, an apoptosis signaling pathway, a Tau/beta signaling pathway, a pro-inflammatory pathway, a differentiation signaling pathway, a T-cell receptor pathway, a death-receptor signaling pathway, a survival signaling pathway, a MAPK signaling pathway, a p38 MAPK signaling pathway, a G protein coupled receptor signaling pathway, a SAPK/JNK signaling pathway, an insulin receptor signaling pathway, a Wnt signaling pathway, a c-Kit pathway, a c-kit signaling pathway, a B-cell antigen signaling pathway, or a Jak/Stat signaling pathway. 38. The method of claim 1, wherein the activity state is phosphorylation of the signaling proteins and measuring comprises determining a ratio of the amount of phosphorylated signaling proteins to the total amount of signaling proteins. 39. The method of claim 1 further comprising repeating the steps of claim 1 for additional diseased cells obtained from the patient during or following administration of the combination to the patient and combining at least one additional therapeutic agent with the combination to make a second combination, wherein the at least one additional therapeutic agent in the second combination reduces a difference in the activity state that was detected by repeating the steps of claim 1. 40. The method of claim 1, wherein the difference in activity states detected is an increase in the activity state of an individual signaling protein from the diseased cells in comparison to the same signaling protein in the reference cells, and the therapeutic agents are selected to counteract the increase in the activity state of the individual signaling protein from the diseased cells. 41. The method of claim 40, wherein the increase in activity state of the individual signaling protein from the diseased cells is an increase in phosphorylation and the therapeutic agents are selected to counteract the increase in phosphorylation of the individual signaling protein from the diseased cells. 42. The method of claim 1, wherein the difference in activity states detected is a decrease in the activity state of an individual signaling protein from the diseased cells in comparison to the same signaling protein in the reference cell, and the therapeutic agents are selected to counteract the decrease in the activity state of the individual signaling protein from the diseased cells. 43. The method of claim 42, wherein the decrease in activity state of the individual signaling protein from the diseased cells is a decrease in phosphorylation and the therapeutic agents are selected to counteract the decrease in phosphorylation of the individual signaling protein from the diseased cells. 44. The method of claim 1, wherein the differences detected are a concordant increase in phosphorylation of a protein belonging to the c-kit family of proteins and an estrogen receptor in the diseased cell relative to the reference cell, and the combination comprises an aromatase inhibitor and STI-572. 45. A method for treatment of a patient having a disease caused by an abnormal network of interconnected cell signaling proteins leading to an aberrant cellular response in the patient, comprising: measuring activity states of a plurality of different signaling proteins extracted from diseased cells obtained from the patient; comparing the activity states of the signaling proteins extracted from the diseased cells to the activity states of the corresponding signaling proteins in reference cells to detect differences between the activity states of individual signaling proteins from the diseased cells and the activity states of the corresponding individual signaling proteins in the reference cells, thereby providing a snapshot of the signaling proteins in the diseased cells that form an abnormal network of interconnected cell signaling proteins that leads to the aberrant cellular response in the patient; selecting a combination of at least two therapeutic agents for administration to the patient, wherein the agents reduce the difference that was detected in the activity states of two or more of the signaling proteins in the network of interconnected cell signaling proteins in the diseased cells compared to the activity states of the corresponding individual signaling proteins in the reference cells; and administering the combination to the patient. 46. A method for screening a combination of drugs for treatment of a disease in a patient caused by an abnormal network of interconnected cell signaling proteins that leads to an aberrant cellular response in the patient, comprising: measuring activity states of a plurality of different signaling proteins extracted from diseased cells obtained from the patient; comparing the activity states of the signaling proteins extracted from the diseased cells to the activity states of the corresponding signaling proteins in reference cells to detect differences between the activity states of individual signaling proteins from the diseased cells and the activity states of the corresponding individual signaling proteins in the reference cells, thereby providing a snapshot of the signaling proteins in the diseased cells that form an abnormal network of interconnected cell signaling proteins that leads to the aberrant cellular response in the patient; administering a combination of at least two drugs to the diseased cells; and determining if the drugs reduce the difference that was detected in the activity states of two or more of the signaling proteins in the network of interconnected cell signaling proteins in the diseased cells compared to the activity states of the corresponding individual signaling proteins in the reference cells, wherein drugs that reduce the difference are candidates for treatment of the disease. 47. The method of claim 46 further comprising treating the diseased cells with each of the drugs in the selected combination separately and at substantially the same dose as in the combination, examining the activity states of the signaling proteins after treatment with each of the drugs separately, and comparing such activity states to the activity states produced by the combination to determine if the combination synergistically reduces the difference that was detected in the activity states of two or more of the signaling proteins in the network of interconnected cell signaling proteins from the activity states of the corresponding signaling proteins in the reference cells. 48. The method of claim 47, wherein the activity states are determined and compared using pattern recognition. 49. The method of claim 46, wherein the activity states comprise phosphorylation. 50. A method for selecting a combination of therapeutic agents for individualized treatment of a disease in a patient caused by an abnormal network of interconnected cell signaling proteins that leads to an aberrant cellular response in the patient, comprising: measuring activity states for a plurality of different signaling proteins extracted from diseased cells obtained from a tissue of the patient; determining whether the activity states measured for the plurality of signaling proteins extracted from the diseased cells are different than activity states for corresponding signaling proteins from reference cells to detect differences between the activity states of individual signaling proteins from the diseased cells and the activity states of the corresponding individual signaling proteins from the reference cells, thereby providing a snapshot of the signaling proteins in the diseased cells that form an abnormal network of interconnected cell signaling proteins that leads to the aberrant cellular response in the patient, wherein measuring the activity states of the plurality of signaling proteins comprises using reverse phase protein microarray analysis of phosphorylated signaling proteins using antibodies that specifically bind to a particular phosphorylated signaling protein; and selecting a combination of at least two different therapeutic agents for the patient to reduce the difference that was detected in the activity states of two or more of the signaling proteins in the network of interconnected cell signaling proteins from the activity states of the corresponding signaling proteins in the reference cells, and wherein the combination of therapeutic agents provides a synergistic improvement in efficacy of treatment of the aberrant cellular response when compared to the combined efficacies of the agents administered alone at the same dose. 51. The method of claim 50, wherein the diseased cells are obtained from tissue of the patient, the method further comprising isolating the diseased cells from the tissue of the patient. 52. The method of claim 51, wherein isolating the diseased cells comprises microdissection of the diseased cells from the tissue. 53. The method of claim 52, wherein microdissection comprises laser capture microdissection. 54. The method of claim 50, further comprising measuring activity states for a plurality of different signaling proteins extracted from the reference cells. 55. A method for selecting a combination of therapeutic agents for individualized treatment of a disease in a patient caused by an abnormal network of interconnected cell signaling proteins that leads to an aberrant cellular response in the patient, comprising: measuring post-translational modifications of a plurality of different signaling proteins extracted from diseased cells obtained from a tissue of the patient; detecting differences between the post-translational modifications of individual signaling proteins from the diseased cells and the post-translational modifications of the corresponding individual signaling proteins from reference cells, thereby providing a snapshot of the signaling proteins in the diseased cells that form an abnormal network of interconnected cell signaling proteins that leads to the aberrant cellular response in the patient; and selecting a combination of at least two different therapeutic agents for the patient to reduce the difference that was detected in the post-translational modifications of two or more of the signaling proteins in the network of interconnected cell signaling proteins from the activity states of the corresponding signaling proteins in the reference cells and wherein the combination of therapeutic agents at the same or a lower dose provides a synergistic improvement in efficacy of treatment of the aberrant cellular response when compared to the combined efficacies of the agents administered alone. 56. The method of claim 55, wherein measuring the post-translational modifications of a plurality of different signaling proteins extracted from the diseased or reference cells comprises measuring the phosphorylation state of the plurality of signaling proteins. 57. The method of claim 55, wherein measuring the post-translational modifications of the plurality of signaling proteins extracted from the diseased cells and the reference cells comprises using reverse phase protein microarray analysis. 58. The method of claim 1, wherein measuring the activity states of the signaling proteins comprises determining a presence, absence, or amount of the signaling proteins. 59. A method for eliminating a combination of drugs from consideration for treatment of a disease in a patient caused by an abnormal network of interconnected cell signaling proteins that leads to an aberrant cellular response in the patient, comprising: measuring activity states of a plurality of different signaling proteins extracted from diseased cells obtained from the patient; comparing the activity states of the signaling proteins extracted from the diseased cells to the activity states of the corresponding signaling proteins in reference cells to detect differences between the activity states of individual signaling proteins from the diseased cells and the activity states of the corresponding individual signaling proteins in the reference cells, thereby providing a snapshot of the signaling proteins in the diseased cells that form an abnormal network of interconnected cell signaling proteins that leads to the aberrant cellular response in the patient; administering a combination of at least two drugs to the diseased cells; determining if the drugs reduce the difference that was detected in the activity states of two or more of the signaling proteins in the network of interconnected cell signaling proteins in the diseased cells compared to the activity states of the corresponding individual signaling proteins in the reference cells; and eliminating the combination of drugs if it does not reduce the difference. 60. The method of claim 1, wherein the aberrant cellular response comprises abnormal growth, apoptosis, proliferation, or angiogenesis. 61. The method of claim 60, wherein measuring the activity states of the signaling proteins comprises measuring the phosphorylation of the signaling proteins. 62. The method of claim 2, wherein measuring the activity states of the signaling proteins comprises measuring the phosphorylation of the signaling proteins. 63. The method of claim 5, wherein measuring the activity states of the signaling proteins comprises measuring the phosphorylation of the signaling proteins. 64. The method of claim 11, wherein measuring the activity states of the signaling proteins comprises measuring the phosphorylation of the signaling proteins. 65. A method for selecting a combination of therapeutic agents for individualized treatment of a disease in a human caused by abnormal cellular growth, comprising: measuring the phosphorylation of a plurality of different signaling proteins extracted from diseased cells obtained from a tissue of the human; detecting differences between the phosphorylation of individual signaling proteins from the diseased cells and the phosphorylation of corresponding individual signaling proteins from reference cells thereby providing a snapshot of the signaling proteins in the diseased cells that form an abnormal network of interconnected cell signaling proteins that leads to the abnormal cellular growth in the human; and selecting a combination of at least two different therapeutic agents for the human to reduce differences that were detected in the phosphorylation of two or more of the signaling proteins in the network of interconnected cell signaling proteins compared to the phosphorylation of the corresponding signaling proteins in the reference cells. 66. The method of claim 65, wherein the abnormal cellular growth is cancer, the reference cells are obtained from a person other than the human being treated, and measuring the phosphorylation comprises determining the ratio of the amount of phosphorylated signaling protein to the total amount of signaling protein. 67. The method of claim 66, wherein the cancer is breast cancer and the reference cells are obtained from one or more samples from a population of breast cancer patients. 68. The method of claim 66, wherein the cancer is breast cancer and the reference cells are normal cells. 69. The method of claim 45, wherein the combination of therapeutic agents provides a synergistic improvement in efficacy of treatment of the aberrant cellular response when compared to the combined efficacies of the agents administered alone at the same dose. 70. The method of claim 45, wherein the combination of therapeutic agents comprises two or more of drugs that separately target a combination of EGFr dimerization, EGFr phosphorylation, AKT phosphorylation, non-voltage gated calcium ion channels, cyclooxygenase-1, cyclooxygenase-2, MEK-1, NFKB/IKB, and P38. 71. The method of claim 45, wherein the combination prevents shunting to or around a signaling pathway. 72. The method of claim 71, wherein the combination includes a drug that inhibits MEK phosphorylation of ERK kinase, and shunting occurs via activation and phosphorylation of CREB. 73. The method of claim 45, wherein the combination comprises a prostaglandin pathway effector and a non-voltage gated calcium influx channel effector. 74. The method of claim 45, wherein the combination comprises CaI and a specific COX-2 inhibitor. 75. The method of claim 74, wherein the specific COX-2 inhibitor comprises Rofecoxib, Celecoxib or LM-1685. 76. The method of claim 45, wherein the combination comprises an AKT kinase inhibitor and either an EGFR dimerization inhibitor or an EGF kinase inhibitor. 77. The method of claim 76, wherein the EGF dimerization inhibitor comprises Trastuzumab and the EGF kinase inhibitor comprises IRESSA. 78. The method of claim 45, wherein the combination comprises a PKCalpha agonist resulting in phosphorylation and activation of PKCalpha. 79. The method of claim 76, wherein the combination comprises an AKT kinase inhibitor and Trastuzumab. 80. The method of claim 45, wherein the aberrant cellular response comprises abnormal growth, apoptosis, proliferation, or angiogenesis. 81. The method of claim 45, wherein the disease is cancer. 82. The method of claim 45, wherein measuring the activity states of the signaling proteins comprises determining a post-translational modification. 83. The method of claim 82, wherein the post-translational modification comprises phosphorylation. 84. The method of claim 45, wherein the abnormal cell signaling pathway comprises the Akt signaling pathway. 85. The method of claim 45, wherein the combination of at least two therapeutic agents comprises an Akt kinase inhibitor and an EGF kinase inhibitor. 86. A method for treating a human having a disease caused by abnormal cellular growth, comprising: measuring the phosphorylation of a plurality of different signaling proteins extracted from diseased cells obtained from the human; comparing the phosphorylation of the signaling proteins extracted from the diseased cells to the phosphorylation of the corresponding signaling proteins in reference cells to detect differences between the phosphorylation of individual signaling proteins from the diseased cells and the phosphorylation of the corresponding individual signaling proteins in the reference cells thereby providing a snapshot of the signaling proteins in the diseased cells that form an abnormal network of interconnected cell signaling proteins that leads to the abnormal cellular growth in the human; selecting a combination of at least two therapeutic agents for administration to the human, wherein the agents reduce the difference that was detected in the phosphorylation of the individual signaling proteins in the network of interconnected cell signaling proteins in the diseased cells compared to the phosphorylation of the corresponding individual signaling proteins in the reference cells; and administering the combination to the human. 87. The method of claim 86, wherein the abnormal cellular growth is cancer and the reference cell is obtained from a person other than the human being treated. 88. The method of claim 87, wherein the cancer is breast cancer and the reference cell is obtained from one or more samples from a population of breast cancer patients. 89. The method of claim 87, wherein the cancer is breast cancer and the reference cell is a normal cell. 90. The method of claim 1, further comprising the step of measuring activity states for a plurality of different signaling proteins extracted from the reference cells.

Details for Patent 8,168,568

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	09/25/1998		2023-03-10
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	02/10/2017		2023-03-10
Genentech, Inc.	HERCEPTIN HYLECTA	trastuzumab and hyaluronidase-oysk	Injection	761106	02/28/2019		2023-03-10