Amyotrophic lateral sclerosis, commonly referred to as Lou Gehrig's disease, is characterized by selective, premature degeneration and death of motor neurons in the motor cortex, brain stem and spinal cord. The loss of motor neurons causes progressive muscle paralysis ultimately leading to death from respiratory failure. Approximately 90% of all amyotrophic lateral sclerosis cases are sporadic amyotrophic lateral sclerosis, without a family history of the disease, and the other approximately 10 percent of cases are cases of familial amyotrophic lateral sclerosis. Despite significant efforts to identify risk factors and potential susceptibility genes, the etiology of sporadic amyotrophic lateral sclerosis remains largely unknown.
Various rodent models carrying dominant mutations of the human superoxide dismutase (SOD1) that is causative in about 20 percent of familial amyotrophic lateral sclerosis cases, have been instrumental to model motor neuron toxicity in amyotrophic lateral sclerosis. These models have demonstrated that not only motor neurons, but also non-neuronal cell types including microglia and astrocytes play a significant role in disease onset and progression. Studies have identified microglia as mediators of motor neuron death in amyotrophic lateral sclerosis by a yet undetermined inflammatory mechanism. Insight into the mechanisms underlying motor neuron death in amyotrophic lateral sclerosis as a result of neuroinflammatory effects is pertinent for the development of successful therapies for amyotrophic lateral sclerosis.
Accordingly, the present inventors have discovered that the mechanism underlying motor neuron death as a result of neuroinflammation is activation of NF-κB in microglia, and have used this knowledge to develop therapies for amyotrophic lateral sclerosis. The pharmaceutical compositions, methods and uses, and kits described herein can be used to treat sporadic or familial amyotrophic lateral sclerosis.
Several embodiments of the invention are described by the following enumerated clauses:
1. A method for treating a patient with amyotrophic lateral sclerosis by decreasing the expression of NF-κB in the patient, the method comprising the steps of                administering to the patient a composition comprising an effective amount of a compound that decreases the expression of NF-κB in microglia or macrophages of the patient; and        inhibiting motor neuron death in the patient.        
2. The method of clause 1 wherein the expression of NF-κB is decreased in microglia.
3. The method of clause 1 wherein the expression of NF-κB is decreased in macrophages.
4. The method of any one of clauses 1 to 3 wherein the decrease in expression of NF-κB in microglia is effective for reducing the symptoms of amyotrophic lateral sclerosis.
5. The method of any one of clauses 1 to 4 wherein a decrease in the level of expression of NF-κB in astrocytes is not effective for reducing the symptoms of amyotrophic lateral sclerosis.
6. The method of any one of clauses 1 to 5 wherein the composition comprises an aqueous solution.
7. The method of any one of clauses 1 to 6 wherein the compound is selected from the group consisting of a drug, a peptide, and a nucleic acid.
8. The method of clause 7 wherein the compound is a nucleic acid.
9. The method of clause 8 wherein the nucleic acid functions by RNA interference or is an antisense RNA molecule.
10. The method of clause 8 wherein the nucleic acid is selected from the group consisting of an siRNA, an miRNA, and an shRNA.
11. The method of clause 10 wherein the nucleic acid is an shRNA.
12. The method of any one of clauses 8 to 11 wherein the nucleic acid is delivered to the patient in a bacterial vector or in a viral vector.
13. The method of any one of clauses 8 to 11 wherein the nucleic acid has the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
14. The method of any one of clauses 12 to 13 wherein the vector is a viral vector.
15. The method of any one of clauses 1 to 14 wherein the amyotrophic lateral sclerosis is sporadic amyotrophic lateral sclerosis.
16. The method of any one of clauses 1 to 14 wherein the amyotrophic lateral sclerosis is familial amyotrophic lateral sclerosis.
17. The method of any one of clauses 1 to 16 wherein the amount of the compound is in the range of about 1 ng/kg of patient body weight to about 1 mg/kg of patient body weight.
18. The method of any one of clauses 1 to 17 wherein the amount of the compound is in the range of about 1 ng/kg of patient body weight to about 500 ng/kg of patient body weight.
19. The method of any one of clauses 1 to 18 wherein the amount of the compound is in the range of about 1 ng/kg of patient body weight to about 100 ng/kg of patient body weight.
20. The method of any one of clauses 1 to 19 wherein the composition further comprises a carrier, an excipient, or a diluent, or a combination thereof.
21. The method of clause 20 wherein the composition comprises a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is a liquid carrier.
22. The method of clause 21 wherein the liquid carrier is selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof.
23. The method of any one of clauses 1 to 22 wherein the composition is administered in a single-dose or a multiple-dose regimen.
24. A method for treating amyotrophic lateral sclerosis by inhibiting the activity of NF-κB in microglia or macrophages of a patient, the method comprising the step of                administering to the patient a composition comprising an effective amount of a compound that inhibits the activity of NF-κB in microglia or macrophages of the patient; and        inhibiting motor neuron death in the patient.        
25. The method of clause 24 wherein the activity of NF-κB is decreased in microglia.
26. The method of clause 24 wherein the activity of NF-κB is decreased in macrophages.
27. The method of any one of clauses 24 to 26 wherein the decrease in activity of NF-κB in microglia is effective for reducing the symptoms of amyotrophic lateral sclerosis.
28. The method of any one of clauses 24 to 27 wherein a decrease in the level of activity of NF-κB in astrocytes is not effective for reducing the symptoms of amyotrophic lateral sclerosis.
29. The method of any one of clauses 24 to 28 wherein the composition comprises an aqueous solution.
30. The method of any one of clauses 24 to 29 wherein the compound is selected from the group consisting of a drug, a peptide, and a nucleic acid.
31. The method of clause 30 wherein the compound is a nucleic acid.
32. The method of clause 31 wherein the nucleic acid is delivered to the patient in a bacterial vector or in a viral vector.
33. The method of clause 32 wherein the vector is a viral vector.
34. The method of clause 33 wherein the vector is selected from the group consisting of a lentiviral vector, an adeno-associated virus vector, and an adenovirus vector.
35. The method of any one of clauses 31 to 34 wherein the nucleic acid has the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
36. The method of any one of clauses 24 to 35 wherein the amyotrophic lateral sclerosis is sporadic amyotrophic lateral sclerosis.
37. The method of any one of clauses 24 to 35 wherein the amyotrophic lateral sclerosis is familial amyotrophic lateral sclerosis.
38. The method of any one of clauses 24 to 37 wherein the amount of the compound is in the range of about 1 ng/kg of patient body weight to about 1 mg/kg of patient body weight.
39. The method of any one of clauses 24 to 38 wherein the amount of the compound is in the range of about 1 ng/kg of patient body weight to about 500 ng/kg of patient body weight.
40. The method of any one of clauses 24 to 39 wherein the amount of the compound is in the range of about 1 ng/kg of patient body weight to about 100 ng/kg of patient body weight.
41. The method of any one of clauses 24 to 40 wherein the composition further comprises a carrier, an excipient, or a diluent, or a combination thereof.
42. The method of clause 41 wherein the composition comprises a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is a liquid carrier.
43. The method of clause 42 wherein the liquid carrier is selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof.
44. The method of any one of clauses 24 to 43 wherein the composition is administered in a single-dose or a multiple-dose regimen.
45. A pharmaceutical composition comprising a dosage form of a compound effective to decrease the expression of NF-κB in the microglia or macrophages of a patient with amyotrophic lateral sclerosis.
46. The composition of claim 45 wherein the expression of NF-κB in microglia is decreased.
47. The composition of claim 45 wherein the expression of NF-κB in macrophages is decreased.
48. The composition of any one of clauses 45 to 47 wherein the compound is selected from the group consisting of a drug, a peptide, and a nucleic acid.
49. The composition of clause 48 wherein the compound is a nucleic acid.
50. The composition of clause 49 wherein the nucleic acid is selected from the group consisting of siRNA, an miRNA, and an shRNA.
51. The composition of clause 50 wherein the compound is an antisense RNA molecule.
52. The composition of clause 50 wherein the nucleic acid is an shRNA.
53. The composition of any one of clauses 49 to 52 wherein the nucleic acid has the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
54. The composition of any one of clauses 45 to 53, wherein the composition further comprises one or more carriers, diluents, or excipients, or a combination thereof.
55. The composition of clause 54 wherein the composition comprises a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is a liquid carrier.
56. The composition of clause 55 wherein the liquid carrier is selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof.
57. The composition of any one of clauses 45 to 56 wherein the purity of the compound is at least 98 percent based on weight percent.
58. The composition of any one of clauses 45 to 57 wherein the composition is in an ampoule or a sealed vial.
59. The composition of any one of clauses 45 to 54 or 57 to 58 in the form of a reconstitutable lyophilizate.
60. A pharmaceutical composition comprising a dosage form of a compound effective to decrease the activity of NF-κB in the microglia or macrophages of a patient with amyotrophic lateral sclerosis.
61. The composition of claim 60 wherein the expression of NF-κB in microglia is decreased.
62. The composition of claim 60 wherein the expression of NF-κB in macrophages is decreased.
63. The composition of any one of clauses 60 to 62 wherein the compound is selected from the group consisting of a drug, a peptide, and a nucleic acid.
64. The composition of clause 63 wherein the compound is a nucleic acid.
65. The composition of clause 64 wherein the nucleic acid is selected from the group consisting of siRNA, an miRNA, and an shRNA.
66. The composition of any one of clauses 63 to 65 wherein the compound is an antisense RNA molecule.
67. The composition of clause 65 wherein the nucleic acid is an shRNA.
68. The composition of clause 67 wherein the nucleic acid has the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
69. The composition of any one of clauses 60 to 68, wherein the composition further comprises one or more carriers, diluents, or excipients, or a combination thereof.
70. The composition of clause 69 wherein the composition comprises a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is a liquid carrier.
71. The composition of clause 70 wherein the liquid carrier is selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof.
72. The composition of any one of clauses 60 to 71 wherein the purity of the compound is at least 98 percent based on weight percent.
73. The composition of any one of clauses 60 to 72 wherein the composition is in an ampoule or a sealed vial.
74. The composition of any one of clauses 60 to 69 or 72 to 73 in the form of a reconstitutable lyophilizate.
75. The method or pharmaceutical composition of any one of clauses 1 to 74 wherein the composition is in a dosage form selected from the group consisting of an inhalation dosage form, an oral dosage form, and a parenteral dosage form.
76. The method or pharmaceutical composition of clause 75 wherein the composition is in a parenteral dosage form and the parenteral dosage form is selected from the group consisting of an intradermal dosage form, a subcutaneous dosage form, an intramuscular dosage form, an intraperitoneal dosage form, an intravenous dosage form, and an intrathecal dosage form.
77. The composition of clause 59 or 74 in the form of a lyophilizate.
78. The composition of any one of clauses 45 to 54, 57 to 69, or 72 to 77 in the form of a solid.
79. A kit comprising a sterile vial, the composition of any one of clauses 45 to 78, and instructions for use describing use of the composition for treating a patient with amyotrophic lateral sclerosis.
80. The kit of clause 79 wherein the compound or composition is in the form of a reconstitutable lyophilizate.
81. The kit of clause 79 or 80 wherein the dose of the compound is in the range of 1 to 5 μg/kg of patient body weight.
82. The kit of any one of clauses 79 to 81 wherein the purity of the compound is at least 99 percent based on weight percent.
83. The kit of any one of clauses 79 to 82 wherein the compound or the composition is in a parenteral dosage form.
84. The kit of clause 83 wherein the parenteral dosage form is selected from the group consisting of an intradermal dosage form, a subcutaneous dosage form, an intramuscular dosage form, an intraperitoneal dosage form, an intravenous dosage form, and an intrathecal dosage form.
85. The kit of any one of clauses 79 to 84 wherein the composition further comprises a pharmaceutically acceptable carrier.
86. The kit of clause 85 wherein the pharmaceutically acceptable carrier is a liquid carrier selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof.
87. Use of the composition of any one of clauses 45 to 78 for the manufacture of a medicament for treating amyotrophic lateral sclerosis.
88. The pharmaceutical composition of any one of clauses 45 to 78 for use in treating amyotrophic lateral sclerosis.
89. The method of any one of clauses 12, 33, or 34 wherein the nucleic acid is delivered to the patient in a viral vector and the viral vector is a lentiviral vector.
90. The method or composition of any one of clauses 1 to 89 wherein administration of the composition increases the survival of the patient by 90 days or greater.
91. The method or composition of any one of clauses 1 to 90 wherein the patient has a mutation in the SOD1 gene.
92. The method or composition of any one of clauses 1 to 91 wherein the purity of the compound is at least 90 percent based on weight percent.
93. The method of any one of clauses 1 to 44 wherein the expression of a proinflammatory marker is decreased.
94. The method of clause 93 wherein the proinflammatory marker is selected from the group consisting of CD68, CD86, and NOS.
95. A method for inhibiting the expression or the activity of NF-κB in microglia or macrophages to inhibit motor neuron death, the method comprising the steps of                contacting the microglia or macrophages with a composition comprising an effective amount of an exogenous compound that decreases the expression or the activity of NF-κB in the microglia or the macrophages; and        inhibiting motor neuron death.        
96. The method of clause 95 wherein the expression of NF-κB is decreased in microglia.
97. The method of clause 95 wherein the expression of NF-κB is decreased in macrophages.
98. The method of any one of clauses 95 to 97 wherein the composition comprises an aqueous solution.
99. The method of any one of clauses 95 to 98 wherein the compound is selected from the group consisting of a drug, a peptide, and a nucleic acid.
100. The method of clause 99 wherein the compound is a nucleic acid.
101. The method of clause 100 wherein the nucleic acid functions by RNA interference or is an antisense RNA molecule.
102. The method of clause 100 wherein the nucleic acid is selected from the group consisting of an siRNA, an miRNA, and an shRNA.
103. The method of clause 100 wherein the nucleic acid is an shRNA.
104. The method of any one clauses 100 to 103 wherein the nucleic acid is delivered in a bacterial vector or in a viral vector.
105. The method of any one of clauses 100 to 104 wherein the nucleic acid has the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
106. A method for treating a patient with amyotrophic lateral sclerosis, the method comprising the steps of                administering to the patient a first composition comprising an effective amount of a first compound that decreases the expression or the activity of NF-κB in microglia or macrophages of the patient;        administering to the patient a second composition comprising an effective amount of a second compound that decreases the expression of SOD-1 in astrocytes, motor neurons, neurons, and/or oligodendrocytes of the patient; and        inhibiting motor neuron death in the patient.        
107. A method for inhibiting the expression or activity of NF-κB in microglia or macrophages of a patient with amyotrophic lateral sclerosis and for inhibiting the expression of SOD-1 in the patient, the method comprising the steps of                contacting the microglia or macrophages in the patient with a first composition comprising an effective amount of a first compound that decreases the expression or the activity of NF-κB in the microglia or the macrophages in the patient;        contacting the astrocytes in the patient with a second composition comprising an effective amount of a second compound that decreases the expression of SOD-1 in astrocytes, motor neurons, neurons, and/or oligodendrocytes of the patient; and        inhibiting motor neuron death.        
108. The method of clause 106 or 107 wherein the expression of NF-κB is decreased in microglia.
109. The method of clause 106 or 107 wherein the expression of NF-κB is decreased in macrophages.
110. The method of any one of clauses 106 to 109 wherein the decrease in expression of NF-κB in microglia is effective for reducing the symptoms of amyotrophic lateral sclerosis.
111. The method of any one of clauses 106 to 110 wherein a decrease in the level of expression of NF-κB in astrocytes is not effective for reducing the symptoms of amyotrophic lateral sclerosis.
112. The method of any one of clauses 106 to 111 wherein the composition comprises an aqueous solution.
113. The method of any one of clauses 106 to 112 wherein the first compound is selected from the group consisting of a drug, a peptide, and a nucleic acid.
114. The method of any one of clauses 106 to 113 wherein the second compound is selected from the group consisting of a drug, a peptide, and a nucleic acid.
115. The method of clause 113 or 114 wherein the compound is a nucleic acid.
116. The method of clause 115 wherein the nucleic acid functions by RNA interference or is an antisense RNA molecule.
117. The method of clause 116 wherein the nucleic acid is selected from the group consisting of an siRNA, an miRNA, and an shRNA.
118. The method of clause 117 wherein the nucleic acid is an shRNA.
119. The method of clause 118 wherein the nucleic acid is delivered to the patient in a bacterial vector or in a viral vector.
120. The method of clause 119 wherein the nucleic acid has the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
121. The method of clause 119 wherein the vector is a viral vector.
122. The method of any one of clauses 106 to 121 wherein the amyotrophic lateral sclerosis is sporadic amyotrophic lateral sclerosis.
123. The method of any one of clauses 106 to 121 wherein the amyotrophic lateral sclerosis is familial amyotrophic lateral sclerosis.
124. The method of any one of clauses 106 to 123 wherein the amount of the compound is in the range of about 1 ng/kg of patient body weight to about 1 mg/kg of patient body weight.
125. The method of any one of clauses 106 to 124 wherein the amount of the compound is in the range of about 1 ng/kg of patient body weight to about 500 ng/kg of patient body weight.
126. The method of any one of clauses 106 to 125 wherein the amount of the compound is in the range of about 1 ng/kg of patient body weight to about 100 ng/kg of patient body weight.
127. The method of any one of clauses 106 to 126 wherein the composition further comprises a carrier, an excipient, or a diluent, or a combination thereof.
128. The method of clause 127 wherein the composition comprises a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is a liquid carrier.
129. The method of clause 128 wherein the liquid carrier is selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof.
130. The method of any one of clauses 106 to 129 wherein the composition is administered in a single-dose or a multiple-dose regimen.
131. The method of any one of clauses 106 to 130 wherein the composition is in a dosage form selected from the group consisting of an inhalation dosage form, an oral dosage form, and a parenteral dosage form.
132. The method of clause 131 wherein the composition is in a parenteral dosage form and the parenteral dosage form is selected from the group consisting of an intradermal dosage form, a subcutaneous dosage form, an intramuscular dosage form, an intraperitoneal dosage form, an intravenous dosage form, and an intrathecal dosage form.
133. The method of any one of clauses 106 to 132 wherein administration of the first or the second composition increases the survival of the patient by 90 days or greater.
134. The method of any one of clauses 106 to 133 wherein the patient has a mutation in the SOD1 gene.
135. The method of any one of clauses 106 to 134 wherein the purity of the first or the second compound is selected from the group consisting of at least 90 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, and at least 99.5 percent.
136. The method of any one of clauses 1 to 44, 75, 76, or 90 to 105 wherein the purity of the compound is selected from the group consisting of at least 90 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, and at least 99.5 percent.
137. The method of any one of clauses 106 to 135 wherein the first composition and/or the second composition comprises an aqueous solution.
138. The method of any one of clauses 1 to 44, 75, 76, or 90 to 137 wherein the compound comprises an adeno-associated virus vector.
139. The method of any one of clauses 1 to 44, 75, 76, or 89 to 105 wherein the administration of the compound increases the survival of the patient for a number of days selected from the group consisting of at least 20 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 80 days, at least 85 days, at least 90 days, at least 95 days, at least 100 days, at least 150 days, at least 200 days, at least 250 days, and at least 300 days compared to a patient who is not treated with the compound.
140. The method of any one of clauses 106 to 132, 134 to 135, or 137 wherein the administration of the first compound or the second compound increases the survival of the patient for a number of days selected from the group consisting of at least 20 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 80 days, at least 85 days, at least 90 days, at least 95 days, at least 100 days, at least 150 days, at least 200 days, at least 250 days, and at least 300 days compared to a patient who is not treated with the first or the second compound.