US2015094267A1PendingUtilityA1
Succinimide-activated nitroxyl compounds and methods for the use thereof for nitroxylation of proteins
Individually held — no corporate assignee on recordPriority: Apr 3, 2012Filed: Mar 15, 2013Published: Apr 2, 2015
Est. expiryApr 3, 2032(~5.7 yrs left)· nominal 20-yr term from priority
A61P 41/00A61P 9/14A61P 9/10A61P 39/02A61P 43/00A61P 7/06A61P 9/04A61P 7/08A61P 9/08A61P 7/02A61P 7/00A61P 7/04A61P 9/00A61P 27/02A61P 31/04A61P 33/06A61P 35/00A61P 1/16A61P 1/04A61P 15/00A61P 11/00A61P 17/02A61P 25/00A61P 1/00C07D 207/416C07D 401/14C07D 403/12C07K 14/765A61K 38/42C07K 14/805C07D 401/12
47
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Claims
Abstract
The present invention relates to succinimide-activated nitroxyl compounds and methods for the synthesis of such compounds. The present invention also relates to the use of succinimide-activated nitroxyl compounds to prepare nitroxylated proteins, for example nitroxylated heme proteins (e.g., nitroxylated hemoglobin and nitroxylated myoglobin). The nitroxylated proteins are optionally also conjugated to a polyalkylene oxide (PAO), for example to a polyethylene glycol (PEG). Polynitroxylated heme proteins are useful as oxygen therapeutic agents (OTAs). The invention further relates to pharmaceutical compositions of the nitroxylated proteins and methods for the use of nitroxylated proteins in the treatment of various conditions.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A nitroxylating agent of formula (I):
wherein:
each of R 1 , R 2 , R 3 , and R 4 is independently C 1 -C 4 alkyl;
X is oxygen, sulfur, nitrogen, phosphorus, or silicon;
Y is CH 2 ;
n is 0 or 1; and
m is 0 or 1.
2 . A method for preparing the nitroxylating agent of the formula (II)
comprising reacting a compound having the formula (III)
with N,N′-disuccinimidyl carbonate (DSC) in the presence of an organic base;
wherein each of R 1 , R 2 , R 3 , and R 4 is independently C 1 -C 4 alkyl;
X is oxygen, sulfur, nitrogen, phosphorus, or silicon;
Y is CH 2 ; and
m is 0 or 1.
3 . A method for preparing a nitroxylating agent of the formula (IV)
comprising reacting a compound having the formula (V)
with N-hydroxysuccinimide (NHS) in the presence of N,N′-dicyclohexylcarbodiimide (DCC);
wherein
each of R 1 , R 2 , R 3 , and R 4 is independently C 1 -C 4 alkyl;
Y is CH 2 ; and
m is 0 or 1.
4 . A nitroxylated protein having the structure (VI):
wherein
Z represents the protein;
each of R 1 , R 2 , R 3 , and R 4 are independently C 1 -C 4 alkyl;
X is oxygen, sulfur, nitrogen, phosphorus, or silicon;
Y is CH 2 ;
m is 0 or 1;
n is 0 or 1;
p is the average number of activated-PEG polymers conjugated to the protein; and
N is a nitrogen of the protein.
5 . A method for preparing a nitroxylated protein comprising reacting the protein with a nitroxylating agent of formula (II)
wherein
each of R 1 , R 2 , R 3 , and R 4 is independently C 1 -C 4 alkyl;
X is oxygen, sulfur, nitrogen, phosphorus, or silicon;
Y is CH 2 ; and
m is 0 or 1.
6 . A method for preparing a nitroxylated protein comprising reacting the protein with a nitroxylating agent of formula (IV):
wherein each of R 1 , R 2 , R 3 , and R 4 is independently C 1 -C 4 alkyl;
Y is CH 2 ; and
m is 0 or 1.
7 . A nitroxylating agent, nitroxylating protein, or method of any one of claims 1 - 6 , wherein each of R 1 , R 2 , R 3 , and R 4 is —CH 3 .
8 . A nitroxylating agent of claim 1 or 7 , wherein n is 1.
9 . A nitroxylating agent of claim 8 , wherein X is oxygen or sulfur.
10 . A nitroxylating agent of claim 8 , wherein X is oxygen.
11 . A nitroxylating agent of claim 8 , wherein X is oxygen and each of R 1 , R 2 , R 3 , and R 4 is —CH 3 .
12 . A nitroxylating agent of claim 1 or 7 , wherein n is 0.
13 . A nitroxylating agent, nitroxylating protein, or method of any one of claims 1 - 12 , wherein m is 0.
14 . A nitroxylating agent, nitroxylating protein, or method of any one of claims 1 - 12 , wherein m is 1.
15 . A nitroxylating agent of claim 1 selected from the group consisting of:
16 . A nitroxylating agent of claim 1 wherein the nitroxylating agent is:
17 . A method of any one of claims 2 , 7 , 13 and 14 , wherein the organic base comprises triethylamine (TEA), N,N-diisopropylethylamine, 4-dimethylaminopyridine, pyridine, N-methylpiperidine, or a combination thereof.
18 . A method of claim 17 , wherein the organic base comprises triethylamine.
19 . A method of claim 18 , wherein the compound of formula (III), the N,N′-disuccinimidyl carbonate, and the triethylamine are present in a ratio of about 1:2:3.
20 . A method of any one of claims 2 , 7 , 13 , 14 and 17 - 19 , wherein the reaction is carried out at a temperature of about 2° C. to about 30° C.
21 . A method of claim 20 , wherein the reaction is carried out at a temperature of about 15° C. to about 25° C.
22 . A method of any one of claims 2 , 7 , 13 , 14 and 17 - 21 , wherein the reaction is carried out at a temperature of about 4° C.
23 . A method of claim 21 , wherein the reaction is carried out at a temperature of about 20° C.
24 . A method of any one of claims 2 , 7 , 13 , 14 and 17 - 23 , wherein the reaction is allowed to proceed for about three to about six hours.
25 . A method of any one of claims 2 , 7 , 13 , 14 and 17 - 24 , wherein the reaction is carried out in a polar aprotic solvent.
26 . A method of claim 25 , wherein the polar aprotic solvent comprises acetonitrile (ACN), tetrahydrofuran (THF), ethyl acetate (EtOAc), acetone, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or a combination thereof.
27 . A method of claim 26 , wherein the polar aprotic solvent comprises acetonitrile.
28 . A method of any one of claims 3 , 7 , 13 and 14 , wherein the compound of formula (IV), the N-hydroxysuccinimide, and the N,N′-dicyclohexylcarbodiimide are present in a molar ratio of about 1:1.1:1.1.
29 . A method of any one of claims 1 - 28 , wherein m is 0.
30 . A method of any one of claims 1 - 28 , wherein m is 1.
31 . A method of any one of claims 3 , 7 , 13 , 14 , and 28 - 30 , wherein the reaction is carried out at a temperature of about 2° C. to about 30° C.
32 . A method of claim 31 , wherein the reaction is carried out at a temperature of about 15° C. to about 25° C.
33 . A method of claim 31 , wherein the reaction is carried out at a temperature of about 4° C.
34 . A method of claim 31 , wherein the reaction is carried out at a temperature of about 20° C.
35 . A method of any one of claims 3 , 7 , 13 , 14 , and 28 - 34 , wherein the reaction is allowed to proceed for about 6 to about 24 hours.
36 . A method of any one of claims 3 , 7 , 13 , 14 , and 28 - 35 , wherein the reaction is carried out at a pH of about 7.2 to about 7.6.
37 . A method of any one of claims 3 , 7 , 13 , 14 , and 28 - 36 , wherein the reaction is carried out at a pH of about 7.4.
38 . A nitroxylated protein of any one of claims 4 , 7 , 13 , 14 , 29 , and 30 , wherein the N-terminal amino group of the protein is nitroxylated.
39 . A nitroxylated protein of any one of claims 4 , 7 , 13 , 14 , 29 , and 30 , wherein at least one epsilon (ε)-amino group of a lysine residue is nitroxylated.
40 . A nitroxylated protein of any one of claims 4 , 7 , 13 , 14 , 29 , 30 , 38 and 39 , wherein p is about 1 to about 25.
41 . A nitroxylated protein of any one of claims 4 , 7 , 13 , 14 , 29 , 30 , 38 and 39 , wherein p is at least about 2.
42 . A nitroxylated protein of claim 41 , wherein p is at least about 10.
43 . A nitroxylated protein of claim 40 , wherein p is about 15 to about 20.
44 . A nitroxylated protein of any one of claims 4 , 7 , 13 , 14 , 29 , 30 , and 38 - 43 , wherein the protein comprises a hemoglobin (Hb) α-subunit, a hemoglobin β-subunit, a hemoglobin tetramer, a myoglobin, or an albumin.
45 . A nitroxylated protein of claim 44 , wherein the protein comprises a serum albumin.
46 . A nitroxylated protein of claim 45 , wherein the serum albumin comprises human serum albumin (HSA).
47 . A nitroxylated protein of claim 44 , wherein the protein comprises a hemoglobin α-subunit or a hemoglobin β-subunit or hemoglobin tetramer.
48 . A nitroxylated protein of claim 44 , wherein the protein comprises a human hemoglobin α-subunit, an human hemoglobin β-subunit, or a hemoglobin tetramer comprising human hemoglobin α-subunits and β-subunits.
49 . A nitroxylated protein of any one of claim 44 , 47 or 48 , wherein the hemoglobin tetramer comprises a cross-linked αα dimer or a cross-linked ββ dimer.
50 . A nitroxylated protein of claim 49 , wherein the nitroxylated protein comprises a human hemoglobin α-subunit.
51 . The nitroxylated protein of claim 50 , wherein the human hemoglobin α-subunit is nitroxylated at the α-amino group of the N-terminal valine residue.
52 . The nitroxylated protein of claim 50 or 51 , wherein the human hemoglobin α-subunit is nitroxylated at the ε-amino group of a lysine residue selected from the group consisting of lysine-7, lysine-11, lysine-16, lysine-40, lysine-56, lysine-60, lysine-61, lysine-90, lysine-99, lysine-127, lysine-139, and a combination thereof.
53 . A nitroxylated protein of claim 50 , wherein the nitroxylated protein comprises a human hemoglobin β-subunit.
54 . A nitroxylated protein of claim 53 , wherein the human hemoglobin β-subunit is nitroxylated at the α-amino group of the N-terminal valine residue.
55 . A nitroxylated protein of claim 53 or 54 , wherein the human hemoglobin β-subunit is nitroxylated at the ε-amino group of a lysine residue selected from the group consisting of lysine-8, lysine-17, lysine-59, lysine-61, lysine-65, lysine-66, lysine-82, lysine-95, lysine-120, lysine-132, lysine-144, and a combination thereof.
56 . A nitroxylated protein of any one claims 44 - 55 , wherein the protein comprises hemoglobin tetramer and the hemoglobin tetramer comprises about seventeen nitroxylated amino groups.
57 . A nitroxylated protein of any one of claims 4 , 7 , 13 , 14 , 29 , 30 and 38 - 56 , wherein the nitroxylated protein is conjugated to a polyalkylene oxide (PAO).
58 . A nitroxylated protein of claim 57 , wherein the PAO is a polyethylene glycol (PEG).
59 . A nitroxylated protein of claim 58 , wherein the PEG has an average molecular weight of about 2,000 to about 20,000 Daltons.
60 . A nitroxylated protein of claim 58 , wherein the PEG has an average molecular weight of about 3,000 to about 10,000 Daltons.
61 . A nitroxylated protein of claim 58 , wherein the PEG has an average molecular weight of about 4,000 to about 6,000 Daltons.
62 . A nitroxylated protein of claim 61 , wherein the PEG has an average molecular weight of about 5,000 Daltons.
63 . A nitroxylated protein of any one of claims 58 - 62 , wherein the PEG is a maleimide-PEG.
64 . A nitroxylated protein of claim 63 , wherein the maleimide is linked to the PEG via an alkylene or phenylene linker.
65 . A nitroxylated protein of claim 64 , wherein the alkylene linker is an ethylene linker.
66 . A nitroxylated protein of any one of claims 63 - 65 , wherein the maleimide-PEG is conjugated to a thiol moiety of the protein selected from the group consisting of an intrinsic thiol moiety of a cysteine residue of the protein, a thiol moiety of a thiolated lysine residue of the protein, and a combination thereof.
67 . A nitroxylated protein of claim 66 , wherein the maleimide-PEG conjugated to an intrinsic thiol moiety of a cysteine residue or conjugated to a thiol moiety of a thiolated lysine residue has the structure (VIII)
wherein
Z represents the protein,
S is a thiol of the protein,
R 3 is an alkylene or phenylene group,
X is a terminal group,
m is the average number of activated-PEG polymers conjugated to the protein, and
n represents the average number of oxyethylene units of a PEG having an average molecular weight of about 2,000 to about 20,000 Daltons.
68 . A nitroxylated protein of claim 67 , wherein R 3 is ethylene.
69 . A nitroxylated protein of claim 67 or 68 , wherein m is about 6 to about 10.
70 . A nitroxylated protein of any one of claims 67 - 69 , wherein X is methoxy (—OCH 3 ) or carboxylate (—COOH).
71 . A nitroxylated protein of any one of claims 63 - 70 , wherein the maleimide-PEG is conjugated to a thiol moiety of a cysteine-93 residue of a hemoglobin β-subunit.
72 . A nitroxylated protein of any one of claims 63 - 71 , wherein the maleimide-PEG is conjugated to a thiol moiety of a thiolated lysine residue of a hemoglobin α-subunit or β-subunit.
73 . A nitroxylated protein of claim 72 , wherein the thiolated lysine residue is a thiolated lysine residue of a human hemoglobin α-subunit selected from the group consisting of lysine-7, lysine-11, lysine-16, lysine-40, lysine-56, lysine-60, lysine-61, lysine-90, lysine-99, lysine-127, lysine-139, and a combination thereof.
74 . A nitroxylated protein of claim 72 , wherein the thiolated lysine residue is a thiolated lysine residue of a human hemoglobin β-subunit selected from the group consisting of lysine-8, lysine-17, lysine-59, lysine-61, lysine-65, lysine-66, lysine-82, lysine-95, lysine-120, lysine-132, lysine-144, and a combination thereof.
75 . A nitroxylated protein of any one of claims 58 - 62 , wherein the PEG is a succinimidyl valerate PEG (SVA-PEG).
76 . A nitroxylated protein of claim 75 , wherein the SVA-PEG is conjugated to an amino moiety of the protein selected from an ε-amino moiety of a lysine residue of the protein, an α-amino moiety of a terminal valine residue of the protein, or a combination thereof.
77 . A nitroxylated protein of claim 76 , wherein the SVA-PEG conjugated to an ε-amino moiety of a lysine residue of the protein or an α-amino moiety of a terminal valine residue of the protein has the structure (IX)
wherein:
Z is the protein,
N is an amino group of the protein,
X is a terminal group,
m is the number of activated-PEG polymers conjugated to the protein, and
n is the average number of oxyethylene units of a PEG having an average molecular weight of from about 2,000 to about 20,000 Daltons.
78 . A nitroxylated protein of claim 77 , wherein X is methoxy (—OCH 3 ) or carboxylate (—COOH).
79 . A nitroxylated protein of claim 77 or 78 , wherein m is on average from about 6 to about 10 PAO molecules per tetramer.
80 . A nitroxylated protein of any one of claims 75 - 79 , wherein the SVA-PEG is conjugated to an ε-amino moiety of a lysine residue of a hemoglobin α-subunit or β-subunit.
81 . A nitroxylated protein of any one of claims 75 - 80 , wherein the SVA-PEG is conjugated to an α-amino moiety of a terminal valine residue of a hemoglobin α-subunit or β-subunit.
82 . A nitroxylated protein of claim 80 or 81 , wherein the lysine residue is a lysine residue of a human hemoglobin α-subunit selected from the group consisting of lysine-7, lysine-11, lysine-16, lysine-40, lysine-56, lysine-60, lysine-61, lysine-90, lysine-99, lysine-127, lysine-139, and a combination thereof.
83 . A nitroxylated protein of claim 80 or 81 , wherein the lysine residue is a lysine residue of a human hemoglobin β-subunit selected from the group consisting of lysine-8, lysine-17, lysine-59, lysine-61, lysine-65, lysine-66, lysine-82, lysine-95, lysine-120, lysine-132, lysine-144, and a combination thereof.
84 . A hemoglobin tetramer comprising at least one α-subunit of any one of claims 44 , 47 , 48 , 50 - 52 , 57 - 70 , 72 , 73 , and 75 - 82 or at least one β-subunit of any one of claims 44 , 47 , 48 , 53 - 55 , 57 - 72 , 74 - 81 , and 83 .
85 . The hemoglobin tetramer of claim 84 , comprising at least one α-subunit of any one of claims 44 , 47 , 48 , 50 - 52 , 57 - 70 , 72 , 73 , and 75 - 82 and at least one β-subunit of any one of claims 44 , 47 , 48 , 53 - 55 , 57 - 72 , 74 - 81 , and 83 .
86 . The hemoglobin tetramer of claim 85 , comprising two α-subunits of any one of claims 44 , 47 , 48 , 50 - 52 , 57 - 70 , 72 , 73 , and 75 - 82 and two β-subunits of any one of claims 44 , 47 , 48 , 53 - 55 , 57 - 72 , 74 - 81 , and 83 .
87 . A hemoglobin tetramer of any one of claims 84 - 86 , wherein the hemoglobin is conjugated to on average 5 to 10 PAO molecules per tetramer.
88 . A hemoglobin tetramer of claim 87 , wherein the hemoglobin is conjugated to on average 7.1 to 8.9 PAO molecules per tetramer.
89 . A hemoglobin tetramer of any one of claims 84 - 88 , wherein the hemoglobin is oxygenated.
90 . A hemoglobin tetramer of any one of claims 84 - 89 , wherein the hemoglobin is deoxygenated.
91 . A hemoglobin tetramer of any one of claims 84 - 90 , wherein the hemoglobin is liganded with CO, NO, or a mixture of CO and NO.
92 . A method of any one of claims 5 - 7 , 13 , 14 , 29 , and 30 , wherein the ratio of the nitroxylating agent is present at about a 5-fold to about 100-fold molar excess over the protein.
93 . A method of any one of claims 5 - 7 , 13 , 14 , 29 , 30 and 92 , wherein the protein comprises an α- or β-subunit of a hemoglobin tetramer.
94 . A method of any one of claims 5 - 7 , 13 , 14 , 29 , 30 , 92 and 93 , wherein the protein comprises a hemoglobin tetramer.
95 . A method of claim 94 , wherein the hemoglobin tetramer is a non-oxygenated hemoglobin tetramer.
96 . A method of claim 95 , wherein the non-oxygenated hemoglobin tetramer is a CO-liganded hemoglobin tetramer.
97 . A method of claim 95 , wherein the hemoglobin tetramer is a deoxygenated hemoglobin tetramer.
98 . A method of any one of claims 5 - 7 , 13 , 14 , 29 , 30 , 92 - 97 , wherein the reaction is carried out at a temperature of about 2° C. to about 30° C.
99 . A method of claim 98 , wherein the reaction is carried out at a temperature of about 15° C. to about 25° C.
100 . A method of claim 98 , wherein the reaction is carried out at about 2° C. to about 8° C.
101 . A method of claim 98 , wherein the reaction is carried out at a temperature of about 4° C.
102 . A method of claim 98 , wherein the reaction is carried out at a temperature of about 20° C.
103 . A method of any one of claims 5 - 7 , 13 , 14 , 29 , 30 , 92 - 102 , wherein the reaction is allowed to proceed for about three to about 20 hours.
104 . A method of claim 103 , wherein the reaction is allowed to proceed for about three to about six hours.
105 . A method of claim 103 , wherein the reaction is allowed to proceed for about 16 hours.
106 . A method of any one of claims 5 - 7 , 13 , 14 , 29 , 30 , 92 - 105 , wherein the reaction is carried out in an aqueous solvent.
107 . A method of any one of claims 5 - 7 , 13 , 14 , 29 , 30 , 92 - 106 , wherein the reaction is carried out at a pH of about 6.5 to about 8.5.
108 . A method of claim 107 , wherein the reaction is carried out at a pH of about 7.5.
109 . A method of claim 107 , wherein the reaction is carried out at a pH of about 7.2.
110 . A method of any one of claims 5 - 7 , 13 , 14 , 29 , 30 , 92 - 109 , wherein the protein comprises a hemoglobin tetramer, the reaction is carried out at a pH of about 7.2 and a temperature of about 2° C. to about 8° C. and is allowed to proceed for about sixteen hours, and wherein the method yields a nitroxylated hemoglobin tetramer having about 17 nitroxylated amino groups.
111 . A method of any one of claims 5 - 7 , 13 , 14 , 29 , 30 , and 92 - 110 , wherein the protein comprises a hemoglobin tetramer, and the nitroxylating agent is present at about a 10- to about 100-fold molar excess over the hemoglobin tetramer.
112 . A method of any one of claims 5 - 7 , 13 , 14 , 29 , 30 , and 92 - 111 , wherein the product of the reaction is a nitroxylated protein of any one of claims 4 , 7 , 13 , 14 , 29 , 30 and 38 - 83 or a hemoglobin tetramer of any one of claims 84 - 91 .
113 . A method of any one of claims 5 - 7 , 13 , 14 , 29 , 30 , and 92 - 112 , further comprising conjugating the protein to a polyalkylene oxide (PAO).
114 . A method of claim 113 , further comprising:
adding succinimidyl valerate PAO to the protein in the aqueous diluent to form a PAO-valerate conjugated protein.
115 . A method of claim 113 , further comprising:
mixing the protein with 2-iminothiolane (2-IT) in an aqueous diluent to form a thiolated protein; and adding PAO-maleimide to the thiolated protein in the aqueous diluent to form a PAO-maleimide conjugated protein.
116 . A method of any one of claims 113 - 115 , wherein the protein comprises a hemoglobin tetramer.
117 . A method of claim 113 or 116 , wherein the hemoglobin tetramer comprises a cross-linked αα dimer or a cross-linked ββ dimer.
118 . The method of any one of claims 115 - 117 , wherein the 2-iminithiolane is present at a concentration of between about 7- and about 15-fold molar excess over the protein concentration.
119 . The method of any one of claims 115 - 117 , wherein the 2-iminothiolane is present at a concentration of between about 7- and about 8-fold molar excess over the protein concentration.
120 . The method of any one of claims 115 - 117 , wherein the 2-iminothiolane is present at a concentration of between about 7-0.5-fold molar excess over the protein concentration.
121 . The method of any one of claims 115 - 120 , wherein the PAO-maleimide is present at a concentration of between about 9- and about 20-fold molar excess over the protein concentration.
122 . The method of any one of claims 115 - 120 , wherein the PAO-maleimide is present at a concentration of between about 9- and about 15-fold molar excess over the protein concentration.
123 . The method of any one of claims 115 - 120 , wherein the PAO-maleimide is present at a concentration of between about 12-fold molar excess over the protein concentration.
124 . The method of any one of claims 115 - 123 , wherein the thiolation step is carried out at a pH of between about 7 and about 9.
125 . The method of any one of claims 115 - 123 , wherein the thiolation step is carried out at a pH of about 8.5.
126 . The method of any one of claims 115 - 125 , wherein the step of adding the PAO-maleimide to the thiolated protein to form a PAO-maleimide conjugated protein is carried out at a pH of between about 6.5 and about 8.5.
127 . The method of any one of claims 115 - 125 , wherein the step of adding the PAO-maleimide to the thiolated protein to form a PAO-maleimide conjugated protein is carried out at a pH of about 7.5.
128 . The method of any one of claims 113 - 127 , wherein the protein is conjugated to PAO prior to nitroxylation of the protein.
129 . The method of claim 115 - 128 , wherein the step of adding the PAO-maleimide to the thiolated protein to form a PAO-maleimide conjugated protein is performed concurrently with nitroxylation of the protein.
130 . The method of claim 114 - 129 , wherein the step of adding the succinimidyl valerate PAO to the protein to form a PAO-valerate conjugated protein is performed concurrently with nitroxylation of the protein.
131 . A pharmaceutical composition comprising a nitroxylated protein of any one of claims 4 , 7 , 13 , 14 , 29 , 30 , and 38 - 83 or a hemoglobin tetramer of any one of claims 84 - 91 and a pharmaceutically acceptable carrier.
132 . A pharmaceutical composition of claim 131 , wherein the composition is normooncotic with blood.
133 . A pharmaceutical composition of claim 131 or 132 , wherein the composition is hyperoncotic as compared to blood.
134 . A pharmaceutical composition of any one of claims 131 - 133 , wherein the pharmaceutically acceptable carrier comprises an aqueous diluent.
135 . A pharmaceutical composition of claim 134 , wherein the aqueous diluent comprises an aqueous solution of a colloid or an aqueous solution of a non-oxygen carrying component.
136 . A pharmaceutical composition of claim 134 or 135 , wherein the aqueous diluent comprises an aqueous cell-free solution.
137 . A pharmaceutical composition of any one of claims 134 - 136 , wherein the aqueous diluent comprises an aqueous solution of proteins, an aqueous solution of glycoproteins, an aqueous solution of polysaccharides, or a combination thereof.
138 . A pharmaceutical composition of any one of 134 - 137 , wherein the aqueous diluent comprises an aqueous cell-free solution of albumin.
139 . A pharmaceutical composition of any one of claims 131 - 138 , wherein the pharmaceutically acceptable carrier comprises physiological saline, a saline-glucose mixture, Ringer's solution, lactated Ringer's solution, Locke-Ringer's solution, Krebs-Ringer's solution, Hartmann's balanced saline, heparinized sodium citrate-citric acid-dextrose solution, an acetate solution, a multiple electrolyte solution, a lactiobionate solution, a polymeric plasma substitute, or a combination thereof.
140 . A pharmaceutical composition of claim 139 , wherein the polymeric plasma substitute comprises polyethylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol, an ethylene oxide-propylene glycol condensate, or a combination thereof.
141 . The pharmaceutical composition of any one of claims 131 - 140 , wherein the pharmaceutically acceptable carrier comprises a filler, a salt, a physiological buffer, a carbohydrate, an alcohol, a poly alcohol, an anti-oxidant, an anti-bacterial agent, an oncotic pressure agent, a reducing agent, or a combination thereof.
142 . The pharmaceutical composition of claim 141 , wherein the reducing agent comprises, ascorbic acid, glutathione, N-acetyl cysteine, or a combination thereof.
143 . A pharmaceutical composition of any one of claims 131 - 142 , for use in the treatment of acute liver failure, beta thalassemia, a burn, chronic critical limb ischemia, carbon dioxide or cyanide poisoning, chronic obstructive pulmonary disease (COPD), congestive heart failure, hypoxia, malaria, organ ischemia, peripheral vascular disease, porphyria, pre-eclampsia in pregnancy, sepsis, sickle cell disease, retinal disease, an intra-ocular condition, testicular torsion, trauma, shock, traumatic brain injury, ulcers, vasospasm, or a combination thereof.
144 . A pharmaceutical composition of claim 143 , wherein the organ ischemia comprises acute bowel ischemia (torsion), acute bowel ischemia (embolism), cardiogenic shock, acute vascular organ ischemia, stroke, myocardial infarction, or severe cardiac ischemia.
145 . A pharmaceutical composition of any one of claims 131 - 142 , for use in the treatment of non-traumatic hemorrhagic shock, pre-hospital setting trauma, traumatic hemorrhagic shock, acute lung injury, adult respiratory distress syndrome, traumatic brain injury, stroke, solid tumor cancer, organ degradation (ex-vivo), organ degradation (in recipient), severe sepsis, septic shock, myocardial infarction, cardiac ischemia, cardiogenic shock, acute heart failure, pulmonary embolism, or a combination thereof.
146 . A pharmaceutical composition of any one of claims 131 - 142 , for use as an adjunct to angioplasty, as an adjunct for plastic surgery, or as an adjunct in implanting a ventricular assist device; as a blood substitute, a cardioprotectant, a cryopreservative, a hemodialysis adjunct, an oncology agent, an organ preservative, a performance enhancement agent, a surgery adjunct, or a wound healing agent; in imaging; to improve lung function; or a combination thereof.
147 . A pharmaceutical composition of any one of claims 131 - 142 , for veterinary treatment of of loss of blood due to injury, hemolytic anemia, infectious anemia, bacterial infection, Factor IV fragmentation, hypersplenation and splenomegaly, hemorrhagic syndrome in poultry, hypoplastic anemia, aplastic anemia, idiopathic immune hemolytic conditions, iron deficiency, isoimmune hemolytic anemia, microangiopathic hemolytic anemia, parasitism, or surgical-anesthesia induced brain damage.
148 . A method of treatment comprising administering a hemoglobin tetramer of any one of claims 84 - 91 or a pharmaceutical composition of any one of claims 131 - 147 to a subject in need thereof.
149 . A method of treatment of claim 148 , wherein the subject is an animal.
150 . A method of treatment of claim 149 , wherein the subject is a human.
151 . A method of treatment of any one of claims 148 - 150 , wherein the method is a method for the treatment of acute liver failure, beta thalassemia, a burn, chronic critical limb ischemia, carbon dioxide or cyanide poisoning, chronic obstructive pulmonary disease (COPD), congestive heart failure, hypoxia, malaria, organ ischemia, peripheral vascular disease, porphyria, pre-eclampsia in pregnancy, sepsis, sickle cell disease, retinal disease, an intra-ocular condition, testicular torsion, trauma, shock, traumatic brain injury, ulcers, vasospasm, or a combination thereof.
152 . The method of treatment of claim 151 , wherein the organ ischemia comprises acute bowel ischemia (torsion), acute bowel ischemia (embolism), cardiogenic shock, acute vascular organ ischemia, stroke, myocardial infarction, or severe cardiac ischemia.
153 . A method of treatment of any one of claims 148 - 150 , wherein the method is a method for the treatment of non-traumatic hemorrhagic shock, pre-hospital setting trauma, traumatic hemorrhagic shock, acute lung injury, adult respiratory distress syndrome, traumatic brain injury, stroke, solid tumor cancer, organ degradation (ex-vivo), organ degradation (in recipient), severe sepsis, septic shock, myocardial infarction, cardiac ischemia, cardiogenic shock, acute heart failure, pulmonary embolism, or a combination thereof.
154 . A method of treatment of any one of claims 148 - 150 , wherein the hemoglobin tetramer or pharmaceutical composition is administered as an adjunct to angioplasty, as an adjunct for plastic surgery, or as an adjunct in implanting a ventricular assist device; as a blood substitute, a cardioprotectant, a cryopreservative, a hemodialysis adjunct, an oncology agent, an organ preservative, a performance enhancement agent, a surgery adjunct, or a wound healing agent; in imaging; to improve lung function; or a combination thereof.
155 . A method of treatment of any one of claims 148 - 150 , wherein the hemoglobin tetramer or pharmaceutical composition is administered as an adjunct to angioplasty, as an adjunct to thoracic aortic repairs, as an adjunct to cardiopulmonary bypass, or as a priming solution for cardiopulmonary bypass.
156 . A method of treatment of claim 148 or 149 , wherein the subject is a non-human animal and the method is a method for veterinary treatment of loss of blood due to injury, hemolytic anemia, infectious anemia, bacterial infection, Factor IV fragmentation, hypersplenation and splenomegaly, hemorrhagic syndrome in poultry, hypoplastic anemia, aplastic anemia, idiopathic immune hemolytic conditions, iron deficiency, isoimmune hemolytic anemia, microangiopathic hemolytic anemia, parasitism, or surgical-anesthesia induced brain damage.
157 . A method of delivering oxygen, nitric oxide, carbon monoxide or mixtures thereof to tissue and reducing nitrite to nitric oxide (NO) in the microvasculature, the method comprising administering the nitroxylated protein of any one of claims 4 , 7 , 13 , 14 , 29 , 30 and 38 - 83 or the pharmaceutical composition of any one of claims 131 - 147 to a subject in need thereof, wherein following administration, the hemoglobin becomes unliganded and converts nitrite to nitric oxide in the microvasculature.Join the waitlist — get patent alerts
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