Hemoglobin Derivative Co-conjugated with Fatty Acid-linked PEG and Alkoxy PEG as a Blood Substitute

Abstract

The invention relates to hemoglobin derivative, particularly hemoglobin which is co-conjugated with both fatty acid-linked polyethylene glycol (FA-PEG) derivatives and alkoxy polyethylene glycol (alkoxy-PEG) derivatives, and a method for making such hemoglobin derivative. Various embodiments of the invention include crosslinked hemoglobin which is co-conjugated with both FA-PEG derivatives and alkoxy-PEG derivatives. Such hemoglobin derivative according to the invention exhibit non-toxicity and extended intravascular retention time.

Claims

1. A hemoglobin derivative in which hemoglobin (Hb) or crosslinked hemoglobin (xHb) is conjugated with a fatty acid-PEG (FA-PEG) derivative and an alkoxy-PEG derivative.

2. The hemoglobin derivative according to claim 1, wherein the alkoxy-PEG is methoxy-PEG.

3. The hemoglobin derivative according to claim 1 or 2, wherein the hemoglobin derivative is represented by the following formula (I): [FA-PEG].sub.p-Hb-[alkoxy-PEG].sub.q where p=1˜10, and q=1˜20; or the following formula (II): [FA-PEG].sub.p-xHb-[alkoxy-PEG].sub.q where p=1˜10, and q=1˜20.

4. The hemoglobin derivative according to any one of claims 1 to 3, wherein the FA-PEG derivative is selected from the group consisting of FA-PEG acetaldehyde, FA-PEG propionaldehyde, FA-PEG butyraldehyde, FA-PEG maleimide, FA-PEG succinimidyl carbonate, FA-PEG succinimidyl carboxymethyl, FA-PEG succinimidyl glutarate, FA-PEG succinimidyl propionate, FA-PEG succinimidyl succinate, and FA-PEG succinimidyl carboxymethyl ester.

5. The hemoglobin derivative according to any one of claims 1 to 4, wherein the alkoxy-PEG derivative is selected from the group consisting of alkoxy-PEG acetaldehyde, alkoxy-PEG propionaldehyde, alkoxy-PEG butyraldehyde, alkoxy-PEG maleimide, alkoxy-PEG succinimidyl carbonate, alkoxy-PEG succinimidyl carboxymethyl, alkoxy-PEG succinimidyl glutarate, alkoxy-PEG succinimidyl propionate, and alkoxy-PEG succinimidyl succinate.

6. The hemoglobin derivative according to any one of claims 1 to 5, wherein the fatty acid is saturated fatty acid or unsaturated fatty acid.

7. The hemoglobin derivative according to claim 6, wherein the saturated fatty acid or the unsaturated fatty acid has the number of carbons from 6 to 24.

8. The hemoglobin derivative according to any one of claims 1 to 7, wherein the PEG has molecular weight of 1,000˜100,000 Da.

9. The hemoglobin derivative according to any one of claims 1 to 8, wherein the hemoglobin derivative is represented by the following formula (III) or the following formula (IV): ##STR00012## where p=1˜10, q=1˜20, n and m=20˜2,000, R.sub.1 is C.sub.6-24 alkyl or C.sub.6-24 alkenyl, R.sub.2 is C.sub.1-6 alkoxy, L is each independently NH or S, and X is each independently a divalent linker group with at least one amide, carbamate, carbonate, ester, ether, carbonyl, urethane, or succinimidyl; ##STR00013## where p=1˜10, q=1˜20, n and m=20˜2,000, R.sub.1 is C.sub.6-24 alkyl or C.sub.6-24 alkenyl, R.sub.2 is C.sub.1-6 alkoxy, L is each independently NH or S, and X is each independently a divalent linker group with at least one amide, carbamate, carbonate, ester, ether, carbonyl, urethane, or succinimidyl.

10. The method for preparing the hemoglobin derivative according to any one of claims 1 to 9, comprising reacting hemoglobin (Hb) or crosslinked hemoglobin (xHb) with the FA-PEG derivatives and the alkoxy-PEG derivatives to provide the hemoglobin derivative conjugated with FA-PEG derivatives and alkoxy-PEG derivatives.

11. The method according to claim 10, wherein said reaction is performed in a buffer solution at a temperature of 4 to 35° C. and a pH of 6 to 9.

12. A blood substitute composition comprising the hemoglobin derivative according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

13. A pharmaceutical composition comprising the hemoglobin derivative according to any one of claims 1 to 9 for treating, preventing, or alleviating a disease selected from the group consisting of hypoxia, ischemia, sepsis, sickle cell disease, retinal disease, diabetes, myocardial infarction, hemorrhagic shock, trauma, traumatic brain injury, brain stroke, tumor, and cancer.

14. A method of treating, preventing, or alleviating a disease selected from the group consisting of hypoxia, ischemia, sepsis, sickle cell disease, retinal disease, diabetes, myocardial infarction, hemorrhagic shock, trauma, traumatic brain injury, brain stroke, tumor, and cancer, comprising administering the hemoglobin derivative according to any one of claims 1 to 9 to a subject in need thereof.

15. Use of the hemoglobin derivative according to any one of claims 1 to 9 for treating, preventing, or alleviating a disease selected from the group consisting of hypoxia, ischemia, sepsis, sickle cell disease, retinal disease, diabetes, myocardial infarction, hemorrhagic shock, trauma, traumatic brain injury, brain stroke, tumor, and cancer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0079] The present invention relates to a hemoglobin derivative in which hemoglobin (Hb) or crosslinked hemoglobin (xHb) is conjugated with fatty acid-PEG (FA-PEG) derivatives and alkoxy-PEG derivatives.

[0080] Hemoglobin can be isolated from red blood cells (RBC) of human blood, bovine blood, porcine blood, blood from any land or oceanic animal species, or recombinant, or from red blood cells produced by stem cell technology. The human blood can be collected from donor blood that has passed its shelf-life. The animal blood can be collected from live or freshly slaughtered donors. The blood should be collected in a sanitary manner.

[0081] The present invention may comprise crosslinked hemoglobin as well as native hemoglobin.

[0082] The crosslinked hemoglobin may be intramolecularly-crosslinked. More specifically, the crosslinked hemoglobin may be β,β-intramolecularly-crosslinked or α,α-intramolecularly-crosslinked by conventional methods known in the art.

[0083] The crosslinked hemoglobin may be produced by reacting hemoglobin with a crosslinking agent such as bis(3,5-dibromosalicyl) fumarate or bis(3,5-dibromosalicyl) succinate.

[0084] The crosslinking of hemoglobin may allow stabilization of the tetrameric structure of the hemoglobin with its subunits linked together by linkers and protection of hemoglobin from dissociating into non-functional subunits.

[0085] In the context of the present invention, the crosslinked hemoglobin may be applied to all embodiments of hemoglobin.

[0086] Polyethylene glycol when conjugated to hemoglobin may advantageously elicit pharmacokinetic changes such as diminished immunogenic reactions, increased intravascular retention time, and increased water solubility, and while maintaining oxygen delivery capability.

[0087] Fatty acid of the present invention may be effective in increasing the intravascular retention time of the fatty acid-linked PEG-conjugated hemoglobin(FA-PEG-Hb). This is made possible because fatty acids are known to readily bind with human serum albumin FA-PEG-Hb intravenously injected may come in contact with albumin that is abundantly present in human blood. FA-PEG-Hb bound with one or more molecules of albumin will possess larger molecular weight and larger molecular radius, which will in turn manifest increased intravascular half-life. As albumin is also known to have anti-oxidant activity, the presence of albumin bound to FA-PEG-Hb may deter the rate of naturally-occurring oxidation of hemoglobin.

[0088] The hemoglobin or crosslinked hemoglobin may be reduced or deoxygenated prior to the conjugation with FA-PEG derivatives and alkoxy-PEG derivatives.

[0089] In one embodiment, the fatty acid-PEG derivatives or alkoxy-PEG derivatives may be covalently conjugated to hemoglobin or crosslinked hemoglobin.

[0090] The hemoglobin or crosslinked hemoglobin may be conjugated via a biologically stable, nontoxic, covalent linkage to alkoxy-PEG or fatty acid-PEG. Such linkages may include, but are not limited to, urethane linkages, carbamate linkages, carbonate linkages, ester linkages, carbonyl linkages, succinimidyl linkages, secondary amine linkages or amide linkages. In one embodiment, the one side or both sides of terminal end-groups in an alkoxy-PEG derivative and an FA-PEG derivative may be modified to contain a reactive functional group, and specifically, electrophilic functional group to be readily conjugated with hemoglobin or crosslinked hemoglobin.

[0091] The fatty acid-PEG derivative and the alkoxy-PEG derivative may be conjugated with hemoglobin or crosslinked hemoglobin by reacting the reactive functional group of the fatty acid-PEG derivative and the alkoxy-PEG derivative with a functional group of hemoglobin or crosslinked hemoglobin.

[0092] A fatty-acid PEG derivative and an alkoxy-PEG derivative can be conjugated to the surface amino acid side chains such as cysteine residues, lysine residues, or the terminal valine residue of hemoglobin or crosslinked hemoglobin using known methods, specifically nucleophilic substitution reaction.

[0093] The reactive functional group of the derivatives serves to link fatty acid-PEG and alkoxy-PEG to hemoglobin or crosslinked hemoglobin, and is non-reactive in vivo after the linkage.

[0094] The fatty acid-PEG derivative may be prepared by nucleophilic substitution reaction. More specifically, the fatty acid-PEG may be formed by nucleophilic substitution reaction in which a fatty acid having an electrophilic functional group is attacked by a nucleophilic group of a PEG derivative (e g amine or thiol), resulting in the conjugation reaction between the fatty acid and the nucleophilic atoms of a PEG derivative (e.g. nitrogen of amine or sulfur of thiol), while the electrophilic functional group from a fatty acid is detached as a leaving group.

[0095] The fatty acid having an electrophilic functional group may be formed by substitution reaction of a carboxyl group of a fatty acid, resulting in substitution of a a hydroxyl group of a carboxyl group with electrophilic functional group.

[0096] In the preferred embodiment, the alkoxy-PEG is a methoxy-PEG.

[0097] In one embodiment, the hemoglobin derivative may be represented by the following formula (I): [FA-PEG].sub.p-Hb-[alkoxy-PEG].sub.q, where p=1˜10, and q=1˜20; or the following formula (II): [FA-PEG].sub.p-xHb-[alkoxy-PEG].sub.q, where p=1˜10, and q=1˜20.

[0098] Formula (I) refers to the hemoglobin derivative in which hemoglobin (Hb) is conjugated with fatty acid-PEG (FA-PEG) derivatives and alkoxy-PEG derivatives, and formula (II) refers to the hemoglobin derivative in which crosslinked hemoglobin (xHb) is conjugated with fatty acid-PEG (FA-PEG) derivatives and alkoxy-PEG derivatives.

[0099] The mPEG-SS used in this Example is just one example of many PEG derivatives with varying molecular weight that can be used. Many other PEG derivatives that are reactive towards the amine on the surface of Hb can be used.

[0100] In one embodiment, the FA-PEG derivatives may be, but not limited to, any derivatives which comprise fatty acid-PEG and can be readily reacted with hemoglobin or crosslinked hemoglobin to be conjugated therewith. The FA-PEG derivatives may be selected from the group consisting of FA-PEG acetaldehyde, FA-PEG propionaldehyde, FA-PEG butyraldehyde, FA-PEG maleimide, FA-PEG succinimidyl carbonate, FA-PEG succinimidyl carboxymethyl, FA-PEG succinimidyl glutarate, FA-PEG succinimidyl propionate, FA-PEG succinimidyl succinate, and FA-PEG succinimidyl carboxymethyl ester. More preferably, the FA-PEG derivatives may be FA-PEG succinimidyl carboxymethyl ester. The structural forms of the FA-PEG can be also diverse, and the examples include, but are not limited to, linear FA-PEG, branched FA-PEG, or FA-PEG with degradable linkages built within the backbone.

[0101] In one embodiment, the alkoxy-PEG derivatives may be, but are not limited to, any derivatives which comprise alkoxy-PEG and can be readily reacted with hemoglobin or crosslinked hemoglobin to be conjugated therewith. The alkoxy-PEG derivatives may be selected from the group consisting of alkoxy-PEG acetaldehyde, alkoxy-PEG propionaldehyde, alkoxy-PEG butyraldehyde, alkoxy-PEG maleimide, alkoxy-PEG succinimidyl carbonate, alkoxy-PEG succinimidyl carboxymethyl, alkoxy-PEG succinimidyl glutarate, alkoxy-PEG succinimidyl propionate, and alkoxy-PEG succinimidyl succinate. More preferably, the alkoxy-PEG derivatives may be alkoxy-PEG succinimidyl succinate, and most preferably, the alkoxy-PEG derivatives may be methoxy-PEG succinimidyl succinate. The structural forms of the alkoxy-PEG can be also diverse, and the examples include, but are not limited to, linear alkoxy-PEG, branched alkoxy-PEG, or alkoxy-PEG with degradable linkages built within the backbone.

[0102] In one embodiment, the fatty acid may be saturated fatty acid or unsaturated fatty acid.

[0103] The saturated fatty acid or the unsaturated fatty acid may have the number of carbons from 6 to 24, preferably from 10 to 24.

[0104] The saturated fatty acids can be such as, but are not limited to, stearic acid, palmitic acid, myristic acid, lauric acid, capric acid, or arachidic acid. The unsaturated fatty acids can be such as, but are not limited to, oleic acid, linoleic acid, myristoleic acid, palmitoleic acid, or arachidonic acid.

[0105] The fatty acid which is used to form the fatty acid-PEG derivatives may have a molecular weight of about 60˜400 Da, preferably 80˜340 Da.

[0106] In one embodiment, the PEG has molecular weight of about 100˜100,000 Da. Exemplary molecular weights of PEG include about to about 1,000˜100,000 Da; about 1,000 to about 80,000 Da; about 1,000 to about 70,000 Da; preferably, about 1,000 to about 50,000 Da; and more preferably, about 2,000 to about 10,000 Da.

[0107] In a specific embodiment, the hemoglobin derivative is represented by the following formula (III) or following formula (IV):

##STR00001##

where p=1˜10, q=1˜20, n and m=each independently 20˜2,000, R.sub.1 is C.sub.6-24 alkyl or C.sub.6-24 alkenyl, R.sub.2 is C.sub.1-6 alkoxy, L is each independently NH or S, and X is each independently a divalent linker group with at least one amide, carbamate, carbonate, ester, ether, carbonyl, urethane, or succinimidyl;

##STR00002##

where p=1˜10, q=1˜20, n and m=each independently 20˜2,000, R.sub.1 is C.sub.6-24 alkyl or C.sub.6-24 alkenyl, R.sub.2 is C.sub.1-6 alkoxy, L is each independently NH or S, and X is each independently a divalent linker group with at least one amide, carbamate, carbonate, ester, ether, carbonyl, urethane, or succinimidyl.

[0108] Formula (III) refers to the hemoglobin derivative in which hemoglobin (Hb) is conjugated with fatty acid-PEG (FA-PEG) derivatives and alkoxy-PEG derivatives, and formula (IV) refers to the hemoglobin derivative in which crosslinked hemoglobin (xHb) is conjugated with fatty acid-PEG (FA-PEG) derivatives and alkoxy-PEG derivatives.

[0109] In one embodiment, PEG of fatty acid-PEG and alkoxy-PEG may be covalently attached via an amino reactive moiety or sulfur reactive moiety of an amino acid side chain on the hemoglobin or crosslinked hemoglobin. The amino reactive moiety or sulfur reactive moiety may be linked to the PEG by group X. That is, X refers to the divalent linker group which links alkoxy-PEG and hemoglobin or crosslinked hemoglobin, or links fatty acid-PEG and hemoglobin or crosslinked hemoglobin.

[0110] In a specific embodiment, X is selected from the group consisting of amide, carbamate, carbonate, ester, ether, carbonyl, urethane, succinimidyl, C.sub.1-6 alkylene, C.sub.1-6 alkylene-amide, C.sub.1-6 alkylene-carbamate, C.sub.1-6 alkylene-carbonate, C.sub.1-6 alkylene-ester, C.sub.1-6 alkylene-ether, C.sub.1-6 alkylene-carbonyl, C.sub.1-6 alkylene-urethane, C.sub.1-6 alkylene-succinimidyl, amide-C.sub.1-6 alkylene-carbonyl, carbamate-C.sub.1-6 alkylene-carbonyl, carbonate-C.sub.1-6 alkylene-carbonyl, ester-C.sub.1-6 alkylene-carbonyl, ether-C.sub.1-6 alkylene-carbonyl, carbonyl-C.sub.1-6 alkylene-carbonyl, urethane-C.sub.1-6 alkylene-carbonyl, and succinimidyl-C.sub.1-6 alkylene-carbonyl.

[0111] More preferably, X of FA-PEG derivatives may be amide-C.sub.1-6 alkylene-carbonyl and X of alkoxy-PEG derivatives may be carbonyl-C.sub.1-6 alkylene-carbonyl.

[0112] X may be derived from the FA-PEG derivatives and alkoxy-PEG derivatives, and L may be derived from the hemoglobin or crosslinked hemoglobin.

[0113] In one embodiment, linkage between L and X may be formed by a nucleophilic substitution reaction in which an FA-PEG derivative or an alkoxy-PEG derivative having an electrophilic functional group is attacked by the nucleophilic groups of hemoglobin or crosslinked hemoglobin (e g amines or thiols), resulting in the conjugation reaction between the FA-PEG derivative or alkoxy-PEG derivative and the nucleophilic atoms of hemoglobin (e.g. nitrogen of amines or sulfur of thiols), while the functional group from an FA-PEG derivative or an alkoxy-PEG derivative is detached as a leaving group.

[0114] In one embodiment, in the case that L is S, X is succinimidyl or C.sub.1-6 alkylene-succinimidyl.

[0115] In the formula (III), p denotes the number of FA-PEGs conjugated to hemoglobin, and q denotes the number of alkoxy-PEGs conjugated to hemoglobin.

[0116] In the formula (IV), p denotes the number of FA-PEGs conjugated to crosslinked hemoglobin, and q denotes the number of alkoxy-PEGs conjugated to crosslinked hemoglobin.

[0117] The number p is preferably 1 to 8, more preferably, 1 to 6, and most preferably, 1 to 4. The number q is preferably 2 to 18, more preferably, 4 to 16, and most preferably, 10 to 16.

[0118] In the formulas (III) and (IV), n and m are each independently the average number of oxyethylene units of a PEG, and preferably, n and m are each independently 20 to 1,800, more preferably, 50 to 1,800, and most preferably, 100 to 1,500.

[0119] R.sub.1 corresponds to a hydrocarbon chain which is comprised in a fatty acid. In one embodiment, R.sub.1 is C.sub.6-24 alkyl or C.sub.6-24 alkenyl, preferably, R.sub.1 is C.sub.10-24 alkyl or C.sub.10-24 alkenyl, and more preferably, R.sub.1 is C.sub.10-22 alkyl or C.sub.10-22 alkenyl. In one embodiment, in the case that R.sub.1 is C.sub.6-24 alkenyl, R.sub.1 may have 1 to 5 unsaturated bonds.

[0120] In one embodiment, R.sub.2 is alkoxy, preferably, R.sub.2 is alkoxy, and most preferably, R.sub.2 is methoxy.

[0121] The present invention further relates to the method for preparing the hemoglobin derivative, comprising reacting hemoglobin (Hb) or crosslinked hemoglobin (xHb) with the FA-PEG derivatives and the alkoxy-PEG derivatives to provide hemoglobin derivative conjugated with FA-PEG derivatives and alkoxy-PEG derivatives.

[0122] In one embodiment, reaction of FA-PEG derivatives and reaction of alkoxy-PEG derivatives may be progressed simultaneously or progressed in turns.

[0123] In one specific embodiment, hemoglobin derivative may be prepared by reaction of hemoglobin or crosslinked hemoglobin with both FA-PEG derivatives and alkoxy-PEG derivatives simultaneously in one single step.

[0124] In other specific embodiment, hemoglobin derivative may be prepared by reaction of hemoglobin or crosslinked hemoglobin first with FA-PEG derivatives, followed by reaction with alkoxy-PEG derivatives, or may be prepared by reaction of hemoglobin or crosslinked hemoglobin first with alkoxy-PEG derivatives, followed by reaction with FA-PEG derivatives.

[0125] In the case of the method for preparing the hemoglobin derivative from crosslinked hemoglobin, firstly, crosslinked hemoglobin may be prepared by reacting hemoglobin with a crosslinking agent such as bis(3,5-dibromosalicyl) fumarate or bis(3,5-dibromosalicyl) succinate prior to undergoing a reaction with FA-PEG derivatives and alkoxy-PEG derivatives.

[0126] In one embodiment, the reaction is performed in a buffer solution at the temperature of 4 to 35° C. and pH of 6 to 9.

[0127] In a specific embodiment, the reaction may be performed in a buffer solution at a temperature of 4° C. to 35° C., preferably at a temperature of 10° C. to 30° C., and most preferably at a temperature of 15° C. to 28° C.

[0128] In an additional specific embodiment, the reaction may be performed in a buffer solution at a pH of 6 to 9, preferably at a pH of 6.5 to 9, preferably at a pH of 7 to 9, preferably at a pH of 7 to 8.5, and preferably at a pH of 7.5 to 8.5.

[0129] The reaction of FA-PEG derivatives and the reaction of alkoxy-PEG derivatives respectively may be performed under different conditions or may be performed in the same conditions.

[0130] After the reaction, the product may be diafiltered (e.g. using a diafiltration membrane device) and/or passed through chromatography (e.g. ion-exchange chromatography, affinity chromatography or size exclusion chromatography) to separate the desired product from a reactant, such as unreacted hemoglobin, unreacted FA-PEG derivatives, and unreacted alkoxy-PEG derivatives, and to only collect the desired product.

[0131] The present invention further relates to a blood substitute composition comprising the hemoglobin derivative.

[0132] The hemoglobin derivative can be used as a blood substitute in that they are hemoglobin-based and are capable of delivering carbon monoxide as well as oxygen to tissues in a subject.

[0133] The present invention further relates to a pharmaceutical composition comprising the hemoglobin derivative for treating, preventing, or alleviating a disease selected from the group consisting of hypoxia, ischemia, sepsis, sickle cell disease, retinal disease, diabetes, myocardial infarction, hemorrhagic shock, trauma, traumatic brain injury, brain stroke, tumor, and cancer.

[0134] The blood substitute composition and the pharmaceutical composition can comprise a pharmaceutically acceptable carrier as well as the hemoglobin derivative.

[0135] Examples of a pharmaceutically acceptable carrier comprise any diluent, such as a protein, a glycoprotein, a polysaccharide, and other colloids, any salt that is pharmaceutically acceptable for delivery to a mammal, such as KCl, NaCl, NaHCO.sub.3, NaH.sub.2PO.sub.4.2H.sub.2O, MgSO.sub.4.2H.sub.2O, CaCl.sub.2.2H.sub.2O, cysteine, and dextrose.

[0136] The compositions of the invention may be isotonic, hypertonic, or hypotonic. In various embodiments, the composition is isotonic. In an exemplary embodiment, the composition includes a sufficient amount of salts to render it isotonic. In other embodiments, the diluent is isotonic phosphate buffered saline.

[0137] The composition can additionally comprise pharmaceutically-acceptable fillers and other materials well-known in the art, the selection of which depends on the dosage form, the condition being treated, the particular purpose to be achieved according to the determination of the ordinarily skilled artisan in the field and the properties of such additives. For example, the composition can include a physiological buffer, a carbohydrate (e.g. glucose, mannitol, or sorbitol), alcohol or poly alcohol, a pharmaceutically acceptable salt (e.g., sodium or potassium chloride), a surfactant (e.g., polysorbate 80), an anti-oxidant, an anti-bacterial agent, an oncotic pressure agent (e.g. albumin or polyethylene glycol), or a stabilizer (e.g., ascorbic acid, glutathione, or N-acetyl cysteine).

[0138] In a preferred embodiment, the composition may include stabilizers such as, but not limited to cysteine, N-acetyl cysteine, glutathione, or ascorbate.

[0139] In a preferred embodiment, the pharmaceutical composition may be formulated in an aqueous saline solution, specifically, an aqueous isotonic saline solution, and more specifically, a physiologically acceptable electrolyte-containing aqueous isotonic saline solution.

[0140] A composition of the present invention can be administered by injecting the composition directly and/or indirectly into the circulatory system of the subject by one or more injection methods.

[0141] Examples of direct injection methods may include intravascular injections, such as intravenous and intra-arterial injections, and intracardiac injections. Examples of indirect injection methods include intraperitoneal injections, subcutaneous injections, such that the hemoglobin derivative will be transported by the lymph system into the circulatory system, or injections into the bone marrow by means of a trocar or catheter. The compositions can also be administered by gavage. A preferred injection method may be an intravascular injection.

[0142] In one embodiment, the composition is administered simultaneously, separately, or sequentially in combination with one or more additional therapeutic agents to a subject in need thereof.

[0143] The present invention also relates to a method of treating, preventing, or alleviating a disease selected from the group consisting of hypoxia, ischemia, sepsis, sickle cell disease, retinal disease, diabetes, myocardial infarction, hemorrhagic shock, trauma, traumatic brain injury, brain stroke, tumor, and cancer, comprising administering the hemoglobin derivative to a subject in need thereof.

[0144] The hemoglobin derivative can be used as a therapeutic agent for various types of tumors and cancers. More effectively, the hemoglobin derivative can be used to oxygenate the solid tumors and cancers that are known to be hypoxic and thus resistant to chemotherapy and radiotherapy. Oxygenation of hypoxic tumor or cancer cells is known to render such cells more susceptible to therapy.

[0145] Generally an effective administration amount of a hemoglobin derivative of the invention will depend on the relative efficacy of the hemoglobin derivative chosen, the severity of the disorder being treated and/or prevented, and the weight of the subject.

[0146] The present invention also relates to use of the hemoglobin derivative as a medicament.

[0147] The present invention relates to use of the hemoglobin derivative for treating, preventing, or alleviating a disease selected from the group consisting of hypoxia, ischemia, sepsis, sickle cell disease, retinal disease, diabetes, myocardial infarction, hemorrhagic shock, trauma, traumatic brain injury, brain stroke, tumor, and cancer.

[0148] The hemoglobin derivative and compositions of the present invention can be used during various surgical procedures. For example, they can be used as an adjunct to angioplasty, thoracic aortic repairs, during a cardiopulmonary bypass procedure, or as a cardiopulmonary priming solution.

[0149] The present invention is additionally explained below by means of examples. These explanations must by no means be interpreted as a limitation to the scope of the invention as defined in the claims.

Examples

Example 1: Preparation of Native Pure Hemoglobin

[0150] Hemoglobin can be isolated from red blood cells (RBC) of human blood, bovine blood, porcine blood, or blood from other animal species. The human blood can be collected from donated blood that has passed its shelf-life. The animal blood can be collected from live or freshly slaughtered donors. The blood should be collected in a sanitary manner.

[0151] The methods are those known in the art for the process of isolation and purification of hemoglobin from blood, and these methods were generally applicable to the compositions of the present invention. The description following herein is illustrative and not limiting.

[0152] At the time of blood collection, the blood was mixed with an anticoagulant to prevent blood coagulation. Blood anticoagulants are well known in the art and include, for example, sodium citrate, ethylenediamine tetra-acetic acid, and heparin.

[0153] The collected blood was centrifuged to separate plasma-borne proteins from red blood cells (RBC). The RBCs were obtained as a precipitate in the centrifuge tube by draining the top fluid.

[0154] The collected RBCs were then mixed with isotonic saline, and subsequently washed with isotonic saline by a suitable means, such as by diafiltration, to separate RBCs from residual blood-borne plasma proteins, such as serum albumins or antibodies. The diafiltration was continued until several volumes of filtrate solution were consumed to achieve 99% removal of plasma proteins. In general, the diafiltration of RBCs utilizing 6 volumes of isotonic solution may remove about 99% of plasma protein and antibodies.

[0155] After the washing of RBCs, the Hb was extracted from the RBCs. Extraction can be performed by various methods including lysis and hypo-osmotic swelling and compression of the RBCs. Various RBC lysis methods exist, such as mechanical lysis, chemical lysis, and hypotonic lysis.

[0156] Following the lysis, the lysed RBC solution was then ultrafiltered to remove debris, such as red cell membrane particles. An appropriate ultrafiltration device is one that can separate native hemoglobin from large cell debris, and such a device may have molecular weight cut-off of 100,000 Da so that native hemoglobin having molecular weight of 65,000 Da can be separated from large particles. Other methods for separating Hb from the lysed cell debris can be used, including centrifugation, sedimentation, and microfiltration, or a combination of two or more methods.

[0157] The collected Hb solution was concentrated to be suitable for the next step by using an ultrafiltration device having molecular weight cut-off of 30,000 Da or lower in order to retain and collect the Hb in a solution while squeezing the excess water out.

[0158] The concentrated Hb solution was directed into an ion-exchange chromatography to purify the Hb by removing residual contaminants such as plasma proteins, antigens, antibodies, endotoxins, membrane lipids, and phospholipids. Further purification can be achieved by additional chromatography employing size exclusion, ion-exchange, or hydrophobic-interaction chromatography, or sequential combination of two or more types of chromatography.

[0159] The purified Hb solution was then treated with reduction methods to prevent Hb from oxidation. The native Hb without the protection of the RBC membrane tends to oxidize such that the ferrous iron (Fe.sup.++) residing inside the heme pocket of Hb was liable to oxidize to become ferric iron (Fe.sup.+++). The Hb with ferric iron was called Met-Hb. Ferric iron does not bind with oxygen, and thus the Met-Hb loses its oxygen carrying capability.

[0160] The stabilization of Hb can be achieved by deoxygenation or by addition of chemical reductants, or both. The Hb exposed to atmosphere is readily oxygenated to oxy-Hb which becomes liable for oxidation to Met-Hb. To deoxygenate, the oxy-Hb was directed through courses of a gas exchange device utilizing extra-luminal pressure of inert gases such as nitrogen, argon, or helium. Nitrogen is often the most safe and inexpensive choice. Deoxygenation of oxy-Hb can also be aided by addition of reductants such as, but not limited to, cysteine, N-acetyl-L-cysteine (NAC), glutathione, dithionate, or ascorbate.

[0161] The deoxygenated Hb solution was then directed to a condition of a thermal viral deactivation process. The thermal viral deactivation process relates to exposing the solution to a temperature that was raised sufficiently and for a time sufficient to deactivate essentially all viral activity in said solution. The thermal viral deactivation process is well known in the art, and it often employs the condition of raising the temperature of said solution to 60° C. and sustaining the temperature for 4 hours or more. An alternative to the thermal viral deactivation process is using a virus filtration device, and having the said solution pass through a virus filtration device. After the virus deactivation, the purified Hb solution is refrigerated or frozen for storage purposes.

Example 2: Preparation of a Stearic-PEG Derivative

[0162] In this exemplary embodiment, stearic acid is selected as a representative example of a saturated fatty acid.

[0163] In this exemplary embodiment, as an example of an FA-PEG derivative, stearic-PEG-succinimidyl carboxymethyl ester was prepared.

[0164] In the first step of synthesis, stearic acid in an organic solvent of methylene chloride was reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxy succinimide (NHS) to obtain succinimidyl stearate. The reaction mechanism is illustrated below.

##STR00003##

[0165] In the second step of synthesis, PEG (PEG-diol) was first reacted in organic solvent of methylene chloride (MC) and triethylamine and tosyl chloride with a subsequent addition of 28% ammonia water. In the exemplary embodiment, PEG with a molecular weight of 5,000 Da is used. The intermediate PEG compounds thus formed were a mixture of HO-PEG-OH (PEG-diol), amine-PEG-OH (amine PEG-alcohol), and amine-PEG-amine (PEG-diamine), shown as compounds (1) in the illustration below. This mixture was then directed to pass through an ion-exchange chromatography to separate and to only obtain amine-PEG-OH, shown as compound (2) of the illustration below. The amine-PEG-OH was then reacted in organic solvent of MC with triethylamine and di-tert-butyl dicarbonate (BOC.sub.2O) to obtain BOC-PEG-OH, shown as compound (3) in the illustration below. The BOC-PEG-OH was then reacted with ethylisocyanoacetate (OCNCH.sub.2CO.sub.2-ethyl) and triethanolamine (TEA) with the addition of 1N NaOH to obtain BOC-PEG-urethane acetic acid, which was then directed to pass through an ion-exchange chromatography to obtain further purified BOC-PEG-urethane acetic acid, shown as compound 4 in the illustration below. The BOC-PEG-urethane acetic acid was reacted with trifluoroacetic acid (CF.sub.3COOH) to remove the BOC group and finally to obtain the amine-PEG-urethane acetic acid, shown as compound (5) in the illustration below.

##STR00004##

[0166] In the third step of synthesis, the succinimidyl stearate (from the first step) was reacted with amine-PEG-urethane acetic acid (from the second step) in the organic solvent of methylene chloride (MC) with the addition of base N,N-diisopropylethylamine (DIEA) under a room temperature (RT) for 16 hours to obtain the stearic-PEG-acid, as illustrated below. This compound has an acid end group, which is derivatized in the next step.

##STR00005##

[0167] In the fourth step of synthesis, the stearic-PEG-acid obtained above was reacted in the organic solvent of MC along with N-hydroxy succinimide (NHS) and N,N-dicyclohexylcarbodiimide (DCC) under RT for 16˜20 hours to obtain stearic-PEG-urethane-succinimidyl carboxymethyl ester as illustrated below. This stearic-PEG-urethane-succinimidyl carboxymethyl ester (Stearic-PEG-SCM) will be used to conjugate with Hb in Example 3.

##STR00006##

Example 3: Preparation of [Stearic-PEG].SUB.p.-Hb-[mPEG].SUB.q

[0168] [Stearic-PEG].sub.p-Hb-[mPEG].sub.q is a Hb conjugated with both stearic-PEG and mPEG.

[0169] The pure Hb obtained from Example 1 was first reacted with stearic-PEG-urethane succinimidyl carboxymethyl ester of MW 5,000 Da (Stearic-PEG-SCM) obtained from Example 2 to yield [Stearic-PEG].sub.p-Hb in the following process.

[0170] The Hb solution was prepared in an isotonic buffer solution at 22° C. at pH 8.2 with Hb concentration at 5% (w/v). Into the Hb solution was added 5 molar equivalents of stearic-PEG-SCM and well agitated for 2 hours. As a result, [Stearic-PEG].sub.p-Hb was obtained. The schematics of the reaction is illustrated below.

##STR00007##

[0171] After the conjugation reaction, [Stearic-PEG].sub.p-Hb was diafiltered against diluent of an isotonic buffer to remove unreacted Hb and unreacted stearic-PEG. The diafiltration may use a diafiltration membrane device having molecular weight cut-off of 50,000 Da to separate the [Stearic-PEG].sub.p-Hb from unreacted Hb and unreacted stearic-PEG.

[0172] After the diafiltration, the [Stearic-PEG].sub.p-Hb solution was further directed to pass through an ion-exchange chromatography to separate out and eliminate the undesired portion of the [Stearic-PEG].sub.p-Hb and to only collect the desired portion of the [Stearic-PEG].sub.p-Hb. The conjugate was a mixture of different molecular weights including [Stearic-PEG].sub.1-Hb, [Stearic-PEG].sub.2-Hb, [Stearic-PEG].sub.3-Hb, and so on. Using proper chromatography, one may separate and obtain a desired portion.

[0173] The purified [Stearic-PEG].sub.p-Hb was then reacted with methoxy PEG-succinimidyl succinate of MW 5,000 Da (mPEG-SS). The [Stearic-PEG].sub.p-Hb was prepared in isotonic buffer at pH 8.2 and at 22° C. with a Hb concentration of 5% (w/v). Into the [Stearic-PEG].sub.p-Hb solution was added an amount of mPEG-SS at 15 molar equivalents and well agitated for 2 hours. As a result, [Stearic-PEG].sub.p-Hb-[mPEG].sub.q was obtained. The schematics of the reaction is illustrated below.

##STR00008##

[0174] After pegylation with mPEG-SS, the resulting compound, [Stearic-PEG].sub.p-Hb-[mPEG].sub.q, was directed through diafiltration followed by chromatography in order to remove unreacted mPEG-SS and obtain a portion of the final compound with a desired molecular weight. The final compound may be formulated into a physiologically acceptable electrolyte-containing aqueous isotonic saline solution, and optionally stabilizers such as, but not limited to, cysteine, N-acetyl cysteine, glutathione, or ascorbate, can be added.

[0175] In this example, [Stearic-PEG].sub.p-Hb-[mPEG].sub.q was prepared by pegylation of Hb first with an FA-PEG derivative, followed by pegylation with a mPEG derivative. However, the sequence of pegylation may be reversed in that [Stearic-PEG].sub.p-Hb-[mPEG].sub.q may be prepared by pegylation of Hb first with a mPEG derivative, followed by pegylation with an FA-PEG derivative, to achieve an identical result.

Example 4: Preparation of [Stearic-PEG].SUB.p.-xHb-[mPEG].SUB.q

[0176] [Stearic-PEG].sub.p-xHb-[mPEG].sub.q is a xHb (crosslinked Hb) pegylated with both stearic-PEG and mPEG. First, Hb is crosslinked to make xHb (crosslinked Hb). This xHb is next conjugated with stearic-PEG-SCM and mPEG-SS.

[0177] The xHb was prepared by reacting Hb with bis(3,5-dibromosalicyl) fumarate. A Hb solution was prepared at 5% (w/v) concentration, a pH of 8.2, and a temperature of 22° C. in an isotonic buffer. The amount of bis(3,5-dibromosalicyl) fumarate added was 2 molar equivalents with respect to Hb. The reaction solution was well agitated for 2 hours to obtain the xHb solution. The schematics of the reaction is illustrated below.

##STR00009##

[0178] Next, xHb was prepared in an aqueous solution containing isotonic buffers at 22° C. with a pH of 8.2 and a Hb concentration at 5% (w/v). Into the xHb solution, 5 molar equivalents of stearic-PEG-SCM was added, which was well agitated for 2 hours to obtain [Stearic-PEG].sub.p-xHb. The schematics of the reaction is illustrated below.

##STR00010##

[0179] After the conjugation, the [Stearic-PEG].sub.p-xHb solution was diafiltered against diluent of an isotonic buffer to remove unreacted Hb and unreacted stearic-PEG. Such diafiltration may use a diafiltration membrane device with molecular weight cut-off of 50,000 Da. The 50,000 Da diafiltration membrane device may be effective in terms of separating the [Stearic-PEG].sub.p from unreacted stearic-PEG.

[0180] After the diafiltration, the [Stearic-PEG].sub.p-xHb solution was further directed to pass through ion-exchange chromatography to separate out and eliminate the undesired portion of the [Stearic-PEG].sub.p-xHb, and to only collect the desired portion of the [Stearic-PEG].sub.p-xHb. The conjugate was a mixture of different molecular weights, including [Stearic-PEG].sub.1-xHb, [Stearic-PEG].sub.2-xHb, [Stearic-PEG].sub.3-xHb, and so on. Using appropriate chromatography, one may separate and obtain the desired portion.

[0181] Next, [Stearic-PEG].sub.p-xHb was prepared in an aqueous solution containing isotonic buffers at 22° C. along with a pH of 8.2 and a Hb concentration of 5% (w/v). Into the [Stearic-PEG].sub.p-xHb solution, 15 molar equivalents of mPEG-SS was added, which was well agitated for 2 hours, to obtain [Stearic-PEG].sub.p-xHb-[mPEG].sub.q. The schematics of the reaction is illustrated below.

##STR00011##

[0182] After the reaction with mPEG-SS, the resulting compound, [Stearic-PEG].sub.p-xHb-[mPEG].sub.q, was directed through diafiltration followed by chromatography in order to remove unreacted mPEG-SS to obtain desired a portion of the final compound with a desired molecular weight. The final compound may be formulated into a physiologically acceptable electrolyte-containing aqueous isotonic saline solution, along with optionally adding stabilizers.

[0183] In this example, [Stearic-PEG].sub.p-xHb-[mPEG].sub.q was prepared by pegylation of xHb first with a FA-PEG derivative, followed by pegylation with an mPEG derivative. However, the sequence of pegylation may be reversed in that [Stearic-PEG].sub.p-xHb-[mPEG].sub.q may be prepared by pegylation of xHb first with an mPEG derivative, followed by pegylation with a FA-PEG derivative, to achieve an identical result.

Example 5: Analysis of [Stearic-PEG].SUB.p.-Hb-[mPEG].SUB.q

[0184] The [Stearic-PEG].sub.p-Hb-[mPEG].sub.q obtained from Example 3 was analyzed. The protein concentration of [Stearic-PEG].sub.p-Hb-[mPEG].sub.q as represented by the concentration of Hb was analyzed by CO-OX SC80 Blood Gas Analyzer to be 4.5% (w/v).

[0185] Homogeneity and molecular weight of the [Stearic-PEG].sub.p-Hb-[mPEG].sub.q were analyzed by high performance liquid chromatography (HPLC) with an absorbance reading at 280 nm. The chromatogram showed a single peak with a main peak fraction greater than 99%.

[0186] The analysis to determine the number of PEGs attached to the [Stearic-PEG].sub.p-Hb and [Stearic-PEG].sub.p-Hb-[mPEG].sub.q was performed by TNBS (2,4,6-trinitrobenzene sulfonic acid) assay method that is known in the art (Reference: Analytical Biochemistry 14, 328-336 (1966) “Determination of Free Amino Groups in the Proteins by Trinitrobezenesulfonic Acid”).

[0187] As the embodiment of Example 3 was prepared sequentially in that Hb had been treated first with an FA-PEG derivative to produce [Stearic-PEG].sub.p-Hb, a sample of this intermediate product [Stearic-PEG].sub.p-Hb was collected and assayed by the TNBS method. The number of FA-PEGs attached on Hb was 2. The [Stearic-PEG].sub.p-Hb, as per Example 3, had been treated with mPEG derivatives to produce [Stearic-PEG].sub.p-Hb-[mPEG].sub.q, which was analyzed by TNBS method. The number of total PEGs attached to Hb was 14. Therefore, it is deduced that the number of mPEGs attached to Hb was 12.

[0188] The molecular weight of the [Stearic-PEG].sub.p-Hb-[mPEG].sub.q can now be calculated to be 135,000 Da, with the molecular weight of each PEG being 5,000 Da and the molecular weight of Hb being 65,000 Da.

[0189] Thus, the product from Example 3 can now be expressed as [Stearic-PEG].sub.2-Hb-[mPEG].sub.12.

Example 6: Analysis of [Stearic-PEG].SUB.p.-xHb-[mPEG].SUB.q

[0190] The [Stearic-PEG].sub.p-xHb-[mPEG].sub.q obtained by Example 4 was analyzed. The protein concentration of [Stearic-PEG].sub.p-xHb-[mPEG].sub.q as represented by the concentration of Hb was analyzed by the CO-OX SC80 Blood Gas Analyzer to be 4.2% (w/v).

[0191] Homogeneity and molecular weight of the [Stearic-PEG].sub.p-xHb-[mPEG].sub.q were analyzed by high performance liquid chromatography (HPLC) with absorbance reading at 280 nm. The chromatogram showed a single peak with a main peak fraction greater than 97%.

[0192] The number of PEGs on the [Stearic-PEG].sub.p-xHb-[mPEG].sub.q was determined by the TNBS assay method.

[0193] As the embodiment in Example 4 was prepared sequentially in that xHb had been treated first with an FA-PEG derivative to produce [Stearic-PEG].sub.p-xHb, a sample of this intermediate product [Stearic-PEG].sub.p-xHb was collected and assayed by the TNBS method. The number of FA-PEGs attached on xHb was 2. The [Stearic-PEG].sub.p-xHb, as per Example 4, had been treated with mPEG derivatives to produce [Stearic-PEG].sub.p-xHb-[mPEG].sub.q, which was analyzed by the TNBS method. The number of total PEGs attached to Hb was 14. Therefore, it is deduced that the number of mPEGs attached to Hb was 12.

[0194] The molecular weight of the [Stearic-PEG].sub.p-xHb-[mPEG].sub.q can now be calculated to be 135,000 Da, with the molecular weight of each PEG being 5,000 Da and the molecular weight of Hb being 65,000 Da.

[0195] Thus, the product from Examples 4 can now be expressed as [Stearic-PEG].sub.2-xHb-[mPEG].sub.12.

Example 7: Toxicity of [Stearic-PEG].SUB.2.-Hb-[mPEG].SUB.12 .in Animals

[0196] Purpose of this test was to investigate the acute toxicity of the [Stearic-PEG].sub.2-Hb-[mPEG].sub.12 solution within animals. Eight-week old Sprague-Dawley male rats weighing 300±30 g were chosen and acclimated for a week. The animals were injected with 15 mL/kg of test materials, and were observed in terms of their survival for 7 days. The study period was up to 7 days, after which all animals were euthanized.

[0197] As the test material, a solution containing 4.5% of [Stearic-PEG].sub.2-Hb-[mPEG].sub.12 having a molecular weight of 135,000 Da, along with a pH of 7.2 was prepared.

[0198] For the test group, 5 rats received intravenously 15 mL/kg (equivalent to 675 mg/kg) of a [Stearic-PEG].sub.2-Hb-[mPEG].sub.12 solution via a jugular vein at a controlled rate of 0.1 mL/min using a syringe pump. For the control group, rats received 15 mL/kg of Ringers Lactate via the same route and manner. All animals were weighed and observed for behavior and survival every day for 7 days. As a result, all animals in both groups survived and were determined to be healthy at day 7. This study demonstrated the safety or non-toxicity of [Stearic-PEG].sub.2-Hb-[mPEG].sub.12 in rats when tested under a single intravenous bolus injection model at a dosage of 15 mL/kg (equivalent to 675 mg/kg).

Example 8: Pharmacokinetics of [Stearic-PEG].SUB.2.-xHb-[mPEG].SUB.12

[0199] The purpose of this test was to investigate the pharmacokinetics of [Stearic-PEG].sub.2-xHb-[mPEG].sub.12 in animals. Eight-week old Sprague-Dawley male rats weighing approximately 300 g were chosen and acclimated for a week. After injection of a [Stearic-PEG].sub.2-xHb-[mPEG].sub.12 solution, blood samples were collected at planned time intervals and analyzed for the Hb concentration in the plasma. The Hb present in the plasma portion of the collected blood is interpreted as the concentration of [Stearic-PEG].sub.2-xHb-[mPEG].sub.12 retained in the intravasculature.

[0200] As the test material, a solution containing 4.5% of [Stearic-PEG].sub.2-xHb-[mPEG].sub.12 having a molecular weight of 135,000 Da along with a pH of 7.2 was prepared.

[0201] Three animals received 15 mL/kg (equivalent to 675 mg/kg) of [Stearic-PEG].sub.2-xHb-[mPEG].sub.12 intravenously via a jugular vein at a controlled rate of 0.1 mL/min using a syringe pump.

[0202] Blood samples were collected via a caudal vein at time points of 0 (immediately before injection), 1, 2, and 4 hours after injection, and at 1, 2, 3, 4, 7, and 14 days after injection. Blood samples were collected into EDTA-treated micro-containers, which were immediately centrifuged at 3,500 rpm for 15 minutes at 4° C. to separate the plasma from the precipitate. The collected plasma samples were analyzed for total Hb concentration using the CO-OX SC80 Blood Gas Analyzer. The total Hb concentration in the plasma samples indicates the presence and quantity of [Stearic-PEG].sub.2-xHb-[mPEG].sub.12. The [Stearic-PEG].sub.2-xHb-[mPEG].sub.12 concentration data was plotted on a time scale, which yielded pharmacokinetic curves.

[0203] From the pharmacokinetic data, it was determined that the intravascular half-life of 4.5% solution of [Stearic-PEG].sub.2-xHb-[mPEG].sub.2 having a molecular weight of 135,000 Da, when tested in rats at the dosage of 15 mL/kg (equivalent to 675 mg/kg), was 22 hours.

TABLE-US-00001 U.S. Pat. No. Documents 5,234,903 August 1993 Nho et al. 5,386,014 January 1995 Nho et al. 5,478,806 December 1995 Nho et al. 6,844,317 January 2005 Winslow et al. 7,501,499 March 2009 Acharya et al. 7,625,862 December 2009 Winslow et al. 7,989,414 August 2011 Winslow et al. 8,021,858 September 2011 Vandegriff et al. 8,377,868 February 2013 Winslow et al. 8,609,815 December 2013 Vandegriff et al. 9,241,979 January 2016 Winslow et al. 10,029,001 July 2018 Vandegriff et al. 10,080,782 September 2018 Abuchowski et al. 10,172,950 January 2019 Abuchowski et al. 10,821,158 November 2020 Malavalli et al.

OTHER PUBLICATIONS

[0204] Walder, J. A. et al., Biochemistry, Vol 18, No 20, 4265-4270, 1979, “Diaspirins That Cross-Link β Chains of Hemoglobin: Bis(3,5-dibromosalicyl) Succinate and Bis(3,5-dibromosalicyl) Fumarate” [0205] Habeeb, A. F. Anal Biochem 14(3):328-336, 1966 “Determination of Free Amino Groups in the Proteins by Trinitrobezenesulfonic Acid