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WO1999018979A1 - Composition d'hemoglobine stable lors de son stockage - Google Patents

Composition d'hemoglobine stable lors de son stockage Download PDF

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Publication number
WO1999018979A1
WO1999018979A1 PCT/US1998/019768 US9819768W WO9918979A1 WO 1999018979 A1 WO1999018979 A1 WO 1999018979A1 US 9819768 W US9819768 W US 9819768W WO 9918979 A1 WO9918979 A1 WO 9918979A1
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WO
WIPO (PCT)
Prior art keywords
hemoglobin
composition
dithiol
disulfide
solution
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Application number
PCT/US1998/019768
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English (en)
Inventor
Ton That Hai
Deanna J. Nelson
David E. Pereira
Original Assignee
Baxter International Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Baxter International Inc. filed Critical Baxter International Inc.
Priority to AU94030/98A priority Critical patent/AU9403098A/en
Publication of WO1999018979A1 publication Critical patent/WO1999018979A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/41—Porphyrin- or corrin-ring-containing peptides
    • A61K38/42—Haemoglobins; Myoglobins

Definitions

  • the present invention relates to a storage-stable hemoglobin composition and a method for inhibiting degradation of a hemoglobin composition during storage. More specifically, the present invention relates to a hemoglobin composition that can be stored at room temperature while remaining physiologically active.
  • Hemoglobin solutions are known to lose their ability to function as blood substitutes during storage. To improve shelf life, the loss of function is generally delayed by refrigerating or freezing the solutions, or controlling the oxygenation state of the hemoglobin within the solution.
  • a hemoglobin solution loses its ability to function as a blood substitute because of spontaneous transformation of oxyhemoglobin in the solution to methemoglobin.
  • Methemoglobin is physiologically inactive — it does not bind oxygen and does not function as a blood substitute by releasing oxygen into a patient's bloodstream.
  • Methemoglobin is formed by an autoxidation reaction in which an electron is transferred from a heme-bound ferrous atom (Fe 2+ ) to bound oxygen to provide hemoglobin having a ferric atom (Fe 3+ ) and a superoxide radical anion (Winterbourn et al., "Reactions Involving Superoxide and Normal and Unstable Hemoglobins," Biochem. J., 155:493-502, 1976; Watkins et al., "Autoxidation Reactions of Hemoglobin A Free from Other Red Cell Components: A Minimal Mechanism," Biochem. Biophvs. Res. Commun., 132:742-748, 1985).
  • Room-temperature storage is most desirable so that hemoglobin solutions can be immediately administered to patients. Immediate use can be a life-saving measure in emergency medical situations such as trauma, stroke, shock and cardiac arrest. Stability during room-temperature storage also prevents mistakes in handling the solution such as inadvertent or prolonged unrefrigeration or inappropriate thawing which may cause the hemoglobin to become physiologically inactive.
  • U.S. Patent No. 5,352,773 describes a storage method that enables storage of a therapeutic hemoglobin solution at room temperature by filling an oxygen- impermeable plastic container with the solution in a low oxygen environment and storing the solution in the container.
  • oxyhemoglobin in the solution autoxidizes to methemoglobin
  • oxygen binds to deoxyhemoglobin to form oxyhemoglobin for autoxidation
  • the methemoglobin content of the solution increases.
  • the methemoglobin in the solution is nearly depleted
  • the methemoglobin spontaneously reduces to deoxyhemoglobin and the methemoglobin content of the solution decreases during storage to less than 50% and preferably less than 15% methemoglobin.
  • the solution is stored for a prolonged time at a temperature between -270°C and 45°C before being administered to a patient as a blood substitute.
  • methemoglobin under anaerobic conditions
  • methemoglobin is reduced very slowly by thiols such as cysteine under anaerobic conditions via a mechanism involving a thiyl radical.
  • a thiyl radical may react with a globin or a porphyrin ligand to generate a thiol-modified globin or a sulfhemoglobin, respectively.
  • the modified globin may degrade, denature, or increase the immunogenicity of the hemoglobin composition (Riechlin, Adv. Immunol.. 220:71-132, 1975).
  • a sulfhemoglobin can bind and release oxygen, it does so at rates that may be a hundred-fold or more slower than those of native hemoglobin.
  • the peroxide anion (HOO-) can react catalytically with iron to generate a hydroxyl radical.
  • the oxygen generated in scheme (2) can bind to hemoglobin and continue such undesirable radical chain reactions.
  • Hemoglobin having at least one thiol group can also self-polymerize, generating hemoglobin disulfides and polymers of hemoglobin covalently joined by disulfide bonds, as shown in the following reaction scheme:
  • the hemoglobin disulfides are susceptible to oxidation to methemoglobin and polymerization, and the polymers are susceptible to denaturation and precipitation.
  • therapeutic hemoglobin solutions may degrade, denature or form precipitates during storage because of hemoglobin self-polymerization and other undesirable hemoglobin reactions.
  • an anti-oxidant or reducing agent to a hemoglobin solution will improve storage stability.
  • not all reducing agents can reduce hemoglobin and some may even act as hemoglobin oxidants.
  • some anti-oxidants or reducing agents are unsuitable for therapeutic use, may be effective only within a pH range that is not tolerated well physiologically, or are toxic to mammals in the concentrations required to show effectiveness as an anti-oxidant.
  • WO 96/34889 reports that a hemoglobin composition containing a partially deoxygenated hemoglobin solution (i.e., a solution containing less than 5000 parts per million of oxygen) and less than 4 moles of a reducing agent, such as dithionite, sodium borohydride, ascorbate, ferrous salts, or ⁇ -tocopherol, per mole of hemoglobin is storage stable for at least three months when stored at temperatures less than about 40 °C.
  • a reducing agent such as dithionite, sodium borohydride, ascorbate, ferrous salts, or ⁇ -tocopherol
  • N-acetyl-L-cysteine a recognized anti-oxidant
  • N-acetyl-L-cysteine a recognized anti-oxidant
  • a hemoglobin composition that is stable after storage at temperatures up to about 40°C; the provision of a hemoglobin composition that remains physiologically active throughout storage and later therapeutic use; and the provision of a hemoglobin composition that can be stored at room temperature so that it is immediately available for use.
  • the present invention is directed to a hemoglobin composition including deoxyhemoglobin, oxyhemoglobin in an amount less than about 10% based upon the total hemoglobin content of the composition, and a dithiol or a disulfide in an amount effective to reduce the oxyhemoglobin to deoxyhemoglobin.
  • the present invention is also directed to a hemoglobin composition containing between about 80% and 100% deoxyhemoglobin, up to about 10% oxyhemoglobin, and at least about 0.1 mole of a dithiol or a disulfide per mole of hemoglobin in the composition.
  • Another embodiment of the invention is a process for preparing a hemoglobin composition by providing a hemoglobin composition containing deoxyhemoglobin and up to about 10% oxyhemoglobin based upon the total hemoglobin content of the composition, and admixing a dithiol or a disulfide with the hemoglobin composition.
  • Yet another embodiment of the invention is directed to a method for inhibiting degradation of a hemoglobin composition during storage by storing a hemoglobin composition containing deoxyhemoglobin and a dithiol or a disulfide in an oxygen- impermeable container at a temperature less than about 40 °C.
  • FIG. 1 is a plot of the methemoglobin content of partially oxygenated hemoglobin solutions as a function of time during storage at room temperature wherein - ⁇ - represents the control DCLHb ® solution, - ⁇ - represents a solution having an N-acetyl-L-cysteine (NAC) to DCLHb ® molar ratio of 1 :1 , and -O- represents a solution having an NAC/DCLHb ® molar ratio of 2:1 ;
  • NAC N-acetyl-L-cysteine
  • FIG. 2 is a plot of the methemoglobin content of partially oxygenated hemoglobin solutions as a function of time during storage at room temperature wherein - ⁇ - represents the control DCLHb ® solution, - ⁇ - represents a solution having a dihydrolipoic acid (DHLA) to DCLHb ® molar ratio of 1 :1 , and -O- represents a solution having a DHLA/DCLHb ® molar ratio of 2:1 ; and
  • DHLA dihydrolipoic acid
  • FIG. 3 is a plot of the methemoglobin content of partially oxygenated hemoglobin solutions as a function of time during storage at room temperature wherein - ⁇ - represents the control DCLHb ® solution, - ⁇ - represents a solution having a DHLA/DCLHb ® molar ratio of 3:1 , and -O- represents a solution having a DHLA DCLHb ® molar ratio of 4:1.
  • a dithiol or disulfide to a partially oxygenated hemoglobin composition reduces oxyhemoglobin to deoxyhemoglobin to deplete oxyhemoglobin in the composition.
  • the reduction reaction maintains the biological activity of the composition by eliminating the autoxidation of oxyhemoglobin to physiologically- inactive methemoglobin.
  • an excess of the dithiol or disulfide accelerates the rate of autoreduction of methemoglobin to deoxyhemoglobin, and can also minimize or prevent hemoglobin self-polymerization to significantly reduce formation of precipitates in the hemoglobin composition during storage.
  • a deoxygenated hemoglobin composition or a partially oxygenated hemoglobin composition remains therapeutically useful throughout storage at temperatures up to about 40 °C if a dithiol or disulfide is added to the composition and the composition is stored in an oxygen-impermeable container.
  • the hemoglobin composition of the invention contains deoxyhemoglobin and a dithiol, a disulfide, or a mixture of a dithiol and a disulfide. If the composition is partially oxygenated when it is manufactured (i.e., it includes oxyhemoglobin in an amount less than about 10% oxyhemoglobin based upon the total hemoglobin content of the composition, preferably less than about 5%, and more preferably, less than about 2%), the dithiol or disulfide or mixture thereof is present in an amount sufficient to reduce oxyhemoglobin in the composition to deoxyhemoglobin and thereby eliminate any autoxidation of oxyhemoglobin to methemoglobin.
  • the dissolved oxygen content of the hemoglobin composition is preferably less than about 10 ppm, more preferably less than about 1 ppm, and most preferably less than about 0.150 ppm.
  • the composition can contain an excess of a dithiol, a disulfide, or a mixture of a dithiol and a disulfide to accelerate the rate at which methemoglobin in the composition is reduced to deoxyhemoglobin and to decrease degradation of the composition during storage so that the hemoglobin composition remains useful for its intended purpose.
  • the dithiol or disulfide is present in an amount sufficient to decrease degradation during storage so that the composition maintains its ability to transport and deliver oxygen to cells.
  • degradation includes any measurable indication of hemoglobin deterioration such as oxidation to methemoglobin, precipitate formation, and less readily measured hemoglobin degradation such as undesirable hemoglobin polymerization, undesirable globin modification, and sulfhemoglobin formation. While not wishing to be bound by any particular theory, it is believed that oxyhemoglobin is reduced by a dithiol, for example, to provide deoxyhemoglobin, a cyclic disulfide and water as shown in the reaction scheme below:
  • Reduction of methemoglobin in the hemoglobin composition then proceeds once oxyhemoglobin is eliminated.
  • Residual dithiol or disulfide in the composition provides sacrificial thiol that is consumed by reaction before thiol within hemoglobin reduces and hemoglobin self-polymerized as shown in reaction scheme (3).
  • the residual dithiol or disulfide minimizes and may prevent undesirable polymerization and precipitation.
  • the hemoglobin composition contains between about 0.1 mole and about 10 moles of dithiol per mole of hemoglobin in the composition, preferably between about 0.2 moles and about 5 moles of dithiol per mole of hemoglobin, more preferably between about 0.2 moles and about 2 moles of dithiol per mole of hemoglobin, and most preferably, between about 0.2 moles and about 1 mole of dithiol per mole of hemoglobin.
  • the dithiol preferably has the formula:
  • n is an integer from 0 to 12
  • R 2 is hydrogen or alkyl
  • R 3 is hydrogen or alkyl
  • R 4 is hydrogen or alkyl.
  • a particularly preferred dithiol is dihydrolipoic acid, which has the formula HS-
  • the hemoglobin composition contains between about 0.1 mole to about 10 moles of disulfide per mole of hemoglobin in the composition, preferably between about 0.5 mole and about 6 moles of disulfide per mole of hemoglobin, more preferably between about 0.5 mole and about 2 moles of disulfide per mole of hemoglobin, and most preferably, between about 0.6 mole and about 1.5 moles of disulfide per mole of hemoglobin.
  • the disulfide preferably has the formula:
  • R 5 is -(CH 2 ) n -C(O)OR 2) -(CH 2 ) n -C(O)NR 3 R 4 , or -(CH 2 ) n -C(O)O ⁇ or a salt thereof, n is an integer from 0 to 12, R 2 is hydrogen or alkyl, R 3 is hydrogen or alkyl, and R 4 is hydrogen or alkyl.
  • a particularly preferred disulfide is lipoic acid, which has the formula
  • the hemoglobin composition is prepared by deoxygenating the composition until a desired oxyhemoglobin content is obtained, and adding a dithiol or disulfide to the composition.
  • the composition is deoxygenated by any known method such as gas-gas separation, gas-liquid separation or sorption processes, and is then maintained in an oxygen-free atmosphere throughout its preparation.
  • the oxyhemoglobin content of the hemoglobin composition can be determined in any known manner such as by spectroscopy or by use of a device for measuring dissolved oxygen content in solution (e.g., a MOCON analyzer as manufactured by Mocon, Minneapolis, MN).
  • a dithiol or disulfide is added to the hemoglobin composition in an amount at least sufficient to reduce the oxyhemoglobin present in the composition to deoxyhemoglobin.
  • An excess of the dithiol or disulfide can be added to increase the rate of methemoglobin reduction in the hemoglobin composition.
  • the composition After the addition of the dithiol or disulfide, the composition is maintained in an inert atmosphere (i.e., an atmosphere free of oxygen) by any known method, such as by packaging the composition in an oxygen-impermeable container as described in U.S. Patent No. 5,352,773.
  • an inert atmosphere i.e., an atmosphere free of oxygen
  • the hemoglobin compositions of the invention can be stored at temperatures less than about 40°C for periods of several months or longer.
  • the hemoglobin composition is a pharmacologically acceptable solution to which is slowly added an aqueous solution of a dithiol or disulfide which has been degassed using nitrogen or argon. The solution is stirred after addition to ensure homogeneity, and then apportioned into oxygen-impermeable containers for storage.
  • compositions may be formulated for subcutaneous, intravenous, intra- arterial, intra-peritoneal or intramuscular injection or infusion, arterial cannulization, or topical or oral administration in small or large volumes to mammals such as humans.
  • Therapeutic hemoglobin compositions are preferably administered systemically.
  • the compositions can be administered in a single dose, or in a series of multiple subdoses.
  • the single dose or each of the multiple subdoses can be administered by slow continuous infusion.
  • the hemoglobin compositions of the present invention can be prepared for many prophylactic or therapeutic uses in both clinical and veterinary medicine.
  • Such uses include, but are not limited to, use as a blood substitute for oxygen delivery or volume expansion; use in treating and/or preventing and/or reducing the severity of anemia, angina, ischemia, myocardial infarction, cerebral infarction, shock such as hemorrhagic, septic, anaphylactic, allergic, burn or cardiogenic shock, stroke, subarachnoid hemorrhage, cerebral vasospasms, thalassemia, cardiac arrest, cytopenia, cachexia, sepsis, hemorrhage, hypotension, edema, congestive heart failure, sickling crisis, reperfusion injury, stenosis, restenosis, respiratory disease, schistosomiasis, schizophrenia, contusions, Alzheimer's disease, amyotrophic lateral sclerosis, malaria, muscular dystrophies, muscle contraction, fatigue or spasm, depression, diabetes, gastric mucosa, head injury, hyperemia, hyperlipidemia, hypoxia, infection, thrombosis or
  • the methemoglobin content of the hemoglobin composition is preferably less than 50%, more preferably less than 30%, and most preferably less than 15% based on the total hemoglobin content of the composition.
  • some uses such as treatment of carbon monoxide poisoning, may necessitate greater methemoglobin content.
  • the hemoglobin composition is administered in dosages between about 10 milligrams and about 20 grams or more per kilogram of body weight over a period of seconds to hours.
  • the dosage, period of administration, and manner of dosing the patient depend upon the condition being treated, the form of the hemoglobin composition, the condition of the patient, and other factors from which one of ordinary skill in the medical arts can determine an appropriate dosage.
  • the term "hemoglobin” includes all proteins containing globin or globin-like polypeptides and heme.
  • the term “hemoglobin” includes all naturally- and non-naturally-occurring hemoglobin.
  • hemoglobin preparation includes hemoglobin, which is capable of transporting and releasing oxygen to cells, tissues or organs when introduced into the blood stream of a mammal, in a physiologically compatible carrier or as lyophilized and reconstituted with a physiologically compatible carrier, but does not include whole blood, red blood cells or packed red blood cells.
  • hemoglobin composition does not include whole blood, red blood cells, or packed red blood cells.
  • deoxyhemoglobin includes all deoxygenated hemoglobin
  • oxygenated hemoglobin includes all oxygenated hemoglobin.
  • metalhemoglobin includes all hemoglobin containing iron in the ferric (Fe 3+ ) state.
  • Naturally-occurring hemoglobin includes any hemoglobin identical to hemoglobin naturally existing within a cell. Naturally-occurring hemoglobin is predominantly wild-type hemoglobin, but also includes naturally-occurring mutant hemoglobin. Wild-type hemoglobin is hemoglobin most commonly found within natural cells. Wild-type human hemoglobin includes hemoglobin A, the normal adult human hemoglobin having two ⁇ - and two ⁇ -globin chains. Mutant hemoglobin has an amino-acid sequence that differs from the amino-acid sequence of wild-type hemoglobin as a result of a mutation, such as a substitution, addition or deletion of at least one amino acid.
  • adult human mutant hemoglobin has an amino-acid sequence that differs from the amino-acid sequence of hemoglobin A.
  • Naturally-occurring mutant hemoglobin has an amino-acid sequence that has not been modified by humans.
  • the naturally-occurring hemoglobin of the present invention is not limited by the methods by which it is produced. Such methods typically include, for example, erythrocytolysis and purification, recombinant production, and protein synthesis.
  • Non-naturally-occurring hemoglobin includes mutant hemoglobin having an amino-acid sequence different from the amino-acid sequence of hemoglobin naturally existing within a cell, and chemically-modified hemoglobin.
  • Such non- naturally-occurring mutant hemoglobin is not limited by its method of preparation, but is typically produced using one or more of several techniques known in the art, including, for example, recombinant DNA technology, transgenic DNA technology, protein synthesis, and other mutation-inducing methods.
  • Chemically-modified hemoglobin is a natural or non-natural hemoglobin molecule which is bonded to or encapsulated by another chemical moiety.
  • a hemoglobin molecule can be bonded to pyridoxal-5'-phosphate, or other oxygen-affinity-modifying moiety to change the oxygen-binding characteristics of the hemoglobin molecule, to crosslinking agents to form crosslinked or polymerized hemoglobin, or to conjugating agents to form conjugated hemoglobin.
  • Conjugated, polymerized and crosslinked hemoglobins generally exhibit longer intravascular retention times than unmodified hemoglobin.
  • hemoglobin modification technology which can be used in the practice of the present invention have been described in the scientific literature (reviewed by R. M. Winslow (1992) in Hemoglobin-Based Red Cell Substitutes, The Johns Hopkins University Press, Baltimore, MD). Some representative methods of preparing chemically-modified hemoglobin for use in the invention are described below.
  • Hemoglobin can be modified to improve its oxygen-binding affinity.
  • Reagents that bind to the 2,3-diphosphoglycerate binding site of a hemoglobin molecule, reduce the oxygen affinity of the hemoglobin molecule, and prolong intravascular retention are described in U.S. Patent Nos. 4,529,719 and 5,380,824 (pyridoxal-5'- phosphate), U.S. Patent No. 4,600,531 (carboxyl-, phosphonate-, phosphate-, sulfonate- or sulfate-phenyl ester-containing compounds such as mono(3,5- dibromosalicyl)fumarate), U.S. Patent No.
  • the hemoglobin has a P 50 of between about 20 and about 45 mm Hg.
  • An encapsulated hemoglobin is hemoglobin surrounded by a material which retains the hemoglobin within the material yet allows smaller molecules to pass through the material to react with hemoglobin and reaction products to pass out of the material.
  • Materials for encapsulating hemoglobin are described in U.S. Patent No. 4,343,715 (polyurethane, acrylic gels, maleic anhydride polymers, epoxy polymers, glutaronic aldehyde polymers), U.S. Patent Nos. 5,061 ,688, 5,217,648 and 5,438,041 (oil emulsion), U.S. Patent No. 4,911 ,929 (liposomes), and U.S. Patent Nos.
  • a conjugated hemoglobin is at least one non-hemoglobin molecule covalently or ionically bound to a hemoglobin.
  • the non-hemoglobin molecule can also form an intermolecular crosslink between hemoglobin molecules.
  • Conjugating materials and methods for preparing hemoglobin conjugates are described in WO 91/07190 (polyalkylene glycol), U.S. Patent Nos. 4,670,417, 5,091 ,176, 5,219,564, 5,234,903, 5,312,808 and 5,386,014, WO 94/04193, WO 94/09027 and Japanese Patent Nos.
  • Crosslinked hemoglobin is intramolecularly linked between globin or globin- like protein subunits by a crosslinking agent.
  • a subunit is one globin or globin-like protein of a hemoglobin molecule. Intramolecular crosslinking prevents dissociation of globin or globin-like proteins when hemoglobin is administered in vivo.
  • Hemoglobin A can dissociate into two ⁇ - ⁇ globin dimers if the dimers are not crosslinked.
  • Crosslinked hemoglobins and methods for their preparation are described in U.S. Patent Nos. 4,529,719 and 4,600,531 ( ⁇ - ⁇ linkage using diphenyl ester derivatives such as bis(3,5-dibromosalicyl)fumarate), U.S. Patent Nos. 4,001 ,401 and 4,053,590 ( ⁇ - ⁇ globin linkage using halogenated cycloalkanes, diepoxides, and diazobenzidines), U.S. Patent No. 4,857,636 (aldehyde derived from oligosaccharide), U.S.
  • Patent No. 5,334,705 (benzenetricarboxylate), WO 94/21682 ( ⁇ - ⁇ globin linkage using di- or trisaccharide), U.S. Patent No. 5,290,919 and 5,387,672 (di- or trivalent compounds), U.S. Patent No. 5,334,707 ( ⁇ - ⁇ or ⁇ - ⁇ linkage using acyl phosphate ester), U.S. Patent No. 5,362,885 and WO 92/09630 (imidoesters, such as dimethyl adipimidate or dimethyl suberimidate), U.S. Patent No. 5,514,780 (polycarboxylic acid), U.S. Patent No. 5,399,671 and WO 90/13309 ( ⁇ - ⁇ linkage), and U.S. Patent No. 4,473,496 (dialdehyde).
  • a polymerized hemoglobin is intermolecularly linked between hemoglobin molecules. Polymerization generally increases the molecular weight of the hemoglobin, which improves its intravascular half-life. Polymerization agents for preparing polymerized hemoglobin are described in pending U.S. applications Serial Nos. 08/149,679, 08/173,882, 08/480,593, and 08/473,459, U.S. Patent No. 4,777,244 (aliphatic dialdehyde), U.S. Patent No. 5,349,054 (benzenepentacarboxylate), WO 94/14460 (transglutaminase), and EP 201618 (glutaraldehyde).
  • Hemoglobins can also be modified by a combination of the methods described above, for example, as described in Japanese Patent Nos. 59-089629, 59- 103322, and 59-104323 (pyridoxal-5'-phosphate modification and polyethylene glycol conjugation of hemoglobin), U.S. Patent No. 5,248,766 (crosslinking and polymerization of tetrameric hemoglobins with oxiranes), U.S. Patent Nos.
  • Recombinantiy-produced hemoglobin is produced by recombinant DNA methodologies, for example, by site-directed mutagenesis, gene fusion, or transfecting a genetically engineered plasmid into a microorganism such as a bacterium or yeast, a cultured cell such as an insect cell, a mammalian cell, or plant cell, a transgenic plant, a transgenic animal, or any other host cell or organism, where the plasmid includes a nucleic acid polymer (e.g., cDNA) which encodes a globin protein, a fusion protein, or a protein similar to globin that can reversibly bind oxygen.
  • a nucleic acid polymer e.g., cDNA
  • Pyrogen-free hemoglobin is hemoglobin that is absolutely free of fever-producing contaminants, or hemoglobin that contains amounts of fever-producing contaminants that are physiologically acceptable to patients to which the hemoglobin will be administered.
  • Bacterial endotoxins can contaminate hemoglobin derived from erythrocytes.
  • Recombinant hemoglobin produced in non-erythrocyte host cells such as bacteria can also become contaminated with cellular components such as proteins, toxins, or polysaccharides that can elicit toxic or pyrogenic responses when administered to mammals (Rietschel et al. (1992) Scientific American 267:54-61 ; Suff redini et al. (1989) New Eng. J. Med. 321 :280-287).
  • Hemoglobins for use in the present invention are also stroma-free.
  • Stroma the insoluble cell membrane fragments that contaminate hemoglobin derived from lysed erythrocytes, is toxic and has been reported to cause dyspnea, bronchospasm, hypotension, arrhythmia, disseminated intravascular coagulation, activation of complement, and renal, myocardial, and hepatic changes associated with ischemia and acute inflammation (Feola (1988) Surgery, Gynecology & Obstetrics 166:211- 222; MacDonaid et al. (1988) F.A.S.E.B. J. 2(6) Abstr. 8217; Stone et al.
  • stroma-free hemoglobin is hemoglobin, as defined herein, which is either absolutely free of stroma, or which contains stroma at concentrations that are physiologically acceptable (i.e., do not cause adverse side effects) in a patient.
  • Stroma-free hemoglobin that is absolutely free of stroma includes recombinant hemoglobin produced in a non-erythrocyte.
  • Stroma-free hemoglobin that contains stroma at physiologically acceptable levels includes, for example, purified hemoglobin derived from erythrocytes.
  • the hemoglobin can be dialyzed or exchanged by ultrafiltration into a physiologically acceptable solution preferably to between about 1 and about 20 g/dl hemoglobin.
  • the solution generally comprises a physiologically compatible electrolyte vehicle isosmotic with whole blood and which maintains the reversible oxygen-carrying and delivery properties of the hemoglobin.
  • the physiologically acceptable solution can be, for example, 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, and polymeric plasma substitutes, such as polyethylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol and ethylene oxide-propylene glycol condensates.
  • Such solutions can be administered parenterally, for example by intravenous or intra-arterial injection or infusion (i.e., systemic administration), without adverse side effects.
  • the hemoglobin can also be lyophilized for storage and reconstituted prior to use. Methods for preparing such solutions or lyophilized powders are known in the art.
  • the composition can include other components such as salts (e.g., sodium chloride, potassium chloride), buffers (e.g., lactate, gluconate, phosphate), surfactants or chelating agents (e.g., ethylenediamine pentaacetic acid) and the like.
  • the pH of the hemoglobin composition is preferably from about 6 to about 8 at 37°C.
  • a preferred hemoglobin for use in the present invention is hemoglobin crosslinked with bis(3,5-dibromosalicyl) fumarate to create a fumarate crosslink between the two ⁇ subunits (DCLHb ® , manufactured by Baxter Healthcare, Deerfield, Illinois).
  • This crosslinked hemoglobin is more fully described, together with methods for its preparation, in U.S. Patents Nos. 4,598,064, 4,600,531 , and RE 34,271 , omitting the chromatography step.
  • This hemoglobin is preferably manufactured under the conditions disclosed in U.S. Patent Nos. 4,831 ,012, 4,861 ,867, 5,128,452 and 5,281 ,579 and U.S. patent application serial no. 07/207,346.
  • a preferred DCLHb ® solution contains 10 g/dl of modified tetrameric hemoglobin in a balanced electrolyte solution.
  • the product is prepared from units of human red cells from volunteer donors which have been tested and found negative for HbsAg, HIV-1 and 2, and HCV. During manufacture, the red cells are osmotically lysed to release hemoglobin. After ultrafiltration, the stroma-free hemoglobin is reacted with the diaspirin crosslinking agent to produce a stabilized tetrameric hemoglobin having a fumaryl moiety linking the two ⁇ subunits.
  • Control solution DCLHb ® (1.55 mM) in lactated electrolyte solution, pH 7.5.
  • DHLA Dihydrolipoic Acid
  • Each deoxyDCLHb solution was prepared as follows.
  • the DCLHb ® solution (1.55 mM in lactated electrolyte solution) was deoxygenated at 25°C by passing the solution through a membrane oxygenator charged with oxygen-free nitrogen gas. Deoxygenation was stopped when the level of oxyhemoglobin was about 10%.
  • the appropriate additive N-acetylcysteine, gluconate or dihydrolipoic acid
  • All manipulations were performed under nitrogen.
  • the solutions were stored at room temperature; in addition, the DHLA solution was kept in the dark.
  • each of the deoxyDCLHb solutions was passed though a 0.2 ⁇ pore-size filter, and the filtrate was analyzed by on-line, multi-wavelength, UV-visible spectroscopic analysis (Van Assendeift et al., Analvt. Biochem.. 69:43-48, 1975), followed by application of the algebraic formulae for the determination of methemoglobin, oxyhemoglobin, and deoxyhemoglobin percentages and total hemoglobin content.
  • the methemoglobin content of each of the solutions was about 28%.
  • the methemoglobin content of the control solution increased to about 55% methemoglobin and then decreased gradually to about 36% methemoglobin by day 28, as shown in FIGs. 1 and 2.
  • the test solution containing 3 mM gluconate showed an increase to about 40% methemoglobin and then a gradual decrease to about 36% methemoglobin.
  • gluconate appeared to prevent hemoglobin oxidation but had no other observable effects.
  • the changes in solution turbidity were used as a surrogate monitor for precipitate formation.
  • the turbidity of the solutions was determined using a Hach 2100AN turbidimeter.
  • the turbidity results were reported in Normalized Turbidity Units (NTUs) corresponding to the difference between filtered (0.2 ⁇ pore-size filter) and unfiltered samples.
  • NTUs Normalized Turbidity Units
  • the properties of the deoxyDCLHb in solution were assessed by thiol determination, size-exclusion chromatography (SEC), reversed-phase high performance liquid chromatography (RP-HPLC), and SDS-PAGE electrophoresis.
  • SEC was performed using SuperdexTM 200 column (Pharmacia), a mobile phase consisting of 50 mM phosphate, pH 7.0, containing 0.15 M NaCI, delivered at a flow rate of 0.7 mlJmin., and analyte detection at 215 nm.
  • materials elute from the stationary phase in the order from largest to smallest in size, i.e., larger entities elute with shorter retention times and smaller entities elute with longer retention times.
  • RP-HPLC was performed using a Vydac Protein C-18 column, with elution using mobile phases (A) and (B) delivered at 1 mL/min. as a linear gradient having the following compositions over time: 1 ) 0% B to 40% B over 60 minutes; 2) 40% B to 78% B over 20 minutes; 3) hold at 78% B for 2 minutes; 4) 78% B to 100% B over 10 minutes.
  • Mobile phase (A) consisted of CH 3 CN/H 2 O/TFA, 60:40:0.1 , by volume.
  • Mobile phase (B) consisted of CH 3 CN/H 2 O/TFA, 60:40:0.1 , by volume. Analytes were monitored at 215 nm. SDS-PAGE electrophoresis was completed under reducing and non-reducing conditions. Thiol determination was completed by the Neis method (Neis et al., Toxicology. 31 :319-329, 1984).
  • DHLA eliminated the hemoglobin oxidation phase, significantly accelerated methemoglobin reduction, and decreased precipitate formation.
  • Control solution DCLHb ® (1.55 mM) in lactated electrolyte solution, pH 7.5.
  • the DCLHb ® control solution (1.55 mM in lactated electrolyte solution) was deoxygenated at 25°C by passing the solution through a membrane oxygenator charged with oxygen-free nitrogen gas. Deoxygenation was stopped when the level of oxyhemoglobin was about 10%.
  • DHLA was added to provide solutions 2 and 3. All manipulations were performed under nitrogen. The solutions were stored in darkness at room temperature. At intervals, each deoxyDCLHb solution was passed though a 0.2 ⁇ pore-size filter, and the filtrate was analyzed to determine methemoglobin content as described in Example 1 . The change in methemoglobin content during storage at 25°C is shown in FIG. 3. The small fluctuations in methemoglobin content observed between day 0 and 1 were within the experimental error of the method. DHLA eliminated the hemoglobin oxidation phase and significantly accelerated methemoglobin reduction.
  • a DCLHb ® solution (1 mM in a 0.1 M HEPES solution at pH 8.0) was deoxygenated at 25°C by passing the solution through a membrane oxygenator charged with oxygen-free nitrogen gas. Deoxygenation was stopped when the level of oxyhemoglobin was about 10%.
  • DHLA was added to provide solutions having a molar ratio of DHLA:DCLHb ® of 0.5:1 , 1 :1 , 1.5:1 and 2:1. All manipulations were performed under nitrogen. The solutions were stored in darkness at room temperature. At intervals, each deoxyDCLHb solution was passed though a 0.2 ⁇ pore-size filter, and the filtrate was analyzed to determine methemoglobin content as described in Example 1. The change in methemoglobin content during storage under anaerobic conditions at 5°C is shown in Table 3 below.
  • DHLA eliminated the hemoglobin oxidation phase and accelerated the methemoglobin reduction rate more rapidly at greater molar ratios of DHLA:DCLHb ® .
  • Lipoic acid (LA) was added to the DCLHb ® solution of Example 3 to provide solutions having a molar ratio of LA:DCLHb ® of 0.5:1 , 1 :1 , 1.5:1 and 2:1.
  • the solutions were stored in darkness at room temperature.
  • each deoxyDCLHb solution was passed though a 0.2 ⁇ pore-size filter, and the filtrate was analyzed to determine methemoglobin content as described in Example 1.
  • the change in methemoglobin content during storage under anaerobic conditions at 5°C is shown in Table 4 below.
  • Lipoic acid eliminated the hemoglobin oxidation phase. Methemoglobin reduction proceeded at a slower rate as compared to solution containing DHLA.
  • Baseline measurements were obtained in each animal prior to treatment, and additional sampling was performed immediately post-treatment and at 0.5, 1 , 8, 24 and 48 hours post-infusion. About 48 hours after dosing, the animals were sacrificed and necropsied. The heart, brain, lung, liver, kidneys, spleen, pancreas, and gastrointestinal tract were collected and fixed for histopathoiogical examination by a veterinary pathologist. The physiological parameters monitored included heart rate; mean, systolic and diastolic blood pressures; hematocrit; and plasma hemoglobin and methemoglobin content.
  • the effects of the LA-DCLHb were similar to those of the reference control as observed in this study and as reported in previous studies. Increased MAP between 40-50 mm Hg above baseline, increased activities of AST, LDH and CK, minor gastrointestinal distress (emesis and/or diarrhea), and minimal to moderate microscopic lesions of recent myocardial necrosis were observed.
  • the presence of minor unilateral or bilateral, focal cortical and/or proximal tubular necrosis in three of the four LA-DCLHb-treated animals was attributed to the lipoic acid, since these changes were not observed in this study or previous studies in pigs receiving DCLHb ® alone. No effects were observed for the other physiological or hematological parameters or blood chemistries monitored.
  • lipoic acid did not significantly or adversely affect the biocompatibility of DCLHb ® .
  • EXAMPLE 6 Six domestic crossbred barrows of 10-14 kg body weight were catheterized surgically and then allowed to stabilize for 24 hours. 20 mL/kg body weight of a 10 g/dl DCLHb ® in lactated electrolyte solution containing 1.28 g of lipoic acid per liter of DCLHb ® (LA-DCLHb) were administered to four barrows as a topload infusion at a rate of 1 mL/kg/min. The units of LA-DCLHb were covered with foil during infusion because lipoic acid is photosensitive. 20 mL kg body weight of a 10 g/dl deoxyDCLHb solution were administered to one barrow as a reference control at a rate of 1 mL kg/min. The remaining barrow was an untreated control.
  • LA-DCLHb lactated electrolyte solution containing 1.28 g of lipoic acid per liter of DCLHb ®
  • Baseline measurements were obtained in each animal prior to treatment, and additional sampling was performed immediately post-treatment and at 0.5, 1 , 8, 24 and 48 hours post-infusion. About 48 hours after dosing, the animals were sacrificed and necropsied. The heart, brain, lung, liver, kidneys, spleen, pancreas, and gastrointestinal tract were collected and fixed for histopathoiogical examination by a veterinary pathologist.
  • the physiological parameters monitored included heart rate; mean, systolic and diastolic blood pressures; hematocrit; and plasma hemoglobin and methemoglobin content.
  • the blood chemistries monitored included BUN, lactate, creatinine, electrolytes, total protein, albumin, non-heme globulin, osmolality, and the enzymes amylase, lipase, alanine, ALT, AST, LDH, and CK(total and isoenzymes).
  • Hematologicai parameters monitored included total and differential white blood cell counts, PT, APTT, fibrinogen, and fibrin degradation products.
  • the effects of the LA-DCLHb were similar to those of the reference control as observed in this study and as reported in previous studies. Increased activities of AST, LDH and CK, minor gastrointestinal distress (emesis and/or diarrhea), and minimal to moderate microscopic lesions of recent myocardial necrosis were observed. The presence of minor unilateral or bilateral, focal cortical and/or proximal tubular necrosis in all of the LA-DCLHb-treated animals was attributed to the lipoic acid, since these changes were not observed in this study or previous studies in pigs receiving DCLHb ® alone. No effects were observed for the other physiological or hematological parameters or blood chemistries monitored.
  • lipoic acid did not significantly or adversely affect the biocompatibility of deoxyDCLHb.

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Abstract

Cette composition d'hémoglobine comprend de la désoxyhémoglobine, de l'oxyhémoglobine dont la teneur est inférieure à environ 10 % de la teneur totale en hémoglobine, ainsi qu'un dithiol ou un disulfure, en quantité suffisante pour réduire l'oxyhémoglobine en désoxyhémoglobine.
PCT/US1998/019768 1997-10-10 1998-09-22 Composition d'hemoglobine stable lors de son stockage WO1999018979A1 (fr)

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EP1191037A4 (fr) * 1999-07-01 2003-05-02 Int Reagents Corp Moyens pour stabiliser les hemoglobines
WO2010144629A1 (fr) * 2009-06-09 2010-12-16 Prolong Pharmaceuticals, LLC Compositions d'hémoglobine
US9657271B2 (en) 2006-01-24 2017-05-23 The Board Of Trustees Of The University Of Illinois Method of isolating cells from a tissue in a mammal
US10172950B2 (en) 2009-06-09 2019-01-08 Prolong Pharmaceuticals, LLC Hemoglobin compositions
US10172949B2 (en) 2009-06-09 2019-01-08 Prolong Pharmaceuticals, LLC Hemoglobin compositions
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JP5410647B2 (ja) * 1999-07-01 2014-02-05 シスメックス株式会社 グリコヘモグロビン分析装置用の標準物質及びコントロール物質
EP1191037A4 (fr) * 1999-07-01 2003-05-02 Int Reagents Corp Moyens pour stabiliser les hemoglobines
KR100674690B1 (ko) * 1999-07-01 2007-01-25 시스멕스 가부시키가이샤 헤모글로빈 안정화수단
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US7435795B2 (en) 2001-04-18 2008-10-14 Northfield Laboratories, Inc. Stabilized hemoglobin solutions
WO2002085111A1 (fr) * 2001-04-18 2002-10-31 Northfield Laboratories Systeme de contenant souple servant a entreposer des solutions d'hemoglobine stabilisees
US9657271B2 (en) 2006-01-24 2017-05-23 The Board Of Trustees Of The University Of Illinois Method of isolating cells from a tissue in a mammal
CN102458451A (zh) * 2009-06-09 2012-05-16 普罗朗制药有限责任公司 血红蛋白组合物
WO2010144629A1 (fr) * 2009-06-09 2010-12-16 Prolong Pharmaceuticals, LLC Compositions d'hémoglobine
US10080782B2 (en) 2009-06-09 2018-09-25 Prolong Pharmaceuticals, LLC Hemoglobin compositions
US10172950B2 (en) 2009-06-09 2019-01-08 Prolong Pharmaceuticals, LLC Hemoglobin compositions
US10172949B2 (en) 2009-06-09 2019-01-08 Prolong Pharmaceuticals, LLC Hemoglobin compositions
US10772937B2 (en) 2009-06-09 2020-09-15 Prolong Pharmaceuticals, LLC Hemoglobin compositions
US10780148B2 (en) 2009-06-09 2020-09-22 Prolong Pharmaceuticals, LLC Hemoglobin compositions
US10786613B2 (en) 2016-04-26 2020-09-29 Gambro Lundia Ab Apparatus and method for determining a parameter indicative of the progress of an extracorporeal blood treatment
US11311655B2 (en) 2016-04-26 2022-04-26 Gambro Lundia Ab Apparatus and method for determining a parameter indicative of the progress of an extracorporeal blood treatment

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