WO1992008478A1 - Procede d'amelioration de la stabilite du stockage a long terme de produits d'hemoglobine - Google Patents

Procede d'amelioration de la stabilite du stockage a long terme de produits d'hemoglobine Download PDF

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Publication number
WO1992008478A1
WO1992008478A1 PCT/US1991/008622 US9108622W WO9208478A1 WO 1992008478 A1 WO1992008478 A1 WO 1992008478A1 US 9108622 W US9108622 W US 9108622W WO 9208478 A1 WO9208478 A1 WO 9208478A1
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WO
WIPO (PCT)
Prior art keywords
hemoglobin
pharmaceutical composition
permeable membrane
polyalkylene oxide
inert gas
Prior art date
Application number
PCT/US1991/008622
Other languages
English (en)
Inventor
Kwang Nho
Original Assignee
Enzon, 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.)
Filing date
Publication date
Application filed by Enzon, Inc. filed Critical Enzon, Inc.
Publication of WO1992008478A1 publication Critical patent/WO1992008478A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol

Definitions

  • the present invention relates to a method for ⁇ * s enhancing the stability of hemoglobin products comprising
  • ⁇ stroma-free hemoglobin was observed to be rapidly eliminated by the kidneys, with a transfusion half-life of only about 100 minutes.
  • stroma-free hemoglobin A number of chemical modifications have been introduced into stroma-free hemoglobin in attempts to increase the P 5 _ and to render the hemoglobin more stable. Perhaps the most widely used chemical modification of stroma-free hemoglobin utilizes pyridoxal 5'-phosphate and sodium or potassium borohydride to increase the P 5Q (Benesch et al., 1972, Biochem. 11:3576) .
  • hemoglobin has been linked to other macromolecules, such as dextran (Chang, J. E. et al., 1977, Can. J. Biochem. 55:398), hydroxyethyl starch (DE OS No. 2,616,085), gelatin (DE AS 2,449,885), albumin. (DE AS 2,449,885), and polyethyleneglycol (PEG) (DE 3026398; U. S. Patent No. 4,670,417; U. S. Patent No. 4,412,989; U. S. Patent No.
  • stroma-free hemoglobin included a method for decreasing the rate of methemoglobin formation using NADH and NADPH (Sehgal et al., 1981, J. Surg. Res. 3_1:13-17).
  • Keipert and Chang (1985, Biomater. Med. Devices Artif. Organs 13_:156) tested the efficacy of pyridoxal phosphate treated polyhemoglobin in resuscitating rats acutely bled to 67 percent of total blood volume, and found it comparable to whole blood in providing for long- term survival.
  • glycerol interacts favorably with water, by entering into the water lattice and strengthening solvent structure, its presence in the aqueous medium could increase the hydrophobicity of the protein.
  • the nonpolar groups on the protein surface may react unfavorably to contact with the mixed solvent. For example, surface hydrophobic groups may prefer to migrate into the interior of the protein in order to avoid contact with glycerol/water solvent, thereby distorting protein structure.
  • sucrose The alteration in protein-solvent interactions in the presence of sucrose also has been studied (Lee and Timasheff, 1981, J. Biol. 256:7193-78201) .
  • Sucrose was not observed to induce a conformational change in the proteins studied.
  • the enthalpy of thermal unfolding showed little dependence on the concentration of sucrose, while the apparent activation energy of the unfolding process was increased by the addition of sucrose.
  • sucrose was preferentially excluded from the protein domain, thereby increasing the free energy of the system. Thermodynamically, this may lead to protein stabilization since the unfolded state of the protein may become thermodynamically less favorable in the presence of sucrose.
  • sucrose from the protein domain appeared to be related to higher cohesive force of the sucrose-water solvent system.
  • a major factor in the stabilization is thought to have been the free energy required to form a cavity in the solvent needed for accommodating the protein molecule, the stabilization, perhaps, being conferred on the protein by the increase in the solvent cohesive force when sucrose was added.
  • Glucose and maltose have also been added to aqueous hemoglobin solutions in attempts to minimize the rate of methemoglobin (MetHb) formation. According to a study (Iwasaki et al. U.S. Patent No.
  • Lyophilization of hemoglobin has also been utilized in efforts toward long-term preservation.
  • the process of freeze-drying is harsh enough to generate unacceptable levels of oxidized hemoglobin.
  • Additives, including cryoprotectors such as sugars that appear to protect the integrity of proteins during lyophilization have been tested on hemoglobin solutions with some degree of success.
  • cryoprotectors have appeared to significantly reduce the amount of MetHb generated during the lyophilization process, the MetHb in the final product has remained undesirably high.
  • the present invention relates to a method for enhancing the stability of hemoglobin products comprising deoxygenating hemoglobin by gas exchange through a permeable membrane. It is based, in part, on the observation that hemoglobin was rendered significantly more stable upon deoxygenation; methemoglobin formation from hemoglobin processed by the methods of the invention was surprisingly low.
  • the stable hemoglobin-based products are deoxygenated prior to storage, so as to prolong shelf-life.
  • hemoglobin may be deoxygenated prior to chemical treatment, including but not limited to conjugation to polyalkylene oxide.
  • the present invention offers the advantages of decreasing the rate of conversion of hemoglobin to methemoglobin in solution, and diminishes the need for chemical reducing agents or stabilizing agents, such as sugars. Because the presence of chemical reducing agents or sugars may be clinically problematic, the present invention provides for hemoglobin pharmaceutical preparations of superior purity.
  • FIGURES Figure 1 Diagram of a hemoglobin solution-containing reactor connected to a gas exchange device for deoxygenation of hemoglobin.
  • hemoglobin products may be construed to refer to hemoglobin which is in solution and which may or may not be chemically modified, as well as to hemoglobin within living cells.
  • Chemically modified hemoglobin may include but not be limited to hemoglobin which has been cross-linked, which has been treated with pyridoxal phosphate, or which has been conjugated to polyalkylene oxide.
  • Hemoglobin products may be derived from a human or non-human source or by genetic engineering methods.
  • the hemoglobin product is deoxygenated by exposing the hemoglobin to an inert gas via a gas permeable membrane, such that the inert gas must pass through the membrane in order to come in contact with the hemoglobin.
  • any membrane which is gas permeable but hemoglobin impermeable, and which does not chemically react with hemoglobin may be utilized.
  • Membranes used according to the invention may preferably be easily sterilized.
  • gas permeable membranes which may be used according to the invention include, but are not limited to, polypropylene and cellulose acetate membranes.
  • membranes may be utilized such that a large surface area is available for gas exchange. Accordingly, it may be desirable to provide a large surface area of membrane for a relatively small volume of blood.
  • gas permeable membranes may be assembled into long cylindrical shapes, and groups of these cylinders may be assembled together; a hemoglobin solution may be passed through these cylinders while an inert gas is circulated outside the cylinders, or, alternatively, an inert gas may be passed through the cylinders while hemoglobin solution is circulated outside the cylinders [FIGURE l] .
  • whole blood, red blood cells, or hemoglobin products, including chemically modified hemoglobin may also be deoxygenated in this manner.
  • the membrane configuration should permit the passage of whole cells without lysis or accumulation of cells.
  • gas-permeable membranes designed and manufactured so as to generate a large surface area of gas/liquid contact, such as those produced by Hoechst-Celanese, Celgard G-240/11, polypropylene fiber diameter 240 micron, surface area 11 square feet or CD Medical, Inc., Cell-Pharm Hollow Fiber Oxygenators, or any structurally and functionally equivalent apparatus, may be used.
  • Hemoglobin products may be circulated over the gas permeable membrane using any method known in the art so as to create a flow rate which permits effective gas exchange.
  • Appropriate circulation methods would include but not be limited to those associated with gravity, a peristaltic pump, capillary action, pressure differentials or centrifugal force.
  • the Celgard G-240/11 gas exchange device may be used, and hemoglobin may be circulated through the device at a flow rate of about 500 ml/min or at such a flow rate that deoxygenation may be achieved after 10-15 minutes under constant inert gas flow at about 5-10 p.s.i.
  • the exterior space of the fibers may be desirably under constant inert gas (e.g. nitrogen) flow at about 5-10 psi, in which case complete deoxygenation may be achieved after about 10-15 minutes.
  • the gas used to deoxygenate hemoglobin may be any gas 0 which does not react with the hemoglobin in solution, including, but not limited to, nitrogen, helium, argon, and carbon dioxide gas.
  • a Radiometer OSM3 Hemoximeter In preferred embodiments of the invention, at least about 90 percent of the hemoglobin in solution may be deoxygenated.
  • the present invention provides for pharmaceutical compositions comprising hemoglobin deoxygenated according to the invention.
  • the hemoglobin is in aqueous solution at concentrations ranging from about 5-15%.
  • the hemoglobin compositions of the invention differ from those produced by other known methods in that the rate of methemoglobin formation is extremely low in the absence of stabilizing compounds, such as sugars, as well as in the presence of extremely low concentrations of sugar, such as dextrose at a concentration of less than five percent g/dl, or at a concentration of one percent (see Example Sections 6 and 7, infra) .
  • the reactor was connected to a gas-exchange device, Celgard
  • hemoglobin deoxygenated according to the methods of the invention was significantly more stable, and formed only 5.9% methemoglobin after ten months of cold storage. While the addition of dextrose improved stability somewhat, its effect was relatively minor, decreasing methemoglobin formation by only about 1.0 percent.
  • SC-PEG was prepared according to the method described in U. S. patent application serial number
  • TNBS trinitrobenzene sulfonic acid
  • Non-deoxygenated PEG-Hb was included in storage studies as a control. As shown in Table II, PEG-Hb deoxygenated by the methods of the invention was significantly more stable with respect to methemoglobin formation than non-deoxygenated PEG-Hb. Furthermore, the addition of dextrose resulted in a relatively minor improvement in stability.

Abstract

Procédé d'amélioration de la stabilité de produits d'hémoglobine consistant à désoxygéner l'hémoglobine par échange gazeux à travers une membrane perméable. Le procédé est basé, en partie, sur l'observation que l'hémoglobine est rendue sensiblement plus stable par désoxygénation; la formation de méthémoglobine à partir d'hémoglobine traitée selon les procédés de l'invention s'est avérée étonnamment faible. Dans certains modes de réalisation de l'invention, les produits à base d'hémoglobine stable sont désoxygénés avant le stockage de manière à prolonger la durée de conservation. Dans d'autres modes de réalisation de l'invention, l'hémoglobine peut être désoxygénée avant le traitement chimique, y compris la conjugaison avec l'oxyde de polyalkylène, entre autres. L'invention offre l'avantage de réduire la vitesse de conversion d'hémoglobine en méthémoglobine en solution, et elle réduit les besoins en agents réducteurs chimiques ou en agents stabilisateurs tels que des sucres. Du fait que la présence d'agents réducteurs chimiques ou de sucres peut être cliniquement problématique, l'invention permet d'obtenir des préparations pharmaceutiques d'hémoglobine de pureté supérieure.
PCT/US1991/008622 1990-11-20 1991-11-19 Procede d'amelioration de la stabilite du stockage a long terme de produits d'hemoglobine WO1992008478A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US61612890A 1990-11-20 1990-11-20
US616,128 1990-11-20

Publications (1)

Publication Number Publication Date
WO1992008478A1 true WO1992008478A1 (fr) 1992-05-29

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WO (1) WO1992008478A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0665850A1 (fr) * 1992-10-13 1995-08-09 Enzon, Inc. Fractionnement de solutions d'hemoglobine conjuguees avec de l'oxyde de polyalkylene
US5650388A (en) * 1989-11-22 1997-07-22 Enzon, Inc. Fractionated polyalkylene oxide-conjugated hemoglobin solutions
AU705225B2 (en) * 1995-03-23 1999-05-20 Biopure Corporation Stable polymerized hemoglobin blood substitute
US5929031A (en) * 1995-05-02 1999-07-27 Baxter Biotech Technology Sarl Storage stable hemoglobin solutions
WO2001017549A1 (fr) * 1999-09-07 2001-03-15 Japan Science And Technology Corporation Stockage stable d'une transfusion d'hydrogene
US7494974B2 (en) 2006-10-24 2009-02-24 Ikor, Inc. Carboxymethylated cross-linked tetrameric hemoglobin
US7504377B2 (en) 2006-10-23 2009-03-17 Ikor, Inc. Nitric oxide-blocked cross-linked tetrameric hemoglobin

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4670417A (en) * 1985-06-19 1987-06-02 Ajinomoto Co., Inc. Hemoglobin combined with a poly(alkylene oxide)
US4777244A (en) * 1981-11-11 1988-10-11 Biotest-Serum-Institut Gmbh Crosslinked hemoglobin of extended shelf life and high oxygen transport capacity and process of preparing same
US4826811A (en) * 1986-06-20 1989-05-02 Northfield Laboratories, Inc. Acellular red blood cell substitute
US4831012A (en) * 1984-03-23 1989-05-16 Baxter International Inc. Purified hemoglobin solutions and method for making same
US4857636A (en) * 1987-05-05 1989-08-15 Hsia Jen Chang Pasteurizable, freeze-driable hemoglobin-based blood substitute
US4920194A (en) * 1986-10-28 1990-04-24 Wolfgang Feller Blood substitute

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4777244A (en) * 1981-11-11 1988-10-11 Biotest-Serum-Institut Gmbh Crosslinked hemoglobin of extended shelf life and high oxygen transport capacity and process of preparing same
US4831012A (en) * 1984-03-23 1989-05-16 Baxter International Inc. Purified hemoglobin solutions and method for making same
US4670417A (en) * 1985-06-19 1987-06-02 Ajinomoto Co., Inc. Hemoglobin combined with a poly(alkylene oxide)
US4826811A (en) * 1986-06-20 1989-05-02 Northfield Laboratories, Inc. Acellular red blood cell substitute
US4920194A (en) * 1986-10-28 1990-04-24 Wolfgang Feller Blood substitute
US4857636A (en) * 1987-05-05 1989-08-15 Hsia Jen Chang Pasteurizable, freeze-driable hemoglobin-based blood substitute

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5650388A (en) * 1989-11-22 1997-07-22 Enzon, Inc. Fractionated polyalkylene oxide-conjugated hemoglobin solutions
EP0665850A1 (fr) * 1992-10-13 1995-08-09 Enzon, Inc. Fractionnement de solutions d'hemoglobine conjuguees avec de l'oxyde de polyalkylene
EP0665850A4 (fr) * 1992-10-13 1996-07-24 Enzon Inc Fractionnement de solutions d'hemoglobine conjuguees avec de l'oxyde de polyalkylene.
AU705225B2 (en) * 1995-03-23 1999-05-20 Biopure Corporation Stable polymerized hemoglobin blood substitute
EP1093720A1 (fr) * 1995-03-23 2001-04-25 Biopure Corporation Succédané de sang stable à base d' hémoglobine polymérisée
US5929031A (en) * 1995-05-02 1999-07-27 Baxter Biotech Technology Sarl Storage stable hemoglobin solutions
WO2001017549A1 (fr) * 1999-09-07 2001-03-15 Japan Science And Technology Corporation Stockage stable d'une transfusion d'hydrogene
US6864094B2 (en) 1999-09-07 2005-03-08 Japan Science And Technology Corporation Method of preserving oxygen infusions
US7504377B2 (en) 2006-10-23 2009-03-17 Ikor, Inc. Nitric oxide-blocked cross-linked tetrameric hemoglobin
US8129338B2 (en) 2006-10-23 2012-03-06 Ikor, Inc. Nitric oxide-blocked cross-linked tetrameric hemoglobin
US7494974B2 (en) 2006-10-24 2009-02-24 Ikor, Inc. Carboxymethylated cross-linked tetrameric hemoglobin

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