# US 5,349,054 A · Activated benzenepentacarboxylate-crosslinked low oxygen affinity hemoglobin > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,349,054 A - Document ID: US5349054A - Status: Granted - Kind: A - Application number: 07/745,587 - Filed: 1991-08-15 - Granted: 1994-09-20 - Earliest claimed: 1991-08-15 - Assignees: Duke University - Inventors: Joseph Bonaventura; Marius Brouwer; Robert E. Cashon - Web page: https://patentgrep.com/patent/US5349054A ## CPC - A61K 38/42: Haemoglobins; Myoglobins - C07K 14/805: Haemoglobins; Myoglobins - A61M 1/3693: {using separation based on different densities of components, e.g. centrifuging} - A61M 2202/0433: Free haemoglobin - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - A61M: DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY (introducing media into or onto the bodies of animals A61D7/00; means for inserting tampons A61F13/26; devices for administering food or medicines orally A61J; containers for collecting, storing or administering blood or medical fluids A61J1/05); DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY (surgery A61B; chemical aspects of surgical articles A61L); DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR {(Electrotherapy, e.g. producing anaesthesia by the use of alternating or intermittent currents A61N1/36021)} ## Abstract The invention is directed to a stroma-free tetrameric mammalian hemoglobin which is crosslinked with benzenepentacarboxylate, in which the crosslinking is carried out by a method comprising the step of activating at least two carboxylate groups of the benzenepentacarboxylate with an activating agent prior to reaction with the hemoglobin as well as methods for its production. Crosslinked stroma-free hemoglobin produced by methods of the present invention may be used in applications requiring physiological oxygen carriers such as in blood substitute solutions, or as in a plasma expander. ## Claims ### Claim 1 Stroma-free tetrameric mammalian hemoglobin having an affinity for oxygen which is lower than that of non-crosslinked hemoglobin A, and which is crosslinked with benzenepentacarboxylate, in which at least two carboxylate groups have been activated with an activating agent prior to reaction with the hemoglobin. ### Claim 2 (depends on 1) The stroma-free tetrameric hemoglobin according to claim 1 in which the hemoglobin is derived from a human. ### Claim 3 (depends on 1) The stroma-free tetrameric hemoglobin according to claim 1 in which the activating agent of the carboxylate is a carbodiimide. ### Claim 4 (depends on 3) The stroma-free tetrameric hemoglobin according to claim 3 in which the carbodiimide is 1-ethyl-3-(-3-dimethylaminopropyl)-carbodiimide. ### Claim 5 (depends on 1) The stroma-free tetrameric hemoglobin according to claim 1 in which the P.sub.50 is at least twice as high as the P.sub.50 of hemoglobin A when both of said P.sub.50 values are measured under substantially the same conditions. ### Claim 6 Stroma-free tetrameric mammalian hemoglobin having an affinity for oxygen which is lower than that of non-crosslinked hemoglobin A, and which is crosslinked with benzenepentacarboxylate, in which from about two to three carboxylate groups have been activated with a carbodiimide prior to reaction with the hemoglobin. ### Claim 7 (depends on 6) The stroma-free tetrameric hemoglobin according to claim 6 in which the hemoglobin is derived from a human. ### Claim 8 (depends on 6) The stroma-free tetrameric hemoglobin according to claim 6 in which the carbodiimide is 1-ethyl-3-(-3-dimethyl-aminopropyl)-carbodiimide. ### Claim 9 A method for producing stroma-free crosslinked hemoglobin having an affinity for oxygen which is lower than that of non-crosslinked hemoglobin A, comprising: (a) crosslinking hemoglobin with benzenepentacarboxylate, in which at least two carboxylate groups have been activated with an activating agent prior to the crosslinking; and (b) isolating the crosslinked hemoglobin of step (a). ### Claim 10 A method for producing stroma-free crosslinked hemoglobin having an affinity for oxygen which is lower than that of non-crosslinked hemoglobin A, comprising: (a) crosslinking hemoglobin with benzenepentacarboxylate, in which about two to three of the carboxylate groups have been activated with a carbodiimide prior to the crosslinking; and (b) isolating the crosslinked hemoglobin of step (a). ### Claim 11 (depends on 10) The method according to claim 10 in which the activation with a carbodiimide is done in the presence of catalase and EDTA. ### Claim 12 (depends on 9) The method according to claim 9 in which the activating agent is 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. ### Claim 13 (depends on 10) The method according to claim 10 in which the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. ### Claim 14 (depends on 11) The method according to claim 11 in which the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. ### Claim 15 (depends on 4) The stroma-free tetrameric hemoglobin according to claim 4 in which the hemoglobin is derived from a human. ### Claim 16 (depends on 8) The stroma-free tetrameric hemoglobin according to claim 8 in which the hemoglobin is derived from a human. ### Claim 17 (depends on 12) The method according to claim 12 in which the hemoglobin is derived from a human.