Intramolecularly cross-linked hemoglobin and method of preparation

• US 4,584,130 A
• Filed: 03/29/1985
• Issued: 04/22/1986
• Est. Priority Date: 03/29/1985
• Status: Expired due to Term

• Current Assignee: University of Maryland
• Original Assignee: University of Maryland
• Inventors: Bucci, Enrico, Hosmane, Ramachandra, Fronticelli-Bucci, Clara
• Primary Examiner(s): SCHAIN, HOWARD
• Application Number:
  US06/717,783
• Time in Patent Office: 389 Days
• Field of Search: 260/112 B, 260/112.5 R, 260/115, 424/101, 514/6
• US Class Current: 530/385
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07C 317/00 Sulfones; Sulfoxides
• CPC Class Codes: C07D 231/12 with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
• CPC Class Codes: C07D 233/56 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, attached to ring carbon atoms
• CPC Class Codes: C07D 249/08 1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
• CPC Class Codes: C07F 9/4012 substituted by B, Si, P or a metal C07F9/4025 takes precedence
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins

Abstract

Stroma-free hemoglobin cross-linked with reagents that mimic 2,3 diphosphoglycerate and transform stroma-free hemoglobin into a physiologically competent oxygen carrier which is retained in vivo for adequate periods of time and thus can be used in fluids for transporting oxygen; reagents for cross-linking stroma-free hemoglobin and method for cross-linking stroma-free hemoglobin.

Claims

1. Cross-linked stroma-free hemoglobin obtained by cross-linking stroma-free hemoglobin with at least one cross-linking reagent selected from the group consisting of compounds of the general formulae (I) and (II):

##STR16## wherein one of R₁ or R₂ represents an electron-withdrawing atom or group, and the other of R₁ or R₂ represents a leaving atom or group or an electron-withdrawing group containing a leaving atom or group;

one of R₃ or R₄ represents an electron-withdrawing atom or group, and the other of R₃ or R₄ represents a leaving atom or group or an electron-withdrawing group containing a leaving atom or group;

R₅ and R₁₀, which may be the same or different, each represents an electron-withdrawing atom or group or an electron-withdrawing group containing a leaving atom or group;

one of R₆ or R₇ represents a hydrogen atom, an alkyl group, an aryl group, or a heterocyclic group, and the other of R₆ or R₇ represents a leaving atom or group;

one of R₈ or R₉ represents a hydrogen atom, an alkyl group, an aryl group or a heterocylic group, and the other of R₈ or R₉ represents a leaving atom or group;

X represents a linking atom or group, n=0 to 4, m=0 to 4 and l=0 to 4.

2. The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein the electron-withdrawing group represented by R₁, R₂, R₃, R₄, R₅ and R₁₀ is a member selected from the group consisting of CN, NO₂, halogen, S(O)R₁₁, S(O₂)R₁₁, C(O)R₁₁, wherein R₁₁ is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C₁ -C₁₂ alkyl group, a substituted or unsubstituted mono- or bicyclic aryl group, and a substituted or unsubstituted heterocyclic group.

3. The cross-linked stroma-free hemoglobin as claimed in claim 2, wherein said substituents on said alkyl group, aryl group, and heterocyclic group are selected from the group consisting of a halogen atom, CN, a C₁ -C₁₂ alkyl group, a substituted or unsubstituted phenyl group, NO₂, OH, and a C₁ -C₁₂ alkoxy group.

4. The cross-linked stroma-free hemoglobin as claimed in claim 2, wherein said heterocyclic group is selected from the group consisting of substituted or unsubstituted imidazolyl and substituted or unsubstituted pyrazinyl.

5. The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein R₁, R₂, R₃, R₄, R₅, and R₁₀ are selected from the group consisting of CN and SO₂ R₁₁.

6. The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein the electron-withdrawing group containing a leaving group represented by R₁, R₂, R₃, R₄, R₅ and R₁₀ is a member selected from the group consisting of R₁₂ P(O)OR₁₂, P(O)(OR₁₂)₂, OP(O)(OR₁₂)₂ and CO₂ R₁₂, wherein R₁₂ is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C₁ -C₁₂ alkyl group, a substituted or unsubstituted mono- or bicyclic aryl group, a substituted or unsubstituted heterocyclic group and a halogen atom and P(O)(R₁₃)₂, S(O)R₁₃, S(O₂)R₁₃ and C(O)R₁₃, wherein R₁₃ is a halogen atom.

7. The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein the group other than a leaving group represented by R₆, R₇, R₈ and R₉ is selected from the group consisting of a hydrogen atom, a C₁ -C₁₂ alkyl group, an aryl group, and a heterocyclic group.

8. The cross-linked stroma-free hemoglobin as claimed in claim 7, wherein R₆, R₇, R₈ and R₉ are selected from the group consisting of C₂ H₅, a phenyl group, or an imidazolyl group.

9. The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein the leaving group represented by R₁, R₂, R₃, R₄, R₆, R₇, R₈ and R₉ is selected from the group consisting of a halogen atom and OR₁₁, wherein R₁₁ is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C₁ -C₁₂ alkyl group, a substituted or unsubstituted mono- or bicyclic aryl group and a substituted or unsubstituted heterocyclic group.

10. The cross-linked stroma-free hemoglobin as claimed in claim 9, wherein R₇ and R₉ are selected from the group consisting of CH₃, C₂ H₅ and CH₂ C₆ H₅.

11. The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein X is selected from the group consisting of SO₂, SO, P(O)(OR₁₁), CO, Se, Te and Sr, wherein R₁₁ is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C₁ -C₁₂ alkyl group, a substituted or unsubstituted mono- or bicyclic aryl group and a substituted or unsubstituted heterocyclic group.

12. The cross-linked stroma-free hemoglobin as claimed in claim 11, wherein X is selected from the group consisting of SO₂, SO, P(O)(OR₁₁) and CO, wherein R₁₁ is as defined in claim 11.

13. The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein n=0 to 2, n=0 to 2, and l=0 to 2.

14. The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein said reagent is selected from the group consisting of ethyl-2,2'"'"'-sulfonyl-bis-cyanoacetate, diethyl-2,2'"'"'-sulfonyl-bis-malonate, ethyl-2,2'"'"'-sulfonyl-2,2'"'"'-benzene sulfonyl-bis-acetate, 2,2'"'"'-sulfonyl-2,2'"'"'-imidazole-N-sulfonyl-bis-acetonitrile, methyl 2,2'"'"'-sulfonyl-bis-cyanoacetate, 2,2'"'"'-methoxymethylene-2,2'"'"'-sulfonyl-bis-acetonitrile and dimethyl 2,2'"'"'-methoxyphosphinylidene-2-2'"'"'-methoxymethylene-bis-methanephosphonate.

15. The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein said stroma-free hemoglobin is selected from the group consisting of human, equine, porcine, ovine, bovine, simian and fish hemoglobin.

16. The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein said stroma-free hemoglobin is bovine hemoglobin.

17. The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein said stroma-free hemoglobin is human hemoglobin.

18. The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein said cross-linking is carried out in the presence of oxygen.

19. The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein said cross-linking is carried out in the absence of oxygen.

20. A method of producing cross-linked stroma-free hemoglobin, comprising:

(a) cross-linking stroma-free hemoglobin with at least one cross-linking reagent selected from the group consisting of compounds of the general formulae (I) and (II);

##STR17## wherein one of R₁ or R₂ represents an electron-withdrawing atom or group, and the other of R₁ or R₂ represents a leaving atom or group or an electron-withdrawing group containing a leaving atom or group;

one of R₃ or R₄ represents an electron-withdrawing atom or group, and the other of R₃ or R₄ represents a leaving atom or group or an electron-withdrawing group containing a leaving atom or group;

R₅ and R₁₀, which may be the same or different, each represents an electron-withdrawing atom or group or an electron-withdrawing group containing a leaving atom or group;

one of R₆ or R₇ represents a hydrogen atom, an alkyl group, an aryl group, or a heterocyclic group, and the other of R₆ or R₇ represents a leaving atom or group;

one of R₈ or R₉ represents a hydrogen atom, an alkyl group, an aryl group or a heterocyclic group, and the other of R₈ or R₉ represents a leaving atom or group;

X represents a linking atom or group, n=0 to 4, m=0 to 4 and l=0 to 4; and

(b) purifying the resulting cross-linked hemoglobin.

21. The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein the electron-withdrawing group represented by R₁, R₂, R₃, R₄, R₅ and R₁₀ is a member selected from the group consisting of CN, NO₂, halogen, S(O)R₁₁, S(O₂)R₁₁, C(O)R₁₁, wherein R₁₁ is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C₁ -C₁₂ alkyl group, a substituted or unsubstituted mono- or bicyclic aryl group, and a substituted or unsubstituted heterocyclic group.

22. The method of producing cross-linked stroma-free hemoglobin as claimed in claim 21, wherein said substituents on said alkyl group, aryl group, and heterocyclic group are selected from the group consisting of a halogen atom, CN, a C₁ -C₁₂ alkyl group, a substituted or unsubstituted phenyl group, NO₂, OH, and a C₁ -C₁₂ alkoxy group.

23. The mthod of producing cross-linked stroma-free hemoglobin as claimed in claim 21, wherein said heterocyclic group is selected from the group consisting of substituted or unsubstituted imidazolyl and substituted or unsubstituted pyrazinyl.

24. The method of producing cross-linked stroma-free hemoglobin as claimed in claim 21, wherein R₁, R₂, R₃, R₄, R₅, and R₁₀ are selected from the group consisting of CN and SO₂ R₁₁.

25. The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein the electron-withdrawing group containing a leaving group represented by R₁, R₂, R₃, R₄, R₅ and R₁₀ is a member selected from the group consisting of R₁₂ P(O)OR₁₂, P(O)(OR₁₂)₂, OP(O)(OR₁₂)₂, and CO₂ R₁₂, wherein R₁₂ is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C₁ -C₁₂ alkyl group, a substituted or unsubstituted mono- or bicyclic aryl group, a substituted or unsubstituted heterocyclic group and a halogen atom and P(O)(R₁₃)₂, S(O)R₁₃, S(O₂)R₁₃ and C(O)R₁₃, wherein R₁₃ is a halogen atom.

26. The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein the group other than a leaving group represented by R₆, R₇, R₈ and R₉ is selected from the group consisting of a hydrogen atom, a C₁ -C₁₂ alkyl group, an aryl group, and a heterocyclic group.

27. The method of producing cross-linked stroma-free hemoglobin as claimed in claim 26, wherein R₆, R₇, R₈ and R₉ are selected from the group consisting of C₂ H₅, a phenyl group, or an imidazolyl group.

28. The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein the leaving group represented by R₁, R₂, R₃, R₄, R₆, R₇, R₈ and R₉ is selected from the group consisting of a halogen atom and OR₁₁, wherein R₁₁ is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C₁ -C₁₂ alkyl group, a substituted or unsubstituted mono- or bicyclic aryl group and a substituted or unsubstituted heterocyclic group.

29. The method of producing cross-linked stroma-free hemoglobin as claimed in claim 28, wherein R₁₁ is selected from the group consisting of CH₃, C₂ H₅, CH₂ C₆ H₅, and C₆ H₅.

30. The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein X is selected from the group consisting of SO₂, SO, P(O)(OR₁₁), CO, Se, Te and Sr, wherein R₁₁ is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C₁ -C₁₂ alkyl group, a substituted or unsubstituted mono- or bicyclic aryl group and a substituted or unsubstituted heterocyclic group.

31. The method of producing cross-linked stroma-free hemoglobin as claimed in claim 30, wherein X is selected from the group consisting of SO₂, SO, P(O)(OR₁₁) and CO, wherein R₁₁ is as defined above.

32. The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein n=0 to 2, n=0 to 2, and l=0 to 2.

33. The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein said reagent is selected from the group consisting of ethyl-2,2'"'"'-sulfonyl-bis-cyanoacetate, diethyl-2,2'"'"'-sulfonyl-bis-malonate, ethyl-2,2'"'"'-sulfonyl-2,2'"'"'-benzene sulfonyl-bis-acetate, 2,2'"'"'-sulfonyl-2,2'"'"'-imidazole-N-sulfonyl-bis-acetonitrile, methyl 2,2'"'"'-sulfonyl-bis-cyanoacetate, 2,2'"'"'-methoxymethylene-2,2'"'"'-sulfonyl-bis-acetonitrile and dimethyl 2,2'"'"'-methoxyphosphinylidene-2-2'"'"'-methoxymethylene-bis-methanephosphonate.

34. The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein said stroma-free hemoglobin is selected from the group consisting of human, equine, porcine, ovine, bovine, simian and fish hemoglobin.

35. The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein said stroma-free hemoglobin is bovine hemoglobin.

36. The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein said stroma-free hemoglobin is human hemoglobin.

37. The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein said cross-linking is carried out in the presence of oxygen.

38. The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein said cross-linking is carried out in the absence of oxygen.

39. The method of producing cross-linked hemoglobin as claimed in claim 20, wherein said cross-linking is carried out at about 10°

C. to 37°

C.

40. The method of producing cross-linked hemoglobin as claimed in claim 39 wherein said cross-linking is carried out at about 25°

C. to 30°

C.

41. The method of producing cross-linked hemoglobin as claimed in claim 20, wherein the molar ratio of said reagent to said hemoglobin is 1:

1 to 1;

10.

42. The method of producing cross-linked hemoglobin as claimed in claim 41, wherein the molar ratio of said reagent to said hemoglobin is 1:

2 to 1;

6.