Hemoglobin mutants with increased soluble expression and/or reduced nitric oxide scavenging

• US 6,455,676 B1
• Filed: 04/25/2000
• Issued: 09/24/2002
• Est. Priority Date: 05/02/1997
• Status: Expired due to Fees

• Current Assignee: Baxter Biotech Technology SARL (Baxter International Inc.), Rice University
• Original Assignee: Baxter Biotech Technology SARL (Baxter International Inc.), Rice University
• Inventors: Weickert, Michael J., Mathews, Antony J., Doherty, Daniel H., Olson, John S., Lemon, Douglas D., Glascock, Christopher B.
• Primary Examiner(s): CARLSON, KAREN C
• Application Number:
  US09/403,208
• Time in Patent Office: 882 Days
• Field of Search: 530/385
• 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: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 7/08 Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock artificial tears A61P27/04
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/08 Vasodilators for multiple indications
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins

Abstract

The invention relates to novel recombinant hemoglobins having reduced nitric oxide scavenging and/or increased high soluble expression. The invention further relates to methods of increasing the soluble expression of recombinant hemoglobin by adding exogenous hemin in molar excess of the heme binding sites of recombinant hemoglobin.

Claims

1. A hemoglobin mutant with reduced rate of reaction with nitric oxide selected from the following mutation in the alpha subunit:

E11(Val→

Leu) and E7(His→

Gln);

E11(Val→

Phe or Trp) and E7(His→

Gln);

E11(Val→

Phe or Trp or Leu) and E7(His→

Gln) and G8(Leu→

Phe or Trp);

B10(Leu→

Phe) and E4(Val→

Leu);

B10(Leu→

Trp) and E4(Val→

Leu);

B10(Leu→

Trp) and E7(His→

Gln);

B10(Leu→

Trp) and E11(Val→

Phe);

B10(Leu→

Trp) and E11(Val→

Trp);

B10(Leu→

Trp) and E11(Val→

Leu) and G8(Leu→

Trp);

B10(Leu→

Trp) and E11(Val→

Leu) and G8(Leu→

Phe);

B10(Leu→

Trp) and E11(Val→

Phe) and G8(Leu→

Trp);

B10(Leu→

Trp) and E11(Val→

Phe) and G8(Leu→

Ilc);

B10(Leu→

Trp) and E7(His→

Gln) and E11(Val→

Leu) and G8(Leu→

Trp);

B10(Leu→

Trp) and E11(Val→

Trp) and G8(Leu→

Trp);

E11(Val→

Leu) and G8(Leu→

Phe);

E11(Val→

Leu) and G8(Leu→

Trp);

B13(Met→

Phe or Trp);

G12(Leu→

Phe or Trp);

or

B14(Phe→

Trp).

2. The hemoglobin mutant of claim 1, wherein mutations in the alpha subunit are B10(Leu→

Trp) and E7(His→

Gln).

3. The hemoglobin mutant of claim 1, wherein mutations in the alpha subunit are B10(Leu→

Phe), E7(His→

Gln) and G8(Leu→

Trp).

4. The hemoglobin mutant of claim 1, wherein mutations in the alpha subunit are B10(Leu→

Trp), E7(His→

Gln), E11(Val→

Leu) and G8(Leu→

Trp).

5. The hemoglobin mutant of claim 1, wherein mutations in the alpha subunit are B10(Leu→

Trp) and E11(Val→

Phe).

6. The hemoglobin mutant of claim 1, wherein mutations in the alpha subunit are B10(Leu→

Trp) and E11(Val→

Trp).

7. The hemoglobin mutant of claim 1, wherein mutations in the alpha subunit are B10(Leu→

Trp), E11(Val→

Phe) and G8(Leu→

Ile).

8. A hemoglobin mutant with reduced rate of reaction with nitric oxide selected from the following mutation in the beta subunit:

B13(Leu→

Phe or Trp);

G12(Leu→

Phe or Trp);

B10(Leu→

Phe) and E4(Val→

Leu);

B10(Leu→

Trp) and E4(Val→

Leu);

B14(Leu→

Phe or Trp);

G8(Leu→

Phe) and G12(Leu→

Trp);

E11(Val→

Leu) and G8(Leu→

Trp);

E11(Val→

Trp) and G8(Leu→

Met);

E11(Val→

Leu) and G8(Leu→

Phe);

E11(Val→

Leu) and G8(Leu→

Met);

E11(Val→

Phe) and G8(Leu→

Ile);

E11(Val→

Phe) and G8(Leu→

Phe);

E11(Val→

Phe) and G8(Leu→

Trp);

E11(Val→

Phe) and G8(Leu→

Met);

E11(Val→

Met) and G8(Leu→

Trp);

E11(Val→

Met) and G8(Leu→

Trp) and E7(His→

Gln);

E11(Val→

Trp) and G8(Leu→

Ile);

E7(His→

Gln) and E11(Val→

Trp);

E7(His→

Gln) and E11(Val→

Leu);

E7(His→

Gln) and E11(Val→

Phe);

E7(His→

Gln) and E11(Val→

Phe) and G8(Leu→

Phe or Trp);

E7(His→

Gln) and E11(Val→

Leu or Trp) and G8(Leu→

Phe or Trp);

E11(Val→

Trp or Phe) and G12(Leu→

Trp or Met);

E11(Val→

Trp or Phe) and B13(Leu→

Trp or Met);

B10(Leu→

Trp) and B13(Leu→

Trp or Met);

B10(Leu→

Phe) and B13(Leu→

Trp);

B10(Leu→

Trp or Phe) and G12(Leu→

Trp);

B10(Leu→

Phe) and G12(Leu→

Met);

G8(Leu→

Trp) and G12(Leu→

Trp or Met);

or

G8(Leu→

Trp) and B13(Leu→

Trp or Met).

9. The hemoglobin mutant of claim 8, wherein mutations in the beta subunit are E11(Val→

Met) and G8(Leu→

Trp).

10. The hemoglobin mutant of claim 8, wherein mutations in the beta subunit are E11(Val→

Met), G8(Leu→

Trp) and E7(His→

Gln).

11. The hemoglobin mutant of claim 8, wherein mutations in the beta subunit are E11(Val→

Leu) and G8(Leu→

Trp).

12. A hemoglobin mutant having the following mutations:

α

B10(Leu→

Trp), α

E7(His→

Gln) and β

E11(Val→

Trp);

or

α

B10(Leu→

Phe), α

E7(His→

Gln) and β

G8(Leu→

Trp).