MODIFIED HAEMOGLOBIN PROTEINS

• US 20200131247A1
• Filed: 03/13/2018
• Published: 04/30/2020
• Est. Priority Date: 03/13/2017
• Status: Active Application

• Current Assignee: University Of Essex Enterprises Limited
• Original Assignee: University Of Essex Enterprises Limited
• Inventors: Cooper, Chris E., Reeder, Brandon, Silkstone, Gary
• Application Number:
  US16/493,654
• Publication Number:
  US 20200131247A1
• Time in Patent Office: Days
• 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: A61K 38/42 Haemoglobins; Myoglobins
• 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 present invention relates to modified proteins e.g. oxygen-carrying proteins, with improved or enhanced, in comparison to a reference protein, reduction of a metal ion associated with the modified protein. The present invention also relates to methods of using such modified proteins and compositions comprising such proteins e.g. in therapy.

Claims

1. A modified oxygen-carrying protein comprising at least one insertion of at least one redox-active amino acid residue and/or at least one substitution of an amino acid residue of the modified protein with at least one redox-active amino acid residue,

wherein said insertion and/or substitution introduces or enhances reduction of at least one iron ion and increases a rate at which an oxidised form of the modified oxygen-carrying protein is capable of re-oxygenation to an oxygen-binding form as compared to a reference protein,

wherein the at least one iron ion is associated with the modified protein and the modified protein exhibits enhanced reduction of a ferric (Fe³⁺) ion to a ferrous (Fe²⁺) ion as compared to a reference protein.

2. The modified protein as claimed in claim 1, wherein the at least one iron ion is located within at least one porphyrin molecule bound to the modified protein.

3. The modified protein as claimed in claim 2, wherein the least one porphyrin molecule is a haem, optionally wherein the at least one redox-active amino acid is located at about 10 Å

from an outer boundary of the at least one haem.

4. The modified protein as claimed in claim 1, wherein the modified protein is:

(i) a globin protein;

(ii) a haemoglobin or a haemoglobin chain subunit;

(iii) a mammalian haemoglobin or haemoglobin chain subunit;

or

(iv) a human haemoglobin or haemoglobin chain subunit.

8. The modified protein as claimed in claim 4, wherein the haemoglobin chain subunit is selected from at least one or more of a beta, a gamma or a delta chain subunit.

10. The modified protein as claimed in claim 1, wherein the at least one redox-active amino acid residue is selected from at least one or more of tyrosine, histidine and/or tryptophan, preferably tyrosine.

12. The modified protein as claimed in claim 1, wherein the at least one modification is selected from one or more of:

(a) β

T84Y, β

F85Y or a combination thereof;

(b) γ

1F85Y or a combination thereof;

(c) γ

2F85Y or a combination thereof;

(d) γ

1L96Y or a combination thereof; and

/or

(e) γ

2L96Y or a combination thereof.

13. The modified protein as claimed in claim 1, wherein:

(i) the modified protein further comprises at least one further modification which reduces the nitric oxide reactivity of the modified protein, or

(ii) the modified protein is conjugated to at least one protecting group, optionally wherein the at least one protecting group is at least one antioxidant enzyme or polyalkylene glycol.

17. A modified protein as claimed in claim 1, comprising an amino acid sequence which has at least 80% sequence identity to an amino acid sequence selected from:

a. the amino acid sequence as set forth in SEQ. ID. No. 10; and

/or

b. the amino acid sequence as set forth in SEQ. ID. No. 13.

18. A multimeric protein, comprising at least one modified protein as claimed in claim 1.

19. A multimeric protein as claimed in claim 18, wherein the multimer is cross-linked.

23. The composition as claimed in claim 20, comprising at least one modified protein as claimed in claim 1.

29. The method as claimed in claim 28, the method comprising administering a pharmaceutically effective amount of a modified protein as claimed in claim 1 and/or a composition as claimed in claim 21.

30. The modified protein as claimed in claim 1 or the multimeric protein as claimed in claim 18, or the composition as claimed in claim 20 for use as a medicament, oxygen therapeutic, or treatment of ischemia and/or hypoxia.

33. The method as claimed in claim 32, comprising administering a pharmaceutically effective amount of a modified oxygen-carrying protein as claimed in claim 1 or the multimeric protein as claimed in claim 18.

5-7. -7. (canceled)

9. (canceled)

11. (canceled)

14-16. -16. (canceled)

20. A composition comprising:

a modified oxygen-carrying protein, wherein the protein comprises at least one modification which introduces or enhances reduction of at least one metallic ion and increases a rate at which an oxidised form of the modified oxygen-carrying protein is capable of re-oxygenation to an oxygen-binding form as compared to a reference protein; and

a pharmaceutically acceptable carrier or diluent.

21. The composition as claimed in claim 20, further comprising at least one reductant, optionally wherein the at least one reductant is ascorbate.

24. The composition as claimed in claim 20, wherein the composition is a blood substitute composition.

22. (canceled)

25-27. -27. (canceled)

28. A method of treating and/or preventing ischemia and/or hypoxia, the method comprising administering a pharmaceutically effective amount of a pharmaceutical composition comprising a modified oxygen-carrying protein, wherein the protein comprises at least one modification which introduces or enhances reduction of at least one metallic ion and increases a rate at which an oxidised form of the modified oxygen-carrying protein is capable of re-oxygenation to an oxygen-binding form as compared to a reference protein.

31. (canceled)

32. A method of treating and/or preventing ischemia and/or hypoxia, the method comprising administering a pharmaceutically effective amount of a modified oxygen-carrying protein, wherein the protein comprises at least one modification which introduces or enhances reduction of at least one metallic ion and increases a rate at which an oxidised form of the modified oxygen-carrying protein is capable of re-oxygenation to an oxygen-binding form as compared to a reference protein.

34. (canceled)