Beta-lactamases with improved properties for therapy

Patent metadata

Field	Value
Publication number	US-9783797-B1
Application number	US-201715611881-A
Country	US
Kind code	B1
Filing date	Jun 2, 2017
Priority date	Apr 17, 2014
Publication date	Oct 10, 2017
Grant date	Oct 10, 2017

Abstract

This invention relates to, in part, compositions of beta-lactamases and methods of using these enzymes in, for example, gastrointestinal tract (GI tract) disorders such as C. difficile infection (CDI).

First claim

What is claimed is:

1. A beta-lactamase comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1 and the following mutations according to Ambler classification:
a polar and neutral hydrophilic residue other than aspartate (D) at position 276, and
one or more mutations selected from the group consisting of: an aromatic hydrophobic residue other than phenylalanine (F) at position 33; a polar and neutral hydrophilic residue other than serine (S) at position 240, a polar and neutral hydrophilic residue other than alanine (A) at position 238; a polar and neutral hydrophilic residue other than serine (S) at position 266; and an aliphatic hydrophobic residue other than threonine (T) at position, 243.

2. The beta-lactamase of claim 1 , wherein the beta-lactamase has a D276N mutation, a S240P mutation, and a F33Y mutation according to Ambler classification.

3. The beta-lactamase of claim 1 , wherein the beta-lactamase has a D276N mutation, a S238T mutation, and a F33Y mutation according to Ambler classification.

4. The beta-lactamase of claim 1 , wherein the beta-lactamase has a D276N mutation, a S266N mutation, and a T243I mutation according to Ambler classification.

5. The beta-lactamase of claim 1 , wherein the polar and neutral hydrophilic residue is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).

6. The beta-lactamase of claim 1 , wherein the aromatic hydrophobic residue is selected from tryptophan (W) and tyrosine (Y).

7. The beta-lactamase of claim 1 , wherein the aliphatic hydrophobic residue is selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V).

8. The beta-lactamase of claim 1 , wherein the beta-lactamase hydrolyzes one or more of penicillins and cephalosporins.

9. The beta-lactamase of claim 8 , wherein the penicillin is ampicillin.

10. The beta-lactamase of claim 8 , wherein the cephalosporin is selected from ceftriaxone, cefotaxime, cefozolin, cefoperazone, cefepime, cefuroxime, and ceftazidime.

11. The beta-lactamase of claim 1 , wherein the beta-lactamase has improved catalytic efficiency against cephalosporins as compared to SEQ ID NO: 1.

12. The beta-lactamase of claim 11 , wherein the beta-lactamase has about a 10 fold to about a 1000 fold improved catalytic efficiency against a cephalosporin as compared to SEQ ID NO: 1.

13. The beta-lactamase of claim 12 , wherein the cephalosporin is selected from ceftriaxone, cefotaxime, cefozolin, cefoperazone, cefepime, cefuroxime, and ceftazidime.

14. The beta-lactamase of claim 1 , wherein the beta-lactamase has improved catalytic efficiency against a cephalosporin as compared to P3A.

15. The beta-lactamase of claim 14 , wherein the cephalosporin is selected from cefotaxime, cefepime, and ceftriaxone.

16. The beta-lactamase of claim 1 , wherein the beta-lactamase is active in the GI tract.

17. The beta-lactamase of claim 16 , wherein the beta-lactamase is stable in the small intestine, optionally selected from one or more of the duodenum, jejunum, and ileum.

18. A polynucleotide comprising a polynucleotide sequence encoding the beta-lactamase of claim 1 .

19. A host cell comprising the polynucleotide of claim 18 .

20. A pharmaceutical composition, comprising the beta-lactamase of claim 1 and a pharmaceutically acceptable carrier or excipient.

21. The pharmaceutical composition of claim 20 , wherein the composition is formulated for oral administration, optionally selected from a tablet, multi-particulate sprinkle, and a multi-particulate capsule.

22. A method for preventing an antibiotic-induced adverse effect in the gastrointestinal (GI) tract, comprising administering an effective amount of a beta-lactamase to a patient in need thereof, wherein the beta-lactamase comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1 and the following mutations according to Ambler classification:
a polar and neutral hydrophilic residue other than aspartate (D) at position 276, and
one or more mutations selected from the group consisting of: an aromatic hydrophobic residue other than phenylalanine (F) at position 33; a polar and neutral hydrophilic residue other than serine (S) rat position 240, a polar and neutral hydrophilic residue other than alanine (A) at position 238; a polar and neutral hydrophilic residue other than serine (S) at position 266; and an aliphatic hydrophobic residue other than threonine (T) at position 243.

23. The method of claim 22 , wherein the polar and neutral hydrophilic residue is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).

24. The method of claim 22 , wherein the aromatic hydrophobic residue is selected from tryptophan (W) and tyrosine (Y).

25. The method of claim 22 , wherein the aliphatic hydrophobic residue is selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V).

26. The method of claim 22 , wherein the subject is being administered or will be administered an antibiotic.

27. A method for treating or preventing an antibiotic induced C. difficile infection (CD) and/or a C. difficile -associated disease, comprising administering an effective amount of a beta-lactamase to a patient in need thereof; wherein the beta-lactamase comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1 and the following mutations according to Ambler classification:
a polar and neutral hydrophilic residue other than aspartate (D) at position 276, and
one or more mutations selected from the group consisting of: an aromatic hydrophobic residue other than phenylalanine (F) at position 33; a polar and neutral hydrophilic residue other than serine (S) at position 240, a polar and neutral hydrophilic residue other than alanine (A) at position 238; a polar and neutral hydrophilic residue other than serine (S) at position 266; and an aliphatic hydrophobic residue other than threonine (T) at position 243.

28. The method of claim 27 , wherein the polar and neutral hydrophilic residue is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).

29. The method of claim 27 , wherein the aromatic hydrophobic residue is selected from tryptophan (W) and tyrosine (Y).

30. The method of claim 27 , wherein the aliphatic hydrophobic residue is selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V).

31. The method of claim 27 , wherein the subject is being administered or will be administered an antibiotic.

32. The method of claim 27 , wherein the C. difficile -associated disease is antibiotic-associated diarrhea (AAD).

Assignees

• Synthetic Biologics Inc

Inventors

• Kaleko Michael
• Connelly Sheila

Classifications

• A61P39/02

  Antidotes · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61K9/0053

  Mouth and digestive tract, i.e. intraoral and peroral administration · CPC title

• C12N9/86 Primary

  acting on amide bonds in cyclic amides, e.g. penicillinase {(3.5.2)} · CPC title

• A61K45/06

  Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca · CPC title