# US 2019/0169590 A1 · Modified beta-lactamases and methods and uses related thereto

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- Number: US 2019/0169590 A1
- Document ID: US20190169590A1
- Status: Published application
- Kind: A1
- Application number: 16/279,547
- Filed: 2019-02-19
- Published: 2019-06-06
- Earliest claimed: 2016-02-26
- Assignees: Synthetic Biologics, Inc.
- Applicants: Synthetic Biologics, Inc.
- Inventors: Pertti KOSKI; Ulla AIRAKSINEN; Katja VALIMAKI
- Web page: https://patentgrep.com/patent/US20190169590A1

## CPC

- C12N 9/86: acting on amide bonds in cyclic amides, e.g. penicillinase {(3.5.2)}
- A61K 9/0053: {Mouth and digestive tract, i.e. intraoral and peroral administration}
- A61K 9/4891: {Coated capsules; Multilayered drug free capsule shells (with drug coating for immediate release A61K9/4808; osmotic devices A61K9/0004)}
- A61K 31/43: Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula [IMAGE cpc-sch-A61K-0952.gif], e.g. penicillins, penems
- A61K 31/545: Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula: [IMAGE cpc-sch-A61K-0953.gif] , e.g. cephalosporins, {cefaclor, or cephalexine}
- A61K 38/50: acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 1/08: for nausea, cinetosis or vertigo; Antiemetics
- A61P 1/12: Antidiarrhoeals
- A61P 7/10: Antioedematous agents; Diuretics
- A61P 17/00: Drugs for dermatological disorders
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- C12Y 305/02006: Beta-lactamase (3.5.2.6)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Y: ENZYMES

## Abstract

The present invention relates to pharmaceuticals and modified beta-lactamases. Specifically, the invention relates to novel recombinant beta-lactamases and pharmaceutical compositions comprising the beta-lactamases. Also, the present invention relates to methods for modifying a beta-lactamase, producing the beta-lactamase and treating or preventing beta-lactam antibiotic induced adverse effects. Furthermore, the present invention relates to the beta-lactamase for use as a medicament and to the use of the beta-lactamase in the manufacture of a medicament for treating or preventing beta-lactam antibiotics induced adverse effects. Still further, the invention relates to a polynucleotide and a host cell comprising the polynucleotide.

## Claims

### Claim 1

A beta-lactamase comprising an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 1 and having a hydrophilic amino acid residue at a position of SEQ ID NO: 1 corresponding to position 276 according to Ambler classification, or a variant or fragment thereof.

### Claim 2

A method of modifying a beta-lactamase comprising an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 1, characterized in that an amino acid of the beta-lactamase at a position of SEQ ID NO: 1 corresponding to position 276 according to Ambler classification is replaced with a hydrophilic amino acid.

### Claim 3 (depends on 1, 2)

The beta-lactamase according to claim 1 or the method according to claim 2, wherein the beta-lactamase comprises an amino acid sequence having at least 68% sequence identity with SEQ ID NO:1, or a variant or fragment thereof having beta-lactamase activity and including a hydrophilic amino acid residue other than aspartic acid (D) at a position corresponding to position 276 according to Ambler classification.

### Claim 4 (depends on 1, 2)

The beta-lactamase according to claim 1 or the method according to claim 2, wherein the beta-lactamase comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO:1, and wherein the hydrophilic amino acid residue at a position corresponding to position 276 according to Ambler classification is other than aspartic acid (D).

### Claim 5 (depends on 1, 2)

The beta-lactamase according to claim 1 or the method according to claim 2, characterized in that the hydrophilic amino acid is selected from polar and positively charged hydrophilic amino acids from the group consisting of arginine (R), histidine (H) and lysine (K).

### Claim 6 (depends on 1, 2)

The beta-lactamase according to claim 1 or the method according to claim 2, characterized in that the hydrophilic amino acid is selected from polar and neutral of charge hydrophilic amino acids from the group consisting of asparagine (N), glutamine (Q), serine (S) and threonine (T).

### Claim 7 (depends on 6)

The beta-lactamase according to claim 6, characterized in that the amino acid at the position of SEQ ID NO: 1 corresponding to position 276 is asparagine.

### Claim 8 (depends on 5)

The beta-lactamase according to claim 5, characterized in that the amino acid at the position of SEQ ID NO: 1 corresponding to position 276 is arginine.

### Claim 9

The beta-lactamase or the method according to any one of the previous claims, characterized in that the hydrophilic amino acid at the position of SEQ ID NO: 1 corresponding to position 276 locates in an alpha helix.

### Claim 10

The beta-lactamase or the method according to any one of the previous claims, characterized in that the beta-lactamase further comprises at least one amino acid selected from the group consisting of Leu220 and Arg244 according to Ambler classification.

### Claim 11

The beta-lactamase or the method according to any one of the previous claims, characterized in that the beta-lactamase hydrolyses penicillins and/or cephalosporins.

### Claim 12

The beta-lactamase or the method according to any one of the previous claims, characterized in that the beta-lactamase has improved catalytic efficiency on cephalosporins compared to wild type beta-lactamases.

### Claim 13 (depends on 11, 12)

The beta-lactamase or the method according to claim 11 or 12, characterized in that the cephalosporins are selected from the group consisting of cefoperazone, ceftriaxone and cefazoline.

### Claim 14 (depends on 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

A method of producing the beta-lactamase according to any one of claims 1 or 3-13, characterized in that the method comprises the following steps:
v) providing a gene encoding the beta-lactamase according to any one of claims 1 or 3-13;
vi) transforming a host cell with the gene;
vii) obtaining a host cell that produces the beta-lactamase;
viii) recovering the beta-lactamase.

### Claim 15 (depends on 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

A method of treating or preventing beta-lactam antibiotic induced adverse effects in the gastro-intestinal tract, characterized by administering beta-lactamase according to any one of claims 1 or 3-13 simultaneously or sequentially with a beta-lactam antibiotic to a subject.

### Claim 16 (depends on 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

A polynucleotide, characterized in that it comprises a sequence of any one of SEQ ID NO:s 2 or 4 or a degenerate thereof, or it encodes the beta-lactamase according to any one of claims 1 or 3-13.

### Claim 17 (depends on 16)

A host cell comprising the polynucleotide according to claim 16.

### Claim 18 (depends on 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

A pharmaceutical composition comprising the beta-lactamase according to any one of claims 1 or 3-13.

### Claim 19 (depends on 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

The beta-lactamase according to any one of claims 1 or 3-13 for use as a medicament.

### Claim 20 (depends on 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

Use of the beta-lactamase according to any one of claims 1 or 3-13 in the manufacture of a medicament for treating or preventing beta-lactam antibiotics induced adverse effects in the gastro-intestinal tract.

### Claim 21 (depends on 15, 20)

The method according to claim 15 or the use according to claim 20, characterized in that the beta-lactamase(s) is administered orally.

### Claim 22 (depends on 15, 20)

The method according to claim 15 or the use according to claim 20, characterized in that the beta-lactamase(s) is administered directly to the gastro-intestine of a patient.

### Claim 23 (depends on 15, 20, 21, 22)

The method according to any one of claims 15 or 21-22 or the use according to any one of claims 20-22, characterized in that the beta-lactamase(s) and the beta-lactam antibiotic are administered together in the form of an enteric coated pellet to a subject.