Bacterial hyaluronidase and process for its production

Patent metadata

Field	Value
Publication number	US-2018362949-A1
Application number	US-201715728959-A
Country	US
Kind code	A1
Filing date	Oct 10, 2017
Priority date	Jun 17, 2013
Publication date	Dec 20, 2018
Grant date	—

Abstract

The present invention relates to a hyaluronidase from S. koganeiensis , applications thereof and a method for the production thereof.

First claim

1 - 13 . (canceled)

14 . Method for the production of hyaluronidase from Streptomyces koganeiensis ATCC 31394 comprising the amino acid sequence shown in SEQ. ID. No. 21 comprising the following steps:
a) inoculating a bacterial culture medium in a bioreactor with a inoculum of recombinant cells that contain at least one vector comprising the sequence shown as SEQ ID No. 41; b) subjecting the content of the bioreactor of step a) to fermentation at a pH between 6.7 and 7.1 in the presence of a nourishment solution; c) adding an inducer of the lac genes to the mixture of step b); d) subjecting the mixture of step c) to an induction period of between 8 and 24 hours; e) centrifuging the bacterial cells obtained in step d); f) re-suspending the pellets obtained in step e) and subjecting the resulting suspension to osmotic shock; g) extracting the periplasmic proteins by centrifugation of the suspension of step f); h) purifying the protein fraction having hyaluronidase enzymatic activity obtained in step g) by a sequence of: i. strong ion-exchange chromatography and isolation of the hyaluronidase enzymatic activity fraction; ii. weak cation-exchange chromatography and isolation of the hyaluronidase enzymatic activity fraction; and iii. aromatic hydrophobic interaction chromatography and isolation of the hyaluronidase enzymatic activity fraction.

15 . Method according to claim 14 , wherein the recombinant cell of step a) is selected from a cell of Escherichia coli and one of Bacillus subtilis.

16 . Method according to claim 14 , wherein in step b), a glycerol solution is used as nourishment solution.

17 . Hyaluronidase from Streptomyces koganeiensis ATCC 31394 in purified form and comprising the amino acid sequence in SEQ. ID. No. 21, wherein said hyaluronidase is free from cysteine and has an endotoxin content<0.5 U/mg.

18 . A composition suitable for pharmaceutical or cosmetic use comprising the hyaluronidase according to claim 17 .

19 . The hyaluronidase according to claim 17 , for the treatment and/or prevention of a disease or disorder, optionally in combination with at least another active principle.

20 . The hyaluronidase according to claim 19 , wherein the disorder or disease are at least one selected from the group consisting of edemas, inflammatory conditions, chilblains, solid tumors, IgE-mediated allergies, diseases of the oral cavity, spontaneous vitreous haemorrhages, arteriosclerosis, blood pressure disorders, cardio-cerebro vascular disorders such as brain arterial stenosis or stroke or bovine mastitis.

21 . The hyaluronidase according to claim 17 , for cosmetic applications and/or to improve the aesthetic appearance.

22 . A hyaluronidase from Streptomyces koganeiensis ATCC 31394 comprising the amino acid sequence in SEQ. ID. No. 21 obtainable by the method of claim 14 .

23 . The hyaluronidase obtainable by the method of claim 14 , for the treatment and/or prevention of a disease or disorder, optionally in combination with at least another active principle.

24 . The hyaluronidase according to claim 23 , wherein the disorder or disease is at least one selected from the group consisting of edemas, inflammatory conditions, chilblains, solid tumors, IgE-mediated allergies, diseases of the oral cavity, spontaneous vitreous haemorrhages, arteriosclerosis, blood pressure disorders, cardio-cerebro vascular disorders such as brain arterial stenosis or stroke or bovine mastitis.

25 . The hyaluronidase obtainable by the method of claim 14 , for cosmetic applications and/or to improve the aesthetic appearance.

26 . A method for preventing and/or treating at least one disorder selected from the group consisting of oedemas, inflammatory conditions, chilblains, solid tumours, IgE-mediated allergies, diseases of the oral cavity, spontaneous vitreous haemorrhages, arteriosclerosis, blood pressure disorders, cardio-cerebro vascular disorders such as brain arterial stenosis or stroke or bovine mastitis, comprising administering to a subject in need thereof the hyaluronidase according to claim 17 .

27 . A method for preventing and/or treating at least one disorder selected from the group consisting of oedemas, inflammatory conditions, chilblains, solid tumours, IgE-mediated allergies, diseases of the oral cavity, spontaneous vitreous haemorrhages, arteriosclerosis, blood pressure disorders, cardio-cerebro vascular disorders such as brain arterial stenosis or stroke or bovine mastitis, comprising administering to a subject in need thereof the hyaluronidase obtainable with the method according to claim 14 .

Assignees

• Fidia Farm Spa

Inventors

• Messina Luciano
• Musumeci Luca
• Vaccaro Susanna

Classifications

• C12Y302/01035

  Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase · CPC title

• C12N9/2474 Primary

  Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase · CPC title

• A61K38/00

  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 title