Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases

• US 20110008309A1
• Filed: 06/15/2010
• Published: 01/13/2011
• Est. Priority Date: 03/05/2003
• Status: Active Grant

• Current Assignee: Halozyme, Inc. (Halozyme Therapeutics, Inc.)
• Original Assignee: Halozyme, Inc. (Halozyme Therapeutics, Inc.)
• Inventors: Bookbinder, Louis H., Frost, Gregory I., Kundu, Anirban
• Granted Patent: US 8,105,586 B2
• Time in Patent Office: Days
• US Class Current: 424/94.3
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 38/47 acting on glycosyl compounds (3.2), e.g. cellulases, lactases
• CPC Class Codes: A61K 39/395 Antibodies agglutinins A61K38/36 ; as drug carriers A61K47/50; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 27/12 for cataracts
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/06 for tuberculosis
• CPC Class Codes: A61P 31/08 for leprosy
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 33/02 Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
• CPC Class Codes: A61P 33/04 Amoebicides
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• 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: C12N 9/2408 : acting on alpha -1,4-glucosidic bonds
• CPC Class Codes: C12Y 302/01035 : Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase

Abstract

The invention relates to the discovery of novel soluble neutral active Hyaluronidase Glycoproteins (sHASEGPs), methods of manufacture, and their use to facilitate administration of other molecules or to alleviate glycosaminoglycan associated pathologies. Minimally active polypeptide domains of the soluble, neutral active sHASEGP domains are described that include asparagine-linked sugar moieties required for a functional neutral active hyaluronidase domain. Included are modified amino-terminal leader peptides that enhance secretion of sHASEGP. The invention further comprises sialated and pegylated form of a recombinant sHASEGP to enhance stability and serum pharmacokinetics over naturally occurring slaughterhouse enzymes. Further described are suitable formulations of a substantially purified recombinant sHASEGP glycoprotein derived from a eukaryotic cell that generate the proper glycosylation required for its optimal activity.

Claims

1. A pharmaceutical composition, comprising:

a) a hyaluronidase glycoprotein that is active at neutral pH and contains at least one sugar moiety that is covalently attached to an asparagine (N) residue of the hyaluronidase polypeptide, wherein;

(i) the hyaluronidase glycoprotein consists of the sequence of amino acids set forth as amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, or 36-483 of SEQ ID NO;

1;

or

(ii) the hyaluronidase glycoprotein contains amino acid substitutions in the sequence of amino acids set forth as amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, or 36-483 of SEQ ID NO;

1, whereby the amino acid-substituted hyaluronidase glycoprotein consists of a sequence of amino acids that has at least 95% amino acid sequence identity with the sequence of amino acids set forth as amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, or 36-483 of SEQ ID NO;

1; and

b) a steroidal anti-inflammatory drug selected from among alclomethasones, algestones, beclomethasones, betamethasones, budesonides, clobetasols, clobetasones, clocortolones, cloprednols, corticosterones, cortisones and hydrocortisones, cortivazols, deflazacorts, desonides, desoximetasones, dexamethasones, difluorosones, diflucortolones, difluprednates, enoxolones, fluazacorts, flucloronides, flumethasones, flunisolides, fluocinolones, fluocinonides, fluocortins, fluocortolones, fluorometholones, fluperolones, fluprednidenes, fluprednisolones, flurandrenolides, fluticasones, formocortals, halcinonides, halobetasols, halometasones, halopredones, hydrocortamates, hydrocortisones, loteprednol etabonate, mazipredones, medrysones, meprednisones, methylprednisolones, mometasone furoate, paramethasones, prednicarbates, prednisolones, prednisones, prednivals, prednylidenes, rimexolones, tixocortols, triamcinolones.

2. The composition of claim 1, wherein the steroidal anti-inflammatory drug is a dexamethasone.

3. The composition of claim 1, wherein the steroidal anti-inflammatory drug is selected from among hydrocortisone acetate, dexamethasone 21-phosphate, prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate, triamcinolone acetonide, triamcinolone benetonide, and triamcinolone hexacetonide.

4. A composition of claim 1, wherein the hyaluronidase glycoprotein is modified with a polymer.

5. A composition of claim 4, wherein the polymer is a dextran or a pegylation moiety.

6. The composition of claim 4, wherein the hyaluronidase glycoprotein is pegylated.

7. A composition of claim 1, wherein the hyaluronidase glycoprotein consists of amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:

1.

8. A pharmaceutical composition of claim 1 that comprises a polypeptide that consists of amino acids 36-482 of SEQ ID NO:

1.

9. A pharmaceutical composition of claim 1, wherein the hyaluronidase glycoprotein is modified with a polymer.

10. A pharmaceutical composition of claim 9, wherein the polymer is a pegylation moiety or dextran.

11. The composition of claim 1, wherein the hyaluronidase glycoprotein is pegylated.

12. A pharmaceutical composition of claim 1, wherein the composition is formulated for subcutaneous administration.

21. The pharmaceutical composition of claim 1, wherein the hyaluronidase glycoprotein is produced by expression of a nucleic acid molecule that encodes amino acids 36-482 of SEQ ID NO:

1 in a mammalian cell.

22. The pharmaceutical composition of claim 21, where the mammalian cell is a CHO cell.

25. The pharmaceutical composition of claim 1, wherein the hyaluronidase glycoprotein is secreted when expressed in CHO cells.

13. A combination, comprising:

a) a first composition that comprises a hyaluronidase glycoprotein that is active at neutral pH and contains at least one sugar moiety that is covalently attached to an asparagine (N) residue of the hyaluronidase polypeptide, wherein;

(i) the hyaluronidase glycoprotein consists of the sequence of amino acids set forth as amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, or 36-483 of SEQ ID NO;

1;

or

(ii) the hyaluronidase glycoprotein contains amino acid substitutions in the sequence of amino acids set forth as amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, or 36-483 of SEQ ID NO;

1, whereby the amino acid-substituted hyaluronidase glycoprotein consists of a sequence of amino acids that has at least 95% amino acid sequence identity with the sequence of amino acids set forth as amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, or 36-483 of SEQ ID NO;

1; and

b) a second composition that comprises a steroidal anti-inflammatory drug selected from among beclomethasones, betamethasones, budesonides, clobetasols, clobetasones, clocortolones, cloprednols, corticosterones, cortisones and hydrocortisones (e.g. hydrocortisone acetate), cortivazols, deflazacorts, desonides, desoximetasones, dexamethasones (e.g. dexamethasone 21-phosphate), difluorosones, diflucortolones, difluprednates, enoxolones, fluazacorts, flucloronides, flumethasones, flunisolides, fluocinolones, fluocinonides, fluocortins, fluocortolones, fluorometholones, fluperolones, fluprednidenes, fluprednisolones, flurandrenolides, fluticasones, formocortals, halcinonides, halobetasols, halometasones, halopredones, hydrocortamates, hydrocortisones, loteprednol etabonate, mazipredones, medrysones, meprednisones, methylprednisolones, mometasone furoate, paramethasones, prednicarbates, prednisolones (e.g. prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate), prednisones, prednivals, prednylidenes, rimexolones, tixocortols, triamcinolones (e.g. triamcinolone acetonide, triamcinolone benetonide, and triamcinolone hexacetonide.

14. A combination of claim 13, wherein the hyaluronidase glycoprotein consists of amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:

1.

15. A combination of claim 13, that comprises a polypeptide that consists of amino acids 36-482 of SEQ ID NO:

1.

16. A combination of claim 13, wherein the hyaluronidase glycoprotein is modified with a polymer.

17. A combination of claim 16, wherein the polymer is PEG or dextran.

18. A combination of claim 13, wherein the hyaluronidase glycoprotein is pegylated.

19. The combination of claim 13 that is packaged as a kit.

20. The combination of claim 18, wherein the hyaluronidase glycoprotein is produced by expression of a nucleic acid molecule that encodes amino acids 36-482 of SEQ ID NO:

1 in a mammalian cell.

23. The combination of claim 13, wherein the hyaluronidase glycoprotein is produced by expression of a nucleic acid molecule that encodes amino acids 36-482 of SEQ ID NO:

1 in a mammalian cell.

24. The combination of claim 23, where the mammalian cell is a CHO cell.