Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof

• US 8,431,380 B2
• Filed: 02/20/2009
• Issued: 04/30/2013
• 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, Kundu, Anirban, Frost, Gregory I.
• Primary Examiner(s): Kosson, Rosanne
• Application Number:
  US12/378,984
• Publication Number:
  US 20090181032A1
• Time in Patent Office: 1,530 Days
• Field of Search: 435/201, 435/69.1, 424/94.62, 530/350
• US Class Current: 435/201
• CPC Class Codes: A61K 2039/505 comprising antibodies
• 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 38/47 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 9/0048 Eye, e.g. artificial tears
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/24 Antidepressants
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• 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 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: A61P 33/04 Amoebicides
• CPC Class Codes: A61P 33/06 : Antimalarials
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/04 : Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: A61P 7/10 : Antioedematous agents; Diuretics
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 : Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• 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: A61P 9/12 : Antihypertensives
• CPC Class Codes: C12N 9/2474 : Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
• CPC Class Codes: C12Y 302/01035 : Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to the discovery of novel soluble neutral active Hyaluronidase Glycoproteins (sHASEGP'"'"'s), 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 forms 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 substantially purified hyaluronidase polypeptide selected from:

a) a polypeptide that consists of the sequence of amino acids set forth as amino acid residues 36-482 of SEQ ID NO;

1, or

b) a polypeptide that consists of a sequence of amino acids that has at least 99% sequence identity with the polypeptide of a), wherein;

the hyaluronidase polypeptides of a) and b) contain at least one sugar moiety that is covalently attached to an asparagine residue of the hyaluronidase polypeptide; and

the polypeptide is catalytically active.

33. A liposome, comprising the polypeptide of claim 1.

2. A substantially purified hyaluronidase polypeptide selected from:

a) a hyaluronidase polypeptide that comprises residues 36-464 of SEQ ID NO;

1, and is C-terminally truncated so that it does not include the full-length of the polypeptide whose sequence is set forth in SEQ ID NO;

1, or

b) a hyaluronidase polypeptide that contains amino acid substitutions in the sequence of amino acids of the C-terminally truncated polypeptide of a), whereby the amino acid-substituted hyaluronidase polypeptide consists of a sequence of amino acids that has at least 91% amino acid sequence identity to a polypeptide with a sequence of a), wherein;

the hyaluronidase polypeptide contains at least one sugar moiety that is covalently attached to an asparagine residue of the hyaluronidase polypeptide;

the polypeptide is catalytically active; and

the polypeptide is truncated at an amino acid residue selected from among residues 477, 478, 479, 480, 481, 482 and 483 of SEQ ID NO;

1.

3. The substantially purified hyaluronidase polypeptide of claim 2, wherein the polypeptide is secreted when produced in CHO cells.

4. A substantially purified hyaluronidase polypeptide of claim 2, wherein the polypeptide is modified with a polymer.

5. A substantially purified hyaluronidase polypeptide of claim 4, wherein the polymer is a polyethylene glycol (PEG) or a dextran.

6. A pharmaceutical composition, comprising a substantially purified hyaluronidase polypeptide of claim 2.

7. The pharmaceutical composition of claim 6, wherein the polypeptide is produced by expression in mammalian cells of a nucleic acid molecule that encodes amino acids 36-482 of SEQ ID NO:

1.

8. The pharmaceutical composition of claim 7, wherein the mammalian cell is a CHO cell.

9. The pharmaceutical composition of claim 6, further comprising a pharmaceutically active agent.

10. The pharmaceutical composition of claim 9, wherein the pharmaceutically active agent is a small molecule.

11. The pharmaceutical composition of claim 9, wherein the pharmaceutically active agent is selected from among a chemotherapeutic agent, an analgesic agent, an anti-inflammatory agent, an antimicrobial agent, an amoebicidal agent, a trichomonocidal agent, an anti-parkinson agent, an anti-malarial agent, an anti-convulsant agent, an anti-depressant agent, and antiarthritics agent, an anti-fungal agent, an antihypertensive agent, an antipyretic agent, an anti-parasite agent, an antihistamine agent, an alpha-adrenargic agonist agent, an alpha blocker agent, an anesthetic agent, a bronchial dialator agent, a biocide agent, a bactericide agent, a bacteriostat agent, a beta adrenergic blocker agent, a calcium channel blocker agent, a cardiovascular drug agent, a contraceptive agent, a decongestant agent, a diuretic agent, a depressant agent, a diagnostic agent, a electrolyte agent, a hypnotic agent, a hormone agent, a hyperglycemic agent, a muscle relaxant agent, a muscle contractant agent, an ophthalmic agent, a parasympathomimetic agent, a sedative agent, a sympathomimetic agent, a tranquilizer agent, a viricide agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an angiotensin converting enzyme inhibitor agent, and a sleep inducer.

12. The pharmaceutical composition of claim 11, wherein the chemotherapeutic agent is a toxin or a tumor necrosis factor.

13. The pharmaceutical composition of claim 11, wherein the anesthetic agent is lidocaine or bupivacaine.

14. The pharmaceutical composition of claim 9, further comprising a hormonal agent.

15. The pharmaceutical composition of claim 14, wherein the hormonal agent is epinephrine.

16. The pharmaceutical composition of claim 9, wherein the pharmaceutically active agent is selected from among an insulin, a cytokine, an antibody and a monoclonal antibody.

17. A conjugate, comprising:

a polypeptide of claim 2 linked directly or indirectly via a linker to a targeting agent.

18. A kit, comprising:

a first composition, comprising the hyaluronidase polypeptide of claim 2; and

a second composition, comprising a pharmaceutically active agent, wherein the first and second compositions are separate or combined to form a single composition.

19. The kit of claim 18, wherein the pharmaceutically active agent is a small molecule.

20. The kit of claim 18, wherein the pharmaceutically active agent is selected from among a chemotherapeutic agent, an analgesic agent, an anti-inflammatory agent, an antimicrobial agent, an amoebicidal agent, a trichomonocidal agent, an anti-parkinson agent, an anti-malarial agent, an anticonvulsant agent, an anti-depressant agent, and antiarthritics agent, an anti-fungal agent, an antihypertensive agent, an antipyretic agent, an anti-parasite agent, an antihistamine agent, an alpha-adrenargic agonist agent, an alpha blocker agent, an anesthetic agent, a bronchial dilator agent, a biocide agent, a bactericide agent, a bacteriostat agent, a beta adrenergic blocker agent, a calcium channel blocker agent, a cardiovascular drug agent, a contraceptive agent, a decongestant agent, a diuretic agent, a depressant agent, a diagnostic agent, a electrolyte agent, a hypnotic agent, a hormone agent, a hyperglycemic agent, a muscle relaxant agent, a muscle contractant agent, an ophthalmic agent, a parasympathomimetic agent, a sedative agent, a sympathomimetic agent, a tranquilizer agent, a viricide agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an angiotensin converting enzyme inhibitor agent and a sleep inducer.

21. The kit of claim 18, wherein the pharmaceutically active agent is selected from among insulin, a cytokine, an antibody and a monoclonal antibody.

22. The substantially purified hyaluronidase polypeptide of claim 2, wherein the amino-acid substituted polypeptide of b) consists of a sequence of amino acids that has at least 95% amino acid sequence identity with a sequence of the polypeptide of a).

23. The substantially purified hyaluronidase polypeptide of claim 2 that contains amino acid substitutions in the sequence of amino acids of the C-terminally truncated polypeptide of a), whereby the amino acid-substituted hyaluronidase polypeptide consists of a sequence of amino acids that has at least 91% amino acid sequence identity with to a polypeptide of a).

24. The hyaluronidase polypeptide of claim 2 that is produced and secreted from a mammalian expression system.

25. The pharmaceutical composition of claim 9, wherein the pharmaceutically active agent is a corticosteroid.

26. The kit of claim 18, wherein the pharmaceutically active agent is a corticosteroid.

27. A liposome, comprising the polypeptide of claim 2.

28. The pharmaceutical composition of claim 6 that comprises a slow release delivery vehicle.

29. The pharmaceutical composition of claim 28, wherein the slow release delivery vehicle comprises a lipid-polymer conjugate.

30. The pharmaceutical composition of claim 8, where in the slow release delivery vehicle comprises a colloidal dispersion system.

31. The pharmaceutical composition of claim 30, wherein the colloidal dispersion system is a liposome or a microsphere.

32. The pharmaceutical composition of claim 28, wherein the slow release delivery vehicle comprises a biodegradable hydrogel matrix, a dendritic polymer or a multivesicular liposome.