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

• US 7,767,429 B2
• Filed: 03/05/2004
• Issued: 08/03/2010
• Est. Priority Date: 03/05/2003
• Status: Active Grant

• Current Assignee: Halozyme, Inc. (Halozyme Therapeutics, Inc.)
• Original Assignee: Halozyme, Inc. (Halozyme Therapeutics, Inc.)
• Inventors: Frost, Gregory I., Kundu, Anirban, Bookbinder, Louis H.
• Primary Examiner(s): Carlson; Karen Cochrane
• Assistant Examiner(s): Kosson; Rosanne
• Application Number:
  US10/795,095
• Publication Number:
  US 20040268425A1
• Time in Patent Office: 2,342 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 glycoprotein that consists of the sequence of amino acids set forth as amino acids 1-477, 36-477, 1-478, 36-478, 1-479, 36-479, 1-480, 36-480, 1-481, 36-481, 1-482, 36-482, 1-483 or 36-483 of SEQ ID NO:

1, or contains amino acid substitutions in the sequence of amino acids set forth as amino acids 1-477, 36-477, 1-478, 36-478, 1-479, 36-479, 1-480, 36-480, 1-481, 36-481, 1-482, 36-482, 1-483 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 1-477, 36-477, 1-478, 36-478, 1-479, 36-479, 1-480, 36-480, 1-481, 36-481, 1-482, 36-482, 1-483 or 36-483 of SEQ ID NO;

1, wherein the glycoprotein contains at least one sugar moiety that is covalently attached to an asparagine (N) residue of the polypeptide.

2. The hyaluronidase glycoprotein of claim 1 that 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 set forth in SEQ ID NO:

1 or the glycoprotein that 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.

3. The hyaluronidase glycoprotein of claim 1 that 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 set forth in SEQ ID NO:

1.

4. The hyaluronidase glycoprotein of claim 1, wherein the sugar moiety is covalently attached to an asparagine (N) residue selected from among amino acid residues 82, 166, 235, 254, 368 and 393 as set forth in SEQ ID NO:

1.

5. A pharmaceutical composition, comprising a hyaluronidase glycoprotein of claim 1, wherein the hyaluronidase glycoprotein is selected from among a polypeptide that consists of the sequence of amino acids set forth as amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 and 36-483 of SEQ ID NO:

1.

11. A composition, comprising a hyaluronidase glycoprotein of claim 1.

6. A substantially purified hyaluronidase glycoprotein, wherein the hyaluronidase glycoprotein:

is neutral active;

contains at least one sugar moiety that is covalently attached to an asparagine (N) residue of the polypeptide; and

consists of the sequence of amino acids set forth as amino acids 1-477, 1-478, 1-479, 1-480, 1-481, 1-482 or 1-483 set forth in SEQ ID NO;

1 or 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 1-477, 1-478, 1-479, 1-480, 1-481 or 1-482 or 1-483 of SEQ ID NO;

1.

7. A substantially purified hyaluronidase glycoprotein, wherein the hyaluronidase glycoprotein:

is soluble;

is neutral active;

contains at least one sugar moiety that is covalently attached to an asparagine (N) residue of the polypeptide; and

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 set forth in SEQ ID NO;

1 or 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.

8. The hyaluronidase glycoprotein of claim 7 that consists of the sequence of amino acids set forth in SEQ ID NO:

4.

9. The hyaluronidase glycoprotein of claim 7, wherein the hyaluronidase glycoprotein is modified with a polymer.

10. The hyaluronidase glycoprotein of claim 9, wherein the polymer is PEG or dextran.

12. A composition, comprising a hyaluronidase glycoprotein of claim 7.

13. A composition, comprising a substantially purified hyaluronidase glycoprotein of claim 7 that is produced in and secreted from CHO cells by a method comprising:

introducing an expression vector comprising a polynucleotide that consists of a sequence of nucleotides set forth as nucleotides 106-1446 of SEQ ID NO;

6 or degenerates thereof or the sequence of nucleotides set forth in SEQ ID NO;

48 or degenerates thereof inserted between flanking sequences in the vector into a CHO cell;

culturing the CHO cell under conditions whereby an encoded hyaluronidase glycoprotein polypeptide of claim 7 is expressed and secreted by the cell; and

recovering a composition comprising the expressed and secreted hyaluronidase glycoprotein.

14. A pharmaceutical composition, comprising the hyaluronidase glycoprotein of claim 7 that consists of amino acids 36-482 of SEQ ID NO:

1.

15. A pharmaceutical composition, comprising the hyaluronidase glycoprotein of claim 7 and a pharmaceutical carrier.

16. The pharmaceutical composition of claim 15, wherein the concentration of the hyaluronidase glycoprotein is about 80 Units/ml.

17. The pharmaceutical composition of claim 15, wherein the concentration of the hyaluronidase glycoprotein is between about 1 Unit/ml and 5000 Units/ml.

18. The pharmaceutical composition of claim 15, wherein the concentration of the hyaluronidase glycoprotein is between about 1 Unit/ml and 300 Units/ml.

19. The pharmaceutical composition of claim 15, wherein the concentration of the hyaluronidase glycoprotein is between about 1 Unit/ml and 150 Units/ml.

20. The pharmaceutical composition of claim 15, further comprising calcium ions or magnesium ions.

21. The pharmaceutical composition of claim 15 that is formulated as a timed release composition or that is released upon contact with the site of application or targeted tissue.

22. The pharmaceutical composition of claim 15 that is formulated as a spray, foam, aerosol, liquid suspension or solution.

23. The pharmaceutical composition of claim 15 that is formulated as a tablet or capsule.

24. The pharmaceutical composition of claim 23, further comprising an enteric coating.

25. The pharmaceutical composition of claim 24, wherein the coating is selected from among cellulose acetate phthalate, polyethylene glycol, polyoxyethylene sorbitan, castor oil, ethyl cellulose pseudolatex, phenyl salicylate, n-butyl stearate, stearic acid and carnauba wax.

26. The pharmaceutical composition of claim 15 that is a lyophilized powder.

27. The pharmaceutical composition of claim 15 that is formulated for topical, local, enteric, parenteral, intracystal, intracutaneous, intravitreal, subcutaneous, intramuscular or intravenous administration.

28. The pharmaceutical composition of claim 15, further comprising a pharmaceutically active agent.

29. The pharmaceutical composition of claim 28, 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, an anti-arthritic 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, a cytokine, an insulin, a sleep inducer and an antibody or antigen-binding fragment thereof.

30. The pharmaceutical composition of claim 29, comprising a chemotherapeutic agent that is a toxin or a tumor necrosis factor.

31. The pharmaceutical composition of claim 29, wherein the anesthetic agent is lidocaine or bupivacaine.

32. The pharmaceutical composition of claim 29, wherein the hormone agent is epinephrine.

33. The pharmaceutical composition of claim 28, wherein the pharmaceutically active agent is selected from among a cytokine and an antibody, wherein the antibody is a monoclonal antibody.

34. The pharmaceutical composition of claim 28, wherein the pharmaceutically active agent is an insulin.

35. The pharmaceutical composition of claim 15, wherein the pharmaceutical carrier comprises a stabilizing solution.

36. The pharmaceutical composition of claim 35, wherein the stabilizing solution comprises NaCl, and CaCl₂ or MgCl₂.

37. The pharmaceutical composition of claim 35, wherein the stabilizing solution comprises ethylenediamine tetra-acetic acid (EDTA).

38. The pharmaceutical composition of claim 35, wherein the stabilizing solution comprises a carrier selected from among albumin, detergent and a surfactant.

39. The pharmaceutical composition of claim 35, wherein the stabilizing solution comprises phenol red, human serum albumin, HEPES, NaCl and a metal ion that is calcium ion or magnesium ion.

40. A sterile vial containing the pharmaceutical composition of claim 15.

41. The sterile vial of claim 40, wherein the sterile vial contains an amount of the pharmaceutical composition that is for single dose administration.

42. The sterile vial of claim 40, wherein the sterile vial further contains an amount of sterile water for injection;

and the concentration of the glycoprotein in the sterile vial is between about 1 and 5000 Units/ml.

43. A kit, comprising the combination of claim 40 and at least one of:

(a) a packaging material;

or

(b) instructions for using the kit for use of the pharmaceutical composition.

44. A sterile syringe containing the pharmaceutical composition of claim 27.

45. The sterile syringe of claim 44, wherein the concentration of the glycoprotein in the sterile syringe is between about 1 and about 5000 units/ml.

46. The sterile syringe of claim 44, wherein the pharmaceutical composition is dissolved in about 5 μ

l to about 50 μ

l of sterile water.

47. The sterile syringe of claim 44, wherein the sterile syringe has a volume of between about 5 to 50 μ

l.

48. A kit, comprising the sterile syringe of claim 44 and one or more of the following:

(a) packaging material; and

(b) instructions for using the kit for the use of the pharmaceutical composition.

49. A combination, comprising the sterile syringe of claim 44, and further comprising a second syringe containing a pharmaceutically active agent.

50. The combination of claim 49, 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, an anti-arthritic 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, a cytokine, an insulin, a sleep inducer and an antibody or antigen-binding fragment thereof.

51. The combination of claim 49, wherein the pharmaceutically active agent is selected from among a cytokine and an antibody.

52. The combination of claim 49, wherein the pharmaceutically active agent is an insulin.