Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases

• US 7,871,607 B2
• Filed: 02/23/2005
• Issued: 01/18/2011
• Est. Priority Date: 03/05/2003
• Status: Active Grant

• Current Assignee: Halozyme, Inc. (Halozyme Therapeutics, Inc.)
• Original Assignee: Halozyme, Inc. (Halozyme Therapeutics, Inc.)
• Inventors: Frost, Gregory I., Kundu, Anirban, Bookbinder, Louis H.
• Primary Examiner(s): Nashed; Nashaat T
• Application Number:
  US11/065,716
• Publication Number:
  US 20050260186A1
• Time in Patent Office: 2,155 Days
• Field of Search: None
• US Class Current: 424/94.62
• 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 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 method for increasing the diffusion of an antibiotic in a tissue of a subject, comprising:

administering to the tissue of a subject a composition containing a hyaluronidase glycoprotein in an amount sufficient to increase diffusion of the antibiotic, wherein;

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 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;

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

the glycoprotein is active at neutral pH; and

administering to the tissue an antibiotic.

2. The method of claim 1, wherein the antibiotic is a small molecule or is a protein.

3. The method of claim 1, wherein the antibiotic is a polypeptide.

4. The method of claim 1, wherein the antibiotic agent or combination of agents is selected from among Aminoglycosides;

Amphenicols;

Ansamycins;

Carbacephems;

Carbapenems;

Cephalosporins;

Cephems;

Cephamycins;

Clavams;

Cyclic lipopeptides;

Diaminopyrimidines;

Ketolides;

Lincosamides;

Macrolides;

Monobactams;

Nitrofurans;

Oxacephems;

Oxazolidinones;

Penems;

thienamycins;

beta-lactams;

Penicillins;

Polypeptides antibiotics;

Quinolones;

Sulfonamides;

Sulfones;

Tetracyclines;

Clofoctols;

Fusidic acids;

Hexedines;

Methenamines;

Nitrofurantoins;

Nitroxolines;

Ritipenems;

Taurolidines and Xibomols.

5. The method of claim 1, wherein the glycoprotein is administered at a dose between 1 and 5,000,000 turbidity reducing units (TRU).

6. The method of claim 1, wherein the glycoprotein consists of the sequence of amino acids set forth in SEQ ID NO. 4.

7. The method of claim 1, wherein the tissue is skin, the subcutaneous layer of the skin or a muscle.

8. The method of claim 1, wherein the glycoprotein is modified with a polymer.

9. The method of claim 8, wherein the polymer is PEG or dextran.

10. The method of claim 1, wherein the glycoprotein is encoded by a nucleic acid molecule that encodes amino acids 36-482 of SEQ ID NO:

1.

11. The method of claim 1, wherein the hyaluronidase glycoprotein in the composition consists of the sequence of amino acids set forth as amino acids 36-482 of SEQ ID NO. 1.

12. The method of claim 1, wherein the sequence of amino acids residues of the glycoprotein is selected from among 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 in SEQ ID NO:

1.

13. The method of claim 1, wherein the antibiotic agent is selected from among Ampicillins, Azithromycins, Becaplermins, Cefazolins, Cefepimes, Cefotetans, Ceftazidimes, Ceftriaxones, Clavulanic Acids, Clindamycins, Gatifloxacins, Levofloxacins, Meropenems, Piperacillins, Sulbactams, Tazobactams, Ticarcillins, and Vancomycins.

14. The method of claim 13, wherein the glycoprotein is modified with a polymer.

15. The method of claim 14, wherein the polymer is PEG or dextran.

16. The method of claim 13, wherein the sequence of amino acid residues of the glycoprotein is set forth in SEQ ID NO:

4.

17. The method of claim 13, wherein the glycoprotein is encoded by a nucleic acid molecule that encodes amino acids 36-482 of SEQ ID NO:

1.

18. The method of claim 13, wherein the hyaluronidase glycoprotein in the composition consists of the sequence of amino acid residues 36-482 of SEQ ID NO. 1.

19. The method of claim 13, wherein the sequence of amino acids residues of the glycoprotein is selected from among 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 in SEQ ID NO:

1.

20. The method of claim 1, wherein the glycoprotein is administered separately or is co-formulated with the antibiotic.

21. The method of claim 1, wherein the glycoprotein is administered prior to, simultaneously with or following administration of the antibiotic.

22. The method of claim 1, wherein the glycoprotein and antibiotic are co-formulated and administered as an aerosol.

23. The method of claim 1, wherein the antibiotic is selected from among a Aclacinomycins;

Aclarubicins;

Actinomycins;

Adriamycins;

Alatrofloxacins;

Ambomycins;

Ametantrones;

Amikacins;

Anthramycins;

Asperlins;

Azotomycins;

Bacitracins;

Bleomycins;

Cactinomycins;

Capreomycins;

Carubicins;

Cefotaximes;

Cefoxitins;

Ceftizoximes;

Cefuroximes;

Chloramphenicols;

Chromomycins;

Ciprofloxacins;

Cirolemycins;

Dactinomycins;

Daunorubicins;

Daunomycins;

Doxorubicins;

Doxorubicin liposomals;

Doxycyclines;

Duazomycins;

Elsamitrucins;

Epirubicins;

Ertapenems;

Erythromycins;

Esorubicins;

Fepirubicins;

Fostriecins;

Galarubicins;

Gentamicins;

Guamecyclines;

Idarubicins;

Kanamycins;

Levamisoles;

Lincomycins;

Mithramycins;

Minocyclines;

Mitocarcins;

Mitocromins;

Mitomalcins;

Mitomycin C;

Mitoxantrones;

Nafcillins;

Nemorubicins;

Nogalamycins;

Olivomycins;

Oxacillinsl ;

Oxytetracyclines;

Peliomycins;

Peplomycins;

Pirarubicins;

Plicamycins;

Polymyxin b;

Porfiromycins;

Puromycins;

Pyrazofurins;

Rifampins;

Rufocromomycins;

Sabarubicins;

Sparsomycins;

Streptonigrins;

Spectinomycins;

Streptomycins;

Streptozocins;

Talisomycins;

Tubercidins;

Tobramycins;

Valrubicins;

Vinblastines;

Vincristines;

Xanthomycin A;

Zinostatins; and

Zorubicins.

24. The method of claim 23, wherein the glycoprotein is modified with a polymer.

25. The method of claim 24, wherein the polymer is PEG or dextran.

26. The method of claim 23, wherein the sequence of amino acid residuess of the glycoprotein is set forth in SEQ ID NO:

4.

27. The method of claim 23, wherein the glycoprotein is encoded by a nucleic acid molecule that encodes amino acids 36-482 of SEQ ID NO:

1.

28. The method of claim 23 , wherein the hyaluronidase glycoprotein in the composition consists of the sequence of amino acids 36-482 of SEQ ID NO. 1.

29. The method of claim 23, wherein the sequence of amino acid residuess of the glycoprotein is selected from among 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 in SEQ ID NO:

1.

30. The method of claim 1, wherein the hyaluronidase glycoprotein is produced in and secreted from a mammalian cell.

31. The method of claim 30, wherein the mammalian cell is a CHO cell.

32. The method of claim 1, wherein the glycoprotein and antibiotic are co-formulated and administered as a liquid solution.