Non-immunogenic polypeptides

• US 4,179,337 A
• Filed: 07/28/1977
• Issued: 12/18/1979
• Est. Priority Date: 07/20/1973
• Status: Expired due to Term

• Current Assignee: Research Corporation Technologies Incorporated A Not-For-Profit Non-Stock Corporation of de
• Original Assignee: Frank F. Davis, Nicholas C. Palczuk, Theodorus Van Es
• Inventors: Palczuk, Nicholas C., Davis, Frank F., Van Es, Theodorus
• Primary Examiner(s): Naff, David M.
• Application Number:
  US05/819,831
• Time in Patent Office: 873 Days
• Field of Search: 195/63, 195/68, 195/DIG. 11, 424/94, 424/177, 424/178, 424/78, 260/112.5, 260/112 R, 260/6, 260/8, 260/112.7
• US Class Current: 435/181
• 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 38/44 Oxidoreductases (1)
• CPC Class Codes: A61K 38/446 Superoxide dismutase (1.15)
• CPC Class Codes: A61K 38/45 Transferases (2)
• CPC Class Codes: A61K 38/47 acting on glycosyl compounds (3.2), e.g. cellulases, lactases
• CPC Class Codes: A61K 38/4826 Trypsin (3.4.21.4) Chymotrypsin (3.4.21.1)
• CPC Class Codes: A61K 38/51 Lyases (4)
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: C07K 14/62 Insulins
• CPC Class Codes: C08G 65/329 with organic compounds
• CPC Class Codes: C12N 11/089 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
• CPC Class Codes: Y10S 530/816 Attached to the carrier via a bridging agent

Abstract

Polypeptides such as enzymes and insulin are coupled to polyethylene glycol or polypropropylene glycol having a molecular weight of 500 to 20,000 daltons to provide a physiologically active non-immunogenic water soluble polypeptide composition. The polyethylene glycol or polypropylene glycol protect the polypeptide from loss of activity and the composition can be injected into the mammalian circulatory system with substantially no immunogenic response.

Claims

1. A physiologically active, substantially non-immunogenic water soluble polypeptide composition comprising, a physiologically active polypeptide coupled with a coupling agent to

at least one substantially linear polymer having a molecular weight of between about 500 to about 20,000 daltons selected from the group consisting of polyethylene glycol and polypropropylene glycol

wherein the polymer is unsubstituted or substituted by alkoxy or alkyl groups, said alkoxy or alkyl group possessing less than 5 carbon atoms.

2. The polypeptide composition of claim 1 wherein the polypeptide is selected from the group consisting of insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, etythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalmic releasing factors, antidiuretic hormones, thyroid stimulating hormone and prolactin.

3. The polypeptide composition of claim 2 wherein the polypeptide is insulin.

4. The polypeptide composition of claim 1 wherein the polymer has a molecular weight of between about 750 and 2,000 daltons.

5. The polypeptide composition of claim 4 which comprises between 10 and 100 polymer moieties per molecule of insulin.

6. The polypeptide composition of claim 1 wherein the polymer is polyethylene glycol.

7. The polypeptide composition of claim 4 wherein the polypeptide is insulin.

8. The polypeptide composition of claim 6 wherein the polypeptide is insulin.

9. The polypeptide composition of claim 1 wherein the group between the polypeptide and the polymer has the formula selected from the group consisting of ##STR14## wherein the bond designated--·

of each group is attached to the polypeptide provided that in the case where the group is ##STR15## portion is part of the polypeptide carboxy group.

10. The polypeptide composition of claim 9 wherein the polypeptide is insulin.

11. The polypeptide composition of claim 10 wherein the group is ##STR16##

12. The polypeptide composition of claim 11 wherein the polymer is polyethylene glycol.

13. A substantially non-immunogenic insulin-containing composition comprising the insulin composition of claim 3 and a pharmaceutically acceptable injectable carrier.

14. A process of preparing a physiologically active, substantially non-immunogenic water soluble polypeptide composition from a polypeptide;

originally having physiological activity and immunogenicity which comprises the steps of;

(a) reacting at least one terminal carbon atom bearing a hydroxy group, of a substantially straight chain polymer selected from the group consisting of polyethylene glycol and polypropylene glycol having a molecular weight of from about 500 to about 20,000 daltons said polymer being unsubstituted or substituted by alkoxy or alkyl groups, said alkoxy or alkyl groups having less than 5 carbon atoms with a coupling agent to provide an activated polymer containing a reactive terminal group, and;

(b) reacting a physiologically active immunogenic poly peptide with from 10 to 100 moles of said activated polymer per mole of poly peptide by coupling said polypeptide to the reactive terminal group of the polymer to provide said water soluble polypeptide composition.

15. A process of claim 14 wherein the coupling agent is cyanuric chloride.

16. A process of claim 15 wherein the polypeptide is insulin.

17. A physiologically active substantially non-immunogenic water soluble enzyme composition comprising a physiologically active enzyme coupled with a coupling agent to

at least one substantially linear polymer selected from the group consisting of polyethylene glycol and polypropylene glycol having molecular weight of between 500 and 20,000 daltons

wherein the polymer moiety is unsubstituted or substituted by alkoxy or alkyl groups, said alkoxy or alkyl group possessing less than 5 carbon atoms.

18. The enzyme composition of claim 17 wherein the polymer has a molecular weight of between about 500 and about 5,000 daltons.

19. The enzyme composition of claim 17 which comprises between 10 and 100 polymer moieties per molecule of enzyme.

20. The enzyme composition of claim 17 wherein the polymer is polyethylene glycol.

21. The enzyme composition of claim 17 wherein the enzyme is selected from the group consisting of oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases.

22. The enzyme composition of claim 17 wherein the group between the enzyme and the polymer has the formula selected from the group consisting of ##STR17## wherein the bonds designated--·

of each group is attached to the enzyme provided that in the case where the group is ##STR18## portion is part of the polypeptide carboxy group.

23. A process of preparing a physiologically active, substantially non-immunogenic water soluble enzyme composition from an enzyme originally having physiological activity and immunogenicity which comprises the steps of:

(a) reacting at least one terminal carbon atom bearing a hydroxy group, of a substantially straight chain polymer selected from the group consisting of polyethylene glycol and polypropylene glycol having a molecular weight of from about 500 to about 20,000 daltons said polymer being unsubstituted or substituted by alkoxy or alkyl groups, said alkoxy or alkyl groups having less than 5 carbon atoms with a coupling agent to provide an activated polymer containing a reactive terminal group, and

(b) reacting a physiologically active immunogenic enzyme with from 10 to 100 moles of said activated polymer per mole of enzyme by coupling the enzyme to the reactive terminal group of said polymer to provide said water soluble enzyme composition.

24. The process of claim 23 wherein the enzyme is selected from the group consisting of oxidoreductases, transrerases, hydrolases, lysases, isomerases, and ligases.

25. A process of claim 24 wherein the coupling moiety is cyanuric chloride.

26. A process of claim 24 wherein the polymer is polyethylene glycol.

27. A process of claim 26 wherein the coupling moiety is cyanuric chloride.