Insoluble crosslinked cytotoxic oxidase-peroxidase system

• US 4,486,408 A
• Filed: 04/07/1981
• Issued: 12/04/1984
• Est. Priority Date: 04/07/1981
• Status: Expired due to Fees

• Current Assignee: United States Of America As Represented By The Secretary Of The Air Force
• Original Assignee: Intech Development, Inc.
• Inventors: Kiel, Johnathan L.
• Primary Examiner(s): Rosen, Sam
• Application Number:
  US06/251,694
• Time in Patent Office: 1,337 Days
• Field of Search: 424/94, 424/78, 435/174, 435/175, 435/177, 435/180, 435/181, 435/182
• US Class Current: 424/94.4
• 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 38/385 Serum albumin
• CPC Class Codes: A61K 38/44 Oxidoreductases (1)
• CPC Class Codes: A61K 38/443 acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
• CPC Class Codes: A61K 47/42 Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein oligopeptides having up to five amino acids A61K47/183; polyamino acids A61K47/34
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: A61K 9/06 Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels composition of ointments, creams or gels A61K47/00
• CPC Class Codes: A61K 9/14 Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles microspheres A61K9/16; microcapsules A61K9/50; nanocapsules, nanoparticles of the matrix type A61K9/51
• CPC Class Codes: A61K 9/146 with organic macromolecular compounds
• CPC Class Codes: A61K 9/19 lyophilised , i.e. freeze-dried, solutions or dispersions lyophilised products with subsequent particle size reduction A61K9/14; granules or pellets made by lyphilisation A61K9/1682; solid oral dosage forms made by lyophilisation A61K9/2095; lyophilisation additives A61K47/00
• CPC Class Codes: C12N 11/18 Multi-enzyme systems
• CPC Class Codes: C12Y 101/03004 Glucose oxidase (1.1.3.4)
• CPC Class Codes: C12Y 111/01007 Peroxidase (1.11.1.7), i.e. horseradish-peroxidase

Abstract

A therapeutic preparation, and a process of using the preparation, for the control of tumor growth and immune diseases. The preparation is an immobilized cytotoxic enzyme system which is carried by an insoluble substrate that is administered into the mammalian subject by injection or implantation. The preferred embodiment of the preparation uses a polymerized albumin carrier containing immobilized glucose oxidase and peroxidase. The immobilized enzyme system is prepared in the form of small particles that can be administered either by implantation or by injection into tumor-bearing mammals. To date, antibacterial activity has been demonstrated only in vitro. Intraperitoneal injection into rats bearing Novikoff hepatomas or into mice bearing B16-F1 melanomas or L1210 (Lymphoid) tumors has resulted in the selected killing of tumor tissues. Several daily administrations are required to achieve complete destruction of the tumor. Additionally, the preparation is also active as a stimulant of the specific immune response to neoplasia. Its effect on the immune system could be useful in the stimulation of the immune system in diseases where the action of the endogenous immune system is insufficient.

Claims

1. A process for preparing a composition of matter possessing cytotoxic activity toward prokaryotic and eukaryotic cells comprising the steps of:

a. mixing in a buffer solution;

a first enzyme which is an oxidase;

a second enzyme which is a peroxidase, wherein said second enzyme is capable of chemically cross-linking with said first enzyme, and wherein both enzymes remain active; and

a support;

b. adding a polymerizing agent to said solution; and

c. allowing said solution to polymerize; and

wherein the final composition of said composition of matter forms a cross-linked composition.

2. A process, as set forth in claim 1, wherein said process comprises the additional steps of:

a. cooling said polymerized solution until a gel is formed;

B. removing any soluble material in said gel, thereby resulting in an insoluble gel; and

c. drying said insoluble gel.

3. A process as set forth in claim 1, wherein said first enzyme is a glucose oxidase.

4. A process, as set forth in claim 1, wherein said second enzyme is horseradish peroxidase.

5. A process, as set forth in claim 1, wherein said second enzyme is lacto peroxidase.

6. A process, as set forth in claim 1, wherein said support is bovin serum albumin.

7. A process, as set forth in claim 1, wherein said support is human serum albumin.

8. A process, as set forth in claim 2, wherein said process further includes the additional steps of:

a. freezing said insoluble gel; and

b. lyophilizing said frozen insoluble gel.

9. A process, as set forth in claim 2, wherein said step of removing any soluble material in said gel comprises the constituent steps of:

a. soaking said gel in liquid water to remove any soluble material in said gel, thereby resulting in a mixture of an insoluble gel in a liquid medium;

b. filtering said mixture from said soaking to separate said insoluble gel from said liquid medium.

10. A process, as set forth in claim 8, wherein said process further includes the additional steps of:

a. grinding said lyophilized insoluble gel; and

b. mixing said ground lyophilized insoluble gel in a saline solution.

11. A composition of matter possessing cytotoxic activity toward prokaryotic and eukaryotic cells comprising:

a. a first enzyme which is an oxidase

b. a second enzyme which is a peroxidase, wherein said second enzyme is chemically cross-linked with said first enzyme, and wherein both enzymes remain active;

c. a support;

wherein said first enzyme, said second enzyme, and said support are mixed in a buffer solution; and

d. a polymerizing agent which is added to said solution, whereby said solution is polymerized; and

wherein the final composition of said composition of matter forms a cross-linked composition of matter.

12. A composition of matter, set forth in claim 11, wherein said polymerized solution is cooled to form a gel, and the gel is dryed, and soluble material is removed from said dried gel.

13. A composition of matter, as set forth in claim 11, wherein said first enzyme is a glucose oxidase.

14. A composition of matter, as set forth in claim 11, wherein said second enzyme is horseradish peroxidase.

15. A composition of matter, as set forth in claim 11, wherein said second enzyme is lacto peroxidase.

16. A composition of matter, as set forth in claim 11, wherein said support is bovin serum albumin.

17. A composition of matter, as set forth in claim 11, wherein said support is human serum albumin.

18. A composition of matter possessing cytotoxic activity toward prokaryotic and eukaryotic cells comprising:

a. a first enzyme which is an oxidase;

b. a second enzyme which is a peroxidase; and

c. a support;

wherein said first enzyme, said second enzyme, and said support are chemically cross-linked to form an insoluble material.

19. A composition of matter, as set forth in claim 18, wherein said composition comprises a solid polymer.

20. A composition of matter, as set forth in claim 18, wherein said first enzyme is a glucose oxidase.

21. A composition of matter, as set forth in claim 18, wherein said second enzyme is horseradish peroxidase.

22. A composition of matter, as set forth in claim 18, wherein said second enzyme is lacto peroxidase.

23. A composition of matter, as set forth in claim 18, wherein said support is bovine serum albumin.

24. A composition of matter, as set forth in claim 18, wherein said support is human serum albumin.

25. A process for cytolysis of prokaryotic and eukaryotic cells in living organisms comprising the steps of:

a. preparing a mixture of a support and a composition of matter possessing cytolysis activity toward prokaryotic and eukeryotic cells in living organisms, wherein said composition of matter is cross-linked with said support and is formed by;

mixing in a buffer solution a first enzyme which is an oxidase, a second enzyme which is a peroxidase, and said support; and

polymerizing said composition of matter by adding a polymerizing agent thereto such that the final composition of said composition of matter forms a cross-linked composition; and

b. inserting said mixture into a living organism.

26. A process, as set forth in claim 25, wherein after performing the step of preparing a mixture of a support and of a composition of matter and prior to performing the step of inserting said mixture into a living organism, the following additional steps are performed:

a. cooling said composition of matter to form a gel;

b. drying said gel; and

c. removing all soluble material from said gel.

27. A process, as set forth in claim 25, wherein said living organism is selected from the group of animals known as mammals.

28. A process, as set forth in claim 25, wherein said mammal is a human.

29. A process, as set forth in claim 25, wherein said cytolytic activity is directed against neoplasia.

30. A process, as set forth in claim 25, wherein the step of inserting said mixture into a living organism is performed by infusing said mixture into said living organism, such that said infused mixture stimulates the activity of the endogeneous immune system of said living organism.

31. A composition of matter possessing cytotoxic activity toward prokaryotic and eukaryotic cells comprising:

a. a peroxidase selected from the group consisting of horseradish peroxidase, lactoperoxidase, myeloperoxidase and snake skin peroxidase;

b. an enzyme that produces hydrogen peroxide or superoxide in sufficient amounts and for a sufficient period of time as to maintain the cytotoxicity of said peroxidase; and

c. a support;

wherein said peroxidase, said enzyme, and said support form a cross-linked composition.

32. A composition of matter, as set forth in claim 31, wherein said support comprises a protein.

33. A composition of matter, as set forth in claim 32, wherein said protein is albumin.

34. A composition of matter, as set forth in claim 31, wherein said enzyme is glucose oxidase.

35. A composition of matter, as set forth in claim 31, wherein said enzyme is xanthine oxidase.

36. A composition of matter, as set forth in claim 31, wherein said peroxidase, said enzyme, and said support are dissolved in a buffer solution, and a polymerizing agent is added to said solution, whereby said solution is polymerized.

37. A composition of matter, as set forth in claim 36, wherein said polymerized solution is cooled to form a gel, and the gel is dried, and soluble material is removed from said dried gel.

38. A solid composition of matter possessing cytotoxic activity against tumor cells upon administration to a living tumor-bearing subject comprising:

a. a first enzyme selected from the group of enzymes which in the presence of hydrogen peroxide and a halide ion possess cytolytic activity toward prokaryotic and eukaryotic cells;

b. a second enzyme selected from the group consisting of enzymes that produce hydrogen peroxide or superoxide in sufficient amounts and for a sufficient period of time as to maintain the cytotoxicity of said first enzyme; and

c. a support wherein said first and second enzymes are crosslinked to said support.

39. A composition of matter, as set forth in claim 38, wherein said first enzyme is horseradish peroxidase.

40. A composition of matter, as set forth in claim 38, wherein said first enzyme is lactoperoxidase.

41. A composition of matter, as set forth in claim 38, wherein said first enzyme is myeloperoxidase.

42. A composition of matter, as set forth in claim 38, wherein said first enzyme is snake skin peroxidase.

43. A composition of matter, as set forth in claim 38, wherein said first enzyme is peroxidase.

44. A composition of matter, as set forth in claim 38, wherein said second enzyme is glucose oxidase.

45. A composition of matter, as set forth in claim 38, wherein said support is a protein.

46. A composition of matter, as set forth in claim 45, wherein said protein is albumin.