Claims for Patent: 5,187,260

Summary for Patent: 5,187,260

Title:	Process for the preparation of a high purity protamine-DNA complex and process for use of same
Abstract:	A process is disclosed in which high purity protamine-DNA complexes are prepared by collecting nucleoprotamines specific developmental stages of a life form, specifically, amphibian, egg by low temperature processing. The process also includes the steps of sequential homogenization in a high concentration aqueous salt solution at a buffered low pH, followed by ultracentrifugation to remove insoluble matter. Either a crude mixture or pure isolate of the complexes may be produced. Pure isolates require aqueous chloroform extraction to isolate protein and to remove lipids. Lyophilization then removes chloroform and excess water. The isolate is then fractionated by single pass alumina chromatography. Dialysis against pure water removes salts. Repeated lyophilization removes excess water and concentrates single protamines and protamine-like proteins. The mixture may then be reconstituted with 5% weight/volume heterologous or homologous DNA, in order to shield from charge toxicity. Crude mixtures may be produced by precipitating the supernate of ultracentrifugation in pure water, followed by ultracentrifugation to sediment in solids. Lyophilization then removes any water from the damp solids. The crude solids are suitable for oral use, especially if utilized in gelatin capsules. Sterile filtration to injection quality aqueous form. Following isolation of the protamine-DNA complex, encapsulation of the prepared solid or aqueous protamine-DNA complexes in a specific carrier substance may be accomplished, depending upon the target tissue for the protamine. Several encapsulation carriers are known from prior art literature, such as, for example, liposomes and nanoparticles. The protamine-DNA complexes of the present invention are useful in inhibiting tumor growth, among other uses.
Inventor(s):	Karali; Sharifa (Flushing, NY), Barberii; John K. (Tucson, AZ)
Assignee:
Application Number:	07/685,593
Patent Claims:	1. A process for providing a high-purity protamine-DNA complex, consisting essentially of the sequential steps of: collecting and treating a nucleoprotamine from a developmental stage of a life form by homogenization in an aqueous buffered salt solution at a pH of approximately 2.2 to obtain a mixture; removing insoluble matter from the mixture of said collecting and treating step; isolating protein from the mixture by a first aqueous chloroform extraction; removing lipids from the isolated protein by a second chloroform aqueous extraction; performing dialysis of the protein obtained by the second extraction, against sterile water, to remove excess salt; and reconstituting the dialyzed protein with 5% weight/volume heterologous or 5% weight/volume homologous DNA; and, sterile filtration to obtain an aqueous protamine-DNA complex. 2. The process according to claim 1, further consisting essentially of the step of separating of discrete protein peaks by chromatography. 3. The process according to claim 1, wherein said developmental stage of said life form is fertilized egg. 4. The process according to claim 1, wherein said removing of insoluble matter from said mixture occurs by ultracentrifugation. 5. The process according to claim 1, further consisting essentially of the step of encapsulating said aqueous protamine-DNA complex in a carrier substance. 6. The process according to claim 1, further consisting essentially of the steps of: precipitating said aqueous protamine-DNA complex in water; and, lyophilizing to remove said water and to produce a solid form of the protamine-DNA complex. 7. The process according to claim 6, further consisting essentially of the step of encapsulating said solid protamine-DNA complex in a carrier substance. 8. A process for providing a high-purity protamine-DNA complex, consisting essentially of the sequential steps of: collecting and treating a nucleoprotamine from any developmental stage of a life form by homogenization in a buffered aqueous 4M salt solution at a pH of approximately 2.2 to obtain a mixture; removing insoluble matter from the mixture of said collecting and treating step; isolating protein from the mixture by a first aqueous chloroform extraction; removing lipids from the isolated protein by a second aqueous chloroform extraction; reconstituting the protein obtained by the second extraction with heterogenous DNA of a target tissue; performing dialysis of the reconstituted protein, against sterile water, to remove excess salt; and, sterile filtration to obtain an aqueous protamine-DNA complex. 9. The process according to claim 8, further consisting essentially of the step of separating of discrete protein peaks by chromatography. 10. The process according to claim 9, wherein said chromatography is alumina and is developed by an aqueous K.sub.2 HPO.sub.4 buffer. 11. The process according to claim 8, wherein said developmental stage of said life form is egg. 12. The process according to claim 8, wherein said salt solution is sodium chloride and said buffer is sodium citrate. 13. The process according to claim 8, wherein said removing of insoluble matter from said mixture occurs by ultracentrifugation. 14. The process according to claim 8, further consisting essentially of the step of encapsulating said aqueous protamine-DNA complex in a carrier substance. 15. The process according to claim 8, further consisting essentially of the steps of: precipitating said aqueous protamine-DNA complex in water; and, lyophilizing to remove said water and to produce a solid form of the protamine-DNA complex. 16. The process according to claim 15, further consisting essentially of the step of encapsulating said solid protamine-DNA complex in a carrier substance. 17. A process for providing a high-purity protamine-DNA complex, consisting essentially of the sequential steps of: collecting and treating a nucleoprotamine from a developmental stage of a life form by homogenization in an aqueous buffered salt solution to obtain a mixture; removing insoluble matter from the mixture of said collecting and treating step; isolating protein and removing lipids from the mixture by a single aqueous chloroform extraction; performing dialysis of the protein obtained by the second extraction, against sterile water, to remove excess salt; and, reconstituting the dialyzed protein with 5% weight/volume heterologous or homologous DNA; and, sterile filtration to obtain an aqueous protamine-DNA complex. 18. The process according to claim 17, further consisting essentially of the step of separating of discrete protein peaks by chromatography. 19. The process according to claim 17, wherein said developmental stage of said life form is fertilized egg. 20. The process according to claim 17, wherein said removing of insoluble matter from said mixture occurs by ultracentrifugation. 21. The process according to claim 17, further consisting essentially of the step of encapsulating said aqueous protamine-DNA complex in a carrier substance. 22. The process according to claim 17, further consisting essentially of the steps of: precipitating said aqueous protamine-DNA complex in water; and, lyophilizing to remove said water and to produce a solid form of the protamine-DNA complex. 23. The process according to claim 22, further consisting essentially of the step of encapsulating said solid protamine-DNA complex in a carrier substance. 24. A process for providing a high-purity protamine-DNA complex, consisting essentially of the sequential steps of: collecting and treating a nucleoprotamine from any developmental stage of a life form by homogenization in a buffered aqueous salt solution, to obtain a mixture; removing insoluble matter from the mixture of said collecting and treating step; isolating protein and removing lipids from the mixture by a single aqueous chloroform extraction; reconstituting the protein obtained by the second extraction with heterogenous DNA of a target tissue; performing dialysis of the reconstituted protein, against distilled water, to remove excess salt; and, sterile filtration to obtain an aqueous protamine-DNA complex. 25. The process according to claim 24, further consisting essentially of the step of separating of discrete protein peaks by chromatography. 26. The process according to claim 25, wherein said chromatography is alumina and is developed by an aqueous K.sub.2 HPO.sub.4 buffer. 27. The process according to claim 24, wherein said developmental stage of said life form is egg. 28. The process according to claim 24, wherein said salt solution is sodium chloride and said buffer is citric acid. 29. The process according to claim 24, wherein said removing of insoluble matter from said mixture occurs by ultracentrifugation. 30. The process according to claim 24, further consisting essentially of the step of encapsulating said aqueous protamine-DNA complex in a carrier substance.

Details for Patent 5,187,260

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	ZOSTAVAX	zoster vaccine live	For Injection	125123	05/25/2006		2010-02-16