Claims for Patent: 8,389,492

Summary for Patent: 8,389,492

Title:	Lyophilized DNA formulations for enhanced expression of plasmid DNA
Abstract:	The present invention provides for a method of treating or preventing ischemic or liver disease in a subject by administering a composition reconstituted from a lyophilized hepatocyte growth factor (HGF) DNA formulation, where the DNA formulation comprises an HGF plasmid DNA, salt and a carbohydrate. The invention further provides for a method of making such a lyophilized DNA formulation that preserves or enhances gene expression both in vitro and in vivo, thus maintaining or stimulating the biological activity of the expressed protein. The invention also provides for the DNA formulation, or the lyophilized DNA formulation according to the methods disclosed.
Inventor(s):	Kim; Jong-Mook (Seoul, KR), Kim; Sujeong (Seoul, KR), Hahn; Woong (Goyang-si, KR), Yoo; WonSun (Seoul, KR)
Assignee:	ViroMed Co., Ltd. (KR)
Application Number:	13/045,460
Patent Claims:	1. A method of administering a DNA composition to a subject comprising administering to said subject a composition reconstituted from a lyophilized DNA formulation comprising, prior to lyophilization, (a) a plasmid DNA comprising a hybrid HGF construct wherein the hybrid HGF construct comprises: (i) a first cDNA which has the same sequence as exons 1-4 of the human HGF gene wherein said exons 1-4 are arranged in sequential order without an intron therebetween, or degenerates thereof which do not alter the amino acid sequence encoded by said first cDNA, (ii) a polynucleotide that has the same sequence as intron 4 of a HGF gene or a fragment thereof, and (iii) a second cDNA which has the same sequence as exons 5-18 of the human HGF gene wherein said exons 5-18 are arranged in sequential order without an intron therebetween, or degenerates thereof which do not alter the amino acid sequence encoded by said second cDNA, wherein (ii) is located between (i) and (iii) and the hybrid HGF construct simultaneously encodes two heterotypes of human HGF, wherein said plasmid DNA is not complexed to a liposome, (b) a salt and (c) a carbohydrate, wherein said administration is intramuscular and HGF expression from the reconstituted lyophilized DNA formulation is higher than expression from said hybrid HGF construct in non-lyophilized form. 2. The method of claim 1, wherein said DNA composition is administered to said subject for treating or preventing an ischemic disease or a liver disease in said subject. 3. The method of claim 1, wherein said carbohydrate is a mono-, oligo-, or polysaccharide selected from the group consisting of sucrose, glucose, lactose, trehalose, arabinose, pentose, ribose, xylose, galactose, hexose, idose, mannose, talose, heptose, fructose, gluconic acid, sorbitol, mannitol, methyl a-glucopyranoside, maltose, lactone, sorbose, glucaric acid, erythrose, arabinose, allose, altrose, gulose, erythrulose, ribulose, xylulose, psicose, tagatose, glucuronic acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, neuraminic acid, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans, levan, fucoidan, carrageenan, galactocarolose, pectins, pectic acids, amylose, pullulan, glycogen, amylopectin, cellulose, dextran, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xantham gum, starch, and admixtures thereof. 4. The method of claim 3, wherein said carbohydrate is selected from the group consisting of sucrose, mannitol, and admixtures thereof. 5. The method of claim 1, wherein said carbohydrate is in an amount of between about 0.05% to about 30%. 6. The method of claim 1, wherein said salt is selected from the group consisting of NaCl, KCl, and admixtures thereof. 7. The method of claim 1, wherein said salt is in an amount of between about 0.01% and 10%. 8. The method of claim 1, wherein said hybrid HGF construct is selected from the group consisting of HGF-X2, HGF-X3, HGF-X6, HGF-X7, and HGF-X8. 9. The method of claim 8, wherein said hybrid HGF construct is HGF-X7. 10. The method of claim 1, wherein said plasmid DNA is selected from the group consisting of: pCK-HGF-X2, pCK-HGF-X3, pCK-HGF-X6, pCK-HGF-X7, pCK-HGF-X8, pCP-HGF-X2, pCP-HGF-X3, pCP-HGF-X6, pCP-HGF-X7, and pCP-HGF-X8. 11. The method of claim 1, wherein said plasmid DNA of the reconstituted composition is at a concentration of from about 1 ng/mL to about 30 mg/mL. 12. The method of claim 1, wherein said lyophilized DNA formulation is reconstituted in a pharmaceutically acceptable solution. 13. The method of claim 12, wherein said pharmaceutically acceptable solution is selected from the group consisting of water, PBS, TE, Tris buffer, and normal saline. 14. The method of claim 1, wherein said plasmid DNA is a naked DNA. 15. The method of claim 1, wherein said reconstituted composition is administered by direct injection to the muscle. 16. The method of claim 2, wherein the ischemic disease is coronary artery disease (CAD) or peripheral artery disease (PAD). 17. A method of administering a DNA composition to a subject comprising administering to said subject a composition reconstituted from a lyophilized DNA formulation comprising, prior to lyophilization, (a) a plasmid DNA comprising a hybrid HGF construct wherein the hybrid HGF construct comprises: (i) a first cDNA which has the same sequence as exons 1-4 of the human HGF gene wherein said exons 1-4 are arranged in sequential order without an intron therebetween, or degenerates thereof which do not alter the amino acid sequence encoded by said first cDNA, (ii) a polynucleotide that has the same sequence as intron 4 of a HGF gene or a fragment thereof, and (iii) a second cDNA which has the same sequence as exons 5-18 of the human HGF gene wherein said exons 5-18 are arranged in sequential order without an intron therebetween, or degenerates thereof which do not alter the amino acid sequence encoded by said second cDNA, wherein (ii) is located between (i) and (iii) and the hybrid HGF construct simultaneously encodes two heterotypes of human HGF, wherein said plasmid DNA is non-complexed, (b) a salt selected from the group consisting of NaCl and KCl in an amount of between about 0.01% and 10%, (c) a carbohydrate selected from the group consisting of sucrose, glucose, lactose, trehalose, arabinose, pentose, ribose, xylose, galactose, hexose, idose, mannose, talose, heptose, fructose, gluconic acid, sorbitol, mannitol, methyl a-glucopyranoside, maltose, lactone, sorbose, glucaric acid, erythrose, arabinose, allose, altrose, gulose, erythrulose, ribulose, xylulose, psicose, tagatose, glucuronic acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, neuraminic acid, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans, levan, fucoidan, carrageenan, galactocarolose, pectins, pectic acids, amylose, pullulan, glycogen, amylopectin, cellulose, dextran, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xantham gum, and starch in an amount of between about 0.05% to about 30%, wherein said administration is intramuscular and HGF expression from the reconstituted lyophilized DNA formulation is higher than expression from said hybrid HGF construct in non-lyophilized form. 18. The method of claim 17, wherein said hybrid HGF construct is selected from the group consisting of HGF-X2, HGF-X3, HGF-X6, HGF-X7, and HGF-X8. 19. The method of claim 17, wherein said DNA composition is administered to said subject for treating or preventing an ischemic disease or a liver disease in said subject. 20. The method of claim 17, wherein said reconstituted composition is administered by direct injection to the muscle.

Details for Patent 8,389,492

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2028-04-09
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2028-04-09
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2028-04-09