Pharmaceutical composition for stimulation of angiogenesis

• US 9,616,103 B2
• Filed: 08/02/2013
• Issued: 04/11/2017
• Est. Priority Date: 08/31/2012
• Status: Active Grant

• Current Assignee: NextGen Company Limited (Human Stem Cells Institute OJSC)
• Original Assignee: NextGen Company Limited (Human Stem Cells Institute OJSC)
• Inventors: Deev, Roman Vadimovich, Kiselev, Sergej L'vovich, Vorob'ev, Ivan Ivanovich, Orlova, Nadezhda Aleksandrovna, Isaev, Artur Aleksandrovich
• Primary Examiner(s): Marvich, Maria
• Application Number:
  US14/423,532
• Publication Number:
  US 20150335711A1
• Time in Patent Office: 1,348 Days
• Field of Search: None
• CPC Class Codes: A61K 38/18 Growth factors; Growth regulators
• CPC Class Codes: A61K 38/1866 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: A61K 47/26 Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/515 Angiogenesic factors; Angiogenin

Abstract

Provided is a pharmaceutical composition for growth induction in blood vessel tissue which contains purified plasmid DNA encoding a vascular endothelial growth factor (VEGF) and pharmaceutically acceptable excipients that include a cryoprotectant as a vehicle and/or a pH stabilizer for stabilizing the pH in the range of 7.0 to 9.0, in effective quantities. Also provided is a storage method of plasmid DNA which encodes a VEGF comprising mixing the purified plasmid DNA with a solution of at least one cryoprotectant having properties of a vehicle and/or a pH stabilizer in the pH range of 7.0-9.0. The solution is lyophilized and stored at +2 to +8° C. Supercoiled DNA pCMV-VEGF165 may be used which is produced by culturing Esherichia coli strain TOP10/pCMV-VEGF165. The pharmaceutical composition is administered to a human in quantities sufficient to provide a necessary therapeutic effect. The provided pharmaceutical composition of plasmid DNA pCMV-VEGF165 does not change significantly properties of the active substance when stored for a long time at a temperature from +2 to +8° C.

Claims

1. A pharmaceutical composition for induction of blood vessel tissue growth, the composition comprising:

purified plasmid DNA encoding a human recombinant vascular endothelial growth factor (VEGF), wherein the VEGF encoding sequences are operatively linked to expression control sequences to provide gene expression in human cells, and from 200 to 400 mM of glucose and from 3 to 30 mM of sodium phosphate, wherein the pharmaceutical composition is an isotonic solution, and wherein a concentration of the purified plasmid DNA in the composition is from 0.1 to 10 mg/ml, and a pH of the composition is form 7.0 to 9.0.

2. The pharmaceutical composition according to claim 1, wherein the pH of the composition is from 7.8 to 9.0.

3. The pharmaceutical composition according to claim 2, comprising from 5 to 20 mM of the sodium phosphate.

4. The pharmaceutical composition according to claim 1, wherein the pH of the composition is from 7.4 to 8.5.

5. The pharmaceutical composition according to claim 1, wherein the pH of the composition is from 7.4 to 8.2.

6. The pharmaceutical composition according to claim 1, wherein the concentration of the purified plasmid DNA in the composition is from 0.5 to 4 mg/ml.

7. The pharmaceutical composition according to claim 1, wherein the concentration of the purified plasmid DNA in the composition is from 0.8 to 1.2 mg/ml.

8. The pharmaceutical composition according to claim 1, wherein the purified plasmid DNA is pCMV-VEGF165.

9. The pharmaceutical composition according to claim 1, wherein the purified plasmid DNA comprises the polynucleotide sequence of SEQ ID NO:

1.

10. The pharmaceutical composition according to claim 1, wherein not less than 70% of the purified plasmid DNA is present in a supercoiled form.

11. The pharmaceutical composition according to claim 1, wherein the the purified plasmid DNA comprises

not more than 5% of impurities selected from the group consisting of a relaxed ring form, a linear plasmid form, and a mixture thereof, not more than 1% of genomic DNA, not more than 1% of RNA, not more than 0.1% of total protein, and

not more than 50 EU of an endotoxin per 1 mg of an active substance.

12. A pharmaceutical composition for induction of blood vessel tissue growth the composition comprising

from 0.8 to 1.2 mg/ml of the purified plasmid DNA comprising the polynucleotide sequence of SEQ ID NO:

1;

from 280 to 320 mM of dextrose; and

from 8 to 12 mM of sodium phosphate,

wherein the pH of the composition is from 7.4 to 8.2.

13. A method for storage of purified plasmid DNA which encodes a human recombinant vascular endothelial growth factor (VEGF), the method comprising:

adding from 200 to 400 mM of glucose and from 3 to 30 mM of sodium phosphate to the purified plasmid DNA, thereby obtaining an isotonic solution,

subsequently lyophilizing the isotonic solution, thereby obtaining a lyophilizate, and

storing the lyophilizate at a temperature of from +2°

C. to +8°

C.,

wherein a concentration of the purified plasmid DNA in the isotonic solution prior to the lyophilizing is from 0.1 to 10 mg/ml, and a pH of the isotonic solution is from 7.0 to 9.0.

14. The method according to claim 13, wherein a mass ratio of glucose to the purified plasmid DNA in the isotonic solution is 1:

50.

15. The method according to claim 14, wherein a proportion of relaxed plasmid DNA in the lyophilizate stored for 3 months is not more than 10%.

16. The method according to claim 14, wherein the at comprising from 5 to 20 mM of the sodium phosphate and the pH of the isotonic solution is from 7.8 to 9.0.

17. The method of claim 13, wherein the purified plasmid DNA comprises the polynucleotide sequence of SEQ ID NO:

1.

18. A pharmaceutical composition comprising:

purified plasmid DNA encoding a human recombinant vascular endothelial growth factor (VEGF), wherein the VEGF encoding sequences are operatively linked to expression control sequences to provide gene expression in human cells, and wherein the purified plasmid DNA comprises the polynucleotide sequence of SEQ ID NO;

1, and

from 3 to 30 mM of sodium phosphate and from 200 to 400 mM of glucose to make the pharmaceutical composition isotonic, and

wherein a concentration of the purified plasmid DNA in the composition is from 0.1 to 10 mg/ml, and a pH of the composition is form 7.4 to 9.0.

19. The method of claim 18, wherein the pH of the composition is from 7.8 to 9.0.