Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes

• US 6,051,698 A
• Filed: 07/21/1997
• Issued: 04/18/2000
• Est. Priority Date: 06/06/1997
• Status: Expired due to Term

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: NeXstar Pharmaceuticals, Inc. (Gilead Sciences Inc.)
• Inventors: Janjic, Nebojsa, Vargeese, Chandra, Schmidt, Paul, Gold, Larry
• Primary Examiner(s): ZITOMER, STEPHANIE W
• Application Number:
  US08/897,351
• Time in Patent Office: 1,002 Days
• Field of Search: 536/24.31, 435/6, 514/44, 935/77, 935/78
• US Class Current: 536/24.31
• 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 47/544 Phospholipids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C12N 15/115 Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: C12N 2310/3515 Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules

Abstract

This invention discloses a method for preparing a complex comprised of a VEGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound by identifying a VEGF Nucleic Acid Ligand by SELEX methodology and associating the VEGF Nucleic Acid Ligand with a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound. The invention further discloses Complexes comprising one or more VEGF Nucleic Acid Ligands in association with a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound. The invention further includes a Lipid construct comprising a VEGF Nucleic Acid Ligand or Complex and methods for making the same.

Claims

1. A purified and isolated non-naturally occurring RNA ligand to Vascular Endothelial Growth Factor wherein said ligand is comprised of 2'"'"'fluoro (2'"'"'F)-modified nucleotides.

3. The RNA ligand to VEGF of claim 1 wherein said ligand is selected from the group consisting of the sequences set forth in Tables 1-4 (SEQ ID NOS:

10-86).

4. The RNA ligand of claim 3 wherein said ligand is substantially homologous to and has substantially the same ability to bind VEGF as a ligand selected from the group consisting of the sequences set forth in Tables 1-4 (SEQ ID NOS:

10-86).

5. The RNA ligand of claim 3 wherein said ligand is substantially the same structure and substantially the same ability to bind VEGF as a ligand selected from the group consisting of the sequences set forth in Tables 1-4 (SEQ ID NOS:

10-86).

6. The RNA ligand to VEGF of claim 1 identified according to the method comprising:

a) contacting a Candidate Mixture of RNA with VEGF, wherein the RNA having an increased affinity to VEGF relative to the Candidate Mixture may be partitioned from the remainder of the Candidate Mixture;

b) partitioning the increased affinity RNA from the remainder of the Candidate Mixture; and

c) amplifying the increased affinity RNA to yield a mixture of RNA enriched for RNA having an increased affinity for VEGF, whereby RNA Ligands of VEGF are identified.

7. The RNA ligand to VEGF of claim 3 identified according to the method comprising:

a) contacting a Candidate Mixture of RNA with VEGF, wherein the RNA having an increased affinity to VEGF relative to the Candidate Mixture may be partitioned from the remainder of the Candidate Mixture;

b) partitioning the increased affinity RNA from the remainder of the Candidate Mixture; and

c) amplifying the increased affinity RNA to yield a mixture of RNA enriched for RNA having an increased affinity for VEGF;

whereby RNA Ligands of VEGF are identified.

8. A Complex comprised of the RNA ligand to VEGF of claim 1 and a Non-Immunogenic, High Molecular Weight Compound.

9. The Complex of claim 8 further comprising a Linker between said ligand and said Non-Immunogenic, High Molecular Weight Compound.

10. The Complex of claim 8 wherein said Non-Immunogenic, High Molecular Weight Compound is a Polyalkylene Glycol.

11. The Complex of claim 10 wherein said Polyalkylene Glycol is polyethylene glycol.

12. The Complex of claim 11 wherein said polyethylene glycol has a molecular weight of about 10-80 Kd.

13. The Complex of claim 11 wherein said polyethylene glycol has a molecular weight of about 20-45 Kd.

14. The Complex of claim 11 wherein said Complex is ##STR9##

15. The Complex of claim 11 wherein said Complex is

2. A method for improving the pharmacokinetic properties of VEGF Nucleic Acid Ligand in an occular application comprising covalently linking a Non-Immunogenic, High Molecular Weight compound to a VEGF Nucleic Acid Ligand to form a complex comprised of a VEGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound.

16. The method of claim 2 wherein said Non-Immunogenic, High Molecular Weight Compound is a Polyalkylene Glycol.

17. The method of claim 16 wherein said Polyalkylene Glycol is polyethylene glycol.

18. The method of claim 17 wherein said polyethylene glycol has a molecular weight of about 10-80 Kd.

19. The method of claim 17 wherein said polyethylene glycol has a molecular weight of about 20-45 Kd.

20. The method of claim 19 wherein said complex has the structure

21. The complex of claim 19 wherein said complex is