Vascular endothelial growth factor (VEGF) Nucleic Acid Ligand Complexes

• US 6,168,778 B1
• Filed: 06/06/1997
• Issued: 01/02/2001
• Est. Priority Date: 06/11/1990
• Status: Expired due to Fees

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: NeXstar Pharmaceuticals, Inc. (Gilead Sciences Inc.)
• Inventors: Janjic, Nebojsa, Gold, Larry, Vargeese, Chandra, Schmidt, Paul
• Primary Examiner(s): ZITOMER, STEPHANIE W
• Application Number:
  US08/870,930
• Time in Patent Office: 1,306 Days
• Field of Search: 536/24.3, 536/24.31, 536/25.4, 435/6, 435/91.2, 424/1.73
• US Class Current: 424/1.73
• 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: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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 14/001 by chemical synthesis
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C12N 15/1048 SELEX
• 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 2310/13 Decoys
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3515 Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: F02B 2075/027 four
• CPC Class Codes: G01N 2333/16 : HIV-1, HIV-2
• CPC Class Codes: G01N 2333/163 : Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx
• CPC Class Codes: G01N 2333/503 : basic FGF [bFGF]
• CPC Class Codes: G01N 2333/8125 : Alpha-1-antitrypsin
• CPC Class Codes: G01N 2333/96433 : Serine endopeptidases (3.4.21)
• CPC Class Codes: G01N 2333/966 : Elastase
• CPC Class Codes: G01N 2333/974 : Thrombin
• CPC Class Codes: G01N 33/531 : Production of immunochemical test materials
• CPC Class Codes: G01N 33/532 : Production of labelled immunochemicals
• CPC Class Codes: G01N 33/535 : with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/56988 : HIV or HTLV
• CPC Class Codes: G01N 33/68 : involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/76 : Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Abstract

This invention discloses a method for preparing a complex comprised of a VEGF Nucleic Acid Ligand and a Lipophilic Compound by identifying a VEGF Nucleic Acid Ligand by SELEX methodology and associating the VEGF Nucleic Acid Ligand with a Lipophilic Compound. The invention further discloses Complexes comprising one or more VEGF Nucleic Acid Ligands in association with a 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 Complex comprised of a VEGF Nucleic Acid Ligand comprising 2′

F-modified nucleotides and a Lipophilic Compound.

2. The Complex of claim 1 wherein said Lipophilic Compound is a glycerol lipid.

3. The Complex of claim 2 wherein said Lipophilic compound is a glycerol lipid having the structure

where R¹, R², and R³ are independently selected from the group consisting of CH₃(CH₂)_n—

O(PO₃)—

CH₂—

; and

CH₃(CH₂)_n—

CONH₂—

CH₂—

, CH₃(CH₂)_nO—

, CH₃(CH₂)_nOCH₂—

, CH₃(CH₂)_n(CO)OCH₂—

, CH₃(CH₂)_n(CO)O— and

X—

, wherein at least one must be X—

, and X is independently selected from the group consisting of (PO₄), and O, and wherein n=0-30, preferably 10-20.

4. The Complex of claim 3 wherein said glycerol lipid is a phospholipid wherein one of R¹, R², or R³ is CH₃(CH₂)_n—

O(PO₃)—

CH₂—

.

5. The Complex claim 4 wherein R¹ is CH₃(CH₂)_n—

O(PO₃)—

CH₂—

, R² is CH₃(CH₂)_n—

O(PO₃)—

CH₂—

, and R³ is X—

, and wherein n=17.

6. The Complex of claim 5 wherein X is PO₄.

7. The Complex of claim 3 wherein said glycerol lipid is a glycerol amide lipid wherein one of R¹, R² or R³ is CH₃(CH₂)_n—

CONH₂—

CH₂—

.

8. The Complex of claim 7 wherein R¹ is CH₃(CH₂)_n—

CONH₂—

CH₂—

, R² is CH₃(CH₂)_n—

CONH₂—

CH₂—

, and R³ is X—

, and wherein n=16.

9. The Complex of claim 8 wherein X is PO₄.

10. The Complex of claim 8 wherein X is O.

11. A Lipid Construct comprising the Complex of claim 1.

12. A Lipid Construct comprising the Complex of claim 3.

13. A Lipid Construct comprising the Complex of claim 4.

14. A Lipid Construct comprising the Complex of claim 5.

15. A Lipid Construct comprising the Complex of claim 6.

16. The Lipid Construct of claim 15 wherein said Complex is

Ligand Component=fCmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfUfUmAfUmAfCmAfUfCfCmG-3′

3′

-dT (VEGF ligand) (SEQ ID NO;

5).

17. A Lipid Construct comprising the Complex of claim 7.

18. A Lipid Construct comprising the Complex of claim 8.

19. A Lipid Construct comprising the Complex of claim 9.

20. The Lipid Construct of claim 19 wherein said Complex is

Ligand Component=fCmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfUfUmAfUmAfCmAfUfCfCmG-3′

3′

-dT (VEGF ligand) (SEQ ID NO;

6).

21. A Lipid Construct comprising the Complex of claim 10.

22. The Lipid Construct of claim 21 wherein said Complex is

Ligand Component=fCmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfUfUmAfUmAfCmAfUfCfCmG-3′

3′

-dT (VEGF ligand) (SEQ ID NO;

7).

23. A method for the preparation of a Complex comprised of a VEGF Nucleic Acid Ligand comprising 2′

-F-modified nucleotides and a Non-Immunogenic, High Molecular Weight Com-pound of Lipophilic Compound, said method comprising;

a) identifying a VEGF Nucleic Acid Ligand from a Candidate Mixture of Nucleic Acids by the method comprising;

(i) contacting the Candidate Mixture with VEGF, wherein Nucleic Acids having increased affinity to VEGF relative to the Candidate Mixture may be partitioned from the remainder of the Candidate Mixture;

(ii) partitioning the increased affinity VEGF Nucleic Acids from the remainder of the Candidate Mixture;

(iii) amplifying the increased affinity VEGF Nucleic Acids to yield a ligand-enriched mixture of Nucleic Acids; and

b) forming a covalent bond between said identified VEGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound of Lipophilic Compound.

24. The method of claim 23 wherein said Complex is further associated with a Lipid Construct.

25. The method of claim 24 wherein said Lipid Construct is a Liposome.

26. The method of claim 25 wherein said Complex is comprised of a Nucleic Acid Ligand and a Lipophilic Compound and wherein said Complex is associated with the bilayer of said Liposomes by the method comprising the steps of:

a) forming a liposome; and

b) mixing said Complex comprised of a Nucleic Acid Ligand and a Lipophilic Compound with the Liposomes of step a) whereby the Nucleic Acid Ligand Component of said Complex becomes associated with the bilayer of the Liposome and projects from the exterior of the Lipid bilayer.

27. The method of claim 23 wherein said Non-Immunogenic, High Molecular Weight Compound is Polyalkylene Glycol.

28. The method of claim 27 wherein said Polyalkylene Glycol is polyethylene glycol.

29. The method of claim 28 wherein said polyethylene glycol has a molecular weight of about between 10-80 K.

30. The method of claim 29 wherein said polyethylene glycol has a molecular weight of about 20-45 K.

31. The method of claim 30 wherein said Complex has the structure

Ligand Component=fCmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfUfUmAfUmAfCmAfUfCfCmG-3′

3′

-dT (VEGF ligand) (SEQ ID NO;

     8).

32. The method of claim 30 wherein said Complex has the structure

Ligand Component=fCmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfUfUmAfUmAfCmAfUfCfCmG-3′

3′

-dT (VEGF ligand) (SEQ ID NO;

     9).

33. The method of claim 24 wherein said Complex is

Ligand Component=fCmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfUfUmAfUmAfCmAfUfCfCmG-3′

3′

-dT (VEGF ligand) (SEQ ID NO;

     5).

34. The method of claim 24 wherein said Complex is

Ligand Component=fCmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfUfUmAfUmAfCmAfUfCfCmG-3′

3′

-dT (VEGF ligand) (SEQ ID NO;

6).

35. The method of claim 24 wherein said Complex is

Ligand Component=fCmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfUfUmAfUmAfCmAfUfCfCmG-3′

3′

-dT (VEGF ligand) (SEQ ID NO;

     7).