Radiolabeled DNA carrier, method of preparation and therapeutic uses thereof

• US 20040067197A1
• Filed: 02/02/2001
• Published: 04/08/2004
• Est. Priority Date: 11/26/1997
• Status: Abandoned Application

• Current Assignee: Angiogene, Inc., Centre De Recherche Du Centre Hospitalier De L'Universite De Montreal
• Original Assignee: Angiogene, Inc., Centre De Recherche Du Centre Hospitalier De L'Universite De Montreal
• Inventors: Leclerc, Guy, Martel, Remi
• Application Number:
  US09/775,479
• Publication Number:
  US 20040067197A1
• Time in Patent Office: Days
• 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 51/0491 Sugars, nucleosides, nucleotides, oligonucleotides, nucleic acids, e.g. DNA, RNA, nucleic acid aptamers
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/351 Conjugate

Abstract

The present invention relates to a radiolabeled DNA carrier, a method of preparation thereof and the therapeutic uses of this substance to prevent uncontrolled cellular proliferation. The invention also relates to devices incorporating the above radiolabeled DNA carrier (such as an oligonucleotide) for the therapeutic treatment of uncontrolled cellular proliferation. More specifically, the present invention is concerned with the prevention of restenosis by intravascular delivery of radiolabeled DNA carrier at a dilatation site of an artery. This invention is also directed to a method of treatment of vascular proliferative diseases and/or other proliferative disorders such as cancer and related metastasis. More particularly, the invention relates to the preparation of DNA sequences carrying one or several radioisotopes, located within the DNA sequence, and which are able to prevent cell proliferation in vitro and, pursuant to local drug delivery and/or systemic drug delivery, are able to prevent cell proliferation in vivo, more particularly restenosis and malignant tumors. In other words, the invention relates to the synthesis process, the stability data of the radiolabeled DNA carrier, the efficacy of the invention in vitro, in cell culture, and the in vivo delivery of the molecule.

Claims

1. An anti-proliferative substance for preventing uncontrolled cellular proliferation, which comprises a radiolabeled DNA carrier, wherein a radioisotope is located internally within the DNA sequence, at 5′

end or at 3′

end, and wherein said radiolabeled DNA carrier penetrates cell membrane and is retained intracellularly for a time sufficient for the radioisotope to effect an efficient dose therapy.

2. The anti-proliferative substance according to claim 1, wherein said carrier is an oligonucleotide.

3. The anti-proliferative substance according to claim 2, wherein said oligonucleotide is linear.

4. The anti-proliferative substance according to claim 1, wherein said carrier is a plasmid.

5. The anti-proliferative substance according to claim 4, wherein said plasmid is circular.

6. The anti-proliferative substance according to claim 5, wherein said plasmid is of viral or bacterial origin.

7. The anti-proliferative substance according to claim 1, wherein said radioisotope is selected from the group consisting of ³²P, ³³P, ¹²⁵I, ¹³¹I, ³⁵S, ¹⁹⁸AU, ⁹⁰Y, ⁸⁹SR, ¹⁸⁶Re, ⁴⁵Ca and ¹⁵³Sm.

8. The anti-proliferative substance according to claim 3, wherein said oligonucleotide is a double-stranded DNA sequence or a single-stranded DNA sequence.

9. The anti-proliferative substance according to claim 3, wherein said oligonucleotide is conjugated with an antibody for cell-specific delivery.

10. The anti-proliferative substance according to claim 8, wherein said DNA oligonucleotide sequence is a single-stranded sense DNA sequence for hybridization to a specific genetic target.

11. The anti-proliferative substance according to claim 8, wherein said DNA oligonucleotide sequence is a single-stranded antisense DNA sequence for hybridization to a specific genetic target.

12. The anti-proliferative substance according to claim 1, which comprises DNA sequences of at least about 2 to about 2000 nucleotides.

13. The anti-proliferative substance according to claim 12, wherein the DNA sequence is selected from the group consisting of

14. The anti-proliferative substance according to claim 3, wherein the oligonucleotide is conjugated to at least one selected from the group consisting of a stent surface, cholesterol, oleic acid, linoleic acid, TGFα

, antibody, TGFβ

, cytokines and growth factors.

15. The anti-proliferative substance according to claim 13, wherein the radioisotope is selected from the group consisting of ³²P, ³³P, ¹²⁵I, ¹³¹I, ³⁵S, ¹⁹⁸AU, ⁹⁰Y, ⁸⁹SR, ¹⁸⁶Re, ⁴⁵Ca and ¹⁵³Sm.

18. The method of claim 12, which further include a step f) after step e) to obtain a double-stranded carrier having both strand radiolabeled, which comprises:

f) hybridizing together complementary radiolabeled single-stranded DNA carrier of step e).

19. The method of claim 18, wherein said radioisotope is selected from the group consisting of ³²P, ³³P, ¹²⁵I, ¹³¹I, ³⁵S, ¹⁹⁸AU, ⁹⁰Y, ⁸⁹SR, ¹⁸⁶Re, ⁴⁵Ca and ¹⁵³Sm.

20. The method of claim 18, wherein said two parts of step a) form an antisense sequence and said sequence capable of hybridizing of step c) is a corresponding sense sequence.

21. The method of claim 18, wherein said two parts of step a) form a sense sequence and said sequence capable of hybridizing of step c) is a corresponding antisense sequence.

22. Method for the prevention of uncontrolled cell proliferation in a mammal, which comprises delivering to said mammal a therapeutic substance according to claim 1 in situ where said uncontrolled cell proliferation takes place.

23. Method according to claim 22, wherein said uncontrolled cell proliferation is a restenosis following angioplasty, and said therapeutic substance is delivered by site-specific intravascular delivery.

24. Method according to claim 23, wherein the therapeutic substance is coupled to an antibody.

25. Method according to claim 22, wherein said uncontrolled cell proliferation is cancer or a malignant tumor, and said therapeutic substance is coupled to a peptide moiety.

26. Method according to claim 25, wherein said peptide moiety is selected from the group consisting of an antibody, TGFα

, TGFβ

, cytokines and any growth factors.

16. A method for preparing a radiolabeled DNA carrier sequence wherein a radioisotope is located internally within the DNA sequence, which comprises the steps of:

a) synthesizing a DNA sequence in at least two parts;

b) labeling the 5′

end of one of said two parts with a radioisotope;

c) hybridizing said two parts of step b) with a sequence capable of hybridizing under stringent conditions; and

d) ligating together said hybridized two parts.

17. The method of claim 16, which further include a step e) after step d) to obtain a single-stranded radiolabeled DNA carrier, which comprises

e) separating the hybridized DNA and recovering the radiolabeled DNA carrier sequence.