Method of phosphate ester hydrolysis

• US 5,763,172 A
• Filed: 06/07/1995
• Issued: 06/09/1998
• Est. Priority Date: 01/21/1992
• Status: Expired due to Fees

• Current Assignee: Pharmacyclics Incorporated (Abbvie Incorporated), Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Pharmacyclics Incorporated (Abbvie Incorporated), Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Dow, William C., Miller, Richard A., Sessler, Jonathan L., Magda, Darren, Wright, Meredith
• Primary Examiner(s): Marschel, Ardin H.
• Assistant Examiner(s): Riley, Jezia
• Application Number:
  US08/486,962
• Time in Patent Office: 1,098 Days
• Field of Search: 435/6, 536/22.1, 536/23.1, 536/24.3, 536/25.32, 514/1, 514/44
• US Class Current: 435/6.12
• CPC Class Codes: A61K 41/0038 Radiosensitizing, i.e. administration of pharmaceutical agents that enhance the effect of radiotherapy radiotherapy per se A61N5/10
• CPC Class Codes: A61K 47/546 Porphyrines; Porphyrine with an expanded ring system, e.g. texaphyrine
• CPC Class Codes: A61K 49/0021 the fluorescent group being a small organic molecule
• CPC Class Codes: A61K 49/0054 Macromolecular compounds, i.e. oligomers, polymers, dendrimers
• CPC Class Codes: B01J 2531/025 Ligands with a porphyrin ring system or analogues thereof, e.g. phthalocyanines, corroles
• CPC Class Codes: B01J 2531/38 Lanthanides other than lanthanum
• CPC Class Codes: B01J 31/061 Chiral polymers
• CPC Class Codes: B01J 31/1658 immobilised by covalent linkages, i.e. pendant complexes with optional linking groups, e.g. on Wang or Merrifield resins
• CPC Class Codes: B01J 31/1815 with more than one complexing nitrogen atom, e.g. bipyridyl, 2-aminopyridine
• CPC Class Codes: C07D 487/22 in which the condensed system contains four or more hetero rings
• 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

Abstract

A method of phosphate ester hydrolysis including incubating a solution of an aqueous phosphate ester with a metallotexaphyrin complex under conditions and for a time sufficient to hydrolyze the phosphate ester. The metal is a metal cation having catalytic activity for ester bond hydrolysis in aqueous solution. Phosphate ester substrates include nucleic acid such as RNA, phosphoric anhydrides, phospholipids, and alkyl phosphate esters.

Claims

1. A method for enhancing the therapeutic activity of an oligonucleotide in a cell, the method comprising:

contacting a targeted intracellular RNA in a cell with a metallotexaphyrin-oligonucleotide conjugate, the contact being under physiologic conditions and for a time sufficient to hydrolyze the phosphate ester bond of said targeted intracellular RNA, wherein the metallotexaphyrin of said metallotexaphyrin-oligonucleotide conjugate has catalytic activity for phosphate ester bond hydrolysis and the oligonucleotide of said metallotexaphyrin-oligonucleotide conjugate has complementary binding affinity to said targeted intracellular RNA.

2. A method for enhancing the therapeutic activity of an oligonucleotide in a cell, the method comprising:

contacting a targeted intracellular RNA in a cell with a metallotexaphyrin-oligonucleotide conjugate, the contact being under physiologic conditions and for a time sufficient to hydrolyze the phosphate ester bond of said targeted intracellular RNA, wherein the metallotexaphyrin of said metallotexaphyrin-oligonucleotide conjugate has catalytic activity for phosphate ester bond hydrolysis and the oligonucleotide of said metallotexaphyrin-oligonucleotide conjugate has complementary binding affinity to said targeted intracellular RNA;

where the metallotexaphyrin-oligonucleotide conjugate has the following structure;

##STR6## M is a divalent or a trivalent metal cation catalyzing phosphate ester bond hydrolysis in aqueous solution;

R₁ -R₄, R₇ and R₈ are independently hydrogen, halide, hydroxyl, alkyl, aryl, haloalkyl, nitro, formyl, acyl, hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, saccharide, carboxy, carboxyalkyl, carboxyamide, carboxyamidealkyl, aminoalkyl, sulfonatoalkyl, amidealkyl, aryl, a site-directed molecule, a catalytic group, or a couple to a site-directed molecule or to a catalytic group;

R₆ and R₉ are independently selected from the groups of R₁ -R₄, R₇ and R₈, with the proviso that the halide is other than iodide and the haloalkyl is other than iodoalkyl;

R₅ and R₁₀ -R₁₂ are independently hydrogen, alkyl, aryl, hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, carboxyalkyl, carboxyamidealkyl or a couple to a saccharide to a site-directed molecule or to a catalytic group; and

Z is less than or equal to 5;

where at least one of R₁ -R₁₂ is a site-directed molecule or a couple to a site-directed molecule, and the site-directed molecule is an oligonucleotide having binding affinity to said targeted intracellular RNA.

3. The method of claim 2 wherein the catalytic group is imidazole, guanidine, an amino acid, an amino acid derivative, a polyamino acid, an amine-substituted saccharide or a metallotexaphyrin complex.

4. The method of claim 2 wherein the site-directed molecule is an oligonucleotide, a hormone, an antibody, a molecule having affinity for a biological receptor, or a sapphyrin molecule.

5. The method of claim 2 wherein the oligonucleotide is an oligodeoxyribonucleotide.

6. The method of claim 2 wherein the oligonucleotide is further conjugated to a catalytic group.

7. The method of claim 6 wherein the catalytic group is imidazole, guanidine, an amino acid, an amino acid derivative, a polyamino acid, an amine-substituted saccharide or a metallotexaphyrin complex.

8. The method of claim 6 wherein the catalytic group is a metallotexaphyrin complex.

9. The method of claim 2 wherein the oligonucleotide has complementary binding affinity for oncogenes.

10. The method of claim 2 wherein the oligonucleotide has complementary binding affinity for the targeted RNA in a region proximal to the phosphate ester bond being hydrolyzed.

11. The method of claim 2 wherein the oligonucleotide is an antisense oligonucleotide.

12. The method of claim 2 wherein one of R₃, R₇, and R₈ is an oligonucleotide or a couple to an oligonucleotide.

13. The method of claim 2 wherein one of R₃, R₇ or R₈ is O(CH₂)_n CO-oligonucleotide where n is 1-7.

14. The method of claim 2 wherein M is a lanthanide cation.

15. The method of claim 2 wherein M is Eu(III), Tb(III) or Dy(III).

16. The method of claim 2 wherein the targeted RNA is selected from the group consisting of viral, including retroviral, RNA;

messenger RNA (mRNA);

ribosomal RNA;

RNA cofactors;

transfer RNA;

small nuclear RNA; and

small cytoplasmic RNA.

17. The method of claim 2 wherein the targeted RNA is mRNA.

18. The method of claim 2 where the oligonucleotide is an oligoribonucleotide.

19. The method of claim 2 where the oligonucleotide is a derivatized oligonucleotide or an oligonucleotide analog.

20. The method of claim 2 where the oligonucleotide is a derivatized oligonucleotide and is selected from the group consisting of methylphosphonates, phosphotriesters, phosphorothioates, and phosphoramidates.

21. The method of claim 2 where the oligonucleotide is 2'"'"'-O-alkyl oligoribonucleotide.

22. The method of claim 2 where R₁ is CH₂ CH₃ or CH₂ CH₂ CH₂ OH;

each of R₂ and R₃ is CH₂ CH₃ ;

R₄ is CH₃ ;

each of R₅, R₆, and R₉ -R₁₂ is H;

R₇ is H, CH₃, OCH₃, O(CH₂)_n COOH, OCH₂ CH₂ CH₂ OH or O(CH₂ CH₂ O)_n CH₃ where n is 1-7; and

R₈ is an oligonucleotide or a couple to an oligonucleotide.

23. The method of claim 22 where R₈ is O(CH₂)_n CO-oligonucleotide where n is 1-7.

24. The method of claim 2 where M is a lanthanide metal cation;

R₁ is CH₂ CH₃ or CH₂ CH₂ CH₂ OH;

each of R₂ and R₃ is CH₂ CH₃ ;

R₄ is CH₃ ;

each of R₅, R₆, and R₉ -R₁₂ is H R₇ is H, CH₃, OCH₃, O(CH₂)_n COOH, OCH₂ CH₂ CH₂ OH or O(CH₂ CH₂ O)_n CH₃ where n is 1-7; and

R₈ is an oligonucleotide or a couple to an oligonucleotide.

25. The method of claim 24 where R₈ is O(CH₂)_n CO-oligonucleotide where n is 1-7.

26. The method of claim 24 where the targeted RNA is mRNA.

27. The method of claim 24 where the oligonucleotide is an antisense oligonucleotide.

28. The method of claim 2 where M is Y(III).