Compositions and use of EPAS1 inhibitors

• US 20100010071A1
• Filed: 04/03/2009
• Published: 01/14/2010
• Est. Priority Date: 04/04/2008
• Status: Active Grant

• Current Assignee: Calando Pharmaceuticals, Inc. (Arrowhead Pharmaceuticals, Inc.)
• Original Assignee: Calando Pharmaceuticals, Inc. (Arrowhead Pharmaceuticals, Inc.)
• Inventors: Liu, Joanna Yi-Ching, Heidel, Jeremy D., Davis, Mark E.
• Granted Patent: US 8,114,983 B2
• Time in Patent Office: Days
• US Class Current: 514/44.R
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• 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 2310/14 interfering N.A.

Abstract

The present invention relates to inhibitors of endothelial PAS domain protein 1 (EPAS1), and methods and compositions related to the EPAS1 inhibitors. In certain embodiments, the EPAS1 inhibitors include nucleic acids, such as for example siRNAs.

Claims

1. A nucleic acid comprising:

(i) a first strand of about 15 to about 30 nucleotides in length that comprises a sequence selected from SEQ ID NOs;

2-4, and

(ii) a second strand of about 15 to about 30 nucleotides in length,

wherein at least 12 nucleotides of the first and second strands are complementary to each other and form a double-stranded nucleic acid under physiological conditions, and wherein the double-stranded nucleic acid can reduce the expression of endothelial PAS domain protein 1 (EPAS1) in a cell by an RNA interference mechanism.

2. The nucleic acid of claim 1, further characterized by one or more of the following:

the nucleic acid is a double-stranded RNA;

wherein the double-stranded portion is optionally about 15 to about 30 nucleotides in length;

the nucleic acid is a hairpin RNA;

wherein the hairpin RNA comprises a loop region optionally having about 4 to about 10 nucleotides in length;

the first strand is a DNA polynucleotide and the second strand is an RNA polynucleotide;

the first and/or second strand further comprises a 3′

overhang region, a 5′

overhang region, or both 3′ and

5′

overhang regions;

wherein the overhang region optionally contains about 1 to about 10 nucleotides in length;

the nucleic acid comprises one or more modified backbone or base moieties;

wherein the modified backbone or base moieties optionally comprise one or more of the following;

alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxylmethyl esters, carbonates, and phosphate triesters;

wherein the modified backbone or base moieties optionally comprise at least one 2′

-O-alkylated ribonucleotide;

the first strand comprises a sequence selected from SEQ ID NOs;

5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, and 81;

the second strand comprises a sequence selected from SEQ ID NO;

6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82;

the nucleic acid inhibits EPAS1 expression in cells by 50% or greater, when contacted with the cells under physiological conditions at a concentration of 10 nanomolar.

5. A pharmaceutical composition comprising the nucleic acid according to any one of claims 1-4, and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5, further characterized by one or more of the following:

the pharmaceutically-acceptable carrier includes a cationic polymer;

the pharmaceutically acceptable carrier includes a cyclodextrin polymer;

wherein the cyclodextrin structure is optionally im-CDP;

the pharmaceutical composition comprises a particle that includes a cyclodextrin polymer and the nucleic acid and is PEGylated;

wherein the particle further optionally comprises adamantane;

the pharmaceutical composition further comprises a ligand that targets a particular tissue or cell type;

wherein the ligand optionally comprises transferrin;

the pharmaceutical composition comprises nanoparticles, wherein the nanoparticles are from about 10 to about 100 nm in diameter;

wherein the nanoparticles are optionally about 50 to about 70 nm in diameter;

wherein the nanoparticles are optionally about 50 nm in diameter;

the pharmaceutically acceptable carrier comprises;

an imidazole-modified cyclodextrin-containing cationic polymer, and

a targeting moiety comprising adamantane-PEG-ligand,

wherein the polymer and targeting moiety form nanoparticles that encapsulate the nucleic acid;

wherein the nanoparticles optionally have a diameter of about 50 to about 120 nm;

wherein the nanoparticles optionally have a diameter of about 50 to about 100 nm;

wherein the nanoparticles optionally have a diameter of about 50 to about 70 nm;

wherein the nanoparticles optionally have a diameter of about 50 nm;

wherein the targeting ligand optionally comprises galactose;

wherein the targeting ligand optionally comprises transferrin.

7. Use of the nucleic acid according to any one of claims 1-4 in the manufacture of a medicament for the treatment of a disease or condition associated with unwanted proliferation of cells.

8. The use according to claim 7, characterized by one or more of the following:

the cells are cancerous or tumor cells;

the cells are pathogen cells;

the cells are normal cells, the unwanted proliferation of which leads to the disease or condition.

9. A method for treating a patient having a cancer comprising administering to the patient a therapeutically effective amount of the double-stranded nucleic acid of claim 1 or 3.

10. The method according to claim 9, further characterized by one or more of the following:

the method further comprises at least one additional anti-cancer chemotherapeutic agent that inhibits proliferation of cancer cells in an additive or synergistic manner with the nucleic acid;

wherein the chemotherapeutic agent is optionally fluorouracil (5FU);

the nucleic acid is formulated with a pharmaceutically acceptable carrier;

the nucleic acid is formulated with a ligand targeting the cancer cell;

wherein the cancer cell is optionally a clear-cell renal cell carcinoma and the ligand optionally comprises tranferrin;

wherein the ligand is optionally galactose;

the nucleic acid is formulated as a component of a polymeric nanoparticle;

wherein the nanoparticle is optionally from about 10 to about 120 nm in diameter;

wherein the nanoparticle is optionally from about 50 to about 120 nm in diameter;

wherein the nanoparticle is optionally from about 50 to about 100 nm in diameter;

wherein the nanoparticle is optionally about 50 nm in diameter;

the therapeutically effective amount of the nucleic acid is administered systemically;

the cancer cell expresses a higher level of EPAS1 compared to a noncancerous cell from a comparable tissue.

3. An isolated nucleic acid comprising a sequence that hybridizes to a region of an EPAS1 transcript corresponding to nucleotides 655-718, 2878-2929, or 4978-5039 of SEQ ID NO:

1 under physiological conditions and decreases the expression of EPAS1 in a cell.

4. The isolated nucleic acid of claim 3, further characterized by one or more of the following:

the nucleic acid comprises a sequence that hybridizes to a region of an EPAS1 transcript corresponding to nucleotides 665-708, 2888-2919, or 4988-5029 of SEQ ID NO;

1;

the nucleic acid comprises a sequence that hybridizes to a region of an EPAS1 transcript corresponding to nucleotides 670-703, 2893-2914, or 4992-5024 of SEQ ID NO;

1;

the nucleic acid comprises at least 10 consecutive nucleotides that are complementary to one of said regions of EPAS1.

the nucleic acid is from about 14 to about 50 nucleotides in length;

the nucleic acid is single-stranded;

the nucleic acid is double-stranded;

the nucleic acid is a DNA molecule, optionally comprising one or more modified backbone or base moieties;

the nucleic acid is an RNA molecule, optionally comprising one or more modified backbone or base moieties;

the nucleic acid comprises a DNA strand and an RNA strand and optionally comprises one or more modified backbone or base moieties;

the nucleic acid is an RNAi construct;

wherein the RNAi construct is optionally a dsRNA, comprising, for example, one or more modified backbone or base moieties;

wherein the RNAi construct is optionally a hairpin RNA, comprising, for example, one or more modified backbone or base moieties;

wherein the duplex portion of the RNAi construct is from about 15 to about 30 nucleotides in length;

wherein the RNAi construct comprises a sequence selected from SEQ ID NOs;

2-4, optionally comprising one or more modified backbone or base moieties;

wherein the RNAi construct comprises a sequence selected from SEQ ID NOs;

5-82, optionally comprising one or more modified backbone or base moieties;

wherein the RNAi construct optionally comprises one or more modified backbone or base moieties;

further comprising at least one internucleotide linkage selected from, for example, alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxylmethyl esters, carbonates, and phosphate triesters;

further optionally comprising at least one 2′

-O-alkylated ribonucleotide;

the nucleic acid is an enzymatic nucleic acid;

wherein the enzymatic nucleic acid is optionally a ribozyme;

wherein the enzymatic nucleic acid is optionally a DNA enzyme;

the nucleic acid inhibits EPAS1 expression in cells by 50% or greater, when contacted with the cells under physiological conditions at a concentration of 10 nanomolar.

11. A method of treating a patient having a cancer comprising administering to the patient a therapeutically effective amount of a double-stranded nucleic acid to reduce expression of EPAS1 by an RNAi mechanism and one or more anti-cancer agents that induce EPAS1 expression.

12. The method of claim 11, further characterized by one or more of the following:

the nucleic acid is formulated with a pharmaceutically acceptable carrier;

the nucleic acid is formulated with a ligand targeting the cancer cell;

wherein the cancer cell is optionally a clear-cell renal cell carcinoma and the ligand optionally comprises tranferrin;

wherein the ligand is optionally galactose;

the nucleic acid is formulated as a component of a polymeric nanoparticle;

wherein the nanoparticle is optionally from about 10 to about 120 nm in diameter;

wherein the nanoparticle is optionally from about 50 to about 120 nm in diameter;

wherein the nanoparticle is optionally from about 50 to about 100 nm in diameter;

wherein the nanoparticle is optionally about 50 nm in diameter;

the therapeutically effective nucleic acid is administered systemically.

13. The method according to claims 9 or 11, further characterized by one or more of the following:

the method further comprising determining the level of von Hippel-Lindau (VHL) protein activity in the patient prior to administering the nucleic acid;

wherein said activity is optionally determined by measuring a decrease in RNA or protein level of VHL in the patient as compared to said levels in a healthy subject;

wherein said activity is optionally determined by a genetic screening to identify one or more mutations in the VHL gene, said one or more mutations comprising one or more of the following;

a truncation-causing mutation, a mutation that causes allelic loss, a nonsense mutation, a frameshift mutation, a promoter mutation, an enhancer mutation, splice site mutation, null mutation, and poly-A tail mutation.