WO2011133142A1

WO2011133142A1 - Treatment of vhl-negative tumors

Info

Publication number: WO2011133142A1
Application number: PCT/US2010/031768
Authority: WO
Inventors: Paula J. Bates; Md. Ashraful Islam
Original assignee: University Of Louisville
Priority date: 2010-04-20
Filing date: 2010-04-20
Publication date: 2011-10-27

Abstract

The present invention relates to methods of treating VHL-negative tumors (e.g., VHL-negative renal cell carcinomas, such as clear-cell renal cell carcinomas, VHL-negative kidney adenocarcinomas, VHL-negative hemangioblastomas of the central nervous system, VHL-negative pheochromocytomas, VHL-negative endolymphatic sac tumors of the inner ear, VHL-negative epididymal and broad ligament cystadenomas, and VHL-negative islet-cell tumors of the pancreas) in a subject in need of treatment comprising administering to the subject a therapeutically effective amount of a G-rich oligonucleotide (GRO).

Description

TREATMENT OF VHL-NEGATIVE TUMORS

BACKGROUND OF THE INVENTION

Von Hippel-Lindau (VHL) disease is an autosomal dominant familial cancer syndrome that predisposes affected individuals to a variety of tumors. VHL kindreds have germline mutations in the VHL gene, and somatic inactivation or loss of the remaining wild-type VHL allele is linked to tumor formation. More specifically, the VHL gene is a tumor suppressor gene and its functional inactivation stimulates tumor formation in a variety of ways, such as by increasing the stability of Hypoxia Inducible Factor- 1 (HIF-1). The most common tumors resulting from inactivation of the VHL gene are hemangioblastomas of the central nervous system, pheochromocytomas and renal cell carcinomas (RCC).

Of these common VHL-negative tumors, renal cell carcinomas has proven most refractory to standard therapies. In fact, (RCC) is estimated to have caused 13,010 deaths in 2008 (Jemal et al. (2008) CA Cancer J. Clin. 58: 71-96) in the United States alone. Use of chemotherapy for the treatment of RCC has

demonstrated only low levels of antitumor activity. Immunotherapy has long been the standard of care for the treatment of RCC. For example, interferon (IFN) alpha has produced response rates of up to 15% with modest to no prolongation of overall survival versus inactive controls. In addition, while interleukin (IL)-2 is noteworthy for a small but real percentage of durable complete remissions, it can only be applied to a select minority or RCC patients, because of significant side effects including capillary leak syndrome that necessitates intensive blood pressure monitoring and in some instances the need for vasopressors.

In view of the above, new agents targeting VHL-negative tumors such as renal cell carcinomas are needed.

SUMMARY OF THE INVENTION

The present invention relates to methods of treating VHL-negative tumors (e.g., VHL-negative renal cell carcinomas, such as clear-cell renal cell carcinomas, VHL-negative kidney adenocarcinomas, VHL-negative hemangioblastomas of the central nervous system, VHL-negative pheochromocytomas, VHL-negative endolymphatic sac tumors of the inner ear, VHL-negative epididymal and broad ligament cystadenomas, and VHL-negative islet-cell tumors of the pancreas) in a subject in need of treatment.

In a first embodiment, the present invention is a method of treating a VHL- negative tumor in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a G-rich oligonucleotide. In a particular aspect, the VHL-negative tumor is selected from the group consisting of: VHL-negative renal cell carcinomas, VHL-negative kidney adenocarcinomas, VHL-negative hemangioblastomas of the central nervous system, VHL-negative

pheochromocytomas, VHL-negative endolymphatic sac tumors of the inner ear, VHL-negative epididymal and broad ligament cystadenomas, and VHL-negative islet-cell tumors of the pancreas. In a more particular aspect, the VHL-negative tumor is a renal cell carcinoma, such as a VHL-negative clear-cell renal cell carcinoma. In a most particular aspect, the VHL-negative tumor is a renal cell carcinoma and the subject is a human.

In a second embodiment, the present invention is a method of treating a VHL-negative tumor in a subject in need thereof comprising the steps of determining that the subject has a VHL-negative tumor; and administering to the subject having the VHL-negative tumor a therapeutically effective amount of a G- rich oligonucleotide. In a particular aspect, the VHL-negative tumor is selected from the group consisting of: VHL-negative renal cell carcinomas, VHL-negative kidney adenocarcinomas, VHL-negative hemangioblastomas of the central nervous system, VHL-negative pheochromocytomas, VHL-negative endolymphatic sac tumors of the inner ear, VHL-negative epididymal and broad ligament

cystadenomas, and VHL-negative islet-cell tumors of the pancreas. In a more particular aspect, the VHL-negative tumor is a renal cell carcinoma, such as a VHL- negative clear-cell renal cell carcinoma. In a most particular aspect, the VHL- negative tumor is a renal cell carcinoma and the subject is a human.

In a third embodiment, the present invention is a method of treating a VHL- negative renal cell carcinoma in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of a G-rich oligonucleotide. In a particular aspect, the VHL-negative renal cell carcinoma is a clear-cell renal cell carcinoma. In a most particular embodiment, the subject is a human.

In a fourth embodiment, the present invention is a method of inhibiting

(either in vivo or in vitro) proliferation of a VHL-negative tumor comprising contacting the tumor with a therapeutically effective amount of a G-rich

oligonucleotide. In a particular aspect, the VHL-negative tumor is selected from the group consisting of: VHL-negative renal cell carcinomas, VHL-negative kidney adenocarcinomas, VHL-negative hemangioblastomas of the central nervous system, VHL-negative pheochromocytomas, VHL-negative endolymphatic sac tumors of the inner ear, VHL-negative epididymal and broad ligament cystadenomas, and VHL- negative islet-cell tumors of the pancreas. In a more particular aspect, the VHL- negative tumor is a renal cell carcinoma, such as a VHL-negative clear-cell renal cell carcinoma. In a most particular aspect, the VHL-negative tumor is a renal cell carcinoma and contacting is done in vivo.

In a fifth embodiment, the present invention is a method of enhancing apoptosis of a VHL-negative tumor in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a G-rich oligonucleotide. In a particular aspect, the VHL-negative tumor is selected from the group consisting of: VHL-negative renal cell carcinomas, VHL-negative kidney adenocarcinomas, VHL-negative hemangioblastomas of the central nervous system, VHL-negative pheochromocytomas, VHL-negative endolymphatic sac tumors of the inner ear, VHL-negative epididymal and broad ligament cystadenomas, and VHL- negative islet-cell tumors of the pancreas In a more particular aspect, the VHL- negative tumor is a renal cell carcinoma, such as a VHL-negative clear-cell renal cell carcinoma. In a most particular aspect, the VHL-negative tumor is a renal cell carcinoma and the subject is a human.

In a sixth embodiment, the present invention is a method of inhibiting angiogenesis of a VHL-negative tumor in a subject in need thereof comprising administering to the said a therapeutically effective amount of a G-rich

oligonucleotide. In a particular aspect, the VHL-negative tumor is selected from the group consisting of: VHL-negative renal cell carcinomas, VHL-negative kidney adenocarcinomas, VHL-negative hemangioblastomas of the central nervous system, VHL-negative pheochromocytomas, VHL-negative endolymphatic sac tumors of the inner ear, VHL-negative epididymal and broad ligament cystadenomas, and VHL- negative islet-cell tumors of the pancreas. In a more particular aspect, the VHL- negative tumor is a renal cell carcinoma, such as a VHL-negative clear-cell renal cell carcinoma. In a most particular aspect, the VHL-negative tumor is a renal cell carcinoma and the subject is a human.

The G-rich oligonucleotide administered in any of the first through sixth embodiments above or any of the aspects thereof comprises a sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 18 (e.g., SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18). For example, the G- rich oligonucleotide administered in any of the first, second or third embodiments or any of the aspects thereof is SEQ ID NO: 1. BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.

FIG. 1 A is a plot of cell culture absorbance as a function of concentration of AS 1411, showing VHL-positive and VHL-negative RCC4 cell density during the MTT ("method of transcriptional and translational") cell proliferation assays.

FIG. IB is a plot of cell culture absorbance as a function of concentration of AS 1411, showing VHL-positive and VHL-negative ACHN cell density during the MTT ("method of transcriptional and translational") cell proliferation assays.

FIG. 2 A depicts the results of a clonogenic assay showing effects of AS 1411 on VHL-negative and VHL-positive RCC4 cells.

FIG. 2B depicts the results of a clonogenic assay showing effects of AS 1411 on VHL-negative and VHL-positive ACHN cells. FIG. 3A depicts the results of a FACS flow cytometry analysis of the cell cycle of the VHL-negative and VHL-positive RCC4 cells in the presence of AS1411.

FIG. 3B depicts the results of a FACS flow cytometry analysis of the cell cycle of the VHL-negative and VHL-positive ACHN cells in the presence of AS1411.

DETAILED DESCRIPTION OF THE INVENTION

VHL-Negative Tumors

As used herein, the term "VHL-Negative Tumor" refers to any tumor characterized by tumor cells in which the von Hippel-Lindau tumor suppressor (VHL) gene is inactive (i.e., fails to express the gene product pVHL) or expresses mutant pVHL gene product. VHL-negative tumors include, but are not limited to, VHL-negative renal cell carcinomas, such as VHL-negative clear-cell renal cell carcinomas, VHL-negative kidney adenocarcinomas, VHL-negative

hemangioblastomas of the central nervous system, VHL-negative

pheochromocytomas, VHL-negative endolymphatic sac tumors of the inner ear, VHL-negative epididymal and broad ligament cystadenomas, and VHL-negative islet-cell tumors of the pancreas.

Renal cell carcinomas (RCC) are the most common form of kidney cancer and the majority of these (75%) have biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) by mutation or less commonly by hypermethylation. The VHL gene product, pVHL, has multiple functions including ubiquitination of HIF transcription factors, regulation of endocytosis, and control of microtubule orientation. The present invention is based on the discovery that the antiproliferative effect of G-rich oligonucleotides (GROs) on renal cancer cell lines is enhanced where the cell line does not express Von-Hippel-Lindau (VHL) factor, i.e. is VHL- negative.

Determination that a tumor cell is VHL-negative can be accomplished by any standard technique, such as by determining the absence of a pVHL gene product by means of immunoblotting (Western blotting) or immunoprecipitation, or by determining the absence of mRNA by means of Northern blotting, or determining chromosomal deletion or rearrangements by means of Southern blotting. (See e.g., F. Latif et al., Science, Vol. 260, 28 May 1993, p. 1317.) Examples of VHL- negative cancers include, but are not limited to, renal cell carcinomas, such as clear- cell renal cell carcinomas.

As used herein, the term "cancer" refers to the uncontrolled growth of abnormal cells that have mutated from normal tissues. A cancerous tumor

(malignancy) is of potentially unlimited growth and expands locally by invasion and systemically by metastasis. Cancers that can be treated by the methods of the present invention include cancers characterized by VHL-negative tumors (e.g., renal cell carcinomas).

G-Rich Oligonucleotides (GRO)

The term "G-rich oligonucleotide (GRO)" as used herein refers to oligonucleotides rich in guanosine. "Rich in guanosine", means comprising 50% or more guanosine nucleosides. Preferably, the G-rich oligonucleotides useful in embodiments of the present invention are primarily comprised of guanosine with at least one contiguous guanosine repeat. GROs useful in the methods described herein consist of 4 to 100 nucleotides, for example from 14 to 56, 10 to 30, or 25 to 32 nucleotides. The oligonucleotides have anti-proliferative activity against VHL- negative tumors.

As used herein, the term "oligonucleotide" is defined as a molecule comprising two or more deoxyribonucleotides or ribonucleotides with DNA, RNA, 2'-0-methyl, phosphorothioate or other chemically similar backbones. In referring to "bases" or "nucleotides" the terms include both deoxyribonucleic acids and ribonucleic acids.

The GROs useful in embodiments of the present invention are capable of forming G-quartet structures comprising a hydrogen bonded arrangement of four guanines (G-quartets are also referred to as G-tetrads). G-quartets can be formed intra- or intermolecularly, and GROs which form G-quartets can further associate into quadruplexes formed from one, two, three, or more G-quartets stacked on top of each other. Representative G-quartet-forming sequences have been described in Burge et al, Nucleic Acids Research, 2006, Vol. 00, No. 00 1-14: (i) "G_mX_nG_mX₀G_mX_pG_m" for unimolecular (i.e., intramolecular) quadruplexes; (ii) X_nG_mX₀G_mX_p for bimolecular quadruplexes; and (iii) X_nG_mX₀ or G_mX_nG_m, wherein m is the number of G residues which are usually involved in the G-tetrad, and the G- tracts do not have to be of equal length.

In some embodiments, the GROs useful in embodiments of the present invention bind to nucleolin. Binding to nucleolin can be assessed using routine techniques known in the art, for example, an electrophoretic mobility shift assay (EMSA) (see WO 2000/61597).

Nucleolin is an abundant multifunctional 110 kDa phosphoprotein thought to be located predominantly in the nucleolus of proliferating cells and which has been implicated in many cellular activities (Tuteja et al. (1998) Crit. Rev. Biochem. Mol. Biol. 33,407-436; Ginisty et al. (1999) J. Cell Sci. 112,761-772; Ginisty et al. (1998) EMBO J. 17, 1476-1486;. Kibbey et al. (1995) J. Neurosci. Res. 42,314-322; Lee et al. (1998) J. Biol. Chem. 273,7650-7656; Waggoner et al. (1998) J. Virol. 72,6699- 6709, Gotzmann et al. (1997) Electrophoresis 18,26452653, Leger-Silvestre et al.

(1997) Chromosoma 105,542-52, Tuteja et al. (1995) Gene 160,143-148). Levels of nucleolin are known to relate to the rate of cellular proliferation (Derenzini et al. (1995) Lab. Invest. 73,497-502; Roussel et al. (1994) Exp. Cell Res. 214,465-472.), being elevated in rapidly proliferating cells, such as malignant cells, and lower in more slowly dividing cells. Nucleolin is overexpressed in the cytoplasm and on the surface of cancer cells (Ireson et al, Mol Cancer Ther. 2006; 5: 2957-62; Dapic et al, Nucleic Acids Res. 2003; 31 : 2097-107; Bates et al J Biol Chem. 1999; 274: 26369- 77).

In some embodiments of the present invention the G-rich oligonucleotides are primarily comprised of thymidine and guanosine with at least one contiguous guanosine repeat and contain one or more GGT motifs.

Examples of GROs useful in the methods of the present invention include, but are not limited to, those in Table 1, as well as oligonucleotides having a sequence identity of at least about 80 to 99 % with any of SEQ ID NO: 1 to 18. As used herein, "at least about 80 to 99%" is specifically intended to individually disclose 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%.

Table 1. SEQ ID No : 1 : 5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-3' (alternatively known as AS1411, GR026B and AGRO100)

SEQ ID No : 2 : 5' GTTGTTTGGGGTGG-3' (GR014A)

SEQ ID No : 3 : 5'-GTTGTTTGGGGTGGT-3' (GR015A)

SEQ ID No : 4 : 5'-GGTTGGGGTGGGTGGGGTGGGTGGG-3' (GR025A)

SEQ ID No : 5 : 5' -TTTGGTGGTGGTGGTTGTGGTGGTGGTG- 3 ' (GR028A)

SEQ ID No : 6 : 5' -TTTGGTGGTGGTGGTTGTGGTGGTGGTGG- 3 ' (GR029A)

SEQ ID No : 7 : b'- 1 1 1 G 1 G 1 G 1 G 1 1 1 1 G 1 G 1 G 1 G-3' (GR029-2)

SEQ ID No : 8 : 5' -TTTGGTGGTGGTGGTGGTGGTGGTGGTGG- 3 ' (GR029-3)

SEQ ID No : 9 : 5'-TTTGGTGGTGGTGGTTTGGGTGGTGG TGG-3' (GR029-5)

SEQ ID No : 10 : 5' -TGGTGGTGGTGGT- 3 ' (GR029-13)

SEQ ID No : 11 : 5'-GGTGGTGGTGG-3' (GR011A)

SEQ ID No : 12 : 5'-GGTGGTTGTGGTGG- 3' (GR014C)

SEQ ID No : 13

5'-GGTGGTGG TGGTTGTGGTGGTGGTGGTTGTGGTGGTGGTGGTTGTGGTGGTGGTGG-3' (GR056A)

SEQ ID No : 14 : 5'-GGTGGTTGTGGTGGTTGTGGTGGTTGTGGTGG-3' (GR032A)

SEQ ID No : 15 : 5' -TTTGGTGGTGGTGGTTGTGGTGGTGGTGGTTT- 3 ' (GR032B)

SEQ ID No : 16 : 5' -GGTGGTGGTGGTTGTGGTGGTGGTGGTTT- 3 ' (GR029-6)

SEQ ID No : 17 : 5'-TTTGGTGGTGGTGGTGTGGTGGTGGTGG-3' (GR028B)

SEQ ID No : 18 : 5' -TGGTGGTGGT- 3 ' (GR013A).

Preferably, the G-rich oligonucleotide has the sequence of SEQ ID NO: 1, also referred to as AS 1411, GR026B and AGRO100.

In one embodiment, the G-rich oligonucleotide has a 3 ' end and a 5 ' end, and one or both of the 3 ' and 5 ' ends have been modified to alter a property of the G-rich oligonucleotide. Modifications at the 3' end include the addition of a propylamine group which has been found to increase the stability of the oligonucleotide to serum nucleases. Other modifications that are well known in the art include 3' and 5' modifications, for example, the binding of cholesterol, and backbone modifications, for example, phosphorothioate substitution and/or 2'-0-methyl DNA.

In one embodiment of the invention, a method of treating VHL-negative tumor in a subject in need thereof comprises administering to the subject a

therapeutically effective amount of a G-rich oligonucleotide that comprises the sequence of one of SEQ IDs Nos. 1 to 18. In a particular embodiment, the G-rich oligonucleotide is selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 18. Preferably, the G-rich oligonucleotide is that of SEQ ID NO: 1.

Administration

The GROs of the present invention can be administered to a patient or subject either alone or as part of a pharmaceutical composition. The GROs can be administered to patients either orally, rectally, parenterally (intravenously, intramuscularly, or subcutaneously), intracisternally, intravaginally, intraperitonally, intravesically, locally (powders, ointments, or drops), or as a buccal or nasal spray.

As used herein, the terms "administer" or "administering" refer to either directly administering a compound in accordance with the present teachings or a pharmaceutical composition containing the compound, or administering the compound or pharmaceutical composition indirectly via a prodrug derivative or analog which will form an equivalent amount of the active compound or substance within the body. The methods also can include identifying a subject in need of such treatment, and administering a compound disclosed herein to the subject in need thereof.

As used herein, the term "treating" refers to eliciting a desirable biological activity or effect. In some embodiments of the present invention, the desired biological response is a reduction (complete or partial) of at least one symptom associated with a VHL-negative tumor (e.g., renal cell carcinoma, such as clear-cell renal cell carcinoma). In yet another embodiment, the desired biological response is inhibition of cell proliferation in culture.

As used herein, "reducing (completely or partially) at least one symptom associated with a VHL-negative tumor" includes achieving, partially or

substantially, one or more of the following: arresting the growth or spread of a VHL- negative tumor, reducing the extent of a VHL-negative tumor (e.g., reducing size of a tumor or reducing the number of affected sites), inhibiting the growth rate of a - negative tumor, and ameliorating or improving a clinical symptom or indicator associated with a VHL-negative tumor (such as tissue or serum components). The term "treating" as used herein is intended to encompass curing as well as

ameliorating at least one symptom of the condition or disease. For example, in the case of cancer, a response to treatment includes a reduction in cachexia, increase in survival time, elongation in time to tumor progression, reduction in tumor mass, reduction in tumor burden and/or a prolongation in time to tumor metastasis, time to tumor recurrence, tumor response, complete response, partial response, stable disease, progressive disease, progression free survival, overall survival, each as measured by standards set by the National Cancer Institute and the U.S. Food and Drug Administration for the approval of new drugs (see Johnson et al. (2003) J. Clin. Oncol. 21(7): 1404-1411).

The term "therapeutically effective amount" means the amount of an oligonucleotide, that when administered to the subject is a proper doing regimen is sufficient to treat (i.e., elicit the desirable biological response) a VHL-negative tumor (e.g., a renal cell carcinoma). The therapeutically effective amount may be empirically determined by a skilled person such as a clinician based on the patient's clinical parameters including, but not limited to the stage of disease, age, gender, histology, and likelihood for tumor recurrence.

When administered for the treatment or inhibition of a particular disease state or disorder, it is understood that an effective dosage can vary depending upon the particular compound utilized, the mode of administration, and severity of the condition being treated, as well as the various physical factors related to the individual being treated. In therapeutic applications, a compound of the present teachings can be provided to a patient already suffering from a disease in an amount sufficient to treat the symptoms of the disease and its complications. The dosage to be used in the treatment of a specific individual typically must be subjectively determined by the attending physician. The variables involved include the specific condition and its state as well as the size, age and response pattern of the patient.

Exemplary dose ranges for the GROs for use according to the methods described herein are as follow:

The GROs can be administered at doses from 4 to 80 mg/kg/day (e.g., 40 mg/kg/day) for multiple days, for example, up to seven days per cycle (typically for a total of 10 to 560 mg/kg per cycle). For example, the GROs can be administered at 40 mg/kg/day on days 1-4 of a 28 day cycle for up to 2 cycles. The administration can be by continuous iv infusion. For example, the GROs (e.g., AS 1411) can be administered at 40 mg/kg/day by continuous iv infusion for days 1-4 of a 28-day cycle for up to 2 cycles.

The actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. For convenience, the total daily dosage may be divided and administered in portions during the day if desired. Intermittent therapy (e.g., one week out of three weeks or three out of four weeks) may also be used.

The G-rich oligonucleotides of the invention can be used in combination (i.e., coadministered) with other chemotherapuetic agents. Suitable

chemotherapeutics agents for use in combination with the G-rich oligonucleotides include, but are not limited to, DNA methyltransferase (DNMT) inhibitors, clofarabine, cytarabine, cis-platin, mitoxantrone, etoposide, camptothecin, 5- fluorouracil, vinblastine, paclitaxel, docetaxel, mithramycin A, dexamethosone, interferon alpha and inter leukin 2.

Coadminstration of the G-rich oligonucleotide with another

chemotherapeutic agent can take place sequentially in any order, simultaneously or a combination thereof. For example, administration of G-rich oligonucleotide, can take place prior to administration of the other chemotherapeutic agent, after the other chemotherapeutic agent, at the same time as the other chemotherapeutic agent or a combination thereof. For example, a total treatment period can be decided for the G-rich oligonucletoide. The other chemotherapeutic agent can be administered prior to onset of treatment with the G-rich oligonucleotide, or following treatment with the G-rich oligonucleotide. In addition, the other chemotherapeutic agent can be administered during the period of GRO administration but does not need to occur over the entire inhibitor treatment period.

Also, in general, the GROs do not have to be administered in the same pharmaceutical composition, and may be administered by different routes. The appropriate pharmaceutical composition and mode of administration (e.g., i.v., oral) may be readily determined by one skilled in the art. Pharmaceutical compositions comprising at least one compound described herein and one or more

pharmaceutically acceptable carriers, excipients, or diluents. Examples of such carriers are well known to those skilled in the art and can be prepared in accordance with acceptable pharmaceutical procedures, for example, those described in Remington's Pharmaceutical Sciences, 17th edition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, PA (1985), the entire disclosure of which is incorporated by reference herein for all purposes. As used herein, "pharmaceutically acceptable" refers to a substance that is acceptable for use in pharmaceutical applications from a toxico logical perspective and does not adversely interact with the active ingredient. Accordingly, pharmaceutically acceptable carriers are those that are compatible with the other ingredients in the formulation and are biologically acceptable. Supplementary active ingredients can also be incorporated into the pharmaceutical compositions. Compounds of the present teachings can be administered orally or parenterally, neat or in combination with conventional pharmaceutical carriers. Applicable solid carriers can include one or more substances which can also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet- disintegrating agents, or encapsulating materials. The compounds can be formulated in conventional manner, for example, in a manner similar to that used for known anti-inflammatory agents. Oral formulations containing an active compound disclosed herein can comprise any conventionally used oral form, including tablets, capsules, buccal forms, troches, lozenges and oral liquids, suspensions or solutions. In powders, the carrier can be a finely divided solid, which is an admixture with a finely divided active compound. In tablets, an active compound can be mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets can contain up to about 99% or greater of the active compound.

Capsules can contain mixtures of active compound(s) with inert filler(s) and/or diluent(s) such as the pharmaceutically acceptable starches (e.g., corn, potato or tapioca starch), sugars, artificial sweetening agents, powdered celluloses (e.g., crystalline and microcrystalline celluloses), flours, gelatins, gums, and the like.

Useful tablet formulations can be made by conventional compression, wet granulation or dry granulation methods and utilize pharmaceutically acceptable diluents, binding agents, lubricants, disintegrants, surface modifying agents (including surfactants), suspending or stabilizing agents, including, but not limited to, magnesium stearate, stearic acid, sodium lauryl sulfate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, microcrystalline cellulose, sodium carboxymethyl cellulose, carboxymethylcellulose calcium,

polyvinylpyrrolidine, alginic acid, acacia gum, xanthan gum, sodium citrate, complex silicates, calcium carbonate, glycine, sucrose, sorbitol, dicalcium

phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, low melting waxes, and ion exchange resins. Surface modifying agents can include nonionic and anionic surface modifying agents. Representative examples of surface modifying agents include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidol silicon dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum silicate, and triethanolamine. Oral formulations herein can utilize standard delay or time-release formulations to alter the absorption of the active compound(s). The oral formulation can also consist of administering an active compound in water or fruit juice, containing appropriate solubilizers or emulisifiers as needed.

Liquid carriers can be used in preparing solutions, suspensions, emulsions, syrups, and elixirs. An active compound described herein can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, or a mixture of both, or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers, and osmo-regulators. Examples of liquid carriers for oral and parenteral administration include, but are not limited to, water (particularly containing additives as described above, e.g., cellulose derivatives such as a sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration, the carrier can be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are used in sterile liquid form compositions for parenteral

administration. The liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellants. Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intrathecal, intramuscular,

intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously. Compositions for oral administration can be in either liquid or solid form.

Preferably the pharmaceutical composition is in unit dosage form, for example, as tablets, capsules, powders, solutions, suspensions, emulsions, granules, or suppositories. In such form, the pharmaceutical composition can be sub-divided in unit dose(s) containing appropriate quantities of the active compound. The unit dosage forms can be packaged compositions, for example, packeted powders, vials, ampoules, prefilled syringes or sachets containing liquids. Alternatively, the unit dosage form can be a capsule or tablet itself, or it can comprise the appropriate number of any such compositions in package form. Such unit dosage form may contain from about 1 mg/kg of active compound to about 500 mg/kg of active compound, and can be given in a single dose or in two or more doses. Such doses can be administered in any manner useful in directing the active compound(s) to the recipient's bloodstream, including orally, via implants, parenterally (including intravenous, intraperitoneal and subcutaneous injections), rectally, vaginally, and transdermally. Such administrations can be carried out using the compounds of the present teachings including pharmaceutically acceptable salts thereof, in lotions, creams, foams, patches, suspensions, solutions, and suppositories (e.g., rectal and vaginal).

In some cases, it may be desirable to administer a compound directly to the airways of the patient in the form of a dry powder or an aerosol. For administration by intranasal or intrabronchial inhalation, the compounds of the present teachings can be formulated, for example, into an aqueous or partially aqueous solution.

Compounds described herein can be administered enterally or parenterally (such as, without limitation, interperitoneal, intramuscular, intravascular, intrathecal, intra- articular or subcuteaneous injection or infusion). Solutions or suspensions of these active compounds or pharmaceutically acceptable salts thereof can be prepared in water suitably mixed with a surfactant such as hydroxyl-propylcellulose.

Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations typically contain a preservative to inhibit the growth of

microorganisms.

The pharmaceutical forms suitable for injection can include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In preferred embodiments, the form is sterile and its viscosity permits it to flow through a syringe. The form preferably is stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

Compounds described herein can be administered transdermally, i.e., administered across the surface of the body and the inner linings of bodily passages including epithelial and mucosal tissues. Such administration can be carried out using the compounds of the present teachings including pharmaceutically acceptable salts thereof, in lotions, creams, foams, patches, suspensions, solutions, and suppositories (e.g., rectal and vaginal). Topical formulations that deliver active compound(s) through the epidermis can be useful for localized treatment of inflammation and arthritis.

Transdermal administration can be accomplished through the use of a transdermal patch containing an active compound and a carrier that can be inert to the active compound, can be non-toxic to the skin, and can allow delivery of the active compound for systemic absorption into the blood stream via the skin. The carrier can take any number of forms such as creams and ointments, pastes, gels, and occlusive devices. The creams and ointments can be viscous liquid or semisolid emulsions of either the oil-in-water or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active compound can also be suitable. A variety of occlusive devices can be used to release the active compound into the blood stream, such as a semi-permeable membrane covering a reservoir containing the active compound with or without a carrier, or a matrix containing the active compound. Other occlusive devices are known in the literature.

Compounds described herein can be administered into a body cavity, (e.g., rectally or vaginally) in the form of a conventional suppository. Suppository formulations can be made from traditional materials, including cocoa butter, with or without the addition of waxes to alter the suppository's melting point, and glycerin. Water-soluble suppository bases, such as polyethylene glycols of various molecular weights, can also be used.

Lipid formulations or nanocapsules can be used to introduce compounds of the present teachings into host cells either in vitro or in vivo. Lipid formulations and nanocapsules can be prepared by methods known in the art. For example, the compounds described herein can be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances, and are formed by mono or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any nontoxic, pharmacologically acceptable lipid capable of forming liposomes can be used.

The term "subject" and "patient" are used interchangeably and mean all mammals including humans. The term "mammal" refers to any warm blooded species, such as a human, and examples of subjects include humans, cows, dogs, cats, goats, sheep, and pigs. Subjects can be adult or pediatric individuals. A human pediatric individual is a human individual at any age between the day of its birth (i.e., zero (0) years of age) and 21 years of age. A human pediatric individual includes a "neonate" or "newborn" which is a human individual at any age between the day of its birth (i.e., zero (0) years of age) and 30 days of age; an "infant" which is a human individual at any age between 31 days and two years of age; a "child" which is an individual at any age between two and twelve years of age; an

"adolescent" which is an individual at any age between twelve and twenty-one years of age. A human adult is an individual older than twenty-one years of age.

Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes are described as having, including, or comprising specific process steps, it is contemplated that compositions of the present teachings also can consist essentially of, or consist of, the recited components, and that the processes of the present teachings also consist essentially of, or consist of, the recited processing steps.

In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be

understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.

The use of the singular herein includes the plural (and vice versa) unless specifically stated otherwise. In addition, where the use of the term "about" is before a quantitative value, the present teachings also include the specific quantitative value itself, unless specifically stated otherwise.

Aspects of the present teachings can be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.

EXEMPLIFICATION

In the following examples, AS 1411 is a single-stranded phosphodiester DNA oligonucleotide 26 nucleotides in length with a molecular weight 8272 Da and which corresponds to SEQ ID NO: 1.

In the examples below, the effects of AS 1411 treatment for two matched pairs of RCC cell lines that are positive or negative for functional VHL have been investigated. The cell lines are described in Table 2.

Table 2: Cell lines used in this study.

VHL-positive VHL-negative

RCC4 "RCC+" "RCC-"

RCC4 stably transfected with Parental RCC 4 cells, which have wild type VHL. mutant, inactive VHL.

ACHN "ACHN+" "ACHN-"

Parental AcHN cells, which ACHN stably transfected with shRNA have wild type VHL. targeting VHL. Example 1 AS 1411 Selectively Inhibits Proliferation of VHL-negative Renal Cell Carcinoma Cells Lines

Renal cell carcinoma cells (see Table 2) were plated at 1 ,200 cells per well in 96-well plates. The following day, AS1411 (active aptamer) or CRO (inactive control oligonucleotide) was added directly to wells to give a final concentration of 0-12 μΜ. The relative number of viable cells in each well was determined after a further 5 days using the MTT cell proliferation assay. {See, e.g., Mosmann

(December 1983). "Rapid colorimetric assay for cellular growth and survival:

application to proliferation and cytotoxicity assays." J. of Imm. Meth. 65 (1-2): 55- 63 and Cory et al., (July 1991) "Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture". Cane. Comm. 3 (7): 207-12.) Each experiment was performed in triplicate, and bars shown are standard error of the mean. Experiments were repeated to confirm reproducibility and representative results are shown.

The results (FIG. 1A and FIG. IB) show that the antiproliferative effect of

AS 1411 is enhanced in both VHL-negative RCC4 cell line and VHL-negative ACHN cell line in comparison to the matched VHL-positive cell lines.

Example 2 AS1411 Selectively Inhibits Clonogenicity of VHL-negative Renal Cell Carcinoma Cell Lines

Renal cell carcinoma cells (see Table 2) were plated at 300 cells per 60 mm tissue culture dish. The following day, AS 1411 (active aptamer) or CRO (inactive control oligonucleotide) was added and cells were incubated in the presence of drug for 10 days. The colonies were fixed and stained with crystal violet. Each experiment was performed at least three times and representative results are shown.

The results (FIG. 2A and FIG. 2B) show that colonogenicity of both VHL- negative RCC4 cell line and VHL-negative ACHN cell line is inhibited in the presence of AS 1411.

Example 3 AS 1411 Induces S and G2/M Cell Cycle Arrest Only in VHL-negative Renal Cell Carcinoma Cell Lines Renal cell carcinoma cells (see Table 2) were treated for three or four days (as indicated) with 10 μΜ CRO (inactive control oligonucleotide) or were left untreated. Cells were harvested by trypsinization, fixed and stained with propidium iodide using the Cycle Test Plus™ kit (Becton Dickinson®). Cells were then analyzed using a FACScan cytometer. The experiments were repeated to verify the results and representative histograms are shown.

The results (FIG. 3 A and FIG. 3B) show that AS 1411 induces S and G2/M cell cycle arrest in both the VHL-negative RCC4 cells and the VHL-negative ACHN cells. More specifically, cell cycle analysis shows that VHL-negative RCC4 cells treated with AS 1411 accumulate predominantly in S phase after 72 hours, followed by S and G2/M phase of cell cycle after 96 hour treatment. In contrast, there was no perturbation of the cell cycle in the VHL-positive RCC4/VHL cells.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS is claimed is:

A method of treating a VHL-negative tumor in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a G-rich oligonucleotide.

The method of Claim 1, wherein the VHL-negative tumor is selected from the group consisting of: VHL-negative renal cell carcinomas, VHL-negative kidney adenocarcinomas, VHL-negative hemangioblastomas of the central nervous system, VHL-negative pheochromocytomas, VHL-negative endolymphatic sac tumors of the inner ear, VHL-negative epididymal and broad ligament cystadenomas, and VHL-negative islet-cell tumors of the pancreas.

The method of Claim 2, wherein the VHL-negative tumor is a VHL-negative renal cell carcinoma.

The method of Claim 3, wherein the VHL-negative renal cell carcinoma is a clear-cell renal cell carcinoma.

The method of any one of Claims 1-4, wherein the G-rich oligonucleotide comprises a sequence selected from the groups consisting of SEQ ID NO: 1 through SEQ ID NO: 18.

The method of any one of Claims 1-4, wherein the G-rich oligonucleotide selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 18.

7. The method of any one of Claims 1-4, wherein the G-rich oligonucleotide comprises a sequence of SEQ ID NO: 1. The method of any one of Claims 1-4, wherein the G-rich oligonucleotide consists of the sequence of SEQ ID NO: 1.

A method of treating a VHL-negative renal cell carcinoma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of AS 1411.

The method of Claim 9, wherein the VHL-negative renal cell carcinoma is a clear-cell renal cell carcinoma.

The method of Claim 9 or Claim 10, wherein the subject is a human.

The method of Claim 11, wherein the AS 1411 is administered at a dose of from 4 mg/kg/day to 80 mg/kg/day.

The method of Claim 12, wherein the AS 1411 is administered for multiple continuous days.

The method of Claim 13, wherein the AS 1411 is administered for up to seven days.

15. The method of Claim 12, wherein the AS 1411 is administered at

40mg/kg/day on days 1-4 of a 28 day cycle.

The method of Claim 15, wherein the AS 1411 is administered by continuous IV infusion.

17. A method of enhancing apoptosis of a VHL-negative tumor in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a G-rich oligonucleotide.

18. The method of Claim 17, wherein the VHL-negative tumor is a VHL- negative renal cell carcinoma. The method of Claim 18, wherein the VHL-negative renal cell carcinoma is a clear-cell renal cell carcinoma.

The method of any one of Claims 17-19, wherein the G-rich oligonucleotide comprises a sequence selected from the groups consisting of SEQ ID NO: 1 through SEQ ID NO: 18.

A method of inhibiting angiogenesis in a VHL-negative tumor in a subject in need thereof comprising administering to the subject therapeutically effective amount of a G-rich oligonucleotide.

The method of Claim 21, wherein the VHL-negative tumor is a VHL- negative renal cell carcinoma.

The method of Claim 22, wherein the VHL-negative renal cell carcinoma is a clear-cell renal cell carcinoma.

24. The method of any one of Claims 21-23, wherein the G-rich oligonucleotide comprises a sequence selected from the groups consisting of SEQ ID NO: 1 through SEQ ID NO: 18.