Abstract:
It is becoming increasingly clear that the genome and epigenome of another particular cancer cell represents the end product of a vast array of selection events, creating an extremely heterogeneous metabolic landscape. Since genomic and epigenomic variability exists between different cells in a tumor, the target complexity becomes even more extreme when the entire cancer cell population is considered. The only way to overcome this target complexity is by developing complex treatment modalities capable of simultaneously interdicting multiple pathways critical to the growth and survival of the cancer cell population. We have developed a method to couple suppression of NADPH levels (by inhibition of the Pentose Phosphate Pathway) with a system of antisense oligonucleotides targeting NADPH-dependent enzymes critical to the cancer cell. In the preferred embodiment of this invention, the PPP inhibitor is administered systemically, and the antisense oligonucleotides locally, directly to the tumor.

Description:
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH OR DEVELOPMENT 
       [0001]    Although the applicant has received several federal research grants in the past (e.g., RO1 CA47217; R29 CA47217), the work underlying this invention is unrelated to the work conducted under such grants. 
     
    
     CROSS REFERENCE TO RELATED PATENTS 
       [0002]    None 
       SEQUENCE LISTING 
       [0003]    File Attached. All sequences containing ADENINE are understood to exist in both unmethylated adenine and methylated adenine (6meA) forms, and in both RNA and DNA forms, both single and double stranded. Similarly, because some dogs may exhibit hypersensitivity to unmethylated CG dinucleotides, all sequences in the listing containing CG dinucleotides are understood to exist in both methylated and unmethylated forms. 
       PRIOR DISCLOSURES 
       [0004]    There have been no prior disclosures of this invention 
       BACKGROUND OF THE INVENTION 
       [0005]    1. Field of the Invention 
         [0006]    The field is directed to compositions and methods of treating canine cancer comprising a system of antisense oligonucleotides designed to destroy specific messenger RNA (mRNA) targets of NADP-dependent enzymes or pathways, combined with an inhibitor of NADPH production, in order to synergize the anticancer effect of such combination. 
         [0007]    2. Description of Related Art 
         [0008]    Approximately 1.7 million Americans are diagnosed with cancer each year, and approximately 600,000 will die each year from cancer (National Cancer Institute, Annual Cancer Statistics, United States, http://seer.cancer.gov/statfacts/). Worldwide, about 14 million new cases of cancer are diagnosed annually, and more than 8 million die from cancer (World Cancer Research Fund International, http://www.wcrf.org/int/cancer-facts-figures/worldwide-data). It has been authoritatively stated that more than 100 years of research into cancer treatment has resulted in an increased life span of as little as 3 months for many, if not most forms of cancer. (See Siddhartha Mukherjee , The Emperor of All Maladies, a Biography of Cancer; Simon &amp; Schuster, 2010). In dogs, the situation is even worse due to their increased cancer risk as compared to humans. (See Torres de la Riva, G et al, PLoS One, 2013, 8(2):e55937). There thus remains a critical need to augment current treatment modalities, and to devise new, better treatment modalities for both human and veterinary cancer. 
         [0009]    (0003) Many of the targets of classical chemotherapy drugs, for example methotrexate (CAS 59-05-2; MTX) target an enzyme requiring NADPH as cofactor. In the case of MTX, this enzyme is dihydrofolate reductase (CAS 9002-0303; DHFR). DHFR is the rate limiting step in the one carbon pool, or folate pathway ( FIG. 1 a, b   ). This pathway was deemed an appropriate target for chemotherapy design because it produces virtually all of the cancer cell&#39;s purines, pyrimidines, and S-adenosylmethionine (SAM), all of which play critical roles in intermediary metabolism and cell division. However, MTX is a quite toxic molecule when it is delivered systemically. In some therapeutic regimens, MTX is administered in lethal doses, and then the patient is rescued with folinic acid (leucovorin; CAS 1492-18-8). Leucovorin bypasses the DHFR inhibition by MTX, and replenishes the folate pathway and all of its products. 
         [0010]    Another NADPH-dependent enzyme is ribonucleotide reductase (CAS 9047-64-7sy; RNR). RNR is a critical enzyme of deoxynucleotide synthesis, converting ribonucleotides to their corresponding deoxyribonucleotides. It directly requires NADPH reducing equivalents to effect this reaction ( FIG. 2 ). Hydroxyurea (CAS 127-07-1) is a classical RNR inhibitor, first synthesized in 1869. HU shows efficacy in a variety of hematologic and other malignancies, but resistance can develop by down regulation of the M2 component of RNR. (See McClarty, G A et. al., Biochemical and Biophysical Research Communications 154(3):975-981, 1988). (0005) Gemcitabine (CAS 95058-81-4), another inhibitor of RNR, has found use in the treatment of a variety of human cancers, but the development of resistance is a recurring problem (See Kim, M P and Gallick, E, Clinical Cancer Res 14:2247, 2008). Clearly, new methods to inhibit RNR are needed that can avoid the development of resistance that appear to inhibit the effectiveness of the small molecule approach. 
         [0011]    Thymidine kinase is found to be upregulated in many different kinds of cancer, especially lymphoma. (See Alegre, M M et al, Cancer and Clinical Oncology 2(1):159, 2013) Thymidine kinase is also an NADPH-dependent enzyme because its substrate, thymidine, requires folate pathway products for its synthesis, and the folate pathway is highly NADPH-dependent, as noted above. Although TK is recognized as a viable target for anticancer therapy (See Byun, Y, Doctoral Dissertation, The Ohio State University, 2006) and much work is ongoing attempting to develop small molecule TK inhibitors, none have yet been developed. 
         [0012]    (0007) Like TK, deoxycytidine kinase (CAS 9002-06-6; dCK) is also NADPH dependent because its substrate is produced in part using products of the folate pathway. dCK phosphorylates deoxycytidine, deoxyguanosine and deoxyadenosine to their monophosphate forms, and so is an important enzyme in nucleotide biosynthesis and cell division. 2′-chloro-2′-deoxyadenosine (Cladribine, CAS 4291-63-8) is an inhibitor of dCK with activity in leukemia, but clearly, the small molecule approach has not produced much in the way of active inhibitors of this enzyme critical to the cancer cell. One reason for this may be that dCK is required to activate some commonly used anticancer drugs such as gemcitabine and cytarabine (cytosine arabinoside, CAS). (See Staub, M and Eriksson, S, Cancer Drug Discovery and Development, Peters, G J ed., Humana Press, Inc., Totowa, N.J.). Our antisense oligonucleotides should not be used in settings where use of agents that require activation by dCK. However, treatment of most solid tumors do not use such agents. 
         [0013]    Thymidylate Synthetase (CAS 9031-61-2; TS) is another indirectly NADPH-dependent enzyme, because its substrate, dUMP, is also dependent upon folate pathway products for its synthesis. Several current and classical drugs target TS, including 5-Fluorouracil (CAS 51-21-8), and its prodrug, capecitabine. The utility of these TS inhibitors is reduced substantially by their toxicity profile, which can be life threatening in some individuals (Saif, M W, Cancer Genomics Proteomics. 2013 March-April; 10(2):89-92). 
         [0014]    Adenosine receptors are dependent upon NADPH because the purine backbone required for the synthesis of adenosine is manufactured in the folate pathway. Since adenosine, through its receptors, primarily the adenosine A2A and A1 receptors, appears to play a fundamental role in cancer (Fishman, P, Handbook Exp Pharmacol 193:399-441, 2013; doi: 10.1007/978-3-540-89616-9_14), for example as a main tumor effector of escape from host immune control, novel methods of inhibiting adenosine receptor function in cancer are needed. 
         [0015]    Tyrosine kinases such as c-kit are NADPH dependent because they are regulated by NADPH Oxidase (Paletta-Silva, R, Int J Mol Sci 14(2):3683-3704, 2013). Palladia is a c-kit inhibitor that has found some use in the treatment of canine mast cell cancer, but the upregulation of c-kit in a variety of tumor types clearly indicates the need for additional c-kit inhibitor strategies. 
         [0016]    One characteristic feature of cancer is dramatically enhanced de novo fatty-acid synthesis. (See Swierczynski, J et al, World J Gastroenterology 20(9):2279-2303, 2014). Fatty acid synthesis is heavily NADPH dependent, and the FASN gene is frequently found to be significantly upregulated in cancer (See Flavin, R et al, Future Oncol 6(4):551-562, 2010). Nevertheless, no reliable treatment based upon the inhibition of the FASN gene product has been discovered. 
         [0017]    Farnesyl transferase (FT) and Geranylgeranyl transferase (GGT) enzymes, critical for the isoprenylation of Ras, Rab and other oncoproteins, are potently dependent upon NADPH because their substrates are products of the mevalonate pathway, the rate limiting enzyme of which is HMG CoA reductase. HMG CoA reductase is extremely rare in intermediary metabolism in requiring 2 mols of NADPH for each mol of mevalonic acid product produced (Schultz, S and Nyce, J W, cancer Res 51:6563-6567, 1991). Although there are several statin drugs available that inhibit HMG CoA reductase, methods to specifically inhibit only portions of this pathway, e.g., farnesylation of Ras, Geranylation of Rab, are sorely needed. 
         [0018]    The inactivation of tumor suppressor genes by aberrant DNA methylation is a well-known problem and rivals mutation as a mechanism of carcinogenesis. (See Robert, M F, Nat Genet 33(1):61-5, 2003). Most of this aberrant DNA methylation is thought to be induced by one of the primary DNA methylation gene products, DNMT1. While demethylating agents do exist, e.g., 5-azacytidine (CAS 320-67-2) and 5-aza-2′-deoxycytidine (CAS 2353-33-5), they must be incorporated into DNA to exert cytotoxicity, which can be a source of mutation in normal tissues. Is DNA methylation an NADPH-dependent process? Yes, it is. The methyl donor in all such methylation reactions is SAM, another product of the heavily NADPH-dependent folate pathway. Without SAM, DNMT1 cannot function. New ways to reverse the inactivation of tumor suppressor genes by aberrant methylation of them by DNMT1, methods that do not involve mutation-inducing events like incorporation into the DNA, are greatly needed. 
         [0019]    BCL2, an anti-apoptotic gene upregulated in many different cancer types, especially hematopoietic malignancies, is another NADPH-dependent enzyme. Thus, it has been shown that down regulation of the folate pathway reduces BCL2 activity, releasing its block on apoptosis. (See Lin, H. L. et al, British J Nutrition 95:870-878, 2006). Although a number of BCL2 inhibitors have reached clinical trials, none have yet shown an efficacy to toxicity ratio acceptable to the FDA. 
         [0020]    EZH2 is a Polycomb group gene the product of which is responsible for mono-methylation, di-methylation and tri-methylation at histone H3K27. EZH2 is overexpressed in many tumors, and is well known to suppress the expression of tumor suppressor genes (Liu, T. P. et al, Anti-Cancer Drugs 26(2):139-147, 2015). EZH2 is NADPH-dependent by virtue of its requirement for SAM as cofactor in its methylation reactions. Compositions and methods to inhibit EZH2 in tumors could be very important clinically. 
         [0021]    S-Adenosylhomocysteine hydrolase (SAHH) converts S-Adenosyl-L-homocysteine to adenosine and homocysteine. Since the adenosine and homocysteine moieties involved in this reversible reaction are products of the folate pathway, SAHH is an NADPH-dependent enzyme. It is a major source of adenosine, which inactivates the host immune response. SAHH is not considered a good target for small molecule approaches to cancer chemotherapy because systemic administration would have severe pathological consequences. New methods to overcome or mitigate toxicity when SAHH is targeted are urgently needed. 
         [0022]    Alkaline phosphatase (CAS 9001-78-9; ALPP) is another NADPH-dependent enzyme because its substrates, deoxyribonucleotides, have their structural backbones formed as a result of folate pathway activity. ALPP can metabolize adenosine nucleotides into adenosine. Methods to inhibit ALPP and thereby reduce or prevent defeat of immune surveillance by adenosine secretion into the tumor microenvironment would be important additions to the cancer chemotherapy armamentarium. 
         [0023]    Phosphoribosylpyrophosphate Synthase (CAS 9031-46-3; PRPS), a critical pathway toward the de novo synthesis of purines and pyrimidines required for cell division, is heavily NADPH dependent because its substrate PRPP is produced by the Pentose Phosphate Pathway (PPP). Inhibition of either glucose-6-phosphate dehydrogenase (CAS 9001-40-5; G6PD) or 6-Phosphogluconate dehydrogenase (CAS 9001-82-5; PGD) (or both), two major enzymes of the PPP, leads to PRPP depletion and interruption of nucleotide biosynthesis. Two isoforms of the PRPS gene exist. Compositions and methods to inhibit PRPP would represent novel and potentially very therapeutic additions to cancer chemotherapy. 
         [0024]    Amidophosphoribosyl transferase (EC 2.4.2.14; APRT) represents the committed step toward purine biosynthesis. Like PRPS, it is an NADPH-dependent enzyme because PRPP is produced by the PPP, and would be rendered inactive by any diminution in the biosynthesis of NADPH. No inhibitors of APRT have yet been introduced to the clinic. 
         [0025]    IMP Dehydrogenase (CAS, 9028-93-7; IMPD) is an important enzyme in the biosynthesis of both adenine and guanine nucleotides. Its substrate, inosine monophosphate, is the product of de novo purine biosynthesis in which the purine ring structure is synthesized in the NADPH-dependent folate pathway. While IMPD is a recognized target for immunosuppressive drugs, it has not made headway as an anti-cancer target because of toxicity when delivered systemically. Methods to overcome this toxicity are greatly needed. 
         [0026]    Polymerase alpha (CAS 9012-90-2; Pol a) is expressed in high amounts in cancer cells to maintain their rates of cell division. Pol α is of course NADPH-dependent because all the deoxyribonucleotides used as its substrate are derived from the NADPH-dependent folate pathway. Like many of the DNA synthesis enzymes discussed above, Pol α represents a good target because it is highly expressed in replicating cancer cells, and not expressed in normal, fully differentiated cells. Yet no inhibitors of this important target have yet found clinical utility. 
         [0027]    Antisense oligonucleotides represent a promising alternative to antibody and small molecule approaches to the treatment of disease, as the present author (Nyce, J and Metzger, J M, Nature 385:721-725, 1997) and others (See Pirollo. K F et al, Pharmacol &amp; Therapeutics 99(1):55-77, 2003) have demonstrated. However, their successful application to the field of cancer has been extremely limited. Methods to enhance their activity would have great therapeutic potential. 
         [0028]    Antisense oligonucleotides comprise short (generally 15-20 base), single-stranded DNA or RNA, with (generally) chemically modified backbone to reduce nuclease degradation, that are complimentary to some region of a target mRNA (See Geary, RS et al Adv Drug Deliv Rev. 2015). There are several forms of backbone modification, including the exchange of one of the oxygen molecules for sulfur to create phosphorothioate oligonucleotides. Phosphorothioate and most (but not all) of the other chemistries permit facile hybridization to the target mRNA, with the result that the RNA:DNA hybrid becomes a substrate for RNAase H, which degrades the mRNA, allowing the antisense oligonucleotide to diffuse away with the potential of binding to another target mRNA and repeating the process. Locked nucleic acid (LNA) technology permit shorter 12-14 base long antisense oligonucleotides, and these are assumed in our listings. (See Straarup, E M et al, Nucleic Acids Res 38(20):7100-7111, 2012). 
         [0029]    Backbone chemistries to prevent/decrease nuclease degradation envisioned in this patent include, for either DNA or RNA oligonucleotides, phosphorothiate, phosphorodithioate, 2′-O-methyl, 2′-Omethoxy-ethyl, 2′-F-arabino nucleic acid, Peptide nucleic acid, Morpholino phosphoroamidate, Bridged (locked) nucleic acid, cyclohexene nucleic acid, and hexitol nucleic acid. (See  FIG. 3 ). 
         [0030]    Antisense oligonucleotides present several advantages over the small molecule approach to cancer therapeutics. For example, it is possible to locally deliver antisense oligonucleotides either by direct injection, inhalation, and other methods. While small molecules can be and are injected, inhaled, etc., they readily diffuse out of the injection/inhalation focus creating systemic toxicity. Oligonucleotides, on the other hand, diffuse much less readily, and in many local delivery scenarios may never leave the tumor. Toxicity can thus be limited to the tumor and tumor associated non-neoplastic cells, e.g., blood vessels, etc. 
         [0031]    Another advantage of antisense technology is that specific targets associated with neoplastic and not differentiated normal cells can be selected for attack. For example, normal differentiated cells express very minor amounts of such DNA synthesis genes as ribonucleotide reductase, thymidine kinase, nucleoside kinases, etc, while cancer cells generally express high levels of these enzymes as a requirement for their more rapid rates of replication. Thymidine kinase, for example, is expressed at high levels needed for cell division in cancer cells, while differentiated normal cells, in general, express the much reduced amounts needed only for DNA repair. 
         [0032]    Local delivery of antisense oligonucleotides may be the key for their successful use, as envisioned in this invention (see below). Certainly, systemic delivery of antisense oligonucleotides has been shown to have many difficulties. As an example, consider an antisense oligonucleotide designated G3139 developed by Genta, Inc to target Bcl-2. This antisense oligonucleotide was called Genasense, and was designed to be complementary to the first 6 codons of Bcl-2 mRNA. While initial results in Phase I/II trials for lymphoma were promising, a disappointing melanoma trial caused Genta to close its doors in 2012. 
         [0033]    One of the present inventors has several patents in relevant fields. 
         [0034]    U.S. Pat. No. 6,040,296 A (Nyce, J W, 2000) teaches the use of antisense oligonucleotides targeting adenosine receptors for treatment of disorders associated with bronchoconstriction and lung inflammation. 
         [0035]    U.S. Pat. No. 5,994,315 A (Nyce, J W 1999) teaches the use of antisense oligonucleotides with low adenosine content to treat lung diseases. 
         [0036]    U.S. Pat. No. 6,025,339 A (Nyce, J W, 2000) teaches the use of antisense oligonucleotides for the treatment of bronchoconstriction and lung inflammation. 
         [0037]    U.S. Pat. No. 6,670,349 B1 (Nyce, J W, 2003) teaches the use of Dehydroepiandrosterone to lower adenosine levels to treat asthma. 
         [0038]    U.S. Pat. No. 7,456,161 B2 (Nyce, J W, 2008), teaches the use of dehydroepiandrosterone to treat chronic obstructive pulmonary disease by reducing levels of adenosine in bronchial mucosa. 
         [0039]    U.S. Pat. No. 8,951,527 B2 (Isenberg, J S et al, 2015) and EU Patent application WO 2012170250 (Moulton, J 2011) both inform the use as radiation protectants of morpholino antisense oligonucleotides targeting the human CD47 sequence, in which the inhibition of CD47 allows normal tissue within a radiation beam to survive otherwise damaging or lethal doses of radiation. 
         [0040]    U.S. Pat. No. 8,710,020 B2 (Gleave, M E and Cormack, S D, 2014) teaches a method for using antisense oligonucleotides to reduce the expression of clusterin and thereby treat cancers in which clusterin play a major role. 
         [0041]    Application WO2014153209 Al (Erin, L O et al (2014) teaches the use of antisense oligonucleotides targeting non-coding chimeric mitochondrial DNA in the treatment or prevention of relapsing or metastatic cancer. 
         [0042]    U.S. Pat. No. 7,402,574 B2 (Iverson, P L et al, 2008) teaches the use of antisense oligonucleotides targeting the SNAIL transcript. 
         [0043]    U.S. Pat. No. 7,569,551. B2 (Gleave, M et al, 2009) teaches the use of antisense oligonucleotides targeting Testosterone-repressed prostate message 2 (TRPM-2) 
         [0044]    U.S. Pat. No. 5,641,754 A (Iversen, P L, 1997) teaches the cornbination of an antisense oligonucleotide and a hydroxyl-radical upregulator to treat cancer. 
         [0045]    U.S. Pat. No. 6,727,230 B1 (Hutcherson, S. L., Glover, J M, 2004) teaches the use of phosphorothioate oligonucleotide analogs to stimulate an immune response. 
         [0046]    EU Patent EP 1009822 A1, (Higenbottom, T, 2000) teaches the use of antisense oligonucleotides targeting ET-1 for the treatment of pulmonary hypertension. 
         [0047]    U.S. Pat. No. 7,585,968 B2 (James G. Karras, Kenneth W. Dobie , 2009) teaches the use of antisense oligonucleotides targeting TARC for the treatment of airway hyper responsiveness and pulmonary inflammation. 
         [0048]    U.S. Pat. No. 7,001,890 B1 (Wagner, H et al, 2006) teaches the combination of an oligonucleotide and a protein/peptide antigen for the purpose of vaccination. 
         [0049]    Application WO2001025422 A2 (Bartelmez, S H and Iversen, PL (2001) teaches a method to treat cancer which comprises using an antisense oligonucleotide to increase the number of lineage committed progenitor cells. 
         [0050]    Application WO1997011170 A1 (Zamecnik, P A, 1997) teaches the use of prostate-specific antisense oligonucleotides to treat prostate cancer. 
         [0051]    Application WO2008058225 A2 (Brown, B D, 2008) teaches the use of an antisense oligonucleotide targeting BCL-2 for the treatment of cancer. This antisense did not work in clinical trials and represents a good example of why antisense oligonucleotides have experienced difficulty in their clinical development. Administered systemically, and without any method to secondarily interfere with the target protein (and not just the mRNA), these molecules cannot overcome the heterogeneity and plasticity of tumor cell populations. 
         [0052]    U.S. Pat. No. 5,801,154 A (Baracchini, E et al, 1998) teaches the use of antisense oligonucleotides targeting the Multi-drug resistance-associated protein to treat or prevent the occurrence of multi-drug resistance during chemotherapy. 
         [0053]    U.S. Pat. No. 6,017,898 A (Pietrzkowski, Z et al, 2000) teaches the use of antisense oligonucleotides targeting IL-8 and the IL-8 receptor for the treatment of cancer. 
         [0054]    Chinese patent CN1120179 C (         , 2003) teaches the use of an antisense oligonucleotide targeting c-raf to treat cancer. 
         [0055]    Application CA2471967 A1, (Lopez-Bernstein et al, 2003) teaches the use of antisense oligonucleotides targeting Wt1 for the treatment of cancer. 
         [0056]    U.S. Pat. No. 5,691,317 A, (Yoon, S C C, 1997) teaches the use of an antisense oligonucleotide targeting the RI-α subunit of cyclic AMP-dependent protein kinase. This is an early entry into the field of antisense oligonucleotides targeting cancer genes. 
         [0057]    U.S. Pat. No. 7,704,968 B2 (Nerenberg, M I, 2010) teaches the suppression of nuclear factor-kB for treating inflammatory conditions. 
         [0058]    A major flaw in all of these patents and applications, a flaw which places severe restraints upon their clinical utility, is that they depend upon an antisense oligonucleotide alone to reduce the impact of the target upon the disease process. What is needed to move antisense technology forward is a method or methods to reduce the production of the target protein using antisense technology, preferably by local administration directly to the tumor, while simultaneously inhibiting the protein target, preferably by a non-toxic, systemic route of administration. While this could be effected using local administration of antisense oligonucleotides and systemic administration of classical chemotherapy agents, such a method would still suffer from the systemic toxicity inherent in the chemotherapy drugs. A better approach would be to find a class of cancer targets that all have one common dependency, for example, targets that all require the same cofactor, reductant, vitamin, etc., and exploit that dependency by causing its depletion, so that upon application of antisense oligonucleotides against the targets, such targets are simultaneously suppressed at both the protein and the mRNA levels. 
       BRIEF SUMMARY OF THE INVENTION 
       [0059]    This invention comprises a system of antisense oligonucleotides targeting canine genes whose enzyme products are NADPH-dependent, combined with one or more inhibitors of the pentose phosphate pathway (PPP), the major source of cellular NADPH. The object is to synergize the inhibition of target gene products by (a) reducing the amount of target protein via antisense oligonucleotide interference with its translation/production, and (b) inhibiting remaining target protein via NADPH-depletion. An additional goal is to maximize tumor toxicity and minimize systemic toxicity by delivering the antisense oligonucleotides locally, as, for example, with direct injection into the tumor, while the PPP inhibitor can be delivered systemically or locally. 
         [0060]    The NADPH-dependent genes targeted by this system include thymidine kinase (TK), thymidylate synthetase (TS), dihydrofolate reductase (DHFR), ribonucleotide reductase (RNR), c-kit tyrosine kinase, fatty acid synthase (FASN), the anti-apoptotic gene BCL2, the polycomb gene EZH2, the DNA methyltransferase gene DNMT1, farnesyl transferase (FT), geranylgeranyl transferase (GGT), Phosphoribosylpyrophosphate Synthase (PRPS), Amido-phosphoribosyl transferase (APRT), IMP Dehydrogenase (IMPD), and Polymerase a (Pol α). 
         [0061]    The PPP inhibitors include Dehydroepiandrosterone (DHEA) and any of its congeners, analogs, precursors, products, and salts; and/or antisense oligonucleotides targeting Glucose-6-phosphate dehydrogenase (G6PD) and/or 6-Phosphogluconate Dehydrogenase (PGD). 
         [0062]    Where DHEA is used as the PPP inhibitor, it may be necessary to reconstitute certain metabolites depleted as a result of PPP inhibition, as described in a parallel United States Patent Application filed this same date. Such depleted metabolites that may need to be reconstituted include tetrahydrobiotperin, N-6-isopentenyladenosine, a nitric oxide donor such as potassium nitrate, folinic acid (or products of the folate pathway), ubiquinone +tocotrienols, and certain monoamine precursors and cofactors including L-DOPA, 5-Hydroxytryptophan, pyridoxine, SAMe, ascorbate, pantothenic acid, and zinc. 
         [0063]    Where DHEA or one of its congeners is the PPP inhibitor, it may be administered by any of several techniques, but injected, inhaled, transdermal and oral methods are preferred. The antisense oligonucleotides may be administered by any of several techniques, but injected, inhaled and transdermal methods are preferred. 
         [0064]    It may sometimes be necessary to select sequences that are not 100% complimentary to the human version of the respective genes so as to avoid the possibility of human exposure during development and/or clinical usage, and such selection for canine-specific antisense oligonucleotides that will not interact with human mRNAs (and thereby represent a threat to humans exposed to it) represents one aspect of this invention. 
         [0065]    In addition to DNA based antisense oligonucleotides, this invention also envisions the use of small interfering RNA (RNAi), Dicer-substrate siRNA, and microRNA oligonucleotides based upon the sequences in our listing. Accordingly, even though the sequences listed are single-stranded, if they are used in some of the RNA procedures, it is understood that the listed sequences can represent one half of a double stranded nucleic acid the bases of which are participating in hydrogen bonding between the counter opposing strands. 
     
    
     
       BRIEF DESCRIPTION OF FIGURES AND TABLES 
         [0066]      FIG. 1( a )  is a representation of the DHFR pathway illustrating the dependency of DHFR upon NADPH, 1 (b) illustrates how folate intermediates are required for purine and pyrimidine biosynthesis, for DNA, RNA protein and lipid methylation reactions, etc. 
           [0067]      FIG. 2  is a representation of the RNR pathway illustrating the dependency of RNR upon NADPH. 
           [0068]      FIG. 3  is a detailed illustration of the various oligonucleotide chemistries available. The present invention envisions using any or all of these chemistries. 
           [0069]      FIG. 4 . (a) Effects of adenosine A1 receptor antisense oligodeoxynucleotide upon PC50 values in asthmatic Rabbits. PC50 adenosine values were determined before and after intratracheal administration of aerosolized antisense oligonucleotide targeting the adenosine A2 receptor (A2AS), or a control oligonucleotide in which the A1 receptor sequence was randomized (A1MM, A1MM2)). After a two-week rest period between parts of the experiment, rabbits were then crossed over, with those that had received the A1 antisense oligonucleotide now receiving the randomized oligonucleotide, and those that had received the randomized oligonucleotide now receiving the A1 antisense oligonucleotide. A1MM2 animals constituted a separate group. (b), Data summary. Results are presented as the mean±s.e.m. Significance was determined by repeated measures ANOVA and Tukey&#39;s protected t test. Asterisks indicate a significant difference from all other groups, P&lt;0.01. PC50 adenosine, the amount of adenosine required to reduce lung compliance by 50%. 
           [0070]      FIG. 5 . Specificity of action of Adenosine Al receptor antisense oligodeoxynucleotide A1AS. Airway smooth muscle tissue was dissected from rabbits administered a total of 20 mg A1AS or A1MM in four divided doses over 48 hr. Plasma membrane fractions were prepared. (a), Saturation isotherm of [ 3 H]DPCPX (a potent selective antagonist of the adenosine Al receptor) binding to allergic rabbit lung plasma membrane from A1AS (filled circles) and A1MM (open circles) allergic rabbits showing an approximate 75% decrease in adenosine A1 receptor number in airway smooth muscle from A1AS-treated animals. (b), Scatchard plot saturation isotherm from (a) indicating a single class of binding sites; A1AS (filled circles), A1MM (Open circles). Please note that the error bars are an artifact of the printing program; Scatchard plots have no error bars. (c), Saturation isotherm of [ 3 H]NPC17731 (a selective bradykinin B2 receptor antagonist) binding to allergic rabbit lung plasma membrane from AlAS (open squares) and A1MM-treated (filled squares) allergic rabbit showing no change in Bradykinin B2 receptor number in airway smooth muscle of A1AS-treated animals. (d), Scatchard plot of saturation isotherm from c indicating a single class of binding sites. A1AS (open squares), A1MM (filled squares). Error bars are an artifact of the drawing program. 
           [0071]      FIG. 6 . Specificity of action of bradykinin B2 receptor antisense oligonucleotide B2AS. Airway smooth muscle tissue was dissected from rabbits administered 20 mg B2AS or B2MM in four divided doses over 48 hr. Plasma membrane fractions were prepared. (a), Saturation isotherm of [ 3 H]NPC17731 binding to allergic rabbit lung plasma membrane from B2ASO (open triangles) and B2MM-treated (filled triangles) allergic rabbits showing an approximate 40% decrease in bradykinin B2 receptor number in airway smooth muscle from B2AS-treated animals. (b), Scatchard plot of saturation isotherm from a indicating a single class of binding sites; B2AS (open triangles), B2MM (filled triangles). (c), Saturation isotherm of [ 3 H]DPCPX binding to allergic rabbit lung plasma membrane from B2AS (open diamonds) and B2MM (filled diamonds) allergic rabbits showing no change in adenosine A1 receptor number. (d), Scatchard plot of saturation isotherm from c indicating a single class of binding sites; B2AS (open diamonds), B2MM (filled diamonds). 
           [0072]      FIG. 7 . The effect of antisense and mismatched/randomized oligodeoxynucleotides on allergen-induced airway obstruction and bronchial hyper responsiveness in allergic rabbits. The allergen employed was house dust mite allergen ( Dermatophagoides farina ). (a), Effect of AlAS antisense oligodeoxynucleotide upon allergen-induced airway obstruction. Allergen only (filled circles); allergen+antisense (open circles). As calculated from the area under the curve, A1as significantly inhibited allergen-induced airway obstruction (55%, P&lt;0.05; repeated measures ANOVA and Tukey&#39;s protected t test. (b), Lack of effect of mismatch control A1MM on allergen-induced airway obstruction. Allergen only (filled circles); allergen=mismatch control (open circles). (c), Effect of A1AS antisense oligodeoxynucleotide on allergen-induced bronchial hyperresponsiveness. As calculated from the PC50 histamine, A1AS significantly inhibited allergen-induced bronchial hyperresponsiveness in allergic rabbits (61%, P&lt;0.05; repeated measures ANOVA and Tukey&#39;s protected t test). (d), Lack of effect of A1MM mismatch control on allergen-induced bronchial hyperresponsiveness. Dynamic compliance (Cdyn) is the change in the volume of the lungs divided by the change in the alveolar-distending pressure during the course of a breath. 
       
    
    
       [0073]    Table 1. Binding characteristics of the adenosine A1 receptor-selective ligand [ 3 H]DPCPX and the bradykinin B2-selective ligand [ 3 H]NPC17731 in membranes isolated from airway smooth muscle of A1 adenosine receptor and B2 Bradykinin receptor antisense- and mismatch control-treated allergic rabbits. Treatment values refer to total oligonucleotide administered in four equivalently divided doses over a 48 hour period. Significance was determined by repeated measures ANOVA and Tukey&#39;s protected t test; N=4-7 for all groups. All assays were performed in triplicate. *Significantly different from mismatch control and saline-treated groups, P&lt;0.001. ** Significantly different from mismatch control and saline treated groups, P&lt;0.05. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0074]    A preferred embodiment of the invention is a pharmaceutical composition comprising one or more antisense oligonucleotides drawn from the sequence listing, combined with an inhibitor of the PPP. The NADPH-dependent targets identified in the sequence listing include thymidine kinase (TK), thymidylate synthetase (TS), dihydrofolate reductase (DHFR), ribonucleotide reductase (RNR), the adenosine receptors A1 and A2A, c-kit tyrosine kinase, fatty acid synthase (FASN), the anti-apoptotic gene BCL2, the polycomb gene EZH2, the DNA methyltransferase gene DNMT1, farnesyl transferase (FT), geranylgeranyl transferase (GGT), S-Adenosylhomo-cysteine hydrolase (SAHH), alkaline phosphatase (ALP), Phosphoribosylpyro-phosphate Synthase (PRPS), Amidophosphoribosyl transferase (APRT), IMP Dehydrogenase (IMPD), and Polymerase a (pol α). 
         [0075]    If sequencing of the tumor is performed before treatment, drivers may be identified and groups selected for antisense targeting accordingly. If sequencing is not performed prior to treatment, a series of treatments in which one or more antisense oligonucleotides is administered alone or in combination, may be used to address NADPH-dependent drivers. 
         [0076]    The sequence list is comprised of NADPH-dependent drivers found in whole or in part in most neoplasms, including mast cell tumors, mammary gland tumors, soft tissue sarcomas, anal gland tumors, osteosarcomas (primary and metastatic), lung tumors (primary and metastatic), melanomas (primary and metastatic), fibrosarcomas, hemangiosarcomas (primary and metastatic), nasal cavity cancers, squamous cell carcinomas, tumors of the CNS, gastric tumors, intestinal cancers, hepatic cancers, kidney cancers, prostate cancers, urinary tract cancers, testicular cancers, pancreatic cancers, anal gland tumors, and lymphosarcoma. 
         [0077]    The backbone chemistries to prevent/slow nuclease degradation envisioned in this patent include for either DNA or RNA or DNA/RNA mixed oligonucleotides, phosphorothiate, 2′-O-methyl, 2′-O-methoxy-ethyl, 2′-F-arabino nucleic acid, Peptide nucleic acid, Morpholino phosphoroamidate, Bridged (locked) nucleic acid, cyclohexene nucleic acid, and hexitol nucleic acid. (See  FIG. 1 ). These chemistries can be applied to all the nucleotides in the sequence, or to a more limited number, even as few as 1. These chemistries can also be mixed, such that portions of the antisense oligonucleotide can utilize one chemistry, and other portions another chemistry, in any combination or sequence desired. 
         [0078]    In addition to DNA based antisense oligonucleotides, this invention also envisions the use of small interfering RNA (RNAi), Dicer-substrate siRNA , and microRNA oligonucleotides based upon the sequences in our listing. For the purposes of this invention, then, the words antisense oligonucleotide are meant to imply the use of any or all of these technologies. 
         [0079]    A preferred embodiment of an antisense oligonucleotide targeting canine TK-1 can be drawn from sequence ID number 283, having the sequence 5′-gcaggttgat gcagctcatg g-3′, but other of the TK-1 antisense sequences in the list may be used as well. 
         [0080]    A preferred embodiment of an antisense oligonucleotide targeting canine TS can be drawn from sequence ID number 1119, having the sequence 5′-gggcgggcat ggcgcgggcg g-3′, but other of the TS antisense sequences in the list may be used as well. 
         [0081]    A preferred embodiment of an antisense oligonucleotide targeting canine DHFR can be drawn from sequence ID number 327, having the sequence 5′-ggctgcggcc ccatttcatg t-3′, but other of the DHFR antisense sequences in the list may be used as well. 
         [0082]    A preferred embodiment of an antisense oligonucleotide targeting canine RNRM1 can be drawn from sequence ID number 741, having the sequence 5′-cgcttgatca cgtgcatcgc g-3′, but other of the RNR antisense sequences in the list may be used as well. 
         [0083]    A preferred embodiment of an antisense oligonucleotide targeting canine RNRM2 can be drawn from sequence ID number 792, having the sequence 5′-cgcggacgga gagcatggcg g-3′, but other of the RNR antisense sequences in the list may be used as well. 
         [0084]    A preferred embodiment of an antisense oligonucleotide targeting canine RNRM3 can be drawn from sequence ID number 893, having the sequence 5′-cctgtttata cattttccaa a-3′, but other of the RNR antisense sequences in the list may be used as well. 
         [0085]    A preferred embodiment of an antisense oligonucleotide targeting canine adenosine A2a receptor can be drawn from sequence ID number 107, having the sequence 5′-gcccatggtg gacatggctg c-3′, but other of the adenosine A2a receptor antisense sequences in the list may be used as well. 
         [0086]    A preferred embodiment of an antisense oligonucleotide targeting canine adenosine A1 receptor can be drawn from sequence ID number 468, having the sequence 5′-ggcgggcggc atggcaggcg c-3′, but other of the adenosine A1 receptor antisense sequences in the list may be used as well. 
         [0087]    A preferred embodiment of an antisense oligonucleotide targeting canine c-kit tyrosine kinase can be drawn from sequence ID number 249, having the sequence 5′-gcgagcgcct ctcatcgcgg t-3′, but other of the c-kit tyrosine kinase antisense sequences in the list may be used as well. 
         [0088]    A preferred embodiment of an antisense oligonucleotide targeting canine FASN can be drawn from sequence ID number 1015, having the sequence 5′-cctcctccat ggctgctctg c-3′, but other of the FASN antisense sequences in the list may be used as well. 
         [0089]    A preferred embodiment of an antisense oligonucleotide targeting canine BCL2 can be drawn from sequence ID number 571, having the sequence 5′-gcccagcgtg cgccatcctc c-3′, but other of the BCL2 antisense sequences in the list may be used as well. 
         [0090]    A preferred embodiment of an antisense oligonucleotide targeting canine EZH2 can be drawn from sequence ID number 672, having the sequence 5′-ggcccatgat tattctgcgc c-3′, but other of the EZH2 antisense sequences in the list may be used as well. 
         [0091]    A preferred embodiment of an antisense oligonucleotide targeting canine DNMT1 can be drawn from sequence ID number 671, having the sequence 5′-ggcccatgat tattctgcgc cc-3′, but other of the DNMT1 antisense sequences in the list may be used as well. 
         [0092]    A preferred embodiment of an antisense oligonucleotide targeting canine FT can be drawn from sequence ID number 684, having the sequence 5′-cacgaactcc atgctggcgg c-3′, but other of the FT antisense sequences in the list may be used as well. 
         [0093]    A preferred embodiment of an antisense oligonucleotide targeting canine ALPP (alkaline phosphatase) can be drawn from sequence ID number 993, having the sequence 5′-gggtcaggag cattgcaggg c-3′, but other of the ALPP antisense sequences in the list may be used as well. 
         [0094]    A preferred embodiment of an antisense oligonucleotide targeting canine GGT 1α can be drawn from sequence ID number 1077, having the sequence 5′-gcgcccgtgc atggtgccgg c-3′, but other of the GGT antisense sequences in the list may be used as well. 
         [0095]    A preferred embodiment of an antisense oligonucleotide targeting canine GGT 1β can be drawn from sequence ID number 1063, having the sequence 5′-cgccgctcct ctacatcgaa c-3′, but other of the GGT antisense sequences in the list may be used as well. 
         [0096]    A preferred embodiment of an antisense oligonucleotide targeting canine SAHH can be drawn from sequence ID number 940, having the sequence 5′-cagtttgtcc gacatgctgg c-3′, but other of the SAHH antisense sequences in the list may be used as well. 
         [0097]    A preferred embodiment of an antisense oligonucleotide targeting canine PRPS1 can be drawn from sequence ID number 1046, having the sequence 5′-cggcatcttg ggtgcctacc c-3′, but other of the PRPS antisense sequences in the list may be used as well. 
         [0098]    A preferred embodiment of an antisense oligonucleotide targeting canine PRPS2 can be drawn from sequence ID number 1039, having the sequence 5′-ccgatgacat ccttctccga t-3′, but other of the PRPS2 antisense sequences in the list may be used as well. 
         [0099]    A preferred embodiment of an antisense oligonucleotide targeting canine APRT can be drawn from sequence ID number 1130, having the sequence 5′-cctccagctc catgtcgctg cc-3′, but other of the APRT antisense sequences in the list may be used as well. 
         [0100]    A preferred embodiment of an antisense oligonucleotide targeting canine IMPD can be drawn from sequence ID number 1135, having the sequence 5′-gcggcccctc catgcggagg c-3′, but other of the IMPD antisense sequences in the list may be used as well. 
         [0101]    A preferred embodiment of an antisense oligonucleotide targeting canine IMPD2 can be drawn from sequence ID number 1160, having the sequence 5′-cgcatgcgca aagcgcgccg t-3′, but other of the IMPD antisense sequences in the list may be used as well. 
         [0102]    A preferred embodiment of an antisense oligonucleotide targeting canine Pol a can be drawn from sequence ID number 1023, having the sequence 5′-cgccgtctac cagcgccatg g-3′, but other of the Pol α antisense sequences in the list may be used as well. 
         [0103]    In a preferred embodiment of this invention, the antisense oligonucleotides can be administered in doses ranging from, preferentially, 0.1 mg/kg to 50 mg/kg; more preferentially 1 mg to 30 mg/kg; and most preferentially, 2 mg to 20 mg/kg. In a preferred embodiment of the invention the antisense oligonucleotides are administered locally, for example by direct injection into a tumor; by inhalation when tumors exist in the lung; by means of a transdermal cream if injection is not possible, for example in tumors at risk of catastrophic bleeding such as hemangioendotheliomas; and by intrathecal injection for tumors in the brain. 
         [0104]    A variety of microorganisms do not methylate the majority of CpG dinucleotides in their DNA. Bacterial genomes also contain 6-methyladenine replacing adenine in some sites in their DNA. These non-self “signals” activate Toll receptors and initiate an immune response directed to the vicinity where they are detected. A preferred embodiment of this invention therefore involves the substitution of any number of adenosine moieties with 6-methyladenosine moieties to stimulate a local immune response. Because some dogs may show heightened response to unmethylated CpGs, a preferred embodiment of this invention, for use in dogs with hypersensitivity to unmethylated CpGs, is the substitution of 5 mC for C in such unmethylated CpGs, 
         [0105]    A preferred embodiment of the PPP inhibitor is DHEA, and/or any of its congeners capable of inhibiting the PPP, such as DHEA sulfate, DHEA sulfatide, any DHEA salt, 7-keto-DHEA, 3 acetyl-7-oxo DHEA, DHEA acetate, the DHEA metabolites androstenediol and androstendione, and the less metabolizable analogs fluasterone (CAS 112859-71-9) and the brominated analog of fluasterone. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0106]    An additional preferred embodiment of the PPP inhibitor is an antisense oligonucleotide targeting canine G6PD, which antisense oligonucleotide can be drawn from sequence ID number 720, having the sequence 5′-cccgccggct catttaacca g-3′, but other of the G6PD antisense sequences in the list may be used as well. 
         [0107]    An additional preferred embodiment of the PPP inhibitor is an antisense oligonucleotide targeting canine PGD, which antisense oligonucleotide can be drawn from sequence ID number 504, having the sequence 5′-cggcctcggc catggcggcg g-3′, but other of the PGD antisense sequences in the list may be used as well. 
         [0108]    An additional preferred embodiment of the PPP inhibitor is a combination of a canine G6PD antisense oligonucleotide with a canine PGD antisense oligonucleotide, such that both distal steps in the PPP are inhibited. Also, DHEA or one of its PPP-inhibiting congers can be administered systemically, and either G6PD antisense oligonucleotide or PGD antisense oligonucleotide, or both, can be administered locally, so as to maximize inhibition of the PPP. 
         [0109]    Where systemic DHEA or a congener is used to inhibit the PPP, an additional preferred embodiment of the invention is supplementation of DHEA-treated dogs with metabolites that may, as a consequence of DHEA-mediated NADPH-depletion, become depleted physiologically and which may need to be replenished to maintain optimum health in the treated dog. Such supplementation may include tetrahydrobiotperin (BH4); isopentenyladenosine (IPA); folinic acid or certain products of the folate pathway (e.g., SAM, adenine, guanine, and uracil and/or any of their nucleosides or nucleotides); a nitric oxide donor such as Potassium Nitrate; ubiquinone and/or tocotrienols to maintain healthy ubiquinone levels; and certain monoamine precursors and cofactors such as hydroxytryptophan, L-DOPA, melatonin, pyridoxine, ascorbate, and pantothenic acid. 
         [0110]    A preferred embodiment of the invention is target selection from the sequence list based upon Next Generation RNA sequencing such that those targets on the list that are expressed in aberrant, greater than normal levels are identified. Such sequencing uses reverse transcription of RNA isolated from canine tumors (and control tissue, where available), a panel of PCR primers specific for the listed targets to identify and quantitate their contribution to the transcriptome, and annealing of barcode DNA to individual tumor samples so that the transcriptomes from several different animals can be multiplexed, i.e., mixed together during the sequencing run. Output is then extracted for each animal by barcode decoding, and the contribution of different targets to the transcriptome is analyzed. Where such RNA sequencing is not possible, various antisense oligonucleotides selected from the list may be used sequentially or in groups to treat the cancer in a blind fashion. 
         [0111]    A preferred embodiment of the invention is the local administration of the antisense oligonucleotide or nucleotides, in order to reduce systemic toxicity, and the systemic administration of DHEA or its congeners, although local routes of DHEA administration may be preferred in some situations. If one of the G6PD antisense oligonucleotides, and/or one of the PGD antisense oligonucleotides are selected for use, these will be administered locally, by inhalation, injection (intratumor, intrathecal, suppository, subcutaneous), or transdermal routes. 
         [0112]    Preparation of antisense oligonucleotides is accomplished by any of several published techniques. A variety of chemical modifications can be used to inhibit nuclease degradation in vivo. For phosphorothioate oligonucleotides we use standard phosphoramidite chemistry, substituting iodine reagent with either Beaucage reagent or Sulfurizing Reagent 2 (Glen Research, Sterling, Va.), using an ABI Expedite 8909 High Throughput DNA Synthesizer equipped with a 16 column MOSS (Multiple Oligonucleotide Synthesis System) unit. Purification is on 20 gram GlenPak cartridges as described (Glen Research). 
         [0113]    Phosphonoacetate monomers may be included in phosphorothioate oligonucleotides, or used as a stand alone chemistry. In fully modified oligos, the non-aqueous oxidizer camphorsulfonyloxaziridine is used, and oxidation precedes capping in the synthesis cycle. The use of such monomers greatly enhances the uptake of oligonucleotides into cells, and accelerates RNAase H degradation. The standard protocol for cleavage and deprotection requires a two-step method with pretreatment using 1,8-diazabicyclo[5.4.0]undec-7-ene (to deprotect the dimethylcyanoethyl protecting groups and prevent alkylation of the bases during deprotection) and subsequent cleavage using methylamine. 
         [0114]    Methyl Phosphonamidite chemistry may be used in DNA synthesizers following conventional CE Phosphoramidite protocols to produce oligonucleotides containing one or more methyl phosphonate linkages. 
         [0115]    Replacing two non-bridging oxygen atoms with sulfur atoms in a DNA phosphodiester linkage creates a phosphorodithioate (PS2) linkage. Like natural DNA, the phosphorodithioate linkage is achiral at phosphorus. This analog is completely resistant to nuclease degradation but forms complexes with DNA and RNA with somewhat reduced stabilities. 
         [0116]    After purification, the antisense oligonucleotides useful in this invention can be formulated as dry powders; as micronized or otherwise manipulated dry powders to enhance their formulation, encapsulation, compression into tablets, uptake or delivery; as liquids, with or without flavor enhancers and stabilizers; as semi-liquid mixtures, as for example in gel caps; as respirable particles; and the like. They can be administered orally, as a liquid, a lozenge, a capsule or a tablet. Flavor additives, stabilizers, solubilizing agents and flow enhancers and the like can be used to modify its flavor, activity, solubility and compressibility. They may also be administered non parenterally by any number of methods including transdermally; as a suppository; by inhalation of respirable formulations either to the lung or nasal cavities; by way of eye drops; sublingually; and by injection by any of the following routes: subcutaneous, intravenous, intraperitoneal, intratumor, intracranial or intrathecal. 
         [0117]    The antisense oligonucleotides useful for this invention can be formulated either alone or in combination with any or all of the other antisense oligonucleotides of this invention. 
         [0118]    As used in the context of this invention, co-administration refers to temporal proximity. Thus, the agents described as “co-administered” may be administered exactly together; they may be delivered one or more before the other(s); they may be delivered one or more after the other(s); or some combination of the above. 
         [0119]    Antisense oligonucleotides can be supplied for clinical use in dried form, to be taken up in a suitable buffer depending upon the route of administration, immediately before use. Alternatively, they can be shipped in sterile buffered aqueous solution, but a pH above 7.0 (e.g., 7.5-8.0) is needed to maintain stability. Depending upon the sequence structures, if several antisense oligonucleotides are administered together in the same solution, structures with extensive regions of base pairing (&gt;30-40%) may require heating to destabilize hydrogen bonding. One procedure is to rapidly heat the mixture of oligonucleotides to 100° C., then plunge them rapidly into ice water. This procedure should be sufficient to destabilize most hydrogen bond-induced aggregation in mixtures of antisense oligonucleotides with moderate base pairing potential. 
         [0120]    As an example of local administration of an antisense oligonucleotide, we utilized a phosphorothioate antisense oligonucleotide targeting the adenosine Al receptor delivered by inhalation to rabbits with well-quantitated bronchoconstriction upon challenge with adenosine (Nyce and Metzger, Nature 385:721-725, 1997). The antisense oligonucleotide targeted the same region of the target mRNA, centering on the initiation codon, as all of our selected antisense oligonucleotides in the sequence listing.  FIGS. 4-7  and Table 1 show results of studies which show selective and potent inhibition of the adenosine Al receptor, and inhibition of adenosine Al receptor-mediated bronchoconstriction in these animals. The lung is an excellent local target for aerosolized antisense oligonucleotides because it is lined with surfactant, a material that appears to facilitate the pulmonary distribution and intracellular uptake of respired oligonucleotides. 
         [0121]    In separate studies we showed that adenosine Al receptors in other tissues outside the lung were unaffected by the adenosine Al receptor antisense applied to the lung by inhalation. 
         [0122]    These studies show one mechanism by which antisense oligonucleotides can be applied locally to an organ or tissue, for example a dog with lung cancer, avoiding systemic toxicity. 
         [0123]    While the antisense oligonucleotide is applied locally, here via inhalation, the PPP inhibitor can be applied systemically where that is of advantage. For example, in neutered dogs with cancer, where DHEA is the PPP inhibitor used, DHEA has the added advantage of replenishing steroid hormone levels in the dog via Extra Gonadal Steroid Synthesis (EGSS). We have shown that EGSS can dramatically improve the quality of life in neutered dogs, both those with cancer and those without. 
         [0124]    Inhalation is not the only method by which local delivery of antisense oligonucleotides can be made. In a separate series of dogs we inoculated tumors directly with antisense oligonucleotides drawn from our sequence list, and delivered DHEA as the PPP inhibitor either orally or by transdermal application. Results were positive. In at least 50% of treated dogs, with a variety of different tumor types, we observed what appeared to be complete tumor regression. 
         [0125]    Current thinking about cancer suggests that tumors are distinguished by their genetic and epigenetic signatures, that is, both the mutations (genetic) and the changes in gene expression (epigenetic) that have occurred within the tumor cell population. It is likely that many drivers, both genetic and epigenetic, are involved in each neoplasm, whether in a dog or human. The present invention provides a unique method to approach a large number of targets simultaneously while confining most toxicity to the tumor volume itself. 
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         WO2014153209 A1, Eriz, L O et al, 2014, (Abstract: The invention provides oligonucleotides complementary to a non-coding chimeric mitochondrial RNA as well as compositions and kits comprising the same, and their use in treating and preventing metastasis or relapse of a cancer in an individual previously treated for cancer with a therapy. The invention also provides oligonucleotides complementary to a non-coding chimeric mitochondrial RNA as well as compositions and kits comprising the same, and their use in treating a refractory cancer (e.g., a refractory HPV-associated cancer). 
       
     
         [0153]    WO 1997011170 A1, Zamecnik, P A, 1997, Antisense oligonucleotide chemotherapy for benign hyperplasia or cancer of the prostate. (Abstract: Methods of selectively inhibiting the growth of or killing prostatic cells, using antisense oligonucleotides to prostate specific genes, are disclosed. The oligonucleotides may have natural nucleic acid structures or may be modified oligonucleotides with enhanced stability or tissue specific targeting. The prostate specific genes to which the antisense may be directed include the AR and the aFGF gene. Pharmaceutical compositions including such antisense oligonucleotides are also described for use in the methods. The methods and products are of particular utility in the treatment of benign prostatic hyperplasia or prostate cancer. 
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