WO2018200145A1 - Association synergique d'oligonucléotides d'acides nucléiques et d'agents chimiothérapeutiques à analogues nucléosidiques - Google Patents

Association synergique d'oligonucléotides d'acides nucléiques et d'agents chimiothérapeutiques à analogues nucléosidiques Download PDF

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WO2018200145A1
WO2018200145A1 PCT/US2018/026177 US2018026177W WO2018200145A1 WO 2018200145 A1 WO2018200145 A1 WO 2018200145A1 US 2018026177 W US2018026177 W US 2018026177W WO 2018200145 A1 WO2018200145 A1 WO 2018200145A1
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sirna
nucleic acid
madd
shrna
gene
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PCT/US2018/026177
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Sidney HOPPS
Aditi MATHUR
Bellur Prabhakar
Fei YUE
Shikha SAINI
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Jivana Biotechnology Inc.
The Board Of Trustees Of The University Of Illinois
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Publication of WO2018200145A1 publication Critical patent/WO2018200145A1/fr
Priority to US16/662,588 priority Critical patent/US11273172B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides

Definitions

  • the present invention is concerned with pharmaceutical compositions and methods for treating cancers comprising administering a combination of antineoplastic agents, wherein the combination comprises nucleoside analog chemotherapeutics and one or more nucleic acid molecules capable of down- regulating expression of at least one splice variant of the Insulinoma-Glucagonoma (IG20) gene, and wherein not all splice variants of the IG20 gene are down- regulated.
  • IG20 Insulinoma-Glucagonoma
  • the splice variant of the IG20 gene is the MADD splice variant and the nucleic acid molecules capable of down-regulating expression of at least one splice variant of the IG20 gene are selected from siRNA, shRNA and antisense oligonucleotides, wherein the siRNA, shRNA and antisense
  • oligonucleotides comprise a nucleic acid sequence which is complementary to a nucleic acid sequence of exon 13L of the MADD splice variant and/or to an mRNA transcript of exon 13L of the MADD splice variant.
  • the invention encompasses methods of treating cancers which include combination therapy with nucleic acid molecules capable of down-regulating the expression of at least one splice variant of the IG20 gene, and nucleoside analog chemotherapeutics.
  • the IG20 gene plays an important role in cancer cell proliferation, apoptosis and survival (Chow VT. Lee SS. (1996). DMA Seq 6: 263-273, Chow VT. Lim KM. Lim D. (1998). Genome 41: 543-552; Schievella AR. Chen JH. Graham JR. Lin LL. (1997). J Biol Chem 272: 12069-12075; Brinkman BM. Telliez JB. Schievella AR. Lin LL. Goldfeld AE. (1999). J Biol Chem 274: 30882-30886; Murakami-Mori K. Mori S. Bonavida B. Nakamura S. (1999). J Immunol 162: 3672-3679; Telliez JB. Bean KM. Lin LL. (2000). Biochem Biophys Acta 1478: 280-288; Al-Zoubi AM.
  • US Patent No. 8,722,637 describes "IG20 and IG20-[SV2], and the previously reported KIAA0358, MADD, and DENN-SV are splice variants of the IG20 gene, which is localized to chromosome 11p11 and consists of 36 exons.
  • the IG20, MADD, SV2 and DENN-SV isoforms may be considered to be described in the article: Contrasting Effects of IG20 and Its Splice Isoforms, MADD and DENN-SV, on Tumor Necrosis Factor a-induced Apoptosis and Activation of Caspase-8 and -3, Adeeb M. Al-Zoubi. Maria V. Efimova. Shashi
  • Efimova describes that "all seven variants of IG20 identified to date arise from alternative splicing of exons 13L, 16, 21 , 26 and 34.
  • the full-length cDNA of IG20 (IG20-FL) (accession number AF440100) is 5995 base pairs (bps) long, consists of all 36 exons and represents the longest variant.
  • IG20-FL accession number AF440100
  • bps base pairs
  • Figure 1 graphically displays human IG20 splice variants generated by alternative mRNA splicing. The cDNA sequence homology among the seven IG20 splice variants is shown. Solid bars represent regions of complete homology between all variants. Empty areas indicate exons 13L, 16, 21 , 26 and 34, which, when spliced in different combinations, produce the seven splice variants shown on the left. Splicing of exon 34 in KIAA0358 and IG20-SV4 induces an early stop codon in exon 35. Shown also are different 5' untranslated regions (UTRs) for different splice variants.
  • UTRs 5' untranslated regions
  • DENN-SV Overexpression of DENN-SV was associated with enhanced cell replication and resistance to apoptosis induced by chemotherapy. '637 at column 5, lines 4-6.
  • KIAA0358 cancer cell survival (i.e. MADD) and proliferation (i.e. DENN-SV).
  • the patent provides a functional characterization of IG20 isoforms and explains the complexity in devising strategies which may selectively modulate various isoform expression while avoiding unintended lethal consequences.
  • the IG20pa splice variant is pro-apoptotic, anti-proliferative, and renders cells more susceptible to induced cell death (i.e. is a tumor suppressor).
  • IG20pa, or a fragment thereof may be over expressed to control cell proliferation, cell cycle, and to render cells more susceptible to chemotherapy, radiation therapy or death receptor mediated cell death.
  • DENN-SV expression can be down modulated to reduce cell
  • IG20pa and DENN-SV splice variants renders cells either more susceptible or resistant to induced cell death respectively, and the pro-apoptotic property of IG20pa variant may be exploited to render tumor cells that are otherwise chemotherapeutic resistant to become susceptible to killing by TRAIL and/or chemotherapeutic agents.
  • IG20pa was over-expressed in cells, the cells showed significantly reduced proliferation and were much more susceptible to spontaneous, TNFa and TRAIL induced apoptosis.
  • the pro-apoptotic property of the IG20pa splice variant may be exploited to render tumor cells which are otherwise resistant to become susceptible to killing by TRAIL and/or chemotherapeutic agents.
  • IG20 splice variants with siRNA Down modulation of expression of IG20 splice variants with siRNA was evaluated using siRNA targeting the Mid region of the IG20 mRNA, specifically exon 15, having the sequence (S'-GTACCAGCTTCAGTCTTTC-S') and siRNA targeting the Death Domain (DD) region of IG20 mRNA. Both the Mid region and the DD region are present in all IG20 splice variants.
  • siRNA to down modulate DENN-SV expression, specifically, without affecting expression of IG20 splice variants which are necessary for normal cell function, in particular, neuronal function and survival (KIAA0358 and IG20-SV4 splice variants), is desired.
  • oligodeoxynucleotides which take into account the research of the '637 patent may be optimized to bind to particular exons which are differentially expressed in different isoforms. Knockdown using such engineered oligodeoxynucleotides may knock down only those isoforms in which that particular targeted exon is expressed, allowing for selective knockdown of intended isoforms and reducing unintended negative effects associated with knockdown of critical isoforms.
  • Map kinase Activating Death Domain (MADD) containing protein a product of the MADD splice variant of the IG20 gene, is essential for cancer cell survival. MADD is expressed at much higher levels in cancer cells and tissues relative to their normal counterparts. MADD has been shown to bind to death receptor-4 (DR4) and death receptor-5 (DR5) and to confer resistance to TRAIL induced apoptosis in thyroid, ovarian and cervical cancer cell lines (Mulherkar N. Prasad KV. Prabhakar BS. M ADD/DENN splice variant of the IG20 gene is a negative regulator of caspase-8 activation.
  • DR4 death receptor-4
  • DR5 death receptor-5
  • M ADD/DENN splice variant of the IG20 gene is a negative regulator of caspase-8 activation.
  • Knockdown enhances TRAIL-induced apoptosis of cancer cells J Biol Chem 282: 11715-11721 (2007): Subramanian M. Pilli T. Bhattacharva P. Pacini F. Nikiforov YE. et al.. Knockdown of IG20 gene expression renders thyroid cancer cells susceptible to apoptosis, J Clin Endocrinol Metab 94: 1467-1471 (2009); Prabhakar BS. Mulherkar N. Prasad KV. Role of IG20 splice variants in TRAIL resistance,. Clin Cancer Res 14: 347-351 (2008); Li LC. Javaram S. Ganesh L. Qian L. Rotmensch J. et al.. Knockdown of MADD and c-FLIP overcomes resistance to TRAIL-induced apoptosis in ovarian cancer cells, Am J Obstet Gynecol 205: 362 e312-325 (2011)).
  • MADD may render cancer cells more susceptible to spontaneous as well as TRAIL (tumor necrosis factor-related apoptosis-inducing ljgand)-induced apoptosis.
  • TRAIL tumor necrosis factor-related apoptosis-inducing ljgand
  • Spontaneous as well as TRAIL-induced apoptosis in cells devoid of MADD may be inhibited by expression of CrmA or dominant-negative FADD, thereby suggesting that endogenous MADD may interfere with caspase-8 activation.
  • MADD can directly interact with death receptors, but not with either caspase-8 or FADD, but nonetheless inhibits caspase-8 activation.
  • MADD has been shown to interfere with recruitment of FADD to the cytoplasmic domain of death receptors (Mulherkar N. Prasad K and Prabhakar B. M ADD/DENN Splice Variant of the IG20 Gene is a Negative Regulator of Caspase-8 Activation, Journal of Biological Chemistry, Vol. 282, no. 16, 11715- 11721 (2007). This demonstrates the importance of MADD in the control of cancer cell survival/death and in conferring resistance to TRAIL-induced apoptosis.
  • the ERK (extracellular signal-related kinase) pathway is a drug target for cancer chemotherapy since, in approximately one-third of all human cancers, there is deregulation of the mammalian mitogen-activated protein kinase (MAPK) pathways leading to ERK activation.
  • MAPKs are serine/threonine-specific protein kinases which respond to extracellular stimuli (mitogens) and regulate several important and critical cellular functions required for cell homeostasis like metabolism, cell cycle progression, expression of cytokines, motility and adherence.
  • MAPKs influence cell survival, proliferation, differentiation, development and apoptosis.
  • Extracellular stimuli such as cytokines, growth factors and environmental stresses lead to the sequential activation of a signaling cascade composed of
  • cytoplasmic substrates involved in a multitude of cellular processes including transcriptional factors, signaling proteins, kinases and phosphatases, cytoskeletal proteins, apoptotic proteins and proteinases.
  • the ERK pathway may be activated by numerous extracellular signals, the pathways whereby cytokines and growth factors activate ERK signaling are of particular relevance to cancer.
  • TNF-a a cytokine rich in tumor stroma binds to TNF receptor 1 (TNFR1 ) which is present on cancer cells and potently activates ERK MAPKs.
  • TNFR1 TNF receptor 1
  • this pro-survival signaling pathway may be converted into an apoptotic signaling pathway leading to cancer cell death (Kurada BRWSN. Li LC.
  • Miiherkar N. Subramanian M. Prasad KV. Prabhakar BS. MADD a Splice Variant of IG20, is Indispensable for MAPK Activation and Protection against Apoptosis upon Tumor Necrosis Factor-a Treatment, (2009), Journal of Biological Chemistry 284:13533- 13541).
  • An extrinsic cell death inducing signaling pathway may be initiated upon death Ikjand (e.g. TRAIL) binding to its cognate death receptors.
  • the death receptors undergo trimerization and recruit FADD resulting in subsequent caspase-8 activation followed by executioner caspase-3 activation leading to apoptosis.
  • TRAIL normally binds to death receptors-4 (DR4) and -5 (DR5) on cancer cells resulting in death receptor (DR) oligomerization and subsequent recruitment of FADD and procaspase-8 to the DRs (Bodmer JL. Holler N. Reynard S. Vinciauerra P. Schneider P. et al..
  • TRAIL receptor-2 signals apoptosis through FADD and caspase-8, Nat Cell Biol 2: 241-243 (2000); Sprick MR. Weiaand MA. Rieser E. Rauch CT. Juo P. et al.. FADD/MORT1 and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2, Immunity 12: 599-609 (2000); Kischkel FC. Lawrence PA. Chuntharapai A. Schow P. Kim KJ. et al.. Apo2L/TRAIL- dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5, Immunity 12: 611-620 (2000)).
  • Procaspase-8 then undergoes proximity induced cleavage and activation forming caspase-8 which then activates executioner caspase-3 which causes apoptotic cell death.
  • MADD binds to DR4 and DR5 and prevents FADD recruitment to the DRs.
  • FADD is more readily recruited to the DRs, resulting in enhanced apoptosis (Mulherkar N. Prasad KV. Prabhakar BS.
  • MADD/DENN splice variant of the IG20 gene is a negative regulator of caspase-8 activation. Knockdown enhances TRAIL-induced apoptosis of cancer cells, J Biol Chem 282: 11715-11721 (2007): Mulherkar N. Ramaswamv M. Mordi DC. Prabhakar BS. MADD/DENN splice variant of the IG20 gene is necessary and sufficient for cancer cell survival, Oncogene 25: 6252-6261 (2006)).
  • TRAIL is unique in that it generally does not adversely affect normal cells or tissues (Keane MM. Ettenberg SA. Nau MM. Russell EK. Lipkowitz S.
  • MADD is one such anti-apoptotic protein demonstrating the utility of MADD down- regulation in rendering cancer cells susceptible to cell death (Mulherkar N.
  • MADD may have a dual function in regulating apoptosis depending on its phosphorylation by Akt.
  • the tumor suppressor PTEN phosphatase and tensin homolog deleted on chromosome 10
  • PTEN phosphatase and tensin homolog deleted on chromosome 10
  • PI3K phosphatidylinositol 3-kinase
  • MADD can act as a pro-apoptotic factor to initiate apoptosis when its phosphorylation is attenuated by PTEN (Javarama S. Li L. Ganesh L. Mardi D. Kanteti P. Hav N. Li P. and Prabhakar BS. J Cell Biochem. 2014, 115(2):261-270).
  • TRAIL induces an up-regulation of PTEN with a concomitant reduction in MADD phosphorylation.
  • Down-regulation of PTEN interferes with TRAIL-induced reduction in pMADD levels.
  • MADD translocates from the plasma membrane to cytoplasm where it binds to 14-3-3 protein and displaces 14-3-3 associated Bax, which Bax translocates to mitochondria resulting in cytochrome-c release.
  • PTEN can convey the death signal by preventing MADD phosphorylation by Akt.
  • the extrinsic apoptotic pathway may be abrogated by phosphorylated MADD.
  • Endogenous MADD is phosphorylated at three highly conserved sites by Akt, and only the phosphorylated MADD can directly interact with the TRAIL receptor DR4 thereby preventing FADD recruitment.
  • TRAIL induces a reduction in MADD phosphorylation levels resulting in MADD dissociation from, and FADD association with DR4, which allows death- inducing signaling complex (DISC) formation leading to apoptosis (Li P. Javarama S. Ganesh L. Mordi D. Carr R. Kanteti P. Hav N. and Prabhakar BS. J Biol Chem.
  • Akt pro-survival function of MADD is dependent upon its phosphorylation by Akt. Because Akt is active in most cancer cells and phosphorylated MADD confers resistance to TRAIL-induced apoptosis, co- targeting the Akt-MADD axis is likely to increase efficacy of therapeutics which involve DR4/5 binding, including TRAIL-based therapies.
  • the intrinsic apoptotic pathway is initiated when a death signal induces the release of mitochondrial pro-apoptotic proteins such as cytochrome c (Li P.
  • Cytochrome-c forms a complex with Apaf-1 and procaspase-9 resulting in the activation of caspase-9.
  • Smac/Diablo can associate with Inhibitor of Apoptosis Proteins (lAPs) and counteract their caspase inhibitory effects.
  • the intrinsic pathway is regulated by the Bcl-2 family members.
  • Bcl-2 and Bcl-xL can associate with Bax and Bad thereby preventing them from inducing death (Antignani A. Youle RJ.
  • MADD cDNA sequence is available on the GenBank database under accession number: NM_130470, and is represented herein by the nucleotide sequence of SEQ ID NO:11 and the polypeptide sequence of SEQ ID NO: 12.
  • Interfering RNAs which down-regulate MADD including siRNA, shRNA and
  • antisense oligonucleotides are designed to target a nucleic acid sequence of exon 13L of a splice variant of the IG20 gene, and include any allelic variants and naturally occurring mutants of MADD, and polymorphisms which occur in the MADD splice variant which may be found in a particular segment of the population.
  • sequences which are highly similar e.g., about 95% at the amino acid level and about 75% at the nucleic acid level
  • MADD splice variant variations in the MADD splice variant are within the scope of the disclosure, wherein the siRNA, shRNA and antisense oligonucleotides disclosed herein are capable of down-regulating the expression of such sequences.
  • Exon 13L of the MADD splice variant may comprise a nucleotide sequence represented by nucleotides 2699 to 2827 of SEQ ID NO: 11. Nucleic acid sequences which are about 80% or 90% or 95% similar at the nucleic acid level to the MADD sequence disclosed herein may also be down-regulated.
  • Nucleic acid sequences which generate siRNA and shRNA which comprise nucleic acid sequences complementary to a nucleic acid sequence of exon 13L of the MADD splice variant of the IG20 gene and/or an mRNA transcript of exon 13L of the MADD splice variant, as well as nucleic acid variations which may occur within the exon 13L target region are within the scope of the instant disclosure.
  • antisense oligonucleotides which comprise nucleic acid sequences complementary to a nucleic acid sequence of exon 13L of the MADD splice variant of the IG20 gene and/or an mRNA transcript of exon 13L of the MADD splice variant, as well as nucleic acid variations which may occur within the exon 13L target region are within the scope of the instant disclosure
  • Methods for specifically down-regulating the expression of a splice variant of an IG20 gene have been shown to include: (a) obtaining a nucleic acid molecule which is capable of down-regulating MADD expression, wherein the nucleic acid molecule or a transcription product thereof is capable of selectively binding to an mRNA molecule, the mRNA molecule which includes a nucleic acid sequence of a MADD splice variant of the IG20 gene; and (b) contacting a cell which expresses the MADD splice variant of the IG20 gene with the nucleic acid molecule, wherein the nucleic acid molecule down-regulates the expression of the MADD splice variant.
  • nucleic acids selected from siRNA, expressed shRNAs, and antisense oligonucleotides bind to target exon 13L mRNA and lead to degradation of the target 13L mRNA which down-regulates expression of the MADD splice variant.
  • down-regulating MADD expression has been shown to be a substantial downregulation, for example, more than 90% or 95% reduction of the endogenous MADD expression.
  • downregulation of, for example, at least 40%, at least 50%, at least 60%, at least 70%, and at least 80% of endogenous MADD expression is desirable.
  • siRNA, shRNA and antisense oligonucleotides targeting exon 16 could knockdown IG20pa, only the siRNA, shRNA and antisense oligonucleotides targeting exon 13L could cause cancer cell death. This indicated for the first time that specifically MADD might be critical for cancer cell survival. An additional line of evidence which shows MADD to be essential and sufficient for cancer cell survival is demonstrated in cells transfected with Mid-shRNA resistant IG20 splice variants in which the 3rd base of the triple codons was replaced in the Mid-shRNA targeted region of the cDNA constructs, which DMA substitutions will not alter the amino acid sequence but renders them resistant to Mid-shRNA).
  • US Patent No. 7,910,723 describes nucleic acid molecules which target exon 13L of the IG20 gene and the use of encoded siRNA, shRNA and antisense oligonucleotides in down-regulating expression of MADD protein.
  • the patent describes how the nucleic acid selection may be optimized to achieve effective down- regulation of MADD expression, including selecting for nucleic acid molecules which consist essentially of a nucleotide sequence CGGCGAATCTATGACAATC (SEQ ID NO:1), and transcribed products thereof, encoding nucleic acid molecules consisting essentially of a nucleotide sequence CGGCGAAUCUAUGACAAUC (SEQ ID NO:2).
  • Species nucleic acid molecules representative of such strategy include siRNA, shRNA and antisense oligonucleotides, which comprise less than 50% GC nucleotide content, high AU nucleotide content towards the 3' end and no inverted repeats within the siRNA region, which oligonucleotides constitute an array of nucleic acid molecules which may span the exon 13L nucleotide sequence.
  • These encoded nucleic acid molecules and encoded siRNA, shRNA and antisense oligonucleotides are demonstrated to be sufficient to down-regulate the expression of MADD splice variants. Natural variations of MADD including specific SNPs, allelic variants, or mutations which may appear in one or more of sub-groups of cancer types may be targeted by such nucleic acid molecules.
  • Methods for down-regulating expression of MADD include: (a) obtaining a nucleic acid molecule which selectively down-regulates MADD expression, wherein the nucleic acid molecule is capable of selectively binding to an mRNA molecule of a MADD splice variant of the IG20 gene; and (b) contacting a cancer cell which expresses the MADD splice variant of the IG20 gene with the nucleic acid molecule, wherein the nucleic acid molecule down-regulates the expression of the MADD splice variant in the cancer cell.
  • nucleic acid molecules which are shown to down-regulate the MADD splice variant of IG20, allelic variations thereof,
  • polymorphisms thereof, and genetic mutations thereof include siRNA, shRNA, and anti-sense nucleic acid molecules which may comprise a nucleotide sequence
  • the siRNA may be in the form of a duplex with the cognate antisense nucleic acid, which cognate antisense nucleic acid is complementary to a target MADD exon 13L nucleotide sequence and/or mRNA transcripts of MADD exon 13L.
  • X encoding siRNA
  • X includes or consists essentially of a nucleic acid having the sequence CGGCGAATCTATGACAATC (SEQ ID NO:1 ).
  • the nucleic acid is transcribed to form shRNA and may be cleaved to form siRNA which may ultimately inhibit MADD expression.
  • RNA molecules which are transcribed in vitro or in vivo, e.g., in a cancer cell or tumors to form shRNA and siRNA are also included.
  • An exemplary dsDNA nucleic acid sequence encoding an shRNA inhibiting MADD expression may be
  • double stranded RNA or dsRNA refers to a double stranded RNA which matches a predetermined gene sequence which is capable of activating cellular enzymes which degrade corresponding messenger RNA transcripts of the gene.
  • dsRNAs comprise nucleic acid molecules which may be short interfering RNA (siRNA) and may be used to inhibit gene expression.
  • double stranded RNA or “dsRNA” as used herein refers to a double stranded RNA molecule capable of RNA interference "RNAi,” including short interfering RNA “siRNA.”
  • siRNA, shRNA and antisense oligonucleotide molecules as described herein may also include nucleic acid modifications known in the art to enhance stability and to enhance cleavage destruction of the target mRNA.
  • a representative example may comprise a 19- base core which includes 2 or 3 nucleotide overhanding 3 * ends, such as a 3' terminal thymidines (TT). The overhangs may play a structural role for presenting a
  • the siRNA, shRNA and antisense oligonucleotide molecules may also be chemically synthesized de novo.
  • the synthesized nucleic acid molecules may be in the form of a single-stranded nucleic acid molecule or may be in the form of a duplex with the cognate antisense nucleic acid molecule.
  • the siRNA, shRNA and antisense oligonucleotides comprise a nucleic acid sequence which is
  • RNA interference is a conserved pathway found in most eukaryotes where double-stranded RNAs (dsRNAs) down-regulate expression of genes with complementary sequences.
  • dsRNAs double-stranded RNAs
  • siRNAs small interfering RNAs
  • siRNAs are usually around 21 base pairs (bp) long with a central 19 bp duplex and 2 -base 3' -overhangs (this could be TT) (Elbashir. SM. Lendeckel W and Tuschl T.
  • RNA interference is mediated by 21- and 23-nucleotide RNAs, Genes & Development, 2001 , 15:188-200).
  • Dicer processing occurs in a multiprotein complex with the RNA -binding protein TRBP.
  • the nascent siRNA associates with Dicer, TRBP, and Argonaute 2 (Ago2) to form the RNA -Induced Silencing Complex (RISC).
  • RISC RNA -Induced Silencing Complex
  • one strand of the siRNA (the passenger strand/the strand that has the same sequence as the target mRNA) is degraded or discarded while the other strand (the guide strand/strand that is complementary to the targeted mRNA) remains to direct sequence specificity of the silencing complex.
  • the Ago2 component of RISC is a ribonuclease that will cleave the target RNA under direction of the guide strand.
  • RNA duplexes as their RNAi reagents, which mimic the natural siRNAs that result from Dicer processing of long substrate RNAs. These synthetic siRNA duplexes are transfected into cell lines where they mimic in vivo Dicer products.
  • MADD expression and/or downregulation of MADD expression utilizing siRNA, shRNA or antisense oligonucleotides may augment traditional cancer therapies.
  • cancer chemotherapy has advanced dramatically in recent years. Numerous cancer chemotherapy substances have been identified which are effective in treating cancer. Nonetheless, many cancer chemotherapies are characterized by toxic side effects which are often encountered with administration of particular chemotherapeutics.
  • Chemotherapeutics such as nucleoside analogs, are synthetic, chemically modified compounds that mimic their physiological counterparts and thereby, interfere with the synthesis of nucleic acids by incorporating into DNA and RNA. They can also interact and inhibit essential enzymes such as DNA-dependent DNA polymerase, RNA-dependent DNA polymerase, RNA-dependent RNA polymerase, kinases, DNA methyltransferases, pyrimidine and purine nucleoside phosphorylase etc.
  • Nucleoside analogues exert cytotoxic effect by being incorporated into and altering the DNA and RNA macromolecules themselves, by interfering with various enzymes involved in synthesis of nucleic acids, or by modifying the
  • Nucleoside analogues such as Gemcitabine, 5- Fluorouracil and Cytarabine, are used in the treatment of breast cancer, non-small cell lung cancer, pancreatic cancer, bladder cancer, biliary tract cancer and colon cancer, esophageal cancer, stomach cancer, pancreatic cancer, breast cancer, cervical cancer, and other types of solid tumor cancers.
  • the triphosphate analogue of gemcitabine replaces cytidine during DNA replication leading to arrest of tumor growth.
  • 5-Fluorouracil acts as a thymidylate synthase inhibitor leading to blockage of synthesis of thymidine, which eventually inhibits DNA replication.
  • Nucleoside analogues also present certain unwanted side effects such as nausea, vomiting, poor appetite, low blood counts.
  • the present inventors have now identified novel methods of administering nucleoside analogs which, when administered in combination with down-regulation of the MADD splice variant, result in dramatic synergistic effects including increased efficacy as well as reduced side-effects.
  • the present inventors have conceived and demonstrate for the first time that the clinical combination of one or more nucleic acid molecules capable of down- regulating expression of at least one splice variant of the IG20 gene, wherein not all splice variants of the IG20 gene are down-regulated, with conventional
  • chemotherapeutics such as nucleoside analogs
  • chemotherapeutics is an unexpectedly valuable pharmacotherapeutic approach to treating various forms of cancer.
  • the present inventors demonstrate that, when administered in combination to subjects suffering from cancers such as those of ovary, breast, lung, pancreas, bladder, cervix, prostate, melanoma, esophageal and other solid tumors including Kaposi's sarcoma, the effects of siRNA, shRNA and antisense oligonucleotides, wherein the siRNA, shRNA and antisense oligonucleotides comprise a nucleic acid sequence which is complementary to a nucleic acid sequence of exon 13L of the MADD splice variant or to an mRNA transcript of exon 13L of the MADD splice variant, and
  • chemotherapeutics such as nucleoside analogs is of unexpected benefit and, at least over a period of time results in an unexpectedly superadditive relief of symptoms, evidenced by dramatic reduction in, or absence of, symptoms, tumor growth and tumor survival, and in this way will be particularly beneficial in the treatment of multiple cancers.
  • oligonucleotides comprise a nucleic acid sequence which is complementary to a nucleic acid sequence of exon 13L of the MADD splice variant or to an mRNA transcript of exon 13L of the MADD splice variant, and chemotherapeutics such as nucleoside analogs may, for the first time, show promise in providing complete remission from multiple cancers, as well as enhanced margin of safety and tolerance.
  • It is an object of the present invention to provide novel combination antineoplastic treatments comprising administering representative chemotherapeutics in combination with nucleic acid molecules capable of down-regulating the
  • An additional object of the invention is the provision of a process for producing targeted formulations and therapeutic delivery procedures for the combination antineoplastic treatments. Yet additional objects will become apparent hereinafter, and still further objects will be apparent to one skilled in the art.
  • a combination of antineoplastic agents useful for treating cancer comprising an effective amount of one or more nucleic acid molecules capable of down-regulating expression of at least one splice variant of the IG20 gene, wherein not all splice variants of the IG20 gene are down-regulated, and one or more nucleoside analog chemotherapeutic.
  • the at least one splice variant of the IG20 gene is selected from a MADD splice variant, SNPs, allelic variations thereof, polymorphisms thereof, and genetic mutations thereof.
  • nucleic acid molecules capable of down-regulating expression of the at least one splice variant of the IG20 gene is selected from siRNA, shRNA and antisense oligonucleotides.
  • siRNA, shRNA and antisense oligonucleotides comprise nucleic acids which are complementary to a nucleic acid sequence of exon 13L of a MADD splice variant, SNPs, allelic variations thereof, polymorphisms thereof, and genetic mutations thereof, and/or an mRNA transcript thereof.
  • nucleic acid molecule capable of down-regulating expression of at least one splice variant of the IG20 gene is comprised in an siRNA or shRNA.
  • siRNA and shRNA is encoded by a nucleic acid molecule which includes the structure:
  • Xsense - hairpin ⁇ - Xanti-eense wherein X includes or consists essentially of a nucleic acid sequence
  • siRNA or shRNA comprises a nucleic acid having the sequence CGGCGAAUCUAUGACAAUC (SEQ ID NO:2).
  • siRNA or shRNA comprises a nucleic acid having the sequence CGGCGAAUCUAUGACAAUC (SEQ ID NO:2) and is in the form of a duplex with a cognate nucleic acid having the sequence
  • CGAATCTATGACAATCTTCAAGAGAGATTGTCATAGATTCGCCG (SEQ ID NO:3), wherein a hairpin loop region is from positions 20-28 of the sequence.
  • siRNA, shRNA and antisense oligonucleotide comprising nucleic acids which are complementary to a nucleic acid sequence of exon 13L of a MADD splice variant of the IG20 gene and/or an mRMA transcript thereof comprises a nucleic acid having the sequence selected from GAUUGUCAUAGAUUCGCCGTT (SEQ ID NO:4) and
  • nucleic acid molecules capable of down-regulating expression of the at least one splice variant of the IG20 gene is comprised in a drug delivery system.
  • nucleoside analog chemotherapeutic is selected from Gemcitabine, 5-Fluorouracil and Cytarabine.
  • nucleoside analog chemotherapeutic is in the form of a pharmaceutically acceptable salt.
  • cancer is selected from breast cancer, non-small cell lung cancer, pancreatic cancer, bladder cancer, biliary tract cancer and colon cancer, esophageal cancer, stomach cancer, cervical cancer, and other types of solid tumor cancers.
  • a method of treating cancers selected from breast cancer, non-small cell lung cancer, pancreatic cancer, bladder cancer, biliary tract cancer and colon cancer, esophageal cancer, stomach cancer, pancreatic cancer, breast cancer, cervical cancer, and other types of solid tumor cancers in a subject in need thereof comprising administering an effective amount of a combination of antineoplastic agents comprising an effective amount of one or more nucleic acid molecules capable of down-regulating expression of at least one splice variant of the IG20 gene, wherein not all splice variants of the IG20 gene are down-regulated, and one or more nucleoside analog chemotherapeutic.
  • nucleic acid molecules capable of down-regulating expression of at least one splice variant of the IG20 gene is selected from siRNA, shRNA and antisense oligonucleotides.
  • oligonucleotides comprise nucleic acids which are complementary to a nucleic acid sequence of exon 13L of a MADD splice variant, SNPs, allelic variations thereof, polymorphisms thereof, and genetic mutations thereof, of the IG20 gene and/or an mRNA transcript thereof.
  • nucleic acid molecule capable of down- regulating expression of at least one splice variant of the IG20 gene is comprised in siRNA or shRNA.
  • siRNA and shRNA is encoded by a nucleic acid molecule which includes the structure:
  • Xsense - hairpin ⁇ - Xarrti-eense wherein X includes or consists essentially of a nucleic acid sequence
  • siRNA and shRNA comprises a nucleic acid having the sequence CGGCGAAUCUAUGACAAUC (SEQ ID NO:2).
  • siRNA and shRNA comprises a nucleic acid having the sequence CGGCGAAUCUAUGACAAUC (SEQ ID NO:2) and is in the form of a duplex with a cognate nucleic acid having the sequence
  • shRNA is encoded by a nucleic acid having the sequence CGAATCTATGACAATCTTCAAGAGAGATTGTCATAGATTCGCCG (SEQ ID NO: 3), wherein a hairpin loop region is from positions 20-28 of the
  • oligonucleotides comprising nucleic acids which are complementary to a nucleic acid sequence of exon 13L of a MADD splice variant of the IG20 gene and/or an mRNA transcript thereof comprise a nucleic acid having the sequence selected from
  • Such a method wherein the one or more siRNA, shRNA and antisense oligonucleotides is administered in the form of a liposomal formulation or by lentivirus transfection.
  • Such a method wherein the one or more siRNA, shRNA and antisense oligonucleotides is administered as an adjuvant.
  • chemotherapeutic is selected from Gemcitabine, 5-Fluorouracil and Cytarabine.
  • chemotherapeutic is administered in the form of a pharmaceutical composition further comprising one or more pharmaceutically acceptable diluents, excipients, or carriers.
  • chemotherapeutic or is administered simultaneously with the one or more nucleoside analog chemotherapeutic.
  • Such a method wherein the one or more siRNA, shRNA and antisense oligonucleotides is administered in the form of a pharmaceutical composition further comprising one or more pharmaceutically acceptable diluents, excipients, or carriers.
  • a pharmaceutical composition further comprising one or more pharmaceutically acceptable diluents, excipients, or carriers.
  • Such a method wherein the one or more siRNA, shRNA, and antisense oligonucleotides and the one or more nucleoside analog chemotherapeutic are formulated in a dosage pack and are administered according to a selected treatment regime.
  • FIG. 1 Human I G20 splice variants generated by alternative mRNA splicing. The cDNA sequence homology among the seven IG20 splice variants is shown. Solid bars represent regions of complete homology between all variants. Empty areas indicate exons 13L, 16, 21 , 26 and 34, which, when spliced in different combinations, produce the seven splice variants shown on the left. Splicing of exon 34 in KIAA0358 and IG20-SV4 induces an early stop codon in exon 35. Shown also are different 5' untranslated regions (UTRs) for different splice variants.
  • UTRs 5' untranslated regions
  • the instant invention provides a novel drug combination useful for treating, preventing, arresting, delaying the onset of and/or reducing the risk of developing, or reversing at least one symptom of a cancer selected from ovarian, breast, lung, pancreas, bladder, cervical, prostate, melanoma, esophageal and other solid tumors including Kaposi's sarcoma, in a mammal comprising administering to said mammal an amount of siRNA, shRNA and/or antisense oligonucleotides capable of down-regulating the expression of the MADD splice variant of an IG20 gene, wherein the splice variant is MADD, SNPs, allelic variations thereof, polymorphisms thereof, and genetic mutations thereof, and wherein not all splice variants of IG20 are down-regulated, and nucleoside analogs at therapeutically effective dosages which, when combined, provide a beneficial effect.
  • a cancer selected from ovarian, breast, lung, pancrea
  • compositions comprising both drugs of the invention (i.e., one or more siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene, wherein not all splice variants of IG20 are down-regulated, and one or more nucleoside analogs) or two separate pharmaceutical compositions (formulations), each comprising a single drug of the invention (i.e., one or more siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene, wherein not all splice variants of IG20 are down- regulated and one or more nucleoside analogs), to be administered conjointly or in a pretreatment/treatment protocol.
  • drugs of the invention i.e., one or more siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an
  • the term “conjoint administration” is used to refer to administration of the one or more siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene, wherein not all splice variants of IG20 are down-regulated and one or more nucleoside analogs simultaneously in one composition, or simultaneously in different compositions, or sequentially in different compositions.
  • the sequential administration to be considered “conjoint", however, the administration of the one or more siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene, wherein not all splice variants of IG20 are down-regulated and one or more nucleoside analogs must be administered separated by a time interval which still permits the resultant beneficial effect of conjoint treatment for treating, preventing, arresting, delaying the onset of and/or reducing the risk of developing a cancer in a mammal.
  • the one or more siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene, wherein not all splice variants of IG20 are down- regulated and one or more nucleoside analogs may be administered sequentially.
  • siRNA may be administered 8-72 hours (hrs) before the administration of the nucleoside analog so as to have the MADD expression down-modulated prior to treatment with the chemotherapeutic.
  • the one or more siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene is used as an adjuvant.
  • An "adjuvant" in the context of the present description refers to an enhancer of the specific nucleoside analog response.
  • Using the one or more siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene means including the one or more siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene in a pre-treatment prior to the chemotherapy agent, or in combination with the chemotherapeutic agent for simultaneous delivery.
  • An adjuvant such as the one or more siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene in combination with a chemotherapeutic agent provides synergistic cell death.
  • the one or more siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene are administered to mammals exhibiting cancers, including humans, activate the death of cancerous cells.
  • the one or more siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene and one or more chemotherapeutic agent may also be used to support apoptotic pathways in a situation of increased susceptibility to developing tumors, such as in the case of patients in remission.
  • the term “treat” is used herein to mean to relieve or alleviate at least one symptom of a disease in a subject.
  • the term “treat” may mean to relieve or alleviate tumor growth or symptoms associated with the cancer and/or cause tumor regression.
  • the term “treat” may also denote to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease.
  • the term “protect” is used herein to mean prevent delay or treat, or all, as appropriate, development or continuance or aggravation of a disease in a subject.
  • the cancer is associated with clinical manifestations, including without limitation drug induced undesirable side-effects.
  • a prophylactic administration of one or more siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene and nucleoside analogs may protect a recipient subject to risk of developing cancer (e.g., individuals having elevated levels of CA125, individuals, who exhibit histopathologic cancer markers; see also genetic screening and clinical analysis described in oncology literature for standard screening for various cancers).
  • a therapeutic administration of one or more siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene and nucleoside analogs can lead to slow-down in the development of clinical symptoms or even regression of symptoms.
  • siRNA refers to RNA molecules which are capable of interfering with a particular gene transcription, thereby silencing the gene expression of a target protein.
  • Representative mechanisms of this process may comprise administration of nucleic acid molecules comprising siRNAs, dsRNAs, short hairpin RNAs (shRNA) and/or antisense oligonucleotides complementary to a nucleic acid sequence of exon 13L of the MADD splice variant mRNA transcript.
  • dsRNA and short hairpin RNAs are cleaved by an endo-ribonuclease Dicer, which cuts the dsRNA or shRNA into constituent siRNA.
  • the siRNA operates through the formation of RNA-induced Silencing Complexes or RISCs.
  • the RISC complex unwinds the siRNA to form single stranded siRNA.
  • the RISC comprising single stranded siRNAs binds to the target mRNA, cleaving the mRNA, rendering it unrecognizable and thereby silencing the production of the intended protein.
  • siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene are used to refer to drugs, which target messenger RNAs.
  • Embodiment siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene of the invention may be siRNA derivatives such as modified siRNAs, shRNAs and/or antisense oligonucleotides comprise nucleic acids which are complementary to the nucleic acid sequence of exon 13L of the MADD splice variant mRNA
  • siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene may be accomplished through techniques which have been established.
  • the siRNA, shRNA and antisense oligonucleotides used to target MADD mRNA transcripts were obtained from Dharmacon (Lafayette, Colo.). The most suitable sequences were sorted out based on less than 50% GC nucleotide content, high AU nucleotide content towards the 3' end and no inverted repeats within the siRNA region
  • Antisense oligonucleotides refers to a nucleic acid molecule which binds to target mRNA by means of RNA-RNA or RNA-DNA or RNA-PNA (peptide nucleic acid) interactions and alters the activity of the target mRNA.
  • antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule.
  • antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex.
  • the antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA.
  • Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof.
  • nucleic acid molecules or antisense molecules which interact with target RNA molecules and down-regulate MADD activity are expressed from transcription units inserted into DNA or RNA vectors.
  • the recombinant vectors may be DNA plasmids or viral vectors.
  • Enzymatic nucleic acid molecule or antisense expressing viral vectors can be constructed based on adeno- associated virus, retrovirus, adenovirus, or alphavirus.
  • the recombinant vectors capable of expressing the nucleic acid molecules are delivered as described herein, and persist in target cells.
  • viral vectors can be used that provide for transient expression of shRNA/siRNA/anti-sense nucleic acid molecules.
  • nucleic acid molecule or antisense expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells explanted from the patient or subject followed by reintroduction into the patient or subject, or by any other means that would allow for introduction into the desired target cell.
  • Antisense DMA can also be expressed via the use of a single stranded DNA intracellular expression vector.
  • oligonucleotides are designed accordingly based on the nucleotide sequence of the exon 13L of the MADD splice variant.
  • Exon 13L of the MADD splice variant exhibits the nucleotide sequence such as that defined as nucleotides 2699 to 2827 of SEQ ID NO:11.
  • the siRNA, shRNA and antisense oligonucleotides comprise
  • oligonucleotides having a 19-base core nucleotide sequence which specifically targets exon 13L of the MADD splice variant may construct siRNA, shRNA and antisense oligonucleotides which comprise less than 50% GC nucleotide content, high AU nucleotide content towards the 3' end and no inverted repeats, which oligonucleotides constitute an array of oligonucleotides which may span exon 13L nucleotide sequence.
  • siRNA, shRNA and antisense oligonucleotides which comprise less than 50% GC nucleotide content, high AU nucleotide content towards the 3' end and no inverted repeats, which oligonucleotides constitute an array of oligonucleotides which may span exon 13L nucleotide sequence.
  • oligonucleotides may further comprise 2 or 3 nucleotide overhanding 3' ends, such as a terminal TT to enhance cleavage destruction of the target mRNA.
  • dTdT is selected because it can confer nuclease resistance to oligonucleotides.
  • UU overhangs or overhangs that are complementary to the authentic mRNA target may be added to the 19-base core of the oligonucleotides.
  • the siRNA, shRNA and antisense oligonucleotide comprises a nucleic acid having the sequence GAUUGUCAUAGAUUCGCCGTT (SEQ ID NO:4).
  • the siRNA and shRNA may be in the form of a duplex.
  • the siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene, wherein not all splice variants of the IG20 gene are down-regulated comprises a nucleic acid having the sequence GAUUGUCAUAGAUUCGCCG (SEQ ID NO: 10).
  • the siRNA or shRNA may be in the form of a duplex.
  • the siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene, wherein not all splice variants of the IG20 gene are down-regulated consists essentially of a nucleic acid having the sequence GAUUGUCAUAGAUUCGCCG (SEQ ID NO: 10).
  • the siRNA or shRNA may be in the form of a duplex
  • the antisense oligonucleotide capable of down-regulating expression of at least one splice variant of the IG20 gene, wherein not all splice variants are down- regulated may be a single-stranded nucleic acid molecule which exhibits a nucleic acid sequence which is complementary to the nucleic acid sequence, or a portion thereof, of exon 13L of the MADD splice variant and/or an mRNA transcript thereof.
  • the single-stranded nucleic acid molecule may be in the form of RNA or DNA.
  • the antisense oligonucleotide comprises the sequence
  • nucleoside analog is used herein to refer to a drug which exhibits an anti-cancer chemotherapeutic effect.
  • the term encompasses synthetic, chemically modified compounds that mimic their physiological counterparts and thereby, interfere with the synthesis of nucleic acids by incorporating into DNA and RNA. They can also interact and inhibit essential enzymes such as DNA-dependent DNA polymerase, RNA-dependent DNA polymerase, RNA-dependent RNA
  • Nucleoside analogues exert cytotoxic effect by being incorporated into and altering the DNA and RNA macromolecules themselves, by interfering with various enzymes involved in synthesis of nucleic acids, or by modifying the
  • Nucleoside analogues such as Gemcitabine, 5- Fluorouracil and Cytarabine, are used in the treatment of breast cancer, non-small cell lung cancer, pancreatic cancer, bladder cancer, biliary tract cancer and colon cancer, esophageal cancer, stomach cancer, cervical cancer, and other types of solid tumor cancers.
  • analog or “derivative” is used herein in the conventional pharmaceutical sense, to refer to a molecule which structurally resembles a reference molecule (such as nucleoside analogs), but has been modified in a targeted and controlled manner to replace one or more specific substituents of the referent molecule with an alternate substituent, thereby generating a molecule which is structurally similar to the reference molecule.
  • Synthesis and screening of analogs e.g., using structural and/or biochemical analysis, to identify slightly modified versions of a known compound which may have improved or biased traits (such as higher potency and/or selectivity at a specific targeted receptor type, greater ability to penetrate mammalian blood-brain barriers, fewer side effects, etc.) is a drug design approach which is well known in pharmaceutical chemistry.
  • salts and isomers can include addition salts of pharmaceutically acceptable free acids or free bases.
  • acids which may be employed to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric, sulfuric, or phosphoric acid, and organic acids such as acetic, maleic, succinic, or citric acid, etc. All of these salts (or other similar salts) may be prepared by conventional means.
  • the nature of the salt or isomer is not critical, provided that it is non-toxic and does not substantially interfere with the desired pharmacological activity.
  • terapéuticaally effective applied to a dose or amount refers to that quantity of a compound or pharmaceutical composition which is sufficient to result in a desired activity upon administration to a mammal in need thereof.
  • pharmaceutical compositions comprising an siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene and nucleoside analogs, the term
  • chemotherapeutically effective amount/dose refers to the amount/dose of a compound or pharmaceutical composition which is sufficient to produce an effective chemotherapeutic response upon administration to a mammal. Note that when a combination of active ingredients is administered the effective amount of the combination may or may not include amounts of each ingredient which are
  • the term "subthreshold” referring to the amount of an active ingredient means an amount inadequate to produce a response, i.e., an amount below the minimum effective amount.
  • the term “suboptimal” in the same context means an amount of an active ingredient which produces a response but not to its full extent, which would be achieved with a higher amount.
  • the phrase "synergistic effect”, “synergistic”, “synergy”, “synergism”, as used in connection with the combination therapy of the invention, refers to the cooperative action of two or more stimuli that when combined produce an effect which is greater than the sum of the effect of the contributions of each individual stimulus, i.e., more than an additive effect.
  • the stimuli for example, may be an agent which down-regulates expression of MADD and at least one therapeutic agent.
  • compositions of the invention refers to molecular entities and other ingredients of such compositions which are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal ⁇ e.g., human).
  • pharmaceutically acceptable refers to molecular entities and other ingredients of such compositions which are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal ⁇ e.g., human).
  • the term "pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.
  • carrier applied to pharmaceutical compositions of the invention refers to a diluent, excipient, or vehicle with which an active compound (e.g., an siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene and nucleoside analogs) is administered.
  • active compound e.g., an siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene and nucleoside analogs
  • Such pharmaceutical carriers may be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • suitable carriers may comprise liposomal formulations. Suitable pharmaceutical carriers may also be described in "Remington's Pharmaceutical Sciences”
  • subject refers to a mammal (e.g., rodent such as mouse or rat). In particular, the term refers to humans.
  • the term “about” or “approximately” usually means within 20%, within 10%, and optionally within 5% of a given value or range. Alternatively, especially in biological systems, the term “about” means within about a log ⁇ i.e., an order of magnitude) optionally within a factor of two of a given value.
  • compositions comprising a therapeutically effective amount of an siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene and/or a therapeutically effective amount of an nucleoside analog as well as, optionally, an additional carrier or excipient (all pharmaceutically acceptable).
  • siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene and nucleoside analogs may be either formulated as a single composition or as two separate compositions, which may be administered conjointly.
  • compositions are formulated as a single composition or as two separate compositions, which are optionally administered sequentially or simultaneously.
  • the compositions may be formulated for once-a-day administration or twice-a-day administration, as well as dosage regimens typical in the respective therapies.
  • the instant combinations may be formulated such that they may be administrated in a titration regimen such that the patient may acclimate to the effects of the nucleoside analog and/or the clinician may titrate up the siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene so that the nucleoside analog may be down titrated to significantly lower dosages so as to minimize toxic side effects of the nucleoside analog and/or accommodate for dosing difficulties associated with the nucleoside analog.
  • both the siRMA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene and nucleoside analogs are present in therapeutically effective amounts.
  • the optimal therapeutically effective amount should be
  • both the siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene and nucleoside analogs are administered in suitable form in doses ranging from those understood in the art.
  • the siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene are administered at therapeutic doses; the nucleoside analogs are administered at suboptimal or lowered doses. It may also be desirable in certain cases to administer one or the other of the active ingredients in a suboptimal or subthreshold amount, and such administration would also be within the scope of the invention.
  • the invention also provides a method for preparing pharmaceutical compositions comprising admixing an siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene and nucleoside analogs in therapeutically effective amounts, and optionally one or more physiologically acceptable carriers and/or excipients and/or auxiliary substances.
  • the active agents of the present invention may be administered intradermally, parenterally, intranasally or intra-tumorally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers with or without a targeting molecule which would selectively deliver the active agent to a particular cell type or tissue.
  • the intradermal administration could involve using transdermal patches, microabrasion or nanoemulsions.
  • administered medicaments may be administered in the form of an injection, a time-controlled release vehicle, including diffusion-controlled systems, osmotic devices, dissolution-controlled matrices, and erodible/degradable matrices.
  • the active drug component may be combined with non-toxic, pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, sucrose, glucose, mamitol, sorbitol and other reducing and non-reducing sugars, microcrystalline cellulose, calcium sulfate, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica, steric acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate, and the like); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate), coloring and flavoring agents, gelatin, sweeteners, natural and synthetic gums (such as acacia,
  • binding agents e.g., pregelatinized maize star
  • the drug components may be combined with non-toxic, pharmaceutically acceptable inert carriers (e.g., ethanol, glycerol, water), suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils), preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid), and the like.
  • Stabilizing agents such as antioxidants (BHA, BUT, propyl gallate, sodium ascorbate, citric acid) can also be added to stabilize the dosage forms.
  • the tablets may be coated by methods well known in the art.
  • the compositions of the invention may be also introduced in microspheres or
  • microcapsules e.g., fabricated from polyglycolic acid/lactic acid (PGLA) (see, e.g., U.S. Patents No. 5,814,344; 5,100,669 and 4,849,222; PCT Publications No.
  • PGLA polyglycolic acid/lactic acid
  • Liquid preparations for oral administration can take the form of, for example, solutions, syrups, emulsions or suspensions, or they may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Preparations for oral administration may be suitably formulated to give controlled or postponed release of the active compound.
  • Drug delivery systems known in the art are specialized technologies for the targeted delivery and/or controlled release of therapeutic agents.
  • the drug delivery systems deploy medications intact to specifically targeted parts of the body through a medium that can control the therapy's administration.
  • Such drug delivery systems may include micro- and nanotechnology.
  • the nucleic acid molecules capable of down-regulating expression of the at least one splice variant of the IG20 gene selected from siRNA, shRNA and antisense oligonucleotides complementary to the nucleotide sequence the MADD splice variant of the IG20 gene mRNA transcript may be incorporated into drug delivery systems known in the art and which may include polymeric microspheres, polymer micelles, and hydrogel-type materials, which drug delivery systems are understood in the art to be effective in enhancing drug targeting specificity, lowering systemic toxicity, improving treatment absorption rates, and providing protection for pharmaceuticals against biochemical degradation.
  • drug delivery systems may include lentivirus-mediated transduction of nucleic acids encoding nucleic acid molecules capable of down- regulating expression of the at least one splice variant of the IG20 gene are selected from siRNA, shRNA and antisense oligonucleotides complementary to the nucleotide sequence the MADD splice variant of the IG20 gene mRNA transcript.
  • the active drugs can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes may be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines, as is well known.
  • siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene may be bound in the form of a targeted liposome formulation.
  • Representative assemblies may include encapsulation within self-assembled engineered proteins which provide for efficient packaging, binding, assembly and delivery of such oligonucleotides.
  • the constituents of such engineered proteins may be selected from peptides which actively target tumor cells through attachment to selected cell surface receptors, peptides which facilitate receptor-mediated endocytosis and peptides which provide for active release of the transported oligonucleotides.
  • Such self-assembled protein transport molecules comprising the oligonucleotides of the instant invention, may be assembled in the form of nanoparticles ( ⁇ 50nm) comprising two components: the engineered polypeptide (targeting peptide, membrane penetration peptide,
  • oligonucleotide capturing peptide oligonucleotide capturing peptide
  • Drugs of the invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.
  • Active drugs may also be coupled with soluble polymers as targetable drug carriers.
  • soluble polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxy- propyl methacrylamide-phenol, polyhydroxy-ethyl-aspartamide-phenol, or
  • active drug may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid,
  • polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogets.
  • the therapeutics according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the formulations of the invention may be delivered parenterally, i.e.. by intravenous (i.v.), intracerebroventricular (i.c.v.), subcutaneous (s.c), intraperitoneal (i.p.), intramuscular (i.m.), subdermal (s.d.), intratumoral (i.t.) or intradermal (i.d.) administration, by direct injection, via, for example, bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • compositions can take such forms as excipients, suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • compositions of the present invention can also be formulated for rectal administration, e.g., as suppositories or retention enemas (e.g., containing
  • conventional suppository bases such as cocoa butter or other glycerides.
  • siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene and nucleoside analogs may be mixed with excipients which are
  • the preparations may also include minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or agents which enhance the effectiveness of the pharmaceutical composition.
  • auxiliary molecules may be delivered systemically or locally as proteins or by expression of a vector which codes for expression of the molecule.
  • the techniques described above for the delivery of siRNA, shRNA and antisense oligonucleotide capable of down- regulating the expression of the MADD splice variant of an IG20 gene and nucleoside analogs can also be employed for the delivery of auxiliary molecules.
  • the active agents of the present invention may be any active agents of the present invention.
  • a single daily dose of each of the siRNA, shRNA and antisense oligonucleotide capable of down- regulating the expression of the MADD splice variant of an IG20 gene and nucleoside analogs, with a single daily dose of both agents in one composition or in two separate compositions administered simultaneously is an embodiment.
  • the instant invention also encompasses a process for preparing pharmaceutical compositions comprising combining siRNA, shRNA and antisense oligonucleotides capable of down-regulating the expression of the MADD splice variant of an IG20 gene and nucleoside analogs with a pharmaceutically acceptable carrier and/or excipient.
  • Specific amounts of the siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene which may be used in unit dosage amounts of the invention may be readily ascertainable to those skilled in the art.
  • Specific amounts of the nucleoside analog which may be used in reduced unit dosage amounts of the invention include, for example, the nucleoside analog used at 3/4, 1/2, 1/3"*, 1/4 th , 1/5 th 1/10 th 1/20" 1 of the recommended dose.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers containing one or more of the ingredients of the formulations of the invention.
  • the present invention provides a kit for the preparation of the pharmaceutical compositions of the invention, said kit comprising a formulation of one or more siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene in a first container or multiple containers, and one or more nucleoside analog in a second container or multiple containers, and, optionally, instructions for admixing the two drugs and/or for administration of the drugs in therapeutically meaningful regimens.
  • Each container of the kit may also optionally include one or more physiologically acceptable carriers and/or excipients and/or auxiliary substances.
  • Associated with such container(s) may be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • the pharmaceutical compositions described herein are administered to a patient at therapeutically effective doses, in an embodiment, with minimal toxicity other than required for the therapeutic purpose of the combination.
  • the Section entitled “Definitions” provides definitions for the terms “chemotherapeutically effective dose” and “therapeutically effective dose”.
  • the siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene and nucleoside analogs are each used at a dosage which, when combined, provide an enhanced effect, for example, an effect not observed upon administration of each agent alone.
  • both the siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene and nucleoside analogs are administered at "suboptimal” or “subthreshold” doses, which doses, in combination, provide for a superadditive effect with surprising reduction in unwanted side effects.
  • the efficacy of the siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene of the invention may be determined using such in vitro pharmacological tests such as measurements of the levels of mRNA using quantitative Reverse
  • Q-RT-PCR Transcriptase-Polymerase Chain Reaction
  • RT-PCR Transcriptase-Polymerase Chain Reaction
  • the efficacy of the nucleoside analogs of the invention may be determined in vitro using methods known to those skilled in the art, for example, cell cytotoxicity assays, MTT assay, apoptosis assays, cell migration assays, etc.
  • the therapeutically effective dose may be estimated initially from animal models to achieve a circulating plasma concentration range which includes the ICso (i.e., the concentration of the test compound which achieves a half-maximal). Dose- response curves derived from animal systems may then be used to determine testing doses for the initial clinical studies in humans. In safety determinations for each composition, the dose and frequency of administration should meet or exceed those anticipated for use in the clinical trial.
  • ICso i.e., the concentration of the test compound which achieves a half-maximal
  • the dose of the components in the compositions of the present invention is determined to ensure that the dose administered
  • a specific dose naturally varies depending on the dosage procedure, the conditions of a patient or a subject animal such as age, body weight, sex, sensitivity, feed, dosage period, drugs used in combination, seriousness of the disease.
  • the appropriate dose and dosage times under certain conditions may be determined by the test based on the above-described indices but may be refined and ultimately decided according to the judgment of the practitioner and each patient's
  • an appropriate dose of an siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene is generally ascertainable to those skilled in the art, and an appropriate dose of nucleoside analog is generally ascertainable to those skilled in the art.
  • the dosage of siRNA to be administered may range from 1 to 10 mg per kilogram of body weight. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. In an embodiment, a single dose of each drug may be administered daily.
  • Toxicity and therapeutic efficacy of the compositions of the invention may be determined by standard pharmaceutical procedures in experimental animals, e.g., by determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population).
  • the dose ratio between therapeutic and toxic effects is the therapeutic index and it may be expressed as the ratio ED50/LD50. Formulations/combinations which exhibit large therapeutic indices are preferred.
  • the data obtained from animal studies may be used in formulating a range of doses for use in humans.
  • the therapeutically effective doses of siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene and nucleoside analogs in humans lay within a range of circulating concentrations which include the ED50 with little or no toxicity other than therapeutically necessary.
  • such therapeutically effective circulating concentration for siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene generally ascertainable to those skilled in the art.
  • the dosage of siRNA, shRNA and antisense oligonucleotide capable of down-regulating the expression of the MADD splice variant of an IG20 gene to be administered may range from 1 to 10 mg per kilogram of body weight.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. Ideally, a single dose of each drug should be used daily.
  • the drug combination of the invention is not only highly effective at relatively low doses but also possesses low toxicity other than therapeutically necessary and produces few side effects.
  • the only common side effects for the nucleoside analogs of the invention are those for which the instant combination therapy has been designed to alleviate, while the most common side effect resulting from the use of siRNA, shRNA and antisense oligonucleotide capable of down- regulating the expression of the MADD splice variant of an IG20 gene of the invention is that associated with injection of RNA such as transient heightened inflammatory response including increased interferon production.
  • results demonstrate a synergistic effect of the combination of siRNA knockdown of MADD and Gemcitabine treatment on the cell death of lung cancer cells, wherein the amount of cell death from the combination of siRNA knockdown of MADD and Gemcitabine treatment exceeds the additive effect of monotherapy with either MADD siRNA knockdown or Gemcitabine monotherapy.
  • the results demonstrate a synergistic effect of the combination of siRNA knockdown of MADD and Gemcitabine treatment on the cell death of hepatic cancer cells, wherein the amount of cell death from the combination of siRNA knockdown of MADD and Gemcitabine treatment exceeds the additive effect of monotherapy with either MADD siRNA knockdown or Gemcitabine monotherapy.
  • the results demonstrate a synergistic effect of the combination of siRNA knockdown of MADD and Gemcitabine treatment on the cell death of ovarian cancer cells, wherein the amount of cell death from the combination of siRNA knockdown of MADD and Gemcitabine treatment exceeds the additive effect of monotherapy with either MADD siRNA knockdown or Gemcitabine monotherapy.
  • results demonstrate a synergistic effect of the combination of siRNA knockdown of MADD and Gemcitabine treatment on the cell death of gastric adenocarcinoma cells, wherein the amount of cell death from the combination of siRNA knockdown of MADD and Gemcitabine treatment exceeds the additive effect of monotherapy with either MADD siRNA knockdown or Gemcitabine monotherapy.
  • results demonstrate a synergistic effect of the combination of siRNA knockdown of MADD and 5-flurouracil treatment on the cell death of gastric adenocarcinoma cells, wherein the amount of cell death from the combination of siRNA knockdown of MADD and 5-flurouracil treatment exceeds the additive effect of monotherapy with either MADD siRNA knockdown or 5-flurouracil monotherapy.
  • Cancer cell lines representative of various cancers which may be susceptible to nucleoside analog treatment include 8505C (thyroid carcinoma), HTH7 (thyroid carcinoma), C643 (human thyroid carcinoma), AsPC-1 (pancreatic cancer), SU.86.86 (pancreatic cancer), CFPAC-1 (pancreatic cancer), MCF7
  • SK-BR-3 breast cancer
  • OVCAR3 ovarian cancer
  • SKOV3 ovarian cancer
  • NCI-H522 non-small cell lung cancer
  • NCI-H2122 non-small cell lung cancer
  • NCI-H2227 small cell lung cancer
  • HepG2 liver hepatocellular carcinoma
  • PLC/PRF/5 liver hepatoma
  • AGS gastric
  • adenocarcinoma adenocarcinoma
  • K562 chronic myelogenous leukemia
  • Cell lines may be obtained from the National Cancer Institute, Bethesda, MD or American Type Culture Collection, or similar organizations in other countries. These cell lines may be selected because they all express higher levels of the MADD splice variant, are derived from different types of cancers, all which are in need of new modalities of treatment, exhibit unique growth properties and have differential susceptibility to different modalities of treatment with chemotherapeutic agents due to underlying different mutations. In spite of their heterogeneity, these cell lines may be rendered susceptible to therapeutic treatment upon MAOD knockdown, and thus show the potential beneficial effects of MADD knockdown in a range of different cancers.
  • 8505C cells are cultured in RPMI-1640 culture medium and
  • HTH7 cells are cultured in RPMI-1640 culture medium and
  • C643 cells are cultured in RPMI-1640 culture medium and
  • AsPC-1 cells are cultured in RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate, for use in incubators using 5% CO2 in air. Additional sodium bicarbonate may be required for use in incubators containing higher percentages of CO2.
  • Base medium is supplemented with 10% fetal bovine serum and antibiotics and anti-mycotic agents.
  • SU.86.86 cells are cultured in RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate, for use in incubators using 5% CO2 in air. Additional sodium bicarbonate may be required for use in incubators containing higher percentages of CO2.
  • Base medium is supplemented with 10% fetal bovine serum and antibiotics and anti-mycotic agents.
  • CFPAC-1 cells are cultured in Iscove's Modified Dulbecco's Medium (I M DM) containing 4 mM L-glutamine, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate. This base medium is supplemented with 10% fetal bovine serum and antibiotics and anti-mycotic agents.
  • I M DM Iscove's Modified Dulbecco's Medium
  • MCF7 cells are cultured in Eagle's minimum essential medium modified to contain Earle's Balanced Salt Solution, non-essential amino acids, 2 mM L- glutamine, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate. This base medium is supplemented with 0.01 mg/ml human recombinant insulin; fetal bovine serum to a final concentration of 10%.
  • SK-BR-3 cells are cultured in McCoy's 5A Medium modified to contain 1.5 mM L-glutamine and 2200 mg/L sodium bicarbonate. This base medium is supplemented with 10% fetal bovine serum and antibiotics and anti-mycotic agents.
  • OVCAR3 cells are cultured in RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate, for use in incubators using 5% CO2 in air.
  • This base medium is supplemented with 0.01 mg/ml bovine insulin, antibiotic and anti-mycotic agents and fetal bovine serum to a final concentration of 20%.
  • SKOV3 cells are cultured in McCoy's 5A Medium is modified to contain 1.5 mM L-glutamine and 2200 mg/L sodium bicarbonate. This base medium is supplemented with 10% fetal bovine serum and antibiotics and anti-mycotic agents.
  • NCI-H522 cells are cultured in RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate, for use in incubators using 5% CO2 in air.
  • This base medium is supplemented with 10% fetal bovine serum and antibiotics and anti- mycotic agents.
  • NCI-H2122 cells are cultured in RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate, for use in incubators using 5% CO2 in air.
  • This base medium is supplemented with 10% fetal bovine serum and antibiotics and anti- mycotic agents.
  • NCI-H2227 cells are cultured in DMEM:F12 Medium containing 0.005 mg/ml Insulin, 0.01 mg/ml Transferrin, 30nM Sodium selenite, 10 nM Hydrocortisone, 10 nM beta-estradiol, extra 2mM L-glutamine (for final cone, of 4.5 mM), 5% fetal bovine serum.
  • HepG2 cells are cultured in Eagle's Minimum Essential Medium
  • EMEM fetal bovine serum
  • PLC/PRF/5 cells are cultured in Eagle's Minimum Essential Medium (EMEM) and supplemented with 10% fetal bovine serum and 1%
  • K562 cells are cultured in Iscove's Modified Dulbecco's Medium (IMDM) and supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.
  • IMDM Iscove's Modified Dulbecco's Medium
  • HCT116 cells are cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.
  • AGS cells are cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.
  • N87 cells are cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.
  • Nucleoside analog chemotherapeutics may be obtained commercially as FDA approved drugs from pharmacies with a doctor's prescription.
  • concentrations of each drug are as follows:
  • Cytotoxicity of the respective cell line models may be evaluated based on the compared cytotoxicity as measured utilizing the MTT assay.
  • MTT (3-[4,5-Dimethylthiazol-2- yl]-2,5-diphenyltetrazolium bromide; Thiazolyl blue) may be purchased from Sigma Aldrich (Cat number: M5655) and dissolved in sterile DPBS at a concentration of 5mg/ml.
  • Dimethylformamide is added and the plates are incubated overnight at 37° C in the humidified CO2 incubator. After overnight incubation, plates are kept on shaker for 10 minutes for compete dissolution of crystals and absorbance is recorded at 595 nm using Bio-Rad iMark microplate reader.
  • Percent cell survival is calculated using MS excel. GraphPad prism software is used to calculate ECso. The MTT assay indicates the overall cell death.
  • nucleoside analog chemotherapeutic exhibits a dose response profile measurable based on the death of the cancer cells.
  • Example 2 Map kinase Activating Death Domain containing protein (MADD) siRNA Transfection
  • siRNA, shRNA and/or antisense oligonucleotides comprising nucleic acid molecules which target exon 13L of the MADD splice variant may be designed.
  • siRNAs comprising a central 19 bp duplex with 2 -base 3'-overhangs are
  • siRNAs double stranded duplex
  • MADD siRNA 5 * - G AUUG UCAUAG AU UCG CCGTT - 3' (SEQ ID NO:4)
  • Scramble siRNA 5' - UUGCUAAGCG UCGG UCAAUTT - 3' (SEQ ID NO:5)
  • Lipofectamine RNAimax (transfection mediating reagent) is a cationic unilamellar liposomal structure with a positive surface charge in water.
  • the lipid cationic charges interacts with negative phosphate group of nucleic acids in siRNA and forms a liposome/siRNA transfection complex with the cell membrane. This complex can easily fuse with a cell membrane and siRNA is delivered inside the cell via endocytosis.
  • siRNA and transfection reagents needs to be determined for each cell line.
  • the manufacturer's recommended volume of transfection reagent per mm 3 area of tissue culture plate is used, generally, and the siRNA concentration may vary. For most cells, 10 nM of siRNA is sufficient to cause MADD knock-down within 48 hours of transfection.
  • RNAiMax Thermo Fisher Scientific reagent is mixed in 4.7 ⁇ OPTI-MEM in tube B. Contents from tube A and B are mixed and incubated for 15 minutes at room temperature. Reaction mix is added to the plates and incubated for 24h, 48h and 72h at 37° C. Media is changed after 48 hours.
  • RNA is extracted using Trizol reagent (Ambion; Cat number:
  • Chloroform (Fisher; Cat number: C606-1 ) is added to the cell suspension. Samples are incubated at room temperature for 3 minutes. Tubes are centrifuged at 10,000 rpm for 15 minutes at 4°C. The top layer is transferred to a fresh tube and 600 ⁇ of isopropanol (Fisher; Cat number: A451-1) is added. The tube is incubated at room temperature for 10 minutes. The tube is subsequently centrifuged at 10,000 rpm for 10 minutes at 4°C. Supernatant is discarded and the pellet is washed with 1 ml of 75% ethanol (Decon; Cat number: DSP-AZ-1 ). The tube is again centrifuged at 7500 rpm for 5 minutes at 4° C.
  • Decon Cat number: DSP-AZ-1
  • RNA is quantified by using Thermo Fisher Scientific NanoDrop One. A260/280 and A260/230 are used to validate the purity of sample.
  • MADD and ⁇ -actin (internal loading control) primers are used.
  • the sequences of primers used are: MADD 13L Forward: 5'- AGC CCC AAT ATG GCT TTC CC-3' (SEQ ID NO:6)
  • ⁇ -actin forward 5'- ATCTG GCACCACACCTTCT ACAATG AG CTG CG-3' (SEQ ID NO:8)
  • RT_PCR kit (Cat number: 210212) and BioRaD T100 thermocycler are used. The description of RT-PCR is given below. Table 3.
  • Amplification protocol for RT-PCR is:
  • Example 3 Effect off nucleoside analog chemotherapeutics on cell death in cancer cells with and without MADD knockdown
  • cytotoxic effects of nucleoside analog chemotherapeutics on cancer cells in the presence or absence of MADD down-regulation may be
  • Cells are cultured and maintained at 37°C in a humidified atmosphere containing 5% CO2 as described above. On Day 0, Cells are seeded in 100 mm 3 tissue culture dishes (Corning; Cat Number: 353003) to attain 60-70 % confluence. In parallel, cells are also seeded in one 96-well plate (Corning; Cat number: 353072) for analysis of survival before reseeding.
  • siRNAs are reconstituted to 100 ⁇ concentration in 1x siRNA dissolution buffer (GE Healthcare; Cat number: B-002000-UB-700) and stored at -20° C.
  • reaction mix per plate is prepared by adding 30 ⁇ lipofectamine RNAimax (Invitrogen; Cat number 13778-150), 1ml Opti-MEM (Gibco; Cat number: 31985062), and an aliquot of the stock siRNA to obtain a final concentration of 10 nM siRNA per plate or wells of a plate containing 70% confluent cells. Reaction mix is incubated at room temperature for 10 minutes. Transfection mixture is added dropwise on to the plate containing 70% confluent cells in RPMI media with 10% FBS but no antibiotics. The transfection mix is added in parallel to cells plated in a 96-well plate.
  • Equal number of cells [Control (untreated), Scramble and MADD siRNA transfected] are seeded in 96-well plates. On the same day (after 48 hours of transfection), an MTT assay is carried out to determine the relative cell survival before reseeding.
  • An MTT assay is performed to determine relative survival after drug treatment as described above.
  • MTT Staining for Metabolic Activity MTT (3-[4,5-Dimethylthiazol-2- yl]-2,5-diphenyltetrazolium bromide; Thiazolyl blue) is purchased from Sigma Aldrich (Cat number: M5655). MTT is dissolved in sterile DPBS at the concentration of 5mg/ml and filtered through 0.45 ⁇ syringe filter (Corning; Cat number: 431220). 10 ⁇ of MTT solution is added per well of 96-well plate. Plates are incubated for 2 hours at 37° C in the humidified CO2 incubator. After 2 hours, media is aspirated using vacuum inside the biosafety hood.
  • Each data set takes into account any spontaneous cell death which is observed upon transfection with Scramble siRNA; the amount of spontaneous cell death from Scramble siRNA transfection (baseline) is subtracted from the amount of cell death observed in each siRNA transfection reaction to yield a Net Percent (Net %) cell death after accounting for Scrambled siRNA-induced cell death.
  • 25 nM Gemcitabine was added as monotherapy to H2227 lung cancer cells. Moreover, cells were transfected with MADD siRNA as monotherapy. In combination, 25 nM Gemcitabine was added to cells transfected with MADD siRNA. The amount of cell death in the cell cultures of the treatment paradigm represented by untreated control cells, control cells transfected with
  • 0.03125 ⁇ Gemcitabine was added as monotherapy to PLC/PRF/5 hepatic cancer cells. Moreover, cells were transfected with MADD siRNA as monotherapy. In combination, 0.03125 ⁇ Gemcitabine was added to cells transfected with MADD siRNA. The amount of cell death in the cell cultures of the treatment paradigm represented by untreated control cells, control cells transfected with Scrambled siRNA, cells transfected with MADD siRNA, cells treated with 0.03125 ⁇ Gemcitabine, and cells transfected with MADD siRNA and treated with 0.03125 ⁇ Gemcitabine, was evaluated at 24 hours post Gemcitabine treatment. Table 8.
  • 0.0125 ⁇ Gemcitabine was added as monotherapy to O VCAR3 ovarian cancer cells. Moreover, cells were transfected with MADD siRNA as monotherapy. In combination, 0.0125 ⁇ Gemcitabine was added to cells transfected with MADD siRNA. The amount of cell death in the cell cultures of the treatment paradigm represented by untreated control cells, control cells transfected with Scrambled siRNA, cells transfected with MADD siRNA, cells treated with 0.0125 ⁇ Gemcitabine, and cells transfected with MADD siRNA and treated with 0.0125 ⁇ Gemcitabine, was evaluated at 72 hours post Gemcitabine
  • 1 ⁇ Gemcitabine was added as monotherapy to AGS gastric adenocarcinoma cells. Moreover, cells were transfected with MADD siRNA as monotherapy. In combination, 1 ⁇ Gemcitabine was added to cells transfected with MADD siRNA. The amount of cell death in the cell cultures of the treatment paradigm represented by untreated control cells, control cells transfected with Scrambled siRNA, cells transfected with MADD siRNA, cells treated with 1 ⁇ Gemcitabine, and cells transfected with MADD siRNA and treated with 1 ⁇
  • 25 ⁇ 5-flurouracil was added as monotherapy to AGS gastric adenocarcinoma cells. Moreover, cells were transfected with MADD siRNA as monotherapy. In combination, 25 ⁇ 5-flurouracil was added to cells transfected with MADD siRNA. The amount of cell death in the cell cultures of the treatment paradigm represented by untreated control cells, control cells transfected with Scrambled siRNA, cells transfected with MADD siRNA, cells treated with 25 ⁇ 5-flurouracil, and cells transfected with MADD siRNA and treated with 25 ⁇ 5- flurouracil, was evaluated at 72 hours post 5-flurouracil treatment. Table 12.
  • the instant assay demonstrates that combination of nucleoside analog chemotherapeutic treatment and MADD knock-down results in surprisingly synergistic effects on inducing cancer cell death.
  • the results demonstrate that the combination nucleoside analog chemotherapy and MADD knock down results in more than an additive effect on cell death.
  • dosages of the nucleoside analog chemotherapeutic may be lowered to a level which was previously considered to be non-therapeutic, thereby providing a cancer therapy which exhibits an unexpected margin of safety and reduction of unwanted side effects.
  • the present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

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Abstract

L'invention concerne des compositions comprenant une association d'agents antinéoplasiques, l'association comprenant des agents chimiothérapeutiques à analogues nucléosidiques et une ou plusieurs molécules d'acides nucléiques pouvant réguler à la baisse l'expression d'au moins un variant d'épissage du gène IG20, et tous les variants d'épissage du gène IG20 n'étant pas régulés à la baisse. Le variant d'épissage du gène IG20 est au moins un variant d'épissage MADD et les molécules d'acides nucléiques sont des ARN interférents courts, des ARN courts en épingles à cheveux et/ou des oligonucléotides antisens qui comprennent une séquence d'acide nucléique complémentaire d'une séquence d'acide nucléique de l'exon 13L du variant d'épissage MADD ou d'un produit de transcription d'ARN messager de l'exon 13L du variant d'épissage MADD. L'invention concerne également des méthodes de traitement de cancers par l'administration desdites compositions.
PCT/US2018/026177 2017-04-24 2018-04-05 Association synergique d'oligonucléotides d'acides nucléiques et d'agents chimiothérapeutiques à analogues nucléosidiques WO2018200145A1 (fr)

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US20090075929A1 (en) * 2006-01-19 2009-03-19 The Board Of Trustees Of The University Of Illinois Ig20 splice variants therapeutics for cancer

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US20090075929A1 (en) * 2006-01-19 2009-03-19 The Board Of Trustees Of The University Of Illinois Ig20 splice variants therapeutics for cancer

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