WO2000015242A1 - REGULATION DE L'EXPRESSION D'ONCOGENE HER2/neu A L'AIDE DE POLYAMIDES SYNTHETIQUES - Google Patents

REGULATION DE L'EXPRESSION D'ONCOGENE HER2/neu A L'AIDE DE POLYAMIDES SYNTHETIQUES Download PDF

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WO2000015242A1
WO2000015242A1 PCT/US1999/020971 US9920971W WO0015242A1 WO 2000015242 A1 WO2000015242 A1 WO 2000015242A1 US 9920971 W US9920971 W US 9920971W WO 0015242 A1 WO0015242 A1 WO 0015242A1
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polyamide
correspond
composition
her2
polyamides
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PCT/US1999/020971
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Peter B. Dervan
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California Institute Of Technology
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Priority to CA002343289A priority Critical patent/CA2343289A1/fr
Priority to AU64965/99A priority patent/AU6496599A/en
Priority to EP19990952908 priority patent/EP1112080A4/fr
Priority to JP2000569826A priority patent/JP2002524525A/ja
Publication of WO2000015242A1 publication Critical patent/WO2000015242A1/fr

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    • 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
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention is directed generally to methods and compositions for the modulation, or regulation, of gene expression by the use of polyamides that bind DNA.
  • the methods and compositions result in inhibition, or down-regulation, of gene expression or overexpression by interactions between the polyamides and the minor groove of double-stranded DNA (dsDNA).
  • the polyamides of these methods and compositions bind predetermined target nucleic acid sequences located within the promoter region of genes to be down-regulated or inhibited. Inhibition or down- regulation of target oncogenes that are expressed, or overexpressed, at undesirable levels is one application of the invention.
  • the invention is directed to reducing the expression or overexpression of target endogenous cellular oncogenes.
  • the tyrosine kinase membrane growth factor receptor HER2/neu also known as pl85HER2 ; is encoded by a cellular oncogene of the same name that is overexpressed and amplified in 20 to 30% of human breast cancers, among others, including other human gynecologic adenocarcinomas, such as those of the ovary, endometrium, fallopian tube, and cervix.. See Baert, J.-L. et al., Int. J.
  • Her-2/neu protein likely plays a role in cell motility and hence in metastasis.
  • inhibition of Her-2/neu gene expression by direct interference at the DNA level may be a potent therapeutic approach for metastatic disease.
  • transcription factors such as ESX, AP-2, and the TATA binding protein ("TBP") - play an important role in the regulation of the expression of the gene for the HER2/neu growth factor receptor. See Baert, J.-L. et al., Benz, C, et al; supra; Chang, C- H., et al., supra; Bosher, J. M., et al., Proc. Natl. Acad. Sci. USA 92, 744-747 (1995). These transcription factors activate the expression of pl85"ER2 U p on binding to sites within the HER2/neu promoter.
  • the nucleotide sequence of the HER2/neu promoter and a schematic representation are shown in Figure 1.
  • TBP is ubiquitous transcription factor that is involved in the activation of most protein-encoding genes.
  • TBP is a DNA-binding protein that interacts to the minor groove of DNA. It should be noted that, apart from ESX, AP-2, and TBP, there are other potential transcription factor binding sites within the HER2/neu promoter.
  • the agent must not possess any general cell toxicity; second, the agent must be cell-permeable and, in the case of the DNA-binding agents, the compounds must transit to the nucleus and bind their target sequence with high affinity and specificity in the context of cellular chromatin; and, third, binding of the agent to its DNA target sequence must interfere with gene transcription.
  • the potential approaches listed above has its own peculiar limitations. For example, while triple helix-forming oligonucleotides have the potential for sequence selectivity and can effectively inhibit transcription in vitro, these molecules suffer from poor cell permeability and permeabilized cells need to be used for effective gene inhibition.
  • calicheamicin oligosaccharides are sufficiently hydrophobic to pass through cell membranes, but these molecules possess severely limited sequence specificity (4 bp) and bind DNA with very low affinities (100 ⁇ M or higher required for inhibition of protein-DNA interactions).
  • sequence specificity 4 bp
  • affinities 100 ⁇ M or higher required for inhibition of protein-DNA interactions
  • Another approach utilizes cell-permeable small molecules that target particular DNA sequences. These molecules would be useful for the regulation of gene expression.
  • the design of small synthetic DNA-binding ligands that recognize specific sequences in the DNA double helix has been a long standing goal of chemistry. Oligodeoxynucleotides that recognize the major groove of double-helical DNA via triple- helix formation bind to a broad range of sequences with high affinity and specificity. Although oligonucleotides and their analogs have been shown to interfere with gene expression, the triple helix approach is limited to purine tracks and suffers from poor cellular uptake. Other small molecules have also been of interest as DNA-binding ligands. Wade, et al.
  • polyamides have been synthesized to recognize much longer sequences. For example, a twelve-ring double hairpin polyamide has been designed to target a 12 bp site and binding is again observed with nanomolar affinity. Such a sequence would be predicted to occur at random only once every 16 million base pairs, or only 125 times in the human genome. Such molecules thus have the potential to act as specific inhibitors of gene transcription in vivo and as human therapeutic agents if the conditions outlined above can be met.
  • the present invention relates to and includes methods and compositions for the modulation, or regulation, of gene expression or overexpression by reducing the transcription of genes.
  • the transcription of specific individual target genes is reduced.
  • Such reductions result from the application of polyamides that bind or interact with the minor groove of double-stranded DNA (dsDNA) within the promoter regions of target genes.
  • the binding or interaction is with a predetermined target nucleic acid sequence within the promoter regions to inhibit or down-regulate transcription.
  • the present invention reduces gene expression and overexpression by use of sequence-specific DNA-binding small molecules that are cell-permeable and capable of inhibiting gene transcription. Appropriate application of such molecules may inhibit overexpression of endogenous oncogenes to provide a fundamentally new therapeutic strategy for the treatment of various diseases, including cancer.
  • the small molecules of the invention are polyamides that bind to or interact with nucleic acid sequences within the promoter region of target genes. Preferably, these sequences are recognized, or proximal to those that are recognized, by one or more transcription factors.
  • the polyamides bind to the minor groove of double-stranded DNA in a promoter region that controls the transcription and expression of a gene.
  • the transcription of the gene is inhibited by modulating the binding of a protein transcription factor to dsDNA.
  • the transcription factors are ESX, ETS, and TBP.
  • both classes of transcription factors can be inhibited by polyamides that contact or bind the minor groove of dsDNA.
  • DNA complexation of proteins contacting the minor groove may be inhibited by direct steric hinderance, repulsion, or exclusion or, alternatively, byallosteric efforts.
  • the binding of major groove binding proteins may be suppressed by a polyamide-induced change of the DNA conformation.
  • inhibition can also be achieved other ways, for example, by conjugating a DNA cleavage agent to a polyamide targeted to a desired site, or by chemically modifying DNA.
  • the expression or overexpression of oncogenes is targeted.
  • the oncogenes are endogenous cellular oncogenes involved in cancer, particularly human breast cancer.
  • One oncogene target of the invention is the HER-2/neu gene, which may be down-regulated or inhibited by the use of polyamides that bind to target sequences within the HER-2/neu promoter region.
  • these sequences are, or are proximal to, transcription factor binding sites within the HER2/neu promoter. Interactions or binding between the polyamide and the target sequence can inhibit the transcription of the HER2/neu gene.
  • the degree of inhibition of HER2/neu expression may be extensive and includes the inhibition of HER2/neu overexpression.
  • the invention further encompasses application of polyamides for the treatment of various tumors or cancers, including breast cancer.
  • Suitable polyamides have a binding affinity at the dsDNA target sequence of at least 10 9 M "1 and a selectivity of at least about two. Selectivity is defined as the ratio of the binding affinity for the identified dsDNA target sequence to the binding affinity for a single base-pair mismatch dsDNA sequence. In preferred embodiments, selectivity against at least 90% of single base mismatch sequences is greater than about 10.
  • compositions that comprise a pharmaceutically acceptable excipient and a transcription-inhibiting amount of at least one polyamide of the invention.
  • Each polyamide contains at least four complementary pairs of aromatic carboxamide residues, which pairs are selected to correspond to an identified nucleotide sequence of a dsDNA target.
  • the polyamides additionaly comprise at least two aliphatic amino acid residues chosen from the group consisting of glycine, ⁇ -alanine, ⁇ -aminobutyric acid, R-2,4-diaminobutyric acid, and 5 -amino valeric acid, and at least one terminal alkylamino residue, the polyamide having a binding affinity at the target dsDNA sequence of at least 10 9 M "! and a selectivity of at least about two, selectivity being defined as the ratio of the binding affinity for the identified target dsDNA sequence to the binding affinity for a single base- pair mismatch dsDNA sequence.
  • the invention further provides methods suitable for treating a subject having a condition associated with abnormal expression of a cellular oncogene.
  • the subject is preferably a human patient and, more particularly, one afflicted with breast cancer or other diseases or conditions associated with aberrant Her-2/neu oncogene expression.
  • Figure 1 depicts the HER2/neu promoter, showing the nucleotide sequence in A, including binding sites of Ets, AP-2, and TBP ("TATA") transcription factors and the "CCAAT box", and in B, a schematic diagram, not to scale.
  • Figure 2 A is a graphical representation of the results of a DNase I footprint titration of polyamide HER2-1 (left) and the mismatch polyamide ImPy- ⁇ -Pylm- ⁇ - PyPyPyPyPy- ⁇ -Dp (right) and 2B, the schematic structures and association constants of the polyamides, where the polyamides are represented by closed circles for imidazole rings, open circles for pyrrole rings, curved lines for ⁇ -aminobutyric acid, diamonds for ⁇ - alanine, and a half circle with a positive charge for dimethylaminopropylamide.
  • Figure 3 compares the sequence of the HER2/neu promoter and polyamide structures and binding sites; the binding site for the TATA binding protein (TBP) is indicated along with the structures of the polyamides HER2-A, HER2-1, 70, and the mismatch polyamide 86.
  • TATA binding protein TATA binding protein
  • Figure 4 is a graphical representation of the results of experiments showing the effects of polyamides Her2-1 (A) and 70 on TBP binding.
  • Figure 5 is a graphical representation of the results of experiments showing the effects of the polyamide HER2-1 on HER2/neu transcription in vitro in a cell free system.
  • Figure 6 is a graphical representation of the results of experiments showing the effects of the polyamides HER2-1 and 70 on HER2/neu mRNA production in the human breast cancer cell line SK-BR-2.
  • the present invention is directed to methods and compositions for modulating or regulating gene expression or overexpression by reducing gene transcription.
  • the methods and compositions are preferably directed toward the inhibition of oncogene transcription, especially of oncogenes involved in cancer, particularly human cancer and especially breast cancer.
  • the reductions in gene transcription result from binding or other interactions between polyamides and the minor groove of dsDNA within the promoter regions of target genes.
  • the polyamides bind or interact with specific target nucleic acid sequences within the promoter regions to inhibit or down-regulate transcription.
  • the sequences are recognized, or proximal to those that are recognized, by one or more transcription factors.
  • the polyamides are preferably cell-permeable and capable of inhibiting gene transcription in vivo, in vitro, or in cell free systems. Appropriate application of such polyamide molecules may be used to inhibit expression or overexpression of endogenous oncogenes as a treatment of various diseases, including cancer.
  • the polyamides bind to the minor groove of double stranded DNA in a promoter region that controls the transcription and expression of a target gene.
  • Preferred target genes are endogenous oncogenes involved in cancer formation or progression.
  • the transcription of the gene is inhibited by modulating the binding of a protein, such a transcription factor, to the same promoter region with which the polyamide binds or interacts.
  • the transcription factors are one or more of the following: ESX; ETS; and TBP.
  • the present invention includes the use of polyamides that inhibit or modulate the activity of both TBP and Ets transcription factors.
  • the invention may affect transcription factor activity by use of one or more polyamides that contact or bind the minor groove of dsDNA. Such contact or binding may inhibit formation of DNA-transcription factor complexes in the minor groove by direct steric repulsion, allosteric effects, or other mechanisms (e.g., cleavage or chemical modification of the dsDNA). This is possibly in contrast to major groove DNA binding proteins, such as TBP, which may be inhibited by a polyamide- induced change in DNA conformation.
  • the expression or overexpression of oncogenes is targeted.
  • the oncogenes are those implicated in human breast cancer, and their expression or overexpression is inhibited by polyamides that contact or bind the minor groove in the region of the oncogene promoter.
  • the contacted or bound portions of the promoter region are, or are proximal to, transcription factor binding sites.
  • the degree of inhibition is preferably large and more preferably enough to inhibit even overexpression of the oncogene, in when the copy number of the gene increases.
  • One oncogene target of the invention is the HER-2/neu gene, which may be down-regulated or inhibited by the use of polyamides that bind to target sequences within the HER-2/neu promoter region. Preferably, these sequences are, or are proximal to, transcription factor binding sites within the HER2/neu promoter. These transcription factors include TBP, ESX and AP-2. Interactions or binding between the polyamide and the target sequence result in inhibition of the HER2/neu gene transcription.
  • a polyamide was designed to bind immediately downstream of the TATA element found in the human Her-2/neu breast cancer oncogene promoter.
  • This polyamide, Her2-1 of composition ImPy- ⁇ -Pylm- ⁇ -PyPy- ⁇ -PyPy- ⁇ -Dp, binds the sequence 5'-AGAATGA-3' (where the 5' A of this sequence is the 3' A of the TATA element) with an apparent dissociation constant of 200 pM.
  • Her2-1 is an effective inhibitor of TBP binding and transcription.
  • the present invention includes compositions comprising a pharmaceutically acceptable excipient and a transcription-inhibiting amount of at least one polyamide for the inhibition of gene expression or overexpression. These compositions may also be used for the treatment of various tumors or cancers, including breast cancer.
  • the invention further provides methods of administering such compositions to result in inhibition of gene expression or overexpression.
  • the methods and compositions are preferably suited for treating a subject having a condition associated with abnormal expression of a cellular oncogene.
  • the subject is preferably a human patient particularly one afflicted with cancer, especially breast cancer.
  • Polyamides of the invention are preferably a human patient particularly one afflicted with cancer, especially breast cancer.
  • the polyamides used in the present invention comprise N-methylimidazole and N-methylpyrrole carboxamides. These polyamides generally have a crescent-shaped structure that permits interaction and complexation the minor groove of double-stranded DNA. NMR studies have confirmed that these compounds can bind to DNA in a 2:1 ratio by a motif in which two polyamide ligands are arranged in an antiparallel way, side- by-side to each other (Pelton, J., et al., Proc. Natl. Acad. Sci. USA 86, 5723-5727 (1986); Mrksich, M., et al., Proc. Natl. Acad. Sci. USA, 89, 7586-7590 (1992); Wade, W.
  • polyamides are called "hairpin polyamides", as they adopt a hairpin-like conformation in the DNA complex.
  • the sequence of the imidazole and the pyrrole carboxamides in the polyamide determines the DNA sequence specificity of the ligand, according to the scheme of carboxamide pairs that recognize nucleotide pairs described above.
  • polyamides comprising N-methylimidazole and N-methylpyrrole carboxamides can inhibit gene expression in eukaryotic cells (Gottesfeld, J.M., et al. Nature 387, 203-205 (1997)).
  • polyamides containing a new aromatic amino acid 3- hydroxy-N-methylpyrrole (Hp), paired opposite Py, have the ability to discriminate A»T nucleotide pairs from T «A nucleotide pairs in DNA sequences.
  • Hp 3- hydroxy-N-methylpyrrole
  • the replacement of a single hydrogen atom on the pyrrole with a hydroxyl group in a Hp/Py pairing affects the affinity and specificity of a polyamide by an order of magnitude.
  • polyamides can selectively distinguish all four Watson-Crick base pairs in the minor groove of double stranded DNA.
  • the invention encompasses the use of improved polyamides for binding to the minor groove of DNA in methods and compositions for reducing gene expression or overexpression.
  • the preparation and use of polyamides for binding in the minor groove of DNA are described in the art.
  • Included in the invention is an improvement of the existing technology which utilizes 3-hydroxy-N-methylpyrrole to provide carboxamide binding pairs for DNA binding polyamides.
  • the improvement relates to the inclusion of a binding pair of Hp/Py carboxamides in the polyamide to bind to a T «A base pair in the minor groove of DNA or Py/Hp carboxamide binding pair in the polyamide to bind to an A*T base pair in the minor groove of DNA.
  • the polyamides used in the invention have at least four carboxamide binding pairs that will distinguish A*T, T «A, C «G, and G*C base pairs in the minor groove.
  • the polyamides may also have ⁇ -aminobutyric acid or another turn unit to form a hairpin-loop with a member of each carboxamide pairing on each side of it.
  • the invention also includes polyamides containing a ⁇ -alanine substituted for a Py residue that would ordinarily be used in a carboxamide binding pair to match a particular nucleotide pair.
  • the ⁇ -alanine is referred to in formulas of this invention as ⁇ .
  • the ⁇ -alanine becomes a member of a carboxamide binding pair, and serves to optimize hydrogen bonding of neighboring amino acid moieties to nucleotide base pairs.
  • the invention further includes the substitution of a ⁇ * ⁇ binding pair for a non-Hp containing binding pair.
  • binding pairs in addition to the Hp/Py and Py/Hp are Py/Py, Im/Py, Py/Im, Im/ ⁇ , ⁇ Im, Py/ ⁇ , ⁇ /Py, and ⁇ / ⁇ .
  • the polyamides of the invention are suitable for inhibiting the transcription of a gene, preferably an oncogene.
  • the polyamides consist of at least four complementary pairs of aromatic carboxamide residues, which pairs are selected to correspond to the nucleotide sequence of a dsDNA target.
  • These polyamides contain at least two aliphatic amino acid residues chosen from the group consisting of glycine, ⁇ - alanine, ⁇ -aminobutyric acid, and 5-aminovaleric acid, and at least one terminal alkylamino residue.
  • the complementary pairs of aromatic carboxamide residues selected to correspond to the nucleotide sequence of an identified dsDNA target are chosen from the group consisting of Im/Py to correspond to the nucleotide pair G/C, Py/Im to correspond to the nucleotide pair C/G, Py/Py to correspond to the nucleotide pair A/T, Py/Py to correspond to the nucleotide pair T/A, Hp/Py to correspond to the nucleotide pair T/A, and Py/Hp to correspond to the nucleotide pair A/T, where Im is N-methyl imidazole, Py is N-methyl pyrrole and Hp is 3-hydroxy N-methyl pyrrole.
  • Application of the above principles permits the design of specific polyamides that bind or interact with specific target nucleic acid sequences for use in reducing gene expression or overexpression.
  • Preferred polyamides contain at least one ⁇ -alanine as an aliphatic amino acid residue.
  • the terminal alkylamino residue is a N,N- dimethylaminopropyl residue.
  • corresponding pairs can be formed between aliphatic amino acids and aromatic carboxamides, such as Im/ ⁇ , ⁇ /Im, Py/ ⁇ and ⁇ /Py.
  • a hairpin molecule is formed by inclusion an aliphatic amino acid residue such as ⁇ -aminobutyric acid.
  • at least one Py of a carboxamide pair is replaced by a ⁇ - alanine.
  • Suitable polyamides have a binding affinity at the dsDNA target sequence of at least 10 9 M " ' and a selectivity of at least about two. Selectivity is defined as the ratio of the binding affinity for the identified dsDNA target sequence to the binding affinity for a single base-pair mismatch dsDNA sequence. In preferred embodiments, selectivity against at least 90% of single base mismatch sequences is greater than about 10.
  • Each polyamide used in the compositions of the invention preferably contains at least four complementary pairs of aromatic carboxamide residues, which pairs are selected to correspond to an identified nucleotide sequence of a dsDNA target.
  • the polyamides also preferably contain at least two aliphatic amino acid residues chosen from the group consisting of glycine, ⁇ -alanine, ⁇ -aminobutyric acid, R-2,4-diaminobutyric acid, and 5-aminovaleric acid, and at least one terminal alkylamino residue.
  • the polyamides also preferably have a binding affinity at the target dsDNA sequence of at least 10 9 M "1 and a selectivity of at least about two, selectivity being defined as the ratio of the binding affinity for the identified target dsDNA sequence to the binding affinity for a single base-pair mismatch dsDNA sequence.
  • Polyamide Her2-1 was designed to bind to the DNA sequence 5'-AGAATGA-3', which, as discussed above, is immediately adjacent to the TATA box of the HER2/neu promoter. DNAse 1 footprint analysis confirms that this polyamide binds to the desired sequence with a dissociation constant (Kd) of about 0.2 nM. Polyamide 70 also binds adjacent to, and partially overlaps, the HER2/neu TATA box. These polyamides targeted to the DNA sequences flanking or overlapping the Her-2/neu TATA element were synthesized by solid phase methods. Polyamide Her2-A, of sequence composition Imlm- ⁇ -Pylm- ⁇ -PyPy- ⁇ -PyPy- ⁇ -Dp
  • a mismatch polyamide of sequence composition Imlm- ⁇ -Imlm- ⁇ -PyPy- ⁇ -PyPy- ⁇ -Dp was also used in these studies.
  • Polyamide 70 of sequence composition ImPyPyPy- ⁇ -PyPyPyPy- ⁇ -Dp, binds the sequence 5'-AGTATA-3' overlapping the TATA box, while polyamide 86, of sequence composition ImPylmPy- ⁇ - PyPyPyPy- ⁇ -Dp, is a mismatch polyamide, with a single atom substitution from polyamide 70.
  • Figure 1 shows the sequence of the Her-2/neu promoter region and the binding sites of several transcription factors.
  • the hairpin polyamide ImPy- ⁇ -Pylm- ⁇ -PyPy- ⁇ - PyPy- ⁇ -Dp was synthesized to bind immediately downstream of the TBP binding site ( Figure 2B, left).
  • polyamides are represented schematically between the two DNA strands at their respective binding sites. Shaded and unshaded circles represent imidazole (Im) and pyrrole (Py) rings, respectively; curved lines represent ⁇ -aminobutyric acid ( ⁇ ); diamonds represent ⁇ -alanine ( ⁇ ); and Dp represents dimethylaminopropylamide.
  • the apparent binding affinities for each of these polyamides was determined by quantitative DNase I footprint titrations.
  • Polyamide Her2-A binds its match site with a K, of ⁇ 10 8 M "1 while polyamide Her2-1 binds with a K a of 5 x 10 9 M "1 .
  • compositions may be formulated into pharmaceutical or therapeutic compositions, formulations, or preparations.
  • Pharmaceutically acceptable salts of the polyamide compounds of the invention are formed where appropriate with strong or moderately strong, non-toxic, organic, or inorganic acids or bases by methods known in the art.
  • Exemplary of the salts that are included in this invention are maleate, fumarate, lactate, oxalate, methanesulfonate, ethanesulfonate, benzenesulfonate, tartrate, citrate, hydrochloride, hydrobromide, sulfate, phosphate, and nitrate salts.
  • the polyamide compounds of the invention possess the ability to inhibit gene expression or overexpression, properties that are exploited in the treatment of any of a number of diseases or conditions, most notably cancer and especially breast cancer.
  • a composition of this invention may be active er se, or may act as a "pro-drug" that is converted in vivo to an active form.
  • the compounds of the invention may be incorporated into convenient dosage forms, such as capsules, impregnated wafers, tablets, or injectable preparations.
  • Solid or liquid pharmaceutically acceptable carriers may be employed.
  • Pharmaceutical compositions designed for timed release may also be formulated.
  • the compounds of the invention are administered systemically, e.g., by injection.
  • injection may be by any known route, preferably intravenous, subcutaneous, intramuscular, intracranial, or intraperitoneal.
  • injectables can be prepared in conventional forms, either as solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid.
  • Liquid carriers include syrup, peanut oil, olive oil, saline, water, dextrose, glycerol and the like.
  • the carrier or diluent may include any prolonged release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, liquid containing capsule, sterile injectable liquid (e.g., a solution), such as an ampoule, or an aqueous or nonaqueous liquid suspension.
  • sterile injectable liquid e.g., a solution
  • an ampoule or an aqueous or nonaqueous liquid suspension.
  • the pharmaceutical preparations are made following conventional techniques of pharmaceutical chemistry involving such steps as mixing, granulating and compressing, when necessary for tablet forms, or mixing, filling and dissolving the ingredients, as appropriate, to give the desired products for oral or parenteral, including topical, transdermal, intravaginal, intranasal, intrabronchial, intracranial, intraocular, intraaural and rectal administration.
  • the pharmaceutical compositions may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and so forth.
  • the pharmaceutical composition may be administered topically or transdermally, e.g., as an ointment, cream or gel, orally, rectally, e.g., as a suppository, parenterally, by injection or continuously by infusion, intravaginally, intranasally, intrabronchially, intracranially intra-aurally, or intraocularly.
  • the composition may be incorporated into topically applied vehicles such as a salve or ointment.
  • the carrier for the active ingredient may be either in sprayable or nonsprayable form.
  • Non-sprayable forms can be semi-solid or solid forms comprising a carrier indigenous to topical application and having a dynamic viscosity preferably greater than that of water.
  • Suitable formulations include, but are not limited to, solution, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like.
  • auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers, or salts for influencing osmotic pressure and the like.
  • Preferred vehicles for non-sprayable topical preparations include ointment bases, e.g., polyethylene glycol-1000 (PEG-1000), conventional creams such as HEB cream, gels, as well as petroleum jelly and the like.
  • sprayable aerosol preparations wherein the compound, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant.
  • the aerosol preparations can contain solvents, buffers, surfactants, perfumes, and/or antioxidants in addition to the compounds of the invention.
  • compositions of the invention can also be administered in combination with one or more additional compounds that are used to treat the disease or condition.
  • the polyamides and derivatives are given in combination with anti-tumor agents, such as mitotic inhibitors, e.g., vinblastine; alkylating agents, e.g., cyclophosphamide; folate inhibitors, e.g., methotrexate, pritrexim or trimetrexate, antimetabolites, e.g., 5-fluorouracil and cytosine arabinoside, intercalating antibiotics, e.g., adriamycin and bleomycin, enzymes or enzyme inhibitors, e.g., asparaginase, topoisomerase inhibitors, e.g., etoposide, or biological response modifiers, e.g., interferon.
  • anti-tumor agents such as mitotic inhibitors, e.g., vinblastine; alkylating agents, e.g., cyclophosphamide; folate inhibitors, e.g., methotrexate,
  • Typical single dosages of the compounds of this invention are between about 1 ng and about 10 g/kg body weight.
  • the dose is preferably between about O.Olmg and about lg/kg body wt. and, most preferably, between about 0.1 mg and about lOOmg/kg body wt.
  • dosages in the range of about 0.01-20% concentration of the compound, preferably 1-5% are suggested.
  • a total daily dosage in the range of about 1-500 mg is preferred for oral administration.
  • the foregoing ranges are, however, suggestive, as the number of variables in regard to an individual treatment regime is large, and considerable excursions from these recommended values are expected and may be routinely made by those skilled in the art.
  • Effective amounts or doses of the compound for treating a disease or condition can be determined using recognized in vitro systems or in vivo animal models for the particular disease or condition.
  • many art-recognized models are known and are representative of a broad spectrum of human tumors.
  • the compositions may be tested for inhibition of tumor cell growth in culture using standard assays with any of a multitude of tumor cell lines of human or nonhuman animal origin. Many of these approaches, including animal models, are described in detail in Geran, R.I. et al., "Protocols for Screening Chemical Agents and Natural Products against Animal Tumors and Other Biological Systems (Third Edition)", Cane. Chemother. Reports, Part 3, 3:1- 112. Administration methods
  • the treatment methods of the invention are directed to the administration of polyamide-containing compositions.
  • the polyamide-containing preparations of the invention may be administered systemically or locally and may be used alone or as components of mixtures.
  • the route of administration may be topical, intravenous, oral, or by use of an implant.
  • polyamides may be administered by means including, but not limited to, topical preparations, intravenous injection or infusion, oral intake, or local administration in the form of intradermal injection or an implant. Additional routes of administration are subcutaneous, intramuscular, or intraperitoneal injections of the polyamides in conventional or convenient forms. Liposomal or lipophilic formulations may also be used when desired.
  • the polyamides may be in standard topical formulations and compositions including lotions, suspensions or pastes. Oral administration of suitable formulations may also be appropriate in those instances where the polyamides may be readily administered to the target cells or tissues via this route.
  • polyamides may be optimized by the skilled artisan depending on factors such as, but not limited to, the polyamides chosen, the physical delivery system in which it is carried, the individual subject, and the judgment of the skilled practitioner. Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.
  • Electrophoretic Mobility Shift Assays were performed to determine whether the addition of various concentrations of polyamides specific for the sequences flanking the TATA box of the HER2/neu promoter could interfere with the DNA binding activity of the TATA binding protein (TBP).
  • Oligonucleotides corresponding to the HER2/neu TATA box and the adjacent sequences were synthesized.
  • the first oligonucleotide, HERTATA1 has the sequence:
  • the complementary oligonucleotide, HERTATA2 has the sequence: 5'-CTTCACAACTTCATTCTTATACTTCCTCAAGCAGC-3'.
  • These complementary 35 base oligonucleotides were 5' end-labeled with ⁇ - 32 P-ATP and T4 polynucleotide kinase and then annealed to give a double-stranded 35 base pair oligonucleotide.
  • This oligonucleotide was then used in electrophoretic mobility shift assays employing 5% nondenaturing polyacrylamide gels (29:1 acrylamide to bisacrylamide) containing 4 mM MgCl 2 and 0.02% (v/v) NP-40 nonionic detergent along with 44 mM Tris-borate, pH 8.3, 1 mM EDTA.
  • the labeled oligo at a concentration of 0.1 nM, was reacted with 1 nM final concentration of TBP (Promega) in a reaction volume of 20 ⁇ l, containing 10% glycerol (v/v), 20 mM HEPES-OH, pH 7.9, 25 mM KCl, 0.025%) NP-40 (v/v), 100 ⁇ g/ml bovine serum albumin, 0.5 mM dithiothreitol, 0.8 mM spermidine, 0.1 mM EDTA, 2 mM MgCl 2 .
  • RPR indicates the presence of a charged arginine-proline-arginine tail on the polyamide.
  • the restriction endonuclease Dra I was used to linearize the plasmid pGEM/HNP, containing the HER2/neu promoter (Ebbinghaus, et al. (1993)), to produce a template for transcription.
  • This template contained 270 base pairs of the HER2/neu promoter as well as 400 base pairs of downstream sequence. Transcription reactions were performed in 20 ⁇ l reactions containing lOOng of template DNA and 2 ⁇ l of a HeLa cell nuclear extract in a reaction volume of 25 ⁇ l as recommended by the supplier (Promega).
  • RNAzol as recommended by the supplier (Teltest) and subjected to denaturing polyacrylamide gel electrophoresis on 8% polyacrylamide gels containing 8.3 M urea and 88 mM Tris-borate, pH 8.3, 2 mM EDTA.
  • the cell line SK-BR-3 was treated with polyamides for 6 days.
  • polyamide HER2-1 and polyamide 70 in separate experiments were added to the cell culture media for a final concentration of 0.5 ⁇ M. After 3 days of incubation, fresh media and fresh polyamide were added to the cells. These cells were incubated for an additional 3 days and then harvested for RNA extraction.
  • the polyamide- treated breast cancer cells were harvested by treating the adherent cells with 2 ml of 0.05% trypsin-0.53 mM EDTA (Gibco BRL) to detach the cells from the culture flask. These cells were collected and pelleted in a clinical centrifuge at 5000 rpm (IEC ). The cells were rinsed with cold lx phosphate buffered saline (PBS) and pelleted in the clinical centrifuge. Total RNA was extracted from the cells using RNAzol (Teltest). To a packed cell volume of approximately 100 ⁇ l, 1ml of RNAzol and lOO ⁇ l of chloroform were added.
  • RT reverse transcriptase
  • PCR reverse transcriptase
  • HER2/neu mRNA levels should correlate with the amount of transcription from the HER2/neu promoter, allowing the determination of whether polyamide HER2-1 has any effect on transcription in vivo.
  • the PCR product will correspond to HER2/neu cDNA, reflecting the relative levels of HER2/neu mRNA.
  • the PCR primers were: (Her2A) 5'- GCTGGCCCGATGTATTTGATGGT-3' and (Her2B) 5'-
  • the relative amounts of HER2/neu mRNA from the various cells can be determined using reverse transcriptase (RT)-polymerase chain reaction (PCR).
  • PCR reverse transcriptase
  • concentration of total RNA is determined by spectrophotometry (using the optical density at 260 nM) for each different cell type and polyamide concentration.
  • An equal amount of total RNA (10 ng) is used for each RT-PCR.
  • RT-PCR was carried out using the Reverse Transcription System kit (Promega).
  • cDNAs are synthesized from the mRNA templates by the enzyme reverse transcriptase at 42° C for 25 min, as recommended. These cDNAs are then used as templates for PCR.
  • PCR was carried out at 26 cycles of denaturation at 94° C for 45 seconds, annealing at 60° C for 45 seconds, and extension at 72° C for 2 minutes. Five ⁇ Ci of the radioactive nucleotide ⁇ - 32 P-dATP is included in the PCR step to produce a radiolabeled PCR product which can be analyzed on an acrylamide gel and visualized by autoradiography.
  • the relative amount of PCR product can be quantitated using a Phosphorimager (Molecular Dynamics).
  • the level of HER2/neu mRNA from cells which have not been treated with polyamide are the positive control and are given a value of 1.0 and the HER2/neu mRNA levels for the polyamide- treated samples are given a value relative to the value for untreated cells.

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Abstract

La présente invention concerne des méthodes et des compositions comprenant des polyamides pouvant se lier au sillon mineur d'ADN double brin destinées à inhiber ou à réduire la transcription et l'expression de gène. Ces polyamides comprennent au moins quatre paires de résidus carboxamides aromatiques choisis afin de contacter et/ou de se lier spécifiquement à la séquence de nucléotides d'une cible ADN à double brin dans la région promoteur du gène de la cible. Ces méthodes, compositions et polyamides sont destinés à l'inhibition de la transcription et de l'expression d'oncogène aussi bien qu'au traitement de cancer.
PCT/US1999/020971 1998-09-11 1999-09-10 REGULATION DE L'EXPRESSION D'ONCOGENE HER2/neu A L'AIDE DE POLYAMIDES SYNTHETIQUES WO2000015242A1 (fr)

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CA002343289A CA2343289A1 (fr) 1998-09-11 1999-09-10 Regulation de l'expression d'oncogene her2/neu a l'aide de polyamides synthetiques
AU64965/99A AU6496599A (en) 1998-09-11 1999-09-10 Regulation of her2/neu oncogene expression by synthetic polyamides
EP19990952908 EP1112080A4 (fr) 1998-09-11 1999-09-10 REGULATION DE L'EXPRESSION D'ONCOGENE HER2/neu A L'AIDE DE POLYAMIDES SYNTHETIQUES
JP2000569826A JP2002524525A (ja) 1998-09-11 1999-09-10 合成ポリアミドによる発癌遺伝子ＨＥＲ／２ｎｅｕ発現の制御

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US60/099,906 1998-09-11

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PCT/US1999/020971 WO2000015242A1 (fr) 1998-09-11 1999-09-10 REGULATION DE L'EXPRESSION D'ONCOGENE HER2/neu A L'AIDE DE POLYAMIDES SYNTHETIQUES

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WO2018044817A1 (fr) * 2016-08-29 2018-03-08 California Institute Of Technology Compositions et méthodes de traitement du cancer de la prostate
US10723716B2 (en) 2016-12-21 2020-07-28 New York University Alpha-helix mimetics as modulators of Abeta self-assembly

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EP1470119A4 (fr) 2001-06-13 2005-10-19 Genesoft Pharmaceuticals Inc Composes benzothiophene presentant une activite anti-infectieuse
WO2002101007A2 (fr) 2001-06-13 2002-12-19 Genesoft Pharmaceuticals, Inc Composes benzamides anti-pathogenes
US6777425B2 (en) 2001-06-13 2004-08-17 Genesoft Pharmaceuticals, Inc. Isoquinoline compounds having antiinfective activity
DE60326735D1 (de) 2002-08-02 2009-04-30 Genesoft Pharmaceuticals Inc Biaryl-verbindungen mit antiinfektiver wirkung
US7265129B2 (en) 2002-10-25 2007-09-04 Genesoft Pharmaceuticals, Inc. Anti-infective biaryl compounds
JP2006509027A (ja) 2002-12-10 2006-03-16 オーシェント ファーマシューティカルズ コーポレーション （ピロールカルボキサミド）−（ベンズアミド）−（イミダゾールカルボキサミド）モチーフを有する抗菌化合物
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Cited By (4)

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WO2002010368A1 (fr) * 2000-07-21 2002-02-07 Chiba Prefecture Promoteurs specifiques de tumeur
US7030099B2 (en) 2000-07-21 2006-04-18 Research Corporation Technologies, Inc. Tumor specific promoters of the midkine gene that allow for selective expression in P53-inactivated cells
WO2018044817A1 (fr) * 2016-08-29 2018-03-08 California Institute Of Technology Compositions et méthodes de traitement du cancer de la prostate
US10723716B2 (en) 2016-12-21 2020-07-28 New York University Alpha-helix mimetics as modulators of Abeta self-assembly

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AU6496599A (en) 2000-04-03
JP2002524525A (ja) 2002-08-06
EP1112080A4 (fr) 2002-10-24
WO2000015209A2 (fr) 2000-03-23
EP1112080A1 (fr) 2001-07-04
WO2000015209A3 (fr) 2000-05-25
AU6034199A (en) 2000-04-03
CA2343289A1 (fr) 2000-03-23

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