WO1995004749A1 - Oligomers for modulating metabolic function - Google Patents

Oligomers for modulating metabolic function Download PDF

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Publication number
WO1995004749A1
WO1995004749A1 PCT/US1994/009026 US9409026W WO9504749A1 WO 1995004749 A1 WO1995004749 A1 WO 1995004749A1 US 9409026 W US9409026 W US 9409026W WO 9504749 A1 WO9504749 A1 WO 9504749A1
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seq
oligomer
group
alkyl
alkoxy
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PCT/US1994/009026
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French (fr)
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Clarence F. Bennett
Christopher K. Mirabelli
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Isis Pharmaceuticals, Inc.
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Priority to AU75598/94A priority Critical patent/AU7559894A/en
Publication of WO1995004749A1 publication Critical patent/WO1995004749A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3181—Peptide nucleic acid, PNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention is directed to compounds that are not polynucleotides yet which bind in a complementary fashion to DNA and RNA strands.
  • the invention concerns compounds wherein naturally-occurring nucleobases or other nucleobase-binding moieties are covalently bound to a polyamide backbone. These compounds are useful for therapeutic and other applications directed to modulating metabolic processes.
  • PNAs Peptide Nucleic Acids
  • mRNA messenger RNA
  • tRNA transfer RNAs
  • RNA- synthesizing enzyme RNA polymerase
  • this recognition is preceded by sequence-specific binding of protein transcription factors to the promoter.
  • Other proteins which bind to the promoter, but whose binding prohibits action of RNA polymerase are known as repressors.
  • gene activation is typically regulated positively by transcription factors and negatively by repressors.
  • Most conventional drugs function by interaction with and modulation of one or more targeted endogenous proteins, e.g., enzymes.
  • drugs are typically not specific for targeted proteins but interact with other proteins as well.
  • a relatively large dose of drug must be used to effectively modulate a targeted protein.
  • Typical daily doses of drugs are from 10 " -10 " millimoles per kilogram of body weight or 10 " -10 millimoles for a 100 kilogram person. If this modulation could instead be effected by interaction with and inactivation of mRNA, a dramatic reduction in the necessary amount of drug could likely be achieved, along with a corresponding reduction in adverse side effects. Further reductions could be achieved if such interaction could be rendered site-specific.
  • Oligodeoxynucleotides offer such opportunities.
  • synthetic oligodeoxynucleotides have been used as antisense probes to block and eventually lead to the breakdown of mRNA.
  • It also may be possible to modulate the genome of an animal by, for example, triple helix formation using oligonucleotides or other DNA recognizing agents.
  • triple helix formation there are a number of drawbacks associated with triple helix formation. For example, it can only be used for homopurine sequences and it requires unphysiologically high ionic strength and low pH.
  • Unmodified oligonucleotides are impractical both in the antisense approach and in the triple helix approach because they have short in vivo half-lives. They are also poor penetrators of the cell membrane. These problems have resulted in an extensive search for improvements and alternatives. For example, the problems arising in connection with double-stranded DNA (dsDNA) recognition through triple helix formation have been diminished by a clever "switch back" chemical linking whereby a sequence of polypurine on one strand is recognized, and by "switching back", a homopurine sequence on the other strand can be recognized. Also, competent helix formation has been obtained by using artificial bases, thereby improving binding conditions with regard to ionic strength and pH.
  • dsDNA double-stranded DNA
  • PNAs denominated peptide nucleic acids
  • ligands are linked to a polyamide backbone through aza nitrogen atoms.
  • peptide nucleic acids are disclosed in which their recognition moieties are linked to the polyamide backbone additionally through amido and/or ureido tethers.
  • PCT/EP 92/01219 filed May 22, 1992 also discloses protein nucleic acids.
  • peptide nucleic acids are synthesized by adaptation of certain peptide synthesis procedures, either in solution or on a solid phase.
  • the synthons used are certain monomer amino acids or their activated derivatives, protected by standard groups.
  • These oligonucleotide analogs also can be synthesized by using the corresponding diacids and diamines.
  • Peptide nucleic acid oligomers have been found to be superior to prior reagents in that they have significantly higher affinity for complementary single stranded DNA (ssDNA) . These compounds are also able to form triple helices wherein a first PNA strand binds with RNA or ssDNA and a second PNA strand binds with the resulting double helix or with the first PNA strand.
  • PNAs generally possess no significant charge and are water soluble, which facilitates cellular uptake.
  • PNAs contain amides of non- biological amino acids, making them biostable and resistant to enzymatic degradation, for example, by proteases.
  • PNAs can ideally be used to target RNA and ssDNA to produce antisense-type gene regulating moieties.
  • Reagents that bind sequence-specifically to dsDNA, RNA, or ssDNA have applications as gene targeted drugs useful for modulating metabolic processes such as metabolic regulatory dysfunctions, such as cancer.
  • PNAs can also be useful in diagnostics, as for example, as probes for specific mRNAs.
  • Human intercellular adhesion molecule-1 (ICAM-1) is encoded by a 3.3 kb mRNA resulting in the synthesis of a
  • ICAM- 1 55,219 dalton (Da) cell surface transme brane protein.
  • ICAM- 1 is heavily glycosylated through N-linked glycosylation sites.
  • the mature protein has an apparent molecular mass of 90 kDa as determined by SDS-polyacryla ide gel electrophoresis. Staunton et al., Cell 1988, 52 , 925-933.
  • the primary binding site for ICAM-1 is lymphocyte-associated antigen-1 (LFA-1) .
  • ICAM-1 can be regulated on vascular endothelial cells, fibroblasts, keratinocytes, astrocytes and several cell lines by treatment with bacterial lipopolysaccharide and cytokines such as interleukin-1, tumor necrosis factor, gamma-interferon, and lymphotoxin. See, e . g. , Frohman et al., J. Neuroimmunol . 1989, 23 , 117-124.
  • ICAM-1 plays a role in adhesion of neutrophils to vascular endothelium, as well as adhesion of onocytes and lymphocytes to vascular endothelium, tissue fibroblasts and epidermal keratinocytes. ICAM-1 also plays a role in T-cell recognition of antigen presenting cell, lysis of target cells by natural killer cells, lymphocyte activation and proliferation, and maturation of T cells in the thymus. In addition, recent data have demonstrated that ICAM-1 is the cellular receptor for the major serotype of rhinovirus, which account for greater than 50% of common colds. Staunton et al., Cell 1989, 56 , 849-853; Greve et al. , Cell 1989, 56 , 839-847.
  • ICAM-1 ICAM-1-associated fibroblasts
  • inflammatory skin disorders such as allergic contact dermatitis, fixed drug eruption, lichen planus, and psoriasis; Ho et al., J. Am . Acad . Dermatol . 1990, 22 , 64-68; Griffiths and Nickoloff, Am . J . Pathology 1989, 135 , 1045- 1053; Lisby et al., Br . J . Dermatol . 1989, 120 , 479-484; Shiohara et al., Arch. Dermatol . 1989, 125 , 1371-1376.
  • ICAM-1 expression has been detected in the synovium of patients with rheumatoid arthritis; Hale et al., Arth . Rheum . 1989, 32 , 22-30, pancreatic B-cells in diabetes; Campbell et al., Proc . Natl . Acad . Sci . U.S .A . 1989, 86 , 4282-4286; thyroid follicular cells in patients with Graves' disease; Weetman et al., J . Endocrinol .
  • Endothelial leukocyte adhesion molecule-1 (ELAM-1) is a 115-kDa membrane glycoprotein which is a member of the selectrin family of membrane glycoproteins. Bevilacqua et al., Science 1989, 243 , 1160-1165.
  • the amino terminal region of ELAM-1 contains sequences with homologies to members of lectin-like proteins, followed by a domain similar to epidermal growth factor, followed by six tandem 60-amino acid repeats similar to those found in complement receptors 1 and 2.
  • ELAM-1 is encoded for by a 3.9 kb mRNA.
  • the 3'-untranslated region of ELAM-1 mRNA contains several sequence motifs ATTTA which are responsible for the rapid turnover of cellular mRNA consistent with the transient nature of ELAM-1 expression.
  • ELAM-1 is primarily involved in the adhesion of neutrophils to vascular endothelial cells. ELAM-1 exhibits a limited cellular distribution in that it has only been identified on vascular endothelial cells. Like ICAM-1, ELAM-1 is inducible by a number of cytokines including tumor necrosis factor, interleukin-1 and lymphotoxin and bacterial lipopolysaccharide. In contrast to ICAM-1, ELAM-1 is not induced by gamma-interferon. Bevilacqua et al., Proc . Natl . Acad . Sci . USA 1987, 84 , 9238-9242; Wellicome et al., J . Immunol . 1990, 144 , 2558-2565.
  • Vascular cell adhesion molecule-1 (VCAM-1) is a 110-kDa membrane glycoprotein encoded by a 3.2 kb mRNA.
  • VCAM-1 appears to be encoded by a single-copy gene. Osborn et al., Cell 1989, 59 , 1203-1211. Like ICAM-1, VCAM-1 is a member of the immunoglobulin supergene family, containing six immunoglobulin-like domains of the H type. The receptor for VCAM-1 is proposed to be CD29 as demonstrated by the ability of monoclonal antibodies to CD29 to block adherence of Ramos cells to VCAM-1. VCAM-1 is expressed primarily on vascular endothelial cells. Like ICAM-1 and ELAM-1, expression of VCAM-1 on vascular endothelium is regulated by treatment with cytokines.
  • VCAM-1 primarily binds T and B lymphocytes. In addition, VCAM-1 may play a role in the metastasis of melanoma, and possibly other cancers.
  • Inhibitors of ICAM-1, ELAM-1, and VCAM-l expression would provide a novel therapeutic class of anti-inflammatory agents with activity towards a variety of inflammatory diseases or diseases with an inflammatory component such as asthma, rheumatoid arthritis, allograft rejections, various dermatological conditions, and psoriasis.
  • inhibitors of ICAM-1 may also be effective in the treatment of colds due to rhinovirus infection, AIDS, and some cancers and their metastasis.
  • the use of neutralizing monoclonal antibodies against ICAM-1 in animal models provide evidence that such inhibitors if identified would have therapeutic benefit for asthma; Wegner et al..
  • the present invention provides oligomers comprising peptide nucleic acids (PNAs) , that bind complementary ssDNA and RNA strands through their oligoribonucleotide ligands which are linked to a peptide backbone.
  • PNAs peptide nucleic acids
  • the sequence of the oligoribonucleotide ligands specifies the target to which they bind.
  • These PNA oligomers are useful as therapeutic agents for treating diseases like cancer, AIDS and genetic and metabolic diseases. These compositions are also useful in diagnostic applications and as research tools.
  • Oligomers of the present invention comprise oligomers wherein at least one subunit of the oligomer is a peptide nucleic acid subunit of the formula:
  • L is one of the adenine, thymine, cytosine or guanine heterocyclic bases of the oligomer
  • C is (CR R ) where R is hydrogen and R is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R and R are independently selected from the group consisting of hydrogen, (C 2 -C 6 )alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C,-C 6 ) alkoxy, (C.,-
  • C 6 )alkylt s independently selected from the group consisting of hydrogen, (C,-C 4 )alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C.,-C 4 ) alkyl, hydroxy, alkoxy, alkylthio and amino; and R is hydrogen, (C 1 -C 6 )alkyl, hydroxy-, alkoxy-, or alkylthio- substituted (C 1 -C 6 )alkyl, or R and R taken together complete an alicyclic or heterocyclic system; D is (CR R ) z where R and R are as defined above; each of y and z is zero or an integer from 1 to 10, the sum y + z being greater than 2 but not more than 10;
  • G is -NR 3 CO-, -NR 3 CS-, -NR 3 S0- or -NR 3 S0 2 -, in either orientation, where R is as defined above; each pair of A and B is selected such that:
  • A is a group of formula (Ila) , (lib) or (lie) and B is N or R N + ; or
  • A is a group of formula (lid) and B is CH;
  • X is 0, S, Se, NR , CH 2 or C(CH 3 ) 2 ;
  • Y is a single bond, 0, S or NR 4; each of p and q is zero or an integer from 1 to 5, the sum p+q being not more than 10; each of r and s is zero or an integer from 1 to 5, the sum r+s being not more than 10; each R 1 and R2 is independently selected from the group consisting of hydrogen, (C.-C ⁇ )alkyl which may be hydroxy- or alkoxy- or alkylthio- substituted, hydroxy, alkoxy, alkylthio, amino and halogen.
  • Subunits refers to basic unit which are chemically similar and which can form polymers. Repeating basic units form polymers referred to as "oligomers". Oligomers of the present invention may thus refer to oligomers in which substantially all subunits of the oligomer are subunits as described in Formula I. Oligomers of the present invention may also comprise one or more subunits which are naturally occuring nucleotides or nucleotide analogs as long as at least one subunit satisfies Formula I. Thus, oligomers as used herein may refer to a range of oligomers from oligomers comprising only one PNA subunit as defined in Formula I to oligomers in which every subunit is a PNA subunit as defined in Formula I.
  • Those subunits which are not PNA subunits comprise naturally occuring bases, sugars, and intersugar (backbone) linkages as well as non-naturally occurring portions which function similarly to naturally occurring portions. Sequences of oligomers of the present invention are defined by reference to the L group (for PNA subunits) or nucleobase (for nucleotide subunits) at a given position.
  • the nomeclature is modeled after traditional nucleotide nomenclature, identifying each PNA subunit by the identity of its L group such as the heterocycles adenine (A) , thymine (T) , guanine (G) and cytosine (C) and identifying nucleotides or nucleosides by these same heterocycle residing on the sugar backbone.
  • L group such as the heterocycles adenine (A) , thymine (T) , guanine (G) and cytosine (C) and identifying nucleotides or nucleosides by these same heterocycle residing on the sugar backbone.
  • the sequences are conveniently provided in traditional 5' to 3' or amino to carboxy orientation.
  • Oligomers of the present invention may range in size from about 5 to about 50 subunits in length. In other embodiments of the present invention, oligomers may range in size from about 10 to about 30 subunits in length. In still other embodiments of the present invention oligomers may range in size from about 10 to about 25 subunits in length. In yet further embodiments of the present invention, oligomers may range in size from about 12 to about 20 subunits in length.
  • the preparation of protein nucleic acid oligomers is known in the art, such as is described in PCT/EP 92/01219 filed May 22, 1992, which is incorporated by reference herein in its entirety.
  • Solid- Phase Synthesis the principle of anchoring molecules onto a solid matrix, which helps in accounting for intermediate products during chemical transformations, is known as Solid- Phase Synthesis or Merrifield Synthesis (see, e . g. , Merrifield, J. Am . Chem . Soc . , 1963, 85, 2149 and Science , 1986, 232 , 341) .
  • Established methods for the stepwise or fragmentwise solid-phase assembly of amino acids into peptides normally employ a beaded matrix of slightly cross- linked styrene-divinylbenzene copolymer, the cross-linked copolymer having been formed by the pearl polymerization of styrene monomer to which has been added a mixture of divinylbenzenes. A level of 1-2% cross-linking is usually employed.
  • Such a matrix also can be used in solid-phase PNA synthesis in accordance with the present invention.
  • benzhydrylamino functionality (Pietta, et al . , J. Chem . Soc , 1970, 650) are the most widely applied. Regardless of its nature, the purpose of the functionality is normally to form an anchoring linkage between the copolymer solid support and the C-terminus of the first amino acid to be coupled to the solid support. As will be recognized, anchoring linkages also can be formed between the solid support and the amino acid N-terminus. It is generally convenient to express the "concentration" of a functional group in terms of millimoles per gram (mmol/g) . Other reactive functionalities which have been initially introduced include 4-methylbenzhydrylamino and 4-methoxybenzhydrylamino.
  • Preferred methods for PNA synthesis employ aminomethyl as the initial functionality, in that aminomethyl is particularly advantageous with respect to the incorporation of "spacer” or “handle” groups, owing to the reactivity of the amino group of the aminomethyl functionality with respect to the essentially quantitative formation of amide bonds to a carboxylic acid group at one end of the spacer-forming reagent.
  • spacer- or handle-forming bifunctional reagents have been described (see, Barany, et al . , Int . J . Peptide Protein Res . , 1987, 30 , 705), especially reagents which are reactive towards amino groups such as found in the aminomethyl function.
  • bifunctional reagents include 4- (haloalkyl)aryl-lower alkanoic acids such as 4- (bromomethyl)phenylacetic acid, Boc-aminoacyl-4- (oxymethyl)aryl-lower alkanoic acids such as Boc-aminoacyl-4- (oxy ethyl)phenylacetic acid, N-Boc-p-acylbenzhydrylamines such as N-Boc-p-glutaroylbenzhydrylamine, N-Boc-4'-lower alkyl-p-acylbenzhydrylamines such as N-Boc-4'-methyl-p- glutaroylbenzhydrylamine, N-Boc-4'-lower alkoxy-p-acylbenz- hydrylamines such as N-Boc-4'-methoxy-p-glutaroyl-benzhy- drylamine, and 4-hydroxymethylphenoxyacetic acid.
  • One type of spacer group particularly relevant within the context of the present invention is the phenylacetamidomethyl (Pam) handle (Mitchell and Merrifield, J . Org . Chem . , 1976, 41 , 2015) which, deriving from the electron withdrawing effect of the 4-phenylacetamidomethyl group, is about 100 times more stable than the classical benzyl ester linkage towards the Boc-amino deprotection reagent trifluoroacetic acid (TFA) .
  • Pam phenylacetamidomethyl
  • spacer or handle groups An alternative strategy concerning the introduction of spacer or handle groups is the so-called "preformed handle” strategy (see, Tarn, et al . , Synthesis , 1979, 955- 957) , which offers complete control over coupling of the first amino acid, and excludes the possibility of complications arising from the presence of undesired functional groups not related to the peptide or PNA synthesis.
  • spacer or handle groups of the same type as described above, are reacted with the first amino acid desired to be bound to the solid support, the amino acid being N-protected and optionally protected at the other side-chains which are not relevant with respect to the growth of the desired PNA chain.
  • the first amino acid to be coupled to the solid support can either be coupled to the free reactive end of a spacer group which has been bound to the initially introduced functionality (for example, an aminomethyl group) or can be reacted with the spacer- forming reagent.
  • the space-forming reagent is then reacted with the initially introduced functionality.
  • Other useful anchoring schemes include the "multidetachable" resins (Tarn, et al . , Tetrahedron Lett . , 1979, 4935 and J. Am . Chem . Soc , 1980, 102 , 611; Tam, J. Org . Chem . , 1985, 50 , 5291), which provide more than one mode of release and thereby allow more flexibility in synthetic design.
  • Suitable choices for N-protection are the tert- butyloxycarbonyl (Boc) group (Carpino, J . Am . Chem . Soc , 1957, 79 , 4427; McKay, et al . , J. Am . Chem . Soc , 1957, 79 , 4686; Anderson, et al . , J . Am . Chem . Soc , 1957, 79 , 6180) normally in combination with benzyl-based groups for the protection of side chains, and the 9-fluorenylmethyloxy- carbonyl (Fmoc) group (Carpino, et al . , J . Am . Chem .
  • amino protecting groups particularly those based on the widely-used urethane functionality, successfully prohibit racemization (mediated by tautomerization of the readily formed oxazolinone (azlactone) intermediates (Goodman, et al . , J. Am . Chem . Soc , 1964, 86 , 2918)) during the coupling of most ⁇ -amino acids.
  • racemization mediated by tautomerization of the readily formed oxazolinone (azlactone) intermediates (Goodman, et al . , J. Am . Chem . Soc , 1964, 86 , 2918)
  • a whole range of otherwise "worthless" nonurethane- type of amino protecting groups are applicable when assembling PNA molecules, especially those built from achiral units.
  • amino protecting groups are useful within the context of the present invention, but virtually any amino protecting group which largely fulfills the following requirements: (1) stability to mild acids (not significantly attacked by carboxyl groups) ; (2) stability to mild bases or nucleophiles (not significantly attacked by the amino group in question) ; (3) resistance to acylation (not significantly attacked by activated amino acids) . Additionally: (4) the protecting group must be close to quantitatively removable, without serious side reactions, and (5) the optical integrity, if any, of the incoming amino acid should preferably be highly preserved upon coupling.
  • side-chain protecting groups in general, depends on the choice of the amino protecting group, since the protection of side-chain functionalities must withstand the conditions of the repeated amino deprotection cycles. This is true whether the overall strategy for chemically assembling PNA molecules relies on, for example, differential acid stability of amino and side-chain protecting groups (such as is the case for the above- mentioned “Boc-benzyl” approach) or employs an orthogonal, that is, chemoselective, protection scheme (such as is the case for the above-mentioned "Fmoc-tBu” approach) ,
  • Novel monomer synthons may be selected from the group consisting of amino acids, diacids and diamines having general formulae:
  • the temporary protecting group, such as a Boc or Fmoc group, on the last-coupled amino acid is quantitatively removed by a suitable treatment, for example, by acidolysis, such as with trifluoroacetic acid, in the case of Boc, or by base treatment, such as with piperidine, in the case of Fmoc, so as to liberate the N-terminal amine function.
  • a suitable treatment for example, by acidolysis, such as with trifluoroacetic acid, in the case of Boc, or by base treatment, such as with piperidine, in the case of Fmoc, so as to liberate the N-terminal amine function.
  • the next desired N-protected amino acid is then coupled to the N-terminal of the last-coupled amino acid. This coupling of the C-terminal of an amino acid with the N- ter inal of the last-coupled amino acid can be achieved in several ways.
  • the incoming amino acid in a form with the carboxyl group activated by any of several methods, including the initial formation of an active ester derivative such as a 2,4,5- trichlorophenyl ester (Pless, et al . , Helv . Chim . Acta , 1963, 46, 1609), a phthalimido ester (Nefkens, et al . , J. Am . Chem . Soc , 1961, 83 , 1263), a pentachlorophenyl ester (Kupryszewski, Rocz . Chem .
  • an active ester derivative such as a 2,4,5- trichlorophenyl ester (Pless, et al . , Helv . Chim . Acta , 1963, 46, 1609), a phthalimido ester (Nefkens, et al . , J. Am . Chem . Soc , 1961,
  • anhydride such as a symmetrical anhydride (Wieland, et al . , Angew. Chem . , Int . Ed . Engl . , 1971, 10 , 336).
  • carboxyl group of the incoming amino acid can be reacted directly with the N-terminal of the last-coupled amino acid with the assistance of a condensation reagent such as, for example, dicyclohexylcarbodiimide (Sheehan, et al. , J. Am . Chem . Soc , 1955, 77 , 1067) or derivatives thereof.
  • Benzotriazolyl N- oxytrisdimethylaminophosphonium hexafluorophosphate BOP
  • Castro's reagent "Castro's reagent” (see, e .g . , Rivaille, et al . , Tetrahedron , 1980, 36 , 3413) is recommended when assembling PNA molecules containing secondary amino groups.
  • activated PNA monomers analogous to the recently-reported amino acid fluorides (Carpino, J. Am. Chem . Soc , 1990, 212, 9651) hold considerable promise to be used in PNA synthesis as well.
  • the next step v/ill normally be deprotection of the amino acid moieties of the PNA chain and cleavage of the synthesized PNA from the solid support.
  • These processes can take place substantially simultaneously, thereby providing the free PNA molecule in the desired form.
  • stepwise chain building of achiral PNAs such as those based on aminoethylglycyl backbone units can start either from the N-terminus or the C-terminus, because the coupling reactions are free of racemization.
  • syntheses commencing at the C-terminus typically employ protected amine groups and free or activated acid groups
  • syntheses commencing at the N-terminus typically employ protected acid groups and free or activated amine groups.
  • PEPS polyethylene
  • PS polystyrene
  • This matrix is comprised of a polyethylene (PE) film with pendant long-chain polystyrene (PS) grafts (molecular weight on the order of 10 ) .
  • the loading capacity of the film is as high as that of a beaded matrix, but PEPS has the additional flexibility to suit multiple syntheses simultaneously.
  • the PEPS film is fashioned in the form of discrete, labeled sheets, each serving as an individual compartment. During all the identical steps of the synthetic cycles, the sheets are kept together in a single reaction vessel to permit concurrent preparation of a multitude of peptides at a rate close to that of a single peptide by conventional methods. It was reasoned that the PEPS film support, comprising linker or spacer groups adapted to the particular chemistry in question, should be particularly valuable in the synthesis of multiple PNA molecules, these being conceptually simple to synthesize since only four different reaction compartments are normally required, one for each of the four "pseudo- nucleotide" units.
  • the PEPS film support has been successfully tested in a number of PNA syntheses carried out in a parallel and substantially simultaneous fashion.
  • the yield and quality of the products obtained from PEPS were comparable to those obtained by using the traditional po- lystyrene beaded support.
  • experiments with other geometries of the PEPS polymer such as, for example, non- woven felt, knitted net, sticks or microwellplates have not indicated any limitations of the synthetic efficacy.
  • Two other methods proposed for the simultaneous synthesis of large numbers of peptides also apply to the preparation of multiple, different PNA molecules. The first of these methods (Geysen, et al . , Proc . Natl . Acad . Sci .
  • spacer molecules are typically added via the beta alanyl group, followed thereafter by the amino acid residue subunits.
  • the beta alanyl-containing monomer can be replaced with an acryloyl sarcosine monomer during polymerization to form resin beads.
  • the polymerization is followed by reaction of the beads with ethylenediamine to form resin particles that contain primary amines as the covalently linked functionali ⁇ ty.
  • the polyacrylamide-based supports are relatively more hydrophilic than are the polystyrene-based supports and are usually used with polar aprotic solvents including dimethyl- formamide, dimethylacetamide, N-methylpyrrolidone and the like (see Atherton, et al . , J . Am . Chem .
  • a second group of solid supports is based on silica-containing particles such as porous glass beads and silica gel.
  • silica-containing particles such as porous glass beads and silica gel.
  • One example is the reaction product of trich- loro-[3-(4-chloromethyl)phenyl]propylsilane and porous glass beads (see Parr and Grohmann, Angew . Chem . Internal . Ed . 1972, 11 , 314) sold under the trademark "PORASIL E” by Waters Associates, Framingham, MA, USA.
  • solid- phase PNA synthesis in the context of the present invention is normally performed batchwise. However, most of the syn ⁇ theses may equally well be carried out in the continuous-flow mode, where the support is packed into columns (Bayer, et al . , Tetrahedron Lett . , 1970, 4503 and Scott, et al . , J. Chromatogr. Sci . , 1971, 9 , 577) .
  • the rigid poly(dimethylacrylami- de)-Kieselguhr support Atherton, et al . , J . Chem . Soc Chem . Commun .
  • PNA molecules may be assembled enzymatically by enzymes such as proteases or derivatives thereof with novel specificities (obtained, for example, by artificial means such as protein engineering) .
  • PNA ligases for the condensation of a number of PNA fragments into very large PNA molecules; (6) since antibodies can be generated to virtually any molecule of interest, the recently developed catalytic antibodies (abzymes) , discovered simultaneously by the groups of Lerner (Tramantano, et al . , Science , 1986, 234 , 1566) and of Schultz (Pollack, et al . , Science , 1986, 234 , 1570), should also be considered as potential candidates for assembling PNA molecules.
  • there has been considerable success in producing abzymes catalyzing acyl-transfer reactions see for example Shokat, et al .
  • Peptide nucleic acid oligomers hybridizable with, or targeted to, metabolic targets are provided by the present invention.
  • hybridizable is meant that at least 70% sequence homology is present.
  • peptide nucleic acid oligomers have at least 85% sequence homology to a desired target.
  • peptide nucleic acid oligomers of the present invention are at least 95% homologous to a target of interest.
  • Oligomers of the present invention comprising PNA subunits can be used in diagnostics, therapeutics and as research reagents and kits.
  • Diagnostic and research reagents may be employed by contacting a cell or other biological sample such as blood, urine, cerebral fluid, ascites, etc. with oligomers of the present invention in vitro .
  • Oligomers of the invention can be formulated in a pharmaceutical composition, which can include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the oligomer.
  • Pharmaceutical compositions also can include one or more active ingredients such as antimicrobial agents, anti- inflammatory agents, anesthetics, and the like in addition to oligomer.
  • the pharmaceutical composition can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration can be topically (including opthalmically, vaginally, rectally, intranasally) , orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • Formulations for topical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Coated condoms may also be useful.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets.
  • Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • Formulations for parenteral administration can include sterile aqueous solutions which also can contain buffers, diluents and other suitable additives.
  • Dosing is dependent on severity and responsiveness of the condition to be treated, but will normally be one or more doses per day, with course of treatment lasting from several days to several months or until a cure is effected or a diminution of disease state is achieved. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates.
  • oligomers targeted to the translation initiation codon (AUG) , 5' untranslated region (5' UTR) , 5' CAP region, coding region, translation termination region or 3' untranslated region (3' UTR) of ICAM-1 were identified having specific sequences. These oligomers will be useful for the treatment of conditions modulated by or associated with ICAM-1 such as asthma, rheumatoid arthritis, allograft rejection, and psoriasis. These oligomers will also be useful as diagnostic and research reagents. The sequences, SEQ ID numbers and targets of these oligomers are shown in Table 1. SEQUENCE
  • Endothelial Leukocyte Adhesion Molecule-l (ELAM-1)
  • oligomers targeted to the translation initiation codon (AUG) , 5' untranslated region (5' UTR), coding region, intron/exon (I/E) junction, or 3' untranslated region (3' UTR) of ELAM-1 were identified having specific sequences. These oligomers will be useful for the treatment of conditions modulated by or associated with ELAM- 1 such as asthma, rheumatoid arthritis, allograft rejection, and psoriasis.
  • the sequences, SEQ ID numbers and targets of these oligomers are shown in Table 2.
  • CAATCATGACTTCAAGAGTTCT 30 ACCACACTGGTATTTCACAC 31 GTATGGAAGATTATAATATAT 32 CACAATCCTTAAGAACTCTTT 33 ACCTCTGCTGTTCTGATCCT 34 ' CTGCTGCCTCTGTCTCAGGT 35 GGTATTTGACACAGC 36
  • AATCATGACTTCAAGAGTTCT 37 TGAAGCAATCATGACTTCAAG 38 TATAGGAGTTTTGATGTGAA 39 ACAATGAGGGGGTAATCTACA 40
  • GACAATATACAAACCTTCCAT 41 ACGTTTGGCCTCATGGAAGT 42 GGAATGCAAAGCACATCCAT 43
  • GGGCCAGAGACCCGAGGAGA 44 TTCCCCAGATGCACCTGTTT 45
  • CTGATTCAAGGCTTTGGCAG 46 CCAAAGTGAGCTGAGAGA 47
  • ACAGGATCTCTCAGGTGGGT 48 GAAGTCAGCCAAGAACAGCT 49 TCACTGCTGCCTCTGTCTCAGG 50 TGATTCTTTTGAACTTAAAAGGA 51 TTAAAGGATGTAAGAAGGCT 52
  • CATAAGCACATTTATTGTC 53 TTGGGAAGCAGTTGTTCA 54
  • AACTGTGAAGCAATCATGACT 55 CCTTGAGTGGTGCATTCAACCT 56
  • oligomers targeted to the translation initiation codon (AUG) , 5' untranslated region (5' UTR) , coding region, exon/intron (E/I) junction, translation termination region or 3' untranslated region (3' UTR) of VCAM-l were identified having specific sequences. These oligomers will be useful for the treatment of conditions modulated by or associated with VCAM-l such as asthma, rheumatoid arthritis, allograft rejection, and psoriasis.
  • the sequences, SEQ ID numbers and targets of these oligomers are shown in Table 3.
  • Example 1 The following examples are provided for illustrative purposes only and are not intended to limit the invention.
  • Example 1 The following examples are provided for illustrative purposes only and are not intended to limit the invention.
  • PNA subunits for oligomers of the invention are prepared generally in accordance with the methods disclosed by WO 92/20702, incorporated by reference herein in its entirety.
  • Benzyhydrylamine resin (initially loaded 0.28 mmol/gm with Boc-L-Lys(2-chlorobenyloxycarbonyl) ) is swollen in DMF and an excess of a monomer to be coupled is added, followed by dicyclohexylcarbodiimide (0.15M in 50% DMF in dichloromethane) .
  • the Boc deprotection is accomplished by trifluoroacetic acid treatment. The progress of the coupling reactions is monitored by quantitative ninhydrin analysis.
  • PNA is released from the resin using anhydrous HF under standard conditions.
  • the products are purified using HPLC with acetonitrile-water (0.1%TFA) gradient and structure confirmed by fast atom bombardment mass spectro etry.
  • PNA homopolymer has the structure:
  • k is 1; is 1; 1 is 1; p is 0; R is OH; R 1 is H; and n is the number of bases in the oligomer sequence minus 1.
  • the effect of the addition of PNA oligomer on the expression of ICAM-1, VCAM-l and ELAM-1 on the surface of cells can be quantitated using specific monoclonal antibodies in an ELISA.
  • Cells are grown to confluence in 96 well microtiter plates. The cells are stimulated with either interleukin-1 or tumor necrosis factor, after pretreatment in the presence or absence of PNA oligomers for 4 to 8 hours to quantitate ELAM-1 and 8 to 24 hours to quantitate ICAM-1 and VCAM-l.
  • TCTGTAAGTCTGTGGGCCTC (SEQ ID NO: 23), AGTCTTGCTCCTTCCTCTTG (SEQ ID NO: 24), CTCATCAGGCTAGACTTTAA (SEQ ID NO: 25), TGTCCTCATGGTGGGGCTAT (SEQ ID NO: 26), TCTGAGTAGCAGAGGAGCTCGA (SEQ ID NO: 27), TCTGAGTAGCAGAGGAGCTC-K (SEQ ID NO: 28) TCTGAGTAGCAGAGGAGCTCGA (SEQ ID NO: 29) , ACCACACTGGTATTTCACAC (SEQ ID NO: 31), GTATGGAAGATTATAATATAT (SEQ ID NO: 32), CACAATCCTTAAGAACTCTTT (SEQ ID NO: 33), ACCTCTGCTGTTCTGATCCT (SEQ ID NO: 34), CTGCTGCCTCTGTCTCAGGT (SEQ ID NO: 35), AATCATGACTTCAAGAGTTCT (SEQ ID NO: 37) , TGAAGCAATCAT
  • GACAATATACAAACCTTCCAT (SEQ ID NO: 41), ACGTTTGGCCTCATGGAAGT (SEQ ID NO: 42), GGAATGCAAAGCACATCCAT (SEQ ID NO: 43), GGGCCAGAGACCCGAGGAGA (SEQ ID NO: 44), TTCCCCAGATGCACCTGTTT (SEQ ID NO: 45), CTGATTCAAGGCTTTGGCAG (SEQ ID NO: 46), CCAAAGTGAGAGCTGAGAGA (SEQ ID NO: 47), ACAGGATCTCTCAGGTGGGT (SEQ ID NO: 48), GAAGTCAGCCAAGAACAGCT (SEQ ID NO: 49), GCCTGGGAGGGTATTCAGCT (SEQ ID NO: 79) , GGCATTTTAAGTTGCTGTCG (SEQ ID NO: 80) , TGAACATATCAAGCATTAGC (SEQ ID NO: 81) , CGAATCTTGCTATGGCATAA (SEQ ID NO: 82) , AACCCAGTGCTC
  • the cells are gently washed three times with a buffered isotonic solution containing calcium and magnesium such as Dulbecco's phosphate buffered saline (D-PBS) .
  • D-PBS Dulbecco's phosphate buffered saline
  • the cells are then directly fixed on the microtiter plate with 1 to 2% paraformaldehyde diluted in D-PBS for 20 minutes at 25°C.
  • the cells are washed again with D-PBS three times. Nonspecific binding sites on the microtiter plate are blocked with 2% bovine serum albumin in D-PBS for 1 hour at 37°C.
  • Cells are incubated with the appropriate monoclonal antibody diluted in blocking solution for 1 hour at 37°C.
  • Unbound antibody is removed by washing the cells three times with D-PBS.
  • Antibody bound to the cells is detected by incubation with a 1:1000 dilution of biotinylated goat anti- mouse IgG (Bethesda Research Laboratories, Gaithersberg, MD) in blocking solution for 1 hour at 37°C.
  • Cells are washed three times with D-PBS and then incubated with a 1:1000 dilution of streptavidin conjugated to ⁇ -galactosidase (Bethesda Research Laboratories) for 1 hour at 37°C.
  • the cells are washed three times with D-PBS for 5 minutes each.
  • the concentration of the product is determined by measuring the absorbance at 575 nm in an ELISA microtiter plate reader
  • a second cellular assay which can be used to demonstrate the effects of PNA oligomer on ICAM-1, VCAM-l or ELAM-1 expression is a cell adherence assay.
  • Target cells are grown as a monolayer in a ultiwell plate, treated with the aforementioned PNA oligomers, directed to the adhesion molecule of choice, followed by cytokine.
  • the adhering cells are then added to the monolayer cells and incubated for 30 to 60 minutes at 37°C and washed to remove nonadhering cells.
  • Cells adhering to the monolayer may be determined either by directly counting the adhering cells or prelabeling the cells with a radioisotope such as 51Cr and quantitatmg the radioactivity associated with the monolayer as described. Dustin and Springer, J . Cell Biol . , 1988, 107 , 321-331. It is expected to observe the inhibition or reduction of cell adherence upon treatment of cells with PNA oligomers directed to the adhesion
  • the human lung carcinoma cell line A549 is obtained from the American Type Culture Collection (Bethesda MD) . Cells are grown in Dulbecco's Modified Eagle's Medium (Irvine Scientific, Irvine CA) containing 1 gm glucose/liter and 10% fetal calf serum (Irvine Scientific) . Human umbilical vein endothelial cells (HUVEC) (Clonetics, San Diego CA) are cultured in EGM-UV medium (Clonetics) . HUVEC are used between the second and sixth passages. Human epidermal carcinoma A431 cells are obtained from the American Type
  • Opti-MEM Gibcadel-containing either 10 ⁇ g/ml N-[l- (2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA, Bethesda Research Labs, Bethesda MD) in the case of HUVEC cells or 20 ⁇ g/ml DOTMA in the case of A549 cells is added to each well.
  • PNA oligomers directed to the adhesion molecules are sterilized by centrifugation through 0.2 ⁇ m Centrex cellulose acetate filters (Schleicher and Schuell, Keene, NH) .
  • PNA oligomers are added as 2Ox stock solution to the wells and incubated for 4 hours at 37°C. Medium is removed and replaced with 150 ⁇ l of the appropriate growth medium containing the indicated concentration of PNA oligomer. Cells are incubated for an additional 3 to 4 hours at 37°C then stimulated with the appropriate cytokine for 14 to 16 hours, as indicated.
  • ICAM- 1, ELAM-1, and VCAM-l expression are determined as described in Example 3.

Abstract

Oligomers useful for modulating metabolic processes are disclosed, for example oligomers directed against Human Intercellular Adhesion Molecule-1 (ICAM-1). These oligomers are comprised of subunits, at least one of which is a protein nucleic acid subunit. Therapeutic and diagnostic methods are also provided.

Description

OLIGOMERS FOR MODULATING METABOLIC FUNCTION
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part PCT/US91/02628 filed April 17, 1991 which is a continuation- in-part of U.S. Serial No. 516,969 filed April 30, 1990. This application is also a continuation-in-part of U.S. Serial No. 08/007,997 filed January 20, 1993, which is a continuation-in-part of 939,855 filed September 2, 1992 which is a continuation of PCT/US91/05209 filed July 23, 1991 and U.S. Serial No. 567,286 filed August 14, 1990. This application is also a continuation-in-part of PCT/US92/10785 filed December 16, 1992 which is a continuation-in-part of U.S. Serial No. 814,963 filed December 24, 1991. These applications are assigned to the assignee of this invention. The entire disclosure of each is incorporated herein by reference.
FIELD OF THE INVENTION
This invention is directed to compounds that are not polynucleotides yet which bind in a complementary fashion to DNA and RNA strands. In particular, the invention concerns compounds wherein naturally-occurring nucleobases or other nucleobase-binding moieties are covalently bound to a polyamide backbone. These compounds are useful for therapeutic and other applications directed to modulating metabolic processes. BACKGROUND OF THE INVENTION Peptide Nucleic Acids (PNAs)
Genes function by transferring information to a messenger RNA (mRNA) molecule, a process referred to as transcription. The interaction of mRNA with the riboso al complex directs the synthesis of a protein encoded within its sequence. This synthetic process is known as translation and requires the presence of various co-factors and building blocks, the amino acids, and their transfer RNAs (tRNA) , all of which are present in normal cells.
The initiation of transcription requires specific recognition of a promoter DNA sequence by the RNA- synthesizing enzyme, RNA polymerase. In many cases in prokaryotic cells, and most likely in all cases in eukaryotic cells, this recognition is preceded by sequence-specific binding of protein transcription factors to the promoter. Other proteins which bind to the promoter, but whose binding prohibits action of RNA polymerase, are known as repressors. Thus, gene activation is typically regulated positively by transcription factors and negatively by repressors.
Most conventional drugs function by interaction with and modulation of one or more targeted endogenous proteins, e.g., enzymes. However, such drugs are typically not specific for targeted proteins but interact with other proteins as well. Thus, a relatively large dose of drug must be used to effectively modulate a targeted protein. Typical daily doses of drugs are from 10" -10" millimoles per kilogram of body weight or 10" -10 millimoles for a 100 kilogram person. If this modulation could instead be effected by interaction with and inactivation of mRNA, a dramatic reduction in the necessary amount of drug could likely be achieved, along with a corresponding reduction in adverse side effects. Further reductions could be achieved if such interaction could be rendered site-specific. Given that a functioning gene continually produces mRNA throughout the life of the cell, it would thus be even more advantageous if gene transcription could be arrested in its entirety. Oligodeoxynucleotides offer such opportunities. For example, synthetic oligodeoxynucleotides have been used as antisense probes to block and eventually lead to the breakdown of mRNA. It also may be possible to modulate the genome of an animal by, for example, triple helix formation using oligonucleotides or other DNA recognizing agents. However, there are a number of drawbacks associated with triple helix formation. For example, it can only be used for homopurine sequences and it requires unphysiologically high ionic strength and low pH.
Unmodified oligonucleotides are impractical both in the antisense approach and in the triple helix approach because they have short in vivo half-lives. They are also poor penetrators of the cell membrane. These problems have resulted in an extensive search for improvements and alternatives. For example, the problems arising in connection with double-stranded DNA (dsDNA) recognition through triple helix formation have been diminished by a clever "switch back" chemical linking whereby a sequence of polypurine on one strand is recognized, and by "switching back", a homopurine sequence on the other strand can be recognized. Also, competent helix formation has been obtained by using artificial bases, thereby improving binding conditions with regard to ionic strength and pH. In order to improve half life as well as membrane penetration, a large number of variations in polynucleotide backbones has been undertaken. These variations include the use of ethylphosphonates, monothiophosphates, dithiophos- phates, phosphoramidates, phosphate esters, bridged phosphoro-amidates, bridged phosphorothioates, bridged methylene-phosphonates, dephospho internucleotide analogs with siloxane bridges, carbonate bridges, carboxymethyl ester bridges, acetamide bridges, carba ate bridges, thioether, sulfoxy, sulfono bridges, various "plastic" DNAs, -anomeric bridges, and borane derivatives.
The great majority of these modifications has led to decreased stability for hybrids formed between the modified oligonucleotide and its complementary, native oligonucleotide, as assayed by measuring Tm values. Consequently, it is generally understood in the art that backbone modifications destabilize such hybrids, i.e., result in lower Tm values, and should be kept to a minimum.
In WO 92/20702, moieties denominated peptide nucleic acids (PNAs) are disclosed wherein ligands are linked to a polyamide backbone through aza nitrogen atoms. In U.S. Serial No. 08/054,363 filed April 26, 1993, peptide nucleic acids are disclosed in which their recognition moieties are linked to the polyamide backbone additionally through amido and/or ureido tethers. PCT/EP 92/01219 filed May 22, 1992 also discloses protein nucleic acids.
These peptide nucleic acids are synthesized by adaptation of certain peptide synthesis procedures, either in solution or on a solid phase. The synthons used are certain monomer amino acids or their activated derivatives, protected by standard groups. These oligonucleotide analogs also can be synthesized by using the corresponding diacids and diamines.
Peptide nucleic acid oligomers have been found to be superior to prior reagents in that they have significantly higher affinity for complementary single stranded DNA (ssDNA) . These compounds are also able to form triple helices wherein a first PNA strand binds with RNA or ssDNA and a second PNA strand binds with the resulting double helix or with the first PNA strand. PNAs generally possess no significant charge and are water soluble, which facilitates cellular uptake. Moreover, PNAs contain amides of non- biological amino acids, making them biostable and resistant to enzymatic degradation, for example, by proteases.
Accordingly, PNAs can ideally be used to target RNA and ssDNA to produce antisense-type gene regulating moieties. Reagents that bind sequence-specifically to dsDNA, RNA, or ssDNA have applications as gene targeted drugs useful for modulating metabolic processes such as metabolic regulatory dysfunctions, such as cancer. PNAs can also be useful in diagnostics, as for example, as probes for specific mRNAs.
Adhesion Molecules
Human intercellular adhesion molecule-1 (ICAM-1) is encoded by a 3.3 kb mRNA resulting in the synthesis of a
55,219 dalton (Da) cell surface transme brane protein. ICAM- 1 is heavily glycosylated through N-linked glycosylation sites. The mature protein has an apparent molecular mass of 90 kDa as determined by SDS-polyacryla ide gel electrophoresis. Staunton et al., Cell 1988, 52 , 925-933. The primary binding site for ICAM-1 is lymphocyte-associated antigen-1 (LFA-1) . The expression of ICAM-1 can be regulated on vascular endothelial cells, fibroblasts, keratinocytes, astrocytes and several cell lines by treatment with bacterial lipopolysaccharide and cytokines such as interleukin-1, tumor necrosis factor, gamma-interferon, and lymphotoxin. See, e . g. , Frohman et al., J. Neuroimmunol . 1989, 23 , 117-124.
ICAM-1 plays a role in adhesion of neutrophils to vascular endothelium, as well as adhesion of onocytes and lymphocytes to vascular endothelium, tissue fibroblasts and epidermal keratinocytes. ICAM-1 also plays a role in T-cell recognition of antigen presenting cell, lysis of target cells by natural killer cells, lymphocyte activation and proliferation, and maturation of T cells in the thymus. In addition, recent data have demonstrated that ICAM-1 is the cellular receptor for the major serotype of rhinovirus, which account for greater than 50% of common colds. Staunton et al., Cell 1989, 56 , 849-853; Greve et al. , Cell 1989, 56 , 839-847. Expression of ICAM-1 has been associated with a variety of inflammatory skin disorders such as allergic contact dermatitis, fixed drug eruption, lichen planus, and psoriasis; Ho et al., J. Am . Acad . Dermatol . 1990, 22 , 64-68; Griffiths and Nickoloff, Am . J . Pathology 1989, 135 , 1045- 1053; Lisby et al., Br . J . Dermatol . 1989, 120 , 479-484; Shiohara et al., Arch. Dermatol . 1989, 125 , 1371-1376. In addition, ICAM-1 expression has been detected in the synovium of patients with rheumatoid arthritis; Hale et al., Arth . Rheum . 1989, 32 , 22-30, pancreatic B-cells in diabetes; Campbell et al., Proc . Natl . Acad . Sci . U.S .A . 1989, 86 , 4282-4286; thyroid follicular cells in patients with Graves' disease; Weetman et al., J . Endocrinol . 1989, 122 , 185-191; and with renal and liver allograft rejection; Faull and Russ, Transplantation 1989, 48 , 226-230; Adams et al., Lancet 1989, 1122-1125. Endothelial leukocyte adhesion molecule-1 (ELAM-1) is a 115-kDa membrane glycoprotein which is a member of the selectrin family of membrane glycoproteins. Bevilacqua et al., Science 1989, 243 , 1160-1165. The amino terminal region of ELAM-1 contains sequences with homologies to members of lectin-like proteins, followed by a domain similar to epidermal growth factor, followed by six tandem 60-amino acid repeats similar to those found in complement receptors 1 and 2. These features are also shared by GMP-140 and MEL-14 antigen, a lymphocyte homing antigen. ELAM-1 is encoded for by a 3.9 kb mRNA. The 3'-untranslated region of ELAM-1 mRNA contains several sequence motifs ATTTA which are responsible for the rapid turnover of cellular mRNA consistent with the transient nature of ELAM-1 expression.
ELAM-1 is primarily involved in the adhesion of neutrophils to vascular endothelial cells. ELAM-1 exhibits a limited cellular distribution in that it has only been identified on vascular endothelial cells. Like ICAM-1, ELAM-1 is inducible by a number of cytokines including tumor necrosis factor, interleukin-1 and lymphotoxin and bacterial lipopolysaccharide. In contrast to ICAM-1, ELAM-1 is not induced by gamma-interferon. Bevilacqua et al., Proc . Natl . Acad . Sci . USA 1987, 84 , 9238-9242; Wellicome et al., J . Immunol . 1990, 144 , 2558-2565.
Vascular cell adhesion molecule-1 (VCAM-1) is a 110-kDa membrane glycoprotein encoded by a 3.2 kb mRNA.
VCAM-1 appears to be encoded by a single-copy gene. Osborn et al., Cell 1989, 59 , 1203-1211. Like ICAM-1, VCAM-1 is a member of the immunoglobulin supergene family, containing six immunoglobulin-like domains of the H type. The receptor for VCAM-1 is proposed to be CD29 as demonstrated by the ability of monoclonal antibodies to CD29 to block adherence of Ramos cells to VCAM-1. VCAM-1 is expressed primarily on vascular endothelial cells. Like ICAM-1 and ELAM-1, expression of VCAM-1 on vascular endothelium is regulated by treatment with cytokines. Rice and Bevilacqua, Science 1989, 246 , 1303- 1306; Rice et al., J". Exp. Med . 1990, 171 , 1369-1374. Increased expression appears to be due to induction of the mRNA. VCAM-1 primarily binds T and B lymphocytes. In addition, VCAM-1 may play a role in the metastasis of melanoma, and possibly other cancers.
Inhibitors of ICAM-1, ELAM-1, and VCAM-l expression would provide a novel therapeutic class of anti-inflammatory agents with activity towards a variety of inflammatory diseases or diseases with an inflammatory component such as asthma, rheumatoid arthritis, allograft rejections, various dermatological conditions, and psoriasis. In addition, inhibitors of ICAM-1 may also be effective in the treatment of colds due to rhinovirus infection, AIDS, and some cancers and their metastasis. To date, there are no known therapeutic agents which effectively prevent the expression of the cellular adhesion molecule ICAM-1. The use of neutralizing monoclonal antibodies against ICAM-1 in animal models provide evidence that such inhibitors if identified would have therapeutic benefit for asthma; Wegner et al.. Science 1990, 247 , 456-459 and renal allografts; Cosimi et al., J. Immunol . 1990, 144 , 4604-4612. The use of a soluble form of ICAM-1 molecule was also effective in preventing hinovirus infection of cells in culture. Marlin et al.. Nature 1990, 344 , 70-72. Accordingly, methods of modulating the expression of adhesion molecules ICAM-1, ELAM-1 and VCAM- 1 are highly desireable. Highly specific binders of ICAM-1, ELAM-1 and VCAM-l mRNA would also be desireable for use in diagnostics and as research reagents. SUMMARY OF THE INVENTION
The present invention provides oligomers comprising peptide nucleic acids (PNAs) , that bind complementary ssDNA and RNA strands through their oligoribonucleotide ligands which are linked to a peptide backbone. The sequence of the oligoribonucleotide ligands specifies the target to which they bind. These PNA oligomers are useful as therapeutic agents for treating diseases like cancer, AIDS and genetic and metabolic diseases. These compositions are also useful in diagnostic applications and as research tools.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Oligomers of the present invention comprise oligomers wherein at least one subunit of the oligomer is a peptide nucleic acid subunit of the formula:
Figure imgf000010_0001
(I) wherein:
L is one of the adenine, thymine, cytosine or guanine heterocyclic bases of the oligomer;
C is (CR R ) where R is hydrogen and R is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R and R are independently selected from the group consisting of hydrogen, (C2-C6)alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C,-C6) alkoxy, (C.,-
3 hio, NRR4 and SR5, where each of R and R4 i.
C6)alkylt s independently selected from the group consisting of hydrogen, (C,-C4)alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C.,-C4) alkyl, hydroxy, alkoxy, alkylthio and amino; and R is hydrogen, (C1-C6)alkyl, hydroxy-, alkoxy-, or alkylthio- substituted (C1-C6)alkyl, or R and R taken together complete an alicyclic or heterocyclic system; D is (CR R )z where R and R are as defined above; each of y and z is zero or an integer from 1 to 10, the sum y + z being greater than 2 but not more than 10;
G is -NR3CO-, -NR3CS-, -NR3S0- or -NR3S02-, in either orientation, where R is as defined above; each pair of A and B is selected such that:
(a) A is a group of formula (Ila) , (lib) or (lie) and B is N or R N+; or
(b) A is a group of formula (lid) and B is CH;
Figure imgf000011_0001
(Ila) (lib)
Figure imgf000011_0002
(lie) (lid) where:
X is 0, S, Se, NR , CH2 or C(CH3)2;
Y is a single bond, 0, S or NR 4; each of p and q is zero or an integer from 1 to 5, the sum p+q being not more than 10; each of r and s is zero or an integer from 1 to 5, the sum r+s being not more than 10; each R 1 and R2 is independently selected from the group consisting of hydrogen, (C.-C^)alkyl which may be hydroxy- or alkoxy- or alkylthio- substituted, hydroxy, alkoxy, alkylthio, amino and halogen.
Subunits, as used herein, refers to basic unit which are chemically similar and which can form polymers. Repeating basic units form polymers referred to as "oligomers". Oligomers of the present invention may thus refer to oligomers in which substantially all subunits of the oligomer are subunits as described in Formula I. Oligomers of the present invention may also comprise one or more subunits which are naturally occuring nucleotides or nucleotide analogs as long as at least one subunit satisfies Formula I. Thus, oligomers as used herein may refer to a range of oligomers from oligomers comprising only one PNA subunit as defined in Formula I to oligomers in which every subunit is a PNA subunit as defined in Formula I.
Those subunits which are not PNA subunits comprise naturally occuring bases, sugars, and intersugar (backbone) linkages as well as non-naturally occurring portions which function similarly to naturally occurring portions. Sequences of oligomers of the present invention are defined by reference to the L group (for PNA subunits) or nucleobase (for nucleotide subunits) at a given position. Thus, for a given oligomer, the nomeclature is modeled after traditional nucleotide nomenclature, identifying each PNA subunit by the identity of its L group such as the heterocycles adenine (A) , thymine (T) , guanine (G) and cytosine (C) and identifying nucleotides or nucleosides by these same heterocycle residing on the sugar backbone. The sequences are conveniently provided in traditional 5' to 3' or amino to carboxy orientation.
Oligomers of the present invention may range in size from about 5 to about 50 subunits in length. In other embodiments of the present invention, oligomers may range in size from about 10 to about 30 subunits in length. In still other embodiments of the present invention oligomers may range in size from about 10 to about 25 subunits in length. In yet further embodiments of the present invention, oligomers may range in size from about 12 to about 20 subunits in length. The preparation of protein nucleic acid oligomers is known in the art, such as is described in PCT/EP 92/01219 filed May 22, 1992, which is incorporated by reference herein in its entirety.
Briefly, the principle of anchoring molecules onto a solid matrix, which helps in accounting for intermediate products during chemical transformations, is known as Solid- Phase Synthesis or Merrifield Synthesis (see, e . g. , Merrifield, J. Am . Chem . Soc . , 1963, 85, 2149 and Science , 1986, 232 , 341) . Established methods for the stepwise or fragmentwise solid-phase assembly of amino acids into peptides normally employ a beaded matrix of slightly cross- linked styrene-divinylbenzene copolymer, the cross-linked copolymer having been formed by the pearl polymerization of styrene monomer to which has been added a mixture of divinylbenzenes. A level of 1-2% cross-linking is usually employed. Such a matrix also can be used in solid-phase PNA synthesis in accordance with the present invention.
Concerning the initial functionalization of the solid phase, more than fifty methods have been described in connection with traditional solid-phase peptide synthesis (see, e . g . , Barany and Merrifield in "The Peptides" Vol. 2, Academic Press, New York, 1979, pp. 1-284, and Stewart and Young, "Solid Phase Peptide Synthesis", 2nd Ed., Pierce Chemical Company, Illinois, 1984) . Reactions for the introduction of chloromethyl functionality (Merrifield resin; via a chloromethyl methyl ether/SnCl4 reaction) , aminomethyl functionality (via an N-hydroxymethylphthalimide reaction; see , Mitchell, et al . , Tetrahedron Lett . , 1976, 3795) , and benzhydrylamino functionality (Pietta, et al . , J. Chem . Soc , 1970, 650) are the most widely applied. Regardless of its nature, the purpose of the functionality is normally to form an anchoring linkage between the copolymer solid support and the C-terminus of the first amino acid to be coupled to the solid support. As will be recognized, anchoring linkages also can be formed between the solid support and the amino acid N-terminus. It is generally convenient to express the "concentration" of a functional group in terms of millimoles per gram (mmol/g) . Other reactive functionalities which have been initially introduced include 4-methylbenzhydrylamino and 4-methoxybenzhydrylamino. All of these established methods are in principle useful within the context of the present in¬ vention. Preferred methods for PNA synthesis employ aminomethyl as the initial functionality, in that aminomethyl is particularly advantageous with respect to the incorporation of "spacer" or "handle" groups, owing to the reactivity of the amino group of the aminomethyl functionality with respect to the essentially quantitative formation of amide bonds to a carboxylic acid group at one end of the spacer-forming reagent. A vast number of relevant spacer- or handle-forming bifunctional reagents have been described (see, Barany, et al . , Int . J . Peptide Protein Res . , 1987, 30 , 705), especially reagents which are reactive towards amino groups such as found in the aminomethyl function. Representative bifunctional reagents include 4- (haloalkyl)aryl-lower alkanoic acids such as 4- (bromomethyl)phenylacetic acid, Boc-aminoacyl-4- (oxymethyl)aryl-lower alkanoic acids such as Boc-aminoacyl-4- (oxy ethyl)phenylacetic acid, N-Boc-p-acylbenzhydrylamines such as N-Boc-p-glutaroylbenzhydrylamine, N-Boc-4'-lower alkyl-p-acylbenzhydrylamines such as N-Boc-4'-methyl-p- glutaroylbenzhydrylamine, N-Boc-4'-lower alkoxy-p-acylbenz- hydrylamines such as N-Boc-4'-methoxy-p-glutaroyl-benzhy- drylamine, and 4-hydroxymethylphenoxyacetic acid. One type of spacer group particularly relevant within the context of the present invention is the phenylacetamidomethyl (Pam) handle (Mitchell and Merrifield, J . Org . Chem . , 1976, 41 , 2015) which, deriving from the electron withdrawing effect of the 4-phenylacetamidomethyl group, is about 100 times more stable than the classical benzyl ester linkage towards the Boc-amino deprotection reagent trifluoroacetic acid (TFA) .
Certain functionalities (e . g . , benzhydrylamino, 4- methylbenzhydrylamino and 4-methoxybenzhydrylamino) which may be incorporated for the purpose of cleavage of a synthesized PNA chain from the solid support such that the C-terminal of the PNA chain is in amide form, require no introduction of a spacer group. Any such functionality may advantageously be employed in the context of the present invention.
An alternative strategy concerning the introduction of spacer or handle groups is the so-called "preformed handle" strategy (see, Tarn, et al . , Synthesis , 1979, 955- 957) , which offers complete control over coupling of the first amino acid, and excludes the possibility of complications arising from the presence of undesired functional groups not related to the peptide or PNA synthesis. In this strategy, spacer or handle groups, of the same type as described above, are reacted with the first amino acid desired to be bound to the solid support, the amino acid being N-protected and optionally protected at the other side-chains which are not relevant with respect to the growth of the desired PNA chain. Thus, in those cases in which a spacer or handle group is desirable, the first amino acid to be coupled to the solid support can either be coupled to the free reactive end of a spacer group which has been bound to the initially introduced functionality (for example, an aminomethyl group) or can be reacted with the spacer- forming reagent. The space-forming reagent is then reacted with the initially introduced functionality. Other useful anchoring schemes include the "multidetachable" resins (Tarn, et al . , Tetrahedron Lett . , 1979, 4935 and J. Am . Chem . Soc , 1980, 102 , 611; Tam, J. Org . Chem . , 1985, 50 , 5291), which provide more than one mode of release and thereby allow more flexibility in synthetic design.
Suitable choices for N-protection are the tert- butyloxycarbonyl (Boc) group (Carpino, J . Am . Chem . Soc , 1957, 79 , 4427; McKay, et al . , J. Am . Chem . Soc , 1957, 79 , 4686; Anderson, et al . , J . Am . Chem . Soc , 1957, 79 , 6180) normally in combination with benzyl-based groups for the protection of side chains, and the 9-fluorenylmethyloxy- carbonyl (Fmoc) group (Carpino, et al . , J . Am . Chem . Soc , 1970, 92 , 5748 and J. Org . Chem . , 1972, 37 , 3404), normally in combination with tert-butyl (tBu) for the protection of any side chains, although a number of other possibilities exist which are well known in conventional solid-phase peptide synthesis. Thus, a wide range of other useful amino protecting groups exist, some of which are Adoc (Hass, et al . , J . Am . Chem . Soc , 1966, 88 , 1988), Bpoc (Sieber, Helv. Chem . Acta . , 1968, 51 , 614), Mcb (Brady, et al . , J. Org. Chem . , 1977, 42 , 143), Bic (Kemp, et al . , Tetrahedron , 1975, 4624), the o-nitrophenylsulfenyl (Nps) (Zervas, et al . , J. Am . Chem . Soc , 1963, 85 , 3660), and the dithiasuccinoyl (Dts) (Barany, et al . , J . Am . Chem . Soc , 1977, 99 , 7363). These amino protecting groups, particularly those based on the widely-used urethane functionality, successfully prohibit racemization (mediated by tautomerization of the readily formed oxazolinone (azlactone) intermediates (Goodman, et al . , J. Am . Chem . Soc , 1964, 86 , 2918)) during the coupling of most α-amino acids. In addition to such amino protecting groups,a whole range of otherwise "worthless" nonurethane- type of amino protecting groups are applicable when assembling PNA molecules, especially those built from achiral units. Thus, not only the above-mentioned amino protecting groups (or those derived from any of these groups) are useful within the context of the present invention, but virtually any amino protecting group which largely fulfills the following requirements: (1) stability to mild acids (not significantly attacked by carboxyl groups) ; (2) stability to mild bases or nucleophiles (not significantly attacked by the amino group in question) ; (3) resistance to acylation (not significantly attacked by activated amino acids) . Additionally: (4) the protecting group must be close to quantitatively removable, without serious side reactions, and (5) the optical integrity, if any, of the incoming amino acid should preferably be highly preserved upon coupling. Finally, the choice of side-chain protecting groups, in general, depends on the choice of the amino protecting group, since the protection of side-chain functionalities must withstand the conditions of the repeated amino deprotection cycles. This is true whether the overall strategy for chemically assembling PNA molecules relies on, for example, differential acid stability of amino and side-chain protecting groups (such as is the case for the above- mentioned "Boc-benzyl" approach) or employs an orthogonal, that is, chemoselective, protection scheme (such as is the case for the above-mentioned "Fmoc-tBu" approach) ,
Following coupling of the first amino acid, the next stage of solid-phase synthesis is the systematic elaboration of the desired PNA chain to incorporate additional subunits using monomer synthons. Novel monomer synthons may be selected from the group consisting of amino acids, diacids and diamines having general formulae:
L __ L
I I I
A A A
E^ -F E^C- -E ' V
(II) (III) (IV) wherein L, A, B, C and D are as defined above, except that any amino groups therein may be protected by amino protecting groups; E is COOH, CSOH, SOOH, S020H or an activated derivative thereof; and F is NHR or NPgR , where R is as defined above and Pg is an amino protecting group. This elaboration involves repeated deprotection/coupling cycles. The temporary protecting group, such as a Boc or Fmoc group, on the last-coupled amino acid is quantitatively removed by a suitable treatment, for example, by acidolysis, such as with trifluoroacetic acid, in the case of Boc, or by base treatment, such as with piperidine, in the case of Fmoc, so as to liberate the N-terminal amine function. The next desired N-protected amino acid is then coupled to the N-terminal of the last-coupled amino acid. This coupling of the C-terminal of an amino acid with the N- ter inal of the last-coupled amino acid can be achieved in several ways. For example, it can be bound by providing the incoming amino acid in a form with the carboxyl group activated by any of several methods, including the initial formation of an active ester derivative such as a 2,4,5- trichlorophenyl ester (Pless, et al . , Helv . Chim . Acta , 1963, 46, 1609), a phthalimido ester (Nefkens, et al . , J. Am . Chem . Soc , 1961, 83 , 1263), a pentachlorophenyl ester (Kupryszewski, Rocz . Chem . , 1961, 35 , 595), a pentafluoro- phenyl ester (Kovacs, et al . , J . Am . Chem . Soc , 1963, 85 , 183) , an o-nitrophenyl ester (Bodanzsky, Nature , 1955, 175 , 685), an imidazole ester (Li, et al . , J. Am . Chem . Soc , 1970, 92 , 7608), and a 3-hydroxy-4-oxo-3,4-dihydroquinazoline (Dhbt-OH) ester (Konig, et al . , Chem . Ber . , 1973, 103 , 2024 and 2034) , or the initial formation of an anhydride such as a symmetrical anhydride (Wieland, et al . , Angew. Chem . , Int . Ed . Engl . , 1971, 10 , 336). Alternatively, the carboxyl group of the incoming amino acid can be reacted directly with the N-terminal of the last-coupled amino acid with the assistance of a condensation reagent such as, for example, dicyclohexylcarbodiimide (Sheehan, et al. , J. Am . Chem . Soc , 1955, 77 , 1067) or derivatives thereof. Benzotriazolyl N- oxytrisdimethylaminophosphonium hexafluorophosphate (BOP) , "Castro's reagent" (see, e .g . , Rivaille, et al . , Tetrahedron , 1980, 36 , 3413) is recommended when assembling PNA molecules containing secondary amino groups. Finally, activated PNA monomers analogous to the recently-reported amino acid fluorides (Carpino, J. Am. Chem . Soc , 1990, 212, 9651) hold considerable promise to be used in PNA synthesis as well. Following assembly of the desired PNA chain, including protecting groups, the next step v/ill normally be deprotection of the amino acid moieties of the PNA chain and cleavage of the synthesized PNA from the solid support. These processes can take place substantially simultaneously, thereby providing the free PNA molecule in the desired form. Alternatively, in cases in which condensation of two separately synthesized PNA chains is to be carried out, it is possible by choosing a suitable spacer group at the start of the synthesis to cleave the desired PNA chains from their respective solid supports (both peptide chains still incorporating their side-chain protecting groups) and finally removing the side-chain protecting groups after, for example, coupling the two side-chain protected peptide chains to form a longer PNA chain.
In the above-mentioned "Boc-benzyl" protection scheme, the final deprotection of side-chains and release of the PNA molecule from the solid support is most often carried out by the use of strong acids such as anhydrous HF (Sakakibara, et al . , Bull . Chem . Soc . Jpn . , 1965, 38 , 4921), boron tris (trifluoroacetate) (Pless, et al . , Helv. Chim . Acta , 1973, 46, 1609), and sulfonic acids such as trifluoromethanesulfonic acid and methanesulfonic acid (Yajima, et al . , J. Chem . Soc , Chem . Comm . , 1974, 107) . This conventional strong acid (e . g . , anhydrous HF) deprotection method, produces very reactive carbocations that may lead to alkylation and acylation of sensitive residues in the PNA chain. Such side-reactions are only partly avoided by the presence of scavengers such as anisole, phenol, dimethyl sulfide, and mercaptoethanol and, therefore, the sulfide-assisted acidolytic SN2 deprotection method (Tam, ei al . , J . Am . Chem . Soc , 1983, 105 , 6442 and J. Am . Chem . Soc , 1986, 108 , 5242), the so-called "low", which removes the precursors of harmful carbocations to form inert sulfonium salts, is frequently employed in peptide and PNA synthesis, either solely or in combination with "high" methods. Less frequently, in special cases, other methods used for deprotection and/or final cleavage of the PNA-solid support bond are, for example, such methods as base-catalyzed alcoholysis (Barton, et al . , J . Am . Chem . Soc , 1973, 95 , 4501) , and ammonolysis as well as hydrazinolysis (Bodanszky, et al . , Chem . Ind . , 1964 1423), hydrogenolysis (Jones, Tetrahedron Lett . 1977 2853 and Schlatter, et al . , Tetrahedron Lett . 1977 2861)), and photolysis (Rich and Gurwara, J. Am . Chem . Soc , 1975 97 , 1575)).
Finally, in contrast with the chemical synthesis of "normal" peptides, stepwise chain building of achiral PNAs such as those based on aminoethylglycyl backbone units can start either from the N-terminus or the C-terminus, because the coupling reactions are free of racemization. Those skilled in the art will recognize that whereas syntheses commencing at the C-terminus typically employ protected amine groups and free or activated acid groups, syntheses commencing at the N-terminus typically employ protected acid groups and free or activated amine groups.
Based on the recognition that most operations are identical in the synthetic cycles of solid-phase peptide synthesis (as is also the case for solid-phase PNA synthesis) , a new matrix, PEPS, was recently introduced (Berg, et al . , J . Am . Chem . Soc , 1989, 111 , 8024 and International Patent Application WO 90/02749) to facilitate the preparation of large numbers of peptides. This matrix is comprised of a polyethylene (PE) film with pendant long-chain polystyrene (PS) grafts (molecular weight on the order of 10 ) . The loading capacity of the film is as high as that of a beaded matrix, but PEPS has the additional flexibility to suit multiple syntheses simultaneously. Thus, in a new configuration for solid-phase peptide synthesis, the PEPS film is fashioned in the form of discrete, labeled sheets, each serving as an individual compartment. During all the identical steps of the synthetic cycles, the sheets are kept together in a single reaction vessel to permit concurrent preparation of a multitude of peptides at a rate close to that of a single peptide by conventional methods. It was reasoned that the PEPS film support, comprising linker or spacer groups adapted to the particular chemistry in question, should be particularly valuable in the synthesis of multiple PNA molecules, these being conceptually simple to synthesize since only four different reaction compartments are normally required, one for each of the four "pseudo- nucleotide" units. Thus, the PEPS film support has been successfully tested in a number of PNA syntheses carried out in a parallel and substantially simultaneous fashion. The yield and quality of the products obtained from PEPS were comparable to those obtained by using the traditional po- lystyrene beaded support. Also, experiments with other geometries of the PEPS polymer such as, for example, non- woven felt, knitted net, sticks or microwellplates have not indicated any limitations of the synthetic efficacy. Two other methods proposed for the simultaneous synthesis of large numbers of peptides also apply to the preparation of multiple, different PNA molecules. The first of these methods (Geysen, et al . , Proc . Natl . Acad . Sci . USA, 1984, 81 , 3998) utilizes acrylic acid-grafted polyethylene- rods and 96-microtiter wells to immobilize the growing peptide chains and to perform the compartmentalized synthesis. While highly effective, the method is only applicable on a microgram scale. The second method (Houghten, Proc . Natl . Acad . Sci . USA, 1985, 82 , 5131) utilizes a "tea bag" containing traditionally-used polymer beads. Other relevant proposals for multiple peptide or PNA synthesis in the context of the present invention include the simultaneous use of two different supports with different densities (Tregear, in "Chemistry and Biology of Peptides" , J. Meienhofer, ed., Ann Arbor Sci. Publ., Ann Arbor, 1972 pp. 175-178) , combining of reaction vessels via a manifold (Gorman, Anal . Biochem . , 1984, 136 , 397), multicolumn solid- phase synthesis (e.g. Krchnak, et al . , Int . J . Peptide Protein Res . , 1989, 33 , 209), and Holm and Meldal, in "Proceedings of the 20th European Peptide Symposium" , G. Jung and E. Bayer, eds., Walter de Gruyter & Co., Berlin, 1989 pp. 208-210), and the use of cellulose paper (Eichler, et al . , Collect . Czech . Chem . Commun . , 1989, 54 , 1746). While the conventional cross-linked styrene/divinylbenzene copolymer matrix and the PEPS support are presently preferred in the context of solid-phase PNA synthesis, a non-limiting list of examples of solid supports which may be of relevance are: (1) Particles based upon copolymers of dimethylacrylamide cross-linked with N,N'- bisacryloylethylenediamine, including a known amount of N- tertbutoxycarbony1-beta-alany1-N'- acryloylhexamethylenedia ine. Several spacer molecules are typically added via the beta alanyl group, followed thereafter by the amino acid residue subunits. Also, the beta alanyl-containing monomer can be replaced with an acryloyl sarcosine monomer during polymerization to form resin beads. The polymerization is followed by reaction of the beads with ethylenediamine to form resin particles that contain primary amines as the covalently linked functionali¬ ty. The polyacrylamide-based supports are relatively more hydrophilic than are the polystyrene-based supports and are usually used with polar aprotic solvents including dimethyl- formamide, dimethylacetamide, N-methylpyrrolidone and the like (see Atherton, et al . , J . Am . Chem . Soc , 1975, 97 , 6584, Bioorg . Chem . 1979, 8, 351), and J.C.S. Perkin I 538 (1981)); (2) a second group of solid supports is based on silica-containing particles such as porous glass beads and silica gel. One example is the reaction product of trich- loro-[3-(4-chloromethyl)phenyl]propylsilane and porous glass beads (see Parr and Grohmann, Angew . Chem . Internal . Ed . 1972, 11 , 314) sold under the trademark "PORASIL E" by Waters Associates, Framingham, MA, USA. Similarly, a mono ester of 1,4-dihydroxymethylbenzene and silica (sold under the trademark "BIOPAK" by Waters Associates) has been reported to be useful (see Bayer and Jung, Tetrahedron Lett . , 1970, 4503) ; (3) a third general type of useful solid supports can be termed composites in that they contain two major ingredients: a resin and another material that is also substantially inert to the organic synthesis reaction conditions employed. One exemplary composite (see Scott, et al . , J . Chrom . Sci . , 1971, 9 , 577) utilized glass particles coated with a hydrophobic, cross-linked styrene polymer containing reactive chloromethyl groups, and was supplied by Northgate Laboratories, Inc., of Hamden, CT, USA. Another exemplary composite contains a core of fluorinated ethylene polymer onto which has been grafted polystyrene (see Kent and Merrifield, Israel J . Chem . 1978, 17 , 243) and van
Rietschoten in "Peptides 1974 " , Y . Wolman, Ed., Wiley and Sons, New York, 1975, pp. 113-116) ; and (4) contiguous solid supports other than PEPS, such as cotton sheets (Lebl and Eichler, Peptide Res . 1989, 2, 232) and hydroxypropyla- crylate-coated polypropylene membranes (Daniels, et al . , Tetrahedron Lett . 1989, 4345), are suited for PNA synthesis as well.
Whether manually or automatically operated, solid- phase PNA synthesis in the context of the present invention is normally performed batchwise. However, most of the syn¬ theses may equally well be carried out in the continuous-flow mode, where the support is packed into columns (Bayer, et al . , Tetrahedron Lett . , 1970, 4503 and Scott, et al . , J. Chromatogr. Sci . , 1971, 9 , 577) . With respect to continuous- flow solid-phase synthesis, the rigid poly(dimethylacrylami- de)-Kieselguhr support (Atherton, et al . , J . Chem . Soc Chem . Commun . , 1981, 1151) appears to be particularly successful, but another valuable configuration concerns the one worked out for the standard copoly(styrene-l%-divinylbenzene) support (Krchnak, et al . , Tetrahedron Lett . , 1987, 4469). While the solid-phase technique is presently preferred in the context of PNA synthesis, other methodologies or combinations thereof, for example, in combination with the solid-phase technique, apply as well:
(1) the classical solution-phase methods for peptide synthesis (e . g. , Bodanszky, "Principles of Peptide Synthesis" , Springer-Verlag, Berlin-New York 1984), either by stepwise assembly or by segment/fragment condensation, are of particular relevance when considering especially large scale productions (gram, kilogram, and even tons) of PNA compounds;
(2) the so-called "liquid-phase" strategy, which utilizes soluble polymeric supports such as linear polystyrene
(Shemyakin, et al . , Tetrahedron Lett . , 1965, 2323) and polyethylene glycol (PEG) (Mutter and Bayer, Angew . Chem . , Int . Ed. Engl . , 1974, 13 , 88), is useful; (3) random polymerization (see, e . g. , Odian, "Principles of Polymerization" , McGraw-Hill, New York (1970)) yielding mixtures of many molecular weights ("polydiεperse") peptide or PNA molecules are particularly relevant for purposes such as screening for antiviral effects; (4) a technique based on the use of polymer-supported amino acid active esters (Fridkin, et al . , J . Am . Chem . Soc , 1965, 87 , 4646), sometimes referred to as "inverse Merrifield synthesis" or "polymeric reagent synthesis", offers the advantage of isolation and purification of intermediate products, and may thus provide a particularly suitable method for the synthesis of medium-sized, optionally protected, PNA molecules, that can subsequently be used for fragment condensation into larger PNA molecules; (5) it is envisaged that PNA molecules may be assembled enzymatically by enzymes such as proteases or derivatives thereof with novel specificities (obtained, for example, by artificial means such as protein engineering) . Also, one can envision the development of "PNA ligases" for the condensation of a number of PNA fragments into very large PNA molecules; (6) since antibodies can be generated to virtually any molecule of interest, the recently developed catalytic antibodies (abzymes) , discovered simultaneously by the groups of Lerner (Tramantano, et al . , Science , 1986, 234 , 1566) and of Schultz (Pollack, et al . , Science , 1986, 234 , 1570), should also be considered as potential candidates for assembling PNA molecules. Thus, there has been considerable success in producing abzymes catalyzing acyl-transfer reactions (see for example Shokat, et al . , Nature , 1989, 338 , 269) and references therein). Finally, completely artificial enzymes, very recently pioneered by Stewart's group (Hahn, et al . , Science , 1990, 248 , 1544), may be developed to suit PNA synthesis. The design of generally applicable enzymes, ligases, and catalytic antibodies, capable of mediating specific coupling reactions, should be more readily achieved for PNA synthesis than for "normal" peptide synthesis since PNA molecules will often be comprised of only four different amino acids (one for each of the four native nucleobases) as compared to the twenty natural by occurring (proteinogenic) amino acids constituting peptides. In conclusion, no single strategy may be wholly suitable for the synthesis of a specific PNA molecule, and therefore, sometimes a combination of methods may work best.
Peptide nucleic acid oligomers hybridizable with, or targeted to, metabolic targets are provided by the present invention. By hybridizable is meant that at least 70% sequence homology is present. In preferred embodiments of the present invention, peptide nucleic acid oligomers have at least 85% sequence homology to a desired target. In still more preferred embodiments of the present invention, peptide nucleic acid oligomers of the present invention are at least 95% homologous to a target of interest.
Oligomers of the present invention comprising PNA subunits can be used in diagnostics, therapeutics and as research reagents and kits. . Diagnostic and research reagents may be employed by contacting a cell or other biological sample such as blood, urine, cerebral fluid, ascites, etc. with oligomers of the present invention in vitro .
Oligomers of the invention can be formulated in a pharmaceutical composition, which can include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the oligomer. Pharmaceutical compositions also can include one or more active ingredients such as antimicrobial agents, anti- inflammatory agents, anesthetics, and the like in addition to oligomer.
The pharmaceutical composition can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration can be topically (including opthalmically, vaginally, rectally, intranasally) , orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
Formulations for topical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms may also be useful.
Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets.
Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
Formulations for parenteral administration can include sterile aqueous solutions which also can contain buffers, diluents and other suitable additives.
Dosing is dependent on severity and responsiveness of the condition to be treated, but will normally be one or more doses per day, with course of treatment lasting from several days to several months or until a cure is effected or a diminution of disease state is achieved. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates.
Such methodologies will be useful for targeting the following targets for treatment of metabolic disfunctions.
Intercellular Adhesion Molecule-1 (ICAM-1)
A series of oligomers targeted to the translation initiation codon (AUG) , 5' untranslated region (5' UTR) , 5' CAP region, coding region, translation termination region or 3' untranslated region (3' UTR) of ICAM-1 were identified having specific sequences. These oligomers will be useful for the treatment of conditions modulated by or associated with ICAM-1 such as asthma, rheumatoid arthritis, allograft rejection, and psoriasis. These oligomers will also be useful as diagnostic and research reagents. The sequences, SEQ ID numbers and targets of these oligomers are shown in Table 1. SEQUENCE
TGGGAGCCATAGCGAGGC GAGGAGCTCAGCGTCGACTG GACACTCAATAAATAGCTGGT GAGGCTGAGGTGGGAGGA CGATGGGCAGTGGGAAAG GGGCGCGTGATCCTTATAGC CATAGCGAGGCTGAGGTTGC CGGGGGCTGCTGGGAGCCAT AGAGCCCCGAGCAGGACCAG TGCCCATCAGGGCAGTTTGA GGTCACACTGACTGAGGCCT CTCGCGGGTGACCTCCCCTT TCAGGGAGGCGTGGCTTGTG CCTGTCCCGGGGATAGGTTCA CCCCCACCACTTCCCCTCTC TTGAGAAAGCTTTATTAACT AGCCATAGCGAGGC CCATAGCGAGGC ATAGCGAGGC TGGGAGCCATAGCGAG GGAGCCATAGCGAGGC GCCCAAGCTGGCATCCGTCA TCTGTAAGTCTGTGGGCCTC AGTCTTGCTCCTTCCTCTTG CTCATCAGGCTAGACTTTAA TGTCCTCATGGTGGGGCTAT TCTGAGTAGCAGAGGAGCTCGA TCTGAGTAGCAGAGGAGCTC-K TCTGAGTAGCAGAGGAGCTCGA
Figure imgf000027_0001
Endothelial Leukocyte Adhesion Molecule-l (ELAM-1)
A series of oligomers targeted to the translation initiation codon (AUG) , 5' untranslated region (5' UTR), coding region, intron/exon (I/E) junction, or 3' untranslated region (3' UTR) of ELAM-1 were identified having specific sequences. These oligomers will be useful for the treatment of conditions modulated by or associated with ELAM- 1 such as asthma, rheumatoid arthritis, allograft rejection, and psoriasis. The sequences, SEQ ID numbers and targets of these oligomers are shown in Table 2.
SEQUENCE SEQ ID NO:
CAATCATGACTTCAAGAGTTCT 30 ACCACACTGGTATTTCACAC 31 GTATGGAAGATTATAATATAT 32 CACAATCCTTAAGAACTCTTT 33 ACCTCTGCTGTTCTGATCCT 34 ' CTGCTGCCTCTGTCTCAGGT 35 GGTATTTGACACAGC 36
AATCATGACTTCAAGAGTTCT 37 TGAAGCAATCATGACTTCAAG 38 TATAGGAGTTTTGATGTGAA 39 ACAATGAGGGGGTAATCTACA 40 GACAATATACAAACCTTCCAT 41 ACGTTTGGCCTCATGGAAGT 42 GGAATGCAAAGCACATCCAT 43 GGGCCAGAGACCCGAGGAGA 44 TTCCCCAGATGCACCTGTTT 45 CTGATTCAAGGCTTTGGCAG 46 CCAAAGTGAGAGCTGAGAGA 47 ACAGGATCTCTCAGGTGGGT 48 GAAGTCAGCCAAGAACAGCT 49 TCACTGCTGCCTCTGTCTCAGG 50 TGATTCTTTTGAACTTAAAAGGA 51 TTAAAGGATGTAAGAAGGCT 52 CATAAGCACATTTATTGTC 53 TTTTGGGAAGCAGTTGTTCA 54 AACTGTGAAGCAATCATGACT 55 CCTTGAGTGGTGCATTCAACCT 56 AATGCTTGCTCACACAGGCATT 57 CTCTCAGGTGGGTATCACTG
Figure imgf000028_0001
58 Vascular Cell Adhesion Molecule-l (VCAM-l)
A series of oligomers targeted to the translation initiation codon (AUG) , 5' untranslated region (5' UTR) , coding region, exon/intron (E/I) junction, translation termination region or 3' untranslated region (3' UTR) of VCAM-l were identified having specific sequences. These oligomers will be useful for the treatment of conditions modulated by or associated with VCAM-l such as asthma, rheumatoid arthritis, allograft rejection, and psoriasis. The sequences, SEQ ID numbers and targets of these oligomers are shown in Table 3.
SEQUENCE
GCCTGGGAGGGTATTCAGCT GGCATTTTAAGTTGCTGTCG TGAACATATCAAGCATTAGC GCAATCTTGCTATGGCATAA AACCCAGTGCTCCCTTTGCT GGCCACATTGGGAAAGTTGC CCCGGCATCTTTACAAAACC AACATCTCCGTACCATGCCA CCTGTGTGTGCCTGGGAGGG CAGCCTGCCTTACTGTGGGC CTTGAACAATTAATTCCACCT GTCTTTGTTGTTTTCTCTTCC CTGTGTCTCCTGTCTCCGCT CGATGCAGATACCGCGGAGT TTACCATTGACATAAAGTGTT CCAGGCATTTTAAGTTGCTGT CCTGAAGCCAGTGAGGCCCG GATGAGAAAATAGTGGAACCA CTGAGCAAGATATCTAGAT CTACACTTTTGATTTCTGT TTGAACATATCAAGCATTAGCT TTTACATATGTACAAATTATGT AATTATCACTTTACTATACAAA AGGGCTGACCAAGACGGTTGT CCATCTTCCCAGGCATTTTA
Figure imgf000030_0001
The following examples are provided for illustrative purposes only and are not intended to limit the invention. Example 1
General Method for the Synthesis of PNA Oligomers
PNA subunits for oligomers of the invention are prepared generally in accordance with the methods disclosed by WO 92/20702, incorporated by reference herein in its entirety. Benzyhydrylamine resin (initially loaded 0.28 mmol/gm with Boc-L-Lys(2-chlorobenyloxycarbonyl) ) is swollen in DMF and an excess of a monomer to be coupled is added, followed by dicyclohexylcarbodiimide (0.15M in 50% DMF in dichloromethane) . The Boc deprotection is accomplished by trifluoroacetic acid treatment. The progress of the coupling reactions is monitored by quantitative ninhydrin analysis. The PNA is released from the resin using anhydrous HF under standard conditions. The products are purified using HPLC with acetonitrile-water (0.1%TFA) gradient and structure confirmed by fast atom bombardment mass spectro etry. PNA homopolymer has the structure:
Figure imgf000032_0001
wherein k is 1; is 1; 1 is 1; p is 0; R is OH; R1 is H; and n is the number of bases in the oligomer sequence minus 1.
Example 2 Determination of Adhesion Molecule Expression
The effect of the addition of PNA oligomer on the expression of ICAM-1, VCAM-l and ELAM-1 on the surface of cells can be quantitated using specific monoclonal antibodies in an ELISA. Cells are grown to confluence in 96 well microtiter plates. The cells are stimulated with either interleukin-1 or tumor necrosis factor, after pretreatment in the presence or absence of PNA oligomers for 4 to 8 hours to quantitate ELAM-1 and 8 to 24 hours to quantitate ICAM-1 and VCAM-l. PNA oligomers prepared in accordance with Example 1, having the following oligomer sequences: TGGGAGCCATAGCGAGGC (SEQ ID NO: 1), GAGGAGCTCAGCGTCGACTG (SEQ ID NO: 2), GACACTCAATAAATAGCTGGT (SEQ ID NO: 3), GAGGCTGAGGTGGGAGGA (SEQ ID NO: 4) , CGATGGGCAGTGGGAAAG (SEQ ID NO: 5), GGGCGCGTGATCCTTATAGC (SEQ ID NO: 6) , CATAGCGAGGCTGAGGTTGC (SEQ ID NO: 7), CGGGGGCTGCTGGGAGCCAT (SEQ ID NO: 8), AGAGCCCCGAGCAGGACCAG (SEQ ID NO: 9) , TGCCCATCAGGGCAGTTTGA (SEQ ID NO: 10), GGTCACACTGACTGAGGCCT (SEQ ID NO: 11), CTCGCGGGTGACCTCCCCTT (SEQ ID NO: 12), TCAGGGAGGCGTGGCTTGTG (SEQ ID NO: 13), CCTGTCCCGGGGATAGGTTCA (SEQ ID NO: 14), CCCCCACCACTTCCCCTCTC (SEQ ID NO: 15), TTGAGAAAGCTTTATTAACT (SEQ ID NO: 16) , AGCCATAGCGAGGC (SEQ ID NO: 17) , TGGGAGCCATAGCGAG (SEQ ID NO: 20), GGAGCCATAGCGAGGC (SEQ ID NO: 21), GCCCAAGCTGGCATCCGTCA (SEQ ID NO: 22),
TCTGTAAGTCTGTGGGCCTC (SEQ ID NO: 23), AGTCTTGCTCCTTCCTCTTG (SEQ ID NO: 24), CTCATCAGGCTAGACTTTAA (SEQ ID NO: 25), TGTCCTCATGGTGGGGCTAT (SEQ ID NO: 26), TCTGAGTAGCAGAGGAGCTCGA (SEQ ID NO: 27), TCTGAGTAGCAGAGGAGCTC-K (SEQ ID NO: 28) TCTGAGTAGCAGAGGAGCTCGA (SEQ ID NO: 29) , ACCACACTGGTATTTCACAC (SEQ ID NO: 31), GTATGGAAGATTATAATATAT (SEQ ID NO: 32), CACAATCCTTAAGAACTCTTT (SEQ ID NO: 33), ACCTCTGCTGTTCTGATCCT (SEQ ID NO: 34), CTGCTGCCTCTGTCTCAGGT (SEQ ID NO: 35), AATCATGACTTCAAGAGTTCT (SEQ ID NO: 37) , TGAAGCAATCATGACTTCAAG (SEQ ID NO: 38), TATAGGAGTTTTGATGTGAA (SEQ ID NO: 39),
GACAATATACAAACCTTCCAT (SEQ ID NO: 41), ACGTTTGGCCTCATGGAAGT (SEQ ID NO: 42), GGAATGCAAAGCACATCCAT (SEQ ID NO: 43), GGGCCAGAGACCCGAGGAGA (SEQ ID NO: 44), TTCCCCAGATGCACCTGTTT (SEQ ID NO: 45), CTGATTCAAGGCTTTGGCAG (SEQ ID NO: 46), CCAAAGTGAGAGCTGAGAGA (SEQ ID NO: 47), ACAGGATCTCTCAGGTGGGT (SEQ ID NO: 48), GAAGTCAGCCAAGAACAGCT (SEQ ID NO: 49), GCCTGGGAGGGTATTCAGCT (SEQ ID NO: 79) , GGCATTTTAAGTTGCTGTCG (SEQ ID NO: 80) , TGAACATATCAAGCATTAGC (SEQ ID NO: 81) , CGAATCTTGCTATGGCATAA (SEQ ID NO: 82) , AACCCAGTGCTCCCTTTGCT (SEQ ID NO: 83) are employed in the determination of adhesion molecule expression. Following the appropriate incubation time with the cytokine, the cells are gently washed three times with a buffered isotonic solution containing calcium and magnesium such as Dulbecco's phosphate buffered saline (D-PBS) . The cells are then directly fixed on the microtiter plate with 1 to 2% paraformaldehyde diluted in D-PBS for 20 minutes at 25°C. The cells are washed again with D-PBS three times. Nonspecific binding sites on the microtiter plate are blocked with 2% bovine serum albumin in D-PBS for 1 hour at 37°C. Cells are incubated with the appropriate monoclonal antibody diluted in blocking solution for 1 hour at 37°C. Unbound antibody is removed by washing the cells three times with D-PBS. Antibody bound to the cells is detected by incubation with a 1:1000 dilution of biotinylated goat anti- mouse IgG (Bethesda Research Laboratories, Gaithersberg, MD) in blocking solution for 1 hour at 37°C. Cells are washed three times with D-PBS and then incubated with a 1:1000 dilution of streptavidin conjugated to β-galactosidase (Bethesda Research Laboratories) for 1 hour at 37°C. The cells are washed three times with D-PBS for 5 minutes each. The amount of β-galactosidase bound to the specific monoclonal antibody is determined by developing the plate in a solution of 3.3 mM chlorophenolred-β-D-galactopyranoside, 50 mM sodium phosphate, 1.5 mM MgCl2; pH=7.2 for 2 to 15 minutes at 37°C. The concentration of the product is determined by measuring the absorbance at 575 nm in an ELISA microtiter plate reader
It is expected to observe the down-regulation of ICAM-1, VCAM-l and ELAM-1 cell surface expression as a result of treating the cells with PNA oligomers directed to the aformentioned adhesion molecules. Thus the concentration of product in the ELISA assay determined by the absorbance at 575 nm will be expected to be reduced.
Example 3
Cell Adherence Assay
A second cellular assay which can be used to demonstrate the effects of PNA oligomer on ICAM-1, VCAM-l or ELAM-1 expression is a cell adherence assay. Target cells are grown as a monolayer in a ultiwell plate, treated with the aforementioned PNA oligomers, directed to the adhesion molecule of choice, followed by cytokine. The adhering cells are then added to the monolayer cells and incubated for 30 to 60 minutes at 37°C and washed to remove nonadhering cells. Cells adhering to the monolayer may be determined either by directly counting the adhering cells or prelabeling the cells with a radioisotope such as 51Cr and quantitatmg the radioactivity associated with the monolayer as described. Dustin and Springer, J . Cell Biol . , 1988, 107 , 321-331. It is expected to observe the inhibition or reduction of cell adherence upon treatment of cells with PNA oligomers directed to the adhesion molecules.
Example 4 Cell Culture and Treatment with PNA Oligomers Directed to Adhesion Molecules
The human lung carcinoma cell line A549 is obtained from the American Type Culture Collection (Bethesda MD) . Cells are grown in Dulbecco's Modified Eagle's Medium (Irvine Scientific, Irvine CA) containing 1 gm glucose/liter and 10% fetal calf serum (Irvine Scientific) . Human umbilical vein endothelial cells (HUVEC) (Clonetics, San Diego CA) are cultured in EGM-UV medium (Clonetics) . HUVEC are used between the second and sixth passages. Human epidermal carcinoma A431 cells are obtained from the American Type
Culture Collection and cultured in DMEM with 4.5 g/1 glucose. Primary human keratinocytes are obtained from Clonetics and grown in KGM (Keratinocyte growth medium, Clonetics) .
Cells grown in 96-well plates are washed three times with Opti-MEM (GIBCO, Grand Island, NY) prewarmed to 37°C. 100 μl of Opti-MEM containing either 10 μg/ml N-[l- (2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA, Bethesda Research Labs, Bethesda MD) in the case of HUVEC cells or 20 μg/ml DOTMA in the case of A549 cells is added to each well. PNA oligomers directed to the adhesion molecules, as described in Example 2, are sterilized by centrifugation through 0.2 μm Centrex cellulose acetate filters (Schleicher and Schuell, Keene, NH) . PNA oligomers are added as 2Ox stock solution to the wells and incubated for 4 hours at 37°C. Medium is removed and replaced with 150 μl of the appropriate growth medium containing the indicated concentration of PNA oligomer. Cells are incubated for an additional 3 to 4 hours at 37°C then stimulated with the appropriate cytokine for 14 to 16 hours, as indicated. ICAM- 1, ELAM-1, and VCAM-l expression are determined as described in Example 3.

Claims

WHAT IS CLAIMED IS:
1. An oligomer hybridizable to the AUG region, coding region, 5' untranslated region, or 3' untranslated region of ICAM-1 and comprising at least one peptide nucleic acid subunit.
2. The oligomer of claim 1 wherein the sequence of the oligomer is selected from the group consisting of:
TGGGAGCCATAGCGAGGC (SEQ ID NO: 1) ;
GAGGAGCTCAGCGTCGACTG (SEQ ID NO: 2); GACACTCAATAAATAGCTGGT (SEQ ID NO: 3);
GAGGCTGAGGTGGGAGGA (SEQ ID NO: 4) ;
CGATGGGCAGTGGGAAAG (SEQ ID NO: 5) ;
GGGCGCGTGATCCTTATAGC (SEQ ID NO: 6) ;
CATAGCGAGGCTGAGGTTGC (SEQ ID NO: 7); CGGGGGCTGCTGGGAGCCAT (SEQ ID NO: 8);
AGAGCCCCGAGCAGGACCAG (SEQ ID NO: 9) ;
TGCCCATCAGGGCAGTTTGA (SEQ ID NO: 10)
GGTCACACTGACTGAGGCCT (SEQ ID NO: 11)
CTCGCGGGTGACCTCCCCTT (SEQ ID NO: 12) TCAGGGAGGCGTGGCTTGTG (SEQ ID NO: 13)
CCTGTCCCGGGGATAGGTTCA (SEQ ID NO: 14);
CCCCCACCACTTCCCCTCTC (SEQ ID NO: 15) ;
TTGAGAAAGCTTTATTAACT (SEQ ID NO: 16);
AGCCATAGCGAGGC (SEQ ID NO: 17); CCATAGCGAGGC (SEQ ID NO: 18);
ATAGCGAGGC (SEQ ID NO: 19);
TGGGAGCCATAGCGAG (SEQ ID NO: 20);
GGAGCCATAGCGAGGC (SEQ ID NO: 21);
GCCCAAGCTGGCATCCGTCA (SEQ ID NO: 22) TCTGTAAGTCTGTGGGCCTC (SEQ ID NO: 23)
AGTCTTGCTCCTTCCTCTTG (SEQ ID NO: 24)
CTCATCAGGCTAGACTTTAA (SEQ ID NO: 25)
TGTCCTCATGGTGGGGCTAT (SEQ ID NO: 26)
TCTGAGTAGCAGAGGAGCTCGA (SEQ ID NO: 27); TCTGAGTAGCAGAGGAGCTC-K (SEQ ID NO: 28) and
TCTGAGTAGCAGAGGAGCTCGA (SEQ ID NO: 29) .
3. The oligomer of claim 1 wherein the sequence is selected from the group consisting of:
TGGGAGCCATAGCGAGGC (SEQ ID NO: 1) ;
GAGGAGCTCAGCGTCGACTG (SEQ ID NO: 2); GACACTCAATAAATAGCTGGT (SEQ ID NO: 3);
GAGGCTGAGGTGGGAGGA (SEQ ID NO: 4);
CGATGGGCAGTGGGAAAG (SEQ ID NO: 5) ;
GGGCGCGTGATCCTTATAGC (SEQ ID NO: 6);
CATAGCGAGGCTGAGGTTGC (SEQ ID NO: 7) ; CGGGGGCTGCTGGGAGCCAT (SEQ ID NO: 8) ;
AGAGCCCCGAGCAGGACCAG (SEQ ID NO: 9) ;
TGCCCATCAGGGCAGTTTGA (SEQ ID NO: 10);
GGTCACACTGACTGAGGCCT (SEQ ID NO: 11) ;
CTCGCGGGTGACCTCCCCTT (SEQ ID NO: 12); TCAGGGAGGCGTGGCTTGTG (SEQ ID NO: 13);
CCTGTCCCGGGGATAGGTTCA (SEQ ID NO: 14);
CCCCCACCACTTCCCCTCTC (SEQ ID NO: 15) ;
TTGAGAAAGCTTTATTAACT (SEQ ID NO: 16) ;
AGCCATAGCGAGGC (SEQ ID NO: 17); TGGGAGCCATAGCGAG (SEQ ID NO: 20);
GGAGCCATAGCGAGGC (SEQ ID NO: 21);
GCCCAAGCTGGCATCCGTCA (SEQ ID NO: 22)
TCTGTAAGTCTGTGGGCCTC (SEQ ID NO: 23)
AGTCTTGCTCCTTCCTCTTG (SEQ ID NO: 24) CTCATCAGGCTAGACTTTAA (SEQ ID NO: 25)
TGTCCTCATGGTGGGGCTAT (SEQ ID NO: 26)
TCTGAGTAGCAGAGGAGCTCGA (SEQ ID NO: 27);
TCTGAGTAGCAGAGGAGCTC-K (SEQ ID NO: 28) and
TCTGAGTAGCAGAGGAGCTCGA (SEQ ID NO: 29) .
4. The oligomer of claim 1 wherein substantially all the subunits of the oligomer are peptide nucleic acid subunits.
5. An oligomer of claim 2 incorporated in a pharmaceutically acceptable carrier.
6. An oligomer having a sequence selected from the group consisting of:
TGGGAGCCATAGCGAGGC (SEQ ID NO: 1) ;
GAGGAGCTCAGCGTCGACTG (SEQ ID NO: 2) ; GACACTCAATAAATAGCTGGT (SEQ ID NO: 3);
GAGGCTGAGGTGGGAGGA (SEQ ID NO: 4);
CGATGGGCAGTGGGAAAG (SEQ ID NO: 5) ;
GGGCGCGTGATCCTTATAGC (SEQ ID NO: 6) ;
CATAGCGAGGCTGAGGTTGC (SEQ ID NO: 7) ; CGGGGGCTGCTGGGAGCCAT (SEQ ID NO: 8) ;
AGAGCCCCGAGCAGGACCAG (SEQ ID NO: 9) ;
TGCCCATCAGGGCAGTTTGA (SEQ ID NO: 10);
GGTCACACTGACTGAGGCCT (SEQ ID NO: 11) ;
CTCGCGGGTGACCTCCCCTT (SEQ ID NO: 12) ; TCAGGGAGGCGTGGCTTGTG (SEQ ID NO: 13);
CCTGTCCCGGGGATAGGTTCA (SEQ ID NO: 14);
CCCCCACCACTTCCCCTCTC (SEQ ID NO: 15) ;
TTGAGAAAGCTTTATTAACT (SEQ ID NO: 16);
AGCCATAGCGAGGC (SEQ ID NO: 17); CCATAGCGAGGC (SEQ ID NO: 18);
ATAGCGAGGC (SEQ ID NO: 19);
TGGGAGCCATAGCGAG (SEQ ID NO: 20);
GGAGCCATAGCGAGGC (SEQ ID NO: 21);
GCCCAAGCTGGCATCCGTCA (SEQ ID NO: 22) TCTGTAAGTCTGTGGGCCTC (SEQ ID NO: 23)
AGTCTTGCTCCTTCCTCTTG (SEQ ID NO: 24)
CTCATCAGGCTAGACTTTAA (SEQ ID NO: 25)
TGTCCTCATGGTGGGGCTAT (SEQ ID NO: 26)
TCTGAGTAGCAGAGGAGCTCGA (SEQ ID NO: 27); TCTGAGTAGCAGAGGAGCTC-K (SEQ ID NO: 28) and
TCTGAGTAGCAGAGGAGCTCGA (SEQ ID NO: 29); and wherein at least one subunit of the oligomer is a peptide nucleic acid subunit of the formula: A
Figure imgf000040_0001
(i) wherein:
L is one of the adenine, thymine, cytosine or guanine heterocyclic bases of the oligomer;
A 7 A 7 C is (CR R ) where R is hydrogen and R is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R and R are independently selected from the group consisting of hydrogen, (C2-C6)alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C1-C6)alkoxy, (C,- C6)alkylthιo, NR 3R4 and SR5, where each of R3 and R4 i.s independently selected from the group consisting of hydrogen, (C1-C4)alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C,-C4)alkyl, hydroxy, alkoxy, alkylthio and amino;, and R is hydrogen, (C1-C6)alkyl, hydroxy-, alkoxy-, or alkylthio- substituted (C1-C6)alkyl, or R and R taken together complete an alicyclic or heterocyclic system; D is (CR R )z where R and R are as defined above; each of y and z is zero or an integer from 1 to 10, the sum y + z being greater than 2 but not more than 10; G is -NR3CO-, -NR3CS-, -NR3SO- or -NR3S02~, in either orien¬ tation, where R is as defined above; each pair of A and B is selected such that: (a) A is a group of formula (Ila) , (lib) or (lie) and B is N or R N+; or (b) A is a group of formula (lid) and B is CH;
Figure imgf000041_0001
(Ila) (Hb)
Figure imgf000041_0002
(lie) (lid) where:
X is O, S, Se, NR , CH2 or C(CH3)2;
Y is a single bond, O, S or NR 4; each of p and q is zero or an integer from 1 to 5, the sum p+q being not more than 10; each of r and s is zero or an integer from 1 to 5, the sum r+s being not more than 10;
1 2 . each R and R is independently selected from the group consisting of hydrogen, (C.,-^) alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen.
7. The oligomer of claim 6 wherein A is -CH2CO-, B is N, C is CH2CH2 and D is CH2.
8. The oligomer of claim 6 wherein all of the subunits are peptide nucleic acid subunits; said oligomer including a group Q on one end of said oligomer and a group I on the other end of said oligomer; Q is -C02H, -CONR'R", -S03H or -S02NR'R" or an activated derivative of -C02H or -S03H; and
I is -NHR'"R"" or -NR" 'C(0)R" " , where R' , R", R" ' and R"" are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides and soluble and non-soluble polymers.
9. An oligomer hybridizable to the AUG region, coding region, 5' untranslated region, or 3' untranslated region of ELAM-1 and comprising at least one peptide nucleic acid subunit.
10. The oligomer of claim 9 wherein the sequence of the oligomer is selected from the group consisting of: CAATCATGACTTCAAGAGTTCT (SEQ ID NO: 30);
ACCACACTGGTATTTCACAC (SEQ ID NO: 31) ;
GTATGGAAGATTATAATATAT (SEQ ID NO: 32) ;
CACAATCCTTAAGAACTCTTT (SEQ ID NO: 33) ;
ACCTCTGCTGTTCTGATCCT (SEQ ID NO: 34); CTGCTGCCTCTGTCTCAGGT (SEQ ID NO: 35);
GGTATTTGACACAGC (SEQ ID NO: 36) ;
AATCATGACTTCAAGAGTTCT (SEQ ID NO: 37) ;
TGAAGCAATCATGACTTCAAG (SEQ ID NO: 38) ;
TATAGGAGTTTTGATGTGAA (SEQ ID NO: 39) ; ACAATGAGGGGGTAATCTACA (SEQ ID NO: 40) ;
GACAATATACAAACCTTCCAT (SEQ ID NO: 41) ;
ACGTTTGGCCTCATGGAAGT (SEQ ID NO: 42)
GGAATGCAAAGCACATCCAT (SEQ ID NO: 43)
GGGCCAGAGACCCGAGGAGA (SEQ ID NO: 44) TTCCCCAGATGCACCTGTTT (SEQ ID NO: 45)
CTGATTCAAGGCTTTGGCAG (SEQ ID NO: 46)
CCAAAGTGAGAGCTGAGAGA (SEQ ID NO: 47)
ACAGGATCTCTCAGGTGGGT (SEQ ID NO: 48)
GAAGTCAGCCAAGAACAGCT (SEQ ID NO: 49) TCACTGCTGCCTCTGTCTCAGG (SEQ ID NO: 50);
TGATTCTTTTGAACTTAAAAGGA (SEQ ID NO: 51) ;
TTAAAGGATGTAAGAAGGCT (SEQ ID NO: 52);
CATAAGCACATTTATTGTC (SEQ ID NO: 53);
TTTTGGGAAGCAGTTGTTCA (SEQ ID NO: 54); AACTGTGAAGCAATCATGACT (SEQ ID NO: 55); CCTTGAGTGGTGCATTCAACCT (SEQ ID NO: 56) ; AATGCTTGCTCACACAGGCATT (SEQ ID NO: 57); and CTCTCAGGTGGGTATCACTG (SEQ ID NO: 58) .
11. An oligomer of claim 9 wherein the sequence of the oligomer is selected from the group consisting of:
ACCACACTGGTATTTCACAC (SEQ ID NO: 31);
GTATGGAAGATTATAATATAT (SEQ ID NO: 32);
CACAATCCTTAAGAACTCTTT (SEQ ID NO: 33);
ACCTCTGCTGTTCTGATCCT (SEQ ID NO: 34); CTGCTGCCTCTGTCTCAGGT (SEQ ID NO: 35);
AATCATGACTTCAAGAGTTCT (SEQ ID NO: 37);
TGAAGCAATCATGACTTCAAG (SEQ ID NO: 38) ;
TATAGGAGTTTTGATGTGAA (SEQ ID NO: 39);
GACAATATACAAACCTTCCAT (SEQ ID NO: 41); ACGTTTGGCCTCATGGAAGT (SEQ ID NO: 42)
GGAATGCAAAGCACATCCAT (SEQ ID NO: 43)
GGGCCAGAGACCCGAGGAGA (SEQ ID NO: 44)
TTCCCCAGATGCACCTGTTT (SEQ ID NO: 45)
CTGATTCAAGGCTTTGGCAG (SEQ ID NO: 46) CCAAAGTGAGAGCTGAGAGA (SEQ ID NO: 47)
ACAGGATCTCTCAGGTGGGT (SEQ ID NO: 48) ; and
GAAGTCAGCCAAGAACAGCT (SEQ ID NO: 49)
12. The oligomer of claim 9 wherein substantially all the subunits of the oligomer are peptide nucleic acid subunits.
13. An oligomer of claim 10 incorporated in a pharmaceutically acceptable carrier.
14. An oligomer having a sequence selected from the group consisting of: CAATCATGACTTCAAGAGTTCT (SEQ ID NO: 30); ACCACACTGGTATTTCACAC (SEQ ID NO: 31) ; GTATGGAAGATTATAATATAT (SEQ ID NO: 32); CACAATCCTTAAGAACTCTTT (SEQ ID NO: 33) ; ACCTCTGCTGTTCTGATCCT (SEQ ID NO: 34);
CTGCTGCCTCTGTCTCAGGT (SEQ ID NO: 35);
GGTATTTGACACAGC (SEQ ID NO: 36);
AATCATGACTTCAAGAGTTCT (SEQ ID NO: 37); TGAAGCAATCATGACTTCAAG (SEQ ID NO: 38) ;
TATAGGAGTTTTGATGTGAA (SEQ ID NO: 39);
ACAATGAGGGGGTAATCTACA (SEQ ID NO: 40) ;
GACAATATACAAACCTTCCAT (SEQ ID NO: 41);
ACGTTTGGCCTCATGGAAGT (SEQ ID NO: 42) GGAATGCAAAGCACATCCAT (SEQ ID NO: 43)
GGGCCAGAGACCCGAGGAGA (SEQ ID NO: 44)
TTCCCCAGATGCACCTGTTT (SEQ ID NO: 45)
CTGATTCAAGGCTTTGGCAG (SEQ ID NO: 46)
CCAAAGTGAGAGCTGAGAGA (SEQ ID NO: 47) ACAGGATCTCTCAGGTGGGT (SEQ ID NO: 48)
GAAGTCAGCCAAGAACAGCT (SEQ ID NO: 49)
TCACTGCTGCCTCTGTCTCAGG (SEQ ID NO: 50) ;
TGATTCTTTTGAACTTAAAAGGA (SEQ ID NO: 51) ;
TTAAAGGATGTAAGAAGGCT (SEQ ID NO: 52); CATAAGCACATTTATTGTC (SEQ ID NO: 53);
TTTTGGGAAGCAGTTGTTCA (SEQ ID NO: 54);
AACTGTGAAGCAATCATGACT (SEQ ID NO: 55);
CCTTGAGTGGTGCATTCAACCT (SEQ ID NO: 56);
AATGCTTGCTCACACAGGCATT (SEQ ID NO: 57) ; and CTCTCAGGTGGGTATCACTG (SEQ ID NO: 58); and wherein at least one subunit of the oligomer is a peptide nucleic acid subunit of the formula:
Figure imgf000044_0001
( I ) wherein:
L is one of the adenine, thymine, cytosine or guanine heterocyclic bases of the oligomer; C is (CR R ) where R is hydrogen and R is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R and R are independently selected from the group consisting of hydrogen, (C2-C6)alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C1-C6)alkoxy, (C,- C6)alkylthio, NR3RA and SR5, where each of R3 and R is independently selected from the group consisting of hydrogen, (C1-C4)alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C,-C4)alkyl, hydroxy, alkoxy, alkylthio and amino;, and R is hydrogen, (C1-C6)alkyl, hydroxy-, alkoxy-, or alkylthio- substituted (C,-C6)alkyl, or R and R taken together complete an alicyclic or heterocyclic system; D is (CR R )z where R and R are as defined above; each of y and z is zero or an integer from 1 to 10, the sum y + z being greater than 2 but not more than 10; G is -NR3CO-, -NR3CS-, -NR3SO- or -NR3S02-, in either orien¬ tation, where R is as defined above; each pair of A and B is selected such that:
(a) A is a group of formula (Ila) , (lib) or (lie) and B is N or R3N+; or (b) A is a group of formula (lid) and B is CH;
Figure imgf000046_0001
(Ila) ( Ilb)
Figure imgf000046_0002
(lie) (lid) where :
X is 0, S, Se, NR , CH2 or C(CH3)2; Y is a single bond, O, S or NR ; each of p and q is zero or an integer from 1 to 5, the sum p+q being not more than 10; each of r and s is zero or an integer from 1 to 5, the sum r+s being not more than 10; each R and R is independently selected from the group consisting of hydrogen, (C,-C4)alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen.
15. The oligomer of claim 14 wherein A is -CH2C0-, B is N, C is CH2CH2 and D is CH2.
16. The oligomer of claim 14 wherein all of the subunits are peptide nucleic acid subunits; said oligomer including a group Q on one end of said oligomer and a group I on the other end of said oligomer;
Q is -C02H, -CONR'R", -S03H or -S02NR'R" or an activated derivative of -C02H or -S03H; and
I is -NHR'"R"" or -NR"'C(0)R" " , where R' , R" , R" ' and R' ' ' ' are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides and soluble and non-soluble polymers.
17. An oligomer hybridizable to the AUG region, coding region, 5' untranslated region, exon/intron junction region, or 3' untranslated region of VCAM-l and comprising at least one peptide nucleic acid subunit.
18. The oligomer of claim 17 wherein the sequence of the oligomer is selected from the group consisting of:
GCCTGGGAGGGTATTCAGCT (SEQ ID NO: 59)
GGCATTTTAAGTTGCTGTCG (SEQ ID NO: 60)
TGAACATATCAAGCATTAGC (SEQ ID NO: 61)
GCAATCTTGCTATGGCATAA (SEQ ID NO: 62) AACCCAGTGCTCCCTTTGCT (SEQ ID NO: 63)
GGCCACATTGGGAAAGTTGC (SEQ ID NO: 64)
CCCGGCATCTTTACAAAACC (SEQ ID NO: 65)
AACATCTCCGTACCATGCCA (SEQ ID NO: 66)
CCTGTGTGTGCCTGGGAGGG (SEQ ID NO: 67) CAGCCTGCCTTACTGTGGGC (SEQ ID NO: 68)
CTTGAACAATTAATTCCACCT (SEQ ID NO: 69);
GTCTTTGTTGTTTTCTCTTCC (SEQ ID NO: 70) ;
CTGTGTCTCCTGTCTCCGCT (SEQ ID NO: 71) ;
CGATGCAGATACCGCGGAGT (SEQ ID NO: 72); TTACCATTGACATAAAGTGTT (SEQ ID NO: 73);
CCAGGCATTTTAAGTTGCTGT (SEQ ID NO: 74) ;
CCTGAAGCCAGTGAGGCCCG (SEQ ID NO: 75);
GATGAGAAAATAGTGGAACCA (SEQ ID NO: 76);
CTGAGCAAGATATCTAGAT (SEQ ID NO: 77); CTACACTTTTGATTTCTGT (SEQ ID NO: 78) ;
TTGAACATATCAAGCATTAGCT (SEQ ID NO: 79);
TTTACATATGTACAAATTATGT (SEQ ID NO: 80);
AATTATCACTTTACTATACAAA (SEQ ID NO: 81);
AGGGCTGACCAAGACGGTTGT (SEQ ID NO: 82); and CCATCTTCCCAGGCATTTTA (SEQ ID NO: 83) .
19. The oligomer of claim 17 wherein the sequence is selected from the group consisting of:
GCCTGGGAGGGTATTCAGCT (SEQ ID NO: 59)
GGCATTTTAAGTTGCTGTCG (SEQ ID NO: 60) TGAACATATCAAGCATTAGC (SEQ ID NO: 61)
GCAATCTTGCTATGGCATAA (SEQ ID NO: 62)
AACCCAGTGCTCCCTTTGCT (SEQ ID NO: 63)
GGCCACATTGGGAAAGTTGC (SEQ ID NO: 64)
CCCGGCATCTTTACAAAACC (SEQ ID NO: 65) AACATCTCCGTACCATGCCA (SEQ ID NO: 66)
CCTGTGTGTGCCTGGGAGGG (SEQ ID NO: 67)
CAGCCTGCCTTACTGTGGGC (SEQ ID NO: 68)
CTTGAACAATTAATTCCACCT (SEQ ID NO: 69);
GTCTTTGTTGTTTTCTCTTCC (SEQ ID NO: 70); CTGTGTCTCCTGTCTCCGCT (SEQ ID NO: 71);
CGATGCAGATACCGCGGAGT (SEQ ID NO: 72); and
TTACCATTGACATAAAGTGTT (SEQ ID NO: 73) .
20. The oligomer of claim 17 wherein substantially all the subunits of the oligomer are peptide nucleic acid subunits.
21. An oligomer of claim 18 incorporated in a pharmaceutically acceptable carrier.
22. An oligomer having a sequence selected from the group consisting of: GCCTGGGAGGGTATTCAGCT (SEQ ID NO: 59)
GGCATTTTAAGTTGCTGTCG (SEQ ID NO: 60)
TGAACATATCAAGCATTAGC (SEQ ID NO: 61)
GCAATCTTGCTATGGCATAA (SEQ ID NO: 62)
AACCCAGTGCTCCCTTTGCT (SEQ ID NO: 63) GGCCACATTGGGAAAGTTGC (SEQ ID NO: 64)
CCCGGCATCTTTACAAAACC (SEQ ID NO: 65)
AACATCTCCGTACCATGCCA (SEQ ID NO: 66)
CCTGTGTGTGCCTGGGAGGG (SEQ ID NO: 67)
CAGCCTGCCTTACTGTGGGC (SEQ ID NO: 68) CTTGAACAATTAATTCCACCT (SEQ ID NO: 69) ;
GTCTTTGTTGTTTTCTCTTCC (SEQ ID NO: 70) ;
CTGTGTCTCCTGTCTCCGCT (SEQ ID NO: 71);
CGATGCAGATACCGCGGAGT (SEQ ID NO: 72); TTACCATTGACATAAAGTGTT (SEQ ID NO: 73) ;
CCAGGCATTTTAAGTTGCTGT (SEQ ID NO: 74);
CCTGAAGCCAGTGAGGCCCG (SEQ ID NO: 75);
GATGAGAAAATAGTGGAACCA (SEQ ID NO: 76);
CTGAGCAAGATATCTAGAT (SEQ ID NO: 77); CTACACTTTTGATTTCTGT (SEQ ID NO: 78);
TTGAACATATCAAGCATTAGCT (SEQ ID NO: 79);
TTTACATATGTACAAATTATGT (SEQ ID NO: 80);
AATTATCACTTTACTATACAAA (SEQ ID NO: 81) ;
AGGGCTGACCAAGACGGTTGT (SEQ ID NO: 82) ; and CCATCTTCCCAGGCATTTTA (SEQ ID NO: 83); and wherein at least one subunit of the oligomer is a peptide nucleic acid subunit of the formula:
Figure imgf000049_0001
(I) wherein: L is one of the adenine, thymine, cytosine or guanine heterocyclic bases of the oligomer;
C is (CR R ) where R is hydrogen and R7 is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R and R are independently selected from the group consisting of hydrogen, (C2-C6) alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C1-C6) alkoxy, (C.,- C6)alkylthio, NR3R4 and SR5, where each of R3 and R4 is independently selected from the group consisting of hydrogen, (C1-C4) alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C.,-C4)alkyl, hydroxy, alkoxy, alkylthio and amino;, and R5 is hydrogen, (C1-C6) alkyl, hydroxy-, alkoxy-, or alkylthio- substituted (C1-C6)alkyl, or R and R taken together complete an alicyclic or heterocyclic system;
D is (CR R )z where R and R are as defined above; each of y and z is zero or an integer from 1 to 10, the sum y + z being greater than 2 but not more than 10;
G is -NR3CO-, -NR3CS-, -NR3SO- or -NRS02~, in either orien¬ tation, where R is as defined above; each pair of A and B is selected such that:
(a) A is a group of formula (Ila) , (lib) or (lie) and B is N or R3N+; or
(b) A is a group of formula (lid) and B is CH;
Figure imgf000050_0001
(Ila) (lib)
Figure imgf000050_0002
(He) (lid) where: X is O, S, Se, NR3, CH-. or C(CH3)2; Y is a single bond, 0, S or NR ; each of p and q is zero or an integer from 1 to 5, the sum p+q being not more than 10; each of r and s is zero or an integer from 1 to 5, the sum r+s being not more than 10;
1 2 . each R and R is independently selected from the group consisting of hydrogen, (C1-C4)alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen.
23. The oligomer of claim 22 wherein A is -CH2CO-, B is N, C is CH2CH2 and D is CH2.
24. The oligomer of claim 22 wherein all of the subunits are peptide nucleic acid subunits; said oligomer including a group Q on one end of said oligomer and a group I on the other end of said oligomer;
Q is -C02H, -CONR'R", -S03H or -SO-NR'R" or an activated derivative of -C02H or -S03H; and
I is -NHR'"R"" or -NR" 'C(0)R'"' , where R' , R", R'' ' and R" " are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides and soluble and non-soluble polymers.
25. A method of modulating a metabolic process comprising contacting a cell with an oligomer comprising a sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 83 and having at least one peptide nucleic acid subunit.
26. The method of claim 25 wherein substantially all of the subunits are peptide nucleic acid subunits.
27. A method of modulating a metabolic process comprising contacting a cell with an oligomer comprising a sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 83 wherein at least one subunit of the oligomer is a peptide nucleic acid subunit of the formula:
Figure imgf000051_0001
(I) wherein:
L is one of the adenine, thymine, cytosine or guanine heterocyclic bases of the oligomer;
C is (CR R )y where R is hydrogen and R is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R and R are independently selected from the group consisting of hydrogen, (C2-C6)alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C,,-C6)alkoxy, (C.,-
C6)alkylthio, NR 3R4 and SR5, where each of R3 and R4 is independently selected from the group consisting of hydrogen, (C1-C4)alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C1-C4)alkyl, hydroxy, alkoxy, alkylthio and amino;, and R is hydrogen, (C1-C6)alkyl, hydroxy-, alkoxy-, or alkylthio- substituted (C1-C6)alkyl, or R and R taken together complete an alicyclic or heterocyclic system;
D is (CR R )z where R and R are as defined above; each of y and z is zero or an integer from 1 to 10, the sum y + z being greater than 2 but not more than 10; G is -NR3C0-, -NR3CS-, -NR3SO- or -NR3S02~, in either orien- tation, where R is as defined above; each pair of A and B is selected such that:
(a) A is a group of formula (Ila) , (lib) or (lie) and B is
N or R3N+; or
(b) A is a group of formula (lid) and B is CH;
Figure imgf000052_0001
(Ila) (lib)
Figure imgf000052_0002
Figure imgf000052_0003
(lie) (lid) where:
X is O, S, Se, NR3, CH2 or C(CH3)2;
4
Y is a single bond, 0, S or NR ; each of p and q is zero or an integer from 1 to 5, the sum p+q being not more than 10; each of r and s is zero or an integer from 1 to 5, the sum r+s being not more than 10; each R 1 and R2 is independently selected from the group consisting of hydrogen, (C1-C4)alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen.
28. The method of claim 27 wherein A is -CH2C0-, B is N, C is CH2CH2 and D is CH2.
29. The oligomer of claim 27 wherein all of the subunits are peptide nucleic acid subunits; said oligomer including a group Q on one end of said oligomer and a group I on the other end of said oligomer;
Q is -C02H, -CONR'R", -S03H or -SO-NR' R" or an activated derivative of -C02H or -S03H; and I is -NHR'"R"" or -NR" 'C(O)R" " , where R' , R", R" ' and R' ' " are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides and soluble and non-soluble polymers.
30. A method of treating a mammal having a disease characterized by a metabolic disfunction comprising admininstering to said mammal an oligomer comprising a sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 83 and having at least one peptide nucleic acid subunit.
31. The method of claim 30 wherein the substantially all of the subunits of the oligomer are peptide nucleic acid subunits.
32. A method of treating a mammal having a disease characterized by a metabolic disfunction comprising admininstering to said mammal an oligomer comprising a sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 83 wherein at least one subunit of the oligomer is a peptide nucleic acid subunit of the formula:
r\
Figure imgf000054_0001
(I) wherein:
L is one of the adenine, thymine, cytosine or guanine heterocyclic bases of the oligomer;
C is (CR R ) where R is hydrogen and R is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R and R are independently selected from the group consisting of hydrogen, (C2-C6)alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C.-C6)alkoxy, (C.,- C6)alkylthio, NR3R4 and SR5, where each of R3 and R4 is independently selected from the group consisting of hydrogen, (C|-C4)alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C1-C4)alkyl, hydroxy, alkoxy, alkylthio and amino;, and R is hydrogen, (C1-C6)alkyl, hydroxy-, alkoxy-, or alkylthio-
6 7 substituted (C1-C6)alkyl, or R and R taken together complete an alicyclic or heterocyclic system;
D is (CR R )z where R and R are as defined above; each of y and z is zero or an integer from 1 to 10, the sum y + z being greater than 2 but not more than 10; G is -NR3C0-, -NR3CS-, -NR3S0- or -NR3S02~, in either orien- tation, where R is as defined above; each pair of A and B is selected such that:
(a) A is a group of formula (Ila) , (lib) or (lie) and B is N or R N+; or
(b) A is a group of formula (lid) and B is CH;
Figure imgf000055_0001
(Ila) (lib)
Figure imgf000055_0003
Figure imgf000055_0002
(IIC) (lid) where:
X is O, S, Se, NR , CH2 or C(CH3)2;
Y is a single bond, O, S or NR 4; each of p and q is zero or an integer from 1 to 5, the sum p+q being not more than 10; each of r and s is zero or an integer from 1 to 5, the sum r+s being not more than 10; each R 1 and R2 is independently selected from the group consisting of hydrogen, (C1-c4)alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen.
33. ' The method of claim 32 wherein A is CH2CO-, B is N, C is CH2CH2 and D is CH2.
34. The oligomer of claim 32 wherein all of the subunits are peptide nucleic acid subunits; said oligomer including a group Q on one end of said oligomer and a group I on the other end of said oligomer; Q is -C02H, -CONR'R", -S03H or -S02NR'R" or an activated derivative of -C02H or -S03H; and
I is -NHR'"R"" or -NR" 'C(0)R"" , where R' , R", R" ' and R'"' are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides and soluble and non-soluble polymers.
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US6133467A (en) * 1997-06-25 2000-10-17 Hoechst Marion Roussel, Inc. 2,6-di-t-butyl-4-[(dimethyl-4-methoxyphenylsilyl)-methyl-oxy]phenol and 2,6-di-t-butyl-4-[(dimethyl-2-methoxy-phenylsilyl)methyloxy]phenol
US6989270B1 (en) 1997-10-17 2006-01-24 Mayo Foundation For Medical Education And Research Using polyamide nucleic acid oligomers to engender a biological response
US6743627B1 (en) 1997-10-17 2004-06-01 Mayo Foundation For Medical Education And Research Using polyamide nucleic acid oligomers to engender a biological response
US6472209B1 (en) 1997-10-17 2002-10-29 Mayo Foundation For Medical Education And Research Using polyamide nucleic acid oligomers to engender a biological response
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EP1071699A1 (en) * 1998-04-17 2001-01-31 Isis Pharmaceuticals, Inc. Enhanced antisense modulation of icam-1
EP1080225A1 (en) * 1998-05-21 2001-03-07 Isis Pharmaceuticals, Inc. Compositions and methods for the pulmonary delivery of nucleic acids
US6723560B2 (en) 1998-10-08 2004-04-20 Mayo Foundation For Medical Education And Research Using polyamide nucleic acid oligomers to engender a biological response
WO2001079487A3 (en) * 2000-04-18 2002-06-20 Klaus Karl Degitz Polydesoxyribonucleotides for inhibiting the expression of the icam-1-gene
WO2001079487A2 (en) * 2000-04-18 2001-10-25 Klaus Karl Degitz Polydesoxyribonucleotides for inhibiting the expression of the icam-1-gene
EP1292606A2 (en) * 2000-06-23 2003-03-19 Yissum Research Development Company of the Hebrew University of Jerusalem Positively-charged peptide nucleic acid analogs with improved properties
EP1292606A4 (en) * 2000-06-23 2004-03-24 Yissum Res Dev Co Positively-charged peptide nucleic acid analogs with improved properties
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GB2558788A (en) * 2017-01-06 2018-07-18 Atlantic Pharmaceuticals Holdings Ltd New therapeutic uses
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