Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
  • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/16—Purine radicals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/16—Purine radicals
  • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

• the present invention relates to novel 5'-substituted nucleosides and to oligonucleotide analogs prepared therefrom having from 2 to about 60 bases and having an intemucleoside backbone containing one or more 3'-0-P ⁇ 2H-0-5'-CR ⁇ R2 intemucleoside linkages instead of the naturally occurring backbone of phosphodiester intemucleoside linkages.
• the present invention also relates to a method of synthesizing oligonucleotide compounds having from 2 to about 60 bases and having an intemucleoside backbone containing one or more 3'-0-P02H-0-5'-CR ⁇ R2 intemucleoside linkages instead of the naturally occurring backbone of phosphodiester intemucleoside linkages, this process comprising preparation of 5'-substituted nucleoside compounds, for example, as illustrated in Scheme 1 , and utilizing them as synthons in automated DNA synthesizers. Oligonucleotide analogs of the present invention are useful as nuclease resistant, sequence specific antisense compounds.
• An antisense compound binds to or hybridizes with a nucleotide sequence in a nucleic acid (RNA or DNA) to inhibit the function (or synthesis) of the nucleic acid. Because they can hybridize with both RNA and DNA, antisense compounds can interfere with gene expression at the level of transcription, RNA processing or translation.
• oligonucleotide compounds such as antisense DNA are susceptible to steric reconfiguration around chiral phosphorous centers. This results in stability problems, too, whether the compounds are free within the body or hybridized to target nucleic acids.
• oligonucleotide analogs To overcome the stability and drug delivery limitations, various oligonucleotide analogs have been investigated. In order to be of practical utility, such analogs should have good cell penetration properties, be resistant to nuclease degradation, have good sequence specific hybridization to target nucleic acids, and be synthesized by chemical methods that are not too difficult or costly.
• modified phosphodiester linkages that have been reported are phosphorothioates, alkylphosphotriesters, methylphosphonates and alkylphosphoramidates.
• non-ionic oligonucleotide analogs sequences containing non-phosphate intemucleoside linkages such as carbonate, acetate, carbamate, sulfone, sulfoxide, sulfonamide and dialkyl- or diaryl- silyl derivatives have been synthesized and reported.
• chimeric oligonucleotide analogs comprising nucleoside linkages containing two carbon atoms and one nitrogen atom or one oxygen atom, as well as those containing three carbon atoms, have been reported. See, e.g., International Patent Publication WO 9202534.
• the prior art describes 1 -mononucleosides and mononucleotides substituted at the 5'- position by a variety of substituents, 2- dinucleosides phosphates substituted at the 5'-position of the 3'-terminal nucleoside by methyl, ethyl, propyl and ally I ; it also discloses the corresponding pairs of stereoisomers (Padyukov, A, et al.
• the present invention provides novel ⁇ '-substituted nucleosides, as well as oligonucleotide analogs of two bases and longer derived therefrom which are resistant to nucleases and will bind in a sequence specific manner to complementary nucleic acid sequences. Also provided is a method of synthesizing such oligonucleotide analogs using the nucleoside derivatives described in this specification. Another advantageous feature of the present invention is the relative ease with which optically pure isomers of the 5' 1 substituted nucleoside analogs of the invention as compared to phosphorothioate, methylphosphonate and phosphoroamidate nucleosides used in the prior art to synthesize oligonucleotide analogs.
• the present invention provides novel ⁇ '-substituted nucleoside analogs and oligonucleotide analogs of 2 to about 60 bases containing 3'-0- P ⁇ 2H-0- ⁇ '-CR ⁇ R2-substituted intemucleoside linkages instead of the naturally occurring phosphodiester intemucleoside linkages.
• the present invention provides novel nucleoside analogs having the structure of Formula I below:
• Q is selected from the group consisting of H, OH, NHR, CHO, phosphate, lower-alkyl, lower alkenyl, protected 0-, protected N-, lower alkoxy, lower alkenyloxy, benzyloxy, dimethoxytrityloxy, amino-lower alkyl, amino-lower alkoxy, N3, epoxyethyl, halogen, phosphonium salt and phosphonate;
• L is selected from the group consisting of -OP(OCH2CH2CN)(N-iPr 2 ), H, OH, NHR, phosphate, lower alkyl, lower alkenyl, lower alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl, phosphonium salt, phosphonate and t-butyldimethylsilyloxy; each R is independently selected from the group consisting of H, OZ, SZ and NHZ; each R 1 and R2 is independently selected from the group consisting of H, OH, lower alkyl, lower alkenyl, lower cycloalkyl, epoxyethyl, amino lower alkyl, amino lower alkoxy, lower alkoxy and lower alkenyloxy; each R3 and R4 is independently selected from the group consisting of H, lower alkyl, lower alkenyl, lower alkoxy and lower alkenyloxy: each Z is independently selected
• Q is selected from the group consisting of H, OH, NHR, CHO, phosphate, lower-alkyl, lower alkenyl, protected O-, protected N-, lower alkoxy, lower alkenyloxy, benzyloxy, dimethoxytrityloxy, amino-lower alkyl, amino-lower alkoxy, N3, epoxyethyl, halogen, phosphonium salt and 0 phosphonate;
• L is selected from the group consisting of -OP(OCH 2 CH 2 CN)(N-iPr2), H, OH, NHR, phosphate, lower alkyl, lower alkenyl, lower alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl, phosphonium salt, phosphonate and t-butyldimethylsilyloxy; each R is independently selected from the group consisting of H, OZ,
• each R-i and R 2 is independently selected from the group consisting of H, OH, lower alkyl, lower alkenyl, lower cycloalkyl, epoxyethyl, amino lower alkyl, amino lower alkoxy, lower alkoxy and lower alkenyloxy; 0 each R3 and R4 is independently selected from the group consisting of
• each Z is independently selected from the group consisting of H, lower alkyl, lower alkenyl, lower alkoxy and lower alkenyloxy: each Z is independently selected from the group consisting of H, lower alkyl, lower alkenyl, aryl, acetyl and protecting groups for 0-, S-, and N-; each E is independently selected from the group consisting of ⁇ -OP(OCH 2 CH 2 CN)(N-iPr 2 ), H, OH, NHR, lower alkyl, lower alkenyl, lower alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl, phosphonium salt and phosphonate; each n is independently 0 or an integer from 1 to 4 each B is independently selected from the group consisting of adenine, cytosine, guanine, thymine, uracil or a modification thereof that does not substantially interfere with the affinity of an oligon
• the term 'oligonucleotide' means nucleic acid compounds which contain only 'natural' phosphodiester intemucleoside linkages.
• the term 'chimeric oligonucleotide analogs' means compounds that comprise sequences containing both 'synthetic' 5 oligonucleoside linkages and oligonucleotide linkages.
• 'oligonucleotide analogs,' we mean both oligonucleotide analogs that contain only synthetic (as opposed to the naturally occurring phosphodiester) intemucleoside linkages and chimeric oligonucleotide analogs.
• the present invention also provides a method of synthesizing 0 oligonucleotide compounds having from 2 to about 60 bases and having an intemucleoside backbone containing one or more 3'-0-PO2H-0-5'-CR-
• nucleoside analogs of the present invention have the structure of Formula I below:
• Q is selected from the group consisting of H, OH, NHR, CHO, phosphate, lower-alkyl, lower alkenyl, protected 0-, protected N-, lower alkoxy, lower alkenyloxy, benzyloxy, dimethoxytrityloxy, amino-lower alkyl, amino-lower alkoxy, N3, epoxyethyl, halogen, phosphonium salt and phosphonate;
• L is selected from the group consisting of -OP(OCH2CH 2 CN)(N-iPr 2 ), H, OH, NHR, phosphate, lower alkyl, lower alkenyl, lower alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl, phosphonium salt, phosphonate and t-butyldimethylsilyloxy; each R is independently selected from the group consisting of H, OZ, SZ and NHZ; each R1 and R2 is independently selected from the group consisting of H, OH, lower alkyl, lower alkenyl, lower cycloalkyl, epoxyethyl, amino lower alkyl, amino lower alkoxy, lower alkoxy and lower alkenyloxy; each R3 and R4 is independently selected from the group consisting of H, lower alkyl, lower alkenyl, lower alkoxy and lower alkenyloxy: each Z is independently
• oligonucleotide analogs of the invention have the structure of Formula II below:
• Q is selected from the group consisting of H, OH, NHR, CHO, phosphate, lower-alkyl, lower alkenyl, protected O-, protected N-, lower alkoxy, lower alkenyloxy, benzyloxy, dimethoxytrityloxy, amino-lower alkyl, amino-lower alkoxy, N3, epoxyethyl, halogen, phosphonium salt and phosphonate;
• L is selected from the group consisting of -OP(OCH 2 CH 2 CN)(N-iPr 2 ), H, OH, NHR, phosphate, lower alkyl, lower alkenyl, lower alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl, phosphonium salt, phosphonate and t-butyldimethylsilyloxy; each R is independently selected from the group consisting of H, OZ, SZ and NHZ; each R1 and R 2 is independently selected from the group consisting of H, OH, lower alkyl, lower alkenyl, lower cycloalkyl, epoxyethyl, amino lower alkyl, amino lower alkoxy, lower alkoxy and lower alkenyloxy; each R3 and R4 is independently selected from the group consisting of H, lower alkyl, lower alkenyl, lower alkoxy and lower alkenyloxy: each Z is independently
• Alkyl means a saturated aliphatic hydrocarbon which may be either straight- or branched-chain. Preferred groups have no more than about 12 carbon atoms and may be methyl, ethyl and structural isomers of propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl.
• “Lower alkyl” means an alkyl group as above, having 1 to 7 carbon atoms.
• Suitable lower alkyl groups are methyl, ethyl, n-propyl, isopropyl, butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, and n-heptyl.
• Aryl means phenyl, naphthyl, substituted phenyl and substituted naphthyl.
• Substituted phenyl (or naphthyl) means a phenyl (or naphthyl) group in which one or more of the hydrogens has been replaced by the the same or different substituents selected from halo, lower alkyl, nitro, amino, acylamino, hydroxyl, lower alkoxy, aryl, heteroaryl, lower alkoxy, alkylsulfonyl, and trifluoromethyl.
• Heteroaryl group means, pyridyl, furyl, thienyl, or imidazolyl.
• Substituted heteroaryl means a heteroaryl group in which one or more of the hydrogens has been replaced by the the same or different substituents selected from halo, lower alkyl, nitro, amino, acylamino, hydroxyl, lower alkoxy, aryl, heteroaryl, lower alkoxy, alkylsulfonyl, and trifluoromethyl.
• “Lower alkenyl” means an unsaturated aliphatic hydrocarbon having 2 to 8 carbon atoms, such as ethylene, propylene, butylene, isobutylene, ⁇ etc., including all structural and geometrical isomers thereof.
• Halo means bromo, chloro or fluoro.
• An "0-, S-, or N-protecting group” is a radical attached to an oxygen, sulfur, or nitrogen atom, respectively, which radical serves to protect the oxygen, sulfur, or nitrogen functionality against undesired reaction.
• 0 protecting groups are well known in the art; many are described in "The Peptides.” E. Gross and J. Meienhofer, Eds. Vol 3 Academic Press, NY (1981 ).
• the N-protecting groups can be N-acyl, N- alkoxycarbonyl, N-arylmethoxy-carbonyl, trifluoromethylacyl and N- arylsulfonyl protecting groups.
• Suitable O-protecting groups include benzyl, ⁇ tert-butyl, methyl, tosyl, dimethoxytrityl, tert-butyl-dimethylsilyl, and carbobenzoxy groups.
• S-Protecting groups include methyl, tert-butyl, benzyl, and carbobenzoxy groups.
• the present invention provides a method of synthesizing 0 oligonucleotide compounds having from 2 to about 60 bases and having an intemucleoside backbone containing one or more 3'-0-P02H-0-5'-CR-
• Scheme 1 illustrates the preparation of various ⁇ -substituted nucleoside analogs of the invention which, in turn, are useful for the preparation of 3'-0-P02H -0- ⁇ '-C R ⁇ R 2 linked dinucleosides and oligonucleotide analogs of the invention.
• 3'-t-butyldimethylsilyloxy-2'-deoxy- ⁇ '-formyl- ⁇ '-deoxy-thymidine 2 is a key intermediate for the preparation of various ⁇ '-substituted nucleoside analogs of the invention, including ones containing the ⁇ '-methyl group 5, the 5'-nitromethyl group 8, the ⁇ '-aminomethyl group 9, the ⁇ '-epoxy group 7, and the ⁇ '-azidomethyl group 13.
• reaction of methylmagnesium bromide with the aldehyde 2 yields the ⁇ '-methyl thymidine 3 which, upon treatment with dimethyltrityl chloride followed by desilylation and reaction with chloro-2-cyanoethyl-N,N-diisopropyl-phosphoramidite, affords the ⁇ '- substituted nucleoside synthon 5.
• the synthon 5 may be utilized for the synthesis of ⁇ '-modified DNA employing a DNA synthesizer such as ABI 380B Oligomer synthesizer.
• the ⁇ '-epoxy compound 7 may be prepared by the reaction of the aldehyde 2 with diazomethane, whereas the ⁇ '-nitromethyl compound 8 may be prepared by the reaction of the aldehyde 2 with nitromethane.
• the chemical reduction of the nitro group with lithium aluminum hydride affords the desired ⁇ '-aminomethyl compound 9, which may be acylated to yield the trifluoroacetyl amide 10.
• azide ring opening of the epoxy compound 7 provides the corresponding azidomethyl analog 12.
• the amide 11 and azido analog 13 may be prepared, respectively, from compounds 10 and 12 via deprotection of t-butyldimethylsilyloxy group followed by reaction with chloro-2-cyanoethyl- N,N-diisopropyl-phosphoramidite.
• This invention also contemplates pharmaceutically acceptable salts of the compounds of Formula I. It is well known in the pharmacological arts that nontoxic addition salts of pharmacologically active amine compounds do not differ in activities from their free base. All stereoisomers as ⁇ well as optical isomers related to the novel antisense agents described in this disclosure are also considered to be within the scope of this invention.
• Pharmaceutically acceptable salts include both acid and base addition salts.
• “Pharmaceutically acceptable salt” refers to those salts which retain the biological effectiveness and properties of the free bases and which 0 are not biologically or otherwise undesirable.
• Suitable pharmaceutically acceptable acid addition salts can be formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, and p-toluenesulfonic acid, and the like.
• Pharmaceutically acceptable base addition salts include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the 0 like. Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts.
• Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, ⁇ tripropylami ne , ethanolami ne , 2-diethylami noethano l , 2- dimethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procain, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, peperizines, piperidine, polyamine resins and the like.
• Particularly preferred organic non- 0 toxic bases are isopropylamine, diethylamine, ethanol-amine and dicyclohexylamine.
• the compounds of the present invention comprise oligomeric antisense agents, as shown in Formula II, of about 6 to about 60 bases, preferably from about 9 to about ⁇ O bases, more preferably ⁇ from about 12 to about 2 ⁇ bases, most preferably from 1 ⁇ to 18 bases.
• oligomeric antisense agents as shown in Formula II, of about 6 to about 60 bases, preferably from about 9 to about ⁇ O bases, more preferably ⁇ from about 12 to about 2 ⁇ bases, most preferably from 1 ⁇ to 18 bases.
• novel ⁇ '-substituted nucleosides of the invention may be incorporated at multiple sites within an oligonucleotide analog, and have no appreciable effect on the hybridization stability of the resulting (antisense) ⁇ oligonucleotide analog to its natural 'sense' target oligonucleotide when compared to the hybridization stability to the same 'sense' target of the corresponding unmodified antisense oligonucleotide.
• antisense agents can be formulated into compositions together with one or more non-toxic physiologically acceptable carriers, 0 adjuvants or vehicles which are collectively referred to herein as carriers, for parenteral injection or oral administration, in solid or liquid form, for rectal or topical administration, or the like.
• compositions can be administered to humans and animals either orally, rectally, parenterally (intravenous, intramuscularly or ⁇ subcutaneously), intracistemally, intravaginally, intraperitoneally, locally (powders, ointments or drops), or as a buccal or nasal spray.
• compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution 0 into sterile injectable solutions or dispersions.
• suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
• Proper fluidity can be maintained, for ⁇ example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
• compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal 0 agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents that delay absorption, for example, aluminum monostearate and gelatin. ⁇ Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
• the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or
• fillers or extenders as for example, starches, lactose, sucrose, glucose, mannitol and silicic acid
• binders as for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia
• humectants as for example, glycerol
• disintegrating agents as for example, agar-agar, calcium ⁇ carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate
• solution retarders as for example paraffin
• absorption accelerators as, for example, quaternary ammonium compounds
• wetting agents as for example, cetyl alcohol and glycerol monostearate
• wetting agents as for example, cetyl alcohol and glycerol monostearate
• wetting agents as
• Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules, using such excipients as lactose ⁇ or milk sugar as well as high molecular weight polyethyleneglycols, and the like.
• Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain opacifying agents, and 0 can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner.
• coatings and shells such as enteric coatings and others well known in the art. They may contain opacifying agents, and 0 can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner.
• embedding compositions which can be used are polymeric substances and waxes.
• the active compounds can also be in micro-encapsulated form, ⁇ if appropriate, with one or more of the above-mentioned excipients.
• Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
• the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other 0 solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1 ,3-butyleneglycol, dimethylformamide, oils, particularly cottonseed oil, ground-nut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and ⁇ fatty acid esters of sorbitan or mixtures of these substances, and the like.
• inert diluents commonly used in the art, such as water or other 0 solvent
• the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
• adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
• Suspensions in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of ⁇ these substances, and the like.
• compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary 0 temperatures but liquid at body temperature and, therefore, melt in the rectum or vaginal cavity and release the active component.
• suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary 0 temperatures but liquid at body temperature and, therefore, melt in the rectum or vaginal cavity and release the active component.
• Dosage forms for topical administration include ointments, powders, sprays and inhalants.
• the active component is admixed under sterile conditions with a physiologically acceptable carrier and any ⁇ preservatives, buffers or propellants as may be required.
• Opthalamic formulations, eye ointments, powders and solutions are also contemplated.
• Actual dosage levels of the active ingredient in the compositions may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and 0 method of administration.
• the selected dosage level therefore depends upon the desired therapeutic effect, on the route of administration, on the desired duration of treatment and other factors.
• the total daily dose of the active ingredients administered to a host in single or divided doses may be in amounts, for example, of from about ⁇ O. ⁇ mg to about 10 mg per kilogram of body weight.
• Dosage unit compositions may contain such amounts or such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of 0 administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.
• the present invention is further directed to a method of inhibiting the expression of a gene that comprises administering to a host mammal in need of such inhibition an inhibition-effective amount of a compound of ⁇ Formula II, in which that compound hybridizes to a nucleotide sequence of the gene.whose expression is to be inhibited.
• the compound of Formula II is dissolved or dispersed in a physiologically tolerable carrier.
• inhibition of the expression of a gene can be effected by interfering with transcription, translation, or RNA processing.
• the activity of an antisense molecule can be at the level of messenger RNA or genomic DNA.
• an antisense ⁇ molecule when an antisense ⁇ molecule hybridizes to messenger RNA, translation is inhibited. When an antisense molecule hybridizes to genomic DNA, transcription is inhibited.
• An antisense molecule may also bind to other nucleic acid species in a cell, including heterogeneous nuclear RNA (hnRNA) and pre-messenger RNA.
• a host mammal in need of the inhibition of the expression of a 0 gene suffers from a disease state in which the expression of the gene is implicated.
• disease states include a variety of cancers, in which the expression of an oncogene or oncogenes is implicated, cystic fibrosis, Huntington's chorea, and other such disease states in which the aberrant expression of a normal gene or the expression of an abnormal gene is ⁇ responsible, in whole or in part, for the disease condition.
• an “inhibition-effective amount” is the amount of a compound of the present invention which is sufficient to inhibit the expression of the gene whose expression is to be inhibited. Means for determining an inhibition-effective amount will depend, as is well known in 0 the art, on the nature of the gene to be inhibited, the type of inhibition desired (i.e., inhibition of translation or transcription or both), the mass of the subject being treated, and the like.
• the compound of the Formula II used in the inhibition of the expression of a gene must hybridize to a sequence of ⁇ that gene in such a way as the expression of that gene is inhibited. That is, the nucleotide bases used to make a compound of the Formula II (B in Formula II as defined above) must hybridize to the nucleotide sequence of the gene whose expression is to be inhibited. Such sequence can readily be ascertained from the known sequence of that gene, and the appropriate 0 antisense molecule of Formula II can therefore be prepared. Hybridization of greater than about 90 percent homology (identity), and more preferably about 99 percent homology, is contemplated in the present invention.
• 4,4-Dimethoxytrityl chloride (0.992 mM, 336 mg) was added to a ⁇ solution of N 6 -benzoyl-3'-t-butyldimethylsilyloxy-2'-deoxy- ⁇ '(RS)-methyl- adenosine (0.824 mM, 400 mg), triethylamine (1.12 mM, 1 ⁇ 6 ⁇ l) and 4- dimethylaminopyridine (0.04 mM, 4.88 mg) in pyridine/ethylene chloride (1 :1 ;
• the reaction mixture was poured over 1 ⁇ 0 ml of ethyl acetate and the organic layer was separated, washed with saturated sodium bicarbonate solution (2x150 ml), dried over anhydrous sodium sulfate and concentrated in vacuo.
• the crude aldehyde in 5 600 ml of benzene was azeotroped (Dean Stark) and the solvent was removed in vacuo to afford 4 g of the crude aldehyde.
• Tetra-n-butyl ammonium fluoride (1.1 M in THF; 0.68 mM, 0.9 ml) was added dropwise via syringe to a solution of 3'-t-butyldimethylsilyloxy-2'- deoxy- ⁇ '-dimethoxytrityl- ⁇ '(RS)-methyl-thymidine (0.68 mM, 460 mg) in anhydrous THF (3.44 ml). The reaction was stirred under nitrogen for 4 hours at room temperature. After a standard aqueous work-up the crude product was purified by flash chromatography (silica gel; 60 - 7 ⁇ % EtOAc/Hexane). Yield: 340 mg (92%).
• Example 8 2'-Deoxy-5'-dimethoxytrityl-5' S methyl-thvmidine-3'-0-(RSW2- cyanoethyl-N.N-diisopropyl-phosphoramidite)
• the product was purified by flash chromatography (silica gel; ⁇ 0%
• the synthesis of the oligomers listed in Table 1 was conducted 5 using an ABI 380B synthesizer in accordance with the manufacturer's protocols. Synthesis of strands at one micromole scale using monomer (e.g., 2'-deoxy- ⁇ '-dimethoxytrityl- ⁇ '(RS)-methyl-thymidine-3'-0-(RS)-(2-cyanoethyl- N,N-diisopropyl-phosphoramidite) was carried out. Monomer was dried just before DNA synthesis. The purification of DNA product was conducted with 0 DMT + reverse phase HPLC with slicing through major peak.
• monomer e.g., 2'-deoxy- ⁇ '-dimethoxytrityl- ⁇ '(RS)-methyl-thymidine-3'-0-(RS)-(2-cyanoethyl- N,N-diisopropyl-phosphoramidite
• oligonucleotides were labelled at the ⁇ ' terminus with 32 P by using T4 polynucleotide kinase and ⁇ standard end-labelling procedures. Unincorporated 32 P-ATP was removed by passing over a NucTrap column followed by purification over Sephadex G- 2 ⁇ . A trace (1-1 O ⁇ M) of 32 P-labelled oligonucleotide was combined with unlabelled oligomer at a concentration of 1 ⁇ M. These conditions minimize any variation incurred due to differences in oligomer labelling efficiencies and simulate concentrations used in typical antisense experiments.
• the oligomer was added to Dulbecco's minimal essential medium (DMEM) cell culture media containing 20% fetal calf serum (FCS) which serves as a source of 3' ⁇ exonuclease activity. After incubation at 37°C for 2. ⁇ hours, the mixture was denatured for 2 minutes at 90°C and analyzed by denaturing polyacrylamide gel electrophoresis (PAGE; 20% polyacrylamide/8 M urea; 19:1 acrylamide:bis-acrylamide; 89 mM Tris/89 mM boric acid/2 mM EDTA). As a 0 hour control, the oligonucleotide was added to serum-free DMEM media prior 0 to PAGE analysis.
• DMEM Dulbecco's minimal essential medium
• FCS fetal calf serum
• Oligodeoxynucleotide #1 ( ⁇ '-TT TTT TTT TT * T-3'; SEQ ID NO:1 ; containing a 3'-end cap modification with a methyl group at the ⁇ ' carbon of 0 the sugar at the penultimate thymidylate residue) was added to a final concentration of 3 nM to tubes on ice containing RPMI 1640 media with L- glutamine (GIBCO). Also added were: HEPES (GIBCO) to a final concentration of 20 mM, and fetal bovine serum to 10%. Total reaction volume was 400 ⁇ l. Tubes which were incubated for 0 time (controls) did not ⁇ receive serum.
• the results of the assay show that the parent oligomer (Oligomer #1 , an 11- mer (retention time of 41.76 min)), was rapidly digested, by 3'-> ⁇ ' exonucleolytic activity present in the 10% fetal bovine serum.
• the product was an extremely stable, N-1 reaction product, a 10-mer (retention time of 0 38.64 min), the result of cleavage of the 3'-terminal thymidylate residue by the enzyme.
• the 10-mer remained undigested for up to 120 minutes in the presence of serum, based on the resulting peak areas.
• the 3'-modification provides protection to the remaining oligomer against further digestion by inhibiting the activity of the 3'-> ⁇ ' exonuclease present in the serum. It is concluded that the methyl group present at the ⁇ ' position of the sugar interferes with the hydrolysis of the phosophodiester bond by the nuclease enzyme.
• the T m for the sense and antisense control (normal, unmodified 0 phosphodiester [PDE]) strands annealed together is 68 +/- O. ⁇ degrees.
• the T m for each of Oligomers 6 (SEQ ID NO:6) and 7 (SEQ ID NO:7) was 68°C.
• the Tm observed for Oligomer # 8 (SEQ ID NO:8) annealed with sense PDE strand was 67.8 degrees.
• the T m for Oligomers 8 (SEQ ID NO:8) and 9 (SEQ ID NO:9) annealed together is 66.8 degrees.
• the results show that there is ⁇ virtually no loss of duplex stability when the normal PDE bond is replaced with this substituted PDE bond.
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)

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