WO1994015619A1 - OLIGONUCLEOTIDES MODIFIES POUR AMELIORER LA STABILITE A pH ACIDE - Google Patents

OLIGONUCLEOTIDES MODIFIES POUR AMELIORER LA STABILITE A pH ACIDE Download PDF

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Publication number
WO1994015619A1
WO1994015619A1 PCT/US1994/000157 US9400157W WO9415619A1 WO 1994015619 A1 WO1994015619 A1 WO 1994015619A1 US 9400157 W US9400157 W US 9400157W WO 9415619 A1 WO9415619 A1 WO 9415619A1
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Prior art keywords
oligomer
methylphosphonate
units
percent
alkyl
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PCT/US1994/000157
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English (en)
Inventor
Paul Scott Miller
Paul On-Pong Ts'o
Lionel Norton Simon
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The Johns Hopkins University
Genta Incorporated
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Application filed by The Johns Hopkins University, Genta Incorporated filed Critical The Johns Hopkins University
Priority to KR1019950702783A priority Critical patent/KR960700062A/ko
Priority to AU59924/94A priority patent/AU692143B2/en
Priority to JP6516188A priority patent/JPH08505396A/ja
Priority to EP94906041A priority patent/EP0690716A4/fr
Publication of WO1994015619A1 publication Critical patent/WO1994015619A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • 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
    • 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
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical

Definitions

  • the present invention is directed to methods of providing Oligomers which exhibit improved stability at acid pH, to methods of delivering such Oligomers to their sites of action and to their use in formulations for oral administration or other dosage forms where acid resistance is advantageous.
  • RNA has been reported to be significantly more stable to depurination under acidic conditions than its DNA counter- part reportedly because of the apparent stabilizing effect of the 2' hydyoxyl on the glycosidic bond between sugar and the base (Hevesi, L., et al. , supra) .
  • RNA may be resistant to depurination under acid conditions, its sensitivity to ubiquitous nucleases present in biological materials limits its therapeutic usefulness. Furthermore, the use of oligoribonucleotides as a drug in its unmodified form is not feasible because of the inherent instability of the molecule to neutral to mildly basic conditions. Unfortunately, protection of the R ⁇ A against nucleases by replacing the phosphate linkages with methylphosphonates is not possible because the 2'- hydroxyl of the sugar rapidly cleaves the methylphosphonate backbone.
  • the present invention is directed to methods of providing Oligomers which comprise nucleosidyl units having a preselected base sequence in an acid resistant form.
  • Oligomers are provided wherein the nucleosidyl units have a sugar moiety which is a 2'-0-alkyl ribosyl group.
  • the Oligomer is substantially neutral.
  • Oligomers having methylphosphonate internucleosidyl linkages more preferably from about 50 percent to about 100 percent of the internucleosidyl linkages are methylphosphonate linkages.
  • Preferred 2'-0- alkyl ribosyl groups include 2'-0-methyl ribosyl groups.
  • the Oligomers provided in acid resistant form comprise methylphosphonate internucleosidyl linkages, preferably from about 50 percent to about 100 percent methylphosphonate linkages.
  • the present invention is directed to methods of preparing an Oligomer which comprises nucleosidyl units having a preselected base sequence which Oligomer is suitable for oral administration and exhibits resistance to acid degradation.
  • this method comprises synthesizing the Oligomer using nucleosidyl units having a sugar moiety which is a 2' -O-alkylribosyl group, more preferably a 2'-0-methyl ribosyl group.
  • the Oligomer is substantially neutral. More preferably the Oligomer is synthesized to have methylphosphonate internucleosidyl units.
  • the method comprises synthesizing the Oligomer using nucleosidyl units having methylphosphonate internucleosidyl linkages.
  • Particularly preferred are Oligomers having from about 50 percent to about 100 percent methylphosphonate internucleosidyl units.
  • the present invention is directed to a method of orally delivering an Oligomer to a mammal for therapeutic purposes wherein said Oligomer comprises a nucleosidyl unit having a purine base which method comprises administration of an acid resistant Oligomer.
  • the acid resistant Oligomer comprises nucleosidyl units having a sugar moiety which comprises a 2'-0-alkyl ribosyl group, preferably a 2' -O-methylribosyl group.
  • the Oligomer is substantially neutral.
  • Preferred acid resis ⁇ tant Oligomers include Oligomers having methylphosphonate internucleosidyl linkages, more preferably from about 50 percent to about 100 percent of the internucleosidyl linkages are methylphosphonate linkages.
  • the acid resistant Oligomer is administered in a controlled-rate release form.
  • the present invention is based on our surprising finding that Oligomers which are synthesized to incorporated nucleosidyl units having a sugar moiety which is a 2'-0-alkyl ribosyl group or which incorporate methylphosphonate internucleosidyl linkages exhibit advantageous resistance to acid catalyzed depurination and subsequent hydrolysis.
  • the Oligomers which comprise nucleosidyl units having a 2' -O-alkylribosyl group and methylphosphonate internucleosidyl linkages appear to form more stable duplexes with an RNA target molecule than do the corresponding 2' -deoxy-ribonucleoside methylphospho- nates.
  • the present invention is directed to pharmaceutical compositions which comprise an acid resistant Oligomer of the present invention in a controlled-rate release form.
  • pharmaceu ⁇ tical compositions comprising an acid resistant Oligomer are provided which compositions are acidic themselves or which may be exposed to acidic conditions during manufacture or storage.
  • purine or “purine base” includes not only the naturally occurring adenine and guanine bases, but also modifications of those bases such as bases substi- tuted at the 8-position, or guanine analogs modified at the 6-position or the analog of adenine, 2-amino purine, as well as analogs of purines having carbon replacing nitrogen at the 9-position such as the 9-deaza purine derivatives and other purine analogs.
  • nucleoside includes a nucleosidyl unit and is used interchangeably therewith, and refers to a subunit of a nucleic acid which comprises a 5-carbon sugar and a nitrogen-containing base.
  • nucleoside also includes other analogs of such subunits, including those which have modified sugars such as 2'-0-alkyl ribose.
  • R is hydrogen or an alkyl or aryl group.
  • Suitable alkyl or aryl groups include those which do not sterically hinder the phosphonate linkage or interact with each other.
  • the phosphonate group may exist in either an "R” or an "S” configuration.
  • Phosphonate groups may be used as internucleosidyl phosphorus group linkages (or links) to connect nucleosidyl units.
  • phosphodiester groups may be used as internucleosidyl phosphorus group linkages (or links) to connect nucleosidyl units.
  • non-nucleoside monomeric unit refers to a monomeric unit wherein the base, the sugar and/or the phosphorus backbone has been replaced by other chemical moieties.
  • nucleoside/non-nucleoside polymer refers to a polymer comprised of nucleoside and non-nucleoside monomeric units.
  • oligonucleoside or “Oligomer” refers to a chain of nucleosides which are linked by internucleoside linkages which is generally from about 4 to about 100 nucleosides in length, but which may be greater than about 100 nucleosides in length. They are usually synthesized from nucleoside monomers, but may also be obtained by enzymatic means.
  • the term "Oligomer” refers to a chain of oligonucleosides which have internucleosidyl linkages linking the nucleoside monomers and, thus, includes oligonucleotides, nonionic oligonucleoside alkyl- and aryl-phosphonate analogs, alkyl- and aryl- phosphonothioates, phosphorothioate or phosphorodithioate analogs of oligonucleotides, phosphoramidate analogs of oligonucleotides, neutral phosphate ester oligonucleoside analogs, such as phosphotriesters and other oligonucleoside analogs and modified oligonucleosides, and also includes nucleoside/non-nucleoside polymers.
  • nucleoside/nucleotide polymers wherein one or more of the phosphorus group linkages between monomeric units has been replaced by a non-phosphorous linkage such as a formacetal linkage, a thioformacetal linkage, a sulfamate linkage, or a carbamate linkage. It also includes nucleoside/non-nucleoside polymers wherein both the sugar and the phosphorous moiety have been replaced or modified such as morpholino base analogs, or polyamide base analogs.
  • nucleoside/non- nucleoside polymers wherein the base, the sugar, and the phosphate backbone of the non-nucleoside are either replaced by a non-nucleoside moiety or wherein a non- nucleoside moiety is inserted into the nucleoside/non- nucleoside polymer.
  • said non-nucleoside moiety may serve to link other small molecules which may interact with target sequences or alter uptake into target cells.
  • alkyl- or aryl-phosphonate Oligomer refers to Oligomers having at least one alkyl- or aryl- phosphonate internucleosidyl linkage.
  • Suitable alkyl- or aryl- phosphonate groups include alkyl- or aryl- groups which do not sterically hinder the phosphonate linkage or interact with each other.
  • Preferred alkyl groups include lower alkyl groups having from about 1 to about 6 carbon atoms.
  • Suitable aryl groups have at least one ring having a conjugated pi electron system and include carbocyclic aryl and heterocyclic aryl groups, which may be optionally substituted and preferably having up to about 10 carbon atoms.
  • methylphosphonate Oligomer (or “MP- Oligomer”) refers to Oligomers having at least one methylphosphonate internucleosidyl linkage.
  • neutral Oligomer refers to Oligomers which have nonionic internucleosidyl linkages between nucleoside monomers (i.e., linkages having no positive or negative ionic charge) and include, for example, Oligomers having internucleosidyl linkages such as alkyl- or aryl- phosphonate linkages, alkyl- or aryl-phosphonothioates, neutral phosphate ester linkages such as phosphotriester linkages, especially neutral ethyltriester linkages; and non-phosphorus-containing internucleosidyl linkages, such as sulfamate, morpholino, formacetal, thioformacetal, and carbamate linkages.
  • a neutral Oligomer may comprise a conjugate between an oligonucleoside or nucleoside/non-nucleoside polymer and a second molecule which comprises a conjugation partner.
  • conjugation partners may comprise intercalators, alkylating agents, binding substances for cell surface receptors, lipophilic agents, nucleic acid modifying groups including photo- cross-linking agents such as psoralen and groups capable of cleaving a targeted portion of a nucleic acid, and the like.
  • conjugation partners may further enhance the uptake of the Oligomer, modify the interaction of the Oligomer with the target sequence, or alter the pharmacokinetic distribution of the Oligomer.
  • the essential requirement is that the oligonucleoside or nucleoside/non-nucleoside polymer that the Oligomer conjugate comprises be substantially neutral.
  • substantially neutral in referring to an Oligomer refers to those Oligomers in which at least about 80 percent of the internucleosidyl linkages between the nucleoside monomers are nonionic linkages.
  • neutral alkyl- or aryl- phosphonate Oligomer refers to neutral Oligomers having neutral internucleosidyl linkages which comprise at least one alkyl- or aryl- phosphonate linkage.
  • neutral methylphosphonate Oligomer refers to neutral Oligomers having internucleosidyl linkages which comprise at least one methylphosphonate linkage.
  • the term "acid resistant” refers to Oligomers which are resistant, in comparison to deoxyribooligo- nucleotides, to acid-catalyzed depurination by hydrolysis of the N-glycosyl bond.
  • the term “triplet” or “triad” refers a hydrogen bonded complex of the bases of three nucleosides between a base (if single stranded) or bases (if double stranded) of a target sequence, a base of a Second Strand and a Third Strand (if a single stranded target sequence) or a base of a Third Strand (if a double-stranded target) .
  • Figure 1 depicts a plot of the percent intact adenine versus time for Oligomers which comprise RNA, DNA, a methylphosphonate Oligomer having a 2'-0-methyl ribosylant and a methylphosphonate Oligomer.
  • Figure 2 depicts a plot of the log of the percent intact adenine versus time for the same Oligomers as plotted in Figure 1.
  • Figure 3 depicts a plot of the percent depurination versus time at 37°C for Oligomers which comprise RNA, DNA, a methylphosphonate Oligomer having a 2' -O-methylribosyl units and a metholphosphonate Oligomer.
  • Figure 4 depicts a plot of the log of the percent depurination versus time at 37°C for the same Oligomers as plotted in Figure 3.
  • Figure 5 depicts a plot of percent intact backbone versus time for a methylphosphonate Oligomer having 2'-0- methyl ribosyl units at 37°C and pH 1.
  • Figure 6 depicts a melting curve for hybridization of Oligomers which comprise an oligodeoxyribonucleotide (1) , a methylphosphonate Oligomer having 2' -O-methylribosyl units (2 . ) and a methylphosphonate Oligomer having deoxyribosyl units (3_) with a DNA target.
  • Figure 7 depicts a melting curve for hybridization of the same Oligomers as Figure 6 with an RNA target .
  • Figure 8 depicts examples of dimers which comprise
  • the Oligomers of the present invention comprise nucleosidyl units having a sugar moiety which is an independently selected 2' -O--alkyl ribosyl group. Suitable are alkyl groups of 1 to 5 carbon atoms. Especially preferred nucleosides have a 2'-0-methyl ribosyl group.
  • Oligomer Strands having the selected internucleoside linkages may be conveniently prepared according to synthetic techniques known to those skilled in the art. For example, commercial machines, reagents and protocols are available for the synthesis of Oligomers having phosphodiester and certain other phosphorus-containing internucleoside linkages. See also Gait, M.J., Oligonucleotide Synthesis: A Practical Approach (IRL Press, 1984) ; Cohen, Jack S., Oligodeoxynucleotides Antisense Inhibitors of Gene Expression. (CRC Press, Boca Raton, FL, 1989) ; and Oligonucleotides and Analogues : A Practical Approach. (F. Eckstein, ed., 1991) . Preparation of Oligomers having certain non-phosphorus-containing internucleoside linkages is described in United States Patent No. 5,142,047, the disclosure of which is incorporated herein by reference.
  • Oligomers that are substantially neutral. According to an especially preferred aspect, these Oligomers have methylphosphonate internucleosidyl linkages. More preferably all the internucleosidyl linkages are methylphosphonate linkages. Oligomers having a mixture of methylphosphonate internucleosidyl linkage and other nucleosidyl linkages may be preferable for certain therapeutic indications and are intended to be within the scope of the present invention.
  • the Oligomer comprise from about 4 to about 40 nucleosides, more preferably, from about 6 to 30 nucleosides . Especially preferred are Oligomer of about 8 to about 20 nucleosides.
  • the Oligomers provided herein may form a high affinity complex with a target sequence such as a nucleic acid or a protein with a high degree of selectivity.
  • a target sequence such as a nucleic acid or a protein with a high degree of selectivity.
  • derivatized Oligomers may be used to bind with and then irreversibly modify a target site in a nucleic acid by cross-linking (psoralens) or cleaving one or both strands (EDTA) .
  • psoralens cross-linking
  • EDTA cleaving one or both strands
  • the Oligomers provided herein may be derivatized to incorporate a nucleic acid reacting or modifying group which can be caused to react with a nucleic acid segment or a target sequence thereof to irreversibly modify, degrade or destroy the nucleic acid and thus irreversibly inhibit its functions.
  • Oligomers may be used to inactivate or inhibit or alter expression of a particular gene or target sequence of the same in a living cell, allowing selective inactivation or inhibition or alteration of expression.
  • the target sequence may be DNA or RNA, such as a pre-mR ⁇ A or an mR ⁇ A.
  • mR ⁇ A target sequences include an initiation codon region, a polyadenylation region, an mR ⁇ A cap site or a splice junction.
  • These Oligomers could also be used to permanently inactivate, turn off or destroy genes which produced defective or undesired products or if activated caused undesirable effects.
  • Oligomers provided herein may form duplexes or triple helix complexes or other forms of stable association with transcribed regions of nucleic acids, ⁇ these complexes are useful in "antisense” or triple strand therapy.
  • Antisense therapy as used herein is a generic term which includes the use of specific binding Oligomers to inactivate undesirable DNA or RNA sequences in vitro or in vivo.
  • Antisense therapy includes targeting a specific D ⁇ A or R ⁇ A target sequence through complementarity or through any other specific binding means, in the case of the present invention by formation of duplexes or triple helix complexes.
  • the Oligomers for use in the instant invention may be administered singly, or combinations of Oligomers may be administered for adjacent or distant targets or for combined effects of antisense mechanisms with the foregoing general mechanisms.
  • the Oligomers can be formulated for a variety of modes of administration, including oral, topical or localized administration. It may be beneficial to have pharmaceutical formulations containing acid resistant Oligomers that may come in contact with acid conditions during their manufacture or when such formulations may themselves be made acidic, to some extent, in order to more compatible with the conditions prevailing at the site of application, e.g., the acid mantle of the skin. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, latest edition.
  • the Oligomer active ingredient is generally combined with a carrier such as a diluent or excipient which may include fillers, extenders, binding, wetting agents, disinte- grant ⁇ , surface-active agents, erodable polymers or lubricants, depending on the nature of the mode of administration and dosage forms.
  • a carrier such as a diluent or excipient which may include fillers, extenders, binding, wetting agents, disinte- grant ⁇ , surface-active agents, erodable polymers or lubricants, depending on the nature of the mode of administration and dosage forms.
  • Typical dosage forms include tablets, powders, liquid preparations including suspensions, emulsions and solutions, granules, and capsules.
  • the Oligomers of the present invention are parti ⁇ cularly suited for oral administration which may require exposure of the drug to acidic conditions in the stomach for up to about 4 hours under conventional drug delivery conditions and for up to about 12 hours when delivered in a sustained release from.
  • These Oligomers may be particularly suited for formulation in preparations for topical administration, since the skin has an acid mantle, formulations including these acid resistant Oligomers may prove advantageous. This also can be advantageous in light of the finding that these Oligomers will cross skin and mucous membranes as described in U.S. Patent Application Serial No. 07/707,879 which is incorporated by reference. Also it may be desirable to provide formulations which include acidic media.
  • the Oligomers for use in the invention are formulated into ointments, salves, eye drops, gels, or creams, as is generally known in the art.
  • Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, bile salts and fusidic acid derivatives for transmucusal administration.
  • detergents may be used to facilitate per ⁇ meation.
  • Transmucosal administration may be through use of nasal sprays, for example, as well as formulations suitable for administration by inhalation, or supposi ⁇ tories.
  • 5' -O-Dimethoxytrityl-2' -O-methyl-N-benzoyladenosine (0.75 g; 1.09 mmoles) (Barry Associates, Inc.) was co- evaporated 3 times with anhydrous l/l acetonitrile/ diisopropylethylamine. The nucleoside was then dis ⁇ solved in 30 ml anhydrous acetonitrile.
  • the tetramen was synthesized and deprotected using standard phosphoramidite procedures (see, e.g.. Gait, M.J., Oligonucleotide Synthesis A Practical Approach, 1984 (IRL Press) on a Milligen 8750 DNA synthesizer.
  • the compound was purified using reverse-phase HPLC on a Whatman RAC II analytical column and a gradient of acetonitrile ("ACN") in 0.1 M triethylammonium acetate (0-30% ACN over 40 minutes at a flow of 1 ml/minute) .
  • ACN acetonitrile
  • OXIDIZER reagent 25 g/L iodine in 0.25% water, 25% 2,6- lutidine, 72.5% tetrahydrofuran.
  • CAP A 10% acetic anhydride in acetonitrile.
  • CAP B 0.625% N,N- dimethylaminopyridine in pyridine. The coupling time was extended to 4 minutes. The dimethoxytriyl group was removed from the oligonucleotide at the end of the synthesis.
  • the oligonucleotide was then cleaved from the support and deprotected.
  • the support bound oligonucleo ⁇ tide was removed from the synthesis cartridge and placed in a glass 1 dram vial with a screw top.
  • the support was treated for 30 minutes at room temperature with 1 ml of a solution of acetonitrile/ethanol/NH 4 OH (9/9/1) .
  • 1 ml of ethylenediamine was added to the reaction vessel and the reaction allowed 6 hours to go to comple ⁇ tion.
  • the supernatant containing the oligonucleotide was then removed from the support and the support rinsed twice with 2 ml of l/l acetonitrile/water, when combined with the supernatant .
  • the combined solution was diluted to 30 ml total volume with water and neutralized with approximately 4 ml of 6 N HCL.
  • the neutralized solution was desalted using a Waters C-18 Sep-Pak cartridge which was pre-equilibrated with 10 ml acetonitrile, 10 ml of 50% acetonitrile/100 mM triethylammonium bicarbonate, and 10 ml of 25 mM triethylammonium bicarbonate, sequentially. After the reaction solution was passed through the column it was washed with 30 ml of water. The product was then eluted with 5 ml of l/l acetonitrile/water.
  • the oligonucleotide was purified by HPLC on a reverse phase column (Whatman RAC II) using a gradient of acetonitrile in 50 mM triethylammonium acetate.
  • Compound 4 was synthesized, deprotected, and purified as described for Compound 2 using the 2'-0- methyl adenosine monomer of Example 1(B) with the exception that the coupling time was extended to 3 minutes to allow adequate coupling of the more sterically hindered 2' -O-methyl monomer reagent.
  • Compound 4 was synthesized on support bound deoxyadenosine.
  • the oligoribonucleotide tetramer (Compound 3) was synthesized using 5' -O-dimethoxytrityl-2' -O-tert-butyl- dimethylsilyl-3' -0-N,N-diisopropyl-/3-cyanoethylphos- phoramidite adenosine (Millipore) .
  • the synthesis was done on a 1 ⁇ mole scale with a Milligen 8750 automated DNA synthesizer using standard Milligen phosphoramidite procedures with the exception that the coupling times were extended to 12 minutes to allow adequate time for the more sterically hindered 2' -0- tert-butyldimethyl- silyl RNA monomer to react .
  • the syntheses were begun on control-pore glass bound 2' -0-tert-butyldimethylsilyl adenosine (Pennisula Laboratories) . All other oligo ⁇ nucleotide synthesis reagents were as described in Milligen' s standard protocols.
  • the oligoribonucleotides were handled under sterile, RNases- free conditions. Water was sterilized by overnight treatment with 0.5% diethylpyrocarbonate followed by autoclaving. All glassware was baked for at least 4 hours at 300° C. The oligonucleotides were deprotected and cleaved from support by first treating the support bound oligomer with 3/1 ammonium hydroxide/ethanol for 15 hours at 55° C. The supernatant, which contained the oligonucleotide, was then decanted and evaporated to dryness.
  • the reverse phase HPLC analyses were performed on a Beckman System Gold HPLC Model 126 pumps, Model 168 photodiode array detector, and Model 507 autoinjector.
  • a Whatman RAC II ODS 3 (100 x 4.6 mm) analytical column was used for the analyses.
  • the solvent system used was a gradient of acetonitrile in 0.1 M aqueous triethylammonium acetate, pH 7. The gradient was 0 to 2% acetonitrile over 10 minutes followed by 2 to 60% acetonitrile over 10 additional minutes. The gradient then returns to 0% acetonitrile to equilibrate the column for the next injection.
  • the flow rate was 1 ml/minute. This gradient cleanly resolved adenine from the starting reagent and various side-products of the acid digestion.
  • Figure 1 depicts a plot of the percent intact adenosine versus time for the tetramers.
  • Figure 2 is a plot of the log of the percent intact adenosine versus time. From the log plot the k A ' s and half-lives of the rates of depurination of the tetramers were calculated. Table 1 contains those figures, as well as the relative rates of depurination as compared to the phosphodiester control (compound 1) .
  • Figure 3 depicts the rate of depurination of the adenosinyl tetramers at pH 1.0 and 37°C plotted as percent depurination versus time.
  • Figure 4 depicts the rate of depurination of adenosinyl tetramers at pH 1.0 and 37°C plotted as log percent depurination versus time.
  • Figure 5 depicts stability of the methylphosphonate backbone to acidic conditions at pH 1.0 and 37°C for the (2'-0-methyl A) 3 (dA) - methylphosphonate Oligomer. The conditions were 0.3 mM Oligomer in 0.1 M HCl, pH 1.0 at 37°C. Limit of detection was 0.2%; error was ⁇ 2%.
  • Rate was determined by increase of free adenine compared with total adeninyl bearing species, which was followed by HPLC. Data were corrected for depurination, and cleavage of 3 '-terminal deoxyadenosine for the 2' -O-methyladenosinyl methylphosphonate Oligomer.
  • oligo-2' -O-methylribonucleoside methylphosphonate was prepared using suitably protected 2' -O-methylribonucleoside methylphosphonamidite synthone.
  • W indicates a 2'-0- methylribonucleoside
  • the underline indicates the positions of the methylphosphonate linkages.
  • the 5'- internucleotide bond of the Oligomer is a phosphodiester linkage.
  • the Oligomer was deprotected by sequential treatment with hydrazine hydrate in pyridine buffered with acetic acid, followed by treatment with a solution of ethylenedia ine in 95% ethanol (1:1 v/v) .
  • the Oligomer was purified by DEAE cellulose chromatography, followed by preparative HPLC on a C-18 reversed phase column. r-AUTMA m GG m A m UTMU , TrG ⁇ Trc A d-ATAGGATTGTC B d-ATAGGATTTGTC C
  • Oligomer A was phosphorylated using polynucleotide kinase and gamma- [ 32 P] -ATP. The phosphorylated Oligomer was then incubated with 0.1 N HCl or 1.0 N HCl at 37°C overnight (-14 hours) . The Oligomer was then analyzed by polyacrylamide gel electrophoresis under denaturing conditions. No degradation of the Oligomer was detected under these conditions. According to our experience, incubation of the deoxyribonucleoside methylphosphonate (B) with 0.1 N HCl overnight at 37°C or with 1.0 N HCl at 37°C for several hours would result in considerable depurination at the G and A nucleosides.
  • B deoxyribonucleoside methylphosphonate
  • oligo-2'-0- methylribonucleoside methylphosphonate 2 .
  • This behavior is in contrast to that of the oligodeoxyribonucleoside methylphosphonate, 3_, which forms a stable duplex with the DNA target, but only a very weak duplex with the RNA target.
  • oligo-2'-0- methylribonucleoside methylphosphonates may form more stable hybrids with RNA than do oligodeoxyribonucleoside methylphosphonates and thus they be more effective antisense reagents when targets against cellular or viral RNAs.

Abstract

Des oligonucléotides acido-résistants modifiés par alkylation au niveau de la position 2'-O peuvent être administrés par voie orale. Sont également décrites des formulations acceptables pour usage oral, préparées avec, comme principes actifs, les oligonucléotides modifiés.
PCT/US1994/000157 1993-01-06 1994-01-05 OLIGONUCLEOTIDES MODIFIES POUR AMELIORER LA STABILITE A pH ACIDE WO1994015619A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
KR1019950702783A KR960700062A (ko) 1993-01-06 1994-01-05 산성 pH에서의 안정성을 개선시키기 위해 변형된 올리고누클레오티드(OLIGONUCLEOTIDES MODIFIED TO IMPROVE STABILITY AT ACIC pH)
AU59924/94A AU692143B2 (en) 1993-01-06 1994-01-05 Oligonucleotides modified to improve stability at acid pH
JP6516188A JPH08505396A (ja) 1993-01-06 1994-01-05 酸性ｐＨにおいて安定性を増加するよう修飾されたオリゴヌクレオチド
EP94906041A EP0690716A4 (fr) 1993-01-06 1994-01-05 OLIGONUCLEOTIDES MODIFIES POUR AMELIORER LA STABILITE A pH ACIDE

Applications Claiming Priority (2)

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US110993A 1993-01-06 1993-01-06
US08/001,109 1993-01-06

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WO1994015619A1 true WO1994015619A1 (fr) 1994-07-21

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EP (1) EP0690716A4 (fr)
JP (1) JPH08505396A (fr)
KR (1) KR960700062A (fr)
CN (1) CN1118140A (fr)
AU (1) AU692143B2 (fr)
CA (1) CA2152672A1 (fr)
IL (1) IL108206A0 (fr)
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US5591721A (en) * 1994-10-25 1997-01-07 Hybridon, Inc. Method of down-regulating gene expression
US5859228A (en) * 1995-05-04 1999-01-12 Nexstar Pharmaceuticals, Inc. Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes
US5869248A (en) * 1994-03-07 1999-02-09 Yale University Targeted cleavage of RNA using ribonuclease P targeting and cleavage sequences
US6011020A (en) * 1990-06-11 2000-01-04 Nexstar Pharmaceuticals, Inc. Nucleic acid ligand complexes
US6015886A (en) * 1993-05-24 2000-01-18 Chemgenes Corporation Oligonucleotide phosphate esters
US6025133A (en) * 1996-12-30 2000-02-15 Gen-Probe Incorporated Promoter-sequestered oligonucleoside and method of use
US6051698A (en) * 1997-06-06 2000-04-18 Janjic; Nebojsa Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes
US6057153A (en) * 1995-01-13 2000-05-02 Yale University Stabilized external guide sequences
US6087112A (en) * 1998-12-30 2000-07-11 Oligos Etc. Inc. Arrays with modified oligonucleotide and polynucleotide compositions
WO2000040591A1 (fr) * 1998-12-30 2000-07-13 Oligos Etc. Inc. Acides nucleiques protones/acidifies, et methodes d'utilisation correspondantes
WO2000040714A2 (fr) * 1998-12-30 2000-07-13 Oligos Etc. Inc. Inhibiteurs de la phosphodiesterase utilises a des fins therapeutiques
WO2000057890A1 (fr) * 1999-03-31 2000-10-05 Oligos Etc. Inc. Administration par voie pulmonaire d'acides nucleiques protones/acidifies
US6147204A (en) * 1990-06-11 2000-11-14 Nexstar Pharmaceuticals, Inc. Nucleic acid ligand complexes
WO2000070093A1 (fr) * 1999-05-13 2000-11-23 Oligos Etc. Inc. Jeu d'echantillons comprenant des compositions d'oligonucleotides et de polynucleotides modifies
US6229002B1 (en) 1995-06-07 2001-05-08 Nexstar Pharmaceuticlas, Inc. Platelet derived growth factor (PDGF) nucleic acid ligand complexes
US6426335B1 (en) 1997-10-17 2002-07-30 Gilead Sciences, Inc. Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes
US6465188B1 (en) 1990-06-11 2002-10-15 Gilead Sciences, Inc. Nucleic acid ligand complexes
US6608035B1 (en) 1994-10-25 2003-08-19 Hybridon, Inc. Method of down-regulating gene expression
US6610478B1 (en) 1996-08-16 2003-08-26 Yale University Phenotypic conversion of cells mediated by external guide sequences
US6627215B1 (en) 1998-12-30 2003-09-30 Oligos Etc. Inc. Devices for improved wound management
US6645943B1 (en) 1994-10-25 2003-11-11 Hybridon, Inc. Method of down-regulating gene expression
US6844151B1 (en) * 1999-09-29 2005-01-18 Oligos Etc. Inc. Methods for production of arrays with modified oligonucleotide and polynucleotide compositions
CN101824063A (zh) * 2010-04-16 2010-09-08 北京欧凯纳斯科技有限公司 一种用烷基修饰的核酸及其修饰方法和应用
US7868162B2 (en) 1998-12-30 2011-01-11 Lakewood-Amedex, Inc. Antimicrobial and antiviral compounds and methods for their use
US8071737B2 (en) 1995-05-04 2011-12-06 Glead Sciences, Inc. Nucleic acid ligand complexes
US8183361B2 (en) 2001-07-10 2012-05-22 Lakewood-Amedex, Inc. Oligonucleotide-containing pharmacological compositions and their use
US8853376B2 (en) 2002-11-21 2014-10-07 Archemix Llc Stabilized aptamers to platelet derived growth factor and their use as oncology therapeutics
WO2014165814A1 (fr) 2013-04-04 2014-10-09 Georgia State University Research Foundation, Inc. Détection par micropuce à arn utilisant l'amplification de signal assistée par nanoparticule
US9381208B2 (en) 2006-08-08 2016-07-05 Rheinische Friedrich-Wilhelms-Universität Structure and use of 5′ phosphate oligonucleotides
US9399658B2 (en) 2011-03-28 2016-07-26 Rheinische Friedrich-Wilhelms-Universität Bonn Purification of triphosphorylated oligonucleotides using capture tags
US9738680B2 (en) 2008-05-21 2017-08-22 Rheinische Friedrich-Wilhelms-Universität Bonn 5′ triphosphate oligonucleotide with blunt end and uses thereof
US10059943B2 (en) 2012-09-27 2018-08-28 Rheinische Friedrich-Wilhelms-Universität Bonn RIG-I ligands and methods for producing them

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Publication number Priority date Publication date Assignee Title
US6011020A (en) * 1990-06-11 2000-01-04 Nexstar Pharmaceuticals, Inc. Nucleic acid ligand complexes
US6465188B1 (en) 1990-06-11 2002-10-15 Gilead Sciences, Inc. Nucleic acid ligand complexes
US6147204A (en) * 1990-06-11 2000-11-14 Nexstar Pharmaceuticals, Inc. Nucleic acid ligand complexes
US6015886A (en) * 1993-05-24 2000-01-18 Chemgenes Corporation Oligonucleotide phosphate esters
US5869248A (en) * 1994-03-07 1999-02-09 Yale University Targeted cleavage of RNA using ribonuclease P targeting and cleavage sequences
US6936593B1 (en) 1994-10-25 2005-08-30 Hybridon, Inc. Method of down-regulating gene expression
US6608035B1 (en) 1994-10-25 2003-08-19 Hybridon, Inc. Method of down-regulating gene expression
US6645943B1 (en) 1994-10-25 2003-11-11 Hybridon, Inc. Method of down-regulating gene expression
US5591721A (en) * 1994-10-25 1997-01-07 Hybridon, Inc. Method of down-regulating gene expression
US6057153A (en) * 1995-01-13 2000-05-02 Yale University Stabilized external guide sequences
US5859228A (en) * 1995-05-04 1999-01-12 Nexstar Pharmaceuticals, Inc. Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes
US8071737B2 (en) 1995-05-04 2011-12-06 Glead Sciences, Inc. Nucleic acid ligand complexes
US6229002B1 (en) 1995-06-07 2001-05-08 Nexstar Pharmaceuticlas, Inc. Platelet derived growth factor (PDGF) nucleic acid ligand complexes
US7879993B2 (en) 1995-06-07 2011-02-01 Gilead Sciences, Inc. Platelet derived growth factor (PDGF) nucleic acid ligand complexes
US6582918B2 (en) 1995-06-07 2003-06-24 Gilead Sciences, Inc. Platelet derived growth factor (PDGF) nucleic acid ligand complexes
US6610478B1 (en) 1996-08-16 2003-08-26 Yale University Phenotypic conversion of cells mediated by external guide sequences
US6962784B2 (en) 1996-10-25 2005-11-08 Gilead Sciences, Inc. Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes
US6025133A (en) * 1996-12-30 2000-02-15 Gen-Probe Incorporated Promoter-sequestered oligonucleoside and method of use
US6051698A (en) * 1997-06-06 2000-04-18 Janjic; Nebojsa Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes
US6426335B1 (en) 1997-10-17 2002-07-30 Gilead Sciences, Inc. Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes
US7939654B2 (en) 1997-12-16 2011-05-10 Gilead Sciences, Inc. Platelet derived growth factor (PDGF) nucleic acid ligand complexes
AU781577B2 (en) * 1998-12-30 2005-06-02 Lakewood-Amedex, Inc. Therapeutic phosphodiesterase inhibitors
WO2000040714A2 (fr) * 1998-12-30 2000-07-13 Oligos Etc. Inc. Inhibiteurs de la phosphodiesterase utilises a des fins therapeutiques
US6562569B1 (en) 1998-12-30 2003-05-13 Oligos Etc. Inc. Arrays with modified oligonucleotide and polynucleotide compositions
US6627215B1 (en) 1998-12-30 2003-09-30 Oligos Etc. Inc. Devices for improved wound management
US8435960B2 (en) 1998-12-30 2013-05-07 Lakewood-Amedex, Inc. Devices for improved wound management
WO2000040714A3 (fr) * 1998-12-30 2000-11-02 Oligos Etc Inc Inhibiteurs de la phosphodiesterase utilises a des fins therapeutiques
US6087112A (en) * 1998-12-30 2000-07-11 Oligos Etc. Inc. Arrays with modified oligonucleotide and polynucleotide compositions
WO2000040591A1 (fr) * 1998-12-30 2000-07-13 Oligos Etc. Inc. Acides nucleiques protones/acidifies, et methodes d'utilisation correspondantes
US7868162B2 (en) 1998-12-30 2011-01-11 Lakewood-Amedex, Inc. Antimicrobial and antiviral compounds and methods for their use
WO2000057890A1 (fr) * 1999-03-31 2000-10-05 Oligos Etc. Inc. Administration par voie pulmonaire d'acides nucleiques protones/acidifies
WO2000070093A1 (fr) * 1999-05-13 2000-11-23 Oligos Etc. Inc. Jeu d'echantillons comprenant des compositions d'oligonucleotides et de polynucleotides modifies
US6844151B1 (en) * 1999-09-29 2005-01-18 Oligos Etc. Inc. Methods for production of arrays with modified oligonucleotide and polynucleotide compositions
US8183361B2 (en) 2001-07-10 2012-05-22 Lakewood-Amedex, Inc. Oligonucleotide-containing pharmacological compositions and their use
US8188259B2 (en) 2001-07-10 2012-05-29 Lakewood-Amedex, Inc. Oligonucleotide-containing pharmacological compositions and their use
US9567584B2 (en) 2001-07-10 2017-02-14 Lakewood Amedex, Inc. Oligonucleotide—containing pharmacological compositions and their use
US8916529B2 (en) 2001-07-10 2014-12-23 Lakewood-Amedex, Inc. Oligonucleotide-containing pharmacological compositions and their use
US8853376B2 (en) 2002-11-21 2014-10-07 Archemix Llc Stabilized aptamers to platelet derived growth factor and their use as oncology therapeutics
US9381208B2 (en) 2006-08-08 2016-07-05 Rheinische Friedrich-Wilhelms-Universität Structure and use of 5′ phosphate oligonucleotides
US10238682B2 (en) 2006-08-08 2019-03-26 Rheinische Friedrich-Wilhelms-Universität Bonn Structure and use of 5′ phosphate oligonucleotides
US9738680B2 (en) 2008-05-21 2017-08-22 Rheinische Friedrich-Wilhelms-Universität Bonn 5′ triphosphate oligonucleotide with blunt end and uses thereof
US10036021B2 (en) 2008-05-21 2018-07-31 Rheinische Friedrich-Wilhelms-Universität Bonn 5′ triphosphate oligonucleotide with blunt end and uses thereof
US10196638B2 (en) 2008-05-21 2019-02-05 Rheinische Friedrich-Wilhelms-Universität Bonn 5′ triphosphate oligonucleotide with blunt end and uses thereof
CN101824063B (zh) * 2010-04-16 2013-07-24 北京欧凯纳斯科技有限公司 一种用烷基修饰的核酸及其修饰方法和应用
CN101824063A (zh) * 2010-04-16 2010-09-08 北京欧凯纳斯科技有限公司 一种用烷基修饰的核酸及其修饰方法和应用
US9399658B2 (en) 2011-03-28 2016-07-26 Rheinische Friedrich-Wilhelms-Universität Bonn Purification of triphosphorylated oligonucleotides using capture tags
US9896689B2 (en) 2011-03-28 2018-02-20 Rheinische Friedrich-Wilhelms-Universität Bonn Purification of triphosphorylated oligonucleotides using capture tags
US10059943B2 (en) 2012-09-27 2018-08-28 Rheinische Friedrich-Wilhelms-Universität Bonn RIG-I ligands and methods for producing them
US10072262B2 (en) 2012-09-27 2018-09-11 Rheinische Friedrich-Wilhelms-Universität Bonn RIG-I ligands and methods for producing them
US11142763B2 (en) 2012-09-27 2021-10-12 Rheinische Friedrich-Wilhelms-Universität Bonn RIG-I ligands and methods for producing them
WO2014165814A1 (fr) 2013-04-04 2014-10-09 Georgia State University Research Foundation, Inc. Détection par micropuce à arn utilisant l'amplification de signal assistée par nanoparticule

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CN1118140A (zh) 1996-03-06
JPH08505396A (ja) 1996-06-11
KR960700062A (ko) 1996-01-19
AU5992494A (en) 1994-08-15
EP0690716A1 (fr) 1996-01-10
EP0690716A4 (fr) 1997-05-02
IL108206A0 (en) 1994-04-12
CA2152672A1 (fr) 1994-07-21
NZ261253A (en) 1997-07-27
AU692143B2 (en) 1998-06-04

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