EP1713509A2 - Procede pour produire des conjugues constitues de polysaccharides et de polynucleotides - Google Patents

Procede pour produire des conjugues constitues de polysaccharides et de polynucleotides

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Publication number
EP1713509A2
EP1713509A2 EP05715273A EP05715273A EP1713509A2 EP 1713509 A2 EP1713509 A2 EP 1713509A2 EP 05715273 A EP05715273 A EP 05715273A EP 05715273 A EP05715273 A EP 05715273A EP 1713509 A2 EP1713509 A2 EP 1713509A2
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EP
European Patent Office
Prior art keywords
polynucleotide
aldonic acid
amino group
hydroxyethyl starch
polysaccharide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05715273A
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German (de)
English (en)
Inventor
Klaus Sommermeyer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Supramol Parenteral Colloids GmbH
TME Pharma AG
Original Assignee
Noxxon Pharma AG
Supramol Parenteral Colloids GmbH
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Filing date
Publication date
Application filed by Noxxon Pharma AG, Supramol Parenteral Colloids GmbH filed Critical Noxxon Pharma AG
Publication of EP1713509A2 publication Critical patent/EP1713509A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a method for producing a conjugate from a polynucleotide and a polysaccharide and to the conjugates obtainable by such a method.
  • PEGylation or HESylation of pharmaceutically active compounds such as proteins is, among other things, that by coupling the proteins to the above-mentioned polymers such as polyethylene glycol (PEG) or hydroxyethyl starch (HES), the short ones, which are too short for the development of the full pharmaceutical potential biological half-life can be extended. Coupling can also have a positive effect on the antigenic properties of proteins. In the case of other active pharmaceutical ingredients, the water solubility can be increased considerably by the coupling. Examples of the HESylation of active pharmaceutical ingredients are described, for example, in international patent application WO 02/080979 A2 or in international patent application WO 03/000738 A2.
  • HES is the hydroxyethylated derivative of amylopectin, a glucose polymer that is found in waxy maize starch and is found to be over 95%.
  • Amylopectin consists of glucose units which are present in ⁇ -1,4-glycosidic bonds and have ⁇ -1, 6-glycosidic branches.
  • HES has advantageous rheological properties and is currently used as a volume substitute and clinically used for hemodilution therapy (Sommermeyer et al., Kranlcenhauspharmazie,
  • the present invention has for its object to provide a method for producing a conjugate from a polynucleotide and a polysaccharide.
  • the object is achieved in a first aspect by a method for producing a conjugate from a polynucleotide and a polysaccharide comprising the steps: a) providing an aldonic acid of the polysaccharide or a derivative thereof; b) reacting the aldonic acid with an alcohol derivative, preferably a carbonate derivative of an alcohol, to an aldonic acid ester, preferably to an activated aldonic acid ester; and c) reacting the aldonic acid ester with the polynucleotide, the polynucleotide having a functional ammo group, characterized in that the reaction of the aldonic acid with the alcohol derivative in step b) is carried out in a dry aprotic polar solvent.
  • the solvent is selected from the group comprising dimethyl sulfoxide, dimethylformamide and dimethylacetamide.
  • the aldonic acid ester is purified and then used in step c).
  • reaction mixture from step b) with the aldonic acid ester is used directly in step c).
  • step c) is carried out at a pH value range from 7 to 9, preferably 7.5 to 9 and preferably 8.0 to 8.8.
  • step c) is carried out at a pH of about 8.4.
  • the molar ratio of aldonic acid to alcohol derivative is approximately 0.9 to 1.1, preferably approximately 1.
  • the alcohol is selected from the group comprising N-hydroxy succinimide, sulfonated N-hydroxy succinimide, phenol derivatives and N-hydroxy benzotriazole.
  • the polysaccharide is selected from the group comprising dextran, hydroxyethyl starch, hydroxypropyl starch and branched starch fractions.
  • the polysaccharide is hydroxyethyl starch.
  • the hydroxyethyl starch has a weight average molecular weight of about 3,000 to 100,000 daltons, preferably of about 5,000 to 60,000.
  • the hydroxyethyl starch has a number average molecular weight of approximately 2,000 to 50,000 daltons.
  • the hydroxyethyl starch has a ratio of weight average molecular weight to number average average molecular weight of approximately 1.05 to 1.20.
  • the hydroxyethyl starch has a molar substitution of 0.1 to 0.8, preferably 0.4 to 0.7.
  • the hydroxyethyl starch has a substitution pattern expressed as the C2 / C6 ratio of about 2 to 12, preferably of about 3 to 10.
  • the polynucleotide is a functional nucleic acid.
  • the functional nucleic acid is an aptamer or a mirror bucket.
  • the polynucleotide has a molecular weight of 300 to 50,000 Da, preferably 4,000 to 25,000 Da and preferably 7,000 to 16,000 Da.
  • the functional amino group is a primary or secondary amino group, preferably a primary amino group.
  • the functional amino group is bound to a terminal phosphate of the polynucleotide. In a preferred embodiment it is provided that the functional amino group is bound to the phosphate group via a linker.
  • the functional amino group is a 5-aminohexyl group.
  • the object is achieved according to the invention by a conjugate of a polysaccharide and a polynucleotide, obtainable by a method according to the first aspect of the present invention.
  • the present invention is based on the surprising finding that from hydroxyethyl starch aldonic acids and aldonic acids from other polysaccharides, such as. B. waxy corn starch degradation fractions, in dry aprotic, polar solvents such as dimethylacetamide (DMA), dimethyl sulfoxide (DMSO) or dimethylformamide (DMF), with alcohols, especially with the carbonates of alcohols, i.e. the diesters of carbonic acid with alcohols, such as , B. N-hydroxy succinimides, the corresponding aldonic acid esters could be prepared, which can be implemented in an aqueous environment with nucleophilic amino groups of polynucleotides to more stable amides advantageously. As a side reaction, the aldonic esters are stranded with water to form free aldonic acid and free alcohol.
  • DMA dimethylacetamide
  • DMSO dimethyl sulfoxide
  • DMF dimethylformamide
  • the present invention turns away from the teachings previously described in the prior art or is based on the knowledge that the various methods described in the prior art are not suitable for the efficient production of a conjugate from a polynucleotide and a polysaccharide.
  • EDC can be used in an aqueous medium to couple molecules containing amino functions to the terminal phosphate group of oligonucleotides with the formation of a phosphoramidate bond.
  • the internal phosphate groups do not react under the reaction conditions that are realized. In this way, in particular 5 'phosphate groups can be specifically modified (Bioconjugate Techniques, Greg T. Hermanson, Academic Press, San Diego, New York, Boston, London, Sydney, Tokyo, Toronto (1996) page 52).
  • FIG. 1A The reaction scheme for the production of a conjugate from a polynucleotide and a polysaccharide according to the invention is shown in FIG. 1, FIG. 1A showing the structure of the aldonic acid group of the aldonic acid of the polysaccharide and FIG. IB clarifying the course of the reaction.
  • FIG. 2 The reaction equations in FIG. 2, which are the subject of Examples 4 to 14, summarize the unsuccessful attempts to prepare a conjugate from a polynucleotide and a polysaccharide.
  • hydroxyethyl starch is a particularly preferred polysaccharide.
  • starch derivatives such as e.g. Hydroxyprolyl starch
  • hyperbranched starch fractions described in German patent application 102 17 994 in particular hyperbranched starch fractions with degrees of branching greater than 10 mol%, preferably greater than 10 mol% and less than 16 mol%, can be used in the context of the present invention.
  • HES is essentially characterized by the weight average molecular weight Mw, the number average molecular weight Mn, the molecular weight distribution and the degree of substitution.
  • Substitution with hydroxyethyl groups in ether linkage is possible at the carbon atoms 2, 3 and 6 of the anhydroglucose units.
  • the substitution pattern is described as the ratio of C2 to C6 substitution (C2 / C6 ratio).
  • the degree of substitution can be described as DS (“for degree of substitution”), which refers to the proportion of substituted glucose molecules of all glucose units, or as MS (for “molar substitution”), which means the average number of hydroxyethyl groups is designated per glucose unit.
  • the aldonic acid esters used according to the invention are prepared by reacting the aldonic acid of the polysaccharide or its derivatives in dry, aprotic solvents such as, for. As dimethylformamide (DMF), dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA) and the carbonates of the alcohol component.
  • aprotic solvents such as, for. As dimethylformamide (DMF), dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA) and the carbonates of the alcohol component.
  • the aldonic acids described herein are known in the prior art and can be prepared, for example, in accordance with the disclosure of German patent application DE 196 28 705.
  • the molar ratio is about 0.9 to 1.1, preferably about 1.0, since with an excess of carbonate, as is the case with the alcohol -
  • selectively activated OH groups of the polysaccharide and excess acid functions are not implemented in the event of a deficit.
  • Particularly preferred alcohols in the context of the present invention are N-hydroxy succinimide, sulfonated N-hydroxy succinimide, phenol derivatives and N-hydroxy-benzotriazole.
  • Suitable phenol derivatives include, inter alia, chlorinated, fluorinated or nitrated compounds, these being able to be activated one or more times, in particular by the electrophilic groups mentioned above. Accordingly, it is within the scope of the present invention to use mono- or polychlorinated phenols, mono- or polyfluorinated phenols or mono- or polynitrated phenols.
  • the aldonic acid esters used according to the invention can be precipitated from the solution in DMF by dry ethanol, isopropanol or acetone and can be purified or enriched by repeating the process several times. Such aldonic acid esters can then be used in bulk for coupling to polysaccharides. However, the solution of the reaction products in inert apolar solvent can also be used directly, without isolation of the active aldonic acid ester, for coupling to polysaccharides.
  • any type of polynucleotide is conjugated to a polysaccharide.
  • the polynucleotide can be produced from L-nucleosides or D-nucleosides or mixtures thereof, whereby these, individually or as a whole, can have further modifications, such as modifications to increase the stability in biological systems.
  • Such a modification represents for example the fluorination at position 2 'of the sugar component of the nucleotides or
  • the sugar components of the nucleotides that make up the polynucleotide can have a sugar other than ribose or deoxyribose.
  • Such sugars can be, for example, other pentoses, such as arabinose, but also hexoses or tetroses.
  • Such sugars can also contain a nitrogen atom or sulfur atom, for example in an aza or thio sugar, and / or the sugar portion of the polynucleotide can be at least partially replaced by a morpholino ring.
  • the polynucleotide can be at least partially designed as a locked nucleic acid (LNA) or peptide nucleic acid (PNA).
  • OH groups of the molecular components making up the backbone of the polynucleotide can be chemically modified by means of suitable NH 2 , SH, aldehyde, carboxyacid, phosphate, iodine, bromine or chlorine groups.
  • the polynucleotide is a ribonucleic acid or a deoxyribonucleic acid or combinations thereof, ie individual or a group of nucleotides are present as RNA and the other nucleotides that make up the nucleic acid are present as DNA and vice versa.
  • L-nucleic acid is used synonymously with the term L-oligonucleotide or L-polynucleotide and denotes, inter alia, both L-deoxyribonucleic acid and L-ribonucleic acid and combinations thereof, ie that individual or a group of nucleotides are present as RNA and the other nucleotides making up the nucleic acid are present as DNA and vice versa. It is also provided that other sugars form the sugar component of the nucleotide instead of deoxyribose or ribose.
  • nucleotides with further modifications at position 2 ' such as NH 2 , OMe, OEt, OAlkyl, NHAlkyl and the use of natural or unnatural nucleobases such as isocytidine, isoguanosine.
  • L-nucleic acid has so-called abasic positions, ie nucleotides that lack the nucleobase. Such abasic positions can be arranged both within the nucleotide sequence of the L-nucleic acid and at one or both of the ends, ie the 5 'and / or the 3' end.
  • polynucleotide is single-stranded, but it is also within the scope of the present invention that it is double-stranded. It is typically the one used according to the invention Polynucleotide around a single-stranded L-nucleic acid, which, however, due to its
  • Primary sequence can form defined secondary structures and also tertiary structures.
  • the conjugated nucleic acids described herein are preferably so-called Spiegelmers.
  • Spiegelmers are functional L-nucleic acids or L-polynucleotides, i.e. H. those nucleic acids that bind to a target molecule or a part thereof, and are the result of contacting a nucleic acid library, in particular a statistical nucleic acid library, with the target molecule.
  • combinatorial DNA libraries are first produced. As a rule, this involves the synthesis of DNA oligonucleotides which contain a central region of 10-100 randomized nucleotides which are flanked by two primer-binding regions 5'- and 3'-terminal.
  • the preparation of such combinatorial libraries is described, for example, in Conrad, RC, Giver, L., Tian, Y. and Ellington, AD, 1996, Methods Enzymol., Vol 267, 336-367.
  • Such a chemically synthesized single-stranded DNA library can be converted into a double-stranded library via the polymerase chain reaction, which library can be used for a selection in itself.
  • the individual strands are separated using suitable methods, so that a single strand library is used again, which is used for the in vitro selection method if it is a DNA selection (Bock, LC, Griffin, LC, Latham, JA, Vermaas, EH and Toole, JJ, 1992, Nature, Vol. 355, 564-566).
  • a DNA selection Bock, LC, Griffin, LC, Latham, JA, Vermaas, EH and Toole, JJ, 1992, Nature, Vol. 355, 564-566.
  • a suitable DNA-dependent polymerase e.g. B. the T7 RNA polymerase, an RNA library.
  • the targets can e.g. B. viruses, proteins, peptides,
  • Nucleic acids small molecules such as metabolites of metabolism, active pharmaceutical ingredients or their metabolites or other chemical, biochemical or biological components such as in Gold, L., Polisky, B., Uhlenbeck, O. and Yarus, 1995, Annu. Rev.
  • Molecules are isolated from the library originally used and, after the selection step, are amplified by means of the polymerase chain reaction. This is the case with RNA selections
  • Upstream amplification step by polymerase chain reaction a reverse transcription.
  • a library enriched after a first round of selection can then be used in a new round of selection, so that the molecules enriched in the first round of selection have the chance to assert themselves again through selection and amplification and to go on with a further round of selection with even more daughter molecules.
  • Molecules prevailed. It has created an enriched pool, whose representatives through
  • Cloning can be isolated and then determined with the usual methods of sequence determination of DNA in its primary structure. The sequences obtained are then checked for their binding properties with respect to the target. The procedure for
  • the best binding molecules can be shortened to their essential binding domain by shortening the primary sequences and can be prepared by chemical or enzymatic synthesis.
  • a special form of aptamers that can be produced in this way are the so-called Spiegelmers, which are essentially characterized in that they are at least partially, preferably completely, composed of the non-natural L-nucleotides.
  • Methods for producing such Spiegelmers are described in PCT / EP97 / 04726, the disclosure of which is hereby incorporated by reference.
  • the peculiarity of the method described therein lies in the generation of enantiomeric nucleic acid molecules, ie of L- Nucleic acid molecules that bind to a native target, that is to say in the natural form or configuration, or to such a target structure.
  • the selection procedure is used to first bind binding nucleic acids or sequences against the enantiomers, i.e. select non-naturally occurring structure of a naturally occurring target, for example in the case that the target molecule is a protein, against a D-protein.
  • the binding molecules obtained in this way (D-DNA, D-RNA or corresponding D-derivatives) are determined in their sequence and the identical sequence is then synthesized with mirror-image nucleotide building blocks (L-nucleotides or L-nucleotide derivatives).
  • Target shape or configuration present.
  • the polynucleotides in particular the functional nucleic acids such as aptamers or Spiegelmers, as are obtained in the selection and shortening processes described herein, have a molecular weight of approximately 300 Da to 50,000 Da. These preferably have a molecular weight of 4,000 Da to 25,000 Da, more preferably 7,000 to 16,000 Da.
  • target molecules described above can be molecules or structures, such as. B. viruses, viroids, bacteria, cell surfaces, cell organelles, proteins, peptides, nucleic acids, small molecules such as metabolites of metabolism, pharmaceutical agents or their metabolites or other chemical, biochemical or biological components.
  • the polynucleotide preferably on a phosphate group of the polynucleotide, has a nucleophilic group with which the aldonic acid ester reacts to form the conjugate. It is particularly preferred in the context of the present invention that this nucleophilic group is a functional amino group, preferably a primary amino group (NH 2 group). It is also within the scope of the invention that the polynucleotide reacted with the aldonic acid ester contains a functional secondary amino group, an imino group. In the context of the present invention, however, it is particularly preferred that the nucleophilic group is a primary amino group which is preferably bound to a phosphate group of the polynucleotide.
  • the amino group on the phosphate group is preferably present at the 5 'or 3' end, that is to say the terminal phosphate groups, of the polynucleotide.
  • the amino group can be bound either directly to the phosphate group or via a linker to the phosphate group.
  • linkers are known in the art.
  • Preferred linkers are alkyl radicals with a length of 1 to 8, preferably 2 to 6, carbon atoms.
  • the nucleophilic groups, such as the purine or pyrimidine base which are still present in the polynucleotide are not converted in the nucleic acids.
  • a polynucleotide as used herein is an oligonucleotide.
  • 1A shows the chemical structure of the aldonic acid group of the HES aldonic acid
  • IB shows a reaction scheme for the activation according to the invention of HES aldonic acid with a carbonate derivative of an alcohol to form an aldonic acid ester and its reaction with a polynucleotide carrying a functional amino group;
  • 2A shows the reaction scheme for the production of conjugates from a polynucleotide and HES aldonic acid according to the prior art, in particular according to Examples 4-9;
  • 2B shows the reaction scheme for the production of conjugates from a polynucleotide and HES aldonic acid according to the prior art, in particular according to example 10
  • 2C shows the reaction scheme for the production of conjugates from a polynucleotide and HES aldonic acid according to the prior art, in particular according to example 11;
  • 2D shows the reaction scheme for the production of conjugates from a polynucleotide and HES aldonic acid according to the prior art, in particular according to Examples 12-13;
  • 2E shows the reaction scheme for the production of conjugates from a polynucleotide and HES according to the prior art, in particular according to example 14;
  • FIG. 3 shows a chromatogram of the result of a reaction mixture for the HESylation of a Spiegelmer according to the present invention, in particular according to Example 1;
  • FIG. 5 shows a diagram of the inhibition of ghrelin-induced calcium 2+ release caused by HESylated mirror bucket or non-HESylated mirror bucket.
  • Example 1 Preparation of a conjugate from a mirror bucket and hydroxyethyl starch
  • a description of the production of the HES acid is disclosed, for example, in German patent application DE 196 28 705.
  • N-hydroxy succinimide ester of the HESylate was prepared as follows:
  • RNA-Spiegelmer 5 mg (corresponding to 1.3 ⁇ mol) 5'-aminohexyl functionalized RNA-Spiegelmer according to Seq. ID. No. 1 are dissolved in 0.7 ml of a 0.3 molar dicarbonate solution with a pH of 8.4.
  • the active ester, prepared as described above, is added directly to this solution and reacted at room temperature for 2 hours.
  • the RNA Spiegelmer has the following sequence:
  • the conjugate is detected by low pressure GPC.
  • the analysis conditions used were the following, the analysis result being shown in FIG. 3:
  • reaction mixture yielded a yield of 62% (with solder precipitation with regard to the chromatogram) or 77% with tailing peak evaluation.
  • Example 3 Comparison of the inhibition of ghrelin-induced calcium release by ghrelin-binding HESylated and non-HESylated Spiegelmers.
  • Stably transfected CHO cells that express the human receptor for ghrelin (GHS-Rla) (obtained from Euroscreen, Gosselies, Belgium) are in a number of 5 - 7 x 10 4 per well of a black 96-well microtiter plate with clear bottom ( Greiner) and sown overnight at 37 ° C and 5% CO 2 in UltraCHO Medium (Cambrex), which also contains 100 units / ml penicillin, 100 ⁇ g / ml streptomycin, 400 ⁇ g / ml geneticin and 2.5 ⁇ g / ml fungi zone , cultivated.
  • GGS-Rla human receptor for ghrelin
  • the cells Before loading with the calcium indicator dye Fluo-4, the cells are washed once with 200 ⁇ l CHO-U +. Then 50 ⁇ l of the indicator dye solution (10 ⁇ M Fluo-4 (Molecular Probes), 0.08% Pluronic 127 (Molecular Probes) in CHO-U + are added and incubated for 60 min at 37 ° C. The cells are then 3 times with 180 ⁇ l CHO -U +, 90 ⁇ l of CHO-U + are then added to each well.
  • the indicator dye solution (10 ⁇ M Fluo-4 (Molecular Probes), 0.08% Pluronic 127 (Molecular Probes) in CHO-U + are added and incubated for 60 min at 37 ° C.
  • the cells are then 3 times with 180 ⁇ l CHO -U +, 90 ⁇ l of CHO-U + are then added to each well.
  • the fluorescence signals are measured at an excitation wavelength of 485 ⁇ m and an emission wavelength of 520 nm in an Fkuostar Optima multi-detection plate reader (BMG).
  • the stimulation solutions are added to the cells for a precise analysis of the time course of the changes in calcium concentrations caused by ghrelin.
  • the wells of a vertical row of a 96 well plate are measured together.
  • three measured values are first recorded every 4 seconds to determine the baseline.
  • the measurement is interrupted, the plate is extended from the reader and 10 ⁇ l of the stimulation solution from the “low profile 96-tube” plate in which the preincubation was carried out is added to the wells of the row to be measured using a multi-channel pipette.
  • the plate is then moved back into the device and the measurement is continued (a total of 20 measurements, each 4 seconds apart).
  • HESylated Spiegelmers cells expressing ghrelin receptor were stimulated with 5 nM ghrelin, or ghrelin which had been preincubated together with various amounts of HESylated or non-HESylated mirror bucket.
  • the measured fluorescence signals were normalized to the signals obtained without a mirror bucket.
  • the HESylated mirror bucket inhibits ghrelin-induced Ca ++ release with an IC 50 of about 6.5 nM, whereas the non-HESylated mirror bucket inhibits with an IC 50 of about 5 nM.
  • the result is shown in FIG. 5.
  • Example 4 Preparation of conjugates from a polynucleotide and HES aldonic acid using methods according to the prior art
  • RNA Spiegelmer according to SEQ. ID. No. 1 given at room temperature. 50 mg of N-ethyl-N '- (3-dimethylaminopropyh-carbodiimide hydrochloride (261 ⁇ mol), dissolved in 1 ml of water, are then added in portions over a period of 2 hours at room temperature. A pH value is obtained by adding hydrochloric acid or sodium hydroxide solution kept constant at 5. After the reaction has ended, the mixture is stirred for a further 2 hours at room temperature, and the reaction mixture was checked by means of low-pressure GPC and the reaction rate of the mirror bucket used was less than 1%.
  • Example 5 Preparation of conjugates from a polynucleotide and HES aldonic acid using methods according to the prior art
  • Example 6 Preparation of conjugates from a polynucleotide xmd HES aldonic acid using methods according to the prior art
  • reaction product After a further 2 hours of reaction, the mixture is analyzed using low-pressure GPC. No reaction product could be detected.
  • Example 7 Preparation of conjugates from a polynucleotide xmd HES aldonic acid using methods according to the prior art
  • Example 8 Preparation of conjugates from a polynucleotide xmd HES aldonic acid using methods according to the prior art
  • Examples 6 and 7 were repeated at pH 4.0 and 6.0.
  • Example 9 Preparation of conjugates from a polynucleotide and HES aldonic acid using methods according to the prior art
  • Example 4 was repeated at reaction temperatures of 4 ° C. and 37 ° C.
  • Example 10 Preparation of conjugates from a polynucleotide and HES aldonic acid using methods according to the prior art
  • RNA Spiegelmer according to Seq. JJD. No. 1 are dissolved in 10 mL water and the pH is adjusted to 8.5 with sodium hydroxide solution or dissolved in 10 mL 0.3 molar bicarbonate buffer of pH 8.4.
  • Example 11 Preparation of conjugates from a polynucleotide and HES aldonic acid using methods according to the prior art
  • Example 12 Preparation of conjugates from a polynucleotide and HES aldonic acid using methods according to the prior art
  • RNA Spiegelmer according to Seq. ID. No. 1 5 mg RNA Spiegelmer according to Seq. ID. No. 1 are dissolved in 5 mL water. 10 mL of the above-mentioned solution of imidazolyl-HES-aldonic acid 10 / 0.4 are added to this solution and the pH is adjusted to 7.5 with sodium hydroxide solution. After stirring at room temperature overnight, the mixture was examined for the reaction product by means of low-pressure GPC. Only traces of the reaction product were found.
  • Example 13 Preparation of conjugates from a polynucleotide and HES aldonic acid using methods according to the prior art
  • RNA Spiegelmer according to Seq. ID. No. 1 5 mg RNA Spiegelmer according to Seq. ID. No. 1 are dissolved in 12.5 mL 0.3 M pH 8.4 bicarbonate buffer. The mixture was cooled to 0 ° C. with ice water and 8.5 ml of the solution of HES 10 / 0.4 - aldonic acid imidazolyl in DMF listed in Example 12 were added. After 2 hours at 0 ° C. and a further 2 hours at room temperature, the mixture was examined for the reaction product. No product could be detected.
  • Example 14 Preparation of conjugates from a polynucleotide and HES using prior art methods 1 g HES 10 / 0.4 (0.25 mmol) are dissolved in 5 mL H 2 O while warm. After cooling, 10 mg (corresponding to 2.5 ⁇ mol) of RNA Spiegelmer according to Seq. ID. No. 1 added and the pH adjusted to 7.5 with sodium hydroxide solution. Then 200 ⁇ l of borane-pyridine complex (Sigma-Aldrich) are added and the mixture is stirred at room temperature in the dark for 10 days. The approach is then examined for possible reaction products by low pressure GPC. Only a conversion ⁇ 3% based on the mirror bucket used could be determined.

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  • Saccharide Compounds (AREA)

Abstract

L'invention concerne un procédé pour produire un conjugué constitué d'un polynucléotide et d'un polysaccharide, ce procédé comprenant les étapes qui consistent : a) à fournir un acide aldonique du polysaccharide ou d'un dérivé correspondant ; b) à faire réagir l'acide aldonique avec un dérivé d'alcool, de préférence un dérivé carbonate d'un alcool, pour obtenir un ester de l'acide aldonique, de préférence un ester activé de l'acide aldonique, et ; c) à faire réagir l'ester de l'acide aldonique avec le polynucléotide, ledit polynucléotide comportant un groupe amino fonctionnel. Cette invention est caractérisée en ce que la réaction entre l'acide aldonique et le dérivé d'alcool au cours de l'étape b) a lieu dans un solvant polaire aprotique sec.
EP05715273A 2004-02-09 2005-02-08 Procede pour produire des conjugues constitues de polysaccharides et de polynucleotides Withdrawn EP1713509A2 (fr)

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DE102004006249 2004-02-09
PCT/EP2005/001252 WO2005074993A2 (fr) 2004-02-09 2005-02-08 Procede pour produire des conjugues constitues de polysaccharides et de polynucleotides

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EP (1) EP1713509A2 (fr)
JP (2) JP5193468B2 (fr)
KR (1) KR101189555B1 (fr)
CN (1) CN1917905B (fr)
AU (1) AU2005210142A1 (fr)
BR (1) BRPI0507540A (fr)
CA (1) CA2555467C (fr)
WO (1) WO2005074993A2 (fr)

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KR20060132906A (ko) 2006-12-22
JP2013056889A (ja) 2013-03-28
CN1917905B (zh) 2012-01-04
CA2555467C (fr) 2012-10-09
JP2007522164A (ja) 2007-08-09
US20090281296A1 (en) 2009-11-12
WO2005074993A3 (fr) 2006-04-20
WO2005074993A2 (fr) 2005-08-18
BRPI0507540A (pt) 2007-07-03
JP5193468B2 (ja) 2013-05-08
CA2555467A1 (fr) 2005-08-18
CN1917905A (zh) 2007-02-21
AU2005210142A1 (en) 2005-08-18
KR101189555B1 (ko) 2012-10-16

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