Classifications

• • 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

Definitions

• the present invention relates to a method for preparing a hydroxyalkyl starch (HAS)- oligonucleotide conjugate as well as to a conjugate obtained or obtainable by said method.
• HAS hydroxyalkyl starch
• HES Hydroxyalkyl starch
• HES hydroxyethyl starch
• HES hydroxyethyl starch
• HES exhibits advantageous biological properties and is used as a blood volume replacement agent and in hemodilution therapy in the clinics (Sommermeyer et al., 1987, Whypharmazie, 8(8), 271-278; Weidler et al., 1991, Arzneistoffforschung/Drug Res., 41, 494-498).
• Amylopectin consists of glucose moieties, wherein in the main chain alpha- 1 ,4-glycosidic bonds are present and at the branching sites alpha- 1,6-glycosidic bonds are found.
• the physico-chemical properties of this molecule are mainly determined by the type of glycosidic bonds. Due to the nicked alpha- 1 ,4-glycosidic bond, helical structures with about six glucose-monomers per turn are produced.
• the physico-chemical as well as the biochemical properties of the polymer can be modified via substitution. The introduction of a hydroxyethyl group can be achieved via alkaline hydroxyethylation.
• polypeptides can be improved and the immune response against these polypeptides is reduced when the polypeptides are coupled to polymeric molecules, i.e. when a conjugate of the polypeptide with the polymeric molecule is formed.
• WO 02/080979 discloses compounds comprising a conjugate of an active agent and a hydroxyalkyl starch wherein the active agent and hydroxyalkyl starch are either linked directly or via a linker compound.
• the conjugation between both components is the result of the reaction of a carboxy group present in the modified hydroxyalkyl starch with free amino groups of the protein.
• the coupling reaction is mediated by the activation agent dicyclohexylcarbodiimide.
• Carbodiimide, as well as the activated carboxy group may also react with any hydroxyl groups present in the hydroxyalkyl starch, potentially leading to an undesired crosslinking of the hydroxyalkyl starch polymer.
• WO 03/074087 discloses hydroxyalkyl starch-protein conjugates in which the bonding between the hydroxyalkyl starch molecule and the protein is a covalent bonding resulting from the coupling of a terminal aldehyde or a functional group resulting therefrom with a functional group of a protein.
• WO 2005/014024 discloses polymers functionalized by an aminooxy group or a derivative thereof, wherein the functionalized polymers are coupled to a protein via the formation of an oxime linking group.
• WO 2005/092928 discloses conjugates of hydroxyalkyl starch and a protein, wherein these conjugates are formed by a reductive amination reaction between at least one aldehyde group of the hydroxyalkyl starch or of a derivative of the hydroxyalkyl starch, and at least one amino group of the protein, so that the hydroxyalkyl starch or the derivative thereof is covalently linked to the protein via an azomethine linkage or an aminomethylene linkage.
• WO 2005/092928 also relates to a method of producing these conjugates and specific uses of the conjugates.
• WO 2004/050710 discloses conjugates of hydroxyalkyl starch and a protein wherein these conjugates are formed by activating HES-aldonic acid derivatives with disuccinimidylcarbonate and reacting the resulting derivatives with free amino groups present in a polypeptide.
• the activation of HES-aldonic acid derivatives with disuccinimidylcarbonate may however yield in undesired crosslinking of the hydroxyalkyl starch polymer as such.
• WO 03/074088 discloses hydroxyalkyl starch conjugates with a low molecular weight compound in which the bonding between the hydroxyalkyl starch and the low molecular weight compound is a covalent bonding resulting from the coupling of a terminal aldehyde or a functional group resulting therefrom with a functional group of the low molecular weight compound.
• WO 2005/074993 describes the conjugation of a hydroxyalkyl starch comprising an NHS ester to a 5' amino functionalized oligonucleotide.
• the present invention relates to a method for preparing a hydroxyalkyl starch (HAS)-oligonucleotide conjugate, the method comprising the steps
• step (c) reacting a hydroxyalkyl starch at its at least one reducing end with a linker L 2 to give a hydroxyalkyl starch derivative comprising at least one functional group W capable of being reacted with the functional group Q; (d) reacting the oligonucleotide derivative according to step (b) with the hydroxyalkyl starch derivative according to step (c) via reaction of the functional group W with the functional group Q, thereby forming a covalent linkage F 3 .
• the present invention relates to the respective conjugates obtained or obtainable by said method.
• hydroxyalkyl starch refers to a starch derivative which has been substituted by at least one hydroxyalkyl group.
• a preferred hydroxyalkyl starch of the present invention has a constitution according to formula (I)
• HAS' is the remainder of the hydroxyalkyl starch molecule and Ri, R 2 and R 3 are independently hydrogen, a linear or branched hydroxyalkyl group or the group
• R 1 , R 2 , R 3 , and R 4 are independently selected from the group consisting of hydrogen, and an alkyl group, preferably hydrogen and a methyl group,
• n 2 to 4, wherein the residues R and R may be the same or different in the m groups CR'R 2 ;
• n is 0 to 20, preferably 0 to 4;
• o is 2 to 20, preferably 2 to 4, wherein the residues R 3 and R 4 may be the same or different in the o groups CR 3 R 4 .
• Ri, R 2 and R 3 are independently a group -(CH 2 CH 2 0) n -H, wherein n is an integer, preferably 0, 1 , 2, 3, 4, 5, or 6, and in particular, Rj, R 2 and R 3 are independently hydrogen or 2-hydroxyethyl.
• HAS' refers to the HAS molecule without the terminal saccharide unit at the reducing end of the HAS molecule. This is meant by the term "remainder of the hydroxyalkyl starch molecule" as used in the context of the present invention.
• hydroxyalkyl starch as used in the present invention is not limited to compounds where the terminal carbohydrate moiety comprises hydroxyalkyl groups Ri, R 2 and/or R 3 as depicted, for the sake of brevity, in formula (I), but also refers to compounds in which at least one hydroxy group which is present anywhere, either in the terminal carbohydrate moiety and/or in the remainder of the hydroxyalkyl starch molecule, HAS', is substituted by a hydroxyalkyl group Ri, R 2 and/or R 3 .
• a hydroxyalkyl starch comprising two or more different hydroxyalkyl groups is also possible.
• the at least one hydroxyalkyl group comprised in HAS may contain one or more, in particular two or more hydroxy groups. According to a preferred embodiment, the at least one hydroxyalkyl group comprised in HAS contains one hydroxy group.
• hydroxyalkyl starch also includes derivatives wherein the alkyl group is mono- or polysubstituted. In this context, it is preferred that the alkyl group is substituted with a halogen, especially fluorine, or with an aryl group. Furthermore, the hydroxy group of a hydroxyalkyl group may be esterified or etherified.
• alkyl instead of alkyl, also linear or branched substituted or unsubstituted alkenyl groups may be used.
• Hydroxyalkyl starch is an ether derivative of starch.
• ether derivatives also other starch derivatives can be used in the context of the present invention.
• derivatives are useful which comprise esterified hydroxy groups. These derivatives may be e.g. derivatives of unsubstituted mono- or dicarboxylic acids with 2- 12 carbon atoms or of substituted derivatives thereof.
• derivatives of unsubstituted monocarboxylic acids with 2-6 carbon atoms especially derivatives of acetic acid.
• acetyl starch, butyryl starch and propionyl starch are preferred.
• derivatives of unsubstituted dicarboxylic acids with 2-6 carbon atoms are preferred.
• the second carboxy group of the dicarboxylic acid is also esterified.
• derivatives of monoalkyl esters of dicarboxylic acids are also suitable in the context of the present invention.
• the substitute groups may be preferably the same as mentioned above for substituted alkyl residues.
• Techniques for the esterification of starch are known in the art (see e.g. Klemm D. et al., Comprehensive Cellulose Chemistry Vol. 2, 1998, Wiley- VCH, Weinheim, New York, especially chapter 4.4, Esterification of Cellulose (ISBN 3-527-29489-9)).
• hydroxyalkyl starch according to above-mentioned formula (I) is employed.
• the other saccharide ring structures comprised in HAS' may be the same as or different from the explicitly described saccharide ring, with the difference that they lack a reducing end.
• Ri, R 2 and R 3 are independently hydrogen or a hydroxyalkyl group, a hydroxyaralkyl group or a hydroxyalkaryl group having of from 2 to 10 carbon atoms in the respective alkyl residue.
• the alkyl, aralkyl and/or alkaryl group may be linear or branched and suitably substituted.
• Hydrogen and hydroxyalkyl groups having of from 2 to 10 carbon atoms are preferred. More preferably, the hydroxyalkyl group has from 2 to 6 carbon atoms, more preferably from 2 to 4 carbon atoms, and even more preferably from 2 to 3 carbon atoms.
• hydroxyalkyl starch is hydroxyethyl starch, in which R l5 R 2 and R 3 are independently hydrogen or a group (CH 2 CH 2 0) n -H, wherein n is an integer, preferably 0, 1, 2, 3, 4, 5, or 6.
• “Hydroxyalkyl starch” therefore preferably comprises hydroxyethyl starch, hydroxypropyl starch and hydroxybutyl starch, wherein hydroxyethyl starch and hydroxypropyl starch are particularly preferred and hydroxyethyl starch is most preferred.
• the present invention also relates to a method and a HAS derivative as described above, wherein Ri, R 2 and R 3 are independently hydrogen or a linear or branched hydroxyalkyl group with from 2 to 6 carbon atoms.
• Ri, R 2 and R 3 preferably may be H, hydroxyhexyl, hydroxypentyl, hydroxybutyl, hydroxypropyl such as 2-hydroxypropyl, 3-hydroxypropyl, 2-hydroxy-isopropyl, hydroxyethyl such as 2-hydroxyethyl, hydrogen and the 2-hydroxyethyl group being especially preferred. Therefore, the present invention also relates to a method and a HAS derivative as described above wherein Ri, R 2 and R 3 are independently hydrogen or a 2-hydroxyethyl group, an embodiment wherein at least one residue Ri, R 2 and R 3 being 2-hydroxyethyl is especially preferred.
• Hydroxyethyl starch is most preferred for all embodiments of the present invention.
• the present invention relates to the method and a HAS derivative as described above, wherein the polymer is hydroxyethyl starch and the derivative is a hydroxyethyl starch (HES) derivative.
• HAS hydroxyethyl starch
• HAS in particular HES, is mainly characterized by the molecular weight distribution, the degree of substitution and the ratio of C 2 : C 6 substitution. There are two possibilities of describing the substitution degree.
• the degree of substitution (DS) of HAS is described relatively to the portion of substituted glucose monomers with respect to all glucose moieties.
• the substitution pattern of HAS, preferably HES can also be described as the molar substitution (MS), wherein the number of hydroxyethyl groups per glucose moiety is counted.
• HAS preferably HES
• MS substitution pattern of HAS, preferably HES
• MS is determined by gas chromatography after total hydrolysis of the HAS molecule, preferably the HES molecule. MS values of the respective HAS starting material, in particular the HES starting material, are given. It is assumed that the MS value is not affected during the derivatization procedure in steps c) and d) of the process of the invention.
• HAS and in particular HES solutions are present as polydisperse compositions, wherein each molecule differs from the other with respect to the polymerization degree, the number and pattern of branching sites, and the substitution pattern.
• HAS and in particular HES is therefore a mixture of compounds with different molecular weight. Consequently, a particular HAS and in particular HES solution is determined by the average molecular weight with the help of statistical means.
• M n is calculated as the arithmetic mean depending on the number of molecules.
• M w (or MW) the weight average molecular weight, represents a unit which depends on the mass of the HAS, in particular HES.
• n is the number of molecules of species i of molar mass Mj.
• M ⁇ indicates that the value is an average, but the line is normally omitted by convention.
• M w is the weight average molecular weight, defined by equation 2: where n; is the number of molecules of species i of molar mass M,.
• A indicates that the value is an average, but the line is normally omitted by w
• the mean molecular weight of hydroxyethyl starch employed is from about 1 to about 1000 kDa, more preferably from about 1 to about 800 kDa, more preferably from about 1 to about 700 kDa, more preferably from about 2 to about 600 kDa, more preferably from about 5 to about 500 kDa, more preferably from about 80 to about 400 kDa, in particular from about 100 to about 350 kDa.
• the molar substitution of HAS and in particular HES is preferably from about 0.1 to about 3, preferably about 0.4 to about 1.3, such as 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, or 1.3.
• HES having a mean molecular weight of about 5 to 500 kDa, preferably 80 to 400 kDa is a HES with a molar substitution of 0.1 to 3, preferably 0.4 to 1.3, such as 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, or 1.3.
• said substitution is preferably in the range of from 2 to 20, more preferably in the range of from 2 to 15 and even more preferably in the range of from 2 to 9.
• oligonucleotide refers to polymers, such as DNA and RNA, of nucleotide monomers or nucleic acid analogs thereof, including double and single- stranded deoxyribonucleotides, ribonucleotides, alpha-anomeric forms thereof, and the like.
• monomers are linked by phosphodiester linkages, wherein the term “phosphodiester linkage” refers to phosphodiester bonds or bonds including phosphate analogs thereof, including associated counterions, e.g., H + , NH 4 + , Na + .
• Oligonucleotides encompassed by the present invention typically range in size from a few monomeric units, e.g.
• the oligonucleotide according to the invention comprises from 5 to 5000, more preferably from 12 to 100, monomeric units.
• ATGCCTG an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG,” it will be understood that the nucleotides are in 5' to 3' order from left to right and that "A” denotes adenosine, “C” denotes cytidine, “G” denotes guanosine, and "T” denotes thymidine, unless otherwise noted.
• Nucleoside refers to a compound consisting of a purine, deazapurine, or pyrimidine nucleobase, e.g., adenine, guanine, cytosine, uracil, thymine, deazaadenine, deazaguanosine, and the like, linked to a pentose at the 1 -position.
• nucleoside base is purine or 7-deazapurine
• the pentose is attached to the nucleobase at the 9-position of the purine or deazapurine
• the nucleobase is pyrimidine
• the pentose is attached to the nucleobase at the 1 -position of the pyrimidine.
• Nucleotide refers to a phosphate ester of a nucleoside, e.g., a triphosphate ester, wherein the most common site of esterification is the hydroxyl group attached to the C-5 position of the pentose.
• a nucleotide is composed of three moieties: a sugar, a phosphate, and a nucleobase (Blackburn, G. and Gait, M. Eds. "DNA and RNA structure” in Nucleic Acids in Chemistry and Biology, 2nd Edition, (1996) Oxford University Press, pp. 15- 81.).
• bases When part of a duplex, nucleotides are also referred to as “bases” or “base pairs”.
• nucleic acid analogs refers to analogs of nucleic acids made from monomeric nucleotide analog units, and possessing some of the qualities and properties associated with nucleic acids.
• nucleic acid analogs comprise modifications in the chemical structure of the base (e.g. C-5-propyne pyrimidine, pseudo-isocytidine and isoguanosine), the sugar (e.g. 2'-0-alkyl ribonucleotides) and/or the phosphate (e.g. 3'-N- phosphoramidate). See for example Englisch, U. and Gauss, D. "Chemically modified oligonucleotides as probes and inhibitors", Angew. Chem. Int. Ed. Engl. 30:613-29 (1991)).
• Nucleotide analogs in particular include, but are not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, and substitution of 5-bromo-uracil; and 2'-position sugar modifications, including but not limited to, sugar-modified ribonucleotides in which the 2 -OH is replaced by a group such as H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 , or CN, wherein R is an alkyl moiety.
• Nucleotide analogs are also meant to include nucleotides with other modified bases, or with different sugars such as 2'-methyl ribose as well as nucleotides having sugars or analogs thereof that are not ribosyl.
• the sugar moieties may be, or be based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4'-thioribose, and other sugars, heterocycles, or carbocycles.
• Nucleotide analogs are also meant to include nucleotides with non-natural linkages such as methylphosphonates, phosphorothioates and peptide linkages. Further the term is meant to include analogs such as locked nucleic acids in which the ribose moiety of the nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon.
• Modified bases refer to nucleotide bases such as, for example, adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups.
• nucleotide bases such as, for example, adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups.
• Some examples of types of modifications that can comprise nucleotides that are modified with respect to the base moieties include but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, individually or in combination.
• More specific examples include, for example, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6- methylguanine, ⁇ , ⁇ -dimethyladenine, 2-propyladenine, 2-propylguanine, 2- aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5-position, 5-(2-amino)propyluridine, 5- halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3- methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2- dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, 6-azothy
• nucleotide is also meant to include species which are known in the art as universal bases.
• universal bases include but are not limited to 3-nitropyrrole, 5- nitroindole, or nebularine.
• nucleotide is also meant to include the N3' to P5 1 phosphoramidate, resulting from the substitution of a ribosyl-3'-oxygen with an amino group.
• nucleotide also includes those species that have a detectable label, such as for example a radioactive or fluorescent moiety, or mass label attached to the nucleotide.
• a class of analogs where the sugar and phosphate moieties have been replaced with a 2- aminoethylglycine amide backbone polymer is peptide nucleic acid PNA (Nielsen, P., Egholm, M., Berg, R. and Buchardt, O. "Sequence-selective recognition of DNA by strand displacement with a thymidine-substituted polyamide", Science 254: 1497-1500 (1991)).
• oligonucleotide further includes polymers comprising mixtures of deoxyribonucleotides and ribonucleotides (DNA/RNA-hybrids). Further the oligonucleotide according to the invention may comprise one or more abasic sites.
• abasic site is meant a monomelic unit contained within an oligonucleotide chain but which does not contain a purine or pyrimidine base.
• the oligonucleotide is an aptamer.
• aptamer refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target such as small molecules, proteins, nucleic acids or other biomolecules. Oligonucleotide aptamers are useful in biotechnological and therapeutic applications as they offer discriminate molecular recognition and elicit little or no immunogenicity in therapeutic applications.
• Aptamers are derived from an in vitro evolutionary process.
• Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
• the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NH2, which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood.
• said oligonucleotide in case the oligonucleotide is an aptamer, said oligonucleotide may for example be a aptamer.
• spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left-handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left- handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
• the oligonucleotide is a siRNA.
• siRNA refers to small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway. These molecules can vary in length (generally between 18-30 basepairs) and contain varying degrees of complementarity to their target mRNA in the antisense strand. Some, but not all, siRNA have unpaired overhanging bases on the 5' or 3' end of the sense strand and/or the antisense strand.
• siRNA includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region.
• the oligonucleotide is a ribozyme.
• ribozyme refers to a synthetic RNA molecule that acts as an enzyme capable of targeting and cleaving particular base sequences in nucleic acids. Ribozymes are used and applied using many of the same principles as antisense nucleic acids. Ribozymes thus also rely on complementary binding to nucleic acids for target recognition. The majority of reactions catalyzed by ribozymes involve ligation and cleavage of nucleic acids.
• the oligonucleotide according to the invention comprises a reactive amino group.
• reactive amino group refers to a functional group comprising an NH group, such as for example, a primary amino group, a secondary amino group, a hydroxylamino group, a hydrazine or a hydrazide, provided that this group is capable of being reacted with the functional group Z ⁇ .
• the "reactive amino group” is not one of the NH groups present in the nucleobases.
• the present inventors have found that despite the presence of NH groups in the nucleobases of oligonucleotides, these groups are not capable of being reacted with the group Zi in the method according to the invention.
• the "reactive amino group” is not a naturally occurring NH group present in the nucleotide bases adenine, guanine, cytosine and thymine as such.
• the "reactive" NH group is a primary or secondary amino group, preferably an aliphatic primary or secondary amino group, more preferably an aliphatic primary amino group, capable of being reacted with the functional group Z ⁇ .
• the "reactive amino group” is a group being attached, preferably via a linker, to the 5' or 3'-end of the oligonucleotide, in particular to one of the terminal phosphate groups.
• said linker is preferably an aliphatic linker, more preferably an alkyl chain comprising 1 to 15, more preferably 1 to 10, most preferably 2 to 6 carbon atoms.
• the "reactive amino group” preferably the aliphatic primary amino group, is positioned at one of the bases, for example a "T", wherein in this case the T is modified with a linker comprising the amino group modification.
• the "reactive amino group” is positioned at the 5'- end of the oligonucleotides.
• the oligonucleotide is a DNA or an RNA or a DNA/RNA hybrid having a structure according to the following formula, with the spacer (linker) being most preferably an alkyl group, in particular a linear alkyl chain having from 1 to 15, more preferably 1 to 10, most preferably 2 to 6 carbon atoms.
• step a) of the method of the invention an oligonucleotide comprising a reactive amino group, as described above, is provided.
• the oligonucleotide is prepared by chemical synthesis using the phosphoramidite method.
• the reactive amino group according to the invention may already be present, preferably in protected form, in at least one of the phosphoramidite building blocks employed in the synthesis.
• building blocks are commercially available.
• the building block comprising a protected reactive amino group, step (a) preferably further comprises the deprotection of said building block.
• the primary amino group may be attached to the oligonucleotide after having completed the phosphoramidite synthesis.
• the DNA is provided by plasmid isolation or PCR and is modified with the reactive amino group in a subsequent step.
• R* is selected from the group consisting of H, methyl, ethyl, propyl, and butyl, more preferably R* is H, methyl or ethyl.
• the functional group Q and the functional group W are thus complementary functional groups.
• complementary functional groups refers to a group capable of reacting with a recited group to form a covalent bond.
• the functional group Q and the functional group W form for example, the linking group F 3 , as further described below.
• the linker may comprise at least one further functional group capable of being attached to a further active agents such as, e.g., a detectable label, such as for example a radioactive or fluorescent moiety, or mass label, under the proviso that this further at least one functional group is not complementary to the functional group W or the reactive amino group, or is, alternatively suitably protected, so that this further functional group does not react with the reactive amino group of the oligonucleotide or the functional group W of the hydroxyalkyl starch derivative thus allowing for a selective reaction between W and Q and Zi and the amino group, respectively.
• a further active agents such as, e.g., a detectable label, such as for example a radioactive or fluorescent moiety, or mass label
• this further at least one functional group is not complementary to the functional group W or the reactive amino group, or is, alternatively suitably protected, so that this further functional group does not react with the reactive amino group of the oligonucleotide or the functional group W of the hydroxyalky
• linker L ⁇ is a bifunctional linker, having s structure according to the following formula (III)
• the present invention also relates to a method as described above, as well as to a conjugate obtainable by said method, wherein the linker Li has a structure according to the formula (III).
• Li' is a linking moiety such as an alkyl, alkenyl, alkylaryl, arylalkyl, aryl or heteroaryl group.
• the term also encompasses alkyl groups which are further substituted by one or more suitable substituent.
• substituted alkyl as used in this context of the present invention preferably refers to alkyl groups being substituted in any position by one or more substituents, preferably by 1 , 2, 3, 4, 5 or 6 substituents, more preferably by 1, 2, or 3 substituents. If two or more substituents are present, each substituent may be the same or may be different from the at least one other substituent. There are in general no limitations as to the substituent.
• the substituents may be, for example, selected from the group consisting of aryl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, amino, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, amidino, nitro, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, s
• cyclopentyl or cyclohexyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl.
• Preferred substituents of such organic residues are, for example, halogens, such as fluorine, chlorine, bromine or iodine, amino groups, hydroxyl groups, carbonyl groups, thiol groups and carboxyl groups.
• alkenyl refers to unsaturated alkyl groups having at least one double bond. The term also encompasses alkenyl groups which are substituted by one or more suitable substituent.
• alkynyl refers to unsaturated alkyl groups having at least one triple bond. The term also encompasses alkynyl groups which are substituted by one or more suitable substituent.
• aryl refers to, but is not limited to, optionally suitably substituted 5- and 6-membered single-ring aromatic groups as well as optionally suitably substituted multicyclic groups, for example bicyclic or tricyclic aryl groups.
• aryl thus includes, for example, optionally suitably substituted phenyl groups or optionally suitably substituted naphthyl groups.
• Aryl groups can also be fused or bridged with alicyclic or heterocycloalkyl rings which are not aromatic so as to form a polycycle, e.g., benzodioxolyl or tetraline.
• heteroaryl as used within the meaning of the present invention includes optionally suitably substituted 5- and 6-membered single-ring aromatic groups as well as substituted or unsubstituted multicyclic aryl groups, for example bicyclic or tricyclic aryl groups, comprising one or more, preferably from 1 to 4, such as 1, 2, 3 or 4, heteroatoms, wherein in case the aryl residue comprises more than 1 heteroatom, the heteroatoms may be the same or different.
• heteroaryl groups including from 1 to 4 heteroatoms are, for example, benzodioxolyl, pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyridazinyl, benzoxazolyl, benzodioxazolyl, benzothiazolyl, benzoimidazolyl, benzothiophenyl, methylenedioxyphenylyl, napthyridinyl, quinolinyl, isoquinolinyl, indolyl, benzofuranyl, purinyl, benzofuranyl, deazapurinyl, or indolizinyl.
• substituted aryl and the term “substituted heteroaryl” as used in the context of the present invention describes moieties having substituents replacing a hydrogen on one or more atoms, e.g. C or N, of an aryl or heteroaryl moiety. Again, there are in general no limitiations as to the substituent.
• the substituents may be, for example, selected from the group consisting of alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, amino, acylamino, including alkylcarbonyl amino, arylcarbonylamino, carbamoyl and ureido, amidino, nitro, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, sul
• cyclopentyl or cyclohexyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl.
• Preferred substituents of such organic residues are, for example, halogens, such as fluorine, chlorine, bromine or iodine, amino groups, hydroxyl groups, carbonyl groups, thiol groups and carboxyl groups.
• Li' comprises the structural unit with n preferably being in the range of from 1 to 10, and wherein R a and R b are independently of each other selected from H and alkyl.
• Li ' has a structure selected from the group consisting of
• n preferably being in the range of from 1 to 10
• R a and R b are independently of each other selected from H and alkyl
• m preferably being in the range of from 1 to 10
• o preferably being in the range of from 1 to 10
• p preferably being in the range of from 1 to 3
• R c , Rj, R e and Rf are, independently of each other, selected from H or alkyl, and wherein Yi is a functional moiety.
• the repeating units -C(R a R b )- may be the same or may be different from each other.
• the repeating units -C(R c R d )- may be the same or may be different from each other.
• the repeating units -Yi-[C(ReR f ) 0 ]- may be the same or may be different from each other.
• the repeating units -C(ReRf)- may be the same or may be different from each other.
• Li' is R a and R b are preferably independently of each other selected from H and alkyl, wherein each repeating unit -C(R a R b )-, in case n is > 1, may be the same or may be different from each other.
• n is preferably in the range of from 1 to 10, although longer alkyl chains are also encompassed by the present invention.
• the following groups Li' are mentioned: -CH 2 -, -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 - CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -,
• the present invention describes a method, as well as a conjugate obtained or obtainable by said method, wherein Li' is
• R a and R b are H, in particular wherein n is in the range of from 1 to 10, preferably of from 3 to 8.
• L ⁇ has as structure according to the following formula as described above, with m preferably being in the range of from 1 to 10, with o preferably being in the range of from 1 to 10, and with p preferably being in the range of from 1 to 3, and wherein Rc, R d , Rg and Rf are, independently of each other, selected from H or alkyl, and wherein Yi is a functional moiety.
• said functional moiety is preferably selected from the group consisting of -0-, -S-, -NH-, -S-S-, -S-succinimidyl-, -succinimidyl-S-, -NH-
• Yi is -0-, -S- or -NH-.
• p is preferably in the range of from 1 to 10, more preferably in the range of from 1 to 3.
• m and p are most preferably 2.
• Li ' has a structure according to the following formula
• Yi is -0-.
• the present invention also relates to a method, as described above, and a conjugate obtained or obtainable by said method, wherein Li' has a structure according to the following formula with m, o and p, as described above and with Rc, Rj, 3 ⁇ 4, Rf as described above, and wherein the functional moiety Yi is -0-.
• the present invention also relates to a method, as described above, as well as to a conjugate obtained or obtainable by said method, wherein the linker Lj is a bifunctional linker, preferably having a structure according to the following formula (HI)
• Lj ' is a linker moiety preferably being selected from the group consisting of
• n preferably being in the range of from 1 to 10, wherein R a and Rj, are H, with m preferably being in the range of from 1 to 10, more preferably with m being 2, with o preferably being in the range of from 1 to 10, more preferably with o being 2, and with p preferably being in the range of from 1 to 3, and wherein Rc, R ⁇ j, e and Rf are H and wherein Yi is -0-.
• this group is a functional group capable of forming upon reaction with the reactive amino group a bond Fi, as described above.
• Zi is a carboxylic acid group or a reactive carboxy group.
• reactive carboxy group as used in this context of the present invention is intended to mean an activated carboxylic acid derivative that reacts readily with electrophilic groups, such as the reactive amino groups of the oligonucleotide, in contrast to those groups that require a further catalyst, such as a peptide coupling reagent, in order to react.
• activated carboxylic acid derivative preferably refers to acid halides such as acid chlorides and also to activated ester derivatives including, but not limited to, formic and acetic acid derived anhydrides, anhydrides derived from alkoxycarbonyl halides such as isobutyloxycarbonylchloride and the like, isothiocyanates or isocyanates, anhydrides derived from reaction of the carboxylic acid with ⁇ , ⁇ '- carbonyldiimidazole and the like, and esters derived from activation of the corresponding carboxylic acid with a coupling reagent.
• Such coupling reagents include, but are not limited to, HATU (0-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate); HOAt, HBTU (0-benzotriazol-l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate); TBTU (2-(lH-benzotriazo-l-yl)-l,l,3,3- tetramethyluronium hexafluorophosphate); TFFH (N,N',N",N"-tetramethyluronium-2- fluoro-hexafluorophosphate); BOP (benzotriazol-l-yloxytris(dimethylamino) phosphonium hexafluorophosphate); PyBOP (benzotriazol-l -yl-oxy-trispyrroli
• EDC l -ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
• CDC 1- cyclohexyl-3-(2-morpholinoethyl)carbodiimide
• Pyclop T3P, CDI, Mukayama's reagent, HODhbt, HAPyU, TAPipU, TPTU, TSTU, TNTU, TOTU, BroP, PyBroP, BOI, TOO, NEPIS, BBC, BDMP, BOMI, AOP, BDP, PyAOP, TDBTU, BOP-C1 , CIP, DEPBT, Dpp-Cl, EEDQ, FDPP, HOTT, TOTT, PyCloP.
• the linker Li and the oligonucleotides are preferably reacted in the presence of at least one coupling agent, wherein the coupling reagent is preferably selected from the group of coupling reagents mentioned above.
• the coupling reagent most preferably EDC (l -ethyl-3-(3-dimethylaminopropyl) carbodiimide) is used.
• EDC l -ethyl-3-(3-dimethylaminopropyl) carbodiimide
• DMAP 4-(dimethylamino)-pyridine
• the coupling is preferably carried out in the presence of a suitable base, preferably an organic base, most preferably an amino group comprising base, most preferably a base selected from the group consisting of diisopropylamine (DIEA), triethylamine (TEA), N- methylmorpholine, N-methylimidazole, 1 ,4-diazabicyclo[2.2.2]octane (DABCO), N- methylpiperidine, N-methylpyrrolidine, 2,6-lutidine, collidine, pyridine, 4- dimethylaminopyridine, diaza(l ,3)bicyclo[5.4.0]undec-7-ene (DBU).
• a suitable base preferably an organic base, most preferably an amino group comprising base, most preferably a base selected from the group consisting of diisopropylamine (DIEA), triethylamine (TEA), N- methylmorpholine, N-methylimidazole, 1 ,4-diazabic
• the reaction is carried out in an aqueous solvent or in an organic solvent or in a mixture thereof.
• organic solvents solvents such as N-methyl pyrrolidone (NMP), dimethyl sulfoxide (DMSO), acetonitrile, acetone, dimethyl acetamide (DMA), dimethyl formamide (DMF), formamide, tetrahydrofuran (THF), 1 ,4-dioxane, diethyl ether, tert.- butyl methyl ether (MTBE), dichloromethane (DCM), chloroform, tetrachloromethane and mixtures of two or more thereof, are mentioned.
• NMP N-methyl pyrrolidone
• DMSO dimethyl sulfoxide
• DMA dimethyl acetamide
• DMF dimethyl formamide
• THF tetrahydrofuran
• MTBE tert.- butyl methyl ether
• DCM dichloromethane
• the reaction is carried out in DMSO, DMF or acetonitrile.
• the temperature of the coupling reaction is preferably in the range of from 0 to 100 °C, more preferably in the range of from 5 to 50 °C, and especially preferably in the range of from 5 to 30 °C. During the course of the reaction, the temperature may be varied, preferably in the above given ranges, or held essentially constant.
• Z ⁇ is a reactive carboxy group, as described above. In this case, preferably no coupling agent (activating agent) is added in step b).
• the method may comprise a further step, prior to step b), in which a precursor of linker Lj comprising a carboxylic acid group is transformed to the linker Li comprising the above described reactive carboxy group Z ⁇ .
• a precursor of linker Lj comprising a carboxylic acid group is transformed to the linker Li comprising the above described reactive carboxy group Z ⁇ .
• This is preferably achieved by reacting the linker with at least one coupling reagent.
• the so formed linker L ⁇ is optionally purified and isolated from the reaction mixture, prior to step b) of the method of the invention.
• group Z ⁇ of linker Li is a reactive carboxy group, wherein in step b) most preferably no coupling agent is added.
• Z ⁇ is selected from the group consisting of N-hydroxysuccinimideester (NHS-ester), pentafluorophenylester, N- hydroxybenzotriazoleester, and trichlorophenylesters, in particular wherein Z ⁇ is an NHS- ester.
• NHS-ester N-hydroxysuccinimideester
• pentafluorophenylester N- hydroxybenzotriazoleester
• trichlorophenylesters in particular wherein Z ⁇ is an NHS- ester.
• the present invention thus also relates to a method as described above, as well as to a conjugate obtained or obtainable by said method, wherein the functional group Zi is a reactive carboxy group, in particular wherein Z ⁇ is selected from the group consisting of N-hydroxysuccinimideester (NHS-ester), pentafluorophenyl ester, N-hydroxybenz- triazoleester, and trichlorophenylesters, in particular wherein Z ⁇ is a NHS-ester.
• NHS-ester N-hydroxysuccinimideester
• pentafluorophenyl ester pentafluorophenyl ester
• N-hydroxybenz- triazoleester N-hydroxybenz- triazoleester
• trichlorophenylesters in particular wherein Z ⁇ is a NHS-ester.
• the reaction in step b) is preferably conducted in that the oligonucleotide is dissolved in an aqueous medium, preferably in a reaction buffer, and linker L ⁇ is added.
• Preferred reaction buffers are, e.g., sodium citrate buffer, sodium acetate buffer, sodium phosphate buffer, sodium carbonate buffer, sodium borate buffer, water.
• Preferred pH values of the reaction buffers are in the range of from 5 to 10, more preferably of from 7 to 10, more preferably of from 8 to 9.
• linker Li is dissolved in a suitable reaction medium prior to the addition to the reaction medium comprising the oligonucleotide.
• linker Lj is at least partly dissolvable in said medium.
• linker Li is dissolved in a reaction medium comprising an organic reaction medium or a mixture of water with an organic reaction medium.
• Suitable organic solvents are, for example, dichloromethane, DMA, DMF, formamide, NMP, DMSO, THF, acetonitrile and the like.
• the linker is dissolved in DMSO, DMF, acetonitrile, NMP or mixtures thereof.
• further solvents may be added to the solution comprising the oligonucleotide and/or the solution comprising L ⁇ and/or to the mixture comprising Li and the oligonucleotide.
• the final reaction mixture in which Li and the oligonucleotide are coupled with each other comprises, preferably consists of, acetonitrile and an aqueous medium, preferably a buffer.
• a non-nucleophilic organic base is added to the reaction mixture.
• a base is in particular added, in case Z ⁇ is a carboxylic acid and in case step b) is carried out in the presence of an activation agent, as described above.
• bases include, but are not limited to, triethylamine, tripropylamine, ⁇ , ⁇ -diisopropylethylamine, imidazole, N-methylimidazole, N- methylmorpholine, and N-ethylmorpholine.
• the preferred non-nucleophilic organic base is N,N-diisopropylethylamine.
• Z ⁇ is a reactive carboxy group
• most preferably no such base is added and the reaction is carried out in a buffer without further addition of such base, as described above.
• the molar ratio of oligonucleotide to Li is preferably 0.5 to 200, more preferably 0.6 to 100 based on M n of the HAS derivative. Especially preferred molar ratios of oligonucleotide to Li are in the range of from 0.6 to 50, more preferably from 0.9 to 10 and even more preferably from 1 to 5.
• the reaction mixture is stirred at a temperature of about 0 °C to about 100 °C, more preferred about 5 °C to about 40 °C, preferably about 5 °C to about 25 °C.
• the temperature may be varied, preferably in the above-given ranges, or held essentially constant.
• the reaction time for step b) may be adapted to the specific needs and is generally in the range of from 1 min to 24 h, preferably of from 5 min to 60 min and more preferably of from 5 min to 30 min.
• the oligonucleotide derivative obtained is preferably isolated from the reaction mixture by at least one isolation step selected from the group consisting of (RP-)HPLC, ion exchange chromatography, ultrafiltration, dialysis and precipitation.
• the method of the present invention is a one pot process.
• a one pot process is to be understood as a process wherein the reaction mixture of step b) is used as such, i.e. without separation prior to step d).
• the reaction mixture of step b) can be added to the reaction vessel comprising the hydroxyalkyl starch derivative, or the hydroxyalkyl starch derivative can be added to the reaction vessel comprising the reaction mixture of step b).
• step b) and step d) are carried out in the same reaction vessel.
• reaction is carried out as one-pot synthesis, there may be further process steps prior to the reaction of the oligonucleotide derivative with the hydroxyalkyl starch derivative according to step d), such as at least one concentrating step (e.g. to remove part of the reaction solvent) and/or at least one quenching step to destroy unreacted amino-reactive groups.
• at least one concentrating step e.g. to remove part of the reaction solvent
• at least one quenching step to destroy unreacted amino-reactive groups.
• step c) of the method of the invention HAS is reacted at its at least one reducing end with a linker L 2 to give a hydroxyalkyl starch derivative comprising at least one functional group W, said functional group W being capable of being reacted with the functional group Q.
• the HAS is reacted at the at least one reducing end
• the HAS is reacted predominantly via its reducing end.
• This term "predominantly via its reducing end” relates to processes according to which statistically more than 50 %, preferably at least 55 %, more preferably at least 60 %, more preferably at least 65 %, more preferably at least 70 %, more preferably at least 75 %, more preferably at least 80 %, more preferably at least 85 %, more preferably at least 90 %, and still more preferably at least 95 % such as 95 %, 96 %, 97 %, 98 %, or 99 % of the HAS molecules employed for a given reaction are reacted via at least one reducing end per HAS molecule, wherein a given HAS molecule which is reacted via at least one reducing end can be reacted in the same given reaction via at least one further suitable functional group which is comprised in said polymer molecule and which is not a reducing end.
• HAS molecule(s) is (are) reacted via at least one reducing end and simultaneously via at least one further suitable functional group which is comprised in this (these) HAS molecule(s) and which is not a reducing end, statistically preferably more than 50 %, preferably at least 55 %, more preferably at least 60 %, more preferably at least 65 %, more preferably at least 70 %, more preferably at least 75 %, more preferably at least 80 %, more preferably at least 85 %, more preferably at least 90 %, and still more preferably at least 95 % such as 95 %, 96 %, 97 %, 98 %, or 99 % of all reacted functional groups of these HAS molecules, said functional groups including the reducing ends, are reducing ends.
• HAS is selectively reacted via at least one reducing end.
• the term "selectively via at least one reducing end” relates to processes according to which a least 99.5 %, more preferably at least 99.9%, most preferably all reacted functional groups of a given HAS molecule, said functional groups including the reducing ends, are reducing ends.
• reducing end as used in the context of the present invention relates to the terminal aldehyde group of a HAS molecule which may be present as aldehyde group and/or as corresponding hemiacetal form and/or as acetal group, the acetal group having the following structure
• residue -OR 3 according to formula (I) above is -0-CH 2 -CH 2 -OH.
• HAS is in particular not reacted via its oxidized reducing end, that is via any reducing end being oxidized prior to the reaction with linker L 2 .
• step (c) the hydroxyalkyl starch is reacted at its at least one reducing end with a linker L 2 to give a hydroxyalkyl starch derivative comprising at least one functional group W capable of being reacted with the functional group Q.
• said linker is not particularly limited with the proviso that the linker comprises at least a functional group capable of being reacted with the reducing end thereby forming a hydroxyalkyl starch derivative comprising the at least one functional group W or a group capable of being modified to give the functional group W, said functional group W being capable of forming a chemical linkage upon reaction with Q.
• linker L 2 is a bifunctional linker, having s structure according to the following formula (II)
• W preferably comprises the structure -NHR ⁇ , wherein is H or alkyl, preferably H. Most preferably, according to this embodiment, W is -NHR ⁇ .
• a hydroxyalkyl starch derivative is formed having a structure according to the following formula or the formula wherein in the latter case, the reaction between the hydroxyalkyl starch and L 2 is a reductive amination.
• Z 2 is a functional group.
• the hydroxyalkyl starch is reacted at its at least one reducing end with a functional group Z 2 of the linker L 2 to give the hydroxyalkyl starch derivative comprising the at least one functional group W, thereby forming a functional group F 2 , said linker L 2 comprising — the at least one functional group Z 2 capable of being reacted with the reducing end of the hydroxyalkyl starch, and
• the at least one functional group W capable of being reacted with the functional group Q.
• the linker L 2 or a salt thereof is reacted in step c) via the functional group Z 2 with an aldehyde and/or hemiacetal group and/or acetal group of the hydroxyalkyl starch.
• said functional group is a group capable of forming a chemical linkage with the reducing end of hydroxyalkyl starch upon reaction with said reducing end.
• the functional group Q and the functional group W are complementary functional groups.
• the term "complementary functional groups" refers to a group capable of reacting with a recited group to form a covalent bond.
• the functional group Q and the functional group W form for example, the linking group F 3 , as already described above.
• the functional group Z 2 and the reducing end form the covalent linkage F 2 .
• the linker may comprise at least one further functional group capable of being attached to a further active agent such as, e.g., a detectable label, such as for example a radioactive or fluorescent moiety, or mass label, under the proviso that this further at least one functional group is not complementary to the functional group Q and does not react with the reducing end of hydroxyalkyl starch, or is, alternatively suitably protected, so that this further functional group does not react with the reducing end of HAS or the functional group Q of the oligonucleotide derivative thus allowing for a selective reaction between W and Q and Z 2 and the reducing end, respectively.
• a further active agent such as, e.g., a detectable label, such as for example a radioactive or fluorescent moiety, or mass label
• this further at least one functional group is not complementary to the functional group Q and does not react with the reducing end of hydroxyalkyl starch, or is, alternatively suitably protected, so that this further functional group does not react with the reducing end of
• linker L 2 is a bifunctional linker, having the structure according to the following formula (II)
• the present invention also relates to a method as described above, as well as to a conjugate obtained or obtainable by said method, wherein the linker L 2 has a structure according to the formula (II).
• each functional group may be used which is capable of forming a chemical linkage with the reducing end of the hydroxyalkyl starch.
• the functional group Z 2 comprises the chemical structure -NH-. Therefore, the present invention also relates to a method as described above, and a conjugate obtained or obtainable by said method, wherein the functional group Z 2 comprises the structure -NH-.
• the functional group Z 2 is a group having the structure R'-NH- where R' is hydrogen or an alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue where the alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue may be linked directly to the NH group or, according to another embodiment, may be linked by an oxygen bridge to the NH group.
• alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl, or cycloalkylaryl residue may be suitably substituted.
• halogenes such as F, CI or Br may be mentioned.
• Especially preferred residues R' are hydrogen, alkyl and alkoxy groups, and even more preferred are hydrogen and unsubstituted alkyl and alkoxy groups.
• alkyl and alkoxy groups groups with 1, 2, 3, 4, 5, or 6 C-atoms are preferred. More preferred are methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, and isopropoxy groups. Especially preferred are methyl, ethyl, methoxy, ethoxy, and particular preference is given to methyl or methoxy.
• the present invention also relates to a method as described above wherein R' is hydrogen, a methyl or a methoxy group.
• the functional group Z 2 is selected from the group consisting of
• the present invention also relates to a method as mentioned above, and a conjugate obtained or obtainable by said method, wherein the functional group Z 2 is selected from the group consisting of
• linker L 2 is a bifunctional linker, having s structure according to the following formula (Ila) Z 2 L 2 '— W (IIa)
• a hydroxyalkyl starch derivative is formed having a structure according to the following formula wherein F 2 is the functional group linking the reducing sugar moiety and L 2 ', which is formed upon reaction of Z 2 with the reducing end.
• the group Z 2 is selected from the group consisting of NH 2 -, NH 2 -NH- and NH 2 -0-.
• the present invention also describes a hydroxyalkyl starch derivative, which is formed upon reaction of a linker having the structure NH 2 -L 2 '-W with the reducing end, said derivative having a structure according to the following formula
• step c comprises a reducing step, according to the following formula
• the present invention also describes a hydroxyalkyl starch derivative, which is formed upon reaction of a linker having the structure NH2-NH-L 2 '-W with the reducing end, said derivative having a structure according to the following formula
• step c comprises a reducing step, according to the following formula
• the present invention also describes a hydroxyalkyl starch derivative, which is formed upon reaction of a linker having the structure NH 2 -0-L 2 '-W with the reducing end, said derivative having a structure according to the following formula
• step c comprises a reducing step, according to the following formula
• linker moiety L 2 ' said moiety is preferably selected from a linking moiety such as an alkyl, alkenyl, alkylaryl, arylalkyl, aryl or heteroaryl group.
• the linker moiety is substituted preferably with 1 to 10, more preferably with 1 to 4, most preferably with 1 substituent.
• said substituent is selected from the group consisting of C C 6 alkyl, such as methyl, ethyl, propyl, iso-propyl, butyl and t-butyl, chloride, fluoride, bromide, hydroxy and thiol.
• L 2 ' in formula II is selected from the group consisting of
• the repeating units -[C(R m R n )]- may be the same or may be different from each other.
• the repeating units -Y 2 -[C(R m R n ) t ]- may be the same or may be different from each other.
• the repeating units -[C(R j R k )]- may be the same or may be different from each other.
• L 2 ' is R g and R h are preferably independently of each other selected from H and alkyl, preferably H and methyl, wherein each repeating unit -[C(R g Rh)]-, in case r is > 1, may be the same or may be different from each other.
• the integer r is preferably in the range of from 1 to 10, although longer alkyl chains are also encompassed by the present invention.
• the following groups L 2 ' are mentioned: -CH 2 -, -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 - CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH
• the present invention describes a method, as well as a conjugate obtained or obtainable by said method, wherein L 2 ' is wherein both R g and R h are H, in particular wherein r is in the range of from 1 to 10, preferably of from 3 to 8.
• At least one of R g and Rh of at least one repeating unit is an alkyl, preferably methyl, wherein n is in the range of from 1 to 10, preferably of from 3 to 8.
• L 2 ' has as structure according to the following formula with s preferably being in the range of from 1 to 10, more preferably with s being 2, with t preferably being in the range of from 1 to 10, more preferably with t being 2, and with u preferably being in the range of from 1 to 3, and wherein R j , Rk, R m and R n are, independently of each other, selected from H or alkyl, preferably wherein R j , Rk, R m and R n are H, and wherein Y 2 is a functional moiety.
• L 2 ' is, for example, -[C(RjR k )] s - NH-[C(R m R n )],-, -[C(R j Rk)]s-S-S-[C(R m R n )] r , -[CiR j R ls-S-succinimidyl-fCCR ⁇ n)],-,
• Rj, R k , R m and R n are, independently of each other, selected from H or alkyl, preferably wherein R j , Rk, R m and R n are H, and with s preferably being in the range of from 1 to 10 and with t preferably being in the range of from 1 to 10. Most preferably s is in the range of from 2 to 4. Further, t is in particular in the range of from 2 to 4.
• the following groups L 2 ' are, according to this embodiment, particularly preferred: -CH 2 -CH 2 -NH-CH 2 -CH 2 - and -CH 2 -CH 2 -S-S-CH 2 -CH 2 -.
• Y 2 is -0-, -S- or -NH-.
• u is preferably in the range of from 1 to 10, more preferably in the range of from 1 to 3.
• s and t are most preferably 2.
• the following groups are, according to this embodiment, preferred: -[C(R j Rk)] 2 -0- [C(R m R n )] 2 -, -[C(R j Rk)]2-0-[C(R m R n )] 2 -0-[C(R m R n )] 2 -, -[C(R j R k )] 2 -0-[C(R m R n )] 2 -0- [C(R m R n )] 2 -0-[C(R m R n )] 2 -, -[C(R j Rk)] 2 -S-[C(R m R n )] 2 -, -[C(R j R k )] 2 -S-[C(R m R n )] 2 -S- [C(R m R n )] 2 -S- [C(R m R n )]
• Y 2 is most preferably— O-.
• the present invention also relates to a method, as described above, and a conjugate obtained or obtainable by said method, wherein L 2 ' has as structure according to the following formula: with s, t and u, as described above and with R j , R k , R m , R n as described above, and wherein the functional moiety Y 2 is -0-.
• the following groups L 2 ' are mentioned: -CH 2 -CH 2 -0-CH 2 -CH 2 -, -CH 2 -CH 2 -0-CH 2 -CH 2 -0-CH 2 -CH 2 - and -CH 2 -CH 2 -0-CH 2 - CH 2 -0-CH 2 -CH 2 -0-CH 2 -CH 2 -.
• the present invention also relates to a method, as described above, as well as to a conjugate obtained or obtainable by said method, wherein the linker L 2 is a bifunctional linker, preferably having a structure according to the following formula (Ila):
• L 2 ' is a linker moiety preferably being selected from the group consisting of
• R g and R h are H
• s preferably being in the range of from 1 to 10, more preferably with s being 2
• t preferably being in the range of from 1 to 10, more preferably with t being 2
• u preferably being in the range of from 1 to 3
• R j , R ⁇ R m and R n are H and wherein Y 2 is -0-.
• preferred linkers L 2 used in the method of the present invention have, for example, the following structures: NH 2 -CH 2 -W, NH 2 -CH 2 -CH 2 -W, NH 2 -CH 2 -CH 2 -CH 2 -W, NH 2 -CH 2 -CH 2 -CH 2 -CH 2 -W, NH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -W, NH 2 -CH 2 -CH 2 -CH 2 - CH 2 -CH 2 -CH 2 -W, NH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -W, NH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -W, NH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -W,
• HAS is preferably dissolved in an aqueous medium, more preferably in a reaction buffer, and L 2 is subsequently added.
• Preferred reaction buffers are, e.g., sodium citrate buffer, sodium acetate buffer, sodium phosphate buffer, sodium carbonate buffer, sodium borate buffer, water.
• Preferred pH values of the reaction buffers are in the range of from 2 to 9, more preferably of from 3 to 7.
• the linker L 2 is preferably added as saturated solution, wherein the linker is preferably dissolved in an aqueous medium, more preferably in a reaction buffer.
• the reaction mixture is preferably stirred at a temperature in the range of from 0 °C to 100 °C, more preferably at a temperature in the range of from 0 °C to 60 °C.
• the reaction is preferably conducted for a time in the range of from 1 to 48 h, more preferably from 1 to 24 h.
• the hydroxyalkyl starch derivative is precipitated form the reaction mixture, in particular by adding an alcohol, preferably 2-propanol or separated by ultrafiltration and/or lyophilisation.
• the obtained precipitate may be purified with conventional steps, in particular by centrifugation, dialysis, ultrafiltration and/or lyophilisation.
• step c) additionally comprises the reduction of the hydroxyalkyl starch derivative obtained upon reaction of HAS with L 2 prior to step d).
• the functional group obtained upon reaction of Z 2 and the reducing end is reduced to give the functional group F 2 .
• said group is reduced to give the group F 2 with F 2 being -CH 2 -NH-.
• said group is reduced to give the group F 2 with F 2 being -CH 2 -NH-NH 2 -.
• the reduction may be carried out at a temperature of about 0 °C to about 100 °C for about 1 h to about 48 h, such as overnight, in the presence of a suitable reducing agent, such as sodium borohydride, sodium cyanoborohydride, sodium triacetoxy borohydride, organic borane complex compounds such as a 4-(dimethylamino)pyridine borane complex, N-ethyldiisopropylamine borane complex, N-ethylmorpholine borane complex, N-methylmorpholine borane complex, N-phenylmorpholine borane complex, lutidine borane complex, triethylamine borane complex, or trimethylamine borane complex, preferably NaCNBH 3 or NaBH 4 .
• a suitable reducing agent such as sodium borohydride, sodium cyanoborohydride, sodium triacetoxy borohydride, organic borane complex compounds such as a 4-(dimethylamino)pyridine
• the concentration of these reducing agents used for this reaction of the present invention is preferably in the range of from 0.001 to 2.0 mol/1, more preferably in the range of from 0.01 to 1.0 mol/1, and more preferably in the range of from 0.1 to 0.8 mol/1, relating, in each case, to the volume of the reaction solution.
• step c) further comprises the reduction of the linking group, as described above
• the reduction can either be carried out subsequent to the coupling process, in which Z 2 is coupled to the reducing end, optionally after isolating the coupled product prior to the reduction, or it is possible to carry out the same reaction all in one pot, with the coupling to the reducing end and the reduction occurring concurrently.
• Both reactions are referred to in the context of the present invention as reductive amination.
• the present invention also relates to a method as described above, and a conjugate obtained or obtainable by said method, wherein the functional group Z 2 is H 2 N- and the reaction according to step c) is a reductive amination.
• the present invention also relates to a method as described above, and a conjugate obtained or obtainable by said method, wherein the functional group Z 2 is H 2 N-NH- and the reaction according to step c) is a reductive amination.
• the molar ratio of L 2 : HAS is preferably in the range of from 1 :1 to 100:1, more preferably from 2: 1 to 80: 1 , more preferably from 3:1 to 70: 1 , more preferably from 4: 1 to 60: 1 , and more preferably from 5:1 to 50:1.
• the concentration of HAS, preferably HES, in the aqueous system is preferably in the range of from 1 to 50 wt.-%, more preferably from 3 to 45 wt.-%, and more preferably from 5 to 40 wt.-%, relating, in each case, to the weight of the reaction solution.
• step d) of the method of the invention the oligonucleotide derivative according to step b) is reacted with the hydroxyalkyl starch derivative according to step c) via reaction of the functional group W with the functional group Q, thereby forming a covalent linkage F 3 .
• the functional group Q and the functional group W are complementary functional groups which readily form the linking group F 3 when reacted with each other.
• the functional group W and/or the functional group Q may comprise a suitable protecting group when used in step b) and/or step c). In this case, the method further comprises at least one deprotection step prior to step d). Suitable protecting groups are known to those skilled in the art.
• the functional groups W and Q may be suitably chosen.
• one of the groups W and Q, i.e. either W or Q may be chosen from the group consisting of the functional groups according to the following, non-limiting list, while the other group, Q or W, is a complementary group being suitably selected and capable of forming a chemical linkage with W or Q:
• C-C-double bonds or C-C-triple bonds such as alkenyl groups, alkynyl groups or aromatic C-C-bonds, in particular alkynyl groups such as the group -C ⁇ C-H;
• alkyl sulfonic acid hydrazides aryl sulfonic acid hydrazides
• amino groups comprising the structure -NR'R", wherein R' and R" are independently of each other selected from the group consisting of H, alkyl groups, aryl groups, arylalkyl groups and alkylaryl groups, preferably -NH 2 ;
• hydroxylamino groups comprising the structure -O-NR'R", wherein R' and R" are independently of each other selected from the group consisting of H, alkyl groups, aryl groups, arylalkyl groups and alkylaryl groups, preferably -0-NH 2 ;
• oxyamino groups comprising the structure unit -NR'-O-, with R' being selected from the group consisting of alkyl groups, aryl groups, arylalkyl groups and alkylaryl groups, preferably -NH-0-;
• activated esters such as esters of hydroxylamines having an imide structure such as N-hydroxysuccinimide
• carbonyl groups such as aldehyde groups, keto groups, hemiacetal groups or acetal groups
• residues comprising a leaving group such as e.g. halogens or sulfonic esters.
• neither the functional group W nor the functional group Q is a carboxy group or an activated carboxy group.
• the present invention also relates to a method as described above, and a conjugate obtained or obtainable by said method, wherein neither the group Q nor the group W is a carboxy group or an activated carboxy group.
• the functional group Q or the functional group W is selected from the group consisting of -SH, thiol reactive groups, NH comprising groups and carbonyl groups such as aldehyde, keto or hemiacetal groups.
• Q or W is a thiol group.
• the complementary functional group is a thiol-reactive group.
• thiol-reactive group as used in the present invention is a group which readily reacts with a thiol group thereby forming a chemical linkage with the proviso that the thiol-reactive group is not a carboxy group or a reactive carboxy group as described above.
• Thiol reactive groups include, but are not limited to, groups such as pyridyl disulfides, maleimide groups, haloacetyl groups, and vinyl sulfones.
• the thiol-reactive group according to the invention is selected from the group consisting of the following structures wherein Hal is CI, Br, or I.
• the present invention also relates to a method, as described above, and a conjugate obtained or obtainable by said method, wherein the functional group Q or the functional group W is a thiol group and the functional group W or the functional group Q is a thiol- reactive group, wherein the thiol-reactive group is preferably selected from the group consisting of pyridyl disulfides, maleimide groups, haloacetyl groups, and vinyl sulfones, in particular from the rou consistin of wherein Hal is CI, Br, or I.
• the covalent linkage F 3 preferably has one of the following structures: -S-TRG'- or -TRG'-S-, with TRG' being the remainder of the thiol-reactive group which is formed upon reaction of -SH with the thiol reactive group.
• TRG' being the remainder of the thiol-reactive group which is formed upon reaction of -SH with the thiol reactive group.
• F 3 has, e.g., a structure according to the following formulas
• F 3 has, e.g., a structure according to the following formulas
• F 3 has, e.g., the structure
• the functional group W is -SH and the functional group Q is one of the thiol reactive groups mentioned above.
• the present invention also relates to a linker L 2 , comprising a functional group W and a functional group Z 2 , wherein W is -SH, more preferably wherein W is -SH and Z 2 is selected from the group consisting of -NH 2 , NH 2 -NH- and ⁇ 2 -0-, more preferably wherein the linker has a structure selected from the following group: NH 2 -CH 2 -SH, NH 2 - CH2-CH2-SH, NH2-CH2-CH2-CH2-SH, NH2-CH2-CH2-CH2-CH2-SH, NH2-CH2-CH2- CH2-CH2-CH2-SH, NH2-CH 2 -CH 2 -CH 2 -CH2-CH 2 -CH 2 -SH, NH2-CH2-CH2-CH2-CH2- CH 2 -CH 2 -CH 2 -SH, NH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-SH, NH 2 -CH 2 -CH 2 -
• the hydroxyalkyl starch derivative formed in step c) preferably has a structure selected from the following formulas
• L2' being as described above, preferably with L 2 ' being selected from the group consisting of -CH 2 -, -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -, -CH2-CH2-CH2-, -CH 2 -CH 2 -CH 2 - CH 2 -CH 2 -, -CH 2 -CH 2 -CH2-CH2-CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 - CH 2 -CH2-CH2-CH2-CH 2 -CH2-CH 2 -, -CH 2 -CH2-CH2-CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH2-CH2-CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH2-CH2-CH 2 -CH 2 -CH 2 -CH 2 -CH
• the functional group Q is preferably a thiol reactive group, as described above, most preferably a group selected from the group consisting of wherein Hal is CI, Br, or I, preferably Br or I.
• the present invention also relates to a method as described above, and conjugate obtained or obtainable by said method, wherein the functional group Q selected from the group consisting of
• the oligonucleotide derivative formed in step b) preferably has a structure selected from the following formulas
• Li' being as described above, most preferably with Li' being selected from the group consisting of -CH 2 -, -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -, -CH 2 - CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH
• the present invention also relates to conjugates, as described above, as well as to a method for preparing conjugates, as described above, and conjugates obtained or obtainable by this method, said conjugates preferably having a structure according to the following structure
• the functional group W is a thiol reactive group and the functional group Q is an SH-group.
• the present invention also relates to a linker L 2 , comprising a functional group W and a functional group Z 2 , wherein W is a thiol reactive group, more preferably wherein W is a thiol reactive group and Z 2 is selected from the group consisting of -NH 2 , NH 2 - NH- and ⁇ 2 -0-, most preferably wherein L 2 is selected from the group consisting of NH 2 -CH 2 -TRG, NH 2 -CH 2 -CH 2 -TRG, NH 2 -CH 2 -CH 2 -CH 2 -TRG, NH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -TRG, NH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -TRG, NH 2 -CH 2 -CH 2 -CH 2 -CH 2 -TRG, NH 2 -CH 2 -CH 2 -CH 2
• the hydroxyalkyl starch derivative formed in step c) preferably has a structure according to the following formula more preferably a structure, selected from the following formulas
• L 2 ' being as described above, preferably with L 2 ' being selected from the group consisting of -CH 2 -, -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 - CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2
• the functional group W is preferably a thiol reactive group selected from the group consisting of wherein Hal is CI, Br, or I, preferably Br or I.
• the present invention also relates to a method, as described above, wherein the HAS derivative formed in step c) has a structure selected from the group consisting of
• the present invention also relates to a method, as described above, wherein the HAS derivative formed in step c) has a structure selected from the group consisting of
• the present invention also relates to a method, as described above, wherein the HAS derivative formed in step c) has a structure selected from the group consisting of
• the present invention also relates to a method, as described above, wherein the HAS derivative formed in step c) has a structure selected
• the oligonucleotide derivative formed in step b) preferably has a structure according to the following formula
• HN-Oligonucleotide more preferably of the following formula
• Li' being as described above, most preferably with Li' being selected from the group consisting of -CH 2 -, -CH 2 -CH 2 -, -CH 2 - CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH
• the present invention also relates to conjugates, as described above, as well as to a method for preparing conjugates, as described above, and conjugates obtained or obtainable by this method, said conjugates preferably having a structure according to the following s more preferably a structure selected from
• the coupling is preferably carried out in a solvent selected from the group consisting of DMSO, DMF, buffer, water and mixtures thereof.
• the reaction is preferably carried out at a temperature in the range of from 0 to 100° C, more preferably in the range of from 5 to 50 °C and especially preferably in the range of from 5 to 25 °C.
• the temperature may be held essentially constant or may be varied during the reaction procedure.
• the pH value for the reaction according to step (d) may be adapted to the specific needs of the reactants.
• the reaction is carried out at a pH in the range of from 3 to 10, depending on the functional groups W and Q used.
• the pH is preferably in the range of from 6 to 9.
• step (b) and step (d) are carried out as one pot synthesis, the pH may be varied during the course of the reaction.
• the reaction time for the (coupling) reaction between oligonucleotide derivative and hydroxyalkyl starch derivative is generally in the range of from 1 min to 7 days, preferably 5 min to 24 hours, more preferably 5 min to 2 hours.
• the conjugate obtained may be subjected to at least one further isolation and/or purification step such as precipitation, centrifugation, filtration, ultrafiltration, dialysis, centrifugal filtration, pressure filtration, ion exchange chromatography, reversed phase chromatography, HPLC, MPLC, gel filtration and/or lyophilisation.
• the conjugate is first separated off from the reaction mixture by a suitable method such as precipitation, centrifugation, filtration, size exclusion chromatography, ion exchange chromatography, reversed phase chromatography, HPLC, MPLC or the like.
• the separated conjugate may be subjected to a further treatment such as an after-treatment like ultrafiltration, dialysis, centrifugal filtration or pressure filtration, and/or gel filtration.
• the obtained conjugate is further lyophilized until the solvent content of the reaction product is sufficiently low according to the desired specifications of the product.
• composition and Use further relates to a pharmaceutical composition
• a pharmaceutical composition comprising, in a therapeutically effective amount, a hydroxyalkyl starch-oligonucleotide conjugate as described above, or a hydroxyalkyl starch-oligonucleotide conjugate obtained or obtainable by the above described method.
• a "therapeutically effective amount” as used herein refers to that amount which provides therapeutic effect for a given condition and administration regimen.
• the hydroxyalkyl starch- oligonucleotide conjugate as described above, or the hydroxyalkyl starch-oligonucleotide conjugate obtained or obtainable by the above described method may be used in combination with a pharmaceutical excipient, adjuvant or carrier.
• the HAS derivative will be in a solid form which can be combined with a suitable pharmaceutical excipient that can be in either solid or liquid form.
• excipients carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof may be mentioned.
• a carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient.
• Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and
• the pharmaceutical composition according to the present invention may also comprise an antimicrobial agent for preventing or deterring microbial growth, such as, e.g., benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.
• an antimicrobial agent for preventing or deterring microbial growth such as, e.g., benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.
• the pharmaceutical composition according to the present invention may also comprise an antioxidant, such as, e.g., ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.
• an antioxidant such as, e.g., ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.
• the pharmaceutical composition according to the present invention may also comprise a surfactant, such as, e.g., polysorbates, or pluronics sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, acids and fatty esters; steroids, such as cholesterol; and chelating agents, such as EDTA or zinc.
• a surfactant such as, e.g., polysorbates, or pluronics sorbitan esters
• lipids such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, acids and fatty esters
• steroids such as cholesterol
• chelating agents such as EDTA or zinc.
• the pharmaceutical composition according to the present invention may also comprise acids or bases such as, e.g., hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof, and/or sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.
• acids or bases such as, e.g., hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and
• the excipient will be present in a pharmaceutical composition according to the present invention in an amount of 0.001 to 99.999 wt.-%, preferably from 0.01 to 99.99 wt.-%, more preferably from 0.1 to 99.9 wt.-%, in each case based on the total weight of the pharmaceutical composition.
• composition of the invention is preferably used in a formulation suitable for subcutaneous or intravenous or parenteral injection.
• suitable excipients and carriers are e.g. sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, sodium glutamate, mannitol, sorbitol, polysorbate 80, HSA and water for injection.
• a method for preparing a hydroxyalkyl starch (HAS)-oligonucleotide conjugate comprising the steps
• step (d) reacting the oligonucleotide derivative according to step (b) with the hydroxyalkyl starch derivative according to step (c) via reaction of the functional group W with the functional group Q, thereby forming a covalent linkage F 3 .
• the method as described in embodiment 1 wherein in (c) the hydroxyalkyl starch is reacted at its at least one reducing end with a functional group Z 2 of the linker L 2 to give the hydroxyalkyl starch derivative comprising the at least one functional group W, thereby forming a functional group F 2 , said linker L 2 comprising
• HAS' is the remainder of the hydroxyalkyl starch molecule and R 1? R 2 and R 3 are independently hydrogen or a linear or branched hydroxyalkyl group.
• G is O or S and, if present twice, independently O or S, and R' is alkyl, preferably wherein the functional group Z 2 is H 2 N- and the reaction according to step (c) is a reductive amination.
• thiol-reactive group is selected from the group consisting of maleimides, haloacetyl groups, pyridyl disulfides and vinyl sulfones.
• the functional group Q is an aldehyde, keto or hemiacetal group, and the functional group W is selected from the group consisting of
• G is O or S and, if present twice, independently O or S, and R' is alkyl.
• linker L 2 is a bifunctional linker, preferably having a structure according to the following formula (Ila)
• L 2 ' is a linker moiety preferably being selected from the group consisting of
• r and -> s with r preferably being in the range of from 1 to 10, wherein R g and Rh are independently of each other selected from H and alkyl, preferably wherein both R g and Rj, are H,
• s preferably being in the range of from 1 to 10, more preferably with s being 2, with t preferably being in the range of from 1 to 10, more preferably with t being 2, and with u preferably being in the range of from 1 to 3,
• Rj, Rk, R m and R n are, independently of each other, selected from H or alkyl, preferably wherein Rj, Rk, R m and R n are H,
• Li' is a linker moiety preferably being selected from the group consisting of
• n preferably being in the range of from 1 to 10,
• R a and Rb are independently of each other selected from H and alkyl, preferably wherein both R a and Rb are H,
• m preferably being in the range of from 1 to 10, more preferably with m being 2, with o preferably being in the range of from 1 to 10, more preferably with o being 2, and with p preferably being in the range of from 1 to 3,
• Rc, Rd, Re and Rf are, independently of each other, selected from H or alkyl, preferably wherein Rc, Rd, e and Rf are H,
• Y ⁇ is a functional moiety, preferably wherein Yi is O.
• F 3 is the functional group which is formed upon reaction of Q and W,
• Li ' is a linker moiety preferably being selected from the group consisting of
• L 2 ' is a linker moiety preferably being selected from the group consisting of
• R a and Rj are independently of each other selected from H and alkyl, preferably wherein both R a and Rb are H,
• n preferably being in the range of from 1 to 10, more preferably with m being
• o preferably being in the range of from 1 to 10, more preferably with o being 2, and with p preferably being in the range of from 1 to 3,
• Rc, Rj, Re and Rf are, independently of each other, selected from H or alkyl, preferably wherein Rc, R ⁇ j, R_g and Rf are H,
• Yi is a functional moiety, preferably wherein Yj is O,
• r is preferably in the range of from 1 to 10,
• R g and Rj are independently of each other selected from H and alkyl, preferably wherein both R g and Rh are H, with s preferably being in the range of from 1 to 10, more preferably with s being 2, with t preferably being in the range of from 1 to 10, more preferably with t being 2, and with u preferably being in the range of from 1 to 3,
• R j , Rk, R m and R n are, independently of each other, selected from H or alkyl, preferably wherein R j , R k , R m and R n are H,
• the conjugate as described in embodiment 16, having a structure according to the formula (IV) wherein F 2 comprises the structure -CH 2 -NH- or -CH N-,
• F 3 is the functional group which is formed upon reaction of Q and W,
• Li' is a linker moiety preferably being selected from the group consisting of
• L 2 ' is a linker moiety preferably being selected from the group consisting of
• R a and R b are independently of each other selected from H and alkyl, preferably wherein both R a and R b are H,
• n preferably being in the range of from 1 to 10, more preferably with m being
• o preferably being in the range of from 1 to 10, more preferably with o being 2, and with p preferably being in the range of from 1 to 3,
• R c , 3 ⁇ 4, R e and Rf are, independently of each other, selected from H or alkyl, preferably wherein Rc, 3 ⁇ 4, R e and Rf are H,
• Y ⁇ is a functional moiety, preferably wherein Y] is O, and wherein r is preferably in the range of from 1 to 10,
• R g and Rj are independently of each other selected from H and alkyl, preferably wherein both R g and R h are H,
• s preferably being in the range of from 1 to 10, more preferably with s being 2, with t preferably being in the range of from 1 to 10, more preferably with t being 2, and with u preferably being in the range of from 1 to 3,
• R j , R k , R m and R n are, independently of each other, selected from H or alkyl, preferably wherein R j , R ⁇ , R m and R n are H,
• step (e) purifying the conjugate obtained according to step (d) via ion exchange chromatography.
• a pharmaceutical composition comprising in a therapeutically effective amount a conjugate as described in any of embodiments 16 to 19 or of a composition as described in embodiment 21.
• Figure 1 Results of size exclusion chromatography according to example 2. Only the relevant section of the chromatograms at the elution time of HES is shown at a wavelength of 220 nm (X-axis: [min], Y-axis: [mAU]): line solid: chromatogram of HES after mock incubation without modification reagent line dashed: chromatogram of reaction according to example 2 A (4: 1 ratio)
• Figure 3 SE-HPLC chromatograms (monitored at 260 nm, only relevant sections shown) of reaction mixtures according to example 4 (X-axis: [min], Y-axis: [mAU]):
• Figure 4 Results of size exclusion chromatography according to example 5 monitored at 260 nm. Only the relevant section of the chromatogram is shown (X-axis: [min], Y-axis: [mAU]). The unmodified DNA elutes at ⁇ 14 min, the respective HES-DNA conjugate peak is front-shifted to an elution time of 1 1 min (peak max.).
• Approximately 10 mg of the hydroxyalkyl starch derivative were dissolved in 1 ml of the mobile phase and particle filtrated with a syringe filter (0.22 mm, mStarll, CoStar Cambridge, MA). The measurement was carried out at a flow rate of 0.5 ml/min. As detectors a multiple-angle laser light scattering detector and a refractometer maintained at a constant temperature, connected in series, were used. Wyatt Astra software was used to determine the mean M w and the mean M n of the sample. For the calculation dn/dc for the HES derivative or conjugate was determined using Wyatt astra software.
• Example 1 Coupling of an amino-functionalized DNA-oligonucleotide to thiol- functionalized HES via the heterobifunctional linker SIA
• oligonucleotide 1 5'-C6-amino-dUGAGdUGACdUGAC- 3'
• SIA iodoacetic acid N-hydroxysuccinimide ester, CAS 39028-27-8
• linker stock solution 2.5 ⁇ of the oligonucleotide stock solution (corresponding to 5 ⁇ g of oligonucleotide 1) were mixed with 1.85 ⁇ of linker stock solution and 1 ⁇ of a 1M sodiumcarbonate-buffer (pH 8.4).
• the molar ratio of SIA to oligonucleotide 1 was about 5:1. After 10 min incubation at 37 °C, a 20-fold molar excess (calculation based on the M w of oligonucleotide 1 and the M n of the HES-derived polymer) of thiol- functionalized HES (degree of derivatization was about 50 %; solution in 0.2 M sodium carbonate buffer pH 8.4) was added and the reaction was incubated for 1 more hour at room temperature. An aliquot of the resulting reaction mixture was analyzed by size exclusion HPLC. The thiol-functionalized HES (HES 100/1.3) derivative bearing the reactive thiol group was obtained by reductive amination of HES with cystamine and subsequent reduction with DTT.
• Example 2 Treatment of HES with 3-(4-hydroxyphenyl)propionic acid N- hydroxysuccinimide ester (example not according to the invention)
• HES derivatives comprising activated carboxy groups may readily be formed due to undesired inter- and intra-crosslinking reactions.
• Example 3 Control experiment showing the site-specificity of HES coupling to amino-modified nucleic acids (example not according to the invention)
• the oligonucleotide sequence 5'-GGCTACGTCCAGGAGCCACCT-3' was synthesized by MWG Biotech (Ebersberg, Germany) either in its unmodified version or with an aminohexyl linker at the 5' end.
• the lyophilized material was dissolved in deionized water (MilliQ) to yield a stock solution of 2 mg ml which was divided into aliquots, shock-frozen in liquid nitrogen and stored at -20°C until further use.
• N-Succinimidyl iodoacetate was purchased from Sigma, Deisenhofen, Germany (Prod. No. 19760).
• Thio-HES HES 100/1.3 derivative bearing a reactive thiol group was obtained by reductive animation of HES with cystamine and subsequent reduction with DTT.
• a detectable conjugation of a HES derivative to DNA only occurred in case the aminohexyl linker modified DNA was used.
• Example 4 Coupling of an amino-functionalized DNA-oligonucleotide to thiol- functionalized HES using different heterobifunctional linkers
• the oligonucleotide sequence 5'-GGCTACGTCCAGGAGCCACCT-3' was synthesized by MWG Biotech (Ebersberg, Germany) with an aminohexyl linker at the 5' end.
• the lyophilized material was dissolved in deionized water (MilliQ) to yield a stock solution of 2 mg/ml which was divided into aliquots, shock-frozen in liquid nitrogen and stored at -20°C until further use.
• N-Succinimidyl iodoacetate (SIA; Prod. No. 19760), N-succinimidyl 3-(2- pyridyldithio)propionate (SPDP; Prod. No. P3415) and N-succinimidyl bromoacetate (SBrA; Prod. No. B8271) were purchased from Sigma (Deisenhofen, Germany); N-[g- maleimidobutyryloxy]succinimide ester (GMBS/MBNHS; Prod. No. 63175) was from Fluka (Deisenhofen, Germany).
• Thio-HES HES100/1.3 derivative bearing a reactive thiol group was obtained by reductive amination of HES with cystamine and subsequent reduction with DTT.
• oligonucleotide 1 5'-C6-amino-GGC TAC GTC CAG GAG CCA CCT-3'
• MilliQ water 2 mg/ml stock solution of oligonucleotide 1 (5'-C6-amino-GGC TAC GTC CAG GAG CCA CCT-3') in MilliQ water was prepared.
• "SFB" Succinimidyl 4-formylbenzoate, CAS 60444-78-2
• linker stock solution 2.5 ⁇ of the oligonucleotide stock solution (corresponding to 5 ⁇ g of oligonucleotide 1) were mixed with 1.32 ⁇ of linker stock solution and 1.2 ⁇ of a 100 mM sodium carbonate buffer (pH 8.4).
• the molar ratio of SFB to oligonucleotide 1 was about 7:1. After 15 min incubation at room temperature, a 20-fold molar excess (calculation based on the Mw of oligonucleotide 1 and the M n of the HES-derived polymer) of ha-functionalized HES (degree of derivatization was about 50 %; solution in 0.1 M sodium acetate buffer pH 5.0) was added and the reaction was incubated for 2 hours at room temperature. An aliquot of the resulting reaction mixture was analyzed by size exclusion HPLC.
• the ha-functionalized HES (HES 100/1.0) derivative bearing the reactive amino group was obtained by reaction of HES with 0-[2-(2-aminooxyethoxy)- ethyl]hydroxyl amine in 1 M acetate buffer pH 5.0 at room temperature overnight.
• the ha-functionalized HES may also be prepared according to the examples of WO2004/024777, e.g. according to example 2.4).

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