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WO2014147175A1 - Dérivés d'hydroxyalkylamidon utilisés comme réactifs pour couplage à des groupes thiol - Google Patents

Dérivés d'hydroxyalkylamidon utilisés comme réactifs pour couplage à des groupes thiol Download PDF

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Publication number
WO2014147175A1
WO2014147175A1 PCT/EP2014/055598 EP2014055598W WO2014147175A1 WO 2014147175 A1 WO2014147175 A1 WO 2014147175A1 EP 2014055598 W EP2014055598 W EP 2014055598W WO 2014147175 A1 WO2014147175 A1 WO 2014147175A1
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group
derivative
alkyl
groups
range
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Norbert Zander
Sabrina HUMMEL
Ulrike Wedemeyer
Thomas Hey
Dominik Heckmann
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Fresenius Kabi Deutschland GmbH
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Fresenius Kabi Deutschland GmbH
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Priority to CA2907371A priority Critical patent/CA2907371A1/fr
Priority to AU2014234272A priority patent/AU2014234272A1/en
Priority to EP14711748.5A priority patent/EP2976366A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B31/00—Preparation of derivatives of starch
    • C08B31/08—Ethers
    • C08B31/12—Ethers having alkyl or cycloalkyl radicals substituted by heteroatoms, e.g. hydroxyalkyl or carboxyalkyl starch

Definitions

  • the present invention relates to a hydroxyalkyl starch derivative comprising a reactive acetamide group as well as to a method for preparing the same. Further, the invention relates to the use of said hydroxyalkyl starch derivative as reactant for coupling to a thiol group of a further compound. Further, the present invention relates to a hydroxyalkyl starch derivative coupled to a thiol group of a further compound and a method for preparing the same.
  • HES Hydroxyalkyl starch
  • HES hydroxyethyl starch
  • HES is a substituted derivative of the naturally occurring carbohydrate polymer amylopectin, which is present in corn starch at a concentration of up to 95 % by weight, and is degraded by alpha amylases in the body.
  • HES in particular exhibits advantageous biological properties and is used as a blood volume replacement agent and in hemodilution therapy in clinics (Westphal et al., Anesthesiology, 2009, 111 : 187-202).
  • 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.
  • polymeric prodrugs thus drugs coupled to polymeric compounds, were suggested to prolong the circulation lifetime in the body due to the increase in size of the drug-polymer conjugate when compared to the single drug which may prevent a quick removal of the drug by glomerular filtration through the kidneys.
  • a hydroxyalkyl starch derivative for coupling to thiol groups of further compounds such as cysteine groups of proteins
  • WO 02/080979 discloses a method for the preparation of hydroxyalkyl starch derivatives for coupling to thiol groups of DNA, wherein a hydroxyalkyl starch is first oxidized at its reducing end, subsequently modified with an amino group and finally reacted with succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC) to give a maleimide modified HAS derivative.
  • Said HAS derivative is further coupled to a thiol group of DNA resulting in a hydroxyalkyl starch DNA conjugate.
  • Halogenacetamide modified HAS derivatives and their coupling to further compounds are not mentioned.
  • WO 2005/014050 discloses a method for the preparation of hydroxyalkyl starch GCS-F conjugates, wherein e.g. a HAS derivative comprising a maleimide group is disclosed.
  • the hydroxyalkyl starch is first oxidized at its reducing end, subsequently modified with an amino group and finally reacted with N- alpha(maleimidoacetoxy)succinimide ester (AMAS) to give the maleimide modified HAS derivative.
  • AMS N- alpha(maleimidoacetoxy)succinimide ester
  • Said HAS derivative is further coupled to a thiol group of GCS-F.
  • HAS derivatives modified with a reactive acetamide group and their coupling to further compounds are also not mentioned.
  • WO 2005/014050 mentiones, generally, the derivatization of an amino-modfied HAS with a monohalogen-subsituted acetic acids in the presence of an activating agent, such as EDC.
  • Halogenacetyl modified HAS molecules and their coupling to thiol groups of further compounds are further described e.g. in WO2003/070772 and EP 1 398 322 Al .
  • HAS is modified via its oxidized reducing end with a linker compound to give the halogenacetyl modified HAS derivate.
  • a linker is coupled to hydroxyalkyl starch using activated carboxylic acid chemistry
  • a product mixture may potentially be obtained by reactions with the multiple hydroxyl groups.
  • This mixture may contain macromolecules carrying different numbers of linker molecules and/or variations in their attachment position within the macromolecules. So often a lower yield for the desired conjugation reaction between HES and the conjugation partner results and, in particular, an inhomogeneous composition potentially comprising crosslinked polymer side products is obtained.
  • the described oxidation of the reducing end of hydroxyalkyl starch is considered to be not completely selective, thus, also for this reason, potentially inhomogeneous products may result.
  • the maleimide-thio-linkage are considered to have unpleasant side effects such as (unwanted) immunogenicity, a low stability of the thiol- reactive functional group during storage and/or under reductive conditions (such as disulfide bonds) and/or in the conjugation reaction and the like.
  • advantageous hydroxyalkyl starch derivatives for coupling to thiol groups of further compounds which can be formed in a highly selective manner, which highly selectively react with the further compound and/or with which a stable and biocompatible linkage to the further compound can be provided.
  • a selective method for the preparation of such derivatives in which possible side reactions such as inter- and intramolecular crosslinking are significantly diminished or avoided and with which the derivatives can be provided in a high yield and high purity.
  • hydroxyalkyl starch derivatives for coupling to thiol groups of further compounds which derivatives overcome the problems of the prior art as well as a method for preparing the same, in particular which method provides the desired derivatives in high yield and with high specificity and purity. It is a further object of the present invention to provide novel, stable and biocompatible hydroxyalkyl starch derivatives comprising a protein attached to HAS via a thiol group of the protein as well as a method for preparing the same, in particular which method provides the desired derivatives in high yield and with high specificity and purity.
  • the present invention relates to a hydroxyalkyl starch (HAS) derivative of formula (I)
  • X is a leaving group, preferably selected from the group consisting of mesyl, tosyl, CI, Br and I;
  • Fl is a functional group comprising the group -NR'-, with R' being H or alkyl;
  • LI is a spacer bridging Fl and S;
  • L2 is a spacer bridging -NR X - and -NR y -;
  • R x and R y are, independently of each other, H or alkyl, and optionally form together with the moiety N-L2-N a ring;
  • HAS' is the remainder of the HAS molecule
  • R b and R c are -[(CR 1 R 2 ) m O] justify-H and are the same or different from each other
  • R a is -[(CR 1 R 2 ) m O] n -H with HAS' being the remainder of the hydroxyalkyl starch molecule, or R a is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch molecule
  • R 1 and R 2 are independently hydrogen or an alkyl group having from 1 to 4 carbon atoms, m is 2 to 4, wherein R 1 and R 2 are the same or different from each other in the m groups CR R 2
  • n is from 0 to 6. It is to be understood that if R a is HAS", the hydroxyalkyl starch molecule has a branching site at the C6 position of the reducing end.
  • HAS hydroxyalkyl starch
  • Fl is a functional group comprising the group -NR'-, with R' being H or alkyl;
  • LI is a spacer bridging Fl and S;
  • L2 is a spacer bridging -NR X - and -NR y -;
  • R x and R y are, independently of each other, H or alkyl, and optionally form together with the moiety N-L2-N a ring;
  • HAS' is the remainder of the HAS molecule and R b and R c are -[(CR ⁇ OJ n -H and are the same or different from each other; R a is -[(CR 1 R 2 ) m O] justify-H with HAS' being the remainder of the hydroxyalkyl starch molecule, or R a is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch molecule; R 1 and R 2 are independently hydrogen or an alkyl group having from 1 to 4 carbon atoms, m is 2 to 4, wherein R 1 and R 2 are the same or different from each other in the m groups CR J R 2 ; n is from 0 to 6.
  • the present invention also relates to a method for the preparation of a hydroxyalkyl starch (HAS) derivative, and a hydroxyalkyl starch (HAS) derivative obtained or obtainable by said method, said method comprising
  • M comprises the group -NHR', with R' being H or alkyl
  • LI is a spacer bridging M and S;
  • T is H or a thiol protecting group PG
  • HAS' is the remainder of the HAS molecule and R b and R c are -[(CR 1 R 2 ) m O] justify-H and are the same or different from each other; R a is -[(CR 1 R 2 ) m O] n -H with HAS' being the remainder of the hydroxyalkyl starch molecule, or R a is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch molecule; R 1 and R 2 are independently hydrogen or an alkyl group having from 1 to 4 carbon atoms, m is 2 to 4, wherein R 1 and R 2 are the same or different from each other in the m groups CR R 2 ; n is from 0 to 6, thereby obtaining a HAS derivative of formula (lb)
  • L2 is a spacer bridging -NR X - and -NR y -;
  • R x and R y are, independently of each other, H or alkyl, and optionally form together with the moiety N-L2-N a ring; thereby obtaining a HAS derivative of formula (I)
  • X is a leaving group, preferably selected from the group consisting of mesyl, tosyl, CI, Br and I.
  • the resulting derivatives according to formula (IV) prepared using the derivatives of formula (I) were further obtained with surprisingly high yields and/or with a high selectivity (see figure 4), thus they showed a high purity, and/or showed a surprisingly high stability (see e.g. figures 5-7) over a broad pH range, In particular at a physiological pH or higher the resulting derivatives according to formula (IV) showed surprisingly high stability.
  • derivatives according to formula (IV) (which may hereiunder also be referred to as "conjugates”) comprising a protein as compound Q surprisingly showed a comparable activity in biological assays than than protein as such (see e.g. examples C3 to C4 hereinunder).
  • conjugates are highly advantagoues since it is contemplated that the hydroxyalkyl starch prolongs the circulation time of the active agent in the body.
  • Hydroxyalkyl starch is an ether derivative of optionally partially hydrolyzed native starches wherein hydroxyl groups of the starch are suitably hydroxyalkylated.
  • hydroxyalkyl starches hydroxypropyl starch and hydroxyethyl starch are preferred, with hydroxyethyl starch being most preferred.
  • Starch is a well-known polysaccharide according to formula (C 6 Hi 0 O5) n which essentially consists of alpha-D glucose units which are coupled via glycosidic linkages. Usually, starch essentially consists of amylose and amylopectin. Amylose consists of linear chains wherein the glucose units are linked via alpha- 1 ,4-glycosidic linkages. Amylopectin is a highly branched structure with alpha- 1 ,4-glycosidic linkages and alpha- 1,6-glycosidic linkages.
  • Native starches from which hydroxyalkyl starches can be prepared include, but are not limited to, cereal starches and potato starches.
  • Cereal starches include, but are not limited to, rice starches, wheat starches such as einkorn starches, spelt starches, soft wheat starches, emmer starches, durum wheat starches, or kamut starches, corn starches, rye starches, oat starches, barley starches, triticale starches, spelt starches, and millet starches such as sorghum starches or teff starches.
  • Preferred native starches from which hydroxyalkyl starches are prepared have a high content of amylopectin relative to amylose.
  • amylopectin content of these starches is, for example, at least 70 % by weight, preferably at least 75 % by weight, more preferably at least 80 % by weight, more preferably at least 85 % by weight, more preferably at least 90 % by weight such as up to 95 % by weight, up to 96 % by weight, up to 97 % by weight, up to 98 % by weight, up to 99 % by weight, or up to 100 % by weight.
  • Native starches having an especially high amylopectin content are, for example, suitable potato starches such as waxy potato starches which are preferably extracted from essentially amylose-free potatoes which are either traditionally bred (e.g. the natural variety Eliane) or genetically modified amylopectin potato varieties, and starches of waxy varieties of cereals such as waxy corn or waxy rice.
  • a preferred hydroxyalkyl starch of the present invention has a constitution according to formula (la)
  • HAS' is the remainder of the HAS molecule and R b and R c are -[(CR ⁇ O ⁇ -H and are the same or different from each other;
  • R a is -[(CR 1 R 2 ) m O] n -H with HAS' being the remainder of the hydroxyalkyl starch molecule, or R a is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch molecule;
  • R 1 and R 2 are independently hydrogen or an alkyl group having from 1 to 4 carbon atoms, m is 2 to 4, wherein R 1 and R 2 are the same or different from each other in the m groups CR R 2 ;
  • n is from 0 to 6.
  • R 1 , R 2 are, independently of each other, selected from the group consisting of hydrogen and a methyl group, more preferably all of R 1 , R 2 are H.
  • Integer m is of from 2 to 4, such as 2, 3 or 4, preferably m is 2.
  • Integer n is of from 0 to 20, preferably of from 0 to 4, more preferably 0, 1, 2 or 3, most preferably 2.
  • the HAS derivative is a hydroxyethyl starch (HES) derivative.
  • R 1 and R 2 are hydrogen
  • m is 2
  • n is 0 to 6, namely 0, 1, 2, 3, 4, 5, or 6
  • R a , R b R c are the same or different from each other.
  • R b and R c are -[(CR 1 R 2 ) m O] flesh-H and R a is -[(CR 1 R 2 ) m O] sanction-H with HAS * being the remainder of the hydroxyalkyl starch molecule, or R a is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch molecule, with n being 0 to 6, namely 0, 1, 2, 3, 4, 5 or 6, wherein in each group R a , R b R c , and n are the same or different from each other.
  • HAS the reducing end of the starch molecule is shown in the non-oxidized form and the terminal saccharide unit of HAS is shown in the hemiacetal form which depending on e.g. the solvent, may be in equilibrium with the (free) aldehyde form.
  • HAS' as used in the context of the present invention 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 herein.
  • hydroxyalkyl starch within the meaning of the present invention is not limited to compounds where the terminal carbohydrate moiety comprises groups R a , R b and/or R c being -[(CR 1 R 2 ) m O] n -H and/or HAS" as depicted, for the sake of brevity, in formula (la), but refers to compounds in which at least one hydroxy group which is present anywhere else in the hydroxyalkyl starch, i.e. either in the terminal saccharide unit of the hydroxyalkyl starch molecule and/or in the remainder of the hydroxyalkyl starch molecule, HAS', is substituted by a group -[(CR 1 R 2 ) m O] justify-H .
  • the integer m in each group -[(CR 1 R 2 ) m O] n -H present in the HAS molecule may be the same or may be different. The same applies to integer n.
  • the HAS may further contain one or more hydroxyalkyl groups, which comprise more than one hydroxyl group, in particular two or more hydroxyl groups. According to a preferred embodiment, the hydroxyalkyl groups comprised in HAS contain one hydroxy group only.
  • hydroxyalkyl starch according to above-mentioned formula (la) 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.
  • 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.
  • substitution pattern of HAS preferably HES
  • MS molar substitution
  • 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 solution, and preferably a 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 and their molecular weight.
  • the mass distribution in HAS, and in particular HES may be described by the weight average molecular weight M w (or Mw).
  • the parameter M n The number average molecular weight is defined by the following equation:
  • M n ⁇ i 3 ⁇ 4Mi / ⁇ i 3 ⁇ 4 wherein 3 ⁇ 4 is the number of hydroxyalkyl starch molecules of species i having molar mass Mi.
  • the weight average molecular weight is defined by the following equation:
  • M w ⁇ i 3 ⁇ 4Mi 2 / ⁇ i 3 ⁇ 4Mi wherein 3 ⁇ 4 is the number of hydroxyalkyl starch molecules of species i having molar mass Mi.
  • typical M w values are preferably in the range of from 1 to 1000 kDa, more preferably of from 1 to 800 kDa, more preferably of from 1 to 700 kDa, more preferably of from 2 to 600 kDa, more preferably of from 5 to 500 kDa, most preferably of from 25 to 400 kDa.
  • MS molecular substitution
  • the parameter MS can be determined according to Ying-Che Lee et al., Anal. Chem. 55,
  • the third parameter which is referred to as "C2/C6 ratio" describes the ratio of the number of the anhydroglucose units being substituted in C2 position relative to the number of the anhydroglucose units being substituted in C6 position.
  • the C2/C6 ratio can be influenced via the pH used for the hydroxyalkylation reaction. Generally, the higher the pH, the more hydroxyl groups in C6 position are hydroxyalkylated.
  • the parameter C2/C6 ratio can be determined, for example, according to Sommermeyer et al., Rohpharmazie 8 (8), 1987, pp 271-278, in particular page 273.
  • typical values of the C2/C6 ratio are in the range of from 2 to 20, preferably of from 2 to 15, more preferably of from 2 to 12.
  • the compound according to formula (II) is selectively reacted via carbon atom C* of the reducing end, i.e. with the reducing end of HAS.
  • the term "selectively reacted with the reducing end” relates to processes according to which preferably a least 95 %, more preferably at least 98 %, more preferably at least 99 %, more preferably at least 99.5 %, more preferably at least 99.9 % of all reacted HAS molecules are exclusively reacted via their reducing end group.
  • HAS is reacted via its non-oxidized reducing end.
  • step (i) of the method according to the invention the HAS according to formula (la) is reacted via carbon atom C* of the reducing end of the HAS with the functional group M of a crosslinking compound according to formula M-Ll-S-T (II), wherein a HAS derivative according to formula (lb) is obtained, wherein -CH 2 -F1- is the moiety resulting from the reaction of the group M with the HAS via the carbon atom C* of the reducing end, and wherein Fl is a functional group comprising the group -NR'-.
  • M is a functional group comprising the moiety -NHR', with R' being H or alkyl.
  • R' is selected from the group consisting of H, methyl, ethyl and propyl.
  • the present invention also relates to a method for the preparation of a hydroxyalkyl starch derivative, as described above, wherein in step (i), the hydroxyalkyl starch (HAS) of formula (la) is reacted via carbon atom C* of the reducing end of the HAS with the functional group M of a crosslinking compound according to formula (II)
  • HAS hydroxyalkyl starch
  • the present invention relates to a hydroxyalkyl starch (HAS) derivative, as described above, or a HAS derivative obtained or obtainable by the method as described above, wherein Fl is -NH-, and wherein the derivative has a structure according to the following formula:
  • LI is a spacer bridging M and S or bridging Fl and S, respectively.
  • the HAS derivatives described above are prepared by a method comprising the step (i), as described above, thus by reacting the reducing end of HAS with the crosslinking compound according to formula (II), LI is first linking M and S in the crosslinking compound, and, subsequently, after reaction of the crosslinking compound of formula (II) with the reducing end, whereupon Fl is formed, LI is linking the thus obtained functional group Fl and S.
  • LI comprises, more preferably consists of, 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 as or may be different from the at least one other substituent. There are in general no limitations as to the substituent.
  • Suitable substituents in the context of spacer LI are, for example, selected from the group consisting of alkyl, aryl, alkenyl, alkynyl, fluorine, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, nitro, alkylthio, arylthio, amide, sulfate, alkylsulfmyl, sulfonate, sulfonamido, trifluoromethyl, cyano, azido, carboxymethylcarbamoy
  • cycloalkyl such as e.g. cyclopentyl or cyclohexyl
  • heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl.
  • substituents of such organic residues are, for example, alkyl groups, amide groups, hydroxyl 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, methylenedioxyphenyl, napthyridinyl, quinolinyl, isoquinolinyl, indolyl, benzofuranyl, purinyl, benzofuranyl, deazapurinyl, or indolizinyl.
  • the spacer LI comprises the moiety -(C(L'L")) q - with L' and L" in each repeating unit -C(L'L")- being, independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, hydroxyl, fluorine, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, amide, carboxyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, nitro, alkylthio, arylthio, sulfate, alkylsulfinyl,
  • cycloalkyl such as e.g. cyclopentyl or cyclohexyl
  • heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl
  • q preferably being in the range of from 1 to 20, more preferably in the range of from 1 to 10, more preferably in the range of from 2 to 6, more preferably, 2, 3 or 4.
  • L' and L" are, independently of each other, selected from the group consisting of H, alkyl groups (including substituted alkyl groups, in particular including hydroxyalkyl groups), amide groups, hydroxyl groups, and carboxyl groups, wherein the groups L' and L" in each repeating unit may be the same or may differ from each other.
  • L' and L" are, independently of each other, selected from the group consisting of H, amide, carboxyl and alkyl (including substituted alkyl groups, in particular including hydroxyalkyl groups), more preferably L' and L" are H or alkyl, such as H or methyl, wherein the groups L' and L" in each repeating unit may be the same or may differ from each other.
  • LI has a structure selected from the group consisting of
  • L' , L", L* and L** are independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, hydroxyl, fluorine, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, amide, carboxyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, nitro, alkylthio, arylthio, sulfate, alkylsulfmyl, sulfonate, sulfonamid
  • cycloalkyl such as e.g. cyclopentyl or cyclohexyl
  • heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl
  • R is alkyl, preferably methyl, and wherein the groups L' , L", L* and L**, in each repeating unit, may be the same or may differ from each other.
  • LI may have one or more asymmetric centers.
  • the linker may be employed as mixtures of enantiomers and as individual enantiomers, as well as diastereomers and mixtures of diastereomers. All possible stereoisomers, single isomers and mixtures of isomers are included within the scope of the present invention.
  • LI comprises one or more asymmetric centers, LI is preferably employed in enantiomeric or diastereomeric pure form.
  • the repeating units -(C(L'L"))- may be the same or may be different from each other.
  • r is > 1
  • the repeating units -(C(L*L**))- may be the same or may be different from each other.
  • s is > 1, the repeating units -Yi- [C(L*L**)] r - may be the same or may be different from each other.
  • LI has the structure wherein L' and L" are, independently of each other, selected from H and alkyl, wherein in each repeating unit -(C(L'L"))-, in case q is > 1, L' and L" may be the same or may be different from each other.
  • 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 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -
  • L' and L' ' are in each repeating unit H.
  • LI is -CH 2 -CH 2 -
  • LI has as structure according to the following formula with q preferably being in the range of from 1 to 20, with r preferably being in the range of from 1 to 10, with s preferably being in the range of from 1 to 10 and with L', L", L* and L** being as described above.
  • s is 1.
  • q is in the range of from 2 to 4.
  • r is in particular in the range of from 2 to 4.
  • s is > 1, preferably of from 2 to 10.
  • this embodiment includes any spacer LI derived from a peptide, thus having a peptidic backbone.
  • LI is derived from a peptidic crosslinking compound (II)
  • the crosslinking compound (II) is preferably employed in enantiomeric or diastereomeric pure form, more preferably in the natural occurring stereoisomeric form.
  • HAS is preferably dissolved in an aqueous medium or a reaction buffer, and a crosslinking compound according to formula (II) is subsequently added.
  • the reaction in (i) is carried out in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, formamide, water, acetic acid reaction buffers and mixtures of two or more thereof.
  • Preferred reaction buffers are, e.g., sodium citrate buffer, sodium acetate buffer, sodium phosphate buffer, sodium carbonate buffer, or sodium borate buffer.
  • Preferred pH values of the reaction buffers are in the range of from 4 to 9, more preferably of from 5 to 8. The pH values given hereinunder and above refer to pH values determined via a pH sensitive glass electrode.
  • the reaction is carried out in water.
  • the crosslinking compound of formula (II) is preferably used as free amine or as a salt and added as solid.
  • Preferred are salts such as the hydrochloride, sulfate, hemisulfate, acetate or trifluoroacetate salt.
  • step (i) preferably an excess of the crosslinking compound of formula (II) is employed.
  • the minimum amount of crosslinking compound (II) is one molar equivalent with respect to the amount of reducing ends to be reacted.
  • the maximum amount is given by the solubility limit of the crosslinking compound in the particular reaction solvents.
  • the crosslinking compound is employed at a concentration in the range of at least 0.05 mol/1, preferably at least 0.1 mol/1, more preferably in the range of from 0.2 to 4 mol/1, more preferably from 0.5 to 2 mol/1, more preferably 0.9 mol/1 to 1.1 mol/1, most preferably about 1 mol/1..
  • the reaction mixture is preferably stirred at a temperature in the range of from 5 °C to 100 °C, more preferably at a temperature in the range of from 20 °C to 90 °C, more preferably in the range of from 40 °C to 80°C.
  • the temperature may be varied, preferably in the above-given ranges, or held essentially constant.
  • the reaction is preferably conducted for a time in the range of from 1 to 48 h, more preferably from 2 to 36 h, more preferably from 4 to 18 h.
  • M consists of an NH 2 - group
  • the ultrafiltration or dialysis is preferably carried out under neutral conditions, preferably in water.
  • the hydroxyalkyl starch derivative may be precipitated from the reaction mixture, in particular by adding an alcohol, preferably 2- propanol.
  • the obtained precipitate may be collected by filtration or centrifugation and may further be purified using conventional purification protocols, preferably ultrafiltration, dialysis or chromatographic methods, preferably size exclusion chromatography.
  • step (i) preferably additionally comprises the conversion of the, optionally isolated, hydroxyalkyl starch derivative obtained upon reaction of HAS with the crosslinking compound according to formula (II) prior to step (ii).
  • the functional group Fl * obtained upon reaction of M with the reducing end is suitably reduced, to give the functional group Fl .
  • said group is reduced to give the group Fl with Fl being -CH 2 -NH-.
  • said group is reduced to give the group Fl with Fl being -CH 2 -NH-NH 2 -.
  • the reduction is preferably carried out 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 sodium cyanoborohydride.
  • 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-ethyl
  • this reduction is carried out using an excess of reducing agent, so preferably a minimum of one molar equivalent with respect to the amount of reducing ends in HAS is applied.
  • concentration of the reducing agent used for this reaction of the present invention is in the range of from 0.001 to 3.0 mol/1, more preferably in the range of from 0.05 to 2.0 mol/1, more preferably in the range of from 0.1 to 1 mol/1, more preferably in the range of 0.3-0.6 mol/L, relating, in each case, to the volume of the reaction solution.
  • the reduction as described above, can either be carried out subsequently to the coupling process, in which M 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. Most preferably the above mentioned one pot synthesis is carried out. 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 M comprises the group HR'N-, preferably H 2 N-, more preferably consist of the group H 2 N- and the reaction according to step (i) is a reductive amination. Further, the present invention also relates to a method as described above, and a conjugate obtained or obtainable by said method, wherein the functional group M is H 2 N-NH- and the reaction according to step (i) is a reductive amination.
  • the solvent used for the reduction step is preferably also selecetd from the solvents already mentioned above in the context of step (i).
  • the reductive amination is carried out in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, formamide, water, acetic acid reaction buffers and mixtures of two or more thereof.
  • Preferred pH values of are thus in the range of from 4 to 9, more preferably of from 5 to 8.
  • the temperature of the reaction mixture is suitably chosen. Generally, during the reductive amination reaction, the temperature of the reaction mixture is in the range of from 5 to 100 °C such as from 20 to 90 °C or from 40 to 80 °C. Preferably, during the reductive amination reaction, the temperature of the reaction mixture is in the range of from 45 to 75 °C, more preferably from 55 to 65 °C.
  • the reductive amination reaction can be carried out for any suitable time period. Generally, the time period is in the range of from 1 to 48 h such as from 2 to 36 h.
  • the time period is in the range of from 3 to 24 h, more preferably from 6 to 21 h, more preferably from 4 to 18 h.
  • to reductive amination is carried out at a temperature of the reaction mixture in the range of from 40 to 90 °C for a time period of from 1 to 36 h, more preferably at a temperature in the range of from 45 to 80 °C for a time period of from 2 h to 24 h, more preferably at a temperature in the range of from 55 to 65 °C for a time period of from 4 to 18 h.
  • the present invention also relates to a method, as described above, and a HAS derivative obtained or obtainable by said method, wherein the reacting according to step (i) is carried out under reductive amination conditions, preferably at a temperature in the range of from 5 °C to 100 °C and in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, formamide, water, acetic acid, and reaction buffers and mixtures of two or more thereof.
  • a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, formamide, water, acetic acid, and reaction buffers and mixtures of two or more thereof.
  • the concentration of HAS, preferably HES, in the aqueous system is preferably in the range of at least 1 weight-%, more preferably at least 10 weight-% understand more preferably in the range of from 20-40 weight-%nch most preferably around 30 weight-%, based on the total weight of the whole solution.
  • the reaction mixture obtained is preferably subjected to a suitable work up.
  • Such working up may comprise one or more stages wherein preferably at least one stage comprises a purification, preferably a purification by ultrafiltration, precipitation, size exclusion chromatography, and a combination of two or more of these methods, more preferably by ultrafiltration.
  • such working up may comprise at least one stage which comprises a pH adjustment, preferably an adjustment to a pH of at least 8, preferably at least 9, more preferably in the range of from 9 to 11. Adjusting the pH of the reaction mixture to a value of at least 8, preferably at least 9, more preferably from 9 to 11 can be realized, if carried out, according to all conceivable methods.
  • an inorganic base preferably an alkali metal base and/or an alkaline earth metal base, more preferably an alkali metal hydroxide and/or an alkaline earth metal hydroxide, more preferably an alkaline metal hydroxide, more preferably sodium hydroxide is added in a suitable amount.
  • the addition of such a basic compound can be performed at the temperature of the reaction mixture of the reductive amination reaction.
  • the reaction mixture obtained from the reductive amination reaction is cooled before the basic compound is added, preferably to a temperature in the range of from 10 to 35 °C, more preferably from 20 to 30 °C.
  • the mixture can be suitably stirred.
  • the pH is to be understood as the value indicated by a pH sensitive glass electrode without correction.
  • the preferably applied ultrafiltration can be performed according to all suitable methods.
  • the ultrafiltration comprising at least one volume exchange with water, preferably at least five volume exchanges with water, more preferably at least 10 volume exchanges with water.
  • the ultrafiltration does not comprise a volume exchange with an acid.
  • the ultrafiltration does not comprise a volume exchange with a base. More preferably, the ultrafiltration does not comprise a volume exchange with an acid and does not comprise a volume exchange with a base.
  • the purified mixture can be subjected directly, without any further intermediate stage, to step (ii) or optionally to further reducing condition as described hereinunder.
  • step (i) subsequent to the reductive amination conditions, and the optional work-up described above, the HAS derivative is subjected to further reducing conditions.
  • complex hydrides such as borohydrides, especially sodium borohydride, and thiols, especially dithiothreitol (DTT) and dithioerythritol (DTE) or phosphines such as tris-(2-carboxyethyl)phosphine (TCEP) are mentioned.
  • the reduction is preferably carried out using borohydrides, especially sodium borohydride.
  • the reducing agent is used in an excess, more preferably at a concentration in the range of from 0.02 to 1.5 M, more preferably in the range of from 0.05 to 1 M, most preferably in the range of from 0.1 to 0.5 M with respect to the total volume of the reaction solution.
  • this deprotection step is preferably carried out at a temperature in the range of from 0 to 80 °C, more preferably in the range of from 10 to 50 °C and especially preferably in the range of from 15 to 35°C.
  • the temperature may be varied, preferably in the above-given ranges, or held essentially constant.
  • aqueous medium refers to a solvent or a mixture of solvents comprising water in an amount of at least 10 % per weight, preferably at least 20 % per weight, more preferably at least 30 % per weight, more preferably at least 40 % per weight, more preferably at least 50 % per weight, more preferably at least 60 % per weight, more preferably at least 70 % per weight, more preferably at least 80 % per weight, even more preferably at least 90 % per weight or up to 100 % per weight, based on the weight of the solvents involved.
  • the aqueous medium may comprise additional solvents like formamide, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide (DMA), N- methylpyrrolidinone (NMP), alcohols such as methanol, ethanol or isopropanol, acetonitrile, tetrahydrofurane or dioxane.
  • the aqueous solution may also contain a transition metal chelator (disodium ethylenediaminetetraacetate, EDTA, or the like) in a concentration ranging from 0.01 to 100 mM most preferably from 0.1 to 10 mM, such as about 1 mM.
  • the preferred solvent is water.
  • the HAS derivative is reacted in the further reduction step preferably at a concentration in the range of from 1% to 30% weight.-%, more preferably in the range of from 5% to 20% weight.-%o, most preferably 10%> weight.-%> based on the total weight of the solution.
  • sodium borohydride (NaBH 4 ) is employed as reducing agent.
  • the mixture obtained from adding the sodium borohydride comprises the sodium borohydride preferably at a concentration in the range of from 0.05 to 1.5 mol/1, more preferably from 0.05 to 1 mol/1, more preferably from 0.1 to 0.5 mol/1.
  • the mixture contains the hydroxyalkyl starch and the hydroxyalkyl starch derivative at a concentration in the range of from 1 to 40 weight-%, more preferably from 5 to 30 weight-%, more preferably from 10 to 20 weight-%. Subjecting the mixture to these further reducing conditions in (i) can be carried out for any suitable time period.
  • the time period is in the range of from 10 min to 24 h.
  • the time period is in the range of from 0.25 to 4 h, more preferably from 1 to 3 h.
  • the further reduction reaction using sodium boohhydrate can be carried out at every suitable temperature.
  • the reduction with sodium borohydrate is carried out a temperature in the range of from 5 to 40 °C, more preferably from 10 to 35 °C, more preferably from 20 to 30 °C such as at room temperature.
  • subjecting the mixture to the further reducing conditions comprises keeping the mixture at a temperature in the range of from 10 to 35 °C for a period of from 0.25 to 4 h, more preferably at a temperature in the range of from 20 to 30 °C for a period of from 1 to 3 h.
  • this HAS derivative may then preferably be isolated from the reaction mixture by any suitable method, such as ultrafiltration or dialysis, preferably ultrafiltration, more preferably followed by lyophilization of the isolated hydroxyalkyl starch derivative.
  • the ultrafiltration or dialysis is preferably carried out under acidic conditions, more preferably at a pH in the range of from 2 to 6, more preferably in the range of from 3 to 5, and/or in the presence of an ion chelator.
  • the acid if present, is selected from the group consisting of hydrochloric acid, phosphoric acid, trifluoroacetic acid, acetic acid and mixtures of two or more thereof; preferred buffers are selected from the group consisting of acetate, phosphate and citrate buffers.
  • ion chelators EDTA ethylenediamine tetraacetic acid
  • DTPA diethylene triamine pentaacetic acid
  • an acetic acid buffer (in the range of from 0.1 mM to 1 M, more preferably in the range of from 1 to 100 mM, most preferably 10 mM) with pH 4 which more preferably comprises EDTA in the range of from 0.01 to 100 mM (most preferably 1 mM) is used as ultrafiltration / dialysis buffer. More preferably, after removal of the reaction impurities, the ultrafiltration / dialysis buffer is replaced by water in order to remove buffer salts from the product.
  • hydroxyalkyl starch derivative according to formula (lb) may be precipitated from the reaction mixture, in particular by adding an alcohol, preferably 2-propanol.
  • the obtained precipitate may be collected by filtration or centrifugation and further purified using conventional purification protocols, preferably ultrafiltration, dialysis or chromatographic methods, preferably size exclusion chromatography.
  • T is H or a thiol protecting group PG.
  • the present invention also relates to a method as described above, and a conjugate obtained or obtainable by said method, comprising
  • T is H.
  • the present invention also relates to a method as described above, and a conjugate obtained or obtainable by said method, comprising (i) reacting hydroxyalkyl starch (HAS) of formula (la)
  • H 2 N- CH 2 -CH 2 -SH H 2 N-CH 2 -CH 2 -CH 2 -SH, H 2 N-CH 2 -CH 2 -CH 2 -CH 2 -SH, H 2 N-CH 2 -CH 2 -CH 2 - CH 2 -CH 2 -SH, H 2 N-CH(COOH)-CH 2 -SH, H 2 N-CH(COOH)-C(CH 3 ) 2 -SH, H 2 N- CH(CH 2 OH)-CH 2 -SH H 2 N-CH(CH 2 OH)-CH 2 -CH 2 -SH H 2 N-CH(CONH 2 )-C(CH 3 ) 2 -SH, H 2 N-CH(CONH 2 )-CH 2 -SH, H 2 N-CH(COOH)-CH 2 -CH 2 -SH, H 2 N-CH 2 -CH 2 -O-CH 2 -CH 2 - SH,
  • the crosslinking compound according to formula (II) is selected from the group consisting of H 2 N-CH 2 -CH 2 -SH, H 2 N-CH 2 -CH 2 -CH 2 -SH, H 2 N-CH 2 -CH 2 -CH 2 -SH, H 2 N-CH 2 -CH 2 -CH 2 -CH 2 -SH, H 2 N-CH(COOH)-CH 2 -SH and H 2 N-CH(COOH)- C(CH 3 ) 2 -SH, H 2 N-CH(CH 2 OH)-CH 2 -SH H 2 N-CH(CH 2 OH)-CH 2 -CH 2 -SH H 2 N- CH(CONH 2 )-C(CH 3 ) 2 -SH, H 2 N-CH(CONH 2 )-CH 2 -SH H 2 N-CH(COOH)-CH 2 -CH 2 -SH, H 2 N-CH 2 -CH 2 -0-CH 2
  • T is PG, wherein PG may be any suitable SH protecting group known to those skilled in the art.
  • PG is a protecting group forming together with -S- a thioether (e.g. benzyl, allyl, triarylmethyl groups, such as trityl (Trt)), a disulfide (e.g. S-sulfonates, S-tert-butyl, S-(2-aminoethyl), 2- pyridylthio).
  • step (i) further comprises a deprotection step.
  • the protecting group may be removed in situ in step (ii) and the S-H group formed in situ may then directly be reacted with the compound (III).
  • the protecting group PG is removed prior to step (i).
  • the crosslinking compound according to formula (II) M-L1-S-PG is preferably a symmetrical disulfide, with PG having the structure -S-Ll-M or is selected from the group consisting of 2-pyridylthio, -S-S0 3 " , -S-S0 2 -aryl and -S-S0 2 - alkyl.
  • the group S-PG present in the crosslinking compound according to formula (II) is selected from the group consisting of -S-Trt, -S-S-Ll-M, -S-S-tBu, -S-S-(2-pyridyl), -S-S0 3 ⁇ , -S-S0 2 -aryl and -S-S0 2 -alkyl, in particular the group -S-PG is -S-S-Ll-M.
  • the crosslinking compound according to formula (II) is selected from the group consisting of H 2 N-L1 -S-Trt, H 2 N-L1-S-S-L1-NH 2 , H 2 N-L1-S-S- tBu, H 2 N-Ll-S-S-(2-pyridyl), H 2 N-L1-S-S0 3 ⁇ , H 2 N-Ll-S-S0 2 -aryl and H 2 N-L1-S-S0 2 - alkyl , most preferably the crosslinking compound according to formula (II) is H 2 N-L1-S-S-
  • H 2 N-CH 2 -CH 2 -S-Trt H 2 N-CH 2 -CH 2 -CH 2 -S-Trt, H 2 N-CH 2 -CH 2 -CH 2 -CH 2 -S- Trt, H 2 N-CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -S-Trt, H 2 N-CH 2 -CH 2 -S-S-CH 2 -CH 2 -NH 2 , H 2 N-CH 2 - CH 2 -S-S-tBu, H 2 N-CH 2 -CH 2 -CH 2 -S-S-tBu, H 2 N-CH 2 -CH 2 -CH 2 -CH 2 -S-S-tBu, H 2 N- CH(COOH)-CH 2 -S-Trt, H 2 N-CH(COOH)-C(CH 3 ) 2 -S-Trt, H 2
  • the crosslinking compound (II) is cystamine H 2 N-CH 2 - CH 2 -S-S-CH 2 -CH 2 -NH 2 or cystine H 2 N-CH(COOH)-CH 2 -S-S-CH 2 -CH(COOH)-NH 2 , more preferably cystamine.
  • the reaction conditions used in the deprotection step are adapted to the respective protecting group used.
  • the group -S-PG is a disulfide, as described above.
  • the deprotection step comprises the reduction of this disulfide bond to give the respective thiol group.
  • This deprotection step is preferably carried out using specific reducing agents.
  • reducing agents complex hydrides such as borohydrides, especially sodium borohydride, and thiols, especially dithiothreitol (DTT) and dithioerythritol (DTE) or phosphines such as tris-(2-carboxyethyl)phosphine (TCEP) are mentioned.
  • the reduction is preferably carried out using borohydrides, especially sodium borohydride.
  • the reducing agent is used in an excess, more preferably at a concentration in the range of from 0.02 to 1.5 M, more preferably in the range of from 0.05 to 1 M, most preferably in the range of from 0.1 to 0.5 M with respect to the total volume of the reaction solution.
  • this deprotection step is preferably carried out at a temperature in the range of from 0 to 80 °C, more preferably in the range of from 10 to 50 °C and especially preferably in the range of from 15 to 35°C.
  • the temperature may be varied, preferably in the above-given ranges, or held essentially constant.
  • aqueous medium refers to a solvent or a mixture of solvents comprising water in an amount of at least 10 % per weight, preferably at least 20 % per weight, more preferably at least 30 % per weight, more preferably at least 40 % per weight, more preferably at least 50 % per weight, more preferably at least 60 % per weight, more preferably at least 70 % per weight, more preferably at least 80 % per weight, even more preferably at least 90 % per weight or up to 100 % per weight, based on the weight of the solvents involved.
  • the aqueous medium may comprise additional solvents like formamide, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide (DMA), N- methylpyrrolidinone (NMP), alcohols such as methanol, ethanol or isopropanol, acetonitrile, tetrahydrofurane or dioxane.
  • the aqueous solution may also contain a transition metal chelator (disodium ethylenediaminetetraacetate, EDTA, or the like) in a concentration ranging from 0.01 to 100 mM most preferably from 0.1 to 10 mM, such as about 1 mM.
  • the preferred solvent is water.
  • the HAS derivative is reacted in the reduction step at a concentration in the range of from 1% to 30% weight.-%, more preferably in the range of from 5% to 20% weight.-%>, most preferably 10%> weight. -% based on the total weight of the solution.
  • the retentate of the ultrafiltration step is directly used for the reduction with NaBH 4 .
  • the present invention also relates to a method, as described above, and a HAS derivative obtained or obtainable by said method, wherein the removing of the protecting group PG in step (i) is carried out at a temperature in the range of from 0 to 80 °C and in an aqueous solvent system, the group S-PG being a disulfide.
  • the pH value in this deprotection step may be adapted to the specific needs of the reactants for example by using aqueous buffer solutions.
  • aqueous buffer solutions include carbonate, phosphate, borate and acetate buffers as well as tris(hydroxymethyl)aminomethane (TRIS) may be mentioned.
  • the reaction is carried out in water at a pH value in the range of 7 to 14.
  • the deprotection step is preferably conducted for a time in the range of from from 0.25 to 24 h, more preferably of from 0.5 to 18 h; most preferably of from 0.5 to 4 h.
  • HAS derivatives Hthus derivatives obtained upon reacting HAS with compound (II), in which T is H, or HAS derivatives obtained after removal of the protecting group PG, in particular after deprotection under non-reductive conditions.
  • step (i) may be carried out.
  • the HAS derivative obtained in step (i) may e.g. be isolated from the reaction mixture by any suitable method, such as ultrafiltration or dialysis, preferably ultrafiltration followed by lyophilization of the isolated hydroxyalkyl starch derivative.
  • this ultrafiltration or dialysis is preferably performed under acidic conditions, preferably at a pH in the range of from 2 to 6, more preferably in the range of from 3 to 5, and/or in the presence of an ion chelator.
  • an acid selected from the group comprising hydrochloric acid, phosphoric acid, trifluoroacetic acid and acetic acid is employed.
  • Preferred buffers are selected from the group comprising acetate, phosphate and citrate buffers.
  • suitable ion chelators EDTA (ethylenediamine tetraacetic acid), DTPA (diethylene triamine pentaacetic acid) and related compounds may be mentioned.
  • an acetic acid buffer (preferably in the range of from 0.1 mM to 1 M, more preferably in the range of from 1 to 100 mM, most preferably 10 mM) with pH 4 preferably comprising EDTA in the range of from 0.01 to 100 mM (most preferably 1 - 10 mM) is used as ultrafiltration or dialysis buffer. After removal of the reaction impurities, the ultrafiltration / dialysis buffer is replaced by water in order to remove buffer salts from the product.
  • the hydroxyalkyl starch derivative may be precipitated from the reaction mixture, in particular by adding an alcohol, preferably 2-propanol.
  • the obtained precipitate may be collected by filtration or centrifugation and further purified using conventional purification protocols, preferably ultrafiltration, dialysis or chromatographic methods, preferably size exclusion chromatography.
  • the obtained derivative is further lyophilized prior to step (ii) until the solvent content of the reaction product is sufficiently low according to the desired specifications of the derivative.
  • step (ii) of the invention the HAS derivative of formula (lb) obtained in step (i) is reacted with a crosslinking compound of formula (III)
  • X is a leaving group
  • L2 is a spacer bridging -NR X - and -NR y -, wherein R x and R y are, independently of each other, H or alkyl, and optionally form together with the moiety N-L2-N a ring.
  • leaving group is denoted to mean a molecular fragment that departs with a pair of electrons in hetero lytic bond cleavage upon reaction with a thiol group.
  • Examples are, inter alia, halogens or sulfonic esters.
  • Examples for sulfonic esters are, inter alia, the mesyl and tosyl group.
  • X is selected from the group consisting of tosyl (Ts), mesyl (Ms), CI, Br and I. More preferably X is Br or I, most preferably I.
  • L2 is a spacer bridging -NR X - and -NR y
  • R x and R y are, independently of each other, selected from the group consisting of H, methyl, ethyl, propyl or butyl or are forming a ring according to formula (V)
  • R x and R y are forming together with the moiety N-L2-N a ring, the ring preferably being a 5-membered to 7-membered ring. More preferably, in this case, R x and R y , taken together, are -(CH 2 ) 2 - or -(CH 2 )3-.
  • L2 is preferably an alkyl group, preferably an optionally substituted methylene, ethylene, or propylene group, more preferably -CH 2 -, -(CH 2 ) 2 - or -(CH 2 ) 3 -.
  • the moiety -NR x -L2-NR y - preferably has one of the following structures:
  • L2 is -(CH 2 ) 2 - and R x and R y , taken together, are -(CH 2 ) 2 -.
  • R x and R y taken together, are -(CH 2 ) 2 -.
  • R x and R y are, independently of each other, H or alkyl, preferably selected from the group consisting of H, methyl, ethyl, propyl or butyl, more preferably both are H, and R x and R y are not forming a ring together with the moiety N-L2-N.
  • L2 comprises, more preferably consists of, an alkyl, alkenyl, alkylaryl, arylalkyl, aryl or heteroaryl group.
  • 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 as or may be different from the at least one other substituent.
  • 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 as or may be different from the at least one other substituent.
  • alkyl alkenyl
  • alkylaryl arylalkyl
  • aryl aryl
  • heteroaryl include the respective suitable substituted groups.
  • Suitable substituents in the context of spacer L2 are, for example, selected from the group consisting of alkyl, aryl, alkenyl, alkynyl, fluorine, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, nitro, alkylthio, arylthio, amide, sulfate, alkylsulfmyl, sulfonate, sulfonamido, trifluoromethyl, cyano, azido, carboxymethylcarbamo
  • cyclopentyl or cyclohexyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl.
  • Preferred substituents of such organic residues are, for example, alkyl groups (including substituted alkyl groups, in particular including hydroxyalkyl groups), fluorine, hydroxyl groups, keto groups, aldehyde groups and carboxyl groups.
  • the spacer L2 comprises the moiety -(C(L t L tt )) t - with L l and L tt in each repeating unit -C(L l L tt )- being, independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, fluorine, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, amide, carboxyl, keto groups, aldehyde groups, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbonylamino, arylcarbony
  • cyclopentyl or cyclohexyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl, wherein the groups L l and L tt in each repeating unit may be the same or may differ from each other, with t preferably being in the range of from 1 to 20, more preferably in the range of from 1 to 10, more preferably in the range of from 2 to 6, more preferably, 2, 3 or 4.
  • L l and L tt are, independently of each other, selected from the group consisting of H, alkyl groups (including substituted alkyl groups, in particular including hydroxyalkyl groups), fluorine, tertiary amino groups, hydroxyl groups, keto groups, aldehyde groups and carboxyl groups, wherein the groups L l and L tt in each repeating unit may be the same or may differ from each other.
  • L l and L tt are, independently of each other, selected from the group consisting of H, carboxyl and alkyl, more preferably L l and L tt are H or alkyl, wherein the groups L l and L tt in each repeating unit may be the same or may differ from each other.
  • L2 has a structure selected from the group consisting of
  • L l , L tl , L u , and L uu are independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, fluorine, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxyl, keto groups, aldehyde groups, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, amidino, nitro, imin
  • cyclopentyl or cyclohexyl, heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl
  • R Y2 is alkyl, preferably methyl, and wherein the groups L l , L tt , L u , and L uu in each repeating unit, may be the same or may differ from each other. More preferably Y 2 , if present, is -0-.
  • L l , L tt , L u , and L uu are, independently of each other, selected from the group consisting of H, alkyl groups (including substituted alkyl groups, in particular including hydroxyalkyl groups), hydroxyl groups, keto groups, aldehyde groups and carboxyl groups, more preferably selected from the group consisting of H, carboxyl and alkyl, in particular L l , L tt , L u , and L uu are, independently of each other, H or alkyl, and wherein Y 2 is a functional moiety as described above, preferably wherein Y 2 is -0-.
  • the repeating units -C(L l L tt )- may be the same or may be different from each other.
  • u is > 1
  • the repeating units -C(L U L UU )- may be the same or may be different from each other.
  • v is > 1, the repeating units -Y 2 -[C(L U L UU )] U - may be the same or may be different from each other.
  • L2 is a symmetrical spacer.
  • the following groups L2 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 2
  • L l and L tl are, independently of each other, selected from H and alkyl. More preferably L l and L tt in each repeating unit are H. According to a particularly preferred embodiment of the invention, L2 is -CH 2 -CH 2 -.
  • L2 has the structure whererin L l , L tt , L u , and L uu are, independently of each other, selected from the group consisting of H, alkyl groups (including substituted alkyl groups, in particular including hydroxyalkyl groups), hydroxyl groups, keto groups, aldehyde groups and carboxyl groups, more preferably selected from the group consisting of H, carboxyl and alkyl, in particular L l , L tt , L u , and L uu are, independently of each other, H or alkyl.
  • the repeating units -(C(L t L tt ))- may be the same or different from each other.
  • u is > 1
  • the repeating units -(C(L U L UU ))- may be the same or different from each other.
  • the following groups L2 are mentioned: -CH 2 -C ⁇ C-CH 2 -, -CH 2 -CH 2 -C ⁇ C-CH 2 -CH 2 - and -CH 2 -CH 2 -CH 2 -C ⁇ C- CH 2 -CH 2 -CH 2 -, in particular -CH 2 -C ⁇ C-CH 2 -.
  • L2 has as structure according to the following formula with t preferably being in the range of from 1 to 20, with u preferably being in the range of from 1 to 10, with v preferably being in the range of from 1 to 10 and with L l , L tt , L u and L uu being as described above.
  • v is preferably of from 1 to 3.
  • Y 2 is -O- or -N(CH 3 )- and u is of from 1 to 3.
  • Preferred crosslinking compounds according to this embodiment are, for example, -CH 2 - CH 2 -0-CH 2 -CH 2 -, -CH 2 -CH 2 -0-CH 2 -CH 2 -0-CH 2 -CH 2 -, -CH 2 -CH 2 -0-CH 2 -CH 2 -CH 2 -0-CH 2 - CH 2 -0-CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -0-CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -0- CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -0- CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH
  • L2 has one of the following structures: wherein L l , L tt , L u , and L uu are, independently of each other, selected from the group consisting of H, alkyl groups (including substituted alkyl groups, in particular including hydroxyalkyl groups), hydroxyl groups, keto groups, aldehyde groups and carboxyl groups, more preferably selected from the group consisting of H, carboxyl and alkyl, in particular L l , L tt , L u , and L uu are, independently of each other, H or alkyl, in particular H.
  • the repeating units -(C(L t L tt ))- may be the same or different from each other.
  • the repeating units -(C(L U L UU ))- may be the same or different from each other.
  • t and u are, independently of each other 1 or 2, preferably both, t and u, are 1 or both, t and u, are 2.
  • Step (ii) according to the invention is preferably carried out in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, formamide, water, reaction buffers and mixtures of two or more thereof.
  • a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, formamide, water, reaction buffers and mixtures of two or more thereof.
  • Especially preferred solvents are DMSO and DMF.
  • the crosslinking compound of formula (III) is preferably dissolved in the reaction solvent prior to addition of the HAS derivative. Alternatively, the crosslinking compound is added to the HAS derivative solution as a solid.
  • the reaction mixture is preferably stirred at a temperature in the range of from 10 °C to 50 °C, more preferably at a temperature in the range of from 20 to 40 °C, more preferably in the range of from 22 to 27°C.
  • the temperature may be varied, preferably in the above-given ranges, or held essentially constant.
  • the present invention also relates to a method, as described above, and a HAS derivative obtained or obtainable by said method, wherein the reacting according to step (ii) is carried out at a temperature in the range of from 10 °C to 50 °C and in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, formamide, water, reaction buffers and mixtures of two or more thereof.
  • the reacting is carried out under non-aqeous conditions, more preferably in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP and mixtures of two or more thereof.
  • the reaction is preferably conducted for a time in the range of from 30 min to 48 h, more preferably of from 1 h to 24 h, more preferably of from 2 h to 18 h.
  • the crosslinking compound of formula (III) is preferably employed in excess (when compared to the HAS derivative (lb)). Therefore the minimum amount is preferably twice the amount of free thiol groups in the HAS derivative (lb).
  • the crosslinking compound of formula (III) is used at a concentration in the range of from 5 to 1000 mM, more preferably of from 10 to 100 mM, and more preferably of from 30 to 80 mM.
  • the maximum concentration is preferably given by the solubility of the crosslinking compound of formula (III) in the reaction solvent.
  • the concentration of the HAS derivative (lb) in the solvent, preferably the aqueous system is preferably in the range of from 3 to 40 wt.-%, more preferably of from 5 to 30 wt.-%, and more preferably of from 10 to 20 wt.-%, relating, in each case, to the weight of the reaction solution.
  • the hydroxyalkyl starch derivative (I) obtained in step (ii) is isolated from the reaction mixture by a sequence of dilution with water, filtration and either ultrafiltration or dialysis, preferably ultrafiltration followed by lyophilization, or the hydroxyalkyl starch derivative is precipitated from the reaction mixture, in particular by adding an alcohol, preferably 2-propanol or water, and separated by ultrafiltration, dialysis or chromatographic methods, preferably by size exclusion chromatography and/or lyophilization.
  • the derivative (I) is preferably purified by at least one volume exchange with aqueous acid, preferably hydroiodic acid, hydrobromic acid, hydrochloric acid, methylsulfonic acid or toluenesulfonic acid, preferably at a pH of in the range of from 1 to 6, more preferably in the range of from 2 to 4. Residual acid is preferably washed out by at least one volume exchange with water.
  • aqueous acid preferably hydroiodic acid, hydrobromic acid, hydrochloric acid, methylsulfonic acid or toluenesulfonic acid
  • the HAS derivative according to formula (I) is preferably used as reactant for coupling to a thiol group comprising compound Q.
  • thiol group comprising compound Q as used in the context of the present invention relates to any substance comprising a thiol group, preferably to a substance which can affect any physical or biochemical property of a biological organism including, but not limited to, viruses, bacteria, fungi, plants, animals, and humans.
  • thiol group comprising compound Q as used in the context of the present invention relates to a substance intended for diagnosis, cure, mitigation, treatment, or prevention of a disease in humans or animals, or to otherwise enhance physical or mental well-being of humans or animals.
  • thiol group comprising, peptides, polypeptides, enzymes, small molecule drugs, dyes, lipids, nucleosides, nucleotides, nucleotide analogs, oligonucleotides, nucleic acid analogs, cells, viruses, liposomes, microparticles, and micelles.
  • thiol group comprising, peptides, polypeptides, enzymes, small molecule drugs, dyes, lipids, nucleosides, nucleotides, nucleotide analogs, oligonucleotides, nucleic acid analogs, cells, viruses, liposomes, microparticles, and micelles.
  • a biologically active substance according to the present invention contains a native thiol group.
  • such thiol group may also be introduced by methods well known to those skilled in the art.
  • thiol group comprising peptide as used in the context of the present invention is denoted to mean peptides comprising up to 50 natural or unnatural, D- or L-amino acids and comprising at least one thiol group.
  • the thiol group may be part of a cysteine or may be introduced into the peptide by a chemical modification.
  • thiol comprising polypeptide includes all compounds having a peptidic backbone and more than 50 monomer (amino acid) units and which comprise at least one thiol group. This term thus in particular includes proteins.
  • protein as used in the context of the present invention includes natural proteins as well as chemically modified derivatives, mutants and analogs thereof.
  • protein mutant is denoted to mean a protein being modified with at least one natural or unnatural amino acid either at the N- or at the C-terminus of the protein or in which at least one naturally-occurring amino acid within the sequence is replaced with another natural or unnatural amino acid.
  • the thiol group may be present in the wild type protein as such or may be introduced by any suitable method such as by adding (e.g. by recombinant means) a cysteine residue either at the N- or at the C-terminus of the polypeptide or by replacing (e.g. by recombinant means) a naturally-occurring amino acid by cysteine to give a mutant of the protein.
  • any group present in the protein may be chemically modified to give a chemically modified derivative of the protein, such as by addition of a suitable linker compound to the N-terminus or C-terminus or to a side chain either during the synthesis of the protein or to the existing full length protein as such.
  • Preferred examples of peptides include, but are not limited to, peptide hormones and peptide aptamers.
  • Preferred examples of polypeptides or proteins include, but are not limited to, the following proteins, plasma proteins such as immunoglobulins, growth factors, cytokines, coagulation factors including vWF, enzymes and enzyme inhibitors, albumins and binding proteins such as alternative scaffold proteins, antibody fragments and soluble receptors.
  • the term "alternative scaffold protein" as used in the context of the present invention relates to a molecule having binding abilities similar to a given antibody wherein the molecule is based on an alternative non-antibody protein framework (see e.g. Hey, T. et al., 2005, Artificial, non-antibody binding proteins for pharmaceutical and industrial applications, Trends BiotechnoL, 23(10), 514-22).
  • oligonucleotide or “nucleic acids” 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 + .
  • oligonucleotide further includes polymers comprising mixtures of deoxyribonucleotides and ribonucleotides (DNA/RNA-hybrids). Further the term includes derivatives thereof chemically modified derivative of the oligonucleotides.
  • 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). 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.
  • 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 an H, OR, R, halogen, 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 and phosphorothioates.
  • 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.
  • PNA peptide nucleic acid
  • oligonucleotide according to the invention may comprise one or more abasic sites.
  • abasic site is meant a monomeric unit contained within an oligonucleotide chain but which does not contain a purine or pyrimidine base.
  • Preferred examples of oligonucleotides and nucleic acid analogs include, but are not limited to, thiol group comprising, ribonucleic acids, deoxyribonucleic acids, peptide nucleic acids (PNA), locked nucleic acids (LNA).
  • the thiol group present in the oligonucleotides, nucleotides, nucleosides or nucleic acid anaolgs of the invention may be attached by any method known to those skilled in the art, i.e., for example, either by introducing a thiol modified building block during the preparation of the respective compound or by chemical modification to any suitable position of the respective compound, in particular by attaching a thiol group comprising linker in 3'- or 5 '-position.
  • lipids refers to a broad group of naturally occurring and unnatural molecules that include fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E, and K), monoglycerides, diglycerides, triglycerides, phospholipids, sphingolipids, sterols, glycophosopho lipids and others. Lipid compounds are suitable for the transport of biologically active substances or molecules. Polymer conjugated lipid compounds are useful in drug delivery, for example in the form of liposomes. (Immordino et al, IJN, 2006: 1 (3) 297-315).
  • compound Q is selected from the group consisting of, thiol group comprising, peptides, polypeptides, oligonucleotides and nucleic acid analogs, more preferably from the group consisting of peptide hormones, peptide aptamers, plasma proteins (such as immunoglobulins, growth factors, cytokines, coagulation factors including vWF), enzymes, enzyme inhibitors, albumins, natural or artificial binding proteins (such as alternative scaffold proteins, antibody fragments, soluble receptors), ribonucleic acids, deoxyribonucleic acids, peptide nucleic acids (PNA), and locked nucleic acids (LNA).
  • HAS hydroxyalkyl starch
  • the present invention also relates to the use of such HAS derivative as a medicament.
  • compound Q is a, thiol group comprising, peptide or polypeptide.
  • the present invention also relates to a hydroxyalkyl starch (HAS) derivative of formula (IV) as described above, or a salt or solvate therof, wherein Q is a, thiol group comprising, peptide or polypeptide. Further, the present invention also relates to the use of such HAS derivatives as a medicament.
  • HAS hydroxyalkyl starch
  • thiol group comprising, peptides and polypeptides (proteins) include, but are not limited to, the following peptides and proteins, or derivatives thereof: erythropoietin (EPO), such as recombinant human EPO (rhEPO) or an EPO mimetic, colony-stimulating factors (CSF), such as G-CSF like recombinant human G-CSF (rhG-CSF), alpha-Interferon (IFN alpha), beta-Interferon (IFN beta) or gamma-Interferon (IFN gamma), such as IFN alpha, IFN beta and IFN gamma like recombinant human IFN alpha, IFN beta or IFN gamma (rhIFN alpha, rhIFN beta or rhIFN gamma), interleukines, e.g.
  • EPO erythropoietin
  • CSF colony-stimulating factors
  • IL-1 to IL-34 such as IL-2 or IL-3 or IL-11 like recombinant human IL-2 or IL-3 (rhIL-2 or rhIL-3), serum proteins such as coagulation factors II-XIII like factor II, factor III, factor V, factor VI, factor VII, factor Vila, factor VIII, such as full-length FVIII, BDD- FVIII or single-chain FVIII, factor IX, factor X, factor XI, factor XII, factor XIII, von Willebrand factor (vWF), enzymes such as lipases, proteases, peptidases, hydrolases, glycosidases, isomerases, reductases, oxidases, transferases, kinases, phosphatases, serine protease inhibitors such as alpha- 1 -antitrypsin (A1AT), activated protein C (APC), plasminogen activators such as tissue-type plasminogen activator (tPA), such as human
  • immunoglobulins such as IgG, IgE, IgM, IgA, IgD and fragments thereof, such as Fab fragments derived from immunoglobuline G molecules (Fab), di-Fabs, tri-Fabs, scFv, bis-scFv, diabodies, triabodies, tetrabodies, minibodies, domain antibodies, VH domain, VL domain, murine immunoglobuline G (mlgG), shark antibodies (IgNAR) and fragments thereof, camelid immunoglobulins and fragments thereof such as VHH domain, receptor proteins, such as cell surface receptors or soluble receptors, hirudin, tissue-pathway inhibitor, plant proteins such as lectin or ricin, bee-venom, snake -venom, immunotoxins, antigen E, alpha
  • polypeptides used as alternative scaffold proteins are derivatives of Protein A, Protein G, lipocalins, CTLA-4, A domain from LDL- receptor like module, ubiquitin, gamma crystallin, repeat proteins such as ankyrin repeat proteins, leucine-rich repeat proteins, tetratricopeptide repeat proteins, HEAT-like proteins, armadillo repeat protein, transferrin, beta-lactamase, C-type lectin domain, fibronectin type III domain 10 proteins, Kunitz domain, knottins such as Ecballium elaterium trypsin inhibitor II (EETI-II) and the C-terminal domain of the human Agouti-related protein (AGRP), tendamistat, thioredoxin, PDZ domain, zinc finger proteins such as the plant homeodomain (PHD) finger protein, T-cell receptors, green-fluorescent protein, Fyn domain 3, Alphabodies, CH2 or CH3 domains of an antibody Fc part.
  • PDD plant homeodomain
  • compound Q is selected from the group consisting of insulin, glucagon, gastric inhibitory peptides, exendins, ghrelin, PYY and peptide aptamers.
  • compound Q is a growth factor or a cytokine, preferably selected from the group consisting of (EPO), such as recombinant human EPO (rhEPO), colony-stimulating factors (CSF), such as G-CSF like recombinant human G-CSF (rhG-CSF), alpha-Interferon (IFN alpha), beta-Interferon (IFN beta), and gamma-Interferon (IFN gamma), such as recombinant human IFN alpha or IFN beta (rhIFN alpha or rhIFN beta), fibroblast growth factors (FGF), human growth hormone (hGH) like recombinant human growth hormone (rhGH), BMPs (bone morphogenic proteins), interleukines, tumor necrosis factors such as TNF alpha and TNF beta.
  • EPO recombinant human EPO
  • CSF colony-stimulating factors
  • G-CSF G-CSF like recombinant human G-C
  • compound Q is a protein hormone, preferably selected from the group consisting of leptins, follicle stimulating hormon (FSH) and luteinizing hormon (LH).
  • FSH follicle stimulating hormon
  • LH luteinizing hormon
  • compound Q is an enzyme or enzyme inhibitor, preferably selected from the group consisting of alpha- 1 -antitrypsin (A1AT), antithrombin such as AT III, glucocerebrosidase, acid maltase, alpha- galactosidase, iduronidase, iduronate-2-sulfatase, arylsulfatase B, asparaginase, phenylalanine ammonia-lyase, and L-methioninase.
  • A1AT alpha- 1 -antitrypsin
  • compound Q is coagulation factor or a protein involved in hemostasis, preferably selected from the group consisting of factor II, factor III, factor V, factor VI, factor VII, factor Vila, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, von Willebrand factor, tissue factor pathway inhibitor (TFPI) and Protein C such as APC.
  • compound Q is an immunoglobulin or fragment thereof, preferably selected from the group consisting of IgG, Fab fragments, Fc fragments, scFvs and dAbs.
  • compound Q is an artificial binding protein or alternative scaffold protein, preferably selected from the group consisting of ubiquitin, Protein A, lipocalins, transferrin, fibronectins and soluble receptors.
  • compound Q is an oligonucleotide or nucleic acid, such as a DNA or RNA aptamer. It is to be understood that the term oligonucleotide or nucleic acid, such as a DNA or RNA aptamer is denoted to mean thiol comprising derivatives of these compounds. Such thiol modifications are known to those skilled in the art.
  • the present invention also relates to a hydroxyalkyl starch (HAS) derivative of formula (IV) as described above, or a salt or solvate therof, wherein compound Q is an oligonucleotide or nucleic acid, such as a DNA or RNA aptamer. Further, the present invention also relates to the use of such HAS derivatives as a medicament.
  • HAS hydroxyalkyl starch
  • compound Q is selected from the group consisting of ribonucleic acid, deoxyribonucleic acid, peptide nucleic acid (PNA), locked nucleic acid (LNA), antisense RNA, RNAi, siRNA, Spiegelmer, aptamer, ribozyme and phosphorothioate-modified nucleic acid.
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • antisense RNA RNAi
  • siRNA siRNA
  • Spiegelmer aptamer
  • ribozyme phosphorothioate-modified nucleic acid
  • the method according to the invention further comprises
  • reaction product of the hydroxyalkyl starch derivative as obtained in step (ii) with the further compound can be obtained in a high yield and in a high purity.
  • respective derivative according to formula (IV) shows advantageous properties in terms of stability.
  • the solvent is chosen depending on the nature of the compound Q to be coupled.
  • reaction is preferably carried out in a solvent selected from the group consisting of water, reaction buffers, DMSO, DMF, DMA, NMP, formamide, and mixtures of two or more thereof.
  • the reaction is carried out in an aqueous medium.
  • aqueous medium is denoted to mean a solvent comprising water and/or at least one reaction buffer.
  • the solvent comprises only minor amounts of organic solvents such as in an amount in the range of from 0 to 10 % by weight, preferably 0 to 5 % by weight, more preferably 0 to 2 % by weight, most preferably less than 1 % by weight, based on the total weight of the reaction solvent.
  • the reaction solvent may comprise detergents, stabilizers, antioxidants and/or reducing agents, preferably at least one antioxidant and/or at least one reducing agent to avoid oxidation of the free thiol groups.
  • a suitable reducing agent may be TCEP, which does not contain a free thiol group and thus does not compete with the Q in the conjugation reaction.
  • a suitable antioxidant may be EDTA, which acts in an indirect manner by complexing transition metal ions, that can catalyze peroxide formation.
  • the reaction is carried out in the presence of TCEP and/or EDTA.
  • Q is a polypeptide, protein or derivative thereof
  • the polypeptide, protein or derivative thereof is preferably incubated with at least one reducing agent and optionally at least one antioxidant, more preferably with DTT, DTE, beta-mercaptoethanol or TCEP, most preferably with DTT and TCEP prior to the addition to or of the HAS derivative of formula (I).
  • Thiol-containing reducing agents should be carefully removed from the reduced protein by methods known to those skilled in the art to prevent unwanted quenching of the thiol-reactive hydroxyalkyl starch derivative.
  • the reaction mixture is preferably stirred at a temperature in the range of from 0 °C to 50 °C, more preferably at a temperature in the range of from 5 to 40 °C, more preferably in the range of from 5 to 25°C.
  • the temperature may be varied, preferably in the above-given ranges, or held essentially constant.
  • the present invention also relates to a method, as described above, and a HAS derivative obtained or obtainable by said method, wherein the reacting according to step (iii) is carried out at a temperature in the range of from 0 °C to 50 °C and in a solvent selected from the group consisting of water, reaction buffers, DMSO, DMF, DMA, NMP, formamide, and mixtures of two or more thereof.
  • the reaction is preferably conducted for a time in the range of from 5 min to 48 h, more preferably of from 20 min to 24 h, more preferably of from 30 min to 18 h.
  • the molar ratio of compound Q : HAS derivative (I) is preferably in the range of from 1 :0.5 to 1 :100, more preferably of from 1 : 1 to 1 :20, more preferably of from 1 : 1 to 1 :5 and more preferably of from 1 : 1.5 to 1 :3.
  • the concentration of the HAS derivative (I) in the solvent, preferably the aqueous system is preferably in the range of from 0.1 to 50 wt.-%, more preferably of from 1 to 30 wt.-%, and more preferably of from 1 to 10 wt.-%, relating, in each case, to the weight of the reaction solution.
  • the derivatives of formula (IV) described hereinunder and above are stable at a pH in range of from 4.0-8.5, preferably in the range of from 6 to 8.5.
  • stable is denoted to mean that the percent of degradation of 1 mg of the derivative according to formula (IV) in 1 mL buffer solution of a respective pH measured after 20 days of incubation at 40 °C, measured by RP-HPLC as described in example CI is preferably less than 8 %, more preferably less than 4%, more preferably less than 3%.
  • X is a leaving group, preferably selected from the group consisting of Ts, Ms, CI, Br and I;
  • Fl is a functional group comprising the group -NR'-, with R' being H or alkyl;
  • LI is a spacer bridging Fl and S;
  • L2 is a spacer bridging -NR X - and -NR y -;
  • R x and R y are, independently of each other, H or alkyl, and optionally form together with the moiety N-L2-N a ring;
  • HAS' is the remainder of the HAS molecule and R b and R c are -[(CR 1 R 2 ) m O] sanction-H and are the same or different from each other; R a is -[(CR 1 R 2 ) m O] sanction-H with HAS * being the remainder of the hydroxyalkyl starch molecule, or R a is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch molecule; R 1 and R 2 are independently hydrogen or an alkyl group having from 1 to 4 carbon atoms, m is 2 to 4, wherein R 1 and R 2 are the same or different from each other in the m groups CR R 2 ; n is from 0 to 6.
  • Fl is a functional group comprising the group -NR'-, with R' being H or alkyl;
  • LI is a spacer bridging Fl and S;
  • L2 is a spacer bridging -NR X - and -NR y -;
  • R x and R y are, independently of each other, H or alkyl, and optionally form together with the moiety N-L2-N a ring;
  • HAS' is the remainder of the HAS molecule and
  • R b and R c are -[(CR 1 R 2 ) m O] wherever-H and are the same or different from each other;
  • R a is -[(CR 1 R 2 ) m O] wherever-H with HAS * being the remainder of the hydroxyalkyl starch molecule, or
  • R a is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch molecule;
  • R 1 and R 2 are independently hydrogen or an alkyl group having from 1 to 4 carbon atoms, m is 2 to 4, wherein R 1 and R 2 are the same or different from each other in the m groups CR R 2 ;
  • n is from 0 to 6.
  • Q is selected from the group consisting of, thiol group comprising, peptides, polypeptides, enzymes, small molecule drugs, dyes, nucleosides, nucleotides, nucleotide analogs, oligonucleotides, nucleic acid analogs, cells, viruses, liposomes, microparticles, and micelles.
  • HAS hydroxyethyl starch
  • R 1 , R 2 , R 3 , and R 4 are hydrogen
  • n 2;
  • n is 0 to 6. 6.
  • R x and R y are, independently of each other selected from the group consisting of H, methyl, ethyl, propyl and butyl, or are forming a ring according to formula
  • R x and R y taken together, are -(CH 2 ) 2 - or -(CH 2 ) 3 -, preferably wherein R x and R y are H.
  • the spacer LI comprises, preferably consists of the moiety -(C(L'L")) q - with L' and L" in each repeating unit -C(L'L")- being, independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, hydroxyl, fluorine, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, amide, carboxyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amine, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, nitro, alkylthio, aryl
  • cycloalkyl such as e.g. cyclopentyl or cyclohexyl
  • heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl
  • q preferably being in the range of from 1 to 20, more preferably in the range of from 1 to 10, more preferably in the range of from 2 to 6, more preferably, 2, 3 or 4.
  • the spacer L2 comprises the moiety -(C(L t L tt )) r with L l and L tt in each repeating unit -C(L l L tt )- being, independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, fluorine, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, amide, carboxyl, keto groups, aldehyde groups, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbony
  • cyclopentyl or cyclohexyl, heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl
  • the groups L l and L tt in each repeating unit may be the same or may differ from each other, with t preferably being in the range of from 1 to 20, more preferably in the range of from 1 to 10, more preferably in the range of from 2 to 6, more preferably, 2, 3 or 4.
  • the HAS derivative of any one of embodiments 1 to 10, wherein the spacer L2 is -CH 2 -CH 2 -.
  • a method for the preparation of a hydroxyalkyl starch derivative comprising
  • M comprises the group -NHR', with R' H or alkyl
  • Ll is a spacer bridging M and S;
  • T is H or a thiol protecting group PG
  • HAS' is the remainder of the HAS molecule and R b and R c are -[(CR 1 R 2 ) m O] justify- H and are the same or different from each other; R a is -[(CR 1 R 2 ) m O] sanction-H with HAS' being the remainder of the hydroxyalkyl starch molecule, or R a is HAS" with HAS' and HAS" together being the remainder of the hydroxyalkyl starch molecule; R 1 and R 2 are independently hydrogen or an alkyl group having from 1 to 4 carbon atoms, m is 2 to 4, wherein R 1 and R 2 are the same or different from each other in the m groups CR J R 2 ; n is from 0 to 6,
  • L2 is a spacer bridging -NR X - and -NR y -;
  • R x and R y are, independently of each other, H or alkyl, and optionally form together with the moiety N-L2-N a ring; thereby obtaining a HAS derivative of formula (I)
  • X is a leaving group, preferably selected from the group consisting of Ts. Ms, CI, Br and I.
  • step (i) further comprises removing PG from the HAS derivative (lb).
  • PG has the structure -S-Ll-M or -Tit (Trityl), preferably S-Ll-M.
  • HAS hydroxy ethyl starch
  • R 1 , R 2 , R 3 , and R 4 are hydrogen
  • n 0 to 6.
  • R x and R y are, independently of each other selected from the group consisting of H, methyl, ethyl, propyl and butyl, or are forming a ring according to formula wherein R x and R y , taken together, are -(CH 2 ) 2 - or -(CH 2 ) 3 -, preferably wherein R and R y are H.
  • step (i) is carried out under reductive amination conditions, preferably at a temperature 5 °C to 100 °C and in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, water, formamide, buffer and reaction buffers and mixtures of two or more thereof, in particular using NaCNBH 4 .
  • a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, water, formamide, buffer and reaction buffers and mixtures of two or more thereof, in particular using NaCNBH 4 .
  • the HAS derivative is purified, preferably by ultrafiltration, and optionally subjected to further reducing conditions prior to step (ii), in particular by employing NaBH 4 . 22.
  • the spacer LI comprises, preferably consists of the moiety -(C(L'L")) q - with L' and L" in each repeating unit -C(L'L")- being, independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, hydroxyl, fluorine, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, amide, carboxyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amine, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, nitro, alkylthio, arylthio,
  • cycloalkyl such as e.g. cyclopentyl or cyclohexyl
  • heterocycloalkyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl
  • q preferably being in the range of from 1 to 20, more preferably in the range of from 1 to 10, more preferably in the range of from 2 to 6, more preferably, 2, 3 or 4.
  • the spacer L2 comprises the moiety -(C(L t L tt )) t - with L l and L tt in each repeating unit -C(L l L tt )- being, independently of each other, selected from the group consisting of H, alkyl, aryl, alkenyl, alkynyl, fluorine, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, amide, carboxyl, keto groups, aldehyde groups, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbony
  • cyclopentyl or cyclohexyl such as e.g. morpholino, piperazinyl or piperidinyl, alkylaryl, arylalkyl and heteroaryl, wherein the groups L l and L tt in each repeating unit may be the same or may differ from each other, with t preferably being in the range of from 1 to 20, more preferably in the range of from 1 to 10, more preferably in the range of from 2 to 6, more preferably, 2, 3 or 4.
  • spacer L2 is a symmetrical spacer, preferably -CH 2 -CH 2 -.
  • step (ii) is carried out at a temperature in the range of from 10 °C to 50 °C and preferably in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP, formamide, water, reaction buffers and mixtures of two or more thereof, more preferably in DMF and/or DMSO,
  • step (ii) is preferably carried out in non aqueous medium, in particular in a solvent selected from the group consisting of DMSO, DMF, DMA, NMP and a mixture of two or more thereof.
  • Q is selected from the group consisting of, thiol group comprising, peptides, polypeptides, enzymes, small molecule drugs, dyes, nucleosides, nucleotides, nucleotide analogs, oligonucleotides, nucleic acid analogs, cells, viruses, liposomes, microparticles, and micelles.
  • Q is selected from the group consisting of, thiol group comprising, peptides, polypeptides, oligonucleotides and nucleic acid analogs, more preferably from the group consisting of peptide hormones, peptide aptamers, plasma proteins (such as immunoglobulins, growth factors, cytokines, coagulation factors including vWF), enzymes, enzyme inhibitors, albumins, natural or artificial binding proteins (such as alternative scaffold proteins, antibody fragments, soluble receptors), ribonucleic acids, deoxyribonucleic acids, peptide nucleic acids (PNA), and locked nucleic acids (LNA).
  • thiol group comprising, peptides, polypeptides, oligonucleotides and nucleic acid analogs, more preferably from the group consisting of peptide hormones, peptide aptamers, plasma proteins (such as immunoglobulins, growth factors, cytokines, coagulation factors including vWF),
  • a HAS derivative according to embodiment 34 wherein the derivative is at least stable at a pH in range of from 4.0 to 8.5, preferably in the range of from 6 to 8.5.
  • Q is a, thiol group comprising, oligonucleotide or nucleic acid, such as a modified DNA or R A aptamer.
  • Chromatographic conditions were as follows:
  • Eluent B 20 mM acetate, pH 4.0, 1 M NaCl
  • Figure 2 shows a section of the RP-HPLC analysis of an L7-HES-Ubi conjugate that was prepared according to example CI .
  • the section shows a part of the gradient from 28.2% to 34.0% of eluent B (broken line).
  • Chromatographic conditions were as follows:
  • Guard column CI 8 guard cartridges (Phenomenex) in a Security Guar dTM Cartridge
  • Sample load 10 ⁇ g of reaction mixture, diluted in demineralized water to a final protein concentration of 0.1 g/1
  • the L7-HES-Ubi conjugate (c) elutes with a retention time of 11.99 min and is separated from the free Ubi (u) eluting at 13.92 min.
  • Figure 3 shows a section of an SEC analysis of an L7-HES-Ubi conjugate prepared according to example CI and incubated for 20 days at pH 7.0 and 40 °C.
  • Chromatographic conditions were as follows:
  • the L7-HES-Ubi conjugate (c) elutes with a retention time of 10.09 min and is separated from the free Ubi (u) eluting at 14.73 min.
  • Figure 4 shows the coupling to the Cys free Ubi variant (W-Ubi) shown as black bars and IFN-a (contains no single Cys) shown as white bars to different HES derivatives.
  • X-axis 1 : L3-HES, 2: L5-HES, 3(#): L7-HES, 4: L10-HES, 5: Ll l-HES, 6: L13-HES, 7: mi- PEG1, 8: mi-PEG2, 9: mi-HES (For the respective linker structures, see Table 1)
  • y-axis yield of conjugated product.
  • the detection of conjugated product is an indication for unwanted non-specific reactions with functional groups other than thiol groups of cysteines.
  • the stress stability of various conjugates was determined by RP-HPLC or SEC (see Example CI) after 20 days incubation and is shown as percent of conjugate degradation (y) at pH 4.0 (black bars), pH 7.0 (white bars) and pH 8.0 (broken bars) and at 40 °C.
  • the numbers of the bars displayed in figure 5 correspond to the respective linker L having the same number as the bar (see Table 1). Stars indicate linkers or conditions that were not investigated for stress stability analysis.
  • the stress stability of various conjugates was determined by RP-HPLC or SEC (see Example El l) after 20 days incubation and is shown as percent of conjugate degradation (y) at pH 4.0 (black bars), pH 7.0 (white bars) and pH 8.0 (broken bars) and at 40 °C.
  • the numbers of the bars displayed in figure 6 correspond to the respective linker L having the same number as the bar (see Table 1).
  • Stars indicate linkers or conditions that were not investigated for stress stability analysis.
  • This figure clearly demonstrates the high stability of the measured MEP comprising conjugates according to the invention, in particular at physiologically pH or higher pH ranges.
  • Figure 7 Average decomposition rate for conjugates with 3 linkers attached to HES molecules of differing size and different target molecules
  • linker structures LI, L7 and L10 were attached to thiol-modified HES molecules of different size (Mw -30, 100 and 250 kDa). Conjugates with MEP, Ubi and HSA (only largest HES) were prepared and subjected to stress stability as described in examples El l and C6. The average degradation rate in % after 20 days for all conjugates of the respective linker tested is shown in the figure.
  • Figure 8 shows a representative section of the RP-HPLC analysis of HES-Ubi conjugation reactions conducted according to table 10.
  • the section shows a part of the gradient from 28.2% to 34.0% of eluent B (broken line).
  • Chromatographic conditions were as follows:
  • Sample load 10 ⁇ g of reaction mixture, diluted in demineralized water to a final protein concentration of 0.1 g/1
  • the HES-Ubi conjugate eluted as a broad peak with a retention time between 4.5 and 5.5 min, multiple by-products of the conjugation between Ubi and presumably linker-derived impurities were found at 5.5 to 6 min, while free Ubi protein was eluting at 6.2 min.
  • X retention time in min
  • Y arbitrary absorption units at 220 nm
  • RGC reactive group content defined as percentage of HES molecules modified with a certain functional group with respect to all HES molecules (based on M n of the respective HES derivative)
  • a 1 1 three-neck flask was equipped with pressure exchange, magnetic stirring bar and dropping funnel. The flask was loaded with 15.1 g of 2,2 ' -dithiodipyridine, 500 ml of methanol and 50 ⁇ of ⁇ , ⁇ -diisopropylethylamine under inert atmosphere. A solution of 2- [2-(2-mercapto-ethoxy)-ethoxy]-ethanethiol in methanol (2 g/120 ml) was added drop-wise over a period of 30 min.
  • a 50 ml three-neck flask was equipped with two dropping funnels and an internal thermometer.
  • the reactor was charged with 2-(2-aminoethoxy)ethylamine (296 ⁇ ; 2.79 mmol) and chloroform (5 ml).
  • the solution was cooled to -5 °C in an acetone dry ice bath and solutions of iodoacetyl chloride (ABCR, Düsseldorf, Germany, 500 ⁇ ; 5.58 mmol) in 5 ml of chloroform and potassium carbonate (780 mg; 5.64 mmol) in 3 ml of water were added simultaneous over a period of 30 min keeping the internal temperature below 5 °C.
  • a solution of diazomethane in diethyl ether (0.245 mo 1/1) was prepared from DIAZALD as described in T.H. Black, Aldrichimica Acta, 1983, 16, 3-10.
  • a 1 1 one-neck flask was equipped with a magnetic stirring bar. An outside cooling (ice/water) was prepared. The flask was loaded with the diazomethane solution (500 ml). A dropping funnel with pressure exchange was installed. A solution of freshly distilled hexanedioyl-dichloride (4.58 g) in 30 ml diethyl ether was added drop-wise under slight development of gas. The funnel was washed with 20 ml diethyl ether and the mixture was stirred for 1 h at 0 °C. An aqueous HBr-solution (9.5 ml, 62% (w/w)) was poured to this reaction mixture in one portion under a strong development of gas. A precipitate appeared.
  • a 25 ml two-neck flask was equipped with a magnetic stirring bar under inert atmosphere.
  • the flask was loaded with sodium iodide (1.1 g), evacuated and refilled with nitrogen.
  • acetone (7 ml) was added and the mixture was stirred until the salt dissolved.
  • L13 (1 g) was added and the resulting mixture was stirred at room temperature for 3 h. Then a further portion of sodium iodide (0.1 g) was added. The reaction was monitored by GC.
  • the product was purified by size exclusion chromatography using ultrapure water as eluent, a guard column HiTrapTM Desalting 1 *5 ml and a separation column HiPrepTM 26/10 Desalting 53 ml (both GE Healthcare). In each run 5 ml samples were injected. The chromatographic procedure was monitored by UV spectroscopy at the wavelength of 210 nm and 5 ml-fractions were collected. After use the column was equilibrated with 5 column volumes of 0.5M acetic acid and 5 column volumes of ultrapure water. The fraction containing the HES-derivative were pooled and lyophilized.
  • HES-linker derivative was purified by ultrafiltration with a membrane (e.g. MWCO 10 kDa) against 20 times its own volume of ultrapure water and lyophilized.
  • HES-linker derivative was dissolved either in 0.21M phosphate buffer pH 8.5 containing 5 mM EDTA (linker L7-L19) or PBS-buffer pH 6.5 containing 5 mM EDTA (linker L1-L6).
  • One equivalent of thiol T (referred to M n of the HES species; see Table 3) was dissolved in 5 mM EDTA solution pH 6.0 and added to the HES-linker derivative solution.
  • the final concentration of HES-linker derivative in the reaction mixture was 15% (w/v). The reaction mixture was incubated at 21 °C for 2 h.
  • the samples were analyzed by RP-HPLC/UV (conditions see below in Example El lc) at the UV maximum of T (for peptides 280 nm and for PET 254 nm).
  • the RGC was evaluated by comparison of the relative peak area of the conjugate to the sum of all other products.
  • the reaction conditions for the various target molecules were not optimized.
  • Example E10 General procedure for the determination of the mean molecular weight M w M w and M n were determined as described in WO 2012/004007 Al, Example 1.9.
  • Example Ell Stability studies
  • HES-linker derivative defined by the linker and the HES species
  • 2-Mercaptoethyl)pyrazine (20 equivalents referred to M n of the HES species) was dissolved in the same volume of DMF and added to the HES-linker derivative solution.
  • the reaction mixture was incubated at 21 °C for 16-24 h. After incubation the samples were purified by first precipitation and subsequently desalting and isolated by freeze drying as described above in example E8.
  • the conjugates were analyzed by RP-HPLC (for conditions see below). The reaction conditions for the various target molecules were not optimized.
  • the conjugates were dissolved in buffer and diluted to a concentration of 20 mg/ml.
  • the samples were incubated for up to 32 d at 40 °C. Samples were taken after 0, 1, 5, 10 and 20 d. They were analyzed by RP-HPLC/UV at 266 nm (for conditions see below). The decay was evaluated by comparison of the relative peak area of the conjugate to the sum of all decomposition products.
  • the product was analyzed by RP-HPLC using ultrapure water with 0.1% TFA (Uvasol ® , for spectroscopy, Merck, Code No. 1.08262.0100) as eluent A and acetonitrile (Uvasol ® ,
  • Cartridge system Widepore CI 8, ODS, 4 mm L * 3.0 mm ID (Phenomenex, Code No.
  • the aqueous layer was extracted with 2 1 of hot trichloromethane and the extract was combined with the organic phase of the reaction.
  • the organic extracts contained large amounts of a solid A, which was filtered off.
  • the filtrate was dried over sodium sulfate and the solvent was evaporated after filtration at a bath temperature of 60 °C to yield 130 g of crude product, which was crystallized from 850 ml of hot ethanol to yield the first batch of E12 as an off-white solid.
  • the solid A (1.1 kg before drying) was crystallized from 3.25 1 of hot ethanol to yield another batch of E12. Both batches were combined after drying at 10 "3 mbar at room temperature to yield 516.30 g of E12 (1.71 mol; 50.5%).
  • Methyl glycolate (5.0 g) was dissolved in THF (40 ml), triethylamine (9.48 ml) was added and the reaction mixture was cooled to 0 °C. A solution of mesyl chloride (7.9 g) in THF (15 ml) was added drop-wise. The reaction was stirred for 30 minutes at 0 °C and over night at room temperature. The mixture was filtered to remove the triethylamine salts, the solid was washed twice with THF and the solvent was removed in vacuo.
  • the aqueous phase was acidified to pH 2-3 with IN HCl and extracted three times with 200 ml of diethyl ether.
  • the combined organic phases were dried with sodium sulfate, filtered and the solvent was removed in vacuo yielding 1.8 g of a white solid (21% yield based on methyl glycolate).
  • Methyl glycolate (1.5 g) was dissolved in THF (25 ml), triethylamine (2.6 ml) was added and the reaction mixture was cooled to 0 °C.
  • Ubiquitin (Ubi, pdb code: 1UBQ) was selected as model protein for testing reactivity and stability of various thiol-reactive HES derivatives.
  • the protein variant Ubi F45W S57C (with a C-terminal His6 tag, manufactured by Scil Proteins, Halle, Germany) was used allowing site-specific conjugation to the single cysteine residue introduced on position 57 and detection by UV spectroscopy by the tryptophan residue introduced on position 45.
  • DTT DTT is used in a 50fold molar excess and the reduction takes place for 1 hour at 37 °C. Afterwards the DTT was removed by cation exchange procedure on a 1 ml HiTrap SP HP (GE Healthcare). DTT elutes from the column in the flow-through, afterwards Ubi was eluted by a step gradient.
  • a 10% or 40% (w/v) stock solution of the thiol- reactive HES derivative (defined by the linker and the HES species) was prepared.
  • the appropriate amount of HES derivative was weighed into the reaction tubes and dissolved in reaction buffer until a clear solution appeared.
  • the protein solution and the HES derivatives were combined in a specified ratio (Table 5) and mixed thoroughly.
  • the appropriate amount of thiol-reactive HES derivative (see Table 5) was weighed directly in a 15 ml Falcon tube, dissolved in reaction buffer (see Table 5) as described above and mixed thoroughly with the appropriate protein solution.
  • the reaction mixtures were analyzed by RP-HPLC (example see Figure 2), SEC (example see Figure 3) and SDS-PAGE.
  • the chromatogram monitored at a wavelength of 220 nm or 280 nm was integrated and the yield of the conjugation reaction was calculated from the peak areas of the conjugate and the non-modified protein.
  • the coupling procedure was optimized for different pH conditions and various HES : target ratios (Table 5).
  • the preparation of the HES-Ubi conjugates was performed by a cation exchange chromatography. All chromatographic steps were performed at room temperature using an Akta Purifier 100 system (GE Healthcare) and monitored by UV spectroscopy at a wavelength of 220 nm and 280 nm and by conductivity measurements.
  • HES-Ubi conjugates For the preparation of the HES-Ubi conjugates a HiTrap SP HP 1 ml column (GE Healthcare) was used. Eluents were exchanged to eluent A (20 mM acetate, pH 4.0) and eluent B (20 mM acetate, pH 4.0, 1 M NaCl); the column was equilibrated with 10 CV eluent A. The reaction mixture was diluted approximately 20fold using buffer A and loaded onto the column using the sample pump. The flow-through was collected in 50 ml Falcon tubes. Unbound sample was washed out with five CV eluent A and the conjugate was eluted with a flow rate of 1.5 ml/min and a segmented salt gradient (Figure 1).
  • Example C2 Conjugation of thiol-reactive HES and IL1RA (Kineret®)
  • Coupling reactions of IL1RA (pdb code: 1IRA) with HES derivatives were performed as described in example CI (examples listed in Table 5).
  • the reaction mixtures were analyzed by RP-HPLC (as described in example CI) with a Jupiter CI 8 column (300 A, 5 ⁇ , 4.6 x 150 mm, Phenomenex) with a segmented gradient (0-0.2 min: 2% B, 0.2-0.8 min: 2-30% B, 0.8-5.8 min: 30-40% B, 5.8-6.5 min: 98% B, 6.5-7.0 min: 98% B, 7.0-9.0 min: 2% B) and a flow rate of 1 ml/min or by SEC (as described in example CI) with a flow rate of 0.5 ml/min within 60 min on a Superose 6 10/300 GL (GE Healthcare) column.
  • the L7-HES-IL1RA conjugate retained an excellent binding affinity with a K D value of 180 pM in comparison to 90 pM for the unmodified ILIRA and 187 pM for ILIRA that is conjugated to HES via N-terminal coupling.
  • A1AT (pdb code: 1KCT) contains a single cysteine that is partially capped and therefore had to be reduced before starting the coupling procedure by addition of a lOfold molar excess of DTT for one hour at 37 °C. After reduction, the DTT was removed by buffer exchange in a centrifugal concentrator.
  • Coupling reactions of A1AT with HES derivatives were performed as described in example CI (examples listed in Table 5).
  • the reaction mixtures were analyzed by RP- HPLC with a Jupiter C18 column (300 A, 5 ⁇ , 4.6 x 150 mm) as described in example CI with a segmented gradient (0-4 min: 5% B, 4-8 min: 5-46% B, 8-17 min: 46-51% B, 17-22 min: 98% B, 22-25 min: 5% B) and a flow rate of 1 ml/min.
  • the activity of the L7-HES-A1AT conjugate was analyzed in an elastase inhibition assay as described in Beatty et al. (1980, JBC, 255, 9, 3931-3934) and compared to unmodified AIAT.
  • the L7-HES-A1AT conjugate shows 101% of the elastase inhibition activity compared to unmodified AIAT.
  • SlyD D101C (Zoldak and Schmid, 2011, JMB, 406(1), 176-94; pdb code: 2K8I) contains a single additional cysteine that had to be reduced by addition of a lOfold molar excess of DTT for one hour at 37 °C. After reduction, the DTT was removed by buffer exchange in a centrifugal concentrator. A coupling reaction of SlyD with a HES derivative was performed as described in example CI (see Table 5).
  • the reaction mixture was analyzed by RP-HPLC with a Jupiter C18 column (300 A, 5 ⁇ , 4.6 x 150 mm) as described in example CI with a segmented gradient (0-1 min: 2-30% B, 1-6 min: 30-50% B, 6-10 min: 98% B, 10-13 min: 2% B) and a flow rate of 2 ml/min or by SEC (as described in example CI) with a flow rate of 0.5 ml/min within 60 min on a Superose 12 10/300 GL (GE Healthcare) column.
  • Prolylisomerases like SlyD catalyze the trans to cis isomerization of peptidyl prolyl bonds.
  • the prolyl isomerase activities of SlyD and the L7-HES-SlyD conjugate were measured by a prolyl isomerase activity assay according to Zoldak et al. (2009, Biochemistry, 48, 10423-10436).
  • the rate constant of the cis to trans isomerization of the prolyl bond was determined using Origin 8.1.
  • the L7-HES-SlyD conjugate shows an equal activity for prolyl isomerization as unmodified SlyD.
  • HSA pdb code: 1E7H
  • HES derivatives were performed as described in example CI, examples are shown in Table 5.
  • the reaction mixtures were analyzed by SEC (as described in example CI) with a Superose 6 10/300 GL column with a flow rate of 0.5 ml/min within 60 min as described in example C 1.
  • HES HES : target describes the molar ratio of reactive groups of HES to protein concentration (target).
  • the coupling reactions were performed in 0.1 M phosphate with 5 mM EDTA or 0.1 M Tris/HCl depending on the required pH for two hours at 20 or 25 °C (Ubi and Al AT) or over night at 5 °C (all other targets). Stability tests/Analysis of the specificity of the coupling to a thiol group of a protein
  • the conjugates were diluted into final buffer conditions of 20 mM acetate (pH 4.0 and 5.5) or 10 mM phosphate (pH 7.0 and 8.0), 0.5 mM ETDA, 154 mM NaCl to a final concentration of 1 mg/ml.
  • the conjugates were stored at 40 °C for 20 days and analyzed by RP-HPLC (as described in example CI), SEC (as described in example CI) or SDS-PAGE. The results are given in Tables 6 & 8; see also Figures 5-7.
  • HES species with a molecular weight between 60 and 90 kDa see Table 2
  • PEG with a molecular weight of approximately 20 kDa were used.
  • HES : target/ PEG target describes the molar ratio of reactive groups of HES/PEG to protein concentration (target).
  • the coupling reactions were performed in 0.1 M phosphate, pH 8.0 at 20 °C for 72 hours.
  • the Ubiquitin variant contains no additional single cysteine.
  • IFN-a2b lacks a single cysteine for site-specific conjugation.
  • the specificity was analyzed by SDS Page and RP-HPLC (see Table 7 and Figure 4).
  • linker L7 shows a high selectivity towards thiol groups, thus no conjugate formation with W-Ubi has been observed.
  • the solution was dialyzed for 4 days against water (SnakeSkin dialysis tubing 3.5 kD cut off, Thermo Fisher, Lot JE123986) and subsequently lyophilized.
  • Example E18 Preparation of iodoacetamide-HES from iodoacetic acid - Linker "IA- EDC" (not according to the invention) 0.2 g (0.013 mmol) amino-oxo-HES 50/0.7 was dissolved in 1 mL Milli-Q water. 13.5 mg (0.07 mmol) iodoacetic acid (Fluka, Lot BCBC8636V) was dissolved in 85 Milli-Q water and the pH was adjusted to 4.7 with 0.01 mol/L NaOH.
  • IA- EDC Linker
  • Thiol-derivatized HES 10/1.0 (X22) was obtained according to example 7 under reaction conditions listed in table 2.
  • L7-HES10/1.0 was synthesized according to example E8 and analyzed as described in example E9. Specific reaction conditions are given in Table 9.
  • the molecular weight Mn of all HES derivatives tested was in a comparable range, the Mw of the NH2-oxHES based reagents was higher due to a higher polydispersity of the starting material.
  • PBS-buffer 25 mmol/1 sodium phosphate, 135 mmol/1 NaCl
  • 5 mmol/1 EDTA pH 6.5
  • 0.1 mol/1 sodium borate buffer 5 mmol/1 EDTA, pH 8.0

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Abstract

La présente invention porte sur un dérivé d'hydroxyalkylamidon (HAS) de formule (I), dans laquelle X représente un groupe partant, de préférence choisi dans le groupe constitué par Ts, Ms, Cl, Br et I; F1 représente un groupe fonctionnel comprenant le groupe -NR'-, R' représentant H ou un groupe alkyle; L1 représente un espaceur reliant F1 et S; L2 représente un espaceur reliant -NRx- et -NRy-; Rx et Ry représentent, chacun indépendamment de l'autre, H ou un groupe alkyle et forment éventuellement un cycle conjointement avec la fraction N-L2-N; HAS' représente le reste de la molécule d'HAS; Rb et RC représentent chacun -[(CR1R2)mO]n-H et sont identiques l'un à l'autre ou différents l'un de l'autre; Ra représente -[(CR1R2)mO]n-H, HAS' représentant le reste de la molécule d'hydroxyalkylamidon, ou Ra représente HAS'', HAS' et HAS'' représentant ensemble le reste de la molécule d'hydroxyalkylamidon; R1 et R2 représentent chacun indépendamment l'atome d'hydrogène ou un groupe alkyle ayant de 1 à 4 atomes de carbone et m vaut 2 à 4, R1 et R2 étant identiques l'un à l'autre ou différents l'un de l'autre dans les m groupes CR1R2; et n vaut de 0 à 6.
PCT/EP2014/055598 2013-03-20 2014-03-20 Dérivés d'hydroxyalkylamidon utilisés comme réactifs pour couplage à des groupes thiol Ceased WO2014147175A1 (fr)

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Application Number Priority Date Filing Date Title
CA2907371A CA2907371A1 (fr) 2013-03-20 2014-03-20 Derives d'hydroxyalkylamidon utilises comme reactifs pour couplage a des groupes thiol
AU2014234272A AU2014234272A1 (en) 2013-03-20 2014-03-20 Hydroxyalkyl starch derivatives as reactants for coupling to thiol groups
EP14711748.5A EP2976366A1 (fr) 2013-03-20 2014-03-20 Dérivés d'hydroxyalkylamidon utilisés comme réactifs pour couplage à des groupes thiol

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EP13160266 2013-03-20
EP13160266.6 2013-03-20

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WO2014147175A1 true WO2014147175A1 (fr) 2014-09-25

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WO2015132724A1 (fr) 2014-03-05 2015-09-11 Pfizer Inc. Mutéines améliorées du facteur viii de coagulation

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WO2004024761A1 (fr) * 2002-09-11 2004-03-25 Fresenius Kabi Deutschland Gmbh Polypeptides-has, notamment, erythropoietine-has ayant subi une acylation
WO2005014050A2 (fr) * 2003-08-08 2005-02-17 Fresenius Kabi Deutschland Gmbh Conjugues d'amidon hydroxyalkyle et de g-csf
EP1681303A1 (fr) * 2002-09-11 2006-07-19 Fresenius Kabi Deutschland GmbH Polypeptides HASylés, en particulier érythropoiétine HASylée
EP2070950A1 (fr) * 2007-12-14 2009-06-17 Fresenius Kabi Deutschland GmbH Dérivés hydroxyalkylés de l'amidon et leur procédé de préparation
WO2012004008A1 (fr) * 2010-07-09 2012-01-12 Fresenius Kabi Deutschland Gmbh Conjugués comprenant un amidon hydroxyalkylé et un agent cytotoxique et leur procédé de préparation

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WO2004024761A1 (fr) * 2002-09-11 2004-03-25 Fresenius Kabi Deutschland Gmbh Polypeptides-has, notamment, erythropoietine-has ayant subi une acylation
EP1681303A1 (fr) * 2002-09-11 2006-07-19 Fresenius Kabi Deutschland GmbH Polypeptides HASylés, en particulier érythropoiétine HASylée
WO2005014050A2 (fr) * 2003-08-08 2005-02-17 Fresenius Kabi Deutschland Gmbh Conjugues d'amidon hydroxyalkyle et de g-csf
EP2070950A1 (fr) * 2007-12-14 2009-06-17 Fresenius Kabi Deutschland GmbH Dérivés hydroxyalkylés de l'amidon et leur procédé de préparation
WO2012004008A1 (fr) * 2010-07-09 2012-01-12 Fresenius Kabi Deutschland Gmbh Conjugués comprenant un amidon hydroxyalkylé et un agent cytotoxique et leur procédé de préparation

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015132724A1 (fr) 2014-03-05 2015-09-11 Pfizer Inc. Mutéines améliorées du facteur viii de coagulation
US10570189B2 (en) 2014-03-05 2020-02-25 Pfizer Inc. Muteins of clotting factor VIII

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AU2014234272A1 (en) 2015-10-08
CA2907371A1 (fr) 2014-09-25

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