Oligosaccharide comprising aminooxy group and conjugate thereof

Abstract

To provide methods for the synthesis of oligosaccharides comprising an aminooxy group.SOLUTION: The present invention provides a method including the following steps: (a) providing an oligosaccharide comprising a first reactive group; (b) providing an aminooxy compound comprising an aminooxy group and a second reactive group; and (c) reacting the first reactive group of the oligosaccharide with the second reactive group of the aminooxy compound, through which the oligosaccharide comprising the aminooxy group is prepared.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 60 / 885,471, filed Jan. 18, 2007, the disclosure of which is incorporated herein by reference.

[0002] The present invention generally relates to methods for synthesizing oligosaccharides containing aminooxy groups from oligosaccharides containing reactive groups. In another embodiment, the present invention further relates to oligosaccharides containing aminooxy groups. The present invention also relates to methods for conjugating oligosaccharides containing aminooxy groups to proteins, including glycoproteins (e.g., lysosomal enzymes), and compositions of oligosaccharide-protein conjugates, including oligosaccharide-glycoprotein conjugates. Another embodiment of the present invention relates to methods of treating lysosomal storage disorders using such oligosaccharide-lysosomal enzyme conjugates. [Background technology]

[0003] Lysosomal storage disorders (LSDs) are a group of rare metabolic disorders that includes more than 40 genetic disorders involving defective activity of lysosomal hydrolases. The hallmark feature of LSDs is the abnormal accumulation of lysosomal metabolites, leading to the formation of numerous distended lysosomes.

[0004] LSDs can be treated by administering to patients the missing active enzyme, a process called enzyme replacement therapy (ERT). The administered replacement enzyme, which contains terminal mannose-6-phosphate (M6P), is taken up by target cells through cell surface-attached cation-independent M6P receptor (CI-MPR)-mediated endocytosis and directed to lysosomes.

[0005] In general, poorly phosphorylated replacement enzymes are not internalized by the M6P receptor on the cell surface and therefore cannot be directed to the lysosomes where they function. Thus, low levels of mannose phosphorylation can have a significant and detrimental effect on the therapeutic efficacy of replacement enzymes. Summary of the Invention

[0006] Therefore, methods have been developed to increase the M6P content of the supplementary enzyme.For example, US Patent No. 7,001,994 describes a method for conjugating an oligosaccharide containing M6P with a glycoprotein.The oligosaccharide of the glycoprotein is first oxidized by periodate or galactose oxidase to form a carbonyl group, and then chemically conjugated with an oligosaccharide functionalized at the reducing end with a carbonyl reactive group (e.g., hydrazine, hydrazide, aminooxy, thiosemicarbazide, semicarbazide, or amine group, etc.) to produce an oligosaccharide-glycoprotein conjugate.

[0007] A conjugate of the lysosomal enzyme acid alpha-glucosidase (GAA) with bis-M6P oligosaccharides was prepared by the above-mentioned method and found to be more effective in reducing skeletal and cardiac muscle glycogen than recombinant human GAA in a mouse model of Pompe disease, an autosomal recessive muscular disorder resulting from a metabolic deficiency of GAA and characterized by the accumulation of lysosomal glycogen.

[0008] The aminooxy group is a particularly useful carbonyl-reactive group for the conjugation reactions described above because the resulting conjugates contain a relatively stable oxime linkage. There is a need for methods for preparing functional oligosaccharides.

[0009] The present invention provides methods for preparing oligosaccharides containing aminooxy groups. These methods are generally applicable to a wide range of protected and unprotected oligosaccharides, such as branched and unbranched, and phosphorylated and unphosphorylated oligosaccharides. In some embodiments, the oligosaccharides may be disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, heptasaccharides, or more. In some embodiments, the oligosaccharides may contain at least one M6P residue. In some embodiments, the oligosaccharides may contain at least 1, 2, 3, 4, 5, 6, or 7 terminal M6P residues.

[0010] The present invention provides a method for preparing an oligosaccharide containing an aminooxy group from an oligosaccharide containing a reactive group, the method comprising: (a) providing an oligosaccharide comprising a first reactive group; (b) providing an aminooxy compound comprising an aminooxy group and a second reactive group; (c) reacting the first reactive group of the oligosaccharide with a second reactive group of the aminooxy compound, thereby preparing an oligosaccharide containing an aminooxy group.

[0011] The first and second reactive groups can be selected from, for example, hydrazine, hydrazide, thiosemicarbazide, semicarbazide, amine, carboxyl, active ester, acyl halide, acyl azide, alkyl halide, anhydride, isothiocyanate, isocyanate, and sulfonyl halide groups.

[0012] In some embodiments, the aminooxy compound is selected from a compound of formula II: [ka] Chemical formula II wherein Y is a second reactive group, Z is selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl, and P is selected from an amino protecting group (such as a carbamate protecting group). For example, in some embodiments, Y can be a carboxyl, an active ester, an acyl halide (such as an acid fluoride or an acyl chloride), an acyl azide, an alkyl halide, an anhydride, an isothiocyanate, an isocyanate, or a sulfonyl halide (such as a sulfonyl chloride or a sulfonyl bromide). In other embodiments, Y can be, for example, a hydrazine, a hydrazide, a thiosemicarbazide, a semicarbazide, or an amine group.

[0013] In some embodiments, the aminooxy compound of formula II is selected from a compound of formula III: [ka] Chemical formula III wherein Y is a second reactive group, n is selected from an integer ranging from 1 to 10, and P is selected from an amino protecting group.

[0014] In certain embodiments, the aminooxy compound comprises an amino-protecting group and the method further comprises a step (d) of deprotecting the oligosaccharide comprising the aminooxy group.

[0015] The present invention further provides an oligosaccharide comprising (1) an aminooxy group and (2) mannose-6-phosphate. In some embodiments, the oligosaccharide is prepared by the methods described above. For example, in some embodiments, the present invention provides an oligosaccharide comprising an aminooxy group of formula IV: [ka] Chemical formula IV In the formula, m and p are independently selected from integers ranging from 1 to 10.

[0016] In another embodiment, the present invention provides an oligosaccharide of formula V: [ka] Chemical formula V

[0017] In another embodiment, the present invention provides a method for conjugating an oligosaccharide to a protein. In one embodiment, the method comprises: (a) providing an oligosaccharide comprising an aminooxy group; (b) providing a protein having at least one carbonyl group; (c) reacting the aminooxy group of the oligosaccharide with at least one carbonyl group of the protein; thereby attaching the oligosaccharide to the protein.

[0018] In other embodiments, the present invention provides an oligosaccharide-protein conjugate further comprising: (1) a protein; (2) an oligosaccharide; and (3) an oxime group linking the protein and the oligosaccharide. For example, in some embodiments, the present invention provides an oligosaccharide-protein conjugate prepared by the above-described method. In some embodiments, the oligosaccharide-protein conjugate is an oligosaccharide-glycoprotein conjugate. In some embodiments, the oligosaccharide-glycoprotein conjugate is a conjugate of an oligosaccharide comprising at least one M6P and a lysosomal enzyme, such as a lysosomal hydrolase. In some embodiments, the present invention provides a pharmaceutical composition comprising the oligosaccharide-protein conjugate of the present invention.

[0019] Another embodiment of the present invention provides a method of treating a lysosomal storage disorder, e.g., as disclosed in Table 1. In some embodiments, the method comprises administering to a mammal an oligosaccharide-glycoprotein conjugate of the present invention, wherein the oligosaccharide comprises at least one M6P and the glycoprotein is a lysosomal hydrolase. The disclosure further provides for the use of the conjugate of the present invention for treating a lysosomal storage disorder in a patient in need of such treatment, and in the manufacture of a medicament for treating a lysosomal storage disorder. [Brief description of the drawings]

[0020] [Figure 1] 1 is a reaction scheme illustrating an exemplary embodiment of the method of the present invention. Oligosaccharide 1, which has a first reactive group (hydrazide group), is reacted with aminooxy compound 2 in the presence of catalyst 3-hydroxy-1,2,3-benzotriazine-4(3H)-one (DHBt-OH) to produce oligosaccharide 3. The tert-butyloxycarbonyl (t-Boc) amino protecting group of oligosaccharide 3 is then removed with 50% trifluoroacetic acid / dichloromethane (TFA / DCM) to produce oligosaccharide 4.

[0021] [Figure 2A] FIG. 2 shows a series of gel chromatographs of intermediates of the synthetic scheme depicted in FIG 1. FIG 2A is an analytical Dionex chromatograph of the starting oligosaccharide 1. [Figure 2B] FIG. 2B is a series of gel chromatographs of intermediates of the synthetic scheme depicted in FIG 1. FIG 2B is a Dionex analytical chromatograph of oligosaccharide 3. [Figure 2C] FIG. 2C is a series of gel chromatographs of intermediates of the synthetic scheme depicted in FIG 1. FIG 2C is an analytical Dionex chromatograph of oligosaccharide 4.

[0022] [Figure 3A] FIG. 3A is a mass spectrum of oligosaccharide 1 (calculated molecular weight=1250, calculated molecular weight of the sodium salt=1338). [Figure 3B]FIG. 3B is a mass spectrum of oligosaccharide 4 (calculated molecular weight=1323, calculated molecular weight of sodium salt=1411).

[0023] [Figure 4] 1 is a reaction scheme illustrating an exemplary embodiment of the method of the present invention. Oligosaccharide 5 having a first reactive group (carboxyl group) is reacted with aminooxy compound 6 in the presence of coupling agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and catalyst N-hydroxysuccinimide (NHS) to produce aminooxy-containing oligosaccharide 3. The Boc amino protecting group of oligosaccharide 3 is then removed by 50% TFA / DCM to produce oligosaccharide 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] I. Preparation of oligosaccharides containing aminooxy groups A. Oligosaccharides containing reactive groups The methods of the invention can be applied to oligosaccharides containing a wide range of reactive groups. As used herein, oligosaccharide refers to disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, heptasaccharides, or higher oligosaccharides (e.g., oligosaccharides containing 2-50, 2-10, 8-25, or 8-50, etc., sugar units). Thus, in various embodiments, an oligosaccharide can be, for example, a disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, or higher oligosaccharide. An oligosaccharide can be a single-, double-, triple-, quadruple-, or penta-antennary structure. An oligosaccharide can contain zero, one, two, three, four, or more branch points.

[0025] The reactive group on the oligosaccharide is also referred to as the first reactive group, and in some embodiments may be, for example, a hydrazine group, a hydrazide group, a semicarbazide group, a thiosemicarbazide group, or an amine group. In some embodiments, the first reactive group may be, for example, a carboxyl, an ester (such as an active ester), an acyl halide (such as an acid fluoride or an acyl chloride), an acyl azide, an alkyl halide, an anhydride, an isothiocyanate, an isocyanate, or a sulfonyl halide (such as a sulfonyl chloride or a sulfonyl bromide) group.

[0026] The first reactive group may be linked to the reducing end of the oligosaccharide or may be located anywhere within the oligosaccharide. The first reactive group may be linked to the oligosaccharide through one or more linkers in some embodiments. The linker, as used herein, may be selected from, for example, a combination of optionally substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, acyloxy, alkoxy, aryloxy, and heterocyclyloxy groups. The linker may be interrupted or terminated by one or more heteroatoms, such as, for example, nitrogen, sulfur, and oxygen. For example, the linker may include one or more ether, ester, or amide groups in some embodiments.

[0027] Any chemical group of the linker (e.g., alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, acyloxy, alkoxy, aryloxy, and heterocyclyloxy, etc.) may be substituted or unsubstituted unless otherwise specified. Substituents may be selected from, for example, acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amido, amino, aryl, aryloxy, azido, carbamoyl, carboalkoxy, carboxy, cyano, cycloalkyl, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonate, sulfonyl, thio, thioacylamino, thioureido, and ureido. Substituents may themselves be substituted or unsubstituted, and may be interrupted or terminated by one or more heteroatoms, such as, for example, nitrogen, sulfur, and oxygen.

[0028] In certain embodiments, the oligosaccharide may include at least one protecting group. The term "protecting group" refers to any substituent that can be used to prevent a functional group on a molecule (e.g., an amine, carboxyl, hydroxyl, hydrazine, hydrazide, semicarbazide, or thiosemicarbazide group, etc.) from undergoing chemical reaction during a chemical change elsewhere in the molecule. Protecting groups can be removed under appropriate chemical conditions. Numerous protecting groups are known to those of skill in the art, and examples of protecting groups, methods for adding them, and methods for preparing them can be found in the literature. Methods for removing them are described, for example, in Greene et al., Protective Groups in Organic Synthesis, 3 rd ed., John Wiley and Sons: New York, 1999, and Kocienski, Protecting Groups, 3 rded., Georg Thieme Verlag: Stuttgard, Germany, 2005, the disclosures of which are incorporated herein by reference. In certain embodiments, the oligosaccharides may include at least one protecting group selected from a hydroxyl protecting group, a carboxyl protecting group, and an amino protecting group. In other embodiments, the oligosaccharides may be "unprotected" and may not include any protecting groups.

[0029] Oligosaccharides may be isolated from natural sources or may be prepared by chemical or enzymatic synthesis. Oligosaccharides isolated from natural sources may be homogeneous or may be a heterogeneous mixture of related oligosaccharides. In some embodiments, oligosaccharides may be prepared by chemical or enzymatic modification of oligosaccharides isolated from natural sources ("semi-synthetic"). In some embodiments, oligosaccharides may be synthetic oligosaccharides that have the chemical structure of naturally occurring oligosaccharides.

[0030] In some embodiments, the oligosaccharide may comprise a monosaccharide that is recognized by a specific receptor. The monosaccharide that is recognized by a specific receptor may be selected from, for example, galactose, GalNac, mannose, M6P, glucose, GlcNAc, sialic acid, or sulfated sialic acid residues. The oligosaccharide may, in some embodiments, comprise at least one M6P residue, such as, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 M6P residues.

[0031] The monosaccharide recognized by a particular receptor may in some embodiments be the penultimate or terminal monosaccharide. In some embodiments, the monosaccharide recognized by a particular receptor may be a terminal galactose, mannose, M6P, glucose, GlcNAc, or sialic acid residue. The oligosaccharide in some embodiments includes at least 1, 2, 3, 4, 5, 6, 7 terminal M6P residues.

[0032] In certain embodiments, the oligosaccharide containing a reactive group may be an M6P-containing hexasaccharide of formula Ia. [ka] Chemical formula Ia

[0033] The oligosaccharide of formula Ia can be represented as butyrylhydrazin-4-yl 6-O-phosphono-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→6)-α-D-mannopyranosyl-(1→6)-[6-O-phosphono-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→3)]-β-D-mannopyranoside.

[0034] In certain embodiments, the oligosaccharide containing a reactive group may be an M6P-containing hexasaccharide of formula Ib. [ka] Chemical formula Ib

[0035] B. Aminoxy Compounds As used herein, an aminooxy compound can be any compound that includes an aminooxy group and a second reactive group, where the second reactive group can react with the first reactive group on an oligosaccharide to form a covalent bond. For example, in some embodiments, the second reactive group can be a carboxyl, ester (e.g., an active ester, etc.), acyl halide (e.g., an acid fluoride or acyl chloride, etc.), acyl azide, an anhydride, an isothiocyanate, an isocyanate, or a sulfonyl halide (e.g., a sulfonyl chloride or sulfonyl bromide, etc.) group. In other embodiments, the second reactive group can be, for example, a hydrazine group, a hydrazide group, a semicarbazide group, a thiosemicarbazide group, or an amine group.

[0036] In certain embodiments, the nitrogen of the aminooxy group of the aminooxy compound is protected by an amino-protecting group. Numerous amino-protecting groups are known to those of skill in the art, and examples of amino-protecting groups, methods for adding them, and methods for removing them can be found, for example, in Greene et al., Protective Groups in Organic Synthesis, Vol. 13, No. 1, 2002, pp. 111-115, 2002, and in J. Am. Chem. Soc., 1999, 143: 111-115, 2002. Synthesis,3 rd ed., John Wiley and Sons: New York, 1999, pp. 494-653; Kocienski, Protecting Groups, 3 rd Chapter 8 of ed., Georg Thieme Verlag: Stuttgard, Germany, 2005; Bodanszky, Principle of Peptide Synthesis, Springer Verlag: New York, 1993; Lloyd-Williams et al., Chemical Approaches to the Synthesis of Peptides and Proteins, CRC Press: Boca Rat on, FL, 1997; and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co.: Rockford, Ill., 1984, the disclosures of which are incorporated herein by reference.

[0037] In some embodiments, the aminooxy compound is selected from a compound of formula II: [ka] Chemical formula II wherein Y is a second reactive group, Z is selected from alkyl, alkenyl, alkynyl, heteroaryl, aryl, and heterocyclyl, and P is selected from an amino protecting group.

[0038] As used herein, any chemical group on an aminooxy compound (e.g., alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, acyloxy, alkoxy, aryloxy, and heterocyclyloxy, etc.) can be substituted or unsubstituted and can be blocked by one or more chemical groups, unless otherwise specified. Substituents and blocking chemical groups can be selected from, for example, acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amido, amino, aryl, aryloxy, azido, carbamoyl, carboalkoxy, carboxy, cyano, cycloalkyl, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonate, sulfonyl, thio, thioacylamino, thioureido, and ureido. The substituents may themselves be substituted or unsubstituted and may be interrupted or terminated by one or more heteroatoms such as, for example, nitrogen, sulfur, and oxygen.

[0039] In some embodiments, Y is, for example, [ka] where X is selected from halogen, azido, acyloxy, alkoxy, aryloxy, heteroaryloxy, and heterocyclyloxy.

[0040] In some embodiments, the aminooxy compound is an active ester. As used herein, an active ester is an ester that reacts to form an amide bond under mild conditions. Generally, an active ester is an ester of a relatively acidic alcohol. In some embodiments, the aminooxy compound of formula II is a compound of formula [ka] and X is selected from alkoxy, aryloxy, heteroaryloxy, and heterocyclyloxy. For example, X is [ka] [ka] You may choose from.

[0041] In other embodiments, Y is selected from, for example, hydrazide, hydrazine, thiosemicarbazide, semicarbazide, and amine groups.

[0042] In some embodiments, Z may include, for example, a carbonyl, ether, ester, or amide group. In some embodiments, Z may be an alkyl interrupted by one or more heteroatoms, such as, for example, an oligoethylene glycol. For example, Z may be monoethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, or larger oligoethylene glycols.

[0043] In some embodiments, Z may be alkyl, e.g., substituted by oxo and interrupted by one or more heteroatoms, such as an oligopeptide. For example, an oligopeptide may include one, two, three, four, five, six, or more component amino acids. The amino acids may be, e.g., α-amino acids, β-amino acids, γ-amino acids, δ-amino acids, and ω-amino acids. The amino acids may have R or S chirality at any chiral atom. Amino acids include, for example, alanine, β-alanine, α-aminoadipic acid, 2-aminobutanoic acid, 4-aminobutanoic acid, 1-aminocyclopentanecarboxylic acid, 6-aminohexanoic acid, 2-aminoheptanedioic acid, 7-aminoheptanoic acid, 2-aminoisobutyric acid, aminomethylpyrrolecarboxylic acid, 8-amino-3,6-dioxa-octanoic acid, aminopiperidinecarboxylic acid, 3-amino-propionic acid, aminoserine, aminotetrahydropyran-4-carboxylic acid, arginine, asparagine, aspartic acid, azetidinecarboxylic acid, benzothiazolylalanine, butylglycine, carnitine, 4-chlorophenylalanine, citrulline, cyclohexylalanine, cyclohexylstatin, cysteine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid, dihydroxyphenylalanine, dimethylthiazolidine carboxylic acid, glutamine, and 4-hydrazinobenzoic acid, 4-amino ...

[0044] P can be selected from amino protecting groups known to those of skill in the art. In some embodiments, P can be a carbamate protecting group, such as, for example, a (9-fluorenylmethyl)carbamate (Fmoc), (tert-butyloxy)carbamate (t-Boc), (trichloroethyl)carbamate (Troc), or allylcarbamate (Alloc) protecting group. In other embodiments, P can be a non-carbamate protecting group, such as, for example, an amide protecting group, such as a phthalimide or trifluoroacetamide protecting group.

[0045] In some embodiments, the aminooxy compound of formula II is selected from a compound of formula III: [ka] Chemical formula III In the formula, Y and P are as defined above, and n is selected from an integer ranging from 1 to 10.

[0046] In some embodiments, n can be selected from integers ranging from 1 to 4, 2 to 6, 2 to 8, 3 to 6, and 4 to 10. In an exemplary embodiment, n is 1.

[0047] In an exemplary embodiment, the aminooxy compound is t-Boc-aminooxyacetic acid tetrafluorophenyl ester, the structure of which is shown below. [ka]

[0048] In another exemplary embodiment, the aminooxy compound has the structure shown below: [ka]

[0049] C. Methods for preparing oligosaccharides containing aminooxy groups In another embodiment, the present invention provides a method for preparing an oligosaccharide containing an aminooxy group from an oligosaccharide containing a reactive group, the method comprising: (a) providing an oligosaccharide comprising a first reactive group; (b) providing an aminooxy compound that includes a second reactive group; (c) reacting a first reactive group of the oligosaccharide with a second reactive group of the aminooxy compound; thereby preparing an oligosaccharide containing an aminooxy group.

[0050] The oligosaccharide containing the first reactive compound may be, for example, any oligosaccharide containing a reactive group, as described above. In an exemplary embodiment, the oligosaccharide containing the first reactive group is an oligosaccharide of formula Ia or an oligosaccharide of formula Ib. The aminooxy compound containing the second reactive group may be any aminooxy compound containing a reactive group, as described above.

[0051] The terms "first reactive group" and "second reactive group" as used herein do not indicate any particular experimental order. That is, step (c) of reacting the first reactive group of the oligosaccharide with the second reactive group of the aminooxy compound may be accomplished by adding the reactants in any order. For example, an oligosaccharide containing a first reactive group may be added to an aminooxy compound containing a second reactive group, or vice versa. In other examples, both the oligosaccharide and the aminooxy compound may be added to the reaction vessel simultaneously.

[0052] Step (c) can be carried out under any suitable conditions known to those skilled in the art, such as solvent and temperature. In some embodiments, one or more additional reagents may be present during step (c), such as, for example, a coupling reagent and a catalyst. A coupling reagent, as used herein, is a reagent that may be used to form a covalent bond between a first reactive group and a second reactive group.

[0053] In some embodiments, the reaction conditions may include a coupling reagent, such as when the first or second reactive group is a carboxyl group. The coupling reagent may be selected from phosphonium coupling reagents such as BOP (benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate), PyBOP® (benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate), and PyBroP® (bromo-tris-pyrrolidino-phosphonium hexafluorophosphate), and HBTU ( The coupling reagent can be selected from aminium (uronium) coupling reagents such as 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), HATU (2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate). The coupling reagent can also be selected from carbodiimide coupling reagents such as, for example, DIC (1,3-diisopropylcarbodiimide), CDI (1,1'carbonyldiimidazole), and EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide). For example, in some exemplary embodiments, the coupling reagent is EDC. In certain embodiments, the reaction conditions include both a coupling reagent and a catalyst.

[0054] The reaction conditions may, in some embodiments, include a catalyst. The catalyst may be selected from any suitable catalyst known to those skilled in the art, such as, for example, DHBt-OH (3-hydroxy-1,2,3-benzotriazine-4(3H)-one), HOBt (N-hydroxybenzotriazole), DMAP (4-dimethylaminopyridine), NHS (N-hydroxysuccinimide), N-hydroxysulfosuccinimide, HONB (N-hydroxy-5-norbornene-endo-2,3-dicarboximide), or tetrabutylammonium salts, such as, for example, TBAI (tetrabutylammonium iodide). In some exemplary embodiments, the reaction conditions include catalytic DHBt-OH or catalytic NHS.

[0055] In some embodiments, the step (c) of reacting the first reactive group of the oligosaccharide with the second reactive group of the aminooxy compound results in the formation of an amide bond. Suitable conditions for the formation of an amide bond are known to those skilled in the art and are described, for example, in Chan et al., eds., Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press: New York, 2000; Bodanszky, Principles of Peptide Synthesis, Springer Verlag: New York, 1993; Llioyd-Williams et al., Chemical Approaches to the Synthesis of Peptides and Proteins, CRC Press: Boca Raton. FL, 1997; and in the catalogue of Novabiochem® (San Diego, CA).

[0056] In certain embodiments, the aminooxy compound comprises an amino-protecting group, and the method comprises the further step (d) of deprotecting the oligosaccharide comprising the aminooxy group and removing the amino-protecting group. Deprotection can occur under any suitable conditions known to those skilled in the art, for example, as described in Greene et al., Protective Groups in Organic Synthesis, 3 rd ed., John Wiley and Sons :New York, 1999, pp. 494-653, and Kocienski, Protecting Groups, 3 rd ed., Georg Thieme Verlag: Stuttgard, Germany, 2005, the inventions of which are incorporated herein by reference.

[0057] An exemplary embodiment of the method of the present invention provides a method for preparing M6P-containing oligosaccharides that contain aminooxy groups, the method comprising: (a) providing an oligosaccharide comprising a first reactive group, the oligosaccharide comprising: [ka] and (b) providing an aminooxy compound comprising a second reactive group, the aminooxy compound being selected from compounds of formula III: [ka] Chemical formula III wherein n is selected from an integer ranging from 1 to 10, P is selected from an amino protecting group, and Y is a second reactive group; (c) reacting a first reactive group of the oligosaccharide with a second reactive group of the aminooxy compound; thereby preparing an oligosaccharide containing an aminooxy group.

[0058] In some embodiments, Y in formula III is [ka] wherein X is selected from hydroxy, aryloxy, heteroaryloxy, and heterocyclyloxy. For example, in certain exemplary embodiments, X is [ka] It is.

[0059] In an exemplary embodiment, the aminooxy compound is: [ka] In certain embodiments, the first reactive group of the oligosaccharide may be reacted with the second reactive group of the aminooxy compound in the presence of a coupling agent, such as EDC, and / or a catalyst, such as DHBt-OH.

[0060] Another exemplary embodiment of the method of the present invention comprises the steps of: (a) providing an oligosaccharide comprising a first reactive group, the oligosaccharide comprising: [ka] and (b) providing an aminooxy compound comprising a second reactive group, the aminooxy compound being selected from compounds of formula III: [ka] Chemical formula III wherein n is selected from an integer ranging from 1 to 10, P is selected from an amino protecting group, and Y is a second reactive group; (c) reacting a first reactive group of the oligosaccharide with a second reactive group of the aminooxy compound; thereby preparing an oligosaccharide containing an aminooxy group.

[0061] In some embodiments, Y in formula III is a hydrazine, hydrazide, aminooxy, thiosemicarbazide, semicarbazide, or amine group. [ka] It is.

[0062] In an exemplary embodiment, the aminooxy compound is: [ka] In certain embodiments, the first reactive group of the oligosaccharide may be reacted with the second reactive group of the aminooxy compound in the presence of a coupling agent, such as EDC, and / or a catalyst, such as NHS.

[0063] II. Oligosaccharides containing aminooxy groups The present invention also provides oligosaccharides comprising aminooxy groups. In some embodiments, the present invention provides oligosaccharides comprising aminooxy groups prepared by the above-mentioned methods. Oligosaccharides comprising aminooxy groups may comprise, for example, at least two, three, four, five, six, or more monosaccharides, including, for example, at least one galactose, GalNac, mannose, M6P, glucose, GlcNAc, sialic acid, or sulfated sialic acid residue. Such oligosaccharides may be mono-, di-, tri-, quadruple-, or penta-antennary in structure and may comprise zero, one, two, three, four, or more branch points.

[0064] In some embodiments, the present invention provides an oligosaccharide comprising (1) an aminooxy group and (2) mannose-6-phosphate. The aminooxy group-containing oligosaccharide may, in some embodiments, comprise, for example, one, two, three, four, five, six, seven, eight, nine, or ten M6P residues. In some embodiments, the aminooxy group-containing oligosaccharide may comprise at least one, two, three, four, or more terminal or penultimate M6P residues.

[0065] The oligosaccharide comprising an aminooxy group is, in one embodiment, selected from the oligosaccharides of formula IV: [ka] Chemical formula IV wherein m and p are independently selected from integers ranging from 1 to 10. For example, in certain embodiments, m and p can be independently selected from integers ranging from 1 to 4, 2 to 6, 2 to 8, 3 to 6, and 4 to 10. In exemplary embodiments, m is 3 and p is 1.

[0066] In other embodiments, the aminooxy group is linked directly to the reducing end of the oligosaccharide. For example, in some embodiments, the oligosaccharide comprising the aminooxy group can be an oligosaccharide of formula V: [ka] Chemical formula V

[0067] III. Conjugation of proteins with oligosaccharides containing aminooxy groups A. Oligosaccharides The oligosaccharides conjugated to the protein can be selected from any oligosaccharide containing a reactive group, as described above, and from any oligosaccharide containing an aminooxy group, as described above. For example, in some embodiments, the oligosaccharide to be conjugated can be an oligosaccharide of Formula Ia, Formula Ib, Formula IV, or Formula V.

[0068] B. Protein The conjugation methods described herein can be broadly applied to any pure, partially purified, or fragment thereof having at least one carbonyl group (wherein the carbonyl group is a ketone or aldehyde), including isolated and recombinantly or synthetically produced proteins. The terms "pure," "purified," and "isolated" refer to a molecule that is substantially free from its natural environment. For example, a pure protein is substantially free from cellular material and / or other proteins from the cell or tissue source from which it is derived. The terms refer to preparations that are, for example, at least 70%-80%, 80%-90%, 90-95%, or at least 95%, 96%, 97%, 98%, 99%, or 100% (w / w) pure.

[0069] In other embodiments, the protein may be an enzyme that has an optimal activity, as measured by an activity assay, at a pH in the range of 1 to 7, e.g., 1 to 3, 2 to 5, 3 to 6, 4 to 5, 5 to 6, or 4 to 6. For example, the enzyme may have an optimal pH in the range of 4 to 6.

[0070] In some embodiments, the protein may be an enzyme with an isoelectric point (pI) in the range of 1 to 8, such as, for example, 1 to 3, 2 to 5, 3 to 8, 4 to 5, 5 to 6, 4 to 6, 5 to 8, 6 to 8, or 7 to 8. The pI of a protein can be measured using, for example, isoelectric focusing, gel electrophoresis.

[0071] In some embodiments, proteins containing carbonyl groups are prepared using methods such as those described in, for example, Wang et al. This can be achieved by using an expression system with an extended genetic code, as described in (E. al., Proc. Natl. Acad. Sci. USA 100:56-61 (2003). In such cases, the carbonyl group may be located on the amino acid side chain when translated.

[0072] In some embodiments, the protein having at least one carbonyl group is a protein having at least one oligosaccharide (i.e., a glycoprotein). For example, the glycoprotein having at least one carbonyl group may be obtained by oxidation of the glycoprotein by any means known to those skilled in the art. In some embodiments, for example, the glycoprotein having at least one carbonyl group may be obtained by oxidation of the glycoprotein by periodate (e.g., sodium periodate) or galactose oxidase. In such cases, the carbonyl group may be located at a glycosylation site of the protein.

[0073] In some embodiments, the protein having at least one carbonyl group is a glycoprotein, such as a therapeutic glycoprotein. The therapeutic glycoprotein may be targeted to the lysosome by conjugation with an oligosaccharide containing mannose-6-phosphate. For example, the glycoprotein may be a lysosomal enzyme, including an ERT enzyme. The enzyme may be a lysosomal hydrolase, including those listed in Table 1. In some embodiments, the lysosomal hydrolase is selected from, for example, α-glucosidase, α-galactosidase A, and acid sphingomyelinase. In some embodiments, the lysosomal hydrolase is GAA. Table 1: Examples of LSDs and corresponding lysosomal hydrolases [Table 1]

[0074] In certain embodiments, a glycoprotein may be a glycoprotein having at least one, two, three, four, five, or more N-linked or O-linked glycosylated amino acid residues, in other embodiments, a protein may have one, two, three, four, five, or more consensus sites for N-linked or O-linked glycosylation, at least one of which is glycosylated.

[0075] In certain embodiments, the protein may be a ligand for a receptor. For example, in some embodiments, the protein may be a glycoprotein that binds to a receptor that recognizes a sugar, such as, for example, mannose or mannose-6-phosphate. In some embodiments, the glycoprotein may bind to, for example, the asialoglycoprotein receptor, the cation-dependent mannose-6-phosphate receptor, the insulin-like growth factor type II / cation-independent mannose-6-phosphate receptor, or the macrophage mannose receptor.

[0076] In certain embodiments, the protein is a glycoprotein that, when conjugated to an oligosaccharide containing mannose-6-phosphate, is internalized (e.g., via CI-MPR-mediated endocytosis) by a target cell more efficiently than the corresponding unconjugated glycoprotein. For example, a conjugated glycoprotein is internalized (e.g., via CI-MPR-mediated endocytosis) more efficiently by a target cell than the corresponding unconjugated glycoprotein, e.g., at least 10%, 15%, 20% more efficiently in a given period of time. , 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90% (w / w) of a conjugated glycoprotein can be internalized more efficiently than an unconjugated glycoprotein. In other embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, or even 10 times (w / w) of a conjugated glycoprotein can be internalized compared to an unconjugated glycoprotein in a given period of time. The reference period can be, for example, 10, 30, 45 minutes, or 1, 2, 3, 5, 6, 12, 24, 48, or 72 hours or more.

[0077] C. Methods for Conjugating Oligosaccharides to Proteins The present invention provides a method for conjugating an oligosaccharide to a protein, e.g., a glycoprotein. In one embodiment, the method comprises: (a) providing an oligosaccharide comprising an aminooxy group; (b) providing a protein having at least one carbonyl group; (c) reacting the aminooxy group of the oligosaccharide with at least one carbonyl group of the protein; thereby attaching the oligosaccharide to the protein.

[0078] In some embodiments, the method further comprises adding a reducing agent to the bound lysosomal enzyme. The reducing agent may be any reducing agent known to those skilled in the art, such as, for example, sodium cyanoborohydride or sodium triacetoxyborohydride (STAB).

[0079] IV. Oligosaccharide-Protein Complexes The invention further provides an oligosaccharide-protein conjugate comprising (1) a protein, (2) an oligosaccharide, and (3) an oxime group linking the protein and the oligosaccharide. In some embodiments, the invention provides an oligosaccharide-protein conjugate prepared by the above-described method. The oligosaccharide and protein components of the conjugate can be, for example, any of the oligosaccharides and proteins described herein, and the conjugate comprises an oxime group, as shown below (the oxime group shown below is formally derived by reaction of an aminooxy group with an aldehyde group, and is also encompassed by the invention as an oxime group formally derived by reaction of an aminooxy group with a ketone group). [ka]

[0080] In certain embodiments, the oligosaccharide-protein conjugate is an oligosaccharide-glycoprotein conjugate. In certain embodiments, the oligosaccharide-protein conjugate is a conjugate of an oligosaccharide comprising at least one M6P and a lysosomal hydrolase.

[0081] V. Pharmaceutical Compositions The present disclosure provides the use of the conjugates of the present invention in the manufacture of a medicament for treating a lysosomal storage disorder. Also provided are pharmaceutical compositions comprising the oligosaccharide-protein conjugates of the present invention. In some embodiments, the pharmaceutical compositions of the present invention comprise a conjugate of at least one oligosaccharide comprising M6P and a lysosomal enzyme.

[0082] The pharmaceutical composition of the present invention may include one or more suitable pharmaceutical excipients. Standard pharmaceutical formulation techniques and excipients are known to those skilled in the art (see, for example, 2005 Physicians' Desk Reference®, Thomson Healthcare: Montvale, NJ, 2004; Remington: The Science and Practice of Pharmacy, 20th ed., Gennado et.al., Eds. Lippincott Williams & Wilkins: Philadelphia, PA, 2000). The composition may or may not include a preservative. In some embodiments, the pharmaceutical composition comprising α-galactosidase A complex may include one or more excipients, such as, for example, mannitol, sodium phosphate monobasic monohydrate, and / or sodium phosphate dibasic heptahydrate. In some embodiments, the pharmaceutical composition comprising a complex of α-glucosidase may include one or more excipients, such as, for example, mannitol, polysorbate 80, sodium phosphate dibasic heptahydrate, and / or sodium phosphate monobasic monohydrate.

[0083] The pharmaceutical composition may comprise any of the conjugates described herein as a single active compound or in combination with other compounds, compositions, or biological materials.For example, the pharmaceutical composition may also comprise one or more small molecules useful for treating LSD and / or treating side effects associated with LSD.In some embodiments, the composition may comprise miglustat and / or one or more compounds described, for example, in U.S. Patent Application Publication Nos. 2003 / 0050299, 2003 / 0153768, 2005 / 0222244, and 2005 / 0267094.

[0084] The formulation of the pharmaceutical composition may vary based on the intended route of administration and other parameters (see, for example, Rowe et al., Handbook of Pharmaceutical Excipients, 4th ed., APha Publications, 2003). In some embodiments, the composition may be a sterile, non-pyrogenic, white or off-white lyophilized cake or powder administered by intravenous injection upon reconstitution with Sterile Water for Injection, USP.

[0085] Administration of the pharmaceutical compositions of the present invention is not limited to any particular delivery system and may include, but is not limited to, parenteral (including subcutaneous, intravenous, intracranial, intraspinal, intraarticular, intramuscular, intrathecal, or intraperitoneal injection), transdermal, or oral (e.g., capsules, suspensions, or tablets) administration. Administration to an individual may occur in single or multiple doses, and in any of a variety of physiologically acceptable salt forms, and / or with acceptable pharmaceutical carriers and / or excipients as part of a pharmaceutical composition.

[0086] The conjugates described herein are administered in a therapeutically effective amount. Generally, the therapeutically effective amount may vary depending on the age, health, and sex of the patient, as well as the severity of the patient's condition. The dosage may be determined by the physician and may be adjusted as necessary to suit the observed outcome of the treatment. The toxicity and therapeutic effectiveness of such compounds may be determined, for example, by the LD 50 (lethal dose for 50% of the population), and ED 50 The LD (the dose that is therapeutically effective in 50% of the population) can be determined by standard formulation procedures in vitro (i.e., in cell culture) or in vivo (i.e., in experimental animal models). The dose ratio between toxic and therapeutic effects is the therapeutic index (or therapeutic ratio), and is known as the LD 50 and ED 50 Conjugates described herein exhibit therapeutic indices of at least 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 20. Conjugates exhibiting large therapeutic indices are preferred.

[0087] The data obtained from in vitro assays and animal studies can be used, for example, to quantify a range of dosages for use in humans. Doses of such compounds are administered at levels that result in low, little, or no toxicity. 50 Preferably, the circulating concentration of the compound is within a range including the IC50, IC60, IC70, IC80, IC90, IC10, IC11, IC12, IC13, IC24, IC15, IC25, IC26, IC31, IC42, IC16, IC27, IC17, IC28, IC31, IC29, IC28, IC32, IC41, IC50, IC61, IC12, IC24, IC15, IC25, IC16, IC26, IC17, IC27, IC18, IC28, IC31, IC19, IC29, IC29, IC31, IC41, IC50, IC61, IC27, IC12, IC28, IC13, IC29, IC29, IC31, IC29, IC31, IC41, IC50, IC61, IC27, IC14, IC28, IC15, IC29, IC29, IC31, IC41, IC50, IC61, IC29, IC10, IC28, IC11, IC29, IC29, IC31, IC29, IC31, IC41, IC50, IC61, IC29, IC10, IC29, IC29, IC31, IC41, IC50, IC61, IC29, IC10, IC29, IC29, IC31, IC29, IC10 ...41, IC50, IC61, IC29, IC10, IC29, IC29, IC31, IC29 50 The compound may be formulated in animal models to achieve a circulating plasma concentration range that includes (i.e., the concentration of the test conjugate that achieves a half-maximal inhibition of symptoms). Plasma levels may be measured, for example, by high performance liquid chromatography or a suitable enzyme activity assay. The effect of any particular dosage can be monitored by a suitable bioassay endpoint.

[0088] Unless otherwise specified, the complexes of the invention may be administered at a dose of about 1 μg / kg to 500 mg / kg, depending on the severity of the condition and progression of the disease. For example, the proteinaceous compounds may be administered on an outpatient basis by slow intravenous infusion, for example every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days, or for example, weekly, biweekly, monthly or bimonthly administration. The appropriate therapeutically effective dose of the compound is selected by the treating clinician and may range from about 1 μg / kg to 500 mg / kg, 1 μg / kg to 10 mg / kg, 1 μg / kg to 1 mg / kg, 10 μg / kg to 100 μg / kg, 100 μg / kg to 1 mg / kg, 500 μg / kg to 5 mg / kg.

[0089] For example, the α-galactosidase A conjugate may be administered by intravenous infusion at an infusion rate of, for example, 10, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 mg / h or less, for example, at a dose of 1.0 mg / kg of body weight every two weeks. In other examples, the α-glucosidase conjugate may be administered by intravenous injection at a dose of, for example, 20 mg / kg or 40 mg / kg every two weeks, for example, over about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In some embodiments, the administration rate of α-glucosidase may begin at, for example, 1 mg / kg / h and then be increased by 2 mg / kg / h every 30 minutes to a maximum of, for example, 7 mg / kg / h after determining patient tolerance to the infusion rate. Additionally, examples of specific dosages can be found in the Physicians' Desk Reference®.

[0090] VI. METHODS FOR TREATING LYSOSOME STORAGE DISORDERS The present invention provides methods for treating lysosomal storage disorders, for example, as disclosed in Table 1. In some embodiments, the present invention provides the use of a conjugate described herein for treating a lysosomal storage disorder in a patient in need of such treatment. The present invention further provides methods for targeting proteins to lysosomes by conjugation to oligosaccharides comprising mannose-6-phosphate.

[0091] In one embodiment, the method comprises administering to a mammal having a lysosomal storage disorder a therapeutically effective amount of an oligosaccharide-glycoprotein conjugate of the invention. The oligosaccharide-glycoprotein conjugate can be a conjugate of a lysosomal enzyme, such as a lysosomal enzyme listed in Table 1, with an oligosaccharide comprising mannose-6-phosphate. In one embodiment, the method comprises administering to a patient in need of treatment a pharmaceutical composition comprising at least one of the conjugates described herein.

[0092] In certain embodiments, the conjugates of the invention are administered in combination with one or more additional therapies. The one or more additional treatments may be administered simultaneously (including concurrent administration as a combined preparation), prior to, or following administration of the conjugate of the invention.

[0093] In some embodiments, the patient may be treated (before, after, or during treatment with the conjugates of the invention) with antipyretics, antihistamines, and / or immunosuppressants. In some embodiments, the patient may be treated with antipyretics, antihistamines, and / or immunosuppressants before treatment with the oligosaccharide-glycoprotein conjugates of the invention to reduce or prevent reactions associated with infusion administration. For example, the patient may be pretreated with one or more of acetaminophen, azathioprine, cyclophosphamide, cyclosporine A, methotrexate, mycophenolate mofetil, oral steroids, or rapamycin.

[0094] In some embodiments, the patient may be treated with one or more of acetaminophen, azathioprine, cyclophosphamide, cyclosporine A, methotrexate, mycophenolate mofetil, oral steroids, or rapamycin, e.g., for the first one, two, three, four, five, six, seven, eight, nine, ten or more occurrences of treatment with a conjugate of the invention, e.g., at or about t=0 (the time of administration of the conjugate of the invention) and / or t=12, 24, 36, 48, 60, 72, 96, 120, and 144 hours. For example, in some embodiments, a patient with Fabry or Pompe disease may be treated with methotrexate (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 25, 30, 35, 40, 50, 60, 70, 80 mg / kg or more of methotrexate) at or about t=0, 24, and 48 hours, e.g., for the first 1, 2, 3, 4, 5, 6, 7, 8 weeks of treatment with a conjugate of the invention. In some embodiments, immune tolerance to the conjugates of the invention may be induced in a patient with a lysosomal storage disorder, e.g., mucopolysaccharidosis type I, by treatment with cyclosporine A and azathioprine. For example, patients can be treated with cyclosporine A and azathioprine, as described in Kakkis et al., Proc. Natl. Acad. Sci USA 101:829-834 (2004).

[0095] In some embodiments, the patient may be treated (before, after, or during treatment with the complex of the present invention) with small molecule therapy and / or gene therapy, including, for example, small molecule therapy and gene therapy for the treatment of lysosomal storage disorders. Small molecule therapy may include administration of one or more compounds described, for example, in U.S. Patent Application Publication Nos. 2003 / 0050299, 2003 / 0153768, 2005 / 0222244, and 2005 / 0267094. Gene therapy may be performed, for example, as described in U.S. Patent Application Publication Nos. 5,952,516, 6,066,626, 6,071,890, and 6,287,857, and U.S. Patent Application Publication No. 2003 / 0087868.

[0096] The terms "treatment," "method of treatment," and their cognate terms refer to both therapeutic therapies and prophylactic / preventative measures. Thus, those in need of treatment can include those already with a particular lysosomal storage disease, as well as those at risk for the disease (i.e., those who may eventually suffer from the disorder or suffer from a particular symptom of the disorder).

[0097] A treatment method will prevent or improve symptoms or otherwise result in a desired biological outcome, which can be assessed by amelioration of clinical signs or delay in onset of disease, an increase in metabolically defective enzyme activity, and / or a decrease in the accumulated level of a substrate for the metabolically defective enzyme.

[0098] The complexes of the present invention are useful for treating various lysosomal storage disorders in humans or animals. For example, administration of the complexes can be used to increase the enzyme activity that is deficient in the patient, for example by at least 10%. The increased enzyme activity can be determined, for example, by a reduction in clinical symptoms or by a suitable clinical or biological assay.

[0099] GAA complexes may be administered to treat Pompe disease (also known as acid alpha-glucosidase deficiency, acid maltase deficiency, glycogen storage disease type II, glycogen storage disease type II, and lysosomal alpha-glucosidase deficiency). Increased GAA activity may be determined by biochemical (see, e.g., Zhu et al., J. Biom. Chem. 279:50336-50341 (2004)) or histological observations of decreased lysosomal glycogen storage, for example, in cardiac myocytes, skeletal muscle cells, or skin fibroblasts. GAA activity may also be assayed, for example, in muscle biopsies, cultured skin fibroblasts, lymphocytes, and dried blood spots. Dried blood spot assays are described, for example, in Umpathysivam et al., Clin. Chem. 47:1378-1383 (2001), and Li et al., Clin. Chem. 50:1785-1796 (2004). Treatment of Pompe disease may also be assessed, for example, by serum levels of creatine kinase, increases in motor function (e.g., as assessed by the Alberta Infant Motor Scale), changes in left ventricular mass index measured by echocardiogram, and cardiac electrical activity measured by electrocardiogram. Administration of GAA complexes may result in a reduction in one or more symptoms of Pompe disease, such as cardiac hypertrophy, cardiomyopathy, daytime somnolence, exertional dyspnea, failure to thrive, difficulty feeding, "hypotonia," gait disturbances, headaches, hypotension, organomegaly (e.g., enlargement of the heart, tongue, liver), lordosis, loss of balance, low back pain, morning headaches, muscle weakness, respiratory insufficiency, scapular winging, scoliosis, decreased deep tendon reflexes, sleep apnea, susceptibility to respiratory infections, and vomiting.

[0100] In another embodiment, a complex of α-galactosidase A and an oligosaccharide containing M6P is administered to treat Fabry disease. Fabry disease, or Anderson-Fabry disease, is a rare X-linked lysosomal storage disorder characterized by a deficiency of α-galactosidase A, which leads to the accumulation of globotriaosylceramide (GL3) and other neutral glycosphingolipids in the lysosomes of visceral tissues, endothelium, perithelium, and muscle cells. The accumulation of neutral glycosphingolipids in the vasculature leads to narrowing and dilation of blood vessels, ultimately leading to ischemia and infarction.

[0101] Administration of α-galactosidase A complex may result in, for example, acrotactic dysesthesias, angina, angiokeratoma, arrhythmias, gait ataxia, burning and / or tingling pain in the hands and feet, cataracts, cold intolerance, conduction abnormalities, corneal whorling, coronary artery disease, dementia, depression, diarrhea, dilated ventricles, dizziness, cardiac hypertrophy, cardiomyopathy, diplopia, dysarthria, fatigue, fever due to elevated erythrocyte sedimentation rate, hearing problems, heart disease, heart valve problems, heat intolerance, hemiparesis, The administration of α-galactosidase A complex may result in a reduction in one or more clinical symptoms of Fabry disease, including ataxia, hemiparesis, reduced sweating, impaired sweating, infarction, ischemia, joint pain, kidney disease, left ventricular hypertrophy, lens abnormalities, lens opacity, lipuria, muscle weakness, myocardial infarction, nausea, nystagmus, pain (e.g., severe pain that spreads throughout the body), polydipsia, proteinuria, postprandial pain, renal failure, retinal abnormalities, tinnitus, stomach pain, ST-T changes, stroke, uremia, valvular disease, dizziness, vomiting, and weakness. The administration of α-galactosidase A complex may result in an increase in α-galactosidase A activity, for example, in plasma, tears, white blood cells, tissue biopsies, or cultured skin fibroblasts. The administration of α-galactosidase A complex may also result in a histological finding of a reduction (e.g., at least 10%) or lack of increase in birefringent lipid globules. It may also result in a reduction in lipid globules in the urinary sediment, improved renal function as measured by serum creatinine concentration levels or creatinine clearance, and a reduction in proteinuria. Administration of α-galactosidase A complex has been shown to improve kidney, heart, and skin function. This may result in a decrease in GL3 contamination in the capillary endothelium of the skin. Additional assays for measuring the effectiveness of treatments for Fabry disease can be found, for example, in MacDermotto et al., J. Med. Genet. 38:750-760 (2001).

[0102] In yet another aspect, the complex of acid sphingomyelinase is administered to treat Niemann-Pick disease or acid sphingomyelinase deficiency.The administration of acid sphingomyelinase complex can result in the reduction of one or more clinical symptoms of Niemann-Pick disease, including, for example, abnormal cholesterol level, abnormal lipid level, ataxia, blood abnormality, cherry red spots in the eye, frequent lung infections, growth retardation, hepatosplenomegaly, decreased platelets, lymphadenopathy, peripheral neuropathy, problems with lung function, shortness of breath, changes in skin pigmentation, or xanthomas.In some embodiments, the complex can be administered intracranially.

[0103] An alternative embodiment relates to the treatment of mucopolysaccharidosis type I (including, for example, Hurler and Hurler / Scheie types of MPS I) with a conjugate comprising α-L-iduronidase. Administration of an α-L-iduronidase conjugate may result in a reduction in one or more clinical symptoms of MPS I including, for example, aortic regurgitation, aortic stenosis, carpal tunnel syndrome, chronic rhinitis, conductive hearing loss, constipation, corneal opacity, developmental delay, diarrhea, abdominal distension, lumbar kyphosis, dorsal deformity, hepatosplenomegaly, hydrocephalus, inguinal hernia, kyphosis, mental retardation, mitral regurgitation, mitral stenosis, night blindness, open-angle glaucoma, hand dysfunction, progressive arthropathy, recurrent respiratory infections, respiratory failure, retinal degeneration, scoliosis, sensorineural hearing loss, severe back pain, rhinorrhea, sleep apnea, spinal cord compression, thenar atrophy, umbilical hernia, and upper respiratory tract complications.

[0104] The foregoing and following descriptions are exemplary and explanatory and are not intended to be limiting of the invention described.

[0105] Examples 1-4 below represent the synthetic route depicted in Figure 1. Compounds 1, 2, 3, and 4, as used below, have the chemical structures depicted in Figure 1. EXAMPLES

[0106] Synthesis of oligosaccharide 3 100 mg of oligosaccharide 1 (MW = 1250; bisM6P-hydrazide (Biomira 100 mg of t-Boc-aminooxyacetic acid tetrafluorophenyl ester 2 (Invitrogen Corp.; Carlsbad, CA; Cat. No. B3030) was dissolved in 7.5 ml of DMSO. 15 ml of the oligosaccharide solution was then mixed with 7.5 ml of 2 in a glass bottle such that the molar ratio of compound 2:compound 1 in the resulting solution was 4:1. 744 μl of DHBt-OH (from a 32.06 mg / ml stock in DMSO) was added to the reaction mixture in the glass bottle such that the final ratio of compound 2:DHBt-OH was 1:0.5. The mixture was gently shaken at room temperature (25° C.) at 100 RPM overnight for approximately 18 hours.

[0107] The following morning, 10 μl of the reaction mixture was removed for Dionex analysis to confirm completion of the reaction, which showed 100% conversion of 1 to 3, as shown in FIG. EXAMPLES

[0108] Purification of oligosaccharide 3 Method A The reaction mixture was diluted with an equal volume of HO and dialysis tubes with a molecular weight cut-off of 1000 Daltons (SpectraPor) were placed in 4 L of HO for at least 3 h at 4 °C. The samples were then dialyzed twice in PBS (Inc.) and lyophilized.

[0109] Method B A Sephadex G-10 gel permeation chromatography column with a bed volume of 225 ml was filled and equilibrated with deionized water. The reaction mixture was loaded onto the column, drained by gravity, and then eluted with deionized water at a flow rate of 75 ml per hour. Fractions of 4.5 ml were collected by a fraction collector. Fractions 10-23, containing oligosaccharide 3, were collected, combined, and lyophilized. Other small molecules, including t-Boc-AOAA, DHBt-OH, and DMSO, were eluted in the latter fractions and discarded. EXAMPLES

[0110] Deprotection of oligosaccharide 3 The t-Boc groups of the lyophilized samples were deprotected in 5 ml of 50% trifluoroacetic acid (TFA) in dichloromethane (DCM) with gentle shaking at 100 RPM in a glass vial for 30 minutes, and the TFA / DCM was then removed by a stream of N2 in a chemical hood. EXAMPLES

[0111] Purification of oligosaccharide 4 Method A After removing the TFA / DCM, the residue was dissolved in 10 ml of 0.5 M sodium acetate buffer at pH 5 and transferred to a dialysis tubing with a molecular weight cut-off of 1000 Daltons. The bottle was washed with 4 ml of the same buffer and then transferred to the dialysis tubing. The sample was dialyzed twice against 3 L of 25 mM sodium acetate buffer at pH 7 for at least 3 hours and then transferred to 4 L of ice-cold H2O for overnight dialysis. The sample was returned from the dialysis tubing and lyophilized.

[0112] Method B After removing TFA / DCM, the residue was dissolved in 5 ml of 0.5 M sodium acetate buffer, pH 7.5, and loaded onto a Sephadex G-10 gel permeation chromatography column as in Example 2, Method B. The reaction mixture was loaded onto the column, drained by gravity, and then eluted with deionized water at a flow rate of 75 ml per hour. Fractions of 4.5 ml were collected by a fraction collector. Fractions 10-23, containing purified oligosaccharide 4, were collected, combined, and lyophilized. The yield of oligosaccharide 4 was higher with purification by Method B than with Method A.

[0113] The final product obtained by method B was analyzed by Dionex chromatography (Figure 2C) and the identity of the product was confirmed by mass spectrometry (Figure 3B). Some impurities were present in the spectra of Figures 2C and 3B. EXAMPLES

[0114] Binding of oligosaccharide 4 to GAA Oxidation of GAA Lyophilized recombinant human GAA (rhGAA) was reconstituted in H2O and dialyzed four times against 4 L of 100 mM acetate buffer (pH 5.6) to completely remove mannitol. After dialysis, rhGAA was oxidized with 7.5 mM sodium periodate from a 100 mM stock solution in 100 mM acetate buffer. After 30 min on ice at 4°C, glycerol was added and the sample was mixed on ice for 10 min to decompose excess sodium periodate. The oxidized material was then dialyzed overnight against an aqueous buffer (e.g., 100 mM sodium acetate).

[0115] join A solution of oligosaccharide 4 in an aqueous buffer (e.g., 100 mM sodium acetate, pH 5.6) was mixed with oxidized GAA and incubated at 37° C. for 4 hours to produce oligosaccharide-GAA conjugate 5. The reaction mixture was then diafiltered against 25 mM sodium phosphate buffer, pH 6.25 to remove unconjugated bisM6P glycans, and then adjusted with GAA formulation buffer (25 mM sodium phosphate buffer, pH 6.25, 2% mannitol, 0.005% Tween-80). EXAMPLES

[0116] Characterization of the GAA complex Detection of M6P The degree of oligosaccharide conjugation was measured by assaying complex 5 for binding to an M6P receptor column to which glycoprotein-starved M6P does not bind. Five micrograms of complex 5 was loaded onto a pre-equilibrated CI-MPR-Sepharose column (prepared by binding CI-MPR isolated from fetal bovine serum to Affigel-10), then washed with CI-MPR binding buffer for 11 x 2 ml fractions and eluted with CI-MPR binding buffer containing 5 mM M6P for 7 x 2 ml fractions. A total of 18 fractions were collected and assayed for enzyme activity.

[0117] Monosaccharide Analysis Conjugate 5 is treated with 4 N trifluoroacetic acid to hydrolyze oligosaccharides, followed by high pH anion exchange chromatography analysis with pulsed amperometric detection (PAD) using a BioLC liquid chromatography system (Dionex). Monosaccharide content is estimated from a monosaccharide standard curve using premixed monosaccharide standards (Dionex).

[0118] specific activity GAA activity is measured using a fluorometric assay in black 96-well microplates using 4-methylumbelliferyl-α-D-glucoside as substrate. Dilutions of complex 5 are added to triplicate microtiter plates. 4-methylumbelliferyl-α-D-glucoside is added to each sample. The 96-well plates are incubated for 30 minutes in a 37° C. incubator. Product release is detected fluorometrically and compared to a standard curve generated by measuring the fluorescence of known standard amounts. The reaction is quenched by adding 125 μL of 1.0 M glycine-carbonate buffer, pH 10.5, to all wells. Specific activity is defined as nmoles of product released per hour and per mg.

[0119] Internalization by L6 myoblasts Cells (ATCC CRL-1458) were cultured at 5.0 × 10 5 Cells / well were seeded in 6-well plates and grown to confluency. Cells were cultured with 0-100 nM GAA (complex 5 or uncomplexed rhGAA) for 16 hours in DMEM + 1% heat-inactivated FBS + 10 mM Hepes pH 6.7. After loading, cells were washed with 3x PBS containing 5 mM M6P and lysed with 0.25% Triton X-100 for 1 hour on ice. Lysates were centrifuged at 18000 g for 5 minutes and tested for specific activity. See, e.g., Zhu et al., J. Biol. Chem. 279:50336-50341 (2004); Zhu et al., Biochem. J. 389:619-628 (2005).

[0120] All references cited herein are hereby incorporated by reference in their entirety. Publications and patents or patent applications incorporated by reference To the extent that any provision herein conflicts with any invention contained herein, the present specification is intended to supersede and / or take precedence over any such conflicting matter.

[0121] All numerical values ​​expressing amounts of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending on the desired properties sought to be obtained by the present invention. At the very least, and without intending to limit the application of the term equivalents to the scope of the claims, each numerical parameter should be construed in light of significant digits and ordinary rounding approaches.

[0122] Numerous modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are provided by way of example only and are not intended to be limiting in any way. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

1. 1. A method for preparing oligosaccharides containing aminooxy groups, comprising the steps of: (a) providing an oligosaccharide comprising a first reactive group; (b) providing an aminooxy compound comprising an aminooxy group and a second reactive group; (c) reacting the first reactive group of the oligosaccharide with the second reactive group of the aminooxy compound; thereby preparing said aminooxy group-containing oligosaccharide.

2. 2. The method of claim 1, wherein the first and second reactive groups are selected from hydrazine, hydrazide, semicarbazide, thiosemicarbazide, amine, carboxyl, ester, acyl halide, acyl azide, alkyl halide, anhydride, isothiocyanate, isocyanate, and sulfonyl halide groups.

3. The method of claim 2 , wherein the first reactive group is a hydrazide group.

4. The method of claim 2 , wherein the first reactive group is a carboxyl group.

5. The aminooxy compound is selected from compounds of formula II: 【Chemistry 1】 Chemical formula II 2. The method of claim 1, wherein Y is the second reactive group, Z is selected from alkyl, alkenyl, alkynyl, heteroaryl, aryl, and heterocyclyl, and P is selected from an amino protecting group.

6. The method of claim 5 , wherein P is selected from a carbamate protecting group.

7. The method of claim 6, wherein P is tert-butyoxycarbonyl.

8. Y is, 【Chemistry 2】 【Chemistry 3】 6. The method of claim 5, wherein X is selected from halogen, azido, acyloxy, alkoxy, aryloxy, heteroaryloxy, and heterocyclyloxy.

9. Y is (b). 【Chemistry 4】 The method according to claim 8, wherein

10. 10. The method of claim 9, wherein X is selected from aryloxy, heteroaryloxy, and heterocyclyloxy.

11. X is, 【Chemistry 5】 The method according to claim 10, wherein

12. The aminooxy compound is selected from compounds of formula III: 【Chemistry 6】 Chemical formula III 6. The method of claim 5, wherein Y is a second reactive group, n is selected from an integer ranging from 1 to 10, and P is selected from an amino protecting group.

13. 13. The method of claim 12, wherein n is 1.

14. 13. The method of claim 12, wherein Y is a carboxyl, ester, acyl halide, acyl azide, alkyl halide, anhydride, isothiocyanate, isocyanate, or sulfonyl halide group.

15. The aminooxy compound is 【Chemistry 7】 The method of claim 14, wherein

16. 13. The method of claim 12, wherein Y is a hydrazine, hydrazide, semicarbazide, thiosemicarbazide, or amine group.

17. The aminooxy compound is 【Chemistry 8】 17. The method of claim 16, wherein:

18. The oligosaccharide is 【Chemistry 9】 The method of claim 1, wherein

19. The oligosaccharide is 【Chemistry 10】 The method of claim 1, wherein

20. 2. The method of claim 1, wherein the aminooxy compound comprises an amino protecting group, the method further comprising the step of (d) deprotecting the oligosaccharide comprising the aminooxy group.

21. 2. An oligosaccharide comprising an aminooxy group produced by the method of claim 1.

22. 1. A method for preparing oligosaccharides containing aminooxy groups, comprising the steps of: (a) providing an oligosaccharide comprising a first reactive group, said oligosaccharide comprising: 【Chemistry 11】 and (b) providing an aminooxy compound comprising an aminooxy group and a second reactive group, said aminooxy compound being selected from compounds of formula III: 【Chemistry 12】 Chemical formula III wherein n is selected from an integer ranging from 1 to 10, P is selected from an amino protecting group, and Y is said second reactive group; (c) reacting the first reactive group of the oligosaccharide with the second reactive group of the aminooxy compound; thereby preparing an oligosaccharide comprising said aminooxy group.

23. Y is, 【Chemistry 13】 23. The method of claim 22, wherein X is selected from hydroxy, aryloxy, heteroaryloxy, and heterocyclyloxy.

24. X is, 【Chemistry 14】 24. The method of claim 23, wherein:

25. The aminooxy compound is 【Chemistry 15】 25. The method of claim 24, wherein:

26. 26. The method of claim 25, wherein step (c) comprises reacting the first reactive group of the oligosaccharide with the second reactive group of the aminooxy compound in the presence of a coupling reagent and / or a catalyst.

27. The catalyst is 【Chemistry 16】 27. The method of claim 26, wherein:

28. 1. A method for preparing oligosaccharides containing aminooxy groups, comprising the steps of: (a) providing an oligosaccharide comprising a first reactive group, said oligosaccharide comprising: 【Chemistry 17】 and (b) providing an aminooxy compound comprising an aminooxy group and a second reactive group, said aminooxy compound being selected from compounds of formula III: 【Chemistry 18】 Chemical formula III wherein n is selected from an integer ranging from 1 to 10, P is selected from an amino protecting group, and Y is said second reactive group; (c) reacting the first reactive group of the oligosaccharide with the second reactive group of the aminooxy compound; thereby preparing an oligosaccharide comprising said aminooxy group.

29. 29. The method of claim 28, wherein Y is a hydrazine, hydrazide, aminooxy, thiosemicarbazide, semicarbazide, or amine group.

30. The aminooxy compound is 【Chemistry 19】 30. The method of claim 29, wherein:

31. 31. The method of claim 30, wherein step (c) comprises reacting the first reactive group of the oligosaccharide with the second reactive group of the aminooxy compound in the presence of a coupling reagent and / or a catalyst.

32. 32. The method of claim 31, wherein the coupling reagent is 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and the catalyst is N-hydroxysuccinimide (NHS).

33. 1. A method for conjugating an oligosaccharide to a protein, comprising the steps of: (a) providing an oligosaccharide comprising an aminooxy group; (b) providing a protein having at least one carbonyl group; (c) reacting the aminooxy group of the oligosaccharide with the at least one carbonyl group of the protein; thereby attaching the oligosaccharide to the protein, and the aminooxy group-containing oligosaccharide comprises 【Chemistry 20】 or 【Chemistry 21】 That is, the method.

34. 34. The method of claim 33, wherein the protein is a glycoprotein.

35. 35. The method of claim 34, wherein the glycoprotein is a lysosomal enzyme.

36. 36. The method of claim 35, wherein the lysosomal enzyme is acid α-glucosidase, α-galactosidase A, acid sphingomyelinase, or α-L-iduronidase.

37. 37. The method of claim 36, wherein the lysosomal enzyme is acid alpha-glucosidase.

38. The oligosaccharide containing an aminooxy group is 【Chemical 22】 34. The method of claim 33, wherein:

39. The oligosaccharide containing an aminooxy group is 【Chemistry 23】 34. The method of claim 33, wherein:

40. 34. An oligosaccharide-protein conjugate produced by the method of claim 33.

41. An oligosaccharide of formula IV: 【Chemistry 24】 Chemical formula IV wherein m and p are independently selected from integers ranging from 1 to 10.

42. 42. The oligosaccharide of claim 41, wherein m is 3.

43. 42. The oligosaccharide of claim 41, wherein p is 1.

44. 42. The oligosaccharide of claim 41, wherein m is 3 and p is 1.

45. An oligosaccharide of formula V. 【Chemistry 25】 Chemical formula V

46. 1. An oligosaccharide-protein conjugate comprising: (1) a protein; (2) an oligosaccharide; and (3) an oxime group linking the protein and the oligosaccharide, wherein the oligosaccharide is an oligosaccharide of formula VI: 【Chemistry 26】 Chemical formula VI

47. The oligosaccharide-protein conjugate of claim 46, wherein the protein is a glycoprotein.

48. The oligosaccharide-protein conjugate of claim 47, wherein the glycoprotein is a lysosomal enzyme.

49. The oligosaccharide-protein conjugate of claim 48, wherein the lysosomal enzyme is acid α-glucosidase, α-galactosidase A, acid sphingomyelinase, or α-L-iduronidase.

50. The oligosaccharide-protein conjugate of claim 49, wherein the lysosomal enzyme is acid α-glucosidase.

51. A pharmaceutical composition comprising the oligosaccharide-glycoprotein conjugate of claim 48 and an excipient.

52. 50. A method for treating a lysosomal storage disorder, comprising administering to a mammal the oligosaccharide-glycoprotein conjugate of claim 48, wherein the protein is a lysosomal enzyme.

53. 53. The method of claim 52, further comprising the step of administering methotrexate to said mammal before, after, or during treatment with said oligosaccharide-glycoprotein conjugate.

54. 49. Use of the conjugate of claim 48 for treating a lysosomal storage disorder in a patient in need of such treatment.

55. 49. Use of the conjugate of claim 48 in the manufacture of a medicament for treating a lysosomal storage disorder.

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