U.S. patent application number 15/280864 was filed with the patent office on 2017-01-19 for oligosaccharides comprising an aminooxy group and conjugates thereof.
The applicant listed for this patent is Genzyme Corporation. Invention is credited to Luis Z. AVILA, Seng H. CHENG, Canwen JIANG, Yunxiang ZHU.
Application Number | 20170014520 15/280864 |
Document ID | / |
Family ID | 39473250 |
Filed Date | 2017-01-19 |
United States Patent
Application |
20170014520 |
Kind Code |
A1 |
ZHU; Yunxiang ; et
al. |
January 19, 2017 |
OLIGOSACCHARIDES COMPRISING AN AMINOOXY GROUP AND CONJUGATES
THEREOF
Abstract
The invention provides methods for the synthesis of
oligosaccharides comprising an aminooxy group. The invention
further provides oligosaccharides comprising an aminooxy group,
methods for coupling oligosaccharides comprising an aminooxy group
to glycoproteins, and oligosaccharide-protein conjugates. Also
provided are methods of treating a lysosomal storage disorder in a
mammal by administration of an oligosaccharide-protein
conjugate.
Inventors: |
ZHU; Yunxiang; (Bridgewater,
NJ) ; CHENG; Seng H.; (Natick, MA) ; JIANG;
Canwen; (Southborough, MA) ; AVILA; Luis Z.;
(Bridgewater, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genzyme Corporation |
Cambridge |
MA |
US |
|
|
Family ID: |
39473250 |
Appl. No.: |
15/280864 |
Filed: |
September 29, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14272960 |
May 8, 2014 |
9469850 |
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15280864 |
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12523631 |
Aug 20, 2009 |
8759501 |
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PCT/US2008/051429 |
Jan 18, 2008 |
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14272960 |
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60885471 |
Jan 18, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07H 15/04 20130101;
C12N 9/16 20130101; A61K 38/47 20130101; C12Y 302/01076 20130101;
A61P 3/00 20180101; C12N 9/2408 20130101; A61P 21/00 20180101; A61P
43/00 20180101; C12Y 302/01022 20130101; C12N 9/96 20130101; A61K
47/549 20170801; C12Y 301/04012 20130101; C12Y 302/0102
20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; C12N 9/26 20060101 C12N009/26; C12N 9/96 20060101
C12N009/96; A61K 38/47 20060101 A61K038/47 |
Claims
1-55. (canceled)
56. A method of coupling an oligosaccharide to a protein,
comprising: (a) providing an oligosaccharide comprising an aminooxy
group; (b) providing a protein having at least one carbonyl group;
and (c) reacting the aminooxy group of the oligosaccharide with the
at least one carbonyl group of the protein, thereby coupling the
oligosaccharide to the protein, wherein the oligosaccharide
comprising an aminooxy group is ##STR00027## wherein m is an
integer from 1 to 10, and p is an integer from 1 to 10.
57. The method of claim 56, wherein the protein is a glycoprotein
and the at least one carbonyl group is obtained by oxidation of the
glycoprotein with periodate.
58. The method of claim 56, wherein the protein is a lysosomal
enzyme.
59. The method of claim 58, wherein the lysosomal enzyme is acid
.alpha.-glucosidase, .alpha.-galactosidase A, acid sphinogyelinase,
or .alpha.-L-iduronidase.
60. The method of claim 58, wherein the lysosomal enzyme is acid
.alpha.-glucosidase.
61. The method of claim 56, wherein m is 3.
62. The method of claim 56, wherein p is 1.
63. The method of claim 56, wherein m is 3 and p is 1.
64. The method of claim 63, wherein the protein is acid
.alpha.-glucosidase.
65. An oligosaccharide-protein conjugate produced by the method of
claim 56.
66. A pharmaceutical composition comprising the
oligosaccharide-protein conjugate of claim 65 and an excipient.
Description
[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 invention relates generally to methods for the synthesis
of oligosaccharides comprising an aminooxy group from
oligosaccharides comprising a reactive group. In another
embodiment, the invention further relates to oligosaccharides
comprising an aminooxy group. The invention also relates to methods
of conjugating oligosaccharides comprising an aminooxy group to
proteins, including glycoproteins (such as, e.g., lysosomal
enzymes), and to compositions of oligosaccharide-protein
conjugates, including oligosaccharide-glycoprotein conjugates.
Another embodiment of the invention relates to methods of treating
lysosomal storage disorders using such oligosaccharide-lysosomal
enzyme conjugates.
[0003] Lysosomal storage disorders (LSDs) are a class of rare
metabolic disorders comprising over forty genetic diseases
involving a deficiency in the activity of lysosomal hydrolases. A
hallmark feature of LSDs is the abnormal accumulation of lysosomal
metabolites, which leads to the formation of large numbers of
distended lysosomes.
[0004] LSDs can be treated by administration of the active version
of the enzyme deficient in the patient, a process termed enzyme
replacement therapy (ERT). The administered replacement enzyme
bearing a terminal mannose-6-phosphate (M6P) is taken up by target
cells through cell-surface-associated cation-independent M6P
receptor (CI-MPR)-mediated endocytosis, and directed to the
lysosome.
[0005] In general, poorly phosphorylated replacement enzymes are
not internalized by the M6P receptor on cell surfaces, and
therefore cannot be directed to the lysosome where they function.
Consequently, a low degree of mannose phosphorylation can have a
significant and deleterious effect on the therapeutic efficacy of a
replacement enzyme.
[0006] Methods thus have been developed for increasing the M6P
content of replacement enzymes. U.S. Pat. No. 7,001,994, for
example, describes a method for coupling oligosaccharides
comprising M6P with glycoproteins. The oligosaccharides of the
glycoproteins are first oxidized with periodate or galactose
oxidase to result in the formation of carbonyl groups, which are
then chemically conjugated with an oligosaccharide functionalized
at the reducing end with a carbonyl-reactive group (such as, e.g.,
a hydrazine, hydrazide, aminooxy, thiosemicarbazide, semicarbazide,
or amine group) to yield an oligosaccharide-glycoprotein
conjugate.
[0007] A conjugate of the lysosomal enzyme acid .alpha.-glucosidase
(GAA) with a bis-M6P oligosaccharide was prepared by the
above-described method, and found to be more effective in reducing
skeletal and cardiac muscle glycogen than recombinant human GAA in
a murine model of Pompe disease, an autosomal recessive muscular
disease resulting from a metabolic deficiency of GAA, and
characterized by the accumulation of lysosomal glycogen.
[0008] Aminooxy groups are particularly useful carbonyl-reactive
groups for the conjugation reactions described above, as the
resulting conjugates comprise a relatively stable oxime linkage.
Therefore, there is a need for methods for the preparation of
aminooxy functionalized oligosaccharides.
[0009] The present invention provides methods of preparing
oligosaccharides comprising an aminooxy group. These methods are
generally applicable to a broad range of protected and unprotected
oligosaccharides, such as, e.g., branched and unbranched, and
phosphorylated and unphosphorylated, oligosaccharides. In certain
embodiments, the oligosaccharide may be a disaccharide,
trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide,
heptasaccharide, or greater. The oligosaccharide may, in certain
embodiments, comprise at least one M6P residue. In some
embodiments, the oligosaccharide may comprise at least 1, 2, 3, 4,
5, 6, or 7 terminal M6P residues.
[0010] The invention provides a method of preparing an
oligosaccharide comprising an aminooxy group from an
oligosaccharide comprising a reactive group. The method comprises:
[0011] (a) providing an oligosaccharide comprising a first reactive
group; [0012] (b) providing an aminooxy compound comprising an
aminooxy group and a second reactive group; and [0013] (c) reacting
the first reactive group of the oligosaccharide with the second
reactive group of the aminooxy compound, thereby preparing the
oligosaccharide comprising an aminooxy group.
[0014] The first and second reactive groups may be chosen from,
e.g., hydrazine, hydrazide, thiosemicarbazide, semicarbazide,
amine, carboxyl, activated ester, acyl halide, acyl azide, alkyl
halide, anhydride, isothiocyanate, isocyanate, and sulfonyl halide
groups.
[0015] In some embodiments, the aminooxy compound is chosen from
compounds of Formula II:
##STR00001##
wherein Y is the second reactive group, Z is chosen from alkyl,
alkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl, and P is
chosen from amino protecting groups (such as, e.g., carbamate
protecting groups). For example, in some embodiments, Y may be a
carboxyl, activated ester, acyl halide (such as, e.g., an acyl
fluoride or acyl chloride), acyl azide, alkyl halide, anhydride,
isothiocyanate, isocyanate, or sulfonyl halide (such as, e.g., a
sulfonyl chloride or sulfonyl bromide). In other embodiments, Y may
be, e.g., a hydrazine, hydrazide, thiosemicarbazide, semicarbazide,
or amine group.
[0016] In certain embodiments, the aminooxy compound of Formula II
is chosen from compounds of Formula III:
##STR00002##
wherein Y is the second reactive group, n is chosen from integers
ranging from 1 to 10, and P is chosen from amino protecting
groups.
[0017] In certain embodiments, the aminooxy compound comprises an
amino protecting group, and the method further comprises a step
(d), deprotecting the oligosaccharide comprising an aminooxy
group.
[0018] The invention further provides an oligosaccharide comprising
(1) an aminooxy group and (2) mannose-6-phosphate. In some
embodiments, that oligosaccharide is prepared by the methods
described above. For example, in some embodiments, the invention
provides an oligosaccharide comprising an aminooxy group of Formula
IV:
##STR00003##
wherein m and p are independently chosen from integers ranging from
1 to 10.
[0019] In another embodiment, the invention provides an
oligosaccharide of Formula V:
##STR00004##
[0020] In another embodiment, the invention provides methods of
coupling an oligosaccharide to a protein. In one embodiment, the
method comprises: [0021] (a) providing an oligosaccharide
comprising an aminooxy group; [0022] (b) providing a protein having
at least one carbonyl group; and [0023] (c) reacting the aminooxy
group of the oligosaccharide with the at least one carbonyl group
of the protein, thereby coupling the oligosaccharide to the
protein.
[0024] In other embodiments, the invention further provides an
oligosaccharide-protein conjugate comprising (1) a protein, (2) an
oligosaccharide, and (3) an oxime group connecting the protein and
the oligosaccharide. For example, in some embodiments, the
invention provides an oligosaccharide-protein conjugate prepared by
the methods disclosed above. In certain embodiments, the
oligosaccharide-protein conjugate is an
oligosaccharide-glycoprotein conjugate. In certain embodiments, the
oligosaccharide-glycoprotein conjugate is the conjugate of an
oligosaccharide comprising at least one M6P and of a lysosomal
enzyme such as, e.g., a lysosomal hydrolase. In some embodiments,
the invention provides pharmaceutical compositions comprising an
oligosaccharide-protein conjugate of the invention.
[0025] Another embodiment of the invention provides methods of
treating a lysosomal storage disorder such as, e.g., those
disclosed in Table 1. In some embodiments, the methods comprise
administering to a mammal an oligosaccharide-glycoprotein conjugate
of the invention, wherein the oligosaccharide comprises at least
one M6P and the glycoprotein is a lysosomal hydrolase. This
disclosure further provides the use of a conjugate of the invention
for treating a lysosomal storage disorder in a subject in need
thereof, and in the manufacture of a medicament for treating a
lysosomal storage disorder.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1 is a reaction scheme depicting an illustrative
embodiment of the methods of the invention. Oligosaccharide 1,
having a first reactive group (a hydrazide group), is reacted with
aminooxy compound 2 in presence of the catalyst
3-hydroxy-1,2,3-benzotriazin-4(3H)-one (DHBt-OH), to yield
oligosaccharide 3. The tert-butyloxycarbonyl (t-Boc) amino
protecting group of oligosaccharide 3 is then removed with 50%
trifluoroacetic acid/dichloromethane (TFA/DCM) to yield
oligosaccharide 4.
[0027] FIG. 2 depicts a series of gel chromatographs of
intermediates in the synthetic scheme described in FIG. 1. FIG. 2A
is a Dionex analytical chromatograph of starting oligosaccharide 1.
FIG. 2B is a Dionex analytical chromatograph of oligosaccharide 3.
FIG. 2C is a Dionex analytical chromatograph of oligosaccharide
4.
[0028] FIG. 3A is a mass spectrum of oligosaccharide 1 (calculated
molecular weight=1250; calculated molecular weight of sodium
salt=1338). FIG. 3B is a mass spectrum of oligosaccharide 4
(calculated molecular weight=1323; calculated molecular weight of
sodium salt=1411).
[0029] FIG. 4 is a reaction scheme depicting an illustrative
embodiment of the methods of the invention. Oligosaccharide 5
having a first reactive group (a carboxyl group) is reacted with
aminooxy compound 6 in presence of the coupling agent
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and the
catalyst N-hydroxysuccinimide (NHS), to yield aminooxy-containing
oligosaccharide 3. The Boc amino protecting group of
oligosaccharide 3 is then removed with 50% TFA/DCM to yield
oligosaccharide 4.
I. PREPARATION OF AN OLIGOSACCHARIDE COMPRISING AN AMINOOXY
GROUP
A. Oligosaccharide Comprising a Reactive Group
[0030] The methods of the invention are applicable to a broad range
of oligosaccharides comprising a reactive group. As used herein, an
oligosaccharide refers to a disaccharide, trisaccharide,
tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide,
or larger oligosaccharide (such as, e.g., an oligosaccharide
comprising 2-50, 2-10, 8-25, or 8-50 saccharide units).
Accordingly, in various embodiments, an oligosaccharide may be,
e.g., a disaccharide, trisaccharide, tetrasaccharide, a
pentasaccharide, a hexasaccharide, a heptasaccharide, or a larger
oligosaccharide. An oligosaccharide may be mono-, bi-, tri-,
tetra-, or penta-antennary in structure. An oligosaccharide may
comprise 0, 1, 2, 3, 4, or more branch points.
[0031] The reactive group on the oligosaccharide, also referred to
as a first reactive group, may be, in some embodiments, e.g., a
hydrazine group, hydrazide group, semicarbazide group,
thiosemicarbazide, or amine group. In some embodiments, the first
reactive group may be, e.g., a carboxyl, ester (such as, e.g., an
activated ester), acyl halide (such as, e.g., acyl fluoride or acyl
chloride), acyl azide, alkyl halide, anhydride, isothiocyanate,
isocyanate, or sulfonyl halide (such as, e.g., sulfonyl chloride or
sulfonyl bromide) group.
[0032] The first reactive group may be connected to the reducing
end of the oligosaccharide or may be located anywhere in the
oligosaccharide. The first reactive group may, in certain
embodiments, be connected through one or more linkers to the
oligosaccharide. A linker, as used herein, may be chosen from,
e.g., a combination of optionally substituted alkyl, alkenyl,
alkynyl, aryl, heteroaryl, heterocyclyl, acyloxy, alkoxy, aryloxy,
and heterocyclyloxy groups. A linker may be interrupted or
terminated by one or more heteroatoms such as, e.g., nitrogen,
sulfur, and oxygen. For example, a linker, in some embodiments, may
comprise one or more ether, ester, or amide group.
[0033] Any chemical group of the linker (such as, e.g., alkyl,
alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, acyloxy, alkoxy,
aryloxy, and heterocyclyloxy) may be substituted or unsubstituted,
unless otherwise stated. Substituents may be chosen from, e.g.,
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, e.g., nitrogen, sulfur, and
oxygen.
[0034] In certain embodiments, an oligosaccharide may comprise at
least one protecting group. The term "protecting group" refers to
any substituent that may be used to prevent a functional group
(such as, e.g., an amine group, a carboxyl group, a hydroxyl group,
a hydrazine group, a hydrazide group, a semicarbazide group, or a
thiosemicarbazide group) on a molecule from undergoing a chemical
reaction while chemical change occurs elsewhere in the molecule. A
protecting group can be removed under the appropriate chemical
conditions. Numerous protecting groups are known to those skilled
in the art, and examples of protecting groups, methods for their
addition, and methods for their removal can be found in, e.g.,
Greene et al., Protective Groups in Organic Synthesis, 3.sup.rd
ed., John Wiley and Sons: New York, 1999 and Kocienski, Protecting
Groups, 3.sup.rd ed., Georg Thieme Verlag: Stuttgard, Germany,
2005, the disclosures of which are herein incorporated by
reference. In certain embodiments, the oligosaccharide may comprise
at least one protecting group chosen from hydroxyl protecting
groups, carboxyl protecting groups, and amino protecting groups. In
other embodiments, an oligosaccharide may be "unprotected," and may
not comprise any protecting groups.
[0035] An oligosaccharide may be isolated from a natural source or
may be prepared by chemical or enzymatic synthesis. An
oligosaccharide isolated from a natural source may be homogeneous
or may be a heterogeneous mixture of related oligosaccharides. In
some embodiments, an oligosaccharide may be prepared by chemical or
enzymatic modification of an oligosaccharide isolated from a
natural source ("semi-synthesis"). In some embodiments, the
oligosaccharide may be a synthetic oligosaccharide having the
chemical structure of a naturally occurring oligosaccharide.
[0036] In some embodiments, an oligosaccharide may comprise a
monosaccharide that is recognized by a particular receptor. The
monosaccharide recognized by a particular receptor may be chosen
from, e.g., galactose, GalNAc, mannose, M6P, glucose, GlcNAc,
sialic acid, or sulfated sialic acid residue. An oligosaccharide
may, in certain embodiments, comprise at least one M6P residue,
such as, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 M6P residues.
[0037] The monosaccharide recognized by a particular receptor may
be, in some embodiments, a penultimate monosaccharide or a 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. An oligosaccharide may, in
some embodiments, contain at least 1, 2, 3, 4, 5, 6, 7 terminal M6P
residues.
[0038] In certain embodiments, the oligosaccharide comprising a
reactive group may be an M6P-containing hexasaccharide of Formula
Ia:
##STR00005##
[0039] The oligosaccharide of Formula Ia can be described as
butyrylhydrazine-4-yl
6-O-phosphono-.alpha.-D-mannopyranosyl-(1.fwdarw.2)-.alpha.-D-mannopyrano-
syl-(1.fwdarw.6)-.alpha.-D-mannopyranosyl-(1.fwdarw.6)-[6-O-phosphono-.alp-
ha.-D-mannopyranosyl-(1.fwdarw.2)-.alpha.-D-mannopyranosyl-(1.fwdarw.3)]-.-
beta.-D-mannopyranoside.
[0040] In certain embodiments, the oligosaccharide comprising a
reactive group may be an M6P-containing hexasaccharide of Formula
Ib:
##STR00006##
B. Aminooxy Compound
[0041] As used herein, an aminooxy compound may be any compound
comprising an aminooxy group and a second reactive group, wherein
the second reactive group may react with a first reactive group on
an oligosaccharide to form a covalent bond. For example, in some
embodiments, the second reactive group may be a carboxyl, ester
(such as, e.g., an activated ester), acyl halide (such as, e.g., an
acyl fluoride or acyl chloride), acyl azide, anhydride,
isothiocyanate, isocyanate, or sulfonyl halide (such as, e.g., a
sulfonyl chloride or sulfonyl bromide) group. In other embodiments,
the second reactive group may be, e.g., a hydrazine group,
hydrazide group, semicarbazide group, thiosemicarbazide, or amine
group.
[0042] In certain embodiments, the nitrogen of the aminooxy group
of the aminooxy compound is protected with an amino protecting
group. Numerous amino protecting groups are known to those skilled
in the art, and examples of amino protecting groups, methods for
their addition, and methods for their removal can be found in pp.
494-653 of Greene et al., Protective Groups in Organic Synthesis,
3.sup.rd ed., John Wiley and Sons: New York, 1999; Chapter 8 of
Kocienski, Protecting Groups, 3.sup.rd ed., Georg Thieme Verlag:
Stuttgard, Germany, 2005; Bodanszky, Principles of Peptide
Synthesis, Springer Verlag: New York, 1993; Lloyd-Williams et al.,
Chemical Approaches to the Synthesis of Peptides and Proteins, CRC
Press: Boca Raton, Fla., 1997; and Stewart et al., Solid Phase
Peptide Synthesis, 2nd ed., Pierce Chemical Co.: Rockford, Ill.,
1984, the inventions of which are incorporated herein by
reference.
[0043] In some embodiments, the aminooxy compound is chosen from
compounds of Formula II:
##STR00007##
wherein Y is the second reactive group, Z is chosen from alkyl,
alkenyl, alkynyl, heteroaryl, aryl, and heterocyclyl, and P is
chosen from amino protecting groups.
[0044] As used herein, any chemical group on the aminooxy compound
(such as, e.g., alkyl, alkenyl, alkynyl, aryl, heteroaryl,
heterocyclyl, acyloxy, alkoxy, aryloxy, and heterocyclyloxy) may be
substituted or unsubstituted, and may be interrupted by one or more
chemical groups, unless otherwise stated. Substituents and
interrupting chemical groups may be chosen from, e.g., 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, e.g., nitrogen, sulfur, and oxygen.
[0045] In certain embodiments, Y may be chosen from, for
example:
##STR00008## [0046] wherein X is chosen from halogens, azide,
acyloxy, alkoxy, aryloxy, heteroaryloxy, and heterocyclyloxy.
[0047] In certain embodiments, the aminooxy compound is an
activated ester. As used herein, an activated ester is an ester
that reacts to form an amide bond under mild conditions. In
general, an activated ester is an ester of a relatively acidic
alcohol. In certain embodiments, the aminooxy compound of Formula
II is an activated ester of formula
##STR00009##
and X is chosen from alkoxy, aryloxy, heteroaryloxy, and
heterocyclyloxy. For example, X may be chosen from:
##STR00010## ##STR00011##
[0048] In other embodiments, Y is chosen from, e.g., hydrazide,
hydrazine, thiosemicarbazide, semicarbazide, and amine groups.
[0049] In some embodiments, Z may comprise, for example, a
carbonyl, ether, ester, or amide group. In some embodiments, Z may
be, for example, alkyl interrupted by one or more heteroatoms, such
as an oligoethyleneglycol. For example, Z may be
monoethyleneglycol, diethyleneglycol, triethyleneglycol,
tetraethyleneglycol, or larger oligoethyleneglycol.
[0050] In some embodiments, Z may be, for example, alkyl
substituted with oxo and interrupted by one or more heteroatoms,
such as an oligopeptide. For example, the oligopeptide may comprise
one, two, three, four, five, six, or more component amino acids.
The amino acids may be, for example, .alpha.-amino acids,
.beta.-amino acids, .gamma.-amino acids, .delta.-amino acids, and
co-amino acids. An amino acid may have R or S chirality at any
chiral atom. An amino acid may be chosen from, e.g., alanine,
.beta.-alanine, .alpha.-aminoadipic acid, 2-aminobutanoic acid,
4-aminobutanoic acid, 1-aminocyclopentanecarboxylic acid,
6-aminohexanoic acid, 2-aminoheptanedioic acid, 7-aminoheptanoic
acid, 2-aminoisobutyric acid, aminomethylpyrrole carboxylic acid,
8-amino-3,6-dioxa-octanoic acid, aminopiperidinecarboxylic acid,
3-amino-propionic acid, aminoserine,
aminotetrahydropyran-4-carboxylic acid, arginine, asparagine,
aspartic acid, azetidine carboxylic acid, benzothiazolylalanine,
butylglycine, carnitine, 4-chlorophenylalanine, citrulline,
cyclohexylalanine, cyclohexylstatine, cysteine, 2,4-diaminobutanoic
acid, 2,3-diaminopropionic acid, dihydroxyphenylalanine,
dimethylthiazolidine carboxylic acid, glutamic acid, glutamine,
glycine, histidine, homoserine, hydroxyproline, isoleucine,
isonipecotic acid, leucine, lysine, methanoproline, methionine,
norleucine, norvaline, ornithine, p-aminobenzoic acid,
penicillamine, phenylalanine, phenyiglycine, pipe ridinylalanine,
piperidinylglycine, proline, pyrrolidinylalanine, sarcosine,
selenocysteine, serine, statine, tetrahydropyranglycine,
thienylalanine, threonine, tryptophan, tyrosine, valine,
allo-isoleucine, allo-threonine, 2,6-diamino-4-hexanoic acid,
2,6-diaminopimelic acid, 2,3-diaminopropionic acid, dicarboxidine,
homoarginine, homocitrulline, homocysteine, homocystine,
homophenylalanine, homoproline, and 4-hydrazinobenzoic acid.
[0051] P may be chosen from amino protecting groups known to those
of skill in the art. In some embodiments, P may be a carbamate
protecting group, such as, e.g., a (9-fluorenylmethyl)carbamate
(Fmoc), (tert-butyloxy)carbamate (t-Boc), (trichloroethyl)carbamate
(Troc), or allylcarbamate (Alloc) protecting group. In other
embodiments, P may be a non-carbamate protecting group, such as,
e.g., an amide protecting group such as a phthalimide or a
trifluoroacetamide protecting group.
[0052] In some embodiments, the aminooxy compound of Formula II is
chosen from compounds of Formula III:
##STR00012##
wherein Y and P are as disclosed above, and n is chosen from
integers ranging from 1 to 10.
[0053] In certain embodiments, n may be chosen from integers from
the following ranges: 1-4, 2-6, 2-8, 3-6, and 4-10. In illustrative
embodiments, n is 1.
[0054] In one illustrative embodiment, the aminooxy compound is
t-Boc-aminooxy acetic acid tetrafluorophenyl ester, the structure
of which is depicted below.
##STR00013##
[0055] In another illustrative embodiment, the aminooxy compound
has the structure depicted below.
##STR00014##
C. Methods of Preparing an Oligosaccharide Comprising an Aminooxy
Group
[0056] In another embodiment, the invention provides a method of
preparing an oligosaccharide comprising an aminooxy group from an
oligosaccharide comprising a reactive group. The method comprises:
[0057] (a) providing an oligosaccharide comprising a first reactive
group; [0058] (b) providing an aminooxy compound comprising a
second reactive group; and [0059] (c) reacting the first reactive
group of the oligosaccharide with the second reactive group of the
aminooxy compound, thereby preparing the oligosaccharide comprising
an aminooxy group.
[0060] The oligosaccharide comprising a first reactive compound may
be, e.g., any oligosaccharide comprising a reactive group as
described supra. In illustrative embodiments, the oligosaccharide
comprising a first reactive group is an oligosaccharide of Formula
Ia or an oligosaccharide of Formula Ib. The aminooxy compound
comprising a second reactive group may be any aminooxy compound
comprising a reactive group, as described supra.
[0061] The terms "first reactive group" and "second reactive
group," as used herein, do not denote any particular experimental
sequence. I.e., step (c), reacting the first reactive group of the
oligosaccharide with the second reactive group of the aminooxy
compound, may be accomplished by any order of addition of the
reactants. For example, the oligosaccharide comprising a first
reactive group may be added to the aminooxy compound comprising the
second reactive group, or vice versa. In another example, both the
oligosaccharide and the aminooxy compound may be added
simultaneously to a reaction vessel.
[0062] Step (c) may occur under any suitable conditions (e.g.,
solvent and temperature) known to those of ordinary skill in the
art. In certain embodiments, one or more additional reagents, such
as, e.g., coupling reagents and catalysts, may be present during
step (c). A coupling reagent, as used herein, is a reagent that may
be used to form a covalent bond between the first reactive group
and the second reactive group.
[0063] In some embodiments, such as, e.g., when the first or second
reactive group is a carboxyl group, the reaction conditions may
comprise a coupling reagent. Coupling reagents may be chosen from,
e.g., phosphonium coupling reagents such as, e.g., BOP
(benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium
hexafluorophosphate), PyBOP.RTM.
(benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate), and PyBroP.RTM.
(bromo-tris-pyrrolidino-phosphonium hexafluorophosphate), and from
aminium (uronium) coupling reagents such as, e.g., HBTU
(2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate), HATU
(2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate), TBTU
(2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate). Coupling reagents may also be chosen from,
e.g., carbodiimide coupling reagents such as, e.g., DIC
(1,3-diisopropylcarbodiimide), CDI (1,1' carbonyl diimidazole), and
EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide). For example,
in some illustrative embodiments, the coupling reagent is EDC. In
certain embodiments, the reaction conditions comprise both a
coupling reagent and a catalyst.
[0064] The reaction conditions may, in certain embodiments,
comprise a catalyst. The catalyst may be chosen from any suitable
catalyst known to those of skill in the art, such as, e.g., DHBt-OH
(3-hydroxy-1,2,3-benzotriazin-4(3H)-one), HOBt
(N-hydroxybenzotriazole), DMAP (4-dimethylaminopyridine), NHS
(N-hydroxysuccinimide), N-hydroxysulfosuccinimide, HONB
(N-hydroxy-5-norbornene-endo-2,3-dicarboximide), or a
tetrabutylammonium salt such as, e.g., TBAI (tetrabutylammonium
iodide). In some illustrative embodiments, the reaction conditions
comprise the catalyst DHBt-OH or the catalyst NHS.
[0065] In some embodiments, step (c), 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.
Conditions suitable for the formation of an amide bond are well
known to those of ordinary skill in the art, and are described in,
e.g., 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; Lloyd-Williams et al., Chemical Approaches to the
Synthesis of Peptides and Proteins, CRC Press: Boca Raton, Fla.,
1997; and the Novabiochem.RTM. (San Diego, Calif.) Catalog.
[0066] In certain embodiments, the aminooxy compound comprises an
amino protecting group, and the method comprises a further step
(d), deprotecting the oligosaccharide comprising an aminooxy group
to remove the amino protecting group. Deprotection may occur under
any suitable conditions known to those of skill in the art, such
as, e.g., those taught in pp. 494-653 of Greene et al., Protective
Groups in Organic Synthesis, 3.sup.rd ed., John Wiley and Sons: New
York, 1999 and Kocienski, Protecting Groups, 3.sup.rd ed., Georg
Thieme Verlag: Stuttgard, Germany, 2005, the inventions of which
are incorporated herein by reference.
[0067] An illustrative embodiment of the method of the invention
provides a method of preparing an M6P-containing oligosaccharide
comprising an aminooxy group. The method comprises: [0068] (a)
providing an oligosaccharide comprising a first reactive group,
wherein the oligosaccharide is
[0068] ##STR00015## [0069] (b) providing an aminooxy compound
comprising a second reactive group, wherein the aminooxy compound
is chosen from compounds of Formula III:
[0069] ##STR00016## [0070] wherein n is chosen from integers
ranging from 1 to 10, P is chosen from amino protecting groups, and
Y is a second reactive group; and [0071] (c) reacting the first
reactive group of the oligosaccharide with the second reactive
group of the aminooxy compound, thereby preparing the
oligosaccharide comprising an aminooxy group.
[0072] In certain embodiments, Y in Formula III is
##STR00017##
where X is chosen from hydroxy, aryloxy, heteroaryloxy, and
heterocyclyloxy. For example, in certain illustrative embodiments,
X is
##STR00018##
[0073] In illustrative embodiments, the aminooxy compound is
##STR00019##
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,
e.g., EDC, and/or a catalyst, such as, e.g., DHBt-OH.
[0074] Another illustrative embodiment of the method of the
invention comprises: [0075] (a) providing an oligosaccharide
comprising a first reactive group, wherein the oligosaccharide
is
[0075] ##STR00020## [0076] (b) providing an aminooxy compound
comprising a second reactive group, wherein the aminooxy compound
is chosen from compounds of Formula III:
[0076] ##STR00021## [0077] wherein n is chosen from integers
ranging from 1 to 10, P is chosen from amino protecting groups, and
Y is a second reactive group; and [0078] (c) reacting the first
reactive group of the oligosaccharide with the second reactive
group of the aminooxy compound, thereby preparing the
oligosaccharide comprising an aminooxy group.
[0079] In certain embodiments, Y in Formula III is a hydrazine,
hydrazide, aminooxy, thiosemicarbazide, semicarbazide, or amine
group. In certain embodiments, Y in Formula III is
##STR00022##
In illustrative embodiments, the aminooxy compound is
##STR00023##
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,
e.g., EDC, and/or a catalyst, such as, e.g., NHS.
II. OLIGOSACCHARIDES COMPRISING AN AMINOOXY GROUP
[0080] The present invention also provides oligosaccharides
comprising an aminooxy group. In some embodiments, the invention
provides oligosaccharides comprising an aminooxy group prepared by
the methods disclosed above. The oligosaccharide comprising an
aminooxy group may comprise, for example, at least 2, 3, 4, 5, 6,
or more monosaccharides, including, e.g., at least one galactose,
GalNAc, mannose, M6P, glucose, GlcNAc, sialic acid, or sulfated
sialic acid residue. Such an oligosaccharide may be mono-, bi-,
tri-, tetra-, or penta-antennary in structure, and may contain 0,
1, 2, 3, 4, or more branch points.
[0081] In some embodiments, the present invention provides an
oligosaccharide comprising (1) an aminooxy group and (2)
mannose-6-phosphate. The oligosaccharide comprising an aminooxy
group may, in some embodiments, comprise, e.g., 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10 M6P residues. In some embodiments, the
oligosaccharide comprising an aminooxy group may comprise at least
1, 2, 3, 4, or more terminal or penultimate M6P residues.
[0082] The oligosaccharides comprising an aminooxy group are, in
certain embodiments, chosen from oligosaccharides of Formula
IV:
##STR00024##
wherein m and p are independently chosen from integers ranging from
1 to 10. For example, in certain embodiments, m and p may be
independently chosen from integers selected from the following
ranges: 1-4, 2-6, 2-8, 3-6, and 4-10. In illustrative embodiments,
m is 3 and p is 1.
[0083] In other embodiments, the aminooxy group is directly linked
to the reducing end of the oligosaccharide. For example, in some
embodiments, the oligosaccharide comprising an aminooxy group may
be an oligosaccharide of Formula V:
##STR00025##
III. CONJUGATION OF AN OLIGOSACCHARIDE COMPRISING AN AMINOOXY GROUP
WITH A PROTEIN
A. Oligosaccharide
[0084] The oligosaccharide to be conjugated with a protein may be
chosen from any oligosaccharide comprising a reactive group, as
discussed supra, and from any oligosaccharide comprising an
aminooxy group, as discussed supra. For example, in some
embodiments, the oligosaccharide to be conjugated may be an
oligosaccharide of Formula Ia, Formula Ib, Formula IV or Formula
V.
B. Protein
[0085] The conjugation methods described herein are broadly
applicable to any pure protein, partially purified protein, or
fragment thereof, having at least one carbonyl group (where a
carbonyl group is a ketone or an aldehyde), including isolated
proteins and recombinantly or synthetically produced proteins. The
terms "pure," "purified," and "isolated" refer to a molecule that
is substantially free of its natural environment. For instance, a
pure protein is substantially free of cellular material and/or
other proteins from the cell or tissue source from which it is
derived. The term refers to preparations that are, for example, at
least 70% to 80%, 80% to 90%, 90 to 95%; or at least 95%, 96%, 97%,
98%, 99%, or 100% (w/w) pure.
[0086] In other embodiments, the protein may be an enzyme that has
optimal activity, as measured by an activity assay, at a pH ranging
from 1-7, such as, e.g., 1-3, 2-5, 3-6, 4-5, 5-6, or 4-6. For
example, the enzyme may have a pH optimum at a pH ranging from
4-6.
[0087] In some embodiments, the protein may be an enzyme that has
an isoelectric point (pI), ranging from 1 to 8, such as, e.g., from
1-3, 2-5, 3-8, 4-5, 5-6, 4-6, 5-8, 6-8, or 7-8. The pI of a protein
may be may be measured using, e.g., isoelectric focusing gel
electrophoresis.
[0088] In some embodiments, the protein containing a carbonyl group
is obtained by the use of an expression system having an expanded
genetic code, as described in, e.g., Wang et al., Proc. Natl. Acad.
Sci. USA 100:56-61 (2003). In such a case, the carbonyl group may
be located on amino acid side chain, as translated.
[0089] In certain embodiments, the protein having at least one
carbonyl group is a protein having at least one oligosaccharide
(i.e., a glycoprotein). For example, a glycoprotein having at least
one carbonyl group may be obtained by oxidation of that
glycoprotein by any means known to those of skill in the art. In
some embodiments, e.g., a glycoprotein having at least one carbonyl
group may be obtained by oxidation of that glycoprotein with
periodate (e.g., sodium periodate) or with galactose oxidase. In
such a case, the carbonyl group may be located at a protein
glycosylation site.
[0090] In certain embodiments, the protein having at least one
carbonyl group is a glycoprotein, such as a therapeutic
glycoprotein. A therapeutic glycoprotein may be targeted to the
lysosome by conjugation with an oligosaccharide comprising
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 certain
embodiments, the lyosomal hydrolase is chosen from, e.g.,
.alpha.-glucosidase, .alpha.-galactosidase A, and acid
sphingomyelinase. In certain embodiments, the lyosomal hydrolase is
GAA.
TABLE-US-00001 TABLE 1 Examples of LSDs and Corresponding Lysosomal
Hydrolases Lysosomal Storage Disorder Defective Enzyme Fabry
.alpha.-Galactosidase A Farber Acid ceramidase Fucosidosis Acid
.alpha.-L-fucosidase Gaucher types 1, 2, and 3 Acid
.beta.-glucosidase G.sub.M1 gangliosidosis Acid
.beta.-galactosidase Hunter (Mucopolysaccharidosis
Iduronate-2-sulfatase (MPS) II) Hurler-Scheie, Hurler, Scheie
.alpha.-L-Iduronidase (MPS I) Krabbe Galactocerebrosidase
.alpha.-Mannosidosis Acid .alpha.-mannosidase .beta.-Mannosidosis
Acid .beta.-mannosidase Maroteaux-Lamy (MPS VI) Arylsulfatase B
Metachromatic leukodystrophy Arylsulfatase A Morquio A (MPS IV)
N-Acetylgalactosamine-6-sulfate sulfatase Morquio B (MPS IV) Acid
.beta.-galactosidase Niemann-Pick A and B Acid sphingomyelinase
(ASM) Pompe Acid .alpha.-glucosidase (.alpha.-glucosidase; GAA)
Sandhoff .beta.-Hexosaminidase B Sanfilippo A (MPS III) Heparan
N-sulfatase Sanfilippo B (MPS III) .alpha.-N-Acetylglucosaminidase
Sanfilippo C (MPS III) Acetyl-CoA:.alpha.-glucosaminide
N-acetyltransferase Sanfilippo D (MPS III)
N-Acetylglucosamine-6-sulfate sulfatase Schindler-Kanzaki
.alpha.-N-acetylgalactosaminidase Sialidosis Sialidase Sly (MPS
VII) .beta.-Glucuronidase Tay-Sachs .beta.-Hexosaminidase A
[0091] In certain embodiments, the glycoprotein may be a
glycoprotein having at least 1, 2, 3, 4, 5, or more N-linked or
O-linked glycosylated amino acid residues. In other embodiments,
the protein may have 1, 2, 3, 4, 5 or more consensus sites for
N-linked or O-linked glycosylation, at least one of which is
glycosylated.
[0092] 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, e.g., mannose or mannose-6-phosphate. In some embodiments, the
glycoprotein may bind to, e.g., the asialoglycoprotein receptor,
the cation-dependent mannose-6-phosphate receptor, the insulin-like
growth factor II/cation-independent mannose-6-phosphate receptor,
or the macrophage mannose receptor.
[0093] In certain embodiments, the protein is a glycoprotein that,
when conjugated to an oligosaccharide comprising
mannose-6-phosphate, is internalized more efficiently by a target
cell (e.g., via CI-MPR-mediated endocytosis) than is the
corresponding unconjugated glycoprotein. For example, the
conjugated glycoprotein may be internalized more efficiently than
the unconjugated glycoprotein by, e.g., at least 10%, 15%, 20%,
25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90% (w/w) in a given
time period. In other embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9,
or 10 fold (w/w) as much of the conjugated glycoprotein may be
internalized, relative to the unconjugated glycoprotein, in a given
time period. The referenced time period may be, for example, 10,
30, 45 minutes or 1, 2, 3, 5, 6, 12, 24, 48, or 72 hours, or
more.
C. Methods of Coupling an Oligosaccharide to a Protein
[0094] The invention provides methods of coupling an
oligosaccharide to a protein, such as, e.g., a glycoprotein. In one
embodiment, the method comprises: [0095] (a) providing an
oligosaccharide comprising an aminooxy group; [0096] (b) providing
a protein having at least one carbonyl group; and [0097] (c)
reacting the aminooxy group of the oligosaccharide with the at
least one carbonyl group of the protein, thereby coupling the
oligosaccharide to the protein.
[0098] In certain embodiments, the methods further comprise adding
a reducing agent to the coupled lysosomal enzyme. The reducing
agent may be any reducing agent known to those of skill in the art,
such as, e.g., sodium cyanoborohydride or sodium
triacetoxyborohydride (STAB).
IV. OLIGOSACCHARIDE-PROTEIN CONJUGATES
[0099] The invention further provides an oligosaccharide-protein
conjugate, comprising (1) a protein, (2) an oligosaccharide, and
(3) an oxime group connecting the protein and the oligosaccharide.
In some embodiments, the invention provides an
oligosaccharide-protein conjugate prepared by the methods disclosed
above. The oligosaccharide and protein components of the conjugate
may be, for example, any oligosaccharide and protein described
herein, wherein a conjugate thereof comprises an oxime group, as
depicted below. (The oxime group depicted below is formally derived
by reaction of an aminooxy group and an aldehyde group; oxime
groups formally derived by reaction of an aminooxy group and a
ketone group are also encompassed by this invention.)
##STR00026##
[0100] In certain embodiments, the oligosaccharide-protein
conjugate is an oligosaccharide-glycoprotein conjugate. In certain
embodiments, the oligosaccharide-protein conjugate is the conjugate
of an oligosaccharide comprising at least one M6P and of a
lysosomal hydrolase.
V. PHARMACEUTICAL COMPOSITIONS
[0101] This disclosure provides the use of a conjugate of the
invention in the manufacture of a medicament for treating a
lysosomal storage disorder. It also provides pharmaceutical
compositions comprising an oligosaccharide-protein conjugate of the
invention. In some embodiments, the pharmaceutical compositions of
the invention comprise a conjugate of an oligosaccharide comprising
at least one M6P and a lysosomal enzyme.
[0102] Pharmaceutical compositions of the invention may comprise
one or more suitable pharmaceutical excipients. Standard
pharmaceutical formulation techniques and excipients are well known
to persons skilled in the art (see, e.g., 2005 Physicians' Desk
Reference.RTM., Thomson Healthcare: Montvale, N.J., 2004;
Remington: The Science and Practice of Pharmacy, 20th ed., Gennado
et al., Eds. Lippincott Williams & Wilkins: Philadelphia, Pa.,
2000. The compositions may or may not contain preservatives. In
some embodiments, pharmaceutical compositions comprising
.alpha.-galactosidase A conjugates may comprise one or more
excipients such as, e.g., mannitol, sodium phosphate monobasic
monohydrate, and/or sodium phosphate dibasic heptahydrate. In some
embodiments, pharmaceutical compositions comprising conjugates of
.alpha.-glucosidase may comprise one or more excipients such as,
e.g., mannitol, polysorbate 80, sodium phosphate dibasic
heptahydrate, and sodium phosphate monobasic monhydrate.
[0103] The pharmaceutical composition may comprise any of the
conjugates described herein either as the sole active compound or
in combination with another compound, composition, or biological
material. For example, the pharmaceutical composition may also
comprise one or more small molecules useful for the treatment of a
LSD and/or a side effect associated with the LSD. In some
embodiments, the composition may comprise miglustat and/or one or
more compounds described in, e.g., U.S. Patent Application
Publication Nos. 2003/0050299, 2003/0153768; 2005/0222244;
2005/0267094.
[0104] The formulation of pharmaceutical compositions may vary
depending on the intended route of administrations and other
parameters (see, e.g., Rowe et al. Handbook of Pharmaceutical
Excipients, 4th ed., APhA Publications, 2003.) In some embodiments,
the composition may be a sterile, non-pyrogenic, white to off-white
lyophilized cake or powder to be administered by intravenous
injection upon reconstitution with Sterile Water for Injection,
USP.
[0105] Administration of a pharmaceutical composition of the
invention is not limited to any particular delivery system and may
include, without limitation, parenteral (including subcutaneous,
intravenous, intracranial, intramedullary, intraarticular,
intramuscular, intrathecal, or intraperitoneal injection),
transdermal, or oral (for example, in capsules, suspensions, or
tablets). Administration to an individual may occur in a single
dose or in repeat administrations, and in any of a variety of
physiologically acceptable salt forms, and/or with an acceptable
pharmaceutical carrier and/or additive as part of a pharmaceutical
composition.
[0106] The conjugates described herein are administered in
therapeutically effective amounts. Generally, a therapeutically
effective amount may vary with the subject's age, condition, and
sex, as well as the severity of the medical condition in the
subject. The dosage may be determined by a physician and adjusted,
as necessary, to suit observed effects of the treatment. Toxicity
and therapeutic efficacy of such compounds can be determined by
standard pharmaceutical procedures in vitro (i.e., cell cultures)
or in vivo (i.e., experimental animal models), e.g., for
determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index (or therapeutic
ratio), and can be expressed as the ratio LD.sub.50/ED.sub.50.
Conjugates that exhibit therapeutic indices of at least 1, 1.5, 2,
3, 4, 5, 6, 7, 8, 9, 10, and 20 are described herein. Conjugates
that exhibit a large therapeutic index are preferred.
[0107] The data obtained from in vitro assays and animal studies,
for example, can be used in formulating a range of dosage for use
in humans. The dosage of such compounds lies preferably within a
range of circulating concentrations that include the ED.sub.50 with
low, little, or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any conjugate used in the present
invention, the therapeutically effective dose can be estimated
initially from in vitro assays. A dose may be formulated in animal
models to achieve a circulating plasma concentration range that
includes the IC.sub.50 (i.e., the concentration of the test
conjugate which achieves a half-maximal inhibition of symptoms) as
determined in in vitro experiments. Levels in plasma may be
measured, for example, by high performance liquid chromatography or
by an appropriate enzymatic activity assay. The effects of any
particular dosage can be monitored by a suitable bioassay of
endpoints.
[0108] Unless otherwise indicated, conjugates of the invention may
be administered at a dose of approximately from 1 .mu.g/kg to 500
mg/kg, depending on the severity of the symptoms and the
progression of the disease. For example, proteinaceous compounds
may be administered by slow intravenous infusion in an outpatient
setting every, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days,
or by, e.g., weekly, biweekly, monthly, or bimonthly
administration. The appropriate therapeutically effective dose of a
compound is selected by a treating clinician and would range
approximately from 1 .mu.g/kg to 500 mg/kg, from 1 .mu.g/kg to 10
mg/kg, from 1 .mu.g/kg to 1 mg/kg, from 10 .mu.g/kg to 1 mg/kg,
from 10 .mu.g/kg to 100 .mu.g/kg, from 100 .mu.g to 1 mg/kg, and
from 500 .mu.g/kg to 5 mg/kg.
[0109] For example, conjugates of .alpha.-galactosidase A may be
administered by intravenous infusion at a dose of, e.g., 1.0 mg/kg
body weight every two weeks at an infusion rate of, e.g., less than
or equal to 10, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, or 33 mg/hour). In another example,
conjugates of .alpha.-glucosidase may be administered intravenous
injection at a dose of, e.g., 20 mg/kg or 40 mg/kg every two weeks,
over approximately, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours.
In some embodiments, the rate of administration of
.alpha.-glucosidase may be started at, e.g., 1 mg/kg/hr and then
increased by, e.g., 2 mg/kg/hr every 30 minutes, after establishing
patient tolerance to the infusion rate, until a maximum of, e.g., 7
mg/kg/hr. Additionally, examples of specific dosages may be found
in the Physicians' Desk Reference.RTM..
VI. METHODS OF TREATING LYSOSOMAL STORAGE DISORDERS
[0110] The invention provides methods of treating lysosomal storage
disorders, such as, e.g., those disclosed in Table 1. In some
embodiments, the invention provides the use of a conjugate
described herein for treating a lysosomal storage disorder in a
subject in need thereof. The invention further provides methods of
targeting proteins to the lysosome by conjugation with
oligosaccharides comprising mannose-6-phosphate.
[0111] In one embodiment, the method comprises administering to a
mammal having a lysosomal storage disorder an
oligosaccharide-glycoprotein conjugate of the invention in a
therapeutically effective amount. The oligosaccharide-glycoprotein
conjugate may 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 subject in need thereof a
pharmaceutical composition comprising at least one of the
conjugates described herein.
[0112] In certain embodiments, conjugates of the invention may be
administered with one or more other therapies. The one or more
other therapies may be administered concurrently with (including
concurrent administration as a combined formulation), before, or
after the administration of the conjugates of the invention.
[0113] In some embodiments, a patient may be treated (before,
after, or during treatment with a conjugate of the invention) with
an antipyretic, antihistamine, and/or immunosuppressant. In some
embodiments, a patient may be treated with an antipyretic,
antihistamine, and/or immunosuppressant prior to treatment with an
oligosaccharide-glycoprotein conjugate of the invention in order to
decrease or prevent infusion associated reactions. For example,
patients may be pretreated with one or more of acetaminophen,
azathioprine, cyclophosphamide, cyclosporin A, methotrexate,
mycophenolate mofetil, oral steroids, or rapamycin.
[0114] In some embodiments, patients may be treated with one or
more of acetaminophen, azathioprine, cyclophosphamide, cyclosporin
A, methotrexate, mycophenolate mofetil, oral steroids, or rapamycin
at or about, e.g., 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, e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
incidences of treatment with a conjugate of the invention. For
example, in some embodiments a patient with Fabry disease or Pompe
disease may be treated with methotrexate (e.g., with 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 methotrexate, or more) at or
about, e.g., t=0, 24, and 48 hours for, e.g., the first 1, 2, 3, 4,
5, 6, 7, 8 weeks of treatment with a conjugate of the invention. In
some embodiments, immune tolerance toward conjugates of the
invention may be induced in a patient with a lysosomal storage
disorder such as, e.g., mucopolysaccharidosis I, by treatment with
cyclosporin A and azathioprine. For example, the patient may be
treated with cyclosporine A and azathioprine as described in Kakkis
et al., Proc. Natl. Acad. Sci. U.S.A. 101:829-834 (2004).
[0115] In some embodiments, a patient may be treated (before,
after, or during treatment with a conjugate of the invention) with
e.g., small molecule therapy and/or gene therapy, including small
molecule therapy and gene therapy directed toward treatment of a
lysosomal storage disorder. Small molecule therapy may comprise
administration of one or more compounds described in, e.g., U.S.
Patent Application Publication Nos. 2003/0050299, 2003/0153768;
2005/0222244; and 2005/0267094. Gene therapy may be performed as
described in, e.g., U.S. Pat. Nos. 5,952,516; 6,066,626; 6,071,890;
and 6,287,857 and U.S. Patent Application Publication No.
2003/0087868.
[0116] The terms "treatment," "therapeutic method," and their
cognates refer to both therapeutic treatment and
prophylactic/preventative measures. Thus, those in need of
treatment may include individuals already having a particular
lysosomal storage disease as well as those at risk for the disease
(i.e., those who are likely to ultimately acquire the disorder or
certain symptoms of the disorder).
[0117] A therapeutic method results in the prevention or
amelioration of symptoms or an otherwise desired biological
outcome, and may be evaluated by improved clinical signs or delayed
onset of disease, increased activity of the metabolically defective
enzyme, and/or decreased levels of the accumulated substrate of the
metabolically defective enzyme.
[0118] The conjugates of the present invention are useful to treat
various lysosomal storage disorders in humans or animals. For
example, administration of the conjugates can be used to increase
the deficient enzymatic activity in a patient, for example, by at
least 10%. The increased enzymatic activity may be determined by,
e.g., a reduction in clinical symptoms or by an appropriate
clinical or biological assay.
[0119] GAA conjugates may be administered for the treatment of
Pompe disease (also known as acid .alpha.-glucosidase deficiency,
acid maltase deficiency, glycogen storage disease type II,
glycogenosis II, and lysosomal .alpha.-glucosidase deficiency).
Increased GAA activity may be determined by biochemical (see, e.g.,
Zhu et al., J. Biol. Chem. 279: 50336-50341 (2004)) or histological
observation of reduced lysosomal glycogen accumulation in, e.g.,
cardiac myocytes, skeletal myocytes, or skin fibroblasts. GAA
activity may also be assayed in, e.g., a muscle biopsy sample, in
cultured skin fibroblasts, in lymphocytes, and in dried blood
spots. Dried blood spot assays are described in e.g., 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 by, e.g., serum levels of creatinine kinase, gains in
motor function (e.g., as assessed by the Alberta Infant Motor
Scale), changes in left ventricular mass index as measured by
echocardiogram, and cardiac electrical activity, as measured by
electrocardiogram. Administration of GAA conjugates may result in a
reduction in one or more symptoms of Pompe disease such as
cardiomegaly, cardiomyopathy, daytime somnolescence, exertional
dyspnea, failure to thrive, feeding difficulties, "floppiness,"
gait abnormalities, headaches, hypotonia, organomegaly (e.g.,
enlargement of heart, tongue, liver), lordosis, loss of balance,
lower back pain, morning headaches, muscle weakness, respiratory
insufficiency, scapular winging, scoliosis, reduced deep tendon
reflexes, sleep apnea, susceptibility to respiratory infections,
and vomiting.
[0120] In another aspect, conjugates of .alpha.-galactosidase A
with oligosaccharides comprising M6P are administered for the
treatment of Fabry disease. Fabry disease, or Anderson-Fabry
disease, is a rare, X-linked, lysosomal storage disorder marked by
a deficiency of .alpha.-galactosidase A, and results in
accumulation of globotriaosylceramide (GL3) and other neutral
glycosphingolipids in the lysosomes of visceral tissues and
endothelial, perithelial, and muscle cells. Accumulation of the
neutral glycosphingolipids in the vasculature results in narrowing
and dilatation of the blood vessels, and ultimately to ischemia and
infarction.
[0121] Administration of .alpha.-galactosidase A conjugates may
result in a reduction in one or more clinical symptoms of Fabry
disease including, e.g., acroparesthesia, angina, angiokeratoma,
arrythmia, ataxia of gait, burning and/or tingling pain in the
hands and feet, cataracts, cold intolerance, conduction
abnormalities, corneal whorling, coronary artery disease, dementia,
depression, diarrhea, dilated cardiac chambers, dizziness,
cardiomegaly, cardiomyopathy, diplopia, dysarthria, fatigue, fever
with elevated erythrocyte sedimentation rate, hearing problems,
heart disease, heart valve problems, heat intolerance, hemiataxia,
hemiparesis, hypohidrosis, impaired sweating, infaraction,
ischemia, joint pain, kidney disease, left ventricular hypertrophy,
lenticular abnormalities, lenticular opacity, lipiduria, muscle
weakness, myocardial infarction, nausea, nystagmus, pain (e.g.,
intense pain radiating throughout the body), polydipsia,
proteinuria, post-prandial pain, renal failure, retinal
abnormalities, ringing in ears, stomach pain, ST-T wave changes,
stroke, uremia, valvular disease, vertigo, vomiting, and weakness.
Administration of .alpha.-galactosidase A conjugates may result in
increased .alpha.-galactosidase A activity in, e.g., plasma, tears,
leukocytes, biopsied tissues, or cultured skin fibroblasts.
Administration of .alpha.-galactosidase A conjugates may also
result in a histologic finding of a reduction (e.g., of at least
10%) or lack of increase of birefringent lipid globules. It may
also result in a decrease in lipid globules in urinary sediment,
improved renal function as measured by serum creatinine levels or
creatinine clearance, and reduced proteinuria. Administration of
.alpha.-galactosidase A conjugates may also result in a reduction
in GL3 inclusions in the capillary endothelium of the kidney,
heart, and skin. Additional assays for measuring efficacy of
treatment for Fabry disease can be found in, e.g., MacDermott et
al., J. Med. Genet. 38:750-760 (2001).
[0122] In yet another aspect, conjugates of acid sphingomyelinase
are administered for treatment of Niemann-Pick disease, or acid
sphingomyelinase deficiency. Administration of acid
sphingomyelinase conjugates may result in a reduction in one or
more clinical symptoms of Niemann-Pick disease including, e.g.,
abnormal cholesterol levels, abnormal lipid levels, ataxia, blood
abnormalities, cherry red spots in the eye, frequent lung
infections, growth retardation, hepatosplenomegaly, low numbers of
platelets, lymphadenopathy, peripheral neuropathy, problems with
lung function, shortness of breath, skin pigmentation changes, or
xanthomas. In some embodiments, conjugates may be administered
intracranially.
[0123] An alternative embodiment relates to treatment of
mucopolysaccharidosis I (including, e.g., Hurler and Hurler-Scheie
forms of MPS I) with conjugates comprising .alpha.-L-iduronidase.
Administration of .alpha.-L-iduronidase conjugates may result in a
reduction in one or more clinical symptoms of MPS I including,
e.g., aortic regurgitation, aortic stenosis, carpal tunnel
syndrome, chronic rhinitis, conductive hearing loss, constipation,
corneal clouding, developmental delay, diarrhea, distended abdomen,
dorsolumbar kyphosis, gibbus deformity of the back,
hepatosplenomegaly, hydrocephalus, inguinal hernia, kyphosis,
mental retardation, mitral regurgitation, mitral stenosis,
night-blindness, open-angle glaucoma, poor hand function,
progressive arthropathy, recurrent respiratory infections,
respiratory insufficiency, retinal degeneration, scoliosis,
sensorineural hearing loss, severe back pain, rhinorrhea, sleep
apnea, spinal cord compression, thenar atrophy, umbilical hernia,
and upper airway complications.
[0124] The foregoing and the following description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed.
EXAMPLES
[0125] Examples 1-4 below describe the synthetic route depicted in
FIG. 1. Compounds 1, 2, 3, and 4, as used below, have the chemical
structures depicted in FIG. 1.
Example 1
Synthesis of Oligosaccharide 3
[0126] 100 mg of oligosaccharide 1 (MW=1250; bisM6P-hydrazide,
supplied by Biomira Inc., Edmonton, Canada) was dissolved in 15 ml
of DMSO/H.sub.2O (50:50 in volume), yielding a 5.3 .mu.mol/ml
solution. 100 mg of t-Boc-aminooxy acetic acid tetrafluorophenyl
ester 2 (Invitrogen Corp.; Carlsbad, Calif.; catalog # B3030) was
dissolved in 7.5 ml of DMSO. 15 ml of the oligosaccharide solution
was then mixed with 7.5 ml of the solution of 2 in a glass bottle,
such that the molar ratio of compound 2:compound 1 in the resulting
solution was 4:1. 744 .mu.l of DHBt-OH (from a 32.06 mg/ml stock in
DMSO) was added to the reaction mixture in a glass bottle, such
that the final ratio of compound 2:DHBt-OH is 1:0.5. The mixture
was gently shaken at room temperature (25.degree. C.) at 100 RPM
overnight for about 18 hours.
[0127] The following morning, 10 .mu.l of the reaction mixture was
removed for Dionex analysis to confirm completion of the reaction.
The results, depicted in FIG. 2, indicated 100% conversion from 1
to 3.
Example 2
Purification of Oligosaccharide 3
[0128] Method A.
[0129] The reaction mixture was diluted with an equal volume of
H.sub.2O and dialyzed in dialysis tubing with molecular weight
cutoff of 1000 Dalton (SpectraPor Inc.) twice against 4 L of
H.sub.2O at 4.degree. C. for at least 3 hours each. The samples
were then lyophilized.
[0130] Method B.
[0131] A Sephadex G-10 gel permeation chromatography column with a
bed volume of 225 ml was packed 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. 4.5 ml fractions were collected with a fraction
collector. Fractions 10-23, which contained oligosaccharide 3, were
collected, combined and lyophilized. The other small molecules,
including t-Boc-AOAA, DHBt-OH, and DMSO, eluted in the later
fractions, and were discarded.
Example 3
Deprotection of Oligosaccharide 3
[0132] The t-Boc group of the lyophilized sample was deprotected in
5 ml of 50% trifluoroacetic acid (TFA) in dicholormethane (DCM) in
a glass bottle for 30 min with gentle shaking at 100 RPM. The
TFA/DCM was then removed by a stream of N.sub.2 in a chemical
hood.
Example 4
Purification of Oligosaccharide 4
[0133] Method A.
[0134] After removing the TFA/DCM, the residue was dissolved in 10
ml of 0.5 M sodium acetate buffer, pH 5, and transferred to
dialysis tubing with a molecular weight cutoff of 1000 Dalton. The
bottle was washed with 4 ml of the same buffer, which was then
transferred to the dialysis tubing. The sample was dialyzed twice
against 3 L of 25 mM sodium acetate buffer, pH 7, for at least 3
hours, and then transferred to 4 L ice-cold H.sub.2O for overnight
dialysis. The sample was recovered from the dialysis tubing and
lyophilized.
[0135] Method B.
[0136] After removing the 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. 4.5 ml fractions were collected with a fraction
collector. Fractions 10-23, which contained purified
oligosaccharide 4, were collected and lyophilized. A higher yield
of oligosaccharide 4 was obtained upon purification by Method B
than by Method A.
[0137] The final product obtained either from method B was analyzed
by Dionex chromatography (FIG. 2C), and the identity of the product
was confirmed by mass spectrometry (FIG. 3B). Some impurities were
present in the spectra of FIG. 2C and FIG. 3B.
Example 5
Coupling of Oligosaccharide 4 to GAA
[0138] Oxidation of GAA.
[0139] Lyophilized recombinant human GAA (rhGAA) was reconstituted
in H.sub.2O and dialyzed against 4 L of 100 mM acetate buffer (pH
5.6) 4 times to completely remove mannitol. After dialysis, the
rhGAA was oxidized with 7.5 mM sodium periodate from 100 mM stock
in 100 mM acetate buffer. After 30 minutes at 4.degree. C. on ice,
glycerol was added, and the sample was mixed on ice for 10 minutes
to decompose excess sodium periodate. The oxidized material was
then dialyzed against aqueous buffer (e.g., 100 mM sodium acetate)
overnight.
[0140] Coupling.
[0141] A solution of oligosaccharide 4 in aqueous buffer (e.g., 100
mM sodium acetate, pH 5.6) was mixed with oxidized GAA and
incubated at 37.degree. C. for 4 hours to yield oligosaccharide-GAA
conjugate 5. The reaction mixture was then diafiltered against 25
mM sodium phosphate buffer, pH 6.25, to remove unconjugated bisM6P
glycan, and then adjusted with GAA formulation buffer (25 mM sodium
phosphate buffer, pH 6.25, 2% mannitol, 0.005% Tween-80).
Example 6
Characterization of the GAA Conjugate
[0142] Detection of M6P.
[0143] The extent of oligosaccharide conjugation was measured by
assaying conjugate 5 for binding to a M6P receptor column to which
glycoproteins lacking M6P do not bind. Five micrograms of conjugate
5 were loaded onto a pre-equilibrated CI-MPR-Sepharose column (the
column was prepared by coupling CI-MPR isolated from fetal bovine
serum to Affigel-10), which was then washed with CI-MPR binding
buffer for 11.times.2 mL fractions and eluted with CI-MPR binding
buffer containing 5 mM M6P for 7.times.2 mL fractions. A total of
18 fractions were collected and assayed for enzymatic activity.
[0144] Monosaccharide Analysis.
[0145] Conjugate 5 is treated with 4N trifluoroacetic acid to
hydrolyze the oligosaccharides, followed by high pH anion exchange
chromatography with pulsed amperometric detection (PAD) on a BioLC
liquid chromatography system (Dionex). The monosaccharide content
is extrapolated from a monosaccharide standard curve using premixed
monosaccharide standards (Dionex).
[0146] Specific Activity.
[0147] GAA activity is measured using a fluorometric assay in black
96-well microplates using 4-methylumbelliferyl-.alpha.-D-glucoside
as a substrate. Dilutions of conjugate 5 are added in triplicate to
a microtiter plate. 4-methylumbelliferyl-.alpha.-D-glucoside is
added to each sample. The 96-well plate is incubated in a
37.degree. C. incubator for 30 minutes. The release of product is
detected fluorometrically, and compared to standard curves
generated by measuring the fluorescence of a known quantity of a
standard. The reaction is quenched by the addition of 125 .mu.L of
1.0 M glycine-carbonate buffer, pH 10.5 to all wells. The specific
activity is defined as nmol product released/hr/mg.
[0148] Internalization by L6 Myoblasts.
[0149] Cells (ATCC CRL-1458) were seeded into 6-well plates at
5.0.times.10.sup.5 cells/well in growth media (DMEM+10% FBS) and
grown to confluency. Cells were incubated with 0-100 nM GAA
(conjugate 5 or unconjugated rhGAA) for 16 hours in DMEM+1%
heat-inactivated-FBS+10 mM Hepes pH 6.7. After uptake, cells were
washed with 3.times.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).
[0150] All references cited herein are incorporated herein by
reference in their entirety. To the extent publications and patents
or patent applications incorporated by reference contradict the
invention contained in the specification, the specification is
intended to supercede and/or take precedence over any such
contradictory material.
[0151] All numbers expressing quantities 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 attached
claims are approximations that may vary depending upon the desired
properties sought to be obtained by the present invention. At the
very least, and not as an attempt to limit the application of the
doctrine of equivalents to the scope of the claims, each numerical
parameter should be construed in light of the number of significant
digits and ordinary rounding approaches.
[0152] Many 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 offered by way of example only and are not
meant 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.
* * * * *