Title of Invention

"A METHOD FOR PREPARING A CONJUGATE COMPRISING A PROTEIN AND A POLYMER DERIVATIVE"

Abstract The present invention realtes to a method for preparing a conjugate comprising a protein and a polymer derivative, wherein the polymer is a hydroxyalkyl starch (HAS) and the protein is a granulocyte colony stimulating factor (G-CSF), the method comprising reacting at least one functional group A of a polymer derivative with at least one functional group Z of the protein and thereby forming a covalent linkage, wherein Z is an amino group, and whereinA is selected from the group consisting of an aldehyde group, a keto group or a hemiacetal group, wherein the method comprises introducing A in the polymer to give the polymer derivative by reacting the polymer with an at least bifunctional compound, one functional group M of which, comprising the chemical structure -NH-, reacts with the polymer and at least one other functional group Q of which is an aldehyde group, a keto group or a hemiacetal group, or is a functional group which is chemically modified to give an aldehyde group, a keto group or a hemiacetal group.
Full Text Field of the Invention
The present invention relates to a method for preparing a conjugate comprising a protein and a polymer derivative.
The present invention further relates to conjugates of hydroxyalkyl starch and a granulocyte colony stimulating factor protein (G-CSF) wherein these conjugates are formed by a covalent linkage between the hydroxyalkyl starch or a derivative of the hydroxyalkyl starch and the protein. The present invention also relates to the method of producing these conjugates and the use of these conjugates.
Background of the Invention
It is generally accepted that the stability of proteins can be improved and the immune response against these proteins is reduced when these proteins are coupled to polymeric molecules. WO 94/28024 discloses that physiologically active proteins modified with polyethylene glycol (PEG) exhibit reduced immunogenicity and antigenicity and circulate in the bloodstream considerably longer than unconjugated proteins, i.e. have a reduced clearance rate.
G-CSF is a 21 kDa glycoprotein stabilized by two intrachain disulfide bonds and containing a single 0-linked carbohydrate moiety. Mature G-CSF has 174 amino acids. In the animal body, G-CSF is synthesized by bone marrow stromal cells, macrophages and fibroblasts. It main function is to be a growth and differentiation factor for neutrophils and their precursor cells. However, it is also known in the art that G-CSF activates mature neutrophils. In addition, it stimulates growth/differentiation of various other haemopoietic progenitor cells (in synergy with additional haemopoietic growth factors) and promotes proliferation and migration of endothelial cells. Clinically, G-CSF is administered for the treatment of deficiencies in neutrophil levels (caused, e.g. by aplastic anaemia, myelodysplasia, AIDS, or chemotherapy).
WO 02/09766 discloses, among others, biocompatible protein-polymer compounds which are produced by conjugation of biologically active protein with a biocompatible polymer derivative. The biocompatible polymers used are highly reactive branched polymers, and the resulting conjugates contain a long linker between polymer derivative and protein. As

biocompatible polymers, polymers of formula (P-OCH2CO-NH-CHR-CO~)n-L-Qic-A are described, wherein P and Q are polymeric residues and k may be 1 or 0. For P and Q,
polyethylene glycol, polypropylene glycol, polyoxyethylene, polytrimethylene glycol,
polylactic acid and its derivatives, polyacrylic acid and its derivatives, polyamino acid,
polyvinyl alcohol, polyurethane, polyphosphazene, poly(L-lysine), polyalkylene oxide,
polyacryl amide and water soluble polymers such as dextran or polysaccharide are
mentioned. As proteins, among others, alpha, beta and gamma interferons, blood factors,
cytokines such as interleukins, G-CSF, GM-CSF are mentioned. In the examples of WO
02/09766, only mono-, di- and tri-polyethyleneglycol derivatives are disclosed which are
coupled exclusively to interferon and epidermal growth factor, and human growth
hormone.
WO 94/01483 discloses biocompatible polymer conjugates which are formed by covalently
binding a biologically inactive polymer or polymer derivative to a pharmaceutically pure,
synthetic hydrophilic polymer via specific types of chemical bonds. As naturally occuring
polymers and derivatives thereof, polysaccharides such as hyaluronic acid, proteoglycans
such as chondroitin sulfates A, B and C, chitin, heparin, heparin sulfate, dextrans such as
cyclodextran, hydroxyethyl cellulose, cellulose ether and starch, lipids such as triglycerides
and phospholipids are disclosed. As synthetic polymers, among others, polyethylene and
derivatives thereof are described having an average molecular weight of from about 100 to
about 100,000. As proteins linked to the polymer or the polymer derivative, cytokines and
growth factors are described, including interferons, tumor necrosis factors, interleukins,
colony stimulating factors, growth factors such as osteogenic factor extract, epidermal
growth factor, transforming growth factor, platelet derived growth factor, acidic fibroblast
growth factor and others are disclosed. In all working examples of WO 94/01483,
polyethylene glycols derivatives are used as polymer.
WO 96/11953 discloses N-terminally chemically modified protein compounds and
methods of their production. Specifically, G-CSF compositions are described which result
from coupling a water soluble polymer to the N terminus of G-CSF. In the context of WO
96/11953, also consensus interferone N-terminally coupled to water soluble polymers are
disclosed. While a wide variety of water polymers are listed in WO 96/11953 (e.g.
copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran,
polyvinyl alcohol, polyvinyl pyrrolidone, poly-l,3-dioxolane, poly-l,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random
copolymers), poly(n-vinyl pyrrolidone)polyethylene glycol, polypropylene glycol
homopolymers, polypropylene oxide/ethylene oxide copolymers or polyoxyethylated
polyols), only PEGylated G-CSF or consensus IFN compositions are described in the
working examples of WO 96/11953.
US 6,555,660 B2 discloses polypeptide conjugates comprising a polypeptide exhibiting GCSF
activity and having an amino acid sequence that differs from the amino acid sequence
of human G-CSF in at least one specified introduced and/or removed amino acid residue,
wherein the conjugate comprises an attachment group for a non-polypeptide moiety, and
further comprises at least one non-polypeptide moiety attached to the attachment group of
the polypeptide. The non-polypeptide moiety may be a polymer such as polyethylene
glycol or an oligosaccharide. In US 6,555,660 B2, it is explicitly and unambiguously stated
that PEG is by far the most preferred polymer molecule since it has only few reactive
groups capable of cross-linking compared to porysaccharides such as dextran.
WO 97/30148 relates to polypeptide conjugates with reduced allergenicity comprising a polymeric carrier molecule having two or more potypetide molecules coupled thereto.
These conjugates are preferably part of compositions used in the personal care market.
Said conjugates are produced by activating a polymeric carrier molecule, reacting two or more polypeptide molecules with the activated polymeric carrier molecule and blocking of
residual active groups on the conjugate. As polymeric carrier moelcule, a vast variety is listed in WO 97/30148, including such different groups of compound like natural or
synthetic homopolymers such as polyols, polyamines, polycarboxylic acids and
heteropolymers comprising at least two different attachment groups. Examples are given, which comprise star PEGs, branched PEGs, polyvinyl alcohols, polycarboxylates, polyvinylpyrrolidones and poly-D,L-amino acids. Among others, also dextrans such as carboxymethyl dextran, celluloses such as hydroxyethyl cellulose or hydroxypropyl cellulose, hydrolysates of chitosan, starches such as hydroxyethyl starches or hydroxypropyl starches, glycogen, agarose, guar gum, inulin, pullulan, xanthan gum,
carrageenin, pectin, alginic acid etc. are disclosed. As polypeptides, only some enzymes
are explicitly disclosed.
Baldwin, J.E, et al, Tetrahedron, vol. 27 (1981), pp. 1723 - 1726 describe the chemical modification of dextran and hydroxyethyl starch to give aldehyde substituted polymers which are allowed to react with hemoglobin to give soluble polymer-bound hemoglobins. These were shown to be capable of binding oxygen, but heart perfusion experiments clearly indicated that the polymer-bound hemoglobins were not suitable for use as blood substitutes.
WO 99/49897 describes conjugates of hemoglobin formed by reacting olysaccharides
such as dextrane or hydroxyethyl starch with amino groups of the hemoglobin. As
functional groups of the polysaccharide, aldehyde groups produced by oxidative saccharide
ring-opening are used. As preferred reducing agent used, borane dimethylamine is
disclosed. Moreover, WO 99/49897 is exclusively limited to hemoglobin.
WO 03/074087 relates to a method of coupling proteins to a starch-derived modified
polysaccharide. The binding action between the protein and the polysaccharide,
hydroxyalkyl starch, is a covalent linkage which is formed between the terminal aldehyde
group or a functional group resulting from chemical modification of said terminal aldehyde
group of the hydroxy alkyl starch molecule, and a functional group of the protein. As
reactive group of the protein, amino groups, thio groups and carboxyl groups are disclosed,
and aldehyde groups of the protein are not mentioned. Moreover, while a vast variety of
possibilities of different linkages is given in the form of many lists, including different
functional groups, theoretically suitable different linker molecules, and different chemical
procedures, the working examples describe only two alternatives: first, an oxidized
hydroxyethyl starch is used and coupled directly to proteins using
ethyldimethylaminopropyl carbodiimide (EDC) activation, or a non-oxidized hydroxyethyl
starch is used and coupled directly to a protein forming a Schiff s base which is
subsequently reduced to the respective amine. Thus, the working examples of WO
03/074087 neither disclose a single conjugate coupled via a thio group or a carboxy group
of the protein, nor describe a conjugate comprising hydroxyethyl starch, the protein, and
one or more linker molecules. Additionally, no G-CSF molecule is used in the working
examples.
Therefore, it was an object of the present invention to provide conjugates of hydroxyalkyl
starch, preferably hydroxy ethyl starch, and G-CSF which are not yet described in the prior
art. It is a further object of the present invention to provide methods of producing these conjugates.
Therefore, the present invention relates to a method for preparing a conjugate comprising a
protein and a polymer or a derivative thereof, wherein the polymer is a hydroxyalkyl starch
(HAS) and the protein is a granulocyte colony stimulating factor (G-CSF), the method
comprising reacting at least one functional group A of the polymer or the derivative thereof
with at least one functional group Z of the protein and thereby forming a covalent linkage,
wherein Z is selected from the group consisting of an amino group, a thiol group, an
aldehyde group and a keto group, and
- wherein, in case Z is an aldehyde group or a keto group, A comprises an amino
group forming said linkage with Z, or
- wherein, in case Z is an amino group, A is selected from the group consisting of a
reactive carboxy group and an aldehyde group, a keto group or a hemiacetal
group,
wherein, in case A is an aldehyde group, a keto group or a hemiacetal group,
the method further comprises introducing A in the polymer to give a polymer
derivative
by reacting the polymer with an at least bifunctional compound, one
functional group of which reacts with the polymer and at least one other
functional group of which is an aldehyde group, a keto group or a
hemiacetal group, or is a functional group which is further chemically
modified to give an aldehyde group, a keto group or a hemiacetal
group, or
by oxidizing the polymer to give at least one, in particular at least two
aldehyde groups, or
— wherein, in case A is a reactive carboxy group, the method further comprises
introducing A in the polymer to give a polymer derivative
by selectively oxidizing the polymer at its reducing end and activating
the resulting carboxy group, or
/=
by reacting the polymer at its non-oxidized reducing end with a
carbonic diester, or
- wherein, in case Z is a thiol group, A comprises
a maleimido group or
— a halogenacetyl group
forming said linkage with Z.
Accordingly, the present invention also relates to a conjugate as obtainable by a method as
described above.
The G-CSF can be produced by chemical synthetic procedures or can be of any human (see
e.g. Burgess, A.W. et al. 1977, Stimulation by human placenta! conditioned medium of
hemopoietic colony formation by human marrow cells, Blood 49 (1977), 573-583; Shah,
R.G. et al. 1977, Characterization of colony-stimulating activity produced by human
monocytes and r^ytohemaggtutiirin-stimiilated lymphocytes, Blood 50 (1977), 811) or
another mammalian source mid can be obtained by purification from naturally occurring
sources like human placenta, human blood or human urine. In addition, a lot of Epithelial
i
carcinomas, acute myeloid leukemia cells and various tumor cell lines (bladder
carcinomas, medulloblastomas), are capable to express this factor.
Furthermore, the expression G-CSF encompasses also a G-CSF variant wherein one or
more amino acids (e.g. 1 to 25, preferably 1 to 10, more preferably 1 to 5, most preferred 1
or 2) have been exchanged by another amino acid and which exhibits G-CSF activity (see
e.g. Riedhaar-Olson, J.F. et al. 1996, Identification of residues critical to the activity of
human granulocyte colony-stimulating factor, Biochemistry 35:9034-9041 1996; U.S. Pat.
Nos. 5,581476; 5,214,132; 5,362,853; 4,904,584). The measurement of G-CSF activity is
described in the art (for measurement of G-CSF activity hi vitro see e.g. Shirafuji, N. et
al.1989, A new bioassay for human granulocyte colony-stimulating factor (hG-CSF) using
murine myeloblastic NFS-60 cells as targets and estimation of its levels hi sera from
normal healthy persons and patients with infectious and hematological disorders, Exp.
Hematol. 1989, 17, 116-119; for measurement of G-CSF activity in vivo see e.g. Tanaka,
H. et al. 1991,_Pharmacokinetics of recombinant human granulocyte colony-stimulating
factor conjugated to polyethylene glycol in rats, Cancer Research 51, 3710-3714,1991).
Further publications where tests for the measurement of the activity of G-CSF are U.S. Pat.
_ —7
No. 6,555,660; Nohynek, G.J. et al.1997, Comparison of the potency of glycosylated and
nonglycosylated recombinant human granulocyte colony-stimulating factors in neutropenic
and nonneutropenic CD rats, Cancer Chemother Pharmacol (1997) 39;259-266.
Preferably, the G-CSF is recombinantly produced. This includes prokaryotic or eukaryotic
host expression of exogenous DNA sequences obtained by genomic or cDNA cloning or
by DNA synthesis. Suitable prokaryotic hosts include various bacteria such as E. coli.
Suitable eukaryotic hosts include yeast such as S. cerevisiae and mammalian cells such as
Chinese hamster ovary cells and monkey cells.The recombinant production of a protein is known in the art. In general, this includes the transfection of host cells with an appropriate expression vector, the cultivation of the host
cells under conditions which enable the production of the protein and the purification of
the protein from the host cells. For detailed information see e.g. Souza, L.M.et al. 1986,
Recombinant human granulocyte colony-stimulating factor: effects on normal and
leukemic myeloid cells, Science 1986 232:61-65,1986; Nagata, S. et.al. 1986, Molecular
cloning and expression of cDNA for human granulocyte colony-stimulating factor, Nature
319:415-418, 1986; Komatsu, Y. et al. 1987, Cloning of granulocyte colony-stimulating
factor cDNA from human macrophages and its expression in Escherichia coli, Jpn J Cancer
Res. 1987 78(11):! 179-1181.
In a preferred embodiment, the G-CSF has the amino acid sequence of human mature GCSF
(see e.g.; Nagata, S. et. al.1986, Molecular cloning and expression of cDNA for
human granulocyte colony-stimulating factor, Nature 319:415-418, 1986 ), and may
further contain a methionin at its amino terminus, which then results in a protein of 175
amino acids. Furthermore, instead of the methionine, G-CSF may contain a serine or a
threonine residue.
The G-CSF used in the methods of the present invention and the conjugates according to
the present invention may comprise one carbohydrate side chain attached to the G-CSF via
O-linked glycosylation at the position Thr 133, i.e. the G-CSF is glycosylated (V. Gervais
et aLJEur. J. Biochem. 1997, 247, 386-395). The structure of the carbohydrate side chain
may be NeuNAc(alpha2-3)Gal(betal-3)[NeuNAc(alpha2-6)]GalNAc und (alpha2-
3)Gal(betal-3)GalNAc (NeuNAc == N-acetylneuramic acid, GalNAc = Nacetylgalactosamine).
Modification of G-CSF and other polypeptides so as to introduce at least one additional
carbohydrate chain as compared to the native polypeptide has been suggested (U.S. Pat.
No. 5,218,092). Depending on the host employed, the G-CSF expression product may be glycosylated with mammalian or other eukaryotic carbohydrates. Usually, when G-CSF is produced in eukaryotic cells, the protein is posttranslationally glycosylated. Consequently,
the carbohydrate side chain may have been attached to the G-CSF during biosynthesis hi
mammalian, especially human, insect or yeast cells.
Recombinant human G-CSF (rhG-CSF) is generally used for treating various forms of
leukopenia. Thus, commercial preparations of rhG-CSF are available under the names
filgrastim (Gran® and Neupogen®), lenograstim (Neutrogin® and Granocyte®) and
nartograstim (Neu-up recombinant E. coli cells. Neutrogin® and Granocyte® are glycosylated and produced in
recombinant CHO cells and Neu-up® is non-glycosylated with five amino acids
substituted at the N-tenninal region of intact rhG-CSF produced in recombinant E. coli
cells.
As glycosylated protein, any glycosylated G-CSF such as Granocyte® may be employed.
As non-glycosylated G-CSF, any non-glycosylated G-CSF such as Neupogen® may be employed in the methods and conjugate according to the present invention.
Furthermore, at position -1, G-CSF may contain a methionine amino acid residue, a serine residue, or a threonine residue.
In the context of the present invention, the term "hydroxyalkyl starch" (HAS) refers to a starch derivative which has been substituted by at least one hydroxyalkyl group. A
preferred hydroxyalkyl starch of the present invention has a constitution according to
formula (I)
wherein the reducing end of the starch molecule is shown in the non-oxidized form and the
terminal saccharide unit is shown in the acetal form which, depending on e.g. the solvent,
may be in equilibrium with the aldehyde form.
The term hydroxyalkyl starch as used in the present invention is not limited to compounds
where the terminal carbohydrate moiety comprises hydroxyalkyl groups Ri, RI, and/or Ra
as depicted, for the sake of brevity, hi formula (I), but also refers to compounds hi which at
least one hydroxy group present anywhere, either in the terminal carbohydrate moiety
and/or in the remaining part of the starch molecule, HAS*, is substituted by a hydroxyalkyl
group R1,R2, or R3.
Hydroxyalkyl starch comprising two or more different hydroxyalkyl groups are also
possible.
The at least one hydroxyalkyl group comprised hi HAS may contain two or more hydroxy
groups. According to a preferred embodiment, the at least one hydroxyalkyl group
comprised hi HAS contains one hydroxy group.
The expression "hydroxyalkyl starch" also includes derivatives wherein the alkyl group is
mono- or polysubstituted. hi this context, it is preferred that the alkyl group is substituted
with a halogen, especially fluorine, or with an aryl group. Furthermore, the hydroxy group
of a hydroxyalkyl group may be esterified or etherified.
Furthermore, instead of alkyl, also linear or branched substituted or unsubstituted alkene
groups may be used.
Hydroxyalkyl starch is an ether derivative of starch. Besides of said ether derivatives, also
other starch derivatives can be used in the context of the present invention. For example,
derivatives are useful which comprise esterified hydroxy groups. These derivatives may be
e.g. derivatives of unsubstituted mono- or dicarboxylic acids with 2-12 carbon atoms or of
substituted derivatives thereof. Especially useful are derivatives of unsubstituted
monocarboxylic acids with 2-6 carbon atoms, especially derivatives of acetic acid. In this
context, acetyl starch, butyl starch and propyl starch are preferred.
Furthermore, derivatives of unsubstituted dicarboxylic acids with. 2-6 carbon atoms are
preferred.
In the case of derivatives of dicarboxylic acids, it is useful that the second carboxy group
of the dicarboxylic acid is also esterified. Furthermore, derivatives of monoalkyl esters of
dicarboxylic acids are also suitable in the context of the present invention.
For the substituted mono- or dicarboxylic acids, the substitute groups may be preferably
the same as mentioned above for substituted alkyl residues.
Techniques for the esterification of starch are known in the art (see e.g. KJemm D. et al,
Comprehensive Cellulose Chemistry Vol. 2, 1998, Whiley-VCH, Weinheim, New York,
especially chapter 4.4, Esterification of Cellulose (ISBN 3-527-29489-9).
According to a preferred embodiment of the present invention, hydroxyalkyl starch
according to formula (I) is employed. In formula (I), the saccharide ring described
explicitly and the residue denoted as HAS' together represent the preferred hydroxyalkyl
starch molecule. The other saccharide ring structures comprised in HAS' may be the same
as or different from the explicitly described saccharide ring.
As far as the residues R], Ra and Ra according to formula (I) are concerned there are no
specific limitations. According to a preferred embodiment, RI, R2 and Ra are independently
hydrogen or a hydroxyalkyl group, a hydroxyaryl group, a hydroxyaralkyl group or a
hydroxyalkaryl group having of from 2 to 10 carbon atoms in the respective alkyl residue.
Hydrogen and hydroxyalkyl groups having of from 2 to 10 are preferred. More preferably,
the hydroxyalkyl group has from 2 to 6 carbon atoms, more preferably from 2 to 4 carbon
atoms, and even more preferably from 2 to 4 carbon atoms. "Hydroxyalkyl starch"
therefore preferably comprises hydroxyethyl starch, hydroxypropyl starch and
hydroxybutyl starch, wherein hydroxyethyl starch and hydroxypropyl starch are
particularly preferred and hydroxyethyl starch is most preferred.
The alkyl, aryl, aralkyl and/or alkaryl group may be linear or branched and suitably
substituted.
Therefore, the present invention also relates to a method as described above wherein R1, R2
and RS are independently hydrogen or a linear or branched hydroxyalkyl group with from 1
to 6 carbon atoms.
Thus, RI, Ra and RS preferably may be hydroxyhexyl, hydroxypentyl, hydroxybutyl,
hydroxypropyl such as 2-hydroxypropyl, 3-hydroxypropyl, 2-hydroxyisopropyl,
hydroxyethyl such as 2-hydroxyethyl, hydrogen and the 2-hydroxyethyl group being
especially preferred.
Therefore, the present invention also relates to a method and a conjugate as described
above wherein RI, R2 and Ra are independently hydrogen or a 2-hydroxyethyl group, an
embodiment wherein at least one residue RI, Rj and RS being 2-hydroxyethyl being
especially preferred.
Hydroxyethyl starch (HES) is most preferred for all embodiments of the present invention.
Therefore, the present invention relates to the method and the conjugate as described
above, wherein the polymer is hydroxyethyl starch and the polymer derivative is a
hydroxyethyl starch derivative.
Hydroxyethyl starch (HES) is a derivative of naturally occurring amylopectin and is
degraded by alpha-amylase in the body. HES is a substituted derivative of the carbohydrate
polymer amylopectin, which is present in corn starch at a concentration of up to 95 % by
weight. HES exhibits advantageous biological properties and is used as a blood volume
replacement agent and in hemodilution therapy in the clinics (Sommerrneyer et al., 1987,
Kjarikenhauspharmazie, 8(8), 271-278; and Weidler et al., 1991, Arzneim.-
Forschung/Drug Res., 41,494-498).
Amyiopectin consists of glucose moieties, wherein in the main chain alpha- 1,4-glycosidic
bonds are present and at the branching sites alpha- 1,6-glycosidic bonds are found. The
physical-chemical properties of this molecule are mainly determined by the type of
glycosidic bonds. Due to the nicked alpha-1,4-glycosidic bond, helical structures with
about six glucose-monomers per turn are produced. The physico-chemical as well as the
biochemical properties of the polymer can be modified via substitution. The introduction
of a hydroxyethyl group can be achieved via alkaline hydroxyethylation. By adapting the
reaction conditions it is possible to exploit the different reactivity of the respective hydroxy
group in the unsubstituted glucose monomer with respect to a hydroxyethylation. Owing to
this fact, the skilled person is able to influence the substitution pattern to a limited extent.
HES is mainly characterized by the molecular weight distribution and the degree of
substitution. There are two possibilities of describing the substitution degree:
1. The degree can be described relatively to the portion of substituted glucose
monomers with respect to all glucose moieties.
2. The degree of substitution can be described as the molar substitution, wherein the
number of hydroxyethyl groups per glucose moiety are described.
In the context of the present invention, the degree of substitution, denoted as DS, relates to
the molar substitution, as described above.
HES solutions are present as polydisperse compositions, wherein each molecule differs
from the other with respect to the polymerisation degree, the number and pattern of
branching sites, and the substitution pattern. HES is therefore a mixture of compounds with
different molecular weight. Consequently, a particular HES solution is determined by
average molecular weight with the help of statistical means. In this context, Mn is
calculated as the arithmetic mean depending on the number of molecules. Alternatively,
Mw (or MW), the weight mean, represents a unit which depends on the mass of the HES.
In the context of the present invention, hydroxyethyl starch may preferably have a mean
molecular weight (weight mean) of from 1 to 300 kD. Hydroxyethyl starch can further
exhibit a preferred molar degree of substitution of from 0.1 to 0.8 and a preferred ratio
between €2: Ce substitution in the range of from 2 to 20 with respect to the hydroxyethyl
groups.
The term "mean molecular weight" as used in the context of the present invention relates to
the weight as determined according to Sommermeyer et al., 1987, Krankenhauspharmazie,
8(8), 271-278; and Weidler et al., 1991, Arzneim.-Forschung/Drug Res., 41,494-498.
According to a preferred embodiment of the present invention, the mean molecular weight
of hydroxyethyl starch employed is from 1 to 300 kD, more preferably from 2 to 200 kD,
more preferably of from 4 to 130 kD, more preferably of from 4 to 70 kD.
An example for HES with a mean molecular weight of about 130 kD is Voluven® from
Fresenius. Voluven® is an artifical colloid, employed, e.g., for volume replacement used in
the therapeutic indication for therapy and prophylaxis of hypovolaemia. The characteristics
of Voluven® are a mean molecular weight of 130,000 +/- 20,000 D, a molar substitution of
0.4 and a C2 . C6 ratio of about 9:1.
Therefore, the present invention also relates to a method and to conjugates as described
above wherein the hydroxyalkyl starch is hydroxyethyl starch having a mean molecular
weight of from 4 to 70 kD.
Preferred ranges of the mean molecular weight are, e.g., 4 to 70 kD or 10 to 70 kD or 12 to
70 kD or 18 to 70 kD or 50 to 70 kD or 4 to 50 kD or 10 to 50 kD or 12 to 50 kD or 18 to
50 kD or 4 to 18 kD or 10 to 18 kD or 12 to 18 kD or 4 to 12 kD or 10 to 12 kD or 4 to 10
kD.
According to particularly preferred embodiments of the present invention, the mean
molecular weight of hydroxyethyl starch employed is in the range of from more than 4 kD
and below 70 kD, such as about 10 kD, or in the range of from 9 to 10 kD or from 10 to 11
kD or from 9 to 11 kD, or about 12 kD, or in the range of from 11 to 12 kD or from 12 to
13 kD or from 11 to 13 kD, or about 18 kD, or in the range of from 17 to 18 kD or from 18
to 19 kD or from 17 to 19 kD, or about 50 kD, or in the range of from 49 to 50 kD or from
50 to 51 kD or from 49 to 51 kD.
As far as the degree of substitution (DS) is concerned, DS is preferably at least 0.1, more
preferably at least 0.2, and more preferably at least 0.4. Preferred ranges of DS are from
0.1 to 0.8, more preferably from 0.2 to 0.8, more preferably from 0.3 to 0.8 and even more
preferably from 0.4 to 0.8, still more preferably from 0.1 to 0.7, more preferably from 0.2
to 0.7, more preferably from 0.3 to 0.7 and more preferably from 0.4 to 0.7. Particularly
preferred values of DS are, e.g., 0.1,0.2, 0.3,0.4, 0.5, 0.6,0.7 or 0.8, with 0.2, 0.3,0.4, 0.5,
0.6, 0.7 or 0.8 being more preferred, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 being even more
preferred, 0.4, 0.5, 0.6, 0.7 or 0.8 being still more preferred and, e.g. 0.4 and 0.7 being
particularly preferred.
Particularly preferred combinations of molecular weight of the hydroxyalkyl starch,
preferably hydroxyethyl starch, and its degree of substitution DS are, e.g., 10 kD and 0.4 or
10 kD and 0.7 or 12 kD and 0.4 or 12 kD and 0.7 or 18 kD and 0.4 or 18 kD and 0.7 or 50
kD and 0.4 or 50 kD and 0.7.
In another preferred embodiment of the present invention, the hydroxyethyl starch (as
employed as well as contained in Ac conjugates described herein) has a molecular weight
of from about 20 kD to about 130 kD (i.e. about 40 kD, about 50 kD, about 60 kD, about
70 kD, about 80 kD, about 90 kD, about 100 kD, about 110 kD, about 120 kD, about 130
kD) preferably a mean molecular weight of about 30 kD to about 100 kD, more preferably
from about 40 to about 70 kD and a degree of substitution from 0.4 to 0.8, more preferred
from 0.5 to 0.8.
In this context the term "about 30kD" is understood to relate to a mean molecular weight in
the range of from 25 kD to 34 kD, i.e. including also starches having a mean molecular
weight of 26,27,28,29,31,32,33 or 34 kD.
In this context the term "about 40kD" is understood to relate to a mean molecular weight in
the range of from 35 kD to 44 kD, i.e. including also starches having a mean molecular
weight of 36, 37, 38, 39,41,42,43 or 44 kD.
In this context the term "about 50kD" is understood to relate to a mean molecular weight in
the range of from 45 kD to 54 kD, i.e. including also starches having a mean molecular
weight of 46,47,48,49, 51, 52, 53 or 54 kD.
In this context the term "about 60kD" is understood to relate to a mean molecular weight in
the range of from 55 kD to 64 kD, i.e. including also starches having a mean molecular
weight of 56,57, 58, 59, 61, 62, 63 or 64 kD.
In this context the term "about 70kD" is understood to relate to a mean molecular weight hi
the range of from 65 kD to 74 kD, i.e. including also starches having a mean molecular
weight of 66, 67, 68, 69, 71,72,73 or 74 kD.
In this context the term "about 80kD" is understood to relate to a mean molecular weight in
the range of from 75 kD to 84 kD, i.e. including also starches having a mean molecular
weight of 76, 77,78, 79, 81, 82, 83 or 84 kD.
In this context the term "about 90kD" is understood to relate to a mean molecular weight in
the range of from 85 kD to 94 kD, i.e. including also starches having a mean molecular
weight of 86,87,88,89,91,92,93 or 94 kD.
In this context the term "about lOOkD" is understood to relate to a mean molecular weight
in the range of from 95 kD to 104 kD, Le. including also starches having a mean molecular
weight of 96,97,98,99,101,102,103 or 104 kD.
In this context the term "about 1 lOkD" is understood to relate to a mean molecular weight
in the range of from 105 kD to 114 kD, i.e. including also starches having a mean
molecular weight of 106, 107, 108,109, 111, 112,113 or 114 kD.
In this context the term "about 120kD" is understood to relate to a mean molecular weight
hi the range of from 115 kD to 124 kD, i.e. including also starches having a mean
molecular weight of 116,117,118,119,121,122,123 or 124 kD.
In this context the term "about 130kD" is understood to relate to a mean molecular weight
in the range of from 125 kD to 134 kD, i.e. including also starches having a mean
molecular weight of 126, 127,128,129,131,132,133 or 134 kD.
Accordingly the embodiment described above comprises a hydroxethyl starch (and the
conjugates described herein comprising the hydroxyl ethyl starch as well as the methods
described herein employing hydroxylethyl starch) that has a mean molecular weight of
about 30 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6,
0.7 or 0.8.
Accordingly the embodiment described above also comprises a hydroxethyl starch (and the
conjugates described herein comprising the hydroxyl ethyl starch as well as the methods
described herein employing hydroxyethyl starch) that has a mean molecular weight of
about 40 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6,
0.7 or 0.8.
Accordingly the embodiment described above also comprises a hydroxethyl starch (and the
conjugates described herein comprising the hydroxyl ethyl starch as well as the methods
described herein employing hydroxyethyl starch) that has a mean molecular weight of
about 50 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6,
0.7 or 0.8.
Accordingly the embodiment described above also comprises a hydroxethyl starch (and the
conjugates described herein comprising the hydroxyl ethyl starch as well as the methods
described herein employing hydroxyethyl starch) that has a mean molecular weight of
about 60 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6,
0.7 or 0.8.
Accordingly the embodiment described above also comprises a hydroxethyl starch (and the
conjugates described herein comprising the hydroxyl ethyl starch as well as the methods
described herein employing hydroxyethyl starch) that has a mean molecular weight of
about 70 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6,
0.7 or 0.8.
Accordingly the embodiment described above also comprises a hydroxethyl starch (and the
conjugates described herein comprising the hydroxyl ethyl starch as well as the methods
described herein employing hydroxyethyl starch) that has a mean molecular weight of
about 80 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6,
0.7 or 0.8.
Accordingly the embodiment described above also comprises a hydroxethyl starch (and the
conjugates described herein comprising the hydroxyl ethyl starch as well as the methods
described herein employing hydroxyethyl starch) that has a mean molecular weight of
about 90 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6,
0.7 or 0.8.
Accordingly the embodiment described above also comprises a hydroxethyl starch (and the
conjugates described herein comprising the hydroxyl ethyl starch as well as the methods
described herein employing hydroxyethyl starch) that has a mean molecular weight of
about 100 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6,
0.7 or 0.8.
Accordingly the embodiment described above also comprises a hydroxethyl starch (and the
conjugates described herein comprising the hydroxyl ethyl starch as well as the methods
described herein employing hydroxyethyl starch) that has a mean molecular weight of
about 110 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6,
0.7 or 0.8.
Accordingly the embodiment described above comprises a hydroxethyl starch (and the
conjugates described herein comprising the hydroxyl ethyl starch as well as the methods
described herein employing hydroxyethyl starch) that has a mean molecular weight of
about 120 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6,
0.7 or 0.8.
Accordingly the embodiment described above comprises a hydroxethyl starch (and the
conjugates described herein comprising the hydroxyl ethyl starch as well as the methods
described herein employing hydroxyethyl starch) that has a mean molecular weight of
about 130 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6,
0.7 or 0.8.
An example of HES having a mean molecular weight of about 130 kD is a HES with a
degree of substitution of 0.2 to 0.8 such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8, preferably of
0.4 to 0.7 such as 0.4,0.5, 0.6, or 0.7.
As
far as the ratio of €2: C(, substitution is concerned, said substitution is preferably in the
range of from 2 to 20, more preferably in the range of from 2 to 15 and even more
preferably in the range of from 3 to 12.
According to a further embodiment of the present invention, also mixtures of hydroxyethyl
starches may be employed having different mean molecular weights and/or different
degrees of substitution and/or different ratios of Ca: Ce substitution. Therefore, mixtures of
hydroxyethyl starches may be employed having different mean molecular weights and
different degrees of substitution and different ratios of €2 : €5 substitution, or having
different mean molecular weights and different degrees of substitution and the same or
about the same ratio of Ca : Ce substitution, or having different mean molecular weights
and the same or about the same degree of substitution and different ratios of €2 : Ce
substitution, or having the same or about the same mean molecular weight and different
degrees of substitution and different ratios of €2: C$ substitution, or having different mean
molecular weights and the same or about the same degree of substitution and the same or
about the same ratio of Cj: C$ substitution, or having the same or about the same mean
molecular weights and different degrees of substitution and the same or about the same
ratio of C2: C5 substitution, or having the same or about the same mean molecular weight
and the same or about the same degree of substitution and different ratios of C2 : C6
substitution, or having about the same mean molecular weight and about the same degree
of substitution and about the same ratio of C2: Ce substitution.
In different conjugates and/or different methods according to the present invention,
different hydroxyalkyl starches, preferably different hydroxyethyl starches and/or different
hydroxyalkyl starch mixtures, preferably different hydroxyethyl starch mixtures, may be
employed.
According to one embodiment of the present invention, the functional group Z of the
protein is an aldehyde group or a keto group. Therefore, the present invention relates to a
method and conjugates as described above, wherein the functional group Z of the protein is
an aldehyde group or a keto group.
While there are no general restrictions as to the location of the aldehyde or keto group
within the protein, the aldehyde or keto group is, according to a preferred embodiment of
the present invention, located in a carbohydrate side chain of the protein. Therefore, in the
context of this embodiment, a glycosylated protein is employed.
As glycosylated protein, any glycosylated G-CSF such as Granocyte® may be employed.
In the context of the present invention, the term "carbohydrate side chain" refers to
hydroxyaldehydes or hydroxyketones as well as to chemical modifications thereof (see
R6mpp Chemielexikon, Thieme Verlag Stuttgart, Germany, 9th edition 1990, Volume 9,
pages 2281-2285 and the literature cited therein). Furthermore, it also refers to derivatives
of naturally occuring carbohydrate moieties like, galactose, N-acetylneuramic acid, and Nacetylgalactosamine)
and the like. In case a mutant of G-CSF is employed being Nglycosylated,
a carbohydrate moiety may be mannose.
In an even more preferred embodiment, the aldehyde group or the keto group is part of a
galactose residue of the carbohydrate side chain. This galactose residue can be made
available for reaction with the functional group A comprised in the polymer or polymer
derivative by removal of terminal sialic acids, followed by oxidation, as described
hereinunder.
hi a still further preferred embodiment, the polymer or polymer derivative comprising
functional group A is linked to a sialic acid residue of the carbohydrate side chains,
preferably the terminal sialic acid residue of the carbohydrate side chain.
Oxidation of terminal carbohydrate moieties can be performed either chemically or
enzymatically.
Methods for the chemical oxidation of carbohydrate moieties of polypeptides are known in
the art and include the treatment with periodate (Chamow et al., 1992, J. Biol. Chem., 267,
15916-15922).
By chemically oxidizing, it is in principle possible to oxidize any carbohydrate moiety,
being terminally positioned or not. However, by choosing mild reaction conditions it is
possible to preferably oxidize the terminal sialic acid of a carbohydrate side chain to give
the aldehyde group or the keto group.
According to one embodiment of the present invention, said mild reaction conditions relate
to reacting the protein with a suitable aqueous periodate solution, having a preferred
periodate concentration in the range of from 1 to 50 mM, more perferably of from 1 to 25
mM and especially perferably of from 1 to 10 mM such as about 1 mM, and at a preferred
reaction temperature of from 0 to 40 °C and especially preferably of from 0 to 21 °C such
as about 0 °C, and for a preferred reaction time of from 5 min to 5 h, more preferably from
10 min to 2 h and especially preferably from 10 min. to 1 h such as about 1 h. The
preferred molar ratio of periodate : protein is from 1:200 to 1:1 and more preferably from
1:50 to 1:5. such as about 15 :1.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein, prior to the reaction of the protein and the polymer or polymer derivate, a
glycosylated protein is reacted with a periodate solution to give a protein having an
aldehyde group or a keto group located in the oxidized carbohydrate side chain.
Alternatively, the carbohydrate side chain may be oxidized enzymatically. Enzymes for the
oxidation of the individual carbohydrate side chain are known in the art, e.g. in the case of
galactose the enzyme is galactose oxidase. If h is intended to oxidize terminal galactose
moieties, it will be eventually necessary to remove terminal sialic acids (partially or
completely) if the polypeptide has been produced in cells capable of attaching sialic acids
to carbohydrate chains, e.g. in mammalian cells or in cells which have been genetically
modified to be capable of attaching sialic acids to carbohydrate chains. Chemical or
enzymatic methods for the removal of sialic acids are known in the art (Chaplin and
Kennedy (eds.), 1996, Carbohydrate Analysis: a practical approach, especially Chapter 5
Montreuill, Glycoproteins, pages 175-177; IRL Press Practical approach series (ISBN 0-
947946-44-3)).
According to another preferred embodiment of the present invention, the aldehyde group
or keto group may be located at the N terminus of the protein and is accessible by suitable
oxidation. Especially in the case that a hydroxy group containing amino acid is located at
the N terminus of the protein, such as threonine or serine, oxidation of said N-terminal
amino acid can be carried out leading to said keto group or an aldehyde group. Theronine,
is the N terminal amino acid in human derived G-CSF. An additional N-terminal serine or
threonkie may be introduced in any protein showing G-CSF like activity by molecular
biological methods. This protein or the protein expressing the human amino acid sequence
may be produced by expression in prokaryotic or eukaryotic cells such as bacteria,
mammalian, insect or yeast cells, and which may or may not be glycosylated. As method
for the chemical oxidation of the suitable N-terminal amino acid, any conceivable method
may be applied, with the oxidation with periodate being preferred.
According to a further preferred embodiment of the present invention, said mild reaction
conditions relate to reacting the protein with a suitable aqueous periodate solution, having
a preferred periodate concentration in the range of from 1 to 50 mM, more perferably of
from 1 to 25 mM and especially perferably of from 1 to 10 mM such as about 1 mM, and
at a preferred reaction temperature of from 0 to 40 °C and especially preferably of from 0
to 21 °C such as about 0 °C, and for a preferred reaction time of from 5 min to 5 h, more
preferably from 10 min to 2 h and especially preferably from 10 min. to 1 h such as about 1
h. The preferred molar ratio of periodate: protein is from 1:200 to 1:1 and move preferably
from 1:50 to 1:5. such as about IS: 1.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the aldehyde group or the keto group is located in a carbohydrate side
chain of the protein and/or at the N-terminal group of the protein.
The oligosaccharide pattern of proteins produced in eukaryotic cells thus having been
posttranslationally glycosylated, are not identical to the human derived proteins. Moreover,
many glycosylated proteins do not have the desired number of terminal sialic acid residues
masking a further carbohydrate moiety such as a galactose residue. Those further
carbohydrate moieties such as a galactose residue, however, if not masked, are possibly
responsible for disadvantages such as a shorter plasma half-life of the protein in possible
uses of the protein as a medicament. It was surprisingly found that by providing a protein
conjugate formed by a hydroxyalkyl starch polymer, preferably a hydroxyethyl starch
polymer, which is covalently linked, e.g. via an oxime linkage as disclosed hereinunder, to
a carbohydrate moiety of a carbohydrate side chain of the protein, either directly or via at
least one linker compounds such as one or two linker compounds, it is possible to
overcome at least the above mentioned disadvantage. Hence it is believed that by coupling
a hydroxyalkyl starch polymer or derivative thereof, preferably a hydroxyethyl starch
polymer or a derivative thereof, to at least one carbohydrate side chain of a glycosylated
protein, the lack of suitable terminal carbohydrate residues located at a carbohydrate side
chain is compensated. According to another aspect of the invention, providing the
respective conjugate with a hydroxyalkyl starch polymer or derivative thereof, preferably a
hydroxyethyl starch polymer or a derivative thereof, coupled to the oxidized carbohydrate
moiety as described above, does not only compensate the disadvantage but provides a
protein conjugate having better characteristics in the desired field of use than the respective
naturally occuring protein. Therefore, the respective conjugates according to the invention
have a compensational and even a synergistic effect on the protein. It also possible that
even proteins which are identical to human proteins or which are human proteins do not
have the desired number of suitable masking terminal carbohydrate residues such as silaic
acid residues at naturally occuring carbohydrate moieties. In such cases, providing the
respective conjugate with a hydroxyalkyl starch polymer or derivative thereof, preferably a
hydroxyethyl starch polymer or a derivative thereof, coupled to the oxidized carbohydrate
moiety as described above, does not only overcome and compensate a disadvantage of an
artificially produced protein, but improves the characteristics of the a natural naturally
occuring protein. As to the functional group of the hydroxyalkyl starch, preferably
hydroxyethyl starch, or a derivative thereof, which is coupled to the aldehyde group or keto
group of the oxidized carbohydrate moiety of the protein, reference is made to the
functional groups A as disclosed hereinunder. This general concept is not only applicable
to glycosylated G-CSF, but principally to all glycosylated having said lack of terminal
carbohydrate residues. Among others, erythropoietin (EPO), interferone beta la (IFN beta
la), ATIII, factor VII, factor VIII, factor IX, alphal-antitrypsin (A1AT), htPA, or GMCSF
may be mentioned.
Therefore, the present invention also relates to the use of hydroxyalkyl starch, preferably
hydroxyethyl starch, or a derivative thereof, for compensating the lack of terminal
carbohydrate residues, preferably sialic acid residues, in naturally occuring or
posttranslationally attached carbohydrate moieties of a protein, by covalently coupling the
starch or derivative thereof to at least one oxidized carbohydrate moiety of a protein having
at least one keto or aldehyde group.
Accordingly, the present invention also relates to a method for compensating the lack of
terminal carbohydrate residues, preferably sialic acid residues, in naturally occuring or
posttranslationally attached carbohydrate moieties of a protein, by covalently coupling
hydroxyalkyl starch, preferably hydroxyethyl starch, or a derivative thereof to at least one
oxidized carbohydrate moiety of a protein having at least one keto or aldehyde group,
preferably via an oxime linkage.
Moreover, the present invention also relates to a conjugate formed by covalent linkage of a
hydroxyalkyl starch, preferably hydroxyethyl starch, or a derivative thereof, to at least one
oxidized carbohydrate moiety of a protein, said protein being either isolated from natural
sources or produced by expression in eukaryotic cells, such as mammalian, insect or yeast
cells, said carbohydrate moiety having at least one keto or aldehyde group, wherein the
conjugate has in the desired field of use, preferably the use as medicament, the same or
better characteristics than the respective unmodified protein.
In case functional group Z of the protein is an aldehyde group or a keto group, functional
group A of the polymer or the derivative thereof comprises an amino group according to
the structure -NH-.
Therefore, the present invention also relates to a method and a conjugate as described
above wherein the functional group A capable of being reacted with the optionally
oxidized reducing end of the polymer, comprises an amino group according to structure -
NH-.
According to one preferred embodiment of the present invention, this functional group A is
a group having the structure R'-NH- where R' is hydrogen or a alkyl, cycloalkyl, aryl,
aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue where the cycloalkyl, aryl,
aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue may be linked directly to the NH
group or, according to another embodiment, may be linked by an oxygen bridge to the NH
group. The alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl, or cycloalkylaryl
residues may be suitably substituted. As preferred substituents, halogenes such as F, Cl or
Br may be mentioned. Especially preferred residues R1 are hydrogen, alkyl and alkoxy
groups, and even more preferred are hydrogen and unsubstituted alkyl and alkoxy groups.
Among the alkyl and alkoxy groups, groups with 1, 2, 3, 4, 5, or 6 C atoms are preferred.
More preferred are methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, and
isopropoxy groups. Especially preferred are methyl, ethyl, methoxy, ethoxy, and particular
preference is given to methyl or methoxy.
Therefore, the present invention also relates to a method and a conjugate as described
above wherein R' is hydrogen or a methyl or a methoxy group.
According to another preferred embodiment of the present invention, the functional group
A has the structure R'-NH-R"- where R" preferably comprises the structure unit -NHand/
or the structure unit -(C=G)- where G is O or S, and/or the structure unit -SCVAccording
to more preferred embodiments, the functional group R" is selected from the
group consisting of
(Figure Removed)
where, if G is present twice, it is independently O or S.
Therefore, preferred functional groups A comprising an amino group -NHz, are, (Figure Removed)

wherein G is O or S and, if present twice, independently O or S, and R1 is methyl.
Especially preferred functional groups A comprising an amino group are aminooxy groups
-O- being particularly preferred, and the hydrazido group
(Figure Removed)
where G is preferably O.
Therefore, the present invention also relates to a method as described above, wherein the
functional group Z of the protein is an aldehyde group or a keto group, and the functional
group A is an aminooxy group or a hydrazido group. According to an especially preferred
embodiment of the present invention, A is an aminooxy group.
Thus, the present invention also relates to a conjugate, as described above, wherein the
functional group Z of the protein is an aldehyde group or a keto group, and the functional
group A is an aminooxy group or a hydrazido group. According to an especially preferred
embodiment of the present invention, A is an aminooxy group.
When reacting the aminooxy group of the polymer or polymer derivative with the aldehyde
group or keto group of the protein, an oxime linkage is formed.
Therefore, the present invention also relates to a conjugate as described above, wherein the
covalent linkage between the protein and the polymer or polymer derivative is an oxime
linkage formed by the reaction of functional group Z of the protein, said functional group Z
being an aldehyde group or a keto group, and functional group A of the polymer or
polymer derivative, said functional group A being an aminooxy group.
When reacting the hydrazido group of the polymer or polymer derivative with the aldehyde
group or keto group of the protein, a hydrazone linkage is formed.
Therefore, the present invention also relates to a conjugate as described above, wherein the
covalent linkage between the protein and the polymer or polymer derivative is a hydrazone
linkage formed by the reaction of functional group Z of the protein, said functional group Z
being an aldehyde group or a keto group, and functional group A of the polymer or
polymer derivative, said functional group A being a hydrazido group.
In order to introduce functional group A into the polymer, no specific restrictions exist
given that a polymer derivative results comprising functional group A.
According to a preferred embodiment of the present invention, the functional group A is
introduced in to the polymer by reacting the polymer with an at least bifunctional
compound, one functional group of which is capable of being reacted with at least one
functional group of the polymer, and at least one other functional group of the at least
bifunctional compound being functional group A or being capable of being chemically
modified to give functional group A.
According to a still further preferred embodiment, the polymer is reacted with the at least
bifunctional compound at its optionally oxidized reducing end.
In case the polymer is reacted with its non-oxidized reducing end, the polymer preferably
has the constitution
(Figure Removed)
wherein in formula (I), the aldehyde form of the non-oxidized reducing end is included.
In case the polymer is reacted with its oxidized reducing end, the polymer preferably has
the constitution according to formula (Ha)
HAS'
(Ha)
and/or according to formula (lib)
(Figure Removed)
The oxidation of the reducing end of the polymer, preferably hydroxyethyl starch, may be
carried out according to each method or combination of methods which result in
compounds having the above-mentioned structures (Ha) and/or (lib).
Although the oxidation may be carried out according to all suitable method or methods
resulting in the oxidized reducing end of hydroxyalkyl starch, it is preferably carried out
using an alkaline iodine solution as described, e.g., in DE 1% 28 705 Al the respective
contents of which (example A, column 9, lines 6 to 24) is incorporated herein by reference.
As functional group of the at least bifunctional compound which is capable of being
reacted with the optionally oxidized ra^y^ng end of the polymer, each functional group
may be used which is capable of forming a chemical linkage with the optionally oxidized
reducing end of the hydroxyalkyl starch.
According to a preferred embodiment of the present invention, this functional group
comprises the chemical structure -NH-.
Therefore, the present invention also relates to a method and a conjugate as described
above wherein the functional group of the at least bifunctional compound, said functional
group being capable of being reacted with the optionally oxidized reducing end of the
polymer, comprises the structure -NH-.
According to one preferred embodiment of the present invention, this functional group of
the at least bifunctional compound is a group having the structure R'-NH- where R' is
hydrogen or a alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl
residue where the cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue
may be linked directly to the NH group or, according to another embodiment, may be
linked by an oxygen bridge to the NH group. The alkyl, cycloalkyl, aryl, aralkyl,
arylcycloalkyl, alkaryl, or cycloalkylaryl residues may be suitably substituted. As preferred
substituents, halogenes such as F, Cl or Br may be mentioned. Especially preferred
residues R' are hydrogen, alkyl and alkoxy groups, and even more preferred are hydrogen
and unsubstituted alkyl and alkoxy groups.
Among the alkyl and alkoxy groups, groups with 1, 2, 3, 4, 5, or 6 C atoms are preferred.
More preferred are methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, and
isopropoxy groups. Especially preferred are methyl, ethyl, methoxy, ethoxy, and particular
preference is given to methyl or methoxy.
Therefore, the present invention also relates to a method and a conjugate as described
above wherein R' is hydrogen or a methyl or a methoxy group.
According to another preferred embodiment of the present invention, the functional group
of the at least ^functional compound has the structure R'-NH-R"- where R" preferably
comprises the structure unit -NH- and/or the structure unit -(C=G)- where G is O or S,
and/or the structure unit -SQz-. According to more preferred embodiments, the functional
group R" is selected from the group consisting of
and G
where, if G is present twice, it is independently O or S.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the functional group of the at least bifunctional compound, said functional
group being capable of being reacted with the optionally oxidized reducing end of the
polymer, is selected from the group consisting of
(Figure Removed)
wherein G is O or S and, if present twice, independently O or S, and R is methyl.
According to an even more preferred embodiment of the present invention, the functional
group of the at least bifunctional compound, said functional group being capable of being
reacted with the optionally oxidized reducing end of the polymer and comprising an amino
group, is an aminooxy groups
H2N-O- being particularly preferred, or the hydrazido group
(Figure Removed)
wherein G is preferably O.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the functional group Z of the protein is an aldehyde group or a keto group,
and the functional group of the at least bifunctional compound, said functional group being
capable of being reacted with the optionally oxidized reducing end of the polymer, is an
aminooxy group or a hydrazido group, preferably an aminooxy group.
Thus, the present invention also relates to a conjugate, as described above, wherein the
functional group Z of the protein is an aldehyde group or a keto group, and the functional
group of the at least bifunctional compound, said functional group being capable of being
reacted with the optionally oxidized reducing end of the polymer, is an aminooxy group or
a hydrazido group, preferably an aminooxy group.
According to a still further preferred embodiment of the present invention, the at least
bifunctional compound is reacted with the polymer at its non-oxidized reducing end.
According to yet another preferred embodiment of the present invention, the at least
bifunctional compound which is reacted with the optionally oxidized reducing end of the
polymer, comprises functional group A.
The at least bifunctional compound may be reacted with the polymer first to give a
polymer derivative which is subsequently reacted with the protein via functional group A.
It is also possible to react the at least bifunctional compound via functional group A with
the protein first to give a protein derivative which is subsequently reacted with the polymer
via at least one functional group of the at least bifunctional compound residue comprised hi
the protein derivative.
According to a preferred embodiment of the present invention, the at least bifunctional
compound is reacted with the polymer first.
Therefore, the present invention relates to a method and a conjugate as described above,
said method further comprising reacting the polymer at its non-oxidized reducing end with
an at least bifunctional Unking compound comprising a functional group capable of
reacting with the non-oxidized reducing end of the polymer and a group A, prior to the
reaction of the polymer derivative comprising A and the protein comprising Z.
The functional group of the at least bifunctional linking compound which is reacted with
the polymer and the functional group A of the at least bifunctional linking compound
which is reacted with functional group Z of the protein may be separated by any suitable
spacer. Among others, the spacer may be an optionally substituted, linear, branched and/or
cyclic hydrocarbon residue. Generally, the hydrocarbon residue has up to 60, preferably up
to 40, more preferably up to 20, more preferably up to 10, more preferably up to 6 and
especially preferably up to 4 carbon atoms. If heteroatoms are present, the separating group
comprises generally from 1 to 20, preferably from 1 to 8, more preferably 1 to 6, more
preferably 1 to 4 and especially preferably from 1 to 2 heteroatoms. As heteroatom, O is
preferred. The hydrocarbon residue may comprise an optionally branched alkyl chain or an
aryl group or a cycloalkyl group having, e.g., from 5 to 7 carbon atoms, or be an aralkyl
group, an alkaryl group where the alkyl part may be a linear and/or cyclic alkyl group.
According to an even more preferred embodiment of the present invention, the functional
groups are separated by a linear hydrocarbon chain having 4 carbon atoms. According to
another preferred embodiment of the present invention, the functional groups are separated
by a linear hydrocarbon chain having 4 carbon atoms and at least one, preferably one
heteroatom, particularly preferably an oxygen atom.
According to a further preferred embodiment, the at least bifunctional linking compound is
a homobifunctional linking compound. Therefore, the present invention also relates to a
method of producing a conjugate as described above, wherein the at least bifunctional
linking compound is a homobifunctional compound.
Thus, with regard to the above mentioned preferred functional groups of the linking
compound, said homobifunctional linking compound preferably comprises either two
aminooxy groups tfeN-O or two aminooxy groups R'-O-NH- or two hydrazido groups
H2N-NH-(C=G)-, the aminooxy groups HaN-O- and the hydrazido groups HaN-NH-
(C=O)- being preferred, and the ammooxy groups HbN-O- being especially preferred.
Among all conceivable homobifunctional compounds comprising two hydrazido groups
H2N-NH-(C=O)-, hydrazides are preferred where the two hydrazido groups are separated
by a hydrocarbon residue having up to 60, preferably up to 40, more preferably up to 20,
more preferably up to 10, more preferably up to 6 and especially preferably up to 4 carbon
atoms. More preferably, the hydrocarbon residue has 1 to 4 carbon atoms such as 1,2,3, or
4 carbon atoms. Most preferably, the hydrocarbon residue has 4 carbon atoms. Therefore, a
homobifunctional compound according to formula
(Figure Removed)
is preferred.
According to an even more preferred embodiment of the present invention, the bifunctional
linking compound is carbohydrazide
(Figure Removed)
As described above, the present invention also relates to a method and a conjugate as
described above, wherein the at least bifunctional linking compound is a homobifunctional
compound and comprises two aminooxy groups. Hence, the present invention also relates
to a method and a conjugate as described above, wherein the at least bifunctional linking
compound is a homobifunctional compound and comprises two aminooxy groups
As described above, the polymer is preferably reacted at its reducing end which is not
oxidized prior to the reaction with the bifunctional linking compound. Therefore, reacting
the preferred homobifunctional compound comprising two aminooxy groups HbN-O- with
the polymer results in a polymer derivative comprising an oxime linkage.
Therefore, since functional group Z of the protein is an aldehyde or a keto group which is
preferably reacted with an aminooxy group of the polymer derivative, the present invention
also relates to a conjugate as described above, said conjugate comprising the polymer and
the protein, each being covatently linked to a linking compound by an oxime or a cyclic
linkage.
Among all conceivable homobifunctional compounds comprising two aminooxy groups
KbN-O-, bifunctional compounds are preferred where the two aminooxy groups are
separated by a hydrocarbon residue having from 1 to 60, preferably from 1 to 40, more
preferably from 1 to 20, more preferably from 1 to 10, more preferably from 1 to 6 and
especially preferably 1 to 4 carbon atoms. More preferably, the hydrocarbon residue has 1
to 4 carbon atoms such as 1, 2, 3, or 4 carbon atoms. Most preferably, the hydrocarbon
residue has 4 carbon atoms. Even more preferably, the hydrocarbon residue has at least one
heteroatom, more preferably one heteroatom, and most preferably one oxygen atom. The
compound O-[2-(2-amhiooxy-ethoxy)-ethyl]hydroxyl amine according to formula
is especially preferred.
Therefore, the present invention relates to a conjugate as described above, said conjugate
having a constitution according to formula
(Figure Removed)
HAS' preferably being HES'. Particularly preferred hydroxyethyl starches are, e.g.,
hydroxethyl starches having a mean molecular weight of about 10 kD and a DS of about
0.4 or hydroxethyl starch having a mean molecular weight of about 10 kD and a DS of
about 0.7 or hydroxethyl starch having a mean molecular weight of about 12 kD and a DS
of about 0.4 or hydroxethyl starch having a mean molecular weight of about 12 kD and a
DS of about 0.7 or hydroxethyl starch having a mean molecular weight of about 18 kD and
a DS of about 0.4 or hydroxethyl starch having a mean molecular weight of about 18 kD
and a DS of about 0.7 or hydroxethyl starch having a mean molecular weight of about 50
kD and a DS of about 0.4 or hydroxethyl starch having a mean molecular weight of about
50 kD and a DS of about 0.7.
The reaction of the polymer at its non-oxidized reducing end with the linking compound,
especially in the case said linking compound is a homobifunctional linking compound
comprising two aminooxy groups HaN-O-, is preferably carried out hi an aqueous system.
The term "aqueous system" as used hi the context of the present invention refers to a
solvent or a mixture of solvents comprising water in the range of from at least 10 % per
weight, preferably at least 50 % per weight, more preferably at least 80 % per weight, even
more preferably at least 90 % per weight or up to 100 % per weight, based on the weight of
the solvents involved. The preferred reaction medium is water.
According to another embodiment, at least one other solvent may be used in which HAS,
preferably HES is soluble. Examples of these solvents are, e.g., DMF, dimethylacetamide
orDMSO.
As far as the temperatures which are applied during the reaction are concerned, no specific
limitations exist given that the reaction results in the desired polymer derivative.
In case the polymer is reacted with the homobifunctional linking compound comprising
two aminooxy groups HaN-O-, preferably O-[2-(2-aminooxy-ethoxy)-ethyl]hydroxyl
amine, the temperature is preferably in the range of from 0 to 45 °C, more preferably in the
range of from 4 to 30 °C and especially preferably in the range of from 15 to 25 °C.
The reaction time for the reaction of the polymer with the homobifunctional Unking
compound comprising two aminooxy groups RfeN-O-, preferably O-[2-(2-aminooxyethoxy)-
ethyl]hydroxyl amine, may be adapted to the specific needs and is generally in the
range of from 1 h to 7 d, preferably in the range of from 1 h to 3 d and more preferably of
from 2 h to 48 h.
The pH value for the reaction of the polymer with the homobifunctional linking compound
comprising two aminooxy groups IkN-O-, preferably O-[2-(2-aminooxy-ethoxy)-
ethyl]hydroxyl amine, may be adapted to the specific needs such as the chemical nature of
the reactants. The pH value is preferably in the range of from 4.5 to 9.5, more preferably in
the range of from 4.5 to 6.5.
Specific examples of above mentioned reaction conditions are, e.g., a reaction temperature
of about 25 °C and a pH of about 5.5.
The suitable pH value of the reaction mixture may be adjusted by adding at least one
suitable buffer. Among the preferred buffers, sodium acetate buffer, phosphate or borate
buffers may be mentioned.
Once the polymer derivative comprising the polymer and the bifunctional linking
compound linked thereto is formed, it may be isolated from the reaction mixture by at least
one suitable method. If necessary, the polymer derivative may be precipitated prior to the
isolation by at least one suitable method.
If the polymer derivative is precipitated first, it is possible, e.g., to contact the reaction
mixture with at least one solvent or solvent mixture other than the solvent or solvent
mixture present in the reaction mixture at suitable temperatures. According to a
particularly preferred embodiment of the present invention where an aqueous medium,
preferably water is used as solvent, the reaction mixture is contacted with 2-propanol, at a
temperature, preferably in the range of from -20 to +50 °C and especially preferably in the
range of from -20 to 25 °C.
Isolation of the polymer derivative may be carried out by a suitable process which may
comprise one or more steps. According to a preferred embodiment of the present invention,
the polymer derivative is first separated off the reaction mixture or the mixture of the
reaction mixture with, e.g., aqueous 2-propanol mixture, by a suitable method such as
centrifugation or filtration. In a second step, the separated polymer derivative may be
subjected to a further treatment such as an after-treatment like dialysis, centrifugal
filtration or pressure filtration, ion exchange chromatography, reversed phase
chromatography, HPLC, MPLC, gel filtration and/or lyophilisation. According to an even
more preferred embodiment, the separated polymer derivative is first dialysed, preferably
against water, and then lyophilized until the solvent content of the reaction product is
sufficiently low according to the desired specifications of the product. Lyophilisation may
be carried out at temperature of from 20 to 35 °C, preferably of from 20 to 30 °C.
The thus isolated polymer derivative is then further reacted, via functional group A, with
the functional group Z of the protein, Z being an aldehyde group or a keto group. In the
especially preferred case that A is an aminooxy group HaN-O- to give an oxime linkage
between polymer derivative and protein, the reaction is preferably carried out in an
aqueous medium, preferably water, at a preferred temperature in the range of from 0 to 40
°C, more preferably from 4 to 25 °C and especially preferably from 15 to 25 °C. The pH
value of the reaction medium is preferably in the range of from 4 to 10, more preferably in
the range of from 5 to 9 and especially preferably hi the range of from 5 to 7 . The reaction
time is preferably in the range of from 1 to 72 h, more preferably in the range of from 1 to
48 h and especially preferably in the range of from 4 to 24 h.
The conjugate may be subjected to a further treatment such as an after-treatment like
dialysis, centrifugal filtration or pressure filtration, ion exchange chromatography, reversed
phase chromatography, HPLC, MPLC, gel filtration and/or lyophilisation.
According to another embodiment of the present invention, the functional group Z of the
protein is an amino group. Therefore, the present invention relates to a method and a
conjugate as described above, wherein the functional group Z of the protein is an amino
group.
According to a further preferred embodiment of the present invention, the functional group
A to be reacted with the functional group Z being an amino group is a reactive carboxy
group. Therefore, the present invention also relates to a method and a conjugate as
described above, wherein the functional group Z is an amino group and the functional
group A of the polymer or the polymer derivative is a reactive carboxy group.
According to a first preferred embodiment of the present invention, the reactive carboxy
group is introduced into the polymer by selectively oxidizing the polymer at its reducing
end.
Therefore, the polymer into which the reactive carboxy group is mtroduced preferably has
the constitution according to formula (Ha)
(Figure Removed)
and/or according to formula (lib)
(Figure Removed)
The oxidation of the reducing end of the polymer according to formula (I)
(Figure Removed)
preferably hydroxyethyl starch, may be carried out according to each method or
combination of methods which result in compounds having the above-mentioned structures
(Ha) and/or (lib).
Although the oxidation may be carried out according to all suitable method or methods
resulting hi the oxidized reducing end of hydroxyalkyl starch, it is preferably carried out
using an alkaline iodine solution as described, e.g., in DE 196 28 705 Al the respective
contents of which (example A, column 9, lines 6 to 24) is incorporated herein by reference.
Introducing the reactive carboxy group into the polymer which is selectively oxidized at its
reducing end may carried out by all conceivable methods.
The oxidized polymer may be employed as such or as salt, such as alkali metal salt,
preferably as sodium and/or potassium salt.
According to a preferred method of the present invention, the polymer which is selectively
oxidized at its reducing end is reacted at the oxidized reducing end with at least one
alcohol, preferably with at least one acidic alcohol. Still further preferred are acidic
alcohols having a pKA value in the range of from 6 to 12, more preferably of from 7 to 11
at 25 °C. The molecular weight of the acidic alcohol is preferably in the range of from 80
to 500 g/mole, more preferably of from 90 to 300 g/mole and especially preferably of from
100to200g/mole.
Suitable acidic alcohols are all alcohols H-O-RA having an acidic proton and are capable of
being reacted with the oxidized polymer to give the respective reactive polymer ester,
preferably according to the formula
(Figure Removed)
still more preferably according to formula
(Figure Removed)
Preferred alcohols are N-hydroxy succinimides such as N-hydroxy succinimide or Sulfo-
N-hydroxy succinimide, suitably substituted phenols such as p-nitrophenol, o,pdinitrophenol,
o,o'-dinitrophenol, trichlorophenol such as 2,4,6-trichlorophenol or 2,4,5-
trichlorophenol, trifluorophenol such as 2,4,6-trifluorophenol or 2,4,5-trifluorophenol,
pentachlorophenol, pentafluorophenol, or hydroxyazoles such as hydroxy benzotriazole.
Especially preferred are N-hydroxy succinimides, with N-hydroxy succinimide and Sulfo-
N-hydroxy succinimide being especially preferred. All alcohols may be employed alone or
as suitable combination of two or more thereof. In the context of the present invention, it is
also possible to employ a compound which releases the respective alcohol, e.g. by adding
diesters of carbonic acid..
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the polymer which is selectively oxidised at its reducing end is activated by
reacting the oxidised polymer with an acidic alcohol, preferably with N-hydroxy
succinimide and/or Sulfo-N-hydroxy succinimide.
According to a still further preferred embodiment of the present invention, the polymer
which is selectively oxidized at its reducing end is reacted at the oxidized reducing end
with at least one carbonic diester RB-O-(C=O)-O-Rc, wherein RB and RC may be the same
or different. Preferably, this method gives reactive polymers according to the formula
(Figure Removed)
wherein HAS1 is preferably HES1.
As suitable carbonic diester compounds, compounds may be employed whose alcohol
components are independently N-hydroxy succinimides such as N-hydroxy succinimde or
Sulfo-N-hydroxy succinimide, suitably substituted phenols such as p-nitrophenol, o,pdinitrophenol,
o,o'-dinitrophenol, trichlorophenol such as 2,4,6-trichlorophenol or 2,4,5-
trichlorophenol, trifluorophenol such as 2,4,6-trifluorophenol or 2,4,5-trifluorophenol,
pentachlorophenol, pentafluorophenol, or hydroxyazoles such as hydroxy benzotriazole.
Especially preferred are N,N'-disuccinimidyl carbonate and Sulfo-N,N'-disuccinimidyl
carbonate, withN,N'-disuccinimidyl carbonate being especially preferred.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the polymer which is selectively oxidised at its reducing end is activated by
reacting the oxidised polymer with N,N'-disuccinimidyl carbonate.
The acidic alcohol is reacted with the oxidized polymer or the salt of the oxidized polymer
at a molar ratio of acidic alcohol: polymer preferably of from 5:1 to 50:1, more preferably
of from 8:1 to 20:1, at a preferred reaction temperature of from 2 to 40 °C, more preferably
of from 10 to 30 °C and especially preferably of from 15 to 25 °C. The reaction time is
preferably in the range of from 1 to 10 h, more preferably of from 2 to 5 h, more preferably
of from 2 to 4 h and particularly of from 2 to 3 h.
The carbonic diester compound is reacted with the oxidized polymer or the salt of the
oxidized polymer at a molar ratio of diester compound : polymer preferably of from 1:1 to
3:1, more preferably of from 1:1 to 1.5:1. The reaction time is preferably in the range of
from 0.1 to 12 h, more preferably of from 0.2 to 6 h, more preferably of from 0.5 to 2 h and
particularly of from 0.75 to 1.25 h.
According to a preferred embodiment of the present invention, reacting the oxidized
polymer with acidic alcohol and/or carbonic diester is carried out in at least one aprotic
solvent, particularly preferably in an anhydrous aprotic solvent having a water content of
not more than 0.5 percent by weight, preferably of not more than 0.1 percent by weight.
Suitable solvents are, among others, dimethyl sulfoxide (DMSO), N-methyl pyrrolidone,
dimethyl acetamide (DMA), dimethyl formamide (DMF) and mixtures of two or more
thereof. The reaction temperatures are preferably in the range of from 2 to 40 °C, more
preferably of from 10 to 30 °C.
For reacting the oxidized polymer with the at least one acidic alcohol, at least one
additional activating agent is employed.
Suitable activating agents are, among others, carbonyldiimidazole, carbodiimides such as
diisopropyl carbodiimde (DIG), dicyclohexyl carbodiimides (DCC), l-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC), with dicyclohexyl carbodiimides (DCC) and
l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) being especially preferred.
Therefore, the present invention also relates to a method and a conjugate as described
above, where the polymer which is oxidized at its reducing end, is reacted with an acidic
alcohol in the presence of an additional activating agent to give the reactive polymer ester.
According to an especially preferred embodiment of the present invention, the reaction of
the oxidized polymer with carbonic diester and/or acidic alcohol is carried out at a low
base activity which may be determined by adding the reaction mixture to water with a
volume ratio of water to reaction mixture of 10:1. Prior to the addition, the water which
comprises essentially no buffer, has a pH value of 7 at 25 °C. After the addition of the
reaction mixture and by measuring the pH value, the base activity of the reaction mixture is
obtained, having a value of preferably not more than 9.0, more preferably of nor more than
8.0 and especially preferably of not more than 7.5.
According to preferred embodiment of the present invention, the oxidized polymer is
reacted with N-hydroxy succinimide in dry DMA in the absence of water with EDC to
selectively give the polymer N-hydroxy succinimide ester according to the formula
(Figure Removed)
more preferably with HAS1 being HES1.
Surprisingly, this reaction does not give by-products resulting from reactions of EDC with
OH groups of HES, and the rearrangement reaction of the O-acyl isourea formed by EDC
and the oxidized polymer to the respective N-acyl urea is surprisingly suppressed.
According to another preferred embodiment of the present invention, the oxidized polymer
is reacted with N.N'-disuccinimidyl carbonate in anhydrous DMF and in the absence of an
activating agent to selectively give the polymer N-hydroxy succinimide ester according to
the formula
(Figure Removed)
more preferably with HAS1 being HES1.
The reactive polymer as described above is preferentially further reacted with at least one
amino group of the protein to give an amide linkage. According to a preferred embodiment
of the present invention, the reactive polymer is reacted with one amino group of the
protein.
Therefore, the present relates to a conjugate preferably having a constitution according to
the formula
(Figure Removed)
wherein the N atom of the amide linkage is derived from an amino group of the protein,
more preferably with HAS1 being HES', the hydroxyethyl starch preferably being
hydroxethyl starch having a mean molecular weight of about 10 kD and a DS of about 0.4
or hydroxethyl starch having a mean molecular weight of about 10 kD and a DS of about
0.7 or hydroxethyl starch having a mean molecular weight of about 12 kD and a DS of
about 0.4 or hydroxethyl starch having a mean molecular weight of about 12 kD and a DS
of about 0.7 or hydroxethyl starch having a mean molecular weight of about 18 kD and a
DS of about 0.4 or hydroxethyl starch having a mean molecular weight of about 18 kD and
a DS of about 0.7 or hydroxethyl starch having a mean molecular weight of about 50 kD
and a DS of about 0.4 or hydroxethyl starch having a mean molecular weight of about 50
kD and a DS of about 0.7.
The reaction of the reactive polymer with the protein may be carried out by combining the
reaction mixture of the preparation of the reactive polymer, i.e. without isolation of the
reactive polymer, comprising at least 10, more preferably at least 30 and still more
preferably at least 50 percent by weight reactive polymer, with an aqueous solution of the
protein. Preferred aqueous solutions of the protein comprises of from 0.05 to 10, more
preferably of from 0.5 to 5 and especially preferably of from 0.5 to 2 percent by weight
protein at a preferred pH of from 5.0 to 9.0, more preferably of from 6.0 to 9.0 and
especially preferably of from 7.5 to 8.5.
According to the present invention, it is also possible to purify the reactive polymer by at
least one, preferably multiple precipitation with at least one suitable precipitation agent
such as anhydrous ethanol, isopropanol and/or acetone to give a solid comprising at least
10, more preferably at least 30 and still more preferably at least 50 percent by weight
reactive polymer.
The purified reactive polymer may be added to the aqueous solution of the protein. It is
also possible to add a solution of the purified reactive polymer to the aqueous solution of
the protein.
According to a preferred embodiment of the present invention, the reaction of the reactive
polymer with the protein to give an amide linkage is carried out at a temperature of from 2
to 40 °C, more preferably of from 5 to 35 °C and especially of from 10 to 30 °C and a
preferred pH of from 7.0 to 9.0, preferably of from 7.5 to 9.0 and especially preferably of
from 7.5 to 8.5, at a preferred reaction time of from 0.1 to 12 h, more preferably of from
0,5 to 5 h, more preferably of from 0,5 to 3 h, still more preferably of from 0,5 to 72 h and
especially preferably of from 0,5 to 1 h, the molar ratio of reactive polymer ester : protein
being preferably of from 1:1 to 70:1, more preferably of from 5:1 to 50:1 and especially
preferably of from 10:1 to 50:1.
According to another embodiment of the present invention, the polymer which is
selectively oxidized at its reducing end is reacted at the oxidized reducing end with an
azolide such as carbonyldiimidazole or carbonyl dibenzimidazole to give a polymer having
a reactive carboxy group. In the case of carbonyldiimidazole, a reactive polymer derivative
according to formula
(Figure Removed)
results, wherein HAS1 is preferably HES1. The imidazolide resulting from the reaction of
the polymer with the azolide may be preferentially reacted with an ammo group of the
protein to give an amide linkage. Also possible is a reaction, if present, with a hydroxy
group of the protein to give an ester linkage, or with a thio group of the protein to give a
thioester linkage, or, if present, with a carboxy group of the protein to give a -(C=O)-O-
(C=O> linkage.
According to another embodiment of the present invention, the polymer having a reactive
carboxy group A resulting from the reaction of the selectively oxidized reducing end of the
polymer with one of the above-mentioned compounds, preferably with at least one of the
acidic alcohols and/or at least one of the carbonic diester compounds, may be linked to the
functional group Z of the protein via at least one linker compound. In case a linker
compound is used, said compound is an at least bifunctional compound having at least one
functional group FI capable of being reacted with the functional group A of the polymer
derivative, and at least one functional group ?2 being capable of being reacted with the
functional group Z of the protein or a functional group ¥2 being capable of being
chemically modified to be reacted with the functional group Z of the protein. The chemical
modification may be, e.g., a reaction of the functional group ?2 with a functional group Fa
of a further linker compound or an oxidation or a reduction of a suitable functional group
Fa. In case at least one linker compound is used, the reaction is not restricted to the amino
group of the protein but, depending on the chemical nature of the functional groups of the
linker compound or linker compounds, may be used to form a linkage with each suitable
functional group of the protein, such as a carboxy group, a reactive carboxy group, an
aldehyde group, a keto group, a thio group, an amino group or a hydroxy group. In case
two linker compounds are used, a first linker compound is employed having at least one
functional group FI being capable of being reacted with the reactive carboxy group A of
the polymer, such as an amino group, a thio group, a hydroxy group, or a carboxy group.
Moreover, the first linker compound has at least one other functional group ¥2 which is
capable of being reacted with at least one functional group Fa of the second linker
compound. As to functional group Fa, the following functional groups are to be mentioned,
among others:
C-C-double bonds or C-C-triple bonds or aromatic C-C-bonds;
the thio group or the hydroxy groups;
alkyl sulfonic acid hydrazide, aryl sulfonic acid hydrazide;
1,2-dioles;
1,2-aminoalcohols;
1,2 amino-thioalcohols;
azides;
the amino group -NHa or derivatives of the amino groups comprising the structure
unit -NH- such as aminoalkyl groups, aminoaryl group, ammoaralkyl groups, or
alkarlyaminogroups;
the hydroxylamino group -O-NHa, or derivatives of the hydroxylamino group
comprising the structure unit -O-NH-, such as hydroxylalkylamino groups,
hydroxylarylamino groups, hydroxylaralkylamino groups, or hydroxalalkarylamino
groups;
alkoxyarnino groups, aryloxyamino groups, aralkyloxyamino groups, or
alkaryloxyamino groups, each comprising the structure unit -NH-O-;
residues having a carbonyl group, -Q-C(=G)-M, wherein G is O or S, and M is, for
example,
an alkoxy group, an aryloxy group, an aralkyloxy group, or an alkaryloxy
group;
an alkylthio group, an arylthio group, an aralkylthio group, or an alkarylthio
group;
an alkylcarbonyloxy group, an arylcarbonyloxy group, an aralkylcarbonyloxy
group, an alkarylcarbonyloxy group;
activated esters such as esters of hydroxylamines having imid structure such as
N-hydroxysuccinimide or having a structure unit ON where N is part of a
heteroaryl compound or, with G = O and Q absent, such as aryloxy compounds
with a substituted aryl residue such as pentafluorophenyl, paranitrophenyl or
trichlorophenyl;
wherein Q is absent or NH or a heteroatom such as S or O;
-NH-NH2, or -NH-NH-;
-N02;
the nitril group;
carbonyl groups such as the aldehyde group or the keto group;
the carboxy group;
the -N=C=O group or the -N=C=S group;
vinyl halide groups such as the vinyl iodide or the vinyl bromide group or inflate;
-C=C-H;
-(C=NH2Cl)-OAlkyl
groups -(C=O)-CH2-Hal wherein Hal is Cl, Br, or I;
-CH=CH-SQ2-;
a disulfide group comprising the structure -S-S-;
(Figure Removed)
the group
the group °2N
wherein Fa is a group capable of forming a chemical linkage with one of the abovementioned
groups and is preferably selected from the above-mentioned groups. Moreover,
the second linker compound has at least one functional group which is capable of being
reacted with the functional group Z of the protein, which is, e.g., an amino group, a thio
group, a carboxy group, a reactive carboxy group, an aldehyde group, a keto group, or a
hydroxy group. In case one linking compound is used to covalently link the-polymer and
the protein, the polymer can be reacted with the linking compound and the resulting
polymer derivative is reacted with the protein, or the protein can be reacted with the
linking compound and the resulting protein derivative is reacted with the polymer. In case
two linking compounds LI and L2 are used, it is possible to react the polymer with LI,
react the resulting polymer derivative with L2 and react the resulting polymer derivative
with the protein, or to react the protein with L2, react the resulting protein derivative with
LI and react the resulting protein derivative with the polymer. It is also possible to react
the polymer with LI and react the protein with L2 and react the polymer derivative with
the protein derivative. Furthermore, it is possible to react LI with L2, react the resulting
compound with the polymer and the resulting polymer derivative with the protein.
Furthermore, it is possible to react LI with L2, react the resulting compound with the
protein and the resulting protein derivative with the polymer.
According to a second preferred embodiment of the present invention regarding the
introduction of a reactive carboxy group into the polymer, the reactive carboxy group is
introduced into the polymer whose reducing end is not oxidized, by reacting at least one
hydroxy group of the polymer with a carbonic diester.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein A is a reactive carboxy group, and wherein A is introduced in the polymer
whose reducing end is not oxidized, by reacting at least one hydroxy group of the polymer
with at least one carbonic diester carbonic diester RB-O-(C=O)-O-Rc, wherein RB and RC
may be the same or different.
According to another embodiment of the present invention, the polymer whose reducing
end is not oxidized, is reacted at at least one hydroxy group with an azolide such as
carbonyldiimidazole, carbonyl-di-(l,2,4-triazole) or carbonyl dibenzimidazol to give a
polymer having a reactive carboxy group.
As suitable carbonic diester compounds, compounds may be employed whose alcohol
components are independently N-hydroxy succinimides such as N-hydroxy succinimide or
Sulfo-N-hydroxy succinimide, suitably substituted phenols such as p-nitrophenol, o,pdinitrophenol,
o,o'-dinitrophenol, trichlorophenol such as 2,4,6-trichlorophenol or 2,4,5-
trichlorophenol, trifluorophenol such as 2,4,6-trifluorophenol or 2,4,5-trifmorophenol,
pentachlorophenol, pentafluorophenol, or hydroxyazoles such as hydroxy benzotriazole.
Especially preferred are symmetrical carbonic diester compounds, RB and RC thus being
the same. The alcohol component of the carbonic diester is preferably selected from the
group consisting of N-hydroxy succinimide, sulfonated N-hydroxy succinimide, Nhydroxy
benzotriazole, and nitro- and halogen-substituted phenols. Among others,
nitrophenol, dinitrophenol, trichlorophenol, trifluorophenol, pentachlorophenol, and
pentafiuorophenol are preferred. Especially preferred are N,N'-disuccinimidyl carbonate
and Sulfo-N,N'nlisuccinirnidyl carbonate, with N,N'-disuccinimidyl carbonate being
especially preferred.
Therefore, the present invention also relates to a hydroxyalkyl starch derivative and a
method of producing same, preferably a hydroxyethyl starch derivative, wherein at least
one hydroxy group, preferably at least two hydroxy groups of said starch have been reacted
with a carbonic diester compound to give the respective reactive ester.
According to a preferred embodiment of the present invention, the reaction of the polymer
whose reducing end is not oxidized, with the at least one carbonic diester compound is
carried out at a temperature of from 2 to 40 °C, more preferably of from 10 to 30 °C and
especially of from 15 to 25 °C and at a preferred reaction time of from 0,5 to 5 h, more
preferably of from 1 to 3 h, and especially preferably of from 2 to 3 h.
The molar ratio of carbonic diester and/or azolide, preferably carbonic diester compound :
polymer depends on the degree of substitution of the polymer regarding the number of
hydroxy groups reacted with carbonic diester compound relative to the number of hydroxy
groups present in the non-reacted polymer.
According to one preferred embodiment of the present invention, the molar ratio of
carbonic diester compound : anhydroglucose units are in the range of from 1:2 to 1:1000,
more preferably of from 1:3 to 1:100 and especially preferably of from 1:10 to 1:50, to
give a degree of substitution in the range of from 0,5 to 0,001, preferably of from 0,33 to
0,01 and especially preferably of from 0,1 to 0,02. The degree of substitution is determined
via UV spectroscopy.
According to a preferred embodiment of the present invention, reacting the polymer whose
reducing end is not oxidized, with carbonic diester is carried out in at least one aprotic
solvent, particularly preferably in an anhydrous aprotic solvent having a water content of
not more than 0.5 percent by weight, preferably of not more than 0.1 percent by weight.
Suitable solvents are, among others, dimethyl sulfoxide (DMSO), N-methyl pyrrolidone,
dimethyl
acetamide (DMA), dimethyl formamide (DMF) and mixtures of two or more
thereof.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the reaction of the at least one hydroxy group of the polymer whose
reducing end is not oxidised, with the carbonic diester to give a reactive ester group A is
carried out in an anhydrous aprotic polar solvent, the solvent preferably being dimethyl
acetamide, dimethyl formamide or a mixture thereof.
The reaction of the reactive polymer comprising at least one reactive ester group,
preferably at least two reactive ester groups, with the protein to give at least one amide
linkage, preferably at least two amide linkages, may be carried out by combining the
reaction mixture of the preparation of the reactive polymer, i.e. without isolation of the
reactive polymer, comprising at least 5, more preferably at least 10 and still more
preferably at least 15 percent by weight reactive polymer, with an aqueous solution of the
protein. Preferred aqueous solutions of the protein comprises of from 0,05 to 10, more
preferably of from 0,5 to 5 and especially preferably of from 0,5 to 2 percent by weight
protein at a preferred pH of from 7.0 to 9., more preferably of from 7.5 to 9 and especially
preferably of from 7.5 to 8.5 .
According to the present invention, it is also possible to purify the reactive polymer by at
least one, preferably by multiple precipitation with at least one suitable precipitation agent
such as anhydrous ethanol, isopropanol and/or acetone to give a solid comprising at least
20, more preferably at least 50 and still more preferably at least 80 percent by weight
reactive polymer.
The purified reactive polymer may be added to the aqueous solution of the protein. It is
also possible to add a solution of the purified reactive polymer to the aqueous solution of
the protein.
According to a preferred embodiment of the present invention, the reaction of the reactive
polymer with the protein to give at least one, preferably at least two amide linkages is
carried out at a temperature of from 2 to 40 °C, more preferably of from 5 to 35 °C and
especially of from 10 to 30 °C and a preferred pH of from 7.5 to 9.5, preferably of from 7.5
to 9 and especially preferably of from 7.5 to 8.5, at a preferred reaction time of from 0,5 to
5 h, more preferably of from 0,5 to 3 h and especially preferably of from 0,5 to 1 h, the
molar ratio of reactive polymer ester : protein being preferably of from 1:1 to 70:1, more
preferably of from 5:1 to 50:1 and especially preferably of from 10:1 to 50:1 .
According to a preferred embodiment of the present invention, oligo- or multiproteinsubstituted
polymers are obtained wherein the protein molecules are linked to the polymer
via an amide linkage.
The degree of substitution of the protein molecules (PDS) as used in the context of the
present invention refers to the portion of glucose moieties linked to a protein with respect
to all glucose moieties comprised in HAS, preferably HES.
PDS is in the range of from 0.001 to 1, preferably from 0.005 to 0.5, more preferably from
0.005 to 0.2.
According to another embodiment of the present invention, the polymer having a reactive
carboxy group A resulting from the reaction of at least one hydroxy group of the polymer
with one of the above-mentioned compounds, preferably with at least one of the carbonic
diester compounds, may be linked to the functional group Z of the protein via at least one
linker compound. In case a linker compound is used, said compound is an at least
bifunctional compound having at least one functional group FI capable of being reacted
with the functional group A of the polymer derivative, and at least one functional group Fa
being capable of being reacted with the functional group Z of the protein or a functional
group F2 being capable of being chemically modified to be reacted with the functional
group Z of the protein. The chemical modification may be, e.g., a reaction of the functional
group Fa with a functional group FS of a further linker compound or an oxidation or a
reduction of a suitable functional group F2. In case at least one linker compound is used,
the reaction is not restricted to the amino group of the protein but, depending on the
chemical nature of the functional groups of the linker compound or linker compounds, may
be used to form a linkage with each suitable functional group of the protein, such as a
carboxy group, a reactive carboxy group, an aldehyde group, a keto group, a thio group, an
amino group or a hydroxy group. In case two linker compounds are used, a first linker
compound is employed having at least one functional group FI being capable of being
reacted with the reactive carboxy group A of the polymer, such as an araino group, a thio
group, a hydroxy group, or a carboxy group. Moreover, the first linker compound has at
least one other functional group ?2 which is capable of being reacted with at least one
functional group FS of the second linker compound. As to functional group FI, the
following functional groups are to be mentioned, among others:
C-C-double bonds or C-C-triple bonds or aromatic C-C-bonds;
the thio group or the hydroxy groups;
alkyl sulfonic acid hydrazide, aryl sulfonic acid hydrazide;
1,2-dioles;
1,2-aminoalcohols;
1,2 amino-thioalcohols;
azides;
the amino group -NHa or derivatives of the amino groups comprising the structure
unit ~NH- such as aminoalkyl groups, aminoaryl group, aminoaralkyl groups, or
alkarryaminogroups;
the hydroxylamino group -Q-NHj, or derivatives of the hydroxylamino group
comprising the structure unit -O-NH-, such as hydroxylalkylamino groups,
hydroxylarylamino groups, hydroxylaralkylamino groups, or hydroxalalkarylamino
groups;
alkoxyamino groups, aryloxyaminp groups, aralkyloxyamino groups, or
alkaryloxyamino groups, each comprising the structure unit -NH-O-;
residues having a carbonyl group, -Q-C(=G)-M, wherein G is O or S, and M is, for
example,
-- -OH or -SH;
an alkoxy group, an aryloxy group, an aralkyloxy group, or an alkaryloxy
group;
an alkylthio group, an arylthio group, an aralkylthio group, or an alkarylthio
group;
an alkylcarbonyloxy group, an arylcarbonyloxy group, an aralkylcarbonyloxy
group, an alkarylcarbonyloxy group;
activated esters such as esters of hydroxylamines having imid structure such as
N-hydroxysuccinimide or having a structure unit O-N where N is part of a
heteroaryl compound or, with G = O and Q absent, such as aryloxy compounds
with a substituted aryl residue such as pentafluorophenyl, paranitrophenyl or
trichlorophenyl;
wherein Q is absent or NH or a heteroatom such as S or O;
-NH-NH2,or-NH-NH-;
-N02;
the nitril group;
carbonyl groups such as the aldehyde group or the keto group;
the carboxy group;
the -N=C=O group or the -N=C=S group;
vinyl halide groups such as the vinyl iodide or the vinyl bromide group or triflate;
-OC-H;
-(ONH2Cl)-OAlkyl
groups -(C=O)-CH2-Hal wherein Hal is Cl, Br, or I;
-CH=CH-SO2-;
a disulfide group comprising the structure -S-S-;
(Figure Removed)
the group
the group °2N
wherein Fa is a group capable of forming a chemical linkage with one of the abovementioned
groups and is preferably selected from the above-mentioned groups. Moreover,
the second linker compound has at least one functional group which is capable of being
reacted with the functional group Z of the protein, which is, e.g., an amino group, a thio
group, a carboxy group, a reactive carboxy group, an aldehyde group, a keto group, or a
hydroxy group. In case one linking compound is used to covalently link the polymer and
the protein, the polymer can be reacted with the linking compound and the resulting
polymer derivative is reacted with the protein, or the protein can be reacted with the
linking compound and the resulting protein derivative is reacted with the polymer. In case
two linking compounds LI and L2 are used, it is possible to react the polymer with LI,
react the resulting polymer derivative with L2 and react the resulting polymer derivative
with the protein, or to react the protein with L2, react the resulting protein derivative with
LI and react the resulting protein derivative with the polymer. It is also possible to react
the polymer with LI and react the protein with L2 and react the polymer derivative with
the protein derivative. Furthermore, it is possible to react LI with L2, react the resulting
compound with the polymer and the resulting polymer derivative with the protein.
Furthermore, it is possible to react LI with L2, react the resulting compound with the
protein and the resulting protein derivative with the polymer.
According to an especially preferred embodiment of the present invention, the functional
group Z of the protein is an amino group, and the functional group A of the polymer or
derivative thereof is a aldehyde group, a keto group or a hemiacetal group. According to a
particularly preferred embodiment, functional group Z and functional group A are reacted
via a reductive amination reaction.
The reductive amination reaction according to the invention, wherein the polymer or
polymer derivative is covalently finked via at least one aldehyde group or keto group or
hemiacetal group to at least one amino group of the protein, is preferably carried out at a
temperature of from 0 to 40 °C, more preferably of from 0 to 25 °C and especially
preferably of from 4 to 21 °C. The reaction time preferably ranges of from 0.5 to 72 h,
more preferably of from 2 to 48 h and especially preferably of from 4 to 7 h. As solvent for
the reaction, an aqueous medium is preferred.
Thus, the present invention also relates to a method and a conjugate as described above,
wherein the reductive amination is carried out at a temperature of from 4 to 21 °C.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein reductive amination is carried out in an aqueous medium.
Thus, the present invention also relates to a method and conjugate as described above,
wherein the reductive amination is carried out at a temperature of from 4 to 21 °C in an
aqueous medium.
The term "aqueous medium" as used in the context of the present invention relates to a
solvent or a mixture of solvents comprising water in the range of from at least 10 % per
weight, more preferably at least 20 % per weight, more preferably at least 30 % per weight,
more preferably at least 40 % per weight, more preferably at least 50 % per weight, more
preferably at least 60 % per weight, more preferably at least 70 % per weight, more
preferably at least 80 % per weight, even more preferably at least 90 % per weight or up to
100 % per weight, based on the weight of the solvents involved. The preferred reaction
medium is water.
The pH value of the reaction medium is generally in the range of from 4 to 9 or from 4 to 8
or from 4 to 7.3.
According to a preferred embodiment of the present invention, the pH at which the
reductive amination reaction is carried out, is below 7.3, more preferably smaller or equal
7 and most preferably below 7, i.e. in the acidic range. Preferred ranges are therefore of
from 3 to below 7, more preferably of from 3.5 to 6.5, still more preferably of from 4 to 6,
still more preferably of from 4.5 to 5.5 and especially preferably about 5.0, i.e. 4.6 or 4.7
or 4.8 or 4.9 or 5.0. or 5.1 or 5.2 or 53 or 5.4. Preferred ranges, are among others, 3 to 6.9
or 3 to 6.5 or 3 to 6 or 3 to 5.5 or 3 to 5 or 3 to 4.5 or 3 to 4 or 3 to 3.5 or 3.5 to 6.9 or 3.5
to 6.5 or 3.5 to 6 or 3.5 to 5.5 or 3.5 to 5 or 3.5 to 4.5 or 3. 5 to 4 or 4 to 6.9 or 4 to 6.5 or 4
to 6. or 4 to 5.5 or 4 to 5 or 4 to 4.5 or 4.5 to 6.9 or 4.5 to 6.5 or 4.5 to 6 or 4.5 to 5.5 or 4.5
to 5 or 5 to 6.9 or 5 to 6.5 or 5 to 6 or 5 to 5.5 or 5.5 to 6.9 or 5.5 to 6.5 or 5.5 to 6 or 6 to
6.9 or 6 to 6.5 or 6.5 to 6.9.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the reductive amination is carried out at a pH of 7 or less, more preferably
at a pH of 6 or less.
Thus, the present invention also relates to a method and conjugate as described above,
wherein the reductive amination is carried out at a temperature of from 4 to 21 °C at a pH
of 7 or less, preferably of 6 or less.
Hence, the present invention also relates to a method and conjugate as described above,
wherein the reductive amination is carried out in an aqueous medium at a pH of 7 or less,
preferably of 6 or less.
Accordingly, the present invention also relates to a method and conjugate as described
above, wherein the reductive animation is carried out at a temperature of from 4 to 21 °C in
an aqueous medium at a pH of 7 or less, preferably of 6 or less.
The molar ratio of polymer derivative : protein used for the reaction is preferably in the
range of from 200:1 to 5:1, more preferably of from 100:1 to 10:1 and especially
preferably of from 75:1 to 20:1 .
It was surprisingly found that it was possible, especially at the preferred pH ranges given
above, particularly at a pH below 7 and greater or equal 4, to react the polymer derivative
predominantly with the amino group located at the N terminus of the protein. The term
"predominantly" as used in the context of the present invention relates to an embodiment
where at least 80 %, preferably at least 85 % of the N-terminal amino groups available are
reacted via reductive amination. It is also possible to react at least 90 % or at least 95 % or
at least 96 % or at least 97 % or at least 98 % or at least 99 % of the N-terminal amino
groups available. Although coupling to amino groups other than the N-terminal amino
group could not be ruled out completely, it is believed mat coupling via reductive
amination according to the present invention at a pH of below 7, preferably below 6, took
place essentially selectively at me N-terminal amino group.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the protein comprises the N-terminal amino group and at least one further
amino group, said conjugate comprises the polymer being predominantly coupled to the Nterminal
amino group.
According to an especially preferred embodiment, the present invention relates to a method
of linking aldehyde or keto or hemiacetal functionalized hydroxyalkyl starch or an
aldehyde or keto or hemiacetal functionalized hydroxyalkyl starch derivative
predominantly to the N-terminal amino group of a protein, said method comprising
subjecting said hydroxyalkyl starch or derivative thereof to a reductive amination reaction,
at a pH of 7 or less, preferably at a pH of 6 or less, said reductive amination reaction being
carried out preferably in an aqueous medium.
According to the present invention, aldehyde functionalized hydroxyalkyl starch or an
aldehyde functionalized hydroxyalkyl starch derivative is preferred.
According to a still further preferred embodiment, the present invention relates to a method
of linking aldehyde or keto or hemiacetal functionalized hydroxyethyl starch or an
aldehyde or keto or hemiacetal functionalized hydroxyethyl starch derivative selectively to
the N-terminal amino group of a protein, said method comprising subjecting said
hydroxyalkyl starch or derivative thereof to a reductive amination reaction, at a pH of 7 or
less, preferably at a pH of 6 or less, said reductive amination reaction being carried out
preferably hi an aqueous medium, the hydroxyethyl starch employed preferably being
hydroxethyl starch having a mean molecular weight of about 10 kD and a DS of about 0.4
or hydroxethyl starch having a mean molecular weight of about 10 kD and a DS of about
0.7 or hydroxethyl starch having a mean molecular weight of about 12 kD and a DS of
about 0.4 or hydroxethyl starch having a mean molecular weight of about 12 kD and a DS
of about 0.7 or hydroxethyl starch having a mean molecular weight of about 18 kD and a
DS of about 0.4 or hydroxethyl starch having a mean molecular weight of about 18 kD and
a DS of about 0.7 or hydroxethyl starch having a mean molecular weight of about 50 kD
and a DS of about 0.4 or hydroxethyl starch having a mean molecular weight of about 50
kD and a DS of about 0.7.
The reaction of the polymer derivative and the protein between the aldehyde group or keto
group or hemiacetal group and the ammo group is a reductive amination wherein a Schiff s
base is produced. Subsequently after the reaction, this base may be reduced by at least one
reductive agent to give a stable linkage between the polymer derivative and the protein. It
is also possible to carry out the reaction in the presence of at least one reductive agent.
According to a preferred embodiment, the reductive amination reaction is carried out In the
presence of at least one reductive agent.
Preferred reductive agents are sodium borohydride, sodium cyanoborohydride, organic
borane complex compounds such as a 4-(dimethylamin)pyridine borane complex, Nethyldiisopropylamine
borane complex, N-ethyhnorpholine borane complex, Nmethyhnorpholine
borane complex, N-phenylmorpholine borane complex, lutidine borane
complex, triethylamine borane complex, or trimethylamine borane complex. Particularly
preferred is sodium cyanoborohydride.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the reductive amination is carried out in the presence of NaCNBHa-
Hence, the present invention also relates to a method and conjugate as described above,
wherein the reductive amination is carried out in an aqueous medium at a pH of 7 or less,
preferably of 6 or less in the presence of reductive agent, preferably NaCNBH-j.
Accordingly, the present invention also relates to a method and conjugate as described
above, wherein the reductive amination is carried out at a temperature of from 4 to 21 °C in
an aqueous medium at a pH of 7 or less, preferably of 6 or less in the presence of reductive
agent, preferably NaCNBHs.
The molar ratio of polymer derivative : protein used for the reaction is preferably in the
range of from 200:1 to 10:1 more preferably of from 100:1 to 10:1 and especially
preferably of from 75:1 to 20:1 .
Therefore, the present invention also relates to a method of producing a conjugate, said
method comprising reacting a polymer or a polymer derivative comprising an aldehyde
group in an aqueous medium with an amino group of the protein in the presence of a
reductive agent, said reductive agent preferably being NaCNBHs.
According to the first preferred embodiment of the present invention, according to which
the polymer comprises at least two aldehyde groups which are introducing hi the polymer
by a ring-opening oxidation reaction, the polymer preferably comprises at least one
structure according to formula
According to this embodiment of the present invention, each oxidation agent or
combination of oxidation agents may be employed which is capable of oxidizing at least
one saccharide ring of the polymer to give an opened saccharide ring having at least one,
preferably at least two aldehyde groups. This reaction is illustrated by the following
reaction scheme which shows a saccharide ring of the polymer which is oxidized to give an
opened ring having two aldehyde groups:
(Figure Removed)
Suitable oxidating agents are, among others, periodates such as alkaline metal periodates or
mixtures of two or more thereof, with sodium periodate and potassium periodate being
preferred.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the polymer is subjected to a ring-opening oxidation reaction using a
periodate to give a polymer derivative having at least one, preferably at least two aldehyde
groups.
For this oxidation reaction, the polymer may be employed with its reducing end either in
the oxidized or in the non-oxidized form, the non-oxidized form being preferred.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the polymer is employed with its reducing end in the non-oxidized form.
The reaction temperature is in a preferred range of from 0 to 40 °C, more preferably of
from 0 to 25 °C and especially preferably of from 0 to 5 °C. The reaction tune is in a
preferred range of from 1 min to 5 h and especially preferably of from 10 min to 4 h.
Depending on the desired degree of oxidiation, i.e. the number of aldehyde groups
resulting from the oxidation reaction, the molar ratio of periodate : polymer may be
appropriately chosen.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the ring-opening oxidation reaction is carried out at a temperature of from
0 to 5 °C.
The oxidation reaction of the polymer with periodate is preferably carried out in an
aqueous medium, most preferably in water.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the ring-opening oxidation reaction is carried out in an aqueous medium.
The suitable pH value of the reaction mixture may be adjusted by adding at least one
suitable buffer. Among the preferred buffers, sodium acetate buffer, phosphate or borate
buffers may be mentioned.
The hydroxyethyl starch subjected to said ring-opening oxidation reaction is preferably
hydroxethyl starch having a mean molecular weight of about 10 kD and a DS of about 0.4
or hydroxethyl starch having a mean molecular weight of about 10 kD and a DS of about
0.7 or hydroxethyl starch having a mean molecular weight of about 12 kD and a DS of
about 0.4 or hydroxethyl starch having a mean molecular weight of about 12 kD and a DS
of about 0.7 or hydroxethyl starch having a mean molecular weight of about 18 kD and a
DS of about 0.4 or hydroxethyl starch having a mean molecular weight of about 18 kD and
a DS of about 0.7 or hydroxethyl starch having a mean molecular weight of about 50 kD
and a DS of about 0.4 or hydroxethyl starch having a mean molecular weight of about 50
kD and a DS of about 0.7.
The resulting polymer derivative may be purified from the reaction mixture by at least one
suitable method. If necessary, the polymer derivative may be precipitated prior to the
isolation by at least one suitable method.
If the polymer derivative is precipitated first, it is possible, e.g., to contact the reaction
mixture with at least one solvent or solvent mixture other than the solvent or solvent
mixture present in the reaction mixture at suitable temperatures. According to a
particularly preferred embodiment of the present invention where an aqueous medium,
preferably water is used as solvent, the reaction mixture is contacted with 2-propanol or
with am mixture of acetone and ethanol, preferably a 1:1 mixture (v/v), indicating equal
volumes of said compounds, at a temperature, preferably in the range of from -20 to +50
°C and especially preferably in the range of from -20 to 25 °C.
Isolation of the polymer derivative may be carried out by a suitable process which may
comprise one or more steps. According to a preferred embodiment of the present invention,
the polymer derivative is first separated off the reaction mixture or the mixture of the
reaction mixture with, e.g., aqueous 2-propanol mixture, by a suitable method such as
centrifugation or filtration. In a second step, the separated polymer derivative may be
subjected to a further treatment such as an after-treatment like dialysis, centrifugal
filtration or pressure filtration, ion exchange chromatography, reversed phase
chromatography, HPLC, MPLC, gel filtration and/or lyophilisation. According to an even
more preferred embodiment, the separated polymer derivative is first diarysed, preferably
against water, and then lyophilized until the solvent content of the reaction product is
sufficiently low according to the desired specifications of the product Lyophilisation may
be carried out at temperature of from 20 to 35 °C, preferably of from 20 to 30 °C.
According to a preferred embodiment, the oxidized polymer resulting from the oxidation
reaction is purified using at least one suitable method such as ultrafiltration and/or dialysis
in order to, e.g., remove undesirable low molecular weight salts and polymer components,
thereby also offering a means of controlling the molecular weight range of oxidized
polymer.
The oxidized polymer can be used directly for the reaction with the protein or is suitably
recovered in a first step, e.g. by lyophilization, and redissolved in water for conjugation to
the protein in a second step. As to the coupling of at least one amhio group of the proteb
with at least one aldehyde group of the polymer by reductive amination, reference is made
to the detailed disclosure above concerning the specific reaction parameters of the
reductive amination reaction such as pH or temperature.
According to the second preferred embodiment, the polymer is reacted with an at least
bifunctional compound comprising at least one functional group M capable of being
reacted with the polymer and at least one functional group Q which is an aldehyde group or
a keto group or a hemiacetal group and which is reacted with an amino group of the protein
by reductive amination.
The oxidized polymer can be used directly for the reaction with the protein or is suitably
recovered in a first step, e.g. by lyophilization, and redissolved in water for conjugation to
the protein in a second step. As to the coupling of at least one amino group of the protein
with at least one aldehyde group of the polymer by reductive amination, reference is made
to the detailed disclosure above concerning the specific reaction parameters of the
reductive amination reaction such as pH or temperature. According to especially preferred
embodiments of the present invention, the reductive amination is preferably carried out at a
temperature of from 0 to 5 °C such as about 4 °C at a pH of about 4.5 to 5.5 such as about
5.0 and for a reaction time of about 20 to 30 h such as about 24 h.
According to the second preferred embodiment, the polymer is reacted with an at least
bifunctional compound comprising at feast one functional group M capable of being
reacted with the polymer and at least one functional group Q which is an aldehyde group, a
keto group or a hemiacetal group and which is reacted with an amino group of the protein
by reductive amination.
It is preferred to employ a compound having, apart from the aldehyde group or keto group
or hemiacetal group, at least one carboxy group or at least one reactive carboxy group,
preferably one carboxy group or one reactive carboxy group. The aldehyde group or keto
group or hemiacetal group and the carboxy group or the reactive carboxy group may be
separated by any suitable spacer. Among others, the spacer may be an optionally
substituted, linear, branched and/or cyclic hydrocarbon residue. Generally, the
hydrocarbon residue has from 1 to 60, preferably from 1 to 40, more preferably from 1 to
20, more preferably from 2 to 10, more preferably from 2 to 6 and especially preferably
from 2 to 4 carbon atoms. If heteroatoms are present, the separating group comprises
generally from 1 to 20, preferably from 1 to 8 and especially preferably from 1 to 4
heteroatoms. The hydrocarbon residue may comprise an optionally branched alkyl chain or
an aryl group or a cycloalkyl group having, e.g., from 5 to 7 carbon atoms, or be an aralkyl
group, an alkaryl group where the alkyl part may be a linear and/or cyclic alkyl group.
According to an even more preferred embodiment, the hydrocarbon residue is an aryl
residue having 5 to 7 and preferably 6 carbon atoms. Most preferably, the hydrocarbon
residue is the benzene residue. According to this preferred embodiment, the carboxy group
and the aldehyde group may be located at the benzene ring in 1,4-position, 1,3 -position or
1,2-position, the 1,4-position being preferred.
As reactive carboxy group, a reactive ester, isothiocyanates or isocyanate may be
mentioned. Preferred reactive esters are derived from N-hydroxy succinimides such as Nhydroxy
succinimide or Sulfo-N-hydroxy succinimide, suitably substituted phenols such as
p-nitrophenol, o,p-dinitrophenol, o,o'-dinitrophenol, trichlorophenol such as 2,4,6-
trichlorophenol or 2,4,5-trichlorophenol, trifluorophenol such as 2,4,6-trifluorophenol or
2,4,5-trifluorophenol, pentachlorophenol, pentafluorophenol, or hydroxyazoles such as
hydroxy benzotriazole. Especially preferred are N-hydroxy succinimides, with N-hydroxy
succinimide and Sulfo-N-hydroxy succinimide being especially preferred. All alcohols
may be employed alone or as suitable combination of two or more thereof. As reactive
ester, pentafluorophenyl ester and N-hydroxy succinimide ester are especially preferred.
Thus, according to a preferred embodiment, the present invention relates to a method and a
conjugate as described above, wherein the polymer is reacted with formylbenzoic acid.
According to another preferred embodiment, the present invention relates to a method and
a conjugate as described above, wherein the polymer is reacted with formylbenzoic acid
pentafluorophenyl ester.
According to yet another preferred embodiment, the present invention relates to a method
and a conjugate as described above, wherein the polymer is reacted with formylbenzoic
acid N-hydroxysuccinimide ester.
According to yet another embodiment, the present invention relates to a method and a
conjugate as described above, wherein the polymer is reacted with 4-(4-formyl-3,5-
dimethoxyphenoxy)butyric acid.
The hydroxyethyl starch subjected to the reaction with the compound comprising M, M
preferably being a carboxy group or a reactive carboxy group and Q being an aldehyde
group or a keto group or a hemiacetal group, is most preferably hydroxethyl starch having
a mean molecular weight of about 10 kD and a DS of about 0.7. Also possible are
hydroxethyl starches having a mean molecular weight of about 10 kD and a DS of about
0.4 or hydroxethyl starch having a mean molecular weight of about 12 kD and a DS of
about 0.4 or hydroxethyl starch having a mean molecular weight of about 12 kD and a DS
of about 0.7 or hydroxethyl starch having a mean molecular weight of about 18 kD and a
DS of about 0.4 or hydroxethyl starch having a mean molecular weight of about 18 kD and
a DS of about 0.7 or hydroxethyl starch having a mean molecular weight of about 50 kD
and a DS of about 0.4 or hydroxethyl starch having a mean molecular weight of about 50
kD and a DS of about 0.7. Particularly preferably, the hydroxyalkyl starch and even more
preferably the hydroxyethyl starch is employed with its reducing end in the oxidized form.
The resulting polymer derivative with the aldehyde group or the keto group or the
hemiacetal group is subsequently reacted with an amino group of the protein via reductive
amination. As to the coupling of at least one amino group of the protein with at least one
aldehyde group or keto group or hemiacetal group of the polymer by reductive amination,
reference is made to the detailed disclosure above concerning the specific reaction
parameters of the reductive amination reaction such as pH or temperature. According to an
especially preferred embodiment of the present invention, the reaction with the amino
group of the protein is preferably carried out at a temperature of from 0 to 40 °C, more
preferably of from 0 to 25 °C and especially preferably of from 4 to 21 °C. The reaction
time preferably ranges of from 30 min to 72 h, more preferably of from 2 to 48 h and
especially preferably of from 4 h to 17 h. As solvent for the reaction, an aqueous medium
is preferred. The pH value of the reaction medium is preferably in the range of from 4 to 9,
more preferably of from 4 to 8 and especially preferably of from 4.5 to 5.5.
According to the third preferred embodiment, the polymer is reacted at its optionally
oxidized reducing end with an at least bifunctional compound comprising an amino group
M and a functional group Q, wherein said amino group M is reacted with the optionally
oxidized reducing end of the polymer and wherein the functional group Q is chemically
modified to give an aldehyde functionalized polymer derivative which is reacted with an
amino group of the protein by reductive amination.
As to functional group Q, the following functional groups are to be mentioned, among
others:
C-C~double bonds or C-C-triple bonds or aromatic C-C-bonds;
the thio group or the hydroxy groups;
alkyl sulfonic acid hydrazide, aryl sulfonic acid hydrazide;
1,2-dioles;
1,2 amino-thioalcohols;
azides;
1,2-aminoalcohols;
the amino group -Nfk or derivatives of the amino groups comprising the structure
unit -NH- such as aminoalkyl groups, aminoaryl group, aminoaralkyl groups, or
alkarlyaminogroups;
the hydroxylamino group -O-NHa, or derivatives of the hydroxylamino group
comprising the structure unit -O-NH-, such as hydroxylalkylamino groups,
hydroxylarylamino groups, hydroxylaralkylamino groups, or hydroxaialkarylamino
groups;
alkoxyamino groups, aryloxyamino groups, aralkyloxyamino groups, or
alkaryloxyamino groups, each comprising the structure unit -NH-O-;
residues having a carbonyl group, -Q-C(=G)-M, wherein G is O or S, and M is, for
example,
- -OHor-SH;
an alkoxy group, an aryloxy group, an aralkyloxy group, or an alkaryloxy
group;
an alkylthio group, an arylthio group, an aralkylthio group, or an alkarylthio
group;
an alkylcarbonyloxy group, an arylcarbonyloxy group, an aralkylcarbonyloxy
group, an alkarylcarbonyloxy group;
activated esters such as esters of hydroxylamines having imid structure such as
N-hydroxysuccinimide or having a structure unit O-N where N is part of a
heteroaryl compound or, with G = O and Q absent, such as aryloxy compounds
with a substituted aryl residue such as pentafluorophenyl, paranitrophenyl or
trichlorophenyl;
wherein Q is absent or NH or a heteroatom such as S or O;
-NH-NH2, or -NH-NH-;
-N02;
the nitril group;
carbonyl groups such as the aldehyde group or the keto group;
the carboxy group;
the -N=C=O group or the -N^C^S group;
vinyl halide groups such as the vinyl iodide or the vinyl bromide group or triflate;
-OC-H;
-(C=NH2Cl)-OAlkyl
groups -(C=O)-CH2-Hal wherein Hal is Cl, Br, or I;
-CH=CH-SO2-;
a disulfide group comprising the structure -S-S-;
the group
According to a preferred embodiment of the present invention, the term "functional group
Q" relates to a functional group Q which comprises the chemical structure -NH-.
According to one preferred embodiment of the present invention, the functional group M is
a group having the structure R'-NH- where R' is hydrogen or a alkyl, cycloalkyl, aryl,
aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue where the cycloalkyl, aryl,
aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue may be linked directly to the NH
group or, according to another embodiment, may be linked by an oxygen bridge to the NH
group. The alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl, or cycloalkylaryl
residues may be suitably substituted. As preferred substituents, halogenes such as F, Cl or
Br may be mentioned. Especially preferred residues R1 are hydrogen, alkyl and alkoxy
groups, and even more preferred are hydrogen and unsubstituted alkyl and alkoxy groups.
Among the alkyl and alkoxy groups, groups with 1, 2, 3, 4, 5, or 6 C atoms are preferred.
More preferred are methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, and
isopropoxy groups. Especially preferred are methyl, ethyl, methoxy, ethoxy, and particular
preference is given to methyl or methoxy.
According to another embodiment of the present invention, the functional group M has the
structure R'-NH-R"- where R" preferably comprises the structure unit -NH- and/or the
structure unit -(C=G> where G is O or S, and/or the structure unit -SO2-. Specific
examples for the functional group R" are
(Figure Removed)
and G
where, if G is present twice, it is independently O or S.
Therefore, the present invention also relates to a method and a conjugate as mentioned
above wherein the functional group M is selected from the group consisting of
(Figure Removed)
wherein G is O or S and, if present twice, independently O or S, and R' is methyl.
According to a particularly preferred embodiment of the present invention, the functional
group M is an amino group -NFfe.
The term "amino group Q" relates to a functional group Q which comprises the chemical
structure -NH-.
According to a preferred embodiment of the present invention, the functional group Q is a
group having the structure R'-NH- where R' is hydrogen or a alkyl, cycloalkyl, aryl,
aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue where the cycloalkyl, aryl,
aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue may be linked directly to the NH
group or, according to another embodiment, may be linked by an oxygen bridge to the NH
group. The alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl, or cycloalkylaryl
residues may be suitably substituted. As preferred substituents, halogenes such as F, Cl or
Br may be mentioned. Especially preferred residues R' are hydrogen, alkyl and alkoxy
groups, and even more preferred are hydrogen and unsubstituted alkyl and alkoxy groups.
Among the alkyl and alkoxy groups, groups with 1, 2, 3, 4, 5, or 6 C atoms are preferred.
More preferred are methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, and
isopropoxy groups. Especially preferred are methyl, ethyl, methoxy, ethoxy, and particular
preference is given to methyl or methoxy.
According to another embodiment of the present invention, the functional group Q has the
structure R'-NH-R"- where R" preferably comprises the structure unit -NH- and/or the
structure unit - where G is O or S, and/or the structure unit -SQz-. According to
more preferred embodiments, the functional group R" is selected from the group consisting
(Figure Removed)
and G
where, if G is present twice, it is independently O or S.
Therefore, the present invention also relates to a method and a conjugate as mentioned
above wherein the functional group Q is selected from the group consisting of
(Figure Removed)
wherein G is O or S and, if present twice, independently O or S, and R' is methyl.
According to a particularly preferred embodiment of the present invention, the functional
group Q is an amino group -NH2.
According to a still further preferred embodiment of the present invention, both M and Q
comprise an amino group -NH-. According to a particularly preferred embodiment, both M
and Q are an amino group -
According to a preferred embodiment of the present invention, the compound comprising
M and Q is a homobifunctional compound, more preferably a homobifunctional compound
comprising, as functional groups M and Q, most preferably the amino group -NHa, or
according to other embodiments, the hydroxylamino group -O-NKb or the group
(Figure Removed)
with G preferably being O. Specific examples for these compounds comprising M and Q
(Figure Removed)
The hydroxyethyl starch subjected to the reaction with the compound comprising M, M
preferably being an amino group -NH- and more preferably being an amino group -
still more preferably both M and Q comprising an arnino group -NH- and particularly
preferably both M and Q comprising an amino group -NH2, is preferably hydroxethyl
starch having a mean molecular weight of about 10 kD and a DS of about 0.4 or
hydroxethyl starch having a mean molecular weight of about 10 kD and a DS of about 0.7.
Also possible are or hydroxethyl starches having mean molecular weight of about 12 kD
and a DS of about 0.4 or hydroxethyl starch having a mean molecular weight of about 12
kD and a DS of about 0.7 or hydroxethyl starch having a mean molecular weight of about
18 kD and a DS of about 0.4 or hydroxethyl starch having a mean molecular weight of
about 18 kD and a DS of about 0.7 or hydroxethyl starch having a mean molecular weight
of about 50 kD and a DS of about 0.4 or hydroxethyl starch having a mean molecular
weight of about 5 0 kD and a DS of about 0.7.
In case both M and Q are an amino group -NEb, M and Q may be separated by any suitable
spacer. Among others, the spacer may be an optionally substituted, linear, branched and/or
cyclic hydrocarbon residue. Generally, the hydrocarbon residue has firom 1 to 60,
preferably firom 1 to 40, more preferably firom 1 to 20, more preferably firom 2 to 10, more
preferably from 2 to 6 and especially preferably firom 2 to 4 carbon atoms. If heteroatoms
are present, the separating group comprises generally firom 1 to 20, preferably from 1 to 8
and especially preferably from 1 to 4 heteroatoms. The hydrocarbon residue may comprise
an optionally branched alkyl chain or an aryl group or a cycloalkyl group having, e.g.,
from 5 to 7 carbon atoms, or be an aralkyl group, an alkaryl group where the alkyl part
may be a linear and/or cyclic alkyl group. According to an even more preferred
embodiment, the hydrocarbon residue is an alkyl chain of from 1 to 20, preferably from 2
to 10, more preferably from 2 to 6, and especially preferably from 2 to 4 carbon atoms.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the polymer is reacted with 1,4-diaminobutane, 1,3-diaminopropane or 1,2-
diaminoethane to give a polymer derivative.
The reaction of the at least bifunctional compound comprising M and Q with the polymer
is preferably carried out at a temperature of from 0 to 100 °C, more preferably of from 4 to
80 °C and especially preferably of from 20 to 80 °C; the reaction time preferably ranges of
from 4 h to 7 d, more preferably of from 10 h to 5 d and especially preferably of from 17 to
4 h. The molar ratio of at least bifunctional compound : polymer is preferably hi the range
of from 10 to 200, specially from 50 to 100.
As solvent for the reaction of the at least bifunctional compound with the polymer, at least
one aprotic solvent, particularly preferably an anhydrous aprotic solvent having a water
content of not more than 0.5 percent by weight, preferably of not more than 0.1 percent by
weight is preferred. Suitable solvents are, among others, dimethyl sulfoxide (DMSO), Nmethyl
pyrrolidone, dimethyl acetamide (DMA), dimethyl formamide (DMF) and mixtures
of two or more thereof.
As solvent for the reaction of the at least bifunctional compound with the polymer, also an
aqueous medium may be used.
According to a preferred embodiment, the polymer derivative comprising the polymer and
the at least bifunctional compound is dbonoically modified at tiie firee iw^ctioDal group Q to
give a polymer derivative comprising an aldehyde group or keto group or hemiacetal
group. According to this embodiment, it is preferred to react the polymer derivative with at
least one at least bifunctional compound which comprises a functional group capable of
being reacted with the functional group Q and an aldehyde group or keto group or
hemiacetal group.
As at least bifunctional compound, each compound is suitable which has an aldeyhde
group or keto group or hemiacetal group and at least one functional group which is capable
of forming a linkage with the functional group Q of the polymer derivative. The at least
one functional group is selected from the same pool of functional groups as Q and is
chosen to be able to be reacted with Q. In the preferred case that Q is an amino group -
NHi, it is preferred to employ a compound having, apart from the aldehyde group or keto
group or hemiacetal group, at least one carboxy group or at least one reactive carboxy
group, preferably one carboxy group or one reactive carboxy group. The aldehyde group
group or keto group or hemiacetal group and the carboxy group or the reactive carboxy
group may be separated by any suitable spacer. Among others, the spacer may be an
optionally substituted, linear, branched and/or cyclic hydrocarbon residue. Generally, the
hydrocarbon residue has from 1 to 60, preferably from 1 to 40, more preferably from 1 to
20, more preferably from 2 to 10, more preferably from 2 to 6 and especially preferably
from 2 to 4 carbon atoms. If heteroatoms are present, the separating group comprises
generally from 1 to 20, preferably from 1 to 8 and especially preferably from 1 to 4
heteroatoms. The hydrocarbon residue may comprise an optionally branched alkyl chain or
an aryl group or a cycloalkyl group having, e.g., from 5 to 7 carbon atoms, or be an aralkyl
group, an alkaryl group where the alkyl part may be a linear and/or cyclic alkyl group.
According to an even more preferred embodiment, the hydrocarbon residue is an aryl
residue having 5 to 7 and preferably 6 carbon atoms. Most preferably, the hydrocarbon
residue is the benzene residue. According to this preferred embodiment, the carboxy group
and the aldehyde group may be located at the benzene ring in 1,4-position, 1,3-position or
1,2 -position, the 1,4-position being preferred.
As reactive carboxy group, a reactive ester, isotbiocyanates or isocyanate may be
mentioned. Preferred reactive esters are derived from N-hydroxy succinimides such as Nhydroxy
succinimide or Sulfo-N-hydroxy succinimide, suitably substituted phenols such as
p-nitrophenol, o,p-dinitn>phenol, o,o'-duutrophenol, trichlorophenol such as 2,4,6-
trichlorophenol or 2,4,5-trichlorophenol, trifluorophenol such as 2,4,6-trifluctophenol or
2,4,5-trifluorophenol, pentachlorophenol, pentafluorophenol, or hydroxyazoles such as
hydroxy benzotriazole. Especially preferred are N-hydroxy succinimides, with N-hydroxy
succinimide and Sulfo-N-hydroxy succinimide being especially preferred. All alcohols
may be employed alone or as suitable combination of two or more thereof. As reactive
ester, pentafluorophenyl ester and N-hydroxy succinimide ester are especially preferred.
Thus, according to a preferred embodiment, the present invention relates to a method and a
conjugate as described above, wherein the polymer derivative comprising Q, Q being an
amino group -NHa, is further reacted with formylbenzoic acid.
According to another embodiment, the present invention relates to a method and a
conjugate as described above, wherein the polymer derivative comprising Q, Q being an
amino group, is further reacted with formylbenzoic acid pentafluorophenyl ester.
According to yet another embodiment, the present invention relates to a method and a
conjugate as described above, wherein the polymer derivative comprising Q, Q being an
amino group, is further reacted with formylbenzoic acid N-hydroxysuccinimide ester.
According to yet another embodiment, the present invention relates to a method and a
conjugate as described above, wherein the polymer derivative comprising Q, Q being an
amino group, is further reacted with 4-(4-formyl-3,5-dimethoxyphenoxy)butyric acid.
As solvent for the reaction of the polymer derivative comprising an amino group and, e.g.,
formylbenzoic acid, at least one aprotic solvent, particularly preferably an anhydrous
aprotic solvent having a water content of not more than 0.5 percent by weight, preferably
of not more than 0.1 percent by weight is preferred. Suitable solvents are, among others,
dimethyl sulfoxide (DMSO), N-methyl pyrrolidone, dimethyl acetamide (DMA), dimethyl
formamide (DMF) and mixtures of two or more thereof.
The reaction is preferably carried out at a temperature of from 0 to 40 °C, more preferably
of from 0 to 25 °C and especially preferably of from 15 to 25 °C for a reaction time
preferably of from 0.5 to 24 h and especially preferably of from 1 to 17 h.
According to a preferred embodiment, the reaction is carried out in the presence of an
activating agent. Suitable activating agents are, among others, carbodiimides such as
diisopropyl carbodiimde (DIG), dicyclohexyl carbodiimides (DCC), l-ethyl-3-(3-
dimethylaminopropyl) carbodumide (EDC), with diisopropyl carbodiimde (DIG) being
especially preferred.
The resulting polymer derivative may be purified from the reaction mixture by at least one
suitable method. If necessary, the polymer derivative may be precipitated prior to the
isolation by at least one suitable method.
If the polymer derivative is precipitated first, it is possible, e.g., to contact the reaction
mixture with at least one solvent or solvent mixture other than the solvent or solvent
mixture present in the reaction mixture at suitable temperatures. According to a
particularly preferred embodiment of the present invention where an aqueous medium,
preferably water is used as solvent, the reaction mixture is contacted with 2-propanol or
with am mixture of acetone and ethanol, preferably a 1:1 mixture (v/v), indicating equal
volumes of said compounds, at a temperature, preferably in the range of from -20 to +50
°C and especially preferably in the range of from -20 to 25 °C.
Isolation of the polymer derivative may be carried out by a suitable process which may
comprise one or more steps. According to a preferred embodiment of the present invention,
the polymer derivative is first separated off the reaction mixture or the mixture of the
reaction mixture with, e.g., aqueous 2-propanol mixture, by a suitable method such as
centrifugation or filtration. In a second step, the separated polymer derivative may be
subjected to a further treatment such as an after-treatment like dialysis, centrifugal
filtration or pressure filtration, ion exchange chromatography, reversed phase
chromatography, HPLC, MPLC, gel filtration and/or lyophilisation. According to an even
more preferred embodiment, the separated polymer derivative is first dialysed, preferably
against water, and then lyophilized until the solvent content of the reaction product is
sufficiently low according to the desired specifications of the product. Lyophilisation may
be carried out at temperature of from 20 to 35 °C, preferably of from 20 to 30 °C.
The resulting polymer derivative with the aldehyde group or keto group or hemiacetal
group is subsequently reacted with an ammo group of the protein via reductive animation.
As to the coupling of at least one amino group of the protein with at least one aldehyde
group or keto group or hemiacetal group of the polymer by reductive animation, reference
is made to the detailed disclosure above concerning the specific reaction parameters of the
reductive animation reaction such as pH or temperature. According to an especially
preferred embodiment of the present invention, the reductive amination is carried out at a
temperature of from 0 to 10 °C such as from 1 to 8 °C or from 2 to 6 °C such as about 4 °C
at a pH of about 4.5 to 5.5 such as about 5.0. The reaction time is about 10 to 20 h such as
from 12 to 19 h or from 14 to 18 h such as about 17 h or about 20 to 30 h such as about 24
h.
Thus, according to the above-mentioned preferred embodiments, the present invention also
relates, in case the polymer was reacted via its oxidized reducing end, to a conjugate
according to the formula
(Figure Removed)
—Protein
According to an especially preferred embodiment, the polymer is hydroxyethyl starch, i.e.
HAS1 is HES1, and n = 2, 3, or 4, most preferably 4, as described above. Therefore, in case
the polymer was reacted via its oxidized reducing end, the present invention also relates to
a conjugate according to the formula
(Figure Removed)
According to another preferred embodiment, the present invention also relates, hi case the
polymer was reacted via its oxidized reducing end, to a conjugate according to the formula
(Figure Removed)
wherein n = 2, 3, or 4, R4 being independently hydrogen or a methoxy group, and m = 0 in
case R4 is hydrogen and m = 1 in case R4 is methoxy, HAS preferably being HES1.
In each of the formulae above, the nitrogen attached to the protein derives from the amino
group of the protein the polymer derivative is linked to via the aldehyde group.
With respect to the above-mentioned embodiments according to which the functional
groups M and Q comprise an amino group -NHa, it is also possible that M is an amino
group -NH2 and Q comprises a beta hydroxy amino group -CH(OH)-CH2-NH2 and
preferably is a beta hydroxy amino group.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the amino group Q of the compound comprising two amino groups M and
Q, is a beta hydroxy amino group -CH(OH)-CH2-NH2.
In this case, M and Q may be separated by any suitable spacer. Among others, the spacer
may be an optionally substituted, linear, branched and/or cyclic hydrocarbon residue.
Generally, the hydrocarbon residue has from 1 to 60, preferably from 1 to 40, more
preferably from 1 to 20, more preferably from 2 to 10, more preferably from 1 to 6 and
especially preferably from 1 to 2 carbon atoms. If heteroatoms are present, the separating
group comprises generally from 1 to 20, preferably from 1 to 8 and especially preferably
from 1 to 4 heteroatoms. The hydrocarbon residue may comprise an optionally branched
alkyl chain or an aryl group or a cycloalkyl group having, e.g., from 5 to 7 carbon atoms,
or be an aralkyl group, an alkaryl group where the alkyl part may be a linear and/or cyclic
alkyl group. According to an even more preferred embodiment, the hydrocarbon residue is
an alkyl chain of from 1 to 20, preferably from 1 to 10, more preferably from 1 to 6, more
preferably from 1 to 4 carbon atoms and especially preferably from 1 to 2 carbon atoms.
Still more preferably, M and Q are separated by a methylene group.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the polymer is reacted with l,3-diamino-2-hydroxypropane.
In case the polymer is reacted via its oxidized reducing end, a polymer derivative
according to the formula results
especially preferably with HAS1 = HES'
The reaction of the at least bifunctional compound comprising M and Q, particularly
preferably l,3-diamino-2-hydroxypropane, with the polymer is preferably carried out at a
temperature of from 40 to 120 °C, more preferably of from 40 to 90 °C and especially
preferably of from 60 to 80 °C. The reaction time preferably ranges from 17 to 168 h, more
preferably from 17 to % h and especially preferably from 48 to 96 h. The molar ratio of at
least biftmctional compound : polymer is preferably in the range of from 200:1 to 10:1,
specially from 50:1 to 100:1.
As solvent for the reaction of the at least bifunctional compound with the polymer, at least
one aprotic solvent, preferably an anhydrous aprotic solvent having a water content of not
more than 0.5 percent by weight, preferably of not more than 0.1 percent by weight is
preferred. Suitable solvents are, among others, dimethyl sulfoxide (DMSO), N-methyl
pyrrolidone, dimethyl acetamide (DMA), dimethyl formamide (DMF) and mixtures of two
or more thereof.
The beta hydroxy amino group Q of the polymer derivative generally may be reacted with
an at least bifunctional compound comprising at least one functional group capable of
being reacted with Q and further comprising at least one functional group being an
aldehyde group or keto group or hemiacetal group or a functional group capable of being
modified to give an aldehyde group or keto group or hemiacetal group. According to
another embodiment of the present invention, the beta hydroxy amino group is directly
chemically modified to give an aldehyde group by chemical oxidation.
This oxidation may be carried with all suitable oxidation agents which are capable of
converting the beta hydroxy amino group to an aldehyde group. Preferred oxidation
reagents are periodates such as alkaline metal periodates. Especially preferred is sodium
periodate which is preferably employed as aqueous solution. This solution has a preferred
iodate concentration of from 1 to 50 mM, more preferably from 1 to 25 mM and especially
preferably of from 1 to 10 mM. Oxidation is carried out at a temperature of from 0 to 40
°C, preferably from 0 to 25 °C and especially preferably from 4 to 20 °C.
The resulting polymer derivative may be purified from the reaction mixture by at least one
suitable method. If necessary, the polymer derivative may be precipitated prior to the
isolation by at least one suitable method.
If the polymer derivative is precipitated first, it is possible, e.g., to contact the reaction
mixture with at least one solvent or solvent mixture other than the solvent or solvent
mixture present in the reaction mixture at suitable temperatures. According to a
particularly preferred embodiment of the present invention where an aqueous medium,
preferably water is used as solvent, me reaction mixture is contacted with 2-propanol or
with am mixture of acetone and ethanol, preferably a 1:1 mixture (v/v), indicating equal
volumes of said compounds, at a temperature, preferably in the range of from -20 to +50
°C and especially preferably in the range of from -20 to 25 °C.
Isolation of the polymer derivative may be carried out by a suitable process which may
comprise one or more steps. According to a preferred embodiment of the present invention,
the polymer derivative is first separated off the reaction mixture or the mixture of the
reaction mixture with, e.g., aqueous 2-propanol mixture, by a suitable method such as
centrifugation or filtration. In a second step, the separated polymer derivative may be
subjected to a further treatment such as an after-treatment like dialysis, centrifugal
filtration or pressure filtration, ton exchange chromatography, reversed phase
chromatography, HPLC, MPLC, gel filtration and/or lyophilisation. According to an even
more preferred embodiment, the separated polymer derivative is first dialysed, preferably
against water, and then lyophilized until the solvent content of the reaction product is
sufficiently low according to the desired specifications of the product. Lyophilisation may
be carried out at temperature of from 20 to 35 °C, preferably of from 20 to 30 °C.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the oxidation of the beta hydroxy amino group Q is carried out using a
periodate.
Therefore, the present invention also relates to a method of producing a conjugate,
wherein, in case the polymer was employed with oxidized reducing end, a polymer
derivative having a beta hydroxy amino group, especially preferably
(Figure Removed)
and particularly with HAS* - HES', is oxidized, preferably with a periodate, to a polymer
derivative having an aldehyde group, especially preferably
HAS
and particularly with HAS' = HES'.
The resulting polymer derivative with the aldehyde group A is subsequently reacted with
the protein. Therefore, the present invention also relates to a method of producing a
conjugate, said method comprising reacting a polymer derivative having a beta hydroxy
amino group, hi case the polymer was employed with oxidized reducing end especially
preferably according to the formula
(Figure Removed)
and particularly with HAS' = HES1, with an amino group of the protein.
The resulting polymer derivative with the aldehyde group is subsequently reacted with an
amino group of the protein via reductive animation. As to the coupling of at least one
amino group of the protein with at least one aldehyde group of the polymer by reductive
amination, reference is made to the detailed disclosure above.
Thus, according to the above-mentioned preferred embodiment, the present invention also
relates to a conjugate according to the formula
HAS1
particularly with HAS' = HES1, in case the polymer was employed with oxidized reducing
end. In the formula above, the nitrogen attached to the protein derives from the amino
group of the protein the polymer derivative is linked to via the aldehyde group.
According to a further embodiment of the present invention, the polymer is first reacted
with a suitable compound to give a first polymer derivative comprising at least one reactive
carboxy group. This first polymer derivative is then reacted with a further, at least
bifunctional compound wherein at least one functional group of this further compound is
reacted with at least one reactive carboxy group of the polymer derivative and at least one
other functional group of the further compound is an aldehyde group or keto group or
hemiacetal group or is a functional group which is chemically modified to give an
aldehyde group or keto group or hemiacetal group, and wherein the resulting polymer
derivative comprising said aldehyde group or keto group or hemiacetal group is reacted via
reductive amination, as described above, with at least one amino group of the protein. It is
also possible to alter the sequence of reacting the respective compounds with each other.
According to a first alternative of said further embodiment, the polymer comprising at least
one reactive carboxy group is prepared by selectively oxidizing the polymer at its reducing
end and subsequently reacting the oxidized polymer being a lactone
(Figure Removed)
and/or a carboxylic acid
(Figure Removed)
or a suitable salt of the carboxylic acid such as alkali metal salt, preferably as sodium
and/or potassium salt, and HAS1 preferably being HES1, with a suitable compound to give
the polymer comprising at least one reactive carboxy group.
Oxidation of the polymer, preferably hydroxyethyl starch, may be carried out according to
each method or combination of methods which result in compounds having the abovementioned
structures (Ila) and/or (lib).
Although the oxidation may be carried out according to all suitable method or methods
resulting in the oxidized reducing end of hydroxyalkyl starch, it is preferably carried out
using an alkaline iodine solution as described, e.g., in DE 196 28 705 Al toe respective
contents of which (example A, column 9, lines 6 to 24) is incorporated herein by reference.
Introducing the reactive carboxy group into the polymer which is selectively oxidized at its
reducing end may carried out by all conceivable methods and all suitable compounds.
According to a specific method of the present invention, the polymer which is selectively
oxidized at its reducing end is reacted at the oxidized reducing end with at least one
alcohol, preferably with at least one acidic alcohol such as acidic alcohols having a pKA
value in the range of from 6 to 12 or of from 7 to 1 1 at 25 °C. The molecular weight of the
acidic alcohol may be in the range of from 80 to 500 g/mole, such as of from 90 to 300
g/mole or of from 100 to 200 g/mole.
Suitable acidic alcohols are all alcohols H-OR*. having an acidic proton and are capable of
being with reacted with the oxidized polymer to give the respective reactive polymer ester,
preferably according to the formula
(Figure Removed)
still more preferably according to formula
(Figure Removed)
Preferred alcohols are N-hydroxy succinimides such as N-hydroxy succinimde or Sulfo-Nhydroxy
succinimide, suitably substituted phenols such as p-nitrophenol, o,p-dinitrophenol,
o,o'-dinitrophenol, trichlorophenol such as 2,4,6-trichlorophenol or 2,4,5-trichlorophenol,
trifluorophenol such as 2,4,6-trifluorophenol or 2,4,5-trifluorophenol, pentachlorophenol,
pentafluorophenol, or hydroxyazoles such as hydroxy benzotriazole. Especially preferred
are N-hydroxy succinimides, with N-hydroxysuccinimide and Sulfo-Nhydroxysuccinimide
being especially preferred. All alcohols may be employed alone or as
suitable combination of two or more thereof. In the context of the present invention, it is
also possible to employ a compound which releases the respective alcohol, e.g. by adding
diesters of carbonic acid.
Therefore, the present invention also relates to a method as described above, wherein the
polymer which is selectively oxidised at its reducing end is activated by reacting the
oxidised polymer with an acidic alcohol, preferably with N-hydroxy succinimide and/or
Sulfo-N-hydroxy succinimide.
According to a preferred embodiment of the present invention, the polymer which is
selectively oxidized at its reducing end is reacted at die oxidized reducing end with at least
one carbonic diester RB-O-{C=O)-O-Rc, wherein RB and RC may be fhe same or different
Preferably, this method gives reactive polymers according to the formula
ORB/C
wherein HAS1 is preferably HES'.
As suitable carbonic diester compounds, compounds may be employed whose alcohol
components are independently N-hydroxy succinimides such as N-hydroxy succinimde or
Sulfo-N-hydroXy succinimide, suitably substituted phenols such as p-nitrophenol, o,pdinitrophenol,
o,o'-dinitrophenol, trichlorophenol such as 2,4,6-trichlorophenol or 2,4,5-
trichlorophenol, trifluorophenol such as 2,4,6-trifluorophenol or 2,4,5-trifiuorophenol,
pentachlorophenol, pentafluorophenol, or hydroxyazoles such as hydroxy benzotriazole.
Especially preferred are N,N'-disuccuiimidyl carbonate and Svufo-N,N'-&succiiumidyl
carbonate, with N^N'-disuccinimidyl carbonate being especially preferred.
Therefore, the present invention also relates a method as described above, wherein the
polymer which is selectively oxidised at its reducing end is activated by reacting the
oxidised polymer with N,N'-disuccinimidyl carbonate.
The acidic alcohol is reacted with the oxidized polymer or the salt of the oxidized polymer
at a molar ratio of acidic alcohol: polymer preferably of from 5:1 to 50:1, more preferably
of from 8:1 to 20:1, at a preferred reaction temperature of from 2 to 40 °C, more preferably
of from 10 to 30 °C and especially preferably of from 15 to 25 °C. The reaction time is
preferably in the range of from 1 to 10 h, more preferably of from 2 to 5 h, more preferably
of from 2 to 4 h and particularly of from 2 to 3 h.
The carbonic diester compound is reacted with the oxidized polymer or the salt of the
oxidized polymer at a molar ratio of diester compound : polymer generally of from 1:1 to
3:1, such as of from 1:1 to 1.5:1. The reaction time is generally in the range of from 0.1 to
12 h, like of from 0.2 to 6 h, or of from 0.5 to 2 h or of from 0.75 to 1.25 h.
According to a preferred embodiment of the present invention, reacting the oxidized
polymer with acidic alcohol and/or carbonic diester is carried out in at least one aprotic
solvent, such as in an anhydrous aprotic solvent having a water content of not more than
0.5 percent by weight, preferably of not more than 0.1 percent by weight. Suitable solvents
are, among others, dimethyl sulfoxide (DMSO), N-methyl pyrrolidone, dimethyl acetamide
(DMA), dimethyl formamide (DMF) and mixtures of two or more thereof. The reaction
temperatures are preferably in the range of from 2 to 40 °C, more preferably of from 10 to
30 °C.
For reacting the oxidized polymer with the at least one acidic alcohol, at least one
additional activating agent is employed.
Suitable activating agents are, among others, carbonyldiimidazole, carbodiimides such as
diisopropyl carbodiimde (DIG), dicyclohexyl carbodiimides (DCC), l-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC), with dicyclohexyl carbodiimides (DCC) and
l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) being especially preferred.
Therefore, the present invention also relates to the method as described above, where the
polymer which is oxidized at its reducing end, is reacted with an acidic alcohol hi the
presence of an additional activating agent to give the reactive polymer ester.
According to one embodiment of the present invention, the reaction of the oxidized
polymer with carbonic diester and/or acidic alcohol is carried out at a low base activity
which may be determined by adding the reaction mixture to water with a volume ratio of
water to reaction mixture of 10:1. Prior to the addition, the water which comprises
essentially no buffer, has a pH value of 7 at 25 °C. After the addition of the reaction
mixture and by measuring the pH value, the base activity of the reaction mixture is
obtained, having a value of preferably not more than 9.0, more preferably of nor more than
8.0 and especially preferably of not more than 7.5.
According to another embodiment of the present invention, the oxidized polymer is reacted
with N-hydroxy succinimide in dry DMA in the absence of water with EDC to selectively
give the polymer N-hydroxy swccimmide ester according to the formula
(Figure Removed)
more preferably with HAS' being HES'.
Surprisingly, this reaction does not give by-products resulting from reactions of EDC with
OH groups of HES, and the rearrangement reaction of the O-acyl isourea formed by EDC
and the oxidized polymer to the respective N-acyl urea is surprisingly suppressed.
According to another preferred embodiment of the present invention, the oxidized polymer
is reacted with N,N'-disuccinimidyl carbonate in dry DMF in the absence of water and in
the absence of an activating agent to selectively give the polymer N-hydroxy succinimide
ester according to the formula
(Figure Removed)
more preferably with HAS' being HES1.
According to another embodiment of the present invention, the polymer which is
selectively oxidized at its reducing end is reacted at the oxidized reducing end with an
azolide such as carbonyldiimidazole or carbonyl dibenzimidazole to give a polymer having
a reactive carboxy group. In the case of carbonyldiimidazole, a reactive imidazolide
polymer derivative according to formula
(Figure Removed)
results, wherein HAS' is preferably HES'.
According to a second alternative of said further embodiment of the present invention
regarding the introduction of at least one reactive carboxy group into the polymer, the
reactive carboxy group is introduced into the polymer whose reducing end is not oxidized,
by reacting at least one hydroxy group of the polymer with a carbonic diester.
Therefore, the present invention also relates to a method and conjugates wherein the
reactive carboxy group is introduced in the polymer whose reducing end is not oxidized,
by reacting at least one hydroxy group of the polymer with at least one carbonic diester
carbonic diester RB-O-(C=O)-O-Rc, wherein RB and RC may be the same or different.
According to another embodiment of the present invention, the polymer whose reducing
end is not oxidized, is reacted at at least one hydroxy group with an azolide such as
carbonyldiimidazole, carbonyl-di-(l,2,4-triazole) or carbonyl dibenzimidazol to give a
polymer having a reactive carboxy group.
As suitable carbonic diester compounds, compounds may be employed whose alcohol
components are independently N-hydroxy succinimides such as N-hydroxy succinimde or
Sulfo-N-hydroxy succinimide, suitably substituted phenols such as p-nitrophenol, o,pdinitrophenol,
o,o'-dinitrophenol, trichlorophenol such as 2,4,6-trichlorophenol or 2,4,5-
trichlorophenol, trifluorophenol such as 2,4,6-trifluorophenol or 2,4,5-trifluorophenol,
pentachlorophenol, pentafluorophenol, or hydroxyazoles such as hydroxy benzotriazole.
Especially preferred are symmetrical carbonic diester compounds, RB and RC thus being
the same. The alcohol component of the carbonic diester is preferably selected from the
group consisting of N-hydroxy succinimide, sulfonated N-hydroxy succinimide, Nhydroxy
benzotriazole, and nitro- and halogen-substituted phenols. Among others,
nitrophenol, dinitrophenol, trichlorophenol, trifluorophenol, pentachlorophenol, and
pentafluorophenol are preferred. Especially preferred are NJsT-disuccinimidyl carbonate
and Sulfo-N,N'-disuccinimidyl carbonate, with N^-dis«ccinunidyl carbonate being
especially preferred.
Therefore, the present invention also relates to a hydroxyalkyl starch derivative, preferably
a hydroxyethyl starch derivative, wherein at least one hydroxy group, preferably at least
two hydroxy groups of said starch have been reacted with a carbonic diester compound to
give the respective reactive ester.
According to one embodiment of the present invention, the reaction of the polymer whose
reducing end is not oxidized, with the at least one carbonic diester compound is carried out
at a temperature of from 2 to 40 °C, more preferably of from 10 to 30 °C and especially of
from 15 to 25 °C. A preferred reaction time ranges from 0,5 to 5 h, more preferably from 1
to 3 h, and especially preferably from 2 to 3 h.
The molar ratio of carbonic diester compound : polymer depends on the degree of
substitution of the polymer regarding the number of hydroxy groups reacted with carbonic
diester compound relative to the number of hydroxy groups present in the non-reacted
polymer.
According to one embodiment of the present invention, the molar ratio of carbonic diester
compound : anhydroglucose units of the polymer is in the range of from 1:2 to 1:1000,
more preferably of from 1:3 to 1:100 and especially preferably of from 1:10 to 1:50, to
give a degree of substitution in the range of from 0.5 to 0.001, preferably of from 0.33 to
0.01 and especially preferably of from 0.1 to 0.02
According to one embodiment of the present invention, reacting the polymer whose
reducing end is not oxidized, with carbonic diester is carried out in at least one aprotic
solvent, particularly preferably in an anhydrous aprotic solvent having a water content of
not more than 0.5 percent by weight, preferably of not more than 0.1 percent by weight.
Suitable solvents are, among others, dimethyl sulfoxide (DMSO), N-methyl pyrrolidone,
dimethyl acetamide (DMA), dimethyl formamide (DMF) and mixtures of two or more
thereof.
Therefore, the present invention also relates to a method as described above wherein the
reaction of the at least one hydroxy group of the polymer whose reducing end is not
oxidised, with the carbonic diester to give a reactive carboxy group is carried out in an
anhydrous aprotic polar solvent, the solvent preferably being dimethyl acetamide, dimethyl
formamide or a mixture thereof.
The reactive polymer derivative comprising at least one reactive carboxy group, preferably
resulting from the reaction of the polymer with the acidic alcohol, the carbonate and/or the
azolide, as described above, is further reacted with a further, at least bifunctional
compound wherein at least one functional group FI of this further compound is reacted
with at least one reactive carboxy group of the polymer derivative. As at least one
functional group FI of the further compound no specific limitations exist given that a
reaction with the at least one reactive carboxy group of the polymer is possible. Preferred
functional groups FI are, e.g., an amino group or a hydroxy group or a thio group or a
carboxy group.
The further, at least bifunctional compound comprises at least one other functional group
F2 being an aldehyde group or a functional group F2 being capable of being chemically
modified to give an aldehyde group. The chemical modification may be, e.g., a reaction of
the functional group F2 with a functional group Fa a further linker compound or an
oxidation or a reduction of a suitable functional group F2.
hi case F2 is reacted with a functional group F3 of a further compound, the functional group
F2 may be selected from, among others,
C-C-double bonds or C-C-triple bonds or aromatic C-C-bonds;
the thio group or the hydroxy group;
alkyl sulfonic acid hydrazide, aryl sulfonic acid hydrazide;
1,2-dioles;
1,2-aminoalcohols;
1,2 amino-thioalcohols;
azides;
the ammo group -NH2 or derivatives of the anuno groups comprising the structure
unit -NH- such as aminoalkyl groups, aminoaryl group, aminoaralkyl groups, or
alkarlyarninogroups;
the hydroxylamino group -O-NHa, or derivatives of the hydroxylamino group
comprising the structure unit -O-NH-, such as hydroxylalkylamino groups,
hydroxylarylamino groups, hydroxylaralkylamino groups, or hydroxalalkarylamino
groups;
alkoxyamino groups, aryloxyamino groups, aralkyloxyamino groups, or
alkaryloxyamino groups, each comprising the structure unit -NH-O-;
residues having a carbonyl group, -Q-C(=G)-M, wherein G is O or S, and M is, for
example,
- -OHor-SH;
an alkoxy group, an aryloxy group, an aralkyloxy group, or an alkaryloxy
group;
an alkylthio group, an arylthio group, an aralkylthio group, or an alkarylthio
group;
an alkylcarbonyloxy group, an arylcarbonyloxy group, an aralkylcarbonyloxy
group, an alkarylcarbonyloxy group;
activated esters such as esters of hydroxylamines having imid structure such as
N-hyoVoxysuccinimide or having a structure unit O-N where N is part of a
heteroaryl compound or, with G = 0 and Q absent, such as aryloxy compounds
with a substituted aryl residue such as pentafluorophenyl, paranitrophenyl or
trichlorophenyl;
wherein Q is absent or NH or a heteroatom such as S or O;
-NH-NH2, or -NH-NH-;
the nitril group;
carbonyl groups such as the aldehyde group or the keto group;
the carboxy group;
the -N=OO group or the -N=C=S group;
vinyl halide groups such as the vinyl iodide or the vinyl bromide group or triflate;
-OC-H;
-(C=NH2a)-OAlkyl
groups -(C=O>CH2-Hal wherein Hal is CI, Br, or I;
-CH=CH-S(V;
a disulfide group comprishig the structure -S-S-;
O.
the group
the group °2N
wherein Fj is a group capable of forming a chemical linkage with one of the abovementioned
groups and is preferably selected from the above-mentioned groups. Moreover,
the second linker compound preferably has at least one aldehyde group or keto group or
hemiacetal group which is capable of being reacted with an amino group of the protein via
reductive animation.
The functional group Fj and the aldehyde group or keto group or hemiacetal group of the at
least bifunctional linking compound which is reacted with the polymer, and/or the
functional groups FI and FX of the at least bifimctional linking compound which is reacted
with the polymer, and/or the functional group Fj and the aldehyde group or keto group or
hemiacetal group of the further, at least bifunctional linking compound, may be
independently separated by any suitable spacer. Among others, the spacer may be an
optionally substituted, linear, branched and/or cyclic, aliphatic and/or aromatic
hydrocarbon residue. Generally, the hydrocarbon residue has up to 60, preferably up to 40,
more preferably up to 20, more preferably up to 10 carbon atoms. If heteroatoms are
present, the separating group comprises generally from 1 to 20, preferably from 1 to 8,
more preferably 1 to 6, more preferably 1 to 4 and especially preferably from 1 to 2
heteroatoms. As heteroatom, O is preferred. The hydrocarbon residue may comprise an
optionally branched alkyl chain or an aryl group or a cycloalkyl group having, e.g., from 5
to 7 carbon atoms, or be an aralkyl group, an alkaryl group where the alkyl part may be a
linear and/or cyclic alkyl group.
Examples of a compound with functional groups FI and Fa are, e.g., optionally substituted
diaminoalkane having from 2 to 20 carbon atoms, especially preferably 1,2-diaminoemane,
13-diaKiinopropane, and 1,4-diamiaobutane. Preferred examples of a compound with
functional groups F3 and an aldehyde group or a keto group or a hemiacetal group are, e.g.,
formylbenzoic acid, 4-formylbenzoic acid pentafluorophenyl ester, 4-formylbenzoic acid-
N-hydroxysuc«baiinide ester and 4^4-formyl-3^Hiimettioxvphenoxy)butyric acid.
Therefore, the present invention also relates to a method of producing a conjugate, said
method comprising reacting the polymer, preferably hydroxyethyl starch, at its optionally
oxidized reducing end with a compound, selected from the group consisting of acidic
alcohols, carbonic diesters and azolides, to give a polymer derivative comprising at least
one reactive carboxy group, reacting said polymer derivative with at least one at least
bifunctional compound to give a polymer derivative comprising an aldehyde group or a
keto group or a hemiacetal group or a functional group capable of being chemically
modified to give an aldehyde group or a keto group or a hemiacetal group, optionally
chemically modifying said functional group to give a polymer derivative comprising an
aldehyde group or a keto group or a hemiacetal group, and reacting the polymer derivative
comprising an aldehyde group or a keto group or a hemiacetal group with an ammo group
of a protein via reductive amination.
Accordingly, the present invention also relates to a conjugate comprising a
preferably hydroxyethyl starch, and a protein covalently linked to each other, obtainable by
a method of producing a conjugate, said method comprising reacting the polymer, at its
optionally oxidized reducing end with a compound, selected from the group consisting of
acidic alcohols, carbonic diesters and azolides, to give a polymer derivative comprising at
least one reactive carboxy group, reacting said polymer derivative with at least one at least
bifunctional compound to give a polymer derivative comprising an aldehyde group or a
keto group or a hemiacetal group or a functional group capable of being chemically
modified to give an aldehyde group or a keto group or a hemiacetal group, optionally
chemically modifying said functional group to give a polymer derivative comprising an
aldehyde group or a keto group or a hemiacetal group, and reacting the polymer derivative
comprising an aldehyde group or a keto group or a hemiacetal group with an amino group
of a protein via reductive animation.
A specific example of a compound having a functional group FI and a functional group Fa
which is oxidized to give an aldehyde group is, e.g., a compound having an amino group as
FI and a beta hydroxy amino group as F2- An especially preferred example is 1^-diammo-
2-hydroxypropane. This oxidation may be carried with all suitable oxidation agents which
are capable of converting the beta hydroxy amino group to an aldehyde group. Preferred
oxidation reagents are periodates such as alkaline metal periodates. Especially preferred is
sodium periodate which is preferably employed as aqueous solution. This solution has a
preferred iodate concentration of from 1 to 50 mM, more preferably from 1 to 25 mM and
especially preferably of from 1 to 10 mM. Oxidation is carried out at a temperature of from
0 to 40 °C, preferably from 0 to 25 °C and especially preferably from 4 to 20 °C.
The resulting polymer derivative may be purified from the reaction mixture by at least one
suitable method. If necessary, the polymer derivative may be precipitated prior to the
isolation by at least one suitable method.
If the polymer derivative is precipitated first, it is possible, e.g., to contact the reaction
mixture with at least one solvent or solvent mixture other than the solvent or solvent
mixture present in the reaction mixture at suitable temperatures. According to a
particularly preferred embodiment of the present invention where an aqueous medium,
preferably water is used as solvent, the reaction mixture is contacted with 2-propanol or
with am mixture of acetone and ethanol, preferably a 1:1 mixture (v/v), indicating equal
volumes of said compounds, at a temperature, preferably in the range of from -20 to +50
°C and especially preferably in the range of from -20 to 25 °C.
Isolation of the polymer derivative may be carried out by a suitable process which may
comprise one or more steps. According to a preferred embodiment of the present invention,
the polymer derivative is first separated off the reaction mixture or the mixture of the
reaction mixture with, e.g., aqueous 2-propanol mixture, by a suitable method such as
centrifugation or filtration, hi a second step, the separated polymer derivative may be
subjected to a further treatment such as an after-treatment like dialysis, centrifugal
filtration or pressure filtration, ion exchange chromatography, reversed phase
chromatography, HPLC, MPLC, gel filtration and/or lyophilisation. According to an even
more preferred embodiment, the separated polymer derivative is first dialysed, preferably
against water, and then lyophilized until the solvent content of the reaction product is
sufficiently low according to the desired specifications of the product Lyophilisation,may
be carried out at temperature of from 20 to 35 °C, preferably of from 20 to 30 °C.
According to another preferred embodiment of the present invention, the functional group
Z of the protein to be reacted with functional group A of the polymer or polymer derivative
is a thiol group.
The thiol group may be present hi the protein as such. Moreover, it is possible to introduce
a thiol group into the protein according to a suitable method. Among others, chemical
methods may be mentioned. If a disulfide bridge is present in the protein, it is possible to
reduce the -S-S- structure to get a thiol group. It is also possible to transform an amino
group present in the polypeptide into a SH group by reaction the polypeptide via the amino
group with a compound which has at least two different functional groups, one of which is
capable of being reacted with the amino group and the other is an SH group or a precursor
of an SH group e.g. N-succinimidyl-S-acetylthioacetate, N-succinimidyl-Sacetylthiopropionate
or N-succimmidyl-3-(pyridyldithio)propionate. It is also possible to
introduce an SH group by mutation of the protein such as by introducing an additional
cysteine into the protein, exchanging an amino acid to a cysteine or such as removing a
cystein from the protein so as to disable another cysteine in the protein to form a disulfide
bridge. Most preferably, the polymer is linked to a free cysteine of the protein, especially
preferably Cys 17 or Cys 18, wherein Cys 17 is, e.g., present in Granocyte®, and Cys 18
is, e.g., present in Neupogen®.
According to a first embodiment, the functional group Z of the protein is a thiol group and
functional group A of the polymer is a halogenacetyl group and wherein A is introduced by
reacting the polymer at its optionally oxidized reducing end with an at least bifunctional
compound having at least two functional groups each comprising an amino group to give a
polymer derivative having at least one functional group comprising an amino group and
reacting the polymer derivative with a monohalogen-substituted acetic acid and/or a
reactive monohalogen-substituted acetic acid derivative.
As to the at least bifunctional compound having at least two functional groups each
comprising an amino group, all compounds are conceivable which are capable of being
reacted with the polymer at its optionally reducing end to give a polymer derivative
comprising an amino group which cm be reacted with a monohalpgen-substituted acetic
acid and/or a reactive monohalogen-substituted acetic acid derivative.
According to a preferred embodiment, one functional group of the at least bifunctional
compound, said functional group being reacted with the optionally oxidized reducing end
of the polymer, is selected from the group consisting of
(Figure Removed)
wherein G is O or S and, if present twice, independently O or S, and R is methyl.
According to an especially preferred embodiment of the present invention, the functional
group of the at least bifunctional compound, said functional group being reacted with the
optionally oxidized reducing end, is the amino group -NHa. According to a still further
preferred embodiment, this functional group, most preferably the amino group, is reacted
with the oxidized reducing end of the polymer.
According to a preferred embodiment of the present invention, the functional group of the
at least bifunctional compound, said functional group being reacted with the monohalogensubstituted
acetic acid and/or a reactive monohalogen-substituted acetic acid derivative, is
an amino group -NHj.
The functional groups, preferably both being an amino group -NHb, of the at least
bifunctional compound, said functional groups being reacted with the polymer at its
optionally oxidized reducing end, preferably the oxidized reducing end, and the
monohalogen-substituted acetic acid and/or a reactive monohalogen-substituted acetic acid
derivative, may be separated by any suitable spacer. Among others, the spacer may be an
optionally substituted, linear, branched and/or cyclic hydrocarbon residue. Suitable
substituents are, among others, alkyl, aryl, aralkyL, alkaryl, halogen, carbonyl, acyl,
carboxy, carboxyester, hydroxy, thio, alkoxy and/or alkyhhio groups. Generally, the
hydrocarbon residue has from 1 to 60, preferably from 1 to 40, more preferably from 1 to
20, more preferably from 2 to 10, more preferably from 2 to 6 and especially preferably
from 2 to 4 carbon atoms. If heteroatoms are present, the separating group comprises
generally from 1 to 20, preferably from 1 to 8 and especially preferably from 1 to 4
heteroatoms. The hydrocarbon residue may comprise an optionally branched alkyl chain or
an aryl group or a cycloalkyl group having, e.g., from 5 to 7 carbon atoms, or be an aralkyl
group, an alkaryl group where the alkyl part may be a linear and/or cyclic alkyl group.
According to an even more preferred embodiment, the hydrocarbon residue is an alkyl
chain of from 1 to 20, preferably from 2 to 10, and especially preferably from 2 to 8 carbon
atoms. Thus, preferred at least bifunctional compounds are bifunctional amino compounds,
especially preferably 1,8-diamino octane, 1,7-diamino heptane, 1,6-diamhio hexane, 1,5-
diamino pentane, 1,4-diammo butane, 1,3-diamino propane, and 1,2-diamino ethane.
According to a further preferred embodiment, the at least bifunctional compound is a
diaminopolyethylenglycol, preferably a diaminopolyethylenglycol according to formula
H2N-(CH2-CH2-0)m-CH2-CH2-NH2
wherein m is an integer, m preferably being 1,2,3, or 4.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the polymer is reacted with 1,8-diaminooctane, 1,7-diaminoheptane, 1,6-
diaminohexane, 1,5-diaminopentane, 1,4-diaminobutane, 1,3-diaminopropane, and 1,2-
diaminoethane at its oxidized reducing end with to give a polymer derivative according to
the formula
(Figure Removed)
with n = 2, 3, 4, 5, 6, 7, or 8, and the polymer especially preferably being HES.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the polymer is reacted with HzN^C^a^^Hr^VCHa-CHy-NHj at hs
oxidized reducing end, wherein m is 1, 2, 3, or 4, to give a polymer derivative according to
the formula
HAS1
with m = 1,2,3, or 4, and the polymer especially preferably being HES.
The oxidation of the reducing end of the polymer, preferably hydroxyethyl starch, may be
carried out according to each method or combination of methods which result in
compounds having the structures (Ha) and/or (lib):
(Figure Removed)
Although the oxidation may be carried out according to all suitable method or methods
resulting in the oxidized reducing end of hydroxyalkyl starch, it is preferably carried out
using an alkaline iodine solution as described, e.g., in DE 196 28 705 Al the respective
contents of which (example A, column 9, lines 6 to 24) is incorporated herein by reference.
The polymer derivative resulting from the reaction of the polymer with the at least
bifunctional compound, is further reacted with the monohalogen-substiruted acetic acid
and/or a reactive monohalogen-substituted acetic acid derivative.
As monohalogen-substituted acetic acid or reactive acid, Cl-substituted, Br-substituted and
I-substituted acetic acid are preferred, with acetic acid chloride being particularly
preferred.
If the halogen-substituted acid is employed as such, it is preferred to react the acid with the
polymer derivative in the presence of an activating agent. Suitable activating agents are,
among others, Suitable activating agents are, among others, carbodiimides such as
diisopropyl carbodiimde (DIG), dicyclohexyl carbodiimides (DCC), l-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC), with dicyclohexyl carbodiimides (DCC) and
l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) being especially preferred.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the polymer, preferably HES, is reacted with a diamino compound,
preferably a diaminoalkane with 2 to 8 carbon atoms or H2N-(CH2-CH2-O)m-CH2-CH2-
NH2 with m = 1, 2, 3, or 4, and reacting the resulting polymer derivative with Brsubstituted
and I-substituted acetic acid in the presence of an activating agent, preferably
EDC.
Therefore, the present invention also relates to a polymer derivative according to the
formula
(Figure Removed)
with X = Cl, Br or I, n = 2, 3,4, 5,6,7, or 8, and the polymer especially preferably being
HES, or a polymer derivative according to the formula
(Figure Removed)
with X = Cl, Br or I, m = 1, 2, 3, or 4, and the polymer especially preferably being HES.
The reaction of the polymer derivative with the halogen-substituted acetic acid is
preferably carried out it in an aqueous system, preferably water, at a preferred pH of from
3.5 to 5.5, more preferably from 4.0 to 5.0 and especially preferably from 4.5 to 5.0; and a
preferred reaction temperature of from 4 to 30 °C, more preferably from 15 to 25 and
especially preferably from 20 to 25 °C; and for a preferred reaction time of from 1 to 8 h,
more preferably from 2 to 6 h and especially from 3 to 5 h.
The reaction mixture comprising the polymer derivative which comprises the polymer, the
at least bifunctional compound and the halogen-substituted acetic acid, can be used for the
reaction with the protein as such. According to a preferred embodiment of the present
invention, the polymer derivative is separated from the reaction mixture, preferably by
ultrafiltration, subsequent precipitation, optional washing and drying in vacuo.
The reaction of the polymer derivative with the protein is preferably carried out in an
aqueous system.
The reaction of the polymer derivative with the protein is carried out at a preferred pH of
from 6.5 to 8.5, more preferably from 7.0 to 8.5 and especially preferably from 7.5 to 8.5;
and a preferred reaction temperature of from 4 to 30 °C, more preferably from 15 to 25 and
especially preferably from 20 to 25 °C; and for a preferred reaction time of from 0.5 to 8 b,
more preferably from 1 to 6 h and especially from 2 to 5 h.
The reaction of the polymer derivative with the tbiol group of the protein results in a
thioether linkage between the polymer derivative and the protein.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the polymer, preferably HES, is reacted with a diamino compound,
preferably a diaminoalkane with 2 to 8 carbon atoms or H2N-(CH2-CH2-O)m-CH2-CH2-
NH2 with m = 1, 2, 3, or 4, the resulting polymer derivative is reacted with Br-substituted
and I-substituted acetic acid in the presence of an activating agent, preferably EDC, and the
resulting polymer derivative is reacted with a thiol group of the protein to give a conjugate
comprising a thioether linkage between the protein and the polymer derivative.
Therefore, the present invention also relates to a conjugate according to the formula
(Figure Removed)
with n - 2, 3, 4, 5, 6, 7, or 8, and the polymer especially preferably being HES, or a
conjugate according to the formula
(Figure Removed)
with m = 1, 2, 3, or 4, and the polymer especially preferably being HES. The hydroxyethyl
starch is preferably hydroxethyl starch having a mean molecular weight of about 10 kD
and a DS of about 0.4 or hydroxethyl starch having a mean molecular weight of about 10
kD and a DS of about 0.7 or hydroxethyl starch having a mean molecular weight of about
12 kD and a DS of about 0.4 or hydroxethyl starch having a mean molecular weight of
about 12 kD and a DS of about 0.7 or hydroxethyl starch having a mean molecular weight
of about 18 kD and a DS of about 0.4 or hydroxethyl starch having a mean molecular
weight of about 18 kD and a DS of about 0.7 or hydroxethyl starch having a mean
molecular weight of about 50 kD and a DS of about 0.4 or hydroxethyl starch having a
mean molecular weight of about 50 kD and a DS of about 0.7.
According to a second embodiment, functional group Z of the protein is a thiol group and
functional group A of the polymer comprises a maleimido group.
According to this embodiment, several possibilities exist to produce the conjugate. In
general, the polymer is reacted at its optionally oxidized reducing end with at least one at
least bifunctional compound, wherein this at least bifunctional compound comprises one
functional group which is capable of being reacted with the optionally oxidized reducing
end of the polymer, and at least one functional group which either comprises the
maleimido group or is chemically modified to give a polymer derivative which comprises
the maleimido group. According to a preferred embodiment, said functional group is
chemically modified to give a polymer derivative which comprises the maleimido group.
Therefore, the present invention relates to a method and a conjugate as described above, by
reacting a polymer derivative comprising a maleimido group with a thiol group of the
protein, said method comprising reacting the polymer at its optionally oxidized reducing
end with an at least bifunctional compound comprising a functional group U capable of
reacting with the optionally oxidised reducing end, the at least bifunctional compound
further comprising a functional group W capable of being chemically modified to give a
maleimido group, the method further comprising chemically modifying the functional
group W to give a maleimido group.
As to functional group U, each functional group is conceivable which is capable of being
reacted with optionally oxidised reducing end of the polymer.
According to a preferred embodiment of the present invention, the functional group U
comprises the chemical structure -NH-.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the functional group U comprises the structure -NH-.
According to one preferred embodiment of the present invention, the functional group U is
a group having the structure R'-NH- where R' is hydrogen or a alkyl, cycloalkyl, aryl,
aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue where the cycloalkyl, aryl,
aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue may be linked directly to the NH
group or, according to another embodiment, may be linked by an oxygen bridge to the NH
group. The alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl, or cycloalkylaryl
residues may be suitably substituted. As preferred substituents, halogenes such as F, Cl or
Br may be mentioned. Especially preferred residues R1 are hydrogen, alkyl and alkoxy
groups, and even more preferred are hydrogen and unsubstituted alkyl and alkoxy groups.
Among the alkyl and alkoxy groups, groups with 1, 2, 3, 4, 5, or 6 C atoms are preferred.
More preferred are methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, and
isopropoxy groups. Especially preferred are methyl, ethyl, methoxy, ethoxy, and particular
preference is given to methyl or methoxy.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein R1 is hydrogen or a methyl or a methoxy group.
According to another preferred embodiment of the present invention, the functional group
U has the structure R'-NH-R"- where R" preferably comprises the structure unit -NHand/
or the structure unit -(C=G)- where G is O or S, and/or the structure unit -SC^-.
According to more preferred embodiments, the functional group R" is selected from the
group consisting of
(Figure Removed)
and G
where, if G is present twice, it is independently O or S.
Therefore, the present invention .also relates to a method and a conjugate as described
above, wherein the functional group U is selected from the group consisting of
(Figure Removed)
wherein G is O or S and, if present twice, independently 0 or S, and R1 is methyl.
According to a still more preferred embodiment of the present invention, U comprises an
amino group -NHb-
According to an embodiment of the present invention, the functional group W of the at
least bifunctional compound is chemically modified by reacting the polymer derivative
comprising W with a further at least bifunctional compound comprising a functional group
capable of being reacted with W and further comprising a maleimido group.
As to functional group W and the functional group of said further at least bifunctional
compound which is capable of being reacted with W, the following functional groups are
to be mentioned, among others:
C-C-double bonds or C-C-triple bonds or aromatic C-C-bonds;
the thio group or the hydroxy groups;
alkyl sulfonic acid hydrazide, aryl sulfonic acid hydrazide;
V
1,2-dioks;
1,2-aminoalcohols;
1,2 amino-tbioalcohols;
azides;
the amino group -NH2 or derivatives of the amino groups comprising the structure
unit -NH- such as aminoalkyl groups, aminoaryl group, aminoaralkyl groups, or
alkarlyaminogroups;
the hydroxylamino group -O-NHa, or derivatives of the hydroxylamino group
comprising the structure unit -O-NH-, such as hydroxylalkylamino groups,
hydroxylarylamino groups, hydroxylaralkylamino groups, or hydroxalalkarylamino
groups;
alkoxyarnino groups, aryloxyamino groups, aralkyloxyamino groups, or
alkaryloxyarnino groups, each comprising the structure unit -NH-O-;
residues having a carbonyl group, -Q-C(=G)-M, wherein G is O or S, and M is, for
example,
- -OH or -SH;
an alkoxy group, an aryloxy group, an aralkyloxy group, or an alkaryloxy
group;
an alkylthio group, an arylthio group, an aralkylthio group, or an alkarylthio
group;
an alkylcarbonyloxy group, an arylcarbonyloxy group, an aralkylcarbonyloxy
group, an alkarylcarbonyloxy group;
activated esters such as esters of hydroxylamines having imid structure such as
N-hydroxysuccinimide or having a structure unit O-N where N is part of a
heteroaryl compound or, with G = O and Q absent, such as aryloxy compounds
with a substituted aryl residue such as pentafluorophenyl, paranitrophenyl or
trichlorophenyl;
wherein Q is absent or NH or a heteroatom such as S or O;
-NH-NH2, or -NH-NH-;
the nitril group;
carbonyl groups such as the aldehyde group or the keto group;
the carboxy group;
the -N=C=O group or the -N=C=S group;
vinyl halide groups such as the vinyl iodide or the vinyl bromide group or triflate;
-OC-H;
-(C=NH2Cl)-OAlkyl
groups -(C=O)-CH2-Hal wherein Hal is d, Br, or I;
-CH=€H-SQ2S
a disulfide group comprising the structure -S-S-;
the group
the group °2N
where W and the functional group of the further at least bifunctional compound,
respectively, is a group capable of forming a chemical linkage with one of the abovementioned
groups.
According to a still more preferred embodiment of the present invention, W comprises an
amino group -
According to preferred embodiments of the present invention, both W and the other
functional group are groups from the list of groups given above.
According to one embodiment of the present invention, one of these functional groups is a
thio group. In this particular case, the other functional group is preferably selected from the
group consisting of
(Figure Removed)
wherein Hal is Cl, Br, or I, preferably Br or I.
According to an especially preferred embodiment of the present invention, one of these
functional groups is selected from the group consisting of a reactive ester such as an ester
of hydroxylamines having nnide structure such as N-hydroxysuccinimide or having a
structure unit ON where N is part of a heteroaryl compound or such as an aryloxy
compound with a substituted aryl residue such as pentafluorophenyl, paranitrophenyl or
trichlorophenyl, or a carboxy group which is optionally transformed into a reactive ester.
In this particular case, the other functional group comprises the chemical structure -NH-.
According to an especially preferred embodiment of the present invention, W comprises
the structure -NH- and the further at least bifunctional compound comprises a reactive
ester and the maleimido group.
As to the functional group W comprising the structure -NH-, reference can be made to the
functional group as described above, wherein W may be the same or different from U.
According to a preferred embodiment of the present invention, U and W are the same.
More preferably, both U and W comprise an amino group. Particularly preferred, both U
and W are an amino group -
According to one embodiment of the present invention, the polymer may be reacted with
the at least bifunctional compound comprising U and W at its non-oxidized reducing end in
an aqueous medium. According to a preferred embodiment where U and W both are an
amino group, the reaction is carried out using the polymer with the reducing end in the
oxidized form, in at least one aprotic solvent, particularly preferably in an anhydrous
aprotic solvent having a water content of not more than 0.5 percent by weight, preferably
of not more than 0.1 percent by weight. Suitable solvents are, among others, dimethyl
sulfoxide (DMSO), N-niethyl pyrrolidone, dimethyl acetamide (DMA), dimethyl
formamide (DMF) and mixtures of two or more thereof.
Especially in case both U and W are an amino group -NHz, U and W may be separated by
any suitable spacer. Among others, the spacer may be an optionally substituted, linear,
branched and/or cyclic hydrocarbon residue. Suitable substituents are, among others, alkyl,
aryl, aralkyl, alkaryl, halogen, carbonyl, acyl, carboxy, carboxyester, hydroxy, thio, alkoxy
and/or alkylthio groups. Generally, the hydrocarbon residue has from 1 to 60, preferably
from 1 to 40, more preferably from 1 to 20, more preferably from 2 to 10, more preferably
from 2 to 6 and especially preferably from 2 to 4 carbon atoms. If heteroatoms are present,
the separating group comprises generally from 1 to 20, preferably from 1 to 8 and
especially preferably from 1 to 4 heteroatoms. The hydrocarbon residue may comprise an
optionally branched alkyl chain or an aryl group or a cycloalkyl group having, e.g., from 5
to 7 carbon atoms, or be an aralkyl group, an alkaryl group where the alkyl part may be a
linear and/or cyclic alkyl group. According to an even more preferred embodiment, the
hydrocarbon residue is an alkyl chain of from 1 to 20, preferably from 2 to 10, more
preferably from 2 to 6, and especially preferably from 2 to 4 carbon atoms.
Therefore, the present invention also relates to a method and a conjugate as described
above, wherein the polymer is reacted with its oxidized reducing end with 1,4-
diaminobutane, 1,3-diaminopropane or 1,2-diaminoethane to give a polymer derivative
according to the formula
with n = 2,3, or 4, the polymer preferably being HES.
According to the above-mentioned preferred embodiment, the polymer derivative
comprising an amino group is further reacted with an at least bifunctional compound
comprising a reactive ester group and the maleimido group. The reactive ester group and
the maleimido group may be separated by a suitable spacer. As to this spacer, reference
can be made to the spacer between the functional groups U and W. According to a
preferred embodiment of the present invention, the reactive ester group and the maleimido
group are separated by a hydrocarbon chain having from 1 to 10, preferably from 1 to 8,
more preferably from 1 to 6, more preferably from 1 to 4, more preferably from 1 to 2 and
particularly preferably 1 carbon atom. According to a still further preferred embodiment,
the reactive ester is a succinimide ester, and according to a particularly preferred
embodiment, the at least bifunctional compound comprising the maleimido group and the
reactive ester group is N-(alpha-malemiidoacetoxy)succinimide ester.
Therefore, the present invent also relates to a polymer derivative according to the formula
(Figure Removed)
with n = 2,3, or 4, the polymer preferably being HES.
The polymer derivative comprising the maleimido group is further reacted with the thiol
group of the protein to give a conjugate comprising the polymer derivative linked to the
protein via a thioether group.
Therefore, the present invention also relates to a conjugate, comprising the protein and the
polymer, according to the formula
(Figure Removed)
with n = 2, 3, or 4, preferably 4, the polymer preferably being HES, and wherein the S
atom in the formula above derives from Cysl? or CyslS of the protein. The hydroxyethyl
starch is preferably hydroxethyl starch having a mean molecular weight of about 10 kD
and a DS of about 0.4 or hydroxethyl starch having a mean molecular weight of about 10
kD and a DS of about 0.7 or hydroxethyl starch having a mean molecular weight of about
12 kD and a DS of about 0.4 or hydroxethyl starch having a mean molecular weight of
about 12 kD and a DS of about 0.7 or hydroxethyl starch having a mean molecular weight
of about 18 kD and a DS of about 0.4 or hydroxethyl starch having a mean molecular
weight of about 18 kD and a DS of about 0.7 or hydroxethyl starch having a mean
molecular weight of about 50 kD and a DS of about 0.4 or hydroxethyl starch having a
mean molecular weight of about 50 kD and a DS of about 0.7.
The reaction of the polymer derivative comprising the maleimido group with the thiol
group of the protein is preferably carried in a buffered aqueous system,, at a preferred pH
of fromS.S to 8.5, more preferably from 6 to 8 and especially preferably from 6.5 to 7.5,
and a preferred reaction temperature of from 0 to 40 °C, more preferably from 0 to 25 and
especially preferably from 4 to 21 °C, and for a preferred reaction time of from 0.5 to 24 h,
more preferably from 1 to 20 h and especially from 2 to 17 h. The suitable pH value of the
reaction mixture may be adjusted by adding at least one suitable buffer. Among the
preferred buffers, sodium acetate buffer, phosphate or borate buffers may be mentioned,
containing either urea at a preferred concentration of from 0 to 8 M, more preffered from 2
to 8 M and especially preferred from 4 to 8 M, and/or containing SDS at a preffered
concentration of from 0 to 1% (w/v), more preferred from 0.4 to 1% (w/v) and especially
prefferd from 0.8 to 1% (w/v).
The
conjugate may be subjected to a further treatment such as an after-treatment like
dialysis, centrifugal filtration or pressure filtration, ion exchange chromatography, reversed
phase chromatography, HPLC, MPLC, gel filtration and/or lyophilisation.
Therefore, the present invention also relates to a conjugate as obtainable by a method as
described above.
Therefore, the present invention also relates to a conjugate as obtainable by a method as
described above, wherein A is a reactive carboxy group, and wherein A was introduced in
the polymer whose reducing end was not oxidized, by reacting at least one hydroxy group
of the polymer with a carbonic diester, and wherein, said comprising one polymer
molecule and at least one, in particular of from 1 to 10 protein molecules linked to the
polymer via amide linkages.
The present invention also relates to a conjugate comprising a protein and a polymer or a
derivative thereof, wherein the polymer is a hydroxyalkyl starch (HAS) and the protein is a
granulocyte colony stimulating factor (G-CSF), having a structure according to the formula
(Figure Removed)
wherein RI, Ra and Rj are independently hydrogen or a hydroxyalkyl group, a hydroxyaryl
group, a hydroxyaralkyl group or a hydroxyalkaryl group having of from 1 to 10 carbon
atoms, preferably hydrogen or a hydroxyalkyl group, more preferably hydrogen or a
hydroxyethyl group,
wherein G is selected from the group consisting of O and S, preferably O, and
wherein L is an optionally suitably substituted, linear, branched and/or cyclic hydrocarbon
residue, optionally comprising at least one heteroatom, preferably an alkyl, aryl, aralkyl,
heteroaryl, heteroaralkyl residue having from 2 to 60 carbon atoms.
The abbreviation "Protein" " as used in the formulae above refers to the G-CSF molecule
used for the reaction without the carbon atom of the carbohydrate moiety which is part of
oxime linkage in the N=C double bond.
The present invention also relates to a conjugate as described above, wherein -L- is -
(CH2)n- with n = 2, 3, 4, 5, 6, 7, 8, 9, 10, preferably 2, 3, 4, 5, 6, more preferably 2, 3, 4,
and especially preferably 4.
The present invention also relates to a conjugate comprising a protein and a polymer or a
derivative thereof, wherein the polymer is a hydroxyalkyl starch (HAS) and the protein is a
granulocyte colony stimulating factor (G-CSF), having a structure according to the formula
(Figure Removed)
wherein Rt, R2 and Ra are independently hydrogen or a hydroxyalkyl group, a hydroxyaryl
group, a hydroxyaralkyl group or a hydroxyalkaryl group having of from 1 to 10 carbon
atoms, preferably hydrogen or a hydroxyalkyl group, more preferably hydrogen or a
hydroxyethyl group, and
wherein G is selected from the group consisting of O and S, preferably O, and
The
abbreviation "Protein1 " as used in the formulae above refers to the G-CSF molecule
used for the reaction without the carbon atom of the carbohydrate moiety which is part of
oxime linkage in the N=C double bond.
The present invention also relates to a conjugate comprising a protein and a polymer or a
derivative thereof, wherein the polymer is a hydroxyalkyl starch (HAS) and the protein is a
granulocyte colony stimulating factor (G-CSF), having a structure according to the formula
(Figure Removed)
wherein RI, Ra and Rj are independently hydrogen or a hydroxyalkyl group, a hydroxyaryl
group, a hydroxyaralkyl group or a hydroxyalkaryl group having of from 1 to 10 carbon
atoms, preferably hydrogen or a hydroxyalkyl group, more preferably hydrogen or a
hydroxyethyl group, and
wherein L is an optionally suitably substituted, linear, branched and/or cyclic hydrocarbon
residue, optionally comprising at least one heteroatom, preferably an alkyl, aryl, aralkyl,
heteroaryl, heteroaralkyl residue having from 2 to 60 carbon atoms.
The abbreviation "Protein1" as used in the formulae above refers to the G-CSF molecule
used for the reaction without the carbon atom of the carbohydrate moiety which is part of
oxime linkage in the N=C double bond.
The two structures above describe a structure where the crosslinking compound is linked
via an oxime linkage to the reducing end of HAS where the terminal saccharide unit of
HES is present in the open form, and a structure with the respective cyclic animal form
where the crosslinking compound is linked to the reducing end of HES via an oxyamino
group and where the terminal saccharide unit of HES is present in the cyclic form. Both
structures may be simultaneously present in equilibrium with each other.
The present invention also relates to a conjugate as described above, wherein -L- is
wherein R*; Rb, RC, Rd are independently hydrogen, alkyl, aryl, preferably hydrogen,
wherein G is selected from the group consisting of O and S, preferably O,
and wherein
m 1 , 2, 3 or 4, wherein the residues Ra and Rb may be the same or different in the
m groups C R, R&;
n 0 to 20» preferably 0 to 10, more preferably 1,2, 3,4,5, most preferably lor 2;
o 0 to 20, preferably 0 to 10, more preferably 1, 2, 3, 4, 5, most preferably 1 or 2,
wherein the residues RC and Rd may be the same or different in the o groups C R Rd;
The present invention also relates to a conjugate as described above, wherein R«; Rb, RC, Rd
are hydrogen, m = 2, n = 1, and o = 2.
The present invention also relates to a conjugate comprising a protein and a polymer or a
derivative thereof, wherein the polymer is a hydroxyalkyl starch (HAS) and the protein is a
granulocyte colony stimulating factor (G-CSF), having a structure according to the formula
(Figure Removed)
wherein RI, R2 and RS are independently hydrogen or a hydroxyalkyl group, a hydroxyaryl
group, a hydroxyaralkyl group or a hydroxyalkaryl group having of from 1 to 10 carbon
atoms, preferably hydrogen or a hydroxyalkyl group, more preferably hydrogen or a
hydroxyethyl group.
The abbreviation "Protein' " as used in the formula above refers to the G-CSF molecule
used for the reaction without the nitrogen atom of the amino group which is part the amide
linkage.
The present invention also relates to a conjugate comprising a protein and a polymer or a
derivative thereof, wherein the polymer is a hydroxyalkyl starch (HAS) and the protein is a
granulocyte colony stimulating factor (G-CSF), having a structure according to the formula
(Figure Removed)
wherein RI, R2 and Ra are independently hydrogen or a hydroxyalkyl group, a hydroxyaryl
group, a hydroxyaralkyl group or a hydroxyalkaryl group having of from 1 to 10 carbon
atoms, preferably hydrogen or a hydroxyalkyl group, more preferably hydrogen or a
hydroxyethyl group, and
wherein the linkage -O-(C=Oy- was formed by a reaction of a carfaoxy group or a reactive
carboxy group with a hydroxy group of the HAS molecule.
The abbreviation "Protein' " as used in the formula above refers to the G-CSF molecule
used for the reaction without the nitrogen atom of the amino group which is part the amide
linkage.
The present invention also relates to a conjugate, comprising a protein and a polymer or a
derivative thereof, wherein the polymer is a hydroxyalkyl starch (HAS) and the protein is a
granulocyte colony stimulating factor (G-CSF), having a structure according to the formula
(Figure Removed)
wherein RI, Rj and R3 are independently hydrogen or a hydroxyalkyl group, a hydroxyaryl
group, a hydroxyaralkyl group or a hydroxyalkaryl group having of from 1 to 10 carbon
atoms, preferably hydrogen or a hydroxyalkyl group, more preferably hydrogen or a
hydroxyethyl group, and
wherein L is an optionally substituted, linear, branched and/or cyclic hydrocarbon residue,
optionally comprising at least one heteroatom, having from 1 to 60 preferably from 1 to 40,
more preferably from 1 to 20, more preferably from 1 to 10, more preferably from 1 to 6
more preferably from 1 to 2 carbon atoms and especially preferably 1 carbon atom, L being
in particular
The abbreviation "Protein1 " as used in the formula above refers to the G-CSF molecule
used for the reaction without the nitrogen atom of the amino group which is part the
aminomethyl linkage.
The present invention also relates to a conjugate, comprising a protein and a polymer or a
derivative thereof, wherein the polymer is a hydroxyalkyl starch (HAS) and the protein is a
granulocyte colony stimulating factor (G-CSF), having a structure according to the formula
(Figure Removed)
wherein RI, RI and RS are independently hydrogen or a hydroxyalkyl group, a hydroxyaryl
group, a hydroxyaralkyl group or a hydroxyalkaryl group having of from 1 to 10 carbon
atoms, preferably hydrogen or a hydroxyalkyl group, more preferably hydrogen or a
hydroxyethyl group, and
wherein LI and LZ are independently an optionally substituted, linear, branched and/or
cyclic hydrocarbon residue, optionally comprising at least one heteroatom, comprising an
alkyl, aryl, aralkyl heteroalkyl, and/or heteroaralkyl moiety, said residue having from 1 to
60 preferably from 1 to 40, more preferably from 1 to 20, more preferably from 1 to 10
carbon atoms, and
wherein D is a linkage, preferably a covalent linkage which was formed by a suitable
functional group F2 linked to LI and a suitable functional group Fa linked to L2.
The abbreviation "Protein1 " as used in the formulae above refers to the G-CSF molecule
used for the reaction without the nitrogen atom of the amino group which is part of the
aminomethyl linkage.
The present invention also relates to a conjugate as described above, wherein LI is -
(CH2)n- with n = 2, 3, 4, 5, 6, 7, 8, 9, 10, preferably 2, 3, 4, 5, 6, more preferably 2, 3, 4,
and especially preferably 4.
The present invention also relates to a conjugate as described above, wherein L2 comprises
an optionally suitably substituted aryl moiety, preferably an aryl moiety containing 6
carbon atoms, L2 being especially preferably CeHU, or wherein L2 is -(CH2)n- with n = 2, 3,
4, 5,6, 7, 8,9,10, preferably 2, 3,4, 5,6, more preferably 2, 3,4.
The present invention also relates to a conjugate as described above, wherein is selected
from the group consisting of
C-C-double bonds or C-C-triple bonds or aromatic C-C-bonds;
the thio group or the hydroxy groups;
alkyl sulfonic acid hydrazide, aryl sulfonic acid hydrazide;
1,2-dioles;
12 amino-thioalcohols;
azides;
1,2-aminoalcohols;
the amino group -NH2 or derivatives of the amino groups comprising the
structure unit -NH- such as aminoalkyl groups, aminoaryl group, aminoaralkyl
groups, or alkarlyaminogroups;
the hydroxylamino group -O-NHfe, or derivatives of the hydroxylamino group
comprising the structure unit -O-NH-, such as hydroxylalkylamino groups,
hydroxylarylamino groups, hydroxylaralkylamino groups, or
hydroxalalkarylamino groups;
alkoxyamino groups, aryloxyamino groups, aralkyloxyamino groups, or
alkaryloxyamino groups, each comprising the structure unit -NH-O-;
residues having a carbonyl group, -Q-C(=G)-M, wherein G is O or S, and M is,
for example,
-OH or -SH;
an alkoxy group, an aryloxy group, an aralkyloxy group, or an alkaryloxy
group;
an alkylthio group, an arylthio group, an aralkylthio group, or an
alkarylthio group;
an alkylcarbonyloxy group, an arylcarbonyloxy group, an
aralkylcarbonyloxy group, an alkarylcarbonyloxy group;
activated esters such as esters of hydroxylamines having imid structure
such as N-hydroxysuccinimide or having a structure unit O-N where N is
part of a heteroaryl compound or, with G = O and Q absent, such as
aryloxy compounds with a substituted aryl residue such as
pentafluorophenyl, paranitrophenyl or trichlorophenyl;
wherein Q is absent or NH or a heteroatom such as S or O;
-NH-NH2, or -NH-NH-;
-N02;
the nitril group;
carbonyl groups such as the aldehyde group or the keto group;
the carboxy group;
the -N=O=O group or the -N=C=S group;
vinyl halide groups such as the vinyl iodide or the vinyl bromide group or
triflate;
-OC-H;
-(C=NH2Ci)-OAlkyl
groups -(C=O)-CH2-Hal wherein Hal is Cl, Br, or I;
-CH=CH-SO2-;
a disulfide group comprising the structure -S-S-;
O.
—N
the group
the group
and wherein ¥3 is a functional group capable of forming a chemical linkage with F2
and is preferably selected from the above-mentioned group, F2 preferably comprising
the moiety -NH-, more preferably comprising an ammo group, Fa preferably
comprising the moiety -(C=G)-, more preferably -(C=O)-, more preferably the
moiety -(CK})-G~, still more preferably -(C=O)-G-, and especially preferably -
(O=O)~O, D being particularly preferably an amide linkage.
The present invention also relates to a conjugate comprising a protein and a polymer or a
derivative thereof, wherein the polymer is a hydroxyalkyl starch (HAS) and the protein is a
granulocyte colony stimulating factor (G-CSF), having a structure according to the formula
(Figure Removed)
wherein the carbon atom of the moiety -CH2-N2- is derived from an aldehyde group which
was introduced hi the polymer by a ring-opening oxidation reaction, wherein HAS" relates
to the hydroxyalkyl starch molecule without the aldeyhde group resulting from the ringopening
oxidation and reacted with the amino group of the protein, and wherein the
nitrogen atom is derived from an amino group of the protein.
The abbreviation "Protein* " as used in the formula above refers to the G-CSF molecule
used for the reaction without the nitrogen atom of the amino group which is part the amide
linkage.
The present invention also relates to a conjugate, comprising a protein and a polymer or a
derivative thereof, wherein the polymer is a hydroxyalkyl starch (HAS) and the protein is a
granulocyte colony stimulating factor (G-CSF), having a structure according to the formula
HAS'> "N. jS^ \ ^-»»^\ .-*•— I I 1 -Protein'
wherein RI, R? and Ra are independently hydrogen or a hydroxyalkyl group, a hydroxyaryl
group, a hydroxyaralkyl group or a hydroxyalkaryl group having of from 1 to 10 carbon
atoms, preferably hydrogen or a hydroxyalkyl group, more preferably hydrogen or a
hydroxyethyl group, and
wherein L is an optionally substituted, linear, branched and/or cyclic hydrocarbon residue,
optionally comprising at least one heteroatom, comprising an alkyl, aryl, aralkyl
heteroalkyl,
and/or heteroaralkyl moiety, said residue having from 2 to 60 preferably from
2 to 40, more preferably from 2 to 20, more preferably from 2 to 10 carbon atoms, and
wherein the sulfur atom is derived from a cysteine residue or a disulfide group of the
protein.
The present invention also relates to a conjugate as described above, wherein -L- is
wherein R*; Rb, RC, R wherein G is selected from the group consisting of O and S, preferably O, and
wherein
1 , 2, 3 or 4, most preferably 2, wherein the residues Ra and Rb may be the same
or different in the m groups C RB Rb;
m
n 1 to 20, preferably 1 to 10, most preferably 1, 2, 3, or 4;
o 1 to 20, preferably 1 to 10, more preferably 1, 2, 3, 4, 5, more preferably 1 or 2,
most preferably 1, •wherein the residues R« and R* may be the same or different
in the o groups ORcRd;
or
wherein
n 0, and
o 2 to 20, preferably 2 to 10, more preferably 2, 3, 4, 5, 6, 7, or 8, wherein the
residues R« and R The present invention also relates to a conjugate, comprising a protein and a polymer or a
derivative thereof, wherein the polymer is a hydroxyalkyl starch (HAS) and the protein is a
granulocyte colony stimulating factor (G-CSF), having a structure according to the formula
(Figure Removed)
wherein RI, Ra and Ra are independently hydrogen or a hydroxyalkyl group, a
hydroxyaryl group, a hydroxyaralkyl group or a hydroxyalkaryl group having of
from 1 to 10 carbon atoms, preferably hydrogen or a hydroxyalkyl group, more
preferably hydrogen or a hydroxyethyl group, and
wherein
L is an optionally substituted, linear, branched and/or cyclic hydrocarbon
residue, optionally comprising at least one heteroatom, comprising an alkyl, aryl,
aralkyl heteroalkyl, and/or heteroaralkyl moiety, said residue having from 2 to 60
preferably from 2 to 40, more preferably from 2 to 20, more preferably from 2 to 10
carbon atoms, and
wherein the sulfur atom is derived from a cysteine residue or a disulfide group of the
protein.
The present invention also relates to a conjugate as described above, wherein -L- is
wherein Ra; Rt» Re, Ra are independently hydrogen, alkyl, aryl, preferably hydrogen,
wherein G is selected from the group consisting of O and S, preferably O, and
wherein
m 1, 2, 3 or 4, most preferably 2, wherein the residues Ra and Rb may be the same
or different in the m groups C R* R*;
n 1 to 20, preferably 1 to 10, most preferably 1, 2, 3, or 4;
o 1 to 20, preferably 1 to 10, more preferably 1, 2, 3, 4, 5, more preferably 1 or 2,
most preferably 1, wherein the residues Re and Rj may be the same or different
in the o groups CRcRd;
or
wherein
n 0, and
o 2 to 20, preferably 2 to 10, more preferably 2, 3, 4, 5, 6, 7, or 8, wherein the
residues RC and Ra may be the same or different in the o groups CRcRd.
The present invention also relates to any conjugate as described above, wherein the
hydroxyalkyl starch is hydroxyethyl starch.
The present invention also relates to any conjugate as described above, wherein the
hydroxyethyl starch has a molecular weight of from 2 to 200 kD, preferably of from 4 to
130 kD, more preferably of from 4 to 70 kD.
According to a further aspect, the present invention relates to a conjugate as described
above, or a conjugate, obtainable by a method as described above, for use in a method for
the treatment of the human or animal body.
Furthermore, the present invention relates to a pharmaceutical composition comprising in a
therapeutically effective amount a conjugate as described above or a conjugate, obtainable
by a method as described above.
The term "in a therapeutically effective amount" as used in the context of the present
invention refers to that amount which provides therapeutic effect for a given condition and
administration regimen. The administration is preferably by routes. The specific route
chosen will depend upon the condition being treated. The administration is preferably done
as part of a formulation containing a suitable carrier, such as polysorbat, a suitable diluent,
such as water and/or a suitable adjuvant such as sorbitol. The required dosage will depend
upon the severity of the condition being treated, the patients individual response, the
method of administration used, and the like.
Thus, in a preferred embodiment, the pharmaceutical composition further comprises at
least one pharmaceutically acceptable diluent, adjuvant and/or carrier, especially
preferably useful in G-CSF therapy.
The pharmaceutical composition is preferably used for the treatment of a disorder
characterized by a reduced hematopoietic or immune function or diseases related thereto.
Therefore, the present invention also relates to the use of a pharmaceutical composition as
described above, comprising a conjugate as described above or a conjugate, obtainable by
a method as described above, for the preparation of a medicament for the treatment of a
disorder characterized by a reduced hematopoietic or immune function.
According to a preferred embodiment, the disorder characterized by a reduced
hematopoietic or immune function, is a result of chemotherapy, radiation therapy,
infectious disease, severe chronic neutropenia, or leukemia. Therefore, the present
invention also relates to the use of a pharmaceutical composition as described above,
comprising a conjugate as described above or a conjugate, obtainable by a method as
described above, for the preparation of a medicament for the treatment of a disorder
characterized by a reduced hematopoietic or immune function, wherein the disorder is a
result of chemotherapy, radiation therapy, infectious disease, severe chronic neutropenia,
or leukemia.
The object of the treatment>with the pharmaceutical composition according to the invention
is preferably administered by i.v. or s.c. routes. For this, the pharmaceutical composition
may be administered as a sterile solution.
The invention is further illustrated by the following figures, tables and examples, which are
in no way intended to restrict the scope of the present invention.
Short description of the Figures
Figure la
Figure la shows an SDS page analysis of the HES—G-CSF conjugate, produced according
to Example 2.1 (a), Neupogen®. For gel electrophoresis, a XCell Sure Lock Mini Cell
(Invitrogen GmbH, Karlsruhe, D) and a Consort El43 power supply (CONSORTnv,
Tumhout, B) were employed. A 12% Bis-Tris gel together with a MOPS SDS running
buffer at reducing conditions (both Invitrogen GmbH, Karlsruhe, D) were used according
to the manufactures instruction.
Lane A: Protein marker SeeBlue®Plus2 (Invitrogen GmbH, Karlsruhe, D) Molecular
weight marker from top to bottom: 188 kD, 98 kD, 62 kD, 49 kD, 38 kD, 28
kD, 17 kD, 14 kD, 6 kD, 3 kD.
LaneB: Crude product after conjugation of G-CSF (Neupogen®) with HES as
described in Example 2.1 (a).
Lane C: G-CSF starting material.
Figure Ib
Figure Ib shows an SDS page analysis of the HES—G-CSF conjugate, produced according
to Example 2.1 (a), Granocyte®. For gel electrophoresis, a XCell Sure Lock Mini Cell
(Invitrogen GmbH, Karlsruhe, D) and a Consort E143 power supply (CONSORTnv,
Tumhout, B) were employed. A 12% Bis-Tris gel together with a MOPS SDS running
buffer at reducing conditions (both Invitrogen GmbH, Karlsruhe, D) were used according
to the manufactures instruction.
Lane A: Protein marker SeeBlue®Plus2 (Invitrogen GmbH, Karlsruhe, D). Molecular
weight marker from top to bottom: 188 kD, 98 kD, 62 kD, 49 kD, 38 kD, 28
kD, 17 kD, 14 kD, 6 kD, 3 kD.
LaneB: Crude product after conjugation of G-CSF (Granocyte®) with HES as
described in Example 2.1 (a).
Lane C: G-CSF starting material.
Figure 2
Figure 2 shows an SDS page analysis of the HES—G-CSF conjugate, produced according
to Example 2.1(b), G-CSF from Strathmann Biotec AG, Hamburg, D. For gel
electrophoresis a XCell Sure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and a
Consort El43 power supply (CONSORTnv, Turnhout, B) were employed. A 12% Bis-Tris
gel together with a MOPS SDS running buffer at reducing conditions (both Invitrogen
GmbH, Karlsruhe, D) were used according to the manufactures instruction.
Lane A: Protein marker SeeBlue®Plus2 (Invitrogen GmbH, Karlsruhe, D). Molecular
weight marker from top to bottom: 188 kD, 98 kD, 62 kD, 49 kD, 38 kD, 28
kD, 17 kD, 14 kD, 6 kD, 3 kD
Lane B: Crude product after conjugation of G-CSF with HES10/0.4 in 0.1M NaOAc
buffer pH 5.0.
Lane C: Crude product after conjugation of G-CSF with HES10/0.7 in 0.1M NaOAc
buffer pH 5.0.
Lane D: Crude product after conjugation of G-CSF with HES50/0.4 in 0.1M NaOAc
buffer pH 5.0.
Lane E: Crude product after conjugation of G-CSF with HES50/0.7 in 0.1M NaOAc
buffer pH 5.0.
Lane F: G-CSF starting material.
Figure 3
Figure 3 shows an SDS page analysis of the HES—G-CSF conjugates, produced according
to Example 2.2, G-CSF from Strathmann Biotec AG, Hamburg, D. For gel electrophoresis
a XCell Sure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and a Consort E143 power
supply (CONSORTnv, Turnhout, B) were employed. A 12% Bis-Tris gel together with a
MOPS SDS running buffer at reducing conditions (both Invitrogen GmbH, Karlsruhe, D)
were used according to the manufactures instruction.
Lane A: Protein marker SeeBlue®Plus2 (Invitrogen GmbH, Karlsruhe, D). Molecular
weight marker from top to bottom: 188 kD, 98 kD, 62 kD, 49 kD, 38 kD, 28
kD, 17 kD, 14 kD, 6 kD, 3 kD.
Lane B: Crude product after conjugation of G-CSF with oxidized HES 10/0.7 in 0.1M
NaOAc buffer pH 5.0.
Lane C: Crude product after conjugation of G-CSF with oxidized HES50/0.4 in 0.1M
NaOAc buffer pH 5.0.
Lane D: Crude product after conjugation of G-CSF with oxidized HES50/0.7 in 0.1M
NaOAc buffer pH 5.0.
Lane E: G-CSF starting material.
Figure 4
Figure 4 shows an SDS page analysis of the HES—G-CSF conjugates, produced according
to Example 2.3, G-CSF is Neupogen® or Granocyte®. For gel electrophoresis a XCell
Sure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and a Consort E143 power supply
(CONSORTnv, Turnhout, B) were employed. A 12% Bis-Tris gel together with a MOPS
SDS running buffer at reducing conditions (both Invitrogen GmbH, Karlsruhe, D) were
used according to the manufactures instruction.
Lane A: Protein marker SeeBlue®Plus2 (Invitrogen GmbH, Karlsruhe, D). Molecular
weight marker from top to bottom: 188 kD, 98 kD, 62 kD, 49 kD, 38 kD, 28
kD, 17kD, 14kD,6kD,3kD.
LaneB: Crude product (i-N) according to Example 2.3.
Lane C: Crude product (ii-N) according to Example 2.3.
Lane D: Crude product (iii-N) according to Example 2.3.
Lane E: Crude product (iv-N) according to Example 2.3.
Lane F: Crude product (i-G) according to Example 2.3.
Lane G: Crude product (ii-G) according to Example 2.3.
Lane H: Crude product (iii-G) according to Example 2.3.
Lane I: Crude product (iv-G) according to Example 2.3.
Lane J: Neupogen®.
Figure 5
Figure 5 shows an SDS page analysis of the HES—G-CSF conjugates, produced according
to Example 2.4, G-CSF from Strathmann Biotec AG, Hamburg, D. For gel electrophoresis
a XCell Sure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and a Consort El43 power
supply (CONSORTnv, Turnhout, B) were employed. A 12% Bis-Tris gel together with a
MOPS SDS running buffer at reducing conditions (both Invitrogen GmbH, Karlsruhe, D)
were used according to the manufactures instruction.
Lane A: Protein marker SeeBlue weight marker from top to bottom: 188 kD, 98 kD, 62 kD, 49 kD, 38 kD, 28
kD, 17 kD, 14 kD, 6kD,3kD.
Lane B: Crude product (vi) according to Example 2.4.
Lane C: Crude product (v) according to Example 2.4.
Lane D: G-CSF starting material.
Lane E: Protein marker SeeBlue®Plus2 (Invitrogen GmbH, Karlsruhe, D). Molecular
weight marker from top to bottom: 188 kD, 98 kD, 62 kD, 49 kD, 38 kD, 28
kD, 17 kD, 14 kD, 6 kD, 3 kD.
Lane F: Crude product (ix) according to Example 2.4.
Lane G: Crude product (viii) according to Example 2.4.
Lane H: Crude product (vii) according to Example 2.4.
Lane I: G-CSF starting material.
Figure 6
Figure 6 shows an SDS page analysis of the HES—G-CSF conjugate, produced according
to Example 2.5, G-CSF from Strathmann Biotec AG, Hamburg, D. For gel electrophoresis
a XCell Sure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and a Consort E143 power
supply (CONSORTnv, Turnhout, B) were employed. A 10% Bis-Tris gel together with a
MOPS SDS running buffer at reducing conditions (both Invitrogen GmbH, Karlsruhe, D)
were used according to the manufactures instruction.
Lane A: Protein marker SeeBlue®Plus2 (Invitrogen GmbH, Karlsruhe, D). Molecular
weight marker from top to bottom: 188 kD, 98 kD, 62 kD, 49 kD, 38 kD, 28
kD, 17 kD, 14kD,6kD,3kD.
Lane B: Crude product according to Example 2.5.
Lane C: G-CSF starting material.
Figure 7
Figure 7 shows an SDS page analysis of the HES—G-CSF conjugate, produced according
to Example 3, G-CSF is Neupogen® or Granocyte®. For gel electrophoresis a XCell Sure
Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and a Consort El43 power supply
(CONSORTnv, Turnhout, B) were employed. A 12% Bis-Tris gel together with a MOPS
SDS running buffer at reducing conditions (both Invitrogen GmbH, Karlsruhe, D) were
used according to the manufactures instruction.
Lane A: Protein marker SeeBlue®Plus2 (Invitrogen GmbH, Karlsruhe, D). Molecular
weight marker from top to bottom: 188 kD, 98 kD, 62 kD, 49 kD, 38 kD, 28
kD, 17 kD, 14kD,6kD,3kD
Lane B: Crude product (x) according to Example 3.
Lane C: Crude product (xi) according to Example 3.
Lane D: Crude product (xii) according to Example 3.
Lane E: Crude product (xiii) according to Example 3.
Lane F: Crude product (xiv) according to Example 3.
Lane G: Crude product (xv) according to Example 3.
Figure 8
Figure 8 shows the in vitro results of Example 6.
In the diagram, the x axis shows the concentration in pg/ml, the y axis refers to the number
of cells /100,000. In the diagram, the following abbreviations refer to
G-CSF/A32 G-CSF conjugate as prepared according to Example 2.5
G-CSF/A33 G-CSF starting material, used for the conjugate of Example 2.5
G-CSF/A57 non-modified Neulasta®
G-CSF/A58 non-modified Neupogen®
G-CSF/A60 G-CSF conjugate as prepared according to Example 4.2
Figure 9
Figure 9 shows the HPGPC chromatogram with regard to the crude conjugation reaction
product according to Example 4.1. The following parameters were used in the HPGPC
analysis:
Column: Superose 12 HR 10/30 300 x 10 mm I.D. (Pharmacia)
Eluent: 27.38 mM Na2HPO4; 12.62 mM NaH2PO4; 0.2 M NaCl; 0,005 % NaN3 in 11
of demineralized water
Flux: 0,24 ml/h
Detector 1: MALLS detector
Detector 2: UV (280 nm)
Detector 3: RI (Refractive Index detector)
A represents the result from detector 1, B represents the result from detector 2.
Figure 10
Figure 10 shows the HPGPC chromatogram with regard to the conjugation reaction
product according to Example 4.1 where the content of the mixture regarding reaction byproducts
such as non-reacted oxo-HES and free N-hydroxysuccinirnide as well as solvent
was downgraded using a 10 kD ultrafiltration membrane in a cooling centrifuge.
The following parameters were used in the HPGPC analysis:
Column: Superose 12 HR 10/30 300 x 10 mm I.D. (Pharmacia)
Eluent: 27.38 mM Na2HPO4; 12.62 mM NaH2PO4; 0.2 M NaCl; 0,005 % NaN3 in 11
of demineralized water
Flux: 0,24 ml/h
Detector 1: MALLS detector
Detector 2: UV (280 nm)
Detector 3: RI (Refractive latex, detector)
A represents the result from detector 1, B represents the result from detector 2.
Figure 11
The following parameters were used in the HPGPC analysis:
Column: Superose 12 HR 10/30 300 x 10 mm I.D. (Pharmacia)
Eluent: 27.38 mM Na2HPO4; 12.62 mM NaH2PO4; 0.2 M NaCl; 0,005 % NaN3 in 11
of demineralized water
Flux: 0,24 ml/h
Detector 1: MALLS detector
Detector 2: UV (280 nm)
Detector 3: RI (Refractive Index detector)
A represents the result from detector 1, B represents the result from detector 2.
Figure 12
The following parameters were used in the HPGPC analysis:
Column: Superose 12 HR 10/30 300 x 10 mm I.D. (Pharmacia)
Eluent: 27.38 mM Na2HPO4; 12.62 mM NaH2PO4; 0.2 M NaCl; 0,005 % NaN3 in 11
of demineralized water
Flux: 0,24 ml/h
Detector 1: MALLS detector
Detector 2: UV (280 nm)
Detector 3: RI (Refractive index detector)
A represents the result from detector 1, B represents the result from detector 2.
Figure 13
Figure 13 shows the SDS-PAGE analysis of the flow-through and the eluate of HESmodified
G-CSF (A32) after chromatography on DEAE-Sepharose CL-6B. 1.5% of the
indicated fractions were desalted by ultrafiltration, dried in a SpeedVac and were applied
onto a 12.5% polyacrylamide gel.
Figure 14
Figure 14 shows a MALDFTOF spectrum of the G-CSF starting material (sample A33)
Figure 15
Figure 15 shows a MALDI/TOF spectrum of HES-modified G-CSF (sample A32)
Figure 16
Figure 16 shows a MALDI/TOF spectrum of HES-modified G-CSF (sample A60)
Figure 17
Figure 17 shows the gel elctrophoresis of the reaction mixtures of example 7.2(b).
For gel electrophoresis a XCell Sure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and
a Consort E143 power supply (CONSORTnv, Turnhout, B) were employed. A 12% Bis-
Tris gel together with a MOPS SDS running buffer at reducing conditions (both Invitrogen
GmbH, Karlsruhe, D) were used according to the manufactures instruction. The gel was
stained with Roti-Blue (Carl Roth GmbH + Co.KG, Karlsruhe, D) according to the
manufacturer's instruction.
Lane A: Protein marker Roti-Mark STANDARD (Carl Roth GmbH + Co.KG,
Karlsruhe, D) Molecular weight marker from top to bottom: 200 KD, 119 KD,
66 KD, 43 KD, 29 KD, 20 KD, 14.3 KD
Lane B: Crude product after conjugation of hG-CSF with the HES derivative prepared
in example 7. l(d)
Lane C: Grade product after conjugation of hG-CSF with the HES derivative prepared
in example 7. l(b)
Lane D: Crude product after conjugation of hG-CSF with the HES derivative prepared
in example 7. l(j)
Lane E: Reaction control: HES 50/07
Figure 18
Figure 18 shows the gel electrophoresis of the reaction mixtures of example 7.2(d). For gel
electrophoresis a XCell Sure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and a
Consort El43 power supply (CONSORTnv, Turnhout, B) were employed. A 12% Bis-Tris
gel together with a MOPS SDS running buffer at reducing conditions (both Invitrogen
GmbH, Karlsruhe, D) were used according to the manufactures instruction. The gel was
stained with Roti-Blue (Carl Roth GmbH + Co.KG, Karlsruhe, D) according to the
manufacturer's instruction.
Lane A: Protem marker Rod-Mark STANDARD (Cad Roth GmbH + Co.KG, Karlsruhe,
D) Molecular weight marker from top to bottom: 200 KD, 119 KD, 66 KD, 43
KD, 29 KD, 20 KD, 14.3 KD.
Lane B: hG-CSF after buffer exchange as described in Example 7.2(c).
Lane C: Crude product after conjugation of hG-CSF with the HES derivative prepared as
described in Example 7.1(f).
Lane D: Crude product after conjugation of hG-CSF with the HES derivative prepared as
described in Example 7.1(h).
Figure 19
Figure 19 shows the HPGPC chromatogram with regard to the conjugation reaction
product according to Example 7.3 (MALLS detector: upper chart; UV detector: lower
chart). The following parameters were used in the HPGPC analysis:
Column: Superose 12 HR10/30 300 x 10 mm ID. (Pharmacia)
Eluent: 27.38 mM Na2HPO4; 12.62 mM NaH2PO4; 0.2 M NaCl; 0,005 % NaN3 in 11
of demineralized water
Flux: 0,24 ml/h
Detector 1: MALLS detector
Detector 2: UV (280 nm)
Detector 3: RI (Refractive Index detector)
Figure 20
Figure 20 shows the results of the mitogenicity assay of example 7.4. The Y axis indicates
number of NFS-60-Cells/ml and the X-axis the concentration in pg/ml.
Figure 21
Figure 21 shows the results of the in vivo assay of example 7.5.
Examples
Example 1: Synthesis of aldehyde functionalized hydroxyethyl starch
Example 1.1 (a): Synthesis by periodate oxidation of hydroxyethyl starch
selectively oxidized at its reducing end and incubation at 0 °C
100 mg of Oxo-HESlO/0.4 (MW = 10 kD, DS = 0.4, prepared by Supramol Parenteral
Colloids GmbH, Rosbach-Rodheim, D; according to DE 196 28 705 Al, were dissolved in
5 ml 20 mM sodium phosphate buffer, pH 7.2 and cooled to 0 °C. 21.4 mg sodium
periodate (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) were dissolved in 5 ml
of the same buffer and cooled to 0°C. Both solutions were mixed and after incubation for
10 min at 0°C, 0.73 ml glycerol were added and the reaction mixture was incubated at
21°C for 10 min. The reaction mixture was dialysed for 24 h against water (SnakeSkin
dialysis tubing, 3.5 kD cut off, Perbio Sciences Deutschland GmbH, Bonn, D) and
lyophilized.
Example l.l(b) Synthesis by periodate oxidation of hydroxyethyl starch
selectively oxidized at its reducing end and incubation at 21 °C
100 mg of Oxo-HES 10/0.4 (MW = 10 kD, DS = 0.4, prepared by Supramol Parenteral
Colloids GmbH, Rosbach-Rodheim, D; according to DE 196 28 705 Al) were dissolved in
5 ml 20 mM sodium phosphate buffer, pH 7.2. 21.4 mg sodium periodate (Fluka, Sigma-
Aldrich Chemie GmbH, Taufkirchen, D) were dissolved in 5 ml of the same buffer. Both
solutions were mixed and after incubation for 10 min at 21 °C 0.73 ml glycerol were added
and the reaction mixture was incubated at 21°C for 10 min. The reaction mixture was
dialysed for 24 h against water (SnakeSkin dialysis tubing, 3.5 kD cut off, Perbio Sciences
Deutschland GmbH, Bonn, D) and lyophilized.
Example 1.2(a): Synthesis of aldehyde functionalized hydroxyethyl starch by
periodate oxidation of hydroxyethyl starch with non-oxidized
reducing end and incubation at 0 °C
100 mg of HES 10/0.4 (MW - 10 kD, DS = 0.4, Supramol Parenteral Colloids GmbH,
Rosbach-Rodheim, D) were dissolved in 5 ml 20 mM sodium phosphate buffer, pH 7.2 and
cooled to 0 °C. 21.4 mg sodium periodate (Fluka, Sigma-Aldrich Chemie GmbH,
Taufkirchen, D) were dissolved in 5 ml of the same buffer and cooled to 0°C. Both
solutions were mixed and after incubation for 10 min at 0°C 0.73 ml glycerol were added
and the reaction mixture was incubated at 21°C for 10 min. The reaction mixture was
dialysed for 24 h against water (SnakeSkin dialysis tubing, 3.5 kD cut off, Perbio Sciences
Deutschland GmbH, Bonn, D) and lyophilized.
Example 1.2(b): Synthesis of aldehyde functionalized hydroxyethyl starch by
periodate oxidation of hydroxyethyl starch with non-oxidized
reducing end and incubation at 21 °C
100 mg of HES 10/0.4 (MW = 10 kD, DS = 0.4, prepared by Supramol Parenteral Colloids
GmbH, Rosbach-Rodheim, D) were dissolved in 5 ml 20 mM sodium phosphate buffer, pH
7.2. 21.4 mg sodium periodate (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D)
were dissolved in 5 ml of the same buffer. Both solutions were mixed and after incubation
for 10 min at 21 °C 0.73 ml glycerol were added and the reaction mixture was incubated at
21 °C for 10 min. The reaction mixture was dialysed for 24 h against water (SnakeSkin
dialysis tubing, 3.5 kD cut off, Perbio Sciences Deutschland GmbH, Bonn, D) and
lyophilized.
Example 1.3: Synthesis of aldehyde functionalized hydroxyethyl starch from
amino functionalized hydroxyethyl starch and formylbenzoic acid
Oxo-HESlO/0.4 (MW = 10 kD, DS = 0.4) was prepared by Supramol Parenteral Colloids
GmbH, Rosbach-Rodheim, D; according to DE 196 28 705 Al.
5.1 g (0.51 mmol) of oxo-HES10/0.4 were dissolved in 15 ml anhydrous dimethyl
sulfoxide (DMSO, Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D)) and added
dropwise under nitrogen to a solution of 5.1 ml (51 mmol) 1,4-diaminobutane in 10 ml
anhydrous dimethyl sulfoxide and stirred at 40 °C for 19 h. The reaction mixture was
added to a mixture of 80 ml ethanol and 80 ml acetone. The resulting precipitate was
separated by centrifugation, washed with a mixture of 20 ml ethanol and 20 ml acetone and
re-dissolved in 80 ml water. The solution was dialyzed for 4 days against water (SnakeSkin
dialysis tubing, 3.5 kD cut off, Perbio Science Deutschland GmbH, Bonn, D) and
subsequently lyophilized. The yield was 67 % (3.4 g) amino-HESlO/0.4.
150 mg 4-formylbenzoic acid and 230 mg 1-hydroxy-lH-benzotriazole (both Aldrich,
Sigma-Aldrich Chemie GmbH, Taufkirchen, D) were dissolved in 10 ml N,Ndimethylformamide
(Peptide synthesis grade, Biosolve, Valkenswaard, NL) and 204 ul
N,N'-diisopropylcarbodiimide were added. After incubation at 21 °C for 30 min, 1 g of the
amino-HES 10/0.4 were added. After shaking for 19 h at 22 °C, the reaction mixture was
added to 84 mL of an ice-cold 1:1 mixture of acetone and ethanol (v/v). The precipitated
product was collected by centrifugation at 4 °C, re-dissolved in 50 m water, diarysed for 2
d against water (SnakeSkin dialysis tubing, 3.5 kD cut off, Perbio Sciences Deutschland
GmbH, Bonn, D) and lyophilized.
Example 1.4: Synthesis of aldehyde functionalized hydroxyethyl starch from
hydroxyethyl starch and formylbenzoic acid
Oxo-HES 10/0.7 (MW = 10 kD, DS = 0.7) was prepared by Supramol Parenteral Colloids
GmbH, Rosbach-Rodheim, D; according to DE 196 28 705 Al.
83 mg of 4-formylbenzoic acid and 180 mg 1-hydroxy-lH-benzotriazole (both Aldrich,
Sigma-Aldrich Chemie GmbH, Taufkirchen, D) were dissolved in 5 mL N,Ndimethylformamide
(DMF, Peptide synthesis grade, Biosolve, Valkenswaard, NL) and 78
ul N,N'-diisopropylcarbodiimide were added. After incubation at 21 °C for 30 min, 0.5 g
of oxo-HES 10/0.7 were added. After shaking for 19 h at 22 °C, the reaction mixture was
added to 37.5 ml of an ice-cold 1:1 mixture of acetone and ethanol (v/v). The precipitated
product was collected by centrifugation at 4 °C, re-dissolved in a mixture of 2.5 ml water
and 2.5 ml DMF and precipitated again as described above. The reaction product was
collected by centrifugation as described, re-dissolved in 10 ml water, dialysed for 2 d
against water (SnakeSkin dialysis tubing, 3.5 kD cut off, Perbio Sciences Deutschland
GmbH, Bonn, D) and lyophilized.
Example 1.5: Synthesis of aldehyde functionalized hydroxyethyl starch from
hydroxyethyl starch and formylbenzoic acid
HES10/0.7 (MW = 10 kD, DS = 0.7) was prepared by Supramol Parenteral Colloids
GmbH, Rosbach-Rodheim, D.
50 mg 4-formylbenzoic acid and 108 mg 1-hydroxy-lH-benzotriazole (both Aldrich,
Sigma-Aldrich Chemie GmbH, Taufkirchen, D) were dissolved in 3 ml N,Ndimethylformamide
(Peptide synthesis grade, Biosolve, Valkenswaard, NL) and 47 ul
N,N'-diisopropylcarbodiimide were added. After incubation at 21 °C for 30 min, 0.3 g of
HES 10/0.7 were added. After shaking for 19 h at 22 °C, the reaction mixture was added to
23 ml of an ice-cold 1:1 mixture of acetone and ethanol (v/v). The precipitated product was
collected by centrifugation at 4 °C, re-dissolved in a mixture of 1.5 ml water and 1.5 ml
DMF and precipitated again as described above. The reaction product was collected by
centrifugation as described, re-dissolved in 10 ml water, dialysed for 2 d against water
(SnakeSkin dialysis tubing, 3.5 kD cut off, Perbio Sciences Deutschland GmbH, Bonn, D)
and lyophilized.
Example 1.6: Synthesis of aldehyde functionalized hydroxyethyl starch from
amino functionalized hydroxyethyl starch and formylbenzoic acid
pentafluorophenyl ester
Oxo-HESlO/0.7 (MW = 10 kD, DS = 0.7) was prepared by Supramol Parenteral Colloids
GmbH, Rosbach-Rodheim, D; according to DE 196 28 705 Al.
6.0 g (0.6 mmol) of oxo-HES 10/0.7 were dissolved in 20 ml anhydrous dimethyl sulfoxide
(DMSO, Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D)) and added dropwise
under nitrogen to a solution of 6 ml (60 mmol) 1,4-diaminobutane in 11 ml anhydrous
dimethyl sulfoxide and stirred at 40 °C for 19 h. The reaction mixture was added to a
mixture of 80 ml ethanol and 80 ml acetone. The resulting precipitate was separated by
centrifugation, washed with a mixture of 20 ml ethanol and 20 ml acetone and re-dissolved
in 80 ml water. The solution was dialyzed for 4 days against water (SnakeSkin dialysis
tubing, 3.5 kD cut off, Perbio Science Deutschland GmbH, Bonn, D) and subsequently
lyophilized. The yield was 52 % (3.15 g) amino-HES 10/0.7.
4-formylbenzoic acid pentafluorophenyl ester was synthesized as described in J. S.
Lindsey at al., Tetrahedron 50 (1994) pp. 8941-68, especially p. 8956. 50 mg of amino-
HES 10/0.7 were dissolved in 0.5 ml N,N-dimethylformamide (Peptide synthesis grade,
Biosolve, Valkenswaard, NL) and 15.3 mg 4-formylbenzoic acid pentafluorophenylester
were added. After shaking for 22 h at 22 °C, the reaction mixture was added to 3.5 ml of
ice-cold 2-propanol. The precipitated product was collected by centrifugation at 4 °C,
washed with 4 ml ice-cold 2-propanol, re-dissolved in 50 ml water, dialysed for 2 d against
water (SnakeSkin dialysis tubing, 3.5 kD cut off, Perbio Sciences Deutschland GmbH,
Bonn, D) and lyophilized.
Example 1.7: Synthesis of aldehyde functionalized hydroxyethyl starch from
hydroxyethyl starch and formylbenzoic acid pentafluorophenyl ester
Oxo-HESlO/0.7 (MW = 10 kD, DS = 0.7) was prepared by Supramol Parenteral Colloids
GmbH, Rosbach-Rodheiin, D; according to DE 1% 28 705 AL
4-formylbenzoic acid pentafluorophenyl ester was synthesized as described in J. S.
Lindsey at al., Tetrahedron 50 (1994) pp. 8941-68, especially p. 8956. 200 mg oxo-
HES10/0.7 were dissolved in 2 ml N^N-dimethylfonnamide (Peptide synthesis grade,
Biosolve, Valkenswaard, NL) and 61.2 mg 4-formylbenzoic acid pentafluorophenyl ester
were added. After shaking for 22 h at 22 °C, the reaction mixture was added to 15 mL of
ice-cold 1:1 mixture of acetone and ethanol (v/v). The precipitated product was collected
by centrifugation at 4 °C, re-dissolved in a mixture of 1.4 ml water and 0.7 ml DMF and
precipitated again as described above. The reaction product was collected by centrifugation
as described, re-dissolved hi 10 ml water, dialysed for 2 d against water (SnakeSkin
dialysis tubing, 3.5 kD cut off, Perbio Sciences Deutschland GmbH, Bonn, D) and
lyophilized.
Example 1.8: Synthesis of aldehyde functionalized hydroxyethyl starch from
amino functionalized hydroxyethyl starch and 4-(4-formyl-3,5-
dimethoxyphenoxy)butyric acid
Oxo-HES 10/0.4 (MW = 10 kD, DS = 0.4) was prepared by Supramol Parenteral Colloids
GmbH, Rosbach-Rodheim, D; according to DE 196 28 705 Al.
5.1 g (0.51 mmol) of oxo-HES10/0.4 were dissolved in 15 ml anhydrous dimethyl
sulfoxide (DMSO, Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D)) and added
drop wise under nitrogen to a solution of 5.1 ml (51 mmol) 1,4-diaminobutane in 10 ml
anhydrous dimethyl sulfoxide and stirred at 40 °C for 19 h. The reaction mixture was
added to a mixture of 80 ml ethanol and 80 ml acetone. The resulting precipitate was
separated by centrifugation, washed with a mixture of 20 ml ethanol and 20 ml acetone and
re-dissolved in 80 ml water. The solution was dialyzed for 4 days against water (SnakeSkin
dialysis tubing, 3.5 kD cut off, Perbio Science Deutschland GmbH, Bonn, D) and
subsequently lyophilized. The yield was 67 % (3.4 g) amino-HES 10/0.4.
80.5 mg 4-(4-formyl-3,5-dimethoxyphenoxy)butyric acid (Calbiochem-Novabiochem,
Laufelfingen, CH) and 61 mg 1-hydroxy-lH-benzotriazole (Aldrich, Sigma-Aldrich
Chemie GmbH, Taufkirchen, D) were dissolved in 3 ml N,N-dimethylformamide (Peptide
synthesis grade, Biosolve, Valkenswaard, NL) and 45.4 ul N^'-diisopropylcarbodiimide
were added. After incubation at 21 °C for 30 min, 0.3 g of amino-HES 10/0.4 were added.
After shaking for 22 h at 22 °C, the reaction mixture was added to 23 ml of ice-cold 1:1
mixture of acetone and ethanol (v/v). The precipitated product was collected by
centrifugation at 4 °C, re-dissolved in a mixture of 2 ml water and 1 ml DMF and
precipitated again as described above. The reaction product was collected by centrifugation
as described, re-dissolved in 10 ml water, dialysed for 1 d against water (SnakeSkin
dialysis tubing, 3.5 kD cut off, Perbio Sciences Deutschland GmbH, Bonn, D) and
lyophilized.
Example 2: Synthesis of G-CSF conjugates by reductive animation '
Example 2.l(a): Synthesis of G-CSF-conjugates by reductive amination with
hydroxyethyl starch with non-oxidized reducing end at pH = 7.4
(Comparative Example)
In Example 2.1, it was tried to use the synthesis method of WO 03/074087 (example 12,
page 22 - 23) for the production of a HES-G-CSF conjugate.
To 3.33 \i\ of an aqueous solution of G-CSF (Neupogen® from Amgen, Miinchen, D, or
Granocyte® from Aventis Pharma AG, Zurich, CH, respectively, 3 mg/ml) in 0.1 M
sodium phosphate buffer with pH 7.4, 3.33 ul of a solution of HES10/0.4 (MW = 10 kD,
DS = 0.4, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D, 79 mg/ml) in the
same buffer were added. To this mixture, 3.33 fal of a 60 mM solution of sodium
cyanoborohydride in the same buffer was added, and the resulting mixture was incubated
for 4 h at 22 °C. Subsequently, another 3.33 ul of the freshly prepared 60 mM sodium
cyanoborohydride solution were added. During the incubation time of 30 h, altogether 5
portions of 3.33 ul of a freshly prepared 60 mM sodium cyanoborohydride solution were
added. The reaction mixture was analysed by gel electrophoresis. No reaction was
observed.
Example 2.1(b): Synthesis of G-CSF-conjugates by reductive animation with
bydroxyethyl starch with non-oxidized reducing end at a pH of from
5.0 to 9.2
(Comparative Example)
To 3.33 uL of an aqueous solution of G-CSF (G-CSF from Strathmann Biotec AG,
Hamburg, D, 3 mg/mL) in a given buffer, 3.33 ul of a HES solution (300 mg/ml) in the
same buffer were added. The mixture was cooled to 4 °C, and 3.33 pi of a 60 mM solution
of sodium cyanoborohydride in the same buffer at 4 °C were added, and the resulting
mixture was incubated for 20 h at 4 °C.
The following HES preparations and buffer were employed:
a) Buffer 0.1 M sodium acetate buffer pH 5.0
HES10/0.4 (MW = 10 kD, DS = 0.4, Supramol Parenteral Colloids GmbH, Rosbach-
Rodheim, D)
HES 10/0.7 (MW = 10 kD, DS = 0.7, Supramol Parenteral Colloids GmbH, Rosbach-
Rodheim, D)
HES50/0.4 (MW - 50 kD, DS = 0.4, Supramol Parenteral Colloids GmbH, Rosbach-
Rodheim, D)
HES50/0.7 (MW = 50 kD, DS = 0.7, Supramol Parenteral Colloids GmbH, Rosbach-
Rodheim, D)
b) Buffer: 0.1 M sodium phosphate buffer pH 7.2
HES 10/0.7 (MW = 10 kD, DS = 0.7, Supramol Parenteral Colloids GmbH, Rosbach-
Rodheim, D)
c) Buffer: 0.1 M sodium borate buffer pH 8.3
HES 10/0.7 (MW = 10 kD, DS = 0.7, Supramol Parenteral Colloids GmbH, Rosbach-
Rodheim, D)
d) Buffer: 0.2 M potassium borate buffer pH 9.2
HES 10/0.7 (MW = 10 kD, DS = 0.7, Supramol Parenteral Colloids GmbH, Rosbach-
Rodheim, D)
Each reaction mixture was analysed by gel electrophoresis. No or negligible conjugation
was observed (gel scans for reactions b) to d) not shown).
Example 2.2: Synthesis of G-CSF-conjugates by reductive animation with
hydroxyethyl starch with oxidized reducing end at a pH of 5.0 from
9.2
(Comparative Example)
To 3.33 uL of an aqueous solution of G-CSF (G-CSF from Strathmann Biotec AG,
Hamburg, D, 3 mg/ml) in a given buffer, 3.33 ul of a solution of oxo-HES (300 mg/ml) in
the same buffer were added. The mixture was cooled to 4 °C, and 3.33 ul of a 60 mM
solution of sodium cyanoborohydride in the same buffer at 4 °C were added, and the
mixture was incubated for 17 h at 4 °C.
The following HES preparations and buffer were employed:
a) Buffer 0.1 M sodium acetate buffer pH 5.0
oxo-HESlO/0.7 (MW = 10 kD, DS = 0.7, Supramol Parenteral Colloids GmbH,
Rosbach-Rodheim, D)
oxo-HESSO/0.4 (MW = 50 kD, DS = 0.4, Supramol Parenteral Colloids GmbH,
Rosbach-Rodheim, D)
oxo-HES50/0.7 (MW = 50 kD, DS = 0.7, Supramol Parenteral Colloids GmbH,
Rosbach-Rodheim, D)
b) Buffer: 0.1 M sodium phosphate buffer pH 7.2
HES10/0.7 (MW = 10 kD, DS = 0.7, Supramol Parenteral Colloids GmbH, Rosbach-
Rodheim, D)
c) Buffer: 0.1 M sodium borate buffer pH 8.3
HES10/0.7 (MW = 10 kD, DS - 0.7, Supramol Parenteral Colloids GmbH, Rosbach-
Rodheim, D)
d) Buffer: 0.2 M potassium borate buffer pH 9.2
HES10/0.7 (MW = 10 kD, DS = 0.7, Supramol Parenteral Colloids GmbH, Rosbach-
Rodheim, D)
Each reaction mixture was analysed by gel electrophoresis. No or negligible conjugation
was observed (gel scans for reactions b) to d) not shown).
Oxidation of HES 10/0.4 (MW = 10 kD, DS = 0.4) was carried out by Supramol Parenteral
Colloids GmbH, Rosbach-Rodheim, D; according to DE 196 28 705 Al.
Example 2.3: Synthesis of G-CSF-conjugates by reductive amination with aldehyde
functionalized hydroxyethyl starch synthesized by periodate
oxidation
To 3.33 ul of an aqueous solution of G-CSF (Granocyte® from Aventis Pharma AG,
Zurich, CH, and Neupogen® from Amgen, Munchen, D, respectively, 3 mg/mL) in 0.1 M
sodium acetate buffer pH 5.0, 3.33 ul of a solution of an aldehydo-HES (79 mg/mL) hi the
same buffer were added. To the mixture 3.33 uL of a 60 mM solution of sodium
cyanoborohydride in the same buffer were added and the mixture was incubated for 25 h at
21 °C. The reaction mixture was analysed by gel electrophoresis.
The following aldehyde functionalized HES conjugates were employed:
(i-N) prepared with Neupogen® according to Example 1.1 (a) hereinabove;
(ii-N) prepared with Neupogen® according to Example 1.1 (b) hereinabove;
(iii-N) prepared with Neupogen® according to Example 1.2(a) hereinabove;
(iv-N) prepared with Neupogen® according to Example 1.2(b) hereinabove;
(i-G) prepared with Granocyte® according to Example 1.1 (a) hereinabove;
(ii-G) prepared with Granocyte® according to Example 1.1 (b) hereinabove;
(iii-G) prepared with Granocyte® according to Example 1.2(a) hereinabove;
(iv-G) prepared with Granocyte® according to Example 1.2(b) hereinabove.
Example 2.4: Synthesis of G-CSF-conjugates by reductive amination with aldehyde
functionalized hydroxyethyl starch synthesized by conjugation of
hydroxyethyl starch to a formyl-carboxylic acid
To 3.33 ul of an aqueous solution of G-CSF (G-CSF from Strathmann Biotec AG,
Hamburg, D, 3 mg/ml) in 0.1 M sodium acetate buffer pH 5.0, 3.33 ul of a solution of an
aldehydo-HES (118.5 mg/mL) in the same buffer were added and cooled to 4 °C. To the
mixture 3.33 ul of a 60 mM solution of sodium cyanoborohydride in the same buffer at 4
°C were added and the mixture was incubated for 17 h at 4 °C. The reaction mixture was
analysed by gel electrophoresis.
The following aldehyde functionalized HES conjugates were employed:
(v) prepared according to Example 1.4 hereinabove;
(vi) prepared according to Example 1.5 hereinabove;
(vii) prepared according to Example 1.6 hereinabove;
(viii) prepared according to Example 1.7 hereinabove;
(ix) prepared according to Example 1.8 hereinabove.
Example 2.5: Synthesis of G-CSF-conjugates by reductive animation with aldehyde
functionalized hydroxy ethyl starch synthesized by conjugation of
hydroxyethyl starch to formyl-carboxylic acid
To 2.5 ml of an aqueous solution of G-CSF (G-CSF from Strathmann Biotec AG,
Hamburg, D, 2.27 mg/ml) in 0.1 M sodium acetate buffer pH 5.0, 136 nag aldehydo-
HES 10/0.4, prepared as described in Example 1.3 hereinabove, were added, and the
solution was cooled to 0 °C. To the mixture 2.5 ml of an ice-cold 40 mM solution of
sodium cyanoborohydride in the same buffer were added and the mixture was incubated
for 17 h at 4 °C. The reaction mixture was analysed by gel electrophoresis.
Example 3: Synthesis of G-CSF conjugate* by SH alkylation
Oxo-HES 10/0.7 (MW = 10 kD, DS = 0.7) was prepared by Supramol Parenteral Colloids
GmbH, Rosbach-Rodheim, D; according to DE 196 28 705 Al.
6.0 g (0.6 mmol) of oxo-HES 10/0.7 were dissolved in 20 ml anhydrous dimethyl sulfoxide
(DMSO, Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D)) and added dropwise
under nitrogen to a solution of 6 ml (60 mmol) 1,4-diaminobutane in 11 ml anhydrous
dimethyl sulfoxide and stirred at 40 °C for 19 h. The reaction mixture was added to a
mixture of 80 ml ethanol and 80 ml acetone. The resulting precipitate was separated by
centrifugation, washed with a mixture of 20 ml ethanol and 20 ml acetone and re-dissolved
in 80 ml water. The solution was dialyzed for 4 days against water (SnakeSkin dialysis
tubing, 3.5 kD cut off, Perbio Science Deutschland GmbH, Bonn, D) and subsequently
lyophilized. The yield was 52 % (3.15 g) amino-HES 10/0.7.
To 132 |ig amino-HES 10/0.7, dissolved in 100 ul sodium phosphate buffer (0.1 M, 0.15 M
NaCl, 50 mM EDTA, pH 7.2), 10 ul of a solution of 17.5 mg/ml Nalpha(
maleimidoacetoxy)succinimide ester (AMAS) in dry DMSO (both Fluka, SigmaAldrich
Chemie GmbH, Taufkirchen, D) were added, and the clear solution was incubated
for 80 min at 25 °C and subsequently for 20 min at 40 °C. The excess of AMAS was
removed by centrifugal filtration with a VTVASPIN 0.5 ml concentrator, 5KD MWCO
(VTVASCIENCE, Hannover, D) at 13,000 rpm, washed 2 times for 30 min with 450 ul of
the phosphate buffer and once with 450 ul of a buffer B. To the residual solution, 10 u,g GCSF
(Neupogen® from Amgen, Munchen, D, and Granocyte® from Aventis Pharma AG,
Zurich, CH, respectively, 3 u,g/ul hi phosphate buffer) were added, and the mixture was
incubated for 16h at 25 °C. The reaction mixture was analysed by gel electrophoresis after
concentration in vacuo.
The following methods were chosen:
(x) G-CSF (Granocyte®) employing sodium phosphate buffer (0.1 M, 0.15 M NaCl, 50
mM EDTA, pH 7.2) as buffer B.
(xi) G-CSF (Neupogeo®) employing sodium phosphate buffer (0.1 M, 0.15 M NaCl, 50
mM EDTA, pH 7.2) as buffer B.
(xii) G-CSF (Granocyte®) employing a 1 : 1 (v/v) mixture of sodium phosphate buffer
(0.1 M, 0.15 M NaCl, 50 mM EDTA, pH 7.2) and 8 M urea, 1 % SDS, pH 7.4 as
buffer B.
(xiii) G-CSF (Neupogen®) employing a 1 : 1 (v/v) mixture of sodium phosphate buffer
(0.1 M, 0.15 M NaCl, 50 mM EDTA, pH 7.2) and 8 M urea, 1 % SDS, pH 7.4 as
buffer B.
(xiv) G-CSF (Granocyte®) employing 8 M urea, 1 % SDS, pH 7.4 as buffer B.
(xv) G-CSF (Neupogen®) employing 8 M urea, 1 % SDS, pH 7.4 as buffer B.
Example 4: Synthesis of G-CSF conjugates by reaction of hydroxyethyl
starch having a reactive ester group with G-CSF
Example 4.1:
Oxo-HES 10/0.4 (MW = 10,559 D, DS = 0.4) was prepared by Supramol Parenteral
Colloids GmbH, Rosbach-Rodheim, D; according to DE 196 28 705 Al. The degree of
oxidation of oxo-HES was 95 %
66 mg of oxo-HES 10/0.4 were dissolved in 0.5 ml anhydrous DMF. To this solution, 3.4
mg of N,N'-disuccinimidyl carbonate were added, and the mixture was stirred for 2 h at
room temperature. The resulting solution had a reactive HES concentration of 13 percent
by weight.
A solution of G-CSF (Strathmann Biotec AG, Hamburg, D), having a concentration of
about 0.5 mg G-CSF/ml, was concentrated to a concentration of 10 rag/ml by
ultracentrifugation at a cut-off of 10 kD using a cooling centrifuge.
To 0.5 ml of this concentrated G-CSF solution, 180 ul of a sodium bicarbonate solution
were added. Subsequently, 3 portions (100 ul each) of the reactive HES solution were
added dropwise to the protein solution, until, after about 30 min., the reaction had come to
an end. Thus, the overall molar ratio of reactive HES : G-CSF was 20 : 1. Then, the pH of
the mixture was adjusted to 4.0 using 0.1 N HC1.
A HPGPC analysis (High-Performance Gel Permeation Chromatography) gave a yield of
about 70 %. This result is shown in Fig. 9.
The mixture could be stored at 4 °C at a pH of 4.0 for 4 d and, according to HPGPC
analyses, remained stable, i.e. unchanged.
Downgrading the content of the mixture regarding reaction by-products such as nonreacted
oxo-HES and free N-hydroxysuccinimide as well as solvent using a 10 kD
ultrafiltration membrane in a cooling centrifuge was possible without difficulties. The
results of this downgrading experiment is shown in Fig. 10.
Example 4.2:
Oxo-HES 10/0.4 (MW = 10,559 D, DS = 0.4) was prepared by Supramol Parenteral
Colloids GmbH, Rosbach-Rodheim, D; according to DE 196 28 705 Al. The degree of
oxidation of oxo-HES was 95 %
400 mg of oxo-HES 10/0.4 were dissolved in 1 ml anhydrous DMF. To this solution, 21 mg
of N,N'-disuccinimidyl carbonate were added, and the mixture was stirred for 2 h at room
temperature. The resulting solution had a reactive HES concentration of 40 percent by
weight.
A solution of G-CSF (Strathmann Biotec AG, Hamburg, D), having a concentration of
about 0.5 mg G-CSF/ml, was concentrated to a concentration of 10 mg/ml by
ultracentrifugation at a cut-off of 10 kD using a cooling centrifuge.
To 0.5 ml of this concentrated G-CSF solution, 180 ul of a sodium bicarbonate solution
were added. Subsequently, 3 portions (100 ul each) of the reactive HES solution were
added dropwise to the protein solution, until, after about 30 min., the reaction had come to
an end. Thus, the overall molar ratio of reactive HES : G-CSF was about 50 : 1. Then, the
pH of the mixture was adjusted to 4.0 using 0.1 N HC1.
A HPGPC analysis (High-Performance Gel Permeation Chromatography) gave a yield of
more than 95 %. No non-reacted G-CSF could be detected. This result is shown hi Fig. 11.
The mixture was purified using uhrafihration technology without difficulties. The results
of thes downgrading is shown in Fig. 12.
Examples
5.1. Purification
Purified G-CSF having essentially the same characteristics as the commercial product
Neupogen ® (Amgen) was obtained and one aliquot was kept unmodified as a control.
5.2 Synthesis of conjugates of HES and G-CSF
Conjugates were synthesized essentially as described in Example 4.2, but with Oxo-
HES50/0.7 (sample code A60), or as described in Example 2.5 (sample code A32), and
used for further buffer exchange and purification.
5.3 Buffer exchange of G-CSF and HES-modified G-CSF samples before purification
by anion-exchange chromatography
HES-modified G-CSF samples or unmodified G-CSF (as a control) (0.5 - 5 mg protein )
were subjected to buffer exchange using Vivaspin 6 Concentrator units ( 10.000 MWCO
PES, Vivascience, Cat. Nr. VS0602). Samples were concentrated to 0.5-0.7 ml and were
diluted to 5 ml with 10 mM Na-phosphate buffer pH 7.2. Each sample was subjected 3
times to this concentration/buffer exchange cycle.
5.4 Anion exchange chromatography of G-CSF and HES-modified forms thereof on a
DEAE-Sepharose column
G-CSF samples after HES-modification and, for comparison, samples of unmodified GCSF
were purified and analyzed by anion exchange chromatography at room temperature
by using an AKTA explorer 10 system as described. Aliquots of G-CSF either before or
after HESylation were dialyzed by ultrafiltration against said buffer A (10 mM Naphosphate,
pH 7.2) or were diluted with about 13 volumes of buffer A. The column
containing 2 ml DEAE-Sepharose (DEAE-Sepharose CL-6B, Pharmacia Kat. Nr. 17-0710-
01) was regenerated by applying 5.0 column volumes (CV) of 6.5 M guanidine/HCl, 5.0
CV buffer A, 5.0 CV of buffer C (1.5 M NaCl in 10 mM Na-phosphate, pH 7.2) and then
10 CV of buffer A. The samples (0.8 - 4.5 ml in 10 mM Na-phosphate buffer pH 7.2)
were then injected by using a flow rate of 0.6 ml/min. Following washing of the sample
loop with 10 ml (2 ml sample loop) or 20 ml (5 ml sample loop) buffer A, depending on
the sample applied, the column was further washed with 0-22 CV of buffer A (flow rate =
0.8 ml/min). Elution was performed by applying a linear gradient from 0-100% buffer B
over 5 CV and an isocratic run with 2.5 CV of 100% buffer B using a flow rate of 0.6
ml/min. The column was re-equilibrated with 5 CV of buffer A and was regenerated as
detailed above by using a flow rate of 1 ml/min.
If required, samples were concentrated using a Vivaspin concentrator and buffer exchange
was performed as described above. Samples were stored at 0-8°C in 10 mM Na-Acetat
buffer pH 4.0 before or after sterile filtration using a 0.2 ^m filtrations unit, Coming, Cat.
No. 431215). The following samples were prepared for in-vitro bioassays and for further
analytical analysis. Protein concentration was determined as described in section 6.1
below:
I. 0401-15/A33, 0.44 mg/ml, volume = 500 ^il
G-CSF (E.coli)
II. 0402-03/A60, 0.35 mg/ml, volume = 600 ul
H-G-CSF (G-CSF HES modified, 10/0.4)
HI. 0401-13/A32, 0.28 mg/ml, volume = 900 ul
G-CSF (Ecoli) HES modified, 10/0.4
IV. 0401-28/A58, 0.60 mg/ml, volume = 350 ul
Neupogen
V. 0401-28/A57, 0.50 mg/ml, volume = 400 ul
Neulasta
5.5. Further analysis of G-CSF samples
Aliquots of the samples were analyzed for their protein content and for modifications,
5.5(a) G-CSF protein quantitation by RP-HPLC
G-CSF protein content of the samples was quantitated using the unmodified protein
preparation (concentration : 0.453 mg/ml) as a standard.
A Dionex HPLC system consisting of a pump P 680 A HPG, degassing unit Degasys DG
1210, an autosainpler and injector ASI-100, a sample loop 250 ul, a thermostatted column
department TCC 100 along with a W/Vis-Detektor UVD170U equipped with a Software
Chromeleon Chromatography Management System was used. A precolumn CC 8/4
Nucleosil 120-5 C4, Macherey-Nagel, Cat No. 721889, and a separation column 40 O4
Nucleosil MPN, 5 urn, 125 x 4 mm RP-cohram (Macherey-Nagel, ordering No.7200
45.40) were used. Solvent A was KfeO phis 0,06 % (v/v) trifluoroacetic acid and solvent B
was 90 % acetonhrile inHbO, containing 0,06 % (v/v)trifhK>roacetic acid; flow rate was : 1
ml/min. UV detection was at 214,221,260 and at 280 nm wavelength.
Samples of approximately 10 - 20 ug were injected into a RP-HPLC column. The
following gradient was used:
0-5 min: 0-10 %B
- 17 min: 10-45 %B
- 35 min: 45-80 %B
- 36 min: 80-100 %B
- 38 min: 100 %B
- 39 min: 10 %B
- 45 min: 10 %B
The resulting peak area at the elution position of the standard G-CSF preparation was used
and compared to the reference standard by comparing the peak appearing at around 29min
at 280 nm wavelength.
5.5(b) Reduction + carboxamidomethylation of G-CSF protein
Aliquots from the G-CSF protein samples were reduced and carboxamidomethylated as
described elsewhere (Guillermina Fomo, Mariela Bollati Fogolin, Marcos Oggero,
Ricardo Kratje, Marina Etcheverrigaray, Harald S. Conradt, Manfred Nimtz (2004) N- and
0-linked carbohydrates and glycosylation site occupancy in recombinant human
granulocyte-macrophage colony-stimulating factor secreted by a Chinese hamster ovary
cell line; European J. Biochem, 273(5), 907-919). Carboxamidomethylation leads to
modified cystein residues. Endoproteinase Glu-C digestion of the carboxamidomethylated
protein was performed in 25 mM NItiHCOa containing 1 M urea at pH 7,8 and using an
enzyme /substrate ratio of 0.2:10 for 18 - 24 hours.
5.5(c) Separation of Endo-Glu-C peptides by RP-HPLC
The peptides generated by the Endo-Glu-C digestion were separated on a Dionex HPLC
system consisting of a pump P 680 A HPG, degassing unit Degasys DG 1210, an
autosampler and injector ASI-100, a sample loop 250 ul, a thermostatted column
(Jepartment TCC 100 along with a UV/Vis-Detektor UVD170U equipped with a Software
Chromeleon Chromatography Management System was used. A precolumn CC 8/4
Nucleosil 120-5 C4, Macherey-Nagel, Cat. No. 721889, and a separation column 40 C-4
Nucleosil MPN, 5 urn, 125 x 4 mm RP-cohann (Macherey-Nagel, ordering No.7200
45.40) were used. Solvent A was H2O plus 0,06 % (v/v) trifluoroacetic acid and solvent B
was 90 % acetonitrile inHzO, containing 0,06 % (v/v)trifluoroacetic acid; flow rate was : 1
ml/min. The following gradient was applied:
0-5 min: 10%B
- 17 min: 45 %B
- 65 min: 100 %B
- 67 min: 100 %B
- 69 min: 10 %B
- 75 min: 10 %B
UV detection was at 214 ,221, 260 and at 280 nm wavelength. Peptides generated by the
Endo-Glu-C digestion were separated (data not shown).
5.5(d) Analysis of proteolyric peptides by Matrix-Assisted Laser Desorption/
lonization Time-of-Flight Mass Spectrometry (MALDI/TOF/TOF-MS)
Mass spectrometry was used to detect the intact N-terminus of G-CSF's hi the different
samples prepared. Samples (3-5 ug) resulting from Endoproteinase Glu-C digestions of
reduced and carboxamidomethylated protein samples were used directly for MS-analysis
(without the RP-HPLC of step 6.3) and purified using ZipTip pipette tips containing
Cl 8 reversed-phase material according to the manufacturer's instructions. After washing
with 0.1%(v/v) formic acid, elution of peptides was performed with 10 ul 0.1% (v/v)
formic acid in 60% (v/v) acetonitrile.
Proteolytic (Endo-Glu-C ) peptide fragments were analyzed with a Bruker ULTRAFLEX
time-of-flight (TOF/TOF) instrument in the linear positive ion mode using a matrix of
22.4 mg 3,5-dimethoxy-4-hydroxy-cinnamic acid hi 400 ul acetonitrile and 600 ul 0.1%
(v/v) trifluoroacetic acid in tbO; (glyco)-peptides were measured using a matrix of 19 mg
a-cyano-4-hydroxycinnamic acid hi the same solvent mixture using the reflectron for
enhanced resolution. Sample solutions of 1 ul and an approximate concentration of 1-10
pmol-ul"1 were mixed with equal amounts of the respective matrix. This mixture was
spotted onto a stainless steel target and dried at room temperature before analysis. Spectra
were recorded in the mass range 900 - 5000 dalton. The following Table correlates the
expected masses with the respective G-CSF peptides.
Table: Theoretical (monoisotopic] masses of Endo-Glu-C peptides resulting from (Table Removed)
Cystein residues were carboxamidomethylated; peptides marked as fat were detected in
MALDI/TOF spectrum of the non modified G-CSF.
The N-terminal Endo-Glu-C peptide (MTPLGPASSLPQSFLLKCLE; m/z 2189.1) comprising
position 1 -20 of the protein was detected in MALDI/TOF-MS spectra of samples
after proteolytic treatment of G-CSF with endoproteinase Glu-C as described above.
5.6. Results
5.6(a) Purification of G-CSF and HES modified variants
A32, A60 and non modified G-CSF were subjected to purification using a DEAESepharose
CL-6B column as described under A4.
In the case of the unmodified sample 0401-15/A33, no significant absorption at 280 nm
was detected in the flow-through and the protein eluted at a concentration of 40-50%
buffer B (0.16-020 M NaCl) in a volume of 6 ml, with a specific peak area of 660 mAU x
ml x mg"1 at 280 nm.
The sample 0401-14/A32 (derived from 0401-15/A33; HESylation with AldehydoHES
10/0.4) eluted over a large range of the gradient at a concentration of buffer B from 20-
80% (0.08-0.32 M NaCl) in a volume of 12 ml. About 90% of the total peak area detected
at 280 nm was found in the flow-through, containing about 50% of the total protein with an
apparently slightly higher molecular mass when compared to the eluted protein, as detected
by SDS-PAGE analysis as shown in Figure 13 above.
The sample 0402-03/A60 (HESylated with HES 10/0.4, following the overall procedure of
Example 4.2) eluted in a volume of 10.5 ml at a similar concentration of 20-80% buffer B.
In this case, about 35% of the total peak area detected at 280 nm was found in the flowthrough,
however, by SDS-PAGE analysis, no unbound protein was detected in this
fraction. When compared to the specific peak area of sample 0401-15/A33, the protein
content in the eluate of the sample 0402-03/A60 was 45% higher than the stated protein
amount that was applied to the column.
Recovery of proteins was calculated based on the peak area (A280 nm) of the eluting
fractions compared to the non modified G-CSF protein.
Table 1: Comparison of the peak areas at 280 nm detection
(Table Removed)
** RP-HPLC quantitation of the protein confirmed these results
5.6(b) Analysis of proteins by peptide mapping and MALDI/TOF MS
after treatment with endoproteinase Glu-C
The N-terminal peptide resulting from endoproteinase Glu-C digestion of both the carboxamidomethylated
unmodified G-CSF (Figure 14) and fee market product Neupogen (data
not shown), was clearly detected by MALDI/TOF-MS (MFPLGPASSLPQSFLLKC*LE,
m/z 2189.1; cystein carboxamidomethylated). This signal was absent in samples subjected
to HES-modification by reductive animation (Figure 15) and in Neulasta (data not shown),
indicating modification of mis peptide. In the case of HES-modified G-CSF, wherein
modification was carried out by activated ester chemistry, the N-terminal peptide was
detected at a relative signal intensity comparable to that of the non modified starting
material A32 (Figure 16) indicating that HES modification of this derivatives has been
achieved at different amino acid side chains.
N-terminal sequencing of HES modified G-CSF (sample A3 3 and the market product
Neulasta) revealed a blocked N-terminus suggesting that in fact the N-terminal methionine
residue of this protein derivative is modified by HES derivative. Since the signal
corresponding to the peptide comprising amino acid residues pos.35-47
(KLCATYKLCHPEE; both cysteine residues carboxamidomethylated m/z 1648.78) was
not detected in sample A60, it is concluded that one or both lysine residues (at pos 35 and
pos 41) might be modified by HES.
References:
Guillermina Forno, Mariela Bollati Fogolin, Marcos Oggero, Ricardo Kratje.,
Marina Etcheverrigaray, Harald S. Conradt, Manfred Nimtz (2004) N- and 0-linked
carbohydrates and glycosylation site occupancy in recombinant human granulocytemacrophage
colony-stimulating factor secreted by a Chinese hamster ovary cell line
Eur. J. Biochem, 271 (5), 907-919
Nimtz, M., Grabenhorst, E., Conradt, H.S., Sanz, L. & Calvete, J.J. (1999) Structural
characterization of the oligosaccharide chains of native and crystallized boar seminal
plasma spermadhesin PSP-I and PSP-II glycoforms. Eur. J. Biochem. 265, 703-718.
Nimtz, M., Martin, W., Wray, V., KBppel, K.-D., Agustin, J. & Conradt, H.S. (1993)
Structures of sialylated oligosaccharides of human erythropoietin expressed in recombinant
BHK-21 cells. Eur J. Biochem. 213,39-56
Nimtz, M., Noll G., Paques, E. & Conradt, H.S. (1990) Carbohydrate structures of human
tissue plasminogen activator variant expressed in recombinant Chinese hamster ovary
cells. FEBSLetL 271,14-18.
Schroter, S., Den, P., Conradt, H.S., Nimtz, M., Hale, G. & Kirchhoff, C. (1999) Malespecific
modification of human CD52. J. Biol Chem. 274,29862-29873
E.Grabenhorst and H.S.Conradt (1999)The Cytoplasmic, Transmembrane and the Stem
Regions of Glycosyhransferases specify their in vivo functional sublocalization and
stability in the Golgi
J. BioL Chem., 274,36107-36116
E. Grabenhorst, A.Hoffmann, M.Nimtz, G. ZettlmeiBl and H. S.Conradt (1995)
Construction of stable BHK-21 cells coexpressing human secretory glycoproteins and
human Galfil-4GlcNAc-R D2,6-sialyltransferase: D2,6-linkedNeuAc is preferably
attached to the Gal61-4GlcNAcfll-2ManB 1-3-branch of biantennary oligosaccharides from
secreted recombinant.-trace protein.
Eur.J.Biochem, 232. 718-725
Example 6: In vitro results of the G-CSF-conjugate obtained in Examples
2.5 and 4.2 and purified according to Example 5: Mitogenicity
of G-CSF variants for mouse NFS-60 cells
G-CSF is known for its specific effects on the proliferation, differentiation, an activation of
hematopoietic cells of the neutrophilic granulocyte lineage. The mitogenic capacity of GCSF
variants was tested using mouse NFS-60 cells (N. Shirafuji et al., Exp. Hematol.
1989, 17, 116-119). Cells grown in RPMI medium with 10% fetal calf serum (Gibco
INVITROGEN GmbH, Karlsruhe, D) containing 5-10 % WEHI-3B (DSMZ,
Braunschweig, D; cultivated as described by the DSMZ) conditioned medium as source of
exogenous
IL-3 were harvested by centrifugation, washed and aliquoted at 100,000 cells
per well in a 24-well plate. Cells were allowed to adapt for 1 hour at 37 °C in RPMI
medium without WEHI-3B conditioned media before G-CSF growth factors sample
diluted in the same media were added. NFS-60 cells were exposed to purified G-CSF
variants for 3 days at 37°C and than the cells were electronically counted (Casy TT Cell
Counter, Scharfe System, Reutlingen, D). The results are summarised in Figure 12. As
seen in Figure 12, the different G-CSF variants (0.5 - 50 pg/ml) were able to stimulate an
increase hi the number of cells after 3 days compared to medium that did not contain added
growth factors.
Unmodified control proteins G-CSF/A33 and G-CSF/A58 stimulated cells at a very similar
extend (ED50=5 - 10 pg/ml) while G-CSF conjugates G-CSF/A60 G-CSF/A32 and GCSF/
A57 showed only a minor decrease in activity if compared to the unmodified version
(ED50=10-25pg/ml).
(see Figure 8)
Example 7 Synthesis of G-CSF-Conjugates
Example 7.1. Synthesis of the Aldehydo-HES Derivatives
Example 7.1(a) Synthesis of AminoHESlO/0.4
5.12 g of oxo HES10/0.4 (MW = 10000 D, DS = 0.4, Supramol Parenteral Colloids GmbH,
Rosbach-Rodheim, D according to DE 196 28 705 Al) were heated over night at 80°C in
vacuo and dissolved under nitrogen in 25 mL dry dimethyl sulphoxide (Fluka, Sigma-
Aldrich Chemie GmbH, Taufkirchen, D) and 5.13 mL of 1,4-diaminobutane were added.
After stirring at 40°C for 17 h the reaction mixture was added to 150 mL of an ice-cold 1:1
mixture of acetone and ethanol (v/v). The precipitated product was collected by
centrifugation at 4°C, washed with 40 mL of an ice-cold 1:1 mixture of acetone and
ethanol (v/v) and collected by centrifugation. The crude product was dissolved in 80 mL
water, dialysed for 4 d against water (SnakeSkin dialysis tubing, 3.5 kD cut off, Perbio
Sciences Deutschland GmbH, Bonn, D) and lyophilized. The yield of isolated product was
67%.
Example 7.1(b) Synthesis of AldehydoHES10/0.4
105 mg 4-formylbenzoic acid and 135 mg 1-hydroxy-lH-benzotriazole (both Aldrich,
Sigma-Aldrich Chemie GmbH, Taufkirchen, D) were dissolved in 7 mL N,Ndimethylformamide
(Peptide synthesis grade, Biosolve, Valkenswaard, NL) and 135 uL
N,N'~diisopropylcarbodiimide (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D)
were added. After incubation at 21°C for 30 min, 0.7 g of aminoHES10/0.4 (synthesised as
described in 1.1) were added. After shaking for 18 h at 22°C, the reaction mixture was
added to 42 mL of an ice-cold 1:1 mixture of acetone and ethanol (v/v). The precipitated
product was collected by centrifugation at 4°C, re-dissolved in 5 mL DMF and precipitated
with 42 mL ethanol/ acetone as described above. After centrifugation, the collected
precipitate was dissolved with water, dialysed for 1 d against water (SnakeSkin dialysis
tubing, 3.5 kD cut off, Perbio Sciences Deutschland GmbH, Bonn, D) and lyophilized. The
yield of isolated product was 95%.
Example 7.1(c) Synthesis of AminoHESlO/0.7
6.02 g of oxo-HES 10/0.7 (MW = 10000 D, DS = 0.7, Supramol Parenteral Colloids
GmbH, Rosbach-Rodheim, D, according to DE196 28 705) were dissolved under nitrogen
in 32 mL dry dimethyl sulphoxide (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D)
and 6.03 mL of 1,4-diaminobutane were added. After stirring at 40°C for 17 h the reaction
mixture was added to 150 mL of an ice-cold 1:1 mixture of acetone and ethanol (v/v). The
precipitated product was collected by centrifugation at 4°C, washed with 40 mL of an icecold
1:1 mixture of acetone and ethanol (v/v) and collected by centrifugation. The crude
product was dissolved in 80 mL water, dialysed for 4 d against water (SnakeSkin dialysis
tubing, 3.5 kD cut off, Perbio Sciences Deutschland GmbH, Bonn, D) and lyophilized. The
yield of isolated product was 52%.
Example 7.1 (d) Synthesis of AldehydoHES10/0.7
150 mg 4-formylbenzoic acid and 230 mg 1-hydroxy-lH-benzotriazole (both Aldrich,
Sigma-Aldrich Chemie GmbH, Taufkirchen, D) were dissolved in 10 mL N,Ndimethylformamide
(Peptide synthesis grade, Biosolve, Valkenswaard, NL) and 204 uL
N,N'-diisopropylcarbodiimide (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D)
were added. After incubation at 21°C for 30 min, 1 g of aminoHES10/0.7 (synthesised as
described in 1.3) were added. After shaking for 19 h at 22°C, the reaction mixture was
added to 84 mL of ice-cold 2-propanol. The precipitated product was collected by
centrifugation at 4°C, re-dissolved in 50 mL water, dialysed for 2 d against water
(SnakeSkin dialysis tubing, 3.5 kD cut off, Perbio Sciences Deutschland GmbH, Bonn, D)
and lyophilized. The yield of isolated product was 83%.
Example 7.l(e) Synthesis of ArainoHES30/0.4
5 g of oxo-HES 30/0.4 (MW = 30000 D, DS - 0.4, Supramol Parenteral Colloids GmbH,
Rosbach-Rodheim, D, using molar ratios of the ingredients according to DE 196 28 705
Al) were heated over night at 80°C in vacuo and were then dissolved under nitrogen in 28
mL dry dimethyl sulphoxide (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) and
1.67 mL of 1,4-diaminobutane were added. After stirring at 40°C for 17 h the reaction
mixture was added to 175 mL of an ice-cold 1:1 mixture of acetone and ethanol (v/v). The
precipitated product was collected by centrifugation at 4°C. The crude product was
dissolved in 40 mL water, dialysed for 2 d against water (SnakeSkin dialysis tubing, 3.5
kD cut off, Perbio Sciences Deutschland GmbH, Bonn, D) and lyophilized. The yield of
isolated product was not determined.
Example 7.1(f) Synthesis of AldehydoHES30/0.4
130 mg 4-fonnylbenzoic acid and 153 mg 1-hydroxy-lH-benzotriazole (both Aldrich,
Sigma-Aldrich Chemie GmbH, Taufkirchen, D) were dissolved in 36 mL N,Ndimethylformamide
(Peptide synthesis grade, Biosolve, Valkenswaard, NL) and 110 uL
N,N'-diisopropylcarbodiimide (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D)
were added. After incubation at 21°C for 30 min, 2.61 g of aminoHES30/0.4 (synthesised
as described in 1.5) were added. After shaking for 22.5 h at 22°C, the reaction mixture was
added to 160 mL of an ice-cold 1:1 mixture of acetone and ethanol (v/v). The precipitated
product was collected by centrifugation at 4°C and washed with an ice-cold 1:1 mixture of
acetone and ethanol (v/v). After centrifugation, the precipitate was dissolved in 30 mL
water, dialysed for 1 d against water (SnakeSkin dialysis tubing, 3.5 kD cut off, Perbio
Sciences Deutschland GmbH, Bonn, D) and lyophilized. The yield of isolated product was
81%.
Example 7.1(g) Synthesis of AminoHES30/0.7
5 g of oxo-HES 30/0.7 (MW = 30000 D, DS =* 0.7, Supramol Parenteral Colloids GmbH,
Rosbach-Rodheim, D, using molar ratios of the ingredient according to DE 196 28 705 Al)
were heated over night at 80°C in vacuo and were then dissolved under nitrogen in 28 mL
dry dimethyl sulphoxide (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) and 1.67
mL of 1,4-diaminobutane were added. After stirring at 40°C for 17 h the reaction mixture
was added to 175 mL of an ice-cold 1:1 mixture of acetone and ethanol (v/v). The
precipitated product was collected by centrifugation at 4°C. The crude product was
dissolved in 40 mL water, dialysed for 2 d against water (SnakeSkin dialysis tubing, 3.5
kD cut off, Perbio Sciences Deutschland GmbH, Bonn, D) and lyopbilized. The yield of
isolated product was not determined.
Example 7.1(h) Synthesis of AldehydoHES30/0.7
122 mg 4-formylbenzoic acid and 144 mg 1-hydroxy-lH-benzotriazole (both Aldrich,
Sigma-Aldrich Chemie GmbH, Taufkirchen, D) were dissolved in 34 mL N,Ndimethylformamide
(Peptide synthesis grade, Biosolve, Valkenswaard, NL) and 103 uL
N,N'-diisopropylcarbodiimide (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D)
were added. After incubation at 21°C for 30 min, 2.46 g of aminoHES30/0.7 (synthesised
as described in 1.7) were added. After shaking for 22.5 h at 22°C, the reaction mixture was
added to 160 mL of an ice-cold 1:1 mixture of acetone and ethanol (v/v). The precipitated
product was collected by centrifugation at 4°C and washed with an ice-cold 1:1 mixture of
acetone and ethanol (v/v). After centrirugation, the precipitate was dissolved in 30 mL
water, dialysed for 1 d against water (SnakeSkin dialysis tubing, 3.5 kD cut off, Perbio
Sciences Deutschland GmbH, Bonn, D) and lyophilized. The yield of isolated product was
87%.
Example 7.1(i) Synthesis of AminoHES50/0.7
6.09 g of oxo-HES 50/0.7 (MW = 50000 D, DS = 0.7, Supramol Parenteral Colloids
GmbH, Rosbach-Rodheim, D, using molar rations of the ingredients according to DE 196
28 705 Al) were heated over night at 80°C in vacuo and were then dissolved under
nitrogen in 32 mL dry dimethyl sulphoxide (Fluka, Sigma-Aldrich Chemie GmbH,
Taufkirchen, D) and 1.22 mL of 1,4-diaminobutane were added. After stirring at 40°C for
17 h the reaction mixture was added to 150 mL of an ice-cold 1:1 mixture of acetone and
ethanol (v/v). The precipitated product was collected by centrifugation at 4°C, washed with
40 mL of an ice-cold 1:1 mixture of acetone and ethanol (v/v) and collected by
centrifugation. The crude product was dissolved in 80 mL water, dialysed for 4 d against
water (SnakeSkin dialysis tubing, 3.5 kD cut off, Perbio Sciences Deutschland GmbH,
Bonn, D) and lyophilized. The yield of isolated product was 82%.
Example 7.1(j) Synthesis of AldehydoHES50/0.7
125 mg 4-formylbenzoic acid and 174 mg 1-hydroxy-lH-benzotriazole (both Aldrich,
Sigma-Aldrich Chemie GmbH, Taufkirchen, D) were dissolved in 38 mL N,Ndimethylformaraide
(Peptide synthesis grade, Biosolve, Valkenswaard, NL) and 155 uL
N^N'-diisopropylcarbodiimide (Fluka, Sigma-Aldrich Chemie GmbH, Taufltirchen, D)
were added. After incubation at 21°C for 30 min, 3.8 g of aminoHES50/0.7 (synthesised as
described in 1.9) were added. After shaking for 19 h at 22°C, the reaction mixture was
added to 160 mL of an ice-cold 1:1 mixture of acetone and ethanol (v/v). The precipitated
product was collected by centrifugation at 4°C, re-dissolved in 20 mL N,Ndimethylformamide
and precipitated with 80 mL of an ice-cold 1:1 mixture of acetone and
ethanol (v/v) as described above. After centrifugation, the precipitate was dissolved in 50
mL water, dialysed for 2 d against water (SnakeSkin dialysis tubing, 3.5 kD cut off, Perbio
Sciences Deutschland GmbH, Bonn, D) and lyophilized. The yield of isolated product was
77%.
Example 7.2 Synthesis of the HES-G-CSF Conjugates by reductive animation
Example 7J2(a) Buffer exchange A:
33 mL of a 0.454 mgAnL solution of hG-CSF (XM02, BioGeneriX AG, Mannheim, D) in
10 mM sodium acetate, 50 mg/mL sorbitol and 0.004% Tween 80 at pH 4.0 were
concentrated by diafiltration at 0°C to 4 mL with a Vivaspin 15R concentrator
(VS15RH11,5KD MWCO, Vivascience AG, Hannover, D) and re-diluted to 15 mL with a
0.1 M sodium acetate buffer at pH 5.0. This diafiltration was repeated twice. The final
concentration in the last diafiltration step was 3 mg/mL.
Example 7.2(b) Reaction of hG-CSF with aldehydoHES derivatives of examples
7.1(b), 7.1(d) and 7.10)
To 1.67 mL of a solution of hG-CSF after buffer exchange into 0.1 M sodium acetate
buffer, pH 5.0 (as described in 7.2(a) above) 1.67 mL of a solution of the HES-derivative
and 1.67 mL of a 60 mM solution of sodium cyanoborohydride, both in the same buffer,
were added and the solution was incubated for 15.5 h at 4°C. All solutions were cooled to
0°C before mixing.
The following final HES concentrations were employed:
39.4 mg/mL for the HES derivatives prepared according to example 7.1(b) and 7.1(d).
197 mg/mL for the HES derivative prepared according to example 7.1(j).
197 mg/mL HES50/0.7 (MW = 50000 D, DS = 0.7, Supramol Parenteral Colloids GmbH,
Rosbach-Rodheim, D) as reaction control.
The reaction mixtures were analysed by gel electrophoresis (see figure 17)
Example 7.2(c) Buffer exchange B:
20 mL of a 0.454 mg/mL solution of hG-CSF (XM02, BioGeneriX AG, Mannheim, D) in
10 mM sodium acetate, 50 mg/mL sorbitol and 0.004% Tween 80 at pH 4.0 was
concentrated by diafiltration at 15°C to 4 mL with a Vivaspin 15R concentrator
(VS15RH11, 5KD MWCO, Vivascience AG, Hannover, D) and re-diluted to 15 mL with a
0.1 M sodium acetate buffer at pH 5.0. This diafiltration was repeated twice. The final
concentration in the last diafiltration step was 1.5 mg/mL.
Example 7.2(d) Reaction of hG-CSF with aldehydoHES derivatives of examples
7.1(f) and 7.1(h)
To 3.3 mL of a solution of hG-CSF after buffer exchange into 0.1 M sodium acetate buffer,
pH 5.0 (as described in example 7.2(c) above) 3.3 mL of a solution of 789 mg of the HESderivative
and 3.3 mL of a 60 mM solution of sodium cyanoborohydride, both in the same
buffer, were added and the solution was incubated for 30 h at 4°C. All solutions were
cooled to 0°C before mixing.
After 17 h samples were removed for reaction control. The reaction mixtures were
analysed by gel electrophoresis (see figure 18).
Example 7.3 Synthesis of HES-GCFS conjugates by NN'-succinimidyl
carbonate coupling
Example 7.3(a) Synthesis of G-CSF conjugates by reaction of hydroxyethyl
starch having a reactive ester group with G-CSF
400 mg of oxo-HES 10/0.7 (prepared by Supramol Parenteral Colloids GmbH, Rosbach-
Rodheim, D; according to DE 196 28 705 Al, degree of oxidation of oxo-HES was 95 %)
were dissolved in 1 ml anhydrous DMF. To this solution, 21 mg of N,N'-disuccmimidyl
carbonate were added, and the mixture was stirred for 2 h at room temperature. The
resulting solution had a reactive HES concentration of 40 percent by weight.
A solution of G-CSF (Strathmann Biotec AG, Hamburg, D), having a concentration of
about 0.5 mg G-CSF/ml, was concentrated to a concentration of 10 mg/ml by
ultracentrifugation at a cut-off of 100 kD using a cooling centrifuge.
To 0.5 ml of this concentrated G-CSF solution, 180 ul of a sodium bicarbonate solution
were added. Subsequently, 3 portions (100 ul each) of the reactive HES solution were
added dropwise to the protein solution, until, after about 30 min., the reaction had come to
an end. Thus, the overall molar ratio of reactive HES : G-CSF was about 50 : 1. Then, the
pH of the mixture was adjusted to 4.0 using 0.1 N HC1.
A HPGPC analysis (High-Performance Gel Permeation Chromatography) gave a yield of
more than 95 %. No non-reacted G-CSF could be detected. This result is shown in Fig. 19.
Example 7.4 In vitro assay
Mitogenicity of G-CSF variants for mouse NFS-60 cells
G-CSF is known for its specific effects on the proliferation, differentiation and activation
of hematopoietic cells of the neutrophflic granulocyte lineage. The mitogenic capacity of
G-CSF variants was tested using mouse NFS-60 cells (N. Shirafuji et al., Exp. Hematol.
1989, 17, 116-119). Cells grown in RPM medium with 10% fetal calf serum (Gibco
INVrrROGEN GmbH, Karlsruhe, D) containing 5-10% WEHI-3B (DSMZ,
Braunschweig, D; cultivated as described by the DSMZ) conditioned medium as source of
exogenous IL-3 were harvested by centrifugation, washed and aliquoted at 100,000 cells
per well in a 24-well plate. Cells were allowed to adapt for 1 hour at 37°C in RPMI
medium without WEHI-3B conditioned media before G-CSF growth factor samples
diluted in the same media were added. NFS-60 cells were exposed to purified G-CSF
variants (purification according to examples 5.3, 5.4, protein quantification according to
example 5.5(a)):
Neupogen®, Neulasta® both from Amgen,
"HES-GCFS 10/0.4 conjugate" prepared in example 7.2(b),
"HES-GCFS 10/0.7 conjugate" prepared in example 7.2(b),
"HES-GCFS30/0.4 conjugate" prepared in example 7.2(d),
"HES-GCFS30/0.7 conjugate" prepared in example 7.2(d),
"HES-GCFS50/0.7 conjugate" prepared in example 7.2(b)5
"HES-GCFS 10/0.7 conjugate (Supramol)" prepared according to example 7.3(a),
"Mock incubation"( = reaction control, 197mg/ml HES50/0.7, MW 50000D, DS 7,
Supramol Parenteral Colloids GmbH, Rosbach Rodheim, Germany),
for 3 days at 37°C and than the cells were electronically counted (Casy TT Cell Counter,
Scharfe System, Reutlingen, D). The results are summarised in Table 2 and Figure 20. In
all cases, the amounts of protein given in Table 2 and Figure 20 represent the G-CSF
content of the conjugates only and are based on the concentrations determined by
GlycoThera. As can be seen in Figure 20, all of the different G-CSF variants (2.5 - 250
pg/ml) were able to stimulate an increase in the number of cells after 3 days compared to a
medium that did not contain added growth factors. All variants reached the same maximum
stimulation level at a concentration of 250 pg/ml.
Table 2: Proliferation of mouse NFS-60 cells, induced by G-CSF variants
(Table Removed)
Example 7.5 in vivo biological effects of hG-CSF conjugates in rats
Upon arrival, the rats [male CRL:CD® rats (7 weeks old), Charles River Deutschland
GmbH, Sanghofer Weg 7, D-97633 Sulzfeld)] were randomly sorted into groups of 5.
After 7 days acclimatization, rats in poor condition were excluded and replaced by spare
animals. The weight of the rats upon arrival was 181-203 g.
Each group of five randomly selected rats was then intravenously administered 100 ug
protein per kg body weight (injection speed 15 sec/dosis, vehicle: 5ml PBS/kg
bodyweight) of the following non-conjugated or conjugated G-CSF samples (purified
according to examples 5.3, 5.4, protein quantification according to example 5.5(a)):
Neupogen® and Neulasta®, both from Amgen,
"HES-GCSF10/0.4 conjugate" (10/0.4) prepared in example 7.2(b),
"HES-GCSF10/0.7 conjugate" (10/0.7) prepared in example 7.2(b),
"HES-GCSF30/0.4 conjugate" (30/0.4) prepared in example 7.2(d),
"HES-GCSF30/0.7 conjugate" (30/0.7) prepared in example 7.2(d),
"HES-GCSF50/0.7 conjugate" (50/0.7) prepared in example 7.2(b),
"HES-GCSF 10/0.7 Supramol" (SI0/0.7) prepared according to example 7.3(a)
"Mock incubation"( - reaction control, 197mg/ml HES50/0.7, MW 50000D, DS 7,
Supramol Parenteral Colloids GmbH, Rosbach Rodheim, Germany) and
a vehicle control.
Blood samples from all animals of approx. 200 ul EDTA whole blood were taken from the
retrobulbar venous plexus under light ether anaesthesia. On test day -5 blood was taken
once in the morning from all animals after overnight fasting. On test days 1 to 8 blood was
taken twice daily at an interval of 12 hours. The first blood sample on day 1 was taken prior
to G-CSF/GCSF-conjugate administration.
The pharmaceutical composition comprising a therapeutically effective amount a
conjugate of the present invention is a synergistic composition, exhibits surprising and
unexpected properties.





We Claim:
1. A method for preparing a conjugate comprising a protein and a polymer derivative,
wherein the polymer is a hydroxyalkyl starch (HAS) and the protein is a
granulocyte colony stimulating factor (G-CSF), the method comprising reacting at
least one functional group A of a polymer derivative with at least one functional
group Z of the protein and thereby forming a covalent linkage, wherein Z is an
amino group, and
- whereinA is selected from the group consisting of an aldehyde group, a keto group or a hemiacetal group,
wherein the method comprises introducing A in the polymer to give the
polymer derivative
by reacting the polymer with an at least bifunctional compound, one functional group M of which, comprising the chemical structure -NH-, reacts with the polymer and at least one other functional group Q of which is an aldehyde group, a keto group or a hemiacetal group, or is a functional group which is chemically modified to give an aldehyde group, a keto group or a hemiacetal group.
2. The method as claimed in claim 1 wherein the hydroxyalkyl starch is hydroxyethyl starch.
3. The method as claimed in claim 1, wherein M comprises an amino group.
4. The method as claimed in claim 1, comprising reacting the polymer with a functional group M of the at least bifunctional compound to give the polymer derivative, the at least bifunctional compound comprising at least one other functional group Q which is not an aldehyde group, keto group or hemiacetal group, the method comprising reacting the functional group Q with at least one suitable compound to give the polymer derivative comprising the aldehyde group, keto group or hemiacetal group A.

5. The method as claimed in claim 4, wherein M and Q comprise an amino group.
6. The method as claimed in claim 4 or 5, wherein the at least one suitable compound comprises a carboxy group and an aldehyde group, keto group or hemiacetal group.
7. The method as claimed in claim 6, wherein the at least one suitable compound is formylbenzoic acid or 4-(4-formyl-3,5-dimethoxyphenoxy)butyric acid.
8. The method as claimed in claim 4 or 5 wherein M comprises an amino group and Q comprises a beta hydroxy amino group.
9. The method as claimed in claim 8, wherein the polymer is reacted at its oxidized reducing end with a functional group M of an at least Afunctional compound.
10. The method as claimed in claim 8, comprising oxidizing the beta hydroxyamino group to give the aldehyde group.
11. The method as claimed in claim 10, wherein the oxidation reaction is carried out using a periodate.
12. The method as claimed in any of claims 1 to 11, wherein the reaction of the polymer or the polymer derivative with the protein is a reductive amination.
13. The method as claimed in claim 12, wherein the reductive amination is carried out in the presence of NaCNBH3.
14. The method as claimed in claim 12 or 13, wherein the reductive amination is carried out at a pH of 7 or less.
15. The method as claimed in claim 14, wherein the pH is 6 or less.
16. The method as claimed in any of claims 12 to 15, wherein the reductive amination is carried out at a temperature of from 0 to 25 °C.

17. The method as claimed in any of claims 12 to 16, wherein the reductive amination is carried out in an aqueous medium.
18. A conjugate as obtainable by a method as claimed in any of claims 1 to 17.
19. The conjugate of claim 18, having a structure according to the formula

(Formula Removed)

wherein R1, R2 and R3 are independently hydrogen or a hydroxyalkyl group, a hydroxyaryl group, a hydroxyaralkyl group or a hydroxyalkaryl group having of from 2 to 10 carbon atoms, preferably hydrogen or a hydroxyalkyl group, more preferably hydrogen or a hydroxyethyl group, and
wherein L is an optionally substituted, linear, branched and/or cyclic hydrocarbon residue, optionally comprising at least one heteroatom, having from 1 to 60 preferably from 1 to 40, more preferably from 1 to 20, more preferably from 1 to 10, more preferably from 1 to 6 more preferably from 1 to 2 carbon atoms and especially preferably 1 carbon atom, L being in particular CH2,
wherein X is selected from a group comprising

(Formula Removed)
20. The conjugate as claimed in claim 19, wherein the hydroxyalkyl starch is hydroxyethyl starch.

21. A pharmaceutical composition as and when prepared by using the conjugates as claimed in claims 19 to 20 for the use in a disorder characterized by a reduced hematopoietic or immune function or diseases related thereto.
22. A method for preparing a conjugate comprising a protein and a polymer derivative substantially as herein described with reference to the accompanying drawings and as illustrated in the foregoing examples.

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870-delnp-2006-form-18.pdf

870-DELNP-2006-Form-2-(26-03-2009).pdf

870-delnp-2006-form-2.pdf

870-delnp-2006-form-26.pdf

870-DELNP-2006-Form-3-(26-03-2009).pdf

870-delnp-2006-form-3.pdf

870-delnp-2006-form-5.pdf

870-delnp-2006-gpa.pdf

870-delnp-2006-pct-210.pdf

870-delnp-2006-pct-220.pdf

870-delnp-2006-pct-237.pdf

870-delnp-2006-pct-301.pdf

870-delnp-2006-pct-304.pdf

870-delnp-2006-pct-308.pdf

870-DELNP-2006-Petition-137-(26-03-2009).pdf


Patent Number 234328
Indian Patent Application Number 870/DELNP/2006
PG Journal Number 26/2009
Publication Date 26-Jun-2009
Grant Date 21-May-2009
Date of Filing 20-Feb-2006
Name of Patentee FRESENIUS KABI DEUTSCHLAND GMBH
Applicant Address ELSE-KRONER-STRASSE 1, 61352 BAD HOMBURG V.D.H., GERMANY
Inventors:
# Inventor's Name Inventor's Address
1 FRANK RONALD DR. HEEGBLICK 1,38527 MRINR-HTSDDRL, GERMANY
2 CONFADT, HARALD DR. OKERSTRASSE 11, 38100 BRAUNSCHWEIG, GERMANY
3 GRABENHOTRST, ECKARD PAPPELBERG 57,38104 BRAUNSCHWEIG, GERMANY
4 SOMMERMEYER, KLAUS, DR. IN DER LAUBACH 26,61191 ROSHACH, GERMANY
5 EICHNER, WOLFRAM DR. AN DER LANDWEHR 30,35510 BUTZBACH, GERMANY
6 KNOLLER, HELMUT DR. RITTERSTRASSE 15,61169 FRUDVERG, GERMANY
7 LUTTERBECK, KATHARINA MAINZER TOR ANLAGE 10,61169 FRIEDBERG, GERMANY
8 ZANDER, NORBERT DR. ZELLBERGSHEIDEWEG 45,38527 MEINE, GERMANY
PCT International Classification Number A61K 47/48
PCT International Application Number PCT/EP2004/008818
PCT International Filing date 2004-08-06
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 04005874.5 2004-03-11 U.S.A.
2 60/552,281 2004-03-11 U.S.A.
3 PCT/EP03/08859 2003-08-08 U.S.A.
4 PCT/EP2003/08829 2003-08-08 U.S.A.
5 PCT/EP03/08858 2003-08-08 U.S.A.