DESCRIPTION
TITLE OF THE INVENTION: AMINO SUGAR-CONTAINING GLUCAN, METHOD
FOR PRODUCING SAME, AND USE THEREOF
5
TECHNICAL FIELD
[0001] The present invention relates to a glucan having at
least one (preferably two or more) aminomonosaccharide
residue on each of at least one non-reducing end, a modified
10 product and a conjugate thereof, as well as a method for
producing the same, and utilization of the same. More
preferably, the present invention relates to a glucan having
at least one (preferably two or more) glucosamine residue
on each of at least one non-reducing end, a modified product
15 and a conjugate thereof, as well as a method for producing
the same, and utilization of the same. The glucan, a modified
product and a conjugate thereof of the present invention
can have a function to non-covalently bond to a nucleic acid
to increase the apparent molecular weight of the nucleic
20 acid.
BACKGROUND ART
[0002] A medically effective ingredient of medicaments is
rapidly changing from a chemically synthesized stable
25 low-molecular weight compound to an unstable substance which
is easily degraded in blood, such as a protein, an antibody
and a nucleic acid. For this reason, there is a necessity
of stabilizing these unstable medically effective
ingredients to keep the blood concentration of the medically
30 effective ingredient high for an elongated time. In addition,
in order to decrease side effects of drugs, a necessity of
delivering drugs to a target tissue efficiently has been
increasing. Under such a background, a so-called drug
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delivery system (DDS) technique has been utilized in earnest
(Non-Patent Documents 1 to 4). A DDS technique refers to
a technique and a system for allowing a medically effective
ingredient to act on "a required site" at "a required amount"
for "a required period of time", that is, controlling 5 ng it
with an ideal pharmacokinetics for a drug to maximally exert
the effect.
[0003] In the DDS technique, a modifying material for a
medically effective ingredient is important. The term
10 "modifying material for a medically effective ingredient"
in the present specification refers to a material which
modifies a medically effective ingredient by covalently
binding, or via non-covalent type interaction, with a
medically effective ingredient. By utilizing the modifying
15 material, a variety of properties (for example,
pharmacokinetics (for example, absorption, distribution,
metabolism and excretion), pharmacological effect,
stability and the like) of the medically effective ingredient
can be modified. As a substance which has been used
20 previously as the modifying material for the medically
effective ingredient, there are a variety of substances,
most generally it is a macromolecular material. For example,
polyethylene glycol (PEG) which is a synthetic macromolecule,
and derivatives thereof are widely utilized as a modifying
25 material for medically effective ingredients. Many
medically effective ingredient-modifying materials having
a functional group for binding the medically effective
ingredient on a terminus of a PEG chain have been developed,
and such modifying materials are actually utilized in
30 producing a medicament. Specific application examples
include pegylated interferon 􀁄 (product name: PEGASYS).
Since interferon 􀁄 has a small molecular weight and is easily
excreted into urine, there was a problem that it has a short
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half-life in blood. However, the half-life in blood was
successfully enhanced dramatically by covalently binding
interferon 􀁄 to a PEG chain having a molecular weight of
40,000 to form an interferon 􀁄-conjugate having a high
molecular weight. As described above, the remarkable 5 effect
is recognized in the modification of the medically effective
ingredient or the nanoparticulate carrier for DDSs, with
a macromolecular material.
[0004] However, on the other hand, a problem has been pointed
10 out. For example, when a high-molecular weight synthetic
macromolecule which has no degradability in a living body
and will not undergo renal glomerular filtration is
administered to blood, there is a risk that the macromolecule
is accumulated in a particular organ and a risk that side
15 effects due to the accumulation are generated. The reason
is that a molecule having a molecular weight of a few tens
thousands or less present in blood, undergoes renal
glomerular filtration and is rapidly excreted into urine,
but a molecule having a molecular weight of a few tens thousands
20 or more does not undergo renal glomerular filtration and
its excretion into urine is limited. For this reason, a
modifying material of the medically effective ingredient
which can be safely utilized is expected.
[0005] In addition, when PEG is utilized as a carrier, PEG
25 is usually linked to a medically effective ingredient through
a covalent bond. While a covalent bond is excellent in
stability, since a portion of the structure of the medically
effective ingredient changes, there is a problem that the
physiological activity of the medically effective ingredient
30 changes. In addition, when the medically effective
ingredient has a plurality of functional groups, there is
a problem that groups having different binding position and
binding number of PEG chains are synthesized. Such problems
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become fatal problem in a biomedicine, especially in a
proteinaceous medicament and a peptidic medicament. The
reason is that it is very difficult to produce products of
the same quality reproducibly. Chemical creativity and
5 originality are needed to covalently bind a PEG molecule
to a specific amino acid residue, and there are various
problems that a specific amino acid sequence may be required,
the kind of the medically effective ingredient may be limited,
and the like.
10 [0006] On the other hand, the development of a nucleic acid
medicament such as an siRNA and an RNA aptamer has been
progressing in recent years. Since a DNA and an RNA, which
are components of a nucleic acid medicament, are easily
degraded by DNA degrading enzyme and RNA degrading enzyme,
15 respectively, it is required to prevent degradation using
a suitable carrier. In addition, in the case of a nucleic
acid medicament comprising a low-molecular weight nucleic
acid medically effective ingredient, it is required to bind
it with a macromolecular carrier to extend the blood retention
20 time. Further, it is preferable that these macromolecular
carriers for a nucleic acid can be given a targeting function,
if necessary. A macromolecular carrier which is safe and
has nucleic acid binding property and can solve such problems
has been sought.
25 [0007] Polysaccharides have been used as a food raw material
for a long time and, in recent years, have begun to be paid
attention as a macromolecular material which is environment
friendly, as a safe material having biocompatibility and,
further, as a functional material. The naturally occurring
30 polysaccharides can be classified into neutral
polysaccharides typified by a starch, cellulose, and dextrin;
and acidic polysaccharides typified by alginic acid and
hyaluronic acid. On the other hand, basic polysaccharides
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do not occur naturally. A method for producing basic
polysaccharides artificially includes a method by which
chitin, a naturally derived polysaccharide which is a
homopolymer of N-acetylglucosamine, is chemically
deacetylated. Such 5 a polysaccharide is generally referred
to as chitosan, which is the only basic polysaccharide which
is industrially applicable.
[0008] Chitosan has many amino groups in the molecule, and
strongly binds to a nucleic acid to form a very huge
10 chitosan:nucleic acid associate referred to as polyplex.
There is a problem that such a bond between a polyanion and
a polycation is so strong that the dissociation of them is
difficult.
[0009] Chitosan is produced by a method such as boiling
15 chitin in a concentrated alkali solution to convert the
acetamide group on the carbon at 2-position in a chitin
backbone into a free primary amino group. In this method,
however, chitosan is degraded into molecules having lower
molecular weight by the cleavage of the chitin main chain,
20 and the strict control of a complete deacetylation is
difficult, therefore, a polysaccharide wherein a plurality
of glucosamines and N-acetylglucosamines are randomly
arranged. The number of glucosamines in this polysaccharide
molecule cannot be controlled strictly. In addition, in this
25 method, it is not possible to selectively deacetylate only
the non-reducing ends of this polysaccharide to produce a
polysaccharide wherein glucosamines are bound only to
non-reducing ends. That is, in this method, it is not
possible to control the strength and ease of dissociation
30 of the bond between chitosan and a nucleic acid. In addition,
chitin and chitosan cannot be degraded rapidly in a body.
Therefore, chitin and chitosan, which have molecular weights
not less than the fractionation size of renal glomerular
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filtration, have a risk of accumulating in a body. As
described above, while chitosan, which is the only cationic
polysaccharide, has nucleic acid binding property, it has
a limited utility as a carrier for nucleic acid medicaments.
[0010] An amino sugar-containing glucan wherein 5 amino sugar
residues are bound only to the non-reducing ends has not
been found in nature. Even using a chemical method, the
synthesis of an amino sugar-containing glucan wherein amino
sugar residues are bound only to the non-reducing ends is
10 not possible, and the chemical synthesis of an amino
sugar-containing glucan wherein amino sugar residues are
bound only to the non-reducing ends has not been disclosed.
In addition, a polysaccharide wherein the number of the bound
amino sugar residues can be strictly controlled is not known.
15 [0011] Non-Patent Document 5 discloses that
glucosamine-1-phosphate (GlcN-1-P) can be synthesized by
a chemical reduction reaction of
2-azido-2-deoxy-glucopyranosyl-1-phosphate, and that a
maltooligosaccharide having a glucosamine residue at the
20 non-reducing end is produced by allowing potato-derived
􀁄-glucan phosphorylase to act on a mixture of
glucosamine-1-phosphate and a maltooligosaccharide.
However, Non-Patent Document 5 does not disclose or suggest
applying a similar method to a high-molecular weight glucan
25 and a branched glucan. It does not disclose that the number
of the glucosamine residues in a glucan molecule can be
controlled. Further, it does not disclose or suggest that
controlling the number of the glucosamine residue in a glucan
molecule has an important effect on the strength and form
30 of the bond with a negatively-charged substance such as a
nucleic acid. Non-Patent Document 5 does not suggest or
disclose that a glucan having one glucosamine residue at
the non-reducing end has a binding property to a
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negatively-charged substance such as a nucleic acid, and
that such a glucan has a function to increase the apparent
molecular weight of a negatively-charged substance such as
a nucleic acid. In addition, when glucosamine-containing
glucan 5 is obtained by the method described in Non-Patent
Document 5, it is not possible to separate the
glucosamine-1-phosphate used in the reaction from the product
glucosamine-containing glucan. Since a medicinal material
does not allow the mixing of impurities, a
10 glucosamine-containing glucan containing unremoved
glucosamine-1-phosphate has a critical drawback of
unavailability as a medicament.
[Prior Art Documents]
15 [Non-Patent Documents]
[0012]
[Non-Patent Document 1] Hiroaki Okada, "Drug delivery using
functional DDS carriers" Chapter 1, General Statement:
Kinousei DDS carrier wo mochiita seizai sekkei ni yoru soyaku
20 (Drug Discovery by Preparation Design Using Functional DDS
Carrier), CMC Publishing Co., Ltd., 2008, 1-23
[Non-Patent Document 2] Masayuki Yokoyama, "Polymeric
materials for drug carriers, Special Topic, DDS ni riyou
sareru kobunshi kagaku (Polymer Chemistry Utilized in DDS)",
25 Drug Delivery System 23-6, 2008: 610-617
[Non-Patent Document 3] Maria Laura Immordino et al.,
International Journal of Nanomedicine 2006: 1(3) 297-315
[Non-Patent Document 4] J. Milton Harris and Robert B. Chess,
NATURE REVIEWS, DRUG DISCOVERY, VOLUME 2, MARCH 2003, 214-221
30 [Non-Patent Document 5] Nawaji et al., Carbohydr. Res. 2008,
343, 2692-2696
SUMMARY OF THE INVENTION
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PROBLEMS TO BE SOLVED BY THE INVENTION
[0013] The present invention is intended to solve the
above-mentioned problems.
[0014] An ideal modifying material for the
negatively-charged medically effective ingredient which 5 can
be safely utilized is thought to have the following
characteristics:
(1) The modifying material shall be a macromolecular
material which can be degraded in a living body;
10 (2) The modifying material shall have such a stable
quality that it can be usable as a medicament. That is, the
structure can be specified, and a modifying material having
the same quality shall be able to produce every time;
(3) The modifying material shall be a macromolecular
15 material which can have the arbitrary number of amino groups
which can bind to or interact with a negatively-charged
medically effective ingredient at specific sites in the
molecule.
[0015] A modifying material for a nucleic acid is thought
20 to be preferable to have the following characteristics:
(4) The modifying material shall have a nucleic acid binding
property and have a function to increase the apparent
molecular weight of the nucleic acid.
[0016] The present inventors thought that a macromolecular
25 substance most excellent as the modifying material for the
medically effective ingredient is a glucan (in the present
specification, the glucan refers to an 􀁄-1,4-glucan, and
an 􀁄-1,4-glucan which is branched with an 􀁄-1,6-bond(s)).
Since glycogen or a starch, which is accumulated in an animal's
30 and plant's body as a polysaccharide for storage, is one
kind of glucan, it is a component which is always present
in a body of a human, and is excellent in biocompatibility.
Further, the glucan undergoes hydrolysis by 􀁄-amylase in
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a body, and is converted into glucose or a
maltooligosaccharide being a component which is always
present in a body. For this reason, the glucan can be said
to be the safest macromolecular material.
[0017] The 5 glucan has another advantage that design of
its structure is relatively easy. Methods for controlling
the molecular weight, degree of branching, cyclization and
the like of the glucan are known. Completely linear glucans
in which glucose residues are bound only with an 􀁄-1,4-bond,
10 glucans which are branched at high frequency by some glucose
residues bound with an 􀁄-1,6-bond, and the like are available.
In a branched glucan, every time an 􀁄-1,6-bond is increased,
one new non-reducing end is generated. The number of
􀁄-1,6-bonds can be suitably adjusted according to desire
15 upon synthesis of a glucan molecule.
[0018] On the other hand, the greatest problem when the
glucan is utilized as the modifying material for the medically
effective ingredient is that it does not have a functional
group which can bind to or interact with the medically
20 effective ingredient. It is possible to introduce a cationic
or anionic functional group into a large number of hydroxyl
groups present in a glucan by a chemical reaction. However,
when such a procedure is used, the position of introduction
of the functional group to the glucan is random and it is
25 difficult to obtain a modifying material of the same quality.
Therefore, this procedure is not preferable for utilization
in medicaments.
[0019] For this reason, it is sought to provide a modifying
material suitable for utilizing in medicaments for a
30 medically effective ingredient, especially for a
negatively-charged medically effective ingredient.
MEANS FOR SOLVING THE PROBLEMS
[0020] In order to solve the aforementioned problems, the
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present inventors thought that specific introduction of an
amino sugar (for example, glucosamine) into a non-reducing
end of a high-molecular weight glucan chain is the extremely
ideal method of modifying a glucan. If an amino sugar (for
example, 5 glucosamine) can be selectively introduced into
a non-reducing end of a high-molecular weight glucan, since
the introduction position is not random, the same quality
material can be produced reproductively, and the resulting
product is suitable for utilization in medicaments. In
10 addition, there is no anxiety for toxicity by administering
an amino sugar. The reason is that glucosamine is a
monosaccharide having an amino group, sold as a supplement
or a health food as a single component or as a mixture with
chondroitin (chondroitin sulfate), and has low anxiety for
15 safety. Further, the amino group of an amino sugar (for
example, glucosamine) is available for binding with a
medically effective ingredient. Furthermore, an amino
sugar (for example, glucosamine) becomes a sugar residue
having a positive charge in an aqueous solution. For this
20 reason, by introducing an amino sugar residue into a glucan
chain, it is possible to give a property to interact with
a negatively-charged molecule (for example, a medically
effective ingredient which is negatively charged in an
aqueous solution) to a glucan chain. Example of
25 negatively-charged molecules includes a nucleic acid. A
biodegradable carrier which binds to a molecule negatively
charged in an aqueous solution and has no anxiety for staying
in a body has not been known to date. For this reason, the
glucan of the present invention can be said to be a useful
30 carrier in a bio DDS technique using a nucleic acid which
has been paid attention in recent years.
[0021] The present inventors found that 􀁄-glucan
phosphorylase can utilize as a substrate an amino
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sugar-1-phosphate (for example, glucosamine-1-phosphate)
which is not its original substrate, can transfer an amino
sugar residue to a non-reducing end of a glucan receptor,
and in spite of this, this enzyme can hardly catalyze the
reverse reaction thereof, and completed the present 5 invention
based on this finding. 􀁄-glucan phosphorylase is an enzyme
which, when acts on glucose-1-phosphate which is the original
substrate, catalyzes both a reaction wherein a glucose
residue is transferred to a glucan receptor (glucan
10 synthesizing reaction) and a reaction wherein the glucose
on the non-reducing end of a glucan is phosphorolysed to
generate glucose-1-phosphate (glucan degrading reaction).
However, surprisingly, when this enzyme uses an amino
sugar-1-phosphate (for example, glucosamine-1-phosphate)
15 as a substrate, this enzyme catalyzes the reaction wherein
an amino sugar residue is transferred to a glucan receptor
(glucan synthesizing reaction), but hardly catalyzes the
reaction wherein an amino sugar residue on the non-reducing
end of a glucan is phosphorolysed to generate an amino
20 sugar-1-phosphate (glucan degrading reaction). While
glucan degrading reaction releases again the bound amino
sugar residue, the present inventors found that in the method
of the present invention, 􀁄-glucan phosphorylase hardly
catalyzes the glucan degrading reaction. That is, the
25 catalytic activity for the synthesizing reaction is much
higher than the catalytic activity for the degradation
reaction. Due to this characteristic, if a glucan has a
plurality of non-reducing ends, 􀁄-glucan phosphorylase can
bind amino sugar residues (for example, glucosamine residues)
30 one by one to the plurality of non-reducing ends of the glucan.
By utilizing this characteristic of this enzyme, the amino
sugar-containing glucan of the present invention became
producible for the first time. Especially, it could be
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obtained for the first time by utilizing this characteristic
of this enzyme that a non-reducing end-modified glucan having
high frequency of (for example, 50% or more) binding rate
of amino sugar residues (for example, glucosamine residues)
to the non-reducing ends, starting 5 with a glucan having a
large number of (for example, 5 or more) non-reducing ends.
[0022] In the present specification, an amino sugar means
that it is a sugar having an amino group. The sugar
constructing the backbone monosaccharide of an amino sugar
10 can be a monosaccharide (for example, glucose) or an
oligosaccharide. In the present specification, an
oligosaccharide means a compound wherein 2 or more and 10
or less monosaccharides are linked. The degree of
polymerization of an amino oligosaccharide bound to one
15 non-reducing end of the amino sugar-containing glucan of
the present invention can be, for example, about 2 or more,
about 3 or more, about 4 or more, about 5 or more or the
like, and can be, for example, about 10 or less, about 9
or less, about 8 or less, about 7 or less, about 6 or less,
20 about 5 or less, about 4 or less, about 3 or less, about
2 or less or the like. In one embodiment, the amino
oligosaccharide bound to one non-reducing end of the amino
sugar-containing glucan of the present invention has 2 sugars
(that is, a dimer). In the present specification, an amino
25 monosaccharide refers to an amino sugar wherein the sugar
backbone is a monosaccharide. In the present specification,
an amino oligosaccharide refers to an amino sugar wherein
the sugar backbone is an oligosaccharide.
[0023] In addition, since a bond by an electrostatic
30 interaction does not bind molecules directly, it is thought
that if a glucan is degraded from the conjugate to separate
the medically effective ingredient, the medically effective
ingredient retains the original stereostructure again. For
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this reason, the bond by an electrostatic interaction is
thought as an ideal binding mode between a medically effective
ingredient and a carrier. However, the bond by an
electrostatic interaction is prone to be affected by the
concentration of salts, and expected 5 ed to be unstable in a
living body. Therefore, it is desirable to control strictly
the position and number of amino sugars (for example,
glucosamines) bound to a glucan molecule in order to control
the strength of the bond with a nucleic acid and the form
10 of the associate.
[0024] Further, the present inventors found that a
glucosamine-containing glucan wherein at least one
glucosamine residue is bound to each of one or more (preferably
two or more) non-reducing ends of a glucan has a function
15 to non-covalently bind to a nucleic acid to increase the
apparent molecular weight of the nucleic acid.
[0025] Furthermore, the present inventors found that the
strength of the bond with a nucleic acid and the form of
the associate can be controlled by controlling the number
20 of amino monosaccharide residues such as glucosamine residues
bound to a glucan molecule.
[0026] The present inventors found that a glucan wherein
amino sugar residues are bound only to the ends of a plurality
of glucan chains having appropriate degree of freedom can
25 be produced, and that a medically effective ingredient and
a carrier can be bound via a stable electrostatic interaction,
by development of a novel amino sugar-containing glucan.
[0027] In the amino sugar-containing glucan, a modified
product thereof and a conjugate thereof of the present
30 invention, an amino sugar residue is bound only to a
non-reducing end of the glucan. In preferred embodiments
of the present invention, the amino sugar residue is
glucosamine residue, galactosamine residue or mannosamine
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residue, or an oligomeric (for example, a dimeric) residue
of sole or the combination thereof, and most preferably,
glucosamine residue. In the glucosamine-containing glucan,
a modified product thereof and a conjugate thereof of the
present invention, a glucosamine residue 5 esidue is bound only to
a non-reducing end of the glucan. In the present invention,
transfer of an amino sugar (for example, glucosamine) residue
is performed using 􀁄-glucan phosphorylase (EC 2.4.1.1). For
this reason, binding of an amino sugar (for example,
10 glucosamine) residue to a glucan is an 􀁄-1,4-bond. That is,
the carbon atom at 4-potision of a glucosyl residue at the
end of glucan and the carbon atom at 1-position of an amino
sugar (for example, glucosamine) residue are 􀁄-bound through
an oxygen atom. An enzyme which can transfer an amino sugar
15 (for example, glucosamine) to a glucan directly in a high
yield has not been found other than this enzyme. Since the
present amino sugar-containing glucan, a modified product
thereof and a conjugate thereof have an amino sugar on a
terminus, a glucan terminus comes to be positively charged
20 in an aqueous solution, and a physicochemical nature of the
glucan is changed. The present amino sugar-containing
glucan, a modified product thereof and a conjugate thereof
are expected to be widely utilized in foods, cosmetics,
medicaments and the like.
25 [0028] The introduction amount of amino sugar (for example,
glucosamine) residues to a glucan can be controlled by the
branching frequency of the glucan used and the frequency
of introduction of amino sugar (for example, glucosamine)
residues to a non-reducing end. When one wants to increase
30 an amount of introduction of an amino sugar (for example,
glucosamine) residue into a glucan, it is possible to increase
the introduction amount by using a glucan having a high
branching frequency and increasing the frequency of
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introduction of amino sugars (for example, glucosamines)
to the non-reducing end. When one wants to decrease an amount
of introduction of an amino sugar (for example, glucosamine)
residue into a glucan, it is possible to decrease the
introduction amount by using a glucan having a low 5 branching
frequency or decreasing the frequency of introduction of
amino sugars (for example, glucosamines) to the non-reducing
end. The lower limit of the introduction amount of amino
sugar (for example, glucosamine) residues to a glucan is
10 the state wherein one amino sugar (for example, glucosamine)
residue is introduced per glucan molecule, which can be
attained by introducing an amino sugar (for example,
glucosamine) residue to the non-reducing end of the glucan
having no branching. Further, since the introduction
15 position is at an end, it is thought to exert no influence
on degradability of a glucan by 􀁄-amylase. In the case of
a glucan which is highly branched, since non-reducing ends
are distributed in an outermost layer of a glucan molecule,
introduced amino sugar (for example, glucosamine) residues
20 are distributed in an outermost layer of a glucan molecule
after introduction of an amino sugar (for example,
glucosamine) residue, and this is ideal for interaction and
binding with the medically effective ingredient. As
described above, a glucan which has an amino sugar (for example,
25 glucosamine) residue selectively bound to a non-reducing
end can be an excellent modifying material for the medically
effective ingredient.
[0029] For example, the present invention provides the
followings:
30 (Item 1)
An amino sugar-containing glucan, wherein at least one amino
monosaccharide residue is bound via an 􀁄-1,4-bond to each
of at least one non-reducing end of a glucan, but no amino
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sugar residue is present at the position other than the
non-reducing end of the glucan, wherein the glucan is a
branched 􀁄-1,4-glucan or a linear 􀁄-1,4-grucan, and the
degree of polymerization of the glucan is about 10 or more
and about 1 x 105 5 or less, and preferably about 15 or more
and about 4 x 105 or less.
(Item 2)
The amino sugar-containing glucan according to item 1,
wherein the glucan is a branched 􀁄-1,4-glucan, the glucan
10 has a plurality of non-reducing ends, and at least one amino
monosaccharide residue is bound via an 􀁄-1,4-bond to each
of at least one (preferably two ore more) non-reducing ends
of the branched 􀁄-1,4-glucan. That is, the amino
sugar-containing glucan is an amino sugar-containing
15 branched glucan wherein the glucan has a plurality of
non-reducing ends and at least one amino monosaccharide
residue is bound via an 􀁄-1,4-bond to each of at least one
(preferably two or more) non-reducing ends of the branched
􀁄-1,4-glucan, but no amino sugar residue is present at the
20 position other than the non-reducing end of the branched
􀁄-1,4-glucan, wherein the degree of polymerization of the
branched 􀁄-1,4-glucan is about 10 or more and about 1 x 105
or less, and about 15 or more and about 4 x 105 or less.
(Item 3)
25 The amino sugar-containing glucan according to item 2,
wherein the branched 􀁄-1,4-glucan is selected from the group
consisting of a branched maltooligosaccharide, a starch,
amylopectin, glycogen, dextrin, enzymatically synthesized
branched glucan and highly branched cyclic glucan.
30 (Item 4)
A hydroxyl group-modified product of the amino
sugar-containing glucan according to any one of items 1 to
3, wherein the modification on the hydroxyl group is a
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modification on some or all of alcoholic hydroxyl groups
of the glucan, and the modification on the hydroxyl group
is independently selected from the group consisting of
hydroxyalkylation, alkylation, acetylation,
5 carboxymethylation, sulfation and phosphorylation.
(Item 5)
A reducing end-modified product of the amino sugar-containing
glucan according to any one of items 1 to 3 or a hydroxyl
group-modified product thereof.
10 (Item 6)
An amino group-modified product of the amino sugar-containing
glucan according to any one of items 1 to 3, a hydroxyl
group-modified product thereof or a reducing end-modified
product thereof, wherein the modification on the amino group
15 is a modification on some or all of amino groups of the amino
monosaccharide residues, the modification on the amino group
is attained by a reaction of the amino group and an amino
group-modifying reagent, and the amino group-modifying
reagent has at least one carboxyl group and at least one
20 other functional group.
(Item 7)
A non-reducing end-modified product of the amino
sugar-containing glucan according to any one of items 1 to
3, a hydroxyl group-modified product thereof, a reducing
25 end-modified product thereof or an amino group-modified
product thereof, wherein a targeting molecule is bound to
at least one of non-reducing ends to which the glucosamine
residue of the glucan is not bound, or to the 4-position
of the glucosamine residue, wherein the targeting molecule
30 is selected from the group consisting of mannose, galactose,
glucuronic acid, N-acetylglucosamine, xylose, fucose,
galactosamine, an antibody, an antibody fragment, a receptor,
a receptor fragment and a receptor ligand.
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(Item 8)
A method for producing an amino sugar-containing glucan,
characterized by allowing an 􀁄-glucan phosphorylase to act
on an aqueous solution comprising a glucan and an amino
5 sugar-1-phosphate, wherein the glucan is a branched
􀁄-1,4-glucan or a linear 􀁄-1,4-glucan, and the degree of
polymerization of the glucan is about 10 or more and about
1 x 105 or less, and preferably about 15 or more and about
4 x 105 or less.
10 (Item 9)
The method according to item 8, wherein the 􀁄-glucan
phosphorylase has 95% or more sequence identity with the
amino acid sequence of 􀁄-glucan phosphorylase derived from
Aquifex aeolicus VF5, and has activity of transferring an
15 amino sugar to a non-reducing end of a branched glucan to
form an 􀁄-1,4-bond.
(Item 10)
A medicament comprising the amino sugar-containing glucan
according to any one of items 1 to 3, a hydroxyl group-modified
20 product thereof, a reducing end-modified product thereof,
an amino group-modified product thereof, or a non-reducing
end-modified product thereof, and a medically effective
ingredient.
(Item 11)
25 The medicament according to item 10, wherein the medically
effective ingredient is selected from the group consisting
of a low-molecular weight organic compound, a protein, a
peptide, an antibody, an antibody fragment, a receptor, a
receptor fragment, a DNA, an RNA, a siRNA, an miRNA and an
30 RNA aptamer.
(Item 12)
A composition for clinical diagnosis comprising the amino
sugar-containing glucan according to any one of items 1 to
EG037PCT
3, a hydroxyl group-modified product thereof, a reducing
end-modified product thereof, an amino group-modified
product thereof, or a non-reducing end-modified product
thereof.
5 (Item 13)
A nanoparticulate carrier for a DDS comprising the amino
sugar-containing glucan according to any one of items 1 to
3, a hydroxyl group-modified product thereof, a reducing
end-modified product thereof, an amino group-modified
10 product thereof, or a non-reducing end-modified product
thereof.
(Item 14)
The carrier according to item 13, wherein the nanoparticulate
carrier for a DDS is selected from the group consisting of
15 a liposome, a virus particle, a macromolecule micelle and
a nanogel composed of macromolecule bearing hydrophobic
groups.
(Item 15)
A complex formed with a nucleic acid molecule comprising
20 a gene which can be expressed in a cell and the amino
sugar-containing branched glucan according to item 1.
(Item 16)
The complex according to item 15, wherein the nucleic acid
molecule is selected from the group consisting of a DNA,
25 an RNA, a siRNA, an miRNA and an RNA aptamer.
(Item 17)
A complex formed with the complex carrier according to item
15 or 16, and a cationic polymer or a cationic lipid.
(Item 18)
30 The complex according to item 17, wherein the cationic polymer
comprises at least one cationic polymer selected from the
group consisting of polyethyleneimine, polylysine,
polyarginine, polyamidoamine dendrimer, poly(aminostyrene),
EG037PCT
chitosan, a cationic glucan and DEAE-dextran.
(Item 19)
A method for delivering a nucleic acid molecule into an
isolated cell, comprising contacting the complex according
5 to any one of items 15 to 18 with the cell.
[0030] In one embodiment, the amino sugar is glucosamine,
and in that case, the present invention is as follows:
(Item 1A)
A glucosamine-containing glucan, wherein at least one
10 glucosamine residue is bound via an 􀁄-1,4-bond to each of
at least one non-reducing end of a glucan, but no glucosamine
residue is present at the position other than the non-reducing
end of the glucan, wherein the glucan is a branched
􀁄-1,4-glucan or a linear 􀁄-1,4-grucan, and the degree of
15 polymerization of the glucan is about 10 or more and about
1 x 105 or less, and preferably about 15 or more and about
4 x 105 or less. That is, the glucosamine-containing glucan
is a glucosamine-containing branched glucan wherein the
glucan has a plurality of non-reducing ends and at least
20 one glucosamine residue is bound via an 􀁄-1,4-bond to each
of at least one (preferably two or more) non-reducing ends
of the branched 􀁄-1,4-glucan, but no glucosamine residue
is present at the position other than the non-reducing ends
of the glucan, wherein the degree of polymerization of the
branched 􀁄-1,4-glucan is about 10 or more and about 1 x 105 25
or less, and preferably about 15 or more and about 4 x 105
or less.
(Item 2A)
The glucosamine-containing glucan according to item 1A,
30 wherein the glucan is a branched 􀁄-1,4-glucan, the glucan
has a plurality of non-reducing ends, and at least one
glucosamine residue is bound to each of at least one
non-reducing end of the branched 􀁄-1,4-glucan.
EG037PCT
(Item 3A)
The glucosamine-containing glucan according to item 2A,
wherein the branched 􀁄-1,4-glucan is selected from the group
consisting of a branched maltooligosaccharide, a starch,
5 amylopectin, glycogen, dextrin, enzymatically synthesized
branched glucan and highly branched cyclic glucan.
(Item 4A)
A hydroxyl group-modified product of the
glucosamine-containing glucan according to any one of items
10 1A to 3A, wherein the modification on the hydroxyl group
is a modification on some or all of alcoholic hydroxyl groups
of the glucan, and the modification on the hydroxyl group
is independently selected from the group consisting of
hydroxyalkylation, alkylation, acetylation,
15 carboxymethylation, sulfation and phosphorylation.
(Item 5A)
A reducing end-modified product of the
glucosamine-containing glucan according to any one of items
1A to 3A or a hydroxyl group-modified product thereof.
20 (Item 6A)
An amino group-modified product of the
glucosamine-containing glucan according to any one of items
1A to 3A, a hydroxyl group-modified product thereof or a
reducing end-modified product thereof, wherein the
25 modification on the amino group is a modification on some
or all of amino groups of the glucosamine residues, the
modification on the amino group is attained by a reaction
of the amino group and an amino group-modifying reagent,
and the amino group-modifying reagent has at least one
30 carboxyl group and at least one other functional group.
(Item 7A)
A non-reducing end-modified product of the
glucosamine-containing glucan according to any one of items
EG037PCT
1A to 3A, a hydroxyl group-modified product thereof, a
reducing end-modified product thereof or an amino
group-modified product thereof, wherein a targeting molecule
is bound to at least one of non-reducing ends to which the
glucosamine residue of the glucan is 5 not bound, wherein the
targeting molecule is selected from the group consisting
of mannose, galactose, glucuronic acid, N-acetylglucosamine,
xylose, fucose, galactosamine, an antibody, an antibody
fragment, a receptor, a receptor fragment and a receptor
10 ligand.
(Item 8A)
A method for producing a glucosamine-containing glucan,
characterized by allowing an 􀁄-glucan phosphorylase to act
on an aqueous solution comprising a glucan and
15 glucosamine-1-phosphate, wherein the glucan is a branched
􀁄-1,4-glucan or a linear 􀁄-1,4-glucan, and the degree of
polymerization of the glucan is about 10 or more and about
1 x 105 or less, and preferably about 15 or more and about
4 x 105 or less.
20 (Item 9A)
The method according to item 8A, wherein the 􀁄-glucan
phosphorylase has 95% or more sequence identity with the
amino acid sequence of 􀁄-glucan phosphorylase derived from
Aquifex aeolicus VF5, and has activity of transferring
25 glucosamine to a non-reducing end of a branched glucan to
form an 􀁄-1,4-bond.
(Item 10A)
A medicament comprising the glucosamine-containing glucan
according to any one of items 1A to 3A, a hydroxyl
30 group-modified product thereof, a reducing end-modified
product thereof, or an amino group-modified product thereof,
and a medically effective ingredient.
(Item 11A)
EG037PCT
The medicament according to item 10A, wherein the medically
effective ingredient is selected from the group consisting
of a low-molecular weight organic compound, a protein, a
peptide, an antibody, an antibody fragment, a receptor, a
receptor fragment, a DNA, an RNA, an miRNA and an RNA ap5 tamer.
(Item 12A)
A composition for clinical diagnosis comprising the
glucosamine-containing glucan according to any one of items
1A to 3A, a hydroxyl group-modified product thereof, a
10 reducing end-modified product thereof, or an amino
group-modified product thereof.
(Item 13A)
A nanoparticulate carrier for a DDS comprising the
glucosamine-containing glucan according to any one of items
15 1A to 3A, a hydroxyl group-modified product thereof, a
reducing end-modified product thereof, or an amino
group-modified product thereof.
(Item 14A)
The carrier according to item 13A, wherein the
20 nanoparticulate carrier for a DDS is selected from the group
consisting of a liposome, a virus particle, a macromolecule
micelle and a nanogel composed of macromolecule bearing
hydrophobic groups.
(Item 15A)
25 A complex formed with a nucleic acid molecule comprising
a gene which can be expressed in a cell and the
glucosamine-containing branched glucan according to any one
of items 1A to 3A.
(Item 16A)
30 The complex according to item 15A, wherein the nucleic acid
molecule is selected from the group consisting of a DNA,
an RNA, a siRNA, an miRNA and an RNA aptamer.
(Item 17A)
EG037PCT
A complex formed with the complex carrier according to item
15A or 16A, and a cationic polymer or a cationic lipid.
(Item 18A)
The complex according to item 17A, wherein the cationic
5 polymer comprises at least one cationic polymer selected
from the group consisting of polyethyleneimine, polylysine,
polyarginine, polyamidoamine dendrimer, poly(aminostyrene),
chitosan, a cationic glucan and DEAE-dextran.
(Item 19A)
10 A method for delivering a nucleic acid molecule into a cell,
comprising contacting the complex according to any one of
item 15A to 18A with the cell.
EFFECTS OF THE INVENTION
15 [0031] In the amino sugar-containing glucan, a hydroxyl
group-modified product thereof, a reducing end-modified
product thereof, an amino group-modified product thereof
and a conjugate thereof of the present invention, an amino
sugar residue is bound only to a non-reducing end of the
20 glucan. Since the present amino sugar-containing glucan,
a hydroxyl group-modified product thereof, a reducing
end-modified product thereof, an amino group-modified
product thereof and a conjugate thereof have amino sugar
on a terminus, a glucan terminus comes to be positively charged
25 in an aqueous solution, and a physicochemical nature of the
glucan is changed. The present amino sugar-containing
glucan, a hydroxyl group-modified product thereof, a reducing
end-modified product thereof, an amino group-modified
product thereof and a conjugate thereof are expected to be
30 widely utilized in foods, cosmetics, medicaments and the
like. These glucans have the characteristics that, when used
as a carrier for a medically effective ingredient, due to
the bond by an electrostatic interaction, these glucans do
EG037PCT
not change in quality largely the chemical structure of the
medically effective ingredient, and can bind a plurality
of amino sugars to the non-reducing end per carrier molecule,
therefore, these glucans can retain the medically effective
5 ingredient stably.
[0032] Since the amino sugar-containing glucan, a hydroxyl
group-modified product thereof, a reducing end-modified
product thereof, and an amino group-modified product thereof
of the present invention can increase a half-life in blood
10 than that of an unmodified glucan, and ultimately completely
degraded in a living body and excreted from kidney, they
have extremely high safety. For this reason, they are useful
as a modifying material for a medically effective ingredient,
a clinical diagnostic agent, a contrast agent and a
15 nanoparticulate carrier for DDS. In the amino
sugar-containing glucan, a hydroxyl group-modified product
thereof, a reducing end-modified product thereof, and an
amino group-modified product thereof of the present invention,
since their structure can be controlled by an enzymatic
20 reaction, they are also excellent in quality stability.
[0033] In the amino sugar-containing glucan and a modified
product thereof of the present invention, since an amino
sugar residue is bound only to a non-reducing end of the
glucan, a site which interacts with other molecules and a
25 site which suppresses degradation can be structurally
separated. Further, the amino sugar-containing glucan and
a modified product thereof of the present invention have
been appropriately suppressed the degradation compared to
naturally-occurring glucan, the interaction with other
30 molecules is not too strong, and can appropriately release
other molecule as the glucan moiety is degraded, therefore,
they can give an appropriate degradability to other molecule
by association with the other molecule. In addition, the
EG037PCT
amino sugar-containing glucan and a modified product thereof
and a conjugate thereof of the present invention are degraded
with an enzyme in a body, and therefore it does not cause
the problem of residual property due to staying in a body
5 for an excessively long term.
[0034] When the amino sugar-containing glucan and a modified
product thereof of the present invention are associated with
a negatively-charged substance such as a nucleic acid
molecule, it is possible to control the molecular weight
10 of the associate. Since in view of safety, stability of the
result, and reproducibility, it is preferable that the
molecular weight of an administered product in DDS can be
controlled, the amino sugar-containing glucan and a modified
product thereof of the present invention can effectively
15 be utilized as an excellent carrier for DDS.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035]
[Fig. 1] Fig. 1 is a schematic view of a reaction of
20 transferring a glucosamine residue to a non-reducing end
of a branched glucan. The structure of the part shown with
an asterisk is shown in the square frame.
[Fig. 2] Fig. 2 is results of analysis of an enzymatically
treated product of a branched glucan obtained in Production
25 Example 2, and an enzymatically treated product of the product
obtained by allowing Aquifex aeolicus VF5-derived 􀁄-glucan
phosphorylase in Production Example 1 to act on
glucosamine-1-phosphate and the branched glucan of
Production Example 2 (the product obtained in Example 1),
30 with a HPAEC-PAD apparatus manufactured by DIONEX. The
horizontal axis represents the elusion time (minutes). A
shows the result obtained by analyzing the degradation
product after degrading the branched portions of the branched
EG037PCT
glucan with the sufficient amount (that is, excess amount)
of isoamylase to cleave all the branches. Regarding numbers
above the peaks in Fig. 2A, 6 indicates maltohexaose, and
7 indicates maltoheptaose. The peaks shown with black solid
5 circles thereabove indicate, from left to right,
maltooligosaccharides having the degree of polymerization
of glucose of from 8 to 14. Fig. 2B shows the result obtained
by analyzing the degradation product obtained after degrading
the branched glucan with an excess amount of isoamylase,
10 and then further degrading it with an excess amount of
microorganism-derived 􀁄-glucosidase. Since 􀁄-glucosidase
is an enzyme which degrades glucan in glucose units starting
from the non-reducing end, the branched glucan was completely
degraded and the peak of glucose (shown by "Glucose" in Fig.
15 2B) was detected. Fig. 2C shows the result obtained by
analyzing the degradation product obtained after degrading
the glucosamine-containing branched glucan obtained in the
present Example 1 with an excess amount of isoamylase. While
the peaks shown with black solid circles in Fig. 2C correspond
20 to the peaks detected at the same elusion time as the peaks
shown with black solid circles at the corresponding positions
in Fig. 2A, the peaks shown with "x" having different intervals
therefrom were detected. Fig. 2D shows the result obtained
by analyzing the degradation product obtained after degrading
25 the glucosamine-containing branched glucan obtained in the
present Example 1 with isoamylase and further degrading with
an excess amount of microorganism-derived 􀁄-glucosidase.
The group of peaks shown with black solid circles disappeared
due to the degradation, and as a result, the peak of glucose
30 (shown by "Glucose" in Fig. 2D) appeared, however, the peaks
shown with "x" did not disappeare. That is, the partially
degraded product indicated by the peaks shown with "x"
exhibited resistance to degradation with 􀁄-glucosidase.
EG037PCT
Fig. 2E shows the result obtained by analyzing the degradation
product obtained after degrading the glucosamine-containing
branched glucan obtained in the present Example 1 with an
excess amount of isoamylase, and further degrading with an
excess amount of microorganism-derived 􀁄5 -glucosidase and
an excess amount of 􀁄-amylase simultaneously. As a result,
in addition to the peak of glucose, the peaks shown with
asterisks were detected. The peaks shown with asterisks
indicate glucosamine-containing oligosaccharide
10 (trisaccharide), which is the smallest unit which cannot
be degraded with 􀁄-amylase and 􀁄-glucosidase, and therefore,
it can be understood that a glucan which has a glucosamine
residue bound to the non-reducing end was obtained.
[Fig. 3] Fig. 3 is a figure showing formation of a complex
15 of the glucosamine-containing glucan of Example 1 and a DNA.
Relative to 0.5 􀁐g of a lambda DNA-Hind III fragment,
ion-exchanged water for Sample 1, 0.1 mg of branched glucan
for Sample 2, 0.5 mg of branched glucan for Sample 3, 0.1
mg of glucosamine-containing branched glucan for Sample 4,
20 0.5 mg of glucosamine-containing branched glucan for Sample
5, the same molar concentration of glucosamine as that of
glucosamine unit contained in Sample 4 for Sample 6, the
same molar concentration of glucosamine as that of
glucosamine unit contained in Sample 5 for Sample 7 were
25 added respectively, and they were allowed to stand at room
temperature in 10 􀁐l for 5 minutes, then 1% agarose gel
electrophoresis were performed. While bands (a) were
detected for Samples 1 to 3, DNA did not migrate through
agarose gel when glucosamine-containing branched glucan were
30 added (b). Therefore, it could be understood that the
cationized glucosamine-containing branched glucan can form
a complex with a DNA. In addition, in Samples 6 and 7 to
which glucosamine were added, the mobility were not changed
EG037PCT
as compared with the case where the glucosamine was not added,
and it could be understood that glucosamine has no effect
to increase the molecular weight of a DNA.
[Fig. 4] Fig. 4 is a figure showing the binding force between
oligo(5 dT)-cellulose and the glucosamine-containing
branched glucans of Example 3 (BN5 and BN6) or the
glucosamine-containing branched glucan of Example 4 (PN6).
Oligo(dT)-cellulose was suspended in NaCl solutions of each
concentration (0.1 M, 0.2 M, 0.3 M, 0.5 M, 1 M, or 2 M),
10 and the glucosamine-containing branched glucan obtained in
Example 3 or Example 4 was added and allowed to stand at
room temperature for 30 minutes, then centrifuged, and
measured the amount of glucosamine-containing branched
glucans which were not bound with oligo(dT)-cellulose
15 contained in the supernatant fraction, thereby calculated
the percentage (%) of the amount of bound cationized glucan
(total sugar amount) relative to the amount of added
glucosamine-containing glucan (total sugar amount). The
obtained results are shown as a graph. It can be understood
20 that PN6 separates from a nucleic acid at 1 M or more of
NaCl. It can be understood that BN6 separates from a nucleic
acid at 0.5 M or more of NaCl. It can be understood that
BN5 separates from a nucleic acid at 0.2 or more. There was
a tendency that the more number of glucosamine residues
25 contained in one molecule of glucosamine-containing branched
glucan, the higher the NaCl concentration at which these
residues separates. Therefore, the binding force with a
nucleic acid could be controlled by controlling the frequency
at which glucosamine was bound to a branched glucan. In the
30 case where polylysine was used, polylysine bound with a
nucleic acid strongly, and did not separate from the nucleic
acid even in 2 M of NaCl.
[Fig. 5] Fig. 5 is a figure showing the transfection efficiency
EG037PCT
of macrophage cell line in the case where the
glucosamine-containing branched glucan of Example 4 (PN6)
was used. The glucosamine-containing branched glucan was
incubated with a plasmid containing LUC gene encoding
5 firefly-derived luciferase for 24 hours, and thereafter,
luciferase activity was measured. As shown in Fig. 5, it
can be understood that PN6 shows a transfection function,
and especially, the highest transfection efficiency was shown
when N/P ratio was 200. While polyethyleneimine showed high
10 transfection efficiencies at N/P ratios of 10 and 50, the
efficiency remarkably lowered when N/P ratio was 200.
Polylysine showed the highest transfection efficiency when
N/P ratio was 2, and the efficiency remarkably lowered when
N/P ratio was higher than 10.
15 [Fig. 6] Fig. 6 is a figure evaluating the cytotoxicity in
transfection of macrophage cell line when the
glucosamine-containing branched glucan of Example 4 (PN6)
was used. The protein concentration in the supernatant of
the cell lysate obtained in Example 11 was measured and used
20 as a measure of toxicity. As shown in Fig. 6, it is shown
that PN6 has low toxicity to a cell. When polyethyleneimine
and polylysine were used, remarkable toxicity was observed
at N/P ratios of 50 and 200.
25 MODE FOR CARRYING THE INVENTION
[0036] The present invention will be explained in detail
below.
[0037] Throughout the present specification, it should be
understood that expression in a singular form includes a
30 concept of a plural form thereof, unless otherwise indicated.
In addition, it should be understood that a term used in
the present specification is used in a sense which is usually
used in the art, unless otherwise indicated.
EG037PCT
[0038] (General Techniques)
The molecular biological procedures, biochemical
procedures, and microbiological procedures used in the
present specification are well known and routine in the art,
and described in, for example, Sambrook, J. et al., 5 ., (1989).
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
and its 3rd Ed. (2001); Ausubel, F.M. (1987). Current
Protocols in Molecular Biology, Greene Pub. Associates and
Wiley-Interscience; Ausubel, F.M. (1989). Short Protocols
10 in Molecular Biology: A Compendium of Methods from Current
Protocols in Molecular Biology, Greene Pub. Associates and
Wiley-Interscience; Innis, M.A. (1990). PCR Protocols: A
Guide to Methods and Applications, Academic Press; Ausubel,
F.M. (1992). Short Protocols in Molecular Biology: A
15 Compendium of Methods from Current Protocols in Molecular
Biology, Greene Pub. Associates; Ausubel, F.M. (1995). Short
Protocols in Molecular Biology: A Compendium of Methods from
Current Protocols in Molecular Biology, Greene Pub.
Associates; Innis, M.A. et al., (1995). PCR Strategies,
20 Academic Press; Ausubel, F.M. (1999). Short Protocols in
Molecular Biology: A Compendium of Methods from Current
Protocols in Molecular Biology, Wiley, and annual updates;
Sninsky, J.J. et al., (1999). PCR Applications: Protocols
for Functional Genomics, Academic Press, Bessatsu
25 Jikken-igaku "Idenshidounyu & Hatsugen kaiseki jikkenhou"
(a separate volume of Experimental Medicine "Experimental
methods for gene introduction and expression analysis"),
Yodosha Co., Ltd., 1997, and the like, the related portions
thereof (which can be the whole) are incorporated herein
30 by reference.
[0039] DNA synthetic techniques and nucleic acid
chemistries for producing an artificially-synthesized gene
are described in, for example, Gait, M.J. (1985).
EG037PCT
Oligonucleotide Synthesis: A Practical Approach, IRL Press;
Gait, M.J. (1990). Oligonucleotide Synthesis: A Practical
Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides
and Analogues: A Practical Approac, IRL Press; Adams, R.L.
et al., (1992). The Biochemistry of the Nucleic Acids, Ch5 apman
& Hall; Shabarova, Z. et al., (1994). Advanced Organic
Chemistry of Nucleic Acids, Weinheim; Blackburn, G.M. et
al., (1996). Nucleic Acids in Chemistry and Biology, Oxford
University Press; Hermanson, G.T. (I996). Bioconjugate
10 Techniques, Academic Press, and the like, the related
portions thereof are incorporated herein by reference.
[0040] When a gene is referred to in the present
specification, a "vector" or a "recombinant vector" refers
to a vector which can transfer an objective polynucleotide
15 sequence into an objective cell. Examples of such vectors
include a vector which can autonomously replicate in a host
cell, such as a prokaryotic cell, a yeast, an animal cell,
a plant cell, an insect cell, an animal individual and a
plant individual, or can be incorporated into a chromosome,
20 and has a promoter at a position suitable for transcribing
the polynucleotide of the present invention. Among the
vectors, those vectors which are suitable for cloning are
referred to as "cloning vector". Such a cloning vector
usually comprises a multiple-cloning site containing a
25 plurality of restriction enzyme sites. Such a restriction
enzyme site and a multiple-cloning site are well known in
the art, and those skilled in the art can select as appropriate
and use them in accordance with the purpose. Such techniques
are described in the documents described in the present
30 specification (for example, Sambrook, et al., supra).
Preferred vectors include, but are not limited to, a plasmid,
a phage, a cosmid, an episome, a viral particle or a virus,
and an integratable DNA fragment (that is, a fragment which
EG037PCT
can be integrated into a host genome by homologous
recombination).
[0041] One type of vectors is a "plasmid", which refers to
a circular duplex DNA loop to which an additional DNA segment
can be linked. Another 5 type of vector is a viral vector,
wherein an additional DNA segment can be linked into a viral
genome. Specific vectors (for example, bacterial vector
having a bacterial origin of replication and an episome
mammalian vector) can autonomously replicate in a host cell
10 to which these vectors are introduced. Other vectors (for
example, non-episome mammalian vector) are integrated to
a genome of a host cell upon introduction to the host cell,
and thereby, replicated together with the host genome. In
addition, specific vectors can direct expression of a gene
15 to which these vectors are operably linked. Such vectors
are referred to as "expression vectors" in the present
specification.
[0042] Therefore, in the present specification, an
"expression vector" or an "expression plasmid" refers to
20 a nucleic acid sequence wherein various regulation elements,
in addition to a structural gene and a promoter regulating
its expression, are connected in a cell of a host in an operable
condition. An expression vector is, preferably, a vehicle
which is operably linked to an objective structural gene
25 such that the objective structural gene is transcripted and
translated, and if necessary, contains factors necessary
for replication in the host cell and selection of a recombinant.
Regulation elements can include, preferably, a terminator,
a selectable marker such as a drug resistant gene, and an
30 enhancer. It is well-known to those skilled in the art that
the type of an expression vector of an organism (e.g. mammal),
and the kind of regulation element used can vary depending
on a host cell. When secretion production of an expressed
EG037PCT
product (􀁄-glucan phosphorylase or a medically effective
ingredient) is intended, a polynucleotide encoding a
secretion signal peptide is linked upstream of a DNA coding
for the objective protein in the correct reading frame.
[5 0043] Preferred expression vectors include pTRC99A
(manufactured by Pharmacia) that is also expressible in
Escherichia coli, and the like. In order to operably link
an 􀁄-glucan phosphorylase gene to factors necessary for
transcription and translation in the aforementioned
10 expression vector, an objective 􀁄-glucan phosphorylase gene
should be processed in some cases. Examples include the case
where the distance between a promoter and a coding region
is too long, and reduction in a transcription efficiency
is predicted, the case where the distance between a ribosome
15 binding site and a translation initiation codon is not
suitable, and the like. The process means include digestion
with a restriction enzyme, digestion with an exonuclease
such as Bal31 and ExoIII, or introduction of site-directed
mutagenesis using a single-stranded DNA such as M13 or PCR.
20 [0044] As "recombinant vectors" for a prokaryotic cell which
can be used in the present application, pcDNA3(+),
pBluescript-SK(+/-), pGEM-T, pEF-BOS, pEGFP, pHAT, pUC18,
pFT-DESTTM 42GATEWAY, pENTRTM/D-TOPO (Invitrogen), and the
like are exemplified. A prokaryotic cell can be used in
25 amplification, modification and the like of a gene.
[0045] "Recombinant vectors" for an eukaryote cell which
can be used in the present application include, but are not
limited to, pECFP (Clontech), pAcGFP (Clontech), pEYFP
(Clontech), pDsRED (Clontech), pTRE (Clontech), pCMV
30 (Clontech), pcDNA (Invitrogen), pTarget (Promega), and the
like.
[0046] In the present specification, a "mammalian
expression vector" refers to a nucleic acid sequence wherein
EG037PCT
various regulation elements such as a promoter regulating
expression of the gene of the present invention are operably
linked in a host cell. The term "regulation sequence" used
in the specification of the present application refers to
a DNA sequence having a functional promoter 5 romoter and any related
transcription elements (for example, an enhancer, a CCAAT
box, a TATA box, an SPI site, and the like). The term "operably
linked" used in the specification of the present application
refers that a polynucleotide related to a gene and various
10 regulation elements, such as a promoter and an enhancer,
which regulate expression of the gene are connected in a
host cell in an operable condition such that the gene can
be expressed. A mammalian expression vector can preferably
include a mammalian gene, a promoter, a terminator, a drug
15 resistant gene and an enhancer. It is well-known to those
skilled in the art that the type of expression vector and
the kind of regulation element used can vary depending on
a host cell.
[0047] Mammalian expression vectors as described above can
20 be made using gene recombination techniques well-known to
those skilled in the art. For construction of a mammalian
expression vector, for example, a pECFP-type vector, a
pcDNA-type vector or the like are preferably used, but not
limited to them.
25 [0048] In the present specification, a "terminator" is a
sequence which is situated downstream of a protein coding
region of a gene, and is involved in termination of
transcription upon transcription of a DNA into an mRNA, and
in the addition of a poly A sequence. It is known that a
30 terminator contributes to the stability of an mRNA and
influences the expression level of a gene.
[0049] In the present specification, a "promoter" refers
to a region on a DNA which determines a transcription
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initiation site of a gene, and directly regulates the
transcription frequency, and is a base sequence to which
a RNA polymerase binds, thereby, initiating transcription.
A putative promoter region varies with every structural gene,
and is usually upstream 5 of a structural gene without
limitation, and may be downstream of a structural gene.
While a promoter may be inducible, constitutive,
site-directed, or stage-specific, a constitutive promoter
or an inducible promoter is preferable. Any promoter is
10 possible as long as which can be expressed in a host cell
such as a mammalian cell, Escherichia coli, or a yeast.
[0050] In the present specification, expression of a
promoter is "constitutive" means the nature by which the
promoter is expressed at an almost fixed amount in all the
15 tissues in an organism at whatever stage of
growth/proliferation of the organism. Specifically, in the
definition of the present invention, when analyzed with
Northern blot, if almost the same degree of expression amount
is observed in either of the same or corresponding sites,
20 for example, at any time point (for example, at two or more
points (for example, on day 5 and day 15)), the expression
is said to be constitutive. It is believed that a
constitutive promoter plays a role in maintenance of
homeostasis of an organism in a normal growing environment.
25 "Responsive" expression of the promoter of the present
invention means the nature by which the expression amount
is changed when at least one factor is given to an organism.
Especially, the nature by which expression amount is
increased is referred to be "inducible" by a factor, and
30 the nature by which expression amount is decreased is referred
to be "decreasing" by a factor. Since "decreasing"
expression has a premise that expression is observed under
a normal condition, it is an idea overlapping with
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"constitutive" expression. These natures can be determined
by extracting an RNA from any portion of an organism and
analyzing expression amount by Northern blot analysis, or
quantifying the expressed protein by western blot. A
5 mammalian cell or a mammal (including a specific tissue or
the like) transformed with a vector integrated a promoter
inducible by a factor together with a nucleic acid encoding
the site-directed recombination inducing factor of the
present invention can perform a site-directed recombination
10 of a site-directed recombination sequence under a certain
condition by using a stimulation factor having an inducible
activity to the promoter.
[0051] The polynucleotide of the present invention can be
linked intact or with modification into an appropriate
15 expression vector using a method well-known to those skilled
in the art, and introduced to a host cell by a known gene
recombination technique. The introduced gene exists
integrated to a DNA in the host cell. It is noted that a
DNA in a host cell includes not only a DNA included in a
20 chromosome but also a DNA included in various organelle (for
example, a mitochondrion and the like) contained in a host
cell.
[0052] When Escherichia coli is used as a host cell,
promoters derived from Escherichia coli, phage and the like,
25 such as trp promoter (Ptrp), lac promoter (Plac), PL promoter,
PR promoter, and PSE promoter, SPO1 promoter, SPO2 promoter,
penP promoter, and the like can be included. In addition,
an artificially designed and modulated promoters or the like
such as a promoter consisting of two serially aligned
30 Ptrps(Ptrp x2), tac promoter, lacT7 promoter, let I promoter
can also be used.
[0053] In the present specification, an "origin of
replication" refers to a specific region on a chromosome
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from which DNA replication starts. An origin of replication
can either be provided by constructing the vector so as to
include an endogenous origin, or by a chromosomal replication
mechanism of a host cell. The latter may be sufficient when
the vector is integrated to a host 5 cell chromosome.
Alternatively, those skilled in the art can transform a
mammalian cell by co-transforming with a selectable marker
and the DNA of the present invention, rather than by using
a vector including a viral origin of replication. Examples
10 of suitable selectable markers are dihydrofolate reductase
(DHFR) or thymidine kinase (see U.S. Patent No. 4,399,216).
[0054] In the present specification, "operably linked"
refers to that expression (operation) of a desired sequence
is placed under the control of a transcription and translation
15 regulating sequence (e.g. promoter, enhancer and the like)
or a translation regulating sequence. In order that a
promoter is operably linked to a gene, usually, a promoter
is disposed immediately upstream of the gene, but it is not
necessary that the promoter is disposed adjacent to the gene.
20 [0055] In the present specification, the technique of
introducing a nucleic acid molecule into a cell may be any
technique. Examples of such techniques include, for example,
transformation, transduction, and transfection. Such
techniques of introducing a nucleic acid molecule are
25 well-known in the art, and are conventional, and are described,
for example, in Ausubel F.A. et al., ed. (1988), Current
Protocols in Molecular Biology, Wiley, New York, NY; Sambrook
J et al., (1987) Molecular Cloning: A Laboratory Manual,
2nd Ed. and 3rd Ed., Cold Spring Harbor Laboratory Press,
30 Cold Spring Harbor, NY; Bessatsu Jikken-igaku "Idenshidounyu
& Hatsugen kaiseki jikkenhou" (a separate volume of
Experimental Medicine "Experimental methods for gene
introduction and expression analysis"), Yodosha Co., Ltd.,
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1997 and the like. The introduction of a gene can be confirmed
using the methods described in the present specification
such as Northern blot analysis and Western blot analysis,
or other well-known and conventional techniques.
5 [0056] Further, as a method for introducing a vector, any
method as described above for introducing a DNA to a cell
can be used, the method include, for example, transfection,
transduction, transformation and the like (for example,
calcium phosphate method, liposome method, DEAE-dextran
10 method, electroporation method, method using a particle gun
(a gene gun) and the like).
[0057] In the present specification, a "transformant"
refers to a whole or a part of a living body such as a cell
produced by transformation. Transformants are exemplified
15 by a prokaryotic cell, a yeast, an animal cell, a plant cell,
an insect cell, and the like. A transformant can also be
referred to as a transformated cell, a transformated tissue,
or a transformated host, depending on the subject. The cell
used in the present invention can be a transformant.
20 [0058] When a prokaryotic cell is used in gene manipulation
or the like in the present invention, prokaryotic cells are
exemplified by the prokaryotic cells belonging to a genus
Escherichia, Serratia, Bacillus, Brevibacterium,
Corynebacterium, Microbacterium, Pseudomonas, or the like,
25 for example, Escherichia coli XL1-Blue, Escherichia coli
XL2-Blue, and Escherichia coli DH1.
[0059] As used in the present specification, as a method
for introducing a recombinant vector, any methods which
introduce a DNA can be used, the methods include, for example,
30 calcium chloride method, electroporation method [Methods.
Enzymol., 194, 182 (1990)], lipofection method, spheroplast
method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], lithium
acetate method, and the like.
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[0060] In the present specification, "detection" or
"quantification" of gene expression (for example, mRNA
expression, and polypeptide expression) can be achieved using
a suitable method including, for example, measurement of
mRNA and immunological measuring method. Mol5 ecular
biological measuring methods are exemplified by, for example,
Northern blot method, dot blot method, PCR method and the
like. Immunological measuring methods are exemplified, for
example, ELISA method using a microtiter plate, RIA method,
10 immunofluorescence method, western blot method,
immunohistostaining, and the like, as the method. Further,
quantifying methods are exemplified by ELISA method, RIA
method or the like. It can be done by a gene analyzing method
using an array (for example, a DNA array, and a protein array).
15 A DNA array is widely reviewed in (Shujunsha Ed., Saibo Kogaku
Bessatsu, "DNA Microarray to Saishin PCR Hou"). A protein
array is detailed in Nat Genet. 2002 Dec; 32 Suppl: 526-32.
Methods for analyzing gene expression include, but are not
limited to, RT-PCR, RACE method, SSCP method,
20 immunoprecipitation method, two-hybrid system, in vitro
translation and the like, in addition to the above. Such
additional analysis methods are described in, for example,
Genomu kaiseki jikkenhou, Nakamura Yusuke lab manual
(Understand the Basics of Genomics Experiments - Yusuke
25 Nakamura Laboratory Manual), Editor Yusuke Namamura, Yodosha
Co., Ltd. (2002) and the like, the whole description of which
is incorporated herein by reference.
[0061] (1. Materials)
(1.1) Glucans and modified products of glucan
30 "Glucan", when used in the present specification, is a
polysaccharide having D-glucose as a constituent unit. In
the present invention, it is preferable to use, as the glucan,
an 􀁄-D-glucan. The bonds which link glucose residues in an
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􀁄-D-glucan predominantly consist of an 􀁄-1,4-glucosidic bond,
and can contain an 􀁄-1,6-glucosidic bond. An 􀁄-D-glucan
containing a 􀁄-1,6-glucosidic bond has a branched structure.
A glucan having at least one 􀁄-1,6-glucosidic bond in the
molecule is referred 5 red to as a branched glucan, and a glucan
having no 􀁄-1,6-glucosidic bond in the molecule is referred
to as a linear glucan. In the present specification, unless
otherwise indicated, it is preferable that the weight average
molecular weight of the "glucan" is about 1 x 103 or more.
10 Preferable glucans used in the present invention are a linear
glucan and a branched glucan, more preferably a linear
􀁄-1,4-glucan and an 􀁄-1,4-glucan which is branched with an
􀁄-1,6-bond (also referred to as a branched 􀁄-1,4 glucan).
It is preferable that the glucan used in the present invention
15 does not contain an 􀁄-1,3-bond.
[0062] The linear 􀁄-D-1,4-glucan refers to a polysaccharide
in which two or more saccharide units of D-glucose units
are bound only with an 􀁄-1,4-glucosidic bond(s). In the
present specification, unless otherwise indicated, the
20 linear 􀁄-D-1,4-glucan is referred to as a linear glucan or
a linear 􀁄-1,4-glucan. The linear glucan has one
non-reducing end. Examples of the linear glucan suitably
utilized in the present invention include amylose.
[0063] In the present specification, the term "amylose"
25 refers to a linear molecule constructed of glucose units
connected via 􀁄-1,4-bonds. An amylose is contained in
natural starch. Amylose may be a natural amylose extracted
from a natural starch, or may be an amylose synthesized by
an enzymatic reaction (also referred to as "enzymatically
30 synthesized amylose" in the present specification).
Natural amylose may contain a branched part in some cases,
but enzymatically synthesized amylose does not contain a
branch. Further, natural amyloses have a large
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polydispersity and vary in the molecular weight, but an
enzymatically synthesized amylose (particularly, an
enzymatically synthesized amylose synthesized by the SP-GP
method described in International Publication WO 02/097107
pamphlet) has a small 5 mall polydispersity and has an extremely
uniform molecular weight. For this reason, in the present
invention, it is preferable to use an enzymatically
synthesized amylose. The degree of polymerization of the
amylose used in the present invention is preferably about
10 10 or more, more preferably about 50 or more, still more
preferably about 100 or more, and most preferably about 180
or more. The degree of polymerization of the amylose used
in the present invention is preferably about 1 x 105 or less,
more preferably about 1 x 104 or less, still more preferably
about 1 x 103 15 or less and most preferably about 500 or less.
[0064] In the present specification, the term "branched
􀁄-D-glucan" refers to a glucan in which a linear glucan,
in which D-glucose units are connected with an
􀁄-1,4-glucosidic bond(s), is branched with a bond other than
20 an 􀁄-1,4-glucosidic bond. In the present specification,
unless otherwise indicated, the branched 􀁄-D-glucan is
referred to as a branched glucan. A branching bond is either
an 􀁄-1,6-glucosidic bond, an 􀁄-1,3-glucosidic bond, or an
􀁄-1,2-glucosidic bond, and most preferably is an
25 􀁄-1,6-glucosidic bond. It is preferable that a branched
􀁄-D-glucan used in the present invention does not contain
an 􀁄-1,3-glucosidic bond and an 􀁄-1,2-glucosidic bond. The
branched glucan usually has the same number of non-reducing
ends as the number of branching bonds. When the branched
30 glucan is treated with an enzyme which selectively breaks
only an 􀁄-1,6-glucosidic bond (for example, isoamylase,
pullulanase, or the like), the branched glucan can be degraded
into a mixture of linear 􀁄-1,4-glucans. These are referred
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to as a unit chain of the branched glucan, and the degree
of polymerization thereof is referred to as a unit chain
length.
[0065] Examples of the branched glucan suitably utilized
5 in the present invention include starches, amylopectin,
glycogen, dextrin, enzymatically synthesized branched
glucan and highly branched cyclic glucan.
[0066] In the present specification, the term "starch" is
a mixture of amylose and amylopectin. As a starch, any starch
10 can be used as long as it is commonly commercially available.
The ratio of the amylose and amylopectin contained in a starch
is different depending on the kind of plant producing the
starch. Almost all starches possessed by glutinous rice,
glutinous corn and the like are an amylopectin. On the other
15 hand, a starch consisting only of amyloses, containing no
amylopectin, can not be obtained from a common plant. Starch
is classified into natural starch, a degraded starch and
modified starch.
[0067] Natural starch is classified into tuber starch and
20 cereal starch depending on the raw material. Examples of
tuber starches include potato starch, tapioca starch, sweet
potato starch, kudzu starch, bracken starch and the like.
Examples of cereal starches include corn starch, wheat starch,
rice starch and the like. Examples of natural starches are
25 high amylose starches (for example, high amylose corn starch)
or waxy starches. The starch can be a soluble starch. A
soluble starch refers to a water-soluble starch obtained
by subjecting a variety of treatment on natural starch. The
starch may be selected from the group consisting of soluble
30 starch, waxy starch and high amylose starch. The starch may
be a modified starch.
[0068] The degree of polymerization of the starch used in
the present invention is preferably about 1 x 103 or more,
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more preferably about 5 x 103 or more, still more preferably
about 1 x 104 or more, and most preferably about 2 x 104 or
more. The degree of polymerization of the starch used in
the present invention is preferably about 1 x 107 or less,
more preferably about 3 x 106 or less, 5 s, still more preferably
about 1 x 106 or less and most preferably about 3 x 105 or
less.
[0069] An amylopectin is a branched molecule in which a
glucose unit(s) is bound via an 􀁄-1,6 bond to glucose units
10 which are bound via an 􀁄-1,4 bond(s). An amylopectin is
contained in natural starch. As an amylopectin, for example,
waxy corn starch, which consists of 100% amylopectin, can
be used. The degree of polymerization of the amylopectin
used in the present invention is preferably about 1 x 103
or more, more preferably about 5 x 103 15 or more, still more
preferably about 1 x 104 or more, and most preferably about
2 x 104 or more. The degree of polymerization of the
amylopectin used in the present invention is preferably about
1 x 107 or less, more preferably about 3 x 106 or less, still
more preferably about 1 x 106 20 or less and most preferably
about 1 x 105 or less.
[0070] A glycogen is one kind of glucan constructed of
glucose, and is a glucan having a high frequency of branching.
A glycogen is widely distributed as a storage polysaccharide
25 for animals in almost all cells in the granule state. In
a plant, glycogen is present, for example, in the seed of
sweet corn species of corn. In a glycogen, typically, sugar
chains consisting of glucoses bound via an 􀁄-1,4-bond(s)
which have an average degree of polymerization of 12 to 18
30 are bound by an 􀁄-1,6-bond(s) at a ratio of around one chain
every about 3 units of glucose, to a sugar chain consisting
of glucoses bound via an 􀁄-1,4-bond(s). In addition,
similarly, a sugar chain consisting of glucoses bound by
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an 􀁄-1,4-bond(s) is bound by an 􀁄-1,6-bond to a branch chain
bound by an 􀁄-1,6-bond(s). For this reason, glycogen forms
a network structure. It is also possible to enzymatically
synthesize a glycogen. The degree of polymerization of the
5 glycogen used in the present invention is preferably about
500 or more, more preferably about 1 x 103 or more, still
more preferably about 2 x 103 or more, and most preferably
about 3 x 103 or more. The degree of polymerization of the
glycogen used in the present invention is preferably about
1 x 107 or less, more preferably about 3 x 106 10 or less, still
more preferably about 1 x 106 or less and most preferably
about 3 x 105 or less.
[0071] Dextrin is one kind of glucan constructed of glucose,
and is a glucan having a medium complexity between those
15 of starch and those of maltose. Dextrin is obtained by
partially degrading starch by an acid, an alkyl or an enzyme.
The degree of polymerization of the dextrin used in the present
invention is preferably about 10 or more, more preferably
about 20 or more, still more preferably about 30 or more,
20 and most preferably about 50 or more. The degree of
polymerization of the dextrin used in the present invention
is preferably about 1 x 104 or less, more preferably about
9 x 103 or less, still more preferably about 7 x 103 or less
and most preferably about 5 x 103 or less.
25 [0072] The enzymatically synthesized branched glucan
refers to a branched glucan synthesized using an enzyme.
By adding a branching enzyme to the reaction solution upon
synthesis of amylose by the SP-GP method, the product can
be branched. The extent of branching can be regulated by
30 the added amount of the branching enzyme. Since the
enzymatically synthesized branched glucan has a uniform
structure as compared with a natural branched glucan, it
is very advantageous when used as a pharmaceutical material.
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For example, the degree of polymerization of the
enzymatically synthesized branched glucan used in the present
invention is preferably about 10 or more, more preferably
about 20 or more, still more preferably about 30 or more,
and most preferably about 500 or more. The 5 degree of
polymerization of the enzymatically synthesized branched
glucan used in the present invention is preferably about
2 x 105 or less, more preferably about 1 x 105 or less, still
more preferably about 5 x 104 or less and most preferably
about 3 x 104 10 or less.
[0073] In the present specification, the term "highly
branched cyclic glucan" refers to a glucan having an
internally branched cyclic structural moiety and an
externally branched structural moiety and having a degree
15 of polymerization of 50 or more. The highly branched cyclic
glucan may have at least two non-reducing ends as a whole
molecule. The degree of polymerization of the highly
branched cyclic glucan as a whole molecule that can be used
in the present invention is preferably about 50 or more,
20 more preferably about 60 or more, and still more preferably
about 100 or more. The degree of polymerization of the highly
branched cyclic glucan as a whole molecule that can be used
in the present invention is preferably about 1 x 104 or less,
more preferably about 7 x 103 or less, and still more preferably
about 5 x 103 25 or less.
[0074] The degree of polymerization of the internally
branched cyclic structural moiety present in the highly
branched cyclic glucan is preferably about 10 or more, more
preferably about 15 or more, and further preferably about
30 20 or more. The degree of polymerization of the internally
branched cyclic structural moiety present in the highly
branched cyclic glucan is preferably about 500 or less, more
preferably about 300 or less, and further preferably about
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100 or less.
[0075] The degree of polymerization of the externally
branched structural moiety present in the highly branched
cyclic glucan is preferably about 40 or more, more preferably
about 100 or more, further preferably 5 bly about 300 or more,
and further more preferably about 500 or more. The degree
of polymerization of the externally branched structural
moiety present in the highly branched cyclic glucan is
preferably about 3 x 103 or less, more preferably about 1
x 103 10 or less, further preferably about 500 or less, and further
more preferably about 300 or less.
[0076] The number of 􀁄-1,6-glucosidic bonds in the
internally branched cyclic structural moiety present in the
highly branched cyclic glucan may be at least one, and for
15 example, can be one or more, 5 or more, 10 or more or the
like; the number of 􀁄-1,6-glucosidic bonds in the internally
branched cyclic structural moiety can be, for example, about
200 or less, about 50 or less, about 30 or less, about 15
or less, about 10 or less or the like.
20 The number of branches (that is, the number of
􀁄-1,6-glucosidic bonds) in the externally branched
structural moiety present in the highly branched cyclic
glucan is preferably about 2 or more, more preferably about
3 or more, further preferably about 5 or more, and especially
25 preferably about 10 or more. The number of branches (that
is, the number of 􀁄-1,6-glucosidic bonds) in the externally
branched structural moiety present in the highly branched
cyclic glucan is preferably about 5 x 103 or less, more
preferably about 4 x 103 or less, and further preferably about
3 x 103 30 or less.
[0077] As the highly branched cyclic glucan, a highly
branched cyclic glucan having one kind of a degree of
polymerization may be used alone, or a mixture of highly
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branched cyclic glucans having a variety of degree of
polymerization may be used. Preferably, the degrees of
polymerization of the highly branched cyclic glucan is such
that the ratio of the degrees of polymerization of the maximum
5 degree of polymerization to the minimum degree of
polymerization is about 100 or less, more preferably about
50 or less, and further more preferably about 10 or less.
[0078] The highly branched cyclic glucan is preferably a
glucan having an internally branched cyclic structural moiety
10 and an externally branched structural moiety and having a
degree of polymerization in a range of 50 to 5 x 103, wherein
the internally branched cyclic structural moiety is a cyclic
structural moiety formed with an 􀁄-1,4-glucosidic bond and
an 􀁄-1,6-glucosidic bond, and the externally branched
15 structural moiety is a non-cyclic structural moiety bound
to the internally branched cyclic structural moiety. The
degree of polymerization of each unit chain of this externally
branched structural moiety is, on average, preferably about
10 or more and preferably about 20 or less. The highly
20 branched cyclic glucan and a method for producing the same
are described in detail in Japanese Laid-Open Publication
No. 8-134104 (Japanese Patent No. 3107358), and this glucan
can be produced according to the description of it. The
highly branched cyclic glucan is commercially available,
25 for example, as "Cluster Dextrin" from Ezaki Glico Co., Ltd.
The degree of polymerization of the highly branched cyclic
glucan used in the present invention is preferably about
50 or more, more preferably about 70 or more, further
preferably about 100 or more, most preferably about 150 or
30 more. The degree of polymerization of the highly branched
cyclic glucan used in the present invention is preferably
about 1 x 104 or less, more preferably about 7 x 103 or less,
further preferably about 5 x 103 or less, and most preferably
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about 4 x 103 or less.
[0079] In a specific embodiment, the branched glucan can
be particulate. It is known that particles having a diameter
of about 4 nm or less are excreted from kidney, particles
having a diameter of about 4 nm to about 200 nm are 5 circulated
in blood for a long time, particles having a diameter of
about 200 nm to about 7 􀁐m are captured by a reticuloendothelial
system, and particles having a diameter of about 7 􀁐m or
more obstruct capillary blood vessels. A
10 reticuloendothelial system is distributed in liver and spleen.
For this reason, by controlling the particle size of the
branched glucan, the pharmacokinetics of the amino
sugar-containing glucan and a modified product thereof and
a conjugate thereof of the present invention in vivo can
15 be controlled. When one intends to circulate the particles
in blood for a long time, the particle size of a particulate
branched glucan is, as the diameter, preferably about 4 nm
or more, more preferably about 10 nm or more, preferably
about 200 nm or less, and more preferably about 100 nm or
20 less. The molecular weight of the particulate branched
glucan having such a particle size is preferably about 5
x 105 or more, more preferably about 1 x 106 or more, preferably
about 5 x 107 or less, and more preferably about 2 x 107 or
less. For example, since it is known that particles having
25 a diameter of 20 to 50 nm are accumulated in cancer cells,
when it is intended that the particles are accumulated in
cancer cells, the particle size of the particulate branched
glucan is, as the diameter, preferably about 10 nm or more,
more preferably about 15 nm or more, preferably about 100
30 nm or less, and more preferably about 50 nm or less. The
molecular weight of the particulate branched glucan having
such a particle size is preferably about 5 x 105 or more,
more preferably about 1 x 106 or more, preferably about 2
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x 107 or less, and more preferably about 5 x 106 or less.
[0080] The number of branches in the 􀁄-glucan (i.e. the
number of 􀁄-1,6-glucosidic bonds) is preferably about 1 or
more, more preferably about 10 or more, further preferably
about 30 or more. The number of branches of the 􀁄5 -glucan
(i.e. the number of 􀁄-1,6-glucosidic bonds) is preferably
about 5 x 103 or less, more preferably about 3 x 103 or less,
further preferably about 1 x 103 or less.
[0081] In the branched 􀁄-glucan used in the present
10 invention, the ratio of the number of 􀁄-1,6-glucosidic bonds
relative to the number of 􀁄-1,4-glucosidic bonds ("number
of 􀁄-1,6-glucosidic bonds" : "number of 􀁄-1,4-glucosidic
bonds") is preferably 1 : 1 to 1 : 1 x 103, more preferably
1 : 1.1 to 1 : 500, further preferably 1 : 1.2 to 1 : 100,
15 further more preferably 1 : 1.5 to 1 : 50, and most preferably
1 : 2 to 1 : 20. The ratio may be, in some cases, 1 : 10
to 1 : 5 x 103, 1 : 50 to 1 : 1 x 103, or 1 : 100 to 1 : 500.
In the present invention, the lower limit of the branching
frequency of the branched glucan is preferably 0.2% or more,
20 preferably 1% or more, more preferably 2% or more, and most
preferably 5% or more. While there is no specific upper limit
of the branching frequency, it can be, for example, 99% or
less, 90% or less, 85% or less, 65% or less, 50% or less,
or the like. The branching frequency is calculated by
25 {(number of 􀁄-1,6-bonds)/(sum of 􀁄-1,4-bonds and 􀁄-1,6-bonds
in glucan)} x 100.
[0082] The 􀁄-1,6-glucosidic bonds may be randomly
distributed in the 􀁄-glucan or may be homogeneously
distributed in the 􀁄-glucan. A distribution to such an extent
30 that a linear chain part(s) of 5 or more saccharide units
can be formed in the 􀁄-glucan is preferable.
[0083] In the present invention, a modified product of the
glucan may be used in place of the glucan. Examples of the
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modified product of the glucan include a modified starch
and an esterified product of the glucan explained above.
Furthermore, the modified product of the glucan may be a
hydroxyl group-modified product or a reducing end-modified
product. In 5 addition, as described later, after at least
one amino monosaccharide residue is 􀁄-1,4-bound to each of
at least one non-reducing end of the glucan, a glucan moiety
may be modified.
[0084] The modified starch is a starch which was made to
10 have a nature that it is easier to use by subjecting a natural
starch to treatment such as hydrolysis, esterification or
gelatinization. Wide variety of modified starches having
a variety of combinations of a gelatinization initiation
temperature, a viscosity of a starch paste, a degree of
15 transparency of a starch paste, stability against
retrogradation and the like are available. There are various
types of modified starches. An example of such a starch is
a starch obtained by immersing starch granules in an acid
at a gelatinization temperature or lower of the starch,
20 thereby cutting a starch molecule but not destroying starch
granules.
[0085] Examples of the modified product of the glucan other
than the modified starch include a modified product in which
at least one of alcoholic hydroxyl groups of an unmodified
25 glucan is modified (hereinafter, in the present specification,
referred to as a "hydroxyl group-modified product of glucan"),
a modified product in which some of non-reducing ends of
the glucan is modified (hereinafter, in the present
specification, referred to as a " non-reducing end-modified
30 product of glucan") and a modified product in which the
reducing end of a glucan is modified (hereinafter, in the
present specification, referred to as a "reducing
end-modified product of glucan").
EG037PCT
[0086] Examples of the modification at a hydroxyl group
include hydroxyalkylation, alkylation, acylation,
carboxymethylation, sulfation and phosphorylation. It is
preferable that modification at a hydroxyl group is a
modification which can be removed with an enzyme in a 5 body.
The hydroxyl group-modified product of the glucan is
preferably an acylated glucan, and further preferably an
acetylated glucan. The frequency of introduction of the
modifying group(s) into alcoholic hydroxyl groups can be
10 arbitrarily set at the time of a modification reaction of
the glucan. The frequency of introduction of the modifying
group(s) into alcoholic hydroxyl groups is expressed as DS,
and DS1 means the state where one modifying group per glucose
residue is introduced. DS can be calculated by DS = (number
15 of modifying group)/(number of glucose residue). Since
there is an OH group at the 2-position, the 3-position and
the 6-position in an unmodified glucose residue,
theoretically, maximum 3 modifying groups per glucose residue
can be introduced. For this reason, the upper limit of DS
20 is usually 3. The frequency of introduction of the modifying
group(s) into alcoholic hydroxyl groups is about DS 0.01
or more, more preferably about DS 0.03 or more, further
preferably about DS 0.05 or more, particularly preferably
about DS 0.07 or more, and most preferably about DS 0.1 or
25 more. The frequency of introduction of modifying group(s)
is preferably about DS 1.5 or less, more preferably about
DS 1.3 or less, further preferably about DS 1.1 or less,
particularly preferably about DS 1.0 or less, and most
preferably about DS 0.9 or less. By modifying the glucan,
30 degradation of the glucan in blood or in a body is suppressed.
[0087] Examples of modification at a non-reducing end
include binding with a targeting molecule such as a mannose
residue or a galactose residue. Modification at a
EG037PCT
non-reducing end will be explained in detail in the following
2.6 and 3 sections. A non-reducing end-modified product is
preferably a conjugate with a mannose residue or a conjugate
with a galactose residue.
[5 0088] Examples of modification at a reducing end include
binding with a substance selected from the group consisting
of a monosaccharide, a non-reducing carbohydrate, a
biocompatible macromolecule, a liposome constituent
component, a glycoside and an amine group-containing
10 low-molecular weight substance. Modification at a
non-reducing end will be explained in detail in the following
2.6 and 3 sections.
[0089] (1.2) Amino sugar
Amino sugar is a generic name of sugars wherein at least
15 one hydroxyl group is replaced with an amino group. It is
preferable that the sugar moiety of an amino sugar is a
monosaccharide, and preferably a hexose. When the sugar
moiety is a hexose, the number of amino groups in the amino
sugar can be any integer of 1 to 4, preferably 1 or 2, and
20 most preferably 1. In the present invention, amino sugars
wherein the hydroxyl group at the 2-position of the sugar
is replaced with an amino group are preferable. Example of
representative amino sugars includes glucosamine,
galactosamine, and mannosamine. Glucosamine is the most
25 preferable as the amino sugar in the present invention. A
plural kind of amino sugars may be used in mixed, or one
kind of amino sugar may be used.
[0090] (2.Method for producing an amino sugar-containing
glucan)
30 (2.1) Amino sugar-1-phosphate
In the method of the present invention, amino
sugar-1-phosphate can be used. Preferably,
glucosamine-1-phosphate, galactosamine-1-phosphate or
EG037PCT
mannosamine-1-phosphate is used. The amino
sugar-1-phosphate may be commercially available amino
sugar-1-phosphate, or amino sugar-1-phosphate synthesized
by a chemical method, an enzymatic method, or a biological
method such as fermentation. The method for producing 5 g amino
sugar-1-phosphate is known in the art. For example, a method
for synthesizing glucosamine-1-phosphate is disclosed in,
for example, Nawaji, et al., Carbohydr. Res. 2008, 343,
2692-2696.
10 [0091] As an amino sugar-1-phosphate, any of amino
sugar-1-phosphate not in a salt form and amino
sugar-1-phosphate in the form of a salt can be used. For
example, as glucosamine-1-phosphate, any of
glucosamine-1-phosphate not in a salt form and
15 glucosamine-1-phosphate in the form of a salt can be used.
For example, a metal salt of glucosamine-1-phosphate can
be used, and an alkali metal salt of glucosamine-1-phosphate
(for example, disodium glucosamine-1-phosphate and
dipotassium glucosamine-1-phosphate) can be used.
20 [0092] (2.2) 􀁄-Glucan phosphorylase
In the present specification, the term "􀁄-glucan
phosphorylase" means an enzymes having 􀁄-glucan
phosphorylase activity. 􀁄-Glucan phosphorylase is
classified in EC 2.4.1.1. 􀁄-Glucan phosphorylase activity
25 refers to an activity catalyzing a reaction producing
glucose-1-phosphate and partial degraded products of an
􀁄-1,4-glucan from inorganic phosphate and the 􀁄-1,4-glucan,
or the reverse reaction thereof. 􀁄-Glucan phosphorylase can
also catalyze an 􀁄-1,4-glucan synthesizing reaction which
30 is the reverse reaction relative to phosphorolysis. In which
direction a reaction proceeds depends on the amount of
substrate.
[0093] In the present invention, any 􀁄-glucan phosphorylase
EG037PCT
can be used as long as it has a function to transfer a
glucosamine residue to a non-reducing end of glucan.
􀁄-glucan phosphorylase used in the present invention can
be derived from a bacterium, a yeast, an animal or a plant.
􀁄-glucan phosphorylase 5 rylase of the present invention can be
derived from, for example, potato, sweet potato, Fava bean,
Arabidopsis thaliana, spinach, corn, rice, wheat, Citrus
hybrid cultivar, Aquifex aeolicus, Thermotoga maritima,
Thermococcus zilligii, Thermoanaerobacter pseudethanolicus,
10 or the like.
[0094] It is preferable that 􀁄-glucan phosphorylase used
in the present invention is 􀁄-glucan phosphorylase derived
from Aquifex aeolicus VF5. In a specific embodiment,
potato-derived 􀁄-glucan phosphorylase or Thermococcus
15 zilligii AN1-derived 􀁄-glucan phosphorylase may be used.
[0095] The base sequence of 􀁄-glucan phosphorylases derived
from Aquifex aeolicus VF5 is set forth in SEQ ID NO: 1, and
its amino acid sequence is set forth in positions 1-692 of
SEQ ID NO: 2. The amino acid sequence of 􀁄-glucan
20 phosphorylases derived from Aquifex aeolicus VF5 has about
21% to about 24 % sequence identity with the amino acid sequence
of plant 􀁄-glucan phosphorylases, about 34% sequence identity
with the amino acid sequence of 􀁄-glucan phosphorylases
derived from Thermus thermophilus, and about 38% sequence
25 identity with the amino acid sequence of 􀁄-glucan
phosphorylases derived from Thermococcus litoralis. It has
about 38% sequence identity with the amino acid sequence
of 􀁄-glucan phosphorylases derived from Thermotoga maritima,
about 38% sequence identity with the amino acid sequence
30 of maltodextrin phosphorylases derived from Thermococcus
zilligii, and about 33% sequence identity with those of
Thermoanaerobacter pseudethanolicus.
[0096] The base sequence of type L 􀁄-glucan phosphorylases
EG037PCT
derived from potato is set forth in SEQ ID NO: 3, and its
amino acid sequence is set forth in positions 15-930 of SEQ
ID NO: 4. The base sequence of 􀁄-glucan phosphorylases
derived from Thermococcus zilligii AN1 is set forth in SEQ
ID NO: 5, and its amino acid sequence is set forth in 5 positions
1-717 of SEQ ID NO: 6.
[0097] In the present specification, an enzyme "derived
from" an organism, means not only that the enzyme is directly
isolated from the organism, but also refers to an enzyme
10 obtained by utilizing the organism in any form. For example,
when a gene encoding an enzyme obtained from an organism
is introduced into Escherichia coli, and the enzyme is
isolated from that Escherichia coli, the enzyme is referred
to as being "derived from" the organism.
15 [0098] In the present specification, "identity" of a
sequence (for example, an amino acid sequence, a base sequence
and the like) refers to the degree of occurrence of the same
amino acid (base when base sequences are compared) between
two sequences. Identity can be generally determined by
20 comparing two amino acid sequences or two base sequences,
and comparing these two sequences which are aligned in an
optimal format, which can contain additions or deletions.
[0099] In the present specification, the identity of
sequences is calculated using maximum matching of GENETYX-WIN
25 Ver.4.0 (Genetics Co., Ltd.). This program aligns sequence
data to be analyzed, and sequence data to be compared so
that amino acid pairs matched between sequences become
greatest while substitution and deletion are considered,
and thereupon, gives a score to each of Matches, Mismatches,
30 and Gaps, calculates a sum, outputs alignment at the smallest
sum, and calculates identity thereupon (Reference: Takeishi,
K., and Gotoh, O. 1984. Sequence Relationships among Various
4.5 S RNA Species J. Biochem. 92:1173-1177).
EG037PCT
[0100] For example, the amino acid sequence of 􀁄-glucan
phosphorylases used in the present invention can be same
with SEQ ID NO: 2, 4 or 6, i.e., it can have 100% identity.
In another embodiment, as long as having activity to transfer
glucosamine residue to non-reducing 5 g end of a glucan, this
amino acid sequence may be altered in up to a certain number
of amino acids compared with a reference amino acid sequence.
Such alterations can be selected from the group consisting
of a deletion, a substitution (including conservative
10 substitution and non-conservative substitution), or an
insertion of at least 1 (for example, 1 or several) amino
acids. This alteration may occur at a position of an amino
terminus or a carboxyl terminus of the amino acid sequence
of SEQ ID NO: 2, 4 or 6, or may occur at any position other
15 than these termini. Alteration of an amino acid residue may
be interspersed with one residue, or a few residues may be
contiguous. For example, 􀁄-glucan phosphorylases used in
the present invention may be added with amino acid residues
(preferably about 20 or less residues, more preferably about
20 10 or less residues, and further preferably about 5 or less
residues) at either terminus of the amino acid sequence of
SEQ ID NO: 2, 4 or 6, for the reasons such as to make ease
of purification of the enzyme, to increase stability, or
the like.
25 [0101] The 􀁄-glucan phosphorylase used in the present
invention has an amino acid sequence which has preferably
about 50% or more, more preferably about 60% or more, further
more preferably about 70% or more, still more preferably
about 80% or more, particularly more preferably about 90%
30 or more, and most preferably about 95% or more sequence
identity with the amino acid sequence of SEQ ID NO: 2, 4
or 6 and has an activity transferring a glucosamine residue
to a non-reducing end of the glucan. The 􀁄-glucan
EG037PCT
phosphorylase used in the present invention can have an amino
acid sequence which has about 96 % or more, about 97% or
more, about 98 % or more, or about 99 % more sequence identity
with amino acid sequence of SEQ ID NO: 2, 4 or 6.
[0102] The amount of the 􀁄-glucan phosphorylase 5 ylase contained
in a solution at the start of the reaction is preferably
about 0.01 U/ml or more, more preferably about 0.1 U/ml or
more, particularly preferably about 0.5 U/ml or more, and
most preferably about 1 U/ml or more. The amount of the
10 􀁄-glucan phosphorylase contained in a solution at the start
of the reaction is preferably about 1,000 U/ml or less, more
preferably about 100 U/ml or less, particularly preferably
about 50 U/ml or less, and most preferably about 20 U/ml
or less. If the weight of 􀁄-glucan phosphorylase is too large,
15 it may became easy to aggregate the enzyme denatured during
the reaction. If the amount used is too small, reaction
itself occurred, but the yield of glucan may be lowered.
It is noted that unit amount of 􀁄-glucan phosphorylase is
defined as follows:
20 Regarding one unit of 􀁄-glucan phosphorylase, an 􀁄-glucan
phosphorylase activity which produces 1 􀁐mol inorganic
phosphate (Pi) per one minute shall be one unit (U or Unit).
This measurement of 􀁄-glucan phosphorylase activity
quantitates free inorganic phosphate (Pi) produced from G-1-P.
25 After 200 􀁐l of a reaction solution (containing 12.5 mM G-1-P,
1% dextrin and an enzyme solution in a 100 mM acetate buffer
(pH 6.0)) is incubated at 50°C for 15 minutes, 800 􀁐l of
a molybdenum regent (15 mM ammonium molybdate, 100 mM zinc
acetate) is added, and this is stirred to stop the reaction.
30 200 􀁐l of 568 mM ascorbic acid (pH 5.8) is added, followed
by mixing. After incubation at 30°C for 15 minutes, an
absorbance is measured at 850 nm using a spectrophotometer.
An absorbance is measured similarly using inorganic phosphate
EG037PCT
having the known concentration, and a standard curve is
produced. An absorbance value obtained for a sample is fitted
to this standard curve, and the amount of inorganic phosphate
in the sample is determined. Inorganic phosphate is
quantitated as a phosphoric acid ion. The 5 amount of
glucose-1-phosphate is not quantitated.
[0103] (2.3 Production of 􀁄-glucan phosphorylase)
􀁄-Glucan phosphorylase used in the present invention
can be directly isolated from an organism producing 􀁄-glucan
10 phosphorylase, such as the aforementioned organisms, present
in the natural world. Alternatively, 􀁄-glucan
phosphorylase used in the present invention may be isolated
from a microorganism (for example, bacteria, fungi and the
like) which has been genetically modified with a gene encoding
15 􀁄-glucan phosphorylase isolated from the aforementioned
organism.
[0104] In a preferred embodiment, 􀁄-glucan phosphorylase
derived from Aquifex aeolicus VF5 is produced by chemically
synthesizing a gene fragment of SEQ ID NO: 1, constructing
20 an expression vector containing this gene fragment,
introducing this expression vector into a microorganism to
make a recombinant microorganism, culturing this recombinant
microorganism to produce 􀁄-glucan phosphorylase, and
collecting produced 􀁄-glucan phosphorylase. 􀁄-glucan
25 phosphorylase derived from other organism can also be
produced as well. An enzymatic production method by gene
recombination is well-known to those skilled in the art.
A host microorganism used in the present invention includes
a prokaryote and a eukaryote, and a mesophile is preferable.
30 Examples of a particularly preferred microorganism include,
but not limited to, Escherichia coli.

CLAIMS
Claim 1. A glucosamine-containing branched glucan, wherein
the glucan has a plurality of non-reducing ends and at least
one glucosamine residue is bound via an 􀁄5 -1,4-bond to each
of two or more non-reducing ends of the branched 􀁄-1,4-glucan,
but no glucosamine residue is present at the position other
than the non-reducing ends of the branched 􀁄-1,4-glucan,
and the degree of polymerization of the branched 􀁄-1,4-glucan
is 15 or more and 4 x 105 10 or less.
Claim 2. The glucosamine-containing branched glucan
according to claim 1, wherein the branched 􀁄-1,4-glucan is
selected from the group consisting of a branched
15 maltooligosaccharide, a starch, amylopectin, glycogen,
dextrin, enzymatically synthesized branched glucan and
highly branched cyclic glucan.
Claim 3. A hydroxyl group-modified product of the
20 glucosamine-containing branched glucan according to claim
1, wherein the modification on the hydroxyl group is a
modification on some or all of alcoholic hydroxyl groups
of the glucan, and the modification on the hydroxyl group
is independently selected from the group consisting of
25 hydroxyalkylation, alkylation, acetylation,
carboxymethylation, sulfation and phosphorylation.
Claim 4. A reducing end-modified product of the
glucosamine-containing branched glucan according to claim
30 1 or a hydroxyl group-modified product thereof.
Claim 5. An amino group-modified product of the
glucosamine-containing branched glucan according to claim
EG037PCT
1, a hydroxyl group-modified product thereof or a reducing
end-modified product thereof, wherein the modification on
the amino group is a modification on some or all of amino
groups of the glucosamine residues, the modification on the
amino group is attained by a reaction 5 ion of the amino group
and an amino group-modifying reagent, and the amino
group-modifying reagent has at least one carboxyl group and
at least one other functional group.
10 Claim 6. A non-reducing end-modified product of the
glucosamine-containing branched glucan according to claim
1, a hydroxyl group-modified product thereof, a reducing
end-modified product thereof or an amino group-modified
product thereof, wherein a targeting molecule is bound to
15 at least one of non-reducing ends to which the glucosamine
residue of the branched glucan is not bound, wherein the
targeting molecule is selected from the group consisting
of mannose, galactose, glucuronic acid, N-acetylglucosamine,
xylose, fucose, galactosamine, an antibody, an antibody
20 fragment, a receptor, a receptor fragment and a receptor
ligand.
Claim 7. A method for producing a glucosamine-containing
branched glucan, characterized by allowing an 􀁄-glucan
25 phosphorylase to act on an aqueous solution comprising a
branched 􀁄-1,4-glucan and glucosamine-1-phosphate, wherein
the degree of polymerization of the branched 􀁄-1,4-glucan
is 15 or more and 4 x 105 or less.
30 Claim 8. The method according to claim 7, wherein the
􀁄-glucan phosphorylase has 95% or more sequence identity
with the amino acid sequence of 􀁄-glucan phosphorylase
derived from Aquifex aeolicus VF5, and has activity of
EG037PCT
transferring glucosamine to a non-reducing end of a branched
glucan to form an 􀁄-1,4-bond.
Claim 9. A medicament comprising the
glucosamine-5 containing branched glucan according to claim
1, a hydroxyl group-modified product thereof, a reducing
end-modified product thereof, an amino group-modified
product thereof, or a non-reducing end-modified product
thereof, and a medically effective ingredient.
10
Claim 10. The medicament according to claim 9, wherein the
medically effective ingredient is selected from the group
consisting of a low-molecular weight organic compound, a
protein, a peptide, an antibody, an antibody fragment, a
15 receptor, a receptor fragment, a DNA, an RNA, a siRNA, an
miRNA and an RNA aptamer.
Claim 11. A composition for clinical diagnosis comprising
the glucosamine-containing branched glucan according to
20 claim 1, a hydroxyl group-modified product thereof, a
reducing end-modified product thereof, an amino
group-modified product thereof, or a non-reducing
end-modified product thereof.
25 Claim 12. A nanoparticulate carrier for a DDS comprising
the glucosamine-containing branched glucan according to
claim 1, a hydroxyl group-modified product thereof, a
reducing end-modified product thereof, an amino
group-modified product thereof, or a non-reducing
30 end-modified product thereof.
Claim 13. The carrier according to claim 12, wherein the
nanoparticulate carrier for a DDS is selected from the group
EG037PCT
consisting of a liposome, a virus particle, a macromolecule
micelle and a nanogel composed of macromolecule bearing
hydrophobic groups.
Claim 14. A 5 complex formed with a nucleic acid molecule and
the glucosamine-containing branched glucan according to
claim 1.
Claim 15. The complex according to claim 14, wherein the
10 nucleic acid molecule is selected from the group consisting
of a DNA, an RNA, a siRNA, an miRNA and an RNA aptamer.
Claim 16. A complex formed with the complex carrier according
to claim 14, and a cationic polymer or a cationic lipid.
15
Claim 17. The complex according to claim 16, wherein the
cationic polymer comprises at least one cationic polymer
selected from the group consisting of polyethyleneimine,
polylysine, polyarginine, polyamidoamine dendrimer,
20 poly(aminostyrene), chitosan, a cationic glucan and
DEAE-dextran.
Claim 18. A method for delivering a nucleic acid molecule
into an isolated cell, comprising contacting the complex
25 according to claim 14 with the cell.