DESCRIPTION
TITLE OF THE INVENTION: NON-REDUCING END MODIFIED GLUCAN,
METHOD FOR PRODUCING SAME, AND USE THEREOF
5
TECHNICAL FIELD
[0001] The present invention relates to a glucan in which
at least one (preferably at least two) non-reducing end is
modified, a modified product thereof, as well as a method
10 for producing the same, and utilization of the same. More
preferably, the present invention relates to a glucan in
which at least one residue selected from an
N-acetylglucosamine residue and a galactose residue is bound
via an 􀁄-1,4-bond to each of at least one (preferably at
15 least two) non-reducing end, a modified product thereof,
as well as a method for producing the same, and utilization
of the same.
BACKGROUND ART
20 [0002] A medically effective ingredient of medicaments is
rapidly changing from a chemically synthesized stable
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
25 of stabilizing these unstable medically effective
ingredients to keep the blood concentration of the medically
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
30 increasing. Under such a background, a so-called drug
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
EG038PCT
ingredient to act on "a required site" at "a required amount"
for "a required period of time", that is, controlling it
with an ideal pharmacokinetics for a drug to maximally exert
the effect.
[5 0003] In the DDS technique, a modifying material for a
medically effective ingredient is important. The term
"modifying material for a medically effective ingredient"
in the present specification refers to a material which
modifies a medically effective ingredient by covalently
10 binding, or via non-covalent type interaction, with a
medically effective ingredient. By utilizing the modifying
material, a variety of properties (for example,
pharmacokinetics (for example, absorption, distribution,
metabolism and excretion), pharmacological effect,
15 stability and the like) of the medically effective ingredient
can be modified. As a substance which has been used
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,
20 polyethylene glycol (PEG) which is a synthetic macromolecule,
and derivatives thereof are widely utilized as a modifying
material for medically effective ingredients. Many
medically effective ingredient-modifying materials having
a functional group for binding the medically effective
25 ingredient on a terminus of a PEG chain have been developed,
and such modifying materials are actually utilized in
producing a medicament. Specific application examples
include pegylated interferon 􀁄 (product name: PEGASYS).
Since interferon 􀁄 has a small molecular weight and is easily
30 excreted into urine, there was a problem that it has a short
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
EG038PCT
40,000 to form an interferon 􀁄-conjugate having a high
molecular weight. As described above, the remarkable effect
is recognized in the modification of the medically effective
ingredient or the nanoparticulate carrier for DDSs, with
a 5 macromolecular material.
[0004] However, on the other hand, a problem has been pointed
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
10 administered to blood, there is a risk that the macromolecule
is accumulated in a particular organ and a risk that side
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
15 glomerular filtration and is rapidly excreted into urine,
but a molecule having a molecular weight of a few tens thousands
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
20 which can be safely utilized is expected.
[0005] 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,
25 further, as a functional material.
[0006] An 􀁄-1,4 glucan, represented by a starch, glycogen
or the like, is a safe natural material which is easily degraded
in the environment and also in a body of a human, and is
promising as a material in the industry of a DDS medicament,
30 regenerative medicine, intracorporeal imaging or the like,
which is expected to grow in the future. When the 􀁄-1,4 glucan
is used for such a purpose, it is necessary to impart to
the 􀁄-1,4 glucan a function of interacting with a medically
EG038PCT
effective ingredient and a function of targeting to an organ
and a tissue, and at the same time, it is necessary to control
degradability of the 􀁄-1,4 glucan.
[0007] On the other hand, in the pharmaceutical field, a
vaccine for preventing or treating an infectious 5 disease,
a cancer or the like is being developed. The vaccine is for
administration of an antigen into a body and preventing or
treating a disease utilizing an immune response to the antigen.
Usually, as the antigen, in the case of a vaccine for an
10 infectious disease, a part of a pathogen, a whole inactivated
pathogen, or a surface protein or a peptide of a pathogen
is used. As the antigen, in the case of a cancer vaccine,
a protein or a peptide which is specifically expressed on
a cancer cell surface is used. Generally, since mere
15 administration of an antigen alone does not lead to effective
immunity induction in many cases, a substance which induces
an immune response (adjuvant) is administered into a body
together with the antigen. For developing an effective
vaccine, it is important to develop a safe and effective
20 adjuvant. It is expected an effective and safe adjuvant
effectively functions when a purified protein or peptide
is administered as an antigen.
[0008] In recent years, due to progress of natural science,
a mechanism of immunological system has been revealed. It
25 has been revealed that a dendritic cell or a macrophage which
is an antigen-presenting cell plays a central role of an
immune reaction, and activation of an antigen-presenting
cell and induction of production of an inflammatory cytokine
(IL-2, IL-6, IL-12, TNF-􀁄 and the like) that results from
30 the activation, are necessary for effective immunity
induction. It has also been revealed that a substance which
has been traditionally utilized as an adjuvant is involved
in induction and enhancement of an immune reaction through
EG038PCT
stimulation and activation of an antigen-presenting cell,
and induction of production of an inflammatory cytokine
􀀋􀂳􀀭􀁄􀁑􀁈􀁚􀁄􀁜􀂶􀁖􀀃􀀬􀁐􀁐􀁘􀁑􀁒􀁅􀁌􀁒􀁏􀁒􀁊􀁜􀂴􀀃􀀋􀁗􀁕􀁄􀁑􀁖􀁏􀁄􀁗􀁌􀁒􀁑􀀃􀁘􀁑􀁇􀁈􀁕􀀃􀁖􀁘􀁓􀁈􀁕􀁙􀁌􀁖􀁌􀁒􀁑􀀃
by Takehiko Sasazuki, Nankodo Co., Ltd.) (original book
􀂳􀀭􀁄􀁑􀁈􀁚􀁄􀁜􀂶􀁖􀀃􀀬􀁐􀁐􀁘􀁑􀁒􀁅􀁌􀁒􀁏􀁒􀁊􀁜􀀃􀁖􀁈􀁙􀁈􀁑􀁗􀁋􀀃􀁈5 􀁇􀁌􀁗􀁌􀁒􀁑􀀃Kenneth Murphy,
Paul Travers, Mark Walport 2008 Garlnd Science, Taylar &
􀀩􀁕􀁄􀁑􀁆􀁌􀁖􀀃􀀪􀁕􀁒􀁘􀁓􀀏􀀃􀀯􀀯􀀦􀂴􀀌).
[0009] Conventionally, a variety of adjuvants are known,
but there are few safe and low cost immune adjuvants which
10 can be used in tumor immunotherapy for the purpose of treating
a human tumor, or preventing metastasis and reoccurrence
of a human tumor. For example, in tumor immunotherapy
utilizing a cultured dendritic cell, keyhole limpet
hemocyanin is used as an immune adjuvant (Geiger, J. D.,
15 et al., Cancer Res., 61, pp. 8513-8519, 2001), but keyhole
limpet hemocyanin is expensive. A method of administering
cytokines such as granulocyte-macrophage colony stimulating
factor (sometimes 􀁄􀁅􀁅􀁕􀁈􀁙􀁌􀁄􀁗􀁈􀁇􀀃􀁄􀁖􀀃􀂳􀀪􀀰-􀀦􀀶􀀩􀂴) which directly
activates a dendritic cell as an immune adjuvant has been
20 proposed, but cytokines are further expensive.
[0010] In the production of a vaccine as a countermeasure
for an infectious disease, as a safe and low cost immune
adjuvant, immune adjuvants having insufficient immune
adjuvant activity such as alum (aluminum hydroxide) and
25 􀀩􀁕􀁈􀁘􀁑􀁇􀂶􀁖􀀃􀁌􀁑􀁆􀁒􀁐􀁓􀁏􀁈􀁗􀁈􀀃􀁄􀁇􀁍􀁘􀁙􀁄􀁑􀁗􀀃􀀋􀁖􀁌􀁑􀁆􀁈􀀃􀁗􀁋􀁌􀁖􀀃􀁄􀁇􀁍􀁘􀁙􀁄􀁑􀁗􀀃􀁌􀁖􀀃􀁒􀁌􀁏􀁜􀀏􀀃
toxicity is feared) are used. These are low in toxicity,
but are weak in the immune adjuvant activity, as compared
􀁚􀁌􀁗􀁋􀀃􀀩􀁕􀁈􀁘􀁑􀁇􀂶􀁖􀀃􀁆􀁒􀁐􀁓􀁏􀁈􀁗􀁈􀀃􀁄􀁇􀁍􀁘􀁙􀁄􀁑􀁗􀀃􀁚􀁋􀁌􀁆􀁋􀀃􀁌􀁖􀀃􀁘􀁖ed in an animal
experiment.
30 [0011] As described above, the conventional adjuvant (also
referred to as an immune adjuvant) has a problem in the ability
to stimulate an immune response, safety or both of them.
For this reason, it is very important to develop an adjuvant
EG038PCT
which has a high ability to stimulate an immune response,
that is, the ability to stimulate an antigen-presenting cell,
and is safe.
5 [Prior Art Documents]
[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
10 ni yoru soyaku (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
15 sareru kobunshi kagaku (Polymer Chemistry Utilized in DDS)",
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,
20 NATURE REVIEWS, DRUG DISCOVERY, VOLUME 2, MARCH 2003, 214-221
SUMMARY OF THE INVENTION
PROBLEMS TO BE SOLVED BY THE INVENTION
[0013] The present invention is intended to solve the
25 above-mentioned problems.
[0014] An ideal modifying material for the medically
effective ingredient which can be safely utilized is thought
to have the following characteristics:
(1) The modifying material shall be a macromolecular
30 material which can be degraded in a living body;
(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
EG038PCT
the same quality shall be able to produce every time;
(3) The modifying material shall have a functional group
which can bind to or interact with a medically effective
ingredient; and
(4) The modifying 5 material shall have tissue targeting
functions or cell stimulation functions.
[0015] The present inventors thought that an excellent
macromolecular substance for modifying material for the
medically effective ingredient is a glucan (in the present
10 specification, the glucan refers to an 􀁄-1,4-glucan, and
an 􀁄-1,4-glucan which is branched with an 􀁄-1,6-bond(s)).
Glycogen or a starch is accumulated in an animal's and plant's
body as a polysaccharide for storage. Glycogen or a starch
is one kind of glucan. Thus, glycogen or a starch which is
15 a component that is always present in a body of a human,
and is excellent in biocompatibility. Further, the glucan
undergoes hydrolysis by 􀁄-amylase in 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
20 can be said to be the safest macromolecular material.
[0016] The 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
25 which glucose residues are bound only with an 􀁄-1,4-bond,
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
by one, one new non-reducing end is generated. The number
30 of 􀁄-1,6-bonds can be suitably adjusted as desired upon
synthesis of a glucan molecule.
[0017] On the other hand, the greatest problem when the
glucan is utilized as the modifying material (for example,
EG038PCT
a carrier, a vaccine adjuvant or the like) as a medically
effective ingredient is that the glucan does not have a
functional group which can bind to or interact with the
medically effective ingredient. It is possible to introduce
5 a cationic or anionic functional group into a large number
of hydroxyl groups or aldehyde group on a reducing end present
in a glucan using a known chemical method.
[0018] Further, it is preferable that a carrier has the
tissue targeting function, in terms of utilization of a glucan
10 as a carrier of a medically effective ingredient. As a means
for realizing targeting to an organ and a tissue, a method
of utilizing a saccharide recognizing mechanism by a receptor
or a lectin on a cell surface has been proposed. A receptor
or a lectin on a cell surface does not recognize glucose,
15 but recognizes a monosaccharide such as galactose or mannose.
Therefore, in order to impart the tissue targeting function
to a glucan, it is necessary to bind a monosaccharide having
the targeting function, other than glucose, to a non-reducing
end of the glucan.
20 [0019] In order to utilize a glucan as a vaccine adjuvant,
it is preferable that the glucan has high stimulating activity
on an antigen-presenting cell playing a central role in an
immune response. The antigen-presenting cell (a dendritic
cell, a macrophage, or the like) is not activated by a glucan
25 consisting only of glucose residues, but is activated by
a monosaccharide such as galactose or mannose. Therefore,
in order to impart the high cell stimulating activity to
a glucan, it is necessary to bind a monosaccharide having
a cell stimulating function to a non-reducing end of the
30 glucan. However, a method of binding a monosaccharide other
than glucose via 􀁄-1,4 bond to a non-reducing end of a branched
glucan having at least two non-reducing ends has not been
previously known. For example, in Nawaji et al., Carbohydr.
EG038PCT
Res., 2008, 343, 2692-2696, it is described that
􀁄-D-glucosamine-1-phospate (GlcN-1-P) can be transferred
to a maltooligosaccharide using potato-derived glucan
phosphorylase. However, Nawaji et al. describe on page 2694,
5 right column, lines 3-5 that
N-acetylglucosamine-1-phosphate (GlcNAc-1-P) is not
recognized by phosphorylase. Recognition of a substrate by
an enzyme is extremely strict, and it is a common knowledge
of those skilled in the art that a normal substrate becomes
10 unrecognizable as a substrate in many cases when it is modified
even if only a little. A normal substrate for glucan
phosphorylase is glucose-1-phosphate, and analogues of the
substrate which were known to be recognized by glucan
phosphorylase were only 􀁄-D-xylose-1-phosphate,
15 mannose-1-phosphate, glucosamine-1-phosphate, glucuronic
acid-1-phosphate (GlcA-1-P) and
N-formylglucosamine-1-phosphate (GlcNF-1-P). For this
reason, it was thought that phosphorylase cannot be used
in order to transfer GlcNAc-1-P or galactose-1-phosphate
20 (Gal-1-P) to a glucan.
MEANS FOR SOLVING THE PROBLEMS
[0020] The present inventors intensively studied in order
to solve the aforementioned problems and found that Aquifex
25 aeolicus VF5-derived􀀃􀀃􀁄-glucan phosphorylase can utilize as
a substrate galactose-1-phosphate (Gal-1-P) and
N-acetylglucosamine-1-phosphate (GlcNAc-1-P) which are not
its original substrates, can catalyze a reaction that binds
a galactose residue (Gal residue) or an N-acetylglucosamine
30 residue (GlcNAc residue) via 􀁄-1,4 bond to a non-reducing
end of a 􀁄-1,4 glucan, and in spite of this, this enzyme
can hardly catalyze the reverse reaction thereof, and
completed the present invention based on this finding. In
EG038PCT
addition, the present inventors also found that
potato-derived 􀁄-glucan phosphorylase can use Gal-1-P as
a substrate, and Thermococcus zilligii AN1-derived 􀁄-glucan
phosphorylase can use Gal-1-P and GlcNAc-1-P as a substrate.
These 􀁄5 -glucan phosphorylases are enzymes which, when act
on glucose-1-phosphate which is the original substrate,
catalyze both a reaction wherein a glucose residue is
transferred to a glucan receptor (glucan synthesizing
reaction) and a reaction wherein the glucose on the
10 non-reducing end of a glucan is phosphorolysed to generate
glucose-1-phosphate (glucan degrading reaction). However,
surprisingly, when these enzyme use
N-acetylglucosamine-1-phosphate or galactose-1-phosphate
as a substrate, these enzymes catalyze the reaction wherein
15 the residue of these monosaccharides is transferred to a
glucan receptor (glucan synthesizing reaction), but hardly
catalyze the reaction wherein the residue of these
monosaccharides bound on the non-reducing end of a glucan
is phosphorolysed to generate
20 N-acetylglucosamine-1-phosphate or galactose -1-phosphate
(glucan degrading reaction). While glucan degrading
reaction again releases the bound monosaccharide residue,
the present inventors found that in the method of the present
invention, 􀁄-glucan phosphorylase hardly catalyzes the
25 glucan degrading reaction. That is, the 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 at least one residue
30 selected from N-acetylglucosamine residue and galactose
residue, one by one, to the plurality of non-reducing ends
of the glucan. By utilizing this characteristic of this
enzyme, the non-reducing end-modified glucan of the present
EG038PCT
invention became producible for the first time. Especially,
it could be 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 N-acetylglucosamine residue5 s
or galactose residues (for example, glucosamine residues)
to the non-reducing ends, starting with a glucan having a
large number of (for example, 5 or more) non-reducing ends.
In addition, the present inventors found that when a Gal
10 residue or a GlcNAc residue is bound via 􀁄-1,4 bond to a
non-reducing end of an 􀁄-1,4 glucan, glucoamylase resistance
can be imparted to the 􀁄-1,4 glucan. Further, the present
inventors found that the non-reducing end-modified glucan
of the present invention can be accumulated into a particular
15 tissue (e.g. liver) when it is administered into blood. The
present inventors also found that the non-reducing
end-modified glucan of the present invention has the activity
of stimulating an antigen-presenting cell (e.g. a dendritic
cell or a macrophage) to produce IL-6 (antigen-presenting
20 cell stimulating activity). In addition, the present
inventors found that the non-reducing end-modified glucan
of the present invention is excellent in biodegradability,
and degradation of the non-reducing end-modified glucan in
a living body can be controlled by modifying (e.g.
25 acetylating) a hydroxyl group of the non-reducing
end-modified glucan of the present invention.
[0021] In the non-reducing end-modified branched 􀁄-1,4
glucan (e.g. a branched 􀁄-1,4 glucan in which at least one
residue selected from an N-acetylglucosamine residue and
30 a galactose residue is bound via 􀁄-1,4 bond to each of at
least one (preferably at least two) non-reducing end(s)),
or a hydroxyl group-modified product thereof, a non-reducing
end-modified product thereof and a reducing end-modified
EG038PCT
product thereof of the present invention, when at least one
kind of residue selected from a glucuronic acid residue,
a mannose residue and a xylose residue is further bound to
at least one or more non-reducing ends among the plurality
of non-reducing ends, the antigen-5 -presenting cell
stimulating activity of these glucans can be further
improved.
[0022] In the present invention, since a saccharide (e.g.
N-acetylglucosamine or galactose) residue is transferred
10 using 􀁄-glucan phosphorylase (EC2.4.1.1), linkage of a
saccharide residue to a glucan is an 􀁄-1,4 bond. That is,
a carbon atom at a 4-position of a glucosyl residue at a
non-reducing end of a glucan, and a carbon atom at a 1-position
of a saccharide (e.g. N-acetylglucosamine or galactose)
15 residue are 􀁄-bound via an oxygen atom.
[0023] In the present specification, the glucan of the
present invention in which at least one residue selected
from an N-acetylglucosamine residue and a galactose residue
is bound via 􀁄-1,4 bond to each of at least one non-reducing
20 end of a glucan is also referred to as a 􀂳􀁑􀁒􀁑-reducing
end-􀁐􀁒􀁇􀁌􀁉􀁌􀁈􀁇􀀃􀁊􀁏􀁘􀁆􀁄􀁑􀂴􀀃􀁒􀁉􀀃􀁗􀁋􀁈􀀃􀁓􀁕􀁈􀁖􀁈􀁑􀁗􀀃􀁌􀁑􀁙􀁈􀁑􀁗􀁌􀁒􀁑.
[0024] A saccharide residue to be bound to a non-reducing
end in the non-reducing end-modified glucan of the present
invention may be a residue of a monosaccharide, or may be
25 a residue of an oligosaccharide. That is, the saccharide
residue to be bound to a non-reducing end may be an
N-acetylglucosamine residue or a galactose residue, or an
oligosaccharide residue thereof. In the present
specification, an oligosaccharide means a compound wherein
30 2 or more and 10 or less monosaccharides are bound. The degree
of polymerization of saccharide residue bound to one
non-reducing end of the non-reducing end-modified glucan
of the present invention can be, for example, about 2 or
EG038PCT
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,
about 5 or less, about 4 or less, about 3 or less, about
2 or less or the like. In 5 one embodiment, the saccharide
residue bound to one non-reducing end of the non-reducing
end-modified glucan of the present invention is residue of
2 sugars (that is, a dimer).
[0025] The introduction amount of galactose residues or
10 N-acetylglucosamine residues to a glucan can be controlled
by the branching frequency of the glucan used and the frequency
of introduction of these residues to a non-reducing end.
When one wants to increase an amount of introduction of these
residues into a glucan, using a glucan having a high branching
15 frequency and increasing the frequency of introduction of
these residues to the non-reducing end. When one wants to
decrease an amount of introduction of these residues into
a glucan, using a glucan having a low branching frequency
or decreasing the frequency of introduction of these residues
20 to the non-reducing end. The lower limit of the introduction
amount of these residues to a glucan is the state wherein
one of these residues is introduced per glucan molecule,
which can be attained by introducing a galactose residue
or an N-acetylglucosamine residue to the non-reducing end
25 of the glucan having no branching. In the case of a glucan
which is highly branched, since non-reducing ends are
distributed in an outermost layer of a glucan molecule, it
is considered that introduced either of these residues are
distributed in an outermost layer of a glucan molecule after
30 introduction of either of these residues, and this is ideal
for interaction and binding with the medically effective
ingredient. As described above, a glucan which has either
of these residues selectively bound to a non-reducing end
EG038PCT
has a possibility of being an excellent modifying material
for the medically effective ingredient.
[0026] For example, the present invention provides the
following glucans and the like:
5 (Item 1)
A glucan wherein the glucan has non-reducing ends and at
least one residue selected from an N-acetylglucosamine
residue and a galactose residue is bound via an 􀁄-1,4-bond
to each of at least one of the non-reducing ends of the glucan
10 (preferably two or more of the two or more non-reducing ends),
but neither an N-acetylglucosamine residue nor a galactose
residue is present at the position other than the non-reducing
ends of the glucan, wherein the glucan is a branched 􀁄-1,4
glucan or a linear 􀁄-1,4 glucan, and the degree of
15 polymerization of the branched 􀁄-1,4 glucan or the linear
􀁄-1,4 glucan is preferably 15 or more and 4 x 105 or less.
It is noted that the glucan which has an N-acetylglucosamine
residue or a galactose residue bound thereto of the present
invention is also referred to as the non-reducing
20 end-modified glucan of the present invention.
(Item 2)
The glucan according to item 1, wherein the glucan is a branched
􀁄-1,4-glucan, wherein the branched 􀁄-1,4-glucan has a
plurality of non-reducing ends and at least one residue
25 selected from an N-acetylglucosamine residue and a galactose
residue is bound to each of at least one (preferably two
or more) non-reducing ends of the branched 􀁄-1,4-glucan.
That is, this glucan is a branched 􀁄-1,4-glucan wherein the
branched 􀁄-1,4-glucan has a plurality of non-reducing ends
30 and at least one residue selected from an N-acetylglucosamine
residue and a galactose 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 neither an
EG038PCT
N-acetylglucosamine residue nor a galactose 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 preferably
15 5 or more and 4 x 105 or less.
(Item 3)
The glucan according to item 2, wherein the branched
􀁄-1,4-glucan is selected from the group consisting of a
branched maltooligosaccharide, starch, amylopectin,
10 glycogen, dextrin, enzymatically synthesized branched
glucan and highly branched cyclic glucan.
(Item 4)
A hydroxyl group-modified product of the glucan (non-reducing
end-modified glucan) according to any one of items 1 to 3,
15 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,
20 carboxymethylation, sulfation and phosphorylation.
(Item 5)
A reducing end-modified product of the glucan (non-reducing
end-modified glucan) according to any one of items 1 to 3
or a hydroxyl group-modified product thereof.
25 (Item 6)
A non-reducing end-modified product of the glucan
(non-reducing end-modified glucan) according to any one of
items 2 to 3 or a hydroxyl group-modified product thereof,
or a reducing end-modified product thereof, which is further
30 modified by liking a monosaccharide residue other than an
N-acetylglucosamine residue and a galactose residue via an
􀁄-1,4-bond to at least one non-reducing end of the plurality
of non-reducing ends of the branched 􀁄-1,4 glucan.
EG038PCT
(Item 6B)
The non-reducing end-modified product of the glucan
(non-reducing end-modified glucan) or a hydroxyl
group-modified product thereof, or a reducing end-modified
product thereof according to 5 item 6, wherein the
monosaccharide residue is one kind or two kinds selected
from a glucuronic acid residue, a glucosamine residue, a
mannose residue, and a xylose residue.
(Item 6C)
10 The non-reducing end-modified product of the glucan
(non-reducing end-modified glucan) or a hydroxyl
group-modified product thereof, or a reducing end-modified
product thereof according to item 6, wherein the
monosaccharide residue is one kind or two kinds selected
15 from a glucuronic acid residue and a mannose residue.
(Item 7)
A method for producing a glucan (that is, a non-reducing
end-modified glucan of the present invention) in which at
least one residue selected from an N-acetylglucosamine
20 residue and a galactose residue is bound to each of two or
more non-reducing ends, characterized by allowing an 􀁄-glucan
phosphorylase to act on an aqueous solution comprising a
glucan (preferably a branched 􀁄-1,4-glucan having two or
more non-reducing ends) and
25 N-acetylglucosamine-1-phosphate or galactose-1-phosphate,
wherein the degree of polymerization of the glucan used as
raw material is preferably 15 or more and 4 x 105 or less.
(Item 8)
The method according to item 7, wherein the 􀁄-glucan
30 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
N-acetylglucosamine residue or galactose residue to a
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non-reducing end of a glucan to form an 􀁄-1,4-bond.
(Item 9)
A medicament comprising:
the glucan comprising an N-acetylglucosamine residue
5 or a galactose residue according to any one of items 1 to
3, a hydroxyl group-modified product thereof, or a reducing
end-modified product thereof, and
a medically effective ingredient.
(Item 10)
10 A medicament comprising:
a non-reducing end-modified product of the glucan
comprising an N-acetylglucosamine residue or a galactose
residue according to any one of items 2 to 3 or a hydroxyl
group-modified product thereof, or a reducing end-modified
15 product thereof, which is further modified by liking a
monosaccharide residue other than an N-acetylglucosamine
residue and a galactose residue via an 􀁄-1,4-bond to at least
one non-reducing end of the plurality of non-reducing ends
of the branched 􀁄-1,4 glucan, wherein the monosaccharide
20 residue is one kind or two kinds selected from a glucuronic
acid residue, a glucosamine, a mannose residue, and a xylose
residue; and
a medically effective ingredient.
(Item 11)
25 The medicament according to item 9 or 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 11A)
The medicament according to any one of items 9 to 11, wherein
the medically effective ingredient is an antigen protein
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or a peptide.
(Item 12)
A composition for clinical diagnosis comprising the glucan
comprising an N-acetylglucosamine residue or a galactose
residue according to any one of items 5 ems 1 to 3, a hydroxyl
group-modified product thereof, or a reducing end-modified
product thereof.
(Item 13)
A composition for clinical diagnosis comprising a
10 non-reducing end-modified product of the glucan comprising
an N-acetylglucosamine residue or a galactose residue
according to any one of items 2 to 3 or a hydroxyl
group-modified product thereof, or a reducing end-modified
product thereof, which is further modified by liking a
15 monosaccharide residue other than an N-acetylglucosamine
residue and a galactose residue via an 􀁄-1,4-bond to at least
one non-reducing end of the plurality of non-reducing ends
of the branched 􀁄-1,4 glucan, wherein the monosaccharide
residue is one kind or two kinds selected from the group
20 consisting of a glucuronic acid residue, a glucosamine, a
mannose residue, and a xylose residue.
(Item 14)
A nanoparticulate carrier for a DDS comprising the glucan
comprising an N-acetylglucosamine residue or a galactose
25 residue according to any one of items 1 to 3, a hydroxyl
group-modified product thereof, or a reducing end-modified
product thereof.
(Item 15)
A nanoparticulate carrier for a DDS comprising a non-reducing
30 end-modified product of the glucan comprising an
N-acetylglucosamine residue or a galactose residue according
to any one of items 2 to 3 or a hydroxyl group-modified product
thereof, or a reducing end-modified product thereof, which
EG038PCT
is further modified by liking a monosaccharide residue other
than an N-acetylglucosamine residue and a galactose residue
via an 􀁄-1,4-bond to at least one non-reducing end of the
plurality of non-reducing ends of the branched 􀁄-1,4 glucan,
wherein the monosaccharide 5 accharide residue is one kind or two kinds
selected from the group consisting of a glucuronic acid
residue, a glucosamine, a mannose residue, and a xylose
residue.
(Item 16)
10 The carrier according to item 14 or 15, wherein the
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.
15 (Item 17)
A vaccine adjuvant comprising the glucan comprising an
N-acetylglucosamine residue or a galactose residue according
to any one of items 1 to 3, a hydroxyl group-modified product
thereof, or a reducing end-modified product thereof.
20 (Item 18)
The vaccine adjuvant according to item 17, further modified
by liking a monosaccharide residue other than an
N-acetylglucosamine residue and a galactose residue via an
􀁄-1,4-bond to at least one or more non-reducing end of the
25 plurality of non-reducing ends of the branched 􀁄-1,4 glucan,
wherein the monosaccharide residue is one kind or two kinds
selected from a glucuronic acid residue and a mannose residue.
EFFECTS OF THE INVENTION
30 [0027] It is thought that the non-reducing end-modified
glucan (a glucan in which at least one residue selected from
an N-acetylglucosamine residue and a galactose residue is
bound via an 􀁄-1,4 bond to each of at least one (preferably
EG038PCT
at least two) non-reducing end(s)), a hydroxyl group-modified
product thereof, a non-reducing end-modified product thereof
and a reducing end-modified product thereof of the present
invention can be accumulated into a particular tissue when
administered into blood. Since the non-5 -reducing
end-modified branched 􀁄-1,4 glucan (a branched 􀁄-1,4 glucan
in which at least one residue selected from an
N-acetylglucosamine residue and a galactose residue is bound
via an 􀁄-1,4 bond to each of at least one (preferably at
10 least two) non-reducing end(s)), a hydroxyl group-modified
product thereof, a non-reducing end-modified product thereof
and a reducing end-modified product thereof of the present
invention have the activity of stimulating an
antigen-presenting cell such as a dendritic cell or a
15 macrophage to produce IL-6 (antigen-presenting cell
stimulating activity), they are thought to be usable as a
vaccine adjuvant. In the non-reducing end-modified
branched 􀁄-1,4 glucan (a branched 􀁄-1,4 glucan in which at
least one residue selected from an N-acetylglucosamine
20 residue and a galactose residue is bound via an 􀁄-1,4 bond
to each of at least one (preferably at least two) non-reducing
end(s)), a hydroxyl group-modified product thereof, a
non-reducing end-modified product thereof and a reducing
end-modified product thereof of the present invention, when
25 at least one kind of residue among a glucuronic acid residue,
a mannose residue and a xylose residue is further bound to
at least one or more non-reducing ends among the plurality
of non-reducing ends, the antigen-presenting cell
stimulating activity of these glucans can be further
30 improved.
The non-reducing end-modified glucan, a hydroxyl
group-modified product thereof, a non-reducing end-modified
product thereof and a reducing end-modified product thereof
EG038PCT
of the present invention can have extended blood half life
longer than that of an unmodified glucan, and are very safe
since they are finally completely degraded in a living body
and excreted. For this reason, these glucans and modified
products thereof of the present invention 5 vention are useful as a
modifying material for a medically effective ingredient,
a clinical diagnostic, an imaging agent and a nanoparticulate
carrier for DDS. Since the non-reducing end-modified glucan,
a hydroxyl group-modified product thereof, a non-reducing
10 end-modified product thereof and a reducing end-modified
product thereof of the present invention can be controlled
in their structure by an enzymatic reaction, they are also
excellent in quality stability.
15 BRIEF DESCRIPTION OF THE DRAWINGS
[0028] [Fig. 1] Fig. 1 shows the results of HPAEC-PAD
analysis after digestion of a non-reducing end-modified
branched glucan (B-GlcNAc) or an unmodified branched glucan
(B) with various amylases. See Example 11.
20 [Fig. 2] Fig. 2 shows the results of HPAEC-PAD analysis after
digestion of a non-reducing end-modified branched glucan
(B-Gal) or an unmodified branched glucan (B) with various
amylases. See Example 12.
[Fig. 3] Fig. 3 shows a schematic view of a structure of
25 products which are thought to be obtained when a non-reducing
end-modified branched glucan or an unmodified branched glucan
(B) is degraded with various amylases. See Example 12.
[Fig. 4] Fig. 4 is a graph showing a change in weight average
molecular weight of a product over time when a branched glucan
30 which has an N-acetylglucosamine residue bound thereto is
degraded with 􀁄-amylase. See Example 13.
[Fig. 5] Fig. 5 is a graph showing a change in weight average
molecular weight over time when non-reducing end
EG038PCT
N-acetylglucosamine residue-bound branched glucans having
different acetylation degrees are degraded with 􀁄-amylase.
See Example 14.
[Fig. 6] Fig. 6 is a graph showing a change in weight average
molecular weight of a product over time when a 5 branched glucan
which has a galactose residue bound thereto is degraded with
􀁄-amylase. See Example 15.
[Fig. 7] Fig. 7 is a graph showing a change in weight average
molecular weight over time when non-reducing end galactose
10 residue-bound branched glucans having different acetylation
degrees are degraded with 􀁄-amylase. See Example 16.
[Fig. 8] Fig. 8 is a graph showing glucan residual ratios
of an acetylated branched glucan which has
N-acetylglucosamine residues bound to non-reducing ends and
15 an acetylated non-reducing end-unmodified-branched glucan
(B) in blood. See Example 17.
[Fig. 9] Fig. 9 is a graph showing glucan residual ratios
of an acetylated branched glucan which has galactose residues
bound to non-reducing ends and an acetylated non-reducing
20 end-unmodified-branched glucan (B) in blood. See Example
18.
[Fig. 10] Fig. 10 is a graph showing concentrations of an
acetylated branched glucan which has a galactose residue
bound to a non-reducing end and an acetylated non-reducing
25 end unmodified branched glucan (B) in blood and liver. See
Example 19.
[Fig. 11] Fig. 11 is a graph showing an IL-6 production amount
when various non-reducing end-modified glucans are contacted
with a dendritic cell. See Example 20.
30 [Fig. 12] Fig. 12 is a graph showing an IL-6 production amount
when various non-reducing end-modified glucans are contacted
with a dendritic cell. See Example 22.
[Fig. 13] Fig. 13 is a graph showing an IL-6 production amount
EG038PCT
when various non-reducing end-modified glucans are contacted
with a dendritic cell. See Example 23.
[Fig. 14] Fig. 14 is a graph showing an IL-6 production amount
when various non-reducing end-modified glucans are contacted
with a dendritic cell. See 5 Example 24.
[Fig. 15] Fig. 15 is a graph showing an IL-6 production amount
when various non-reducing end-modified glucans are contacted
with a macrophage. See Example 25.
[Fig. 16] Fig. 16 is a graph showing an IL-6 production amount
10 when various non-reducing end-modified glucans are contacted
with a macrophage. See Example 26.
MODE FOR CARRYING THE INVENTION
[0029] The present invention will be explained in detail
15 below.
[0030] Throughout the present specification, it should be
understood that expression in a singular form includes a
concept of a plural form thereof, unless otherwise indicated.
In addition, it should be understood that a term used in
20 the present specification is used in a sense which is usually
used in the art, unless otherwise indicated.
[0031] (1. Materials)
(1.1) Glucans and modified products of glucan
"Glucan", when used in the present specification, is a
25 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
􀁄-D-glucan predominantly consist of an 􀁄-1,4-glucosidic bond,
and can contain an 􀁄-1,6-glucosidic bond. An 􀁄-D-glucan
30 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 to as a branched glucan, and a glucan
having no 􀁄-1,6-glucosidic bond in the molecule is referred
EG038PCT
to as a linear glucan. 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 5 the
present invention does not contain an 􀁄-1,3-bond.
[0032] 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
10 present specification, unless otherwise indicated, the
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
15 maltooligosaccharide and amylose.
[0033] In the present specification, the term
"maltooligosaccharide" refers to a substance which is
produced by dehydration condensation of about 2 to about
10 D-glucoses, wherein D-glucose units are bound by 􀁄-1,4
20 bond(s). The degree of polymerization of a
maltooligosaccharide is preferably about 3 or more, more
preferably about 4 or more, and further preferably about
5 or more. The degree of polymerization of a
maltooligosaccharide may be, for example, about 10 or less,
25 about 9 or less, about 8 or less, about 7 or less, or the
like. Examples of maltooligosaccharides include
maltooligosaccharides such as maltose, maltotriose,
maltotetraose, maltopentaose, maltohexaose, maltoheptaose,
maltooctaose, maltononaose, and maltodecaose.
30 [0034] In the present specification, the term "amylose"
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
EG038PCT
from a natural starch, or may be an amylose synthesized by
an enzymatic reaction (also referred to as "enzymatically
synthesized amylose" in the present specification).
Natural amylose may contain a branched part in some cases,
5 but enzymatically synthesized amylose does not contain a
branch. Further, natural amyloses have a large
polydispersity and has a variation in the molecular weight,
but an enzymatically synthesized amylose (particularly, an
enzymatically synthesized amylose synthesized by the SP-GP
10 method described in International Publication WO 02/097107
pamphlet) has a small 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
15 amylose used in the present invention is preferably about
2 or more, more preferably about 3 or more, still more
preferably about 10 or more, and most preferably about 30
or more. The degree of polymerization of the amylose used
in the present invention is preferably about 2 x 103 or less,
more preferably about 1 x 103 20 or less, still more preferably
about 700 or less and most preferably about 500 or less.
[0035] 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
25 􀁄-1,4-glucosidic bond(s), is branched with a bond other than
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
30 􀁄-1,2-glucosidic bond, and most preferably is an
􀁄-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
EG038PCT
branched glucan usually has the same number of non-reducing
ends as the number of branching bonds. When the branched
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 de5 graded
into a mixture of linear 􀁄-1,4-glucans. These are referred
to as a unit chain of the branched glucan, and the degree
of polymerization thereof is referred to as a unit chain
length.
10 [0036] Examples of the branched glucan suitably utilized
in the present invention include branched 􀁄-1,4-glucans
having 2 or more non-reducing ends, branched
maltooligosaccharide, starches, amylopectin, glycogen,
dextrin, enzymatically synthesized branched glucan and
15 highly branched cyclic glucan. Branched CD having one
non-reducing end is not preferable.
[0037] In the present specification, the term "branched
maltooligosaccharide" refers to a substance generated by
dehydration condensation of about 3 to about 10 D-glucoses,
20 in which D-glucose units are bound mainly with an 􀁄-1,4 bond(s),
and which contains one or more branching bonds, i.e., 2 or
more non-reducing ends. The degree of polymerization of the
branched maltooligosaccharide is preferably about 4 or more,
more preferably about 5 or more, further preferably about
25 6 or more. The degree of polymerization of the branched
maltooligosaccharide may be, for example, about 10 or less,
about 9 or less, about 8 or less, about 7 or less, or the
like.
[0038] In the present specification, the term "starch" is
30 a mixture of amylose and amylopectin. As a starch, any starch
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
EG038PCT
starch. Almost all starches possessed by glutinous rice,
glutinous corn and the like are an amylopectin. On the other
hand, a starch consisting only of amyloses, containing no
amylopectin, can not be obtained from a common plant. Starch
5 is classified into natural starch, a degraded starch and
modified starch.
[0039] Natural starch is classified into tuber starch and
cereal starch depending on the raw material. Examples of
tuber starches include potato starch, tapioca starch, sweet
10 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
high amylose starches (for example, high amylose corn starch)
or waxy starches. The starch can be a soluble starch. A
15 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
starch, waxy starch and high amylose starch. The starch may
be a modified starch.
20 [0040] The degree of polymerization of the starch used in
the present invention is preferably about 1 x 103 or more,
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 25 or less,
more preferably about 3 x 106 or less, still more preferably
about 1 x 106 or less and most preferably about 3 x 105 or
less.
[0041] An amylopectin is a branched molecule in which a
30 glucose unit(s) is bound via an 􀁄-1,6 bond to glucose units
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
EG038PCT
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 or more, still more
preferably about 1 x 104 or more, and most preferably about
2 x 104 or more. The degree of polymerization 5 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 or less and most preferably
about 3 x 105 or less.
10 [0042] 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
for animals in almost all cells in the granule state. In
a plant, glycogen is present, for example, in the seed of
15 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
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
20 of glucoses bound via an 􀁄-1,4-bond(s). In addition,
similarly, a sugar chain consisting of glucoses bound by
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
25 synthesize a glycogen. The degree of polymerization of the
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
30 glycogen 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 or less and most preferably
about 3 x 105 or less.
EG038PCT
[0043] Dextrin is one kind of glucan constructed of glucose,
and is a glucan having a medium complexity between those
of starch and those of maltose. Dextrin is obtained by
partially degrading starch by an acid, an alkyl or an enzyme.
Dextrin is also obtained 5 tained by partially degrading starch with
a physical treatment such as sonication. 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, and most
10 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.
15 [0044] 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
20 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.
For example, the degree of polymerization of the
25 enzymatically synthesized branched glucan used in the present
invention is preferably about 15 or more, preferably about
20 or more, more preferably about 50 or more, still more
preferably about 100 or more, and most preferably about 200
or more. The degree of polymerization of the enzymatically
30 synthesized branched glucan used in the present invention
is preferably about 4 x 105 or less, 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
EG038PCT
about 3 x 104 or less.
[0045] 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 5 a degree
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
10 in the present invention is preferably about 50 or more,
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 105 or less,
more preferably about 7 x 104 15 or less, and still more preferably
about 5 x 104 or less. The degree of polymerization of the
highly branched cyclic glucan as a whole molecule that can
be used in the present invention may be, for example, about
1 x 104 or less, about 7 x 103 or less, or about 5 x 103 or
less. 20
[0046] 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
25 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
100 or less.
30 [0047] 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 about 300 or more,
EG038PCT
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 or less, further preferably about 500 or less, and 5 further
more preferably about 300 or less.
[0048] 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
10 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.
15 It is preferable that the highly branched cyclic glucan
used in the present application is a highly branched cyclic
glucan in which the number of non-reducing ends in the
externally branched structural moiety is 2 or more. The
number of non-reducing ends (that is, "the number of
20 􀁄-1,6-glucosidic bonds" + 1) 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 4 or more, especially
preferably about 5 or more, and most preferably about 10
25 or more. The number of non-reducing ends 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 or less.
30 [0049] 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
branched cyclic glucans having a variety of degree of
EG038PCT
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
degree of polymerization to the minimum degree of
5 polymerization is about 100 or less, more preferably about
50 or less, and further more preferably about 10 or less.
[0050] The highly branched cyclic glucan is preferably a
glucan having an internally branched cyclic structural moiety
and an externally branched structural moiety and having a
degree of polymerization in a range of 50 to 5 x 10410 , 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
structural moiety is a non-cyclic structural moiety bound
15 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
branched cyclic glucan and a method for producing the same
20 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,
for example, as "Cluster Dextrin" from Ezaki Glico Co., Ltd.
25 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
more. The degree of polymerization of the highly branched
30 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
about 4 x 103 or less.
EG038PCT
[0051] 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 circulated
in blood for a long 5 ng 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
reticuloendothelial system is distributed in liver and spleen.
10 For this reason, by controlling the particle size of the
branched glucan, the pharmacokinetics of the non-reducing
end-modified glucan and a modified product thereof of the
present invention in vivo can be controlled. When one intends
to circulate the particles in blood for a long time, the
15 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 less. The molecular weight
of the particulate branched glucan having such a particle
size is preferably about 5 x 105 20 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 a diameter of 20 to 50
nm are accumulated in cancer cells, when it is intended that
25 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 nm or less, and more preferably
about 50 nm or less. The molecular weight of the particulate
30 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 x 107 or less, and more preferably about
5 x 106 or less.
EG038PCT
[0052] 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 2 or more, further preferably
about 5 or more, and most preferably about 10 or more. The
number of branches in the 􀁄-glucan may be about 30 or 5 more.
The number of branches of the 􀁄-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 2 x 103 or less. The number of branches in the 􀁄-glucan
may be about 1 x 103 10 or less.
[0053] In the branched 􀁄-glucan used in the present
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 10315 , more preferably
1 : 1.1 to 1 : 500, further preferably 1 : 1.2 to 1 : 100,
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.
20 In the present invention, the lower limit of the branching
frequency of the branched glucan is preferably 0.2% or more,
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
25 less, 90% or less, 85% or less, 65% or less, 50% or less,
or the like. The branching frequency is calculated by
{(number of 􀁄-1,6-bonds)/(sum of 􀁄-1,4-bonds and 􀁄-1,6-bonds
in glucan)} x 100.
[0054] The 􀁄-1,6-glucosidic bonds may be randomly
30 distributed in the 􀁄-glucan or may be homogeneously
distributed in the 􀁄-glucan. A distribution to such an extent
that a linear chain part(s) of 5 or more saccharide units
can be formed in the 􀁄-glucan is preferable.
EG038PCT
In a particular embodiment, a branched glucan having
a large number of non-reducing ends is preferable. The more
the number of non-reducing ends in a branched glucan, the
binding amount of a saccharide residue such as an
N-acetylglucosamine residue, a galactose residue, a manno5 se
residue, a glucuronic acid residue, a glucosamine residue,
or a xylose residue per one molecule can be increased. The
number of non-reducing ends of a branched 􀁄-1,4 glucan used
in the present invention is specifically 2 or more, 3 or
10 more, 5 or more, and is preferably about 10 or more, more
preferably about 30 or more, and about 60 or more. The number
of non-reducing ends of a branched 􀁄-glucan is preferably
about 1 x 104 or less, more preferably about 5 x 103 or less.
A branched glucan having a larger number of non-reducing
15 ends is preferable. Branched CD is not preferable. Since
the branched CD has one non-reducing end per one molecule
and has a cyclic backbone consisting of an 􀁄-1,4 glucan having
6 to 8 glucose residues, and is amylase resistant, it is
not preferable.
20 [0055] In the present invention, a modified product of the
glucan may be used in place of the glucan. Examples of the
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
25 hydroxyl group-modified product or a reducing end-modified
product. In addition, as described later, after at least
one residue selected from N-acetylglucosamine residue and
galactose residue is bound to at least one non-reducing end
of the glucan, a glucan moiety may be modified.
30 [0056] The modified starch is a starch which was made to
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
EG038PCT
a variety of combinations of a gelatinization initiation
temperature, a viscosity of a starch paste, a degree of
transparency of a starch paste, stability against
retrogradation and the like are available. There are various
types of modified starches. An example 5 mple of such a starch is
a starch obtained by immersing starch granules in an acid
at a gelatinization temperature or lower of the starch,
thereby cutting a starch molecule but not destroying starch
granules.
10 [0057] 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
glucan is modified (hereinafter, in the present specification,
referred to as a "hydroxyl group-modified product of glucan"),
15 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
product of glucan") and a modified product in which the
reducing end of a glucan is modified (hereinafter, in the
20 present specification, referred to as a "reducing
end-modified product of glucan").
[0058] Examples of the modification at a hydroxyl group
include hydroxyalkylation, alkylation, acylation,
carboxymethylation, sulfation and phosphorylation. It is
25 preferable that modification at a hydroxyl group is a
modification which can be removed with an enzyme in a 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
30 modifying group(s) into alcoholic hydroxyl groups can be
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,
EG038PCT
and DS1 means the state where one modifying group per glucose
residue is introduced. DS can be calculated by DS = (number
of modifying group)/(number of glucose residue). Since
there is an OH group at the 2-position, the 3-position and
5 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
is usually 3. The frequency of introduction of the modifying
group(s) into alcoholic hydroxyl groups is about DS 0.01
10 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
more. The frequency of introduction of modifying group(s)
is preferably about DS 1.5 or less, more preferably about
15 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,
degradation of the glucan in blood or in a body is suppressed.
[0059] Examples of modification at a non-reducing end
20 include binding with a targeting molecule such as a mannose
residue, a molecule for drug binding such as a glucuronic
acid residue and a glucosamine residue, and an
antigen-presenting cell stimulating molecule such as a
mannose residue and a xylose residue. Modification at a
25 non-reducing end will be explained in detail in the following
2.6 and 3. Non-reducing end modification is preferably
modification by binding of a mannose residue, a glucosamine
residue, a glucuronic acid residue, or a xylose residue.
By binding of a glucosamine residue, an amino group is imparted
30 to a non-reducing end of a glucan. Since an amino group has
a positive charge in an aqueous solution, binding by a
non-covalent bond with a drug having a negative charge in
an aqueous solution becomes possible. In addition, an amino
EG038PCT
group can also be utilized for binding via a covalent bond
with a drug. In addition, by binding of a glucuronic acid
residue, a carboxyl group is imparted to a non-reducing end
of a glucan. Since a carboxyl group has a negative charge
in an aqueous solution, binding by a non-5 -covalent bond with
a drug having a positive charge in an aqueous solution becomes
possible. In addition, carboxyl group can also be utilized
for binding via a covalent bond with a drug. In a particular
embodiment, none of a glucosamine residue and a glucuronic
10 acid residue is bound in the non-reducing end-modified
product of the present invention.
[0060] In modification of a non-reducing end, it is
preferable that a saccharide residue such as a mannose residue
or a galactose residue, which activates an antigen-presenting
15 cell such as a dendritic cell or a macrophage, is bound.
It is preferable that at least two or more of an
N-acetylglucosamine residue(s) and a galactose residue(s)
are bound to a non-reducing end(s) of a glucan. Further,
it is more preferable that two or more kinds of monosaccharide
20 residues that are not only an N-acetylglucosamine residue
or a galactose residue, but also include a mannose residue,
a glucuronic acid residue, a glucosamine residue, or a xylose
residue are bound.
[0061] Examples of modification at a reducing end include
25 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
low-molecular weight substance. Modification at a
30 non-reducing end will be explained in detail in the following
2.6 and 3 sections.
[0062] (1.2) Acetylglucosamine or galactose
Acetylglucosamine has the same meaning as
EG038PCT
N-acetylglucosamine, and is indicated by GlcNAc.
Acetylglucosamine is a substance in which an OH group at
a 2-position of glucose is substituted with a NHCOCH3 group.
[0063] Galactose is an epimer of D-glucose, and is a
substance in which 5 h configurations of an OH group and an H
group at a 4-position of glucose are interchanged. Since
galactose is recognized by an asialoglycoprotein receptor
present on a hepatic parenchymal cell surface, it is
particularly effective in the present invention.
10 [0064] In the method of the present invention,
acetylglucosamine-1-phosphate or galactose-1-phosphate is
used. Acetylglucosamine-1-phosphate or
galactose-1-phosphate may be commercially available one,
or may be synthesized by a chemical method, an enzymatic
15 method or a biological method such as fermentation.
Acetylglucosamine or galactose may be used for synthesizing
acetylglucosamine-1-phosphate or galactose-1-phosphate.
[0065] (1.3) Other monosaccharides
In the present invention, monosaccharide-1-phosphates
20 other than acetylglucosamine-1-phosphate or
galactose-1-phosphate can be used. Examples of such a
monosaccharide-1-phosphate include mannose-1-phosphate,
glucuronic acid-1-phosphate, glucosamine-1-phosphate and
xylose-1-phosphate. In order to obtain a glucan having the
25 cell stimulating activity, among these
monosaccharide-1-phosphates, particularly,
mannose-1-phosphate, glucuronic acid-1-phosphate and
xylose-1-phosphate are preferable. These
monosaccharide-1-phosphates can be used in random
30 combination with acetylglucosamine-1-phosphate and
galactose-1-phosphate. As these
monosaccharide-1-phosphates, commercially available ones
may be used, or chemically synthesized or enzymatically
EG038PCT
synthesized ones according to the methods known in the art
may be used.
[0066] (2. Method for producing a glucan in which at least
one residue selected from N-acetylglucosamine residue and
galactose residue is bound via an 􀁄-1,4 bond to 5 non-reducing
end)
(2.1) Acetylglucosamine-1-phosphate or
galactose-1-phosphate
As acetylglucosamine-1-phosphate or
10 galactose-1-phosphate utilized in the present invention,
those synthesized by a chemical method, an enzymatic method,
or a biological method such as fermentation can be used.
As an example of a method of synthesizing
acetylglucosamine-1-phosphate, for example, an enzymatic
15 method is disclosed in Japanese Laid-Open Publication No.
2007-97517. As an example of a method of synthesizing
galactose-1-phosphate, for example, an enzymatic method is
disclosed in Manu R. M. De Groeve et al., Biotechnol. Lett.
2009, 31, 1873-1877.
20 [0067] As acetylglucosamine-1-phospate, either of
acetylglucosamine-1-phospate in a non-salt form and
acetylglucosamine-1-phospate in a salt form can be used.
For example, a metal salt of acetylglucosamine-1-phospate
can be used, and an alkali metal salt of
25 acetylglucosamine-1-phospate (e.g. disodium
acetylglucosamine-1-phosphate and dipotassium
acetylglucosamine-1-phosphate) can be used.
[0068] As galactose-1-phospate, either of
galactose-1-phospate in a non-salt form and
30 galactose-1-phospate in a salt form can be used. For example,
a metal salt of galactose-1-phospate can be used, and an
alkali metal salt of galactose-1-phospate (e.g. disodium
galactose-1-phospate and dipotassium galactose-1-phospate)
EG038PCT
can be used.
[0069] (2.2) 􀁄-Glucan phosphorylase
In the present specification, the term "􀁄-glucan
phosphorylase" means an enzymes having 􀁄-glucan
phosphorylase activity. 􀁄5 -Glucan phosphorylase is
classified in EC 2.4.1.1. 􀁄-Glucan phosphorylase activity
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,
10 or the reverse reaction thereof. 􀁄-Glucan phosphorylase can
also catalyze an 􀁄-1,4-glucan synthesizing reaction which
is the reverse reaction relative to phosphorolysis. In which
direction a reaction proceeds depends on the amount of
substrate.
15 [0070] In the present invention, any 􀁄-glucan phosphorylase
can be used as long as it has a function to transfer a desired
residue (e.g. an N-acetylglucosamine residue, when it is
intended that an N-acetylglucosamine residue is bound to
a glucan; or a galactose residue, when it is intended that
20 a galactose residue is bound to a glucan) 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 of the present invention
can be derived from, for example, potato, sweet potato, Fava
25 bean, Arabidopsis thaliana, spinach, corn, rice, wheat,
Citrus hybrid cultivar, Aquifex aeolicus, Thermotoga
maritima, Thermococcus zilligii, Thermoanaerobacter
pseudethanolicus, or the like.
[0071] It is preferable that 􀁄-glucan phosphorylase used
30 in the present invention is 􀁄-glucan phosphorylase derived
from Aquifex aeolicus VF5, potato-derived 􀁄-glucan
phosphorylase or Thermococcus zilligii AN1-derived 􀁄-glucan
phosphorylase, and more preferably it is 􀁄-glucan
EG038PCT
phosphorylase derived from Aquifex aeolicus VF5. Since
􀁄-glucan phosphorylase derived from Aquifex aeolicus VF5
have high transferring efficiency, it is quite preferable.
[0072] The base sequence of 􀁄-glucan phosphorylases derived
from Aquifex aeolicus VF5 is set forth 5 rth 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
phosphorylases derived from Aquifex aeolicus VF5 has about
21% to about 24 % sequence identity with the amino acid sequence
10 of plant 􀁄-glucan phosphorylases, about 34% sequence identity
with the amino acid sequence of 􀁄-glucan phosphorylases
derived from Thermus thermophilus, and about 38% sequence
identity with the amino acid sequence of 􀁄-glucan
phosphorylases derived from Thermococcus litoralis. It has
15 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
of maltodextrin phosphorylases derived from Thermococcus
zilligii AN1, and about 33% sequence identity with those
20 of Thermoanaerobacter pseudethanolicus.
[0073] The base sequence of type L 􀁄-glucan phosphorylases
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
25 derived from Thermococcus zilligii AN1 is set forth in SEQ
ID NO: 5, and its amino acid sequence is set forth in positions
1-717 of SEQ ID NO: 6.
[0074] In the present specification, an enzyme "derived
from" an organism, means not only that the enzyme is directly
30 isolated from the organism, but also refers to an enzyme
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
EG038PCT
isolated from that Escherichia coli, the enzyme is referred
to as being "derived from" the organism.
[0075] 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 5 occurrence of the same
amino acid (base when base sequences are compared) between
two sequences. Identity can be generally determined by
comparing two amino acid sequences or two base sequences,
and comparing these two sequences which are aligned in an
10 optimal format, which can contain additions or deletions.
[0076] In the present specification, the identity of
sequences is calculated using maximum matching of GENETYX-WIN
Ver.4.0 (Genetics Co., Ltd.). This program aligns sequence
data to be analyzed, and sequence data to be compared so
15 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,
and Gaps, calculates a sum, outputs alignment at the smallest
sum, and calculates identity thereupon (Reference: Takeishi,
20 K., and Gotoh, O. 1984. Sequence Relationships among Various
4.5 S RNA Species J. Biochem. 92:1173-1177).
[0077] 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.
25 In another embodiment, as long as having activity to transfer
a intended residue (i.e., an acetylglucosamine residue, when
it is intended that an acetylglucosamine residue is
transferred; or a galactose residue, when it is intended
that a galactose residue is transferred) to non-reducing
30 end of a glucan to form an 􀁄-1,4 bond, 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
EG038PCT
substitution (including conservative 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, 5 4 or
6, or may occur at any position other 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
10 be added with amino acid residues (preferably about 20 or
less residues, more preferably about 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
15 the enzyme, to increase stability, or the like.
[0078] 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
20 about 80% or more, particularly more preferably about 90%
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 intended residue
(i.e., an acetylglucosamine residue, when it is intended
25 that an acetylglucosamine residue is transferred; or a
galactose residue, when it is intended that a galactose
residue is transferred) to a non-reducing end of the glucan
to form an 􀁄-1,4 bond. The 􀁄-glucan phosphorylase used in
the present invention can have an amino acid sequence which
30 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.
[0079] The amount of the 􀁄-glucan phosphorylase contained
EG038PCT
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
􀁄-glucan phosphorylase contained in 5 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,
10 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:
15 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.
20 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.
25 200 􀁐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
having the known concentration, and a standard curve is
30 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 amount of
EG038PCT
glucose-1-phosphate is not quantitated.
[0080] (2.3 Production of 􀁄-glucan phosphorylase)
􀁄-Glucan phosphorylase used in the present invention
can be directly isolated from an organism producing 􀁄-glucan
phosphorylase, such as the aforementioned organisms, 5 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
10 􀁄-glucan phosphorylase isolated from the aforementioned
organism.
[0081] In a preferable embodiment, 􀁄-glucan phosphorylase
derived from Aquifex aeolicus VF5 is produced by chemically
synthesizing a gene fragment of SEQ ID NO: 1, constructing
15 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
20 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.
25 Examples of a particularly preferable microorganism include,
but not limited to, Escherichia coli.
[0082] 􀁄-glucan phosphorylase having the amino acid
sequence of positions 1-692 of SEQ ID NO: 2, positions 15-930
of SEQ ID NO: 4, or positions 1-717 of SEQ ID NO: 6, or an
30 amino acid sequence having homology thereto and having an
activity transferring an N-acetylglucosamine, galactose,
mannose, glucuronic acid, glucosamine or xylose residue,
and a polynucleotide encoding the 􀁄-glucan phosphorylase
EG038PCT
which are used in the present invention can be produced using
conventional genetic engineering techniques.
[0083] (2.4) Production of a glucan containing
acetylglucosamine residue(s) or galactose residue(s)
The 5 glucan containing an acetylglucosamine residue(s)
or galactose residue(s) of the present invention can be
produced by a method including a step of allowing a reaction
of a solution containing acetylglucosamine-1-phosphate or
galactose-1-phosphate, a glucan, and 􀁄-glucan phosphorylase
10 (for example, 􀁄-glucan phosphorylase derived from Aquifex
aeolicus VF5, 􀁄-glucan phosphorylase derived from potato,
or 􀁄-glucan phosphorylase derived from Thermococcus zilligii
AN1) which can catalyze a reaction of transferring an
acetylglucosamine residue or a galactose residue to a
15 non-reducing end of a glucan. By using a glucan modified
product in place of a glucan in this method, a modified product
of the glucan containing acetylglucosamine residue(s) or
galactose residue(s) can be produced. As an example, a method
using a glucan will be explained below.
20 [0084] First, a reaction solution is prepared. The
reaction solution can be prepared, for example, by adding
acetylglucosamine-1-phosphate or galactose-1-phosphate, a
glucan, and 􀁄-glucan phosphorylase to a suitable solvent.
Either one of acetylglucosamine-1-phosphate and
25 galactose-1-phosphate may be used, or both of
acetylglucosamine-1-phosphate or galactose-1-phosphate may
be used simultaneously. If necessary, any buffer and
inorganic salts for the purpose of adjusting the pH, as far
as an enzymatic reaction is not inhibited, may be added to
30 this reaction solution. If necessary, glucose-1-phosphate
which is an original substrate of 􀁄-glucan phosphorylase
may be added to this reaction solution. In the case of a
reaction where acetylglucosamine-1-phosphate or
EG038PCT
galactose-1-phosphate and glucose-1-phosphate are
coexistent, a reaction of binding a glucose residue to a
non-reducing end of a receptor glucan and a reaction of binding
acetylglucosamine residue or galactose residue are
simultaneously performed. If an acetylglucosamine 5 etylglucosamine residue
or a galactose residue is bound to a non-reducing end of
the glucan, 􀁄-glucan phosphorylase can further transfer a
molecule to the non-reducing end of the acetylglucosamine
residue or the galactose residue, however, the efficiency
10 of the transfer is much lower than the efficiency of
transferring to a glucose residue. However, when a glucose
residue is bound to a non-reducing end of a glucan, 􀁄-glucan
phosphorylase can further transfer a glucose residue or an
acetylglucosamine residue or galactose residue to a
15 non-reducing end of a resulting molecule. For this reason,
when glucose-1-phosphate is coexistent, the chain length
of the glucan can be extended efficiently. Therefore, the
structure of the finally obtained non-reducing end-modified
glucan is controlled by a ratio between added
20 acetylglucosamine-1-phosphate or galactose-1-phosphate and
added glucose-1-phosphate. If necessary, an enzyme
selected from the group consisting of a debranching enzyme,
a branching enzyme, 4-􀁄-glucanotransferase and a glycogen
debranching enzyme may be added to this reaction solution.
25 [0085] By changing the ratio, in a reaction solution, of
the amount of acetylglucosamine-1-phosphate or
galactose-1-phosphate and the number as a population of
non-reducing ends of a glucan, frequency of introduction
of acetylglucosamine residue or galactose residue can be
30 controlled. That is, as increasing (number of molecule of
glucan) x (amount of acetylglucosamine-1-phosphate or
galactose-1-phosphate relative to number of non-reducing
ends in the molecule), the frequency of introduction of
EG038PCT
acetylglucosamine residue or galactose residue can be
increased. In addition, also by regulating the amount of
enzyme added or enzyme reaction time, the frequency of
introduction of acetylglucosamine residue or galactose
5 residue can be regulated.
[0086] When non-reducing end modification of a glucan is
performed with two or more kinds of saccharide residues,
a reaction may be performed by adding two or more kinds of
saccharide-1-phospates, in one kind of them or mixed in the
10 same reaction liquid. For example, desired
saccharide-1-phosphate selected from
N-acetylglucosamine-1-phosphate, galactose-1-phosphate,
glucuronic acid-1-phosphate, glucosamine-1-phosphate,
mannose-1-phosphate, and xylose-1-phosphate may be reacted
15 one kind by one kind. Alternatively, two or more kinds of
them may be reacted simultaneously. For example, when it
is desired that a saccharide residue selected from a
glucuronic acid residue, a glucosamine residue, a mannose
residue and a xylose residue is further bound, glucuronic
20 acid-1-phosphate, glucosamine-1-phosphate,
mannose-1-phosphate, and xylose-1-phosphate are reacted in
one kind of them or a combination of them, with a non-reducing
end of a branched glucan.
[0087] Then, the reaction solution is reacted, if necessary,
25 by heating by a method known in the art. The reaction
temperature can be any temperature, as far as the effect
of the present invention is obtained. The reaction
temperature can be representatively a temperature of about
30oC to about 90oC. It is preferable that the temperature
30 of a solution in this reaction step is such a temperature
that, after a predetermined reaction time, about 50% or more,
more preferably about 80% or more activity of the activity
of the 􀁄-glucan phosphorylase contained in this solution
EG038PCT
before the reaction remains. The reaction temperature is
preferably about 35oC to about 80oC, more preferably about
35oC to about 70oC, and further preferably about 35oC to about
65oC. 􀁄-Glucan phosphorylase derived from Aquifex aeolicus
VF5 is a thermostable 5 table enzyme, and its optimal reaction
temperature is about 80oC to 90oC. From the viewpoint of
the reaction speed, it is preferable that the reaction
temperature is high to some extent. On the other hand, from
the viewpoint of the optimal reaction temperature of 􀁄-glucan
10 phosphorylase derived from Aquifex aeolicus VF5, a reaction
at about 80oC to 90oC is possible. However, from the viewpoint
of stability of the resulting product, stability of
acetylglucosamine-1-phosphate or galactose-1-phosphate and
glucose-1-phosphate, and the like, it is preferable that
15 the reaction temperature is slightly lower than the optimal
reaction temperature of the 􀁄-glucan phosphorylase derived
from Aquifex aeolicus VF5. The reaction temperature is
preferably about 30oC or higher, more preferably about 35oC
or higher, further preferably about 40oC or higher. In a
20 particular embodiment, the reaction temperature may be about
45oC or higher or about 50oC or higher. The reaction
temperature is preferably about 90oC or lower, more preferably
about 80oC or lower, further preferably about 70oC or lower.
In a particular embodiment, the reaction temperature may
be about 65oC or lower or about 60o25 C or lower. In the case
where an enzyme for which an optimum reaction temperature
is lower such as potato-derived 􀁄-glucan phosphorylase is
used, it is preferable that the reaction temperature is set
lower than the case where Aquifex aeolicus VF5-derived
30 􀁄-glucan phosphorylase is used.
[0088] The reaction time can be set in any time period, in
view of the reaction temperature and remaining activity of
an enzyme. The reaction time is representatively about 1
EG038PCT
hour to about 100 hours, more preferably about 1 hour to
about 72 hours, further more preferably about 2 hours to
about 36 hours, and most preferably about 2 hours to about
24 hours. In a particular embodiment, the reaction time may
5 be, for example, about 1 hour or longer, about 2 hours or
longer, about 5 hours or longer, about 10 hours or longer,
about 12 hours or longer, or about 24 hours or longer. In
a particular embodiment, the reaction time may be, for example,
about 100 hours or shorter, about 72 hours or shorter, about
10 60 hours or shorter, about 48 hours or shorter, about 36
hours or shorter, or about 24 hours or shorter.
[0089] Heating may be performed using any means, but it is
preferable that heating is performed with stirring so as
to homogeneously transmit the heat to the whole solution.
15 The solution is stirred by placing it into, for example,
a reaction tank made of stainless steel, provided with a
warm water jacket and a stirring device.
[0090] Furthermore, in the method of the present invention,
at least one of acetylglucosamine-1-phosphate or
20 galactose-1-phosphate , a glucan, and 􀁄-glucan phosphorylase
may be further added to a reaction solution at a stage where
the reaction has proceeded to some extent.
[0091] In this way, a solution containing a non-reducing
end-modified glucan is produced.
25 [0092] After completion of the reaction, in the reaction
solution, if necessary, an enzyme in the reaction solution
can be inactivated by, for example, heating at 100oC for 10
minutes. After completion of the reaction, enzyme may be
removed by making pH of the reaction solution to be acidic
30 to denature the enzyme protein. Alternatively, a post step
may be performed without performing treatment of inactivating
an enzyme. The reaction solution may be stored as it is,
or may be treated in order to isolate the produced non-reducing
EG038PCT
end-modified glucan.
[0093] After completion of the reaction, after the
non-reducing end-modified glucan is purified, or before the
non-reducing end-modified glucan is purified, a hydroxyl
group-5 modified product of the non-reducing end-modified
glucan may be produced by modifying at least one of alcoholic
hydroxyl groups of a glucan moiety of the resulting
non-reducing end-modified glucan. It is preferable that
modification is performed after purification of the
10 non-reducing end-modified glucan. Modification can be
performed according to a method known in the art. Examples
of modification include hydroxyalkylation, alkylation,
acylation, carboxymethylation, sulfation, and
phosphorylation. Acylation is preferable, and acetylation
15 is more preferable. By modifying a reducing end of the glucan
after producing the glucan containing N-acetylglucosamine
residue(s) or galactose residue(s) or a hydroxyl
group-modified product of the glucan containing
N-acetylglucosamine residue(s) or galactose residue(s), a
20 reducing end-modified product of the glucan containing
N-acetylglucosamine residue(s) or galactose residue(s) or
a hydroxyl group-modified product of the glucan containing
N-acetylglucosamine residue(s) or galactose residue(s) may
be produced. Further, a non-reducing end of these glucan
25 moiety to which neither N-acetylglucosamine residue nor
galactose residue is bound, may be modified. Binding of an
acetylglucosamine residue or galactose residue to the glucan,
modification of a hydroxyl group, modification of a reducing
end, and modification of some of non-reducing ends with a
30 modifying group other than an N-acetylglucosamine residue
or galactose residue may be performed in any order.
[0094] (2.5 Purification of a glucan in which at least one
residue selected from an N-acetylglucosamine residue or an
EG038PCT
galactose residue is liked via 􀁄-1,4 bond to the non-reducing
end(s))

The produced non-reducing end-modified glucan (or a
modified product thereof) can be purified as nec5 essary.
Examples of the impurities removed by purification include
inorganic phosphate, N-acetylglucosamine-1-phosphate or
galactose-1-phosphate, inorganic salts and the like.
Examples of a method of purifying a glucan include a method
10 using an organic solvent (T. J. Schoch et al., J. American
Chemical Society, 64, 2957 (1942)) and a method not using
an organic solvent.
[0095] Examples of the organic solvent which can be used
in purification using the organic solvent include acetone,
15 n-amyl alcohol, pentazole, n-propyl alcohol, n-hexyl alcohol,
2-ethyl-1-butanol, 2-ethyl-1-hexanol, lauryl alcohol,
cyclohexanol, n-butyl alcohol, 3-pentanol,
4-methyl-2-pentanol, d,l-borneol, 􀁄-terpineol, isobutyl
alcohol, sec-butyl alcohol, 2-methyl-1-butanol, isoamyl
20 alcohol, tert-amyl alcohol, menthol, methanol, ethanol and
ether.
[0096] As an example of the purification method not using
an organic solvent, there is a method of removing inorganic
phosphate, acetylglucosamine-1-phosphate or
25 galactose-1-phosphate, and inorganic salts by subjecting
a non-reducing end-modified glucan to membrane fractionation
using an ultrafiltration membrane or chromatography, without
precipitating the non-reducing end-modified glucan
dissolved in water, after the non-reducing end-modified
30 glucan production reaction.
[0097] Examples of the ultrafiltration membrane which can
be used in purification include an ultrafiltration membrane
of a molecular weight cut off of about 1 x 103 to about 1
EG038PCT
x 104, preferably about 5 x 103 to about 5 x 104, more preferably
about 1 x 104 to about 3 x 104 (UF membrane unit manufactured
by DAICEL).
[0098] Examples of a support which can be used in
5 chromatography include a support for gel filtration
chromatography, a support for ligand exchange chromatography,
a support for ion-exchange chromatography and a support for
hydrophobic chromatography.
[0099] (2.6) Glucan in which at least one residue selected
10 from N-acetylglucosamine residue and galactose residue is
bound via 􀁄-1,4 bond to non-reducing end(s), and modified
product thereof.
The glucan of the present invention is a glucan in which
at least one residue selected from an N-acetylglucosamine
15 residue and a galactose residue is bound via 􀁄-1,4 bond to
each of at least one non-reducing end of a glucan. The glucan
of the present invention is also referred to as the
non-reducing end-modified glucan of the present invention.
The glucan-modified product of the present invention can
20 be a hydroxyl group-modified product of the non-reducing
end-modified glucan, or a further non-reducing end-modified
product or reducing end-modified product of the present
invention.
The non-reducing end-modified glucan of the present
25 invention is a glucan in which at least one residue selected
from an N-acetylglucosamine residue and a galactose residue
is bound via 􀁄-1,4 bond to each of at least one non-reducing
end of a glucan. To an end of an N-acetylglucosamine residue
or a galactose residue bound to a non-reducing end of this
30 glucan may be further bound a saccharide or the like, if
necessary. When two or more of an N-acetylglucosamine
residue or a galactose residue, or a combination thereof
are bound to one non-reducing end of the non-reducing
EG038PCT
end-modified glucan of the present invention, a structure
is that these monosaccharides are bound and bind to one end.
For example, the structure is that wherein one monosaccharide
residue is bound to one non-reducing end of a glucan, and
the next monosaccharide residue 5 is further bound to the
non-reducing end of the monosaccharide residue. In one
embodiment, only one monosaccharide residue is bound to each
end of non-reducing ends of a glucan.
[0100] In the non-reducing end-modified glucan of the
10 present invention, N-acetylglucosamine residue(s) or
galactose residue(s) or combination thereof may be bound
to all of the non-reducing ends of glucan, or these
monosaccharide residue(s) may be bound to only a part of
the non-reducing ends. That is, some of the non-reducing
15 ends can remain unbound by these monosaccharide residues.
[0101] Conversion ratio based on the number of non-reducing
end(s), that is, the ratio of the number of bond(s) between
monosaccharide(s) selected from N-acetylglucosamine
residue(s) or galactose residue(s) or combination thereof
20 and non-reducing end(s) relative to the number of
non-reducing end(s) present before these monosaccharide(s)
is/are bound, can be selected as appropriate depending on
the application wherein the glucan containing either of these
monosaccharide residues is used or the like, from the ratio
25 close proximity of 0% (for example, about 1%) to 100%. For
example, when it is desired that a small amount of
N-acetylglucosamine residue(s) or galactose residue(s) be
present, a low conversion ratio can be selected, and when
it is desired that a large amount of N-acetylglucosamine
30 residue(s) or galactose residue(s) be present, a high
conversion ratio can be selected.
[0102] By appropriately adjusting the condition of the
enzymatic reaction in producing a non-reducing end-modified
EG038PCT
glucan, for example, by adjusting the reaction time, the
concentration of the substrate or the like of the enzymatic
reaction, a non-reducing end-modified glucan having a
selected conversion ratio can easily be obtained.
5 [0103] Further, the ratio of the total number of
N-acetylglucosamine residue(s) or galactose residue(s)
bound to non-reducing end(s) relative to the number of
non-reducing end(s) present in a glucan before these
monosaccharide residues(s) is/are bound can be used as an
10 indicator for the amount of these monosaccharides. In the
present specification, this ratio is described as binding
ratio. In general, in analyzing a non-reducing end-modified
glucan of the present invention, it is easier to measure
the binding ratio than to measure the above-mentioned
15 conversion ratio, therefore, it is advantageous from the
practical viewpoint to design a non-reducing end-modified
glucan based on binding ratio.
[0104] It is noted that depending on the condition of
enzymatic reaction in synthesizing a non-reducing
20 end-modified glucan, to the end of either of these
monosaccharide residues bound to one non-reducing end, either
of these monosaccharide residues may be further bound in
some cases. That is, in some cases, the structure may be
that wherein two these monosaccharides are serially bound
25 to one non-reducing end (that is, a structure wherein a
disaccharide is bound to a non-reducing end). A compound
having such a structure can also be used as the non-reducing
end-modified glucan of the present invention. The
above-mentioned binding ratio has an advantage that the
30 amount of N-acetylglucosamine residue(s) or galactose
residue(s) can be evaluated even if the amount of
N-acetylglucosamine residue(s) or galactose residue(s) is
increased by binding a plurality of these monosaccharide
EG038PCT
residues to one non-reducing end as described above. For
this reason, in contrast to the above-mentioned conversion
ratio, the binding ratio may have a value greater than 100%
in some cases. The binding ratio can be selected as
5 appropriate depending on the application wherein the
non-reducing end-modified glucan is used or the like. For
example, when it is desired that a small amount of
N-acetylglucosamine residue(s) or galactose residue(s) be
present, a low binding ratio (for example, about 1% to about
10 2%) can be selected, and when it is desired that a large
amount of N-acetylglucosamine residue(s) or galactose
residue(s) be present, a high binding ratio (for example,
bout 80% to about 120%) can be selected.
[0105] When the application of the non-reducing
15 end-modified branched glucan is a nanoparticulate carrier
for a DDS, a total of the binding ratios of the
N-acetylglucosamine residues and galactose residues is
preferably about 2% or more, more preferably about 5% or
more, further preferably about 10% or more, and most
20 preferably about 15% or more; the total of the binding ratios
of the N-acetylglucosamine residues and the galactose
residues is preferably about 200% or less, more preferably
about 150% or less, further preferably about 120% or less,
and most preferably about 100% or less.
25 [0106] When the application of the non-reducing
end-modified branched glucan is an antigen-presenting cell
stimulating molecule, the binding ratio of the
N-acetylglucosamine residues or galactose residues is, in
a total of them, preferably about 5% or more, more preferably
30 about 10% or more, further preferably about 15% or more,
and most preferably about 20% or more; the binding ratio
of the N-acetylglucosamine residues or galactose residues
is, in a total of them, preferably about 200% or less, more
EG038PCT
preferably about 150% or less, further preferably about 120%
or less, and most preferably about 100% or less.
[0107] When the application of the non-reducing
end-modified branched glucan is a DDS carrier, the number
of bonds of the N-acetylglucosamine residues or the ga5 lactose
residues per glucan molecule is, in a total of them, preferably
about 2 or more, more preferably about 5 or more, further
preferably about 10 or more, particularly preferably about
20 or more, and most preferably about 50 or more. The number
10 of bonds of the N-acetylglucosamine residues or the galactose
residues per glucan molecule is, in a total of them, preferably
about 5000 or less, more preferably about 4000 or less, further
preferably about 3000 or less, and most preferably about
2000 or less.
15 [0108] When the application of the non-reducing
end-modified branched glucan is an antigen-presenting cell
stimulating molecule, the number of bonds of the
N-acetylglucosamine residues or the galactose residues per
glucan molecule is, in a total of them, preferably about
20 2 or more, more preferably about 4 or more, further preferably
about 8 or more, particularly preferably about 10 or more,
and most preferably about 15 or more. The number of bonds
of the N-acetylglucosamine residues or the galactose residues
per glucan molecule is, in a total of them, preferably about
25 2000 or less, more preferably about 1000 or less, further
preferably about 500 or less, and most preferably about 200
or less.
[0109] In the non-reducing end-modified glucan of the
present invention, a sugar residue other than
30 N-acetylglucosamine residue or galactose residue may be bound
to the non-reducing end to which neither N-acetylglucosamine
residue nor galactose residue is bound. That is, some of
non-reducing ends may bind with an N-acetylglucosamine
EG038PCT
residue or galactose residue, and the remaining non-reducing
end(s) may bind with a sugar residue other than these
monosaccharides.
[0110] Alternatively, it is also possible to make a structure
wherein some of non-reducing ends bind 5 to N-acetylglucosamine
residue(s) or galactose residue(s), some bind to a sugar
residue other than these monosaccharide, and the remaining
non-reducing end(s) bind to nothing. In this regard, it is
possible that among a plurality of non-reducing ends, the
10 number of end(s) binding to N-acetylglucosamine residue(s)
or galactose residue(s), the number of end(s) binding to
a sugar residue other than these monosaccharides, and the
number of end(s) not binding to a sugar be selected freely.
[0111] In a particular embodiment of the present invention,
15 a saccharide residue bound to a non-reducing end is an
N-acetylglucosamine residue, a galactose residue or a residue
of an oligomer (e.g. a dimer) of one kind of them or a
combination of them.
[0112] The hydroxyl group-modified product is as described
20 above.

CLAIMS
Claim 1. A branched glucan,
wherein the branched glucan has a plurality of
5 non-reducing ends, and
at least one residue selected from an N-acetylglucosamine
residue and a galactose residue is bound via an 􀁄-1,4-bond
to each of two or more non-reducing ends of the branched
􀁄-1,4-glucan,
10 but neither an N-acetylglucosamine residue nor a
galactose 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 or less.
15
Claim 2. The branched glucan according to claim 1, wherein
the branched 􀁄-1,4-glucan is selected from the group
consisting of a branched maltooligosaccharide, starch,
amylopectin, glycogen, dextrin, enzymatically synthesized
20 branched glucan and highly branched cyclic glucan.
Claim 3. A hydroxyl group-modified product of the branched
glucan according to claim 1, wherein the modification on
the hydroxyl group is a modification on some or all of alcoholic
25 hydroxyl groups of the glucan, and the modification on the
hydroxyl group is independently selected from the group
consisting of hydroxyalkylation, alkylation, acetylation,
carboxymethylation, sulfation and phosphorylation.
30 Claim 4. A reducing end-modified product of the branched
glucan according to claim 1 or a hydroxyl group-modified
product thereof.
EG038PCT
Claim 5. A non-reducing end-modified product of the branched
glucan according to claim 1 or a hydroxyl group-modified
product thereof, or a reducing end-modified product thereof,
which is further modified by liking a monosaccharide residue
other than an N-acetylglucosamine residue and a galactos5 e
residue via an 􀁄-1,4-bond to at least one non-reducing end
of the plurality of non-reducing ends of the branched 􀁄-1,4
glucan.
10 Claim 6. The non-reducing end-modified product of the
branched glucan or a hydroxyl group-modified product thereof,
or a reducing end-modified product thereof according to claim
5, wherein the monosaccharide residue is one kind or two
or more kinds selected from a glucuronic acid residue, a
15 glucosamine residue, a mannose residue, and a xylose residue.
Claim 7. The non-reducing end-modified product of the
branched glucan or a hydroxyl group-modified product thereof,
or a reducing end-modified product thereof according to claim
20 5, wherein the monosaccharide residue is one kind or two
or more kinds selected from a glucuronic acid residue, a
mannose residue, and a xylose residue.
Claim 8. A method for producing a branched glucan in which
25 at least one residue selected from an N-acetylglucosamine
residue and a galactose residue is bound to each of two or
more non-reducing ends, characterized by allowing an 􀁄-glucan
phosphorylase to act on an aqueous solution comprising a
branched 􀁄-1,4-glucan having two or more non-reducing ends
30 and N-acetylglucosamine-1-phosphate or
galactose-1-phosphate, wherein the degree of polymerization
of the branched 􀁄-1,4-glucan is 15 or more and 4 x 105 or
less.
EG038PCT
Claim 9. The method according to claim 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
5 transferring N-acetylglucosamine residue or galactose
residue to a non-reducing end of a glucan to form an 􀁄-1,4-bond.
Claim 10. A medicament comprising:
the branched glucan according to claim 1, a hydroxyl
10 group-modified product thereof, or a reducing end-modified
product thereof, and
a medically effective ingredient.
Claim 11. A medicament comprising:
15 a non-reducing end-modified product of the branched
glucan according to claim 1 or a hydroxyl group-modified
product thereof, or a reducing end-modified product thereof,
which is further modified by liking a monosaccharide residue
other than an N-acetylglucosamine residue and a galactose
20 residue via an 􀁄-1,4-bond to at least one non-reducing end
of the plurality of non-reducing ends of the branched 􀁄-1,4
glucan, wherein the monosaccharide residue is one kind or
two kinds selected from a glucuronic acid residue, a
glucosamine residue, a mannose residue, and a xylose residue;
25 and
a medically effective ingredient.
Claim 12. The medicament according to claim 10 or 11, wherein
the medically effective ingredient is selected from the group
30 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 RNA aptamer.
EG038PCT
Claim 13. The medicament according to claim 10 or 11, wherein
the medically effective ingredient is an antigen protein
or a peptide.
Claim 14. A composition for clinical 5 al diagnosis comprising
the branched glucan according to claim 1, a hydroxyl
group-modified product thereof, or a reducing end-modified
product thereof.
10 Claim 15. A composition for clinical diagnosis comprising
a non-reducing end-modified product of the branched glucan
according to claim 1 or a hydroxyl group-modified product
thereof, or a reducing end-modified product thereof, which
is further modified by liking a monosaccharide residue other
15 than an N-acetylglucosamine residue and a galactose residue
via an 􀁄-1,4-bond to at least one non-reducing end of the
plurality of non-reducing ends of the branched 􀁄-1,4 glucan,
wherein the monosaccharide residue is one kind or two kinds
selected from a glucuronic acid residue, a glucosamine
20 residue, a mannose residue, and a xylose residue.
Claim 16. A nanoparticulate carrier for a DDS comprising
the branched glucan according to claim 1, a hydroxyl
group-modified product thereof, or a reducing end-modified
25 product thereof.
Claim 17. A nanoparticulate carrier for a DDS comprising
a non-reducing end-modified product of the branched glucan
according to claim 1 or a hydroxyl group-modified product
30 thereof, or a reducing end-modified product thereof, which
is further modified by liking a monosaccharide residue other
than an N-acetylglucosamine residue and a galactose residue
via an 􀁄-1,4-bond to at least one non-reducing end of the
EG038PCT
plurality of non-reducing ends of the branched 􀁄-1,4 glucan,
wherein the monosaccharide residue is one kind or two kinds
selected from a glucuronic acid residue, a glucosamine
residue, a mannose residue, and a xylose residue.
5
Claim 18. The carrier according to claim 16 or 17, wherein
the 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
10 bearing hydrophobic groups.
Claim 19. A vaccine adjuvant comprising the branched glucan
according to claim 1, a hydroxyl group-modified product
thereof, or a reducing end-modified product thereof.
15
Claim 20. The vaccine adjuvant according to claim 19, wherein
the branched 􀁄-1,4 glucan is further modified by liking a
monosaccharide residue other than an N-acetylglucosamine
residue and a galactose residue via an 􀁄-1,4-bond to at least
20 one non-reducing end of the plurality of non-reducing ends
of the glucan, wherein the monosaccharide residue is one
kind or two kinds selected from a glucuronic acid residue
and a mannose residue.