Patent Application
20130295163
1. Technical Field
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 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 α-1,4-bond to each of at least one (preferably at least two) non-reducing end, a modified product thereof, as well as a method for producing the same, and utilization of the same.
2. Description of the Related Art
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 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 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 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.
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 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, 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, 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 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 α (product name: PEGASYS). Since interferon α has a small molecular weight and is easily 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 α to a PEG chain having a molecular weight of 40,000 to form an interferon α-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 macromolecular material.
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 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 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 which can be safely utilized is expected.
Polysaccharides have been used as a food raw material for a long time and, in recent years, have begun to be paid attention as a macromolecular material which is environment friendly, as a safe material having biocompatibility and, further, as a functional material.
An α-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, regenerative medicine, intracorporeal imaging or the like, which is expected to grow in the future. When the α-1,4 glucan is used for such a purpose, it is necessary to impart to the α-1,4 glucan a function of interacting with a medically 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 α-1,4 glucan.
On the other hand, in the pharmaceutical field, a vaccine for preventing or treating an infectious 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 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 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 adjuvant. It is expected an effective and safe adjuvant effectively functions when a purified protein or peptide is administered as an antigen.
In recent years, due to progress of natural science, a mechanism of immunological system has been revealed. It 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-α and the like) that results from 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 stimulation and activation of an antigen-presenting cell, and induction of production of an inflammatory cytokine (“Janeway's Immunobiology” (translation under supervision by Takehiko Sasazuki, Nankodo Co., Ltd.) (original book “Janeway's Immunobiology seventh edition Kenneth Murphy, Paul Travers, Mark Walport 2008 Garind Science, Taylar & Francis Group, LLC”)).
Conventionally, a variety of adjuvants are known, but there are few safe and low cost immune adjuvants which 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., 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 abbreviated as “GM-CSF”) which directly activates a dendritic cell as an immune adjuvant has been proposed, but cytokines are further expensive.
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 Freund's incomplete adjuvant (since this adjuvant is oily, toxicity is feared) are used. These are low in toxicity, but are weak in the immune adjuvant activity, as compared with Freund's complete adjuvant which is used in an animal experiment.
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 which has a high ability to stimulate an immune response, that is, the ability to stimulate an antigen-presenting cell, and is safe.
The present invention is intended to solve the above-mentioned problems.
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 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 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 material shall have tissue targeting functions or cell stimulation functions.
The present inventors thought that an excellent macromolecular substance for modifying material for the medically effective ingredient is a glucan (in the present specification, the glucan refers to an α-1,4-glucan, and an α-1,4-glucan which is branched with an α-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 a component that is always present in a body of a human, and is excellent in biocompatibility. Further, the glucan undergoes hydrolysis by α-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 can be said to be the safest macromolecular material.
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 which glucose residues are bound only with an α-1,4-bond, glucans which are branched at high frequency by some glucose residues bound with an α-1,6-bond, and the like are available. In a branched glucan, every time an α-1,6-bond is increased by one, one new non-reducing end is generated. The number of α-1,6-bonds can be suitably adjusted as desired upon synthesis of a glucan molecule.
On the other hand, the greatest problem when the glucan is utilized as the modifying material (for example, 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 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.
Further, it is preferable that a carrier has the tissue targeting function, in terms of utilization of a glucan 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, 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.
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 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 glucan. However, a method of binding a monosaccharide other than glucose via α-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. Res., 2008, 343, 2692-2696, it is described that α-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, 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 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 α-D-xylose-1-phosphate, 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 (Gal-1-P) to a glucan.
The present inventors intensively studied in order to solve the aforementioned problems and found that Aquifex aeolicus VF5-derived α-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 residue (GlcNAc residue) via α-1,4 bond to a non-reducing end of a α-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 addition, the present inventors also found that potato-derived α-glucan phosphorylase can use Gal-1-P as a substrate, and Thermococcus zilligii AN1-derived α-glucan phosphorylase can use Gal-1-P and GlcNAc-1-P as a substrate. These α-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 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 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 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, α-glucan phosphorylase hardly catalyzes the 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, α-glucan phosphorylase can bind at least one residue 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 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 residues 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 residue or a GlcNAc residue is bound via α-1,4 bond to a non-reducing end of an α-1,4 glucan, glucoamylase resistance can be imparted to the α-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 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 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. acetylating) a hydroxyl group of the non-reducing end-modified glucan of the present invention.
In the non-reducing end-modified branched α-1,4 glucan (e.g. a branched α-1,4 glucan in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-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 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-presenting cell stimulating activity of these glucans can be further improved.
In the present invention, since a saccharide (e.g. N-acetylglucosamine or galactose) residue is transferred using α-glucan phosphorylase (EC2.4.1.1), linkage of a saccharide residue to a glucan is an α-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) residue are α-bound via an oxygen atom.
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 α-1,4 bond to each of at least one non-reducing end of a glucan is also referred to as a “non-reducing end-modified glucan” of the present invention.
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 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 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 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 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).
The introduction amount of galactose residues or 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 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 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 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 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 has a possibility of being an excellent modifying material for the medically effective ingredient.
For example, the present invention provides the following glucans and the like:
(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 α-1,4-bond to each of at least one of the non-reducing ends of the glucan (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 α-1,4 glucan or a linear α-1,4 glucan, and the degree of polymerization of the branched α-1,4 glucan or the linear α-1,4 glucan is preferably 15 or more and 4×105 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 end-modified glucan of the present invention.
(Item 2)
The glucan according to item 1, wherein the glucan is a branched α-1,4-glucan, wherein the branched α-1,4-glucan has a plurality of non-reducing ends and at least one residue 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 α-1,4-glucan. That is, this glucan is a branched α-1,4-glucan wherein the branched α-1,4-glucan has a plurality of non-reducing ends and at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via an α-1,4-bond to each of at least one (preferably two or more) non-reducing ends of the branched α-1,4-glucan, but neither an N-acetylglucosamine residue nor a galactose residue is present at the position other than the non-reducing ends of the branched α-1,4-glucan, and the degree of polymerization of the branched α-1,4 glucan is preferably 15 or more and 4×105 or less.
(Item 3)
The glucan according to item 2, wherein the branched α-1,4-glucan is selected from the group consisting of a branched maltooligosaccharide, starch, amylopectin, 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, 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, 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.
(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 modified by liking a monosaccharide residue other than an N-acetylglucosamine residue and a galactose residue via an α-1,4-bond to at least one non-reducing end of the plurality of non-reducing ends of the branched α-1,4 glucan.
(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 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)
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 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 residue and a galactose residue is bound to each of two or more non-reducing ends, characterized by allowing an α-glucan phosphorylase to act on an aqueous solution comprising a glucan (preferably a branched α-1,4-glucan having two or more non-reducing ends) and 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 4×105 or less.
(Item 8)
The method according to item 7, wherein the α-glucan phosphorylase has 95% or more sequence identity with the amino acid sequence of α-glucan phosphorylase derived from Aquifex aeolicus VF5, and has activity of transferring N-acetylglucosamine residue or galactose residue to a non-reducing end of a glucan to form an α-1,4-bond.
(Item 9)
A medicament comprising:
(Item 10)
A medicament comprising:
(Item 11)
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 RNA aptamer.
(Item 11A)
The medicament according to any one of items 9 to 11, wherein the medically effective ingredient is an antigen protein 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 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 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 monosaccharide residue other than an N-acetylglucosamine residue and a galactose residue via an α-1,4-bond to at least one non-reducing end of the plurality of non-reducing ends of the branched α-1,4 glucan, wherein the monosaccharide 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 14)
A nanoparticulate carrier for a DDS 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.
(Item 15)
A nanoparticulate carrier for a DDS 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 product thereof, which is further modified by liking a monosaccharide residue other than an N-acetylglucosamine residue and a galactose residue via an α-1,4-bond to at least one non-reducing end of the plurality of non-reducing ends of the branched α-1,4 glucan, wherein the monosaccharide 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)
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.
(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.
(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 α-1,4-bond to at least one or more non-reducing end of the plurality of non-reducing ends of the branched α-1,4 glucan, wherein the monosaccharide residue is one kind or two kinds selected from a glucuronic acid residue and a mannose residue.
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 α-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 can be accumulated into a particular tissue when administered into blood. Since the non-reducing end-modified branched α-1,4 glucan (a branched α-1,4 glucan in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via an α-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 have the activity of stimulating an antigen-presenting cell such as a dendritic cell or a 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 α-1,4 glucan (a branched α-1,4 glucan in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via an α-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 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 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 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 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 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.
The present invention will be explained in detail below.
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 the present specification is used in a sense which is usually used in the art, unless otherwise indicated.
“Glucan”, when used in the present specification, is a polysaccharide having D-glucose as a constituent unit. In the present invention, it is preferable to use, as the glucan, an α-D-glucan. The bonds which link glucose residues in an α-D-glucan predominantly consist of an α-1,4-glucosidic bond, and can contain an α-1,6-glucosidic bond. An α-D-glucan containing a α-1,6-glucosidic bond has a branched structure. A glucan having at least one α-1,6-glucosidic bond in the molecule is referred to as a branched glucan, and a glucan having no α-1,6-glucosidic bond in the molecule is referred to as a linear glucan. Preferable glucans used in the present invention are a linear glucan and a branched glucan, more preferably a linear α-1,4-glucan and an α-1,4-glucan which is branched with an α-1,6-bond (also referred to as a branched α-1,4 glucan). It is preferable that the glucan used in the present invention does not contain an α-1,3-bond.
The linear α-D-1,4-glucan refers to a polysaccharide in which two or more saccharide units of D-glucose units are bound only with an α-1,4-glucosidic bond(s). In the present specification, unless otherwise indicated, the linear α-D-1,4-glucan is referred to as a linear glucan or a linear α-1,4-glucan. The linear glucan has one non-reducing end. Examples of the linear glucan suitably utilized in the present invention include maltooligosaccharide and amylose.
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 α-1,4 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, 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.
In the present specification, the term “amylose” refers to a linear molecule constructed of glucose units connected via α-1,4-bonds. An amylose is contained in natural starch. Amylose may be a natural amylose extracted from a natural starch, or may be an amylose synthesized by an enzymatic reaction (also referred to as “enzymatically synthesized amylose” in the present specification). Natural amylose may contain a branched part in some cases, but enzymatically synthesized amylose does not contain a branch. Further, natural amyloses have a large polydispersity and has a variation in the molecular weight, but an enzymatically synthesized amylose (particularly, an enzymatically synthesized amylose synthesized by the SP-GP method described in International Publication WO 02/097107 pamphlet) has a small polydispersity and has an extremely uniform molecular weight. For this reason, in the present invention, it is preferable to use an enzymatically synthesized amylose. The degree of polymerization of the amylose used in the present invention is preferably about 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 2×103 or less, more preferably about 1×103 or less, still more preferably about 700 or less and most preferably about 500 or less.
In the present specification, the term “branched α-D-glucan” refers to a glucan in which a linear glucan, in which D-glucose units are connected with an α-1,4-glucosidic bond(s), is branched with a bond other than an α-1,4-glucosidic bond. In the present specification, unless otherwise indicated, the branched α-D-glucan is referred to as a branched glucan. A branching bond is either an α-1,6-glucosidic bond, an α-1,3-glucosidic bond, or an α-1,2-glucosidic bond, and most preferably is an α-1,6-glucosidic bond. It is preferable that a branched α-D-glucan used in the present invention does not contain an α-1,3-glucosidic bond and an α-1,2-glucosidic bond. The branched glucan usually has the same number of non-reducing ends as the number of branching bonds. When the branched glucan is treated with an enzyme which selectively breaks only an α-1,6-glucosidic bond (for example, isoamylase, pullulanase, or the like), the branched glucan can be degraded into a mixture of linear α-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.
Examples of the branched glucan suitably utilized in the present invention include branched α-1,4-glucans having 2 or more non-reducing ends, branched maltooligosaccharide, starches, amylopectin, glycogen, dextrin, enzymatically synthesized branched glucan and highly branched cyclic glucan. Branched CD having one non-reducing end is not preferable.
In the present specification, the term “branched maltooligosaccharide” refers to a substance generated by dehydration condensation of about 3 to about 10 D-glucoses, in which D-glucose units are bound mainly with an α-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 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.
In the present specification, the term “starch” is 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 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 is classified into natural starch, a degraded starch and modified starch.
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 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 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.
The degree of polymerization of the starch used in the present invention is preferably about 1×103 or more, more preferably about 5×103 or more, still more preferably about 1×104 or more, and most preferably about 2×104 or more. The degree of polymerization of the starch used in the present invention is preferably about 1×107 or less, more preferably about 3×106 or less, still more preferably about 1×106 or less and most preferably about 3×105 or less.
An amylopectin is a branched molecule in which a glucose unit(s) is bound via an α-1,6 bond to glucose units which are bound via an α-1,4 bond(s). An amylopectin is contained in natural starch. As an amylopectin, for example, waxy corn starch, which consists of 100% amylopectin, can be used. The degree of polymerization of the amylopectin used in the present invention is preferably about 1×103 or more, more preferably about 5×103 or more, still more preferably about 1×104 or more, and most preferably about 2×104 or more. The degree of polymerization of the amylopectin used in the present invention is preferably about 1×107 or less, more preferably about 3×106 or less, still more preferably about 1×106 or less and most preferably about 3×105 or less.
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 sweet corn species of corn. In a glycogen, typically, sugar chains consisting of glucoses bound via an α-1,4-bond(s) which have an average degree of polymerization of 12 to 18 are bound by an α-1,6-bond(s) at a ratio of around one chain every about 3 units of glucose, to a sugar chain consisting of glucoses bound via an α-1,4-bond(s). In addition, similarly, a sugar chain consisting of glucoses bound by an α-1,4-bond(s) is bound by an α-1,6-bond to a branch chain bound by an α-1,6-bond(s). For this reason, glycogen forms a network structure. It is also possible to enzymatically synthesize a glycogen. The degree of polymerization of the glycogen used in the present invention is preferably about 500 or more, more preferably about 1×103 or more, still more preferably about 2×103 or more, and most preferably about 3×103 or more. The degree of polymerization of the glycogen used in the present invention is preferably about 1×107 or less, more preferably about 3×106 or less, still more preferably about 1×106 or less and most preferably about 3×105 or less.
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 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 preferably about 50 or more. The degree of polymerization of the dextrin used in the present invention is preferably about 1×104 or less, more preferably about 9×103 or less, still more preferably about 7×103 or less and most preferably about 5×103 or less.
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 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 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 synthesized branched glucan used in the present invention is preferably about 4×105 or less, preferably about 2×105 or less, more preferably about 1×105 or less, still more preferably about 5×104 or less and most preferably about 3×104 or less.
In the present specification, the term “highly branched cyclic glucan” refers to a glucan having an internally branched cyclic structural moiety and an externally branched structural moiety and having a degree of polymerization of 50 or more. The highly branched cyclic glucan may have at least two non-reducing ends as a whole molecule. The degree of polymerization of the highly branched cyclic glucan as a whole molecule that can be used in the present invention is preferably about 50 or more, 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 1×105 or less, more preferably about 7×104 or less, and still more preferably about 5×104 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 1×104 or less, about 7×103 or less, or about 5×103 or less.
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 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.
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, 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 3×103 or less, more preferably about 1×103 or less, further preferably about 500 or less, and further more preferably about 300 or less.
The number of α-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 example, can be one or more, 5 or more, 10 or more or the like; the number of α-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.
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 α-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 or more. The number of non-reducing ends in the externally branched structural moiety present in the highly branched cyclic glucan is preferably about 5×103 or less, more preferably about 4×103 or less, and further preferably about 3×103 or less.
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 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 polymerization is about 100 or less, more preferably about 50 or less, and further more preferably about 10 or less.
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 5×104, wherein the internally branched cyclic structural moiety is a cyclic structural moiety formed with an α-1,4-glucosidic bond and an α-1,6-glucosidic bond, and the externally branched structural moiety is a non-cyclic structural moiety bound to the internally branched cyclic structural moiety. The degree of polymerization of each unit chain of this externally branched structural moiety is, on average, preferably about 10 or more and preferably about 20 or less. The highly branched cyclic glucan and a method for producing the same are described in detail in Japanese Laid-Open Publication No. 8-134104 (Japanese Patent No. 3107358), and this glucan can be produced according to the description of it. The highly branched cyclic glucan is commercially available, for example, as “Cluster Dextrin” from Ezaki Glico Co., Ltd. The degree of polymerization of the highly branched cyclic glucan used in the present invention is preferably about 50 or more, more preferably about 70 or more, further preferably about 100 or more, most preferably about 150 or more. The degree of polymerization of the highly branched cyclic glucan used in the present invention is preferably about 1×104 or less, more preferably about 7×103 or less, further preferably about 5×103 or less, and most preferably about 4×103 or less.
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 time, particles having a diameter of about 200 nm to about 7 μm are captured by a reticuloendothelial system, and particles having a diameter of about 7 μm or more obstruct capillary blood vessels. A reticuloendothelial system is distributed in liver and spleen. 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 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 5×105 or more, more preferably about 1×106 or more, preferably about 5×107 or less, and more preferably about 2×107 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 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 branched glucan having such a particle size is preferably about 5×105 or more, more preferably about 1×106 or more, preferably about 2×107 or less, and more preferably about 5×106 or less.
The number of branches in the α-glucan (i.e. the number of α-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 α-glucan may be about 30 or more. The number of branches of the α-glucan (i.e. the number of α-1,6-glucosidic bonds) is preferably about 5×103 or less, more preferably about 3×103 or less, further preferably about 2×103 or less. The number of branches in the α-glucan may be about 1×103 or less.
In the branched α-glucan used in the present invention, the ratio of the number of α-1,6-glucosidic bonds relative to the number of α-1,4-glucosidic bonds (“number of α-1,6-glucosidic bonds”:“number of α-1,4-glucosidic bonds”) is preferably 1:1 to 1:1×103, 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 1:5×103, 1:50 to 1:1×103, or 1:100 to 1:500.
In the present invention, the lower limit of the branching frequency of the branched glucan is preferably 0.2% or more, preferably 1% or more, more preferably 2% or more, and most preferably 5% or more. While there is no specific upper limit of the branching frequency, it can be, for example, 99% or less, 90% or less, 85% or less, 65% or less, 50% or less, or the like. The branching frequency is calculated by {(number of α-1,6-bonds)/(sum of α-1,4-bonds and α-1,6-bonds in glucan)}×100.
The α-1,6-glucosidic bonds may be randomly distributed in the α-glucan or may be homogeneously distributed in the α-glucan. A distribution to such an extent that a linear chain part(s) of 5 or more saccharide units can be formed in the α-glucan is preferable.
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 mannose 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 α-1,4 glucan used in the present invention is specifically 2 or more, 3 or 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 α-glucan is preferably about 1×104 or less, more preferably about 5×103 or less. A branched glucan having a larger number of non-reducing 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 α-1,4 glucan having 6 to 8 glucose residues, and is amylase resistant, it is not preferable.
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 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.
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 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 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.
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”), 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 present specification, referred to as a “reducing end-modified product of glucan”).
Examples of the modification at a hydroxyl group include hydroxyalkylation, alkylation, acylation, carboxymethylation, sulfation and phosphorylation. It is preferable that modification at a hydroxyl group is a modification which can be removed with an enzyme in a body. The hydroxyl group-modified product of the glucan is preferably an acylated glucan, and further preferably an acetylated glucan. The frequency of introduction of the modifying group(s) into alcoholic hydroxyl groups can be arbitrarily set at the time of a modification reaction of the glucan. The frequency of introduction of the modifying group(s) into alcoholic hydroxyl groups is expressed as DS, and DS1 means the state where one modifying group per glucose residue is introduced. DS can be calculated by DS=(number of modifying group)/(number of glucose residue). Since there is an OH group at the 2-position, the 3-position and the 6-position in an unmodified glucose residue, theoretically, maximum 3 modifying groups per glucose residue can be introduced. For this reason, the upper limit of DS is usually 3. The frequency of introduction of the modifying group(s) into alcoholic hydroxyl groups is about DS 0.01 or more, more preferably about DS 0.03 or more, further preferably about DS 0.05 or more, particularly preferably about DS 0.07 or more, and most preferably about DS 0.1 or more. The frequency of introduction of modifying group(s) is preferably about DS 1.5 or less, more preferably about DS 1.3 or less, further preferably about DS 1.1 or less, particularly preferably about DS 1.0 or less, and most preferably about DS 0.9 or less. By modifying the glucan, degradation of the glucan in blood or in a body is suppressed.
Examples of modification at a non-reducing end 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 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 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 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-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 acid residue is bound in the non-reducing end-modified product of the present invention.
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 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 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.
Examples of modification at a reducing end include binding with a substance selected from the group consisting of a monosaccharide, a non-reducing carbohydrate, a biocompatible macromolecule, a liposome constituent component, a glycoside and an amine group-containing low-molecular weight substance. Modification at a non-reducing end will be explained in detail in the following 2.6 and 3 sections.
Acetylglucosamine has the same meaning as 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.
Galactose is an epimer of D-glucose, and is a substance in which 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.
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 method or a biological method such as fermentation. Acetylglucosamine or galactose may be used for synthesizing acetylglucosamine-1-phosphate or galactose-1-phosphate.
In the present invention, monosaccharide-1-phosphates 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 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 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 synthesized ones according to the methods known in the art may be used.
(2. Method for Producing a Glucan in which at Least One Residue Selected from N-Acetylglucosamine Residue and Galactose Residue is Bound Via an α-1,4 Bond to Non-Reducing 9)
As acetylglucosamine-1-phosphate or 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 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.
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 acetylglucosamine-1-phospate (e.g. disodium acetylglucosamine-1-phosphate and dipotassium acetylglucosamine-1-phosphate) can be used.
As galactose-1-phospate, either of galactose-1-phospate in a non-salt form and 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) can be used.
In the present specification, the term “α-glucan phosphorylase” means an enzymes having α-glucan phosphorylase activity. α-Glucan phosphorylase is classified in EC 2.4.1.1. α-Glucan phosphorylase activity refers to an activity catalyzing a reaction producing glucose-1-phosphate and partial degraded products of an α-1,4-glucan from inorganic phosphate and the α-1,4-glucan, or the reverse reaction thereof α-Glucan phosphorylase can also catalyze an α-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.
In the present invention, any α-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 a galactose residue is bound to a glucan) to a non-reducing end of glucan. α-glucan phosphorylase used in the present invention can be derived from a bacterium, a yeast, an animal or a plant. α-glucan phosphorylase of the present invention can be derived from, for example, potato, sweet potato, Fava bean, Arabidopsis thaliana, spinach, corn, rice, wheat, Citrus hybrid cultivar, Aquifex aeolicus, Thermotoga maritima, Thermococcus zilligii, Thermoanaerobacter pseudethanolicus, or the like.
It is preferable that α-glucan phosphorylase used in the present invention is α-glucan phosphorylase derived from Aquifex aeolicus VF5, potato-derived α-glucan phosphorylase or Thermococcus zilligii AN1-derived α-glucan phosphorylase, and more preferably it is α-glucan phosphorylase derived from Aquifex aeolicus VF5. Since α-glucan phosphorylase derived from Aquifex aeolicus VF5 have high transferring efficiency, it is quite preferable.
The base sequence of α-glucan phosphorylases derived from Aquifex aeolicus VF5 is set forth in SEQ ID NO: 1, and its amino acid sequence is set forth in positions 1-692 of SEQ ID NO: 2. The amino acid sequence of α-glucan phosphorylases derived from Aquifex aeolicus VF5 has about 21% to about 24% sequence identity with the amino acid sequence of plant α-glucan phosphorylases, about 34% sequence identity with the amino acid sequence of α-glucan phosphorylases derived from Thermus thermophilus, and about 38% sequence identity with the amino acid sequence of α-glucan phosphorylases derived from Thermococcus litoralis. It has about 38% sequence identity with the amino acid sequence of α-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 of Thermoanaerobacter pseudethanolicus.
The base sequence of type L α-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 α-glucan phosphorylases 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.
In the present specification, an enzyme “derived from” an organism, means not only that the enzyme is directly isolated from the organism, but also refers to an enzyme obtained by utilizing the organism in any form. For example, when a gene encoding an enzyme obtained from an organism is introduced into Escherichia coli, and the enzyme is isolated from that Escherichia coli, the enzyme is referred to as being “derived from” the organism.
In the present specification, “identity” of a sequence (for example, an amino acid sequence, a base sequence and the like) refers to the degree of occurrence of the same amino acid (base when base sequences are compared) between two sequences. Identity can be generally determined by comparing two amino acid sequences or two base sequences, and comparing these two sequences which are aligned in an optimal format, which can contain additions or deletions.
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 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, K., and Gotoh, O. 1984. Sequence Relationships among Various 4.5 S RNA Species J. Biochem. 92:1173-1177).
For example, the amino acid sequence of α-glucan phosphorylases used in the present invention can be same with SEQ ID NO: 2, 4 or 6, i.e., it can have 100% identity. In another embodiment, as long as having activity to transfer 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 end of a glucan to form an α-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 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, 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, α-glucan phosphorylases used in the present invention may 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 the enzyme, to increase stability, or the like.
The α-glucan phosphorylase used in the present invention has an amino acid sequence which has preferably about 50% or more, more preferably about 60% or more, further more preferably about 70% or more, still more preferably about 80% or more, particularly more preferably about 90% 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 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 α-1,4 bond. The α-glucan phosphorylase used in the present invention can have an amino acid sequence which has about 96% or more, about 97% or more, about 98% or more, or about 99% more sequence identity with amino acid sequence of SEQ ID NO: 2, 4 or 6.
The amount of the α-glucan phosphorylase contained in a solution at the start of the reaction is preferably about 0.01 U/ml or more, more preferably about 0.1 U/ml or more, particularly preferably about 0.5 U/ml or more, and most preferably about 1 U/ml or more. The amount of the α-glucan phosphorylase contained in a solution at the start of the reaction is preferably about 1,000 U/ml or less, more preferably about 100 U/ml or less, particularly preferably about 50 U/ml or less, and most preferably about 20 U/ml or less. If the weight of α-glucan phosphorylase is too large, 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 α-glucan phosphorylase is defined as follows:
Regarding one unit of α-glucan phosphorylase, an α-glucan phosphorylase activity which produces 1 μmol inorganic phosphate (Pi) per one minute shall be one unit (U or Unit). This measurement of α-glucan phosphorylase activity quantitates free inorganic phosphate (Pi) produced from G-1-P. After 200 μl 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 μl of a molybdenum regent (15 mM ammonium molybdate, 100 mM zinc acetate) is added, and this is stirred to stop the reaction. 200 μA 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 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 glucose-1-phosphate is not quantitated.
α-Glucan phosphorylase used in the present invention can be directly isolated from an organism producing α-glucan phosphorylase, such as the aforementioned organisms, present in the natural world. Alternatively, α-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 α-glucan phosphorylase isolated from the aforementioned organism.
In a preferable embodiment, α-glucan phosphorylase derived from Aquifex aeolicus VF5 is produced by chemically synthesizing a gene fragment of SEQ ID NO: 1, constructing 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 α-glucan phosphorylase, and collecting produced α-glucan phosphorylase. α-glucan 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. Examples of a particularly preferable microorganism include, but not limited to, Escherichia coli.
α-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 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 α-glucan phosphorylase which are used in the present invention can be produced using conventional genetic engineering techniques.
The 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 α-glucan phosphorylase (for example, α-glucan phosphorylase derived from Aquifex aeolicus VF5, α-glucan phosphorylase derived from potato, or α-glucan phosphorylase derived from Thermococcus zilligii AN1) which can catalyze a reaction of transferring an acetylglucosamine residue or a galactose residue to a 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.
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 α-glucan phosphorylase to a suitable solvent. Either one of acetylglucosamine-1-phosphate and 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 this reaction solution. If necessary, glucose-1-phosphate which is an original substrate of α-glucan phosphorylase may be added to this reaction solution. In the case of a reaction where acetylglucosamine-1-phosphate or 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 residue or a galactose residue is bound to a non-reducing end of the glucan, α-glucan phosphorylase can further transfer a molecule to the non-reducing end of the acetylglucosamine residue or the galactose residue, however, the efficiency 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, α-glucan phosphorylase can further transfer a glucose residue or an acetylglucosamine residue or galactose residue to a 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 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-α-glucanotransferase and a glycogen debranching enzyme may be added to this reaction solution.
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 controlled. That is, as increasing (number of molecule of glucan)×(amount of acetylglucosamine-1-phosphate or galactose-1-phosphate relative to number of non-reducing ends in the molecule), the frequency of introduction of 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 residue can be regulated.
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 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 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 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.
Then, the reaction solution is reacted, if necessary, 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 30° C. to about 90° C. It is preferable that the temperature 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 α-glucan phosphorylase contained in this solution before the reaction remains. The reaction temperature is preferably about 35° C. to about 80° C., more preferably about 35° C. to about 70° C., and further preferably about 35° C. to about 65° C. α-Glucan phosphorylase derived from Aquifex aeolicus VF5 is a thermostable enzyme, and its optimal reaction temperature is about 80° C. to 90° C. 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 α-glucan phosphorylase derived from Aquifex aeolicus VF5, a reaction at about 80° C. to 90° C. 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 the reaction temperature is slightly lower than the optimal reaction temperature of the α-glucan phosphorylase derived from Aquifex aeolicus VF5. The reaction temperature is preferably about 30° C. or higher, more preferably about 35° C. or higher, further preferably about 40° C. or higher. In a particular embodiment, the reaction temperature may be about 45° C. or higher or about 50° C. or higher. The reaction temperature is preferably about 90° C. or lower, more preferably about 80° C. or lower, further preferably about 70° C. or lower. In a particular embodiment, the reaction temperature may be about 65° C. or lower or about 60° C. or lower. In the case where an enzyme for which an optimum reaction temperature is lower such as potato-derived α-glucan phosphorylase is used, it is preferable that the reaction temperature is set lower than the case where Aquifex aeolicus VF5-derived α-glucan phosphorylase is used.
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 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 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 60 hours or shorter, about 48 hours or shorter, about 36 hours or shorter, or about 24 hours or shorter.
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. 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.
Furthermore, in the method of the present invention, at least one of acetylglucosamine-1-phosphate or galactose-1-phosphate, a glucan, and α-glucan phosphorylase may be further added to a reaction solution at a stage where the reaction has proceeded to some extent.
In this way, a solution containing a non-reducing end-modified glucan is produced.
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 100° C. for 10 minutes. After completion of the reaction, enzyme may be removed by making pH of the reaction solution to be acidic 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 end-modified glucan.
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-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 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 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 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 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 modifying group other than an N-acetylglucosamine residue or galactose residue may be performed in any order.
The produced non-reducing end-modified glucan (or a modified product thereof) can be purified as necessary. 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 using an organic solvent (T. J. Schoch et al., J. American Chemical Society, 64, 2957 (1942)) and a method not using an organic solvent.
Examples of the organic solvent which can be used in purification using the organic solvent include acetone, 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, α-terpineol, isobutyl alcohol, sec-butyl alcohol, 2-methyl-1-butanol, isoamyl alcohol, tert-amyl alcohol, menthol, methanol, ethanol and ether.
As an example of the purification method not using an organic solvent, there is a method of removing inorganic phosphate, acetylglucosamine-1-phosphate or 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 glucan production reaction.
Examples of the ultrafiltration membrane which can be used in purification include an ultrafiltration membrane of a molecular weight cut off of about 1×103 to about 1×104, preferably about 5×103 to about 5×104, more preferably about 1×104 to about 3×104 (UF membrane unit manufactured by DAICEL).
Examples of a support which can be used in 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.
The glucan of the present invention is a glucan in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-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 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 invention is a glucan in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-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 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 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 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.
In the non-reducing end-modified glucan of the 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 ends can remain unbound by these monosaccharide residues.
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 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 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 residue(s) or galactose residue(s) be present, a high conversion ratio can be selected.
By appropriately adjusting the condition of the enzymatic reaction in producing a non-reducing end-modified 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.
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 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 conversion ratio, therefore, it is advantageous from the practical viewpoint to design a non-reducing end-modified glucan based on binding ratio.
It is noted that depending on the condition of enzymatic reaction in synthesizing a non-reducing 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 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 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 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 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 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.
When the application of the non-reducing 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 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.
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 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 preferably about 150% or less, further preferably about 120% or less, and most preferably about 100% or less.
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 galactose 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 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.
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 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 2000 or less, more preferably about 1000 or less, further preferably about 500 or less, and most preferably about 200 or less.
In the non-reducing end-modified glucan of the present invention, a sugar residue other than 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 residue or galactose residue, and the remaining non-reducing end(s) may bind with a sugar residue other than these monosaccharides.
Alternatively, it is also possible to make a structure wherein some of non-reducing ends bind 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 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.
In a particular embodiment of the present invention, 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.
The hydroxyl group-modified product is as described above.
The non-reducing end-modified product will be explained. When a glucan moiety of the glucan in which at least one residue selected from N-acetylglucosamine residue and galactose residue is bound via α-1,4 bond to the non-reducing end, or a modified product thereof is a branched α-1,4-glucan, and there is a non-reducing end to which neither N-acetylglucosamine residue nor galactose residue is bound, other substances can be bound to a non-reducing end to which neither N-acetylglucosamine residue nor galactose residue is bound. In a preferable embodiment, “a targeting molecule”, “a molecule for drug binding”, or “a cell stimulating molecule” is bound to a non-reducing end to which no amino sugar residue is bound. In the present specification, the term “targeting molecule” refers to a molecule having tissue targeting function. Examples of a targeting molecule include mannose, xylose, fucose, galactosamine, an antibody, an antibody fragment, a receptor, a receptor fragment and a receptor ligand. Since mannose is recognized by a mannose receptor expressed on a variety of macrophages including a Kupffer cell and a sinusoid vascular endothelial cell of liver, it is effective. Mannose can be bound to a non-reducing end of the glucan containing acetylglucosamine residue(s) or galactose residue(s) or a modified product thereof, for example, by allowing an α-glucan phosphorylase to act to mannose-1-phosphate as a substrate. When bound by an enzymatic reaction, mannose is bound at the position 4 of a non-reducing terminal glucose residue to which neither N-acetylglucosamine residue nor galactose residue is bound. For example, it is also possible to combine mannose and galactose at any proportion and bind to a plurality of non-reducing ends of the branched glucan.
In the present specification, the term “molecule for drug binding” refers to a molecule having an anionic or cationic functional group having a function of binding with a medically effective ingredient. Examples of the molecule for drug binding include glucosamine, glucuronic acid, or a derivative thereof. In the case of a conjugate with a glucosamine residue, an amino group is imparted to a non-reducing end. Since an amino group has a positive charge in an aqueous solution, binding with a drug having a negative charge in an aqueous solution by a non-covalent bond becomes possible. In addition, an amino group can also be utilized for binding with a drug via a covalent bond. In addition, it becomes possible to chemically modify an amino group imparted to a non-reducing end of a glucan and introduce a hydrophobic group into a non-reducing end of a glucan. A glucan in which a hydrophobic group is thus introduced into a non-reducing end can be utilized for binding with a protein or a peptide by hydrophobic interaction. In addition, in the case of a conjugate with a glucuronic acid residue, a carboxyl group is imparted to a non-reducing end. Since a carboxyl group has a negative charge in an aqueous solution, binding with a drug having a positive charge in an aqueous solution by a non-covalent bond becomes possible. In addition, a carboxyl group can also be utilized for binding with a drug via a covalent bond. In addition, it also becomes possible to introduce a hydrophobic group into a non-reducing end of a glucan, by chemically modifying a carboxyl group imparted to a non-reducing end of a glucan. A glucan in which a hydrophobic group is thus introduced into a non-reducing end can be utilized for binding with a protein or a peptide by hydrophobic interaction.
In the present specification, the term “antigen-presenting cell stimulating molecule” refers to a molecule having the activity of stimulating a dendritic cell or a macrophage which is an antigen-presenting cell in an innate immune response. The antigen-presenting cell stimulating activity can be measured by a variety of methods. In the present invention, it is preferable that the antigen-presenting cell stimulating activity is measured by a production amount of interleukin-6 (IL-6). Examples of the antigen-presenting cell stimulating molecule include N-acetylglucosamine, galactose, mannose, glucuronic acid, and xylose. By binding of these saccharide residues to a plurality of non-reducing ends, the cell stimulating activity is further enhanced.
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 cell such as a dendritic cell or a macrophage, is bound. It is preferable that at least two or more residues selected from the group consisting of an N-acetylglucosamine residue and a galactose residue are bound to a non-reducing end of a glucan. Further, it is more preferable that at least two or more kinds of monosaccharide residues selected from the group consisting of not only an N-acetylglucosamine residue and a galactose residue, but also a mannose residue, a glucuronic acid residue, a glucosamine residue, or a xylose residue are bound.
In the present specification, the term “adjuvant” refers to a substance which is administered with an antigen, and is used for potentiating the immune response. In “Janeway's Immunobiology” (translated under supervision of Takehiko Sasazuki, Nankodo Co., Ltd.) (original book “Janeway's Immunobiology seventh edition Kenneth Murphy, Paul Travers, Mark Walport 2008 Garlnd Science, Taylar & Francis Group, LLC”), it is described that a dendritic cell or a macrophage produces IL-6 in response to cell infection, and that produced IL-6 activates a lymphocyte and IL-6 potentiates production of an antibody which works in protection against infection. Therefore, those skilled in the art can understand that the immunological function can be potentiated if activation of a dendritic cell or a macrophage can be enhanced and production of IL-6 can be potentiated. As shown in Examples 20 and 22 to 26 of the present specification, the non-reducing end-modified glucan of the present invention could increase IL-6 production of a dendritic cell or a macrophage by contacting with a dendritic cell or a macrophage. For this reason, it is clear that the non-reducing end-modified glucan of the present invention can act as an adjuvant.
Immunostimulating action can be measured by a known method. The immunostimulating action has approximately the same meaning as the cell stimulating activity. As described above, IL-6 production is an indicator of the cell stimulating activity. For example, it can be measured using an action of inducing a cytokine having the immunostimulating action as an indicator. In Examples of the present specification, in order to investigate the immunostimulating action, an indicator of cell stimulation activation of a dendritic cell and a macrophage, which are involved in an immune response, was measured by production of IL-6 which is one of cytokines
The reducing end-modified product will be explained. The “reducing end-modified product of the N-acetylglucosamine residue-containing glucan” refers to a substance in which another substance is bound to a reducing end present in the N-acetylglucosamine residue-containing glucan of the present invention. The “reducing end-modified product of the galactose residue-containing glucan” refers to a substance in which another substance is bound to a reducing end present in the galactose residue-containing glucan of the present invention. In a preferable embodiment, as a method of binding with a different substance at a reducing end, there are the following two methods. The first method is a method of binding a reducing end of a maltooligosaccharide having a degree of polymerization of 2 or more, more preferably a degree of polymerization of 3 or more, further preferably a degree of polymerization of 4 or more to another substance by a known enzymatic procedure or a known chemical procedure and, thereafter, binding acetylglucosamine residue or galactose residue to a non-reducing end of the maltooligosaccharide using the method of the present invention. The second method is a method of binding a reducing end of the glucan containing acetylglucosamine residue(s) or galactose residue(s) of the present invention to another substance by a known enzymatic procedure.
A method of enzymatically binding a maltooligosaccharide to a different substance in the first method is disclosed, for example, in Japanese Laid-Open Publication No. 5-276883, Japanese Laid-Open Publication No. 07-241181 and International Publication No. WO 01/073106.
The method of enzymatically binding a maltooligosaccharide to a different substance in the first method can be used for substances having an amine group. For example, as a method of chemically binding maltopentaose and a substance having an amine group, there are the following three kinds of methods:
(A) A method of binding reducing terminal aldehyde of maltopentaose and a substance having an amine group by reductive amination;
(B) A method of oxidizing reducing terminal aldehyde of maltopentaose into maltotetraosyl gluconic acid and, thereafter, dehydration-condensing this with a substance having an amine group with a condensing agent; and
(C) A method of oxidizing reducing terminal aldehyde of maltopentaose into maltotetraosyl gluconic acid and, thereafter, dehydrating this to prepare maltotetraosyl gluconolactone, and heating this and a substance having an amine group under an anhydrous solvent condition to allow binding. The three kinds of methods (A), (B) and (C) are described in detail in Japanese Patent Application No. 2008-121693. Furthermore, a glucan having high-molecular weight can be bound to a different substance similarly to these methods.
An enzyme utilizable in the second method that is a method of binding a reducing end of the glucan containing acetylglucosamine residue(s) or galactose residue(s) of the present invention to a different substance by an enzymatic procedure can be applied only to carbohydrates and glycosides. As the enzyme, a so-called glucan chain transferring enzyme such as a branching enzyme, CGTase, a D enzyme, amylomaltase or the like is used. These enzymes cut an α-1,4 bond in the glucan containing acetylglucosamine residue(s) or galactose residue(s), and transfer a fragment on its non-reducing end side (fragment containing acetylglucosamine residue(s) or galactose residue(s)) to a receptor molecule (herein, a carbohydrate or a glycoside).
Examples of a substance to be bound include monosaccharides, non-reducing carbohydrates, biocompatible macromolecules, liposome constituent components, glycosides, and amine group-containing low-molecular weight substances.
Examples of the monosaccharides include monosaccharides having a functional group, such as glucosamine, glucuronic acid and gluconic acid. Particularly, glucosamine, glucuronic acid, or gluconic acid is preferable. In the case of a conjugate with a glucosamine residue, an amino group is imparted to a reducing end. Since an amino group has a positive charge in an aqueous solution, binding with a drug having a negative charge in an aqueous solution by a non-covalent bond becomes possible. In addition, an amino group can also be utilized for binding with a drug via a covalent bond. In addition, in the case of a conjugate with a glucuronic acid residue or a gluconic acid residue, a carboxyl group is imparted to a reducing end. Since a carboxyl group has a negative charge in an aqueous solution, binding with a drug having a positive charge in an aqueous solution by a non-covalent bond becomes possible. In addition, a carboxyl group can also be utilized for binding with a drug via a covalent bond. In addition, since a carboxyl group imparted to a reducing end of a glucan can be chemically modified to introduce a cationic group or a hydrophobic group, for example, a carboxyl group can also be utilized for binding with a protein via a non-covalent bond by hydrophobic interaction. Examples of reducing carbohydrates include sorbitol, sucrose, trehalose, dextrin, and amylose. Examples of biocompatible macromolecules include starches, cellulose, chitin, chitosan, dextran, proteins and peptides. Examples of liposome constituent components include phospholipids, fatty acids, ceramides and surfactants. Examples of glycosides include ascorbic acid glucoside, hydroquinone glucoside, hesperidin glucoside, rutin glucoside, para-nitrophenyl maltopentaose, dodecylmaltose, flavonoid glycosides, terpene glycosides, phenol glycosides, chalcone glycosides and steroid glycosides. Examples of the amine group-containing low-molecular weight substances include various amino acids, dodecylamine, ethylenediamine, and diethylaminoethylamine, and the like.
In another embodiment, a reducing end-modified product of a glucan in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-1,4 bond to a non-reducing end (non-reducing end-modified glucan) refers to a product obtained by oxidizing a reducing end aldehyde of a glucan. Oxidation of the reducing end aldehyde is performed by a known chemical procedure such as periodate oxidation. By the oxidation, an aldehyde group is converted into a carboxyl group. An example of oxidation of a reducing end of a molecule in which an acetylglucosamine residue or a galactose residue is bound to a non-reducing end of a linear glucan is shown in the following formula:
wherein n is any integer of 1 or more.
(3. Utilization of Glucan in which at Least One Residue Selected from N-Acetylglucosamine Residue and Galactose Residue is Bound Via α-1,4 Bond to Non-Reducing End, and Modified Product Thereof)
In the glucan in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-1,4 bond to a non-reducing end of the present invention and a modified product thereof, at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound to a non-reducing end, and therefore they can be targeted a particular tissue.
When the non-reducing end-modified glucan of the present invention is utilized as the modifying material of the medically effective ingredient of medicaments, it is necessary to control degradation of the modifying material in a body. When the glucan is a linear α-1,4-glucan, it undergoes rapid degradation with α-amylase, and the time of existence in a body may be too short. In such a case, there is a possibility that an object as the modifying material cannot be attained. Then, by modifying some or all of hydroxyl groups of glucose constituting a glucan, the necessary time for degradation can be controlled. A hydroxyl group-modified product of such a glucan containing acetylglucosamine residue(s) or galactose residue(s) is preferable in the present invention. Modification is etherification or esterification. Etherification is preferably etherification with alkyl halide or an alcohol. The number of carbon atoms of alkyl halide or an alcohol used in etherification is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3. The halogen group can be preferably fluoro, chloro, bromo or iodo. Esterification is preferably esterification with carboxylic acid or acyl halide. The number of carbon atoms of carboxylic acid or acyl halide used in esterification is preferably 1 to 10, more preferably 1 to 5. Modification is desirably esterification. Esterification is more preferably acylation, further preferably acetylation.
As described above, the non-reducing end-modified glucan and a modified product thereof of the present invention can be freely designed for their structure of the glucan and the structure of its non-reducing end and, by utilizing this, the pharmacokinetics of the medically effective ingredient of medicaments can be arbitrary controlled. This is the modifying material of the medically effective ingredient of medicaments, which has a structure completely degradable in a body, and can be safely utilized without anxiety for toxicity due to accumulation.
Therefore, according to the present invention, a medicament containing the non-reducing end-modified glucan or a modified product thereof of the present invention and the medically effective ingredient is provided. In the medicament of the present invention, the medically effective ingredient is preferably 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 RNA aptamer.
According to a particular embodiment, the present invention provides a composition for clinical diagnosis, containing the non-reducing end-modified glucan, a hydroxyl group-modified product thereof, a reducing end-modified product thereof, or a carboxylic acid group-modified product of the present invention.
According to a particular embodiment, the present invention provides a nanoparticulate carrier for a DDS, containing the non-reducing end-modified glucan, a hydroxyl group-modified product thereof, or a reducing end-modified product thereof of the present invention is provided. This nanoparticulate carrier for a DDS is preferably selected from the group consisting of a liposome, a virus particle, a macromolecule micelle and a nanogel composed of macromolecule bearing hydrophobic groups.
(4. The Structure Possessed by Glucan in which at Least One Residue Selected from N-Acetylglucosamine Residue and Galactose Residue is Bound Via α-1,4-Bound to Non-Reducing End and Modified Product Thereof of the Present Invention)
In the present specification, a glucan in which at least one N-acetylglucosamine residue is bound via α-1,4-bound to each of at least one non-reducing end of a glucan, but no N-acetylglucosamine residue is present at the positions other than the non-reducing end of the glucan is referred to as an “N-acetylglucosamine-containing glucan”. In the present specification, a glucan in which at least one galactose residue is bound to each of at least one non-reducing end of a glucan, but no galactose residue is present at the positions other than the non-reducing end of the glucan is referred to as a “galactose-containing glucan”.
It is noted that when two or more N-acetylglucosamine residues are bound to one non-reducing end of a glucan, the structure is that wherein one N-acetylglucosamine residue is bound to one non-reducing end of the glucan and a next N-acetylglucosamine residue is bound to the end of the N-acetylglucosamine residue. In the present specification, a glucan in which at least one N-acetylglucosamine residue is bound via α-1,4 bond to each of at least one non-reducing end of a glucan, but no N-acetylglucosamine residue is present at a position other than a non-reducing end is referred to as “N-acetylglucosamine residue-containing glucan”. These are similarly applied to a galactose residue.
In the present specification, even when a sugar chain consisting of two or more N-acetylglucosamine residues each bound via an α-1,4-bond is bound to a specific non-reducing end of a glucan and no N-acetylglucosamine residue is present at the position other than the non-reducing end of the glucan, it is considered that an N-acetylglucosamine residue is bound only to a non-reducing end and no N-acetylglucosamine residue is present at the position other than the non-reducing end.
In the present specification, also in the case where a saccharide chain consisting of two or more galactose residues which are respectively bound via an α-1,4 bond is bound to a particular non-reducing end of a glucan and no galactose residue is present at a position other than a non-reducing end in the glucan, it is deemed that a galactose residue is bound to only a non-reducing end, and no galactose residue is present at a position other than a non-reducing end.
The non-reducing end-modified glucan of the present invention is a non-reducing end-modified glucan in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-1,4 bond to each of at least one non-reducing end of a glucan, but neither an N-acetylglucosamine residue nor a galactose residue is present at a position other than a non-reducing end of the glucan, wherein the glucan is a branched α-1,4 glucan or a linear α-1,4 glucan. The residue bound to each of the non-reducing ends can be independently selected from an acetylglucosamine residue and a galactose residue. For this reason, as a whole molecule, only an acetylglucosamine residue, a galactose residue, or an acetylglucosamine residue and a galactose residue may be bound to a non-reducing end. As described in another part in the present specification, a residue selected from a glucuronic acid residue, a glucosamine residue, a mannose residue, and a xylose residue may be further bound to a non-reducing end which has no acetylglucosamine residue or a galactose residue bound thereto, among non-reducing ends, and an acetylglucosamine residue or a galactose residue bound to a non-reducing end.
When a glucan moiety of a non-reducing end-modified glucan is a linear glucan, whereby the number of non-reducing ends is one, one N-acetylglucosamine residue or galactose residue is bound to the non-reducing end. When a glucan moiety of a non-reducing end-modified glucan is a branched glucan, whereby there are two or more non-reducing ends, at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound to one or more non-reducing ends among these. In the non-reducing end-modified glucan of the present invention, an N-acetylglucosamine residue or a galactose residue is not present at a position other than a non-reducing end. It is thought that absence of an N-acetylglucosamine residue or a galactose residue at a position other than a non-reducing end can be confirmed by, for example, treating a non-reducing end-modified glucan with an excessive amount of α-amylase and glucoamylase, or isoamylase. Glucoamylase is an enzyme which cuts α-1,4 bonds and α-1,6 bonds from a non-reducing end side, and when it acts on an unmodified branched glucan, the glucan is all degraded into glucose. A glucan in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-1,4 bond to a non-reducing end exhibits resistance to glucoamylase. On the other hand, α-amylase acts on an α-1,4 glucan to produce maltose and glucose, but an α-1,4 glucan chain in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is α-1,4 bound exhibits resistance to α-amylase. A glucan containing an acetylglucosamine residue or a galactose residue only at a non-reducing end, produces N-acetylglucosaminylmaltose or galactosylmaltose and glucose, by treatment with an excessive amount of glucoamylase and α-amylase, and isoamylase. On the other hand, a glucan in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-1,4 bond also to a place other than a non-reducing end produces a saccharide other than N-acetylglucosaminylmaltose or galactosylmaltose, and glucose, by treatment with an excessive amount of α-amylase and isoamylase. By this method, it can be confirmed that an N-acetylglucosamine residue or a galactose residue is not bound to a position other than a non-reducing end.
The glucan of the present invention, a modified product thereof and the like are described in the above 2.6. Herein, a structure which is thought to be possessed by the glucan which has an N-acetylglucosamine residue or a galactose residue bound thereto in accordance with the present invention will be described in the following.
(4.1) Branched Glucan in which at Least One Residue Selected from N-Acetylglucosamine Residue and Galactose Residue is Bound Via α-1,4 Bond to Non-Reducing End, and Modified Product Thereof.
When a glucan moiety contained in the non-reducing end-modified glucan of the present invention is a branched α-1,4 glucan, in the non-reducing end-modified glucan, at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-1,4 bond to at least one of a plurality of non-reducing ends possessed by the branched α-1,4 glucan. According to the present invention, a modified product of the non-reducing end-modified branched glucan is also provided. Modification of the modified product is as detailed above. A substance which is bound in a conjugate is also as detailed above.
A branched glucan moiety contained in the non-reducing end-modified branched glucan, and a modified product thereof of the present invention is a branched α-1,4-glucan having at least 2 or more non-reducing end, and is preferably selected from the group consisting of a branched maltooligosaccharide, a starch, amylopectin, glycogen, dextrin, an enzymatically synthesized branched glucan and highly branched cyclic glucan. A preferable range of these branched glucan moieties is as explained in “(1.1) Glucans and modified products of glucan”.
The molecular weight of the branched α-1,4-glucan in which at least one residue selected from N-acetylglucosamine residue and galactose residue is bound via α-1,4-bound to at least 2 or more non-reducing ends of the present invention is preferably about 1×103 or more, more preferably about 3×103 or more, and further preferably about 5×103 or more. The molecular weight of the branched glucan containing N-acetylglucosamine residue(s) or galactose residue(s) of the present invention is preferably about 1×109 or less, more preferably about 3×108 or less, and further preferably about 1×108 or less.
The molecular weight of the modified product of the non-reducing end-modified branched α-1,4-glucan of the present invention is preferably about 1×103 or more, more preferably about 3×103 or more, and further preferably about 5×103 or more. The molecular weight of the modified product of the non-reducing end-modified branched glucan of the present invention is preferably about 1×109 or less, more preferably about 3×108 or less, and further preferably about 1×108 or less.
In the present invention, a modified product of the branched glucan in which at least one residue selected from N-acetylglucosamine residue and galactose residue bound via α-1,4-bond to non-reducing end is preferable. Modification is preferably acylation or etherification, and more preferably acetylation. The acylation degree is preferably about 0.1 or more, further preferably about 0.2 or more, and particularly preferably about 0.3 or more. The acylation degree is calculated by quantification of acetic acid released by heating under an alkali condition. The etherification degree is preferably about 0.1 or more, further preferably about 0.2 or more, and particularly preferably about 0.3 or more. The etherification degree is calculated by NMR.
The number of the acetylglucosamine residues or the galactose residues bound to the branched glucan and a modified product thereof of the present invention, in which at least one residue selected from N-acetylglucosamine residue and galactose residue bound via α-1,4-bond to non-reducing end, is, respectively independently, preferably 1 or more, more preferably 2 or more, and further preferably 3 or more, per one molecule. The number of N-acetylglucosamine residue(s) or galactose residue(s) bound to the branched glucan or a modified product thereof of the present invention, in which at least one residue selected from N-acetylglucosamine residue and galactose residue bound via α-1,4-bond to non-reducing end is not limited to them, and for example, may be 5 or more, 10 or more, 15 or more, 20 or more, 50 or more, or 100 or more, or the like, per one molecule. The number can be suitably adjusted according to the purpose. The total of the upper limits of the number of acetylglucosamine residue or galactose residue bound to the branched glucan or a modified product thereof of the present invention, in which at least one residue selected from N-acetylglucosamine residue and galactose residue bound via α-1,4-bond to non-reducing end is the number of non-reducing ends of the branched glucan moiety. The number of N-acetylglucosamine residue or galactose residue bound to the branched glucan or a modified product thereof of the present invention, in which at least one residue selected from N-acetylglucosamine residue and galactose residue bound via α-1,4-bond to non-reducing end can be, respectively independently, for example, about 1,000 or less, about 800 or less, about 700 or less, about 600 or less, about 500 or less, about 400 or less, about 300 or less, about 200 or less, about 100 or less, about 50 or less, or the like, per one molecule.
In a particular embodiment, the total number of N-acetylglucosamine residue or galactose residue bound to the branched glucan in which at least one residue selected from N-acetylglucosamine residue and galactose residue bound via α-1,4-bond to non-reducing end and a modified product thereof of the present invention is preferably about 10% or more, more preferably about 20% or more, particularly preferably about 30% or more and, for example, can be about 40% or more, about 50% or more or about 60% or more of the number of non-reducing ends possessed by the branched glucan moiety. In a particular embodiment, the total number of N-acetylglucosamine residue or galactose residue bound to the branched glucan in which at least one residue selected from N-acetylglucosamine residue and galactose residue bound via α-1,4-bond to non-reducing end and a modified product thereof of the present invention is preferably about 100% or less, more preferably about 90% or less, particularly preferably about 80% or less and, for example, can be about 70% or less, about 60% or less, or about 50% or less of the number of non-reducing ends possessed by the branched glucan moiety.
(4.2) Linear Glucan in which at Least One Residue Selected from N-Acetylglucosamine Residue and Galactose Residue Bound Via α-1,4-Bond to Non-Reducing End, and Modified Product Thereof.
In the non-reducing end-modified linear glucan of the present invention, at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-1,4 bond to a non-reducing end of a linear glucan moiety. Since the linear glucan has only one non-reducing end, the non-reducing end-modified linear glucan of the present invention contains only one N-acetylglucosamine residue or galactose residue. According to the present invention, a modified product of the non-reducing end-modified linear glucan is also provided. Modification of the modified product is as detailed above.
In the non-reducing end-modified linear glucan of the present invention, at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-1,4 bond to a non-reducing end of a linear glucan moiety. Since the linear glucan has only one non-reducing end, if an amount of N-acetylglucosamine-1-phosphate or galactose-1-phosphate to be added to a reaction liquid in a reaction of N-acetylglucosamine-1-phosphate or galactose-1-phosphate with a glucan is small, the non-reducing end-modified linear glucan of the present invention contains only one N-acetylglucosamine residue or galactose residue. When an amount of N-acetylglucosamine-1-phosphate or galactose-1-phosphate to be added to a reaction liquid in the reaction of N-acetylglucosamine-1-phosphate or galactose-1-phosphate with a glucan is large, two or more N-acetylglucosamine residues or galactose residues which are bound via α-1,4 bond may be bound to the non-reducing end of the linear glucan. According to the present invention, a modified product of the linear glucan in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-1,4 bond is also provided. According to the present invention, a conjugate of the linear glucan in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-1,4 bond, or a modified product thereof is also provided. Modification of the modified product is as detailed above. A substance bound to an amino sugar-containing glucan or a modified product thereof in a conjugate is also as detailed above.
The linear glucan moiety contained in the linear glucan or a modified product thereof of the present invention, in which at least one residue selected from N-acetylglucosamine residue and galactose residue bound via α-1,4-bond is preferably selected from the group consisting of a maltooligosaccharide, amylose (for example, natural amylose or enzymatically synthesized amylose) and a derivative thereof. A preferable range of these linear glucan moieties is as explained in “(1.1) Glucan and modified product of glucan”.
The molecular weight of the non-reducing end-modified linear glucan of the present invention is preferably about 450 or more, more preferably about 600 or more, and further preferably about 1×103 or more. The molecular weight of the non-reducing end-modified linear glucan of the present invention is preferably about 2×105 or less, more preferably about 1.5×105 or less, and further preferably about 1×105 or less.
The molecular weight of the modified product of the non-reducing end-modified linear glucan of the present invention is preferably about 450 or more, more preferably about 600 or more, further preferably about 1×103 or more. The molecular weight of the modified product of the non-reducing end-modified linear glucan of the present invention is preferably about 2×105 or less, more preferably about 1.5×105 or less, further preferably about 1×105 or less.
In the present invention, a modified product of the non-reducing end-modified linear glucan is preferable. Modification is preferably acylation or etherification, more preferably acetylation. The acylation degree is preferably about 0.1 or more, further preferably about 0.2 or more, and particularly preferably about 0.3 or more. The acylation degree is calculated by quantification of acetic acid released by heating under an alkali condition. The etherification degree is preferably about 0.1 or more, further preferably about 0.2 or more, and particularly preferably about 0.3 or more. The etherification degree is calculated by NMR.
Examples of the non-reducing end-modified linear glucan of the present invention can be represented, for example, by the structure of the following formula 1:
wherein n is an integer of 1 or more. n is preferably about 1 or more, more preferably about 2 or more, further preferably about 3 or more, still further preferably about 5 or more, particularly preferably about 8 or more, and most preferably about 10 or more. n is preferably about 1200 or less, more preferably about 900 or less, and further preferably about 600 or less.
Examples of the non-reducing end-modified linear glucan and the reducing end-modified product thereof of the present invention can be represented, for example, by the structure of the following formula 2:
wherein n is an integer of 1 or more. n is preferably about 1 or more, more preferably about 2 or more, further preferably about 3 or more, still further preferably about 5 or more, particularly preferably about 8 or more, and most preferably about 10 or more. n is preferably about 1200 or less, more preferably about 900 or less, and further preferably about 600 or less. X is selected from the group consisting of hydrogen, monosaccharides, non-reducing carbohydrates, biocompatible macromolecules, liposome constituent components, glycosides, and amine group-containing low-molecular weight substances. X is preferably selected from the group consisting of hydrogen, glucosamine, glucuronic acid, gluconic acid, sorbitol, sucrose, trehalose, dextrin, amylose, starches, cellulose, chitin, chitosan, dextran, proteins, peptides, phospholipids, fatty acids, ceramide, surfactants, ascorbic acid glucosides, hydroquinone glucosides, hesperidin glucosides, rutin glucosides, ceramide, para-nitrophenyl maltopentaose, dodecylmaltose, flavonoid glycosides, terpene glycosides, phenol glycosides, chalcone glycosides, steroid glycosides, amino acids, dodecylamine, ethylenediamine and dimethylaminoethylamine. X is more preferably selected from the group consisting of hydrogen, glucosamine, glucuronic acid, gluconic acid, dextrin and amylose. Particularly, hydrogen, glucosamine, glucuronic acid, gluconic acid and dextrin are preferable. Provided that when X is amine or the like, since the reducing end moiety of the glucan is cleaved, the reducing end-modified product of the present invention can be represented by the following:
When X is hydrogen and Y is an N-acetylglucosamine residue, then this molecule is an N-acetylglucosamine-containing linear glucan, and when X is a substance other than hydrogen and Y is an N-acetylglucosamine residue, then this molecule is a reducing end-modified product of an N-acetylglucosamine-containing linear glucan. When X is hydrogen and Y is a galactose residue, then this molecule is a galactose-containing linear glucan, and when X is a substance other than hydrogen and Y is a galactose residue, then this molecule is a reducing end-modified product of a galactose-containing linear glucan.
An example of the non-reducing end-modified linear glucan, a hydroxyl group-modified product thereof and a reducing end-modified product thereof of the present invention can be represented by, for example, the structure of the following formula 3.
wherein n is an integer of 1 or more. n is preferably about 1 or more, more preferably about 2 or more, further preferably about 3 or more, still further preferably about 5 or more, particularly preferably about 8 or more, and most preferably about 10 or more. n is preferably about 1200 or less, more preferably about 900 or less, and further preferably about 600 or less. X is preferably selected from the group consisting of hydrogen, monosaccharides, non-reducing carbohydrates, biocompatible macromolecules, liposome constituent components, glycosides, and amine group-containing low-molecular weight substances. X is preferably selected from the group consisting of hydrogen, glucosamine, glucuronic acid, gluconic acid, sorbitol, sucrose, trehalose, dextrin, amylose, starches, cellulose, chitin, chitosan, dextran, proteins, peptides, phospholipids, fatty acids, ceramide, surfactants, ascorbic acid glucosides, hydroquinone glucosides, hesperidin glucosides, rutin glucosides, ceramide, para-nitrophenyl maltopentaose, dodecylmaltose, flavonoid glycosides, terpene glycosides, phenol glycosides, chalcone glycosides, steroid glycosides, amino acids and dodecylamine, ethylenediamine and dimethylaminoethylamine. X is more preferably selected from the group consisting of hydrogen, glucosamine, glucuronic acid, gluconic acid, dextrin and amylose. Particularly, hydrogen, glucosamine, glucuronic acid, gluconic acid and dextrin are preferable. Wherein R is preferably independently selected from the group consisting of hydrogen, hydroxyalkyl group, an alkyl group, an acetyl group, a carboxymethyl group, a sulfuric acid group and a phosphoric acid group. Provided that when X is amine or the like, since the reducing end moiety of the glucan is cleaved, the reducing end-modified product of the present invention can be represented by the following:
When X and all of R are hydrogen and Y is an N-acetylglucosamine residue, then this molecule is a N-acetylglucosamine-containing linear glucan; when X is hydrogen, each R is independently hydrogen or another group, provided that at least one of R is a group other than hydrogen and Y is an acetylglucosamine residue, then this molecule is a hydroxyl group-modified product of a N-acetylglucosamine-containing linear glucan; when X is a substance other than hydrogen, all of R is hydrogen, and Y is an N-acetylglucosamine residue, then this molecule is a reducing end-modified product of a N-acetylglucosamine-containing linear glucan; when X is a substance other than hydrogen, each R is independently hydrogen or another group, provided that at least one of R is a group other than hydrogen and Y is an N-acetylglucosamine residue, then this molecule is a reducing end-modified product of a hydroxyl group-modified product of a N-acetylglucosamine-containing linear glucan. When X and all of R are hydrogen and Y is a galactose residue, then this molecule is a galactose-containing linear glucan; when X is hydrogen, each R is independently hydrogen or another group, provided that at least one of R is a group other than hydrogen and Y is a galactose residue, then this molecule is a hydroxyl group-modified product of a galactose-containing linear glucan; when X is a substance other than hydrogen, all of R is hydrogen, and Y is a galactose residue, then this molecule is a reducing end-modified product of a galactose-containing linear glucan; when X is a substance other than hydrogen, each R is independently hydrogen or another group, provided that at least one of R is a group other than hydrogen and Y is a galactose residue, then this molecule is a reducing end-modified product of a hydroxyl group-modified product of a galactose-containing linear glucan.
Examples of the non-reducing end-modified glucan of the present invention can be represented, for example, by the following formula 5:
wherein m is an integer of 1 or more. m is preferably 1 or more, more preferably 2 or more, further preferably 3 or more, still further preferably about 5 or more, particularly preferably about 8 or more, and most preferably about 10 or more, and m is preferably about 1200 or less, more preferably about 900 or less, and further preferably about 600 or less. R1 is independently H, a glucan chain having the structure of formula A or a glucan chain having the structure of formula B. When all of R1 is H, the glucan shown by formula 5 is a linear glucan. When at least one of R1 has the structure of formula A or formula B, the glucan shown by formula 5 is a branched glucan.
in formula A, k is an integer of 1 or more. k is preferably 1 or more, more preferably 2 or more, further preferably 3 or more, still further preferably about 5 or more, particularly preferably about 8 or more, and most preferably about 10 or more, and k is preferably about 100 or less, more preferably about 25 or less, and further preferably about 20 or less. In formula A, each R2 is independently H, a glucan chain having the structure of formula A or a glucan chain having the structure of formula B.
In formula B, s is an integer of 1 or more. s is preferably 1 or more, more preferably 2 or more, further preferably 3 or more, still further preferably about 5 or more, particularly preferably about 8 or more, and most preferably about 10 or more, and s is preferably about 100 or less, more preferably about 25 or less, and further preferably about 20 or less. In formula B, R3 is independently H, a glucan chain having the structure of formula A or a glucan chain having the structure of formula B.
As explained above, in formula 5, R1 can have a structure in which the position of R2 of a group having the structure of formula A or R3 of a group having the structure of formula B is substituted with a group having the structure of formula A or a group having the structure of formula B several times. The total of times of substitutions with formula A and formula B is equal to the number of unit chains of the branched glucan molecule represented by formula 5. The number of unit chain of branched glucan molecule is preferably about 1 or more, more preferably about 10 or more, and further more preferably about 30 or more. The number of unit chain of branched glucan molecule is preferably about 5,000 or less, more preferably about 2,000 or less, and further more preferably about 1,000 or less.
Examples of the non-reducing end-modified glucan or the hydroxyl group-modified product thereof of the present invention can be represented, for example, by the structure of the following formula 6:
wherein m is an integer of 1 or more. m is preferably about 1 or more, more preferably about 2 or more, further preferably about 3 or more, still further preferably about 5 or more, particularly preferably about 8 or more, and most preferably about 10 or more, and m is preferably about 1200 or less, more preferably about 900 or less, and further preferably about 600 or less. R1 is independently H, a hydroxyalkyl group, an alkyl group, an acetyl group, a carboxymethyl group, a sulfuric acid group, a phosphoric acid group, a glucan chain having the structure of formula 6A or a glucan chain having the structure of formula 6B.
in formula 6A, k is an integer of 1 or more. k is preferably 1 or more, more preferably 2 or more, further preferably 3 or more, still further preferably about 5 or more, particularly preferably about 8 or more, and most preferably about 10 or more, and k is preferably about 100 or less, more preferably about 25 or less, and further preferably about 20 or less. In formula 6A, each R2 is independently H, a hydroxyalkyl group, an alkyl group, an acetyl group, a carboxymethyl group, a sulfuric acid group, a phosphoric acid group, a glucan chain having the structure of formula 6A or a glucan chain having the structure of formula 6B.
in formula 6B, s is an integer of 1 or more. s is preferably about 1 or more, more preferably about 2 or more, further preferably about 3 or more, still further preferably about 5 or more, particularly preferably about 8 or more, and most preferably about 10 or more, and s is preferably about 100 or less, more preferably about 25 or less, and further preferably about 20 or less. In formula 6A, each R3 is independently H, a hydroxyalkyl group, an alkyl group, an acetyl group, a carboxymethyl group, a sulfuric acid group and a phosphoric acid group, a glucan chain having the structure of formula 6A or a glucan chain having the structure of formula 6B.
In formula 6, formula 6A and formula 6B, each R4 is independently selected from the group consisting of H, a hydroxyalkyl group, an alkyl group, an acetyl group, a carboxymethyl group, a sulfuric acid group and a phosphoric acid group.
Examples of the non-reducing end-modified glucan, a hydroxyl group-modified product thereof and a reducing end-modified product thereof of the present invention can be represented, for example, by the structure of the following formula 7:
wherein m is an integer of 1 or more. m is preferably about 1 or more, more preferably about 2 or more, further preferably about 3 or more, still further preferably about 5 or more, particularly preferably about 8 or more, and most preferably about 10 or more, and m is preferably about 1200 or less, more preferably about 900 or less, and further preferably about 600 or less. R1 is independently H, a glucan chain having the structure of formula 6A or a glucan chain having the structure of formula 6B. Formula 6A and formula 6B are same with the definition for the aforementioned formula 6.
In formula 7, formula 6A and formula 6B, R4 is independently selected from the group consisting of hydrogen, a hydroxyalkyl group, an alkyl group, an acetyl group, a carboxymethyl group, a sulfuric acid group and a phosphoric acid group,
in formula 7, X is preferably independently selected from the group consisting of monosaccharides, non-reducing carbohydrates, biocompatible macromolecules, liposome constituent components, glycosides, and amine group-containing low-molecular weight substances. X is more preferably selected from the group consisting of hydrogen, glucosamine, glucuronic acid, gluconic acid, sorbitol, sucrose, trehalose, dextrin, amylose, starches, cellulose, chitin, chitosan, dextran, proteins, peptides, phospholipids, fatty acids, surfactants, ascorbic acid glucosides, hydroquinone glucosides, hesperidin glucosides, rutin glucosides, ceramide, para-nitrophenyl maltopentaose, dodecylmaltose, flavonoid glycosides, terpene glycosides, phenol glycosides, chalcone glycosides, steroid glycosides, amino acids and dodecylamine, ethylenediamine, and diethylaminoethylamine. X is more preferably selected from the group consisting of hydrogen, glucosamine, glucuronic acid, gluconic acid, dextrin and amylose. Particularly, hydrogen, glucosamine, glucuronic acid, gluconic acid and dextrin are preferable. Provided that when X is amine or the like, since the reducing end moiety of the glucan is cleaved, the reducing end-modified product of the present invention can be represented as follows:
As described in another place in the present specification, in the non-reducing end-modified glucan of the present invention, at least one kind of residue of a glucuronic acid residue, a mannose residue and a xylose residue may be bound to at least one or more non-reducing end among a plurality of non-reducing ends thereof. For example, in one embodiment, in the case of a glucan of the aforementioned formula 5, 6 or 7, a residue denoted by Y is a residue selected from the group consisting of an N-acetylglucosamine residue, a galactose residue, a glucuronic acid residue, a mannose residue and a xylose residue, provided that at least one Y in a molecule is an N-acetylglucosamine residue or a galactose residue.
Examples of reducing end modification of the non-reducing end-modified glucan of the present invention and the purpose of the modification are summarized in the following Table 1. The structures in the table are merely examples.
Table 1 exemplifies the case where at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound to a non-reducing end. As explained in another part in the present specification, an oligosaccharide residue in which two or more residues selected from an N-acetylglucosamine residue and a galactose residue are bound via α-1,4 bond may be bound to a non-reducing end. In addition, a glucose residue, a mannose residue, a glucuronic acid residue, a glucosamine residue, a xylose residue or an oligosaccharide residue constructed of them or the like may be bound to a 4-position of an N-acetylglucosamine residue or a galactose residue. In addition, for example, in the case of a branched glucan having a plurality of non-reducing ends, a plurality of N-acetylglucosamine residues or galactose residues may be bound to the position of Y shown in Table 1. In addition, one kind or two or more kinds of a glucose residue, a mannose residue, a glucuronic acid residue, a glucosamine residue, a xylose residue and an oligosaccharide residue constructed of them or the like other than an N-acetylglucosamine residue and a galactose residue may be bound.
Since the non-reducing end-modified glucan, a hydroxyl group-modified product thereof, a non-reducing end-modified product and a reducing end-modified product thereof of the present invention have biodegradability, they can be used safely in a living body. In the present specification, the term “biodegradability” refers to that a substance can be degraded in a body to a molecular weight at which a degradation product can be excreted to the outside of the body. The non-reducing end-modified glucan, a hydroxyl group-modified product thereof, a non-reducing end-modified product and a reducing end-modified product thereof of the present invention can be degraded to an oligosaccharide size by combination of amylases in a body.
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 of the present invention are also biocompatible. In the present specification, the term “biocompatibility” refers to absence of toxicity and immunological rejection ability to a living body.
In 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 of the present invention, an N-acetylglucosamine residue and a galactose residue at a non-reducing end have tissue targeting property and organ accumulating property. Therefore, they can be effectively utilized as a DDS carrier. By mixing them with a drug, or by binding them to a drug using a known method, they can be suitably utilized.
Particularly, a non-reducing end-modified product and a reducing end-modified product of a glucan in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-1,4 bond to a non-reducing end of the present invention (non-reducing end-modified glucan) can be utilized as an effective modifying material for a medically effective ingredient, which has two functions of the function of binding a medically effective ingredient and the tissue targeting function.
When a non-reducing end-modified product of the glucan in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-1,4 bond to a non-reducing end of the present invention (non-reducing end-modified glucan) contains, for example, a glucuronic acid residue, a carboxyl group is imparted to a non-reducing end. Since a carboxyl group has a negative charge in an aqueous solution, binding with a drug having a positive charge in an aqueous solution (for example, polypeptide (for example, protein, oligopeptide or the like), cationic polymer, cationic lipid or the like) by a non-covalent bond becomes possible. In addition, a carboxyl group can also be utilized by binding with a drug via a covalent bond. For example, since a carboxyl group imparted to a non-reducing end of a glucan can be chemically modified to introduce a cationic group or a hydrophobic group, for example, a carboxyl group can also be utilized by binding with a protein via a non-covalent bond by hydrophobic interaction. For example, when a glucosamine residue is contained, an amino group is imparted to a non-reducing end. Since an amino group has a positive charge in an aqueous solution, binding with a drug having a negative charge in an aqueous solution (e.g. nucleic acid molecule, anionic polymer, anionic lipid or the like) by a non-covalent bond becomes possible. In addition, an amino group can also be utilized by binding with a drug via a covalent bond.
When a reducing end-modified product of the non-reducing end-modified glucan of the present invention is, for example, a reducing end conjugate with ethylenediamine, an amino group is imparted to a reducing end. Since an amino group has a positive charge in an aqueous solution, binding with a drug having a negative charge in an aqueous solution by a non-covalent bond becomes possible. In addition, an amino group can also be utilized by binding with a drug via a covalent bond.
When a reducing end-modified product of the glucan containing an N-acetylglucosamine residue or a galactose residue of the present invention is, for example, an oxide of a reducing end, a carboxyl group is imparted to a reducing end. Since a carboxyl group has a negative charge in an aqueous solution, binding with a drug having a positive charge in an aqueous solution by a non-covalent bond becomes possible. In addition, a carboxyl group can also be utilized by binding with a drug via a covalent bond. In addition, when a carboxyl group imparted to a non-reducing end of a glucan is chemically modified to introduce a cationic group or a hydrophobic group, for example, this can also be utilized by binding with a protein via a non-covalent bond by hydrophobic interaction.
In 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 of the present invention, an N-acetylglucosamine residue and a galactose residue at a non-reducing end have the activity of stimulating an antigen-presenting cell such as a dendritic cell or a macrophage. Therefore, they can be utilized as a vaccine adjuvant for a medicament. The glucan containing at least two of one kind or two or more kinds of glucuronic acid, glucosamine, mannose, N-acetylglucosamine, galactose and a xylose residue at a non-reducing end, 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 an increased activity of stimulating an antigen-presenting cell such as a dendritic cell or a macrophage. Therefore, they can be utilized as a vaccine adjuvant for a medicament.
According to the present invention, a composition for antigen-presenting cell stimulating activity, including the non-reducing end-modified glucan in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-1,4 bond to a non-reducing end, a hydroxyl group-modified product thereof, a non-reducing end-modified product thereof and a reducing end-modified product thereof is also provided.
The non-reducing end-modified glucan in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-1,4 bond to a non-reducing end, a hydroxyl group-modified product thereof, a non-reducing end-modified product thereof and a reducing end-modified product thereof of the present invention are used as a carrier of a medically effective ingredient. For this reason, a method of therapy including administering a mixture of the carrier and a medically effective ingredient to a subject is also provided. Combination of an objective disease and a medically effective ingredient, and an administration route, an administration frequency and a dose thereof, or the like can be appropriately determined by those skilled in the art.
According to the present invention, a cancer immunotherapy method, an inflammation therapy method or an immunostimulating method, including administering the non-reducing end-modified glucan, in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-1,4 bond to a non-reducing end, a hydroxyl group-modified product thereof, a non-reducing end-modified product thereof and a reducing end-modified product thereof of the present invention, having the antigen-presenting cell stimulating activity to a subject, is also provided.
When a residue or a group capable of being charged negatively, such as a glucuronic acid residue, is bound to the non-reducing end-modified glucan of the present invention, the resulting glucan can be usefully used in antibody therapy, drug therapy and the like. In a particular embodiment of antibody therapy, the method of therapy of the present invention includes administering a complex of such 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 of the present invention, and an antibody protein molecule to a subject.
When a residue or a group capable of being charged positively, such as a glucosamine residue, is bound to the non-reducing end-modified glucan of the present invention, the resulting glucan can be usefully used in gene therapy, drug therapy and the like. In a particular embodiment of gene therapy, the method of therapy of the present invention includes administering a complex of such 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 of the present invention, and a nucleic acid molecule to a subject.
In a particular embodiment, when a residue or a group capable of being charged positively such as a glucosamine residue is bound to the non-reducing end-modified glucan of the present invention, a method including administering a complex formed of a carrier complex of a glucan obtained and a nucleic acid molecule, and a cationic polymer or a cationic lipid to a subject, wherein the nucleic acid molecule includes a gene for therapy is also provided. In a particular embodiment of drug therapy, the method of therapy of the present invention includes administering a complex of such the glucan of the present invention with a medically effective ingredient to a subject.
A nucleic acid molecule including a gene encoding a protein for therapy appropriate for the therapy of an objective disease can also be produced using the conventional genetic engineering technology. The nucleic acid molecule may be an expression vector or a plasmid. A protein appropriate for the therapy of an objective disease, a gene encoding the protein, an expression vector or a plasmid used for preparing or delivering the gene, and an element contained in them are known in the art.
The present invention will be explained below based on examples, but the present invention is not limited to the examples. α-Glucan phosphorylase derived from Aquifex aeolicus VF5 used in the examples is Aquifex aeolicus VF5-derived α-glucan phosphorylase prepared by the following Production Example 1.
Aquifex aeolicus VF5-derived α-glucan phosphorylase was recombination-produced by the following method.
(A) Making of Aquifex aeolicus VF5-Derived α-Glucan Phosphorylase Gene
A nucleic acid (also referred to as an “α-glucan phosphorylase gene”) having a base sequence (base sequence of 491380th to 493458th of ACCESSION No. AE000657 of GenBank base sequence database) encoding the amino acid sequence for Aquifex aeolicus VF5-derived α-glucan phosphorylase gene (the amino acid sequence described in SEQ ID NO: 2 of Sequence Listing; the amino acid sequence obtained by translating the base sequence of 491380th to 493458th (SEQ ID NO: 1) of ACCESSION No. AE000657 of GenBank base sequence database of National Center for Biotechnology Information (NCBI) in the USA) was chemically synthesized by a method well-known to those skilled in the art. An NdeI site was created upstream of a translation initiation codon of this α-glucan phosphorylase gene. In addition, a BamHI site was created downstream of a translation stop codon, and this synthetic gene was cut with NdeI and BamHI, and inserted into plasmid pET11c (manufactured by Novagen) which had been previously cut with NdeI and BamHI to make a plasmid pET-AqGP having an Aquifex aeolicus VF5-derived α-glucan phosphorylase gene.
(B) Expression of Aquifex aeolicus VF5-Derived α-Glucan Phosphorylase Gene in Escherichia coli
Escherichia coli BL21 (DE3) was transformed with this plasmid pET-AqGP according to a conventional method to obtain a transformant. A liquid containing the transformant was diluted and applied on an ampicillin-containing LB agar medium (100 μg/ml ampicillin, 1% tryptone manufactured by Difco, 0.5% yeast extract manufactured by Difco, 0.5% NaCl, 1.5% agar, pH 7.3) so that independent colonies were obtained, and this was cultured at 37° C. overnight. Escherichia coli grown on this ampicillin-containing LB agar medium is a transformant which harbors an introduced plasmid, and can express the introduced plasmid. In such a way, Escherichia coli expressing an α-glucan phosphorylase gene was successfully made.
(C) Preparation of Aquifex aeolicus VF5-Derived α-Glucan Phosphorylase Enzyme
Escherichia coli expressing the Aquifex aeolicus VF5-derived α-glucan phosphorylase gene, made in the aforementioned (B), was inoculated with a LB medium (50 μg/ml ampicillin, 1% tryptone manufactured by Difco, 0.5% yeast extract manufacture by Difco, 0.5% NaCl, pH 7.3), and cultured at 37° C. for 5 hours. Then, IPTG (isopropyl-(3-D-thiogalactopyranoside) and pyridoxine hydrochloride were added to this culture so that the final concentration became 0.1 mM IPTG and 1 mM pyridoxine hydrochloride and, further, this was cultured at 37° C. for 24 hours. Then, bacterial cells were collected by centrifugation of the culture, and washed with 20 mM citrate buffer (pH 6.7) to remove medium components. The bacterial cells after washing were suspended in 20 mM citrate buffer (pH 6.7), crushed with a sonicator, and centrifuged, and the supernatant was used as a bacterial cell extract. The resulting bacterial cell extract was heated at 60° C. for 30 minutes. Then, this bacterial cell extract was loaded on a Q-Sepharose FF column which had been previously equilibrated, and this was eluted at a concentration gradient of from 0.1 M to 0.3 M NaCl, in 20 mM citrate buffer (pH 6.7), to collect a GP-purified enzyme-containing active fraction.
Using about 1 μg of the resulting purified enzyme-containing active fraction, native PAGE (Native polyacrylamide gel electrophoresis) was performed. As a result, in a fraction obtained from Escherichia coli expressing α-glucan phosphorylase, a single band was recognized at the position of the molecular weight of about 150 kDa and, at other places, no band was found. Since the molecular weight of the α-glucan phosphorylase derived from Aquifex aeolicus VF5 is predicted to be about 75 kDa as calculated from their amino acid sequence, as a result of this native PAGE, it is thought that this α-glucan phosphorylase takes a dimer structure. In this manner, it was demonstrated that the α-glucan phosphorylase derived from Aquifex aeolicus VF5 was homogeneously purified.
50 g of a waxy corn starch (manufactured by SANWA CORNSTARCH CO., LTD) was suspended into 1,000 ml of 10 mM sodium phosphate buffer (pH 7.0), and the suspension was heated to about 100° C. to gelatinize 200,000 units of a highly thermostable branching enzyme prepared according to the method described in Example 1 of Japanese Laid-Open Publication No. 2000-316581 was added to the starch paste which had been cooled to about 70° C. to prepare a reaction solution, and then which was allowed to react at 70° C. for 16 hours. After the reaction solution was heated at 100° C. for 20 minutes, the supernatant after centrifuging it at 6,500 rpm for 10 minutes was filtered with a membrane having a pore diameter of 0.8 μm. Then, the filtrate was desalted using a gel filtration chromatography (AKTA purifier) system (column: HiPrep™ 26/10 Desalting manufactured by GE Healthcare) to remove low-molecular weight polysaccharides. 1,000 ml of the filtrate was divided into 7.5 ml of aliquots, and applied to a gel filtration chromatography system, and elution fractions from 2.7 minutes to 3.7 minutes at a flow rate of 10 ml/min were fractionated, respectively. The elution fractions obtained from 1,000 ml of the filtrate were combined, the combined elution fractions was filtered with a membrane having a pore diameter of 0.2 μm, and then lyophilized to obtain about 35 g of a powder of a branched glucan. The weight average molecular weight of the branched glucan was investigated using a high performance liquid chromatography (HPLC) system (column: OHPAK SB-806 MHQ, manufactured by SHOWA DENKO K.K.) equipped with a multiangle laser light scattering detector (DAWN DSP, manufactured by Wyatt Technology Corporation) and a differential refractometer (Shodex RI-71, manufactured by SHOWA DENKO K.K.). 20 mg of a powder of the branched glucan was dissolved in 10 ml of 100 mM aqueous sodium nitrate solution, and the solution was filtered with a membrane having a pore diameter of 0.45 μm to obtain a filtrate. 100 μl of the resulting filtrate was injected into the aforementioned HPLC system. It was shown that the weight average molecular weight of the branched glucan is about 140 K.
When isoamylase was allowed to act on the aforementioned branched glucan, and the reducing power was investigated by the modified Park-Johnson method (Hizukuri et al., Starch, Vol., 35, pp. 348-350, (1983)), it was shown that the unit chain length of branching was about 15 on average, and the number of branching was about 60 on average.
Type L potato α-glucan phosphorylase was recombination-produced by the following method shown in Japanese National Phase PCT Laid-Open Publication No. 2004-526463.
BamHI site was created at the upstream and the downstream of a potato glucan phosphorylase gene (Nakano et al., Journal of Biochemistry 106: 691-695 (1989); SEQ ID NO: 3 encoding the amino acid sequence of SEQ ID NO: 4), this gene was cut with BamHI, and was incorporated into pET3d (manufactured by STRATAGENE) that had been previously cut with BamHI to obtain the plasmid pET-PGP113. In this plasmid, a glucan phosphorylase gene was operably linked under control of an isopropyl-β-D-thiogalactopyranoside (IPTG) inducible promoter. This plasmid was introduced into Escherichia coli TG-1 (manufactured by STRATAGENE) by the competent cell method. This Escherichia coli was plated on a plate containing LB medium (1% tryptone (manufactured by Difco), 0.5% yeast extract (manufactured by Difco), 1% sodium chloride, and 1.5% agar) containing the antibiotic ampicillin, and this was cultured at 37° C. overnight. Escherichia coli grown on this plate was selected to obtain Escherichia coli in which a potato-derived glucan phosphorylase gene had been introduced. It was confirmed by analyzing the sequence of the introduced gene that the resulting Escherichia coli contains the glucan phosphorylase gene. In addition, it was confirmed by activity measurement that the resulting Escherichia coli expresses the glucan phosphorylase.
This Escherichia coli was inoculated in 1 liter of LB medium (1% tryptone (manufactured by Difco), 0.5% yeast extract (manufactured by Difco) and 1% sodium chloride) containing the antibiotic ampicillin, and this was cultured at 37° C. for 3 hours with shaking at 120 rpm. Thereafter, IPTG was added to this medium to 0.1 mM, and pyridoxine was added to this medium to 1 mM, and this was cultured with shaking at 22° C. for a further 20 hours. Then, this culture was centrifuged at 5,000 rpm for 5 minutes to collect the Escherichia coli cells. The resulting cells were suspended in 50 ml of 20 mM Tris-HCl buffer (pH 7.0) containing 0.05% Triton X-100 and, then, this was crushed by sonication to obtain 50 ml of a crushed cell liquid. This crushed liquid contained 4.7 U/mg glucan phosphorylase.
This crushed cell liquid is heated at 55° C. for 30 minutes. After heating, it is centrifuged at 8,500 rpm for 20 minutes to remove insoluble proteins and the like, to obtain the supernatant. The resulting supernatant was applied to an anion exchange resin Q-Sepharose which had been pre-equilibrated, allowing a glucan phosphorylase to be adsorbed onto the resin. The resin was washed with a buffer containing 200 mM sodium chloride to remove impurities. Subsequently, the protein was eluted with a buffer containing 300 mM sodium chloride, to obtain the recombinant potato glucan phosphorylase enzyme solution.
Using a nucleic acid having a base sequence (base sequence of 1st to 2151th described in ACCESSION No. AJ318499 of GenBank base sequence database; SEQ ID NO: 5) encoding the amino acid sequence of Thermococcus zilligii AN1-derived α-glucan phosphorylase (SEQ ID NO: 6) in place of an Aquifex aeolicus VF5-derived α-glucan phosphorylase gene, α-glucan phosphorylase liquid was obtained in the same manner with the Production Example 1.
1 ml of a reaction liquid containing 10 mM maltopentaose (G5), 100 mM sodium acetate buffer (pH 5.5), 10 mM glucose-1-phosphate analogue shown in the following Table 2, and an enzyme (10 U) prepared in any of Production Example 1, 3 or 4 was allowed to act at 50° C. for 24 hours. When each enzyme catalyzes sugar transfer reaction from various glucose-1-phosphate analogues to a non-reducing end of G5, inorganic phosphate is produced. Then, the concentration of inorganic phosphate in the reaction liquid was measured, the binding ratio of a saccharide to G5 was calculated using the following equation, and the results are shown in Table 2. It is noted that since various reactions occurred as side reactions in this sugar transfer reaction, disproportionation of the length of a glucan chain occurred, and as a result, when maltopentaose having a degree of polymerization of 5 was used as a starting material, various saccharide residue-containing maltooligosaccharides having a degree of polymerization of a glucose residue of 3 or more were obtained, but for simplifying calculation, a binding ratio of a saccharide to G5 was calculated.
Binding ratio of saccharide to G5=(concentration of inorganic phosphate produced in reaction liquid (mM)/concentration of G5 in reaction liquid (mM))×100(%)
It is noted that the concentration of inorganic phosphate (mM) was obtained by mixing 800 μl of a molybdenum reagent (15 mM ammonium molybdate, 100 mM zinc acetate) into an aqueous solution (200 μl) containing inorganic phosphate, subsequently, adding 200 μl of a 568 mM aqueous ascorbic acid solution (pH 5.0), stirring the mixture, retaining the mixture at 30° C. for 20 minutes, and measuring an absorbance at 850 nm using a spectrophotometer. An absorbance was similarly measured using inorganic phosphate having a known concentration to prepare a standard curve. By fitting an absorbance obtained in a sample to this standard curve, the concentration of inorganic phosphate (mM) in the reaction liquid was obtained.
Aquifex aeolicus-
Thermococcus zilligii-
As can be understood from Table 2, it was found that Aquifex aeolicus VF5-derived α-glucan phosphorylase acts on glucuronic acid-1-phosphate (GlcA-1-P), glucosamine-1-phosphate (GlcN-1-P), N-acetylglucosamine-1-phosphate (GlcNAc-1-P), mannose-1-phosphate (Man-1-P) and galactose-1-phosphate (Gal-1-P) in addition to glucose-1-phosphate which is an original substrate, and can catalyze a reaction of binding various saccharide residues to a non-reducing end of G5 (that is, these analogues can be utilized as a substrate). However, Aquifex aeolicus VF5-derived α-glucan phosphorylase could not utilize galactosamine-1-phosphate (GalN-1-P), galacturonic acid-1-phosphate (GalA-1-P) and fucose-1-phosphate (Fuc-1-P) as a substrate.
On the other hand, potato-derived α-glucan phosphorylase acted on GlcN-1-P, Man-1-P and Gal-1-P, and could catalyze a reaction of binding various saccharides to a non-reducing end of G5, but could not act on G1cA-1-P, GlcNAc-1-P, GalN-1-P, GalA-1-P and Fuc-1-P. Thermococcus zilligii AN1-derived α-glucan phosphorylase acted on GlcN-1-P, and could catalyze a reaction of binding various saccharides to a non-reducing end of G5. Thermococcus zilligii AN1-derived α-glucan phosphorylase could act slightly on GlcNAc-1-P, GalN-1-P, and Man-1-P, but could not act on G1cA-1-P, GalN-1-P, and GalA-1-P. As described above, it is not the case that α-glucan phosphorylase can utilize any glucose-1-phosphate analogue other than glucose-1-phosphate which is an original substrate. It was revealed that what glucose-1-phosphate analogue can be utilized as a substrate is entirely different depending on an origin of an enzyme to be used.
Aquifex aeolicus VF5-derived α-glucan phosphorylase (50 U) obtained in Production Example 1 was allowed to act on 5 ml of a reaction liquid containing 10 mM maltopentaose (G5), 10 mM N-acetylglucosamine-1-phosphate, and 100 mM sodium acetate buffer (pH 5.5) at 50° C. for 24 hours. As a result, maltooligosaccharides which have an N-acetylglucosamine residue bound thereto were obtained. Since various reactions occurred as side reactions in this sugar transfer reaction, disproportionation of the length of a glucan chain occurred, and as a result, when maltopentaose having the degree of polymerization of 5 was used as a starting material, various maltooligosaccharides which has an N-acetylglucosamine residue bound thereto, in which the degree of polymerization of a glucose residue was 3 or more, were obtained.
The molar concentration of the produced maltooligosaccharides which have an N-acetylglucosamine residue bound thereto is equal to the molar concentration of inorganic phosphate in the reaction liquid. Then, by quantitating the molar concentration of inorganic phosphate in the reaction liquid, the molar concentration of the maltooligosaccharides which has an N-acetylglucosamine residue bound thereto was calculated. Based on this concentration, the amount of maltooligosaccharides which have an N-acetylglucosamine residue bound thereto was calculated. As a result, it was confirmed that 28.3 mg of maltooligosaccharides which have an N-acetylglucosamine residue bound thereto were produced in the aforementioned reaction liquid.
Aquifex aeolicus VF5-derived α-glucan phosphorylase (50 U) obtained in Production Example 1 was allowed to act on 5 ml of a reaction liquid containing 10 mM maltopentaose (G5), 10 mM galactose-1-phosphate, and 100 mM sodium acetate buffer (pH 5.5) at 50° C. for 24 hours. As a result, maltooligosaccharides which have a galactose residue bound thereto were obtained. Since various reactions occurred as side reactions in this sugar transfer reaction, disproportionation of the length of a glucan chain occurred, and as a result, when maltopentaose having the degree of polymerization of 5 was used as a starting material, various maltooligosaccharides which have a galactose residue bound thereto, in which the degree of polymerization of a glucose residue was 3 or more, were obtained.
The molar concentration of the produced maltooligosaccharides which have a galactose residue bound thereto is equal to the molar concentration of inorganic phosphate in the reaction liquid. Then, by quantitating the molar concentration of inorganic phosphate in the reaction liquid, the molar concentration of the maltooligosaccharides which have a galactose residue bound thereto was calculated. Based on this concentration, an amount of the maltooligosaccharides which have a galactose residue bound thereto was calculated. As a result, it was confirmed that 19.8 mg of the maltooligosaccharides which have a galactose residue bound thereto was produced in the reaction liquid.
Aquifex aeolicus VF5-derived α-glucan phosphorylase (50 U) obtained in Production Example 1 was allowed to act on 5 ml of a reaction liquid containing 10 mM maltopentaose (G5), 10 mM glucosamine-1-phosphate, and 100 mM sodium acetate buffer (pH 5.5) at 50° C. for 24 hours. As a result, maltooligosaccharides which have a glucosamine residue bound thereto were obtained. Since various reactions occurred as side reactions in this sugar transfer reaction, disproportionation of the length of a glucan chain occurred, and as a result, when maltopentaose having the degree of polymerization of 5 was used as a starting material, various maltooligosaccharides which have a glucosamine residue bound thereto, in which the degree of polymerization of glucose residue was 3 or more, were obtained.
The molar concentration of the produced glucosamine-bound maltooligosaccharide is equal to the molar concentration of inorganic phosphate in the reaction liquid. Then, by quantitating the molar concentration of inorganic phosphate in the reaction liquid, the molar concentration of the maltooligosaccharides which have a glucosamine residue bound thereto was calculated. Based on this concentration, an amount of the maltooligosaccharides which have a galactose residue bound thereto was calculated. As a result, it was confirmed that 34.2 mg of the maltooligosaccharides which have a glucosamine residue bound thereto was produced in the aforementioned reaction liquid.
Aquifex aeolicus VF5-derived α-glucan phosphorylase (50 U) obtained in Production Example 1 was allowed to act on 5 ml of a reaction liquid containing 110 mg of the branched glucan obtained in Production Example 2, 10 mM N-acetylglucosamine-1-phosphate, and 100 mM sodium acetate buffer (pH 5.5) at 50° C. for 24 hours.
An amount of N-acetylglucosamine transferred to the branched glucan is equal to an amount of inorganic phosphate produced in the reaction liquid. Then, the concentration of inorganic phosphate in the reaction liquid was quantitated, and an amount of N-acetylglucosamine transferred to the branched glucan was obtained from the resulting concentration of inorganic phosphate. Assuming that N-acetylglucosamine was equally bound to the branched glucan in the reaction liquid, an amount of the branched glucan which has an N-acetylglucosamine residue bound thereto was obtained. In the aforementioned reaction liquid, 114.8 mg of the branched glucan which has an N-acetylglucosamine residue bound thereto was produced. It is noted that the binding ratio to a non-reducing end was 46.8%.
Aquifex aeolicus VF5-derived α-glucan phosphorylase (50 U) obtained in Production Example 1 was allowed to act on 5 ml of a reaction liquid containing 110 mg of the branched glucan obtained in Production Example 2, 10 mM galactose-1-phosphate, and 100 mM sodium acetate buffer (pH 5.5) at 50° C. for 24 hours.
An amount of galactose transferred to the branched glucan is equal to an amount of inorganic phosphate produced in the reaction liquid. Then, the concentration of inorganic phosphate in the reaction liquid was quantitated, and from the resulting concentration of inorganic phosphate, an amount of galactose transferred to the branched glucan was obtained. Assuming that galactose was equally bound to the branched glucan in the reaction liquid, an amount of the branched glucan which has a galactose residue bound thereto was obtained. In the aforementioned reaction liquid, 113.1 mg of the branched glucan which has a galactose residue bound thereto was produced. It is noted that the binding ratio of non-reducing end was 38.5%.
Aquifex aeolicus VF5-derived α-glucan phosphorylase (50 U) obtained in Production Example 1 was allowed to act on 5 ml of a reaction liquid containing 110 mg of the branched glucan obtained in Production Example 2, 10 mM glucosamine-1-phosphate, and 100 mM sodium acetate buffer (pH 5.5) at 50° C. for 24 hours.
An amount of glucosamine transferred to the branched glucan is equal to an amount of inorganic phosphate produced in the reaction liquid. Then, the concentration of inorganic phosphate in the reaction liquid was quantitated, and from the resulting concentration of inorganic phosphate, an amount of glucosamine transferred to the branched glucan was obtained. Assuming that glucosamine is equally bound to the branched glucan in the reaction liquid, an amount of the branched glucan which has a glucosamine residue bound thereto was obtained. In the aforementioned reaction liquid, 115.4 mg of the branched glucan which has a glucosamine residue bound thereto was produced. It is noted that the binding ratio to a non-reducing end was 66.8%.
To 10 ml of 5% maltopentaose (G5) was added 10 ml of a 0.2 M I2/MeOH solution, and 4 ml of a 4% KOH/MeOH solution was slowly added dropwise in portions while stirring the liquid at 30° C., to perform an iodic acid oxidation reaction. A 4% KOH/MeOH solution was further added dropwise until the color of iodine disappeared, to perform a reaction for 1 hour while stirring the liquid. Methanol in an amount which is 3-hold of the volume of the resulting reaction liquid was added to precipitate G5 oxide, and the precipitate was collected by centrifugation. About 0.4 g of G5-reducing end-modified product in which a reducing end of G5 was carboxylated was obtained.
In this oxidation reaction, the following reaction occurred.
Next, Aquifex aeolicus VF5-derived α-glucan phosphorylase (50 U) obtained in Production Example 1 was allowed to act on 5 ml of a reaction liquid containing the 10 mM G5-reducing end-modified product obtained above, 10 mM galactose-1-phosphate, and 100 mM sodium acetate buffer (pH 5.5) at 50° C. for 24 hours. As a result, reducing end-modified products of the maltooligosaccharides which have a galactose residue bound thereto were obtained. Since various reactions occurred as side reactions in this sugar transfer reaction, disproportionation of the length of a glucan chain occurred, and as a result, when maltopentaose having the degree of polymerization of 5 was used as a starting material, various reducing end-modified products of the maltooligosaccharides which have a galactose residue bound thereto, in which the degree of polymerization of a glucose residue was 3 or more, were obtained.
The molar concentration of the produced reducing end-modified product of the maltooligosaccharides which have a galactose residue bound thereto is equal to the molar concentration of inorganic phosphate in the reaction liquid. Then, by quantitating the molar concentration of inorganic phosphate in the reaction liquid, the molar concentration of the reducing end-modified products of the maltooligosaccharides which have a galactose residue bound thereto was calculated. Based on this concentration, an amount of the maltooligosaccharides which have a galactose residue bound thereto was calculated. As a result, it was confirmed that 20.1 mg of the reducing end-modified product of the maltooligosaccharide which have a galactose residue bound thereto were produced in the aforementioned reaction liquid.
10 ml of 0.2 M I2/MeOH solution was added to 10 ml of 5% maltopentaose (G5), and 4 ml of 4% KOH/MeOH solution was added dropwise slowly with stirring at 30° C. to conduct iodate oxidation reaction. A 4% KOH/MeOH solution was further added dropwise until the color of iodine disappeared, to perform a reaction for 1 hour while stirring the liquid. Methanol in an amount which is 3-fold of the volume of the resulting reaction liquid was added to precipitate G5 oxide, and the precipitate (about 0.4 g) was collected by centrifugation. The precipitate was maltohexaosyl gluconic acid in which reducing end glucose of maltopentaose is converted into gluconic acid. To a solution wherein 6 g of glucose-1-phosphate and 0.6 mg of maltohexaosyl gluconic acid were dissolved in 100 ml of 0.2 M maleate buffer (pH 6.0), 150 U of Aquifex aeolicus VF5-derived α-glucan phosphorylase produced in Production Example 1 and 2,000 U of highly thermostable branching enzyme prepared according to the method described in Example 1 of Japanese Laid-Open Publication No. 2000-316581 were added to prepare a reaction solution, and thereafter, the reaction was performed for 16 hours with stirring at 50° C. The enzyme was inactivated by heating the solution, and the inactivated enzyme was removed by filtering through a glass filter. Twice the amount of ethanol was added to the filtrate to precipitate the glucan reaction product and then centrifuged. The precipitate was washed twice with 300 milliliters of 1:1 water:ethanol solution to remove the coexisting glucose-1-P, and further washed twice with ethanol, and thereafter lyophilized. The branched glucan which has gluconic acid bound to the reducing end-modified having molecular weight of 120 kDa was obtained in yield of 1.7 g.
Aquifex aeolicus VF5-derived α-glucan phosphorylase (18 Units/ml) prepared in Production Example 1 was allowed to act on each reaction liquid containing 1.6 mg/mL of the aforementioned reducing end-modified branched glucan, 10 mM galactose-1-phosphate or N-acetylglucosamine-1-phosphate, and 100 mM sodium acetate buffer (pH 5.5) at 50° C. for 20 hours to bind a galactose residue or an N-acetylglucosamine residue to a non-reducing end of a branched glucan which has gluconic acid bound to a reducing end. The binding ratio of galactose or N-acetylglucosamine was obtained by the same method as that of Example 3 and was 33% and 32%, respectively. In this manner, a branched glucan which is non-reducing end-modified with a galactose residue or an N-acetylglucosamine residue, in which a reducing end was modified, was obtained.
Aquifex aeolicus VF5-derived α-glucan phosphorylase (50 U) obtained in Production Example 1 was allowed to act on 5 ml of a reaction liquid containing 110 mg of the branched glucan obtained in Production Example 2, 10 mM galactose-1-phosphate, and 100 mM sodium acetate buffer (pH 5.5) at 50° C. for 5 hours.
An amount of galactose transferred to the branched glucan is equal to an amount of inorganic phosphate produced in the reaction liquid. Then, the concentration of inorganic phosphate in the reaction liquid was quantitated, and from the resulting concentration of inorganic phosphate, an amount of galactose transferred to the branched glucan was obtained. Assuming that galactose was equally bound to the branched glucan in the reaction liquid, an amount of the branched glucan which has a galactose residue bound thereto was obtained. 111.7 mg of a galactose-bound branched glucan was produced. It is noted that the binding ratio of a galactose residue to a non-reducing end was 20.8%.
From a reaction liquid containing this branched glucan which has a galactose residue bound thereto, unreacted galactose-1-phophate and inorganic phosphate in the reaction liquid were removed using a PD-10 column (manufactured by GE Healthcare) according to the instructions of the manufacturer. Aquifex aeolicus VF5-derived α-glucan phosphorylase (50 U) obtained in Production Example 1 was allowed to act on 5 ml of a reaction liquid containing 110 mg of the resulting branched glucan which has a galactose residue bound to a non-reducing end, 10 mM glucosamine-1-phosphate, and 100 mM sodium acetate buffer (pH 5.5) at 50° C. for 5 hours. An amount of glucosamine transferred to the branched glucan which has a galactose residue bound thereto was obtained by the same method as that of an amount of galactose transferred to the aforementioned branched glucan. 112.7 mg of the branched glucan which has galactose and a glucosamine residue bound thereto was produced. It is noted that the binding ratio of a glucosamine residue to a non-reducing end was 33.8%.
After a reaction liquid containing 0.5% of the branched glucan which has a saccharide residue bound thereto produced in Example 5, Example 6, or Reference Example 7, 20 mM sodium acetate buffer (pH 5.5), and glucoamylase (17 U/ml; EC3.2.1.3) was incubated at 37° C. for 18 hours, an amount of glucose in the reaction liquid was quantitated by a glucose oxidase method using Glucose CII-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). A ratio of the glucan portion which had been degraded with glucoamylase relative to the whole branched glucan was obtained as a glucoamylase degradation ratio (%), and a ratio of the glucan portion which had not been degraded with glucoamylase relative to the whole branched glucan was obtained as a glucoamylase resistance (%) according to the following equations. The results are shown in Table 3.
Glucoamylase degradation ratio=(amount of glucose in solution after reaction (mg)/amount of glucan in solution before reaction (mg))×100
Glucoamylase resistance=1−glucoamylase degradation ratio
The branched glucan which has an N-acetylglucosamine residue bound thereto and the branched glucan which has a galactose residue bound thereto exhibited resistance to glucoamylase. However, almost all of the branched glucan which has a glucosamine residue bound thereto was degraded with glucoamylase, and did not exhibit glucoamylase resistance. As described above, it was found that glucoamylase resistance can be imparted when galactose or N-acetylglucosamine is bound to a non-reducing end.
The glucan of the present invention exhibits a characteristic nature that it has resistance to glucoamylase alone. However, since a variety of amylases are present in a body, the glucan is thought to be a safe substance excellent in biodegradability, which is finally metabolized in a body and degraded to a size capable of being excreted to the outside of a body even if the glucan has resistance to glucoamylase.
By the same method as that of Example 5, a branched glucan which has an N-acetylglucosamine residue bound to a non-reducing end was produced. First, Aquifex aeolicus VF5-derived α-glucan phosphorylase (50 U) obtained in Production Example 1 was allowed to act on 2.5 ml of a reaction liquid containing 28 mg of the branched glucan obtained in Production Example 2, 15 mM N-acetylglucosamine-1-phosphate, and 100 mM sodium acetate buffer (pH 5.5) at 50° C. for 24 hours. When a ratio of non-reducing end modification was analyzed similarly to Example 5, an amount of N-acetylglucosamine transferred to the branched glucan (ratio of non-reducing end modification) was 62.9%. After the resulting reaction liquid was adjusted to a pH of 3 with a 5 N HCl solution, a precipitate containing a denatured protein was removed by centrifugation. The supernatant was collected and neutralized with a 5 N NaOH solution. Then, using PD-10 column, small molecules were removed and, at the same time, the solution was desalted, this was further filtered with a 0.2 μm filter, and the resulting filtrate was lyophilized, thereby, a powder of a non-reducing end-modified branched glucan which has an N-acetylglucosamine residue bound to a non-reducing end was obtained. Hereinafter, a branched glucan which has an N-acetylglucosamine residue bound to a non-reducing end is also referred to as B-GlcNAc.
As described below, by comparison with the branched glucan produced in Production Example 2 (unmodified branched glucan; B), the structure of a non-reducing end-modified branched glucan (B-GlcNAc) was confirmed.
A powder of the purified non-reducing end-modified branched glucan (B-GlcNAc) obtained as described above was dissolved in 10 mM acetate buffer (pH 5.5) to be 0.2% by weight, various amylases of conditions 1 to 4 of Table 4 were added thereto, and the mixture was incubated at an appropriate temperature, thereby, the glucan was digested. Thereafter, an amount of glucose in the reaction liquid was measured. Since the amount of enzyme shown in Table 4 is an excessive amount, all of bonds that can be cut by the enzyme or combination of enzymes are cut. A glucose oxidase method was used for quantitating glucose.
In addition, the reaction liquids after enzymatic digestion under respective conditions 2 to 4 were analyzed with a HPAEC-PAD apparatus (pumping system: DX300, detector: PAD-2, analysis column: CarboPacPA100) manufactured by DIONEX. Elusion was performed under the conditions of a flow rate: 1 ml/min, a NaOH concentration: 150 mM, a sodium acetate concentration: 0 min-50 mM, 1 min-50 mM, 27 min-350 mM (Gradient curve No. 4), 35 min-850 mM (Gradient curve No. 7), 37 min-850 mM. The results are shown in
Regarding an amount of glucose, calculating the production amount of glucose produced by a reaction under Condition 4 of an unmodified branched glucan (B) as 100%, the glucose production amounts (%) under respective conditions are shown in Table 4. These results will be explained below.
Under Condition 1, glucoamylase which cuts α-1,4 and α-1,6 bonds of a glucan in a glucose unit from a non-reducing end side was allowed to act. For this reason, in the unmodified branched glucan of Production Example 2 constructed only of glucose residues, the amount of glucose was 100%, and all of this unmodified branched glucan was degraded into glucose. However, in B-GlcNAc in which an N-acetylglucosamine residue was bound to a non-reducing end of a glucan, the amount of glucose was 31.2%, and about 68% of bonds in this non-reducing end-modified branched glucan could not be degraded. This ratio was a value near the binding ratio (62.9%) of N-acetylglucosamine to a non-reducing end, which was quantitated from the phosphoric acid concentration shown in Example 5. As a result, it is presumed that an end-modified branched glucan (B-GlcNAc) has a structure on which glucoamylase having the property of cutting an α-1,4 glucosidic bond from a non-reducing end side cannot act and, therefore, an N-acetylglucosamine residue is bound to a non-reducing end part.
Under Condition 2, isoamylase which cuts an α-1,6 bond was allowed to act. Under Condition 2, regarding either of the branched glucan of Production Example 2 and the end-modified branched glucan of Example 5, the amount of glucose was 0.0%.
Further, under Condition 3, isoamylase and glucoamylase were allowed to act in combination. Under Condition 3, an α-1,6 bonds were cut with isoamylase, but regarding either of the branched glucan of Production Example 2 and the end-modified branched glucan of Example 5, the amount of glucose was not changed from that for Condition 1, and was 100% and 31.2%, respectively.
Under Condition 4, isoamylase, glucoamylase, and α-amylase have been allowed to act in combination. α-Amylase has the property of randomly cutting an α-1,4 bond of a glucan. Under Condition 4, all of the unmodified branched glucan of Production Example 2 was degraded into glucose, but only 83.7% of bonds in B-GlcNAc in which an N-acetylglucosamine residue bound to a non-reducing end were degraded. Therefore, in B-GlcNAc, a part of a structure which cannot be degraded with various amylases capable of degrading the unmodified branched glucan of Production Example 2 accounts for 16.3% of the total, and it is presumed that a product having this structure is an oligosaccharide which has an N-acetylglucosamine residue bound to a non-reducing end. (solid star mark in
Aquifex aeolicus VF5-derived α-glucan phosphorylase (50 U) obtained in Production Example 1 was allowed to act on 2.5 ml of a reaction liquid containing 28 mg of the branched glucan obtained in Production Example 2, 15 mM galactose-1-phosphate, and 100 mM sodium acetate buffer (pH 5.5) at 50° C. for 24 hours. An amount of galactose transferred to the branched glucan was 62.2%. By purifying the resulting reaction liquid by the same method as that of Example 11, a powder of the branched glucan which has a galactose residue bound to a non-reducing end was obtained. Hereinafter, the branched glucan which has a galactose residue bound to a non-reducing end is also referred to as B-Gal.
As described below, by comparison with the branched glucan produced in Production Example 2 (unmodified branched glucan; B), the structure of the modified branched glucan which has a galactose residue bound to a non-reducing end (B-Gal) was confirmed.
A powder of the purified non-reducing end branched glucan (B-Gal) obtained as described above was dissolved in 10 mM acetate buffer (pH 5.5) to be 0.2% by weight, various amylases of Conditions 1 to 4 of Table 5 were added thereto, and the mixture was incubated at an appropriate temperature, thereby, the glucan was digested. Thereafter, an amount of glucose in the reaction liquid was measured. Since the enzyme amount shown in Table 5 is an excessive amount, bonds cuttable with the enzyme or combination of enzymes are all cut. A glucose oxidase method was used for quantitating glucose.
In addition, the reaction liquids after enzymatic digestion under respective conditions 2 to 4 were analyzed with HPAEC-PAD by the method of Example 11. The results are shown in
The production amount of glucose produced in a reaction under Condition 4 of the unmodified branched glucan (B) as 100%, the glucose production amounts (%) under respective conditions are calculated and shown in Table 5. These results will be explained below.
Under Condition 1, glucoamylase which cuts α-1,4 and α-1,6 bonds of a glucan in a glucose unit from a non-reducing end side was allowed to act. For this reason, the amount of glucose for the unmodified branched glucan of Production Example 2 constructed only of glucose residues was 100%, and this unmodified branched glucan was all degraded into glucose. However, in B-Gal in which a galactose residue is bound to a non-reducing end of a glucan, the amount of glucose was 30.2%, and about 70% of bonds in this non-reducing end-modified branched glucan, could not be degraded. This ratio was a value near the binding ratio of galactose to a non-reducing end (62.2%), which was quantitated from the phosphoric acid concentration. As a result, it is presumed that the end-modified branched glucan produced in Example 5 has a structure which cannot be acted by glucoamylase having the property of cutting an α-1,4 glucoside bond from a non-reducing end side and, therefore, a galactose residue is bound to a non-reducing end part.
Under Condition 2, isoamylase which cuts an α-1,6 bond was allowed to act. Under Condition 2, concerning the branched glucan of Production Example 2, maltooligosaccharides were produced (
Under Condition 3, when isoamylase and glucoamylase were allowed to act on the non-reducing end-modified glucans produced in Example 5, the peaks with solid circle in
Under Condition 4, isoamylase, glucoamylase, and α-amylase have been allowed to act in combination. α-Amylase has the property of randomly cutting an α-1,4 bond of a glucan. Under Condition 4, the unmodified branched glucan of Production Example 2 was all degraded into glucose, but only 90.9% of bonds in B-Gal in which a galactose residue bound to a non-reducing end were degraded. Therefore, in B-Gal, a part of a structure which cannot be degraded with various amylases capable of degrading the unmodified branched glucan of Production Example 2 accounts for 9.1% of the total, and it is presumed that this product is an oligosaccharide which has a galactose residue bound to a non-reducing end. (solid star mark in
A powder of the branched glucan which has an N-acetylglucosamine residue bound thereto purified in Example 11 was dissolved by adding into 20 mM acetate buffer (pH 5.5) to be 0.5% by weight, to this solution was added 23 U/ml of α-amylase, and the mixture was incubated at 37° C., thereby, the α-amylase was allowed to act, and reduction in the weight average molecular weight of the product was investigated over time.
For the measurement of weight average molecular weight of the product, the method of measuring the weight average molecular weight of branched glucan shown in Production Example 2 was used. The results are shown in Table 6 and
The weight average molecular weight of the branched glucan which has an N-acetylglucosamine residue bound thereto was reduced by the action of α-amylase, and the weight average molecular weight became ½ or less in the reaction time of 10 minutes, and about ⅕ in the reaction time of 30 minutes. At the reaction time of 60 minutes, the branched glucan which has an N-acetylglucosamine residue bound thereto was degraded to a molecule having a molecular weight of 30000 or less. Amylase in blood is α-amylase. Therefore, the non-reducing end-modified branched glucan of the present invention is thought to be degraded with the amylase in blood to a molecule having a molecular weight of 30000 or less. Since molecules having a molecular weight of 30000 or less are thought to be excreted into urine, it is thought that the non-reducing end-modified branched glucan of the present invention is biodegradable.
A powdery branched glucan was produced by acetylating the branched glucan of Production Example 2, and an acetylated branched glucan which has an N-acetylglucosamine residue bound thereto was produced by the same method as that of Example 11. Acetylation of the branched glucan was performed by mixing the branched glucan dissolved in dimethyl sulfoxide (DMSO), DMSO, a 4% aqueous Na2CO3 solution, and 5 M vinyl acetate prepared in DMSO in a composition of Table 7, and allowed to react at 25° C. for 1 hour with stirring. The resulting reaction liquid was diluted 2-fold with ultrapure water, desalting and solvent removal were performed using a gel filtration column (PD-10), and purified acetylated branched glucans were collected with ultrapure water as an aqueous solution. To 300 μl of the collected aqueous solution of purified acetylated branched glucans was added 200 μl of a 5 N aqueous sodium hydroxide solution, and this was heated at 55° C. for 30 minutes, thereby, the acetylated branched glucans were deacetylated. To this reaction liquid was added 300 μl of a 1 N Tris buffer (pH 7) and, further, 200 μl of 5 N hydrochloric acid was added to neutralize the solution. When an amount of glucose in the solution after neutralization was quantitated by a phenol sulfuric acid method, free acetic acid in the solution after neutralization was quantitated with a free acetic acid quantitating kit, and an acetylation degree was obtained, it was confirmed that the acetylation degree of the resulting acetylated branched glucans were 0.1 to 1.3. As described above, three kinds of aqueous solutions containing acetylated glucans were obtained. Each aqueous acetylated glucan solution was lyophilized to obtain each about 300 mg of a powder of the acetylated glucans.
Aquifex aeolicus-derived α-glucan phosphorylase (50 U/ml) prepared in Production Example 1 was added to a reaction liquid containing 2.5% of the resulting acetylated branched glucan powder, 10 mM N-acetylglucosamine-1-phosphate, and 100 mM acetate buffer (pH 5.5) and allowed to react at 50° C. for 18 hours. After the resulting reaction liquid containing a branched glucan which has an N-acetylglucosamine residue bound thereto was adjusted to a pH of 3 with a 5 N HCl solution, a precipitate containing a denatured protein was removed by centrifugation. The supernatant was collected and neutralized with a 5 N NaOH solution. Then, using a gel filtration column (PD-10), small molecules were removed, and at the same time, the solution was desalted, this was further filtered with a 0.2 μm filter, and the resulting filtrate was lyophilized, thereby, a powder of acetylated branched glucans produced by the method of Example 14 was obtained.
23 U/ml α-amylase was added to the resulting reaction liquid containing 0.5% by weight of the acetylated branched glucan which has an N-acetylglucosamine residue bound thereto and 20 mM acetate buffer (pH 5.5), and this was incubated at an appropriate temperature, thereby, α-amylase was allowed to act on the acetylated branched glucans, and reduction in the weight average molecular weight of the products were investigated over time.
Measurement of the molecular weight was performed similarly to Example 13. The results are shown in Table 8 and
The powder of the branched glucan which has a galactose residue bound thereto purified in Example 12 was dissolved by adding to 20 mM acetate buffer (pH 5.5) to be 0.5% by weight, to this solution was added 23 U/ml of α-amylase, and this was incubated at 37° C. to act α-amylase, and reduction in the weight average molecular weight of the product was investigated over time.
For the measurement of weight average molecular weight of a product, the method of measuring the weight average molecular weight of branched glucan shown in Production Example 2 was used. The results are shown in Table 9 and
The weight average molecular weight of the branched glucan which has a galactose residue bound thereto was reduced by the action of α-amylase, and the weight average molecular weight became ⅓ or less in the reaction time of 10 minutes, and about ⅕ in the reaction time of 20 minutes. After the reaction time of 60 minutes, the branched glucan which has a galactose residue bound thereto was rapidly degraded to a molecule having the molecular weight of 30000 or less. Blood amylase is α-amylase. Therefore, the non-reducing end-modified branched glucan of the present invention is thought to be degraded with blood amylase to a molecule having the molecular weight of 30000 or less. Since molecules having a molecular weight of 30000 or less are thought to be excreted into urine, it is thought that the non-reducing end-modified branched glucan of the present invention is biodegradable.
Acetylated branched glucans in which a galactose residue is bound to a non-reducing end of the acetylated branched glucans used in Example 14 were prepared, and control of amylase degradability was studied.
Aquifex aeolicus-derived α-glucan phosphorylase (50 U/ml) prepared by Production Example 1 was added to a reaction liquid containing 2.5% of the resulting acetylated branched glucan, 10 mM galactose-1-phosphate, and 100 mM acetate buffer (pH 5.5), and this was allowed to react at 50° C. for 18 hours. After the resulting reaction liquid containing the branched glucans which have a galactose residue bound thereto was adjusted to a pH of 3 with a 5 N HCl solution, a precipitate containing a denatured protein was removed by centrifugation. The supernatant was collected and neutralized with a 5 N NaOH solution. Then, using PD-10 column, small molecules were removed and, at the same time, the solution was desalted and, further, the solution was filtered with a 0.2 μm filter, thereafter, the resulting filtrate was lyophilized, thereby, a powder in which a galactose residue is bound to a non-reducing end of the acetylated branched glucan produced by the method of Example 14 was obtained.
23 U/ml of α-amylase was added to a reaction liquid containing 0.5% by weight of the resulting branched glucan which has a galactose residue bound thereto and 20 mM acetate buffer (pH 5.5), the mixture was incubated at an appropriate temperature, thereby, α-amylase was allowed to act on the acetylated branched glucans, and reduction in the weight average molecular weight of the product was investigated over time.
For the measurement of weight average molecular weight of a product, the method of measuring the weight average molecular weight of branched glucan shown in Production Example 2 was used. The results are shown in Table 10 and
In vivo biodegradability of a branched glucan which has an N-acetylglucosamine residue bound thereto was investigated. In brief, as an evaluation sample, the branched glucan produced in Production Example 2 and the acetylated branched glucan prepared by the method shown in Example 14 were fluorescently labeled, respectively, and glucan amounts in blood and urine were investigated by a GPC-fluorescent labeling method.
For fluorescent labeling of the glucan, the branched glucan and the acetylated branched glucan having an acetylation degree of 0.5 were prepared in a 50 mg/ml DMSO solution, respectively. 2 ml of each glucan solution in DMSO was put in a test tube and stirred at 90° C. To this was added a 50 mg/ml fluorescein isothiocyanate (FITC) solution in DMSO in an amount of 56 μl and 112 μl, respectively. One drop of pyridine and one drop of di-n-butyltin dilaurate were added dropwise, and the mixture was stirred at 90° C. for 2 hours. After completion of the reaction, to the reaction liquid was added 20 ml of ethanol, the solution was centrifuged at 3500 rpm for 5 minutes, and a precipitate was collected. Further, ethanol was added to perform washing, the precipitate was dissolved in 1 ml of ultrapure water, 1 ml of the solution was applied on a gel filtration column (PD-10), 1.5 ml of ultrapure water was passed thereon. Thereafter, 2 ml was collected with ultrapure water to perform purification. The resulting samples were lyophilized. For a part of the resulting samples, UV absorption at 490 nm was measured. Uranine was used as a standard substance to obtain the FITC introduction amount of a FITC-labeled branched glucan (F-B) and acetylated branched glucan (F-AcB) according to the following equation, it was confirmed that the introduction ratio was 0.86% and 0.82%, respectively.
FITC introduction ratio (W/W %)=((fluorescein concentration of FITC-labeled branched glucan)/(total saccharide amount of FITC-labeled branched glucan))×100
From the resulting two kinds of fluorescein isothiocyanated branched glucans, further F-B-GlcNAc (binding ratio 64.6%) and F-AcB-GlcNAc (binding ratio 51.7%) in which N-acetylglucosamine is bound to a non-reducing end of a glucan, were prepared by the method of Example 3. The resultant product was administered in an amount of 25 mg/kg into tail veins of 9 week old male SD rats (6 animals), and the concentration of the FITC-labeled body in blood after administration was investigated using a high performance liquid chromatography (HPLC) system (column: manufactured by Tosoh Corporation, TSK-gel G4000PWXL, flow rate 1.0 ml/min, eluate 200 mM NaCl 20 mM Tris-HCl buffer (pH 7.8)) equipped with a fluorescent detector (10RF 10AXL manufactured by Shimadzu Corporation). Each 300 μl of blood was collected at each time point from 5 minutes to 48 hours after administration shown in Table 11, and 100 μl of the supernatant was collected after centrifugation at 10,000 rpm for 1 minute. After addition of 10 μl of 30 wt % trifluoroacetic acid, centrifugation at 10,000 rpm was performed for 5 minutes. To the 60 μl of collected supernatant, 60 μl of 200 mM Tris-HCl buffer (pH 7.8) was added. If necessary, the samples were diluted. Thereafter, it this was filtered with a 0.45 μm filter, and 100 μl of the filtrate was poured into the HPLC system.
The results are shown in Table 11 and
In vivo biodegradability of a branched glucan which has a galactose residue bound thereto was investigated.
The FITC-labeled branched glucan (F-B) and acetylated branched glucan (F-AcB) prepared in Example 17 were further treated with the method of Example 4 to prepare F-B-Gal (binding ratio 64.3%) and F-AcB-Gal (binding ratio 51.4%) in which a galactose residue is bound to a non-reducing end of the glucan. The resultant was administered in an amount of 25 mg/kg into tail veins of 9 week old male SD rats (6 animals), and the concentration of the FITC-labeled body in blood after administration was measured by the method of Example 17. The results are shown in Table 12 and
Whether a branched glucan which has a galactose residue bound to a non-reducing end is taken into liver in a larger amount than a branched glucan which has no galactose residue bound thereto or not was investigated.
The acetylated branched glucan (F-AcB 140) produced in Example 14 and the acetylated branched glucan in which a galactose residue is bound to a non-reducing end (F-AcB 140 (Gal)) produced in Example 15 were administered into a tail vein of a 7 week old male ICR mouse (around 30 g), and orbital blood was collected 2 minutes after administration, and whole-blood collection and evisceration were performed 10 minutes, 1 hour, 2 hours, and 6 hours after administration. Serum, and liver homogenate extraction liquid were analyzed by the GPC fluorescent detecting method as in Example 17. The results are shown in Table 13 and
Regarding the serum, 300 μl of blood was centrifuged at 10000 rpm for 1 minute to collect serum, 10 μl of 30% trifluoroacetic acid was mixed therein and, further, the mixture was centrifuged at 10000 rpm for 1 minute to obtain 60 μl of the supernatant. 200 mM Tris-HCl buffer (pH 7.8) was added in an amount of 60 μl. Regarding liver, PBS in a 4-fold amount was added to the liver and this was homogenized, and then the homogenate was centrifuged at 10000 rpm for 5 minutes to obtain 200 μl of the supernatant. 20 μl of 30% trifluoroacetic acid was mixed therein and, further, the mixture was centrifuged at 10000 rpm for 5 minutes to obtain 150 μl of the supernatant, and an equal amount of 200 mM Tris-HCl buffer (pH 7.8) was added to prepare it.
For the branched glucan which has a galactose residue bound thereto, the concentration in blood was significantly decreased and, on the other hand, the concentration in liver was significantly increased. From these results, application to liver targeting was suggested.
After protein removal, desalting, and small molecule removal of the branched glucan of Production Example 2 were performed, filtration with a 0.2 μm filter was performed, and then lyophilization was performed. 50 U of AqGP of Production Method 1 was added to 5 ml of a reaction liquid containing 110 mg of the resulting branched glucan powder, 1, 3, 10, or 15 mM glucose-1-phosphate derivative (N-acetylglucosamine-1-P (GlcNAc-1-P), galactose-1-P (Gal-1-P), mannose-1-P (Man-1-P), glucosamine-1-P (GlcN-1-P), or glucuronic acid-1-P (GlcA-1-P)) and 100 mM sodium acetate buffer (pH 5.5) and this was allowed to react at 50° C. for 24 hours. The reaction liquid was adjusted to a pH of 3 with a 5 N HCl solution, and precipitates containing denatured proteins were removed by centrifugation. The supernatant was collected and neutralized with a 5 N NaOH solution. Then, small molecule removal and desalting of it were performed with a PD-10 column, it was filtrated with a 0.2 μm filter, and then it was lyophilized.
Then, a dendritic cell was stimulated. Details were as follows: For preparing a dendritic cell, bone marrow cells were separated from femur of a C3H/HeJ mouse (7 week old, female, Japan SLC, Inc.), suspended in 10 ml of a RPMI1640 (Sigma) medium containing 10% FBS (bovine fetal serum, Invitrogen), 50 ng/ml rm GM-CSF (PEPROTECH), 25 ng/ml IL-4 (BD Biosciences), and 50 μg/ml gentamycin (Sigma) so as to be 5×106 cells/ml, and cultured with a petri dish in CO2 incubator at 37° C. for 7 days. Each 2.5 ml of the RPMI medium was added on Day 2 and Day 4 from the start of culturing. After culturing for 7 days, the medium was removed, a PBS buffer was put in the petri dish, and suspending cells were removed. Further, ice-cooled PBS Buffer containing 5 mM EDTA was flushed on the petri dish bottom, and adhesive immature dendritic cells were peeled with the water stream. The resulting immature dendritic cell suspension was centrifuged at 4° C. and 1,200 rpm for 5 minutes to collect a precipitate. The resulting dendritic cell was suspended in a RPMI medium containing 10% FBS (bovine fetal serum, Invitrogen) and 50 μg/ml gentamycin (Sigma) so as to be 5×105 cell/ml. The dendritic cell suspension (180 μl) was put in a 96-well plate, and incubated in a CO2 incubator at 37° C. for 1 hour to adhere cells to the plate. INF-γ (10 ng/ml) and various non-reducing end-modified glucans (1 mg/ml) having the binding ratio to a non-reducing end described in Table 14 prepared above were added to this cell, and incubated in a CO2 incubator at 37° C. for 48 hours, thereby, cell stimulation was performed. After cell stimulation, the cell supernatant was collected by centrifugation, the production amount of IL-6 in the supernatant was measured using the OptEIA ELISA kit (BD Biosciences) by the ELISA method, and IL-6 produced by dendritic cell stimulation was calculated.
The results are shown in Table 14 and
Non-reducing end-modified branched glucans were produced in combination of binding saccharides shown in Conditions 1 to 15 of Table 15.
Regarding Conditions 1 to 6, 50 U of AqGP of Production Method 1 was added to 5 ml of a reaction liquid containing 110 mg of the branched glucan powder (which was obtained after protein removal, desalting, and small molecule removal of the branched glucan of Production Example 2, filtration of it with a 0.2 μm filter, and then lyophilization of it), 15 mM glucose-1-phosphate derivative (N-acetylgulcosamine-1-P (GlcNAc-1-P), galactose-1-P (Gal-1-P), mannose-1-P (Man-1-P), glucosamine-1-P (GlcN-1-P), or glucuronic acid-1-P (GlcA-1-P)) and a 100 mM sodium acetate buffer (pH 5.5) and allowed to act at 50° C. for 24 hours. The results of quantitation of inorganic phosphate after the reaction were calculated as amounts of binding to a non-reducing end and shown in the table. The reaction liquid was adjusted to a pH of 3 with a 5 N HCl solution, and a precipitate containing a denatured protein was removed by centrifugation. The supernatant was collected and neutralized with a 5 N NaOH solution. Then, a small molecule removal and desalting of it were performed with a PD-10 column and, further, it was filtered with a 0.2 μm filter. Regarding Conditions 7 to 14, 5 ml of a reaction liquid containing 110 mg of the branched glucan powder of Production Example 2, and 10 mM N-acetylglucosamine-1-P or galactose-1-P was prepared similarly to Conditions 1 to 6. Thereafter, it was subjected to small molecule removal and desalting, and 2 ml of a reaction liquid containing 1 ml of the branched glucan in which a non-reducing end was modified with one kind of saccharide and 10 mM glucuronic acid-1-P, glucosamine-1-P, galactose-1-P, or mannose-1-P was prepared similarly to Conditions 1 to 6. Thereafter, it was subjected to small molecule removal and desalting. Regarding Conditions 16 to 22, 10 ml of a reaction liquid containing 220 mg of the branched glucan powder of Production Example 2, and 10 mM N-acetylglucosamine-1-P or galactose-1-P was prepared similarly to Conditions 1 to 6. Thereafter, it was subjected to small molecule removal and desalting to obtain the branched glucan in which a non-reducing end was modified with one kind of saccharide. 2 ml of a reaction liquid containing 1 ml of the branched glucan in which a non-reducing end was modified with one kind of saccharide, and two kinds of 10 mM glucuronic acid-1-P, glucosamine-1-P, galactose-1-P or mannose-1-P was prepared similarly to Conditions 1 to 6. Thereafter, it was subjected to small molecule removal and desalting. Regarding Condition 23, 5 ml of a reaction liquid containing 110 mg of the branched glucan powder of Production Example 2, 10 mM mannose-1-P, glucuronic acid-1-P, and glucosamine-1-P was prepared similarly to Conditions 1 to 6. Regarding Conditions 1 to 23, all samples were allowed to an enzymatic reaction, and thereafter subjected to degeneration of GP enzyme protein, small molecule removal and desalting, filtered with a 0.2 μm filter, and lyophilized. The resulting various branched glucans were used in Examples 22 to 27.
Various branched glucans (1 mg/ml cell liquid) produced in Example 21 were added to the C3H/HeJ mouse (female, 7 week old)-derived dendritic cells prepared in Example 20, and these were allowed to react in a CO2 incubator at 37° C. for 48 hours. The supernatant of the reaction liquid was collected, and IL-6 produced by cell stimulation was measured by the ELISA method. The results are shown in Table 16 and
Regarding all of branched glucans which has glucuronic acid, N-acetylglucosamine, galactose, or mannose bound to a non-reducing end, the IL-6 production amount was increased as compared with those for the branched glucan of Production Example 2. On the other hand, regarding the branched glucan which has a glucosamine bound thereto, the production amount of IL-6 was decreased than those for the branched glucan of Production Example 2, and there was exhibited a tendency that production of IL-6 is suppressed.
Various branched glucans (1 mg/ml cell liquid) prepared in Example 19 were added to a dendritic cell prepared by the same method as that of Example 20, and they were reacted in a CO2 incubator at 37° C. for 48 hours. The supernatant of the reaction liquid was collected, and IL-6 produced by cell stimulation was measured by the ELISA method. The results are shown in Table 17 and
Regarding the branched glucan which has N-acetylglucosamine bound to a non-reducing end, the IL-6 production amount was increased as compared with those for the branched glucan of Production Example 2. Using N-acetylglucosamine and galactose or mannose in combination, the production amount of IL-6 was further increased. The branched glucan which has N-acetylglucosamine bound to a non-reducing end was recognized to have the activity of stimulating a dendritic cell and, further, the synergistic effect of the cell stimulating activity was recognized by additionally binding galactose or mannose simultaneously with N-acetylglucosamine.
Similarly to Example 21, the IL-6 production amount of branched glucans, in which bindings of galactose and other saccharides were combined, was investigated. The results are shown in Table 18 and
Using C3H/HeJ mouse (female, 7 week old) intraperitoneal cells, the cells were added to various branched glucans prepared in Example 19 (1 mg/ml cell liquid) to allow them to react for 48 hours. The supernatant of the reaction liquid was collected, and IL-6 produced by cell stimulation was measured by the ELISA method. The results are shown in Table 19 and
Regarding the branched glucan which has N-acetylglucosamine bound to a non-reducing end, the IL-6 production amount was larger as compared with those for the branched glucan of Production Example 2, and the production amount of IL-6 was further increased by combining N-acetylglucosamine and galactose or mannose. The branched glucan which has an N-acetylglucosamine bound to a non-reducing end was recognized to have the activity of stimulating a macrophage and, further, the synergistic effect of the cell stimulating activity was recognized by additionally binding galactose or mannose to N-acetylglucosamine.
Similar to Example 23, the IL-6 production amount with branched glucans, in which bindings of galactose and other saccharides were combined, was investigated. The results are shown in Table 20 and
As described above, the present invention has been exemplified using preferable embodiments of the present invention, but the present invention should not be construed so as to be limited to these embodiments. It is understood that the scope of the present invention should be construed only by claims. It is understood that those skilled in the art can carry out an equivalent scope based on the description of the present invention and common technical knowledge, from the description of specific preferable embodiments of the present invention. It is understood that the content of patents, patent applications and references cited in the present specification should be incorporated herein by reference, as if the content itself is specifically described in the present specification.
It is thought that the glucan in which at least one residue selected from an N-acetylglucosamine residue and a galactose residue is bound via α-1,4 bond to a non-reducing end (non-reducing end-modified glucan) of the present invention, a hydroxyl group-modified product thereof and a reducing end-modified product thereof can be accumulated into a particular tissue when administered into blood. For this reason, they are useful in tissue targeting of a medically effective ingredient. Since 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 of the present invention have the activity of stimulating an antigen-presenting cell such as a dendritic cell or a macrophage to produce IL-6 (antigen-presenting cell stimulating activity), they are useful for stimulating an antigen-presenting cell, and further, useful as a vaccine adjuvant. Since 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 of the present invention can have an extended blood half life longer than that of an unmodified glucan, and are finally completely degraded in a living body and excreted, they are extremely highly safe and, for this reason, are useful as a modifying material for a medically effective ingredient, a clinical diagnostic, an imaging agent and a nanoparticulate carrier for DDS. The non-reducing end-modified glucan, a hydroxyl group-modified product thereof and a reducing end-modified product thereof of the present invention can be widely utilized in foods, cosmetics, and medicaments.
SEQ ID NO: 1: Base sequence of the Aquifex aeolicus VF5-derived α-glucan phosphorylase.
SEQ ID NO: 2: Amino acid sequence of the Aquifex aeolicus VF5-derived α-glucan phosphorylase.
SEQ ID NO: 3: Base sequence of the potato-derived α-glucan phosphorylase.
SEQ ID NO: 4: Amino acid sequence of the potato-derived α-glucan phosphorylase.
SEQ ID NO: 5: Base sequence encoding the amino acid sequence of the Thermococcus zilligii AN1-derived α-glucan phosphorylase.
SEQ ID NO: 6: Amino acid sequence of the Thermococcus zilligii AN1-derived α-glucan phosphorylase.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2010-249087 | Nov 2010 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2011/006224 | 11/7/2011 | WO | 00 | 7/18/2013 |
