JP2020143036A - Liposome, and antimalaria agent containing the same - Google Patents

Abstract

To provide a novel liposome, and an antimalaria agent containing the same.SOLUTION: A liposome to which a chondroitin sulfate oligosaccharide containing the A type chondroitin sulfate (CSA) is introduced is prepared, and it is used to provide an antimalaria agent for treating or preventing malaria infection to a pregnant woman.SELECTED DRAWING: None

Description

The present invention relates to liposomes and antimalarial agents containing them.

Chondroitin sulfate (CS) is a type of glycosaminoglycan, which is an acidic polysaccharide. Structurally, CS has a chondroitin (CH) skeleton in which the disaccharide (GlcA-GalNAc) of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) repeats, and the sulfate group is modified to a diverse sugar hydroxy group. ing. GlcA-GalNAc constituting CS is classified as follows according to the method of modifying the sulfate group (Fig. 1). In this specification, chondroitin sulfates having A to E structures are referred to as type A chondroitin sulfate (CSA) to type E chondroitin sulfate (CSE), respectively.

Structure A: GalNAc residue 4-position is sulfated;
B structure: GlcA is replaced with iduronic acid (epimer at C5 position of GlcA);
C structure: GalNAc residue 6 is sulfated;
D structure: Sulfation at 2 positions of GlcA residue and 6 position of GalNAc residue;
E structure: GalNAc residues 4 and 6 are sulfated.

CS has different configurations for CSA to CSE depending on the animal species, organs, growth stage, lesions, and type of core protein. Further, CS is mainly in the form of a proteoglycan bound to a core protein and is widely present in the living body, but the composition of a CS sugar chain bound to one core protein is not uniform.

In vivo CS proteoglycans are involved in important biological phenomena such as tissue morphogenesis / regeneration and cell differentiation during development. For example, it plays an important role in lesions such as joint bone disease, nerve damage, immune disease, and pathogen infection. Their physiological and pharmacological functions are exhibited by their interactions with specific CS-binding molecules, that is, CS receptors, CS-binding cytokines, pathogen-producing proteins, and the like.

By the way, among malaria infections that occur in as many as 500 million patients worldwide every year, infections to pregnant women (called pregnancy malaria or placental malaria) present serious symptoms and threaten the safety of the mother and fetus. Is done. In gestational malaria, the malaria parasite-derived membrane protein VAR2CSA is expressed in malaria-infected erythrocytes and binds to CS proteoglycans in placental vessels within the villi. Pathogens present in bound erythrocytes can hide in the placenta against the bloodstream, escape drugs and biological defense mechanisms, and become a malignant and intractable condition. VAR2CSA interacted specifically with the A structure of the CS sugar chain, and CSA oligosaccharides having 12 or more sugars showed an activity of inhibiting the binding between the immobilized CSA and the VAR2CSA protein (see Non-Patent Document 1). ..

Sugiura et al. Glycoconj J (2016) 33, 985-994

An object of the present invention is to provide a novel liposome and an antimalarial drug containing the same.

So far, it has been clarified that CSA oligosaccharides have an activity of inhibiting the binding between immobilized CSA and VAR2CSA protein in vitro (Non-Patent Document 1), but free sugar chains have physiological activity in the body. Since it is not expressed or has very low bioactivity, in order for CS to have physiological activity in the body, a form bound to some carrier is required. Therefore, the present inventors diligently studied what kind of carrier should be used using CSA oligosaccharides that strongly bind to VAR2CSA, and found that liposomes introduced with CS oligosaccharides having a high proportion of CSA were infected with malaria. It has been clarified that it can bind to erythrocytes remarkably strongly, and by competitively inhibiting the binding between placenta CS proteoglycan and infected erythrocytes, the infected erythrocytes accumulated in the placenta are released, which has a preventive effect as an antimalaria drug. And it is considered that a therapeutic effect can be obtained, and the present invention has been completed.

One embodiment of the present invention is an antimalarial drug for treating or preventing malaria infection in pregnant women, which comprises a liposome into which a chondroitin sulfate oligosaccharide containing type A chondroitin sulfate (CSA) has been introduced. Is. The liposome may contain phospholipids and cholesterol. The average particle size of the liposome may be 50 to 500 nm. In addition, it may contain a therapeutic agent for malaria.

Another embodiment of the present invention is a liposome introduced with a chondroitin sulfate oligosaccharide containing type A chondroitin sulfate (CSA). The chondroitin sulfate oligosaccharide may contain 30% to 100% of CSA. The chondroitin sulfate oligosaccharide may be derived from the cartilage of a marine organism. The average molecular weight of the chondroitin sulfate oligosaccharide may be 1000 to 100,000. The average sugar chain length of the chondroitin sulfate oligosaccharide may be 4 to 400. Any of the liposomes may contain phospholipids and cholesterol. The average particle size of any of the liposomes may be 50 to 500 nm. It may be administered at the same time as the malaria therapeutic agent.

The present invention has made it possible to provide novel liposomes and antimalarial drugs containing them.

It is a figure which shows the structural formula of the structure A to E of chondroitin sulfate. It is a figure which shows the result of the gel filtration chromatography of the CS-liposome and VAR2 protein mixture in one Example of this invention. (A) Ultraviolet absorption (B) VAR2 protein concentration measurement value It is a figure which shows the result of the gel filtration chromatography of the liposome and the VAR2 protein mixed solution in one Example of this invention. (A) Ultraviolet absorption (B) VAR2 protein concentration measurement value It is a figure which shows the inhibitory activity of the binding between the immobilized CS and the VAR2 protein by CS-liposome and CS5k oligosaccharide in one Example of this invention.

The objects, features, advantages, and ideas thereof of the present invention will be apparent to those skilled in the art by the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustration or explanation purposes, and the present invention is described in them. It is not limited. It will be apparent to those skilled in the art that various modifications and modifications can be made based on the description of the present specification within the intent and scope of the present invention disclosed in the present specification.

== CS-Liposome ==
The liposomes of the present disclosure are liposomes into which chondroitin sulfate oligosaccharides containing type A chondroitin sulfate (CSA) have been introduced (hereinafter referred to as CS-liposomes).

The ratio of CSA contained in the chondroitin sulfate oligosaccharide is not particularly limited, but the ratio of CSA to the total number of CS is preferably 30% to 100%, more preferably 50% to 100%, and 70%. It is more preferably contained in% to 100%.

The average sugar chain length of the chondroitin sulfate oligosaccharide is not particularly limited, but is preferably 4 to 400, and more preferably 10 to 40. The average molecular weight of the chondroitin sulfate oligosaccharide is also not particularly limited, but is preferably 1000 to 100,000, and more preferably 2500 to 10000.

Chondroitin sulfate oligosaccharides may be synthesized by a known enzymatic chemical method using a group of CS synthases, or an enzymatically synthesized CS sugar chain library (Sugiura et al. J Biol Chem (2012) 287). , 43390-43400), or sugar chains extracted from natural animal tissues may be used. For example, by enzymatic chemistry, CSA with an average molecular weight of 5,000 and containing about 70% A structure can be synthesized. Examples of natural animal tissues include cartilage of marine organisms known to contain a large amount of CSA. The type of marine organism is not particularly limited, and may be a fish, a mammal, or a mollusk, and may be, for example, a whale, a ray, a shark, a sturgeon, a salmon, or a squid. Further, the cartilage of a terrestrial organism may be used, and for example, a mammalian bovine or pig may be used. To regulate the sugar chain length, a chondroitin polymerase derived from Escherichia coli K4 strain or a gene-modifying enzyme thereof is used to enzymatically regulate the sugar chain length (Sugiura et al. Glycoconj J (2008) 25, 521-. Known methods can be used, such as 530) and a method of partially decomposing a long-chain high molecular weight CS using a testicular hyaluronidase which is a hydrolysis enzyme.

On the other hand, the method for producing liposomes is not particularly limited, and known methods can be used. As the material, it is preferable to use a phospholipid such as phosphatidylcholine, and it is more preferable to use both phospholipid and cholesterol. As the phospholipid, for example, a phospholipid having an amino group such as diacylphosphatidylethanolamine may be used. The size of the liposome is not particularly limited, but the particle size is preferably 50 to 500 nm, more preferably 80 to 200 nm, and further preferably about 100 nm from the viewpoint of compatibility with the living body. The bilayer membrane of the liposome may be multi-layered or single-layered, but is preferably one-layered.

For CS-liposomes, liposomes may be prepared first, and then CS may be bound to the liposomes. For example, the amino group of the lipid constituting the liposome and the hemiacetal group at the sugar chain reducing end of CS can be covalently bonded by a reductive amination method. Alternatively, a condensing agent such as carbodiimide may be used to bond the amino group of the lipid to the carboxy group of GlcA.

Alternatively, CS and a hydrophobic molecule may be covalently bonded in advance, and the CS-hydrophobic molecule conjugate may be introduced into the liposome. CS-liposomes may be prepared by treatment with lipids constituting liposomes and CS-hydrophobic molecular conjugates. As the hydrophobic molecule, fatty acids, triglycerides and the like may be used, but phosphatidylethanolamine, which is a phospholipid having two fatty acid chains and an amino group as a functional group, is preferable.

When phosphatidylethanolamine is used, its fatty acid chain is not particularly limited and may be saturated fatty acid or unsaturated fatty acid, but higher fatty acids having 14 or more carbon atoms that can be easily introduced into liposomes are preferable, and examples thereof include palmitic acid and stearic acid. .. In addition, the complex of phosphatidylethanolamine and CS sugar chain (CS-PE, Sugiura and Kimata Methods Enzymol (1994) 247, 362-373) has a condensation reaction with a lipid amino group with a carboxy group of GlcA and a sugar chain. It can be synthesized by a reduction amination method of a hemiacetal group at the reducing end and a lipid amino group. Alternatively, the reducing end may be oxidized to form a lactone, and a complex may be formed by an acid amide bond with the amino group of the lipid.

The method for preparing CS-liposomes using the CS-hydrophobic molecular conjugate is not particularly limited, but for example, the SUV liposome and the CS-hydrophobic molecular conjugate are mixed and heated at around 37 ° C. for several hours. As a result, hydrophobic molecules are easily incorporated into liposomes, and CS-liposomes can be produced.

== Antimalarial drug ==
The antimalarial drug of the present disclosure is an antimalarial drug for treating or preventing malaria infection in pregnant women, and contains a liposome into which a chondroitin sulfate oligosaccharide containing type A chondroitin sulfate (CSA) has been introduced as an active ingredient. .. That is, the antimalarial drug contains an effective amount of the liposome. The above-mentioned CS-liposomes are used as the liposomes into which chondroitin sulfate oligosaccharides containing type A chondroitin sulfate (CSA) have been introduced.

CS-liposomes have the function of competitively releasing infected red blood cells that accumulate in the placenta, thus alleviating the symptoms of pregnancy malaria (also referred to as placental malaria).

The route of administration of the agents of the present disclosure can be either systemic or topical. The parenteral route is preferable, and intravascular administration such as intravenous administration and intraarterial administration is particularly preferable. Therefore, the agents of the present disclosure are preferably for injection or infusion.

The antimalarial drug of the present disclosure may further contain a commonly used additive, if necessary, in addition to the active ingredient, and in the case of an injectable drug or an infusion drug, for example, one or more kinds of pharmaceutical drugs. It may include acceptable pH regulators, buffers, stabilizers, tonicity agents, local anesthetics, preservatives and the like. Examples of the pH adjuster and buffer include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, ethylenediaminetetraacetic acid (EDTA), thioglycolic acid, thiolactic acid and the like. Examples of the local anesthetic include procaine hydrochloride, lidocaine hydrochloride and the like. Examples of the tonicity agent include sodium chloride and glucose.

The amount of the active ingredient contained in the drug can be appropriately determined depending on the effective dose range, the number of doses, and the like. The dose to be administered is not particularly limited, and is intended for prophylactic or therapeutic use, dosage form, route of administration, type of disease, nature of the subject (weight, age, medical condition and presence or absence of other drugs), and responsibility. It is appropriately selected according to the judgment of the doctor.

As a therapeutic drug, it is administered to pregnant women infected with malaria, and as a preventive drug, it is administered to pregnant women who are not infected with malaria. Prophylactic agents are effective when administered to pregnant women in an environment where there is a high risk of malaria infection.

In addition to CS-liposomes, the antimalarial agents of the present disclosure may further contain known therapeutic agents for malaria other than CS-liposomes. Since CS-liposomes have a function of releasing infected erythrocytes accumulated in the placenta, the effect of the malaria therapeutic agent can be enhanced by simultaneously administering the malaria therapeutic agent. The therapeutic agent for malaria is not particularly limited, and examples thereof include atovaquone / proguanil combination, primaquine, and artemether / lumefantrine combination. Here, "administering at the same time" means that the administration does not have to be performed at exactly the same time, and is administered so that the effect is exhibited while the effect of CS-liposomes is exhibited. Therefore, the malaria therapeutic agent may be administered before or after administration of CS-liposomes. In addition, known therapeutic agents for malaria may be in a dosage form separate from CS-liposomes, or may be encapsulated in CS-liposomes.

[Example 1] Production of CS-PE An aqueous solution of CS prepared by preparing CS derived from whale cartilage (containing 69% A structure and 25% C structure) with testicular hyaluronidase to an average molecular weight of 5,000 was prepared. These CS aqueous solutions were passed through a column packed with Dowex 50 X8 (H + type) cation exchange resin to collect the eluted desalted CS, and immediately added a tetrabutylammonium (TBA) aqueous solution to prepare a CS / TBA salt. This CS / TBA salt solution was freeze-dried and added to dehydrated methanol to dissolve it. 2 times molar amount of dipalmitoylphosphatidylethanolamine (PE) was added to CS, and after stirring at 60 ° C. for 2 hours, 5 times molar amount of sodium cyanoborohydride (NaBH ₃ CN) was added to CS, and the mixture was sealed 60. Stirred overnight at ° C. After concentrating the reaction solution under reduced pressure, a 0.2 M aqueous sodium acetate solution was added, the insoluble matter was removed by centrifugation, a triple volume of 95% ethanol containing 1.3% potassium acetate was added to the supernatant, and 2 at 4 ° C. After allowing to stand for a long time, the generated precipitate was collected by centrifugation. The resulting precipitate was dissolved in a mixed solution of methanol: water (1: 1), butyl cellulofine was added, the suspension was concentrated under reduced pressure to remove methanol, 0.2 M saline was added, and then the column was added. After removing the solution, it was eluted with a 30% methanol water mixture. Methanol was distilled off from the obtained eluate by concentration under reduced pressure, 95% ethanol containing 1.3% potassium acetate was added, and the produced precipitate was collected to obtain PE-bound CS (CS-PE).

[Example 2] Production of CS-liposomes Phosphate buffered saline obtained by heating 100 mg of a fine powder mixture (presome, manufactured by Nippon Seika) of distearoylphosphatidylcholine (DSPC): cholesterol (molar ratio 3: 2) to 70 ° C. Suspended in 5 mL of water (PBS) and passed through a 0.1 μm porous membrane 20 times at 70 ° C. using an extruder (manufactured by Avanti) to prepare small single membrane (SUV) liposomes having a particle size of 100 nm. did.

The CS-liposomes in which CS-PE was introduced onto the surface of SUV liposomes were introduced by mixing 0.4 mL of the SUV liposomes with a PBS solution of CS-PE (0.4 mg / 0.4 mL) and shaking at 37 ° C. for 2 hours. Was produced.

[Example 3] Analysis of interaction between CS-liposomes and malaria protein VAR2 A recombinant protein VAR2 (80 μg / 100 μL) tagged with V5-tag in the CS-binding region of the malaria parasite-producing protein VAR2CSA was used as the CS-liposome of Example 2. Add to (100 μL), shake at 37 ° C. for 2 hours, then inject into a column connected in series with Sephaprotein S500 (10 mm x 300 mm) and Superiore 6 Increse (10 mm x 30 mm) at a flow rate of 0.5 mL / min. PBS was delivered and gel filtration chromatography was performed. The eluate was measured for ultraviolet absorption at a wavelength of 225 nm, and 1 mL was taken every 2 minutes. 50 μL of the separated eluate was placed on a 96-well ELISA plate (manufactured by Sumitomo Bakelite), and a peroxidase-labeled anti-V5 antibody and a peroxidase substrate (SureBlue, manufactured by KPL) were used to quantify VAR2 in the eluate fraction. Was done. The ultraviolet absorption pattern of the eluate and the VAR2 quantitative pattern are shown in FIG. The reaction between the SUV liposome into which CS was not introduced and VAR2 was also performed, and gel filtration chromatography was performed in the same manner. The result is shown in FIG.

From FIG. 2, VAR2 was eluted at the same time as CS-liposomes into a superpolymer fraction (molecular weight of 1,000 kDa or more), whereas in the chromatography of SUV-liposomes without CS and VAR2 mixed solution, the VAR2 protein was a free single substance. It was eluted in the same fraction as the VAR2 protein (molecular weight 120 kDa).

Thus, liposomes do not bind to the VAR2 protein, while CS-liposomes specifically bind to the malaria protein VAR2 and specifically fuse to VAR2CSA-expressed malaria parasite-infected erythrocytes.

[Example 4] Analysis of inhibitory effect on binding between immobilized CS and VAR2 by CS-liposomes TBS + (20 mM Tris-HCl, pH 7.4, 0) of whale cartilage-derived CS (CS-Biotin) biotinylated at the reducing end. .15 M NaCl, 2 mM CaCl ₂ , 2 mM MgCl ₂ containing buffer solution (2 μg / ml) was added to a 96-hole ELISA plate (manufactured by Thermo Fisher) coated with stressed avidin by 50 μL per hole at room temperature. After shaking for 1 hour, the cells were washed 3 times with TBS + to prepare a CS coated plate. V5-labeled VAR2 protein (50 pmol / mL) and CS-lipposite of Example 2 (0.1 to 20 μg / mL) or free CS (average molecular weight 5,000) oligosaccharide (12.5) as a raw material thereof are placed on the CS coat plate. To 5,000 μg / mL) and 50 μL of the mixed solution were added, shaken at room temperature for 1 hour, and then washed 3 times with TBST + (TBS + solution containing 0.05% Tween 20). By quantifying the VAR2 protein bound to the plate using a peroxidase-labeled anti-V5 antibody and a peroxidase substrate (SureBlue, manufactured by KPL), inhibition of binding between immobilized CS and VAR2 by CS-liposomes or free CS. The activity was measured (Fig. 3). As a result, the 50% inhibitory activity (IC ₅₀ ) value of free CS was 3,000 μg / mL, whereas the IC ₅₀ value of CS-liposomes was 5.2 μg / mL. That is, CS showed about 600 times higher binding inhibitory activity in the form of liposomes.

As described above, CS-liposomes and the recombinant protein VAR2 in the CSA-binding region of VAR2CSA bind specifically and very strongly.

CS-liploids have VAR2CSA on the surface and bind to malaria-infected erythrocytes lurking in the placenta of pregnant patients, and competitively inhibit the binding between placenta CS proteoglycan and infected erythrocytes to release infected erythrocytes accumulated in the placenta. It can be an excellent anti-malaria drug having a function of promoting the biological defense mechanism and the effect of the administered malaria agent.

Claims (12)

An antimalarial drug for the treatment or prevention of malaria infection in pregnant women
An antimalarial drug containing a liposome into which a chondroitin sulfate oligosaccharide containing type A chondroitin sulfate (CSA) has been introduced as an active ingredient. The antimalarial drug according to claim 1, wherein the liposome contains phospholipid and cholesterol. The antimalarial drug according to claim 1 or 2, wherein the liposome has an average particle size of 50 to 500 nm. The antimalarial drug according to any one of claims 1 to 3, further comprising a malaria therapeutic drug. Liposomes into which chondroitin sulfate oligosaccharides containing type A chondroitin sulfate (CSA) have been introduced. The liposome according to claim 5, wherein the chondroitin sulfate oligosaccharide contains 30% to 100% of CSA. The liposome according to claim 5 or 6, wherein the chondroitin sulfate oligosaccharide is derived from the cartilage of a marine organism. The liposome according to any one of claims 5 to 7, wherein the chondroitin sulfate oligosaccharide has an average molecular weight of 1000 to 100,000. The liposome according to any one of claims 5 to 8, wherein the average sugar chain length of the chondroitin sulfate oligosaccharide is 4 to 400. The liposome according to any one of claims 5 to 9, which contains phospholipid and cholesterol. The liposome according to any one of claims 5 to 10, which has an average particle size of 50 to 500 nm. The liposome according to any one of claims 5 to 11, which is administered at the same time as a therapeutic agent for malaria.