Composition for preventing or treating pulmonary fibrosis comprising pulmonary surfactant and antifibrotic agent and preparation method therefor

Abstract

The present invention relates to a pulmonary anti-fibrotic composition for inhalation delivery, comprising a pulmonary surfactant and an antifibrotic agent. By encapsulating the antifibrotic agent within the pulmonary surfactant, type II alveolar cells can be effectively targeted, thereby exhibiting excellent antifibrotic effects, as well as providing advantages of low toxicity and superior structural stability.

Description

Composition for preventing or treating pulmonary fibrosis comprising pulmonary surfactant and antifibrotic agent and method for preparing the same

[0001] The present invention relates to a composition for preventing or treating pulmonary fibrosis, comprising a complex of a pulmonary surfactant and an antifibrotic agent, and a method for producing the same.

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and specific form of fibrotic, interstitial lung disease of unknown etiology. It occurs due to damage to alveolar epithelial cells by various exposures, leading to recurrent inflammation and fibrosis. It is known as a disease characterized by collagen deposition in alveoli that fail to recover normally, resulting in worsening lung function.

[0003] Currently, oral medications such as pirfenidone and nintenanib, which are FDA-approved to prevent the progression of fibrosis, are prescribed. However, when these oral medications are actually used to treat lung diseases, the effectiveness of the medications reaches the lungs only slightly, and all prescribed medications cause systemic side effects such as vomiting, nausea, retching, and decreased appetite.

[0004] The technical problem to be achieved by the present invention is to provide a composition for preventing or treating pulmonary fibrosis, comprising a complex of a pulmonary surfactant and an antifibrotic agent.

[0005] Another technical problem to be achieved by the present invention is to provide a method for producing a composition for preventing or treating pulmonary fibrosis, comprising a complex of a pulmonary surfactant and an antifibrotic agent.

[0006] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0007] To solve the above problem, the present invention provides a composition for preventing or treating pulmonary fibrosis, comprising a complex of a pulmonary surfactant and an antifibrotic agent.

[0008] According to one side, the above-mentioned lung surfactant may be of bovine or porcine origin.

[0009] According to one side, the above-mentioned lung surfactant may be poractant alpha.

[0010] According to one aspect, the surfactant and antifibrotic agent in the complex may be included in a ratio of 20:1 to 2:1 (w / w), respectively.

[0011] According to one side, the diameter of the complex may be 100 to 200 nm.

[0012] According to one side, the surface zeta potential of the complex can be -40 to -20 mV.

[0013] According to one side, the complex may be an antifibrotic agent encapsulated inside a liposome manufactured with a lung surfactant.

[0014] According to another embodiment of the present invention,

[0015] A step of preparing a first solution by dissolving an anti-fibrotic agent in distilled water;

[0016] A step of mixing the first solution into the dry powder of a lung surfactant to hydrate it; and

[0017] A method for preparing a composition for preventing or treating pulmonary fibrosis is provided, comprising the step of dialyzing the above-mentioned hydrated solution.

[0018] According to one side, the hydration may be performed at a temperature of 60 to 70°C.

[0019] The complex containing an antifibrotic agent within the pulmonary surfactant of the present invention and the composition for preventing or treating pulmonary fibrosis containing the same can significantly improve the effect of the antifibrotic agent by effectively targeting type II alveolar cells, and also has the effect of low toxicity and excellent structural stability.

[0020] The effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0021] Figure 1 shows the size and surface charge of particles manufactured using DLS (Dynamic Light Scattering) and an electron microscope photograph of the particles.

[0022] Figure 2 shows the pirfenidone loading efficiency and the pirfenidone loading rate after ultrasonic shock through a nebulizer.

[0023] Figure 3 shows the inhibitory effect of the fibrosis factor α-SMA in vitro.

[0024] Figure 4 shows the results of a comparative experiment on particle distribution within the lung organ.

[0025] Figure 5 shows the administration and treatment plan for an animal model for respiratory treatment.

[0026] Figure 6 shows the results of a respiratory therapy experiment using pirfenidone-loaded particles in vivo.

[0027] Figure 7 shows the results of in vivo respiratory delivery of pirfenidone-loaded liposome particles and pirfenidone-free pulmonary surfactant to confirm the inhibitory effect on pulmonary fibrosis.

[0028] In order to solve the above-described problem, the present inventors have anticipated that a surfactant, as a biocompatible material, can be a drug delivery vehicle capable of effectively delivering drugs to the fibrotic internal environment of the lung, and have completed a complex stably containing an antifibrotic agent therein and an antifibrotic composition for inhalation delivery containing the same, and intend to provide this invention.

[0029] More specifically, the present invention provides a composition for the prevention or treatment of pulmonary fibrosis, comprising a complex of a pulmonary surfactant and an antifibrotic agent. The form of the complex is not particularly limited, but specific examples thereof include a liposome form, and a form in which the pulmonary surfactant and the antifibrotic agent are bound via covalent or hydrogen bonds.

[0030] The above-mentioned lung surfactant may comprise mammalian lung surfactant collected from the lung of a mammal and may be composed of or include a lipoprotein complex. Here, the mammal may be a human or an animal other than a human, specifically a pig or a cow, and most preferably a pig.

[0031] The term "lipid" in the above lipoprotein complex generally refers to a naturally occurring, synthetic, or semi-synthetic (i.e., modified natural) compound that is amphiphilic. Lipids typically contain hydrophilic and hydrophobic components. Exemplary lipids include, but are not limited to, phospholipids, fatty acids, fatty alcohols, triglycerides, phosphatides, oils, glycolipids, fatty alcohols, waxes, terpenes, and steroids. The phrase "semi-synthetic (or modified natural)" refers to a natural compound that has been chemically modified in some way.

[0032] Examples of phospholipids include natural and / or synthetic phospholipids.

[0033] Phospholipids that may be used include, but are not limited to, phosphatidylcholine (saturated and unsaturated), phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, sphingolipids, diacylglycerides, cardiolipin, ceramides, and cerebrosides. Exemplary phospholipids include dipalmitoyl phosphatidylcholine (DPPC), dilauryl phosphatidylcholine (DLPC) (C12:0), dimyristoyl phosphatidylcholine (DMPC) (C14:0), distearoyl phosphatidylcholine (DSPC), dipittanoyl phosphatidylcholine, nonadecanoyl phosphatidylcholine, arachidoyl phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) (C18:1), dipalmitoleoyl phosphatidylcholine (C16:1), linoleoyl phosphatidylcholine (C18:2), myristoyl palmitoyl phosphatidylcholine (MPPC), stearoyl myristoyl phosphatidylcholine (SMPC), stearoyl palmitoyl phosphatidylcholine (SPPC), palmitoyloleoyl phosphatidylcholine (POPC), palmitoyl palmitooleoyl Phosphatidylcholine (PPoPC), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyl oleoyl phosphatidylethanolamine (POPE), dioleoyl phosphatidylethanolamine (DOPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), dioleoyl phosphatidylglycerol (DOPG), palmitoyl oleoyl phosphatidylglycerol (POPG), dipalmitoyl phosphatidylglycerol (DPPG), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dimyristoyl phosphatidylserine (DMPS), distearoyl phosphatidylserine (DSPS), palmitoyl oleoyl phosphatidylserine (POPS), soy lecithin, egg yolk lecithin, sphingomyelin, Including, but not limited to, phosphatidylinositol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, and egg phosphatidylcholine (EPC).

[0034] Examples of fatty acids and fatty alcohols include, but are not limited to, sterols, palmitic acid, cetyl alcohol, lauric acid, myristic acid, stearic acid, phytanic acid, and dipalmitic acid. An exemplary fatty acid includes palmitic acid.

[0035] Examples of fatty acid esters include, but are not limited to, methyl palmitate, ethyl palmitate, isopropyl palmitate, cholesteryl palmitate, palmityl palmitate, sodium palmitate, potassium palmitate, and tripalmitin.

[0036] Meanwhile, the pulmonary surfactant comprises a membrane protein, and the presence of the membrane protein enables it to selectively and effectively target type II alveolar cell-derived adenocarcinoma. The membrane protein may comprise one or more natural surfactant polypeptides selected from the group consisting of SP-A, SP-B, SP-C and SP-D, a portion thereof or a mixture thereof. An exemplary peptide may comprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acid fragments of a natural surfactant polypeptide. An exemplary SP-B polypeptide may comprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acid fragments of SP-B. An SP-B peptide may be an amino-terminal peptide or a carboxy-terminal peptide. An exemplary SP-B peptide may be a 25-amino acid amino-terminal peptide.

[0037] The lung surfactant may preferably be of bovine or porcine origin, and most preferably porcine porcine alfa. Porcine alfa is a modified natural surfactant extracted from porcine lungs, which is substantially composed of polar lipids, primarily phospholipids, and proteins SP-B and SP-C, and is commercially available under the trade name CUROSURF®.

[0038] In another embodiment, the lung surfactant may comprise a recombinantly produced surfactant polypeptide. Recombinant SP-A, SPB, SP-C, SP-D, or a portion thereof may be obtained by expressing a DNA sequence encoding SP-A, SP-B, SP-C, SP-D, or a portion thereof in a suitable prokaryotic or eukaryotic expression system using various known techniques. Recombinant vectors readily adapted to contain isolated nucleic acids encoding surfactant polypeptides or portions thereof, host cells containing recombinant vectors, and methods for preparing such vectors and host cells, as well as their use in the production of encoded polypeptides by recombinant techniques, are well known. A nucleic acid encoding a surfactant polypeptide or a portion thereof may be provided in an expression vector comprising a nucleotide sequence encoding the surfactant polypeptide operably linked to at least one regulatory sequence. It should be understood that the design of an expression vector may depend on factors such as the choice of host cell to be transformed and / or the type of protein desired to be expressed. Considerations should also be given to the vector copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector (e.g., antibiotic markers). For example, the nucleic acid of interest can be used to cause expression or overexpression of kinase and phosphatase polypeptides in culture-grown cells to produce proteins or polypeptides, including fusion proteins or polypeptides.

[0039] To express a surfactant polypeptide or a portion thereof, host cells can be transfected with a recombinant gene. The host cell can be any prokaryotic or eukaryotic cell. For example, the polypeptide can be expressed in a bacterial cell, such as E. coli, an insect cell, a yeast cell, or a mammalian cell. In these examples, if the host cell is human, it may or may not be a living organism. Other suitable host cells are known to those skilled in the art. Additionally, the host cell can be supplemented with tRNA molecules not typically found in the host cell to optimize polypeptide expression. Other methods suitable for maximizing polypeptide expression will be known to those skilled in the art.

[0040] Methods for producing polypeptides are well known in the art. For example, host cells transfected with an expression vector encoding a surfactant polypeptide or a portion thereof can be cultured under appropriate conditions that promote expression of the polypeptide. The polypeptide can be secreted and isolated from a mixture of cells and a medium containing the polypeptide. Alternatively, the polypeptide can remain cytoplasmically intact. The cells are then harvested, lysed, and the protein isolated from the cell lysate.

[0041] The surfactant polypeptide and surfactant lipid interact through hydrostatic interactions. Charged amino acids interact with polar head groups of lipids, and hydrophobic amino acids interact with phospholipid acyl side chains. For example, SP-B and SP-C are hydrophobic proteins. Both SP-B and SP-C preferentially bind anionic lipids (e.g., phosphatidylglycerol (PG), but not DPPC). SP-A and SP-D are hydrophilic proteins and interact with a wide range of amphipathic lipids, including glycerophospholipids, sphingophospholipids, sphingoglycolipids, lipid A, and lipoglycans. SP-A binds DPPC. For example, hydrostatic interactions between KL4, an SP-B mimetic, and lipids in natural surfactants or lipids contained in surface active agents have been observed. For example, the lysine residue in the KL4 peptide interacts with the charged head group of DPPC, and the hydrophobic leucine residue interacts with the phospholipid acyl side chain of phosphatidylglycerol.

[0042]

[0043] According to one side, the pulmonary surfactant and the antifibrotic agent in the complex may be included in a ratio of 20:1 to 2:1 (w / w), but is not particularly limited thereto, and the loading amount of the antifibrotic agent may be appropriately adjusted depending on the condition of the patient or the suitable properties of the complex as the final product.

[0044] Additionally, the diameter of the complex may be 100 to 200 nm, preferably 130 to 170 nm, and most preferably 140 to 160 nm, but is not limited thereto.

[0045] According to one aspect, the surface zeta potential of the complex may be -40 to -20 mV, preferably -35 to -25 mV, but is not limited thereto.

[0046] According to one side, the complex may be an antifibrotic agent encapsulated inside a liposome manufactured with a lung surfactant.

[0047] Any known antifibrotic agent may be used without limitation. The antifibrotic agent may be hydrophilic, non-limiting examples of which include nintedanib (Ofev®) and pirfenidone (Esbriet®), with pirfenidone being preferred.

[0048] According to another embodiment of the present invention,

[0049] A step of preparing a first solution by dissolving an anti-fibrotic agent in distilled water;

[0050] A step of mixing the first solution into the dry powder of a lung surfactant to hydrate it; and

[0051] A method for preparing the composition of claim 1, comprising the step of dialyzing the above-mentioned hydrated solution, is provided.

[0052] According to one side, the hydration may be performed at a temperature of 60 to 70°C.

[0053] The complex of the present invention, which encapsulates an antifibrotic agent with a pulmonary surfactant, and the composition for inhalation delivery containing the same are effectively delivered into the body by targeting type 2 alveolar cells through inhalation delivery, and have the effect of low toxicity and excellent structural stability, and such effect is proven by the examples and experimental examples described below.

[0054]

[0055] The terms used in the examples are for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0056] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0057] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0058]

[0059] Example 1. Preparation of drug-loaded surfactant-based particles

[0060] Pirfenidone, a highly hydrophilic, water-soluble drug that has been shown to be effective in treating pulmonary fibrosis, was used as the drug. Surfactant-based particles loaded with the drug were manufactured through the following process. First, pirfenidone powder was dissolved in distilled water at a concentration of 500 μg / ml. The surfactant solution (manufacturer: Chiesi USA / product name: CUROSURF®) was placed in a glass bottle and dried. The pirfenidone solution dissolved as described above was added to the glass bottle containing the dried surfactant to hydrate it. The hydration experiment was performed on a hot plate, and the temperature was maintained at 60-70°C. The particles formed during the hydration process were adjusted to an average diameter of less than 200 nm using an extruder kit. Subsequently, any drug not encapsulated in the particles was separated by dialysis using a 100 kDa membrane for 12 hours. Thus, surfactant particles loaded with a drug (pirfenidone) were manufactured. Afterwards, the amount of pirfenidone loaded into the particles was confirmed, and it was confirmed that approximately 32 μg / ml to 35 μg / ml of pirfenidone was loaded per 1 mg of surfactant (Table 1 below).

[0061] [Table 1]

[0062]

[0063] Example 2. Confirmation of particle characteristics

[0064] The size and surface charge of the particles manufactured in Example 1 were measured using DLS (Dynamic Light Scattering), and the results are shown in Fig. 1. After manufacturing particles corresponding to each ratio shown in Table 1, it was confirmed that the average size was 200 nm or less, and the average surface charge was measured as -27 to -34 mV.

[0065] To assess the stability of the particles, the following experiment was conducted. The particles manufactured using the above method were subjected to ultrasonic shock via a nebulizer, and the changes in particle size and pirfenidone concentration before and after the shock were measured. The differences in particle size and the concentration of the loaded pirfenidone drug before and after the ultrasonic shock were minimal, indicating excellent particle stability (left side of Figure 2).

[0066] Afterwards, for electron microscopy, the particles were mixed in a composition containing glutaraldehyde particles at a concentration of 2.5% (v / v) and fixed at room temperature for 2 hours. Then, the particles were placed on a carbon grid, stained with a 2% PTA solution, and photographed using a transmission electron microscope (TEM). The results are shown on the right side of Fig. 2.

[0067]

[0068] Example 3. In vitro anti-fibrotic effect confirmation

[0069] A cell environment mimicking the fibrotic environment induced by TGF-β treatment was established in MRC-5 (normal lung fibrocytes), and the PSPNVs-3 of Example 1 of the present invention was administered thereto to confirm the antifibrotic effect. The administration was performed at a concentration of 300 μg / ml. The inhibitory effect of the fibrotic factor α-SMA expressed in cells treated with 10 μg / ml of TGF-β was confirmed, and the results are shown in Fig. 3. Fig. 3 A shows the result of immunofluorescence staining, B shows the expression level of α-SMA, and C shows the result of electrophoresis. It was confirmed that the fibrotic factor was significantly inhibited in the group treated with PSPNVs.

[0070]

[0071] Example 4. Comparative evaluation of liposome-based particles and particle distribution in lung organs.

[0072] In the animal experiments below, all procedures for biodistribution, cellular distribution, inhalation toxicity, and inhalation treatment were approved and conducted in accordance with the regulations of the Animal Research Committee of Korea University (IACUC No. KOREA-2022-0031). Sample size estimates were calculated by the IACUC and online software (Open Epi, http: / / www.openepi.com / OE2.3 / Menu / OpenEpiMenu.htm). Male mice (C57BL / 6) were purchased from the Orient Bio Research Institute at 8–10 weeks of age.

[0073] To establish a pulmonary fibrosis (PF) model, C57BL / 6 mice were intratracheally (IT) administered 100 μl of bleomycin (BLM) (2 mg per kg body weight, mg / kg). Twenty-seven mice with PF were randomly divided into nine groups and received inhalation treatment with free SRB, SRB-labeled Arykayce (NV lacking the PS protein), and SRB-labeled NV. To study the temporal biodistribution of each inhaled group, mice were inhaled with 1 mg / ml of SRB-labeled NV at 0.1 ml / min (per mouse) for 10 min using an inExpose nebulizer (SCIREQ, Montreal, Canada). Mice were secured with a nose cone restraint to ensure exposure to the aerosol generated by the nebulizer. According to the manufacturer's instructions, the size of the generated aerosol was approximately 2.5 μm. To determine whether PSNV was cleared from the lungs, SRB-labeled PSNV was administered to mice via inhalation. Mice were sacrificed, and lungs were collected at different time points (1, 6, and 24 hours) after inhalation. Furthermore, lung fluorescence was measured using an IVIS Lumina instrument (PerkinElmer, Waltham, USA) to determine whether PSNV accumulated in the lungs after inhalation. The results are shown in Figure 4.

[0074] When comparing the respiratory delivery group without a fluorescent dye loaded on the particle (Free), the respiratory delivery group loaded with a liposome-based fluorescent dye (SRB-Arikayce), and the respiratory delivery group loaded with a fluorescent dye loaded on a surfactant-based fluorescent dye (SRB-PSNV), it was confirmed that the respiratory delivery group using the surfactant (PSNV) particles used in this study remained in the lungs at a level superior to the other groups in terms of in vivo lung retention. This confirmed that the pirfenidone-loaded surfactant particles formulated by the method of this study were effective in drug delivery to the lungs.

[0075]

[0076] Example 5. Animal testing - particle efficacy evaluation

[0077] Lung fibrosis was induced by intratracheal (IT) administration of 100 μl of BLM (2 mg per kg body weight, mg / kg), and mice were randomly assigned to groups in a blinded manner. The dosing and treatment schedule for the animal model for respiratory therapy with the manufactured particles is shown in Figure 5. Seven days after BLM administration, mice received various treatments, including oral PFD (30 and 300 mg / kg), inhaled PFD (300 μg / kg), Arikace-PFD (300 μg / kg), PSNV, and inhaled PSPNVs (30 and 300 μg / kg) for five consecutive days. Mice were sacrificed on the third day after drug administration, and tissue and serum samples were examined. To assess inflammation, cytokine levels in mouse serum, including TGF-β and IL-6, were assessed using bronchoalveolar lavage (BAL).

[0078] The experiment was conducted according to the above experimental plan, and in an animal model of pulmonary fibrosis, the respiratory treatment of pirfenidone-loaded particles manufactured by this research method showed a much better anti-fibrotic effect than the group that received pirfenidone orally and the group that received pirfenidone not loaded on particles through respiratory delivery, as confirmed through pathological analysis (H&E, MT staining) in Figure 6. In addition, it was confirmed that the inhibitory effect on fibrotic disease factors such as α-SMA was also effective.

[0079]

[0080] Example 6. Animal experiment - Evaluation of anti-fibrotic effect

[0081] The stably manufactured pirfenidone-loaded liposome particles and pirfenidone-free surfactant particles were respirably delivered to confirm the inhibitory effect on pulmonary fibrosis, which is shown in Fig. 7. Pirfenidone-loaded liposome-based particles (Arikayce-PFD) and pirfenidone-free surfactant particles (PSNV) were respirably delivered and tested. In an animal model of bleomycin-induced pulmonary fibrosis, neither group showed an antifibrotic effect, confirming that respirant delivery of surfactant particles alone cannot show an antifibrotic effect. In addition, it was confirmed that respirable delivery of pirfenidone loaded onto general liposome-based particles without surfactant-specific surface proteins did not show an effect.

[0082]

[0083] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0084] Therefore, other implementations, other manufacturing examples and equivalents to the patent claims also fall within the scope of the claims described below.

Claims

1. A composition for preventing or treating pulmonary fibrosis, comprising a complex of a pulmonary surfactant and an antifibrotic agent, which is delivered by inhalation.

2. In paragraph 1, A composition for preventing or treating pulmonary fibrosis, wherein the above-mentioned pulmonary surfactant is derived from cattle or pigs.

3. In paragraph 2, The above pulmonary surfactant is a composition for preventing or treating pulmonary fibrosis, which is a poractant alpine.

4. In paragraph 1, A composition for preventing or treating pulmonary fibrosis, wherein the pulmonary surfactant and the antifibrotic agent are contained in a ratio of 20:1 to 2:1 (w / w) in the complex.

5. A composition for preventing or treating pulmonary fibrosis, wherein the diameter of the complex is 100 to 200 nm in the first paragraph.

6. A composition for preventing or treating pulmonary fibrosis, wherein the surface zeta potential of the complex in paragraph 1 is -40 to -20 mV.

7. A composition for preventing or treating pulmonary fibrosis, wherein the complex is a liposome manufactured with a pulmonary surfactant, in which an antifibrotic agent is encapsulated inside the liposome.

8. A step of preparing a first solution by dissolving an anti-fibrotic agent in distilled water; A step of mixing the first solution into the dry powder of a lung surfactant to hydrate it; and A method for preparing the composition of claim 1, comprising the step of dialyzing the above-mentioned hydrated solution.

9. In paragraph 8, A manufacturing method wherein the above hydration is performed at a temperature of 60 to 70°C.

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