Functionalized biocatalyst composition containing pancreatic enzymes

Abstract

The present invention relates to a composition comprising a solid carrier, an open-structured lipase or fragment thereof immobilized on the surface of the solid carrier, a protease or fragment thereof immobilized on the surface of the solid carrier, an amylase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, a protective layer that protects the lipase or fragment thereof, the protease or fragment thereof, and the amylase or fragment thereof by embedding them, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group.

Description

Technical Field

[0001] The present invention relates to a composition comprising a solid support, a lipase or a fragment thereof having an open structure immobilized on the surface of the solid support, a protease or a fragment thereof immobilized on the surface of the solid support, an amylase or a fragment thereof immobilized on the surface of the solid support, a substance that interacts with the lid domain of the lipase or a fragment thereof, and a protective layer that protects the lipase or a fragment thereof, the protease or a fragment thereof, and the amylase or a fragment thereof by embedding the lipase or a fragment thereof, the protease or a fragment thereof, and the amylase or a fragment thereof, and a functional component immobilized on the surface of the protective layer. The functional component immobilized on the surface of the protective layer is a polymer containing a repeating unit each containing at least one amino group and / or at least one thiol group. The present invention also relates to a method for producing the composition and its use.

Background Art

[0002] Exocrine pancreatic insufficiency (EPI) is a condition in which there is a disorder in the exocrine function of the pancreas and a defect in the ability to effectively deliver digestive enzymes to the duodenum. This is a serious health condition that causes malabsorption of lipids, fat-soluble vitamins, proteins, and to a lesser extent carbohydrates from the gastrointestinal tract.

[0003] The standard medical therapy for treating clinical symptoms and malabsorption is oral pancreatic enzyme replacement therapy (PERT). Currently approved therapies consist of porcine-derived pancreatic enzyme products (lipase, amylase, protease), also called pancreatin. A minimum dose of 4,0000 - 50,000 units of PERT-lipase is recommended for each main meal, and half that amount is recommended for snacks. Such doses lead to a heavy tablet burden for patients.

[0004] However, current treatments are inefficient and exhibit several limitations, including: inefficiency of treatment, persistence of symptoms, short intraluminal survival time of lipase susceptible to protease degradation (failure of fat digestion), potential intolerance to large enzyme loads, and / or gastrointestinal damage due to large protease intake. Therefore, there is a need to provide an effective composition suitable for the treatment of pancreatic exocrine insufficiency (EPI). [Overview of the Initiative]

[0005] The present invention provides a composition comprising a solid carrier, an open-structured lipase or fragment thereof immobilized on the surface of the solid carrier, a protease or fragment thereof immobilized on the surface of the solid carrier, an amylase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, a protective layer that protects the lipase or fragment thereof, the protease or fragment thereof, and the amylase or fragment thereof by embedding them, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group.

[0006] The present invention also provides a method for producing a composition comprising a solid carrier, an open-structured lipase or fragment thereof immobilized on the surface of the solid carrier, a protease or fragment thereof immobilized on the surface of the solid carrier, an amylase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, a protective layer that protects the lipase or fragment thereof, the protease or fragment thereof, and the amylase or fragment thereof by embedding them, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each comprising at least one amino group and / or at least one thiol group, and the method comprises the following steps: (a) A step of providing a solid carrier, (b) A step of providing lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof. (c) A step of providing a substance that interacts with the lid domain of lipase or a fragment thereof. (d) A step of interacting the lipase or fragment thereof from (b) with the substance from (c), (e) A step of immobilizing lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof on a solid carrier. (f) A step of forming a protective layer on the surface of a solid carrier to protect lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof immobilized on the solid carrier, (g) A step of immobilizing a functional component on the surface of a protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group.

[0007] The present invention also provides methods for the prevention, delaying the progression of, or treating exocrine pancreatic insufficiency (EPI), and methods for enzyme replacement therapy (ERT).

[0008] The inventors of this application have surprisingly found that the compositions provided by the present invention, when applied therapeutically, exhibit unexpectedly high in vivo distribution specificity and high bioactivity in vitro and in vivo, low cytotoxicity and remarkably high biocompatibility, do not disrupt the intestinal barrier even when localized in the gastrointestinal tract, restore lipase degradation function in EPI animal models, and therefore are extremely promising for the prevention, delay of progression, or treatment of pancreatic exocrine insufficiency (EPI). [Brief explanation of the drawing]

[0009] [Figure 1]The process for producing the compositions of the present invention is schematically shown: a) A solid carrier is supplied with lipases, proteases, amylases having closed lids, and substances that interact with the lid domains of lipases (shown as full circles) to immobilize the lipases, proteases, and amylases having open lids onto the solid carrier; b) and c) A protective layer is grown around the immobilized lipases, proteases, and amylases having open lids to embed all three enzymes; d) Functional components are immobilized on the surface of the protective layer. [Figure 2A-B] This shows the in vivo distribution (%ID) of 111In-NP-1 (n=5) and 111In-NP-2 (n=4) in miniature pigs. (A) SPECT / CT images were acquired 0.25, 3, 8, and 24 hours after intraduodenal administration of 111In-NP-1 and 111In-NP-2. The graph shows the relative quantification of nanoparticles at imaging time points in the small intestinal compartment (unit: %). (B) The histogram shows the area under the curve (AUC) of residence time in the small intestine for 111In-NP-1 and 111In-NP-2. The histogram shows the relative quantification of radioactivity in the blood compartment. [Figure 2C] This shows the in vivo distribution (%ID) of 111In-NP-1 (n=5) and 111In-NP-2 (n=4) in miniature pigs. Blood samples were collected 0.25, 3, 8, and 24 hours after intraduodenal administration of 111In-NP-1 or free 111In. The histogram shows the relative quantification of radioactivity in the blood compartment. [Figure 3] The activity of lipase and / or fragments thereof, protease and / or fragments thereof, and amylase and / or fragments thereof, composed of pancreatin immobilized and protected on nanoparticles, is shown. (A) Lipase activity of NP-3 after exposure to olive oil (unit: U / g). (B) Protease activity of NP-5 after exposure to casein (unit: U / mg). (C) Amylase activity of NP-3 after exposure to amylase substrate solution (unit: U / g). [Figure 4]This shows the relative quantification of plasma triglycerides (TG) in pancreatic duct ligation (PDL) rats. PDL rats were administered NP-3 (n=1) or NP-4 (n=1) intraduodenally, followed by enteral nutrition of triolein 5 minutes later. Blood samples were collected before the procedure and at 0.25, 0.5, 1, 1.5, 2, 4, and 6 hours later. Plasma extraction and LC-MS analysis were performed. The graph shows the peak area of ​​triolein (TG(54:3)). [Figure 5] This study compares plasma triglyceride (TG) concentrations in healthy miniature pigs treated with NP-3 and miniature pigs that underwent pancreatic duct ligation (PDL). Olive oil was administered to both a healthy miniature pig (n=1) and a PDL-ligated miniature pig (n=1), followed by administration of NP-3 to the PDL-ligated miniature pig. Blood samples were collected before administration and at 0.083, 0.25, 0.5, 1, 2, 3, 4, and 6 hours after administration, and TG analysis was performed. (A) The graph shows the plasma TG concentrations in miniature pigs determined using a Konelab analyzer. (B) The histogram shows the area under the curve (AUC) for plasma TG concentrations in miniature pigs. [Figure 6] This shows the fecal fat content of miniature pigs on a high-fat diet. Healthy miniature pigs and PDL miniature pigs were fed a high-fat diet for 25 days. PDL miniature pigs were administered NP-3 twice daily for 10 days. Feces were collected on days 8, 9, and 10, and fecal homogenates were analyzed by near-infrared spectroscopy to quantify the fat content in the feces. (A) The histogram shows the absolute values ​​of fecal fat. (B) The graph shows the normalized fecal fat homogenate fat content for healthy miniature pigs and untreated PDL miniature pigs. [Figure 7]This demonstrates the in vitro biocompatibility of NP-5 with respect to the intestinal barrier. (A) In vitro evaluation of intestinal barrier integrity by measurement of transepithelial electrical resistance (TEER). Differentiated Caco-2 / HT29-MTX-E12 cocultures were exposed to gradually increasing doses of NP-5 or pancreatin (32.9 to 263.6 U / m2) for 20 hours. TEER data were normalized with the equilibrium value before NP-5 or pancreatin addition set to 100%. The graph shows the time course profile of averaged and normalized TEER data over 20 hours. The dashed line represents the untreated state. (B) Confocal image of tight junctions in a model of the intestinal barrier. Differentiated Caco-2 / HT29-MTX-E12 cocultures were exposed to NP-5 or pancreatin (263.6 U / m2) for 20 hours. Cells were stained with zonula occludens 1 (ZO-1) and evaluated using a confocal microscope (white signals in the image). [Figure 8] This shows the added value of the covalent bond between the enzyme surface and the protective layer. (A) Quantification of protein performed on the reaction supernatant of NP-3 and NP-3(1). (B) Pancreatin loading per dry weight of SNP. (C) SNP activity expressed in μmol / min. (D) Pancreatin specific activity of pancreatin expressed in U / g. [Figure 9] The absorbance of NP-3 and NP-3(1) at a wavelength of 460 nm is shown. [Modes for carrying out the invention]

[0010] The present invention relates to a composition comprising a solid carrier, an open-structured lipase or fragment thereof immobilized on the surface of the solid carrier, a protease or fragment thereof immobilized on the surface of the solid carrier, an amylase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, a protective layer that protects the lipase or fragment thereof, the protease or fragment thereof, and the amylase or fragment thereof by embedding them, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group.

[0011] For the purposes of interpreting this specification, the following definitions apply, and wherever used in the singular, the plural form is also included, and vice versa. It should be understood that the terms used herein are intended solely to describe and not to limit a particular embodiment.

[0012] Features, integers, properties, and compounds described in relation to specific aspects, embodiments, or examples of the present invention should be understood to be applicable to any other aspects, embodiments, or examples described herein, insofar as they do not contradict each other. All features and / or steps of methods or processes disclosed herein (including the claims, abstract, and drawings) can be combined in any combination, except for any combination in which at least some of such features and / or steps are mutually exclusive. The present invention is not limited to the details of the embodiments described above.

[0013] The term "comprise," and its variations such as "comprises" and "comprising," are generally used to mean "include," that is, "to include, but not limited to," meaning to allow the presence of one or more features or components.

[0014] The singular forms "a", "an", and "the" include references to the plural form unless the context clearly dictates otherwise.

[0015] The term "about" refers to a range of values that is ±10% of a specified value. For example, the expression "about 200" includes values that are ±1% of 200, i.e., from 180 to 220.

[0016] As used herein, the term "solid support" typically refers to particles. Preferably, the solid support is monodisperse or polydisperse particles, more preferably monodisperse particles. The solid support typically includes organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, titanium particles, preferably silica particles, more preferably silica nanoparticles (SNP). The particle size of the solid support is typically 1 nm to 1000 μm, preferably 10 nm to 100 μm, particularly about 50 nm.

[0017] The terms "linker" or "crosslinker", used synonymously herein, refer to any linking reagent that includes a group capable of binding to a specific functional group (e.g., primary amine, sulfhydryl, etc.). A linker in the context of the present invention typically connects the surface of a solid support to an enzyme, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof. For example, a linker can be immobilized on the surface of a solid support, such as a silica surface as a support material, and then an enzyme, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof, can be bound to the unoccupied binding site of the linker. Alternatively, the linker can first bind to an enzyme, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof, and then the linker bound to the enzyme, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof, can bind to the solid support at its unoccupied binding site. Various types of linkers are known in the art, including, but not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, peptide linkers, polyether linkers, and linkers known in the art as tags.

[0018] As used herein, the term “protective layer” refers to a layer for protecting the functional properties of proteins or fragments thereof, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, immobilized on the surface of a solid carrier. The protective layer of the present invention is typically constructed of building blocks, at least a portion of which are monomers capable of interacting with each other, usually by covalent bonds, and with proteins or fragments thereof, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, usually immobilized by non-covalent bonds. The protective layer is formed on the surface of a solid carrier to protect the proteins or fragments thereof, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, immobilized on the solid carrier. The protective layer is typically a homogeneous layer in which at least 50%, preferably at least 70%, more preferably at least 90%, of the proteins or fragments thereof, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, are embedded within the protective layer.

[0019] The term “lipase or its fragment” includes naturally occurring lipases or their fragments, as well as artificially engineered lipases or their fragments. Artificially engineered lipases or their fragments are, for example, lipase variants or functionally active fragments. Therefore, in this specification, the terms “lipase fragment,” “its fragment” in relation to lipase, and “functionally active fragment of lipase” are used synonymously. In relation to the lipases of this invention, “variant or functionally active fragment” means that the fragment or variant (such as an analog, derivative, or mutant) is capable of performing the same physiological function as lipase. Such variants include naturally occurring allelic variants and variants that do not exist in nature. Addition, deletion, substitution, and derivatization of one or more amino acids are attempted, provided that the modification does not result in a loss of functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least about 80% sequence identity with the relevant portion of the lipase, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 98%. The lipase fragments as defined herein typically have the same functional properties as the lipase, i.e., the full-length enzyme from which it is derived, and include at least a lid domain and a substrate-binding region. The lipase fragments typically contain 100 to 450 amino acids, preferably 150 to 400 amino acids, and more preferably 200 to 350 amino acids. Preferred lipases or fragments thereof are lipases or fragments thereof extracted from the pancreas, more preferably lipases or fragments thereof extracted from the pancreas of a pig, and even more preferably lipases or fragments thereof composed of pancreatin.

[0020] The term “protease or fragment thereof” includes naturally occurring proteases or fragments thereof, as well as artificially engineered proteases or fragments thereof. Artificially engineered proteases or fragments thereof are, for example, protease variants or functionally active fragments. Accordingly, in this specification, the terms “protease fragment,” “its fragment” in relation to a protease, and “functionally active fragment of a protease” are used synonymously. In this invention, “variant or functionally active fragment thereof” in relation to a protease means that the fragment or variant (such as an analog, derivative, or mutant) is capable of performing the same physiological function as the protease. Such variants include naturally occurring allelic variants and variants that do not exist in nature. Addition, deletion, substitution, and derivatization of one or more amino acids are attempted, provided that the modification does not result in a loss of functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least about 80% sequence identity with the relevant portion of the protease, more preferably at least about 90% sequence identity, even more preferably at least about 95% sequence identity, and most preferably at least about 98% sequence identity. The protease fragments defined herein typically have the same functional properties as the protease from which they are derived. The protease fragments typically contain 50 to 200 amino acids, preferably 75 to 175 amino acids, and more preferably 100 to 150 amino acids. Preferred proteases or fragments thereof are proteases or fragments thereof extracted from the pancreas, more preferably lipases or fragments thereof extracted from the pancreas of a pig, and even more preferably proteases or fragments thereof composed of pancreatin.

[0021] The term “amylase or its fragment” includes naturally occurring amylase or its fragment, as well as artificially engineered amylase or its fragment. Artificially engineered amylase or its fragment is, for example, an amylase variant or a functionally active fragment. Therefore, in this specification, the terms “amylase fragment,” “its fragment” in relation to amylase, and “functionally active fragment of amylase” are used synonymously. In this invention, “variant or functionally active fragment” of amylase means that the fragment or variant (such as an analog, derivative, or mutant) is capable of performing the same physiological function as amylase. Such variants include naturally occurring allele variants and variants that do not exist in nature. Addition, deletion, substitution, and derivatization of one or more amino acids are attempted, provided that the modification does not result in a loss of functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least about 80% sequence identity with the relevant portion of the amylase, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 98%. The amylase fragments defined herein typically have the same functional properties as the amylase from which they are derived. The amylase fragments typically contain 100 to 550 amino acids, preferably 200 to 500 amino acids, and more preferably 300 to 450 amino acids. Preferred amylases or fragments thereof are amylases or fragments thereof extracted from the pancreas, more preferably amylases or fragments thereof extracted from the pancreas of a pig, and even more preferably amylases or fragments thereof composed of pancreatin.

[0022] The term "pancreatin," as used herein and also known as "pancreatic enzymes," refers to pancreatic enzyme preparations derived from the pancreatic glands of pigs, including lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof. The term "pancreatin" as used herein also includes formulated pancreatic enzymes, such as capsules containing pancreatic enzymes, for example, Zenpep®.

[0023] As used herein, the term “partially embedded protein” means that a protein, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, is not completely covered by the protective layer; therefore, the protein, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, is not completely embedded in the protective layer. In one embodiment, less than 50% of the protein, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, is covered by the protective layer, but typically at least 70% is covered, thereby improving the protection of the protein. In a preferred embodiment, at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% of the protein, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, is covered by the protective layer. In another preferred embodiment, about 70% to about 95%, more preferably about 80% to about 95%, even more preferably about 90% to about 95%, most preferably about 90% to about 95%, 96%, 97%, 98%, or 99% of the protein, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, is covered by a protective layer. In a particularly preferred embodiment, about 70%, particularly about 80%, more specifically about 90%, and most specifically about 95% of the protein, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, is covered by a protective layer. In a particularly more preferred embodiment, about 70%, particularly about 80%, more specifically about 90%, and most specifically about 95% of the protein, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, is covered by a protective layer, and the active site is not covered.

[0024] As used herein, the term “fully embedded protein” means that the proteins according to the present invention, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, are completely, i.e., 100%, covered by a protective layer, i.e., the active site is also covered. Preferably, the lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof according to the present invention are completely, i.e., 100%, covered by a protective layer, i.e., the active site is also covered.

[0025] As used herein, the term “at least partially embedded protein” means that a protein, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof, is at least partially embedded by a protective layer, and may be fully embedded. Thus, “at least partially embedded protein” means that the protective layer covers about 30% to 100%, preferably about 50% to about 100%, more preferably about 80% to about 100%, even more preferably about 90% to about 100%, and most preferably about 95% to about 100% of the protein or a fragment thereof, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof, and the active site is preferably covered.

[0026] As used herein, “substances that interact with the lid domain of lipase or its fragments” typically refers to substances that bind to and / or to the region surrounding the lid domain of lipase or its fragments, thereby causing the lid domain to transition the lipase or its fragments to an open structure and / or to maintain the open structure of the lipase or its fragments. The lid domain of lipase is usually amphiphilic; in the closed structure, their hydrophilic side faces the solvent and their hydrophobic side faces directly toward the catalyst pocket (Brocca S., Secundo F., Ossola M., Alberghina L., Carrea G., Lotti M. (2003). Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes. Protein Sci. 12, 2312-2319.10.1110 / ps.0304003). When lipase transitions to an open structure, its hydrophobic surface is exposed and contributes to the substrate-binding region. Preferably, a substance that interacts with the lid domain of the lipase or its fragment locks the lipase or fragment into its active conformation. Once locked into its active conformation, the lipase is usually fully activated. Substances that interact with the lid domain of the lipase or its fragment to cause it to transition to an open structure include collipase or its fragment, collipase-mimicking peptides, and amphiphilic molecules.

[0027] As used herein, the term “amphiphilic molecule” refers to molecules such as chemical compounds that have both a polar (water-soluble) and a nonpolar (water-insoluble) moiety in their structure. The term may also refer to molecules such as chemical compounds that have both hydrophobic and hydrophilic regions. Examples of amphiphilic molecules include bile salts, phospholipids, and nonionic detergents.

[0028] The terms “open structure” or “open structure of lipase or its fragment” as used interchangeably herein refer to a structure of lipase or its fragment in which a substrate can enter and be converted into the active site of the lipase. In a closed structure, the entry of a substrate into the active site of the lipase or its fragment and its conversion are restricted or impossible. The structure of lipase or its fragment, i.e., whether the lipase is in an open or closed structure, can be determined by X-ray crystallography, enzyme activity testing, site-directed spin labeling (SDSL), and electron paramagnetic resonance (EPR).

[0029] As used herein, the term “colipase or fragment thereof” includes naturally occurring colipases or fragments thereof, as well as artificially engineered colipases or fragments thereof. Artificially engineered colipases or fragments thereof are, for example, lipase variants or functionally active fragments. In relation to the colipases of this invention, “variant or functionally active fragment thereof” means that the fragment or variant (such as an analog, derivative, or mutant) is capable of performing the same physiological function as the colipase. Such variants include naturally occurring allelic variants and variants that do not exist naturally. Addition, deletion, substitution, and derivatization of one or more amino acids are intended, provided that the modification does not result in a loss of functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least about 80% sequence identity with the relevant portion of the lipase, more preferably at least about 90% sequence identity, even more preferably at least about 95% sequence identity, and most preferably at least about 98% sequence identity. The collipase fragments defined herein have the same functional properties as the collipase from which they are derived. The preferred collipase is the collipase having uniplot number: P02703.

[0030] As used herein, the term “colipase-mimicking peptide” refers to a peptide consisting of 10 to 40 amino acids, which allows specific amino acid residues to be geometrically positioned in appropriate locations to interact with the amino acids of the pancreatic lipase structure, thereby inducing conformational elongation and lid opening of the lipase, and thereby possessing the same functional properties as a lipase. The lipase-mimicking peptide that can be used in the present invention is preferably the peptide shown in SEQ ID NO: 1.

[0031] As used herein, the term “bile salt” refers to bile acids conjugated with taurine or glycine, and includes sodium taurocholate, sodium glycocholate, sodium glycodeoxycholate, sodium taurodeoxycholate, sodium glycochenodeoxycholate, and sodium taurochenodeoxycholate.

[0032] As used herein, the term “nonionic detergent” refers to a surfactant, and includes tetraethylene glycol monooctyl ether, octyl-bD-glucopyranoside, N,N-dimethyldodecylamine-N-oxide, and b-octylglucomaltoside.

[0033] As used herein, the term “phospholipid” refers to a class of lipids whose molecules have a hydrophilic “head” containing a phosphate group, linked by an alcohol residue (usually a glycerol molecule), and two hydrophobic “tails” derived from fatty acids. Phospholipids include lecithins and lysolecithins.

[0034] As used herein, the term “functional component” refers to a component that, after being immobilized on the surface of a protective layer, retains its characteristic functional properties. In the sense of the present invention, a functional component is a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group.

[0035] As used herein, the term “polymer comprising repeating units, each containing at least one amino group” refers to a polymer comprising a large number of repeating units (monomers), each of which contains at least one amino group. Preferred polymers comprise a large number of repeating units (monomers), each of which contains one amino group, in particular one primary amino group.

[0036] As used herein, the term “polymer comprising repeating units, each containing at least one thiol group” refers to a polymer comprising a large number of repeating units (monomers), each of which contains at least one thiol group. A preferred polymer comprises a large number of repeating units (monomers), each of which contains one thiol group.

[0037] As used herein, the term “polycarbophil-cysteine ​​conjugate” refers to a conjugate containing cysteine ​​covalently bonded to polycarbophil. Such conjugates can be produced, for example, as described in Bernkop-Schnurch and Thaler, 2000, Journal of Pharmaceutical Sciences 89(7):901-9.

[0038] As used herein, the term “polylysine” refers to α-polylysine and / or ε-polylysine (ε-poly-L-lysine, EPL), preferably ε-polylysine. α-Polylysine is a synthetic polymer and may consist of L-lysine or D-lysine. ε-Polylysine (ε-poly-L-lysine, EPL) is typically produced as a homopolypeptide of approximately 25 to 30 L-lysine residues.

[0039] As used herein, the term "polycysteine" may consist of L-cysteine ​​or D-cysteine, preferably L-cysteine, and preferably containing 2 to 30 cysteine ​​residues, more preferably 2 to 5 cysteine ​​residues.

[0040] As used herein, the term “polyglucosamine” refers to a linear aminopolysaccharide composed of D-glucosamine and N-acetyl-D-glucosamine units linked by (1-4) glycosidic bonds. Polyglucosamine contains a free amine (-NH2) group and is characterized by the ratio of N-acetyl-D-glucosamine units to D-glucosamine units, which is expressed as the degree of deacetylation (DDA) of fully acetylated polymer chitin. Preferred polyglucosamines of the present invention are selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan, and dermatan or their derivatives. Most preferred are chitosan or its derivatives.

[0041] As used herein, the term “chitosan or its derivatives” refers to chitosan or chitosan derivatives, including salts, having a molecular weight preferably of 2000 Da or more, preferably in the range of 25000 to 2000000 Da, more preferably in the range of about 50000 to 350000 Da, and most preferably in the range of about 50000 to 190000 Da or 190000 or 310000 Da. The term chitosan derivatives include esters, ethers, or other derivatives formed by the reaction of an acyl group or alkyl group with an OH group. Examples of these are O-alkyl ethers of chitosan and O-acyl esters of chitosan. Suitable derivatives are described, for example, in GAERoberts, Chitin Chemistry, MacMillan Press Ltd, London, 1992. Suitable salts of chitosan include nitrates, phosphates, sulfates, xanthogenic salts, hydrochlorides, glutamates, lactates, and acetates.

[0042] In a first embodiment, the present invention provides a composition comprising a solid carrier, an open-structured lipase or fragment thereof immobilized on the surface of the solid carrier, a protease or fragment thereof immobilized on the surface of the solid carrier, an amylase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, a protective layer that protects the lipase or fragment thereof, the protease or fragment thereof, and the amylase or fragment thereof by embedding them, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each comprising at least one amino group and / or at least one thiol group.

[0043] Proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, can be immobilized on the surface of a solid support by non-covalent or covalent bonds. Non-covalent bonds include electrostatic interactions such as pp (aromatic) interactions, van der Waals interactions, H-bond interactions, and ionic interactions. Preferably, proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, are immobilized on the surface of a solid support by covalent bonds or by linker-mediated covalent bonds.

[0044] Solutions of pancreatin, lipase or its fragments, protease or its fragments, and / or amylase or its fragments typically contain one protein or its fragment or multiple proteins or their fragments in a buffer. Usable buffers are typically phosphates, chlorides, citrates, MES, MOPS, HEPES, PIPES, ACES, or mixtures thereof. In addition, the solution may contain sugar alcohols or nonionic surfactants as described herein. Solutions of pancreatin, lipase or its fragments, protease or its fragments, and / or amylase or its fragments can be prepared, for example, by dissolving one protein or its fragment or multiple proteins or their fragments in water to reconstitute a stock buffer of one protein or its fragment or multiple proteins or their fragments.

[0045] In one embodiment, the solid support is selected from the group consisting of organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, and titanium particles, preferably silica particles, more preferably silica nanoparticles (SNPs). The particle size is usually measured by measuring the diameter of the particles and is typically from 1 nm to 1000 nm, preferably from 10 nm to 100 nm, and particularly about 50 nm. If the solid support is monodisperse particles, its size is typically from 1 nm to 1000 nm, preferably from 10 nm to 100 nm, and particularly about 50 nm. If the solid support is polydisperse particles, its size is typically from 1 nm to 1000 μm, preferably from 10 nm to 100 μm, and particularly 50 nm to 50 μm. In one embodiment, the composition comprises a solid support, which comprises at least 15%, preferably at least 25%, particularly 15% to 40%, more specifically 25% to 35% of immobilized lipases, proteases, and / or amylases, or fragments thereof, per dry weight of the solid support.

[0046] Typically, monodisperse particles or polydisperse particles, preferably monodisperse particles, are used as the solid carrier in the present invention. In a preferred embodiment, the monodisperse particles are spherical monodisperse particles. In a more preferred embodiment, the polydisperse particles are non-spherical polydisperse particles.

[0047] Solid carriers are usually provided in suspension. The suspension of solid carriers can be done, for example, in water, a buffer, a nonionic surfactant, or a mixture thereof, preferably in a mixture of water and a nonionic surfactant. Nonionic surfactants are typically ethoxylated sorbitan esters such as EG-40 diisostearate P-sorbitan, polysorbate 80 (PS80), polysorbate 20 (PS20), polysorbate 40 (PS40), and polysorbate 60 (PS60); block copolymers such as poloxamer 124, poloxamer 188, poloxamer 331, and poloxamer 407; and fatty acid ethoxylated esters such as PEG-5 oleate, PEG-8 stearate, polyoxyl stearate 40, and polyoxyl hydroxystearate 15. The material is selected from the group consisting of silates, fatty alcohol ethoxylates such as steareth 40; fatty acid esters such as ascorbyl palmitate, beeswax, polyglyceryl-3 oleate, propylene glycol monocaprylate, and propylene glycol monolaurate; fatty alcohols such as cetostearyl alcohol, cetyl alcohol, myristic alcohol, and stearyl alcohol; glycerides; pegylated triglycerides; and sugar esters, preferably polysorbates, more preferably polysorbate 80 (PS80).The buffers that can be used in the method of the present invention include phosphates, piperazine-N,N′-bis(2-ethanesulfonic acid), 2-hydroxy-3-morpholinopropanesulfonic acid, N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), (3-(N-morpholino)propanesulfonic acid), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]amino]ethanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 3-(N,N-bis[2-hydroxy These are ethyl]amino)-2-hydroxypropanesulfonic acid, N,N-bis(2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, N-[tris(hydroxymethyl)methyl]glycine, diglycine, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, N,N-bis(2-hydroxyethyl)glycine, N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, and N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid.

[0048] In one embodiment, a substance that interacts with the lid domain of lipase or a fragment thereof is added to a suspension of a solid carrier, preferably together with the lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof, before immobilizing the lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof onto the solid carrier. The lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof may be added to the suspension of a solid carrier in the form of pancreatin before immobilizing the lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof onto the solid carrier.

[0049] The immobilization of lipase or its fragments, protease or its fragments, and amylase or its fragments onto a solid carrier is typically carried out by adding a solution containing lipase or its fragments, protease or its fragments, and amylase or its fragments, or pancreatin or a solution thereof, to a suspension of the solid carrier. In a preferred embodiment, the immobilization of lipase or its fragments, protease or its fragments, and amylase or its fragments onto a solid carrier is carried out by providing a suspension of the solid carrier and a solution containing lipase or its fragments, protease or its fragments, and amylase or its fragments, or pancreatin or a solution thereof, wherein the suspension of the solid carrier is incubated with a solution containing lipase or its fragments, protease or its fragments, and amylase or its fragments, or pancreatin or a solution thereof, so that the lipase or its fragments, protease or its fragments, and amylase or its fragments can bind to the surface of the solid carrier. In a more preferred embodiment, the immobilization of lipase, protease or fragment thereof, and amylase or fragment thereof, or pancreatin or a solution thereof on a solid carrier is carried out by providing a suspension of the solid carrier, a solution containing lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, or pancreatin or a solution thereof, and a solution of a substance that interacts with the lid domain of lipase or fragment thereof, wherein the suspension of the solid carrier is incubated with the solution containing lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, or pancreatin or a solution thereof, and with the solution of a substance that interacts with the lid domain of lipase or fragment thereof, so that the lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof can bind to the surface of the solid carrier.

[0050] In one embodiment, the surface of a solid support is modified to introduce molecules or functional chemical groups as anchor points, i.e., as anchor points for proteins, such as lipase or fragments thereof, proteases or fragments thereof, and amylase or fragments thereof, or as anchor points for linkers connecting proteins, such as lipase or fragments thereof, proteases or fragments thereof, and amylase or fragments thereof, to the solid support. Preferably, the anchor points are amine functional chemical groups or moieties. In a non-limiting example, an amino-modified surface of a solid support, such as an amino-modified silica surface, can be used as a modified solid support. Such an amino-modified surface of a solid support can be obtained by reacting a solid support having a silica surface with an aminosilane, such as APTES. Therefore, in a preferred embodiment, the solid support is a solid support having a silica surface having an amino-modified surface, and more preferably a solid support obtained by reacting a solid support having a silica surface with an aminosilane, such as APTES. Such modified carriers can form amide bonds between proteins, such as lipase or its fragments, proteases or their fragments, and amylases or their fragments, and amine groups on the surface of the carrier material, or between a linker and amine groups on the surface of the carrier material. In one embodiment, the molecules or functional chemical groups introduced as anchor points are uniformly distributed on the surface of the solid carrier.

[0051] In one embodiment, the substance that interacts with the lid domain of lipase or a fragment thereof is selected from the group consisting of colipase or a fragment thereof, a colipase-mimicking peptide, and an amphiphilic molecule. Preferably, the substance that interacts with the lid domain of lipase or a fragment thereof is selected from the group consisting of colipase or a fragment thereof, a colipase-mimicking peptide, and a bile salt; more preferably, it is selected from the group consisting of colipase or a fragment thereof, a colipase-mimicking peptide, and sodium taurocholate; and even more preferably, it is selected from the group consisting of colipase or a fragment thereof, the colipase-mimicking peptide shown in SEQ ID NO: 1, and sodium taurocholate. Most preferably, the substance that interacts with the lid domain of lipase or a fragment thereof is a bile salt, particularly sodium taurocholate.

[0052] In one embodiment, a substance interacting with the lid domain of the lipase or fragment thereof specifically interacts with the lid domain of the lipase or fragment thereof such that the lipase or fragment thereof transitions to an open structure and / or maintains an open structure.

[0053] In one embodiment, about 50% to 100%, preferably about 80% to 100%, more preferably about 90% to 100%, and even more preferably about 100% of the lipase or fragment thereof immobilized on the surface of the solid carrier is in an open structure.

[0054] In some embodiments, the protective layer has a specified thickness of about 1 to about 200 nm, typically about 1 to about 100 nm, preferably about 1 to about 50 nm, more preferably about 1 to about 25 nm, even more preferably about 1 to about 20 nm, and particularly about 1 to about 15 nm. The most preferred specified thickness is about 1 to about 10 nm. In some embodiments, the layer has a specified thickness of about 5 to about 100 nm, preferably about 5 to about 50 nm, more preferably about 5 to about 25 nm, even more preferably about 5 to about 20 nm, and particularly about 5 to about 15 nm. The most preferred specified thickness is about 5 to about 10 nm. The protective layer is usually porous, with a pore size of 1 to 100 nm, preferably 1 to 20 nm.

[0055] In one embodiment, a protein or fragment thereof, such as lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, is partially embedded by a protective layer. In a preferred embodiment, a protein or fragment thereof, such as lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, is at least partially embedded by a protective layer. In a more preferred embodiment, a protein or fragment thereof, such as lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, is completely embedded by a protective layer.

[0056] In one embodiment, a solid carrier is embedded in the protective layer, and proteins or fragments thereof, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, immobilized on the surface of the solid carrier are embedded. In one embodiment, functional components immobilized on the surface of the protective layer are not embedded by the protective layer. Preferably, the solid carrier is completely embedded in the protective layer, and proteins or fragments thereof, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, immobilized on the surface of the solid carrier are completely embedded. More preferably, the solid carrier is completely embedded in the protective layer, and proteins or fragments thereof, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, immobilized on the surface of the solid carrier are completely embedded, and functional components immobilized on the surface of the protective layer are not embedded by the protective layer. When the solid carrier is completely embedded in the protective layer, and the proteins or fragments thereof immobilized on the surface of the solid carrier, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, are completely embedded, i.e., 100%, by the protective layer, i.e., the active sites are also covered, and the solid carrier is completely, i.e., 100%, covered by the protective layer.

[0057] In preferred embodiments, the lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof used in the present invention are composed of pancreatin. In more preferred embodiments, pancreatin is used to immobilize the lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof on the surface of a solid carrier. Accordingly, the present invention provides a composition comprising a solid carrier, pancreatin comprising lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof in an open structure, wherein the lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof are immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, a protective layer that protects the lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof by embedding them, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each comprising at least one amino group and / or at least one thiol group.

[0058] The thickness of the protective layer can be measured using a microscope such as a scanning electron microscope (SEM), transmission electron microscope (TEM), or scanning probe microscope (SPM), or by light scattering or ellipsometry.

[0059] The compositions of the present invention are typically produced in a reaction vessel such as a reactor. The formation of the protective layer is usually carried out by forming each protective layer with building blocks, which construct the protective layer through polycondensation reactions. Polycondensation can be carried out in different solvents, preferably aqueous solutions. Polycondensation can be easily controlled and stopped as needed, making it possible to achieve a specified thickness of the protective layer. The selection of building blocks that can be used to construct the protective layer may depend on the known structures of proteins, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, in order to adapt the affinity of the protective layer according to optimal and / or desired parameters. As building blocks for the protective layer, structural building blocks and protective building blocks for constructing the protective layer are typically used. A structural building block that can be used is, for example, tetraethyl orthosilicate (referred to herein as "TEOS" or "T"). The protective building blocks that can be used may be, for example, 3-aminopropyltriethoxysilane (referred to herein as "APTES" or "A"), propyltriethoxysilane (referred to herein as "PTES" or "P"), isobutyltriethoxysilane (referred to as "IBTES"), hydroxymethyltriethoxysilane (referred to herein as "HTMEOS" or "H"), benzyltriethoxysilane (referred to herein as "BTES"), ureidopropyltriethoxysilane (referred to as "UPTES"), or carboxyethyltriethoxysilane (referred to herein as "CETES"). The structural building blocks are typically precursors of inorganic silica and can form four covalent bonds in the resulting layer. The protective building blocks are typically organosilanes and have an organic moiety capable of interacting with proteins, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof. Preferred structural building blocks are tetravalent silanes, particularly tetraalkoxysilanes. Preferred protective building blocks are trivalent silanes, particularly trialkoxysilanes. More preferred structural building blocks areA mixture of tetravalent and trivalent silanes, particularly a mixture of tetra-alkoxysilane and tri-alkoxysilane. More preferred structural building blocks are selected from the group consisting of tetraethyl orthosilicate, tetra-(2-hydroxyethyl)silane, and tetramethyl orthosilicate. A more preferred protective building block is selected from the group consisting of carboxyethylsilanetriol, benzylsilane, propylsilane, isobutylsilane, n-octylsilane, hydroxysilane, bis(2-hydroxyethyl)-3-aminopropylsilane, aminopropylsilane, ureidopropylsilane, (N-acetylglycyl)-3-aminopropylsilane, and hydroxy(polyethyleneoxy)propyl]triethoxysilane, particularly benzyltriethoxysilane (BTES), propyltriethoxysilane, isobutyltriethoxysilane, n-octyltriethoxysilane, hydroxymethyltriethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, 3-aminopropyltriethoxysilane, ureidopropyltriethoxysilane, and (N-acetylglycyl)-3-aminopropyltriethoxysilane, or benzyltrimethoxysilane, propyltrimeth Selected from xysilane, isobutyltrimethoxysilane, n-octyltrimethoxysilane, hydroxyrunethyltrimethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, ureidopropyltriethoxysilane, (N-acetylglycyl)-3-aminopropyltriethoxysilane, or benzyltrimethoxysilane, propyltrimethoxysilane, isobutyltrimethoxysilane, n-octyltrimethoxysilane, hydroxymethyltrimethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrimethoxysilane, aminopropyltrimethoxysilane, ureidopropyltrimethoxysilane, (N-acetylglycyl)-3-aminopropyltrimethoxysilane, or benzyltrihydroxyethoxysilane,Selected from propyltrihydroxyethoxysilane, isobutyltrihydroxyethoxysilane, n-octyltrihydroxyethoxysilane, hydroxymethyltrihydroxyethoxysilane (hydroxymefilyltrihydroxyethoxysilane), bis(2-hydroxyethyl)-3-aminopropyltrihydroxyethoxysilane, aminopropyltrihydroxyethoxysilane, and ureidopropyltrihydroxyethoxysilane (N-acetylglycyl)-3-aminopropyltrihydroxymethoxysilane.

[0060] Particularly preferred building blocks are TEOS as structural building blocks and APTES, BTES, and / or HTMEOS as protective building blocks, preferably APTES and / or BTES. In particular, TEOS as structural building blocks and APTES and / or BTES as protective building blocks are used to construct the protective layer.

[0061] The reaction time between the building blocks and the solid support bearing the immobilized enzymes may depend on the length of the linker if one is used, and the size of the protein, such as lipase or its fragments, protease or its fragments, and amylase or its fragments. The reaction is usually carried out over a period of 0.5 to 10 hours, preferably 1 to 5 hours, more preferably 1 to 4 hours, and even more preferably 2 to 4 hours, preferably in an aqueous solution, preferably at room temperature at about 5 to about 25°C or about 20°C. The formation of the protective layer can be stopped by actively halting the polycondensation reaction, for example, by removing unreacted building blocks through a washing process, or by the self-termination of the polycondensation reaction caused by a limited amount of building blocks.

[0062] In a more preferred embodiment, proteins, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, are immobilized on a solid carrier by at least partially modifying the surface of the solid carrier by introducing molecules as the aforementioned anchor points for the proteins, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, using linkers, preferably crosslinkers, that bind to anchor points and proteins, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof.

[0063] In one embodiment, the molecules and / or linkers introduced as anchor points are uniformly distributed on the surface of the solid support.

[0064] In preferred embodiments, the crosslinker may be glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated sulfhydryl, sulfhydryl-reactive 2-pyridyldithiol, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2) Selected from the group consisting of HCl), DST (disuccinimidyl tartrate), sulfo-LC-SMPT (4-sulfosuccinimidyl-6-methyl-a-(2-pyridyldithio)toluamide]hexanoic acid), SPDP (N-succinimidyl 3-(2-pyridyldithio)-propionate), LC-SPDP (succinimidyl 6-(3-[2-pyridyldithio]-propionamide)hexanoic acid), SMPT (4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), DDPPB (1,4-di-[3'-(2'-pyridyldithio)-propionamide]butane), DTME (dithio-bismaleimide ethane), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane). More preferably, the crosslinker is selected from glutaraldehyde, disuccinimidyl tartrate, disuccinimidyl suberate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimerimidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated sulfhydryl (e.g., sulfhydryl-reactive 2-pyridyldithio), and a colipase-mimicking peptide, the colipase-mimicking peptide may be functionalized with a chemical group that enables covalent bonding to the surface of a solid support.In a more preferred embodiment, the crosslinker is selected from the group consisting of glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2HCl), DST (disuccinimidyl tartrate), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane). More preferably, the crosslinker is selected from glutaraldehyde, disuccinimidyl tartrate, disuccinimidyl suberate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimerimidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, and activated sulfhydrils (e.g., sulfurhydryl-reactive 2-pyridyldithio). Most preferably, glutaraldehyde is selected.

[0065] After the protective layer is formed, the resulting suspension can be washed to remove any excess material that interacts with the lid domain of the lipase or its fragment. In one embodiment, after the protective layer is formed, a solid support containing the protein, e.g., lipase or its fragment, protease or its fragment, and amylase or its fragment, along with the protective layer, can be stored. Storage is usually achieved, for example, by washing the formed composition with a buffer and storing it suspended or dissolved in that buffer for a desired period of time. In a preferred embodiment, the solid support containing the protein, e.g., lipase or its fragment, protease or its fragment, and amylase or its fragment, along with the protective layer, is stored at a constant temperature between 2 and 25°C. In a more preferred embodiment, the solid support containing the protein, e.g., lipase or its fragment, protease or its fragment, and amylase or its fragment, along with the protective layer, is stored for 5 to 48 hours, preferably 10 to 30 hours. More preferably, the solid carrier comprising a protein, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof, and a protective layer, is stored at a constant temperature between 2 and 25°C, preferably at room temperature, for 10 to 30 hours.

[0066] In one embodiment, the functional component binds to the mucus.

[0067] In one embodiment, a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is a polymer comprising repeating units, each containing at least one amino group.

[0068] In one embodiment, a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is a polymer comprising repeating units, each containing at least one thiol group.

[0069] In one embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is selected from the group consisting of polyglucosamine, polymerized silane-PEG-NH2, and polymerized silane containing an amino group. In a preferred embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is selected from the group consisting of polyglucosamine, polymerized silane-PEG-NH2, and polymerized APTES.

[0070] In a more preferred embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan and dermatan or derivatives thereof; polymerized silane-PEG-NH2; and polymerized silanes containing an amino group, preferably polymerized APTES. In an even more preferred embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is polyglucosamine, preferably polyglucosamine selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan and dermatan or derivatives thereof, and more preferably chitosan or a derivative thereof.

[0071] The preferred polyglucosamine of the present invention is selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan, and dermatan or their derivatives. The most preferred is chitosan or its derivatives. The preferred polymerized silane-PEG-NH2 is selected from the group consisting of silane-PEG4-NH2, silane-PEG2000-NH2, and silane-PEG5000-NH2. The preferred polymerized silane containing an amino group is selected from the group consisting of APTES, amino-butyl-TES, amino-pentyl-TES, amino-hexyl-TES, amino-heptyl-TES, and amino-octyl-TES, with APTES being particularly preferred.

[0072] In further embodiments, polymers comprising repeating units, each containing at least one amino group and / or at least one thiol group, are selected from the group consisting of polyglucosamine, polymerized silane-PEG-NH2, polymerized silane containing an amino group, polymerized silane containing a thiol group, polycarbophil-cysteine ​​conjugate, polymerized silane-PEG-thiol, and polycysteine. In more preferred further embodiments, polymers comprising repeating units, each containing at least one amino group and / or at least one thiol group, are selected from the group consisting of chitin, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratan, and dermatan or derivatives thereof, and are selected from the group consisting of polyglucosamine; polymerized silane-PEG-NH2; polymerized silane containing a thiol group, preferably polymerized MPTS; polycarbophil-cysteine ​​conjugate; polymerized silane-PEG-thiol; and polycysteine. In a more preferred embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is a thiol-containing polyglucosamine or polymerized silane, preferably a polyglucosamine selected from the group consisting of chitin, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratan, and dermatan or derivatives thereof, more preferably chitosan or a derivative thereof, or a thiol-containing polymerized silane, polycarbophil-cysteine ​​conjugate, and polymerized silane-PEG-thiol, preferably a thiol-containing polymerized silane.

[0073] In certain embodiments, polymers comprising repeating units, each containing at least one amino group and / or at least one thiol group, are selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratin, dermatan or derivatives thereof, particularly chitosan or derivatives thereof, polymerized silane-PEG-NH2, polymerized silane-PEG2000-NH2, polymerized silane-PEG5000-NH2, polymerized silanes containing amino groups, preferably polymerized APTES, and polymerized silanes containing thiol groups, preferably polymerized MPTS.

[0074] In one embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is selected from the group consisting of polyglucosamine, polymerized silane-PEG-NH2, polymerized silane containing an amino group, and polymerized silane containing a thiol group. In a preferred embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is selected from the group consisting of polyglucosamine, polymerized silane-PEG-NH2, polymerized APTES, and polymerized MPTS.

[0075] In a more preferred embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is selected from the group consisting of polyglucosamine selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan and dermatan or derivatives thereof; polymerized silane-PEG-NH2; polymerized silane containing an amino group, preferably polymerized APTES; and polymerized silane containing a thiol group, preferably polymerized MPTS. In certain embodiments, polymers comprising repeating units, each containing at least one amino group and / or at least one thiol group, are selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratin, dermatan or derivatives thereof, particularly chitosan or derivatives thereof, polymerized silane-PEG-NH2, polymerized silane-PEG2000-NH2, polymerized silane-PEG5000-NH2, polymerized silanes containing amino groups that are APTES, and polymerized silanes containing thiol groups that are MPTS.

[0076] In one embodiment, the polymer comprising repeating units, each containing at least one thiol group, is selected from the group consisting of polymerized silanes containing thiol groups, polycarbophil-cysteine ​​conjugates, polymerized silane-PEG-thiols, and polycysteine, preferably selected from the group consisting of polymerized silanes containing thiol groups, polycarbophil-cysteine ​​conjugates, and polymerized silane-PEG-thiols, more preferably polymerized silanes containing thiol groups, and most preferably polymerized MPTS. In one embodiment, the polymerized silane containing thiol groups is preferably polymerized MPTS.

[0077] In one embodiment, 5% to 100%, preferably 10% to 100%, and more preferably 50% to 100% of the surface of the protective layer is covered with a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group.

[0078] In one embodiment, the functional component is immobilized on the surface of the protective layer by bonding, preferably covalent bonding. In a preferred embodiment, the functional component is immobilized on the surface of the protective layer by non-covalent bonding, preferably by electrostatic interaction. In a more preferred embodiment, a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is immobilized on the surface of the protective layer by covalent bonding.

[0079] In one embodiment, the functional component is immobilized on the surface of the protective layer using a spacer that binds to the surface of the protective layer and the functional component. Thus, in one embodiment, the present invention comprises a composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment thereof by embedding it, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each comprising at least one amino group and / or at least one thiol group, and the functional component is immobilized on the surface of the protective layer by a spacer. Examples of such spacers include polyethylene such as PEG4, PEG2000, and PEG5000. The functional component immobilized on the surface of the protective layer by a spacer is usually produced by first reacting the spacer and the functional component so that the spacer binds to the functional component, and then the functional component bound to the spacer reacts with the surface of the protective layer.

[0080] The immobilization of functional components onto the surface of the protective layer is usually carried out by suspending a solid support carrying the aforementioned protein, such as an enzyme, embedded in the protective layer in a reaction vessel such as a reactor, for example, in water, a buffer, a nonionic surfactant, or a mixture thereof, preferably in a mixture of water and a nonionic surfactant. Nonionic surfactants are usually ethoxylated sorbitan esters such as EG-40 diisostearate P-sorbitan, polysorbate 80 (PS80), polysorbate 20 (PS20), polysorbate 40 (PS40), and polysorbate 60 (PS60); block copolymers such as poloxamer 124, poloxamer 188, poloxamer 331, and poloxamer 407; and fatty acid ethoxylated surfactants such as PEG-5 oleate, PEG-8 stearate, polyoxyl stearate 40, and polyoxyl hydroxystearate 15. The functional component is selected from the group consisting of silates, fatty alcohol ethoxylates such as steareth 40; fatty acid esters such as ascorbyl palmitate, beeswax, polyglyceryl-3 oleate, propylene glycol monocaprylate, and propylene glycol monolaurate; fatty alcohols such as cetostearyl alcohol, cetyl alcohol, myristic alcohol, and stearyl alcohol; glycerides; pegylated triglycerides; and sugar esters, preferably polysorbates, more preferably polysorbate 80 (PS80). The functional component is then added to the suspension and reacted with the surface of the protective layer, usually under stirring, to immobilize the functional component on the surface of the protective layer. Typically, such compositions obtained are washed and resuspended in water, buffer, or a nonionic surfactant or a mixture thereof. Immobilization is achieved by non-covalent bonds, such as electrostatic bonds, or by covalent bonds of the functional component.Functional components can be immobilized by chemically modifying the surface of the protective layer and the functional components using "click chemistry" such as copper-catalyzed click chemistry (Copper-catalyzed azide-alkyne cycloaddition, see e.g. Kolb et al. (2001) Angew. Chem. 40(11) 2004-2021), or by using click chemistry that does not use copper (Wittig G, A Chem Ber, 1961, 94, 3260), for example, by first reacting a solid support carrying a protein, such as an enzyme, embedded in the aforementioned protective layer with a reactive compound such as an ethynyl compound, modifying the functional components by adding a reactive compound, such as an azide residue, and then reacting the two components to immobilize the functional components on the surface of the protective layer.

[0081] In a further embodiment, the present invention provides the aforementioned compositions for use as pharmaceuticals.

[0082] In a further embodiment, the present invention provides compositions for use in enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, or for use in methods for the prevention, delaying the progression of, or treating pancreatic exocrine insufficiency (EPI).

[0083] In a preferred embodiment, the present invention provides compositions for use in methods for the prevention, delaying the progression of, or treating exocrine pancreatic insufficiency (EPI). In a further preferred embodiment, the present invention provides compositions for use in methods of enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy.

[0084] Furthermore, the use of the composition described herein for the manufacture of a medicament for the prevention, delay of progression, or treatment of exocrine pancreatic insufficiency (EPI) in a subject is provided. Furthermore, the use of the composition described herein for the prevention, delay of progression, or treatment of exocrine pancreatic insufficiency (EPI) in a subject is provided. Furthermore, a method for the prevention, delay of progression, or treatment of exocrine pancreatic insufficiency (EPI) in a subject is provided, comprising administering a therapeutically effective amount of the composition described herein to the subject. Furthermore, the use of the composition described herein for the manufacture of a medicament for a method of enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, is provided. Furthermore, the use of the composition described herein in a method of enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, in a subject is provided. Furthermore, a method for enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, in a subject is provided, comprising administering a therapeutically effective amount of the composition described herein to the subject.

[0085] The compositions according to the present invention are preferably pharmaceutical compositions comprising a therapeutically effective amount of the composition described herein and one or more suitable pharmaceutically acceptable carriers. The pharmaceutical compositions according to the present invention are suitable for oral administration to a subject. Unless otherwise indicated, the pharmaceutical compositions according to the present invention are prepared by known methods.

[0086] The composition of the present invention, for example, a pharmaceutical composition, may be administered over a continuous period of one week or part thereof, two weeks, three weeks, four weeks, five weeks, or six weeks, and then discontinued for one week or part thereof, two weeks, three weeks, four weeks, five weeks, or six weeks. The composition of the present invention, for example, a pharmaceutical composition, can be conveniently administered in unit dose form.

[0087] As used herein, the terms “effective dose” or “therapeutic effective dose” refer to an amount of the composition of the present invention that is capable of producing one or more desired effects in a subject to which it is administered. Determining the therapeutic effective dose is well within the capabilities of those skilled in the art, particularly in light of the detailed disclosure provided herein.

[0088] As used herein, the terms “treatment” and “to treat” include: (1) delaying the onset of clinical symptoms of a condition, disorder, or pathology in animals, particularly mammals, particularly humans, that are suffering from or susceptible to a condition, disorder, or pathology but have not yet experienced or manifested clinical or subclinical symptoms of the condition, disorder, or pathology; (2) inhibiting a condition, disorder, or pathology (e.g., stopping, reducing, or delaying the onset of the disease with respect to at least one clinical or subclinical symptom, or stopping, reducing, or delaying its recurrence in the case of maintenance treatment); and / or (3) reducing a pathology (i.e., causing a regression of the condition, disorder, or pathology, or at least one of its clinical or subclinical symptoms). The benefit to the patient being treated is statistically significant or at least perceptible to the patient or physician. However, it will be understood that when a patient is given medicine to treat a disease, the result is not always an effective treatment.

[0089] As used herein, “delay in progression” means extending the time to the onset of symptoms or delaying the increase in the severity of symptoms. Furthermore, as used herein, “delay in progression” includes setback or inhibition of disease progression. “Inhibition” of disease progression or disease complications in a subject means preventing or mitigating disease progression and / or disease complications in a subject.

[0090] Preventive measures include prophylactic treatment. For preventive use, the drug combination of the present invention is administered to subjects suspected of having or at risk of developing the above-mentioned disease or disorder. For therapeutic use, the drug combination is administered to subjects, for example, patients already suffering from the above-mentioned disease or disorder, in an amount sufficient to cure or at least partially cessate the symptoms of the disease. The effective dose for such use will depend on the severity and course of the disease, previous treatments, the subject's health status and response to the drug, and the judgment of the attending physician.

[0091] If the target condition does not improve, the pharmaceutical combination of the present invention may be administered chronically, i.e., over a long period including the subject's entire life, to improve, or otherwise control or limit the symptoms of the target disease or condition.

[0092] If the patient's condition improves, the drug combination can be administered continuously; alternatively, the dose of the administered drug may be temporarily reduced or temporarily stopped over a specific period (i.e., a “drug-free period”). Once the patient’s condition improves, a maintenance dose of the drug combination of the present invention may be administered as needed. Thereafter, the dose, or the frequency of administration, or both, may be reduced as a function of the symptoms to a level at which the improved disease is maintained.

[0093] In a further embodiment, the present invention provides a method for producing a composition comprising a solid carrier, an open-structured lipase or fragment thereof immobilized on the surface of the solid carrier, a protease or fragment thereof immobilized on the surface of the solid carrier, an amylase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, a protective layer that protects the lipase or fragment thereof, the protease or fragment thereof, and the amylase or fragment thereof by embedding them, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each comprising at least one amino group and / or at least one thiol group, and the method comprises the following steps: (a) A step of providing a solid carrier, (b) A step of providing lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof. (c) A step of providing a substance that interacts with the lid domain of lipase or a fragment thereof. (d) A step of interacting the lipase or fragment thereof from (b) with the substance from (c), (e) A step of immobilizing lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof on a solid carrier. (f) A step of forming a protective layer on the surface of a solid carrier to protect lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof immobilized on the solid carrier, (g) A step of immobilizing a functional component on the surface of a protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group.

[0094] In a preferred embodiment, pancreatin comprising lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof is provided in step b).

[0095] Step (a) is typically carried out by providing the solid carrier suspended in water or a buffer, preferably in water, a nonionic surfactant or buffer or a mixture thereof, preferably in a buffer, more preferably suspended in water and / or a nonionic surfactant, even more preferably suspended in water and / or a nonionic surfactant without a buffer in the suspension, particularly suspended in a mixture of water and a nonionic surfactant, and more specifically suspended in a mixture of water and a nonionic surfactant without a buffer in the suspension. In a preferred embodiment, pancreatin is provided in step b). Steps b) and c) can typically be carried out separately or simultaneously, and for example, lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, and a drug, or pancreatin and the drug, may be supplied in a single solution. In step b) of this method, the immobilization of proteins, such as lipase or its fragments, proteases or their fragments, and amylase or its fragments, onto a solid carrier is usually carried out by adding a solution of the proteins, such as lipase or its fragments, proteases or their fragments, and amylase or its fragments, or pancreatin or its solution, to a suspension of the solid carrier. Preferably, before adding the protein solution to the suspension of the solid carrier, a linker that connects the solid carrier to the proteins, such as lipase or its fragments, proteases or their fragments, and amylase or its fragments, is added to the suspension of the solid carrier.In a preferred embodiment, the immobilization of proteins, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, onto a solid carrier is carried out by providing a suspension of the solid carrier and adding a solution of the proteins, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, or pancreatin or a solution thereof. The suspension containing the solution to which the proteins, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, or pancreatin or a solution thereof has been added is incubated so that the proteins, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, can bind to the surface of the solid carrier.

[0096] In a more preferred embodiment, the immobilization of proteins, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, onto a solid carrier in step (e) is carried out by i) adding a linker to the solid carrier provided in step (a), preferably adding a linker to a suspension of the solid carrier provided in step (a), and ii) adding the proteins, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, provided in step (b), preferably adding a solution of the proteins, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, provided in step (b), to the solid carrier and linker, or to a suspension containing the solid carrier and linker, wherein the linker links the solid carrier to the proteins, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, in step (e). In one embodiment, building blocks for the protective layer, preferably monomers of the building blocks for the protective layer, more preferably organosilanes, even more preferably triethoxysilanes, particularly APTES, are added to a solid carrier and linker, or a suspension containing a solid carrier and linker, before adding solutions of proteins, such as lipase or fragments thereof, proteases or fragments thereof, and amylase or fragments thereof.

[0097] In preferred embodiments, the surface of the solid carrier is at least partially modified to improve the immobilization of proteins, such as lipase or its fragments, proteases or their fragments, and amylase or its fragments, to the solid carrier. In particular, the surface of the solid carrier is at least partially modified before immobilizing proteins, such as lipase or its fragments, proteases or their fragments, and amylase or its fragments. The surface of the solid carrier can be at least partially modified by adding molecules to the surface of the solid carrier as anchor points for proteins, such as lipase or its fragments, proteases or their fragments, and amylase or its fragments.

[0098] The suspension containing the solid carrier is typically incubated after each of the above addition steps so that the proteins, such as lipase or its fragments, proteases or their fragments, and amylase or its fragments, are linked to the solid carrier, preferably the surface of the solid carrier, via linkers, preferably by covalent bonds, thereby immobilizing the proteins, such as lipase or its fragments, proteases or their fragments, and amylase or its fragments, on the solid carrier, for example, by reacting the solid carrier with molecules as anchor points, the solid carrier with linkers, and the solid carrier containing linkers with proteins, such as lipase or its fragments, proteases or their fragments, and amylase or its fragments, respectively.

[0099] In one embodiment, in step (e), the proteins, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof, are immobilized on a solid carrier by linking the proteins, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof via linkers, preferably by linking the proteins, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof via linkers, and the solid carrier is connected to the proteins, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof by covalent bonds between the linker and the solid carrier and between the linker and the proteins, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof. Preferably, i) a linker is added to the solid carrier provided in step (a), and ii) proteins provided in step (b), such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, are added to the solid carrier and the linker, and the linker links the solid carrier to the proteins, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, in step (e). The linker used is as described above and preferably links the surface of the solid carrier to the proteins, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, by covalent bonds.Preferably, the linker is added to the solid carrier provided in step (a) in a molar excess relative to the protein provided in step (b), such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof; preferably, the linker is added to the solid carrier in step (b) in a molar excess of 1 to 1000 times relative to the protein provided in step (b), such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof; preferably, the linker is added to the solid carrier in step (b) in a molar excess relative to the protein provided in step (b), such as lipase or a fragment thereof, and amylase or a fragment thereof. The linker is added in a molar excess of 2 to 300 times relative to the protein provided in step (b), e.g., lipase or its fragments, protease or its fragments, and amylase or its fragments; more preferably, the linker is added to the solid carrier in step (b) in a molar excess of 4 to 250 times relative to the protein provided in step (b), e.g., lipase or its fragments, protease or its fragments, and amylase or its fragments; in particular, the linker is added to the solid carrier in step (b) in a molar excess of 9 times relative to the protein provided in step (b), e.g., lipase or its fragments, protease or its fragments, and amylase or its fragments.

[0100] In a preferred embodiment, in step (e), the linker that did not link the solid carrier to the protein, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof, is present while a protective layer is formed on the surface of the solid carrier in step (f). In a more preferred embodiment, in step (e), the linker, or a portion thereof, that did not link the solid carrier to the protein, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof, is covalently bonded in step (f) to the protective layer and the protein, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof. In an even more preferred embodiment, in step (e), the linker that did not link the solid carrier to the protein, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof, is not removed in step (e) or step (f), or between steps (e) and (f). In certain embodiments, in step (e), linkers that did not link the solid carrier to proteins, such as lipase or its fragments, proteases or their fragments, and amylase or its fragments are not removed in step (e) or step (f), or between steps (e) and (f). In step (e), linkers or a portion thereof that did not link the solid carrier to proteins, such as lipase or its fragments, proteases or their fragments, and amylase or its fragments are covalently bonded in step (f) to a protective layer and proteins, such as lipase or its fragments, proteases or their fragments, and amylase or its fragments. In step (e), after the addition of protein in ii), the amount of linker that did not link the solid carrier to the protein, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, is usually 30% to 70%, preferably 40% to 60%, and more preferably 50%, of the amount of linker added to the solid carrier in step (e). In one embodiment, there is no washing step between adding the linker to the solid carrier provided in step (a) of (i) and adding the protein, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, to the solid carrier and linker in (ii).In one embodiment, there is no washing step between any of steps (a) to (f). In one embodiment, there is no washing step between adding the linker to the solid carrier provided in step (e) of (i) and adding the proteins, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, to the solid carrier and the linker in (ii), and there is no washing step between any of steps (a) to (f).

[0101] In one embodiment, the linker is glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated sulfhydryl, sulfhydryl-reactive 2-pyridyldithiol, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2) A selection is made from the group consisting of HCl, DST (disuccinimidyl tartrate), sulfo-LC-SMPT (4-sulfosuccinimidyl-6-methyl-a-(2-pyridyldithio)toluamide]hexanoic acid), SPDP (N-succinimidyl 3-(2-pyridyldithio)-propionate), LC-SPDP (succinimidyl 6-(3-[2-pyridyldithio]-propionamide)hexanoic acid), SMPT (4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), DDPPB (1,4-di-[3'-(2'-pyridyldithio)-propionamide]butane), DTME (dithio-bismaleimide ethane), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane), preferably glutaldehyde.

[0102] In preferred embodiments, the linker is glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2 It is selected from the group consisting of HCl, DST (disuccinimidyl tartrate), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane), and is preferably glutaldehyde.

[0103] The formation of the protective layer in step (f) of this method is usually carried out by forming each protective layer using building blocks, and the building blocks construct the protective layer by the polycondensation reaction described above. The immobilization of the functional components on the surface of the protective layer in step (g) of this method is usually carried out as described above.

[0104] In one embodiment, the present invention provides a method for producing a composition comprising a solid carrier, an open-structured lipase or fragment thereof immobilized on the surface of the solid carrier, a protease or fragment thereof immobilized on the surface of the solid carrier, an amylase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, a protective layer that protects the lipase or fragment thereof, the protease or fragment thereof, and the amylase or fragment thereof by embedding them, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each comprising at least one amino group and / or at least one thiol group, and the method comprises the following steps: (a) A step of providing a solid carrier, (b) A step of providing lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof. (c) A step of immobilizing lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof onto a solid carrier. (d) A step of providing a substance that interacts with the lid domain of a lipase or a fragment thereof, (e) A step of interacting the lipase or fragment thereof from (b) with the substance from (d), (f) A step of forming a protective layer on the surface of a solid carrier to protect lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof immobilized on the solid carrier, (g) A step of immobilizing a functional component on the surface of a protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group. In a preferred embodiment, pancreatin comprising lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof is provided in step b). Steps (a) to (g) can be carried out as analogous to steps (a) to (g) of the method provided in a further embodiment of the present invention, considering that step (c) of the above method corresponds to step (e) of the method provided in a further embodiment of the present invention.

[0105] In one embodiment, the protective layer is formed by building blocks, where structural building blocks and protective building blocks are used to form the protective layer, the structural building blocks being inorganic silica precursors capable of forming four covalent bonds in the layer being formed, and the protective building blocks being the aforementioned organic silanes.

[0106] In one embodiment, the protective layer is embedded with approximately 30% to 100% of proteins, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof.

[0107] In one embodiment, the solid support is selected from the group consisting of organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, magnetic particles, and titanium particles, preferably silica particles, and more preferably silica nanoparticles (SNPs).

[0108] A preferred method of the present invention is a method for producing a composition comprising a solid carrier, an open-structured lipase or fragment thereof immobilized on the surface of the solid carrier, a protease or fragment thereof immobilized on the surface of the solid carrier, an amylase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, a protective layer that protects the lipase or fragment thereof, the protease or fragment thereof, and the amylase or fragment thereof by embedding them, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, and the method comprises the following steps: (a) A step of providing a solid carrier, wherein the solid carrier is provided in a suspended state, preferably in a suspended state in a buffer, water and / or a nonionic surfactant, and more preferably in a suspended state in a mixture of water and a nonionic surfactant. (b) A step of providing lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof. (c) A step of providing a substance that interacts with the lid domain of lipase or a fragment thereof. (d) A step of interacting the lipase or fragment thereof from (b) with the substance from (c), (e) A step of immobilizing proteins, e.g., lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, onto a solid carrier, preferably wherein the surface of the solid carrier is at least partially modified before immobilizing the proteins, e.g., lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, onto the solid carrier, i) a linker is added to the suspension of the solid carrier provided in step (a), or i) the linker is added to the solid carrier provided in step (a) after at least partial modification of the surface of the solid carrier. ii) A solution of the protein provided in step (b), such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof, preferably a solution of the protein provided in step (b), such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof, is added to the suspension of the solid carrier and the linker, and in step (e), the linker connects the solid carrier to the protein, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof, step, (f) A step of forming a protective layer on the surface of a solid carrier in order to protect lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof immobilized on the solid carrier, wherein in step (e), linkers, or a part thereof, that did not connect the solid carrier to lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, covalently bond the protective layer to lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof. (g) A step of immobilizing a functional component on the surface of a protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group. Alternatively, steps (a) to (g) can be carried out as analogous to steps (a) to (g) of the above method, considering that step (e) of the above method corresponds to step (c) of the above method.

[0109] Furthermore, a composition is provided comprising a solid carrier, an open-structured lipase or fragment thereof immobilized on the surface of the solid carrier, a protease or fragment thereof immobilized on the surface of the solid carrier, an amylase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, a protective layer that protects the lipase or fragment thereof, the protease or fragment thereof, and the amylase or fragment thereof by embedding them, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each comprising at least one amino group and / or at least one thiol group, and this composition can be obtained by this method, particularly by the preferred method of the above invention. [Examples]

[0110] Materials and methods reagent: - Tetraethyl orthosilicate 99% (TEOS), (3-aminopropyl)-triethoxysilane (APTES), ammonium hydroxide (ACS grade, 28-30%), ethanol (ACS grade, anhydrous), glutaraldehyde (grade I, 25% in water), Chelex® 100 sodium form, polysorbate 80, pancreatin (4x USP specification), bovine serum albumin (BSA), monobasic potassium phosphate, dibasic potassium phosphate, sodium taurocholate hydrate, acetic acid, olive oil, acacia gum arabic, sodium chloride, sodium hydroxide, Trizma base, hydrogen chloride, ammonium acetate, sodium acetate, pancreatin, bile salts, butanol, methanol, isopropanol, acetonitrile, NH4 acetate, amylase activity assay kit, 4% buffered formalin, Triton X-100 were purchased from Sigma-Aldrich. BSA and pancreatin were dissolved in water to reconstitute the stock buffer. - The p-SCN-Bn-DOTA was purchased from Macrocyclics. - Benzyltriethoxysilane (B, 96%) was purchased from abcr GmbH. - Chitosan 95 / 500 P was purchased from Heppe Medical Chitosan GmbH. - Indium chloride ( 111 -In) was purchased from Curium. - Animal feeds Altromin 1324 and Altromin 9033 were purchased from Altromine International. - The catheter was purchased from Instech Laboratories. - Caco-2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) were purchased from the European Collection of Authenticated Cell Cultures (ECACC). - ThinCert TM The cell culture insert plates (1.0 μm membrane) were purchased from Greiner bio-one. - Fetal bovine serum, penicillin / streptomycin (10,000 U / ml penicillin / 10,000 μg / ml streptomycin), MEM non-essential amino acids (100x), L-glutamine 200 mM (100x), Dulbecco phosphate-buffered saline (DPBS) (1x), 0.25% Trypsin-EDTA (1x), RPMI 1640 Medium, and DMEM were purchased from Gibco. - The mounting medium, containing anti-zonula occludens 1 (ZO-1) antibody, goat anti-rabbit IgG conjugated to Alexa Fluor 488, and DAPI, was purchased from ThermoFischer Scientific.

[0111] Synthesis of silica nanoparticles (SNPs): Silica nanoparticles (50 nm) were synthesized according to the original Stoeber process as described in International Publication No. 2015 / 014888. Briefly, ethanol, distilled water (6 M), and ammonium hydroxide (0.13 M) were mixed and stirred at 400 rpm for 1 hour. TEOS (0.28 M) was added, and the solution was stirred at 400 rpm at 20°C for 22 hours. The solution was then centrifuged at 20,000 g for 20 minutes and washed sequentially with ethanol and water. Particle size was measured using SEM micrographs acquired at a magnification of 150,000x with image analysis software Olympus Stream Motion.

[0112] Enzyme shielding and protein shielding, and surface functionalization - Generation of NP-1: SNPs (10 mg / mL, 56 nm) in H2O / PS80 (8 mg / L) were mixed with APTES (3.8 mM). The reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Then, DOTA (3.8 mM) was added and the mixture was stirred at 50°C for 1 hour. The DOTA-labeled particles were washed three times by centrifugation in H2O / PS80 (8 mg / L, Chelex) at 20000 rcf for 20 minutes, resuspended in H2O / PS80 (8 mg / L, Chelex), and the tips were sonicated. APTES (3.8 mM) was added to the DOTA-labeled particles. The reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Then, glutaraldehyde (3.8 mM) was added and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. BSA solution was added to achieve a final BSA concentration of 1.42 mg / mL, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized BSA using APTES (7.5 mM) and TEOS (75.4 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were then subjected to H2O / The particles were washed three times in PS80 (8 mg / L) (by centrifugation at 20,000 rcf for 5 minutes) and resuspended in H2O / PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 121 μg / mL. The reaction mixture was reacted at 20°C and 400 rpm for 30 minutes. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-1 was cured overnight in a water bath at 20°C. SNP-BSA-AT was cured overnight at 20°C.

[0113] - Generation of NP-2: Enzyme immobilization and shielding were performed as described in International Publication No. 2015 / 014888. APTES (3.8 mM) was added to SNPs (10 mg / mL, 56 nm) in H2O / PS80 (8 mg / L). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. DOTA (3.8 mM) was then added, and the mixture was incubated at 50°C for 1 hour. The DOTA-labeled particles were washed three times by centrifugation in H2O / PS80 (8 mg / L, Chelex) (20000 rcf for 20 minutes), resuspended in H2O / PS80 (8 mg / L, Chelex), and the tips were sonicated. APTES (3.8 mM) was added to the DOTA-labeled particles. The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. Next, glutaraldehyde (3.8 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. BSA solution was added to achieve a final BSA concentration of 1.42 mg / mL, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized BSA using APTES (7.5 mM) and TEOS (75.4 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were centrifuged at 20000 rcf for 5 minutes and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-2 was cured overnight in a 20°C water bath.

[0114] - Generation of NP-3: SNP (10 mg / mL, 55 nm) and PS80 (8 mg / L) were added to phosphate buffer (20 mM, pH 8), to which APTES (3.9 mM) was added. The reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.9 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.9 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Sodium taurocholate (2 mM) and pancreatin (20 g / L) were added successively, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized pancreatin using APTES (7.7 mM), TEOS (40.4 mM), and benzyltriethoxysilane (35 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were centrifuged (1000 rcf for 5 minutes), washed three times in phosphate buffer (20 mM, pH 8) and PS80 (8 mg / L) (centrifugation at 1000 rcf for 5 minutes), and resuspended in phosphate buffer (20 mM, pH 8) and PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 121 μg / mL. The reaction mixture was reacted at 20°C and 400 rpm for 30 minutes.

[0115] The particles were centrifuged (at 1000 rcf for 5 minutes), washed three times in phosphate buffer (0.120 M, pH 6) and PS80 (8 mg / L), and resuspended in phosphate buffer (0.120 M, pH 6) and PS80 (8 mg / L) to obtain NP-3. NP-3 was cured overnight in a 20°C water bath.

[0116] - Generation of NP-4: SNP (10 mg / mL, 55 nm) and PS80 (8 mg / L) were added to phosphate buffer (20 mM, pH 8), to which APTES (3.9 mM) was added. The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.9 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.9 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. BSA (11.1 g / L) was added, and the reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized BSA using APTES (7.7 mM), TEOS (40.4 mM), and benzyltriethoxysilane (35 mM). The resulting suspension was incubated at 20°C and 400 rpm for 5 hours. The particles were centrifuged (at 1000 rcf for 5 minutes), washed three times in phosphate buffer (20 mM, pH 8) and PS80 (8 mg / L) (by centrifugation at 1000 rcf for 5 minutes), and resuspended in phosphate buffer (20 mM, pH 8) and PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 121 μg / mL. The reaction mixture was incubated at 20°C and 400 rpm for 30 minutes.

[0117] The particles were centrifuged (at 1000 rcf for 5 minutes), washed three times in phosphate buffer (0.120 M, pH 6) and PS80 (8 mg / L), and resuspended in phosphate buffer (0.120 M, pH 6) and PS80 (8 mg / L) to obtain NP-4. NP-4 was cured overnight in a 20°C water bath.

[0118] - Generation of NP-5: SNPs (10 mg / mL, 58 nm) in H2O / PS80 (8 mg / L) were mixed with APTES (3.7 mM). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.7 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.7 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Pancreatin (23.5 mg / mL) containing lipase and / or its fragments, protease and / or its fragments, and amylase and / or its fragments was added, and the reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized lactase using APTES (7.2 mM) and TEOS (75.6 mM). The resulting suspension was incubated at 20°C and 400 rpm for 2 hours. The particles were washed three times in H2O / PS80 (8 mg / L) by centrifugation at 20,000 rcf for 5 minutes, and then resuspended in H2O / PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 103 μg / mL. The reaction mixture was incubated at 20°C and 400 rpm for 30 minutes. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in H2O / PS80 (8 mg / L). NP-5 was cured overnight in a water bath at 20°C.

[0119] Generation of NP-3 variants: The following experiments investigated the effects of covalently bonding enzymes to a protective layer on enzyme stability and enzyme activity.

[0120] In the first comparative experiment, enzyme immobilization and protective layer formation were performed according to International Publication No. 2015 / 014888, and nanoparticles (NP-3(2)) were generated in buffer. The nanoparticles were washed after each chemical step, resulting in the removal of glutaraldehyde. SNP (10 mg / mL, 69 nm) and PS80 (8 mg / L) were added to phosphate buffer (25 mM, pH 7.5), to which APTES (3.1 mM) was added. The reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Then, glutaraldehyde (3.1 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Priming was performed by adding APTES (3.1 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Sodium taurocholate (2 mM) and pancreatin (15 g / L) were added successively, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of immobilized pancreatin using APTES (5.7 mM), TEOS (29.9 mM), and benzyltriethoxysilane (25.9 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). NP-3(1) was cured overnight in a water bath at 20°C.

[0121] In the second experiment, the nanoparticles (NP-3) were not washed between each chemical step in order to retain the excess glutaraldehyde in the reaction mixture, which did not bind to pancreatin, by reacting with the solid support generated in phosphate buffer (20 mM, pH 8) and PS80 (8 mg / L) according to the section titled "NP-3 Generation" above. Therefore, glutaraldehyde remained present during layer growth, causing covalent bonding between the protective layer and pancreatin. Covalent bonding between the protective layer and pancreatin can be observed by the appearance of a yellow / orange color with a maximum absorbance at 460 nm. This color is due to the formation of imine bonds by the reaction of the aldehyde group of the glutaraldehyde linker with the primary amine amino acids of pancreatin and the organosilica layer. The absorbance of nanoparticles NP-3(1) and NP-3 at a wavelength of 460 nm was measured after the formation of the organic silica layer and final particle washing (see Figure 9). The absorbance of NP-3 at 460 nm was significantly higher than that of NP-3(1). NP-3(1) still exhibits some absorbance at this wavelength, which is due to the formation of imine bonds during enzyme immobilization. However, the absorbance of NP-3 is remarkably higher than the others, indicating that further imine bond formation was induced by the covalent bond between the protective layer and pancreatin.

[0122] Indium-111 111 Radioactive labeling by (In): For the labeling of nanoparticles, additional steps were added to the nanoparticle processes described in the sections titled "Enzyme Shielding and Protein Shielding, and Surface Functionalization," "NP-2 Generation," and "NP-1 Generation."

[0123] The nanoparticles were labeled with specific activity levels of 500–400 MBq / g nanoparticles according to the following protocol.

[0124] All buffers used were preprocessed with Chelex®.

[0125] Nanoparticles, 111The nanoparticles were incubated with Lu (0.02 M HCl) and ammonium acetate (1 M, pH 5.4) under continuous stirring at 45°C for 12 hours. The nanoparticles were centrifuged at 5000 g for 5 minutes and resuspended in sodium acetate (20 mM, pH 5) containing polysorbate 80 (8 mg / L). The nanoparticles were then resuspended in DTPA (1 mM, pH 5) and incubated overnight at room temperature (RT) for quenching. The nanoparticles were then washed and resuspended in 0.9% sodium chloride containing polysorbate 80 (8 mg / L).

[0126] Pancreatic lipase activity assay: The olive oil solution was prepared by mixing olive oil / gum arabic / water (1 / 8.25 / 0.75). The buffer solution was prepared by dissolving Trizma base (0.6 g / L) and sodium chloride (2.34 g / L) in nanopure water. The bile salt solution was prepared by dissolving sodium taurocholate (80 g / L) in water.

[0127] This activity assay was performed by mixing olive oil solution (13.8 mL), buffer solution (11 mL), bile salt solution (2.8 mL), and water (12.4 mL) in a bioreactor at 37°C. The pH was adjusted to 9.2 by adding aqueous sodium hydroxide solution (0.1 M). NP-3 (1.4 mL, 10 mg / mL) was washed twice with water and added to the bioreactor. The kinetics of the lipase were monitored for 10 minutes by measuring the amount of sodium hydroxide added to the reaction mixture to maintain the pH at 9.

[0128] Pancreatic protease activity assay: An enzyme sample (50 μL, 0.2 mg / mL) in phosphate buffer (50 mM, pH 7.4) was mixed with casein solution (250 μL, 0.65% w / v). The reaction mixture was incubated at 37°C and 750 rpm for 30 minutes. The sample was centrifuged at 20000 rcf for 5 minutes, and the supernatant was collected. TCA (166.7 μL) was added to the supernatant (200 μL). The sample was incubated at 37°C and 750 rpm for 30 minutes. The sample was centrifuged at 20000 rcf for 5 minutes, and the supernatant was collected. Na2CO3 was added to the supernatant (200 μL). 3( 500 μL (500 mM) and Folin reagent (100 μL, 0.5 M) were added. The sample was incubated at 37°C and 750 rpm for 30 minutes. The absorbance of 200 μL of the resulting solution was measured at 660 nm.

[0129] Pancreatic amylase activity assay: The activity of NP-3 amylase was evaluated using Sigma's amylase assay kit. In a 96-well plate, the enzyme sample (2 μL, 18.2 mg / mL) was mixed with activity buffer (48 μL). Amylase substrate solution (100 μL) was added to the wells, and the kinetics of the sample were monitored with a spectrophotometer at 37°C for 30 minutes at λ=405 nm.

[0130] animal: All animal experiments were conducted under a license approved by the National Animal Testing Service of the Danish Ministry of Food, Agriculture and Fisheries.

[0131] - Rat: This study was conducted on male Wistar rats (8 weeks old) from Janvier, France.

[0132] 〇Food and drinking water: The rats had free access to the pellet-type complete feed "Altromin 1324." The animals also had free access to drinking water.

[0133] 〇 Pancreatic duct ligation (PDL) and duodenal catheterization: The animals were anesthetized with isoflurane (2-4%) in an induction chamber and then transferred to a nose cone containing isoflurane for surgery. The rats were placed supine on a heated table, and the abdomen was incised along the midline. The pancreas was located and gently moved to locate the biliary and pancreatic ducts. The pancreatic tissue surrounding the biliary and pancreatic ducts was bluntly dissected to visualize the ducts. The area near the biliary and pancreatic ducts was ligated to stop the flow of pancreatic enzymes. After ligating the pancreatic ducts, a catheter (C30PU-RDD1444, Instech Laboratories) was placed in the duodenum anticortex near the opening of the biliary and pancreatic ducts. The catheter was ligated to the intestinal wall and exposed by creating a subcutaneous tunnel in the animal's neck. The abdominal and neck incisions were then closed with sutures.

[0134] Evaluation of the effectiveness of NP-3: PDL rats were administered NP-4 (7 mg) or NP-3 (7 mg) via the duodenum, and 5 minutes later, Triolein (10 mg) was administered by oral enteral nutrition. Approximately 150 μL of blood was collected by EDTA at 0.25, 0.5, 1, 1.5, 2, 4, and 6 hours after Triolein administration. Blood samples were centrifuged (10 minutes, 4°C, 2000×g), and at least 50 μL of plasma was transferred to an Eppendorf tube and stored at -80°C until triglyceride content analysis.

[0135] Measurement of plasma triglycerides in rats: Quantification of the target analyte was performed using an LC system: Thermo Vanquish Horizon Binary Pump and mass spectrometer: Thermo Q Exactive. Plasma samples were prepared according to the BUME method. 10 microliters of plasma sample were mixed with 300 μL of 1-butanol / methanol (3:1, v / v). The sample was incubated with agitation (900 rpm) at 20°C for 1 hour. After centrifugation (16,000 g, 10 min, 20°C), 50 μL was transferred to a glass vial and used for LC-MS.

[0136] The injection volume used was 2.5 μL, and the runtime at a flow rate of 1 mL / min was 7.5 minutes. Mobile phase A consisted of 60% acetonitrile, 40% H2O, and 5 mM NH4 acetate, while mobile phase B consisted of 90% isopropanol, 10% acetonitrile, and 5 mM NH4 acetate. Chromatographic separation was performed using a Waters Premier BEH C18 column (50 mm x 2.1 mm) (gradient: 15% B to 99% B).

[0137] Mass spectrometry (MS) was performed using a Thermo Q Exactive mass spectrometer in DDA top5 capture mode. The MS parameters were as follows: MS1 resolution: 70,000 and MS2 resolution: 17,500. HCD fragmentation was performed with normalized stepwise collision energies of 10, 20, and 30. The target extract for EIC was TG 54:3 for triolein and the most abundant triglyceride. Data analysis was performed using Thermo quan Browser software.

[0138] - Miniature pig: This study was conducted on stock female Göttingen minipigs from Ellegaard Göttingen Minipigs A / S in Denmark.

[0139] 〇Food and drinking water: The miniature pigs were regularly fed approximately 250g of the pellet-type complete feed "Altromin 9033" per day. To evaluate the efficacy of NP-3 (chronic administration), the diet was changed to a high-fat diet, and the daily allocation of altromin 9033 was supplemented with olive oil (olive oil:altromin = 1:10) and 100 g of apple sauce. The high-fat diet was started 15 days prior to the start of administration and maintained throughout the chronic administration of the treatment. The animals had free access to high-quality drinking water available at home.

[0140] 〇SPECT / CT imaging SPECT / CT scans (Clinical D670 SPECT / CT, GE) were initiated 15 minutes, 3, 8, 24, 48, and 72 hours (±1 / 2 hours) after administration of In-111-labeled nanoparticles. SPECT / CT scans were not initiated in control animals, and were initiated 15 minutes, 3, 8, and 24 hours after administration of free In-111. SPECT acquisition times were determined based on the counting rate on the scan day. SPECT imaging included two fields of view (FOV) covering the area from the stomach to the rectum. Both FOVs were scanned, with the FOV containing the stomach and duodenum being the first acquisition. Prior to the CT scan procedure, intravenous infusion of iodine-containing contrast agent (Ultravist®, 370 mg / mL, 1 mL / kg, flow rate 2 mL / s) was performed to improve organ visibility for image analysis. The FOV of the CT imaging included the entire animal. The animals were photographed on the spot and then transported from the enclosure to the scanner under anesthesia. To quantify the uptake of In-111-labeled compounds, regions of interest were defined in the relevant areas of the gastrointestinal organs (three compartments: small intestine, colon, and rectum) identified from CT image data. Uptake is expressed as %ID (percentage injected) and SUV (standardized uptake value). All data analysis was performed by a single viewer to avoid intra-observational deviation.

[0141] 〇 Pancreatic duct ligation (PDL) and duodenal catheterization: In the animal lab, the animals were premedicated with a Zoletil mixture (6.25 mL of Rompun Vet (xylazine, 20 mg / mL) + 1.25 mL of Ketaminol Vet (ketamine, 100 mg / mL) + 2.5 mL of Torbugesic Vet (butorphanol, 10 mg / mL)) from one vial of Zoletil Vet50 (125 mg of tyletamine + 125 mg of zolazepam) to be administered via IM / IV injection. After sedation, the pigs were intubated and anesthesia was maintained with inhalation of 2-4% isoflurane. An ear vein catheter was placed on one side. The animals were placed in a left lateral decubitus position, and the right flank was prepared for surgery according to standard procedure.

[0142] An incision was made in the right flank, and the location of the duodenum and pancreas was gently confirmed. Two ligatures were placed around the pancreatic duct at intervals of 2-3 mm, and the pancreatic duct was incised between the ligatures to stop the flow of pancreatic enzymes.

[0143] After ligating the pancreatic duct, a catheter (Dog duodenal catheter 7F, SAI Infusion Technologies) was placed in the duodenum on the antiintestinal side, near the opening of the biliary and pancreatic ducts. The catheter was ligated to the intestinal wall and exposed by creating a subcutaneous tunnel in the animal's back. The flank incision was then sutured. A protective bandage was applied to the abdomen.

[0144] Pre- and post-operative painkillers and antibiotics Animals received prophylactic analgesia in the form of intramuscular NSAIDs prior to surgery. Postoperative analgesia consisted of oral NSAIDs once daily for four days, with the addition of low-dose transdermal opioid patches for 72 hours as needed.

[0145] The antibiotic (amoxicillin) was administered intramuscularly before the surgery, and the treatment was completed with once-daily oral administration for four days after the surgery.

[0146] Evaluation of the effectiveness of NP-3: ■Single dose: PDL miniature pigs were administered NP-4 (1 g) or NP-3 (1 g; 1365 U) via an intraduodenal catheter, followed by administration of olive oil (14 g) via the same catheter. Following the administration of olive oil, 5 mL of blood was collected by EDTA at 0, 0.0833, 0.25, 0.5, 1, 2, 3, 4, and 6 hours.

[0147] Blood samples were centrifuged (10 minutes, 4°C, 2000×g), and at least 500uL of plasma was transferred to an Eppendorf tube and stored at -80°C until triglyceride content analysis.

[0148] ■Chronic administration: PDL miniature pigs were administered NP-4 (1 g) or NP-3 (1 g; 1365 U) twice daily via a duodenal catheter, followed by a diet for 10 days.

[0149] Fecal samples from individual animals were collected over a 72-hour period before treatment (days -3, -2, and -1) and at the end of treatment (days 8, 9, and 10). Fecal samples were stored at -20°C until analysis.

[0150] Measurement of plasma triglycerides in miniature pigs: The quantification of triglycerides in miniature pig plasma was evaluated using the Konelab system according to the supplier's specifications.

[0151] Measurement of fat content in the feces of miniature pigs Fecal fat content was quantified by near-infrared spectroscopy (NIR) using an Impana FT 9700 (Perkin Elmer). For NIR (700-2500 nm) analysis, stool samples were weighed, homogenized, and placed in open glass petri dishes. Measurements were taken at three different spots for each sample. The results were expressed as the average fat concentration (grams of fat per 100 g of wet stool) from the three spot samples.

[0152] Cell culture: In all experiments, cells were cultured at 37°C and 5% CO2.

[0153] Caco2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) cells were cultured in DMEM supplemented with 10% thermo-inactivated fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids, and 100 U / mL penicillin / streptomycin.

[0154] For the development of an intestinal barrier model, a Transwell PET insert (pore size 1 μm) was used to collect 2.6 × 10⁶ of material. 5 cells / cm 2Cells were seeded at the following density. All cell models were used in the experiment on day 21. Caco-2 cells and HT-29-MTX-E12 cells were used in co-culture at a ratio of 75% to 25%.

[0155] Transepithelial electrical resistance Cell barrier integrity was evaluated by measuring transepithelial electrical resistance (TEER) using the CellZscope system (NanoAnalytics). After refreshing the cell medium and treating it with nanoparticles, TEER was automatically measured every 15 minutes for up to 24 hours in the range of 1 Hz to 100,000 Hz.

[0156] Confocal microscope Cells on the insert were washed with phosphate-buffered saline and fixed with 4% buffered formalin. The fixed cells were permeabilized in PBS solution containing 1% Triton-X 100 (reference). Specific binding was blocked using PBS solution containing 3% bovine serum albumin (BSA). The cells were incubated with primary antibody against zonula occludens 1 (ZO-1; 1:100) at RT for 2 hours. After washing, the cells were incubated with the corresponding fluorescently labeled secondary antibody (goat anti-rabbit IgG conjugated to Alexa Fluor 488 (1:1000)) at RT in the dark for 2 hours. For imaging, the membrane was cut from the insert and placed between two coverslips loaded with mounting medium containing DAPI, and imaged using a confocal laser scanning microscope (FluoView, FV3000, Olympus, Tokyo, Japan).

[0157] result: Example 1: In vivo distribution of chitosan-functionalized nanoparticles in miniature pigs To evaluate the advantage of chitosan-functionalized nanoparticles in specifically retaining in the digestive tract of miniature pigs via mucous binding, unfunctionalized radiolabeled nanoparticles (NP-2) and chitosan-functionalized radiolabeled silica nanoparticles (NP-1) were administered endoscopically into the duodenum of miniature pigs. SPECT / CT images were acquired at different time points (15 minutes to 24 hours), and the in vivo distribution of nanoparticles in the digestive tract was analyzed using a three-compartment analysis including the small intestine, colon, and rectal portion of the colon. Quantification of NP-2 and NP-1 in the small intestine is shown in Figure 2A. Comparing the in vivo distribution data obtained from SPECT / CT of animals administered NP-1 and NP-2, it was shown that NP-1 remained in the small intestine 2.7 times longer than the unfunctionalized nanoparticles (AUC: 170.7 vs. 460.9; NP-2 vs. NP-1, respectively) (Figure 2B). These results demonstrate that NP-1 is specifically retained in the digestive tract in vivo. In summary, these in vivo data suggest that surface functionalization of chitosan-shielded nanoparticles may enable specific targeting of intestinal mucus, potentially allowing the nanoparticles to temporarily implant in the intestinal wall.

[0158] Example 2: Biocatalytic activity of NP-3 and NP-5 Exocrine pancreatic insufficiency is a condition in which the exocrine function of the pancreas is impaired, resulting in a deficiency in its ability to effectively deliver digestive enzymes to the duodenum. The standard medical treatment for clinical symptoms and malabsorption is oral pancreatic enzyme replacement therapy (PERT). Currently approved treatments consist of porcine-derived pancreatic enzyme products (lipase, amylase, and protease).

[0159] NP-3 and NP-5 were evaluated for their enzymatic activity. The lipase and amylase enzymatic activity of NP-3 is shown in Figures 3A and 3C. The protease enzymatic activity of NP-5 is shown in Figure 3B.

[0160] These data demonstrate the ability to immobilize multiple enzymes onto nanoparticles while maintaining the complete activity of each enzyme. Verification of these biocatalytic activities in nanoparticles demonstrates the potential for applying strategies for enzyme immobilization and protection to restore digestive function, which may be useful for therapeutic purposes.

[0161] Example 3: In vivo activity of NP-3 in PDL rats Considering the importance of lipid digestion for patients with exocrine pancreatic insufficiency (EPI), we evaluated the validity of NP-3 by focusing on its in vivo lipid digestive capacity using PDL-rats as the first animal model. NP-3 or NP-4 was administered intraduodenally to PDL-rats before enteral nutrition administration of triolein (hereinafter referred to as the administration sequence). After the single-dose administration sequence, PDL-rats administered with NP-3 (active nanoparticles) showed increased plasma TG compared to PDL-rats administered with inactive nanoparticles (NP-4) (Figure 4). This result demonstrates NP-3's ability to restore lipase digestive function in an EPI animal model.

[0162] Example 4: In vivo activity of NP-3 in PDL miniature pigs From an anatomical and physiological standpoint, it is widely recognized that pig models share a high degree of similarity with the human gastrointestinal tract. Therefore, to evaluate the efficacy of NP-3, PDL miniature pigs were generated. As previously described in the PDL rat model, in the initial validation of NP-3, PDL miniature pigs were administered a single dose of nanoparticles followed by olive oil. Compared to healthy miniature pigs, the increase in plasma TG levels in PDL miniature pigs administered NP-3 showed similar dynamics, reaching a peak level 3 hours after feeding (Figure 5A). The area under the curve (AUC) calculation demonstrates an extremely remarkable digestibility of 62% for NP-3 compared to healthy miniature pigs (Figure 5B). These results confirm NP-3's ability to restore lipase digestive function in EPI animal models.

[0163] Example 5: In vivo therapeutic efficacy of NP-3 in PDL miniature pigs To further demonstrate NP-3's ability to restore lipase digestion, PDL miniature pigs fed a fat-rich diet were administered NP-3 daily for 10 days. Since the primary endpoint of current treatments for EPI patients is the quantification of fat absorption, the benefits of NP-3 were evaluated by measuring unabsorbed fat excreted in the feces. Comparison of fecal fat content between healthy and untreated PDL miniature pigs showed that the EPI animal model exhibited a buildup of fat in the feces reflecting a lack of lipase activity (3.42g vs. 5.55g per 100g of feces for healthy and untreated PDL miniature pigs, respectively) (Figure 6A). Importantly, PDL miniature pigs treated with NP-3 had reduced fecal fat content compared to untreated PDL miniature pigs (Figure 6A). Indeed, PDL miniature pigs administered NP-3 daily were able to digest 53.4% ​​of their ingested fat (Figure 6B). These results clearly demonstrate the therapeutic efficacy of NP-3 over pancreatic enzyme replacement therapy.

[0164] Enzyme replacement therapy is available to treat clinical symptoms and malabsorption. A minimum dose of 40,000–50,000 units of PERT-lipase is recommended with each main meal, and half that amount is recommended for snacks. Such doses can be a burden for patients due to the heavy tablets. However, this treatment is not efficient and has several limitations (persistence of symptoms, short duration of intraluminal activity of lipase, potential intolerance due to large enzyme loads, and gastrointestinal damage due to large protease loads).

[0165] The efficacy of NP-3 was compared with that of free pancreatin. The amount of lipase activity administered with NP-3 was calculated based on fat intake (1300 U per dose per 14 g of olive oil), while the amount of lipase activity administered with pancreatin followed standard treatment (40000 U per dose). Surprisingly, administration of NP-3 at a volume of 1300 U twice daily significantly improved digestive efficiency by 25% compared to standard treatment with pancreatin administered at a much higher dose of 40000 U twice daily. In fact, 80000 U of pancreatin per day promoted 43.6% fat digestion, while 2600 U of NP-3 per day achieved 53.4% ​​fat digestion (Figure 6B). NP-3 consists of nanoparticles functionalized with mucosal adhesive components that enable interaction between the nanoparticles and intestinal mucus (PCT / EP2023 / 051194). As a result, NP-3 temporarily engrafts, and its lipase activity in the intestinal wall is sustained. In addition, immobilizing and protecting the lipase on nanoparticles protects the enzyme from external stress (International Publication No. 2022 / 223699) and stabilizes its activity. In short, this series of data demonstrates the added value of NP-3 for lipase digestion and highlights the potential of NP-3 as a treatment for EPI patients.

[0166] Example 6: In vitro biocompatibility of NP-5 Currently, patients with exocrine pancreatic insufficiency are taking enormous amounts of pancreatin (10-20 tablets per day), resulting in a daily intake of large amounts of proteases. Despite their digestive role, gastrointestinal proteases contribute to gut homeostasis. An imbalance in protease levels can lead to gastrointestinal complications (Vergnolle N et al., Gut 2016;65:1215-1224).

[0167] To evaluate the safety of NP-5, the inventors focused on maintaining intestinal barrier integrity in the presence of nanoparticles and compared it with pancreatin (Figure 7A). Transepithelial electrical resistance (TEER) measurements across a monolayer of Caco2-HT29-MTX-E12 cells showed that intestinal epithelial barrier integrity was maintained even after 24 hours of contact with NP-5, while treatment with pancreatin resulted in a dose-dependent loss of barrier integrity. To correlate TEER measurements with the morphological characteristics of the cell monolayer, the tight junction protein Zonula-Occludens-1 (ZO-1) was evaluated by confocal microscopy (Figure 7B). When cultured in the presence of NP-5, ZO-1 staining showed a continuous ring-like appearance at the cell boundaries, while in the presence of pancreatin, ZO-1 staining showed a diffused, punctate protein distribution around intestinal cells. These morphological data confirmed the safety of NP-5 for gastrointestinal application. Furthermore, these data highlight the added value of the inventors' technology compared to pancreatic enzyme replacement therapy.

[0168] Example 7: Improvement of enzyme stability by covalent bonding to a protective layer In the first experiment, nanoparticles NP-3(1) were generated under buffered conditions, followed by washing after each chemical step (i.e., glutaraldehyde removal before layer growth). In the second experiment, nanoparticles NP-3 were generated under unbuffered conditions without an intermediate washing step (i.e., unreacted glutaraldehyde was still present in the reaction mixture during layer growth).

[0169] To determine the pancreatin immobilization yield on NP-3(1) and its surface, protein quantification was performed on the reaction supernatant. Surprisingly, the results showed that enzyme immobilization yield doubled under conditions where glutaraldehyde was maintained (NP-3) (Figure 8A), and consequently, the enzyme load per dry weight of SNP doubled compared to buffer conditions where glutaraldehyde was removed by the washing step (NP-3(1)) (Figure 8B).

[0170] The lipase biocatalytic activity of NP-3(1) and pancreatin immobilized and protected on NP-3 was evaluated. Even more surprising than the increase in enzyme immobilization load while maintaining the presence of glutaraldehyde, the specific activity of the nanoparticles increased 11-fold compared to buffered conditions in which glutaraldehyde was removed by a washing step (Figure 8C). This astonishing 11-fold increase in nanoparticle specific activity is accompanied by an extremely astonishing 7-fold increase in the pancreatin specific activity (units per gram of pancreatin) of the enzyme protected in the presence of glutaraldehyde, compared to buffered conditions in which glutaraldehyde was removed by a washing step (Figure 8D). In summary, covalent bonding of a protective layer to the enzyme surface unexpectedly improves its load, stability, and enzyme specific activity compared to the enzyme protected by an organosilica layer via electrostatic interactions alone.

Claims

1. A composition comprising a solid carrier, an open-structured lipase or fragment thereof immobilized on the surface of the solid carrier, a protease or fragment thereof immobilized on the surface of the solid carrier, an amylase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, a protective layer that protects the lipase or fragment thereof, the protease or fragment thereof, and the amylase or fragment thereof by embedding them, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group.

2. The composition according to claim 1, wherein the substance that interacts with the lid domain of lipase or a fragment thereof is selected from the group consisting of colipase or a fragment thereof, colipase-mimicking peptides, and amphiphilic molecules.

3. The composition according to claim 1 or 2, wherein the substance specifically interacts with the lid domain of the lipase or fragment thereof such that the lipase or fragment thereof transitions to an open structure and / or maintains an open structure.

4. The composition according to any one of claims 1 to 3, wherein about 100% of the lipase or fragment thereof immobilized on the surface of the solid carrier is in an open structure.

5. The composition according to any one of claims 1 to 4, wherein the polymer comprising repeating units, each having at least one amino group and / or at least one thiol group, is a polyglucosamine selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan, and dermatan or derivatives thereof.

6. The composition according to any one of claims 1 to 4, wherein the polymer comprising repeating units, each each having at least one amino group and / or at least one thiol group, is chitosan or a derivative thereof.

7. The composition according to any one of claims 1 to 6, wherein the functional component is immobilized on the surface of the protective layer by non-covalent or covalent bonds.

8. A composition according to any one of claims 1 to 7, comprising a solid carrier, pancreatin comprising lipase or fragment thereof in an open structure, protease or fragment thereof, and amylase or fragment thereof, wherein the lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof are immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, a protective layer that protects the lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof by embedding them, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each comprising at least one amino group and / or at least one thiol group.

9. The composition according to any one of claims 1 to 8, wherein a solid carrier is embedded in the protective layer, lipase or fragments thereof immobilized on the surface of the solid carrier are embedded, protease or fragments thereof immobilized on the surface of the solid carrier are embedded, and amylase or fragments thereof immobilized on the surface of the solid carrier are embedded.

10. The composition according to any one of claims 1 to 9, wherein the functional component immobilized on the surface of the protective layer is not embedded by the protective layer.

11. A composition according to any one of claims 1 to 10 for use as a pharmaceutical.

12. A composition according to any one of claims 1 to 10, for use in a method of enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, or for use in a method for the prevention, delay of progression, or treatment of pancreatic exocrine insufficiency (EPI).

13. A method for producing a composition comprising a solid carrier, an open-structured lipase or fragment thereof immobilized on the surface of the solid carrier, a protease or fragment thereof immobilized on the surface of the solid carrier, an amylase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, a protective layer that protects the lipase or fragment thereof, the protease or fragment thereof, and the amylase or fragment thereof by embedding them, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, and the method is (a) A step of providing a solid carrier, (b) A step of providing lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof. (c) A step of providing a substance that interacts with the lid domain of a lipase or a fragment thereof. (d) A step of interacting the lipase or fragment thereof from (b) with the substance from (c), (e) A step of immobilizing lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof on a solid carrier. (f) A step of forming a protective layer on the surface of a solid carrier to protect the lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof immobilized on the solid carrier, (g) A step of immobilizing a functional component on the surface of a protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer containing repeating units, and each repeating unit contains at least one amino group and / or at least one thiol group. A method that includes this.

14. A method for producing a composition comprising a solid carrier, an open-structured lipase or fragment thereof immobilized on the surface of the solid carrier, a protease or fragment thereof immobilized on the surface of the solid carrier, an amylase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, a protective layer that protects the lipase or fragment thereof, the protease or fragment thereof, and the amylase or fragment thereof by embedding them, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, and the method is (a) A step of providing a solid carrier, (b) A step of providing lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof. (c) A step of immobilizing lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof onto a solid carrier. (d) A step of providing a substance that interacts with the lid domain of a lipase or a fragment thereof, (e) A step of interacting the lipase or fragment thereof from (b) with the substance from (d), (f) A step of forming a protective layer on the surface of a solid carrier to protect the lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof immobilized on the solid carrier, (g) A step of immobilizing a functional component on the surface of a protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer containing repeating units, and each repeating unit contains at least one amino group and / or at least one thiol group. A method that includes this.

15. The method according to claim 14 or 15, wherein in step b), pancreatin comprising lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof is provided.

16. i) a linker is added to a solid carrier provided in step (a), ii) lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof provided in step (b) are added to the solid carrier and the linker, and the linker links the solid carrier to the lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof in step (e), the method according to claim 13.

17. The method according to claim 16, wherein in step (e), a linker that has not linked the solid carrier with lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof is present while in step (f) a protective layer is formed on the surface of the solid carrier.

18. The method according to claim 16, wherein there is no washing step between (i) adding a linker to the solid carrier provided in step (a) and (ii) adding lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof to the solid carrier and the linker.

19. The method according to any one of claims 13 to 18, wherein there is no washing step between any of steps (a) to (f).

20. The method according to any one of claims 13 to 19, wherein in step (e), a linker or a portion thereof that did not link the solid carrier with lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof covalently bonds the protective layer with lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof in step (f).

21. i) a linker is added to a solid carrier provided in step (a), and ii) lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, provided in step (b), are added to the solid carrier and the linker, and the linker links the solid carrier to the lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof in step (c), the method according to claim 14.

22. The method according to claim 21, wherein in step (c), a linker that does not link the solid carrier with lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof is present while in step (f) a protective layer is formed on the surface of the solid carrier.

23. The method according to claim 21, wherein there is no washing step between (i) adding a linker to the solid carrier provided in step (a) and (ii) adding lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof to the solid carrier and the linker.

24. The method according to any one of claims 14 and 21 to 23, wherein there is no washing step between any of steps (a) to (f).

25. The method according to any one of claims 14 and 21 to 24, wherein in step (c), a linker or a portion thereof that did not link the solid carrier with lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof covalently bonds the protective layer with lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof in step (f).

26. The linker is glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2) The method according to any one of claims 13 to 25, selected from the group consisting of HCl, DST (disuccinimidyl tartrate), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane).

27. The method according to any one of claims 13 to 25, wherein the linker is glutaraldehyde.

28. A composition comprising a solid carrier, an open-structured lipase or fragment thereof immobilized on the surface of the solid carrier, a protease or fragment thereof immobilized on the surface of the solid carrier, an amylase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, a protective layer that protects the lipase or fragment thereof, the protease or fragment thereof, and the amylase or fragment thereof by embedding them, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units each containing at least one amino group and / or at least one thiol group, the composition obtained by the method of any one of claims 17 to 20 or 22 to 25.

Images