Functionalized disaccharide-degrading enzyme composition

Abstract

The present invention relates to a method for producing a composition comprising a solid carrier, a proteolytic enzyme or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the proteolytic enzyme 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 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.

Description

Technical Field

[0001] The present invention relates to a composition comprising a solid support, a disaccharide-degrading enzyme or a fragment thereof immobilized on the surface of the solid support, a protective layer that protects the disaccharide-degrading enzyme or a fragment thereof by embedding the disaccharide-degrading enzyme 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.

Background Art

[0002] Disaccharide-degrading enzymes are usually cleaved into monosaccharides by disaccharide-degrading enzymes located on the brush border of small intestinal enterocytes. Undigested disaccharide-degrading enzymes create an osmotic load that draws water and electrolytes into the intestine, causing watery diarrhea. Bacterial fermentation of carbohydrates in the colon generates gas (hydrogen, carbon dioxide, and methane), resulting in excessive flatulence, bloating and distension, and abdominal pain. Diseases associated with disaccharide-degrading enzyme maldigestion are, for example, lactase deficiency and congenital sucrase-isomaltase deficiency (CSID). In the case of lactase deficiency, lactose malabsorption is due to an imbalance between the amount of lactose ingested and the ability of lactase to hydrolyze the disaccharide. It presents as lactose intolerance, which is one or more clinical syndromes of abdominal pain, diarrhea, nausea, flatulence, and / or bloating after ingestion of lactose or lactose-containing food substances. The most common cause is primary (hereditary) lactase deficiency, which results from a relative or absolute lack of lactase that occurs in childhood in different ethnic populations at various ages. Secondary (acquired) lactase deficiency is due to damage to the small intestine, such as acute gastroenteritis, persistent diarrhea, small intestinal overgrowth, cancer chemotherapy, or other causes of damage to the small intestinal mucosa. Congenital lactase deficiency is a very rare autosomal recessive enzyme deficiency that prevents the expression of lactase from birth, requiring complete lactose avoidance.

[0003] In addition to avoiding lactose, which can cause serious health problems, enzyme replacement therapy with exogenous microbial lactose (β-galactosidase derived from Aspergillus orze) added before or to dairy meals is a possible strategy for primary and secondary lactase deficiencies. Results regarding precise efficacy rates are inconsistent.

[0004] CSID is a multifaceted malabsorption disorder of the small intestine characterized by autosomal recessive mutations in the sucrase-isomaltase gene, resulting in complete or near-complete deficiency of sucrose activity and varying degrees of reduced isomaltase activity. The deficiency of this enzyme complex prevents adequate absorption of nutrients from ingested starch and sucrose, leading to clinically apparent symptoms of diarrhea, abdominal pain, and bloating, particularly in children, and contributing to malnutrition. In addition to lifelong dietary therapy, the only effective treatment option is enzyme substitution with sacrosidase, currently approved in the US by the FDA (Sucraid®) but not in Europe. Therefore, there is a need to provide effective treatments for disorders associated with disaccharide-degrading enzyme dysdigestion. [Overview of the project]

[0005] The present invention provides a composition comprising a solid carrier, a disaccharidase or fragment thereof immobilized on the surface of the solid carrier, a protective layer that protects the disaccharidase 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 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, a disaccharidase or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the disaccharidase 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 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, (b) A step of immobilizing a disaccharide-degrading enzyme or a fragment thereof onto a solid carrier. (c) A step of forming a protective layer on the surface of a solid carrier in order to protect the disaccharogenic enzyme or fragment thereof immobilized on the solid carrier. (d) 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 inventors of this application have surprisingly found that the compositions provided by the present invention, when applied therapeutically to the digestion of disaccharidases, exhibit unexpectedly high activity, low cytotoxicity, and do not disrupt the intestinal barrier when localized in the gastrointestinal tract, and therefore are extremely promising for therapeutic use, particularly in the treatment of lactase deficiency, sucrase-isomaltase deficiency, and disaccharidase intolerance. [Brief explanation of the drawing]

[0008] [Figure 1] A schematic representation of a method for producing the composition of the present invention is shown: a) immobilizing a disaccharidase or a fragment thereof (indicated as "protein") onto a solid carrier; b) and c) growing a protective layer around the immobilized disaccharidase or fragment thereof, embedding the immobilized disaccharidase or fragment thereof; and d) immobilizing a functional component onto the surface of the protective layer. [Figure 2]This demonstrates the added value of the covalent bond between the enzyme surface and the protective layer. (A) Quantification of proteins performed on NP-2(1), NP-2(2), and the reaction supernatant of NP-2. (B) Lactase loading per dry weight of SNPs. [Figure 3] The disaccharide-degrading enzyme activity of nanoparticles is shown. (A) Lactase activity of NP-2 after exposure to lactose (unit: U / g). (B) Invertase activity of NP-3 after exposure to sucrose (unit: U / mg). (C) Isomaltase activity of NP-4 after exposure to isomaltose (unit: U / g). (D) Isomaltase and invertase activity of NP-5 after exposure to isomaltose and sucrose, respectively (unit: U / g). [Figure 4] This shows the in vitro biocompatibility of representative nanoparticle models for the intestinal barrier. (A) In vitro evaluation of intestinal barrier integrity by measuring transepithelial electrical resistance (TEER). Differentiated Caco-2 / HT29-MTX-E12 cocultures were exposed to NP-1 (0.5 mg / mL and 1 mg / mL) for 16 hours. TEER data were normalized to a control point consisting of equilibrium values ​​before the addition of NP-1 (defined as control), and set to 100%. The graph shows the time course profile of averaged, normalized TEER data over 16 hours. The dashed line represents the untreated state. (B) In vitro evaluation of inflammatory effects on the intestinal epithelial barrier. Differentiated Caco2-HT29-MTX-E12 and M0-differentiated THP-1 were cocultured and exposed to NP-1 (1 mg / mL) at 37°C for 16 hours. Lipopolysaccharide (LPS) (50 ug / mL and 100 ug / mL) was used as a positive control to induce an inflammatory response. TEER data were normalized with the equilibrium value before NP-1 or LPS addition set to 100%. The graph shows the time-series profile of averaged, normalized TEER data over 16 hours in the presence of NP-1. [Figure 5]This shows the differences in cecal size in rats. Wistar rats were administered NP-2, NP-1, or Vehicle intraduodenally daily for 15 days, followed immediately by enteral nutrition of lactose. Cecal size was evaluated at the end of the study. (A) Photograph of the rat's gastrointestinal tract. The circle indicates the cecum. (B) MRI image of the rat's gastrointestinal tract. The arrow indicates the cecum. (C) Histogram shows the cecal size evaluated by MRI imaging (unit: cm3). *p<0.05, **p<0.01, one-way ANOVA test. [Figure 6] This study demonstrates the in vitro digestion of sucrose in a model of the intestinal barrier. Differentiated Caco-2 / HT29-MTX-E12 cocultures were exposed to different amounts of NP-3 (0.5 mU or 1 mU) in the presence of sucrose at the leading edge of the barrier for 4 hours. Sucrose hydrolysis was evaluated by quantifying glucose at the basal edge of the barrier. The graph shows the time-course profile of glucose accumulation over 4 hours. [Figure 7] The absorbance of NP-2, NP-2(1), and NP-2(2) at a wavelength of 460 nm is shown. [Modes for carrying out the invention]

[0009] The present invention relates to a composition comprising a solid carrier, a disaccharidase or fragment thereof immobilized on the surface of the solid carrier, a protective layer that protects the disaccharidase 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 containing at least one amino group and / or at least one thiol group.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] The singular forms "a," "an," and "the" include references to the plural form unless otherwise explicitly stated.

[0014] The term "approximately" refers to a range of values ​​that is ±10% of a given value. For example, the expression "approximately 200" includes ±10% of 200, i.e., from 180 to 220.

[0015] As used herein, the term “solid carrier” usually refers to particles. Preferably, the solid carrier is monodisperse or polydisperse particles, more preferably monodisperse particles. The solid carrier typically includes 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 of the solid carrier is typically 1 nm to 1000 μm, preferably 10 nm to 100 μm, and particularly about 50 nm.

[0016] The terms “linker” or “crosslinker,” as used synonymously herein, refer to any linking reagent containing a group capable of binding to a specific functional group (e.g., primary amines, sulfhydryls, etc.). Linkers as used in the present invention typically link the surface of a solid support to a disaccharidase. For example, a linker can be immobilized on the surface of a solid support, such as a silica surface as the support material, and then the disaccharidase can be bound to an unoccupied binding site of the linker. Alternatively, the linker can first bind to the disaccharidase, and then the linker bound to the disaccharidase can bind its unoccupied binding site to the solid support. Various types of linkers are known in the art, including, but not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, peptide linkers, polyether linkers, and linkers known in the art as tags.

[0017] As used herein, the term “protective layer” refers to a layer for protecting the functional properties of a disaccharidase or fragment 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 the immobilized disaccharidase, usually by covalent bonds and usually by non-covalent bonds. The protective layer is formed on the surface of a solid carrier to protect the disaccharidase or fragment immobilized on the solid carrier. The protective layer is typically a homogeneous layer in which at least 50%, preferably at least 70%, and more preferably at least 90% of the disaccharidase or fragment is embedded.

[0018] The term "disaccharidase or a fragment thereof" includes naturally occurring disaccharidases or fragments thereof, as well as artificially engineered disaccharidases or fragments thereof. Disaccharidases are glycoside hydrolases, which are enzymes that break down certain sugars called disaccharides into simpler sugars called monosaccharides. In the human body, most disaccharidases are produced in an area called the brush border of the small intestine wall. Disaccharidases include, for example, lactase, maltase, isomaltase, trehalase, sucrase (also called invertase). Artificially engineered disaccharidases or fragments thereof are, for example, variants of disaccharidases or functionally active fragments. Thus, in this specification, the terms "fragment of a disaccharidase", "fragment thereof" related to a disaccharidase, and "functionally active fragment of a disaccharidase" are used synonymously. "Variant or a functionally active fragment thereof" related to the disaccharidase of the present invention means that the fragment or variant (such as an analog, derivative or mutant) can perform the same physiological function as the disaccharidase. Such variants include naturally occurring allelic variants and non-naturally occurring variants. One or more additions, deletions, substitutions and derivatizations of amino acids are contemplated as long as the modification does not result in loss of the functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least about 80% sequence identity, 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 with the relevant part of the disaccharidase. Fragments of disaccharidases as defined herein usually have the same functional properties as the disaccharidase from which they are derived. Fragments of disaccharidases usually contain from 100 to 1000 amino acids, preferably from 300 to 800 amino acids, more preferably from 500 to 700 amino acids.

[0019] As used herein, the term "partially embedded disaccharidase" means that the disaccharidase is not completely covered by the protective layer, and thus the disaccharidase is not completely embedded in the protective layer. In one embodiment, less than 50% of the target disaccharidase is covered by the protective layer, but typically at least 70%, more than that, is covered, thereby improving the protection of the disaccharidase. In a preferred embodiment, at least 70%, more preferably at least 80%, still more preferably at least 90%, and most preferably at least 95% of the target disaccharidase is covered by the protective layer. In another preferred embodiment, from about 70% to about 95%, more preferably from about 80% to about 95%, still more preferably from about 90% to about 95%, and most preferably from about 90% to about 95, 96, 97, 98 or 99% of the disaccharidase is covered by the protective layer. In a particularly preferred embodiment, about 70%, especially about 80%, more particularly about 90%, and most particularly about 95% of the target disaccharidase is covered by the protective layer. In an even more preferred embodiment, about 70%, especially about 80%, more particularly about 90%, and most particularly about 95% of the target disaccharidase is covered by the protective layer, and the active site is not covered.

[0020] As used herein, the term "completely embedded disaccharidase" means that the target disaccharidase according to the present invention is completely, i.e., 100%, covered by the protective layer, i.e., the active site is also covered.

[0021] As used herein, the term "at least partially embedded disaccharidase" means that the disaccharidase is at least partially embedded and may be completely embedded by the protective layer. Thus, "at least partially embedded disaccharidase" means that the protective layer covers from about 30% to 100%, preferably from about 50% to about 100%, more preferably from about 80% to about 100%, still more preferably from about 90% to about 100%, and most preferably from about 95% to about 100% of the disaccharidase or a fragment thereof, and the active site is preferably covered.

[0022] 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.

[0023] 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.

[0024] 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. A preferred polymer comprises a large number of repeating units (monomers), each of which contains one thiol group.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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 “derivatives” in relation to chitosan includes 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.

[0030] In a first aspect, the present invention provides a composition comprising a solid carrier, a disaccharidase or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the disaccharidase 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 containing at least one amino group and / or thiol group.

[0031] A disaccharidase or fragment thereof can be immobilized on the surface of a solid carrier 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, the disaccharidase or fragment thereof is immobilized on the surface of a solid carrier by covalent bonds or by linker-mediated covalent bonds.

[0032] Solutions of disaccharidases or their fragments typically contain the protein or its fragment in a buffer. Commonly usable buffers include 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 disaccharidases or their fragments can be prepared, for example, by dissolving the disaccharidase or its fragment in water to reconstitute a stock buffer for the disaccharidase or its fragment.

[0033] 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, the solid support comprising at least 15%, preferably at least 20%, particularly 15% to 30%, and more specifically 20% to 30%, of immobilized disaccharidase or fragment thereof per dry weight of the solid support.

[0034] 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.

[0035] Solid carriers are usually provided in suspension. The suspension of solid carriers can be done, for example, in water, a buffer, or 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.

[0036] In one embodiment, the surface of a solid support is modified to introduce molecules or functional chemical groups as anchor points, i.e., anchor points for a disaccharide or for a linker connecting a disaccharide 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 a modified support can form amide bonds between a disaccharide and an amine group on the surface of the support material, or between a linker and an amine group on the surface of the support material. In one embodiment, the molecules or functional chemical groups introduced as anchor points are uniformly distributed on the surface of the solid support.

[0037] 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.

[0038] In one embodiment, the disaccharidase or a fragment thereof is partially embedded by the protective layer. In a preferred embodiment, the disaccharidase or a fragment thereof is at least partially embedded by the protective layer. In a more preferred embodiment, the disaccharidase or a fragment thereof is completely embedded by the protective layer.

[0039] In one embodiment, a solid carrier is embedded in the protective layer, and a disaccharidase or fragment thereof immobilized on the surface of the solid carrier is embedded within it. In another embodiment, a functional component immobilized on the surface of the protective layer is not embedded by the protective layer. Preferably, the solid carrier is completely embedded in the protective layer, and the disaccharidase or fragment thereof immobilized on the surface of the solid carrier is completely embedded within it. More preferably, the solid carrier is completely embedded in the protective layer, and the disaccharidase or fragment thereof immobilized on the surface of the solid carrier is completely embedded within it, while a functional component immobilized on the surface of the protective layer is not embedded by the protective layer. When the solid carrier is completely embedded in the protective layer, and the disaccharidase or fragment thereof immobilized on the surface of the solid carrier is completely embedded within it, the disaccharidase or fragment thereof is completely, i.e., 100%, covered by the protective layer, i.e., the active site is also covered, and the solid carrier is completely, i.e., 100%, covered by the protective layer.

[0040] In preferred embodiments, the disaccharide-degrading enzyme or fragment thereof is selected from the group consisting of lactase or fragment thereof, maltase or fragment thereof, isomaltase or fragment thereof, trehalase or fragment thereof, and invertase or fragment thereof, or mixtures thereof, and more preferably from the group consisting of lactase or fragment thereof, isomaltase or fragment thereof, and invertase or fragment thereof, or mixtures thereof. Thus, the composition also comprises two or more, for example, two, three, or four different disaccharide-degrading enzymes or fragment thereof, immobilized and embedded on the surface of a solid carrier. When two or more disaccharide-degrading enzymes or fragment thereof are used, preferably two different disaccharide-degrading enzymes, more preferably invertase and isomaltase, are used. Thus, in particular embodiments, a mixture of isomaltase or fragment thereof and invertase or fragment thereof is immobilized on the surface of the solid carrier of the composition of the present invention.

[0041] In certain preferred embodiments, the disaccharide-degrading enzyme or fragment thereof is selected from the group consisting of lactase or fragment thereof, invertase or fragment thereof, and mixtures thereof. Most preferred is invertase or fragment thereof.

[0042] 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.

[0043] 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 structure of the disaccharide-degrading enzyme to adapt the affinity of the protective layer according to the optimal and / or desired parameters. As building blocks for the protective layer, structural building blocks and protective building blocks are typically used to construct the protective layer. 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 retain an organic moiety that is well-suited to interacting with disaccharide-degrading enzymes. Preferred structural building blocks are tetravalent silanes, particularly tetraalkoxysilanes. Preferred protective building blocks are trivalent silanes, particularly trialkoxysilanes. More preferred structural building blocks are mixtures of tetravalent and trivalent silanes, particularly mixtures of tetraalkoxysilanes and trialkoxysilanes.Further 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, propyltriethoxysilane, isobutyltriethoxysilane, n-octyltriethoxysilane, hydroxymethyltriethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, 3-aminopropyltriethoxysilane, ureidopropyltriethoxysilane, and (N-acetylglycyl)-3-aminopropyltriethoxysilane, or benzyltrimethoxysilane, propyltrimethoxysilane, and isobutyltrimethoxysilane. Selected from (isobutylimethoxysilane), n-octyltrimethoxysilane, hydroxylnetyltrimethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrimethoxysilane, aminopropyltrimethoxysilane, ureidopropyltriethoxysilane(N-acetylglycyl)-3-aminopropyltrimethoxysilane, or selected from benzyltrihydroxyethoxysilane, propyltrihydroxyethoxysilane, isobutyltrihydroxyethoxysilane, n-octyltrihydroxyethoxysilane, hydroxymethyltrihydroxyethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrihydroxyethoxysilane, aminopropyltrihydroxyethoxysilane, ureidopropyltrihydroxyethoxysilane(N-acetylglycyl)-3-aminopropyltrihydroxymethoxysilane.

[0044] Particularly preferred building blocks are TEOS as structural building blocks and APTES, BTES, and / or HTMEOS, preferably APTES, as protective building blocks. In particular, TEOS as structural building blocks and APTES as protective building blocks are used to construct protective layers.

[0045] The reaction time between the building blocks and the solid support depends on the length of the linker, if one is used, and the size of the disaccharide-degrading enzyme. The reaction typically takes place 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.

[0046] In a more preferred embodiment, the disaccharidase is immobilized on a solid carrier by introducing a molecule as an anchor point for the disaccharidase as described above, and by modifying the surface of the solid carrier at least partially by using a linker, preferably a crosslinker that binds to the anchor point and the disaccharidase.

[0047] In one embodiment, the molecules and / or linkers introduced as anchor points are uniformly distributed on the surface of the solid support.

[0048] 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, and activated sulfhydrils (e.g., sulfurhydryl-reactive 2-pyridyldithio).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.

[0049] After the protective layer is formed, the solid carrier containing the disaccharidase and 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 the buffer for a desired period of time. In a preferred embodiment, the solid carrier containing the disaccharidase and the protective layer are stored at a constant temperature of 2 to 25°C. In a more preferred embodiment, the solid carrier containing the disaccharidase and the protective layer are stored for 5 to 48 hours, preferably 10 to 30 hours. More preferably, the solid carrier containing the disaccharidase and the protective layer are stored at a constant temperature between 2 and 25°C, preferably at room temperature, for 10 to 30 hours.

[0050] In one embodiment, the functional component binds to the mucus.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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; polymerized silane containing an amino group, preferably polymerized APTES; and polymerized silane containing a thiol group, preferably polymerized MPTS. In a particular 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, dermatan or derivatives thereof, most specifically chitosan or a derivative thereof.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] In one embodiment, the functional component is immobilized on the surface of a 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 disaccharidase or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the disaccharidase 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.

[0064] The immobilization of functional components onto the surface of the protective layer is typically carried out by suspending a solid carrier supporting the disaccharide-degrading 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 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 lipids 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 fatty acid ethoxylates, 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 polysorbate, 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 a composition obtained is 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 disaccharide-degrading 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.

[0065] In a further embodiment, the present invention provides the aforementioned compositions for use as pharmaceuticals.

[0066] In further embodiments, the present invention provides compositions for use in methods for the prevention, slowing of progression, or treatment of lactase deficiency, sucrase-isomaltase deficiency, and / or disaccharide intolerance. In one embodiment, the present invention provides compositions for use in methods for the prevention, slowing of progression, or treatment of lactase deficiency or sucrase-isomaltase deficiency. Lactase deficiency includes primary (hereditary) lactase deficiency, secondary (acquired) lactase deficiency, and congenital lactase deficiency, preferably secondary lactase deficiency. Sucrase-isomaltase deficiency includes, and is preferred, congenital sucrase-isomaltase deficiency (CSID).

[0067] Furthermore, the use of the compositions described herein for the manufacture of pharmaceuticals for the prevention, delay of progression, or treatment of lactase deficiency, sucrase-isomaltase deficiency, and / or disaccharidase intolerance in subjects is provided. Furthermore, the use of the compositions described herein for the prevention, delay of progression, or treatment of lactase deficiency, sucrase-isomaltase deficiency, and / or disaccharidase intolerance in subjects is provided. Furthermore, a method for the prevention, delay of progression, or treatment of lactase deficiency, sucrase-isomaltase deficiency, and / or disaccharidase intolerance in subjects is provided, comprising administering a therapeutically effective amount of the compositions described herein to the subject.

[0068] 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.

[0069] Exemplary treatment regimens involve administration once daily, twice daily, three times daily, every other day, twice a week, or once a week. The composition, for example, the pharmaceutical composition of the present invention, is usually administered in multiple doses. The interval between doses can be, for example, less than one day, daily, every other day, twice a week, or weekly. The composition, for example, the pharmaceutical composition of the present invention, may be administered as a continuous, uninterrupted treatment. The composition, for example, the pharmaceutical composition of the present invention, may be given in a regimen in which the subject undergoes treatment cycles interrupted by drug-free or non-treatment periods. Thus, the composition, for example, the pharmaceutical composition of the present invention, may be administered according to the selected intervals above over a continuous period of one week or part thereof, two weeks, three weeks, four weeks, five weeks, or six weeks, and then suspended for a period of one week or part thereof, two weeks, three weeks, four weeks, five weeks, or six weeks.

[0070] The compositions of the present invention, such as pharmaceutical compositions, can be conveniently administered in unit dose form. The unit of enzyme activity ("U") can be expressed as the weight or mass of substrate hydrolyzed per unit time. The unit ("U") can be described in umol (or umol / min) of substrate converted per minute. In an exemplary therapeutic regimen, 500 U to 20,000 U of disaccharide-degrading enzyme contained in the composition, such as the pharmaceutical composition of the present invention, can be administered daily.

[0071] 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.

[0072] 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.

[0073] As used herein, “delay in progression” means extending the time until symptoms of treatment for, for example, lactase deficiency, sucrase-isomaltase deficiency, or disaccharide intolerance appear, or features associated with the treatment of, for example, lactase deficiency, sucrase-isomaltase deficiency, or disaccharide intolerance, or slowing the progression of the severity of symptoms of, for example, disaccharide intolerance. 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.

[0074] Preventive measures include prophylactic treatment. For preventive use, the pharmaceutical combination of the present invention is administered to subjects suspected of having or at risk of developing the above-mentioned diseases or disorders, for example, for the treatment of lactase deficiency, sucrase-isomaltase deficiency, or disaccharide intolerance. For therapeutic use, the pharmaceutical combination is administered to subjects, for example patients already suffering from the above-mentioned diseases or disorders, in an amount sufficient to cure or at least partially cessate the symptoms of the disease, for example, for the treatment of lactase deficiency, sucrase-isomaltase deficiency, or disaccharide intolerance. 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.

[0075] 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.

[0076] 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.

[0077] In a further embodiment, the present invention provides a method for producing a composition comprising the aforementioned composition, for example, a solid carrier, a disaccharidase or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the disaccharidase 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 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, (b) A step of immobilizing a disaccharide-degrading enzyme or a fragment thereof onto a solid carrier. (c) A step of forming a protective layer on the surface of a solid carrier in order to protect the disaccharogenic enzyme or fragment thereof immobilized on the solid carrier. (d) 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.

[0078] Step (a) is typically carried out by providing the solid carrier suspended in water, a nonionic surfactant, a buffer, or a mixture thereof, preferably suspended in water and / or a nonionic surfactant, more preferably suspended in water and / or a nonionic surfactant without a buffer in the suspension, even more preferably suspended in a mixture of water and a nonionic surfactant, particularly suspended in a mixture of water and a nonionic surfactant without a buffer in the suspension. The suspension can be stirred, for example, at 400 rpm and 20°C for 30 minutes. Immobilization of the disaccharidase onto the solid carrier in step b) of this method is typically carried out by adding a solution of the disaccharidase to the suspension of the solid carrier. Preferably, a linker connecting the solid carrier and the disaccharidase is added to the suspension of the solid carrier before adding the solution of the disaccharidase to the suspension of the solid carrier. In a preferred embodiment, immobilization of the disaccharidase onto the solid carrier is carried out by providing a suspension of the solid carrier and adding a solution of the disaccharidase, and the suspension to which the solution of the disaccharidase has been added is incubated so that the enzyme can bind to the surface of the solid carrier. In a more preferred embodiment, the immobilization of the disaccharase or fragment thereof onto a solid carrier in step b) 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 disaccharase or fragment thereof, preferably adding a solution of the disaccharase or fragment thereof to the solid carrier and linker, or to a suspension containing the solid carrier and linker, where the linker connects the solid carrier to the disaccharase or fragment thereof. In one embodiment, a protective layer building block, preferably a monomer of the protective layer building block, more preferably organosilane, even more preferably triethoxysilane, particularly APTES, is added to the solid carrier and linker, or to a suspension containing the solid carrier and linker, before adding a solution of the disaccharase or fragment thereof. In a preferred embodiment, the surface of the solid carrier is at least partially modified to improve the immobilization of the disaccharase to the solid carrier.In particular, the surface of the solid carrier is modified at least partially before the disaccharidase is immobilized. The surface of the solid carrier can be modified at least partially by adding molecules to the surface of the solid carrier as anchor points for the disaccharidase, as described above.

[0079] The suspension containing the solid carrier is typically incubated after each of the above addition steps so that the disaccharase or fragment thereof is linked to the solid carrier, preferably the surface of the solid carrier, and to the disaccharase or fragment thereof via a linker, preferably by covalent bond, thereby immobilizing the disaccharase or fragment thereof on the solid carrier, respectively, enabling reactions between, for example, the solid carrier and a molecule as an anchor point, the solid carrier and a linker, and the solid carrier containing the linker and the disaccharase or fragment thereof.

[0080] In one embodiment, in step (b), the disaccharase or fragment thereof is immobilized on a solid carrier by linking the solid carrier and the disaccharase or fragment thereof via a linker, preferably by linking the solid carrier and the disaccharase or fragment thereof via a linker, and the solid carrier is linked to the disaccharase or fragment thereof by covalent bonds between the linker and the solid carrier and between the linker and the disaccharase or fragment thereof. Preferably in step b), i) a linker is added to the solid carrier provided in step (a), and ii) the disaccharase or fragment thereof is added to the solid carrier and the linker, and the linker links the solid carrier and the disaccharase or fragment thereof. The linker used is as described above, and preferably connects the surface of the solid carrier and the disaccharase by covalent bonds. More preferably, the linker is added to the solid carrier in step (b) in a molar excess relative to the disaccharase or its fragment; preferably, the linker is added to the solid carrier in step (b) in a molar excess of 1 to 1000 times relative to the disaccharase or its fragment; more preferably, the linker is added to the solid carrier in step (b) in a molar excess of 2 to 300 times relative to the disaccharase or its fragment; even 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 disaccharase or its fragment, and in particular, the linker is added to the solid carrier in step (b) in a molar excess of 25 times relative to the disaccharase or its fragment.

[0081] In a preferred embodiment, linkers that did not connect the solid carrier to the disaccharase or its fragment in step (b) are present while a protective layer is formed on the surface of the solid carrier in step (c). In a more preferred embodiment, linkers, or a portion thereof, that did not connect the solid carrier to the disaccharase or its fragment in step (b) covalently bond the protective layer to the disaccharase or its fragment in step (c). In an even more preferred embodiment, linkers that did not connect the solid carrier to the disaccharase or its fragment in step (b) are not removed in step (b) or step (c), or between steps (b) and (c). In a particular embodiment, linkers that did not connect the solid carrier to the disaccharase or its fragment in step (b) are not removed in step (b) or step (c), or between steps (b) and (c), and linkers, or a portion thereof, that did not connect the solid carrier to the disaccharase or its fragment in step (b) covalently bond the protective layer to the disaccharase or its fragment in step (c). The amount of linker that does not link the solid carrier and the disaccharase or fragment thereof in step (b) 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 (b). In one embodiment, there is no washing step between (i) adding the linker to the solid carrier provided in step (a) and (ii) adding the disaccharase or fragment thereof to the solid carrier and linker. In one embodiment, there is no washing step between any of steps (a) to (c). In one embodiment, there is no washing step between (i) adding the linker to the solid carrier provided in step (a) and (ii) adding the disaccharase or fragment thereof to the solid carrier and linker, and there is no washing step between any of steps (a) to (c).

[0082] 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.

[0083] 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.

[0084] The formation of the protective layer in step (c) 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 (d) of this method is usually carried out as described above.

[0085] 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.

[0086] In one embodiment, approximately 30% to 100% of the disaccharide-degrading enzyme is embedded in the protective layer.

[0087] 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).

[0088] A preferred method of the present invention is a method for producing a composition comprising a solid carrier, a disaccharidase or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the disaccharidase 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 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 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 immobilizing a disaccharase or fragment thereof on a solid carrier, preferably, the surface of the solid carrier is at least partially modified before the disaccharase or fragment thereof is immobilized on the solid carrier, i) a linker is added to the suspension of the solid carrier, or i) a linker is added to the suspension of the solid carrier after at least partially modification of the surface of the solid carrier, and ii) a solution of the disaccharase or fragment thereof, preferably the disaccharase or fragment thereof, is added to the suspension of the solid carrier and the linker, the linker linking the solid carrier and the disaccharase or fragment thereof, (c) A step of forming a protective layer on the surface of a solid carrier in order to protect a disaccharase or fragment thereof immobilized on the solid carrier, wherein a linker or part thereof that did not connect the solid carrier and the disaccharase or fragment thereof in step (b) covalently bonds the protective layer to the disaccharase or fragment thereof. (d) 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.

[0089] Furthermore, a composition is provided comprising a solid carrier, a disaccharidase or fragment thereof immobilized on the surface of the solid carrier, a protective layer that protects the disaccharidase 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 repeating unit comprising at least one amino group and / or at least one thiol group, and the composition can be obtained by the present invention, particularly by the preferred method of the present invention described above. [Examples]

[0090] Materials and methods reagent: - 99% thraethyl orthosilicate (TEOS), (3-aminopropyl)triethoxysilane (APTES), ammonium hydroxide (ACS grade, 28-30%), ethanol (ACS grade, anhydrous), glutaraldehyde (grade I, 25% in water), polysorbate 80, acetic acid, lactase (USP standard), invertase, bovine serum albumin (BSA), invertase activity assay kit, lactose, lipopolysaccharide (LPS), and phorbol 12-myristo 13-acetate (PMA) were purchased from Sigma-Aldrich. BSA, lactase, and invertase were dissolved in water to reconstitute the stock buffer. - Chitosan 95 / 500 P was purchased from Heppe Medical Chitosan GmbH. 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). - THP-1 (human acute monocytic leukemia cell line) was purchased from LGC. - 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, DMEM, HEPES, sodium pyruvate, D-glucose, and β-mercaptoethanol were purchased from Gibco. - Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free, was purchased from Corning. - Altromin 1319 and AIN93G modified 200g polysaccharides for animal feed were purchased from Altromin International. - The catheter was purchased from Instech Laboratories. - I purchased the TB100 Holtex blood glucose meter kit from MediSafe. - Bifidobacterium adolescentis-derived oligo-α-1,6-glucosidase 13A, recombinant (isomaltase), was purchased from Creative Enzymes at a concentration of 1 mg / mL in 35 mM NaHepes buffer, pH 7.5, 750 mM NaCl, 200 mM imidazole, 3.5 mM CaCl2, 0.02% sodium azide, and 25% (v / v) glycerol.

[0091] Synthesis of silica nanoparticles: 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.

[0092] Enzyme shielding and surface functionalization - Generation of NP-2: SNPs (10 mg / mL, 55 nm) in H2O / PS80 (8 mg / L) were mixed with APTES (3.9 mM). 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. Lactase (7 mg / mL, 0.1 mM) 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 (8.4 mM) and TEOS (125.9 mM). The resulting suspension was incubated at 20°C and 400 rpm for 5 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 121 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 NaCl (0.9%) / PS80 (8 mg / L). NP-2 was cured overnight in a water bath at 20°C.

[0093] - NP-2 variant: The following experiments investigated the effects of covalently bonding enzymes to a protective layer on enzyme stability and enzyme activity.

[0094] In the initial experiment, nanoparticles (NP-2(1)) were generated in H2O / PS80 (8 mg / L). The nanoparticles were washed after each chemical step, resulting in the removal of glutaraldehyde. APTES (3.3 mM) was added to SNP (10 mg / mL, 69 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. The particles were then removed from H2O / The particles were washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Then, glutaraldehyde (3.3 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 PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Priming was performed by adding APTES (3.3 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. / The particles were washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Lactase (5.2 mg / mL, 0.1 mM) was added, 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 lactase using APTES (6.5 mM) and TEOS (93 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were then removed from H2O / The sample was washed three times in PS80 (8 mg / L) by centrifugation at 20,000 rcf for 5 minutes, and then resuspended in H2O / PS80 (8 mg / L). NP-2(1) was cured overnight in a 20°C water bath.

[0095] In the second comparative experiment, enzyme immobilization and protective layer formation were performed according to International Publication No. 2015 / 014888, and nanoparticles (NP-2(2)) were generated in buffer. The nanoparticles were washed after each chemical step, resulting in the removal of glutaraldehyde. APTES (3.3 mM) was added to SNP (10 mg / mL, 69 nm) and PS80 (8 mg / L) in phosphate buffer (25 mM, pH 7.5). 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 (3.3 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 (3.3 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). Lactase (5.2 mg / mL, 0.1 mM) was added, 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 lactase using APTES (6.5 mM) and TEOS (93 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-2(2) was cured overnight in a 20°C water bath.

[0096] In the third experiment, nanoparticles (NP-2) were generated in H2O / PS80 (8 mg / L) according to the section titled "NP-2 Generation" above. The nanoparticles were not washed between each chemical step in order to retain the excess glutaraldehyde in the reaction mixture, which had not bound the solid support to lactase. Therefore, glutaraldehyde remained present during layer growth, causing covalent bond formation between the protective layer and lactase. The covalent bond between the protective layer and lactase 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 the lactase and organosilica layer. The absorbance of nanoparticles NP-2(1), NP-2(2), and NP-2 at 460 nm was measured after the formation of the organic silica layer and after the final particle washing, i.e., after the organic silica layer was formed and the particles were washed three times in H2O / PS80 and resuspended in H2O / PS80 as described in the section titled "NP-2 Generation". NP-2 showed a higher absorbance at 460 nm than NP-2(1) and NP-2(2) (see Figure 7). NP-2(1) and NP-2(2) still showed some absorbance at this wavelength, which is because imine bonds are formed even during enzyme immobilization. However, the absorbance of NP-2 was significantly higher than the others, indicating that further imine bonds were formed by covalent bonding between the protective layer and lactase.

[0097] - Generation of NP-3: 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. Glutaraldehyde (3.8 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 (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Inverdase (1.726 mg / mL, 0.03 mM) 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 inverdase using APTES (5.4 mM) and TEOS (81.3 mM). The resulting suspension was incubated at 20°C and 400 rpm for 5 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 115 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 NaCl (0.9%) / PS80 (8 mg / L). NP-3 was cured overnight in a water bath at 20°C.

[0098] - Generation of NP-4: APTES (3.6 mM) was added to SNPs (10 mg / mL, 59 nm) in H2O / PS80 (8 mg / L). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.6 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 (3.6 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Isomaltase (3.55 mg / mL, 0.05 mM) 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 inverdase using APTES (5.8 mM) and TEOS (88 mM). The resulting suspension was incubated at 20°C and 400 rpm for 5 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 82 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-4 was cured overnight in a water bath at 20°C.

[0099] - Generation of NP-5: APTES (3.6 mM) was added to an SNP (10 mg / mL, 59 nm) in H2O / PS80 (8 mg / L). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.6 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 (3.6 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Isomaltase (1.77 mg / mL, 0.025 mM) and invertase (4.05 mg / mL, 0.07 mM) were 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 enzyme using APTES (5.8 mM) and TEOS (88 mM). The resulting suspension was incubated at 20°C and 400 rpm for 5 hours. The particles were washed three times in H2O / 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 82 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 H2O / PS80 (8 mg / L). NP-5 was cured overnight at 20°C. Production of NP-1:

[0100] APTES (3.8 mM) was added to SNPs (10 mg / mL, 56 nm) in H2O / PS80 (8 mg / L). 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 stirred 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.

[0101] Disaccharide-degrading enzyme activity assay: - Lactase activity assay A suspension of NP-2 (30 μL, 2.3 mg / mL) in phosphate buffer (100 mM, pH 6.5) / MgCl2 (5 mM) was mixed with lactose solution (100 μL, 50 mg / mL). The reaction mixture was incubated in a thermomixer at 37°C and 750 rpm for 20 minutes. Samples were collected every 2.5 minutes, and glucose production was monitored using a blood glucose meter.

[0102] - Invertase activity assay NP-3 activity was evaluated using Sigma's invertase assay kit. 1X reaction buffer (94 μL) was added to NP-3 (94 μL, 147 μg / L). Next, 1X sucrose solution (11.76 μL) was added. The reaction mixture was incubated in a thermomixer at 37°C and 300 rpm for 20 minutes. The sample was centrifuged at m20000 rcf for 5 minutes. The supernatant was collected, and 85 μL was transferred to a 96-well plate. 90 μL of master reaction mix (prepared by mixing enzyme mix, dye reagent, and assay buffer) was added to each well. The reaction mixture was incubated in the dark at room temperature for 20 minutes. Absorbance was measured at λ = 570 nm.

[0103] - Isomaltase activity assay NP-4 activity was evaluated using a blood glucose meter. An isomaltose solution (25 μL, 100 mM) was equilibrated at 37°C and 700 rpm for 5 minutes. Then, NP-4 (25 μL, 67 μg) in 50 mM phosphate buffer, pH 6.8 was added to the isomaltose solution. The reaction mixture was incubated at 37°C and 700 rpm for 10 minutes. Samples were collected at 2 minutes, 5 minutes, and 10 minutes, and glucose concentrations were determined using a blood glucose meter.

[0104] - Co-immobilized isomaltase-invertase activity assay NP-5 isomaltase activity was evaluated using a blood glucose meter. An isomaltose solution (25 μL, 100 mM) was equilibrated at 37°C and 700 rpm for 5 minutes. Then, NP-5 (25 μL, 67 μg) in 50 mM phosphate buffer, pH 6.8 was added to the isomaltose solution. The reaction mixture was incubated at 37°C and 700 rpm for 10 minutes. Samples were collected at 2 minutes, 5 minutes, and 10 minutes, and glucose concentrations were determined using a blood glucose meter.

[0105] NP-5 invertase activity was evaluated using a blood glucose meter. A sucrose solution (25 μL, 100 mM) was equilibrated at 37°C and 700 rpm for 5 minutes. Then, NP-5 (25 μL, 67 μg) in 50 mM phosphate buffer, pH 6.8 was added to the sucrose solution. The reaction mixture was incubated at 37°C and 700 rpm for 10 minutes. Samples were collected at 2 minutes, 5 minutes, and 10 minutes, and glucose concentrations were determined using a blood glucose meter.

[0106] Cell culture: In all experiments, cells were cultured at 37°C and 5% CO2.

[0107] 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.

[0108] THP-1 (human monocytic leukemia cell line) cells were cultured and maintained in RPMI1640 supplemented with 10% thermoactivated fetal bovine serum, 2 mM L-glutamine, and 100 U / mL penicillin / streptomycin.

[0109] To differentiate THP-1 into macrophages, THP-1 cells were cultured in the following differentiation medium: RPMI1640 containing 10% thermoinactivated fetal bovine serum, 2 mM L-glutamine, 100 U / mL penicillin / streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 2.5 g / L glucose, and 50 pM β-mercaptoethanol. THP-1 cells were differentiated into M0 macrophages by incubation with 150 nM phorbol 12-myrisstart 13-acetate (PMA) for 24 hours, followed by incubation in differentiation medium for 24 hours.

[0110] - Intestinal barrier model 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 2 Cells 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%.

[0111] - Immunodeficient intestinal barrier model To develop an immunodeficient intestinal barrier model, M0 differentiated THP-1 cells were added to the intestinal barrier model on day 21. Immune cells were attached to the posterior surface of a Transwell membrane containing a co-culture of pre-differentiated cells with a 25% Matrigel solution via a dropper method.

[0112] 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.

[0113] In vitro digestion of sucrose Differentiated Caco-2 / HT29-MTX-E12 cocultures in PBS were exposed to NP-3 (0.5 mU and 1 mU) in the presence of sucrose for 4 hours at the leading edge of the barrier. At each time point, 150 μL aliquots were withdrawn from the basal end of the intestinal barrier and replaced with the same volume of pre-warmed PBS. The barrier was further incubated at 37°C. The absorbance of the withdrawn aliquot samples was measured at 570 nm to quantify the glucose level.

[0114] 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.

[0115] This study was conducted on male Wistar rats (8 weeks old) from Janvier, France.

[0116] - Food and drinking water: As a maintenance diet, the rats were given free access to a pellet-type complete feed called "Altromin 1319." The animals were also free to drink water.

[0117] From one week before treatment through the experimental period, rats were given ad libitum access to a low-sugar diet (AIN 93G modified 200g polysaccharides).

[0118] - Duodenal catheterization The animals were anesthetized with isoflurane (2-4%) in the induction chamber and then transferred to a nose cone containing isoflurane for surgery. The catheter (C30PU-RDD1444, Instech Laboratories) was placed on the opposite side of the mesenteric duodenum, near the biliary and pancreatic duct opening. The catheter was ligated to the intestinal wall and exposed by creating a subcutaneous tunnel in the animal's neck. The abdominal and cervical incisions were then closed with sutures. The animals were kept warm throughout the procedure and carefully monitored until they had fully recovered from anesthesia.

[0119] - Medication and lactose administration Prior to drug administration and lactose administration, the animals were starved for 4 hours. Then, rats were administered NP-2 (97 U), NP-1 (54 mg), or vehicle (1.5 mL of NaCl 0.9% polysorbate 80 8 mg / mL) intraduodenally, followed immediately by enteral nutrition of 3 g of lactose. The rats received daily drug and enteral nutrition for 15 days.

[0120] - Cecal analysis After the experiment, all animals were scanned under complete anesthesia using a Bruker Pharmascan 7 Tesla with a rat volume coil to assess cecal size (scan time approximately 5 minutes). In all obtained slices, the region of interest in the cecum was depicted. MRI images were used to determine cecal volume.

[0121] During the autopsy, photographs of the digestive tract were taken to visualize the dilation of the cecum.

[0122] result: Example 1: Improvement of enzyme loading by covalent bonding to the protective layer In the first experiment, nanoparticles NP-2(1) were generated under unbuffered conditions, and washing was performed after each chemical step (i.e., glutaraldehyde removal before layer growth). In the second experiment, nanoparticles NP-2(2) were generated under buffered conditions, and washing was performed after each chemical step (i.e., glutaraldehyde removal before layer growth). In the third experiment, nanoparticles NP-2 were generated under unbuffered conditions without an intermediate washing step (i.e., unreacted glutaraldehyde was still present in the reaction mixture during layer growth).

[0123] To determine the lactase immobilization yield on NP-2(1), NP-2(2), and the surface of NP-2, protein quantification was performed on the reaction supernatant. Surprisingly, the results showed that enzyme immobilization yield increased 26-fold under conditions where the presence of glutaraldehyde was maintained (NP-2) (Figure 2A), and consequently, the enzyme load per dry weight of SNP increased 24-fold compared to buffered conditions where glutaraldehyde was removed by washing (NP-2(2)) (Figure 2B). Similarly, enzyme immobilization under conditions where the presence of glutaraldehyde was maintained (NP-2) doubled the enzyme load per dry weight of SNP compared to unbuffered conditions where glutaraldehyde was removed by washing (NP-2(1)) (Figure 2B).

[0124] In summary, covalent bonding of a protective layer to the enzyme surface unexpectedly improves its loading capacity compared to enzymes protected by an organic silica layer via electrostatic interactions alone.

[0125] Example 2: Disaccharide activity of NP-2, NP-3, NP-4, and NP-5 The biocatalytic activity of four immobilized and protected disaccharide enzymes was evaluated. The results shown in Figure 3 report the enzymatic activity of each nanoparticle: lactase activity of NP-2 (Figure 3A), invertase activity of NP-3 (Figure 3B), isomaltase activity of NP-4 (Figure 3C), and dual enzymatic activity (isomaltase and invertase) of NP-5 (Figure 3D). These data indicate that the disaccharide enzymes reach the catalytic site of the enzyme and are cleaved with high enzymatic activity. The verification of biocatalytic activity in NP-2, NP-3, NP-4, and NP-5 confirms the potential for applying immobilization and protection strategies to a wide range of disaccharide enzymes that can be used for therapeutic purposes.

[0126] Example 3: Biocompatibility of nanoparticles for application to the gastrointestinal tract The intestinal mucosa consists of a single layer of epithelial cells tightly bound together by intercellular tight junctions, adjacent to a subepithelial region containing the lamina propria. The intestinal mucosa functions as a barrier between the external environment and the internal environment. Its integrity is a crucial parameter for ensuring the body's protection against undesirable contaminants such as microorganisms. The lamina propria contains diffuse lymphoid tissue composed of immune cells that maintain homeostasis or respond to the breakdown of epithelial protection.

[0127] To evaluate the biocompatibility of nanoparticles, the inventors developed representative models of NP-2 and NP-3. This model consists of NP-1, which is a nanoparticle that has the same functionalized outer surface as NP-2 and NP-3 but lacks enzymatic activity.

[0128] To evaluate the safety of the nanoparticles, the inventors first focused on maintaining the integrity of the intestinal barrier in the presence of NP-1 (Figure 4A). Transepithelial electrical resistance (TEER) measurements across a monolayer of Caco2-HT29-MTX-E12 cells showed that the integrity of the intestinal epithelial barrier remained intact even after 24 hours of contact with NP-1. This result demonstrates the in vitro biocompatibility of NP-1.

[0129] To further characterize the effects of nanoparticles on the intestinal barrier, the inventors evaluated the ability of NP-1 to induce an inflammatory response. For this purpose, the inventors developed an immunodeficient intestinal barrier model and monitored its integrity. Figure 4B shows that barrier integrity is reduced in a dose-dependent manner when treated with LPS, an inflammatory component. Such loss of integrity reveals the recruitment of macrophages from the basal to the apical side of the barrier.

[0130] Most importantly, TEER measurements showed that the integrity of the epithelial barrier was not compromised even in contact with NP-1 (Figure 4B). NP-1 did not stimulate macrophage recruitment at the apical end of the intestinal barrier, leading to the conclusion that NP-1 does not induce inflammation.

[0131] In summary, these results demonstrate the in vitro safety of nanoparticles for gastrointestinal application.

[0132] Example 4: In vivo efficacy of NP-2 Lactose malabsorption is caused by an imbalance between the amount of lactose ingested and the ability of lactase to hydrolyze disaccharides. Lactose digestion and absorption occur in the small intestine. In cases of lactose malabsorption, undigested lactose reaches the large intestine and comes into contact with the gut microbiota. Bacterial fermentation of lactose leads to the production of short-chain fatty acids and gases, resulting in enlargement of the cecum in particular.

[0133] Rats administered high doses of lactose daily via enteral nutrition for 15 days showed increased substrate availability for fermentation in the large intestine and cecal enlargement compared to rats fed a normal diet without additional lactose (referred to as "vehicle" and "lactose-free" conditions, respectively) (Figure 5). Importantly, a significant reduction in cecal size was reported in rats administered NP-2, while administration of inactive nanoparticles (NP-1) had no effect on cecal size reduction. These results demonstrate the in vivo biocatalytic activity of NP-2 for lactose digestion.

[0134] Enzyme replacement therapy using exogenous lactase derived from microorganisms is available, but results regarding the precise efficacy rate are inconsistent (Montalto et al., World J Gastroenterol 2006, Jan 14;12(2):187-91). The rule used to calculate the amount of lactase is 7500 units per 16 grams of lactose. In addition to this close relationship between the amount of lactose hydrolyzed and the required enzyme units, gastric pH and bile salt concentration affect not only the effectiveness of exogenous lactase but also the loss of specific localization of the enzyme in the intestines.

[0135] Figure 5 demonstrates the in vivo efficacy of NP-2 at a dose of 97 units per 3 g of lactose. Compared to the dosages of currently available lactase preparations, this single dose is a remarkable 14.5% lower. In other words, this set of data demonstrates the high value of NP-2 for lactose digestion and highlights its therapeutic potential for patients with disaccharide digestive deficiencies, such as those with lactose malabsorption.

[0136] Example 5: In vitro efficacy of NP-3 Congenital sucrase-isomaltase deficiency (CSID) is characterized by complete or near-complete absence of sucrose activity and varying degrees of reduced isomaltase activity.

[0137] The efficacy of NP-3 sucrose digestion was evaluated in an intestinal barrier model. A monolayer of differentiated Caco2-HT29-MTX-E12 cells was exposed to NP-3 for 4 hours at its apical end in the presence of its substrate. Figure 6 shows the quantification of sucrose hydrolysis products in the basal compartment of the barrier. This graph reports that glucose accumulates in a dose-dependent manner at the basal side of the barrier as the amount of NP-3 present increases, but glucose is not detected in the untreated intestinal barrier. This result demonstrates the in vitro efficacy of NP-3 and clearly indicates its use for therapeutic purposes.

Claims

1. A composition comprising a solid carrier, a disaccharidase or fragment thereof immobilized on the surface of the solid carrier, a protective layer that protects the disaccharidase 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 containing at least one amino group and / or at least one thiol group.

2. The composition according to claim 1, 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, polyglucosaminoglycan, chondroitin, heparin, keratan, and dermatan or derivatives thereof.

3. The composition according to claim 1 or 2, wherein the polymer comprising repeating units, each having at least one amino group and / or at least one thiol group, is chitosan or a derivative thereof.

4. The composition according to any one of claims 1 to 3, wherein the functional component is immobilized on the surface of the protective layer by non-covalent or covalent bonds.

5. The composition according to any one of claims 1 to 4, wherein the disaccharide-degrading enzyme or fragment thereof is selected from the group consisting of lactase or fragment thereof, maltase or fragment thereof, isomaltase or fragment thereof, trehalase or fragment thereof, and invertase or fragment thereof, or a mixture thereof.

6. The composition according to any one of claims 1 to 4, wherein the disaccharide-degrading enzyme or a fragment thereof is selected from the group consisting of lactase or a fragment thereof, and invertase or a fragment thereof, or a mixture thereof.

7. The composition according to any one of claims 1 to 4, wherein the disaccharide-degrading enzyme or a fragment thereof is invertase or a fragment thereof.

8. The composition according to any one of claims 1 to 7, wherein a solid carrier is embedded in the protective layer, and a disaccharogenic enzyme or a fragment thereof fixed on the surface of the solid carrier is embedded.

9. The composition according to any one of claims 1 to 8, wherein the functional component immobilized on the surface of the protective layer is not embedded by the protective layer.

10. A composition according to any one of claims 1 to 9 for use as a pharmaceutical.

11. A composition according to any one of claims 1 to 9 for use in a method for treating lactase deficiency, sucrase-isomaltase deficiency, and disaccharide intolerance.

12. A method for producing a composition, the composition comprising a solid carrier, a disaccharidase or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the disaccharidase 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 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 immobilizing a disaccharide-degrading enzyme or a fragment thereof onto a solid carrier. (c) A step of forming a protective layer on the surface of a solid carrier in order to protect the disaccharogenic enzyme or fragment thereof immobilized on the solid carrier. (d) 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.

13. The method according to claim 12, wherein in step b), i) a linker is added to the solid carrier provided in step (a), and ii) a disaccharase or a fragment thereof is added to the solid carrier and the linker, and the linker connects the solid carrier and the disaccharase or a fragment thereof.

14. The method according to claim 13, wherein a linker that did not connect the solid carrier to the disaccharase or a fragment thereof in step (b) is present during the formation of a protective layer on the surface of the solid carrier in step (c).

15. The method according to claim 13, wherein there is no washing step between (i) adding a linker to the solid carrier provided in step (a) and (ii) adding a disaccharase or a fragment thereof to the solid carrier and the linker.

16. The method according to any one of claims 12 to 15, wherein there is no washing step between any of steps (a) to (c).

17. The method according to any one of claims 13 to 16, wherein a linker, or a part thereof, that did not connect the solid carrier to the disaccharase or a fragment thereof in step (b) covalently bonds the protective layer to the disaccharase or a fragment thereof in step (c).

18. 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 17, selected from the group consisting of HCl, DST (disuccinimidyl tartrate), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane).

19. The method according to any one of claims 13 to 17, wherein the linker is glutaraldehyde.

20. A composition comprising a solid carrier, a disaccharidase or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the disaccharidase 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 composition can be obtained by the method according to any one of claims 14 to 19.

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