Functionalized bienzyme compositions

Abstract

The present invention relates to a composition comprising a solid carrier, a proteinase or a fragment thereof immobilized on the surface of the solid carrier, a protective layer for protecting the proteinase or the fragment thereof by embedding the proteinase or the fragment thereof, and a functional component immobilized on the surface of the protective layer, wherein the functional component fixed on the surface of the protective layer is a polymer comprising repeating units, and each repeating unit comprises at least one amino group and / or at least one mercapto group. The invention also relates to a method for producing said composition.

Description

TECHNICAL FIELD

[0001] The present invention relates to a composition comprising a solid support, a di-saccharidase or a fragment thereof immobilized on the surface of the solid support, a protective layer protecting the di-saccharidase or the fragment thereof by embedding the di-saccharidase or the fragment thereof, and a functional ingredient immobilized on the surface of the protective layer, wherein the functional ingredient immobilized on the surface of the protective layer is a polymer comprising repeating units, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. The present invention further relates to a method of producing said composition. BACKGROUND

[0002] Di-saccharides are generally broken down into monosaccharides by di-saccharidases located in the brush border of the small intestinal epithelial cells. Undigested di-saccharides cause an osmotic load that draws water and electrolytes into the intestine, causing watery diarrhea. Bacterial fermentation of carbohydrates in the colon produces gas (hydrogen, carbon dioxide and methane), leading to excessive gas in the stomach, bloating and abdominal distension, and abdominal pain. Diseases associated with di-saccharide digestion deficiencies are e.g. 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 di-saccharide. It manifests as lactose intolerance, a clinical syndrome of one or more of the following symptoms: abdominal pain, diarrhea, nausea, flatulence and / or bloating after ingestion of lactose or lactose-containing foods. The most common cause is primary (genetic) lactase deficiency, which is due to a relative or absolute lack of lactase in childhood at different ages in different ethnic groups. Secondary (acquired) lactase deficiency is caused by small intestinal damage, such as acute gastroenteritis, persistent diarrhea, small intestinal overgrowth, cancer chemotherapy or other causes of small intestinal mucosal damage. Congenital lactase deficiency is a very rare autosomal recessive enzyme defect that prevents the expression of lactase from birth, requiring complete avoidance of lactose.

[0003] In addition to lactose avoidance, which can cause serious health problems, enzyme replacement therapy with microbial exogenous lactase (beta-galactosidase obtained from Aspergillus oryzae) before or added to dairy meals represents a possible strategy to treat primary and secondary lactase deficiency. Results regarding the exact rate of efficacy are inconsistent. Aspergillus oryzae

[0004] ​CSID is a multifaceted small intestinal absorption disorder in which the sucrase-isomaltase gene is mutated autosomally recessively, characterized by complete or almost complete lack of sucrose activity and varying degrees of reduction in isomaltase activity. When this enzyme complex is lacking, nutrients from ingested starch and sucrose cannot be fully absorbed, and clinically manifest as diarrhea, abdominal pain and bloating, leading to malnutrition, especially in children. In addition to lifelong dietary measures, the only effective treatment option is enzyme replacement with sacrosidase, currently approved by the US Food and Drug Administration as Sucraid®, but not yet in Europe. Therefore, there is a need to provide an effective treatment for diseases related to the poor digestion of disaccharides. SUMMARY

[0005] The present invention provides a composition comprising a solid support, a disaccharidase or a fragment thereof immobilized on the surface of the solid support, a protective layer for protecting the disaccharidase or the fragment thereof by embedding the disaccharidase or the fragment thereof, 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 a repeating unit, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0006] The present invention also provides a method for producing the composition comprising

[0007] a solid support, a disaccharidase or a fragment thereof immobilized on the surface of the solid support, a protective layer for protecting the disaccharidase or the fragment thereof by embedding the disaccharidase or the fragment thereof, 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 a repeating unit, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, the method comprising the following steps: (a) providing a solid support; (b) immobilizing a disaccharidase or a fragment thereof on the solid support; (c) forming a protective layer on the surface of the solid support to protect the disaccharidase or the fragment thereof immobilized on the solid support; (d) immobilizing a functional component on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising a repeating unit, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0008] The inventors of the present application have surprisingly found that the compositions provided by the present application, if applied therapeutically in the digestion of disaccharides, have unexpectedly high activity, show low cytotoxicity, and do not disrupt the intestinal barrier if positioned in the gastrointestinal tract, thus making them extremely promising for therapeutic use, in particular for lactase deficiency, sucrase-isomaltase deficiency and disaccharidoses intolerance. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 ) shows a schematic representation of the method for the production of the compositions of the present application: a) immobilization of the disaccharidase or fragment thereof (indicated as "protein") on a solid support; b) and c) growth of a protective layer around the immobilized disaccharidase or fragment thereof, thus embedding said immobilized disaccharidase or fragment thereof; and d) immobilization of the functional ingredient on the surface of the protective layer.

[0010] Figure 2 ) shows the added value of the covalent binding of the enzyme surface to the protective layer. (A) Protein quantification of the reaction supernatant of NP-2(1), NP-2(2) and NP-2. (B) Lactase loading per unit of dry weight of SNP.

[0011] Figure 3 ) shows the disaccharidase activity of the nanoparticles. (A) Lactase activity of NP-2 (in U / g) after exposure to lactose. (B) Invertase activity of NP-3 (in U / mg) after exposure to sucrose. (C) Isomaltase activity of NP-4 in isomaltulose (in U / g). (D) Isomaltase and invertase activity of NP-5 (in U / g) after exposure to isomaltulose and sucrose, respectively.

[0012] Figure 4 ) shows the representative model of the intestinal barrier on which the nanoparticles were tested. In vitroBiocompatibility. (A) In vitro assessment 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 h. TEER data were normalized relative to a control point, defined as the equilibrium value before NP-1 addition (defined as control), and set to 100%. The figure shows the time-course curve evolution of the mean normalized TEER data over 16 h. The dashed line represents the untreated condition. (B) In vitro evaluation of the effect of inflammation 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 h. Lipopolysaccharide (LPS) (50 and 100 μg / mL) were used as positive controls to induce an inflammatory response. TEER data were normalized to the equilibrium value before NP-1 or LPS addition (set to 100%). This figure shows the evolution of the time-process curve of the averaged and standardized TEER data over 16 hours in the presence of NP-1.

[0013] Figure 5 (A) Photograph of the rat gastrointestinal tract. The cecum is circled. (B) MRI image of the rat gastrointestinal tract. The arrow indicates the cecum. (C) Histogram showing the size of the cecum as assessed by MRI imaging in cm. 3 The cecum size is represented. A one-way ANOVA test was performed. p<0.05, p<0.01.

[0014] Figure 6 This shows the effect of sucrose on the intestinal barrier model. In vitro Digestion. Differentiated Caco-2 / HT29-MTX-E12 cocultures were exposed to varying amounts of NP-3 (0.5 mU or 1 mU) containing sucrose for 4 h at the top of the barrier. Sucrose hydrolysis was evaluated by quantifying glucose at the bottom of the barrier. The figure shows the evolution of the glucose accumulation time-series curves over 4 h.

[0015] Figure 7 The absorbance of nanoparticles NP-2, NP-2(1) and NP-2(2) at 460 nm is shown. Detailed Implementation

[0016] The present invention relates to a composition comprising a solid carrier, a disaccharidase or a fragment thereof immobilized on the surface of the solid carrier, a protective layer for protecting the disaccharidase or the fragment thereof by embedding the disaccharidase or the fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0017] For the purpose of interpreting this specification, the following definitions will be used, and where appropriate, terms used in the singular will also include the plural, and vice versa. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0018] Features, integrals, properties, and compounds described in connection with specific aspects, embodiments, or examples of the invention should be understood to be applicable to any other aspect, embodiment, or example described herein, unless incompatible therewith. All features disclosed in this specification (including any appended claims, abstract, and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except for combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments.

[0019] The term “comprise” and its variations, such as “comprises” and “comprising”, are generally used in the sense of inclusion, that is, “including but not limited to”, meaning that one or more features or components are allowed to be present.

[0020] Unless the context clearly indicates otherwise, the singular forms “a” and “the” include plural references.

[0021] The term “about” refers to a range of ±10% of a specified value. For example, the phrase “about 200” includes ±10% of 200, or 180 to 220.

[0022] As used herein, the term "solid support" generally refers to particles. Preferably, the solid support is monodisperse or polydisperse particles, more preferably monodisperse particles. Solid supports typically include organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, titanium particles, and are preferably silica particles, more preferably silica nanoparticles (SNPs). The particle size of the solid support is typically from 1 nm to 1000 µm, preferably from 10 nm to 100 µm, and particularly about 50 nm.

[0023] As used herein, the terms "linker" or "crosslinker" refer to any linking agent containing a group capable of binding a specific functional group (e.g., primary amine, thiol, etc.). In the context of this invention, a linker typically links 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 as a carrier material on a silica surface, and then the disaccharidase can bind to an unoccupied binding site of the linker. Alternatively, the linker can first bind to the disaccharidase, and then the linker binding the disaccharidase can bind to the solid support at its unoccupied binding site. Various types of linkers are known in the art, including but not limited to linear or branched carbon linkers, heterocyclic carbon linkers, peptide linkers, polyether linkers, and linkers known in the art as labeled linkers.

[0024] As used herein, the term "protective layer" refers to a layer used to protect the functional properties of a disaccharidase or fragment immobilized on the surface of a solid support. The protective layer of the present invention is typically constructed using building blocks, at least a portion of which are monomers capable of interacting with each other typically via covalent bonding and with the immobilized disaccharidase typically via non-covalent bonding. A protective layer is formed on the surface of a solid support to protect the disaccharidase or fragment thereof immobilized on the solid support. The protective layer is typically a homogeneous layer in which at least 50%, preferably at least 70%, more preferably at least 90% of the disaccharidase or fragment thereof is embedded.

[0025] The term "disaccharidase or fragment thereof" includes naturally occurring disaccharidases or fragment thereof, and also includes artificially modified disaccharidases or fragment thereof. Disaccharidases are glycoside hydrolases that break down certain types of sugars called disaccharides into simpler sugars called monosaccharides. In humans, disaccharidases are primarily produced in a region of the small intestinal wall called the brush border. Disaccharidases include, for example, lactase, maltase, isomaltase, trehalase, and sucrase (also known as invertase). Artificially modified disaccharidases or fragments thereof are, for example, variants or functionally active fragments of disaccharidases. Therefore, the terms "fraction of disaccharidase," "fraction thereof associated with a disaccharidase," and "functionally active fragment of a disaccharidase" are used synonymously herein. A "variant or functionally active fragment thereof" related to the disaccharidase of this invention means that the fragment or variant (such as an analog, derivative, or mutant) is capable of performing the same physiological function as the disaccharidase. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Consideration is given to the addition, deletion, substitution, and derivatization of one or more amino acids, provided that the modification does not result in a loss of functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least about 80% sequence identity with the relevant portion of the disaccharidase, 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. Disaccharidase fragments as defined herein generally have the same functional properties as the disaccharidase from which they are derived. Disaccharidase fragments typically contain 100 to 1000 amino acids, preferably 300 to 800 amino acids, more preferably 500 to 700 amino acids.

[0026] As used herein, the term "partially embedded disaccharidase" refers to a disaccharidase that is not completely covered by the protective layer; therefore, 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, although typically more than 70% is covered, thus improving the protection of the disaccharidase. In a preferred embodiment, at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% of the target disaccharidase is covered by the protective layer. In another preferred embodiment, about 70% to about 95%, more preferably about 80% to about 95%, even more preferably about 90% to about 95%, and most preferably about 90% to about 95%, 96%, 97%, 98%, or 99% of the target disaccharidase is covered by the protective layer. In a particularly preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most particularly about 95% of the target disaccharidase is covered by the protective layer. In a more particularly preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most particularly about 95% of the target disaccharidase is covered by a protective layer, wherein the active site is not covered.

[0027] As used herein, the term "fully encapsulated disaccharidase" means that the disaccharidase according to the present invention is completely, i.e., 100% covered by the protective layer, i.e., the active site is also covered.

[0028] As used herein, the term "at least partially embedded disaccharidase" means that the disaccharidase is at least partially embedded and can be completely embedded by the protective layer. Thus, "at least partially embedded disaccharidase" means that the protective layer covers about 30% to 100% of the disaccharidase or fragments thereof, preferably about 50% to about 100%, more preferably about 80% to about 100%, even more preferably about 90% to about 100%, and most preferably about 95% to about 100%, wherein the active site is preferably covered.

[0029] As used herein, the term "functional component" refers to a component that retains its characteristic and functional properties after being fixed to the surface of a protective layer. In the context of this invention, a functional component is a polymer comprising repeating units, wherein each repeating unit contains at least one amino group and / or at least one thiol group.

[0030] As used herein, the term "polymer comprising repeating units, wherein each repeating unit contains at least one amino group" refers to a polymer comprising several repeating units (monomers), wherein each repeating unit contains at least one amino group. Preferred polymers comprise several repeating units (monomers), wherein each repeating unit contains one amino group, particularly a primary amino group.

[0031] As used herein, the term "polymer comprising repeating units, wherein each repeating unit contains at least one thiol group" refers to a polymer comprising several repeating units (monomers), wherein each repeating unit contains at least one thiol. Preferred polymers comprise several repeating units (monomers), wherein each repeating unit contains one thiol group.

[0032] As used herein, the term "polycarbophil-cysteine ​​conjugate" refers to a conjugate containing a cysteine ​​residue covalently linked to polycarbophil. Such conjugates can be generated as described in Bernkop-Schnurch and Thaler, 2000, Journal of Pharmaceutical Sciences 89(7):901-9.

[0033] 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 that can 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.

[0034] As used herein, the term "polycysteine" may consist of L-cysteine ​​or D-cysteine, preferably L-cysteine, and preferably contains 2 to 30 cysteine ​​residues, more preferably 2 to 5 cysteine ​​residues.

[0035] As used herein, the term "polyglucosamine" refers to a linear amino-polysaccharide composed of D-glucosamine and N-acetyl-D-glucosamine units linked by (1-4) glycosidic bonds. Polyglucosamine contains a free amine (-NH2) group and can be characterized by the ratio of N-acetyl-D-glucosamine units to D-glucosamine units, expressed as the degree of deacetylation (DDA) of a fully acetylated polymeric chitosan. Preferred polyglucosamines of this invention are selected from chitosan, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratin, and dermatan, or derivatives thereof. Chitosan or its derivatives are most preferred.

[0036] As used herein, the term "chitosan or a derivative thereof" refers to chitosan or chitosan derivatives thereof, including its salts, preferably having a molecular weight of 2,000 Da or greater, more preferably in the range of 25,000 to 2,000,000 Da, more preferably about 50,000 to 350,000 Da, and most preferably about 50,000 to 190,000 Da or 190,000 to 310,000 Da. The term "derivative" in relation to chitosan includes esters, ethers, or other derivatives formed by the reaction of an acyl or alkyl group with an OH group. Examples are O-alkyl ethers of chitosan and O-acyl esters of chitosan. Suitable derivatives are, for example, shown in GAE Roberts, Chitin Chemistry, MacMillan Press Ltd, London, 1992. Suitable salts of chitosan include nitrates, phosphates, sulfates, xanthates, hydrochlorides, glutamates, lactates, and acetates.

[0037] In a first aspect, the present invention provides a composition comprising a solid carrier, a disaccharidase or a fragment thereof immobilized on the surface of the solid carrier, a protective layer for protecting the disaccharidase or the fragment thereof by embedding the disaccharidase or the fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0038] Disaccharidases or fragments thereof can be immobilized on the surface of a solid support by non-covalent or covalent binding. Non-covalent binding includes pp (aromatic) interactions, van der Waals interactions, H-bonding interactions, and electrostatic interactions, such as ionic interactions. Preferably, disaccharidases or fragments thereof are immobilized on the surface of the solid support by covalent binding or by covalent binding via a linker.

[0039] Solutions of disaccharidases or fragments thereof are typically contained in a buffer solution containing the protein or fragment thereof. Commonly used buffers include phosphates, chlorides, citrates, MES, MOPS, HEPES, PIPES, ACES, or mixtures thereof. The solution may additionally contain sugar alcohols or nonionic surfactants, as described herein. Solutions of disaccharidases or fragments thereof can be prepared, for example, by dissolving the disaccharidase or fragment thereof in water to reconstitute a stock buffer for the disaccharidase or fragment thereof.

[0040] In one embodiment, the solid support is selected from organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, titanium particles, and preferably silica particles, more preferably silica nanoparticles (SNPs). Particle size is typically measured by measuring the diameter of the particles and is typically from 1 nm to 1000 nm, preferably from 10 nm to 100 nm, particularly about 50 nm. When the solid support is a monodisperse particle, the particle size is typically from 1 nm to 1000 nm, preferably from 10 nm to 100 nm, particularly about 50 nm. When the solid support is a polydisperse particle, the particle size is typically from 1 nm to 1000 μm, preferably from 10 nm to 100 μm, particularly 50 nm to 50 μm. In one embodiment, the composition comprises a solid support, wherein the solid support comprises at least 15%, preferably at least 20%, particularly 15% to 30%, more particularly 20% to 30% of an immobilized disaccharidase or a fragment of it / dry weight solid support.

[0041] Typically, monodisperse or polydisperse particles, preferably monodisperse particles, are used as the solid carrier in this invention. In a preferred embodiment, the monodisperse particles are spherical monodisperse particles. In other preferred embodiments, the polydisperse particles are non-spherical polydisperse particles.

[0042] Solid carriers are typically provided in suspension form. The suspension of the solid carrier can be, for example, in water, a buffer solution, or a nonionic surfactant or a mixture thereof, preferably in a mixture of water and a nonionic surfactant. The nonionic surfactant is typically selected from ethoxylated sorbitol esters such as PEG-40 sorbitol diisostearate, polysorbate 80 (PS80), polysorbate 20 (PS20), polysorbate 40 (PS40), and polysorbate 60 (PS60); and block copolymers such as poloxamer 124, poloxamer 188, poloxamer 331, and poloxamer 40. 7. Fatty acid ethoxylates such as PEG-5 oleate, PEG-8 stearate, polyoxyethylene 40 stearate, polyoxyethylene 15 hydroxy stearate; fatty alcohol ethoxylates such as stearyl alcohol polyether (steareth) 40; fatty acid esters such as palmitic acid ascorbate, beeswax, polyglycerol 3-oleate, propylene glycol monocaprylate, propylene glycol monolaurate; fatty alcohols such as cetearyl alcohol, cetyl alcohol, myristyl alcohol, stearyl alcohol; glycerides; polyethylene glycol-modified triglycerides; sugar esters, preferably polysorbate, more preferably polysorbate 80 (PS80). The buffer solutions that can be used in the method of this invention are 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)propyl-2-yl]amino]ethanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 3-(N,N-bis[2-hydroxyethyl]propanesulfonic acid, etc. [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, N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid.

[0043] In one embodiment, the surface of the solid support is modified to introduce molecular or functional chemical groups as anchoring points, i.e., as anchoring points for disaccharidases or as anchoring points for a linker connecting the disaccharidase to the solid support. Preferably, the anchoring points are amine functional chemical groups or portions. As a non-limiting example, an amino-modified surface of the 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 with a silica surface with an aminosilane, such as APTES. Therefore, in a preferred embodiment, the solid support is a solid support with a silica surface containing an amino-modified surface, more preferably a solid support obtained by reacting a solid support with a silica surface with an aminosilane (e.g., with APTES). Such modified supports can form amide bonds between the disaccharidase and the amine groups on the surface of the support material, or between the linker and the amine groups on the surface of the support material. In one embodiment, the introduced molecular or functional chemical groups are uniformly distributed as anchoring points on the surface of the solid support.

[0044] In some embodiments, the protective layer has a defined thickness of about 1 to about 200 nm, typically 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, particularly about 1 to about 15 nm. The most preferred defined thickness is about 1 to about 10 nm. In some embodiments, the layer has a defined 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, particularly about 5 to about 15 nm. The most preferred defined thickness is about 5 to about 10 nm. The protective layer is typically porous with a pore size of 1 to 100 nm, preferably 1 to 20 nm.

[0045] In one embodiment, the disaccharidase or a fragment thereof is partially embedded in the protective layer. In a preferred embodiment, the disaccharidase or a fragment thereof is at least partially embedded in the protective layer. In a more preferred embodiment, the disaccharidase or a fragment thereof is completely embedded in the protective layer.

[0046] In one embodiment, the protective layer embeds a solid carrier and embeds a disaccharidase or fragment thereof immobilized on the surface of the solid carrier. In another embodiment, the functional component immobilized on the surface of the protective layer is not embedded by the protective layer. Preferably, the protective layer completely embeds the solid carrier and completely embeds the disaccharidase or fragment thereof immobilized on the surface of the solid carrier. More preferably, the protective layer completely embeds the solid carrier and completely embeds the disaccharidase or fragment thereof immobilized on the surface of the solid carrier, and the functional component immobilized on the surface of the protective layer is not embedded by the protective layer. If the protective layer completely embeds the solid carrier and completely embeds the disaccharidase or fragment thereof immobilized on the surface of the solid carrier, then the disaccharidase or fragment thereof is completely, i.e., 100% covered by the protective layer, that is, the active site is also covered, and the solid carrier is completely, i.e., 100% covered by the protective layer.

[0047] In a preferred embodiment, the disaccharidase or a fragment thereof is selected from lactase or a fragment thereof, maltase or a fragment thereof, isomaltase or a fragment thereof, trehalase or a fragment thereof, and invertase or a fragment thereof, or a mixture thereof, more preferably selected from lactase or a fragment thereof, isomaltase or a fragment thereof, and invertase or a fragment thereof, or a mixture thereof. Therefore, the composition also contains one or more, for example two, three, or four different disaccharidases or fragments thereof immobilized and embedded on the surface of a solid support. When using one or more disaccharidases or fragments thereof, it is preferable to use two different disaccharidases, more preferably invertase and isomaltase. Therefore, in a specific embodiment, a mixture of isomaltase or a fragment thereof and invertase or a fragment thereof is immobilized on the surface of the solid support of the composition of the present invention.

[0048] In a particularly preferred embodiment, the disaccharidase or a fragment thereof is selected from lactase or a fragment thereof and invertase or a fragment thereof, as well as mixtures thereof. Most preferably, it is invertase or a fragment thereof.

[0049] The thickness of the protective layer can be measured using microscopes such as scanning electron microscopes (SEM), transmission electron microscopes (TEM), scanning probe microscopes (SPM), light scattering methods, or elliptic polarization techniques.

[0050] The compositions of the present invention are typically produced in a reaction vessel such as a reactor. The formation of the protective layer is generally carried out by the formation of a corresponding protective layer from building units, wherein the building units construct the protective layer during a polycondensation reaction. Polycondensation can be carried out in various solvents, preferably in aqueous solutions. If suitable, polycondensation can be easily controlled and stopped, allowing for the achievement of a defined thickness of the protective layer. The selection of building units that can be used to construct the protective layer can depend on the known structure of the disaccharidase in order to adapt the affinity of the protective layer according to optimal and / or desired parameters. As building units for the protective layer, both structural building units and protective building units are typically used to construct the protective layer. A structural building unit that can be used is, for example, tetraethyl orthosilicate (referred to herein as “TEOS” or “T”). The protective building blocks that can be used are, for example, 3-aminopropyltriethoxysilane (referred to herein as “APTES” or “A”), propyltriethoxysilane (referred to herein as “PTES” or “P”), isobutyltriethoxysilane (referred to herein as “IBTES”), hydroxymethyltriethoxysilane (referred to herein as “HTMEOS” or “H”), benzyltriethoxysilane (referred to herein as “BTES”), ureopropyltriethoxysilane (referred to herein as “UPTES”), or carboxyethyltriethoxysilane (referred to herein as “CETES”). The structural building blocks are typically precursors of inorganic silica capable of forming four covalent bonds in the formed layers. The protective building blocks are typically organosilanes carrying an organic moiety conferred with the ability to interact with disaccharidases. Preferred structural building blocks are tetravalent silanes, particularly tetraalkoxysilanes. Preferred protective building blocks are trivalent silanes, particularly trialkoxysilanes. More preferably, the structural building blocks are mixtures of tetravalent and trivalent silanes, particularly mixtures of tetraalkoxysilanes and trialkoxysilanes. Even more preferably, the structural building blocks are selected from tetraethyl orthosilicate, tetra-(2-hydroxyethyl)silane, and tetramethyl orthosilicate.Even more preferred protective building blocks are selected from carboxyethyl silanetriol, benzyl silane, propyl silane, isobutyl silane, n-octyl silane, hydroxy silane, bis(2-hydroxyethyl)-3-aminopropyl silane, aminopropyl silane, ureopropyl silane, and (N-acetylglycyl) silane. -3-aminopropylsilane, hydroxy(polyvinyloxy)propyl]triethoxysilane, especially selected from benzyltriethoxysilane, propyltriethoxysilane, isobutyltriethoxysilane, n-octyltriethoxysilane, hydroxymethyltriethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, 3-aminopropyltriethoxysilane, ureopropyltriethoxysilane, (N-acetylglycyl)-3-aminopropyltriethoxysilane, or selected from benzyltrimethoxysilane, propyltrimethoxysilane, isobutyltrimethoxysilane, n-octyltrimethoxysilane, hydroxymethyltrimethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrimethoxysilane, aminopropyltrimethoxysilane, ureopropyltrimethoxysilane, (N-acetylglycyl) -3-aminopropyltrimethoxysilane, or selected from benzyltrihydroxyethoxysilane, propyltrihydroxyethoxysilane, isobutyltrihydroxyethoxysilane, n-octyltrihydroxyethoxysilane, hydroxymethyltrihydroxyethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrihydroxyethoxysilane, aminopropyltrihydroxyethoxysilane, ureopropyltrihydroxyethoxysilane, (N-acetylglycyl)-3-aminopropyltrihydroxyethoxysilane.

[0051] The particularly preferred building blocks are TEOS as the structural building block, and APTES, PTES, and / or HTMEOS, with APTES being the preferred protective building block. Specifically, TEOS is used as the structural building block and APTES as the protective building block to construct the protective layer.

[0052] If a linker is used, the reaction time between the building blocks and the solid support depends on the length of the linker and the size of the disaccharidase. The reaction typically proceeds for 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 or about 20°C at about 5 to about 25°C. The formation of the protective layer can be stopped by actively halting the polycondensation reaction, for example by removing unreacted building blocks, such as through a washing step; or by self-stopping the polycondensation reaction caused by a limited amount of building blocks.

[0053] In other preferred embodiments, the disaccharidase is immobilized on the solid support by introducing a molecule as described above for the disaccharidase as an anchoring point and by using a linker, preferably a crosslinking agent, that binds to the anchoring point and the disaccharidase to at least partially modify the surface of the solid support.

[0054] In one embodiment, the introduced molecules and / or binders serving as anchoring points are uniformly distributed on the surface of the solid carrier.

[0055] In a preferred embodiment, the crosslinking agent is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide) succinate, dimethyl adipate, dimethyl pimelimidate, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated thiol, thiol-reactive 2-pyridinedithiol, BSOCOES (bis[2-(succinimideoxycarbonyloxy)ethyl] sulfone), DSP (dithiobis[succinimide] propionate), DTSSP (3,3'-dithiobis[succinimide] propionate), DTBP (3,3'-dithiobispropionylimine dimethyl ester·2 HCl), DST (disuccinimidyl tartrate), sulfonyl-LC-SMPT (4-succinimidyl-6-methyl-a-(2-pyridyldithio)toluamide]hexanoate), SPDP (N-succinimidyl-3-(2-pyridyldithio)-propionate), LC-SPDP (succinimidyl-6-(3-[2-pyridyldithio]-propamido)hexanoate), SMPT (4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), DDPPB (1,4-di-[3'-2'-pyridyldithio)-propionamide]butane), DTME (dithio-bismaleimide ethane), BMDB (1,4-bismaleimide-2,3-dihydroxybutane). More preferably, the crosslinking agent is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide succinate), dimethyl adipate, dimethyl heptamethimide, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, and activated thiol groups (e.g., thiol-reactive 2-pyridinedithiol). In a more preferred embodiment, the crosslinking agent is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide succinate), dimethyl adipate, dimethyl heptamethimide, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimideoxycarbonyloxy)ethyl] sulfone), DSP (dithiobis[succinimide] propionate), DTSSP (3,3'-dithiobis[sulfosuccinimide] propionate), DTBP (3,3'-dithiobispropionine dimethyl ester·2HCl), DST (disuccinimide tartrate), and BMDB (1,4-bismaleimide-2,3-dihydroxybutane).More preferably, the crosslinking agent is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide) succinate, dimethyl adipate, dimethyl heptamethimide, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, and activated thiol groups (e.g., thiol-reactive 2-pyridinedithiol). Glutaraldehyde is most preferred.

[0056] After the protective layer is formed, the solid carrier containing the disaccharidase and the protective layer can be stored. Storage is typically accomplished, for example, by washing the formed composition with a buffer and suspending or dissolving it in the buffer for the required time. In a preferred embodiment, the solid carrier containing the disaccharidase and the protective layer is stored at a constant temperature of 2 to 25°C. In other preferred embodiments, the solid carrier containing the disaccharidase and the protective layer is stored for 5 to 48 hours, preferably 10 to 30 hours. More preferably, the solid carrier containing the disaccharidase and the protective layer is stored at a constant temperature of 2 to 25°C, preferably at room temperature, for 10 to 30 hours.

[0057] In one implementation, the functional ingredient binds to the mucus.

[0058] In one embodiment, a polymer comprising repeating units in which each repeating unit contains at least one amino group and / or at least one thiol group is a polymer comprising repeating units in which each repeating unit contains at least one amino group.

[0059] In one embodiment, a polymer comprising repeating units wherein each repeating unit contains at least one amino group and / or at least one thiol group is a polymer comprising repeating units wherein each repeating unit contains at least one thiol group.

[0060] In one embodiment, the polymer comprising repeating units is selected from polyglucosamine, polymeric silane-PEG-NH2, and amino-containing polymeric silanes, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. In a preferred embodiment, the polymer comprising repeating units is selected from polyglucosamine, polymeric silane-PEG-NH2, and polymeric APTES, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0061] In a more preferred embodiment, the polymer comprising repeating units is selected from polyglucosamines such as chitosan, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratin, and dermatan or derivatives thereof; polymeric silanes-PEG-NH2; and amino-containing polymeric silanes, preferably polymeric APTES, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. In an even more preferred embodiment, the polymer comprising repeating units is a polyglucosamine, preferably selected from polyglucosamines such as chitosan, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratin, and dermatan or derivatives thereof, more preferably chitosan or derivatives thereof, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0062] The preferred polyglucosamine of this invention is selected from chitosan, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratin, and dermatan, or derivatives thereof. Chitosan or its derivatives are most preferred. The preferred silane-PEG-NH2 is selected from silane-PEG4-NH2, silane-PEG2000-NH2, and silane-PEG5000-NH2. Preferred amino-containing polysilanes are selected from APTES, amino-butyl-TES, amino-pentyl-TES, amino-hexyl-TES, amino-heptyl-TES, and amino-octyl-TES, especially APTES.

[0063] In other embodiments, the polymer comprising repeating units is selected from polyglucosamine, polysilane-PEG-NH2, amino-containing polysilanes, thiol-containing polysilanes, polycarbofibril-cysteine ​​conjugates, polysilane-PEG-thiols, and polycysteine, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. In other more preferred embodiments, the polymer comprising repeating units is selected from polyglucosamine of chitosan, chitosan, polyglucosamine polysaccharide, chondroitin, heparin, keratin, and dermatan or derivatives thereof; polysilane-PEG-NH2; thiol-containing polysilanes, preferably polyMPTS; polycarbofibril-cysteine ​​conjugates; polysilane-PEG-thiols; and polycysteine, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. In even more preferred embodiments, the polymer comprising the repeating unit is polyglucosamine or a thiol-containing polymeric silane, preferably selected from chitosan, chitosan, polyglucosamine polysaccharide, chondroitin, heparin, keratin and dermatan or derivatives thereof, more preferably chitosan or derivatives thereof or thiol-containing polymeric silanes, polycarbofil-cysteine ​​conjugates and polymeric silane-PEG-thiol, preferably thiol-containing polymeric silanes, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0064] In a specific embodiment, the polymer containing repeating units is selected from chitosan, chitosan, polyglucosamine, chondroitin, heparin, keratin, dermatan or derivatives thereof, particularly chitosan or derivatives thereof, polysilane-PEG-NH2 selected from polysilane-PEG4-NH2, polysilane-PEG2000-NH2, polysilane-PEG5000-NH2, amino-containing polysilanes, preferably polymeric APTES, and thiol-containing polysilanes, preferably polymeric MPTS, wherein each repeating unit contains at least one amino group and / or at least one thiol group.

[0065] In one embodiment, the polymer comprising repeating units is selected from polyglucosamine, polymeric silane-PEG-NH2, amino-containing polymeric silanes, and thiol-containing polymeric silanes, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. In a preferred embodiment, the polymer comprising repeating units is selected from polyglucosamine, polymeric silane-PEG-NH2, polymeric APTES, and polymeric MPTS, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0066] In a more preferred embodiment, the polymer comprising repeating units is selected from chitosan, chitosan, polyglucosamine, chondroitin, heparin, keratin, and dermatan, or polyglucosamine derivatives thereof; polymeric silane-PEG-NH2; amino-containing polymeric silanes, preferably polymeric APTES; and thiol-containing polymeric silanes, preferably polymeric MPTS, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. In a specific embodiment, the polymer comprising repeating units is selected from chitosan, chitosan, polyglucosamine, chondroitin, heparin, keratin, dermatan, or derivatives thereof, most specifically chitosan or derivatives thereof, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0067] In one embodiment, the polymer comprising the repeating unit is selected from thiol-containing polymeric silanes, polycarbofil-cysteine ​​conjugates, polymeric silane-PEG-thiol, and polycysteine, and is preferably selected from thiol-containing polymeric silanes, polycarbofil-cysteine ​​conjugates, and polymeric silane-PEG-thiol, and more preferably thiol-containing polymeric silanes, and most preferably polymeric MPTS, wherein each repeating unit comprises at least one thiol group. In one embodiment, the thiol-containing polymeric silane is preferably polymeric MPTS.

[0068] In one embodiment, 5% to 100%, preferably 10% to 100%, more preferably 50% to 100% of the surface of the protective layer is covered by a polymer containing repeating units, wherein each repeating unit contains at least one amino group and / or at least one thiol group.

[0069] In one embodiment, the functional component is fixed to the surface of the protective layer by bonding, preferably covalent bonding. In a preferred embodiment, the functional component is fixed to the surface of the protective layer by non-covalent bonding, preferably by electrostatic interaction. In a more preferred embodiment, a polymer comprising repeating units is fixed to the surface of the protective layer by covalent bonding, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0070] In one embodiment, a spacer that binds to both the protective layer surface and the functional component is used to immobilize the functional component on the surface of the protective layer. Therefore, in one embodiment, the 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 the disaccharidase or fragment thereof, 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, wherein 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 the spacer is typically produced by first reacting the spacer with the functional component, such that the spacer binds to the functional component, and then reacting the functional component bound to the spacer with the surface of the protective layer.

[0071] The immobilization of functional components onto the surface of the protective layer is typically carried out in a reaction vessel, such as a reactor, by suspending a solid carrier carrying the disaccharidase embedded in the protective layer as described above in, for example, water, a buffer solution, or a nonionic surfactant or a mixture thereof, preferably a mixture of water and nonionic surfactants. Nonionic surfactants are typically selected from ethoxylated sorbitol esters such as PEG-40 sorbitol diisostearate, 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 ethoxylates such as PEG-5 oleate and PEG-8 stearate. Esters, polyoxyethylene 40 stearate, polyoxyethylene 15 hydroxy stearate, fatty alcohol ethoxylates such as stearyl alcohol polyether (steareth) 40; fatty acid esters such as palmitic acid ascorbate, beeswax, polyglycerol 3-oleate, propylene glycol monocaprylate, propylene glycol monolaurate; fatty alcohols such as cetearyl alcohol, cetyl alcohol, myristyl alcohol, stearyl alcohol; glycerides; polyethylene glycol-modified triglycerides; glycol esters, preferably polysorbate, more preferably polysorbate 80 (PS80). The functional component is then added to the suspension and reacts with the surface of the protective layer, usually under stirring, to immobilize the functional component on the surface of the protective layer. The resulting composition is usually washed and resuspended in water, buffer solution, or nonionic surfactant or mixtures thereof. Immobilization is carried out through non-covalent bonding, such as electrostatic bonding, or through covalent bonding of the functional component. Functional components can be immobilized by chemically modifying the protective layer and the surface of the functional component using methods such as "click chemistry" (e.g., copper-catalyzed azide-alkyne cycloaddition, see, for example, Kolb et al., (2001) Angew. Chem. 40(11) 2004-2021) or copper-free click chemistry (Wittig G, A Chem Ber, 1961, 94, 3260). For example, as described above, a solid carrier carrying a disaccharidase embedded in the protective layer is first reacted with a reactive compound such as an acetylenic compound, and the functional component is modified by adding a reactive compound such as an azide residue. The two components are then reacted to immobilize the functional component on the surface of the protective layer.

[0072] In other respects, the present invention provides compositions as described above, which are used as pharmaceuticals.

[0073] In another aspect, the present invention provides compositions for methods of preventing, delaying the progression of, or treating lactase deficiency, sucrase-isomaltase deficiency, and / or disaccharide intolerance. In one embodiment, the present invention provides compositions for methods of preventing, delaying the progression of, or treating 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, preferably, congenital sucrase-isomaltase deficiency (CSID).

[0074] The use of the compositions described herein in the preparation of a medicament for the prevention, delay of development, or treatment of lactase deficiency, sucrase-isomaltase deficiency, and / or disaccharide intolerance in an individual is also provided. The use of the compositions described herein for the prevention, delay of development, or treatment of lactase deficiency, sucrase-isomaltase deficiency, and / or disaccharide intolerance in an individual is also provided. A method for the prevention, delay of development, or treatment of lactase deficiency, sucrase-isomaltase deficiency, and / or disaccharide intolerance in an individual is also provided, the method comprising administering to the individual a therapeutically effective amount of the compositions described herein.

[0075] The compositions according to the invention are preferably pharmaceutical compositions and comprise a therapeutically effective amount of the composition described herein and one or more suitable pharmaceutically acceptable carriers. The pharmaceutical compositions of the invention are suitable for oral administration to an individual. Unless otherwise stated, the pharmaceutical compositions of the invention are prepared in a manner known per se.

[0076] Exemplary treatment regimens require administration once daily, twice daily, three times daily, every two days, twice weekly, or once weekly. Compositions, such as the pharmaceutical compositions of the present invention, are typically administered in multiple sessions. The interval between single doses can be, for example, less than one day, daily, every two days, twice weekly, or weekly. Compositions, such as the pharmaceutical compositions of the present invention, can be given as continuous, uninterrupted treatment. Compositions, such as the pharmaceutical compositions of the present invention, can also be given in a manner in which an individual receives a treatment cycle interrupted by a medication holiday or period of no treatment. Therefore, compositions, such as the pharmaceutical compositions of the present invention, can be administered at the intervals selected above for one week or a portion thereof, for two weeks, three weeks, four weeks, five weeks, or six weeks, followed by a break for one week or a portion thereof, for two weeks, three weeks, four weeks, five weeks, or six weeks.

[0077] Compositions, such as the pharmaceutical compositions of the present invention, can be conveniently administered in unit dosage forms. Units of enzyme activity (“U”) can be described as the weight or mass of substrate hydrolyzed per unit time. Units (“U”) can be described as μmol of substrate converted per minute (or μmol / min). In exemplary treatment regimens, the composition comprises 500 U to 20,000 U of disaccharidase; for example, the pharmaceutical compositions of the present invention can be administered daily.

[0078] As used herein, the term "effective amount" or "therapeutic effective amount" means an amount capable of causing one or more desired effects in an individual receiving a composition of the present invention. The determination of a therapeutically effective amount is entirely within the competence of those skilled in the art, especially based on the detailed disclosure provided herein.

[0079] As used herein, the term “treatment / treating” includes: (1) delaying the onset of clinical symptoms of a state, symptom, or condition in animals, particularly mammals, especially humans, who may have or be susceptible to the state, symptom, or condition but have not yet experienced or exhibited clinical or subclinical symptoms of the state, symptom, or condition; (2) suppressing a state, symptom, or condition (e.g., preventing, reducing, or delaying the development or recurrence of a disease, or at least one clinical or subclinical symptom thereof, in the case of maintenance treatment); and / or (3) alleviating a condition (i.e., causing the resolution of at least one of a state, symptom, or condition, or its clinical or subclinical symptoms). The benefit of treatment to a patient is statistically significant or at least perceptible to the patient or physician. However, it should be understood that when a patient is given medication to treat a disease, the outcome may not always be an effective treatment.

[0080] As used in this article, "delaying progression" means increasing the time to onset of symptoms such as lactase deficiency, sucrase-isomaltase deficiency, or disaccharide intolerance, or increasing markers associated with treatment for such conditions, or slowing the increase in the severity of symptoms such as disaccharide intolerance. Furthermore, "delaying progression" as used in this article includes reversing or inhibiting disease progression. "Inhibiting" disease progression or complications in an individual means preventing or reducing disease progression and / or complications in that individual.

[0081] Preventive therapy includes preventive treatment. In preventive use, the drug combination of the present invention is administered to an individual suspected of having or at risk of developing the aforementioned diseases or conditions, such as lactase deficiency, sucrase-isomaltase deficiency, or disaccharide intolerance. In therapeutic use, the drug combination is administered to an individual in an amount sufficient to cure or at least partially stop the symptoms of the disease, such as a patient already suffering from the aforementioned diseases or conditions, such as lactase deficiency, sucrase-isomaltase deficiency, or disaccharide intolerance. The effective dosage for this use will depend on the severity and course of the disease, prior treatment, the individual's health condition and response to the drug, and the judgment of the treating physician.

[0082] If the condition of any individual does not improve, The drug combination of the present invention can be administered for a prolonged period of time, including the entire duration of an individual's life, to improve or otherwise control or limit the symptoms of an individual's disease or condition.

[0083] In cases where an individual's condition has indeed improved, the drug combination can be administered continuously; alternatively, the dosage of the administered drugs can be temporarily reduced or temporarily suspended for a period of time (i.e., a "drug holiday"). Once the patient's condition has improved, a maintenance dose of the drug combination of the present invention is administered, if necessary. Subsequently, the dosage or frequency of administration, or both, is preferably reduced to a level that maintains the improvement in the disease, depending on the symptoms.

[0084] In another aspect, the present invention provides a method for producing the composition described above, for example, a composition comprising a solid carrier, a disaccharidase or a fragment thereof immobilized on the surface of the solid carrier, a protective layer for protecting the disaccharidase or the fragment thereof by embedding the disaccharidase or the fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group; the method

[0085] Includes the following steps: (a) Provide a solid carrier; (b) Immobilize the disaccharidase or a fragment thereof on the solid support; (c) A protective layer is formed on the surface of the solid support to protect the disaccharidase or fragment thereof immobilized on the solid support; (d) A functional component is fixed to the surface of the protective layer, wherein the functional component fixed to the surface of the protective layer is a polymer containing repeating units, wherein each repeating unit contains at least one amino group and / or at least one thiol group.

[0086] Step (a) is typically performed by providing a solid carrier in a suspension of water, a nonionic surfactant, or a buffer solution, preferably a suspension of water and / or a nonionic surfactant, more preferably a suspension of water and / or a nonionic surfactant (wherein the suspension does not contain a buffer solution), even more preferably a suspension of a mixture of water and a nonionic surfactant, particularly a suspension of a mixture of water and a nonionic surfactant (wherein the suspension does not contain a buffer solution). The suspension can be stirred at 20°C for, for example, 400 rpm for 30 min. In step (b) of this method, the immobilization of the disaccharidase on the solid carrier is typically performed by adding a disaccharidase solution to the solid carrier suspension. Preferably, a linker connecting the solid carrier and the disaccharidase is added to the solid carrier suspension before the disaccharidase solution is added. In a preferred embodiment, the immobilization of the disaccharidase on the solid carrier is performed by providing a suspension of the solid carrier and adding a disaccharidase solution, wherein the suspension containing the added disaccharidase solution is incubated to allow the enzyme to bind to the surface of the solid carrier. In a more preferred embodiment, the immobilization of the disaccharidase or its fragment on the solid support in step b) is performed by: i) adding a linker to the solid support provided in step (a), preferably adding the linker to a suspension of the solid support provided in step a), and ii) adding the disaccharidase or its fragment to the solid support and the linker or a suspension containing the solid support and the linker, preferably adding a solution of the disaccharidase or its fragment to the solid support and the linker or a suspension containing the solid support and the linker, wherein the linker connects the solid support to the disaccharidase or its fragment. In one embodiment, prior to adding the solution of the disaccharidase or its fragment, building blocks of the protective layer, preferably monomers of the building blocks of the protective layer, more preferably organosilanes, even more preferably triethoxysilanes, particularly APTES, are added to the solid support and the linker or to a suspension containing the solid support and the linker. In a preferred embodiment, the surface of the solid support is at least partially modified to improve the immobilization of the disaccharidase on the solid support. In particular, the surface of the solid support is at least partially modified prior to the immobilization of the disaccharidase. As described above, the surface of a solid carrier can be modified at least partially by adding molecules that serve as anchoring sites for disaccharidases to the surface of the solid carrier.

[0087] Typically, after each of the above-mentioned addition steps, a suspension containing a solid carrier is incubated to allow, for example, reactions between the solid carrier and the molecules serving as anchoring points, the solid carrier and the linker, and the solid carrier containing the linker and the disaccharidase or fragment thereof, respectively, such that the disaccharidase or fragment thereof is linked to the surface of the solid carrier, preferably by covalent bonding, via the linker, thereby immobilizing the disaccharidase or fragment thereof on the solid carrier.

[0088] In one embodiment, in step (b), the disaccharidase or its fragment is immobilized on the solid support by linking the solid support to the disaccharidase or its fragment via a linker, preferably by linking the solid support to the disaccharidase or its fragment via a linker, wherein the solid support is linked to the disaccharidase or its fragment by covalent bonding between the linker and the solid support and between the linker and the disaccharidase or its fragment. Preferably, in step b), i) the linker is added to the solid support provided in step (a), and ii) the disaccharidase or its fragment is added to the solid support and the linker, wherein the linker links the solid support to the disaccharidase or its fragment. The linker used is as described above and links the surface of the solid support to the disaccharidase by preferably covalent bonding. More preferably, the linker is added to the solid carrier in step (b) in an amount that is a molar excess relative to the disaccharidase or its fragment. Preferably, the linker is added to the solid carrier in step (b) in an amount that is a molar excess of 1 to 1000 times relative to the disaccharidase or its fragment. More preferably, the linker is added to the solid carrier in step (b) in an amount that is a molar excess of 2 to 300 times relative to the disaccharidase or its fragment. Even more preferably, the linker is added to the solid carrier in step (b) in an amount that is a molar excess of 4 to 250 times relative to the disaccharidase or its fragment. In particular, the linker is added to the solid carrier in step (b) in an amount that is a molar excess of 25 times relative to the disaccharidase or its fragment.

[0089] In a preferred embodiment, the linker that did not link the solid support to the disaccharidase or its fragment in step (b) is present during step (c) when a protective layer is formed on the surface of the solid support. In a more preferred embodiment, the linker or a portion thereof that did not link the solid support to the disaccharidase or its fragment in step (b) covalently binds the protective layer to the disaccharidase or its fragment in step (c). In other preferred embodiments, the linker that did not link the solid support to the disaccharidase or its fragment in step (b) is not removed in or between steps (b) and (c). In a specific embodiment, the linker that did not link the solid support to the disaccharidase or its fragment in step (b) is not removed in or between steps (b) and (c), and the linker or a portion thereof that did not link the solid support to the disaccharidase or its fragment in step (b) covalently binds the protective layer to the disaccharidase or its fragment in step (c). The amount of ligand not used to link the solid carrier to the disaccharidase or its fragment in step (b) is typically 30% to 70% of the amount of ligand added to the solid carrier in step (b), preferably 40% to 60%, more preferably about 50%. In one embodiment, there is no washing step between adding the ligand to the solid carrier provided in step (a) in (i) and adding the disaccharidase or its fragment to the solid carrier and ligand in (ii). In one embodiment, there is no washing step between any of steps (a) to (c). In one embodiment, there is no washing step between adding the ligand to the solid carrier provided in step (a) in (i) and adding the disaccharidase or its fragment to the solid carrier and ligand in (ii), and there is no washing step between any of steps (a) to (c).

[0090] In one embodiment, the linker is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide) succinate, dimethyl adipate, dimethyl pimelimidate, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated thiol, thiol-reactive 2-pyridinedithiol, BSOCOES (bis[2-(succinimideoxycarbonyloxy)ethyl] sulfone), DSP (dithiobis[succinimide] propionate), DTSSP (3,3'-dithiobis[succinimide] propionate), DTBP (3,3'-dithiobispropionylimine dimethyl ester·2 HCl), DST (disuccinimidyl tartrate), sulfonyl-LC-SMPT (4-succinimidyl-6-methyl-α-(2-pyridyldithio)toluamide]hexanoate), SPDP (N-succinimidyl-3-(2-pyridyldithio)-propionate), LC-SPDP (succinimidyl-6-(3-[2-pyridyldithio]-propamido)hexanoate), SMPT (4-succinimidyloxycarbonyl-methyl-α-[2-pyridyldithio]toluene), DDPPB (1,4-di-[3'-2'-pyridyldithio)-propionamido]butane), DTME (dithio-bismaleimide ethane), BMDB (1,4-bismaleimide-2,3-dihydroxybutane), and preferably glutaraldehyde.

[0091] In a preferred embodiment, the binder is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide succinate), dimethyl adipate, dimethyl heptamethimide, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimideoxycarbonyloxy)ethyl] sulfone), DSP (dithiobis[succinimide] propionate), DTSSP (3,3'-dithiobis[sulfosuccinimide] propionate), DTBP (3,3'-dithiobispropionine dimethyl ester·2HCl), DST (disuccinimide tartrate), BMDB (1,4-bismaleimide-2,3-dihydroxybutane), and preferably glutaraldehyde.

[0092] The formation of the protective layer in step (c) of the method according to the invention is generally carried out by forming the corresponding protective layer with building units, wherein the building units construct the protective layer in a polycondensation reaction as described above. The immobilization of the functional components on the surface of the protective layer in step (d) of the method according to the invention is generally carried out as described above.

[0093] In one embodiment, the protective layer is formed by building units, wherein structural building units and protective building units are used to form the protective layer, wherein the structural building units are precursors of inorganic silica capable of forming four covalent bonds in the formed layer, and the protective building units are organosilanes as described above.

[0094] In one embodiment, the protective layer encapsulates about 30% to about 100% of the disaccharidase.

[0095] In one embodiment, the solid support is selected from organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, magnetic particles, and titanium particles, and is preferably silica particles, more preferably silica nanoparticles (SNPs).

[0096] A preferred method of the present invention is a method for producing a composition comprising a solid carrier, a disaccharidase or a fragment thereof immobilized on the surface of the solid carrier, a protective layer for protecting the disaccharidase or the fragment thereof by embedding the disaccharidase or the fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, the method comprising the following steps: (a) Providing a solid carrier, wherein the solid carrier is provided in the form of a suspension, preferably wherein the solid carrier is provided in the form of a suspension in water and / or a nonionic surfactant, more preferably wherein the solid carrier is provided in the form of a suspension in a mixture of water and a nonionic surfactant; (b) Immobilizing a disaccharidase or a fragment thereof on a solid support, wherein preferably the surface of the solid support is at least partially modified before immobilizing the disaccharidase or a fragment thereof on the solid support, wherein i) a linker is added to a suspension of the solid support, or i) a linker is added to a suspension of the solid support after at least partially modifying the surface of the solid support, and ii) a solution of a disaccharidase or a fragment thereof, preferably a disaccharidase or a fragment thereof, is added to a suspension of the solid support and the linker, wherein the linker links the solid support to the disaccharidase or a fragment thereof; (c) A protective layer is formed on the surface of a solid carrier to protect the disaccharidase or a fragment thereof fixed on the solid carrier, wherein the linker or a portion thereof that did not connect the solid carrier to the disaccharidase or a fragment thereof in step (b) covalently binds the protective layer to the disaccharidase or a fragment thereof. (d) A functional component is fixed to the surface of the protective layer, wherein the functional component fixed to the surface of the protective layer is a polymer containing repeating units, wherein each repeating unit contains at least one amino group and / or at least one thiol group.

[0097] A composition is also provided comprising a solid carrier, a disaccharidase or a fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the disaccharidase or the fragment thereof by embedding the disaccharidase or the fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, wherein the composition can be obtained by the method described above, particularly by the preferred method of the present invention as described above.

[0098] Example

[0099] Example

[0100] Materials and methods:

[0101] Reagents:

[0102] - Tetraethyl orthosilicate 99% (TEOS), (3-aminopropyl)-triethoxysilane (APTES), ammonium hydroxide (ACS grade, 28-30%), ethanol (ACS grade, anhydrous), glutaraldehyde (Grade I, 25% aqueous solution), polysorbate 80, acetic acid, lactase (USP reference standard), invertase, bovine serum albumin (BSA), invertase activity assay kit, lactose, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), purchased from Sigma-Aldrich. BSA, lactase, and invertase were dissolved in water to reconstitute the stock buffer.

[0103] Chitosan 95 / 500P, purchased from Heppe Medical Chitosan GmbH

[0104] Caco-2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line), European Collection of Cell Cultures (ECACC).

[0105] -THP-1 (human acute monocytic leukemia cell line) was purchased from LGC.

[0106] -ThinCert™ cell culture inserts (1.0 μm membrane), purchased from Greiner bio-one.

[0107] 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's phosphate-buffered saline DPBS (1X), 0.25% trypsin-EDTA (1X), RPMI 1640 medium, DMEM, HEPES, sodium pyruvate, D-glucose, β-mercaptoethanol, purchased from Gibco.

[0108] -Matrigel® Low Growth Factor (GFR) Basement Membrane Matrix, LDEV-free, purchased from Corning.

[0109] - 200 g of polysaccharide modified with Altromin 1319 and AIN 93G for animal feed, purchased from Altromin International

[0110] - Catheter, purchased from Instech Laboratories

[0111] - TB100 Holtex glucose meter kit, purchased from MediSafe

[0112] - Oligo-α-1,6-glucosidase 13A from Bifidobacterium adolescentis, 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.

[0113] Synthesis of silica nanoparticles: Silica nanoparticles (50 nm) were synthesized according to the original Stöber method described in WO2015 / 014888A1. Briefly, ethanol, distilled water (6 M), and ammonium hydroxide (0.13 M) were mixed and stirred at 400 rpm for 1 h. TEOS (0.28 M) was added, and the solution was stirred at 400 rpm for 22 h at 20 °C. The solution was then centrifuged at 20,000 g for 20 min and washed successively with ethanol and water. Particle size was measured using SEM micrographs obtained at 150,000x magnification using the Olympus flow motion image analysis software.

[0114] Enzyme shielding and surface functionalization

[0115] -Production of NP-2: APTES (3.9 mM) was added to the SNP (10 mg / mL, 55 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.9 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.9 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Lactase (7 mg / mL, 0.1 mM) was added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized lactase surface using APTES (8.4 mM) and TEOS (125.9 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 121 μg / mL. The reaction mixture was reacted at 20 °C and 400 rpm for 30 min. The particles were centrifuged at 20000 rcf for 5 min and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-2 was cured overnight in a 20 °C water bath.

[0116] - NP-2 variants: The following experiments investigated the effects of covalently linking the enzyme to the protective layer on enzyme stability and enzyme activity.

[0117] In the first experiment, nanoparticles (NP-2(1)) were generated in H2O / PS80 (8 mg / L). The nanoparticles were washed after each chemical step to remove glutaraldehyde. APTES (3.3 mM) was added to the 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 min. The particles were washed three times in H2O / 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 min. The particles were washed three times in H2O / PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Initiation was performed by adding APTES (3.3 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. The particles were washed three times in H2O / 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 min. An organosilicon 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 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). NP-2(1) was solidified overnight in a water bath at 20 °C.

[0118] In the second comparative experiment, enzyme immobilization and protective layer formation were performed according to WO2015 / 014888A1 to generate nanoparticles (NP-2(2)) in buffer. The nanoparticles were washed after each chemical step to remove glutaraldehyde. APTES (3.3 mM) was added to SNP (10 mg / mL, 69 nm) in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). The reaction mixture was reacted at 20°C and 400 rpm for 10 min. 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 min. 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). Initiation was performed by adding APTES (3.3 mM), and the reaction mixture was stirred at 20°C and 400 rpm for 10 min. 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). Lactase (5.2 mg / mL, 0.1 mM) was added, and the reaction mixture was reacted at 20°C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized lactase surface using APTES (6.5 mM) and TEOS (93 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 h. 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 water bath at 20°C.

[0119] In the third experiment, nanoparticles (NP-2) were generated in H2O / PS80 (8 mg / L), according to the section titled "Generation of NP-2" above. To maintain an excess of glutaraldehyde that did not bind the solid support to lactase in the reaction mixture, the nanoparticles were not washed between each chemical step. Therefore, the glutaraldehyde remained during layer growth and resulted in the covalent binding of the protective layer to lactase. This covalent binding of the protective layer to lactase can be observed as a yellow / orange appearance with maximum absorbance at 460 nm. This color is due to the formation of imine bonds between the aldehyde functional group of the glutaraldehyde linker and the primary amine of the lactase amino acid and the organosilicon layer. After the formation of the silicone layer and final particle washing, i.e., after the formation of the silicone layer, the absorbance of nanoparticles NP-2(1), NP-2(2), and NP-2 at 460 nm was measured. The particles were washed three times in H2O / PS80 and then resuspended in H2O / PS80, as described in the section titled "NP-2 Generation" above. This showed that the absorbance of NP-2 at 460 nm was higher than that of NP-2(1) and NP-2(2) (see [link]). Figure 7 Since imine bonds are also formed during enzyme immobilization, NP-2(1) and NP-2(2) still show a certain degree of absorbance at this wavelength. However, the absorbance of NP-2 is significantly higher, indicating that the covalent binding of the protective layer with lactase leads to the formation of additional imine bonds.

[0120] - Production of NP-3: APTES (3.8 mM) was added to the SNP (10 mg / mL, 56 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.8 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.8 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Invertase (1.726 mg / mL, 0.03 mM) was added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the surface of the immobilized invertase using APTES (5.4 mM) and TEOS (81.3 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 115 μg / mL. The reaction mixture was reacted at 20 °C and 400 rpm for 30 min. The particles were centrifuged at 20000 rcf for 5 min and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-3 was cured overnight in a 20 °C water bath.

[0121] - Production of NP-4: APTES (3.6 mM) was added to the SNP (10 mg / mL, 59 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.6 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.6 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Isomaltase (3.55 mg / mL, 0.05 mM) was added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the surface of the immobilized invertase using APTES (5.8 mM) and TEOS (88 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 82 μg / mL. The reaction mixture was reacted at 20 °C and 400 rpm for 30 min. The particles were centrifuged at 20000 rcf for 5 min and washed three times in H2O / PS80 (8 mg / L). NP-4 was cured overnight in a water bath at 20 °C.

[0122] - Production of NP-5: APTES (3.6 mM) was added to the SNP (10 mg / mL, 59 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.6 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.6 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Isomaltase (1.77 mg / mL, 0.025 mM) and invertase (4.05 mg / mL, 0.07 mM) were added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the surface of the immobilized enzyme using APTES (5.8 mM) and TEOS (88 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 82 μg / mL. The reaction mixture was reacted at 20 °C and 400 rpm for 30 min. The particles were centrifuged at 20000 rcf for 5 min and washed three times in H2O / PS80 (8 mg / L). NP-5 was cured overnight in a water bath at 20 °C. Production of NP-1: APTES (3.8 mM) was added to the SNP (10 mg / mL, 56 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.8 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.8 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. BSA solution was added to bring the final BSA concentration to 1.42 mg / mL, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized BSA surface using APTES (7.5 mM) and TEOS (75.4 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 121 μg / mL. The reaction mixture was reacted at 20 °C and 400 rpm for 30 min. The particles were centrifuged at 20000 rcf for 5 min and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-1 was cured overnight in a water bath at 20 °C. SNPs-BSA-AT was cured overnight at 20 °C.

[0123] Disaccharidase activity assay: - Lactase activity assay Add lactose solution (100 μL, 50 mg / mL) to the suspension of NP-2 (30 μL, 2.3 mg / mL) in phosphate buffer (100 mM, pH 6.5) / MgCl2 (5 mM). Incubate the reaction mixture in a hot mixer at 37 °C and 750 rpm for 20 min. Collect samples every 2.5 min and monitor glucose formation using a glucometer.

[0124] - Invertase activity assay

[0125] NP-3 activity was assessed using the Sigma invertase assay kit. NP-3 (94 μL, 147 μg / L) was added to 1X reaction buffer (94 μL). Then, 1X sucrose solution (11.76 μL) was added. The reaction mixture was incubated in a hot mixer at 37°C and 300 rpm for 20 min. The sample was centrifuged at 20,000 rcf for 5 min. The supernatant was collected, and 85 μL was transferred to a 96-well plate. 90 μL of the master reaction mixture (prepared by mixing the enzyme mixture, dye reagent, and assay buffer) was added to each well. The reaction mixture was incubated in the dark at room temperature for 20 min. The absorbance was measured at λ = 570 nm.

[0126] - Isomaltulase activity assay

[0127] NP-4 activity was assessed using a glucose meter. Isomaltose solution (25 μL, 100 mM) was equilibrated at 37°C and 700 rpm for 5 min. Then, NP-4 (25 μL, 67 μg) in 50 mM pH 6.8 phosphate buffer was added to the isomaltose solution. The reaction mixture was incubated at 37°C and 700 rpm for 10 min. Samples were collected after 2 min, 5 min, and 10 min, and glucose concentrations were determined using a glucose meter.

[0128] - Co-immobilized isomaltulase-invertase activity assay

[0129] NP-5 isomaltase activity was assessed using a glucose meter. An isomaltose solution (25 μL, 100 mM) was equilibrated at 37°C and 700 rpm for 5 min. Then, NP-5 (25 μL, 67 μg) in 50 mM pH 6.8 phosphate buffer was added to the isomaltose solution. The reaction mixture was incubated at 37°C and 700 rpm for 10 min. Samples were collected after 2 min, 5 min, and 10 min, and glucose concentration was determined using a glucose meter.

[0130] NP-5 convertase activity was assessed using a glucose meter. A sucrose solution (25 μL, 100 mM) was equilibrated at 37°C and 700 rpm for 5 min. Then, NP-5 (25 μL, 67 μg) in 50 mM pH 6.8 phosphate buffer was added to the sucrose solution. The reaction mixture was incubated at 37°C and 700 rpm for 10 min. Samples were collected after 2 min, 5 min, and 10 min, and glucose concentration was determined using a glucose meter.

[0131] Cell culture : For all experiments, cells were cultured at 37°C and 5% CO2.

[0132] Caco2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids and 100 U / mL penicillin / streptomycin.

[0133] THP-1 (human monocytic leukemia cell line) cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine and 100 U / mL penicillin / streptomycin.

[0134] To differentiate THP-1 cells into macrophages, THP-1 cells were cultured in differentiation medium: RPMI 1640 containing 10% heat-inactivated 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 mercaptoethanol. THP-1 cells were differentiated into MO-macrophages by incubation with 150 nM phorbol 12-myristate 13-acetate (PMA) for 24 h, followed by incubation in differentiation medium for 24 h.

[0135] - Intestinal barrier model

[0136] To develop an intestinal barrier model, cells were stored at a density of 2.6 × 10⁻⁶. 5 cells / cm 2 Cells were seeded at a density in Transwell PET inserts (1 μm pores). All cell models were used for experiments on day 21. For co-culture, Caco-2 and HT-29-MTX-E12 cells were used at a ratio of 75%–25%.

[0137] - Immune competent intestinal barrier model

[0138] To develop an immune-active 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 side of the permeable membrane containing a previously differentiated co-culture with 25% Matrigel solution via a dropwise method.

[0139] Trans-epithelial electrical resistance

[0140] Cell barrier integrity was assessed by measuring transepithelial electrical resistance (TEER) using the CellZscope system (NanoAnalytics). TEER was automatically measured every 15 minutes for 24 h, in the range of 1 Hz to 100,000 Hz, after cell culture medium renewal and treatment with nanoparticles.

[0141] In vitro digestion of sucrose

[0142] On the top side of the intestinal barrier, differentiated Caco-2 / HT29-MTX-E12 cocultures in PBS were exposed to NP-3 (0.5 mU and 1 mU) containing sucrose for 4 h. At each time point, 150 μL aliquots were removed from the basal side of the intestinal barrier and replaced with the same volume of preheated PBS. The barrier was further incubated at 37 °C. The absorbance of the removed aliquots was measured at 570 nm to quantify glucose levels.

[0143] Animals:

[0144] All animal experiments were conducted with permission from the National Animal Experiments Inspectorate under the Ministry of Food, Agriculture and Fisheries of Denmark.

[0145] The study was conducted on male Wistar rats (8 weeks old) of the original breed from Janvier, France.

[0146] - Diet and drinking water: To maintain their diet, the rats were fed an readily available complete pelleted diet, "Altromin 1319". They had free access to drinking water.

[0147] One week before treatment and during the experiment, rats were fed a low-sugar diet (200 g polysaccharide modified with AIN 93G) that was readily available.

[0148] - Duodenal catheterization

[0149] The animal was placed in the induction chamber and anesthetized with isoflurane (2-4%), then transferred to a nasal cannula with isoflurane for surgery. A catheter (C30PU-RDD1444, Instech Laboratories) was placed on the opposite side of the duodenal mesentery, near the opening of the bile and pancreatic ducts. The catheter was ligated and secured to the intestinal wall and then tunneled through a subcutaneous tunnel to the animal's neck, exposing it. The abdominal and cervical incisions were then sutured shut. Throughout the procedure, the animal was kept warm and closely monitored until fully recovered from anesthesia.

[0150] - Dosing and lactose administration

[0151] Animals were starved for 4 hours before administration of the drug and lactose. Then, rats were administered NP-2 (97 U), NP-1 (54 mg), or a solvent (1.5 mL NaCl, 0.9% polysorbate 80 mg / mL) via the duodenum, followed immediately by g of lactose via gavage. This administration and gavage were repeated daily for 15 days.

[0152] - Cecal analysis

[0153] At the end of the experiment, all animals were scanned under complete anesthesia (scan time approximately 5 min) using a rat volume coil on a Bruker Pharmascan 7 Tesla to assess cecal size. Target regions were plotted on the cecum in all obtained sections. MRI images were used to determine cecal volume.

[0154] During autopsy, photographs of the gastrointestinal tract are taken to observe the dilation of the cecum.

[0155] result: Example 1: Enhancing enzyme loading by covalent attachment to the protective layer In the first experiment, nanoparticles NP-2(1) were generated under unbuffered conditions, with washing (i.e., removal of glutaraldehyde before layer growth) included after each chemical step. In the second experiment, nanoparticles NP-2(2) were generated under buffered conditions, with washing (i.e., removal of glutaraldehyde before layer growth) included after each chemical step. In the third experiment, nanoparticles NP-2 were generated under unbuffered conditions without any intermediate washing steps (i.e., unreacted glutaraldehyde remained in the reaction mixture during layer growth).

[0156] Protein quantification was performed on the reaction supernatant to determine the lactase immobilization yield on the surfaces of NP-2(1), NP-2(2), and NP-2. The results showed that, surprisingly, enzyme immobilization in the presence of glutaraldehyde (NP-2) increased the enzyme immobilization yield by twenty-sixfold. Figure 2 A), resulting in a 24-fold increase in SNP enzyme loading per unit dry weight compared to buffered conditions where glutaraldehyde is removed via a washing step (NP-2(2)). Figure 2 B). Similarly, enzyme immobilization under conditions maintaining glutaraldehyde (NP-2) resulted in an enzyme loading per unit dry weight of SNP ( Figure 2 B) is 2 times higher than the unbuffered conditions (NP-2(1)) in which glutaraldehyde is removed by a washing step.

[0157] In summary, compared with enzymes protected by an organosilicon layer solely through electrostatic interactions, the covalent connection between the protective layer and the enzyme surface unexpectedly enhanced its loading capacity.

[0158] Example 2:Disaccharidase activity of NP-2, NP-3, NP-4, and NP-5

[0159] The biocatalytic activities of four different immobilized and protected disaccharidases were evaluated. Figure 3 The results shown report the enzyme activity of each nanoparticle: lactase activity of NP-2 ( Figure 3 A), NP-3 convertase activity ( Figure 3 B), NP-4 isomaltase activity ( Figure 3 C) and NP-5 dual enzyme activities (isomaltase and invertase) Figure 3 D). These data indicate that the disaccharide reaches the catalytic site of the enzyme and is cleaved with high enzymatic activity. Validation of the biocatalytic activity of NP-2, NP-3, NP-4, and NP-5 confirms the possibility of applying immobilization and protection strategies to a variety of disaccharidases that can be used for therapeutic purposes.

[0160] Example 3: Biocompatibility of nanoparticles for gastrointestinal applications

[0161] The intestinal mucosa is composed of a single layer of epithelial cells, which are adjacent to each other and contain a lamina propria (Laminaria stenosis). lamina propria The lamina propria, located beneath the epithelium, is a region of tight junctions between cells. It acts as a barrier between the external and internal environments. Its integrity is a key parameter ensuring protection of the body from unwanted contaminants such as microorganisms. The lamina propria comprises diffuse lymphoid tissue composed of immune cells that maintain homeostasis or respond to disruptions of epithelial protection.

[0162] To evaluate the biocompatibility of nanoparticles, we developed representative models NP-2 and NP-3. NP-2 consists of NP-1, a nanoparticle with the same functional outer surface as NP-2 and NP-3 but without enzymatic activity.

[0163] To evaluate the safety of the nanoparticles, we first focused on their effect on maintaining intestinal barrier integrity in the presence of NP-1. Figure 4 A). Transepithelial electrical resistance (TEER) measurements at both ends of a Caco2-HT29-MTX-E12 cell monolayer showed that the integrity of the intestinal epithelial barrier remained intact after 24 h of contact with NP-1. This result demonstrates the effectiveness of NP-1. In vitro Biocompatibility.

[0164] To further characterize the impact of nanoparticles on the intestinal barrier, we evaluated the ability of NP-1 to trigger an inflammatory response. For this purpose, we developed an immune-active intestinal barrier model and monitored its integrity. Figure 4 B shows that barrier integrity decreased in a dose-dependent manner when treated with LPS (a pro-inflammatory component). This loss of integrity reveals the recruitment of macrophages from the basal to the apical side of the barrier.

[0165] Most importantly, TEER measurements showed that the integrity of the epithelial barrier remained intact upon contact with NP-1. Figure 4 B). NP-1 does not stimulate the recruitment of macrophages on the apical side of the intestinal barrier, leading to the conclusion that NP-1 does not induce inflammation.

[0166] In summary, these results demonstrate the potential of nanoparticles in gastrointestinal applications. In vitro Security.

[0167] Example 4: In vivo efficacy of NP-2

[0168] Lactose malabsorption is attributed to 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 lactose fermentation leads to the production of short-chain fatty acids and gases, significantly contributing to cecal enlargement.

[0169] In rats fed a high dose of lactose daily via tube feeding for 15 days, greater substrate availability for fermentation in the large intestine led to cecal enlargement compared to rats fed a normal diet without additional lactose intake (conditions: "solvent" vs. "lactose-free"). Figure 5 Importantly, a significant reduction in cecal size was reported in rats treated with NP-2, while administration of inactive nanoparticles (NP-1) had no effect on the reduction in cecal size. These results demonstrate the effect of NP-2 on lactose digestion. In vivo Biocatalytic activity.

[0170] Enzyme replacement therapy with exogenous lactase from microorganisms is feasible, but results regarding the exact efficacy are inconsistent (Montalto). et al ., World J Gastroenterol 2006, Jan 14;12(2):187-91). The rule for calculating the amount of lactase is 7500 units per 16 grams of lactose. In addition to this close relationship between the amount of lactose to be hydrolyzed and the required enzyme units, gastric pH and bile salt concentration affect the efficacy of exogenous lactase and the lack of specific localization of the enzyme in the intestine.

[0171] exist Figure 5 In the middle, NP-2 with a dose of 3 g lactose at 97 units In vivo The efficacy has been demonstrated. This dose is surprisingly equivalent to 14.5 times the dose of currently available lactase preparations. Therefore, these data demonstrate the high value of NP-2 in lactose digestion and highlight its therapeutic potential for patients with disaccharide digestion deficiencies, such as lactose malabsorption.

[0172] Example 5: In vitro efficacy of NP-3

[0173] Congenital sucrase-isomaltase deficiency (CSID) is characterized by a complete or near-complete lack of sucrose activity and varying degrees of reduction in isomaltase activity.

[0174] The sucrose digestion efficacy of NP-3 was evaluated in an intestinal barrier model. Differentiated Caco2-HT29-MTX-E12 cell monolayers were exposed to NP-3 for 4 h in the presence of their substrate on their apical side. Quantification of sucrose hydrolysates in the basal compartment of the barrier was observed. Figure 6 The figure reports a dose-dependent accumulation of glucose on the lateral aspect of the intestinal barrier with increased NP-3 levels, whereas glucose was not detected in the untreated intestinal barrier. This result demonstrates the role of NP-3 in... In vitro The efficacy was discussed, and the use of NP-3 in therapeutic applications was highlighted.

Claims

1. A composition comprising a solid carrier, a disaccharidase or a fragment thereof immobilized on the surface of the solid carrier, a protective layer for protecting the disaccharidase or the fragment thereof by embedding the disaccharidase or the fragment thereof, 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, wherein each repeating unit comprises 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 is a polyglucosamine selected from chitosan, chitosan, polyglucosamine, chondroitin, heparin, keratin and dermatan or derivatives thereof, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

3. The composition according to any one of claims 1-2, wherein the polymer comprising repeating units is chitosan or a derivative thereof, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

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

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

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

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

8. The composition according to any one of claims 1-7, wherein the protective layer encapsulates the solid carrier and encapsulates the disaccharidase or fragment thereof fixed on the surface of the solid carrier.

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

10. The composition according to any one of claims 1-9, used as a medicine.

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

12. A method of producing a composition comprising a solid support, a disaccharidase or a fragment thereof immobilized on the surface of the solid support, a protective layer protecting the disaccharidase or the fragment thereof by embedding the disaccharidase or the fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, the method comprising the following steps: (a) Provide a solid carrier; (b) Immobilize the disaccharidase or a fragment thereof on the solid support; (c) A protective layer is formed on the surface of the solid support to protect the disaccharidase or fragment thereof immobilized on the solid support; (d) A functional component is fixed on the surface of the protective layer, wherein the functional component fixed on the surface of the protective layer is a polymer containing repeating units, wherein each repeating unit contains at least one amino group and / or at least one thiol group.

13. The method of claim 12, wherein in step (b), i) a linker is added to the solid support provided in step (a), and ii) a disaccharidase or a fragment thereof is added to the solid support and the linker, wherein the linker links the solid support to the disaccharidase or a fragment thereof.

14. The method of claim 13, wherein the linker that did not link the solid support to the disaccharidase or a fragment thereof in step (b) is present during step (c) when a protective layer is formed on the surface of the solid support.

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

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

17. The method according to any one of claims 13-16, wherein the linker or a portion thereof that did not link the solid carrier to the disaccharidase or a fragment thereof in step (b) covalently binds the protective layer to the disaccharidase or a fragment thereof in step (c).

18. The method according to any one of claims 13-17, wherein the binder is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide succinate), dimethyl adipate, dimethyl heptamethimide, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimideoxycarbonyloxy)ethyl] sulfone), DSP (dithiobis[succinimide] propionate), DTSSP (3,3'-dithiobis[sulfosuccinimide] propionate), DTBP (3,3'-dithiobispropionine dimethyl ester·2HCl), DST (disuccinimide tartrate), BMDB (1,4-bismaleimide-2,3-dihydroxybutane).

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

20. A composition comprising a solid carrier, a disaccharidase or a fragment thereof immobilized on the surface of the solid carrier, a protective layer for protecting the disaccharidase or the fragment thereof by embedding the disaccharidase or the fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, wherein the composition is obtained by the method of any one of claims 14-19.

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