Production method for a stabilised diamine oxidase lyophilisate and pharmaceutical dosage form

Abstract

The invention relates to a method for the production of a stabilised lyophilisate with plant diamine oxidase (DAO), comprising the following steps: providing DAO-containing plants or DAO-containing parts thereof; homogenising the DAO-containing plants or DAO-containing parts thereof in aqueous solution in order to obtain an aqueous homogenised product; adding (a) a sugar or sugar alcohol and (b) an amino acid as stabiliser; and lyophilising the aqueous homogenised product or a DAO-containing fraction thereof in the presence of the stabiliser. The invention also relates to a lyophilisate obtained according to this method, and to a solid pharmaceutical dosage form for oral administration, containing said lyophilisate. This dosage form is intended for therapeutic use, preferably in the treatment of histamine-induced disorders, or can be used as a food supplement or dietetic food.

Description

[0001] Manufacturing process for a stabilized diamine oxidase lyophilisate and pharmaceutical dosage form

[0002] The present invention relates to a process for producing a stabilized lyophilisate with plant diamine oxidase (DAO), as well as a stabilized lyophilisate with plant DAO.

[0003] Histamine (2-(4-imidazolyl)ethylamine) is a biogenic amine that is involved in many physiological processes in the body, but is also found in some foods. Histamine can trigger various, often non-specific symptoms in people suffering from histamine intolerance, which frequently resemble allergic reactions.

[0004] High concentrations of free circulating histamine can trigger symptoms such as headache, nasal congestion, airway obstruction, tachycardia, and extrasystoles, as well as gastrointestinal disturbances, which can lead to loose stools and diarrhea, and hypotension. Swelling of the eyelids and, occasionally, urticarial rashes are also often described. Skin reddening, hypotension, and bronchospasms may also occur.

[0005] DAO (EC 1 . 4 . 3 . 22 ) is an enzyme found in various plants, animals, and microorganisms and is responsible for the degradation of biogenic amines, particularly histamine. DAO is also known as histaminase.

[0006] Various DAO-containing preparations are known in the state of the art which can be taken with meals by people suffering from histamine intolerance in order to support the breakdown of histamine from food in the intestine and thereby reduce or completely prevent the undesirable symptoms caused by the histamine ingested with food.

[0007] WO 2006 / 003213 A1 discloses pharmaceutical compositions, food supplements, and cosmetic compositions comprising DAO and their use. In particular, the DAO is formulated to be enteric-coated. Animal sources for the DAO are preferred, especially DAO from pig kidneys.

[0008] However, plant-based DAO preparations have also gained importance in recent years. Among other things, the general acceptance of herbal medicines and dietary supplements is higher, especially among people with ethical or religious concerns about the use of animal products. Furthermore, obtaining DAO from sustainably cultivated plants can be more environmentally and climate-friendly than obtaining it from animal sources.

[0009] For example, US 2014 / 004179 A1 concerns a composition containing DAO for use in the treatment or prevention of fibromyalgia or chronic fatigue syndrome. The DAO can also be derived from plant sources.

[0010] WO 02 / 43745 A2 relates to a histaminase of plant origin for use in the treatment of allergic and septic shock and allergic asthma.

[0011] Proteins, and enzymes in particular, are susceptible to physical and chemical degradation and are therefore inherently unstable over extended storage periods. This also applies to plant-based DAO, where the instability of the DAO enzyme is reflected in the loss of enzymatic activity, which increases over time.

[0012] The production of pharmaceutical dosage forms and dietary supplements containing plant-based DAO therefore requires a suitable stabilization step. A common method for stabilizing enzymes and other biological materials is lyophilization or freeze-drying. This process involves freezing the enzyme-containing solutions, followed by the removal of water by sublimation under vacuum at low temperatures. However, lyophilization can also contribute to the instability of proteins and enzymes because of the physical stress exerted on the proteins due to phase transitions. In addition, excipients that serve to stabilize the protein structure during lyophilization and are therefore added before lyophilization do not necessarily ensure the stability of the proteins during long-term storage (as a lyophilisate) and can sometimes even impair this stability.

[0013] The most common mechanisms leading to long-term protein degradation are hydrolysis, oxidation, and deamination. Stabilizers are often added as countermeasures to protect proteins. The choice of these stabilizers has a major influence on the long-term stability of proteins. It also depends on the protein or enzyme to be stabilized, as they do not necessarily behave the same during lyophilization or subsequent storage. Padma et al. (Process Biochemistry 43.10 (2008): 1019-1032) is a review article on the topic of enzyme stabilization in aqueous and non-aqueous environments.

[0014] Ö' Fägäin (Enzyme and Microbial Technology 33.2-3 (2003) : 137- 149) is another review article on the topic of enzyme stabilization.

[0015] Forney-Stevens , et al. (Journal of Pharmaceutical Sciences 105.2 (2016) : 697-704) is an article in which the use of

[0016] Amino acids were investigated as excipients to improve the stability of lyophilized sucrose-based protein formulations.

[0017] Al emany-Fernes, et al. (bioRxiv (2023): 2023-04) is a preprint investigating the DAO activity of various commercially available over-the-counter (OTC) preparations. To ensure the long-term stability of herbal DAO, suitable stabilizers must be used to provide the best possible protection for the herbal DAO both during the lyophilization process and during long-term storage.

[0018] Megoura et al. (Molecules 28 (2023): 992) investigated improving the stability of DAO from peas (Pisum sativum) using trehalose or sucrose. The DAO was lyophilized in the absence or presence of sucrose or trehalose, and the stability of the resulting preparations was monitored during storage at 4°C and -20°C for 18 months. Tablets were also formulated from the lyophilizates stored at -20°C by direct compression. The tablets were stored at 4°C, and their stability was monitored for 6 months. The DAO powders freeze-dried with sucrose or trehalose stabilizer, or the tablets prepared from them, exhibited higher enzyme activity after storage compared to DAO powders or tablets that did not contain sucrose or trehalose.

[0019] However, this study did not conduct stability tests at room temperature (25°C or higher, e.g., 30°C). However, these storage conditions are of high practical relevance, as the logistical effort associated with the continuous refrigeration of pharmaceuticals or dietary supplements is associated with high costs and is therefore generally unacceptable. In addition, due to non-linear effects on protein stability, quantitative conclusions regarding the effects of stabilizers at room temperature cannot always be drawn from observations made at significantly lower temperatures. Due to the complex interaction between the individual potential stabilizers and the DAO enzyme, predicting the effect an excipient or class of excipients will have on protein stability is difficult.There is therefore still a need for further optimization of the excipients for stabilizing plant DAO during and after lyophilization, especially during subsequent storage at room temperature.

[0020] It is therefore an object of the present invention to provide a process for producing a stabilized lyophilisate with plant DAO and a corresponding stabilized lyophilisate, wherein the lyophilisate should remain as stable as possible even under practically relevant conditions (such as storage at room temperature and / or increased air humidity), i.e. should have the lowest possible loss of activity.

[0021] In a first aspect, the present invention provides a process for the preparation of a stabilized lyophilisate with plant DAO, comprising the following steps:

[0022] - Providing DAO-containing plants or DAO-containing parts thereof (in particular plant shoots);

[0023] - homogenizing the DAO-containing plants or DAO-containing parts thereof in aqueous solution to obtain an aqueous homogenate;

[0024] - adding ( a ) a sugar or sugar alcohol and ( b ) an amino acid (these preferably serve as stabilizers); and

[0025] - Lyophilizing the aqueous homogenate or a DAO-containing fraction thereof in the presence of ( a ) the sugar or sugar alcohol and ( b ) the amino acid .

[0026] Furthermore, the present invention provides a stabilized lyophilisate with plant DAO, which is obtainable by this process.

[0027] In a further aspect, the present invention provides a stabilized lyophilisate with plant-derived DAO, comprising (a) a sugar or sugar alcohol and (b) a free amino acid. These two components preferably serve as stabilizers (in other words, components (a) and (b) are preferably present as stabilizers).

[0028] A free amino acid is an amino acid that is not part of a peptide or a protein.

[0029] Furthermore, a solid pharmaceutical dosage form (in particular a capsule or tablet) is provided which contains one of the above-mentioned lyophilisates according to the invention, optionally in compressed form. This dosage form can be used for therapeutic purposes, preferably in the treatment of histamine-induced disorders, or as a dietary supplement or dietary food.

[0030] Surprisingly, it was found that combinations of a sugar or sugar alcohol with an amino acid (particularly those specifically disclosed below) are particularly well suited for stabilizing plant-derived DAO, especially with regard to long-term storage, and particularly at room temperature (and a certain level of humidity). It has been shown that the stabilization effect resulting from the presence of these two components is additive or even synergistic.

[0031] In a preferred embodiment of the process according to the invention, the amino acid is a free amino acid.

[0032] In a preferred embodiment of the method according to the invention or of the lyophilisate according to the invention, the amino acid or the free amino acid is preferably selected from the group consisting of basic amino acids such as arginine, histidine, and lysine, as well as the amino acids asparagine, glutamine, cysteine, homocysteine, methionine, tryptophan, and tyrosine. Particular preference is given to basic amino acids, in particular arginine, histidine, and lysine, preferably arginine.

[0033] It goes without saying that the free amino acid can also be used according to the invention in the form of a pharmaceutically acceptable salt. For arginine, for example, this is arginine hydrochloride.

[0034] Consequently, in a further preferred embodiment, the amino acid is present (before addition) in a pharmaceutically acceptable salt form, preferably as a hydrochloride, sulfate, acetate, phosphate, tartrate, or citrate salt, in particular arginine hydrochloride, arginine sulfate, arginine acetate, histidine hydrochloride, histidine sulfate, histidine acetate, lysine hydrochloride, lysine sulfate, or lysine acetate. For example, an aqueous solution of the salt form used for addition can be prepared in advance. Arginine hydrochloride is particularly preferred.

[0035] A further preferred embodiment of the process according to the invention or of the stabilized lyophilisate according to the invention provides that the sugar or sugar alcohol is selected from the group consisting of disaccharides such as trehalose, sucrose, lactose, and maltose, trisaccharides such as raffinose, polysaccharides such as maltodextrin, and the sugar alcohols mannitol, sorbitol, glycerol, xylitol, erythritol, and inositol. In a particularly preferred embodiment, the sugar is a disaccharide, in particular trehalose.

[0036] In a further preferred embodiment of the process according to the invention, (a) the sugar or sugar alcohol is preferably added in an amount of at least 10 wt.% of the homogenate, more preferably at least 20 wt.%, even more preferably at least 30 wt.%, in particular at least 40 wt.% or even at least 50 wt.% of the homogenate and / or (b) the amino acid or the free amino acid is preferably added in an amount of at least 20 wt.% of the homogenate, more preferably at least 40 wt.%, even more preferably at least 60 wt.%, in particular at least 80 wt.% or even at least 100 wt.% of the homogenate (on a weight basis) as a stabilizer.In a further preferred embodiment of the stabilized lyophilisate with plant DAO according to the invention, the lyophilisate comprises (a) a sugar or sugar alcohol in a weight ratio of at least 1:1 or even at least 2:1, preferably of at least 4:1 or even at least 7:1, preferably at least 10:1 or even at least 13:1, more preferably at least 16:1 or even at least 19:1, in particular at least 22:1 or even at least 25:1 to the total protein content of the lyophilisate, and / or (b) a free amino acid in a weight ratio of at least 1:1, preferably of at least 4:1 or even at least 7:1, preferably at least 10:1 or even at least 18:1, more preferably at least 25:1 or even at least 32:1, in particular at least 40:1 or even at least 50:1 to the total protein content of the lyophilisate.

[0037] In other words, it is preferred that both the sugar or sugar alcohol and the amino acid be present in excess of the total protein content on a weight basis.

[0038] In a further preferred embodiment of the method according to the invention or of the stabilized lyophilisate according to the invention, the DAO-containing plants originate from the legume family (Leguminosae), and are preferably selected from the genera Pisum, Lens, Cicer, Lathyrus, Phaseolus and Vicia. These plants (in particular their plant shoots) represent good sources of a plant DAO that can be stabilized according to the present invention. Particular preference is given to peas (Pisum sativum), lentils (Lens culinaris), chickpeas (Cicer arietinum), field peas (Lathyrus sativus), beans (Phaseolus vulgaris) and broad beans (Vicia faba), in particular peas (Pisum sativum).

[0039] Plant sprouts are the early growth stage of plants, beginning shortly after seed germination. During this stage, plants have a high concentration of nutrients, vitamins, minerals, and enzymes (including DAO), as they are needed for the rapid growth and development of the young plant. Therefore, DAO-containing plants are preferred as sprouts.

[0040] In a further preferred embodiment of the process according to the invention or of the stabilized lyophilisate according to the invention, the DAO-containing plants are seedlings.

[0041] In a further preferred embodiment of the method according to the invention or of the stabilized lyophilizate according to the invention, at least one further stabilizer or excipient is added. The addition of these additional stabilizers can further improve the stability of the lyophilizate and at the same time reduce possible harmful reactions or oxidation processes that could impair the quality of the final product. Other excipients, in turn, can fulfill a variety of functions, such as protecting the protein during freezing or during the drying process in the lyophilization process or improving lyophilizability or reducing aggregation in the lyophilizate (when it is in ground form). Particularly preferred as additional stabilizers are antioxidants such as lecithin, tocopherol, resveratrol, lactic acid, glutathione and / or ascorbic acid and / or cryoprotectors.The additional stabilizers and / or excipients can be added before or after lyophilization. If the stabilizer is a cryoprotectant, it is added before lyophilization.

[0042] According to a further preferred embodiment of the method according to the invention, the production process for the stabilized lyophilisate additionally comprises grinding after lyophilization. Grinding can be used in particular when the lyophilisate is intended for further processing into capsules and tablets. The finer particle size facilitates processing and enables uniform distribution of the active ingredient in the solid dosage form. This leads to a more consistent release of the active ingredient in the gastrointestinal tract and thus to improved bioavailability.

[0043] After grinding, the lyophilisate can be filled into capsules or used to produce tablets, pellets, micropellets, granules, or jelly beans. In the production of these dosage forms, the ground lyophilisate can be mixed with other excipients, such as binders, flow regulators, or lubricants, and then, if necessary, compressed to achieve the desired size and shape.

[0044] The solid (pharmaceutical) dosage form according to the invention can comprise one or more (pharmaceutically acceptable) excipients, for example a substance selected from the group of polysaccharides, such as cellulose (e.g. microcrystalline cellulose), hydroxypropylmethylcellulose, carboxymethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, hydroxypropylcellulose, cellulose acetylphthalate, hydroxypropylmethylcellulose phthalate, cellulose acetate, cellulose propionate, cellulose acetate butyrate, ethylcellulose, guar flour, alginic acid and / or alginates, pectin, polyvinylpyrrolidone, polyvinyl acetate, polyvinyl alcohol, polyvinyl chloride, polymers of acrylic acid and their salts or polyacrylates or polymethacrylates or copolymers thereof (e.g. Eudragit® E, Eudragit® R, Eudragit® S, Eudragit® NE, Eudragit® RS, Eudragit® RL). , vinylpyrrolidone-vinyl acetate copolymers, shellac, nylon, colloidal silica (e.g. Aerosil®), behenates (e.g.Glyceryl dibehenate), stearates, polyamide, polyacrylamide, polyethylene, polyalkylene glycols such as polyethylene glycol, copolymers of polyalkylene glycols, e.g., polyethylene glycol and polypropylene glycol (Pluronic®, BASF); and mixtures thereof. Particularly when the dosage form is a tablet, it may contain, for example, as excipients, a filler such as lactose, a disintegrant such as microcrystalline cellulose, a flow agent such as colloidal silica, and / or a lubricant such as magnesium stearate.

[0045] According to a preferred embodiment, the dosage form according to the invention is enteric coating to enable targeted release of the DAO in the intestine. Enteric coating can be achieved, for example, with coatings (e.g. of the tablets, capsules or pellets) that only dissolve at a higher pH, such as that found in the intestine. This ensures that the sensitive DAO present in the dosage form passes through the stomach unharmed. Examples of suitable enteric coatings are polymers such as polyvinyl acetate phthalate (PVAP), hydroxypropyl methylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methylcellulose acetate succinate or shellac. Enteric coatings based on methacrylic acid copolymers can also be used, such as methyl acrylate methacrylic acid copolymers or methyl methacrylate methacrylic acid copolymers.Another preferred polymer for an enteric coating of the dosage form according to the invention is ethylcellulose, preferably in combination with alginate (e.g., based on Surelease®, Colorcon Ltd., United Kingdom). The dosage form can, for example, be in an enteric coating, as disclosed, for example, in WO 2006 / 003213 A1.

[0046] In a further preferred embodiment, the dosage form according to the invention is a capsule, a tablet, or a sachet. If the dosage form is a capsule, its shell can, for example, consist essentially of hard gelatin.

[0047] In a further embodiment, the dosage form according to the invention is a sustained-release dosage form (i.e. a dosage form with delayed active ingredient release), such as a sustained-release tablet. How a dosage form in sustained-release form can be formulated is known in the prior art, for example from WO 2010 / 028794 A1. In the embodiment according to the invention, a polymer is preferably used as the sustained-release excipient, which polymer is selected from the neutral homo- and copolymers of (meth)acrylic acid esters, cationic homo- and copolymers of (meth)acrylic acid esters with quaternary ammonium groups, polyvinyl acetate, cellulose acetate, cellulose propionate, cellulose acetate butyrate and ethylcellulose.

[0048] As already mentioned at the beginning, exogenous histamine ingested through food, as well as endogenous histamine, can trigger a variety of disorders due to undesirable reactions, including headaches, rhinitis, tachycardia, gastric and intestinal complaints, and hypotension. The invention further relates to the solid dosage form according to the invention containing the lyophilisate according to the invention for therapeutic use, in particular for use in the treatment of histamine-induced diseases or disorders. The DAO activity (expressed in kilo Histamine Degrading Units (kHDU) / g lyophilisate) can be determined, for example, according to the following

[0049] Measurement method: DAO REA ("DAO specific radioextraction assay"). Particularly preferred is a DAO activity in the lyophilisate according to the invention of at least 5000 kHDU / g, preferably at least 10000 kHDU / g, even more preferably at least 12500 kHDU / g, in particular at least 15000 kHDU / g or even at least 25000 kHDU / g. Usually the activity in the lyophilisate according to the invention is below 100000 kHDU / g, in particular below 80000 kHDU / g or below 70000 kHDU / g.

[0050] The DAO-REA is disclosed, for example, in Mayer et al. (Allergologie 28.1 (2005): 1-8). The DAO-REA is preferably carried out as disclosed in this publication, whereby the unit is convertible to HDU as stated in the next paragraph. The measurement of DAO activity using DAO-REA and expression in HDU is also used in Commission Implementing Regulation (EU) 2023 / 951 of 12 May 2023 amending Implementing Regulation (EU) 2017 / 2470 as regards the specifications of the novel food protein extract from porcine kidney. As stated in this implementing regulation, 48000 HDU in the DAOREA correspond to 1 mU.

[0051] The total protein content in the lyophilisate according to the invention can be determined by calculating the drying yield and determining the protein content in the pea sprout homogenate using known methods, such as the BCA test (bicinchoninic acid) (Smith, PK et al., Measurement of protein using bicinchoninic acid, Analytical Biochemistry 150 (1): 76-85, 1985), the Dumas method (DIN EN ISO 16634-1:2009-07) or the Kjeldahl method.

[0052] The invention further relates to the following embodiments:

[0053] Embodiment 1. A process for the preparation of a stabilized lyophilisate with plant diamine oxidase (DAO), comprising the following steps:

[0054] - Providing DAO-containing plants or DAO-containing parts thereof;

[0055] - homogenizing the DAO-containing plants or DAO-containing parts thereof in aqueous solution to obtain an aqueous homogenate;

[0056] - adding (a) a sugar or sugar alcohol and (b) an amino acid; and

[0057] - Lyophilizing the aqueous homogenate or a DAO-containing fraction thereof in the presence of (a) the sugar or sugar alcohol and (b) the amino acid.

[0058] Embodiment 2. The method according to embodiment 1, wherein the amino acid is a free amino acid. Embodiment 3. The method according to embodiment 1 or 2, wherein the amino acid is selected from the group consisting of basic amino acids such as arginine, histidine, and lysine, and the amino acids asparagine, glutamine, cysteine, homocysteine, methionine, tryptophan, and tyrosine.

[0059] Embodiment 4. The method according to embodiment 3, wherein the amino acid is a basic amino acid.

[0060] Embodiment 5. The method according to embodiment 4, wherein the amino acid is arginine.

[0061] Embodiment 6. Process according to one of embodiments 1 to 5, wherein the sugar or sugar alcohol is selected from the group consisting of disaccharides such as trehalose, sucrose, lactose, and maltose, trisaccharides such as raffinose, polysaccharides such as maltodextrin, and the sugar alcohols mannitol, sorbitol, glycerol, xylitol, erythritol and inositol.

[0062] Embodiment 7. The process according to embodiment 6, wherein the sugar or sugar alcohol is a disaccharide.

[0063] Embodiment 8. The method according to embodiment 7, wherein the sugar or sugar alcohol is trehalose.

[0064] Embodiment 9. Method according to one of embodiments 1 to 8, wherein the DAO-containing plants are members of the legume family (Leguminosae), preferably selected from the genera Pisum, Lens, Cicer, Lathyrus, Phaseolus and Vicia.

[0065] Embodiment 10. Method according to one of embodiments 1 to 9, wherein the DAO-containing plants are selected from peas (Pisum sativum), lentils (Lens culinaris), chickpeas (Cicer arietinum), field peas (Lathyrus sativus), beans (Phaseolus vulgaris) and broad beans (Vicia faba).

[0066] Embodiment 11. The method according to embodiment 10, wherein the DAO-containing plants are peas (Pisum sativum).

[0067] Embodiment 12. The process according to any one of embodiments 1 to 11, wherein the DAO-containing plants are shoots or seedlings. Embodiment 13. The process according to any one of embodiments 1 to 12, wherein the sugar or sugar alcohol is added as a stabilizer in an amount of at least 10 wt.% of the homogenate, more preferably at least 20 wt.%, even more preferably at least 30 wt.%, in particular at least 40 wt.% or even at least 50 wt.%.

[0068] Embodiment 14. Process according to one of embodiments 1 to 13, wherein the amino acid is added as a stabilizer in an amount of at least 20 wt.% of the homogenate, more preferably at least 40 wt.%, even more preferably at least 60 wt.%, in particular at least 80 wt.% or even at least 100 wt.% of the homogenate.

[0069] Embodiment 15. Process according to one of embodiments 1 to 14, wherein at least one further stabilizer such as an antioxidant or a cryoprotectant or an excipient is added, in particular glutathione, lecithin, tocopherol, resveratrol, lactic acid and / or ascorbic acid.

[0070] Embodiment 16. The method according to any one of embodiments 1 to 15, further comprising grinding after lyophilization.

[0071] Embodiment 17. The process according to any one of embodiments 1 to 16, wherein (a) the sugar or sugar alcohol and / or (b) the amino acid is added as stabilizer(s) upon addition.

[0072] Embodiment 18. Process according to one of embodiments 1 to 17, wherein the amino acid, in particular before addition, is present in a pharmaceutically acceptable salt form, preferably as a hydrochloride, sulfate, acetate, phosphate, tartrate or citrate salt, in particular arginine hydrochloride, arginine sulfate, arginine acetate, histidine hydrochloride, histidine sulfate, histidine acetate, lysine hydrochloride, lysine sulfate or lysine acetate.

[0073] Embodiment 19. The process according to embodiment 18, wherein the amino acid is arginine and, in particular prior to addition, is present as arginine hydrochloride. Embodiment 20. A stabilized lyophilisate containing plant-derived DAO, obtainable by the process according to any one of embodiments 1 to 19.

[0074] Embodiment 21. Stabilized lyophilisate with plant DAO, comprising (a) a sugar or sugar alcohol and (b) a free amino acid.

[0075] Embodiment 22. Stabilized lyophilisate according to

[0076] Embodiment 20 or 21, wherein the sugar or sugar alcohol (a) serves as a stabilizer.

[0077] Embodiment 23. Stabilized lyophilisate according to any one of embodiments 20 to 22, wherein the free amino acid (b) serves as a stabilizer.

[0078] Embodiment 24. Stabilized lyophilisate according to one of embodiments 20 to 23, wherein the sugar or sugar alcohol is present in a weight ratio of at least 1:1 or even at least 2:1, preferably of at least 4:1 or even at least 7:1, preferably at least 10:1 or even at least 13:1, more preferably at least 16:1 or even at least 19:1, in particular at least 22:1 or even at least 25:1 to the total protein content of the lyophilisate.

[0079] Embodiment 25. Stabilized lyophilisate according to any one of embodiments 20 to 24, wherein the free amino acid is present in a weight ratio of at least 1:1, preferably of at least 4:1 or even at least 7:1, preferably at least 10:1 or even at least 18:1, more preferably at least 25:1 or even at least 32:1, in particular at least 40:1 or even at least 50:1 to the total protein content of the lyophilisate.

[0080] Embodiment 26. Stabilized lyophilisate according to any one of embodiments 20 to 25, wherein the free amino acid is present in a pharmaceutically acceptable salt form, preferably as a hydrochloride, sulfate, acetate, phosphate, tartrate, or citrate salt, in particular arginine hydrochloride, arginine sulfate, arginine acetate, histidine hydrochloride, histidine sulfate, histidine acetate, lysine hydrochloride, lysine sulfate, or lysine acetate. Embodiment 27. Stabilized lyophilisate according to any one of embodiments 20 to 26, wherein the free amino acid is selected from the group consisting of basic amino acids such as arginine, histidine, and lysine, and the amino acids asparagine, glutamine, cysteine, homocysteine, methionine, tryptophan, and tyrosine.

[0081] Embodiment 28. Stabilized lyophilisate according to any one of embodiments 20 to 27, wherein the free amino acid is a basic amino acid.

[0082] Embodiment 29. Stabilized lyophilisate according to any one of embodiments 20 to 28, wherein the free amino acid is arginine.

[0083] Embodiment 30 . Stabilized lyophilisate according to

[0084] Embodiment 29, wherein arginine is present as arginine hydrochloride.

[0085] Embodiment 31. Stabilized lyophilisate according to one of embodiments 20 to 30, wherein the sugar or sugar alcohol is selected from the group consisting of disaccharides such as trehalose, sucrose, lactose, and maltose, trisaccharides such as raffinose, polysaccharides such as maltodextrin, and the sugar alcohols mannitol, sorbitol, glycerol, xylitol, erythritol and inositol.

[0086] Embodiment 32. Stabilized lyophilisate according to any one of embodiments 20 to 31, wherein the sugar or sugar alcohol is a disaccharide.

[0087] Embodiment 33. Stabilized lyophilisate according to any one of embodiments 20 to 32, wherein the sugar or sugar alcohol is trehalose.

[0088] Embodiment 34. Stabilized lyophilisate according to any one of embodiments 20 to 33, wherein the lyophilisate has a DAO activity of at least 1 kHDU / g, preferably of at least 10 kHDU / g (preferably measured with DAO-REA); preferably wherein the DAO activity in the lyophilisate is at least 5000 kHDU / g, preferably at least 10000 kHDU / g, even more preferably at least 12500 kHDU / g, in particular at least 15000 kHDU / g or even at least 25000 kHDU / g (preferably measured with DAO-REA).

[0089] Embodiment 35. Solid, preferably gastro-resistant dosage form for oral administration, comprising the stabilized lyophilisate according to any one of embodiments 20 to 34, optionally in compressed form.

[0090] Embodiment 36. Solid dosage form according to embodiment 35 for therapeutic use, preferably in the treatment of histamine-induced disorders.

[0091] Embodiment 37. Use of the solid dosage form according to embodiment 35 as a food supplement or dietetic food.

[0092] In the following, the invention is explained in more detail using preferred, non-limiting examples and figures.

[0093] Fig. 1: Residual activity of DAO in a lyophilized pea sprout homogenate stored at 30 ° C, 65% relative humidity in an open container mixed with the additives (each in a weight ratio of additive to homogenate of 1:1): arginine monohydrochloride ("Arginine"), trehalose ("Trehalose"), as well as the calculated residual activity of the lyophilized pea sprout homogenate with the addition of arginine monohydrochloride and trehalose in combination ("Trehalose + Arginine calculated"). In comparison, the expected residual activity of the combination of arginine monohydrochloride and trehalose predicted from the DoE model ("Trehalose + Arginine DoE model prediction"). The error bars indicate the 95% confidence interval. The effect of arginine and trehalose was found to be higher than expected.

[0094] Fig. 2: Stability study of the pea sprout homogenate formulations derived from the DoE model with arginine monohydrochloride in a weight ratio of arginine monohydrochloride to homogenate of 1:1 (“100% arginine”), trehalose (in a weight ratio of trehalose to homogenate of 1:2) and arginine monohydrochloride in a weight ratio of 1:1 to the homogenate (“50% trehalose, 100% arginine”) as well as trehalose (in a weight ratio of trehalose to homogenate of 1:1) and

[0095] Arginine monohydrochloride in a weight ratio of 1:1 to the homogenate ("100% trehalose, 100% arginine"). The data show the monthly course of the DAO activity decrease within a storage period of 6 months ("IM", "2M", "3M", "4M", "5M", "6M") with reference to the starting value ("Start") as 100%. (a) Storage at 25°C, 60% RH, (b) Storage at 30°C, 65% RH.

[0096] Fig. 3: Synergistic effect when using trehalose and arginine (in the form of arginine hydrochloride - "Arginine*HCl") together. Shown is the course of the DAO activity decrease at different time points, with reference to the starting value ("homogenisate before lyophilization") as 100%. In the last group ("Trehalose and Arginine*HCl after lyophilization"), trehalose and arginine were added only after lyophilization. "Lyo" - lyophilization.

[0097] Fig. 4: Addition of additives in different amounts relative to total protein, expressed as the ratio of total protein to trehalose to arginine*HCl (on a weight basis). The curve of DAO activity decrease at different time points is shown, with reference to the starting value as 100%. "Arginine*HCl" - arginine hydrochloride.

[0098] Example 1 - Stability tests

[0099] Stability tests were conducted with plant-based DAO and various potential stabilizers, both with regard to lyophilization and subsequent storage. Selected tests are described below.

[0100] Experiments using sucrose or maltodextrin as sole stabilizers did not show sufficient stabilization of lyophilized DAO from pea shoots ( Pi sum sativum) under long-term storage conditions at 25 ° C and 60% relative humidity ( RH ).

[0101] In another experiment, various amino acids (up to 10% w / w) were added to a DAO-rich pea shoot homogenate fraction containing 10% w / w maltodextrin. Reduced glutathione was also added to act as an antioxidant. The homogenate was then lyophilized. After lyophilization, the lyophilized product contained 3-4 wt% pea shoot dry matter (mainly pea shoot protein, including DAO). Long-term storage (up to six months) at 25°C, 60% RH showed that the addition of the amino acids cysteine ​​or methionine, in combination with maltodextrin, led to improved long-term stability of the DAO in the pea shoot homogenate powder. Outstanding results (up to 60% residual DAO activity after 6 months) were achieved with arginine in combination with the polysaccharide maltodextrin. Particularly good results were also achieved with histidine in combination with maltodextrin.

[0102] In another experiment, trehalose was used as a stabilizer. Using a design of experiments (DoE) approach, its use in combination with arginine and maltodextrin was tested simultaneously, as well as different concentrations of the mixtures. To stabilize the pea sprout homogenate, it was mixed with maltodextrin, trehalose, and arginine in different weight proportions, lyophilized, and stored at 30°C, 65% RH in an open container to determine stability. Formulations with arginine and trehalose, but without maltodextrin, produced the best DAO stability values ​​in this experiment.

[0103] The DoE model showed that this was more than just an additive effect. If one calculates the sum of the residual activity from the use of trehalose after 6 months of storage at 30°C, 65% RH in an open container of 6.9% and the residual activity from the use of arginine after 6 months of storage at 30°C, 65% RH in an open container of 9.7%, the calculated value is 16.6% residual activity for a combination of trehalose and arginine. However, the expected value predicted from the DoE model for the combination of trehalose with arginine is 21.1% (with a 95% confidence interval of 16.9% to 25.2%). Consequently, a synergistic effect is present. This is illustrated in Fig. 1.

[0104] Based on the above results, three formulations were selected, produced, and stored for verification. The above results were thus confirmed. In this experiment, a residual activity after 6 months of approximately 60% was observed at 25°C, 60% RH, and approximately 40% at 30°C, 65% RH, when the formulation was selected as a 1:2 trehalose to pea sprout homogenate and 1:1 arginine (in the form of arginine monohydrochloride) to pea sprout homogenate, or 1:1 trehalose to pea sprout homogenate and 1:1 arginine (in the form of arginine monohydrochloride) to pea sprout homogenate (data based on weight). In comparison, when only 1:1 arginine (in the form of arginine monohydrochloride) was added to the pea sprout homogenate, the DAO activity had already decreased to less than 20% of the initial activity after one month (see Fig. 2 a. and b.).

[0105] Example 2 - Preparation of the dosage form according to the invention

[0106] DAO-containing pea sprouts (Pisum sativum) were provided. To prepare the pea sprout homogenate, the germinated peas were homogenized with a first portion of water in a blender. The homogenate had a DAO activity of approximately 90,000 kHDU / g. The homogenate was then diluted to improve pipetting. To remove fiber particles, the homogenate was centrifuged at 400xg for 15 minutes. The supernatant was centrifuged again at 11,000xg for 1 h 50 min, and the supernatant was sterile-filtered. This sterile-filtered supernatant was the fraction of the homogenate intended for lyophilization. Determination of DAO activity in the respective processing steps showed that the centrifugation and filtration steps did not result in any loss of activity.

[0107] The resulting pea sprout homogenate was then mixed with one part arginine (100% w / w arginine monohydrochloride to pea sprout homogenate) and one-half part trehalose (50% w / w trehalose to pea sprout homogenate). This resulted in a total mixture (by weight) of 2 / 5 pea sprout homogenate, 2 / 5 arginine monohydrochloride, and 1 / 5 trehalose (normalized to the total protein content, the mixing ratio was, by weight: 1 part total protein : 25 parts trehalose : 50 parts arginine hydrochloride). This mixture was frozen, then lyophilized and ground. After lyophilization, the DAO activity of the powder was approximately 60,000 kHDU / g lyophilisate, or, converted back to the pea sprout homogenisate, approximately 90,000 kHDU / g homogenisate.(In a test batch, powder obtained after lyophilization was stored in closed containers at 25°C and 60% RH for 5 months; the DAO activity was still equivalent to over 60,000 kHDU / g homogenate.)

[0108] To produce the dosage form according to the invention, the powder is lyophilized and mixed with the following additives: microcrystalline cellulose, silicon dioxide, crosscarmellose sodium, and magnesium stearate, and then tableted. To prevent inactivation of the enzyme during gastric passage, an enteric coating consisting of shellac is applied.

[0109] Example 3 - Synergistic effect of the additives In order to further shed light on the synergistic effect of trehalose and arginine as lyophilization and storage stabilizers, as characterized in Example 1, further stability tests were carried out with plant-based DAO.

[0110] For this purpose, DAO-rich pea sprout homogenate (DAO activity: 92,000 kHDU / g homogenate) was prepared. The pea sprout homogenate was lyophilized without additives or with trehalose and arginine hydrochloride in the ratio of 1 part total protein : 25 parts trehalose : 50 parts arginine hydrochloride (on a weight basis) or with arginine hydrochloride only (50 parts : 1 part total protein (on a weight basis)) or with trehalose only (25 parts : 1 part total protein (on a weight basis)). In Fig. 3 it is evident that the joint addition of trehalose and arginine hydrochloride leads to a considerably better stabilizing effect on the DAO during lyophilization (98% residual activity of the powder after lyophilization) than without additives (58% residual activity of the powder after lyophilization) or only with individual components (74% residual activity of the powder after lyophilization with only arginine hydrochloride or 46% residual activity after lyophilization with only trehalose).

[0111] As an additional experiment , lyophilized pea sprout homogenate was mixed with trehalose and arginine hydrochloride after lyophilization in a ratio of 1 part total protein : 25 parts trehalose : 50 parts arginine hydrochloride ( on a weight basis ) .

[0112] The powders obtained after lyophilization were subsequently stored in sealed containers at 25 °C and 60% RH for 6 months. The 6-month values ​​clearly showed that the DAO enzyme activity of the lyophilized pea sprout homogenate decreased significantly without additives (12% residual activity compared to before lyophilization), that the addition of arginine hydrochloride or trehalose alone did not result in any improvement, and that the addition of these stabilizers only after lyophilization did not result in any stabilization in this experiment (only 1% residual activity compared to before lyophilization).

[0113] In contrast, the combined addition of arginine hydrochloride and trehalose before lyophilization resulted in a considerable preservation of DAO enzyme activity, both during lyophilization and during storage at 25 °C and 60% RH. This was due to a synergistic effect of the combination of both additives: the combined use of the two additives resulted in a significantly higher residual activity (64% residual activity compared to before lyophilization) than the stabilizing effect of the individual substances added together (only arginine 5% + only trehalose 12% = added 17% residual activity compared to before lyophilization).

[0114] Example 4 - Addition of additives in different

[0115] Amounts relative to total protein

[0116] Further stability studies investigated the effects of adding the additives in different amounts. On the one hand, 1 part total protein was mixed with 12.5 parts trehalose and 25 parts arginine hydrochloride (by weight). On the other hand, 1 part total protein was mixed with twice the amount of additives, i.e., 25 parts trehalose and 50 parts arginine hydrochloride (by weight). Both versions were lyophilized. The resulting powders were stored in sealed containers at 25°C and 60% RH and tested for DAO enzyme activity for 24 months.

[0117] From Fig. 4 it can be seen that the doubling of the additive amount investigated here led to an improvement in the long-term stability of DAO.

Claims

Patent claims 1. A process for the preparation of a stabilized lyophilisate with plant diamine oxidase (DAO), comprising the following steps: - Providing DAO-containing plants or DAO-containing parts thereof; - homogenizing the DAO-containing plants or DAO-containing parts thereof in aqueous solution to obtain an aqueous homogenate; - Adding (a) a sugar or sugar alcohol and (b) an amino acid; and - Lyophilizing the aqueous homogenate or a DAO-containing fraction thereof in the presence of (a) the sugar or sugar alcohol and (b) the amino acid.

2. The method according to claim 1, wherein the amino acid is selected from the group consisting of basic amino acids such as arginine, histidine and lysine, and the amino acids asparagine, glutamine, cysteine, homocysteine, methionine, tryptophan and tyrosine.

3. The method according to claim 2, wherein the amino acid is a basic amino acid, preferably arginine.

4. The method according to any one of claims 1 to 3, wherein the amino acid is in a pharmaceutically acceptable salt form, preferably as a hydrochloride, sulfate, acetate, phosphate, tartrate or citrate salt, in particular arginine hydrochloride, arginine sulfate, arginine acetate, histidine hydrochloride, histidine sulfate, histidine acetate, lysine hydrochloride, lysine sulfate or lysine acetate.

5. The method according to claim 4, wherein the amino acid is arginine and is present as arginine hydrochloride.

6. The method according to any one of claims 1 to 5, wherein the sugar or sugar alcohol is selected from the group consisting of disaccharides such as trehalose, sucrose, lactose, and maltose, trisaccharides such as raffinose, polysaccharides such as maltodextrin, and the sugar alcohols mannitol, sorbitol, glycerol, xylitol, erythritol and inositol.

7. A process according to claim 6, wherein the sugar or Sugar alcohol is a disaccharide, preferably trehalose.

8. The method according to any one of claims 1 to 7, wherein the DAO-containing plants are members of the legume family (Leguminosae), preferably selected from the genera Pisum, Lens, Cicer, Lathyrus, Phaseolus and Vicia.

9. Stabilized lyophilisate containing plant DAO, obtainable by the process according to any one of claims 1 to 8.

10. Stabilized lyophilisate with plant-based DAO, comprising (a) a sugar or sugar alcohol and (b) a free amino acid.

11. Stabilized lyophilisate according to claim 9 or 10, wherein the sugar or sugar alcohol is present in a weight ratio of at least 1:1 or even at least 2:1, preferably of at least 4:1 or even at least 7:1, preferably at least 10:1 or even at least 13:1, more preferably at least 16:1 or even at least 19:1, in particular at least 22:1 or even at least 25:1 to the total protein content of the lyophilisate.

12. Stabilized lyophilisate according to one of claims 9 to 11, wherein the free amino acid is present in a weight ratio of at least 1:1, preferably at least 4:1 or even at least 7:1, preferably at least 10:1 or even at least 18:1, more preferably at least 25:1 or even at least 32:1, in particular at least 40:1 or even at least 50:1 to the total protein content of the lyophilisate.

13. Stabilized lyophilisate according to one of claims 9 to 12, wherein the free amino acid is selected from the group consisting of basic amino acids such as arginine, histidine and lysine, and the amino acids asparagine, glutamine, cysteine, homocysteine, methionine, tryptophan and tyrosine.

14. Stabilized lyophilisate according to claim 13, wherein the free amino acid is arginine.

15. Stabilized lyophilisate according to one of claims 9 to 14, wherein the free amino acid is present in a pharmaceutically acceptable salt form, preferably as a hydrochloride, sulfate, acetate, phosphate, tartrate or citrate salt, in particular arginine hydrochloride, arginine sulfate, arginine acetate, histidine hydrochloride, histidine sulfate, histidine acetate, lysine hydrochloride, lysine sulfate or lysine acetate.

16. Stabilized lyophilisate according to claim 15, wherein the free amino acid is arginine and is present as arginine hydrochloride.

17. Stabilized lyophilisate according to any one of claims 9 to 16, wherein the sugar or sugar alcohol is selected from the group consisting of disaccharides such as trehalose, sucrose, lactose, and maltose, trisaccharides such as raffinose, polysaccharides such as maltodextrin, and the sugar alcohols mannitol, sorbitol, glycerol, xylitol, erythritol and inositol.

18. Stabilized lyophilisate according to claim 17, wherein the sugar or sugar alcohol is a disaccharide, preferably trehalose.

19. Stabilized lyophilisate according to one of claims 9 to 18, wherein the lyophilisate has a DAO activity of at least 1 kHDU / g, preferably of at least 10 kHDU / g; preferably wherein the DAO activity in the lyophilisate is at least 5000 kHDU / g, preferably at least 10000 kHDU / g, even more preferably at least 12500 kHDU / g, in particular at least 15000 kHDU / g or even at least 25000 kHDU / g.

20. A solid, preferably gastro-resistant dosage form for oral administration, comprising the stabilized lyophilisate according to any one of claims 9 to 19, optionally in compressed form.

21. Solid dosage form according to claim 20 for therapeutic use, preferably in the treatment of histamine-induced disorders.

22. Use of the solid dosage form according to claim 20 as a food supplement or dietetic food.

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