Use of cholecalciferol as an active agent in the treatment of celiac disease - Patent Application 20070229333

Abstract

The use of cholecalciferol as an active agent in the treatment of celiac disease, in reducing atrophy of intestinal villi, and in protecting and regenerating the intestinal epithelium is described. Also described are pharmaceutical compositions or dietary supplements, and foods for celiac disease patients, comprising cholecalciferol and suitable excipients for use in treating celiac disease, reducing atrophy of intestinal villi, and protecting and regenerating the intestinal epithelium.

Description

[Technical field]

[0001] The present invention relates to the use of cholecalciferol as an active agent in the treatment of celiac disease. [Background technology]

[0002] Celiac disease is the most prevalent chronic autoimmune enteropathy in Western populations. The condition is triggered by the ingestion of gluten in genetically predisposed subjects.

[0003] Gluten is a polymeric protein complex present in the endosperm of cereal kernels such as wheat, barley, and rye, and is stored in the seed to ensure a steady supply of nutrients that support the germination and growth of the seedling. Gluten-based cereals represent the most important crops in the world and are commonly used in the manufacture of food products such as pasta, bread, and various bakery and pastry products.

[0004] In fact, the viscoelastic and stabilizing properties of gluten favor its use as an additive in cooking operations in industrial ovens. Moreover, gluten combines with atmospheric oxygen and nitrogen to form disulfide bonds that modify the properties of the dough, thus enhancing the palatability of the food.

[0005] Gluten is a compound of two proteins: glutenin and prolamin.

[0006] Prolamins are the protein fractions responsible for the toxic effects on celiac disease patients.

[0007] The prolamins in wheat are called gliadin and make up about 50% of the gluten content. Similar proteins with the same effect on celiac disease are also found in barley (hordein), rye (secalin), emmer, spelt, kamut, triticale and oats (avenin).

[0008] Pepsin-trypsin-resistant gliadin (PT-G) is an undigested fragment of gliadin that contributes to the pathogenesis of celiac disease by altering intercellular tight junctions (TJs) and compromising intestinal barrier function.

[0009] Even very small amounts of gluten can cause problems: if dietary habits are not changed, intestinal villi are lost and the damaged lining of the small intestine can no longer metabolize nutrients adequately, resulting in malnutrition.

[0010] In fact, gluten intolerance causes severe damage to the intestinal mucosa, including atrophy of the intestinal villi.

[0011] Celiac disease manifests in a wide variety of ways: gastrointestinal problems such as diarrhea, bloating, abdominal pain, nausea, and vomiting often, but not always.

[0012] Other common symptoms may also be indicators of possible celiac disease, including weight and energy loss, loss of appetite, iron deficiency with anemia, osteoporosis, unmet desire to become pregnant or spontaneous abortions, and vitamin and / or mineral deficiencies.

[0013] In children, celiac disease develops in the first few years of life, often already after weaning, when they switch from breast milk to foods containing gluten. If not detected in time, children can suffer from poor growth and development. Children with celiac disease are often thin, cry easily and are very sensitive.

[0014] Unfortunately, there is currently no pharmacological treatment available to treat celiac disease, and no medications are on the market that are specifically indicated for this condition.

[0015] There are some preliminary studies in the literature that have described the possible beneficial effects of calcitriol on the intestinal mucosal barrier against PT-G, both in vivo and in vitro, which may have a protective effect on TJs (Non-Patent Document 1). However, the study concluded that further investigations and clinical trials are needed to determine the possible effective use of calcitriol in the treatment of celiac disease.

[0016] Regarding clinical trials, a phase II clinical trial has recently been started in the United States to test the potential use of latiglutenase, a mixture of two specific recombinant gluten proteases capable of degrading gluten into small physiologically irrelevant fragments, as a treatment for celiac disease. This biological product is administered in oral form and is associated with a gluten-free diet.

[0017] A gluten-free diet is strictly enforced for life and is in fact, unfortunately to this day, the only treatment that guarantees perfect health for those with celiac disease.

[0018] There is therefore a strong felt need to develop drugs that can treat celiac disease or at least counteract the deleterious effects caused by celiac disease.

[0019] Therefore, the object of the present invention is to find a therapeutic agent for the treatment of celiac disease that is effective and well tolerated by the body. [Prior art documents] [Non-patent literature]

[0020] [Non-Patent Document 1] Dong et al.,Dig.Dis.Sci.(2018),63:92-104 Summary of the Invention

[0021] This objective was achieved by the use of cholecalciferol as the active agent in the treatment of celiac disease.

[0022] In another aspect, the present invention relates to the use of cholecalciferol as an active agent in reducing atrophy of intestinal villi.

[0023] In a further aspect, the present invention relates to the use of cholecalciferol as an active agent in the protection and regeneration of the intestinal epithelium.

[0024] In a further aspect, the present invention relates to a pharmaceutical composition or dietary supplement comprising cholecalciferol and at least one suitable excipient for use in the treatment of coeliac disease.

[0025] In a further aspect, the present invention relates to a food product for coeliac patients comprising cholecalciferol and at least one suitable food ingredient. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] [Detailed Description of the Invention] Thus, the present invention relates to the use of cholecalciferol as an active agent in the treatment of celiac disease.

[0027] Cholecalciferol (or vitamin D3) is a natural vitamin D compound of animal / human origin. It is a prohormone and a precursor of the two hydroxylated forms of vitamin D [25OHD and 1,25(OH)2D] and therefore must undergo two natural hydroxylation processes to be converted to its metabolically active form.

[0028] For the purposes of the present invention, the term "cholecalciferol" is understood to include all optical isomers, geometric isomers and stereoisomers, and mixtures thereof, such as mixtures of enantiomers, racemic mixtures and diastereoisomeric mixtures, as well as all polymorphic forms thereof, both amorphous and crystalline, and co-crystals, as well as anhydrous, hydrated and solvated forms, pharma- ceutically acceptable salts and mixtures thereof.

[0029] Furthermore, as will become apparent from the examples below, cholecalciferol is shown to be an active agent that can be used to treat celiac disease.

[0030] In particular, cholecalciferol shows remarkable activity of reducing intestinal villi atrophy.Therefore, cholecalciferol can be effectively used as an active agent of reducing intestinal villi atrophy.Therefore, the present invention also relates to the use of a therapeutically effective amount of cholecalciferol as an active agent of reducing intestinal villi atrophy.

[0031] In the examples below, it has been shown that the villi of animals treated with cholecalciferol are indeed longer than those of untreated celiac disease controls. Since a decrease in the length of the villi is an indication of villous atrophy and celiac enteropathy, the results of histomorphometric analysis showing that the villi of animals treated with cholecalciferol are longer than those of untreated celiac disease controls are clearly positive. Furthermore, it has been shown that the increase in the length of the villi is proportional to the dose of cholecalciferol administered.

[0032] Furthermore, cholecalciferol also exhibits significant activity in the protection and regeneration of intestinal epithelium, so cholecalciferol can also be effectively used as an active agent in the protection and regeneration of intestinal epithelium. Therefore, the present invention also relates to the use of a therapeutically effective amount of cholecalciferol as an active agent in the protection and regeneration of intestinal epithelium.

[0033] In some embodiments, the present invention relates to the use of cholecalciferol as an active agent in the treatment of celiac disease, said treatment comprising the administration of a therapeutically effective amount for the reduction of atrophy of the intestinal villi.

[0034] In another embodiment, the present invention relates to the use of cholecalciferol as an active agent in the treatment of celiac disease, said treatment comprising the administration of a therapeutically effective amount for the protection and regeneration of the intestinal epithelium.

[0035] In a further embodiment, the present invention relates to the use of cholecalciferol as an active agent in the treatment of celiac disease, said treatment comprising the administration of a therapeutically effective amount for reducing atrophy of the intestinal villi and for protecting and regenerating the intestinal epithelium.

[0036] Preferably, cholecalciferol is administered at a dose of 0.4 μg / kg / day to 11 μg / kg / day, more preferably 0.8 μg / kg / day to 11 μg / kg / day, and even more preferably 4 μg / kg / day to 11 μg / kg / day.

[0037] In other words, preferably, cholecalciferol is administered at a dose of 16 IU / kg / day to 430 IU / kg / day, more preferably, 30 IU / kg / day to 430 IU / kg / day, and even more preferably, 160 IU / kg / day to 430 IU / kg / day.

[0038] Preferably, cholecalciferol is administered by oral, injection or subcutaneous route, more preferably by oral route.Except for certain clinical conditions (e.g., malabsorption syndrome), oral route is preferred because it is more effective in increasing serum 25OHD than intramuscular preparations.

[0039] In another aspect, the present invention relates to a pharmaceutical composition or dietary supplement comprising cholecalciferol and at least one suitable excipient for use in the treatment of celiac disease.

[0040] In a further aspect, the present invention relates to a pharmaceutical composition or dietary supplement comprising cholecalciferol and at least one suitable excipient for use in reducing atrophy of intestinal villi.

[0041] In a further aspect, the present invention relates to a pharmaceutical composition or dietary supplement comprising cholecalciferol and at least one suitable excipient for use in the protection and regeneration of the intestinal epithelium.

[0042] In some embodiments, the present invention relates to a pharmaceutical composition or dietary supplement comprising cholecalciferol and at least one suitable excipient for use in the treatment of celiac disease, said treatment comprising administration of a pharmaceutical composition or dietary supplement comprising a therapeutically effective amount of cholecalciferol for reducing atrophy of intestinal villi.

[0043] In another embodiment, the present invention relates to a pharmaceutical composition or dietary supplement comprising cholecalciferol and at least one suitable excipient for use in the treatment of celiac disease, said treatment comprising administration of a pharmaceutical composition or dietary supplement comprising a therapeutically effective amount of cholecalciferol for the protection and regeneration of the intestinal epithelium.

[0044] In a further embodiment, the present invention relates to a pharmaceutical composition or dietary supplement comprising cholecalciferol and at least one suitable excipient for use in the treatment of celiac disease, said treatment comprising the administration of a pharmaceutical composition or dietary supplement comprising a therapeutically effective amount of cholecalciferol for reducing atrophy of the intestinal villi and for protecting and regenerating the intestinal epithelium.

[0045] Said pharmaceutical composition or dietary supplement may also advantageously contain at least one probiotic.

[0046] The pharmaceutical compositions and dietary supplements of the present invention can be administered orally, by injection, or subcutaneously.

[0047] In a preferred embodiment, the pharmaceutical composition or dietary supplement is administered by the oral route.

[0048] When the pharmaceutical composition or dietary supplement is administered by the oral route, it is preferably in the form of an orodispersible solid formulation, gel, capsule, tablet, powder, granules, solution, suspension, emulsion or tincture.

[0049] The pharmaceutical composition or dietary supplement of the invention administered by the oral route in the form of an orally dispersible solid preparation, gel, capsule, tablet, powder, granule, solution, suspension, emulsion or tincture is preferably administered to provide a supply of cholecalciferol of 0.4 μg / kg / day to 11 μg / kg / day, preferably 0.8 μg / kg / day to 11 μg / kg / day, and even more preferably 4 μg / kg / day to 11 μg / kg / day.

[0050] In other words, the pharmaceutical composition or dietary supplement of the invention administered by oral route in the form of an orally dispersible solid formulation, gel, capsule, tablet, powder, granule, solution, suspension, emulsion or tincture is preferably administered to provide a cholecalciferol supply of 16 IU / kg / day to 430 IU / kg / day, preferably 30 IU / kg / day to 430 IU / kg / day, more preferably 160 IU / kg / day to 430 IU / kg / day.

[0051] In a preferred embodiment, the pharmaceutical composition or dietary supplement of the invention administered via the oral route in the form of a solution, suspension, emulsion, gel or tincture contains cholecalciferol in a concentration of 10,000 IU / mL to 20,000 IU / mL.

[0052] The pharmaceutical compositions or dietary supplements of the present invention may advantageously be in unit dose form.

[0053] Said pharmaceutical composition or dietary supplement of the present invention, which is in the form of a unit dose, also comprises at least one suitable excipient.The term "excipient" means a compound or a mixture of compounds that is suitable for pharmaceutical or food use, respectively.For example, an excipient for use in pharmaceutical or food formulations usually must not cause adverse reactions in subjects and must not significantly inhibit the effect of cholecalciferol contained therein.

[0054] Suitable excipients are acidulants, pH adjusters, anticaking agents, antioxidants, bulking agents, strengthening agents, gelling agents, coating agents, modified starches, sequestering agents, thickening agents, sweeteners, diluents, aggregation inhibitors, glidants, dyes, binders, lubricants, stabilizers, adsorbents, water retention agents, flavorings, film formers, emulsifiers, wetting agents, sustained release agents, and mixtures thereof.

[0055] Preferably, the excipient is olive oil, mineral oil, liquid paraffin, white petrolatum, polyoxyethylene, emulsifying wax, stearyl alcohol, isostearyl alcohol, cetylstearyl alcohol, stearic acid, glyceryl stearate, sodium lauryl sarcosine, glycerin, diethylene glycol monoethyl ether, polyethylene glycol, polyethylene glycol stearate, carbopol, carbomer, poloxamer 407, macrogol 400, purified bentonite, myristyl propionate, dimethicone, titanium dioxide, anionic, cationic and nonionic surfactants, water, potassium sorbate, sodium benzoate, ε-polylysine, sucralose, maltodextrin, citric acid, sodium carbonate, calcium carbonate, magnesium carbonate, magnesium stearate, native starch, partially hydrolyzed starch, modified starch, lactose, calcium phosphate, calcium carbonate, calcium sulfate, polyvinylpyrrolidone, silica, colloidal silica, precipitated silica, etc. cellulose, magnesium silicate, aluminum silicate, sodium lauryl sulfate, magnesium lauryl sulfate, methacrylic acid copolymer, sodium dehydroacetate, xanthan gum, guar gum, tara gum, carob gum, fenugreek gum, gum arabic, alginic acid, sodium alginate, propylene glycol alginate, croscarmellose sodium, polyvinylpolypyrrolidone, glyceryl behenate, indigo carmine, cellulose, modified cellulose, carboxymethylcellulose calcium, carboxymethylcellulose sodium, microcrystalline cellulose, ethylcellulose, gelatin, hydroxyethylcellulose, hydroxypropylcellulose, polydextrose, carrageenan, methylcellulose, sucrose, sucrose gum esters, sorbitol, xylitol, dextrose, fructose, maltitol, tragacanth gum, pectin, agar, carboxypolymethylene, hydroxypropylmethylcellulose, tragacanth, mannitol, or mixtures thereof.

[0056] In some embodiments, the pharmaceutical composition or dietary supplement of the present invention consists essentially of cholecalciferol and at least one suitable excipient. The expression "consists essentially of" means that cholecalciferol is the only active ingredient in the treatment of celiac disease present in the composition or dietary supplement, while any additional ingredients as the at least one probiotic or excipient do not interfere with its action. It should be understood that all aspects identified above as preferred and advantageous for the use of cholecalciferol, the composition or the dietary supplement are considered to be preferred and advantageous for these embodiments as well.

[0057] In another embodiment, the pharmaceutical composition or dietary supplement of the present invention consists of cholecalciferol, at least one suitable excipient, and optionally at least one probiotic.

[0058] In a further preferred embodiment of the invention, said composition is a food for coeliac patients comprising cholecalciferol for the above mentioned uses and at least one suitable food ingredient.

[0059] Surprisingly, it has been found that the presence of cholecalciferol allows celiac disease patients to feed themselves without necessarily having to resort to gluten-free products.Thus, advantageously, according to the present invention, the term "celiac disease food" refers to any edible product intended for both human and animal consumption, whether or not it contains gluten, such as baked goods, alcoholic and non-alcoholic drinks, energy drinks, diet bars, condiments, so-called "breakfast cereals", fresh and dried pasta, milk and dairy products (such as yogurt and ice cream), fruit juices, confectionery, and animal feed.

[0060] Preferably, the food products for coeliac patients are baked foods, such as pasta, bread, sweet snacks, snacks, biscuits, breadsticks, crackers, desserts, pastries and generally any type of cooked dough containing gluten or not, more preferably containing gluten-free flour.

[0061] Preferably, the food for celiac disease patients contains cholecalciferol in an amount of 200 to 2,000 IU per serving.

[0062] In a preferred embodiment, the food for coeliac patients, when it is a baked food, contains cholecalciferol in an amount of 1-5 IU / g.

[0063] The food for celiac disease patients can be taken with the aim of contributing to the reduction of atrophy of the intestinal villi and to the protection and regeneration of the intestinal villi.

[0064] As mentioned above, said food for coeliac patients may advantageously contain at least one probiotic.

[0065] All the above pharmaceutical compositions or dietary supplements or coeliac foods can be prepared by methods known in the art.

[0066] It is to be understood that all possible combinations of the preferred embodiments of cholecalciferol, products containing it and the respective uses, as indicated above, are also described and therefore likewise preferred.

[0067] It should also be understood that all aspects identified as preferred and advantageous for cholecalciferol and its products are also believed to be preferred and advantageous for its preparation and use.

[0068] The effectiveness of cholecalciferol as an active agent in the treatment of celiac disease will also be apparent from the examples provided below.

[0069] Without being bound by any theory, cholecalciferol is believed to be beneficial in the treatment of celiac disease, as it can affect the profile of the gut microbiota through the modulation of antimicrobial peptides, and thus modulates the gut bacterial community, ensuring homeostasis of beneficial bacterial strains. At the molecular level, this action may be mediated by the vitamin D receptor (VDR).

[0070] Moreover, cholecalciferol is normally stored in adipose tissue, where it forms deposits from which it is slowly released. In particular, for this reason, it has a relatively short half-life in the blood (estimated T 1 / 2 The long functional half-life (relating to sustained release) is much longer (weeks). The long functional half-life (sustained release by adipose tissue) makes cholecalciferol a very flexible and adaptable active agent in clinical practice, even allowing intermittent dosing regimens.

[0071] The following are examples of the present invention provided for illustrative and non-limiting purposes. EXAMPLES

[0072] Example 1 - Evaluating the efficacy of repeated oral administration of cholecalciferol in an in vivo mouse model of celiac disease (NOD mice) For this study, NOD (non-obese diabetic) mice were selected as an experimental model, which, when fed a standard diet containing gluten, can spontaneously develop enteropathy and autoimmune disorders with small intestinal changes similar to those observed in human celiac disease.

[0073] Next, 103 4-week-old female NOD mice were placed in two different enclosures (one for mice on a standard diet and one for mice on a gluten-free diet) and allowed to acclimate for at least 7 days.

[0074] They were then randomly assigned to seven different experimental groups. The development of enteropathy was achieved by feeding NOD mice a standard diet containing gluten and orally administering gliadin at a dose of 10 μg / kg for 10 consecutive days.

[0075] The seven experimental groups are shown in Table 1 below.

[0076] [Table 1]

[0077] At the end of the treatment period, the mice were sacrificed. A gross examination was performed and the small intestine was removed from each mouse and subjected to microscopic examination.

[0078] In particular, histopathological and histomorphometric examinations were performed on 5- to 7-μm-thick intestinal sections embedded in paraffin and stained with hematoxylin-eosin.

[0079] A scoring system described by Marsh et al,Gut 1990 Jan;31(1):111-4 was used to allow comparisons between the different groups based on the degree of inflammatory infiltrate and type of lesions observed under the microscope, resulting in the following classification:

[0080] - Lesion type 0: normal intestinal mucosa; - Lesion type 1 (invasive): normal morphology and structure of the villi, high number of intraepithelial lymphocytes; - Lesion type 2 (hyperplasia): normal morphology and structure of the villi, high number of intraepithelial lymphocytes, hyperplasia of glandular elements; - Lesion type 3 (infiltrative): Varying degrees of villous atrophy with crypt hypertrophy, decreased height of superficial enterocytes, and high numbers of intraepithelial lymphocytes.

[0081] Fifteen histomorphometric measurements of villus height were then performed from each of the five small intestinal sections evaluated for each mouse.

[0082] The results of the microscopic histopathological evaluation are summarized in Table 2 below, including the severity of the four lesions mentioned above and the number of animals in each group that exhibited the various lesion types.

[0083] [Table 2]

[0084] For the small intestine, the most severe histological manifestations were observed in the untreated celiac group, with 5 of 13 animals showing the greatest degree of lesions.

[0085] On the other hand, an improvement in the overall profile of the lesions was observed in treated mice compared to untreated coeliac mice at all cholecalciferol doses tested, which profile was significantly improved when treatment was performed at the highest cholecalciferol dose, i.e., when mice were treated with repeated doses of 50 and 130 μg / kg cholecalciferol.

[0086] The results of the histomorphometric microscopic evaluation of villus length are shown in Table 3 below.

[0087] [Table 3]

[0088] As is evident from the data presented in Table 3, the villi of animals treated with cholecalciferol proved to be longer than the villi of untreated celiac controls.

[0089] Since a decrease in the length of the villi is a sign of villous atrophy and celiac enteropathy, the results of the histomorphometric analysis are clearly positive, demonstrating that the villi of animals treated with cholecalciferol were longer than those of untreated celiac control animals and that the length increased with the dose of cholecalciferol administered (i.e. showing a dose-dependent relationship between the increase in villus length and the dose of cholecalciferol administered).

[0090] Example 2 - Analysis of the expression of some proteins in the small intestinal mucosa of NOD mice by immunohistochemistry (IHC) Small intestine samples were taken from mice belonging to experimental groups 2 (pathological control) and 7 (cholecalciferol 130 μg / kg) of the experiment described in Example 1 and processed to evaluate the expression of three proteins by immunohistochemistry: Vitamin D receptor (VDR); a T cell marker indicative of lymphocytic infiltration (CD3); and the protein of Zonula Occludens 1 or Zonulin-1 (ZO-1), which is normally released in serum and tissues following disruption of the intestinal epithelial barrier, especially the intercellular tight junctions (TJs).

[0091] Samples were washed in xylene, hydrated, and scaled using a solution of ethanol and water, and washed in water. Antigen retrieval was performed in sodium citrate buffer using a standard "pressure cooker" protocol. Once antigen retrieval was complete, samples were left at room temperature for 10' and then washed twice for 2' in PBS tween. Non-specific sites were blocked with 200 μL of T20 starting block.

[0092] Each slide was then dispensed with 200 μL of a solution containing the primary antibody of interest or 200 μL of the respective negative control, and the slides were then incubated overnight at 4° C. in a humidified chamber containing PBS.

[0093] After incubation, repeated washing was performed in washing buffer at room temperature, after which endogenous peroxidase was blocked with 0.3% hydrogen peroxide buffer.

[0094] Subsequently, washing was repeated again in washing buffer at room temperature, and secondary antibody was added and incubated for 1 hour at room temperature.

[0095] Then, washing was repeated again in the washing buffer at room temperature. Subsequently, a DAB solution (3',3'-diaminobenzidine) was added, and the plate was washed at room temperature for blocking.

[0096] Finally, counterstaining was performed with Mayer's hematoxylin, and the reaction was stopped by washing in water.

[0097] The slides were then mounted with two drops of aqueous mounting medium and allowed to dry. Intestinal mucosal atrophy was assessed according to the Corazza-Villanacci classification. Antibody labeling in villi, crypts, and goblet cells was assessed.

[0098] result VDR Reduced atrophy was detected in the villi of animals treated with cholecalciferol compared to vehicle-treated controls.

[0099] In the group treated with cholecalciferol, the villi were more prominent and therefore the expression of VDR was higher, and the same phenomenon was observed in the crypts. As for the goblet cells, the intensity of the marking was higher in the group treated with the active agent, but the number of marked cells was smaller.

[0100] CD3 This protein was less expressed in the group treated with cholecalciferol and did not label the villi and crypts of the same group, indicating the absence of intraepithelial lymphocytic cell infiltration, indicating a lower immune response underlying the pathogenesis of celiac disease.

[0101] Also, goblet cells in the active agent treated group showed a decreased signal compared to the control, and also showed a decreased intensity of marking.

[0102] ZO-1 In the group treated with cholecalciferol, villous atrophy was less pronounced. Villi and crypts were more prominent in the vehicle-treated group.

[0103] Example 3 - Pharmaceutical composition containing cholecalciferol for oral use in the form of a solution To prepare a pharmaceutical composition in the form of an oral solution, cholecalciferol was mixed with suitable excipients.

[0104] The compositions prepared contained unit doses of approximately 50,000 IU and 25,000 IU per bottle. The amounts of excipients contained in each bottle are shown below in Tables 4a and 4b, respectively.

[0105] [Table 4a]

[0106] [Table 4b]

[0107] The refined olive oil was introduced into a dissolution tank heated to 40°C ± 3°C (ensuring that the temperature does not exceed 45°C) and cholecalciferol was added under nitrogen and stirred until completely dissolved (at least 60 minutes). The mixture was then cooled to about 27°C (± 3°C) and filtered under nitrogen in the tank at a maximum pressure of 0.8 atmospheres.

[0108] The filtered solution was finally automatically dispensed into an Amber Type III bottle and sealed therein.

[0109] Example 4 - Pharmaceutical composition containing cholecalciferol for oral use in the form of a hard gelatin capsule To prepare a pharmaceutical composition in the form of oral hard capsules, cholecalciferol was mixed with suitable excipients. The compositions manufactured contained unit doses of approximately 50,000 IU and 25,000 IU per capsule. The amounts of excipients contained in each capsule are shown below in Tables 5a and 5b, respectively.

[0110] [Table 5a]

[0111] [Table 5b]

[0112] Cholecalciferol was added to refined olive oil in a dissolution tank under nitrogen with stirring until completely dissolved (>60 min).

[0113] 200 μL of the cholecalciferol solution thus prepared was filled into capsules under nitrogen by an automatic machine.

[0114] The capsules were then banded and sealed using gelatin bands and then packaged into PVC / PVDC / Al blisters using an automatic blister machine.

[0115] Example 5 - Preparation of cholecalciferol-containing food Focaccia was prepared containing cholecalciferol and the appropriate ingredients as reported in Table 6.

[0116] [Table 6]

[0117] The yeast was dissolved in water in a container and then added to the flour and mixed. Milk was then slowly added to the mixture while stirring.

[0118] Next, some of the olive oil (about a tablespoon) and salt were added to the dough and stirring continued. The dough was placed into a suitable non-stick container, spread out and left to rise for approximately 2 hours.

[0119] The dough was then sprinkled with coarse salt and rosemary and cooked at approximately 220°C for 25 minutes.

[0120] Finally, the underside of the focaccia was drizzled with the remaining spoonful of olive oil in which 2000 IU of cholecalciferol had been dissolved.

[0121] conclusion In conclusion, all the above studies demonstrate that cholecalciferol is an active agent that can be used to treat celiac disease.

[0122] In particular, cholecalciferol exhibits significant activity in reducing atrophy of intestinal villi, and can therefore be effectively used as an active agent for reducing atrophy of intestinal villi.

[0123] Furthermore, cholecalciferol also exhibits significant activity in protecting and regenerating the intestinal epithelium, and can therefore be effectively used as an active agent for protecting and regenerating the intestinal epithelium.

Claims

1. A pharmaceutical composition or dietary supplement for use in reducing intestinal villi atrophy in the treatment of celiac disease comprising cholecalciferol as an active agent.

2. 2. The pharmaceutical composition or dietary supplement for use according to claim 1, wherein the treatment of celiac disease comprises the administration of a therapeutically effective amount for the reduction of atrophy of the intestinal villi.

3. 3. The pharmaceutical composition or dietary supplement for use according to claim 1 or 2, wherein cholecalciferol is administered in a dose of 0.4 μg / kg / day to 11 μg / kg / day or in a dose of 16 IU / kg / day to 430 IU / kg / day.

4. A pharmaceutical composition or dietary supplement for use according to any one of claims 1 to 3, administered by oral, injectable or subcutaneous route.

5. A pharmaceutical composition or dietary supplement for use as described in claim 4, administered by oral route.

6. A pharmaceutical composition or dietary supplement for use according to any one of claims 1 to 5, comprising at least one suitable excipient.

7. 7. The pharmaceutical composition or dietary supplement of claim 6, wherein said pharmaceutical composition or dietary supplement is administered by oral route and is in the form of an orodispersible solid formulation, gel, capsule, tablet, powder, granule, solution, suspension, emulsion or tincture.

8. 8. The pharmaceutical composition or dietary supplement of claim 7 in the form of a solution, suspension, gel, emulsion, or tincture and comprising cholecalciferol in a concentration of 10,000 IU / mL to 20,000 IU / mL.

9. A food for coeliac disease patients for use in reducing atrophy of intestinal villi in the treatment of coeliac disease, comprising cholecalciferol as an active agent and at least one suitable food ingredient.

10. 10. Food product for celiac disease patients according to claim 9, wherein the food product is an edible product intended for both human and animal consumption selected from baked goods, alcoholic beverages, soft drinks, energy drinks, diet bars, condiments, so-called "breakfast cereals", fresh pasta, dried pasta, milk and dairy products, fruit juices and confectionery, and animal feed.

11. 11. Food for coeliac disease patients according to claim 9 or 10, wherein cholecalciferol is present in an amount of 200-2,000 IU per serving or in an amount of 1-5 IU / g of the food.

12. A pharmaceutical composition or a dietary supplement according to any one of claims 1 to 8 or a food product for coeliac patients according to any one of claims 9 to 11, further comprising at least one probiotic.

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