Methods for determining intestinal permeability

Abstract

This invention relates to an in vitro method for evaluating the state of intestinal permeability in a subject and, as a result, diagnosing diseases or dysfunctions associated with increased intestinal permeability. More specifically, this procedure allows for the measurement of the amount of food antigens that can pass through a dysfunctional intestine using common food components. This procedure enables the development of analytical products and analytical processes within the framework of the medical device industry.

Description

Technical Field

[0001] The present invention belongs to the medical field, clinical analysis, the manufacture and commercialization of health products and services, and basic and clinical research related to the digestive system. In particular, the present invention evaluates the intestinal permeation state of a subject, and as a result, diseases or dysfunctions associated with or occurring with an increase in intestinal permeability, such as (enumerated non-exhaustively), celiac disease, Crohn's disease, irritable bowel syndrome, microscopic colitis, functional dyspepsia, chronic / acute villous atrophy caused by chronic treatment with some drugs, and / or an in vitro method for the diagnosis of non-IgE-mediated food allergy.

Background Art

[0002] The intestinal barrier is essential for human health and constitutes the interface between the external environment and the internal environment of the body. A functional intestinal barrier enables the absorption of nutrients and fluids while preventing harmful substances such as toxins, bacteria, or viruses from passing through the intestinal epithelium and damaging the organism. Changes in intestinal permeability are associated with various chronic conditions such as inflammatory bowel disease, irritable bowel syndrome, celiac disease, and other medical conditions originating from the gastrointestinal tract, as well as diseases such as Alzheimer's disease and Parkinson's disease.

[0003] The pathways for the permeation of poorly digested antigens or even bacteria and / or viruses are the paracellular pathway (through the intercellular space) or the transcellular pathway (through the intestinal epithelial cells). An increase in intestinal permeability to macromolecules is associated with various inflammatory conditions.

[0004] The luminal side of the intestine is lined with epithelial cells, which facilitate the absorption of water and nutrients. The epithelium also provides a dynamic, semipermeable barrier between the luminal microbiota and the host. This barrier is formed by the membranes of individual epithelial cells and tight junction proteins that seal the paracellular spaces between adjacent cells. Therefore, the permeability of this barrier is regulated by the integrity of the cell plasma membranes and tight junctions, as well as epithelial cell processes that mediate secretion and absorption. Small molecules (<300 Da) and electrolytes passively traverse the tight junction barrier.

[0005] Membrane permeability can be altered by several physiological and pathological stimuli. Under homeostasis, the intestinal epithelium absorbs nutrients while effectively preventing the migration of intestinal bacteria. However, certain pathological conditions (e.g., enterotoxins or inflammation) can increase paracellular transport and enhance intestinal barrier permeability, posing a risk of nutrient malabsorption and an inability to prevent the migration of intestinal bacteria and their products. This can lead to chronic intestinal diseases and affect distal organs responsible for eliminating and filtering migrated bacteria and associated products.

[0006] Increased intestinal permeability can lead to systemic inflammation, which is associated with a variety of clinical conditions, including: • Food allergies or hypersensitivity; • Gluten sensitivity and celiac disease; • Inflammatory bowel disease (Crohn's disease or ulcerative colitis); • Autoimmune diseases (rheumatoid arthritis, psoriasis, type 1 diabetes, spondylitis, etc.); • Cognitive impairment (anxiety, depression, schizophrenia, etc.); • Neurological conditions (e.g., Guillain-Barré syndrome, multiple sclerosis).

[0007] The following are known methods for determining intestinal permeability.

[0008] 1. The lactulose / mannitol or lactitol / mannitol test has been performed for over 40 years and measures the ability of two sugar molecules, namely lactulose (an isomer of lactose) and mannitol, or their derivatives, to pass through the intestinal epithelium. Mannitol is a small sugar molecule that is easily absorbed, while lactulose is a larger molecule that is not effectively absorbed. After collecting a reference urine sample, the patient ingests lactulose and mannitol. The concentrations of lactulose and mannitol in urine collected over 6 hours after ingestion are then quantified. This method is economical and easy to perform as urine samples can be collected at the patient's home. However, this method has the drawback of measuring the permeability of small sugar molecules that do not possess immunogenic activity. Furthermore, this method cannot analyze the possibility that antigenic macromolecules may pass through the epithelial barrier to cause and exacerbate potential inflammatory conditions and autoimmune diseases. Lactulose is also a low molecular weight substance, and the movement of this substance across the intestinal barrier does not reflect the movement of dietary proteins and the overall immune response. Furthermore, lactulose intake may worsen symptoms associated with small intestinal overgrowth (SIBO) and is considered a prebiotic. Additionally, it requires highly trained professionals and / or techniques involving expensive equipment such as high-performance liquid chromatography (HPLC), gas chromatography, or tandem mass spectrometry (MS / MS). Finally, because plant-based foods (especially fruits, vegetables, and legumes) are naturally rich in mannitol, patients need to follow a complex diet.

[0009] 2. Macromolecular indicators of intestinal permeability that enable the evaluation of molecules that may possess antigenicity. The presence of specific antibodies against such molecules can reveal the possibility that intestinal permeability was increased at some point in the past. These include the following: a. Lipopolysaccharide (LPS) is a large molecule found on the outer membrane of Gram-negative bacteria. LPS is an endotoxin and, upon absorption, triggers a strong immune response. Detection of LPS antibodies suggests that the endotoxin has crossed the intestinal barrier and entered the systemic circulation. b. Ocurdin is a major component of the proteins that maintain tight junctions between intestinal cells. Detection of antibodies against occurdin may indicate disruption of tight junctions (paracellular pathways). c. Zonulin has been proposed to be a protein that regulates intestinal permeability. Detection of antibodies against zonulin indicates impaired tight junction control (paracellular pathway). d. The actomyosin network is a protein complex that regulates the function of the intestinal barrier by maintaining the plasticity of tight junctions. Antibodies against actomyosin are biomarkers of intestinal barrier dysregulation via cell infiltration (transcellular pathway). e.LBP (Lipopolysaccharide-binding protein). This protein has been found to bind to LPS and can be measured in the blood.

[0010] These indicators are suggested to allow for the identification of levels of intestinal permeability and their causes (LPS or bacterial overgrowth versus stress or food sensitivities).

[0011] Generally, the larger the molecule, the less likely it is to reach the circulatory system. Antibody measurements are affected by steroid use. Therefore, patients must discontinue oral or topical steroid use for at least 60 days prior to analysis. Also, the reference values ​​are based only on adults, and their application to pediatric populations is difficult due to the lack of known normal ranges.

[0012] 3. Direct measurement of serum or blood zonulin. Circulating zonulin (compared to antibodies against zonulin) does not show false negatives with the use of oral or topical steroids, but its levels increase with the use of corticosteroids, potentially leading to false positives for intestinal permeability. Zonulin levels are high in obese individuals and those with impaired glucose tolerance, potentially indicating false positives for intestinal permeability.

[0013] Therefore, to report the most important information, a simple method for assessing the state of intestinal permeability should be based on immunogenic proteins or peptides from commonly consumed foods to monitor diseases associated with permeability changes.

[0014] This invention aims to solve the technical problems associated with current methodologies, namely, that they do not measure the ability of food antigenic peptides to enter the body, are more invasive, expensive, indirect, and / or cumbersome, and proposes a novel strategy for evaluating the intestinal permeability status of a subject. [Overview of the Initiative]

[0015] Brief description of the invention The present invention relates to an in vitro method for evaluating the state of intestinal permeability in a subject, and for diagnosing diseases or dysfunctions associated with or exhibiting increased intestinal permeability, such as celiac disease, Crohn's disease, irritable bowel syndrome, microscopic colitis, functional dyspepsia, chronic / acute villous atrophy resulting from chronic treatment with certain drugs, and non-IgE-mediated food hypersensitivity, as well as for evaluating it as a risk factor for several neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, or multiple sclerosis. Currently, it is not possible to adequately determine individual reference values ​​and changes in intestinal permeability for large molecules such as immunogenic proteins or large food peptides. Therefore, the present invention relates to an in vitro method for determining the degree of intestinal permeability by the following general sequential procedure.

[0016] Step 1: Maintain a gluten-free diet for at least 16 hours, preferably 32 hours, and fast for at least the last 8 hours. Step 2: Take a specified amount of gluten powder (at least 0.5g, preferably 10g) in a suspended or hydrolyzed form in a fluid medium (water, shake, yogurt, etc.). Step 3: Collect a total urine sample over a period of at least 6 hours, maintaining a fast for at least 4 hours during that time, and maintaining a gluten-free diet throughout the entire urine collection period. Step 4: Measure the volume of collected urine and the amount of gluten in the collected urine using a procedure to detect gliadin (as a protein used as an analytical standard for gluten) at levels of less than 15 ng / ml in the urine. Step 5: Compare the excreted gluten levels to the internal reference value (of the same individual) or the mean value of healthy individuals, and other reference values ​​when gluten peptide permeability changes. If there are more gluten peptides in the urine than the internal reference value, it means that the individual's intestinal permeability has not improved. If the gluten peptide levels are higher than the normal margin compared to the corresponding mean of the reference study group (age, sex), the individual has increased intestinal permeability. If it is within the reference margin, the intestines are not showing signs of increased permeability.

[0017] The gluten-free diet in Step 1 is performed to ensure that all gluten peptides originate from the gluten ingested in the suspension in Step 2, and not from food consumed in the hours prior to ingestion. An 8-hour fast prior to ingestion also ensures that there is no food residue remaining in the stomach, as well as in the duodenum, the portion of the small intestine connected to the jejunum. Extending the fasting period beyond 8 hours reduces the possibility of interference from other foods in the metabolism of gluten ingested in Step 2. Removing gluten from the diet for at least 24 hours prior to ingestion reduces the likelihood of having gluten residue from other previously consumed foods, but a 16-hour restriction is usually sufficient to remove all detectable gluten from urine using commercially available methods (e.g., Gluten Detect by Biomedal, Seville).

[0018] The administered gluten is provided in an amount of at least 0.5 g to ensure its detection in urine, and is preferably purified in powder or hydrolyzed form (>80%) to enhance the rate and efficiency of digestion and absorption by the intestines due to a high contact surface with hydrolytic factors of the digestive system. To facilitate and make more comfortable intake in the individual being analyzed, the gluten suspension can be prepared in water or in other fluids such as shakes or yogurt, using dispersants and flavorings. Gluten can be hydrolyzed by pepsin-trypsin enzymatic hydrolysis or enzymatic digestion, which allows for the solubilization of the majority of immunogenic gluten peptides (GIPs) without destroying them, which can be detected by commercially available immunological methods. Preferably, 4–40 g of gluten preparations should be used to have sufficiently high urinary gluten peptide levels to quantify GIPs by commercially available methods, e.g., Quantitative GIP Urine Lateral Flow Strip (LFIA) iVYCHECK read by an LFIA reader (Biomedal, Spain), and other methods that can be applied in small clinical laboratories, and even at the point of care.

[0019] It is preferable that patients fast for 2 to 9 hours after gluten intake to promote rapid digestion of the ingested gluten and produce fragmented gluten peptides that travel through the intestines.

[0020] While maintaining a gluten-free diet, all urine collected for at least 8 hours after ingesting gluten preparations should be collected. This period can be extended up to 16 hours if it is necessary to collect the majority of gluten peptides excreted in the urine, and therefore gluten peptides excreted after that time will remain. Ideally, the collection period after ingestion should be from 6-8 AM to 10 PM-12 AM, accompanied by a gluten-free diet for 24-36 hours prior and a minimum of 8 hours of fasting. The amount of urine excreted during this period should be measured using a graduated urine collection container or cylinder. The concentration of gluten peptides in the urine collected over 8-16 hours should be determined using quantitative methods.

[0021] In a preferred embodiment of the present invention, as a method for determining gluten peptides, specific antibodies against gluten proteins already described, particularly those capable of quantitatively determining gluten in food with high sensitivity (detecting gliadin less than 15 ng / ml), and antibodies that have demonstrated the ability to detect gluten peptides in urine, such as G12, A1 (Biomedal, Spain) and R5 (r-Biopharma (Germany) or Eurofins Ingenasa (Spain)) can be used. They can also be obtained by performing the usual procedures for obtaining antibodies and immunizing animals with peptides having sequences that are most abundantly found when gluten peptides in urine are analyzed by liquid chromatography and mass spectrometry (LC / MS) (Table 1).

Table 1

[0022] The resulting antibodies are effective as long as the quantification limit of the analytical procedure is preferably gliadin less than 15 ng / ml. For very low concentrations of gluten peptides in urine, a detection limit of less than 6 ng / ml of peptide sequence number 1 (33-residue α-gliadin) per ml is required so that the levels of specific gluten peptides in urine can be detected after ingestion of 0.5 - 5 g of gluten.

[0023] To prepare urine samples and enhance the detection sensitivity, a urine peptide concentration system can also be applied in one form of the present invention. Some fractionation concentrations of gluten peptides that have been proven to be effective include solid-phase extraction using a matrix with C18, size exclusion chromatography, solvent evaporation, affinity chromatography, and the like.

[0024] A preferred method for detecting gluten in urine is sandwich ELISA, which quantifies the amount of antigen-antibody complex by coupling one of the antibodies specific to an enzyme that can deliver a measurable signal proportional to the formation of an immune complex with gluten peptides using a colorimetric, fluorescent, or chemiluminescent reagent. Fluorescence or chemiluminescence methods are actually more convenient to apply due to their wider dynamic range and typically do not require dilution to fall within the range, as is the case with colorimetric methods.

[0025] In preferred embodiments of the present invention, a lateral flow immunochromatography strip that can be quantified by a strip reader using image analysis with gold particles or colored polystyrene particles is also intended. Furthermore, quantification is possible using a suitable fluorescence analyzer by using conventional or time-resolved fluorescent probes, such as europium particles. By binding a suitable probe to an antibody, other options such as heat sense or quantum dots can be used as signal quantification options.

[0026] In alternative embodiments of the present invention, instead of antibodies, aptamers developed to recognize gluten in a biological matrix, as have already been developed for food applications, may be used.

[0027] The procedure of the present invention can be easily carried out in a preferred form using a kit containing gluten powder containing at least 0.5 g of gluten, preferably 4 to 40 g of gluten, more preferably 8 to 10 g of gluten, in a gelatin capsule or a sachet containing a dispersant and / or flavoring agent. With such an amount of gluten, gluten excretion can be detected by at least one of the commercially available methods. For example, the LFIA method already marketed by Biomedal (iVYCHECK GIP Urine) based on an antibody sensitive and specific to 33-residue α-gliadin achieves urinary levels exceeding the detection limit after 0.5 g intake in most individuals (87%). It has been reported that 0.5g of gluten does not cause symptoms in most cases of celiac disease (Burger, JPW; van Lochem, EG; Roovers, EA; Drenth, JPH; Wahab, PJ, Dose-Escalating (50-500mg) Gluten Administration Leads to Detectable Gluten-Immunogenic-Peptides in Urine of Patients with Coeliac Disease Which Is Unrelated to Symptoms, a Placebo Controlled Trial. Nutrients 2022, 14, 1771. https: / / doi.org / 10.3390 / nu14091771), and therefore these amounts can be used in cases of celiac disease where the appearance of symptoms is a concern. In any case, to maximize the effectiveness of the procedure of the present invention, it is recommended to use at least 4g to obtain a sufficient amount of gluten peptide to facilitate detection in most individuals, regardless of the degree of intestinal permeability, due to the sensitivity requirements for detecting gluten peptides in urine. Population studies of celiac disease have shown that they do not have a higher mortality risk than the normal population.Gluten intake is frequent in most celiac patients, with 67–89% consuming gluten at least once a week or month [Silvester JA, Comino I, Rigaux LN, et al. Exposure sources, amounts and time course of gluten ingestion and excretion in patients with coeliac disease on a gluten-free diet. Aliment Pharmacol Ther. 2020;00:1-11. https: / / doi.org / 10.1111 / apt.16075]. Occasional gluten intake appears to be less harmful than smaller but more frequent intake [Garzon-Benavides M, Ruiz-Carnicer A, Segura V, Fombuena B, Garcia-Fernandez F, Sobrino Rodriguez S, et al. Clinical utility of urinary gluten immunogenic peptides in the follow-up of patients with coeliac disease. Aliment Pharmacol Ther. 2023;00:1-11. https: / / doi.org / 10.1111 / apt.17417].

[0028] Other optional components of the kit may be at least one urine collection container with a maximum capacity of 2 L for at least the next 8 hours, preferably 9–16 hours, after gluten intake. Generally, a total capacity of 1.2 L in all containers should be sufficient for that period (up to 12 hours from gluten intake). Gluten peptides are stable in urine and can be sent to an analytical laboratory at room temperature. Finally, another kit component is a method for determining gluten peptides. The possibility of using LFIA for quantification allows it to be performed as a point-of-care device (POC) in a healthcare center close to the patient with a reader that can be added in the preferred form of the present invention, but quantitative testing methods such as ELISA, Luminex®, CLIA (chemiluminescent immunoassay), and biosensors can be used.

[0029] In more advanced forms of the procedure of the present invention, gluten peptides in urine can be concentrated by solid-phase extraction, precipitation / centrifugation using a specific formulation, or size exclusion chromatography. For example, gluten peptides can be fractionated by size exclusion chromatography (SEC) or by using a molecular size filter, such as Centricon®, Amicron®, or any apparatus capable of separating peptides corresponding to two molecular sizes. Preferably, the procedure will have a minimum of 1 kDa (detectable by immunological methods and containing two epitopes) and a maximum of 30 kDa, which is the size of gliadin. The ratio of total gluten peptides in urine to total gluten peptides in each fraction is a factor that allows us to know the amount of high molecular weight peptides in urine, and thus the degree of intestinal permeability to peptides of a particular size. These further detailed studies can be performed in individuals where more information is needed to determine the progression of intestinal disease or intestinal permeability.

[0030] The amount of gluten peptides in the urine of these individuals is measured by an immunological method using specific antibodies that quantify gluten peptides resistant to intestinal digestion at epitopes detectable in the urine of these individuals. The amount of gluten peptides excreted in the urine can be compared to a reference value. This reference value may be obtained from a study using a population without gastrointestinal conditions or states that impair intestinal permeability. Alternatively, the values ​​obtained in the first assay using individuals undergoing the procedure of the present invention may be an individualized reference value to consider when determining the progression of intestinal permeability in response to the treatment or chronic disease being evaluated or monitored. Detecting a higher amount of gluten peptides compared to baseline or reference values ​​indicates increased intestinal permeability, while a decrease in these values ​​indicates normalization of intestinal permeability.

[0031] Accordingly, the first embodiment of the present invention relates to an in vitro method for evaluating the intestinal permeability of a subject, comprising the steps of: a) orally administering at least 0.5 g of gluten (preferably 0.5 to 40 g of gluten) or a source thereof to the subject after a fasting period of at least 8 hours and a gluten-free diet of at least 16 hours; and b) determining the total amount of gluten peptides present in a whole urine sample obtained from the subject at least 6 hours (preferably 6 to 16 hours) after the administration in step a), and c) a significantly higher amount of gluten determined in step b) than a pre-established reference value indicates increased intestinal permeability.

[0032] A second embodiment of the present invention relates to an in vitro method for diagnosing, prognosticating, or monitoring patients suffering from diseases associated with increased intestinal permeability, selected from the group consisting of leaky bowel syndrome, celiac disease, Crohn's disease, irritable bowel syndrome, microscopic colitis, functional dyspepsia, chronic / acute villous atrophy due to long-term treatment with specific drugs, systemic inflammation, autoimmune diseases, and / or non-IgE-mediated food sensitivities, the method comprising determining the intestinal permeability of the subject according to the method of the first embodiment of the present invention.

[0033] In a preferred embodiment of the present invention, the gluten source is purified and suspended in an aqueous fluid medium.

[0034] In a preferred embodiment of the present invention, the determination of the amount of gluten peptide is performed directly via an immunoassay for the detection of the peptide itself.

[0035] In a preferred embodiment of the present invention, the immunoassay comprises an antibody against a gluten peptide comprising the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and / or derivatives thereof.

[0036] In preferred embodiments of the present invention, the amount of gluten peptide is determined by an immunoassay selected from LFIA, ELISA, CLIA, SIMOA, Luminex, NULISA, turbidimetry, immunochromatography, or biosensors.

[0037] In a preferred embodiment of the present invention, the gluten peptide is concentrated by one of the following types of processes: solid-phase extraction with a hydrophobic resin, size exclusion chromatography, precipitation, or affinity purification.

[0038] A second embodiment of the present invention is a parts kit suitable for carrying out the method of the present invention, The present invention relates to a component kit comprising: a) a formulation containing at least 0.5 g (preferably 0.5 to 40 g) of purified gluten (preferably unhydrolyzed or partially hydrolyzed gluten powder for suspension or a ready-to-use solution or suspension); b) a urine collection container; and c) an immunological test comprising an antibody capable of forming a complex with an immunogenic gluten peptide and detectable by turbidimetry, visual inspection, colorimetric analysis, fluorescence measurement, chemiluminescence, and / or thermal detection.

[0039] In a preferred embodiment of the present invention, the immunological test consists solely of a lateral flow strip having an antibody against an immunogenic gluten peptide.

[0040] In preferred embodiments of the present invention, the immunological test is performed using ELISA, LFIA, CLIA, or magnetic microparticles, fluorescent microparticles, or phosphorescent microparticles.

[0041] A third embodiment of the present invention relates to a reactive antibody against a gluten peptide or a derivative thereof, characterized by comprising or consisting solely of the sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and / or derivatives thereof, for use in an in vitro method for evaluating the intestinal permeability of a subject. Alternatively, the present invention relates to the use of a reactive antibody against a gluten peptide or a derivative thereof, characterized by comprising or consisting solely of the sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and / or derivatives thereof, for in vitro evaluation of the intestinal permeability of a subject.

[0042] In a preferred embodiment of the present invention, the present invention relates to a gluten peptide or antibody for use in an in vitro method for evaluating the intestinal permeability of a subject, the in vitro method comprising (a) orally administering at least 0.5 g of gluten (preferably 0.5 to 40 g of gluten) or a source thereof to a subject after a fasting period of at least 8 hours and a gluten-free diet of at least 16 hours, and (b) determining the amount of gluten peptide present in a urine sample obtained from the subject at least 6 hours (preferably 6 to 16 hours) after gluten intake, wherein a significantly higher amount of gluten determined in step (b) than a pre-established reference value indicates increased intestinal permeability. Alternatively, the present invention relates to the use of a gluten peptide or a reactive antibody to the peptide or its derivatives, characterized by comprising or consisting solely of the sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and / or derivatives thereof, for in vitro evaluation of the intestinal permeability of a subject, wherein the use comprises (a) orally administering at least 0.5 g of gluten (preferably 0.5 to 40 g of gluten) or a source thereof to the subject after a fasting period of at least 8 hours and after a gluten-free diet of at least 16 hours, and (b) determining the amount of gluten peptide present in a urine sample obtained from the subject at least 6 hours (preferably 6 to 16 hours) after gluten intake, wherein a significantly higher amount of gluten determined in step (b) than a pre-established reference value indicates increased intestinal permeability.

[0043] In a preferred embodiment of the present invention, the invention includes a concentration step by size exclusion chromatography, where an increase in the average size of urinary gluten peptides indicates increased intestinal permeability.

[0044] In the context of the present invention, the following terms or expressions are defined for better interpretation of their scope. The term "includes / equips" means that it encompasses, but is not limited to, what follows the word "includes / equips." Therefore, the use of the term "includes / equips" indicates that the enumerated elements are required or mandatory, while the other elements are optional, may or may not be present. The term "consisting of only" means that what follows the phrase "consisting of only" is included and limited to it. Therefore, the phrase "consisting of only" indicates that the listed elements are required or essential, and that other elements are absent. The term “pre-established reference value,” when referring to the amount of gluten determined in this invention, refers to the amount observed in healthy individuals (i.e., subjects without increased intestinal permeability), particularly in the initial state of the same individual before treatment. In a preferred embodiment, the reference value is measured in an initial assay performed on the subject against results from a population without intestinal disease, or against results from a population without intestinal disease in the same age range as the individual whose intestinal permeability is being analyzed. If the amount of gluten determined in the subject’s urine exceeds the “pre-established reference level,” the subject is likely to have increased intestinal permeability with a specific sensitivity and specificity. The “reference” value may be a threshold or cutoff value that can be determined experimentally, empirically, or theoretically. The threshold may also be arbitrarily selected based on existing experimental and / or clinical conditions, as recognized by those skilled in the art. The threshold must be determined to obtain optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical outcomes of false positives and false negatives). Typically, the optimal sensitivity and specificity (and therefore the threshold) can be determined by an ROC (Receiver Operating Characteristic) curve based on experimental data. [Brief explanation of the drawing]

[0045] [Figure 1]This shows a classification of the state of villous atrophy in the intestinal mucosa. VH:CD (villi height, crypt depth) is the mean ratio between the height of the intestinal villi and the depth of the crypts. A ratio of 2 or more is considered to be a Marsh classification of 0-2, indicating no or mild intestinal atrophy. A ratio of less than 2 is considered to be Marsh 3, ranging from 3a to 3c, suggesting severe intestinal atrophy. [Figure 2] This study shows the dependence of urinary immunogenic gluten peptide concentration and the degree of villous atrophy. All patients ingested 3 g of gluten contained in an energy bar. VH:CD is the ratio of villous height to crypt depth. LOQ = limit of quantification (2.5 ng / ml). [Figure 3] Comparison of average GIP excretion in celiac individuals based on the degree of villous atrophy observed in biopsies performed after extending daily intake of 3g of gluten for at least two months. [Figure 4] Excretion kinetics of gluten peptides in individuals without known intestinal disorders after ingesting 8g of powdered refined gluten while fasting. Each color represents a different subject. [Figure 5] Excretion kinetics of lactulose, expressed in ppm, from a total intake (10g) in individuals without known intestinal diseases (n=15). Each color represents a different subject. [Figure 6] Correlation between GIP excretion and lactulose / mannitol excretion ratio in total urine collected 15 hours after ingesting 8g of purified gluten suspension in the aforementioned fasting state. R² = 0.51. [Modes for carrying out the invention]

[0046] Detailed description of the invention The present invention is illustrated by the embodiments set forth below, without the intention of limiting its scope of protection. [Examples]

[0047] Example 1. Variation in urinary gluten peptide concentration in celiac individuals with different degrees of villous atrophy. This example demonstrates that clinical trial results show that the degree of villous atrophy in celiac disease patients increases urinary GIP excretion (u-GIP) with continuous intake, without controlling for the intake of other foods or the timing of urine collection. Statistically significant variations in peptide concentration can be observed depending on the degree of duodenal villous atrophy. In this study, data on the degree of villous atrophy in celiac individuals were obtained by administering the same amount of gluten for several days before or after duodenal biopsy and measuring the mean ratio of villous height to crypt depth (Vh:Cd). Data on the degree of villous atrophy were obtained by administering the same amount of gluten (3g in the form of an energy bar) daily to analytes (celiac patients) for 6 weeks after duodenal biopsy and measuring the mean ratio of villous height to crypt depth. The concentration of detectable gluten immunogenic peptides in the immunoassay was observed to be significantly higher with increasing intestinal mucosal atrophy. Therefore, for the same amount of gluten intake, the greatest determinant of the concentration of gluten peptides excreted in urine was the degree of increased intestinal permeability resulting from the progression of intestinal epithelial deterioration due to gluten intake (Figure 2). The mean urinary gluten concentration in celiac patients with atrophy grades 0–2 was 23.7 ng / ml, and the median was 11.8 ng / ml. For those with Marsh 3, the mean was 45.7 ng / ml, and the median was 29.9 ng / ml. The mean deviation was high in both cases (21 and 33, respectively), but this trend was significant, suggesting higher permeability in those with Marsh 3. Among those with Marsh 3, mean and median values ​​were observed for those with Marsh 3a, Marsh 3b, and Marsh 3c. These results are shown in Table 2 and Figure 3.

[0048] Table 2: GIP concentrations in samples from patients with varying degrees of intestinal atrophy after 3g gluten intake. VH:CD is the ratio of villous height to crypt depth. [Table 2]

[0049] More severe atrophy was associated with higher urinary GIP concentrations, and the difference was particularly significant in cases of severe atrophy (Marsh 3b and 3c). The most plausible explanation was increased paracellular intestinal permeability in intestinal mucosal damage caused by daily gluten intake.

[0050] Example 2. Metabolic variability between individuals This example of the present invention demonstrates that metabolic and excreted gluten values ​​obtained on different days in two different individuals yield similar u-GIP values. For this purpose, individuals 1 and 2 were given a gluten-free diet for 36 hours, fasted for at least 9 hours, and then ingested a sachet of gluten (8g) suspended in 250ml of water. A urinary GIP detection test was performed before gluten ingestion, and a negative result was confirmed. After ingestion, a gluten-free diet was continued for at least 8 hours, and urine was collected for 9 hours. Urinary GIP concentration was measured using a lateral flow test and a quantitative iVYCHECK Reader, and its volume was calculated. In both individuals analyzed, the standard deviation of GIP excretion detected between measurements taken on different days was less than 20% (Table 3), and therefore, since the variation in the absence of disease is not significant, the values ​​for each individual may serve as an internal reference value before treatment to improve the intestinal permeability of a given individual.

[0051] Table 3. Quantification of gluten peptide excretion (μg of gluten excreted in urine within 9 hours after ingestion) in two subjects after ingesting 8g of gluten suspension on different days while fasting. [Table 3]

[0052] The detection ratio of GIP in urine relative to ingested gluten is 14–23 μg for every 8 g of ingested gluten. Approximately 8 g of ingested gluten, using a specific anti-33 residue immunoassay, corresponds to approximately 1–1.33 g of GIP using iVYCHECK urine (Biomedal, Spain). Therefore, the amount excreted is only 0.001–0.002% of the amount ingested, which is why specific and highly sensitive techniques are required to detect this peptide in urine.

[0053] Example 3. Amount of gluten peptide excreted in different individuals compared to lactulose / mannitol permeability. In this example, it can be observed that the excretion of almost all detectable gluten in urine can occur within a few hours after fasting ingestion of a quantitative amount of powdered gluten, and that this correlates better with the excretion of lactulose than with smaller molecules such as mannitol, suggesting a paracellular permeability (lactulose) versus intracellular permeability (mannitol) comparison. Furthermore, it can be observed that the excretion of the majority of detectable gluten (>80%) occurs in the first 9-15 hours, reducing the degree of intra-individual variability for lactulose. Specifically, subjects (n=15) ingested 8g of gluten after at least 8 hours of fasting following at least 36 hours of gluten-free diet in individuals not diagnosed with celiac disease. The gluten consisted of gluten powder with added flavorings (sweeteners and food colorings) contained in single-use, heat-sealed sachets. Simultaneously, subjects ingested 1g of mannitol and 10g of lactulose. Subsequently, various urine samples were collected in separate containers, allowing for individualized measurement of the concentration and total volume of each urine excretion. The amount of gluten peptides present in urine excreted over 15 hours after gluten intake was determined using iVYCHECK urine products and a calibrated lateral flow strip reader, iVYCHECK Reader (Biomedal, Spain). Ingested gluten was mixed with 1 g of mannitol and 10 g of lactulose and used as controls. Mannitol was used to estimate intracellular permeability, and lactulose was used to estimate paracellular permeability; these reference values ​​have already been widely reported in the biomedical literature. The amount of gluten peptides directly correlated with intestinal permeability. While factors such as the rate of gluten digestion may have an influence, it is understood that within the same individual, the reference value can remain constant, as seen in the example above. Considering the variability of gluten metabolism and its excretion dynamics, it was recommended that intake be carried out when the subject has urinated and emptied the bladder as much as possible (early morning), preferably after maintaining a fasting period of at least 8 hours until the ingested gluten is excreted without being affected by other simultaneously consumed foods, in order to promote its metabolism.The obtained values ​​can be compared to reference values ​​obtained by performing the same procedure on individuals with or without intestinal permeability-related intestinal diseases, or they can be used as a reference themselves for subsequent determination of progression to higher gluten peptide excretion (higher permeability) or lower gluten peptide excretion (lower permeability).

[0054] In this example, conducted with 15 asymptomatic subjects who had not been diagnosed with gastrointestinal disorders, basal urine was analyzed. All urine was collected in separate containers over a 15-hour period following ingestion of gluten sachets. The excreted urine was mixed between 0 and 6 hours after ingestion, and then between 2 and 15 hours after ingestion. The concentration of GIP in the excreted urine was measured by both ELISA and LFIA, respectively, between 0 and 6 hours after ingestion, and between 2 and 15 hours after ingestion. The GIP concentration (Figure 4) and lactulose (Figure 5) of each urine sample from each subject were measured, and subsequently, the percentage of excreted GIP relative to ingested GIP and the lactulose / mannitol ratio (Figure 6) were calculated. The correlation between lactulose:mannitol and GIP was determined. The dynamics of lactulose excretion differed among individuals, and excretion occurred beyond 15 hours. It is known that sugars can be reabsorbed into the body. This is why lactulose may have several excretion peaks. Both lactulose and GIP are presumed to travel through the same paracellular pathway. Therefore, the 2–15 hour mixture of GIP is more similar to the 0–6 hour and 2–15 hour mixtures of lactulose.

[0055] The analysis is also performed to detect outliers in each group and exclude them from the correlation.

Claims

1. An in vitro method for evaluating the state of intestinal permeability of a subject, a. A step of orally administering at least 0.5 g of gluten or a source thereof to the subject after a fasting period of at least 8 hours and after a gluten-free diet of at least 16 hours, b. A step of determining the total amount of gluten peptide present in the whole urine sample obtained from the subject at least 6 hours after the administration performed in step a), An in vitro method comprising the above, wherein the amount of gluten determined in step b) is significantly higher than a pre-established reference value, indicating increased intestinal permeability.

2. An in vitro method for diagnosing, prognosticating, or monitoring a patient suffering from a disease associated with increased intestinal permeability, selected from the group consisting of leaky bowel syndrome, celiac disease, Crohn's disease, irritable bowel syndrome, microscopic colitis, functional dyspepsia, chronic / acute villous atrophy due to long-term treatment with specific drugs, systemic inflammation, autoimmune diseases, and / or non-IgE-mediated food sensitivities, comprising determining intestinal permeability in a subject according to the method of claim 1.

3. The in vitro method according to claim 1 or 2, characterized in that the gluten source is purified and suspended in an aqueous fluid medium.

4. The in vitro method according to any one of claims 1 to 3, characterized in that the determination of the amount of gluten peptide is performed directly via an immunoassay for the detection of the peptide itself.

5. The in vitro method according to any one of claims 1 to 4, wherein the immunoassay comprises an antibody against a gluten peptide comprising the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and / or derivatives thereof.

6. The in vitro method according to any one of claims 1 to 5, characterized in that the determination of the amount of gluten peptide is performed by an immunoassay selected from LFIA, ELISA, CLIA, SIMOA, Luminex, NULISA, turbidimetry, immunochromatography, or biosensor.

7. The in vitro method according to any one of claims 1 to 6, characterized in that the gluten peptide is concentrated by one of the following types of processes: solid-phase extraction with a hydrophobic resin, size exclusion chromatography, precipitation, or affinity purification.

8. An in vitro method according to any one of claims 1 to 7, wherein a concentration step is performed by size exclusion chromatography, and an increase in the average size of urinary gluten peptides indicates increased intestinal permeability of the intestinal tract.

9. A parts kit suitable for carrying out the methods described in claims 1 to 8, a. A preparation containing at least 0.5 g of purified gluten, b. Urine collection container and c. A component kit comprising an immunological test comprising an antibody capable of forming a complex with an immunogenic gluten peptide and detectable by turbidimetry, visual inspection, colorimetric methods, fluorescence assays, chemiluminescence assays, and / or thermal detection methods.

10. The component kit according to claim 9, wherein the immunological test consists solely of a lateral flow strip having an antibody against an immunogenic gluten peptide.

11. The component kit according to claim 9 or 10, wherein the immunological test is performed using ELISA, LFIA, CLIA, or magnetic microparticles, fluorescent microparticles, or phosphorescent microparticles.

12. Use of a gluten peptide or a reactive antibody against the peptide or its derivatives, characterized by comprising or consisting solely of the sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and / or derivatives thereof, for in vitro evaluation of the intestinal permeability of a target.

13. Use of gluten peptide or antibody according to claim 12 for use, comprising the steps of (a) orally administering at least 0.5g of gluten or a source thereof to a subject after a fasting period of at least 8 hours and after a gluten-free diet of at least 16 hours, and (b) determining the amount of gluten peptide present in a urine sample obtained from the subject at least 6 hours after the gluten intake, wherein a significantly higher amount of gluten determined in step b) than a pre-established reference value indicates increased intestinal permeability.

14. The use according to claim 12 or 13, wherein a concentration step is performed by size exclusion chromatography, and the increase in the average size of urinary gluten peptides indicates increased intestinal permeability of the intestinal tract.

Images