WO2014044281A1 - Inhibition d'un peptide de 33-mer dérivé de la gliadine pour le traitement du diabète - Google Patents

Inhibition d'un peptide de 33-mer dérivé de la gliadine pour le traitement du diabète Download PDF

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Publication number
WO2014044281A1
WO2014044281A1 PCT/DK2013/050297 DK2013050297W WO2014044281A1 WO 2014044281 A1 WO2014044281 A1 WO 2014044281A1 DK 2013050297 W DK2013050297 W DK 2013050297W WO 2014044281 A1 WO2014044281 A1 WO 2014044281A1
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diabetes
gliadin
mer
inhibitor
cells
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PCT/DK2013/050297
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Karsten Buschard
Knud Josefsen
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Rigshospitalet
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention relates to a method for prevention and/or treatment of diabetes comprising the administration of an inhibitor of a 33-mer gliadin peptide
  • Type 1 diabetes (T1 D) is a common disease, affecting almost 20 million people worldwide. It comes with the burden of daily insulin injections and blood testing, as well as both short- and long-term complications, and this can include premature death.
  • T1 D accounts for approximately 15% of the diabetic population. Insulin deficiency is a result of the autoimmune destruction of the insulin producing pancreatic beta cells; however, clinical onset of diabetes does not occur until a substantial part of these cells have been destroyed.
  • Type 2 diabetes (T2D), formerly known as non-insulin-dependent diabetes mellitus (NIDDM), accounts for the majority of the remainder of the diagnosed cases of diabetes. T2D stems from both a decreased secretion of insulin as well as the body's inability to effectively utilize the insulin produced (insulin resistance). Current research suggests that the disposition to T2D is genetically inherited, with a high concordance rate in identical twins. Also referred to as adult-onset diabetes, T2D generally develops after the age of thirty, and is commonly associated with obesity. However, T2D can develop at an earlier age. Importantly, several immunological components have been identified in the pathogenesis of the disease in many of the sub-groups of diabetes, including LADA, MODY and gestational diabetes.
  • Coeliac disease also known as gluten intolerance, is a common chronic inflammatory enteropathy, caused by dietary gluten or more specifically by gliadin. Gliadin is a glycoprotein component of gluten and is found in wheat and some other grains, including rye, barley, and millet.
  • Type 1 diabetes In animal models, removal of dietary gluten protects against the development of type 1 diabetes (Funda et al. Gluten-free diet prevents diabetes in NOD mice. Diabetes Metab Res Rev. 1999; 15:323-327). In humans, Type 1 diabetes and other autoimmune diseases occur at a lower rate in patients diagnosed with coeliac disease at a younger age, suggesting that early elimination of gluten may also protect against the
  • WO 2009/034110 relates to immunisation against diabetes and discloses that intranasal administration of gliadin delays and decreases diabetes incidence in non- obese diabetic mice (NOD mice). NOD mice are commonly used as an animal model for type 1 diabetes.
  • Mojiban et al. (Diabetes, Vol. 58, August 2009) have studied the T-cell immune response of type 1 diabetes patients to a number of dietary wheat polypeptides, including the gliadin 33-mer peptide. Half of the diabetic patients had T-cell responses against wheat proteins, indicating an impaired tolerance to gluten peptides. The results were interpreted as evidence of gut barrier and immune system dysfunction in some patients with type 1 diabetes.
  • US 2007/0184049 discloses antibodies capable of reacting with gluten or gluten- derived peptides including the 33-mer gliadin peptide for treatment of diseases associated with gluten intolerance, such as coeliac disease.
  • gliadin and the 33-mer gliadin peptide have previously been investigated for their role in coeliac disease and a possible role for gliadin has been suggested in diabetes.
  • the 33-mer has not previous been investigated for a role in diabetes.
  • the present inventors have shown that digested gliadin stimulates insulin secretion of pancreatic beta cells in vitro. Surprisingly, the inventors found that the increased insulin secretion was mediated directly by the 33-mer gliadin peptide, while a 19-mer gliadin peptide had no effect on insulin secretion. Hence, the inventors of the present invention suggest specific inhibition of the 33-mer peptide for treatment of diabetes.
  • the present invention thus relates to a method for prevention and/or treatment of diabetes comprising the administration of an inhibitor of a 33-mer gliadin peptide (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF; herein identified as SEQ ID NO: 1) and/or fragments thereof.
  • the present invention further relates to a method for prevention and/or treatment of diabetes comprising the administration of an inhibitor of the deamidated form of the 33-mer peptide consisting of the amino acid sequence: LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF; herein identified as SEQ ID NO: 6 and/or fragments thereof.
  • the inhibitor may for example be an enzyme capable of cleaving the 33-mer peptide and/or fragments thereof or an antibody capable of binding to the 33-mer peptide and/or fragments thereof.
  • the present invention further relates to an inhibitor of the 33-mer peptide and/or fragments thereof for use in prevention and/or treatment of diabetes.
  • the inhibitor of the present invention may be used in prophylactic, ameliorative and/or curative treatment of all types of diabetes, such as type 1 diabetes, type 2 diabetes, Latent Autoimmune Diabetes of Adults (LADA), Maturity onset diabetes of the young (MODY) and gestational diabetes.
  • type 1 diabetes type 2 diabetes
  • LADA Latent Autoimmune Diabetes of Adults
  • MODY Maturity onset diabetes of the young
  • gestational diabetes such as type 1 diabetes, type 2 diabetes, Latent Autoimmune Diabetes of Adults (LADA), Maturity onset diabetes of the young (MODY) and gestational diabetes.
  • the methods of the present invention represent an additional or alternative treatment to standard diabetes treatments including cumbersome diet restrictions, vigorous exercise programs and/or insulin administration or other anti-diabetic medications.
  • Figure 1 Gliadin increases weight in NOD mice.
  • NOD mice were injected with gliadin (4.5 ⁇ g/mL(1 : 1000 dilution); 450 ⁇ g/mL (1 :10 dilution); controls) five times over two weeks. Blood glucose levels and weight were measured twice a week.
  • gliadin 4.5 ⁇ g/mL(1 : 1000 dilution); 450 ⁇ g/mL (1 :10 dilution); controls
  • FIG. 2 Gliadin increases insulin secretion in INS-1E cells and rat islets during 24 h stimulation.
  • FIG. 5 The 33-mer gliadin fragment inhibits K A T p currents and stimulates insulin secretion in INS-1E cell. Kir6.2 and SUR1 were transiently expressed in HEK293 cells. The cells were incubated with either the 19-mer or 33-mer gliadin fragment in the medium o/n.
  • B Representative currents from a cell incubated with the 33-mer before and after washout of ATP.
  • C The time-dependence of the effect of the 19-mer (black circles) or the 33-mer (grey circles).
  • the representative data points represent max inward current at the start of the ramp protocol during washout of endogenous ATP.
  • D Summarized current densities.
  • FIG. 1 Tissue distribution of 33-mer (A) and 19-mer (B) gliadin peptides 1 hour following oral administration in mice.
  • Gp Parotic Gland
  • Gm Submandibular gland
  • St Stomach
  • Du Duodenum
  • Je Jejunum
  • II Ileum
  • Pa Pancreas
  • Li Liver
  • Gb Gall Bladder
  • Sp Spleen
  • Ki Kidney
  • He Heart
  • Pu Lungs
  • Gt Thyroid Gland.
  • Figure 7 Anatomical localisation of 3H-labeled 33-mer in mouse pancreas.
  • An antibody also known as an immunoglobulin (Ig) with known binding specificity, is a large Y-shaped protein produced by B cells that is used by the immune system to identify and neutralize foreign objects or promote uptake of the objects by phagocytic cells. The antibody recognizes a unique part of the foreign target, called an antigen.
  • Each tip of the "Y" of an antibody contains a paratope (a structure analogous to a lock) that is specific for one particular epitope (similarly analogous to a key) on an antigen, allowing these two structures to bind together.
  • Chemical forces,- such as electrostatic forces, hydrogen bonds, hydrophobic interaction and van der Waals forces - make antigen and antibody stick together.
  • an antibody can tag a target for attack by other parts of the immune system, or can neutralize its target directly, for example by direct blocking of the target's ability to interact with other molecules, thereby inhibiting the biological effect of the target.
  • Inhibitor The term "inhibitor" used in the present application is to be interpreted as any molecule capable of reducing the biological activity of another molecule.
  • an inhibitor according to the present invention may be an enzyme capable of degrading the 33-mer peptide or an antibody capable of binding to the 33-mer thus preventing the 33-mer from exerting its biological effects.
  • Gluten is a protein composite found in foods processed from wheat and related grain species, including barley and rye. It gives elasticity to dough, helping it to rise and to keep its shape, and often gives the final product a chewy texture. Gluten may also be found in some cosmetics or dermatological preparations. Gluten is the composite of a gliadin and a glutelin, which is conjoined with starch in the endosperm of various grass-related grains. Detailed description of the invention
  • the present invention relates to a method for prevention and/or treatment of diabetes comprising the administration of an inhibitor of the 33-mer gliadin peptide:
  • LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF herein identified as SEQ ID NO: 1 and/or fragments thereof.
  • the present invention further relates to a method for prevention and/or treatment of diabetes comprising the administration of an inhibitor of the deamidated form of the 33-mer peptide consisting of the amino acid sequence: LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF; herein identified as SEQ ID NO: 6 and/or fragments thereof.
  • the inhibitor may be an enzyme capable of cleaving the 33-mer peptide and/or fragments thereof or an antibody capable of binding to and neutralizing the 33-mer peptide and/or fragments thereof.
  • the present invention further relates to an inhibitor of the 33-mer peptide and/or fragments thereof for use in prevention and/or treatment of diabetes.
  • the treatment may be curative or ameliorative.
  • the inhibitor of the present invention results in amelioration or alleviation of diabetes symptoms, such as by normalisation of blood glucose and/or insulin levels.
  • the inhibitor of the present invention may be used in prophylactic, ameliorative and/or curative treatment of type 1 diabetes, type 2 diabetes, LADA, MODY and gestational diabetes.
  • the present invention further relates to a method for normalisation of blood glucose and/or insulin levels in a subject in need thereof.
  • the present invention relating to specific inhibition of the 33-mer gliadin peptide provides an alternative and/or additional treatment to standard diabetes treatments, which include insulin administration and radical lifestyle interventions including diet restrictions and exercise.
  • Diabetes mellitus or simply diabetes, is a group of metabolic diseases in which a person has high blood sugar, either because the body does not produce enough insulin, or because cells do not respond to the insulin that is produced. This high blood sugar produces the classical symptoms of polyuria (frequent urination), polydipsia (increased thirst) and polyphagia (increased hunger).
  • Type 1 diabetes mellitus is characterized by loss of the insulin-producing beta cells of the islets of Langerhans in the pancreas, leading to insulin deficiency. This type can be further classified as immune-mediated or idiopathic. The majority of type 1 diabetes is of the immune-mediated nature, in which beta cell loss is a T-cell-mediated
  • Type 1 diabetes can affect children or adults, but was traditionally termed "juvenile diabetes" because a majority of these diabetes cases were in children. Incidence varies from 8 to 17 per 100,000 in Northern Europe and the U.S., with a high of about 35 per 100,000 in Scandinavia, to a low of 1 per 100,000 in Japan and China.
  • Type 2 diabetes mellitus is characterized by insulin resistance, which may be combined with relatively reduced insulin secretion. The defective responsiveness of body tissues to insulin is believed to involve the insulin receptor.
  • the specific defects are not known.
  • the predominant abnormality is reduced insulin sensitivity.
  • hyperglycemia can be reversed by a variety of measures and medications that improve insulin sensitivity or reduce glucose production by the liver.
  • an estimated 346 million people have type 2 diabetes.
  • Latent Autoimmune Diabetes of Adults also known as Diabetes Type 1.5, is slow-onset Type 1 autoimmune diabetes in adults.
  • LADA is a genetically-linked, hereditary autoimmune disorder that results in the body mistaking the pancreas as foreign and responding by attacking and destroying the insulin-producing beta islet cells of the pancreas.
  • Adults with LADA are frequently initially misdiagnosed as having Type 2 diabetes, based on age, not etiology. In a recent survey conducted by
  • Maturity onset diabetes of the young refers to any of several hereditary forms of diabetes caused by mutations in an autosomal dominant gene (sex independent, i.e. inherited from any of the parents) disrupting insulin production.
  • MODY is often referred to as "monogenic diabetes” to distinguish it from the more common types of diabetes (especially type 1 and type 2), which involve more complex combinations of causes involving multiple genes (i.e., "polygenic") and environmental factors.
  • MODY 2 and MODY 3 are the most common forms.
  • Maturity onset diabetes of the young (MODY) is a rare autosomal dominant form of type 2 DM affecting young people with a positive family history.
  • MODY should not be confused with latent autoimmune diabetes of adults (LADA)— a form of type 1 DM, with slower progression to insulin dependence in later life.
  • diabetes All forms of diabetes have been treatable since insulin became available in 1921 , and type 2 diabetes may be controlled with medications. Both types 1 and 2 are chronic conditions that cannot be cured. Pancreas transplants have been tried with limited success in T1 D; gastric bypass surgery has been successful in many with morbid obesity and T2D. Gestational diabetes usually resolves after delivery. Diabetes without proper treatments can cause many complications. Acute complications include hypoglycemia, diabetic ketoacidosis, or nonketotic hyperosmolar coma. Serious long- term complications include cardiovascular disease, chronic renal failure, and diabetic retinopathy (retinal damage). Adequate treatment of diabetes is thus important, as well as blood pressure control and lifestyle factors such as smoking cessation and maintaining a healthy body weight.
  • beta cells The activity of beta cells is important in the development of diabetes, and increased insulin secretion has been correlated to increased diabetes development.
  • the inhibitor of the present invention is used in the treatment of diabetes.
  • Said treatment may be prophylactic, ameliorative or curative.
  • the present invention relates to treatment of type 1 diabetes.
  • the present invention relates to treatment of type 2 diabetes.
  • the present invention relates to treatment of gestational diabetes. In one embodiment, the present invention relates to treatment of LADA. In one embodiment, the present invention relates to treatment of MODY. Metabolic syndrome
  • Metabolic syndrome is a combination of medical disorders that, when occurring together, increase the risk of developing cardiovascular disease and diabetes. Some studies have shown the prevalence in the USA to be an estimated 25% of the population, and prevalence increases with age.
  • Metabolic syndrome is also known as metabolic syndrome X, cardiometabolic syndrome, syndrome X, insulin resistance syndrome, Reaven's syndrome (named for Gerald Reaven), and CHAOS (in Australia).
  • - Reduced HDL cholesterol ⁇ 40 mg/dL (1.03 mmol/L) in males, ⁇ 50 mg/dL (1.29 mmol/L) in females, or specific treatment for this lipid abnormality - Raised blood pressure (BP): systolic BP > 130 or diastolic BP >85 mm Hg, or treatment of previously diagnosed hypertension
  • the inhibitor of the present invention is used in the treatment of metabolic syndrome.
  • Gliadin is a strongly hydrophobic glycoprotein belonging to the prolaminins and together with glutenin, it constitutes gluten. It attributes elasticity to white bread, making it universally present in the western diet. Its low solubility limits its enzymatic degradation, resulting in multiple undigested gliadin fragments of varying lengths in the gut and intestine. Many of these gliadin fragments have been investigated for their role in the development of coeliac disease. For example a 33-mer peptide
  • LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO: 1), a 19-mer gliadin peptide LGQQPFPPQQPYPQPQPF-OH (SEQ ID NO: 2), a 20-mer peptide
  • LGQQQPFPPQQPY SEQ ID NO: 4
  • multiple other peptide fragments of gliadin have been implicated in the pathogenesis of coeliac disease.
  • the 33-mer gliadin peptide is considered to be stable toward breakdown by all gastric, pancreatic, and intestinal brush-border membrane proteases. However, in some instances it may be further degraded into several shorter peptide fragments, such as an 18-mer peptide product (PQLPYPQPQLPYPQPQPF (SEQ ID NO: 5)). The further degradation of the 33-mer may be mediated by gut microorganisms capable of cleaving the 33-mer.
  • the 33-mer also exists in a deamidated form:
  • LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO: 6), to which the present invention also relates.
  • the present inventors have shown that digested gliadin stimulates insulin secretion of pancreatic beta cells in vitro. Surprisingly, the inventors found that the increased insulin secretion was mediated specifically by the 33-mer gliadin peptide, while a 19-mer gliadin peptide had no effect on insulin secretion. Hence, the inventors of the present invention suggest specific inhibition of the 33-mer peptide for treatment of diabetes. Inhibitors of the 33-mer
  • Inhibition of the biological effect of the 33-mer may be performed in any way known to a person skilled in the art, such as by direct enzymatic cleavage of the 33-mer or by antibody-mediated inhibition of the 33-mer.
  • An inhibitor according to the present invention is any molecule capable of interacting with the 33-mer and/or fragments thereof and thereby inhibits the 33-mer and/or fragments thereof from exerting its biological effect in the subject.
  • Inhibitors according to the present invention include but are not limited to: enzymes capable of degrading the 33-mer; naturally occurring or genetically engineered microorganism capable of degrading the 33-mer; molecules capable of preventing intestinal absorption of the 33- mer by direct interaction with the 33-mer in the intestine, such as proteins, nucleic acids, nanoparticles and resins; and antibodies capable of binding to the 33-mer.
  • the inhibitor is an enzyme capable of degrading the 33-mer.
  • Enzymes capable of cleaving the 33-mer peptide may e.g. be derived from
  • microorganisms such as naturally occurring strains of microorganisms or genetically engineered microorganisms.
  • Enzymes capable of cleaving the 33-mer and/or fragments thereof are known in the art from e.g.: Zamakhchari et al., 2011. PLOS one, 6(9), p. 1-10; De Angelis et al., 2010. Appl. Environ. Microbiol, 76(2): 508-18; WO 201 1/1 10884; WO 2011/044365), all of which are incorporated herewith by reference.
  • the inhibitor of the present invention is an enzyme capable of degrading breakdown products of the 33-mer peptide, such as the 18-mer peptide breakdown product.
  • the inhibitor of the 33-mer is a microorganism capable of degrading the 33-mer and/or fragments thereof.
  • the microorganism may be a naturally occurring microorganism or a genetically modified microorganism containing and/or secreting the inhibitor.
  • the inhibitor of the present invention may also be a molecule or compound capable of inhibiting the intestinal absorption of the 33-mer by direct interaction with the the 33- mer in the intestine.
  • Such molecules or compounds include but for example proteins, nucleic acids, nanoparticles and resins.
  • the inhibitor of the present invention is an antibody capable of binding to the 33-mer peptide and/or fragments thereof, thereby reducing or eliminating the biological effect of the 33-mer.
  • Antibodies capable of reacting with the 33-mer peptide are known in the art from e.g. US 2007/0184049, which is hereby incorporated by reference.
  • the inhibitor of the present invention is an antibody capable of binding to breakdown products of the 33-mer peptide, such as the 18-mer peptide.
  • the biological activity of the 33-mer is inhibited by direct immunization with the 33-mer.
  • the 33-mer may be introduced into a subject e.g. by injection to induce an immune response.
  • the immune response will induce tolerance to the 33-mer and may comprise antibody production against the 33- mer and/or T-cell reactions against the 33-mer.
  • the 33-mer may also be administered to a subject by mucosal administration, such as by nasal administration to induce tolerance to the 33-mer.
  • inhibition of the 33-mer may be achieved through direct genetic manipulation of gliadin genes in for example wheat.
  • Inhibition of the 33-mer may also be achieved by addition of an inhibitor of the 33-mer to wheat flour or food-stuffs comprising wheat flour. Administration of the inhibitor
  • the inhibitor of the present invention may be administered in any way known to a person of skill, for example by enteral, parenteral, transdermal or transmucosal administration.
  • the inhibitor is administered orally, such as in tablets, capsules or drops.
  • the inhibitor of the present invention may also be administered as a food additive to gluten-containing foods.
  • the present invention relates to inhibition of the 33-mer gliadin peptide by oral administration of a microorganism capable of degrading the 33- mer and/or a medium fermented by such a microorganism, wherein the peptide degrading activity is stable under low pH and in the presence of digestive enzymes, e.g. as previously described in WO 201 1/110884.
  • WO 2011/1 10884 is incorporated by reference in its entirety.
  • Strains of microorganisms capable of degrading the 33-mer gliadin peptide and/or fragments thereof include strains of Lactobacillus, Streptococcus and Rothia among others.
  • the microorganism may further be a genetically manipulated bacteria which contains and/or secretes the inhibitor.
  • the inhibitor is administered parenterally, such as by intravenous injection.
  • the inhibitor is administered transdermal ⁇ to achieve a systemic distribution of the inhibitor in the subject.
  • the inhibitor is administered transmucosally to achieve a systemic distribution of the inhibitor in the subject. In one embodiment the inhibitor is administered by inhalation of a pharmaceutical composition comprising the inhibitor or through nasal administration.
  • the inhibitor is administered in connection with gluten intake, e.g. the inhibitor is administered simultaneously with gluten intake.
  • the inhibitor may also be administered as a sustained-release formulation for increased compliance.
  • the present invention relates to use of an inhibitor of the 33-mer peptide and/or fragments thereof for the manufacture of a medicament for the prevention and/or treatment of diabetes.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an inhibitor of the 33-mer gliadin peptide or a fragment thereof.
  • the pharmaceutical composition may comprise one or more of: a pharmaceutically acceptable carrier, a diluent, an excipient or an adjuvant.
  • the subject of the present invention may be a human being of any age or gender, such as a child, an adolescent or an adult.
  • the subject of the present invention is a person suffering from clinical diabetes, such as type 1 diabetes, type 2 diabetes, LADA, MODY or gestational diabetes.
  • the subject of the present invention is person with an increased risk of developing diabetes, such as a pre-diabetic person.
  • Pre-diabetes is the state in which some but not all of the diagnostic criteria for diabetes are met. It is often described as the "gray area" between normal blood sugar and diabetic levels. Impaired fasting glycaemia and impaired glucose tolerance are considered symptoms of pre-diabetes. Impaired fasting glycaemia or impaired fasting glucose (IFG) refers to a condition in which the fasting blood glucose is elevated above what is considered normal levels but is not high enough to be classified as diabetes mellitus. It is considered a pre-diabetic state, associated with insulin resistance and increased risk of cardiovascular pathology, although of lesser risk than impaired glucose tolerance (IGT). IFG sometimes progresses to type 2 diabetes mellitus.
  • IFG impaired glucose tolerance
  • Impaired glucose tolerance is a pre-diabetic state of dysglycemia, that is associated with insulin resistance and increased risk of cardiovascular pathology. IGT may precede type 2 diabetes mellitus by many years.
  • the subject of the present invention with an increased risk of developing diabetes has a particular tissue type, which predisposes said subject for development of diabetes.
  • the subject of the present invention has increased blood glucose levels.
  • the subject of the present invention has abnormal (increased) insulin levels.
  • the subject of the present invention with an increased risk of developing diabetes has an abnormal GTT (glucose tolerance test).
  • the subject of the present invention does not have an increased risk of developing diabetes compared to the general background population.
  • the subject of the present invention is a person suffering from metabolic syndrome.
  • Example 1 The 33-mer gliadin peptide induces insulin secretion of beta cells in vitro
  • Gliadin was digested as follows: 250 mg of gliadin was added to 2.5 ml of 0.1 M HCI, and pH was adjusted to 2.0. After addition of 2.5 mg pepsin (Fluka/Sigma-Aldrich), the mixture was incubated at 37 °C for either 5 h or overnight, until all gliadin had been dissolved. Five hundred ⁇ of 50 mM phosphate buffer (pH 7.0) was added, and pH was adjusted to 7.0 using 3 M NaOH.
  • a Spectra/Por® Float-A-Lyzer® G2 dialyser device (MWCO 100-500 Da, Spectrum labs, Collinso Dominguez, CA, USA) was used to dialyze gliadin overnight against phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • high molecular gliadin digest was prepared through filter centrifugation in a Microcon Centrigual Filter Device (MWCO 3000 Da, Merck Millipore, Billerica, MA, USA). The volume was subsequently restored by adding sterile water, and the solution was sterile filtrated (0.2 ⁇ mixed cellulose filter DG2M-1 10, Spectrum) Animal Experiments
  • mice Animal experiments were conducted in accordance with the University of Copenhagen (license no. 2010-561-1851) regulations. Forty-five female NOD mice 7 weeks of age (The Jackson Laboratory, Bar Harbor, Maine) were fed a standard chow, and climatized for one week prior to experiments. Using a 25 G needle, the mice received i.v. injections with 0.15 ml PBS with 0, 4.5 or 450 ⁇ g digested gliadin six times over a period of two weeks (3-4 days between injections). Twice a week, the mice were weighed and blood glucose levels were measured using Abott Freestyle Lite (Abbot, Abbott Park, Illinois). After three subsequent measurements of a blood glucose concentration > 12 mM, the animals were considered diabetic and sacrificed. Blood, pancreas, liver, ileum, jejunum and lungs were sampled for analysis.
  • INS-1 E cells (a pancreatic beta cell line) were grown at 37 °C and 5% C0 2 in RPMI 1640 medium (Lonza, Basel, Switzerland) supplemented with 10% fetal calf serum (FCS, Gibco/Life technologies, Carlsbad, CA, USA), 1 % Na-pyruvate, 1 % HEPES and 50 ⁇ mercaptoethanol in T75 cell culture flasks. Passages 79-90 were used. For the experiments 4x10 5 cells/well were seeded in 12-well plates and 2x10 4 or 4x10 4 cells/well were seeded in 96-well plates. 24 h later, the medium was replaced with
  • RPMI 1640 supplemented with 0.5% FCS and relevant stimulants added from sterile filtrated (0.2 ⁇ Mixed cellulose, DG2m-1 10, Spectrum) stocks: arginine: 100 mM, diazoxide: 10 mM in DMSO, lipopolysaccharide: 5 mg/ml, forskollin: 5 mM in ethanol. Cells were incubated for 24 h.
  • gliadin In pre-incubation experiments, cells were exposed to 30 or 300 ⁇ g/ml gliadin in RPMI for 24 h, then incubated in RPMI with 3 mM glucose and 0.5% FCS for 2 h (also containing gliadin) and finally Ca-5 buffer supplemented with 3 mM glucose for 30 min. Stimulation was performed in Ca-5 buffer supplemented with 3 or 11 mM glucose. In relevant cases, the Ca-5 stimulation medium also contained gliadin or palmitate. For palmitate stimulation, cells were pre-treated as above. 13 mg palmitic acid was added to 500 ⁇ 0.1 M NaOH for a final concentration of 100 mM. The mixture was heated to 70 °C, and added to Ca-5 buffer to a final concentration of 500 ⁇ . After 30 min the supernatant was harvested, centrifuged at 200 x g for 5 min, and stored at -20 °C before insulin ELISA (Mercodia, Uppsala, Sweden) was performed. Gliadin fragment
  • Gliadin 19-mer and de-aminated 33-mer (LGQQQPFPPQQPYPQPQPF-OH (SEQ ID NO: 2) and LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO: 6) were synthesised by Schafer-N (Copenhagen, Denmark), and purity was confirmed by HPLC analysis.
  • the peptides were dissolved in RPMI 1640, supplemented with 0.5% FCS and 1 1 mM glucose at a final concentration of 100 ⁇ . Solutions were sterile filtrated through a low-protein binding PVDF 0.22 ⁇ Millex® filter (Millipore) before being added to cells for 24 h.
  • Islets were isolated from male Lewis Rats (Charles River lab, Wilmington, MA, USA), using collagenase infusion via the pancreatic duct. After isolation, islets from four rats were pooled and cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal calf serum (Gibco) ("growth medium”) at 37°C and 5% C0 2 in 12-well plates. Prior to stimulation, islets were placed in 3 mM glucose in growth medium for 24 h. The media was subsequently changed to RPMI 1640 with 3 mM glucose and 0.5% FCS for 24 h. After 2 h, gliadin and/or glucose were added at relevant concentrations, and islets were incubated for 24 h. Supernatants were stored for insulin ELISA measurements.
  • ATP levels were measured using an adenosine 5-triphosphate (ATP) bioluminescentsomatic cell assay kit (Cat # FLASC) in INS-1 E cells plated in a 96-well plate, at 2x10 4 cells/well following 24 h of stimulation. Chemoluminescence was detected in a Fluoroscan Ascent TL (Thermo Fisher Scientific, Waltham, MA, USA) using an integration time of 3 s.
  • ATP adenosine 5-triphosphate
  • Resazurin dissolved in PBS 0.1 1 mg/ml was added to cells in a final concentration of 1 1 ⁇ g/ml to cell medium for approximately 2 h. Resazurin conversion was detected at 549/585 nm (Fluoroscan Ascent TL, Thermo Scientifics).
  • Endotoxin levels in gliadin and digestion enzymes were quantified using a LAL QCL- 1000 assay (Lonza).
  • siRNA were purchased from Ambion (Austin, TX).
  • MyD88 siRNA cat# 4390771 ID s141418
  • RNA concentration was determined using a Nanodrop 1000 (Thermo Scientific, Waltham, MA) and 500 ng RNA was reverse transcribed to cDNA using qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD). Primers
  • Primers were designed using Primer-3 software and synthesised by Taq Copenhagen (Copenhagen, Denmark). Sequences were as following:
  • GPR40 left 5 ' -CAG AGG CTG GGT GGA TAA CA- 3 ' (SEQ ID NO: 9)
  • GPR40 right 5 ' -AGC CCA CAT AGC AGA AAG CA- 3 ' (SEQ ID NO: 10)
  • MyD88 left 5' -ATC CCA CTC GCA GTT TGT TG- 3' (SEQ ID NO: 1 1)
  • qPCR was carried out on a Lightcycler 2 (Roche, Penzberg, Germany). As standards, PCR products were generated using Premix Taq (Takara, Otsu, Japan).
  • HEK-293 Human embryonic kidney (HEK-293) cells were kept in Dulbecco's modified Eagle's medium (University of Copenhagen, Denmark) supplemented with 10% fetal calf serum (GIBCO, Invitrogen) at 37°C in 5% C0 2 .
  • GIBCO fetal calf serum
  • Enhanced GFP was added for identification of transfected cells.
  • gliadin Invitrogen, Carlsbad, USA) according to the manufacturer's instructions.
  • gliadin To test the effect of gliadin, cells were incubated overnight (ON) in 300 ⁇ g/ml gliadin digest or a comparable volume of enzyme solution for control that was added to the cell medium after transfection.
  • gliadin 19-mer and 33-mer were added at concentrations of 100 ⁇ , and cells were incubated with the peptides ON.
  • Gliadin fragments increase insulin secretion in INS-1E rat insulinoma cells
  • gliadin did not have increased insulin secretion (Fig 2A).
  • a resazurin conversion assay was performed, which showed no difference in the cell mass of cells with and without gliadin (Fig 2D). Incubation with digested ovalbumin did not increase insulin secretion (Fig.
  • Gliadin-induced insulin secretion is mediated by higher molecular weight fragments
  • Amino acids can affect insulin secretion.
  • Arginine for instance, is a positively charged amino acid, which induces insulin secretion rapidly in beta cells by depolarising the cell membrane, leading to opening of voltage-gated calcium channels and triggering of granule fusion.
  • INS-1 E cells we incubated INS-1 E cells with 1 mM arginine for 24 h and saw no difference in insulin secretion (Fig. 2I).
  • dialysis MWCO 100-500 Da
  • filter centrifugation 3000 Da cut-off
  • Gliadin is not affected by MyD88 or FFARI knockdown, but potentiates fatty acid induced insulin secretion
  • GPR40/FFAR1 a fatty acid receptor potentiating insulin secretion by affecting calcium channels.
  • MyD88 a component in the TLR2/4 signalling.
  • Gliadin does not increase intracellular ATP
  • Gliadin potentiates insulin secretion in the presence of forskolin
  • cAMP cyclic AMP
  • INS- 1 E cells adenylate cyclase activator
  • KATP ATP-sensitive potassium channels
  • INS-1 E cells with diazoxide resulted in a significant decrease of insulin secretion in high glucose medium compared to low glucose conditions.
  • ATP-sensitive K+ channels are composed of Kir6.2 pore-forming subunits and sulfonylurea receptor 1 (SUR1) subunits.
  • the SUR1 contains nucleotide binding domains that are critical in sensing the metabolic status of cells.
  • Kir6.2 and SUR1 were expressed in HEK-293 cells. Whole-cell currents were recorded using a 200 ms ramp protocol ranging from -120 mV to +20 mV. For the cells incubated in enzyme mix, the KATP currents were initially almost absent but after washout of endogenous ATP, current levels increased (Fig. 4A).
  • the gliadin 33-mer blocks current through KATP channels and increases insulin secretion in a dose-dependent manner
  • Proline-rich, protease-resistant gliadin fragments have been implicated in the pathogenesis of celiac disease. These fragments include a 33-mer as well as a 19-mer. To test whether the effect of digested gliadin could be mediated by any of these fragments, we investigated the effect of the 19- and the 33-mer on transfected HEK- 293 cells and INS-1 E cells (Fig. 5). Kir6.2 and the SUR1 were expressed in HEK-293 cells and the cells incubated overnight in the presence of the 19- or the 33-mer. Whole- cell currents were compared to controls after washout of endogenous ATP (Fig. 5A-D).
  • HEK-293 cells that had been exposed to 100 ⁇ 19-mer had currents comparable to those of controls, whereas cells exposed to 100 ⁇ 33-mer, currents were significantly reduced up to 10 times (Fig. 5D).
  • the 33-mer can thus affect the KATP channel in the same manner as the gliadin digest.
  • gliadin fragments potentiate insulin secretion in INS-1 E cells and rat islets independently of glucose levels. The effect relies on closure of the ATP-sensitive potassium channel. At present state it is unknown whether the gliadin fragments interact directly with the channel, or via an indirect mechanism such as disruption of the cytoskeleton. Our results indicate that the protease resistant gliadin 33-mer fragment, which is generated in large quantities by enzymatic digestion of gliadin, is the responsible component for the stimulatory effect of gliadin. We also observed weight gain in NOD mice following administration of a gliadin digest, most likely the result of the trophic effect of increased insulin secretion.
  • Gliadin digest injections did not result in accelerated diabetes development in the treated NOD mice. This may be paralleled by the finding that high-dose gliadin does not increase NOD diabetes incidence. Also, the mice were not kept on a gluten-free diet during the study, which could mask effects of the injections on diabetes
  • gliadin fragments have previously been demonstrated in breast milk, suggesting passage through healthy epithelium and not just in patients with celiac disease.
  • the 33-mer is transported across Caco-2 colon carcinoma cells in an un-cleaved form via transcytose, a process which is stimulated by interferon gamma.
  • the 33-mer was also shown to be transported into the early endosomes of duodenal biopsies from patients with active celiac disease, but was not found to associate with the late endosomes, suggesting that the fragments escape lysosomal degradation.
  • gliadin induces zonulin release in two different epithelial intestinal cell lines, resulting in increased monolayer permeability, indicating that transport could occur through the intestinal cells and between them.
  • increased intestinal permeability has been described both in patients with type 1 diabetes and in BB rats, providing a mechanism for increased entry into the blood of diabetes patients. The latter have reduced expression of the tight junction protein claudin-1 compared to the Wistar rat, which correlates to increased intestinal permeability.
  • a recent study suggests that the increased permeability of intestinal epithelium to gliadin in active celiac disease was specific rather than due to general "leakiness".
  • T2D type 2 diabetes
  • a high-fat diet has been shown to increase intestinal permeability, probably by reducing the expression of ZO-1 protein.
  • a study of T2D patients did not find any alterations in intestinal permeability, but does not exclude a role for gliadin since changes in permeability could occur transiently or specifically to gliadin fragments, as seen in celiac disease.
  • gliadin components may contribute to beta cell stress through a direct interaction with beta cells. This might be particularly important in the prediabetic state, where the permeability of the intestine is increased, and which might facilitate the absorption of gliadin into the blood stream.
  • the tissue distribution of 33-mer (A) and 19-mer (B) gliadin peptides 1 hour following oral administration in mice is shown in figure 6.
  • the de-amidated 33-mer H-LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF-OH (SEQ ID NO:6)
  • 19-mer H-LGQQQPFPPQQPYPQPQPF-OH (SEQ ID NO: 2)
  • Iodine was exchanged with tritium using a tritium manifold system (RC Tritec). Specific acitivities of 13.7 and 15.9 Ci/mmol were obtained for the 33-mer and of 28.8 and 29.7 Ci/mmol for the 19-mer.
  • mice intraveneously or 50-400 uCi perorally to adult mice.
  • the mice were sacrificed by cervical dislocation after 1 , 24 ⁇ 4 or 72 ⁇ 4 hr.
  • Heparin plasma was prepared from the blood, and organs were removed.
  • Pieces of 15-150 mg of the organs were placed in Eppendorph tubes for scintillation counting. Other pieces of each tissue were fixed in buffered formalin for later autoradiography.
  • the tissue pieces were weighed and dissolved in Solvable (Perkin Elmer) at 55°C (2-24 hr depending on the specific organ), transferred to scintillation vials and incubated at 55°C with 100-300 ul 30% hydrogen peroxide if necessary to decolorize the solutions. 10 ml of Ultima Gold (Perkin Elmer) was added, the tubes were shaken and counted on a Tri-Carb
  • Liquid scintillation Analyser (Model 1600TR) after >1 hr. They were counted for 2-4 min in the window of 0-18.6 keV. Counts per minute (cpm) were converted to
  • FIG. 7 Anatomical localisation of 3H-labeled 33-mer in mouse pancreas is shown in figure 7. Localisation is seen as silver grain in endocrine tissue, but in particular located in azurophilic granules of the exocrine pancreas. A high concentration is seen in the pancreatic duct.
  • the fixed tissues were dehydrated through a series of ethanol solutions, and finally incubated in xylene to replace the alcohol.
  • the tissues were then embedded in paraffin, and cut I 3 micron slices using a microtome.
  • the slices were deparaffinated, rehydrated and dipped in a mixture of 1 : 1 of Amplify (GE Healthcare) and Kodak NTB emulsion.
  • the slides were developed in Kodak D19 developer and fixed in llford Rapid Fixer.
  • the slides were mounted in Pertex (Histolab) and examined on a Olympus BX51 microscope and photographed using an Olympus Colorview camera.
  • results show localisation of the 33-mer in the endocrine tissue, where the 33-mer may exert an effect on the insulin-producing beta cells.

Abstract

La présente invention concerne une méthode de prévention et/ou de traitement du diabète comprenant l'administration d'un inhibiteur du peptide de 33-mer dérivé de la gliadine, présentant la séquence d'acides aminés LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF et/ou des fragments correspondants. L'inhibiteur peut être une enzyme apte à cliver le peptide de 33-mer ou un anticorps apte à se lier au peptide de 33-mer et à le neutraliser.
PCT/DK2013/050297 2012-09-19 2013-09-19 Inhibition d'un peptide de 33-mer dérivé de la gliadine pour le traitement du diabète WO2014044281A1 (fr)

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Citations (7)

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US20070184049A1 (en) 2005-11-03 2007-08-09 Fox Barbara S Antibody therapy for treatment of diseases associated with gluten intolerance
WO2008090223A2 (fr) * 2007-01-25 2008-07-31 Actogenix N.V. Traitement d'une maladie immunitaire par l'administration mucosale d'antigènes
WO2009034110A1 (fr) 2007-09-11 2009-03-19 Kobenhavns Universitet Prévention du diabète de type 1 par l'administration de gliadine
WO2009137572A2 (fr) * 2008-05-06 2009-11-12 Alba Therapeutics Corporation Inhibition des peptides de la gliadine
WO2011044365A1 (fr) 2009-10-07 2011-04-14 Trustees Of Boston University Glutamine endopeptidases d'espèce rothia et utilisation de celles-ci
WO2011110884A1 (fr) 2010-03-12 2011-09-15 Compagnie Gervais Danone Bactérie de l'acide lactique pour la maladie cœliaque
US20120107329A1 (en) * 2009-06-10 2012-05-03 University Of Maryland, Baltimore EGFR and PAR2 Regulation of Intestinal Permeability

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* Cited by examiner, † Cited by third party
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US20070184049A1 (en) 2005-11-03 2007-08-09 Fox Barbara S Antibody therapy for treatment of diseases associated with gluten intolerance
WO2008090223A2 (fr) * 2007-01-25 2008-07-31 Actogenix N.V. Traitement d'une maladie immunitaire par l'administration mucosale d'antigènes
WO2009034110A1 (fr) 2007-09-11 2009-03-19 Kobenhavns Universitet Prévention du diabète de type 1 par l'administration de gliadine
WO2009137572A2 (fr) * 2008-05-06 2009-11-12 Alba Therapeutics Corporation Inhibition des peptides de la gliadine
US20120107329A1 (en) * 2009-06-10 2012-05-03 University Of Maryland, Baltimore EGFR and PAR2 Regulation of Intestinal Permeability
WO2011044365A1 (fr) 2009-10-07 2011-04-14 Trustees Of Boston University Glutamine endopeptidases d'espèce rothia et utilisation de celles-ci
WO2011110884A1 (fr) 2010-03-12 2011-09-15 Compagnie Gervais Danone Bactérie de l'acide lactique pour la maladie cœliaque

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DE ANGELIS ET AL., APPL. ENVIRON. MICROBIOL, vol. 76, no. 2, 2010, pages 508 - 18
FUNDA ET AL.: "Gluten-free diet prevents diabetes in NOD mice", DIABETES METAB RES REV., vol. 15, 1999, pages 323 - 327
MOJIBAN ET AL., DIABETES, vol. 58, August 2009 (2009-08-01)
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