WO2014044281A1 - Inhibition of a 33-mer gliadin peptide for treatment of diabetes - Google Patents

Description

Inhibition of a 33-mer gliadin peptide for treatment of diabetes

Field of invention

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

(LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF; herein identified as SEQ ID NO:

Background of invention

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.

Even with tight glucose control, there is a significant risk of neuropathy, retinopathy and nephropathy, as well as a 3- fold increase in the risk of severe hypoglycaemia. 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. Patients with type 1 diabetes are at increased risk of other immune mediated diseases. These most commonly include coeliac disease, thyroid disease, autoimmune gastritis and Addison's disease. 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. Although the pathophysiology of coeliac disease is not completely understood, it is clear that the presence of the toxic proteins in the patient's diet causes a total or partial damage of intestinal mucosa leading to severe

malabsorption syndromes, thus causing diarrhoea, vomit, abdominal pain, anorexia, growth retardation, undernutrition and anaemia. Current treatment is by strict lifelong gluten exclusion - a difficult, socially restrictive and expensive therapy. The association between coeliac disease and type 1 diabetes has been studied for almost 50 years. Many studies all over the world have demonstrated increased prevalence of coeliac disease in children, adolescents and adults with type 1 diabetes. Both are autoimmune conditions resulting from a complex interaction between genetic, immunological and environmental factors. It is not yet clear whether the simultaneous occurrence of the two diseases is linked to a common genetic base, or whether one disease in fact predisposes patients to the other; currently it is the first hypothesis that is more widely accepted.

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

manifestation of type 1 diabetes in humans (Cronin et al. Insulin-dependent diabetes mellitus and coeliac disease. Lancet 1997; 349:1096- 1097). Several later studies have confirmed this theory.

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.

A number of enzymes and microorganisms have been identified which are capable of degrading the 33-mer gliadin peptide (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 1/110884; WO 2011/044365). Such enzymes and microorganisms have been suggested for treatment of coeliac disease.

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.

Summary of invention

The role of 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. However, 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.

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.

Description of Drawings

Figure 1 : Gliadin increases weight in NOD mice. NOD mice were injected with gliadin (4.5 μg/mL(1 : 1000 dilution); 450 μg/mL (1 :10 dilution); controls) five times over two weeks. Blood glucose levels and weight were measured twice a week. A.

Comparison of blood glucose levels. A significant difference was observed between the groups (P=0.0003, n=15), but no clear tendencies were observed. B. Diabetes incidence. No difference was observed between groups (P=0.53, n=15, Mantel-Cox). C. Total weight. There was a significantly increased body weight in mice injected with the 1 : 10 (24.9+/- xx g at day 100) (but not 1 : 1000) gliadin solution compared to controls (23.6+/- xx g at day 100) (P<0.0001 , n=15). D. Weight increase over time. Mice injected with the higher gliadin dose obtained a significant higher body weight than mice injected with the lower gliadin dose and controls, in average 5.6 ± 0.8 g for the 1 :10 dilution vs. 4.7±0.5 g for controls at day 99 (P<0.0001 , n=15).

Figure 2: Gliadin increases insulin secretion in INS-1E cells and rat islets during 24 h stimulation. A. Gliadin significantly increased insulin secretion (P<0.001 , n=6). No significant effect was observed when cells were incubated with heat-inactivated digestion enzymes (P=0.27, n=5) or with lipopolysaccharide (P=0.82, n=4). B. In INS- I E cells, insulin release increased dose-dependently compared to glucose alone (1.5±0.29x to 2.5±0.4x (95% CI)), when cells were incubated with various

concentrations of gliadin for 24 h (P<0.0001 , n=6). C. Addition of gliadin to cells in low glucose still increased insulin secretion by 50 % (P=0.01 , n=4). D. Quantification of live cells with resazurin. Data were normalized relative to the number of live cells in RPMI 1640 with 3 mM glucose after 24 h. No difference was observed in cell number between cells stimulated in RPMI 1640 with 11 mM glucose and 1 1 mM glucose + gliadin (p=0.626, n=4). E. Incubation with 1 1 mM glucose and enzymatically digested ovalbumin for 24 h did not increase the insulin secretion significantly compared to controls (P=0.22, n=4). Gliadin significantly increased insulin secretion compared to albumin (1.75x, P=0.034, n=3). F. Gliadin-stimulated insulin secretion in rat islets of Langerhans was significantly increased up to 1.5 times compared to controls (t-test, p=0.015, n=4). G. Stimulation of INS-1 E cells with increasing amounts of 33-mer in 11 mM glucose, resulted in a dose-dependent increase in insulin secretion (P=0.03, n=4) up to 1.7 fold of control, comparable to what was observed for stimulation with gliadin digest. The 19-mer had no effect on insulin secretion at any concentration assayed (P=0.98, n=4). H. During 30 min stimulation with 11 mM glucose in calcium-5 buffer, INS-1 E cells secreted significantly more insulin compared to 3 mM glucose (P<0.001 , n=4). However, co-incubation with gliadin did not increase insulin secretion (P>0.05, n=4), and neither did glucose stimulation after 24 h pre-incubation with gliadin (P>0.05, n=4). I. Arginine (1 mM) had no significant effect on the insulin secretion in INS-1 E cells stimulated for 24 h in medium containing 1 1 mM glucose (p=0.84, n=5). The removal of peptides < 100-500 Da (by dialysis) and 3000 Da (Centrifugal filtration) did not reduce the ability of gliadin to stimulate insulin secretion in INS-1 E cells in the presence of 1 1 mM glucose (dialysis: P=0.026, filtration: P=0.003, n=3). Figure 3. The effect of gliadin is independent of FFAR1 and MyD88, but is abolished by diazoxide treatment A. Fatty acid receptor FFAR1 knockdown using siRNA, did not affect insulin secretion during 24 hours of gliadin stimulation (p=0.48, n=4). Following MyD88 knock-down, glucose-induced insulin secretion was reduced to 50 % (p=0.02, n=4). Though not significant, gliadin was still able to increase insulin secretion for the MyD88 knockdown (p=0.06, n=4). B. No significant cell death was observed after transfections, except for cells treated with FFAR1 siRNA (P=0.05, n=3). Data were normalised to the secretion in the presence of 1 1 mM glucose (A) or 1 1 mM glucose + forskolin (B). C. Efficiency of the MyD88 siRNA silencing was confirmed using qPCR. D. Efficiency of the FFAR1 -siRNA silencing was confirmed using qPCR. In low-glucose medium, FFAR1 expression was increased by 50 % (P<0.01 , n=4). Generally, cell number was reduced to about 85 % of the number of untreated cells, but no difference was detected between groups. E. Cells treated with gliadin for 24 hours before being stimulated with palmitate and glucose for 30 minutes secreted 15 % more insulin than palmitate-stimulated control cells (P=0.04, n=5). F. Gliadin did not increase ATP content in INS-1 E cells compared to controls after 24 h (p=0.34, n=5). G. No significant increase in intracellular ATP was detected in cells stimulated with glucose and gliadin compared to controls (p=0.35, n=4). H. Incubation of I NS-1 E cells for 24 h in RPM I 1640 with 1 1 mM glucose supplemented with 10 μΜ forskolin, resulted in 5-fold higher insulin secretion compared to 1 1 mM glucose alone (p=0.003, n=4). Insulin secretion could be further increased by 20%, when cells were stimulated with both forskolin and gliadin (p=0.03, n=4). I . Addition of 100 μΜ diazoxide to medium with 1 1 mM glucose, resulted in a reduction of insulin secretion to the levels observed for low glucose medium with 3 mM glucose (P=0.0008, n=4). However, when diazoxide was added to cells stimulated with gliadin and glucose, insulin secretion was restored to the levels observed for 1 1 mM glucose alone (p=0.87, n=4).

Figure 4. Gliadin incubation inhibits current through K_ATp- Kir6.2 and SUR1 were transiently expressed in H EK293 cells. The cells were incubated in either 1 %

inactivated enzyme mix or 1 % gliadin o/n. Currents were activated by a ramp protocol every 5 s. A. Representative KATP currents in a cell incubated in 1 % enzyme mix. Trace (1 ): Prior to washout of endogenous ATP, trace (2): after washout of ATP in the continued presence of 1 % enzyme mix. Trace (3): after 2.5 min exposure to 1 % gliadin. B. Representative currents from a cell incubated in 1 % gliadin o/n, in the continued presence of gliadin before and after washout of ATP and after application of enzyme mix. The 3 traces overlay. C. The time-dependence of the effect of 1 % gliadin on KATP currents. Representative data from a cell incubated in 1 % enzyme mix o/n. The data points represent max inward current at the start of the ramp protocol as indicated by the arrow in (A) during washout of endogenous ATP and after application of 1 % gliadin. D. Summarized current densities: +ATP, average current density prior to ATP washout in cells incubated in 1 % enzyme mix o/n (n=7), -ATP, average current density after washout (n=9), Glia: average current density after 2.5 min exposure to 1 % gliadin (n=9). The effect of gliadin was not significant. In cells incubated o/n in 1 % gliadin, 10/12 cells did not express KATP current. In the first bar graph all cells are included; in the second bar graph the 2 cells with current are excluded. For cells incubated in enzyme mix, 3/12 cells had no current and are not included.

Figure 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. A. Representative KATP currents in a cell incubated with the 19-mer. Trace (1): prior to washout of endogenous ATP, trace (2): after washout of ATP. 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. Non-transfected cells (NT, n=3), transfected cells incubated in control medium (KATP, n=7), transfected cells incubated in the 19-mer (KATP, 19, n=9) or transfected cells incubated in the 33- mer (KATP, 33, n=9). Average current density prior to ATP washout and after washout are shown.

Figure 6. 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.

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.

Definitions

Antibody: An antibody (Ab), 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. Using this binding mechanism, 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. For example, 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: 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 term "33-mer peptide" as used herein collectively refers to either of SEQ ID NO:1 and SEQ ID NO:6.

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.

Hence, in one embodiment, 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

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

autoimmune attack. There is no known preventive measure against type 1 diabetes, which causes approximately 15% of diabetes mellitus cases in North America and Europe. Most affected people are otherwise healthy and of a healthy weight when onset occurs. Sensitivity and responsiveness to insulin are usually normal, especially in the early stages. 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. However, the specific defects are not known. In the early stage of type 2, the predominant abnormality is reduced insulin sensitivity. At this stage, hyperglycemia can be reversed by a variety of measures and medications that improve insulin sensitivity or reduce glucose production by the liver. Globally, as of 2012, an estimated 346 million people have type 2 diabetes.

Latent Autoimmune Diabetes of Adults (LADA), 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

Australia's Type 1 Diabetes Network, one third of all Australians with type 1 diabetes reported being initially misdiagnosed as having the more common type 2 diabetes.

Maturity onset diabetes of the young (MODY) 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.

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.

The activity of beta cells is important in the development of diabetes, and increased insulin secretion has been correlated to increased diabetes development.

In one embodiment the inhibitor of the present invention is used in the treatment of diabetes. Said treatment may be prophylactic, ameliorative or curative. In one embodiment, the present invention relates to treatment of type 1 diabetes. In one embodiment, the present invention relates to treatment of type 2 diabetes.

In one embodiment, 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).

The International Diabetes Federation consensus worldwide definition of the metabolic syndrome (2006) is: Central obesity (defined as waist circumference with ethnicity- specific values) AND any two of the following:

- Raised triglycerides: > 150 mg/dL (1.7 mmol/L), or specific treatment for this lipid abnormality

- 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

- Raised fasting plasma glucose (FPG): >100 mg/dL (5.6 mmol/L), or previously diagnosed type 2 diabetes In one embodiment the inhibitor of the present invention is used in the treatment of metabolic syndrome.

Gliadin

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 LGQQQPFPPQQPYPQPQPF-OH (SEQ ID NO: 2), a 20-mer peptide

QQLPQPQQPQQSPFQQQRPF (SEQ ID NO: 3) and a 13-mer peptide

LGQQQPFPPQQPY (SEQ ID NO: 4) and 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.

In one embodiment, 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.

In one embodiment, 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. In one embodiment 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.

In an alternative embodiment, 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.

In one embodiment, 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.

In an alternative embodiment, the biological activity of the 33-mer is inhibited by direct immunization with the 33-mer. For example, 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. In other embodiments, 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. In one preferred embodiment, 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.

In one particular embodiment 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.

In one embodiment, the inhibitor is administered parenterally, such as by intravenous injection.

In one embodiment, the inhibitor is administered transdermal^ to achieve a systemic distribution of the inhibitor in the subject.

In one embodiment, 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.

In one embodiment, 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. In one embodiment, 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.

In one embodiment, the present invention relates to 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.

Patient groups

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.

In one embodiment, 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. In a particularly interesting embodiment, 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. There is a 50% risk over 10 years of progressing to overt diabetes. Impaired glucose tolerance (IGT) 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. In one embodiment, 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. In one embodiment, the subject of the present invention has increased blood glucose levels.

In one embodiment the subject of the present invention has abnormal (increased) insulin levels.

In one embodiment, the subject of the present invention with an increased risk of developing diabetes has an abnormal GTT (glucose tolerance test).

In one embodiment, the subject of the present invention does not have an increased risk of developing diabetes compared to the general background population.

In one embodiment, the subject of the present invention is a person suffering from metabolic syndrome.

Examples

Example 1 : The 33-mer gliadin peptide induces insulin secretion of beta cells in vitro

Materials and methods Chemicals

Unless otherwise is noted, chemicals were purchased from Sigma-Aldrich (St. Louis, Missouri) and cell culture materials from Greiner Cellstar, Radnor, Pennsylvania.

Gliadin digestion

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 μΙ of 50 mM phosphate buffer (pH 7.0) was added, and pH was adjusted to 7.0 using 3 M NaOH. Subsequently, 1.6 mg each of trypsin (Fluka), chymotrypsin (AppliChem, Darmstadt, Germany) and carboxypeptidase, along with 320 μg elastase (Fluka) were added, and the mixture was incubated at 37 °C for 3 h. The enzymes were heat-inactivated at 80 °C for 5 min and the mixture was subsequently sterile filtered (0.2 μηι mixed cellulose filter DG2M-1 10, Spectrum), and stored as aliquots at -20 °C. Protein concentration was determined using a BCA protein assay reagent (Pierce, Rockford, IL, USA). For in vitro experiments, an additional step was introduced, where the digest was centrifuged at 20.000 x g for 20 min before sterile filtration in order to prevent excessive clogging of the sterile filter. As a control, ovalbumin was digested using the same procedure.

Gliadin dialysis and filter centrifugation

A Spectra/Por® Float-A-Lyzer® G2 dialyser device (MWCO 100-500 Da, Spectrum labs, Rancho Dominguez, CA, USA) was used to dialyze gliadin overnight against phosphate buffered saline (PBS). Alternative, 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 μηι mixed cellulose filter DG2M-1 10, Spectrum) Animal Experiments

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 μg 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.

Cell culture

INS-1 E cells (a pancreatic beta cell line) were grown at 37 °C and 5% C0₂ 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 μΜ mercaptoethanol in T75 cell culture flasks. Passages 79-90 were used. For the experiments 4x10⁵ cells/well were seeded in 12-well plates and 2x10⁴ or 4x10⁴ 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 μηι 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.

In pre-incubation experiments, cells were exposed to 30 or 300 μg/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 μί 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 μΜ. 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 fragments

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 μΜ. Solutions were sterile filtrated through a low-protein binding PVDF 0.22 μηι Millex® filter (Millipore) before being added to cells for 24 h.

Islet isolation and culture

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% C0₂ 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

Cellular 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 2x10⁴ 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.

Resazurin cell count

Resazurin dissolved in PBS (0.1 1 mg/ml) was added to cells in a final concentration of 1 1 μg/ml to cell medium for approximately 2 h. Resazurin conversion was detected at 549/585 nm (Fluoroscan Ascent TL, Thermo Scientifics).

Endotoxin

Endotoxin levels in gliadin and digestion enzymes were quantified using a LAL QCL- 1000 assay (Lonza). siRNA knockdown

The following siRNA were purchased from Ambion (Austin, TX).

MyD88 siRNA: cat# 4390771 ID s141418

FFAR1 siRNA: cat# 4390771 ID S153613

Control siRNA: cat# 4390843

For each well, 1 μΙ_ of siRNA was mixed with 49 μΙ_ of optimem (Gibco, Invitrogen) and 2 uL of DharmaFECT2 (Thermo Scientific) was mixed with 48 μΙ_ of optimem. The two mixtures were incubated 5 minutes at room temperature, before being mixed, with subsequent 20 minute incubation at room temperature. 4- 10⁵ INS-1 E cells/well were seeded in a 12-well plate, in 300 μΙ_ RPMI 1640, and the 100 μΙ_ siRNA/dharmafect mix. After 4 hours, 600 μΙ_ standard media was added. On the following day, cells were stimulated with gliadin for 24 hours similar to the other experiments. Experiments were done in triplicates. After stimulation, supernatant was harvested and RNA was isolated using trizol. 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:

Actin left: 5^'-AGC CAT GTA CGT AGC CAT CC- 3^' (SEQ ID NO: 7)

Actin right: 5^'-CTC TCA GCT GTG GTG GTG AA- 3^' (SEQ ID NO: 8)

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)

MyD88 right: 5' -CTC CTG TTT CTG CTG GTT GC- 3' (SEQ ID NO: 12) qPCR

qPCR was carried out on a Lightcycler 2 (Roche, Penzberg, Germany). As standards, PCR products were generated using Premix Taq (Takara, Otsu, Japan).

Fragment's specificity was confirmed by sequencing (GATC biotech). The resulting fragment was diluted 1 : 1000 and subsequently serial diluted 1 : 10 to generate a standard curve spanning from 10^"3 to 10^"10. For each reaction, 5 μΙ_ SYBR Premix Ex Taq II (2X) (Takara), 3 μΙ_ water and 1 μΙ_ primer mix (100 μΜ stock) was added, along with 1 μΙ_ sample or standard. qPCR was run using the following parameters: Initial denaturation 95 °C for 10 sec, followed by a 45 cycle shuttle PCR, run as 95 °C for 10 sec, 58 °C for 5 sec and 72 °C for 15 sec. Following this, a melting curve analysis was made, with temperatures increasing from 65 °C to 95 °C. Data were analysed using the lightcycler software. Expression was normalised to actin expression.

Cell culture and transfections for electrophysiology

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% C0₂. Cells at 80% confluence in a T25 flask were transfected with 0.5 μg hK_ir6.2 (GenBank Acc.No. NM_000525) and 1.5 μg hSUR1 (NM_000352). Enhanced GFP was added for identification of transfected cells.

Transfections were performed using Lipofectamine and Plus reagent (GIBCO,

Invitrogen, Carlsbad, USA) according to the manufacturer's instructions. To test the effect of gliadin, cells were incubated overnight (ON) in 300 μg/ml gliadin digest or a comparable volume of enzyme solution for control that was added to the cell medium after transfection. Using the same protocol, gliadin 19-mer and 33-mer were added at concentrations of 100 μΜ, and cells were incubated with the peptides ON.

Electrophysiology

Whole-cell currents were recorded 1-2 days post transfection using a MultiClamp 700B amplifier and MultiClamp Commander (Axon Instruments, Foster City, CA, USA). The cells were superfused with a solution containing in (mM): 138 NaCI, 5.6 KCI, 1.2 CaCI₂, 2.6 MgCI₂, 10 HEPES, 3 glucose, pH=7.2 with NaOH. Patch pipettes were fabricated from borosilicate glass capillaries (Module Ohm, Herlev, Denmark) and had tip resistances between 1.5 and 2.0 ΜΩ when filled with pipette solution of the following composition (mM): 107 KCI, 2.0 MgCI₂, 1.0 CaCI₂, 10 NaCI, 10 HEPES and 10 EGTA, pH=7.2 with KOH. All experiments were performed at room temperature (20-22 °C). Electronic compensation of series resistance to 70-85% was applied to minimize voltage errors. All analog signals were acquired at 10-50 kHz, filtered at 6 kHz, digitized with a Digidata 1440 converter (Axon Instruments) and stored using pClampI O software (Axon Instruments). Statistics and data analysis

Data was analyzed using Graphpad Prism. Unpaired t-test, One sample t-test, ANOVA and Mantel-Cox was used. For patch-clamp experiments, data were analyzed using pClampI O software (Axon Instruments), and statistical comparisons were made using paired or unpaired Student's t-test. The following symbols were used for the figures: Δ: PO.05, ΔΔ: P<0.01 , ΔΔΔ: P<0.001 relative to medium with 3 mM glucose. *: P<0.05, **: P<0.01 , ***: P<0.001 , relative to medium with 1 1 mM glucose alone. Data were normalised to experiments using 11 mM glucose, unless otherwise noted. Results

Gliadin fragments induce weight gain in NOD mice

Though the average blood glucose levels (Fig 1 A) or diabetes incidence (Fig 1 B) were unchanged following administration of the gliadin digest, we did see a significant weight gain in the group injected with 450 μg gliadin, relative to control mice and relative to mice injected with 4.5 μg (Figure 1 C, P<0.0001) (Figure 1 D, PO.0001). This corresponded to a 25% larger increase at day 99 (5.6 ± 0.8 g for 450 μg gliadin vs. 4.7±0.5 g for controls, n=15, Figure 1 D). The weight increase was observed shortly after the gliadin digest injections started and persisted until the mice were 200 days old.

Gliadin fragments increase insulin secretion in INS-1E rat insulinoma cells

To address whether or not the increased weight could be due to an effect of gliadin on the beta cells, we stimulated INS-1 E cells with gliadin digests. Upon incubation with 300 μg/ml for 24h, we observed a significant increase in insulin secretion, up to 1.7 times the secretion observed for glucose alone (Fig 2A, P<0.0001 , n=6). The effect was dose-dependent as demonstrated by incubating the cells with gliadin

concentrations between 30μg/ml and 600μg/ml (Fig 2B, P< 0.0001 , n=6). Endotoxins present in the digestion enzymes (approximately 15 ng/ml in the stimulation media) were not responsible for this effect, since we showed that cells incubated with a solution of the heat-inactivated digestion enzymes or high doses of endotoxin

(lipopolysaccharide 5μg/ml) did not have increased insulin secretion (Fig 2A). The effect of gliadin was independent of glucose, shown by incubating with gliadin in the presence of 3 mM glucose, which still led to an increase in insulin secretion with 50% (Fig 2C, P=0.01 , n=4). To show that gliadin did not stimulate cell proliferation, which in turn could increase insulin secretion due to increased cell mass, 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. 2E, P=0.02, n=4), suggesting the effect to be specific for digested gliadin. In isolated rat islets of Langerhans, a similar effect was observed, with a 50% increase in insulin secretion after 24h following exposure to 300 μg gliadin/ml compared to glucose alone (Fig. 2F, P=0.015, n=4). Incubation of INS-1 E cells for 24 h with the gliadin 33-mer showed a dose-dependent effect (Fig. 2G, P=0.03, n=4 with up to 70 % more insulin secreted (at 100 μΜ), compared to controls and cells incubated with the 19-mer. This suggests that the 33-mer fragment could be the fragment responsible for the effect of gliadin on KATP and insulin secretion.

Short-term (30 min) exposure to gliadin in calcium-5 with low glucose did not increase basal or stimulated levels of insulin secretion (Fig. 2H, P=0.43, n=4 and Fig. 2H, P=0.31 , n=4, respectively). Likewise, preincubation with gliadin for 24h prior to glucose stimulation in calcium-5 buffer did not increase the glucose-induced insulin secretion (Fig. 2H, P=0.59, n=4). Hence, gliadin has no acute effect on insulin secretion.

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. To investigate whether the effect of gliadin was due to release of amino acids, we incubated INS-1 E cells with 1 mM arginine for 24 h and saw no difference in insulin secretion (Fig. 2I). Furthermore, we used two methods to separate the low- molecular compounds from the gliadin solution: dialysis (MWCO 100-500 Da) and filter centrifugation (3000 Da cut-off). Removal of these fractions did not affect the ability of gliadin to increase insulin secretion (Fig. 2I). All in all, these results suggest that the increase in insulin secretion is not due to amino acids or other low-molecular weight compounds.

Gliadin is not affected by MyD88 or FFARI knockdown, but potentiates fatty acid induced insulin secretion

Two other pathways were singled out as potential targets. One was GPR40/FFAR1 , a fatty acid receptor potentiating insulin secretion by affecting calcium channels. The other was MyD88, a component in the TLR2/4 signalling. As gliadin is highly

hydrophobic, it might interact with receptors recognising fatty acids or LPS, which are ligands for TLR4. When FFAR1 was knocked down, gliadin treatment still significantly increased insulin release to a similar degree as cells transfected with scramble siRNA (Fig. 3A, P=0.48, n=4). In MyD88 knockdowns, although not statistically significant, in all our experiments, gliadin-stimulated cells secreted more insulin than cells treated with glucose alone in (Fig. 3A, P=0.06, n=4). Further, they secreted significantly less insulin upon glucose stimulation, only about 50 % compared to control cells (Fig 3A, P=0.02, n=4). This was not due to cell death during transfection, as no change in number of cells was detected between the two groups (Fig. 3B, P=0.5, n=3). To verify the efficiency of our siRNA, we used qPCR, which showed a 45 % increased expression of FFAR1 in cells treated with low glucose (Fig. 3C, P<0.01 , n=4) and a similar tendency towards increased MyD88 expression (Fig. 3D, P=0.06, n=4). In several experiments, we observed a tendency towards increased GPR40 expression after 24 hours of gliadin treatment, although no significant trend was observed (Fig. 3D, P=0.31 , n=4). To see if cells incubated with gliadin had an increased response to fatty acids, we stimulated cells with gliadin overnight, and subsequently challenged them with fatty acids. Gliadin treatment lead to a 15 % increase in insulin secretion response to fatty acids compared to untreated cells (Fig. 3E, P=0.04, n=5).

Gliadin does not increase intracellular ATP

No significant increase was observed in intracellular ATP content after 24h of incubation with gliadin compared to control (Fig. 3F), suggesting that the cells do not metabolize gliadin, and that gliadin does not contribute to an increased energy level in the cells. Likewise, no increase in intracellular ATP was observed in cells that were incubated with gliadin for 30 minutes in salt-buffer with 1 1 mM glucose compared to controls (Fig. 3G).

Gliadin potentiates insulin secretion in the presence of forskolin

To investigate whether potentiation of the insulin secretion by gliadin is mediated by cyclic AMP (cAMP), which exerts major effects on the insulin secretion pathway, we used the adenylate cyclase activator, forskolin, to increase intracellular cAMP. In INS- 1 E cells, this led to a large increase in insulin secretion (Fig. 3H). During forskolin stimulation, gliadin could however further increase insulin secretion by 25% compared to forskolin alone (Fig. 3H, P=0.03, n=4), indicating an additive effect and suggesting that the effect of gliadin is not mediated through activation of the cAMP pathway.

Gliadin-mediated insulin secretion is abolished by diazoxide treatment

To address whether or not gliadin digest affects the ATP-sensitive potassium channels (KATP) of the insulin secretion pathway, we used the KATP activator diazoxide.

Treatment of INS-1 E cells with diazoxide resulted in a significant decrease of insulin secretion in high glucose medium compared to low glucose conditions. Co-incubation with diazoxide and gliadin digest removed the stimulatory effect of gliadin on insulin secretion (Fig. 3I, P=0.0008, n=4), to a level comparable to cells in 1 1 rtiM. This suggests a dependence on ATP-sensitive K+ channels for the gliadin-induced insulin secretion independent of an increase in cellular ATP levels.

Gliadin incubation inhibits KATP current

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. To evaluate the possible direct effect of gliadin on ATP sensitive K+ channels, 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). In contrast, cells incubated in gliadin digest for 24 h, 10 out of 12 cells did not express KATP currents after ATP washout (Fig. 4B). Acute application of gliadin to the cells resulted in a 6.7±2.6% decrease in current after 2.5 min, but this change was not significant (Fig. 4C). To confirm that the recorded current was mediated by KATP, we added 5 mM BaCI2 at the end of the experiment, which is known to block this type of current. Results are summarized in Fig. 4D.

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 μΜ 19-mer had currents comparable to those of controls, whereas cells exposed to 100 μΜ 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.

Discussion We have demonstrated that 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

development.

Though intravenous injection of gliadin fragments is not physiological, accumulated data suggest that undigested gliadin fragments do in fact cross the intestinal barrier in vivo. First, gliadin fragments have previously been demonstrated in breast milk, suggesting passage through healthy epithelium and not just in patients with celiac disease. Second, 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. Third, 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. Fourth, 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". Direct contact between β-cells and gliadin would also require gliadin to overcome the endothelial barrier of the islet vascular system. Recently, it was demonstrated that a 70 kDa dye was capable of diffusing from the vascular confinement of the islets into the surrounding acinar tissue in the pancreas of prediabetic mice. This was not observed in healthy mice and would suggest that in prediabetic mice, increased endothelial permeability could lead to entry of gliadin fragments into the islets. Likewise, in BB rats, both diabetes prone and diabetes resistant, endothelial permeability was higher in the pancreatic venules, as visualised by injection with the dye Monastral Blue B, than in 3 different control rat strains. This was also seen in the absence of inflammation in the endothelium, and it was hypothesised that an intravascular population of monocytes were responsible for the increase in permeability when stimulated. Finally, in female NOD mice, an increased blood flow was detected through the islets at 10 to 14 weeks of age, compared to males of similar age and female ICR mice. This was mediated by an excessive production of nitric oxide, which could increase exposure of the beta cells to gliadin. Once near the beta cells, gliadin fragments can stress the cells by the mechanism described in this study.

Evidence is accumulating that gliadin influences diabetes development in humans. The most striking case is a recent study, where a 6 year old boy diagnosed with T1 D without celiac disease, was prescribed a gluten-free diet. The boy has subsequently been without need of insulin therapy for 28 months, and has stable fasting blood glucose levels at 4.0-5.0 mmol/L, indicating that a gluten-free diet might be an effective treatment of T1 D patients. Thus, gluten peptides could lower the insulin response to glucose over time, as demonstrated in a group of relatives to T1 D patients who showed improved intravenous glucose tolerance test after 6 months on a gluten-free diet. The improvement was lost upon reintroducing gluten into their diet. The activity of beta cells is important in the development of diabetes, and increased insulin secretion has been correlated to increased diabetes development. First-degree relatives to T1 D patients have been shown to have increased levels of insulin in the blood. While gliadin has primarily been described in connection with T1 D, due to its beta cell stimulatory effect, gliadin could also be implicated in the pathogenesis of type 2 diabetes (T2D). 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. Thus, increased leakiness induced by high-fat diet and inflammation, together with transcellular transport of gliadin peptides, could introduce gliadin fragments into the bloodstream, where gliadin could contribute to beta cell stress. Alternatively, gliadin might contribute to insulin resistance development peripherally. Although neither FFAR1 , nor MyD88 were identified as targets for gliadin, interesting observations were made for both genes. MyD88 has previously been identified as an interesting target for diabetes, and knockout mice for the upstream receptor TLR4 show attenuated inflammation during diabetes. Our finding shows surprisingly that MyD88 is important for the normal insulin response to glucose in INS- 1 E cells, providing a novel role for this protein. This is in contrast with a previous study, which showed that islets from MyD88 -/- mice responded to glucose similarly to wildtype mice, although these mice became more susceptible to hyperglycemia in prediabetic conditions. Our finding that INS-1 E cells treated with gliadin secrete more insulin upon palmitate challenge, shows that gliadin primes beta cells to react stronger to fatty acids. As gliadin and fatty acids is often consumed together, gliadin might make beta cells more susceptible to stress if a high fat diet is consumed. A recent case study, in which an overweight 51 -year old woman with iron deficiency anaemia experienced resolution of metabolic syndrome after 6 months on a gluten-free diet, suggests that gluten might be a contributing factor in the development of metabolic syndrome.

In conclusion our findings indicate that gliadin components, and in particular gliadin 33- mer, 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.

Example 2

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)) and 19-mer (H-LGQQQPFPPQQPYPQPQPF-OH (SEQ ID NO: 2)) were synthesized with incorporation of a di-iodated tyrosine (Y(3,5-I2)) at the marked positions. 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.

The 3H peptide solutions were diluted in PBS and 50-200 uCi were given

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. For scintillation counting, 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

disintegrations per minut (dpm) by use of the spectral index of the external standard (tSIE). Effectivities were calculated from tSIE according to a calibration curve obtained by a series of external standards. Total radioactivity was divided by the weight of the tissue to obtain dpm/mg tissue. Organ radioactivities (dpm/mg) were normalized by the respective blood values. The results show that both the 33-mer and the 19-mer are widely distributed in several different tissues. The tissue distribution pattern of the 33-mer and the 19-mer are quite similar, with the highest levels in stomach, intestine and pancreas.

Interestingly, the highest levels of both the 33-mer and the 19-mer were found in the pancreas, suggesting that the peptides could have a physiological effect in this organ. However, as the results in Example 1 show, only the 33-mer was found to have an effect on insulin secretion of pancreatic beta cells.

Example 3

Autoradiography

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.

For autoradiography 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. Following exposure for 1-3 weeks, 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.

The 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.

Claims

Claims

A method for prevention and/or treatment of diabetes comprising the administration of an inhibitor of a peptide consisting of SEQ ID NO: 1 and or fragments thereof, or an inhibitor of a peptide consisting of SEQ ID NO:6 and/or fragments thereof, to a subject in need thereof.

The method according to claim 1 , wherein said inhibitor is selected from the group consisting of: enzymes; antibodies, naturally occurring or genetically engineered microorganisms; and molecules otherwise capable of preventing the intestinal absorption of the peptide according to claim 1 , such as proteins, nucleic acids, nanoparticles and resins.

The method according to claim 2, wherein said enzyme is capable of cleaving the peptide consisting of SEQ ID NO: 1 or the peptide consisting of SEQ ID NO:6, and/or fragments thereof.

The method according to claim 2, wherein said antibody is capable of binding to the peptide consisting of SEQ ID NO: 1 or the peptide consisting of SEQ ID NO:6, and or fragments thereof.

The method according to any of the preceding claims wherein the diabetes is selected from type 1 diabetes, type 2 diabetes, LADA, MODY and gestational diabetes.

The method according to claim 5, wherein the diabetes is type 1 diabetes.

The method according to claim 3, wherein the diabetes is type 2 diabetes.

The method according to any of the preceding claims, wherein the subject has an increased risk of developing diabetes, such as a pre-diabetic person.

The method according to any of the preceding claims, wherein the inhibitor is administered by enteral, parenteral, transdermal or transmucosal administration.

10. The method according to claim 9, wherein the inhibitor is administered by enteral administration, such as by oral administration.

1. An inhibitor of a peptide consisting of SEQ ID NO: 1 and or fragments thereof, or an inhibitor of a peptide consisting of SEQ ID NO:6 and/or fragments thereof, for use in prevention and/or treatment of diabetes.

2. The inhibitor for use according to claim 1 1 , wherein said inhibitor is selected from the group consisting of: enzymes; antibodies, naturally occurring or genetically engineered microorganisms; and molecules otherwise capable of preventing the intestinal absorption of the peptide according to claim 1 , such as proteins, nucleic acids, nanoparticles and resins.

13. The inhibitor for use according to claim 12, wherein said enzyme is capable of cleaving the peptide consisting of SEQ ID NO: 1 or the peptide consisting of SEQ ID NO:6, and/or fragments thereof.

14. The inhibitor for use according to claim 12, wherein said antibody is capable of binding to the peptide consisting of SEQ ID NO: 1 or the peptide consisting of SEQ ID NO:6, and/or fragments thereof.

15. The inhibitor for use according to any of claims 11-14, wherein the diabetes is selected from type 1 diabetes, type 2 diabetes, LADA, MODY and gestational diabetes.

16. The inhibitor for use according to any of claims 11-15, wherein the inhibitor is used for treatment of a subject having an increased risk of developing diabetes, such as a pre-diabetic person.

17. Use of an inhibitor of a peptide consisting of SEQ ID NO:1 or SEQ ID NO:6 and/or fragments thereof for the manufacture of a medicament for the prevention and/or treatment of diabetes.