The present invention relates to the treatment of type 2 diabetes by decreasing hyperglycemia and improving insulin sensitivity via oral administration of lnterleukin-6 (IL-6). The invention allows the regulation of blood glucose as well as the reduction of side effects associated with diabetes and those associated with known treatments.

IL-6 is one of the most important inflammatory cytokines and mediates cellular communication. IL-6 was identified to be involved in tissue regeneration, inflammation, pathogen defense. However, IL-6 signaling is also a major trigger for the onset and progression of many pathological conditions such as rheumatoid arthritis, inflammatory bowel disease or sepsis. IL-6 can induce signaling via a membrane bound and a soluble receptor, generated by alternative splicing or proteolysis by metalloproteases.

The IL-6/slL-6R-complex can then associate with the ubiquitously expressed transmembrane protein gp130, which activates IL-6-dependent intracellular signaling cascades, such as the JAK/STAT pathway, even in cells lacking the membrane- bound IL-6 receptor. The soluble IL-6R, which is abundant in extracellular fluid, including plasma and cerebrospinal fluid, is rather pro-inflammatory.

From a metabolic point of view, IL-6 is strongly associated with chronic inflammatory states, including the low-grade inflammation associated with obesity and type 2 diabetes. In obesity, adipose tissue immune cells have emerged as the major source of elevated circulating IL-6 levels. IL-6 has long been recognized as an initiator of insulin resistance, particularly because acute peripheral IL-6 infusion can impair insulin action in mice and because neutralization of IL-6 improves insulin resistance in distinct inflammatory mouse models. Despite its suggested detrimental role in glucose homeostasis, a growing body of evidence identifies IL-6 as a homeostatic regulator of energy and glucose metabolism. In support of this, mice lacking IL-6 expression develop systemic insulin resistance and late-onset obesity. Moreover, IL-6 promotes pancreatic alpha-cell expansion during obesity promotes insulin secretion via enhanced glucagon-like peptide (GLP)-1 production, and is a crucial mediator for the regulatory action of glucose-dependent insulinotropic peptide (GIP) in glucose control.

Taking these findings into consideration, elevation of IL-6 during obesity might serve as an adaptive mechanism to increase insulin production and improve glucose tolerance to overcome obesity-associated insulin resistance.

In contrast to the obese state, exercise induces secretion of muscle-derived IL-6, leading to an acute and robust increase in systemic IL-6 up to 100-fold higher than basal levels, which is associated with improved peripheral energy availability and increased insulin sensitivity in the periphery as well as at the level of the hypothalamus.

Furthermore, IL-6 impacts the regulation of energy homeostasis at the level of the central nervous system (CNS). First, IL-6 plays a critical role in the physiology of neuronal tissue homeostasis (neurotrophic factor). Secondly, IL-6 can activate hypothalamic area to increase energy expenditure and decrease body weight gain.

IL-6 is a pleiotropic cytokine with a wide range of biologic effects. In response to prolonged exercise, IL-6 is synthesized by contracting skeletal muscle and released into circulation. Circulating IL-6 is thought to maintain energy status during exercise by acting as an energy sensor for contracting muscle and stimulating glucose production. If tissue damage occurs, immune cells infiltrate and secrete cytokines, including IL-6, to repair skeletal muscle damage. With adequate rest and nutrition, the IL-6 response to exercise is attenuated as skeletal muscle adapts to training. Flowever, sustained elevations in IL-6 due to repeated bouts of unaccustomed activities or prolonged exercise with limited rest may result in untoward physiologic effects, such as accelerated muscle proteolysis and diminished nutrient absorption, and may impair normal adaptive responses to training. Recent intervention studies have explored the role of mixed meals or carbohydrate, protein, w-3 fatty acid, or antioxidant supplementation in mitigating exercise-induced increases in IL-6. Emerging evidence suggests that sufficient energy intake before exercise is an important factor in attenuating exercise-induced IL-6 by maintaining muscle glycogen. The document W001/03725 relates to the production, release and role of IL-6 in obesity and body weight regulation, body fat, alcohol consumption, atherosclerosis, etc. This document indicates that IL-6 production is increased by TNF-alpha, another cytokine believed to play a role in the induction of type 2 diabetes. This document describes the use of IL-6 to prevent the development of obesity and associated disorders such as metabolic syndrome. The mechanism described in this document is the activation of IL-6 receptors. This document is aimed at the treatment of hyperlipidemia, obesity but is silent on the treatment of glycemia as such. Moreover, the animal models tested are“IL-6 KO” mice, i.e. mice whose IL-6 gene has been invalidated to demonstrate the necessity of IL-6 in the described mechanisms. In this document, the animals without gene invalidation have never received any administration of IL-6 in any form. Only the two phenotypes IL-6 ⁺ and IL-6- were used as a basis for comparison. The only compound administration was for leptin to show that the “IL-6 KO” (IL-6 ) mice were not receptive. The measures evaluated were related to weight and body mass index and never focused on glycemia and/or its regulation.

The document W003/03315 describes the use of IL-6 in the treatment of diabetic neuropathies. This document teaches that untreated diabetes can lead to complications, and that neuropathies can also develop beyond these complications and are also multifactorial. This document describes the role of IL-6 in neurological diseases such as diabetic neuropathy via the gp130 receptor signaling pathway, and never addresses the regulation of glycemia or any insulin resistance in the context of diabetes or prediabetes.

The document W02006/025057 describes the use of IL-6 for the treatment of microvascular complications due to various conditions such as lung disease, diabetes, hypertension, ulcers. This document describes the improvement of blood flow after administration of IL-6 but does not describe nor focus on glycemia.

All this indicates a key but ultimately rather confusing role for IL-6 in various metabolic mechanisms and pathologies, but glucose and diabetes metabolism in general is not well addressed.

Diabetes, whether type 1 or 2, is an increasingly prevalent disease. In France, the diabetic population is currently estimated at 3 million. With 400 new cases diagnosed each day, it will be close to 5 million by 2022.

Insulin is an hypoglycemic hormone, produced by the cells of the pancreas. It promotes glucose storage in peripheral tissues (skeletal muscle and adipose tissue) and reduces endogenous glucose production by the liver (neoglucogenesis). In a healthy subject, the peak plasma glucose resulting from the absorption of dietary glucose causes insulin to be released by pancreatic cells. The amount of insulin thus produced quickly reduces glycemia to a normal level by its actions on the liver and the peripheral tissues.

“Pre-diabetic” subjects initially have glucose intolerance due to the progressive development of resistance of the liver and the peripheral tissues to the action of insulin (i.e. insulin resistance). To cope with this loss of sensitivity, the pancreas overproduces insulin, which reduces fasting glycemia to normal values. Parallel to the development of insulin resistance, there is a progressive loss of the functional mass of pancreatic cells. Eventually, the loss of functional mass of cells is such that the pancreas is no longer able to compensate for the loss of sensitivity to insulin, thus leading to the development of type 2 diabetes.

It is therefore clear that, if we consider the natural history of type 2 diabetes, hyperglycemia can be considered as a delayed manifestation of a long pathophysiological process spread over several years, starting from the development of resistance to the action of insulin, through a progressive loss of the functional mass of insulin-secreting cells, and ultimately leading to pancreatic decompensation at the basis of the hyperglycemia and the diabetes diagnosis.

The cardiovascular complications that make the disease so serious occur as early as the pre-diabetes stage, before the appearance of strong hyperglycemia (> 1.26 g/l) indicative of diabetes.

Screening subjects in the pre-diabetes stage and preventing the transition from pre diabetes to type 2 diabetes is therefore a major public health issue.

“Pre-diabetes” is an intermediate state between a homeostasis of normal glucose and proven T2D, including two clinical entities: moderate fasting hyperglycemia (MFH) and/or impaired glucose tolerance (IGT).

Fasting hyperglycemia is defined by the WFIO as fasting glycemia between 1.10 g/l, but <1.26 g/l after an 8-hour fast and checked twice.

Glucose intolerance, in turn, is defined as glycemia, 2h after an oral load of 75 g glucose, comprised between 1.40 g/l and <2 g/l.

The risk of developing type 2 diabetes is higher in patients with MFFI and/or IGT, the risk is highest in those with both abnormalities.

The detection and treatment of pre-diabetic patients are important to avoid their transition to a disease situation of established type 2 diabetes.

Current diabetes medications are able to delay the onset of type 2 diabetes in an at- risk population. Flowever, none of these drugs permanently block the natural progression of the disease and more than half of the population who are truly“pre diabetic” will develop type 2 diabetes after 4 to 5 years of treatment.

Treatments for type 2 diabetes include, as a matter of example, metformin, thiazolidinedione, sulfonylurea, and/or insulin. These treatments are not without side effects. Diabetic patients have many intestinal disorders (diarrhea, constipation, etc.) due in particular to an alteration in the activity of the enteric nervous system, responsible for the secretory and motor functions of the gastrointestinal tract. These disorders, which can affect up to 75% of patients, primarily affect the quality of life of these patients. The consequences of diabetes on the digestive tract and the increased incidence of the disease will lead hepato-gastroenterologists to be increasingly confronted with the digestive consequences of diabetic disease.

There remains a need for an effective and side effect-free treatment which treats diabetes but also its associated gastroenteropathic side effects.

There is also a need for a treatment for hyperglycemia, for fasting hyperglycemia and for glucose intolerance.

Finally, the treatment of pre-diabetic patients is essential to avoid the progression to established diabetes.

The inventors of the present invention have indeed discovered that oral administration of IL-6 reduces duodenal contractility in diabetic mice and thus improves the animal’s diabetic status via a decrease in fasting hyperglycemia, by restoring communication between the intestine-brain axis and the peripheral organs involved in regulating glucose homeostasis.

Thus, the present invention relates to the following embodiments:

1. IL-6 for use to reduce intestinal contractility in the oral treatment of diabetes or pre-diabetes.

Indeed, in one embodiment, the invention lies on the property of IL-6 orally administered and the invention is directed to IL-6 for its use to treat diabetes, by oral administration, through the reduction of intestinal contractility. The invention is also directed to IL-6 for its use to treat prediabetes, by oral administration, through the reduction of intestinal contractility.

2. IL-6 for use according to embodiment 1 , characterized in that the diabetes is a type 2 diabetes.

3. IL-6 for use according to embodiment 1 or 2; characterized in that the amount of

IL-6 for each oral daily administration is comprised between 10 nM and 1 mM.

In the context of the present invention, the dosage corresponds to a daily oral administration of IL-6, comprised between 10 nM and 1 pM and preferably comprised between 25 nM and 750 nM, particularly between 50 nM and 750 nM, and more particularly, at least 100 nM, at least 150 nM, at least 200 nM, at least 250 nM, at least 300 nM, at least 400 nM, at least 500 nM, preferably comprised between 200 nM and 1 pM per day, preferably approximately 400 nM.

The advantage of the present invention also lies in the small amount of active principle delivered. IL-6 administered in a small amount acts in the duodenum by modulating, i.e. decreasing intestinal contractility and is not absorbed into the general bloodstream.

4. IL-6 for use according to one of the preceding embodiments, characterized in that the diabetes is associated with at least one gastroenteropathic disorder and that said disorder is reduced.

5. IL-6 for use according to embodiment 4, characterized in that the gastroenteropathic disorder is selected from transit disorders occurring from the stomach to the colon. In particular, the transit disorder is selected from diarrhea, transit disorders, constipation and combination thereof, for example.

6. IL-6 for use according to one of the preceding embodiments, characterized in that the diabetes is treated with a second antidiabetic molecule, in particular selected from metformin, thiazolidinedione, sulfonylurea, and/or insulin and other known antidiabetic molecules.

7. IL-6 for use according to embodiment 6, characterized in that the side effects associated with the second molecule are reduced. 8. Combination product comprising the following components: a component (A) comprising IL-6 and at least one pharmaceutically acceptable excipient; a component (B) comprising an antidiabetic, preferably selected from metformin, thiazolidinedione, sulfonylurea, and/or insulin, and at least one pharmaceutically acceptable excipient.

9. Product according to embodiment 8, wherein the components (A) and (B) are suitable for simultaneous, separate or sequential administration.

In a particular case, the component (A) is suitable for oral administration. In another particular case, the component (B) is suitable for oral administration.

10. Product according to one of embodiments 8 or 9, for use in reducing intestinal contractility in the treatment of diabetes or pre-diabetes in a patient.

According to one embodiment, the combination product is suitable for oral administration, in particular of IL-6.

1 1 . Oral pharmaceutical composition comprising IL-6 and a pharmaceutically acceptable excipient.

12. Composition according to embodiment 1 1 , characterized in that the pharmaceutically acceptable excipient comprises a gastro-resistant excipient.

A gastro-resistant drug is a drug that does not dissolve in the stomach, and will thus allow the active substance, IL-6, to be released in the duodenum.

Gastro-resistant formulations are designed to release the drug into the intestinal compartment. According to European Pharmacopoeia 8.0, gastro-resistant dosage forms are delayed-release dosage forms designed to resist gastric fluid and release their active ingredient into the intestinal fluid, particularly in the duodenal lumen. Gastro-resistance minimizes the dietary effect of the pharmaceutical composition of the present invention and thus improves the bioavailability of the drug and in the present case allows it to be released where it exerts its effect, i.e. in the duodenum. By way of illustration, the gastro-resistant pharmaceutical composition of the present invention comprises IL-6 molecularly dispersed in a mixture containing an enteric polymer and a non-enteric polymer. The pharmaceutical composition of the present invention may also be in granular form, whereby the granules may be coated with an enteric polymer. It is preferred that the enteric coating of the granules and the enteric polymer constituent of the granules contain the same enteric polymer. According to a preferred embodiment, the granules are composed of IL-6, an enteric polymer and a non-enteric polymer and optionally any other pharmaceutically acceptable excipient.

According to one embodiment, the invention aims at an oral pharmaceutical composition as described above for use to reduce intestinal contractility in the context of the oral treatment of diabetes or pre-diabetes, in particular type 2 diabetes.

The invention also aims at a pharmaceutical composition according to the invention for the oral treatment of diabetes associated with at least one gastroenteropathic disorder and that said disorder is reduced.

The enteropathic disorder can be selected from gastroparesis, diarrhea, transit disorders, constipation, for example.

13. The invention also relates to a method for treating diabetes or pre-diabetes comprising decreasing intestinal contractility via oral administration of IL-6 to a diabetic or pre-diabetic patient, in particular a diabetic or pre-diabetic type 2 patient.

14. In an embodiment, the invention relates to a method for limiting intestinal glucose absorption by reducing intestinal contractility, in particular duodenal contractility, in a diabetic or pre-diabetic patient comprising oral administration of IL-6.

The reduction of intestinal contractility limits glucose absorption in the duodenum and thus reduces glycemia.

The invention thus aims at IL-6 for oral use to reduce intestinal contractility and limit intestinal glucose absorption in a diabetic or pre-diabetic patient, in the treatment of diabetes or prediabetes. The invention also aims at IL-6 for oral use to reduce intestinal contractility and treat and/or prevent glucose intolerance; more particularly in the treatment of diabetes or pre-diabetes, particularly type 2. 15. It is also an object of the present invention to aim at IL-6 for oral use to modulate, in particular to diminish or reduce, intestinal contractility, in particular duodenal contractility.

16. The invention also relates to IL-6 for use according to embodiment 15, characterized in that the modulation consists of a reduction.

17. The invention also aims at providing a method for regulating glycemia in a diabetic or pre-diabetic patient comprising bringing IL-6 into contact with the duodenal mucosa.

18. Method according to embodiment 17, characterized in that the contact is achieved by oral administration.

19. Use of IL-6 for the manufacture of a drug for the oral treatment of diabetes or pre-diabetes, in particular type 2 diabetes.

The invention also relates in a particular embodiment to the use of IL-6 for the manufacture of a drug for reducing intestinal contractility in the context of the oral treatment of diabetes or pre-diabetes, in particular type 2 diabetes.

20. IL-6 for use for the oral treatment of a pathology mediated by an alteration of intestinal contractility, in particular duodenal contractility. In particular, the alteration is a decrease.

21 . IL-6 for use according to embodiment 20, characterized in that the pathology is selected from diabetes, in particular type 2 diabetes, the complications and side effects thereof such as obesity and gastroenteropathic disorders.

22. IL-6 for use to reduce intestinal contractility in the oral treatment of hyperglycemia. In particular, the hyperglycemia is selected in the group comprising fasting hyperglycemia, fed hyperglycemia as well as combination thereof. 23. IL-6 for its separate, simultaneous or sequential use with an antidiabetic compound, in particular selected from the group consisting of biguanides, thiazolidinediones, alpha-glucosidase inhibitors, sulfonylurea, insulin and/or its analogs, other known antidiabetic molecule and combination thereof, for the treatment of hyperglycemia. Suitable biguanide maybe metformin for example.

In the methods and uses as described hereabove according to the invention, the IL-6 is used and administered orally.

The present invention is also and particularly applicable to diabetes in domestic and farm animals.

The term“domestic animal” refers to cats and dogs.

The term“farm animal” refers in particular to cattle, poultry, horse, pork, goat and sheep.

In particular, in cats, which are carnivorous by nature, there is an increasing incidence of diabetic pathologies due mainly to the diet based on dry or reduced- moisture foods rich in polysaccharides. Such a diet results in a carbohydrate ration of about 40% instead of the 10% of a diet based on natural prey. Consequently, the development of hyperglycemia, diabetes and obesity in domestic cats continues to increase.

The same can be said for dogs, although their omnivorous nature somewhat moderates the situation.

Via oral administration, thus targeting neurons of the enteric nervous system, the inventors demonstrated that a decrease in duodenal contractility following oral administration of IL-6 in diabetic mice improves the animal’s diabetic status. This results in 1 ) a decrease in fasting and/or fed hyperglycemia and 2) restoration of the intestine-brain axis to improve insulin sensitivity. This second effect allows a better use of glucose by cells, especially muscle cells, and also contributes to glycemia control.

The molecule according to the invention, IL-6, is a cytokine whose effects on this cellular/molecular target were unknown by the oral route until now. FIGURES

Figure 1 : Measurement of duodenal mechanical contraction amplitude in response to an injection (in organ bath) of Krebs Ringer solution (Vehicle) or IL-6 between 25 and 50nM in HFD45% mice. n=7-8 per group. ^*** p>0.001 and ^**** pO.0001 vs Vehicle.

Figure 2: Measurement of colonic mechanical contraction amplitude in response to an injection (in organ bath) of Krebs Ringer solution (Vehicle) or IL-6 between 25 and 50nM in FIFD45% mice. n=7-8 per group. ^*** p<0.001 and ^**** pO.0001 vs Vehicle.

Figure 3: Measurement of duodenal mechanical contraction amplitude in response to an oral administration of water (Vehicle) or IL-6 12.5nM for one week in FIFD60% mice. n=5 per group. ^** p<0.01 vs Vehicle.

Figure 4: Measurement of colonic mechanical contraction amplitude in response to an oral administration of water (Vehicle) or IL-6 12,5nM for one week in FIFD60% mice. n=5 per group p vs Vehicle.

Figure 5: Effects of an oral administration of water (Vehicle) or IL-6 0.03pg for one week on glycemia in FIFD60% mice, after 6h fasting. n=10 per group. ^* p< 0.05 vs Vehicle.

Figure 6: Effects of an oral administration of water (Vehicle) or IL-6 0.03pg for one week on glycemia in FIFD60% mice, after 12h fasting. n=10 per group. ^* p< 0.05 vs Vehicle.

Figure 7: A - Oral glucose tolerance test (OGTT) in fasted FIFD45% mice, after an oral administration of water (Vehicle) or IL-6 25nM for one week. B - The graph represents the average area under the curve (AUC). n = 9-10 per group. ^** p < 0.01 ,

^*** p < 0.001 vs Vehicle. Figure 8: Effects of an oral administration of water (Vehicle) or IL-6 25nM for one week on body weight gain in HFD45% mice n = 9-10 per group. ^*** p < 0.001 vs Vehicle.

Figure 9: Ex vivo measurement of duodenal mechanical contraction amplitude in response to Krebs Ringer solution (Vehicle), Metformin (100nM) and/or IL-6 25nM. n=7-8 per group. ^* p < 0.05, ^*** p < 0.001 vs Vehicle, ## p < 0.01 , #### p < 0.0001 vs Metformin.

EXEMPLE

Ten-week-old male C57BL/6J mice (Charles River Laboratory, I’Arbresle, France) were housed in specific pathogen free conditions and in controlled environment (room temperature of 23±2°C, 12h daylight cycle) with free access to food and water. Experiments were conducted according to the European Community regulations concerning the protection of experimental animals and were approved by the local Animal Care and Use Committee. Mice were fed on high-fat diet (FIFD) containing 20% protein, 35% carbohydrate, and 45 % or 60% fat (Research Diet, New Brunswick, NJ, USA). These mice became obese and insulin resistant after, respectively, 3 or 2 months of FIFD.

Isotonic contraction

Mice were euthanized in fed conditions, corresponding to the phase where intestinal segmental waves are generated to increase the rate of nutrients absorption. After dissection, duodenum and colon segments were washed and incubated in oxygenated Krebs-Ringer solution for 30 min at 37 °C, attached to the isotonic transducer (MLT7006 Isotonic Transducer, Flugo Basile, Comerio, Italy), and immersed in an organ bath of the same medium maintained at 37 °C. The load applied to the lever was 1 g (10 mN). Isotonic contractions were recorded on Labchart software (AD Instruments) following the transducer displacement. After attaching of intestinal segments, basal contractions were recorded for 10 minutes. For in vivo protocol, the mice were daily treated with an oral gavage of 100mI_ of each drug (Control or IL-6 0.03pg/mouse) during the last week of HFD treatment. . For ex vivo protocol, subsequently, 100 mI_ of IL-6 at 25 and 50 nM were added to the survival medium, and contractions were recorded for 10 min for each dose. Contraction amplitudes are presented as percentage relative to the basal response. In the combination experiments metformin 100nM was added to the survival medium and IL- 6 at 25 nM.

Evaluation of fasted glvcemia

As described above, the mice were daily treated with an oral gavage of 100pL of each drug (vehicle or IL-6 0.03pg) during the last week of FIFD treatment as described in Fournel, A. et al, Gut 66, 258-269 (2017). After six or twelve hours fasting, blood glycemia is evaluated with a gluco-meter (Accu-Chek Active, Roche).

Oral glucose tolerance test

Mice were treated daily with an oral gavage of 100 pL of each drug (vehicle or IL-6 25nM) during the last week of FIFD treatment. Eight-hour-fasted mice were orally loaded with glucose (3g/kg of body weight), 24h after the last oral administration of vehicle or peptide of interest. Glycemia was measured at -30, 0 (time of oral glucose loading), +15, +30, +60, +90 and +120 min with a gluco-meter (Accu-Chek Active, Roche).

RESULTS

Impact of chronic IL-6 treatment on intestinal contractility First, we measured the effect of IL-6 on duodenal contraction in ex vivo condition. Using isotonic sensor, we discovered that IL-6 inhibits the mechanical contractions of the duodenum in mice fed with high fat diet 45% (Figure 1 ). In addition, we measured the colonic contraction and we found that there were slightly decreased in mice after chronic treatment with IL-6 (p=0.0651 ) (Figure 2). The authors confirm these results in duodenum (Figure 3) and colon (Figure 4) in mice fed with high fat diet 60% treated with an oral administration of IL-6 for one week.

Impact of chronic IL-6 treatment on fasted glvcemia

Oral IL-6 treatment for one week decreases blood glucose level in fasted diabetic mice fed with high fat diet 60%, regardless of fasting duration, i.e. 6 hours (Figure 5) or 12 hours (Figure 6).

Impact of chronic IL-6 treatment on glucose tolerance

IL-6 oral treatment for one week improves glucose tolerance (Figure 7) and decreases body weight gain (Figure 8).

Impact of combined IL-6/Metformin treatment on intestinal contractility

The co-treatment of metformin 100nM and increasing doses of IL-6 are tested. At this low dose, metformin increases duodenal hypercontractility of obese/diabetic mice From IL-6 25 nM, the treatment decreases duodenal contractions as IL-6 alone (Figure 9). This suggests that IL-6 can reduce/prevent deleterious effects of metformin .

CONCLUSIONS

For the first time, the authors have demonstrated that IL-6 can modulate duodenal contraction in diabetic mice supplemented with a 45% or 60% high fat diet. Furthermore, IL-6 reduces deleterious hyperglycemia in these mice. A positive correlation exists between intestinal contraction and glucose absorption.

These results demonstrate that the decrease of glycemia induced by oral administration of IL-6 is due to a decrease in intestinal contractility. This would promote reduction of glucose intestinal absorption and restore the "gut-brain" axis in diabetic mice.