Angiotensin converting enzyme inhibitors or angiotensin ii receptor antagonists for use in prevention and / or treatment of gastrointestinal alterations associated with diabetes mellitus

Abstract

The present invention relates to repurposing the use of angiotensin Converting Enzyme Inhibitors (ACEI) and Angiotensin II Receptor Antagonists (ARAs) to prevent and / or treat gastrointestinal complications associated with Diabetes mellitus.

Description

[0001] DESCRIPTION

[0002] ANGIOTENSIN CONVERTING ENZYME INHIBITORS OR ANGIOTENSIN II RECEPTOR ANTAGONISTS FOR USE IN PREVENTION AND / OR TREATMENT OF GASTROINTESTINAL ALTERATIONS ASSOCIATED WITH DIABETES

[0003] MELL ITUS

[0004] Technical field

[0005] The present invention relates to the prevention and / or treatment of gastrointestinal alterations associated with Diabetes Mellitus using Angiotensin Converting Enzymes Inhibitors (ACEI s ) or Angiotensin I I Receptor Antagonists (ARAs ) .

[0006] Background art

[0007] Diabetes Mellitus ( DM) is a complex chronic progressive metabolic disorder, medically incurable , that can af fect almost every organ system with a prevalence in humans of all age groups at alarming proportions worldwide . There are di f ferent animal models of DM, but streptozotocin ( STZ ) has been the agent of choice to chemically induce diabetes in rats and mice , causing the selective destruction of pancreatic p-cells . High doses of STZ are associated with type 1 DM ( T1DM) induction, while multiple low doses are usually associated with a high fat diet to cause insulin resistance , characteristic of type 2 DM ( T2DM) . In this animal model of T1DM structural , functional and biochemical alterations resemble those observed in human diabetic patients . Over time , several investigators have used this model with di f ferent induction times ( raising questions about animal wel fare for longer protocols ) in di f ferent portions of the intestine , making it harder to compare results .

[0008] Gastrointestinal ( GI ) complications of DM are very common and very important as they can be associated with signi ficant morbidity, af fecting up to 75% of patients . Though this high prevalence , the knowledge and treatment options for these complications are considerably scarce .

[0009] DM related complications in intestine and colon result in symptoms like chronic constipation, diarrhea ( occasionally as sociated with fecal incontinence ) , and are associated with worse outcomes in clinical conditions like colorectal cancer and inflammatory bowel disease . Additionally, possible complications like megacolon, pseudo-obstruction, stercoral ulcer or perforation may occur .

[0010] The most common GI complications include esophageal dysmotility, gastroparesis , enteropathy and colonic disorders , such as chronic constipation and diarrhea . Since these symptoms are not considered important causes of mortality in patients with DM they are often neglected . However, it is important to recogni ze that they negatively influence health status and quality of li fe .

[0011] The pathogenesis of diabetic intestinal dys function seems to be multi factorial , related to the accumulation of advanced glycation end-products (AGE ) , inj ury of the Enteric Nervous System (ENS ) or interstitial cells of Caj al , and muscular layers fibrosis . Several known studies also indicate that diabetic autonomic neuropathy causes damage to the ENS and changes the number and si ze of myenteric neurons throughout the entire GI tract in rats (He, C.-L., et al, Gastroenterology 2001; Furlan, M.M.D., et al, Arg. de Neuro-Psiquiatria 2002; Honore, S.M., et al, Auton. Neurosci. 2011; Fregonesi, C.E.P.T., et al, Arq. Neuro-Psiquiatria 2001; Brasileiro, A.D., et al, Brain Res. 2018; Zanoni, J., et al, Arq. Neuropsiquiatr . 1998) . It has also been described as a deficit in the intestine's cholinergic neurotransmission, since the response to exogenous acetylcholine (ACh) seems to be impaired in the ileum (30 days after STZ-induction) and colon of long-term diabetic rats (60 weeks) (Lucas, P.D., et al, Gastroenterology, 1991; Kim, S.J., el al, J. Neurogastroenterol . Motil. 2011) . Mechanical factors can also contribute to intestinal disorders, since DM seems to cause structural remodelling that can affect histomorphometry and biomechanical properties, increasing stiffness, and decreasing the resting compliance and relaxation capacity of the intestinal wall.

[0012] The renin-angiotensin system (RAS) is mostly known for its effects in the cardiovascular and renal systems, regulating blood pressure and fluid homeostasis, but it also has an influence in other systems, such as the GI tract, which expresses all of the RAS components. RAS is however associated with DM since the Angiotensin Converting Enzymes Inhibitors (ACEI) or Angiotensin Type 1 Receptor (ATi) Antagonist (ARAs) have shown to reduce the incidence of vascular complications, nephropathy and cardiovascular disease in these patients.

[0013] Angiotensin II (Ang II) is the major effector peptide of this system, and most of its functions are mediated by the Ang II type 1 receptor (ATiR) , while activation of the Ang II type 2 receptor (AT2R) usually counteracts them. In the colon, Ang II contracts circular and longitudinal smooth muscle in response to direct activation of postj unctional ATiR and indirect activation of pre- unctional ATiR in myenteric and submucosal neurons . Curiously, the human colonic smooth muscle is more sensitive to Ang I I than to acetylcholine (ACh) , but the physiological importance of Ang I I in the GI tract is still not completely understood . Interestingly, there is little information on RAS alterations in the intestine of diabetic individuals , but recently one study concluded that ACE gene polymorphism in patients with T2DM influences intestinal motility, since those patients presented a prevalent genotype that was associated to constipation (Malik et al . , Bi ochem . Genet . , 2020 ) .

[0014] Also , increased levels of tecidual ACE were found in DM patients , resulting in additional formation o f Ang I I . In addition, in patients with type 2 DM ( T2DM) RAS inhibition improves insulin sensitivity allowing better control of glycemic values . In fact , infusion of Ang I I seems to induce insulin resistance . However, to date there is no information involving RAS with diabetic GI alterations .

[0015] There are limited treatment options available for GI complications as sociated with DM, with existing treatments primarily focusing on symptomatic management . Therapeutic considerations depend on the severity of symptoms , the ability of the patient to maintain adequate nutrition and their responsiveness to therapy .

[0016] For gastroparesi s , prokinetics are usually used in order to increase gastric motility and enhance stomach emptying . Medications commonly used in treatment are shown in Table 1 . Table 1 . Drugs used in treatment of diabetic gastroparesis (Krishnan et al . , World. J. Diabetes, 2013)

[0017] Endoscopic and surgical treatments can also be used . For example , endoscopic pyloric inj ections of botul inum toxin have been tried in the management of gastroparesis and shown improved symptoms and accelerated gastric emptying persisting up to 3- 6 months . Surgical treatments are rarely performed and are mainly reserved for patients with refractory gastroparetic symptoms who have fai led to improve with other measures . Completion gastrectomy was shown to give long-term symptom relief in some patients with post-surgical gastroparesis , but data on patients with diabetic gastroparesis are limited .

[0018] The treatment of diabetic diarrhea mainly involves symptom relief , correction of fluid and electrolyte deficits , improvement of nutrition and glycemic control , and management of underlying causes .

[0019] Lactulose and osmotic laxatives may be necessary in severe cases of constipation . Newer drugs for treatment of chronic constipation include prucalopride , a selective 5- HT4 receptor agonist that enhances colonic transit and lubiprostone , which stimulates colonic water and electrolyte secretion through activation of type 2 chloride channels in enterocytes.

[0020] Insulin-like growth factor 1 (IGF-1) treatment can inhibit the DM induced colonic smooth muscle cell apoptosis and may be involved in the alleviation of colonic dysmotility in DM rats. Alpha-glucosidase inhibitors such as acarbose treatment have been demonstrated to increase the content of gut Bifidobacterium longum and partially restore the imbalance of gut microbiota in diabetic patients that also seems to be important in diabetic dysmotility. In contrast, GLP-1 receptor agonists such as liraglutide seem to modulate the composition of the gut microbiota .

[0021] Document "Wagatsuma et al. , Clinical usefulness of the angiotensin II receptor antagonist losartan in patients with portal hypertensive gastropathy, Hepatogastroenterology, 2006" discloses a study involving the administration of the angiotensin II receptor antagonists Losartan, an agent developed as antihypertensive agents, in patients with portal hypertensive gastropathy patients, in order to examine its clinical usefulness.

[0022] Document "Krishnan et at. , Gastrointestinal complications of diabetes mellitus , World J. Diabetes, 2013" discloses an update on the gastrointestinal complications of diabetes, their pathophysiology, diagnostic evaluation and management.

[0023] Document "Dedeli, 0., et al; Prevalence of Gastrointestinal Symptoms and Its Effect on Quality of life among Patients with Diabetes Mellitus ; Am. J. Nurs. Res . 2015" discloses a study to determine the prevalence of gastrointestinal symptoms and its effect on quality of life among patients with diabetes mellitus.

[0024] Document "Werner, C., et al. RAS blockade with ARB and ACE inhibitors: current perspective on rationale and patient selection. Clin Res Cardiol 97, 418-431 (2008)" discloses studies of the influence of ACE inhibitors and ARBs on the number and function of endothelial progenitor cells revealing additional mechanisms of action of these drugs .

[0025] Document "Pushpakom S, et al. Drug repurposing: progressrchallenges and recommendations . Nat Publ Gr . 2018" discloses approaches used for drug repurposing (also known as drug repositioning) , discuss the challenges faced by the repurposing community and recommend innovative ways by which these challenges could be addressed to help realize the full potential of drug repurposing.

[0026] Nowadays, no therapy targets the prevention and / or treatment of gastrointestinal alterations linked to DM, whether in humans or other animals.

[0027] Summary of the invention

[0028] The present invention relates to repurposing the use of angiotensin converting enzyme inhibitors (ACEI) and angiotensin II receptor antagonists (ARAs) to prevent and / or treat gastrointestinal alterations associated with Diabetes mellitus.

[0029] Description of the drawings The following description is made with reference to the accompanying drawings, which are presented for illustrative purposes only, without any limiting character, and wherein:

[0030] Figure 1 - Evaluation during the experimental protocol (14 days) in control (CTRL, n = 8) and streptozotocin- induced diabetic rats (STZ, n = 16-21) of: (a) body weight; (b) water intake; (c) , food intake and (d) fecal excretion. Values are mean ± SEM and unpaired student's t test was used to compare the two experimental groups (CTRL and STZ) . * Statistical difference, p < 0.05.

[0031] Figure 2 - Macroscopic evaluation of the ileum and colon of control (CTRL, black bars, n = 8-12) and streptozotocin-induced diabetic rats (STZ, white bars, n = 11-14) : (a) representative images of the colon length (CTRL, above the scale bar) ; (b) quantitative analysis of colon length (left y axis) and colon length per rat weight (right y axis) ; (c) tissue circumferential perimeter of the colon and ileum and (d) relative weight of intestinal segments (without fecal content) expressed as g of colon or ileum / g of body weight. Values are mean ± SEM and unpaired student's t test was used to compare the two experimental groups (CTRL and STZ) . * Statistical difference, p < 0.05.

[0032] Figure 3 - Representative microscopic photographs of intestinal segments of control (CTRL, a,c,e,g) and streptozotocin-induced diabetic rats (STZ, b,d,f,h) , stained with hematoxylin and eosin: ileum (a,b) ; proximal colon (c,d) ; middle colon (e,f) and distal colon (g,h) . The scale bar (100 pm) is valid for all images. Figure 4 - Morphometric evaluation of intestinal segments (ileum, proximal colon, middle colon and distal colon) of control (CTRL, n = 4) and streptozotocin-induced diabetic (STZ, n = 8) rats: (a) total wall thickness (pm) of each intestinal segment; (b) thickness (pm) of the intestinal layers (longitudinal muscle, circular muscle, submucosa and mucosa) of each intestinal segment. Values are mean ± SEM and a 2-way ANOVA followed by an unpaired t test with Welch' s correction was used to compare the two experimental groups (CTRL and STZ) . * Statistical difference p < 0.05 vs. correspondent control.

[0033] Figure 5 - Contractile response to KC1 (125 mM) in the ileum, proximal colon, middle colon and distal colon of control (CTRL, n = 6) and streptozotocin-induced diabetic rats (STZ, n = 10) . Data are expressed as mN of force per g of fresh tissue (mN / g) . Values represent the median (95% confidence limits) and a Mann-Whitney test was used to compare the two experimental groups (CTRL and STZ) .

[0034] Figure 6 - Concentration-response curves to ACh in the ileum, proximal colon, middle colon and distal colon of control (CTRL, n = 6-7) and streptozotocin-induced diabetic rats (STZ, n = 10) . Data are expressed as mN of force per g of fresh tissue (mN / g) . Values are mean ± SEM.

[0035] Figure 7 - Concentration-response curves to Angiotensin IT in the ileum, proximal colon, middle colon and distal colon of control (CTRL, n = 5-8) and streptozotocin-induced diabetic rats (STZ, n = 5) . Data are expressed as mN of force per g of fresh tissue (mN / g) . Values are mean ± SEM. Figure 8 - Angiotensin IT contractile effect (expressed as percentage of change) in the ileum, proximal colon (PC) , middle colon (MC) and distal colon (DC) of control (CTRL, n = 5-8) and streptozotocin-induced diabetic rats (STZ, n = 4-6) in the presence of the following antagonists: (a) candesartan (ATiR antagonist, 10 nM) and (b) PD123,319 (AT2R antagonist, 100 nM) . Values are mean ± SEM. For statistical analysis, a paired t test was used between the effect in the absence and presence of the antagonist. * p < 0.05 vs. the correspondent response to Angiotensin IT in the absence of the antagonist.

[0036] Figure 9 - Evaluation during the experimental protocol (6 hours) in control (CTRL) and GK rats (GK) of: (a) blood glucose; (b) body weight; and (c) food intake and water intake. Values are mean ± SEM and unpaired student's t test was used to compare the two experimental groups (CTRL and STZ) . * Statistical difference, p < 0.05

[0037] Figure 10 - Morphometric evaluation of intestinal segment (duodenum, jejunum, ileum, cecum, proximal colon and distal colon) of control (CTRL) and GK rats (GK) rats: total wall thickness (pm) of each intestinal segment. Values are mean ± SEM and a 2-way ANOVA followed by an unpaired t test with Welch' s correction was used to compare the two experimental groups (CTRL and STZ) . * Statistical difference p < 0.05 vs. correspondent control.

[0038] Figure 11 - Representative microscopic photographs of intestinal segments of control (CTRL, a,c,e,g,i,k) and GK rats (GK, b, d, f , h, j , 1 ) , stained with hematoxylin and eosin: distal colon (a,b) ; proximal colon (c,d) ; cecum (e, f ) ; ileum (g,h) ; jejunum (i,j) and duodenum (k,l) . The scale bar (100 pm) is valid for all images.

[0039] 1 Figure 12 - Morphometric evaluation of intestinal segments (distal colon, proximal colon, cecum, ileum, jejunum and duodenum) of control (CTRL) and GK rats (GK) : (a,b,c) thickness (pm) of the intestinal layers (longitudinal muscle, circular muscle, submucosa and mucosa) of each intestinal segment; (d,e,f) thickness (pm) of the intestinal layers (longitudinal muscle, circular muscle, submucosa, mucosa, crypts and villi) of each intestinal segment. Values are mean ± SEM and a 2-way ANOVA followed by an unpaired t test with Welch' s correction was used to compare the two experimental groups (CTRL and STZ) . * Statistical difference p < 0.05 vs. correspondent control .

[0040] Figure 13 - Quantitative analysis of nucleus in the muscular layers of intestinal segments (duodenum, jejunum, ileum, cecum, proximal colon and distal colon) of control (CTRL) and GK rats (GK) : (a) Muscular SMCs density (nucleus / lOOpm2) of the intestinal layers of each intestinal segment; (b) representative microscopic photographs of intestinal segments of control rats (CTRL) and GK rats (GK) , stained with hematoxylin and eosin: duodenum. The scale bar (100 pm) is valid for all images.

[0041] Figure 14 - Quantitative analysis of neuronal nucleus in the myenteric plexus (duodenum, jejunum, ileum, cecum, proximal colon and distal colon) of control (CTRL) and GK rats (GK) : (a) Myenteric neuronal density (nucleus / mm2) of the intestinal layers of each intestinal segment; (b) representative microscopic photographs of intestinal segments of control rats (CTRL) and GK rats (GK) , stained with hematoxylin and eosin: ileum. The scale bar (100 pm) is valid for all images.

[0042] 1 Figure 15 - Evaluation during the experimental protocol (14 days) in control (CTRL) , streptozotocin- induced diabetic rats (STZ) and streptozotocin-induced diabetic rats treated with losartan (STZ+LOS) of: (a) glycemia; (b) body weight; (c) water intake and (d) food intake. Values are mean ± SEM and unpaired student's t test was used to compare the three experimental groups (CTRL, STZ and STZ+LOS) . * Statistical difference, p < 0.05.

[0043] Figure 16 - Morphometric evaluation of intestinal segment (colon) of control (CTRL) , streptozotocin-induced diabetic rats (STZ) and streptozotocin-induced diabetic rats treated with losartan (STZ+LOS) : (a) thickness (pm) of the intestinal layers (longitudinal muscle, circular muscle, submucosa and mucosa) of each intestinal segment. Values are mean ± SEM and a 2-way ANOVA followed by an unpaired t test with Welch' s correction was used to compare the two experimental groups (CTRL, STZ and STZ + LOS) . *

[0044] Statistical difference p < 0.05 vs. correspondent control; (b) representative microscopic photographs of intestinal segments of control rats (CTRL) , streptozotocin-induced diabetic rats (STZ) and streptozotocin-induced diabetic rats treated with losartan (STZ+LOS) , stained with hematoxylin and eosin: colon. The scale bar (500 pm) is valid for all images.

[0045] Figure 17 - Morphometric evaluation of intestinal segment (ileum) of control (CTRL) , streptozotocin-induced diabetic rats (STZ) and streptozotocin-induced diabetic rats treated with losartan (STZ+LOS) : (a) thickness (pm) of the intestinal layers (longitudinal muscle, circular muscle, submucosa and mucosa) of each intestinal segment. Values are mean ± SEM and a 2-way ANOVA followed by an

[0046] 1 unpaired t test with Welch' s correction was used to compare the two experimental groups (CTRL, STZ and STZ + LOS) . * Statistical difference p < 0.05 vs. correspondent control; (b) representative microscopic photographs of intestinal segments of control rats (CTRL) , streptozotocin-induced diabetic rats (STZ) and streptozotocin-induced diabetic rats treated with losartan (STZ+LOS) , stained with hematoxylin and eosin: ileum. The scale bar (500 pm) is valid for all images.

[0047] Figure 18 - Ultrasound evaluations of intestinal segments of diabetic pets: (a) stomach - Inter rugal; (b) Stomach - Rugal (c) Duodenum; (d) Jejunum; (e) Ileum; and

[0048] (f) colon.

[0049] Figure 19 - Representative microscopic photographs of intestinal segments (stomach, jejunum, ileum and colon) of diabetic pets showing: collagen deposits (Masson's trichrome staining) in the muscular layers and between mucosal crypts and muscularis mucosa (black dashed circles in the stomach and jejunum) ; and inflammatory infiltrate (hematoxylin and eosin staining, black square in the ileum and colon) . The scale bar (500 pm) is valid for all images.

[0050] Detailed description of the invention

[0051] The present invention relates to the use of ARAs or ACEI to prevent and / or treat diabetic-induced GI alterations, preconizing the use of said drugs to prevent important GI alterations that occur in DM. These drugs are already authorized to control hypertension, heart failure, chronic kidney disease, among other important pathologies, but they were never considered to prevent and / or treat diabetic GI alterations.

[0052] 1 For this, a new research data with diabetic rats was carried out, with the following goals:

[0053] 1) enhancing the evidence supporting the preventive effects of losartan (ARA) on GI alterations occurring in type 1 diabetes mellitus (T1DM) ;

[0054] 2) determining whether losartan (ARA) can prevent and / or treat established GI alterations in diabetic rats; and

[0055] 3) determining whether diabetic rats with type 2 diabetes mellitus (T2DM) present the same GI alterations as the diabetic rats with T1DM.

[0056] The present disclosure involves repurposing these drugs, with the aim of reduction morbidity linked to gastrointestinal alterations in diabetic patients. The advantage lies in the fact that these drugs are already commercially available for treating other conditions.

[0057] In the context of the present invention, the following commercially available drugs were used:

[0058] - Candesartan - lOnM, ATiR antagonist (in vitro)

[0059] - PD123.319 (di (trifluoroacetate) salt hydrate, Sigma- Aldrich, USA) - lOOnM, AT2R antagonist (in vitro) ;

[0060] - Losartan - 20mg / kg,PO, ATiR antagonist (in vivo) .

[0061] Experiment 1 - Evaluation of the histomorphometric impact of diabetes mellitus in the intestine and colon

[0062] The evaluation of the histomorphometric impact of diabetes mellitus in the intestine and colon was carried out with:

[0063] Type 1 DM: Streptozotocin (STZ) induced rats; and

[0064] Type 2 DM: Goto-Kakizaki (GK) rats. 1. Type 1 DM: STZ induced rats

[0065] The STZ induced rats are an animal model of Type 1 DM (T1DM) , resulting from the induction of Streptozotocin (STZ) in order to chemically induce diabetes in rats and mice, causing the selective destruction of pancreatic p- cells .

[0066] Results

[0067] Animal Welfare and Monitorization

[0068] STZ-induced rats had an initial glycemia of 99.30 ± 3.29 mg / dL that increased to 395.09 ± 13.80 mg / dL within 48 hours (p < 0.0001, n = 23) , while control rats had an initial glycaemia of 105.63 ± 6.31 mg / dL that was roughly the same within 48 hours (111.14 ± 5.41 mg / dL; p > 0.05, n = 8) . On d7 and dl4, almost all STZ rats had glycemia above 500 mg / dL, while control animals presented glycemic values of 105.57 ± 4.76 mg / dL (n = 8) on the 14th day.

[0069] The parameters documented during the daily monitorization (body weight, water / food intake and fecal excretion) are shown in Figure 1. In the control group (n = 8) , rats progressively gained weight, their weight being 7.8% ± 0.73% higher by dl4 than on dO (before fasting) . Diabetic rats (n = 21) had a consistent weight loss that was more pronounced on d2 (5% less compared to the previous day) and then maintained that weight for the remainder of the protocol (7.66 ± 1.04% lower at dl4 when compared to the initial weight before fasting) (Figure la) . Water intake was significantly higher in diabetic rats comparing to controls that maintained a constant water intake through

[0070] 1 all the experimental protocol: 37.54 ± 0.53 mL / day (n = 8) . The STZ group drank more water since dl (48.38 ± 1.16 mL) , but their water intake increased progressively throughout the protocol, reaching values 7 times higher than those of control animals at dl4: 264.08 ± 12.18 mL (n = 16) (Figure lb) . Despite the weight loss, STZ rats' food intake was significantly higher than controls after the 3rd day. Diabetic rats started the experimental protocol eating 13.25 ± 1.86 g in the first day, and progressively increased food consumption until the last day, when the intake was 49.08 ± 2.64 g / rat (n = 16) . The control group maintained a constant food intake during the experimental time, with a mean consumption of 22.44 ± 0.38 g / day (n = 8)

[0071] (Figure 1c) .

[0072] The study performed by the Applicant allows the quantification of fecal excretion in STZ-induced diabetic animals. Non-diabetic animals maintained a relatively stable fecal excretion during the entire experimental period (7.75 ± 0.18 g / day / rat, n = 8) , whereas diabetic rats gradually increased their fecal excretion, reaching values 4 times higher than those obtained in the first day (dl: 7.11 ± 0.34 g / rat; dl4: 30.79 ± 0.73 g / rat; p < 0.0001, n = 16) (Figure Id) .

[0073] Ileum and Colon ic Evaluation

[0074] Comparing to control animals, all segments of the intestines of STZ rats seemed enlarged. In addition, upon the opening of the abdomen of STZ-induced rats it was easy to perceive an extremely dilated cecum that produced a "mass effect", pushing the intestine to the side. The colon length was significantly higher in diabetic animals compared to the control group (Figure 2a, b: 25.75 ± 0.77

[0075] 1 cm, n = 14 vs. 19.63 ± 0.47 cm, n = 12, p < 0.05) . Since some animals were heavier than others, colon length per body weight was measured and the difference between the two groups was maintained (Figure 2b) . The circumferential perimeter of the intestinal portions was also measured, being significantly higher in the STZ-induced rats (n = 11) compared to non-diabetic rats (n = 8) both in the colon (15.45 ± 0.58 mm vs. 11 ± 0.46 mm, p < 0.0001, respectively) and ileum (12.55 ± 0.31 mm vs. 9.38 ± 0.32 mm, p < 0.0001, respectively) (Figure 2c) . The relative weight of the whole intestine segment studied (with fecal content) was higher in STZ-induced animals than in controls (2.69 ± 0.10 g / g of body weight, n = 21 vs. 1.80 ± 0.05 g / g of body weight, n = 12; p < 0.0001, respectively) . This increase was also observed at the individual intestinal segments free of fecal content (Figure 2d) . Furthermore, no differences were found between STZ-induced animals and controls in the wet-to-dry ratio of all the segments studied (ileum: 5.23 ± 0.37 vs. 5.61 ± 0.33; PC: 5.17 ± 0.24 vs. 4.52 ± 0.20; MC : 4.84 ± 0.30 vs. 5.16 ± 0.21; DC: 5.07 ± 0.20 vs. 4.86 ± 0.28, respectively, p > 0.05 for all) . The 2-way ANOVA results showed an interaction between the experimental group (control or STZ) and the intestinal segments (p < 0.0001) , in accordance with our visual observation of the marked dilatation of the intestine in STZ-induced animals. The relative fecal content weight was also higher in STZ-induced animals than in controls (7.10 ± 0.15 g / g of body weight, n = 21 vs. 2.66 ± 0.11 g / g of body weight, n = 12; p < 0.0001) .

[0076] Ileum and Colon Microscopic Evaluation

[0077] The results of the histomorphometric evaluation of the intestines of STZ-induced animals (n = 8) were concordant

[0078] 1 with the macroscopic data, showing an increase in the thickness of the intestinal wall of the ileum, proximal colon (PC) , middle colon (MC) and distal colon (DC) compared to controls (n = 4) , as can be observed in Figures 3 and 4a (ileum: 671.64 ± 74.34 pm vs. 404.97 ± 82.04 pm; PC: 666.66 ± 32.340 pm vs. 389.24 ± 39.03 pm; MC : 589.03 ± 17.88 pm vs. 376.06 ± 50.62 pm; DC: 570.93 ± 27.16pm vs. 430.42 ± 26.26pm, respectively, p < 0.01 for all) . The intestinal wall thickness increase was similar for all the intestinal segments, as 2-way ANOVA showed a nonsignificant association (p = 0.1681) between experimental group and intestinal segment. Both ileum (longitudinal muscle: 81.02 ± 7.66 pm vs. 31.18 ± 5.44 pm, circular muscle: 116.12 ± 4.59 pm vs. 44.47 ± 10.40 pm, submucosa: 41.68 ± 1.68 pm vs. 17.47 ± 2.13 pm, mucosa: 432.82 ± 20.59 pm vs. 311.85 ± 24.51 pm, respectively, p < 0.01 for all) and middle colon (longitudinal muscle: 48.93 ± 2.93 pm vs. 29.66 ± 4.25 pm, circular muscle: 142.55 ± 8.37 pm vs. 74.31 ± 10.9 pm, submucosa: 56.39 ± 4.09 pm vs. 35.63 ± 6.47 pm, mucosa: 341.17 ± 13.79 pm vs. 236.46 ± 34.58 pm, respectively, p < 0.05 for all) presented increased thickness of all the intestinal layers assessed in STZ- induced rats compared to controls (Figure 4b) . In the PC, the submucosa was the only layer that presented a similar thickness between STZ-induced animals and controls (50.47 ± 7.33 pm vs. 33.81 ± 6.00 pm, respectively, p = 0.1104) , while all the other segments were thicker in diabetic animals compared to controls (longitudinal muscle: 57.02 ± 6.90 pm vs. 34.64 ± 4.29 pm, circular muscle: 205.2 ± 17.00 pm vs. 90.14 ± 11.33 pm, mucosa: 353.97 ± 14.27 pm vs. 230.64 ± 26.18 pm, respectively, p < 0.05 for all) . DC only showed an increase in the muscle thickness (longitudinal muscle: 52.51 ± 2.72 pm vs. 28.51 ± 1.67 pm, circular muscle: 150.54 ± 14.58 pm vs. 87.21 ± 7.06 pm, p < 0.01 for

[0079] 1 both; submucosa: 66.11 ± 7.70 pm vs. 53.27 ± 7.54 pm and mucosa: 301.77 ± 10.00 pm vs. 261.42 ± 16.49 pm, p > 0.05 for both) (Figure 4b) . The 2-way ANOVA showed an association between the experimental group (control vs. STZ) and the intestinal layers thickness (longitudinal muscle, circular muscle, submucosa and mucosa) for the ileum (p = 0.0058) , PC (p = 0.0002) , MC (p = 0.0027) but not for the DC (p = 0.1109) . Ileum and Colon Functional Evaluation

[0080] To assess whether intestinal muscle contraction is altered in diabetic animals, ileum and colon reactivity to exogenously applied KC1 (Figure 5) , ACh (Figure 6) and Ang II (Figure 7) was evaluated. For the concentration-response curves to ACh and Ang II the results were expressed using two recognized pharmacological concepts: the maximum contractile effect (Emax, expressed in mN / g) and the concentration of agonist capable of causing 50% of the maximal contraction (ECso, expressed in uM) . In all intestinal segments (ileum, PC, MC and DC) the contractile response to 125 mM KC1 (and the ACh concentration-dependent contraction were similar in both control and STZ-induced animals (Figure 6) , with comparable Emax and ECso values, presented in Table 2.

[0081] Table 2. Emax(mN / g) and EC50(pM) values of smooth muscle contraction induced by ACh application in the ileum, proximal colon, middle colon and distal colon of control (CTRL, n = 6—7) and streptozotocin-induced diabetic rats (STZ, n = 10) .

[0082] Ileum Proximal Colon Middle Colon Distal Colon

[0083] Control

[0084] 165.9 [116.4- 141.0 [116.7- 184.7 [68.95- 313.4 [176.2-

[0085] Emax (mN / g)

[0086] 216.0] 278.5] 378.8] 823.1]

[0087] 1 Ileum Proximal Colon Middle Colon Distal Colon

[0088] 0.85 [0.32- 1.15 [0.22- 2.74 [0.94-

[0089] EC50 (pM) 3.41 [1.1-4.8]

[0090] 3.53] 14.70] 7.47]

[0091] STZ

[0092] 79.06 [34.65- 158.0 [75.0- 143.6 [86.56- 271.7 [163.6-

[0093] Emax (mN / g) 338.9] 569.5] 411.3] 370.9]

[0094] 0.82 [0.27- 114.0 [8.31- 18.96 [0.87- 2.94 [0.28-

[0095] EC50 (pM) 1.87] 3408] 75.7] 142.0]

[0096] For comparison between the two experimental groups (CTRL and STZ) we used a Mann-Whitney test. Values are median (95% confidence limits) .

[0097] Regarding reactivity to Ang II, this RAS effector peptide caused a concentration-dependent contraction in control and diabetic animals (Figure 7) . The contractile response to Ang II normalized to the tissue weight was lower (but with the same EC50) in the ileum, PC and MC of STZ-induced animals. Interestingly, the maximum response in the DC was similar between control and STZ-induced animals, but the EC50 of that portion of diabetic colon was significantly lower than that of controls (Table 3) .

[0098] Table 3. Emax (mN / g) and EC50 (pM) values of smooth muscle contraction induced by Angiotensin II application in the ileum, proximal colon, middle colon, and distal colon of control (CTRL, n = 5-8) and streptozotocin-induced diabetic rats (STZ, n = 5) .

[0099] Ileum Proximal Colon Middle Colon Distal Colon

[0100] Control

[0101] 305.3 [138.6- 181.5 [136.0- 276.6 [246.4- 344.4 [222.4-

[0102] Emax (mN / g) 620.5] 297.0] 451.1] 433.5]

[0103] 8.29 [1.24- 1.10 [0.36- 3.80 [1.95- 40.50 [17.08-

[0104] EC50 (pM)

[0105] 24.68] 2.12] 4.76] 309.3]

[0106] STZ

[0107] 71.20 [12.3- 50.46 [15.32- 100.6 [22.86- 263.5 [165.0-

[0108] Emax (mN / g) 100.6] * 78.15] * 163.5] * 415.9] Ileum Proximal Colon Middle Colon Distal Colon

[0109] 7.985 [0.31- 0.59 [0.35- 2.60 [0.89- 4.17 [0.84-

[0110] EC50 ( M)

[0111] 8.89] 14.93] 7.81] 8.38] *

[0112] For comparison between the two experimental groups (CTRL and STZ) we used a Mann-Whitney test. Values are median (95% confidence limits) . * p < 0.05 vs. correspondent control.

[0113] Knowing that the differences observed in the contractile response to Ang II could result from an imbalance between ATiR and AT2R mediated effect, the Applicant decided to further characterize the response to Ang II. The contractile response to Ang II was antagonized by candesartan (10 nM) , an ATiR antagonist, in all four intestinal segments of both control (in mN / g for all, ileum: 54.20 ± 4.50 vs. 2.35 ± 1, 60; PC: 17.37 ± 3.14 vs. 1.07 ± 0.49; MC: 12.42 ± 2.23 vs. 0.28 ± 0.15; DC:15.85 ±

[0114] 1.32 vs. 0.1 6± 0.08; p < 0.05 for all) and STZ-induced rats (ileum: 35,75 ± 11,06 vs. -0,87 ± 2,78; PC: 24.80 ±

[0115] 9.45 vs. 0.7 8± 1.19; MC :95.86 ± 29.03 vs. 5.20 ± 6.39;

[0116] DC: 288.48 ± 49.08 vs. 5.57 ± 5.54; p < 0.05 for all) (Figure 8a) . Differently, PD123319 (AT2R antagonist, 100 nM) decreased the response to Ang II in the ileum (12.43 ± 1.03 mN / g vs. 11.02 ± 1.21 mN / g, p < 0.05) and increased the response in all colonic segments of control animals (in mN / g for all, PC: 19.95 ± 3.34 vs. 22.02 ± 3.45; MC : 14.99 ± 1.97 vs. 17.48 ± 2.44; DC: 19.88 ± 2.82 vs. 23.50 mN / g ± 2.64; p < 0.05 for all) , but was unable to modify Ang II- induced contraction in the ileum (92.58 ± 21.23 mN / g vs. 104.24 ± 23.50 mN / g) , MC (146.13 ± 18.53 mN / g vs. 127.88 ± 21.89 mN / g) and DC of diabetic rats (236.37 ± 19.03 mN / g vs. 248.38 ± 25.64 mN / g; p > 0.05 for all) , decreasing it in the PC (166.14 ± 20.49 vs. 108.45 ± 19.00; p < 0.05) (Figure 8b) . Conclusion

[0117] The obtained results show that early STZ-induced DM is associated with increased thickness of the rat colon and ileum, decreased contractile response to Ang I I and dif ferent responses to AT2R inhibition .

[0118] The results presented in this study demonstrate that it is possible to refine a classic animal model of T1DM, improving animal wel fare . In this early ( two-week evolution) STZ-induced T1DM model we observed :

[0119] 1 ) all the characteristic signs of T1DM (polydipsia , polyuria, polyphagia and body weight loss ) and increased fecal excretion;

[0120] 2 ) increased length, perimeter and weight in the ileum and colon;

[0121] 3 ) increased thickness of several histological intestinal layers ( less evident in CD) of the ileum and colon; and

[0122] 4 ) decreased Ang I l-induced smooth muscle contraction ( less evident in the DC ) associated with altered balance between the function of Ang I I receptors . These reported histomorphometric di f ferences and altered reactivity may help to explain diabetic enteric dysmotility .

[0123] 2 . Type 2 DM: Goto-Kakizaki (GK) rats

[0124] The GK rats are aa animal model of Type 2 DM ( T2DM) , being an established model of non-obese type- I I diabetes that exhibits reduced glucose-stimulated insulin secretion .

[0125] A selective reproduction of non-diabetic Wi star rats with slight glucose intolerance was carried out in order to select non-obese animal model of T2DM . These GK rats present hyperphagia and a lower body weight than Wistar rats, no fat accumulation in adipose tissue and chronic inflammation associated with oxidative stress in adipose tissue. Also, they present glucose intolerance, tissue (skeletal muscle and liver) insulin resistance, reduced p- cell function and number and increased fasting blood glucose levels.

[0126] Results

[0127] Animal Welfare and Monitorization

[0128] Compared to control, GK rats presented elevated fasted glucose concentrations (237.88 ± 81.05mg / dL vs 100 ± 1.73mg / dL) and insulin resistance; lower body weight, although the % of weight gain during the experimental period was roughly the same between the 2 groups (GK rats and control) ; increased water and food intake.

[0129] The parameters documented during the monitorization at 15 min, 30 min, 60 min, 120 min and 6 hours after the insulin injection (blood glucose, body weight and water / food intake) are shown in Figure 9. Insulin resistance and all the above results are consistent with descriptions in the literature and validate GK rats as a non-obese T2D animal model.

[0130] Histomorphometric evaluation

[0131] The structural changes in the intestine of Goto- Kakizaki (GK) rats, a spontaneous experimental model of type 2 DM, were studied. Results of the histomorphometric evaluation of the intestines showed an increase in the total wall thickness of GK rats (p<0.05) compared to controls in the duodenum (1089.02139.19 vs 864.19137.17pm) , ileum (726.29124.75 vs 498.76116.86pm) , cecum (642.24134.15 vs 500.97128.81pm) and distal colon (1211.81+51.32 vs 831.71 + 53.25pm) (Figure 10) .

[0132] The histomorphometric evaluation of GK animals showed an increase in the thickness of the total intestinal wall of the duodenum, ileum, cecum and distal colon compared to controls, while there was no difference between GK and control rats in proximal colon and jejunum.

[0133] The GK rats exhibited mucosa thickening in the duodenum, ileum and distal colon while the muscle layers were increased in all the segments studied, except the duodenum. These data suggests that the development of T2DM in GK rats is associated with intestinal remodeling, that could be related to diabetic dysmotility (data not shown) .

[0134] The muscular layers of the intestinal wall of GK animals increased in all segments compared to controls, except in duodenum. Mucosa was only increased in duodenum, ileum and distal colon (Figure 11, Figure 12) .

[0135] Quantitative analysis of nucleus in the muscular layers

[0136] When performing a quantitative analysis of nucleus in the muscular layers, the number of nuclei of smooth muscle cells was lower in GK in all portions studied compared to controls, except duodenum (Figure 13) . Quantitative analysis of neuronal nucleus in the myenteric plexus

[0137] When performing a quantitative analysis of neuronal nucleus in the myenteric plexus , the number of nuclei per mm2(neuronal density) in the myenteric plexus was lower in GK animals compared to controls in all portions studied ( Figure 14 ) .

[0138] Conclusion

[0139] The typical DM signs of GK rats are hyperglycemia, polydipsia, reduced weight with polyphagia and insulin resistance .

[0140] By analysing the obtained data, it was possible to observe structural di f ferences showing intestinal remodelling, neuronal population reduction within the myenteric plexuses and smooth muscle cells hypertrophy, changes that can be related to diabetic dysmotility .

[0141] These results show that GK rats , a diabetic animal model of T2DM, present similar GT alterations as diabetic rats with T1DM ( STZ rats ) , indicating that this is a transversal problem .

[0142] Experiment 1 - Final Conclusion

[0143] With these two studies - Type 1 DM : STZ induced rats ; and Type 2 DM : Goto-Kaki zaki ( GK) rats , it was demonstrated that structural changes in the intestine associated with diabetes , are evident in both type 1 and type 2 DM . Experiment 2 - Comparison of structural changes in the ileum and colon of early streptozotocin (STZ) -induced diabetic rats treated with Losartan (AT1R blocker) .

[0144] Losartan is an angiotensin II receptor antagonist (ARA) used to treat hypertension and renal diseases, in a dosage well reported in the literature.

[0145] In this experiment, a structural change comparison between the colon of 1-early streptozotocin ( STZ ) -induced diabetic rats (STZ) with 2-STZ rats treated with losartan (ARA) (STZ+LOS) and 3-non-diabet ic Wistar rats (CTRL) was performed .

[0146] In vivo parameters were measured for these three groups, such as glycemia, body weight, water intake and food intake, during the 14 days of the experiment. Figure 15 shows that: all diabetic rats presented hiperglycemia (Figure 15a) , loss of body weight (Figure 15b) and higher water and food intake (Figure 15c, d) , when compared to nondiabetic rats (CTRL) .

[0147] Histomorphometry evaluation of the gut revealed that colonic mucosa (M) and muscular layer (ML, that includes the longitudinal and the circular muscles) in the nontreated diabetic group (STZ, n=4) were significantly thicker compared to control group (CTRL, n=4) (M: CTRL=

[0148] 655.32 ± 45.45pm vs STZ= 786.13 ± 25.80 pm and ML:

[0149] CTRL=217.93 ± 21.45 pm vs STZ=325.72 ± 26.68 pm; p<0.05 for both) . Surprisingly, treatment with Losartan (n=4) prevented the increase in colonic thickness observed in the STZ group (M= 684.34 ± 8.07 pm and ML= 206.63 ± 3.49 pm, p<0.05) , being no different from that of the CTRL group (p>0.05) (Figure 16) . According to the results obtained STZ-rats presented increased thickness of muscle layers and mucosa, that was prevented in diabetic animals treated with Losartan (STZ+LOS) .

[0150] The typical T1DM signs observed in rats are hyperglycemic state, polydipsia, polyphagia and loss of body weight.

[0151] Two weeks after STZ induction, it was possible to observe :

[0152] - Structural differences and increased thickness in both colon and ileum of STZ-rats;

[0153] Muscular layers thickening was prevented by the treatment with Losartan.

[0154] These results show that intestinal remodelling in the ileum and colon of diabetic rats is likely associated with the activation of ATiR.

[0155] Experiment 3 - Assessment of GI alterations in diabetic companion animals

[0156] An assessment was carried out to verify if diabetic companion animals also present GI alterations through: questionnaires to characterize the owner's perception of digestive changes;

[0157] - ultrasound (US) evaluations; and

[0158] - histopathological evaluation.

[0159] In companion animals, DM is one of the most common metabolic diseases, occurring in 1 of every 300 patients (0.21% - 1.24% in cats and 0.34% - 1.2% in dogs) . One of 100 dogs reaching 12 years of age is expected to develop diabetes. Most cases of spontaneous diabetes occur in middle-aged dogs and middle-aged to older cats. In dogs, females are affected twice as often as males, but obese male cats seem to be more commonly affected than females. There are almost no reports of GI changes in diabetic pets.

[0160] According to Kelly Diehl (1995) "although there is considerable information regarding gastrointestinal complications of DM in humans, there is little more than anecdotal information about these problems in dogs and cats". However, DM has been suggested as one of the possible causes of GI dysmotility in critically ill dogs and cats and a thickened small intestine has been associated with intestinal impaction in diabetic dogs. In a review of cases of DM seen at the Colorado State University Veterinary Teaching Hospital (CSU-YTH) about 38% of those dogs and 31% of cats also had gastrointestinal (GI) disease .

[0161] In order to perform the present assessment, 7 domestic spayed diabetic cats (5 males and 2 females) with no history of digestive disease have been studied with the following characteristics:

[0162] • Time since DM diagnosis: 3 days to 5 years

[0163] • Age: 12.85 ± 1.5 years (ranging from 6 to 19 years)

[0164] • Weight: 5.14 ± 0.90 kg (ranging from 2.1 to 9 kg)

[0165] • Glycemia levels: 361 ± 62.11 mg / dL

[0166] All 7 cats exhibited polyuria and polydipsia, 4 of which presented polyphagia and 3 presented weight loss. Diabetic cat owners answered a questionnaires in order to characterize their perception of digestive changes, and the following results have been obtained:

[0167] - Only 1 owner (14%) reported no change in their cat's GI health, and 86% of owners reported at least one alteration;

[0168] - From the group of cats showing GI alterations, 1 cat (17%) had increased defecation frequency; 2 cats (33%) were vomiting at least twice a week and 3 cats (50%) presented changes in elimination behaviours, defecating outside the litter box or meowing while defecating.

[0169] Ultrasound evaluations were conducted on all 7 domestic spayed diabetic cats (5 males and 2 females) with no history of digestive disease. The cats had been diagnosed with DM for a period ranging from 1 month to 5 years. Full-length GI tract was examined, and 3 evaluations were performed per portion (table 4) .

[0170] Table 4 - Ultrasound evaluations (7 cats)

[0171] RV - Reference Value

[0172] All cats exhibited gastric wall thickness, with measurements of rugal fold=5.4010.47mm (reference value, RV=4mm) and inter-rugal=2, 7110.07mm (RV=2mm) ; duodenum=3.1510 .07mm (RV=2.5mm) ; j ejunum=3.1410.15 (RV=2.5mm) . On the other hand, the ileum and colon displayed normal thickness in all cats: 3.0710.11mm (RV=3.2mm) and 2.1310.18mm (RV=2.5mm) , respectively. Ultrasound images from those portions were obtained and are shown in Figure 18.

[0173] Additionally, histopathological evaluation was performed on 2 diabetic cats submitted to necropsy. Inflammatory infiltrate was observed throughout all sections of the GI tract, and collagen deposits were identified (using Masson's trichrome staining) in the muscular layers and between mucosal crypts and muscularis mucosa (Figure 19) . Said Histopathological evaluation shown: inflammatory infiltrate (black square) ; Collagen deposits (black dashed circle) in the muscular layers and between mucosal crypts and muscularis mucosa.

[0174] Experiment 3 - final conclusions

[0175] From the obtained data, it is possible to conclude that :

[0176] - Diabetic cats may experience similar symptoms to those observed in human diabetic patients; Diabetic cats may undergo intestinal remodelling akin to what has been described in humans and laboratory animals ;

[0177] - The gradual decrease in GI thickness in the distal direction aligns with previous observations in diabetic laboratory animals , suggesting that a common mechanism may be involved;

[0178] Diabetic cat ' s gastrointestinal tract presents inflammatory infiltrate and fibrosis .

[0179] General conclusions of experiments

[0180] These findings reveal that diabetic cats undergo intestinal remodelling akin to what has been described in humans and laboratory animals . Plus , the gradual decrease in GI thickness in the distal direction aligns with previous observations in diabetic laboratory animal s , suggesting that a common mechanism may be involved .

[0181] These data show a novel application of ACET or ARAs to prevent and / or treat gastrointestinal changes observed in companion animals af fected by diabetes .

[0182] Therefore , despite these experiments were conducted in companion animals , it is strongly suggested that humans having T1DM or T2DM experiencing GI alterations , wi ll have similar results as obtained by the present experiments , i . e . , treating and / or preventing GI alterations when treated with ARAs and ACEI s .

[0183] From these findings , it is possible to demonstrate that : - the existence of marked gastrointestinal changes in rat animal models of type 1 (STZ model) and type 2 (GK model) Diabetes;

[0184] - Diabetic cats undergo intestinal remodelling similar to what has been described in humans and laboratory animals ;

[0185] - Intestinal alterations in a typel DM rat model are linked to ATi receptors activation by angiotensin II, meaning that blocking these receptors with an ARA (such as losartan) effectively prevents intestinal remodelling.

Claims

CLAIMS1. Angiotensin Converting Enzyme Inhibitors or Angiotensin II Receptor Antagonists for use in the prevention and / or treatment of gastrointestinal alterations associated with diabetes mellitus.

2. Angiotensin Converting Enzyme Inhibitors or Angiotensin II Receptor Antagonists according to claim 1 for use in the preventions and / or treatment of gastrointestinal alterations associated with type-I diabetes mellitus.

3. Angiotensin Converting Enzymes Inhibitors or Angiotensin II Receptor Antagonists according to claim 1 for use in the preventions and / or treatment of gastrointestinal alterations associated with type-II diabetes mellitus.

4. Angiotensin II Receptor Antagonists for use according to any of claims 1-3, wherein the Angiotensin Receptor Antagonists is a type-I or type-II receptor antagonist .

5. Angiotensin II Receptor Antagonists for use according to any of claims 1-3, wherein the Angiotensin Type-I Receptor Antagonists (ARA) is Losartan, but is not limited to it.

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