Angiotensin-converting enzyme inhibitors or angiotensin ii receptor antagonists for use in preventing and / or treating gastrointestinal alterations associated with diabetes

Abstract

The present invention relates to the use of angiotensin converting enzyme inhibitors (ACEI) and angiotensin II receptor antagonists (ARA) for the prevention and / or treatment of gastrointestinal complications associated with diabetes.

Description

Technical Field

[0001] This invention relates to the use of angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II receptor antagonists (ARA) for the prevention and / or treatment of gastrointestinal changes associated with diabetes. Background Technology

[0002] Diabetes mellitus (DM) is a complex, chronic, progressive metabolic disorder that is medically incurable and can affect virtually every organ system, with a staggering prevalence across all age groups worldwide. Different animal models of DM exist, but streptozotocin (STZ) has been the agent of choice for the chemical induction of diabetes in rats and mice, causing selective destruction of pancreatic β-cells. High doses of STZ are associated with type 1 DM (T1DM) induction, while repeated low doses are generally associated with a high-fat diet leading to insulin resistance (a hallmark of type 2 DM (T2DM)). In this T1DM animal model, the structural, functional, and biochemical alterations are similar to those observed in human patients with diabetes. Over time, several researchers have used this model in different parts of the intestine at varying induction times (raising questions about animal welfare for longer-term protocols), making comparisons of outcomes more difficult.

[0003] Gastrointestinal (GI) complications of diabetes mellitus (DM) are very common and important, as they can be associated with a significant morbidity, affecting up to 75% of patients. Despite this high prevalence, knowledge and treatment options for these complications are quite limited.

[0004] DM-related complications in the intestines and colon can lead to symptoms such as chronic constipation and diarrhea (occasionally associated with fecal incontinence), and are associated with worse outcomes in clinical conditions such as colorectal cancer and inflammatory bowel disease. In addition, possible complications may occur, such as megacolon, pseudo-obstruction, fecal ulcers, or perforation.

[0005] The most common complications of GI include esophageal motility disorders, gastroparesis, enteropathy, and colonic disorders such as chronic constipation and diarrhea. Because these symptoms are not considered a significant cause of death in patients with DM, they are often overlooked. However, it is important to recognize their negative impact on health and quality of life.

[0006] The pathogenesis of diabetic bowel dysfunction appears to be multifactorial, involving the accumulation of advanced glycation end products (AGEs), damage to the enteric nervous system (ENS) or Cajal interstitial cells, and muscle layer fibrosis. Several known studies have also shown that diabetic autonomic neuropathy leads to damage to the ENS and alters the number and size of myenteric neurons throughout the GI tract in rats (He, C.-L., et al., Gastroenterology 2001; Furlan, MMD, et al., Arq. de Neuro-Psiquiatria 2002; Honoré, SM, et al., Auton. Neurosci. 2011; Fregonesi, CEPT, et al., Arq. Neuro-Psiquiatria 2001; Brasileiro, AD, et al., Brain Res. 2018; Zaoni, J., et al., Arq. Neuropsiquiatr. 1998). It has also been described as a defect in enterocholinergic neurotransmission, as the response to exogenous acetylcholine (ACh) appears to be impaired in the ileum (30 days after STZ-induction) and colon of long-term diabetic rats (60 weeks) (Lucas, PD, et al., Gastroenterology, 1991; Kim, SJ, et al., J. Neurogastroenterol. Motil. 2011). Mechanical factors may also contribute to intestinal disturbances, as DM appears to induce structural remodeling that may affect histomorphometry and biomechanical properties, increasing stiffness and reducing the resting compliance and relaxation capacity of the intestinal wall.

[0007] The renin-angiotensin system (RAS) is primarily known for its role in the cardiovascular and renal systems (regulating blood pressure and fluid homeostasis), but it also has influences in other systems, such as the GI tract, which expresses all RAS components. However, the RAS is associated with diabetes mellitus (DM) because angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin type 1 receptor (AT1) antagonists (ARAs) have been shown to reduce the incidence of vascular complications, kidney disease, and cardiovascular disease in these patients.

[0008] Angiotensin II (Ang II) is the main effector peptide of this system, and much of its function is mediated by Ang II type 1 receptors (AT1R), while activation of Ang II type 2 receptors (AT2R) usually counteracts them. In the colon, Ang II causes contraction of circular and longitudinal smooth muscle that responds to direct activation of postsynaptic AT1R and indirect activation of presynaptic AT1R in myenteric and submucosal neurons. Unusually, human colonic smooth muscle is more sensitive to Ang II than to acetylcholine (ACh), but the physiological importance of Ang II in the GI tract remains not fully understood. Interestingly, little information is available about changes in the intestinal RAS in individuals with diabetes, but a recent study concluded that ACE gene polymorphisms in patients with T2DM affect intestinal motility because those patients exhibit a prevalent genotype associated with constipation (Malik et al.). Biochem. Genet. (2020).

[0009] Furthermore, elevated tecidal ACE levels have been found in patients with diabetes mellitus (DM), leading to the additional formation of Ang II. Additionally, in patients with type 2 DM (T2DM), RAS suppression improves insulin sensitivity, allowing for better glycemic control. In fact, Ang II infusion appears to induce insulin resistance. However, to date, there is no information regarding the relationship between RAS and diabetic glycemic changes.

[0010] Treatment options for GI complications associated with DM are limited, with existing treatments primarily focusing on symptom management. Treatment considerations depend on the severity of symptoms, the patient's ability to maintain adequate nutrition, and their responsiveness to therapy.

[0011] For gastroparesis, prokinetic agents are typically used to increase gastric motility and enhance gastric emptying. Commonly used medications in treatment are shown in Table 1.

[0012] Table 1. Medications used in the treatment of diabetic gastroparesis (Krishnan et al., World J. Diabetes (2013)

[0013]

[0014] Endoscopic and surgical treatments are also available. For example, endoscopic pyloric injection of botulinum toxin has been tried in the management of gastroparesis and has shown improved symptoms and accelerated gastric emptying, lasting up to 3-6 months. Surgical treatment is rarely performed and is mainly used for patients with refractory gastroparesis symptoms who have not responded to other measures. Total gastrectomy has been shown to provide long-term symptom relief in some patients with postoperative gastroparesis, but data on patients with diabetic gastroparesis are limited.

[0015] Treatment of diabetic diarrhea mainly involves symptom relief, correction of fluid and electrolyte deficiencies, improvement of nutrition and glycemic control, and management of underlying causes.

[0016] In severe cases of constipation, lactulose and osmotic laxatives may be required. Newer medications for treating chronic constipation include prucalopride (a selective 5-HT4 receptor agonist that enhances colonic transport) and rubiprostone (which stimulates colonic water and electrolyte secretion by activating type 2 chloride channels in intestinal epithelial cells).

[0017] Insulin-like growth factor 1 (IGF-1) treatment can inhibit DM-induced colonic smooth muscle cell apoptosis and may participate in the relief of colonic motility disorders in DM rats. α-glucosidase inhibitors such as acarbose have been shown to increase intestinal Bifidobacterium longum (…). Bifidobacterium longum It reduces the levels of GLP-1 receptor agonists and partially restores the imbalance of the gut microbiota in diabetic patients (which also appears to be important in diabetic motility disorders). In contrast, GLP-1 receptor agonists such as liraglutide appear to modulate the composition of the gut microbiota.

[0018] The literature "Wagatsuma et al." Clinical usefulness of the angiotensin II receptor antagonist losartan in patients with portal hypertensive gastropathy The journal Hepatogastroenterology, 2006, published a study involving the administration of the angiotensin II receptor antagonist losartan (developed as an antihypertensive agent) to patients with portal hypertensive gastropathy in order to examine its clinical usefulness.

[0019] The literature "Krishnan et al." Gastrointestinal complications of diabetes mellitus The World Journal of Diabetes, 2013, published the latest advances in the study of gastrointestinal complications of diabetes, their pathophysiology, diagnostic evaluation, and management.

[0020] The reference “Dedeli, O., et al.; Prevalence of Gastrointestinal Symptoms and Its Effect on Quality of Life among Patients with Diabetes Mellitus ; Am. J. Nurs. Res. The 2015 study published a study that identified the prevalence of gastrointestinal symptoms and their impact on quality of life in patients with diabetes.

[0021] The literature "Werner, C., et al." RAS blockade with ARB and ACE inhibitors: current perspective on rationale and patient selection The study “Clin Res Cardiol 97, 418–431 (2008)” published a study on the effects of ACE inhibitors and ARBs on the number and function of endothelial progenitor cells, revealing additional mechanisms of action of these drugs.

[0022] The literature "Pushpakom S, et al." Drug repurposing: progress, challenges and recommendations The Nat Publ Gr. 2018 publication disclosed methods for drug repurposing (also known as drug repositioning), discussed the challenges facing the repurposing community, and recommended innovative ways to address these challenges to help realize the full potential of drug repurposing.

[0023] Currently, there are no therapies available for the prevention and / or treatment of gastrointestinal changes associated with DM, either in humans or other animals. Invention Overview

[0025] This invention relates to the use of altered angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor antagonists (ARA) to prevent and / or treat gastrointestinal changes associated with diabetes. Attached Figure Description

[0026] The following description is made with reference to the accompanying drawings, which are presented for illustrative purposes only and have no limiting features, and wherein:

[0027] Figure 1 – Evaluation of the following during the experimental protocol (day 14) in control (CTRL, n = 8) and streptozotocin-induced diabetic rats (STZ, n = 16–21): (a) body weight; (b) water intake; (c) food intake; and (d) fecal excretion. Values ​​are mean ± SEM, and unpaired Student's t-tests were used to compare the two experimental groups (CTRL and STZ). Statistically different, p < 0.05.

[0028] Figure 2 – Macroscopic evaluation of the ileum and colon in control (CTRL, black bars, n = 8–12) and streptozotocin-induced diabetic rats (STZ, white bars, n = 11–14): (a) representative images of colon length (CTRL, above scale bar); (b) quantitative analysis of colon length (left y-axis) and colon length / rat body weight (right y-axis); (c) tissue circumference of colon and ileum; and (d) relative weight of intestinal segments (without fecal contents), expressed as grams of colon or ileum / grams of body weight. Values ​​are mean ± SEM, and unpaired Student's t-tests were used to compare the two experimental groups (CTRL and STZ). Statistically different, p < 0.05.

[0029] Figure 3– Representative microscopic images of intestinal segments from 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); mid-colon (e, f); and distal colon (g, h). Scale bar (100 µm) is applied to all images.

[0030] Figure 4 – Morphometric evaluation of intestinal segments (ileum, proximal colon, mid-colon, and distal colon) in control (CTRL, n = 4) and streptozotocin-induced diabetic (STZ, n = 8) rats: (a) total wall thickness (μm) for each intestinal segment; (b) thickness of the intestinal layers (longitudinal muscle, circular muscle, submucosa, and mucosa) for each intestinal segment (μm). Values ​​are mean ± SEM, and two-way ANOVA was used, followed by an unpaired t-test with Welch's correction to compare the two experimental groups (CTRL and STZ). The statistical difference compared with the corresponding control was p < 0.05.

[0031] Figure 5 – In the control (CTRL, n = 6) and streptozotocin-induced diabetic rats (STZ, n = Contractile response to KCl (125 mM) in the ileum, proximal colon, mid-colon, and distal colon of 10). Data are expressed as mN / g fresh tissue (mN / g). Values ​​represent the median (95% confidence limit), and the Mann-Whitney test was used to compare the two experimental groups (CTRL and STZ).

[0032] Figure 6 – Concentration-response curves for ACh in the ileum, proximal colon, mid-colon, and distal colon of control (CTRL, n = 6–7) and streptozotocin-induced diabetic rats (STZ, n = 10). Data are expressed as mN / g fresh tissue (mN / g). Values ​​are mean ± SEM.

[0033] Figure 7 – In the control (CTRL, n = 5–8) and streptozotocin-induced diabetic rats (STZ, n = 5) Concentration-response curves of angiotensin II in the ileum, proximal colon, mid-colon, and distal colon. Data are expressed as mN / g fresh tissue (mN / g). Values ​​are mean ± SEM.

[0034] Figure 8 – In the control (CTRL, n = 5–8) and streptozotocin-induced diabetic rats (STZ, n = The vasoconstrictive effects of angiotensin II in the ileum, proximal colon (PC), mid colon (MC), and distal colon (DC) of (4–6) in the presence of the following antagonists (expressed as percentage change): (a) candesartan (AT1R antagonist, 10 nM) and (b) PD123,319 (AT2R antagonist, 100 nM). Values ​​are mean ± SEM. For statistical analysis, paired t-tests were used between effects in the absence and presence of antagonists. Compared to the response to angiotensin II in the absence of an antagonist, p < 0.05.

[0035] Figure 9 – The following were evaluated in control (CTRL) and GK rats (GK) during the experimental protocol (6 hours): (a) blood glucose; (b) body weight; and (c) food intake and water intake. Values ​​are mean ± SEM, and unpaired Student's t-tests were used to compare the two experimental groups (CTRL and STZ). Statistically different, p < 0.05.

[0036] Figure 10 – Morphometric evaluation of intestinal segments (duodenum, jejunum, ileum, cecum, proximal colon, and distal colon) in control (CTRL) and GK rats (GK) rats: total wall thickness (μm) for each segment. Values ​​are mean ± SEM, and two-way ANOVA was used, followed by an unpaired t-test with Welch correction to compare the two experimental groups (CTRL and STZ). The statistical difference compared with the corresponding control was p < 0.05.

[0037] Figure 11 – Representative microscopic images of intestinal segments from control (CTRL, a, c, e, g, i, k) and GK rats (GK, b, d, f, h, j, l), 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). Scale bar (100 µm) is applied to all images.

[0038] Figure 12– Morphometric evaluation of intestinal segments (distal colon, proximal colon, cecum, ileum, jejunum, and duodenum) in control (CTRL) and GK rats (GK): (a, b, c) Thickness (μm) of the intestinal layers (longitudinal muscle, circular muscle, submucosa, and mucosa) in each segment; (d, e, f) Thickness (μm) of the intestinal layers (longitudinal muscle, circular muscle, submucosa, mucosa, crypts, and villi) in each segment. Values ​​are mean ± SEM, and two-way ANOVA was used, followed by an unpaired t-test with Welch correction to compare the two experimental groups (CTRL and STZ). The statistical difference compared with the corresponding control was p < 0.05.

[0039] Figure 13 – Quantitative analysis of nuclei in the muscle layer of intestinal segments (duodenum, jejunum, ileum, cecum, proximal colon, and distal colon) of control (CTRL) and GK rats (GK): (a) Muscle SMC density (nuclei / 100µm) in each intestinal segment 2 (b) Representative microscopic photographs of intestinal segments from control rats (CTRL) and GK rats (GK), stained with hematoxylin and eosin: duodenum. Scale bar (100 µm) applies to all images.

[0040] Figure 14 – Quantitative analysis of neuronal nuclei in the myenteric plexus (duodenum, jejunum, ileum, cecum, proximal colon, and distal colon) of control (CTRL) and GK rats (GK): (a) Density of myenteric neurons in the intestinal layer of each intestinal segment (nuclei / mm²) 2 (b) Representative microscopic photographs of intestinal segments from control rats (CTRL) and GK rats (GK), stained with hematoxylin and eosin: ileum. Scale bar (100 µm) applies to all images.

[0041] Figure 15 – The following were evaluated during the experimental protocol (14 days) in control (CTRL), streptozotocin-induced diabetic rats (STZ), and streptozotocin-induced diabetic rats treated with losartan (STZ+LOS): (a) blood glucose; (b) body weight; (c) water intake; and (d) food intake. Values ​​are mean ± SEM, and unpaired Student's t-tests were used to compare the three experimental groups (CTRL, STZ, and STZ+LOS). Statistically different, p < 0.05.

[0042] Figure 16– Morphometric evaluation of intestinal segments (colon) in control (CTRL), streptozotocin-induced diabetic rats (STZ), and streptozotocin-induced diabetic rats treated with losartan (STZ+LOS): (a) Thickness (μm) of the intestinal layers (longitudinal muscle, circular muscle, submucosa, and mucosa) of each segment. Values ​​are mean ± SEM and were compared between the two experimental groups (CTRL, STZ, and STZ+LOS) using two-way ANOVA followed by an unpaired t-test with Welch correction. (a) Statistical difference compared with the corresponding control (p < 0.05); (b) Representative microscopic images of intestinal segments from control rats (CTRL), streptozotocin-induced diabetic rats (STZ), and streptozotocin-induced diabetic rats treated with losartan (STZ+LOS), stained with hematoxylin and eosin: colon. Scale bar (500 µm) applied to all images.

[0043] Figure 17 – Morphometry of intestinal segments (ileum) in control (CTRL), streptozotocin-induced diabetic rats (STZ), and streptozotocin-induced diabetic rats treated with losartan (STZ+LOS): (a) Thickness (μm) of the intestinal layers (longitudinal muscle, circular muscle, submucosa, and mucosa) of each segment. Values ​​are mean ± SEM and were compared between the two experimental groups (CTRL, STZ, and STZ+LOS) using two-way ANOVA followed by an unpaired t-test with Welch correction. (a) Statistical difference compared with the corresponding control (p < 0.05); (b) Representative microscopic images of intestinal segments from control rats (CTRL), streptozotocin-induced diabetic rats (STZ), and streptozotocin-induced diabetic rats treated with losartan (STZ+LOS), stained with hematoxylin and eosin: ileum. Scale bar (500 µm) applied to all images.

[0044] Figure 18 – Ultrasound evaluation of intestinal segments in diabetic pets: (a) inter-rugal region of the stomach; (b) rugal region of the stomach; (c) duodenum; (d) jejunum; (e) ileum; and (f) colon.

[0045] Figure 19 – Representative microscopic images of intestinal segments (stomach, jejunum, ileum, and colon) in diabetic pets, showing: collagen deposition in the muscular layer and between the mucosal crypts and the muscularis mucosae (Masson's trichrome stain) (black dashed circles in the stomach and jejunum); and inflammatory infiltration (black squares in the ileum and colon, stained with hematoxylin and eosin). Scale bar (500 µm) is applied to all images. Invention Details

[0047] This invention relates to the use of ARAs or ACEIs for the prevention and / or treatment of diabetes-induced GI changes, and discloses the use of these drugs for the prevention of significant GI changes occurring in diabetes mellitus (DM). These drugs have been approved for the control of significant conditions such as hypertension, heart failure, and chronic kidney disease, but they have never been considered for the prevention and / or treatment of diabetic GI changes.

[0048] Therefore, new research data were collected on diabetic rats with the following objectives:

[0049] 1) Enhance the evidence supporting the preventive effect of losartan (ARA) on GI changes occurring in type 1 diabetes mellitus (T1DM);

[0050] 2) To determine whether losartan (ARA) can prevent and / or treat established GI alterations in diabetic rats; and

[0051] 3) Determine whether diabetic rats with type 2 diabetes (T2DM) exhibit the same GI changes as diabetic rats with T1DM.

[0052] This disclosure relates to changing the use of these medications with the aim of reducing the incidence of gastrointestinal changes associated with diabetes in patients. An advantage is that these medications are already commercially available for the treatment of other conditions.

[0053] In the context of this invention, the following commercially available drugs are used:

[0054] – Candesartan - 10 nM, AT1R antagonist (in vitro)

[0055] - PD123.319 (di(trifluoroacetic acid) salt hydrate, Sigma-Aldrich, USA) – 100 nM, AT2R antagonist (in vitro);

[0056] – Losartan – 20 mg / kg, PO, AT1R antagonist (in vivo).

[0057] Experiment 1 - Evaluation of the histomorphometric effects of diabetes in the intestine and colon

[0058] The histomorphometric effects of diabetes in the intestine and colon were evaluated as follows:

[0059] - Type 1 DM: streptozotocin (STZ)-induced rats; and

[0060] - Type 2 DM: Goto–Kakizaki (GK) rat.

[0061] 1. Type 1 DM: STZ-induced rats

[0062] STZ-induced rats are an animal model of type 1 diabetes mellitus (T1DM) induced by streptozotocin (STZ) to chemically induce diabetes in rats and mice, resulting in selective destruction of pancreatic β-cells.

[0063] result

[0064] Animal welfare and monitoring

[0065] STZ-induced rats had an initial blood glucose level of 99.30 ± 3.29 mg / dL, which increased to 395.09 ± 13.80 mg / dL within 48 hours (p < 0.0001, n = 23), while control rats had an initial blood glucose level of 105.63 ± 6.31 mg / dL, which was roughly the same within 48 hours (111.14 ± 5.41 mg / dL; p > 0.05, n = 8). On days 7 and 14, almost all STZ-induced rats had blood glucose levels above 500 mg / dL, while control animals showed a blood glucose level of 105.57 ± 4.76 mg / dL on day 14 (n = 8).

[0066] The parameters recorded during daily monitoring (body weight, water / food intake, and fecal excretion) are shown in the figure. Figure 1 In the control group (n = 8), rats gradually increased their body weight, reaching a 7.8% ± 0.73% increase by day 14 compared to day 0 (before fasting). Diabetic rats (n = 21) exhibited a sustained decrease in body weight, more pronounced on day 2 (a 5% decrease compared to the previous day), and maintained this weight for the remainder of the protocol (a 7.66% ± 1.04% decrease by day 14 compared to their initial body weight before fasting). Figure 1 a) Water intake was significantly higher in diabetic rats compared to the control group (n = 8) which maintained a constant water intake of 37.54 ± 0.53 mL / day. The STZ group consumed more water from day 1 (48.38 ± 1.16 mL), but their water intake gradually increased throughout the protocol, reaching seven times the water intake of the control animals on day 14: 264.08 ± 12.18 mL (n = 16). Figure 1b). Despite weight loss, food intake in STZ rats was significantly higher than in the control group after day 3. Diabetic rats started the experimental protocol with a food intake of 13.25 ± 1.86 g on day 1, and food consumption was gradually increased until the last day, at which point intake was 49.08 ± 2.64 g / rat (n = 16). The control group maintained a constant food intake throughout the experimental period, with an average consumption of 22.44 ± 0.38 g / day (n = 8). Figure 1 c).

[0067] The applicant's study allowed for the quantification of fecal excretion in STZ-induced diabetic animals. Non-diabetic animals maintained relatively stable fecal excretion throughout the experimental period (7.75 ± 0.18 g / day / rat, n = 8), while diabetic rats gradually increased their fecal excretion, reaching a four-fold increase from the first day (Day 1: 7.11 ± 0.34 g / rat; Day 14: 30.79 ± 0.73 g / rat; p < 0.0001, n = 16). Figure 1 d).

[0068] Macroscopic evaluation of ileum and colon

[0069] Compared to control animals, all segments of the intestine in STZ-induced rats appeared enlarged. Furthermore, upon opening the abdomen of STZ-induced rats, an extremely dilated cecum exhibiting a "mass effect" was readily apparent, pushing the intestine to one side. The colon length was significantly greater in diabetic animals compared to the control group. Figure 2 a, b: 25.75 ± 0.77 cm, n = 14 vs. 19.63 ± 0.47 cm, n = 12, p < 0.05). Since some animals were heavier than others, colon length / body weight was measured, and the difference between the two groups was maintained. Figure 2 b). The circumference of the intestinal segments was also measured, and in STZ-induced rats (n = 11), it was significantly higher in both the colon (15.45 ± 0.58 mm vs. 11 ± 0.46 mm, p < 0.0001) and ileum (12.55 ± 0.31 mm vs. 9.38 ± 0.32 mm, p < 0.0001) compared to non-diabetic rats (n = 8). Figure 2c). The relative weight of the entire intestinal segment studied in STZ-induced animals (with fecal contents) was higher than that in the control (2.69 ± 0.10 g / g body weight, n = 21 vs. 1.80 ± 0.05 g / g body weight, n = 12; p < 0.0001). This increase was also observed in individual intestinal segments without fecal contents. Figure 2 d). Furthermore, no difference was found between STZ-induced animals and controls in the wet-to-dry ratio of all segments studied (specifically, 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; for all, p > 0.05). Two-way ANOVA results showed an interaction between the experimental group (control or STZ) and the intestinal segment (p < 0.0001), consistent with our visual observation of significant midgut dilatation in STZ-induced animals. The relative fecal contents weight in STZ-induced animals was also higher than in the control (7.10 ± 0.15 g / g body weight, n = 21 vs. 2.66 ± 0.11 g / g body weight, n = 12; p < 0.0001).

[0070] Microscopic evaluation of ileum and colon

[0071] STZ-induced animals ( n = The results of the histomorphometric evaluation of the intestine (8) were consistent with the macroscopic data, showing a difference compared to the control ( n = 4) Compared to the ileum, the proximal colon (PC), mid-colon (MC), and distal colon (DC) show increased intestinal wall thickness, such as in... Figure 3 and 4 Observable values ​​in a (specifically, ileum: 671.64 ± 74.34 µm vs. 404.97 ± 82.04 µm; PC: 666.66 ± 32.340 µm vs. 389.24 ± 39.03 µm; MC: 589.03 ± 17.88 µm vs. 376.06 ± 50.62 µm; DC: 570.93 ± 27.16 µm vs. 430.42 ± 26.26 µm; for all, p < 0.01). The increase in intestinal wall thickness was similar for all intestinal segments, as two-way ANOVA showed no significant association between the experimental group and the intestinal segment. p = 0.1681). In STZ-induced rats, compared with controls, the ileum (respectively, longitudinal muscle: 81.02 ± 7.66 µm vs. 31.18 ± 5.44 µm, circular muscle: 116.12 ± 4.59 µm vs. 44.47 ± 10.40 µm, submucosa: 41.68 ± 1.68 µm vs. 17.47 ± 2.13 µm, mucosa: 432.82 ± 20.59 µm vs. 311.85 ± 24.51 µm; for all, p < 0.01) and mid-colon (respectively, longitudinal muscle: 48.93 ± 2.93 µm vs. 29.66 ± 4.25 µm, circular muscle: 142.55 ± 8.37 µm vs. 74.31 ± 10.9 µm, submucosa: 56.39 ± 4.09 µm vs. 35.63 ± 6.47 µm, mucosa: 341.17 ± 13.79 µm vs. 236.46 ± 34.58 µm; for all, p < Both showed an increase in the thickness of all assessed intestinal layers (0.05). Figure 4 b). In PC, the submucosa was the only layer to show a similar thickness between STZ-induced animals and controls (50.47 ± 7.33 µm vs. 33.81 ± 6.00 µm, respectively). p = The layer was 0.1104 µm thicker than the control in diabetic animals, while all other segments were thicker (specifically, longitudinal muscle: 57.02 ± 6.90 µm vs. 34.64 ± 4.29 µm, circular muscle: 205.2 ± 17.00 µm vs. 90.14 ± 11.33 µm, mucosa: 353.97 ± 14.27 µm vs. 230.64 ± 26.18 µm; for all, p < 0.05). DC only showed an increase in muscle thickness (longitudinal muscle: 52.51 ± 2.72 µm vs. 28.51 ± 1.67 µm, circular muscle: 150.54 ± 14.58 µm vs. 87.21 ± 7.06 µm, for both, p < 0.01; Submucosal layer: 66.11 ± 7.70 µm compared to 53.27 ± 7.54 µm and mucosa: 301.77 ± 10.00 µm compared to 261.42 ± 16.49 µm, for both, p > 0.05)( Figure 4 b). Two-way ANOVA showed that the experimental group (control compared to STZ) and the ileum ( p = 0.0058), PC ( p = 0.0002), MC ( p = The correlation between intestinal layer thickness (longitudinal muscle, circular muscle, submucosa, and mucosa) was 0.0027, but for DC ( p = 0.1109) No correlation.

[0072] Evaluation of ileal and colonic function

[0073] To assess whether intestinal muscle contraction is altered in diabetic animals, the effects of exogenously administered KCl ( Figure 5 ), Ach ( Figure 6 ) and Ang II ( Figure 7 The ileal and colonic reactivity of ACh and Ang II. For concentration-response curves of ACh and Ang II, the results were expressed using two recognized pharmacological concepts: maximal contractile effect (Econcentration). max (expressed in mN / g) and the agonist concentration that can cause 50% of the maximum contraction (EC). 50 (expressed in μM). In both control and STZ-induced animals, the contractile response to 125 mM KCl (and ACh concentration-dependent contraction) was similar in all intestinal segments (ileum, PC, MC, and DC). Figure 6 ), of which a considerable amount of E max and EC 50 The values ​​are presented in Table 2.

[0074] Table 2. In the control group (CTRL, n = 6–7) and streptozotocin-induced diabetic rats (STZ, n = 10) E-type smooth muscle contractions induced by ACh application in the ileum, proximal colon, mid-colon, and distal colon. max (mN / g) and EC 50 (μM) value.

[0075]

[0076] To compare the two experimental groups (CTRL and STZ), we used the Mann-Whitney test. The value is the median (95% confidence limit).

[0077] Regarding responsiveness to Ang II, this RAS effector peptide caused concentration-dependent contraction in both control and diabetic animals. Figure 7 In the ileum, PC, and MC of STZ-induced animals, the contractile response to Ang II normalized to tissue weight was lower (but with the same EC). 50 Interestingly, the maximum response in DCs was similar between control and STZ-induced animals, but the EC in this part of the diabetic colon was significantly higher. 50 Significantly lower than control EC 50 (Table 3)

[0078] Table 3. In the control group (CTRL, n = 5–8) and streptozotocin-induced diabetic rats (STZ, n = 5) E (electro-induced smooth muscle contraction of the ileum, proximal colon, mid-colon, and distal colon by angiotensin II administration) max (mN / g) and EC 50 (μM) value.

[0079]

[0080] To compare the two experimental groups (CTRL and STZ), we used the Mann-Whitney test. The value is the median (95% confidence limit). Compared with the corresponding control p < 0.05.

[0081] Given that the observed differences in contractile responses to Ang II could be due to an imbalance between AT1R and AT2R mediated effects, the applicant decided to further characterize the response to Ang II. In controls (for all, in mN / g, 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 ± 1.32 vs. 0.16 ± 0.08; for all, p < 0.05) and STZ-induced rats (ileum: 35.75 ± 11.06 vs -0.87 ± 2.78; PC: 24.80 ± 9.45 vs 0.78 ± 1.19; MC: 95.86 ± 29.03 vs 5.20 ± 6.39; DC: 288.48 ± 49.08 vs 5.57 ± 5.54; for all, p < 0.05) In all four intestinal segments of both organisms, the contractile response to Ang II was antagonized by candesartan (10 nM) (an AT1R antagonist). Figure 8 a). Unlike other studies, PD123319 (an AT2R antagonist, 100 nM) reduced the response to Ang II in the ileum of control animals (12.43 ± 1.03 mN / g compared to 11.02 ± 1.21 mN / g). p < 0.05) and increased response in all colonic segments (for all, in mN / g, PC: 19.95 ± 3.34 compared to 22.02 ± 3.45; MC: 14.99 ± 1.97 compared to 17.48 ± 2.44; DC: 19.88 ± 2.82 compared to 23.50 mN / g ± 2.64; for all, p < 0.05), but it did not alter 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 (236.37 ± 19.03 mN / g vs. 248.38 ± 25.64 mN / g) in diabetic rats; for all, p > AngII-induced contraction in 0.05) decreased in PC (166.14 ± 20.49 vs. 108.45 ± 19.00); p < 0.05)( Figure 8 b).

[0082] in conclusion

[0083] The results obtained indicate that early STZ-induced DM is associated with increased thickness of the rat colon and ileum, decreased contractile response to Ang II, and different responses to AT2R inhibition.

[0084] The results presented in this study demonstrate that classic animal models of T1DM can be improved, thereby enhancing animal welfare. In this early (two-week evolution) STZ-induced T1DM model, we observed:

[0085] 1) All characteristic signs of T1DM (polydipsia, polyuria, polyphagia, and weight loss) and increased fecal excretion;

[0086] 2) Increased length, circumference, and weight in the ileum and colon;

[0087] 3) Increased thickness of several histological intestinal layers in the ileum and colon (less pronounced in CD); and

[0088] 4) Ang II-induced reduction in smooth muscle contraction (less pronounced in DCs) is related to a balance between changes in Ang II receptor function. These reported histomorphometric differences and altered responsiveness may help explain diabetic bowel motility disorders.

[0089] 2. Type 2 DM: Goto–Kakizaki (GK) rats

[0090] GK rats are an animal model of type 2 diabetes mellitus (T2DM), an established model of non-obese type 2 diabetes, exhibiting glucose-stimulated insulin secretion.

[0091] Nondiabetic patients with mild glucose intolerance Wistar Selective breeding of rats to select non-obese animal models of type 2 diabetes mellitus (T2DM). Wistar Compared to rats, these GK rats exhibited excessive appetite and lower body weight, no fat accumulation in adipose tissue, and chronic inflammation associated with oxidative stress in adipose tissue. Furthermore, they also showed glucose intolerance, tissue (skeletal muscle and liver) insulin resistance, decreased β-cell function and number, and elevated fasting blood glucose levels.

[0092] result

[0093] Animal welfare and monitoring

[0094] Compared with the control, GK rats showed elevated fasting glucose concentrations (237.88 ± 81.05 mg / dL vs. 100 ± 1.73 mg / dL) and insulin resistance; lower body weight, although the percentage of body weight gain was roughly the same between the two groups (GK rats and control) during the experimental period; and increased water and food intake.

[0095] During the monitoring period, parameters (blood glucose, body weight, and water / food intake) recorded at 15 min, 30 min, 60 min, 120 min, and 6 hours after insulin injection are shown in the figure. Figure 9 The results showed that insulin resistance and all of the above findings were consistent with descriptions in the literature and validated that GK rats are a non-obese T2D animal model.

[0096] Morphometric Evaluation

[0097] The structural changes in the intestine of Goto–Kakizaki (GK) rats (a spontaneous experimental model of type 2 diabetes mellitus) were investigated.

[0098] Histomorphometry of the intestines showed that the total wall thickness of GK rats was increased compared with the control in the duodenum (1089.02±39.19 vs. 864.19±37.17 μm), ileum (726.29±24.75 vs. 498.76±16.86 μm), cecum (642.24±34.15 vs. 500.97±28.81 μm), and distal colon (1211.81±51.32 vs. 831.71±53.25 μm) (p<0.05). Figure 10 ).

[0099] Histomorphometry of GK animals showed an increase in total intestinal wall thickness in the duodenum, ileum, cecum, and distal colon compared to the control, while there was no difference between GK and control rats in the proximal colon and jejunum.

[0100] GK rats exhibited mucosal thickening in the duodenum, ileum, and distal colon, with increased muscle layer in all segments studied except the duodenum. These data suggest that the development of T2DM in GK rats is associated with intestinal remodeling, which may be related to diabetic motility disorders (data not shown).

[0101] Compared to the control, the muscular layer of the intestinal wall in GK animals was increased in all segments except the duodenum. Mucosal increase was only observed in the duodenum, ileum, and distal colon. Figure 11 , Figure 12 ).

[0102] Quantitative analysis of nuclei in muscle layer

[0103] When quantitative analysis of nuclei in the muscle layer was performed, the number of smooth muscle cell nuclei was lower in all parts studied in GK, except for the duodenum, compared to the control. Figure 13 ).

[0104] Quantitative analysis of neuronal nuclei in the myenteric plexus

[0105] When performing quantitative analysis of neuronal nuclei in the myenteric plexus, compared with the control, the number of nuclei / mm in the myenteric plexus in all parts studied in GK animals was [data missing]. 2 (Neuron density) is lower ( Figure 14 ).

[0106] in conclusion

[0107] Typical signs of diabetes mellitus (DM) in GK rats include hyperglycemia, polydipsia, weight loss accompanied by polyphagia and insulin resistance.

[0108] Analysis of the obtained data revealed structural differences, showing changes such as intestinal remodeling, a reduction in neuronal populations within the myenteric plexus, and smooth muscle cell hypertrophy, which may be associated with diabetic motility disorders.

[0109] These results indicate that GK rats (a diabetic animal model of T2DM) exhibit similar GI changes to diabetic rats with T1DM (STZ rats), suggesting that this is a transverse problem.

[0110] Experiment 1 – Final Conclusion

[0111] In the cases of these two studies—type 1 DM: STZ-induced rats; and type 2 DM: Goto–Kakizaki (GK) rats—structural changes in the gut associated with diabetes have been demonstrated to be evident in both type 1 and type 2 DM.

[0112] Experiment 2 – Comparison of structural changes in the ileum and colon of early streptozotocin (STZ)-induced diabetic rats treated with losartan (AT1R blocker).

[0113] Losartan is an angiotensin II receptor antagonist (ARA) used to treat hypertension and kidney disease, and its dosage is well reported in the literature.

[0114] In this experiment, structural changes in the colon were compared between 1-early streptozotocin (STZ)-induced diabetic rats (STZ), 2-STZ rats treated with losartan (ARA) (STZ+LOS), and 3-non-diabetic Wistar rats (CTRL).

[0115] During the 14-day experiment, in vivo parameters such as blood glucose, weight, water intake, and food intake were measured in these three groups. Figure 15 This indicates that all diabetic rats exhibited hyperglycemia compared to non-diabetic rats (CTRL). Figure 15 a), weight loss ( Figure 15 b) and higher water and food intake ( Figure 15 c, d).

[0116] Histomorphometry of the intestines revealed that the colonic mucosa (M) and muscular layer (ML, which includes longitudinal and circular muscles) in the untreated diabetes group (STZ, n=4) were significantly thicker than those in the control group (CTRL, n=4) (M: CTRL = 655.32 ± 45.45 μm vs. STZ = 786.13 ± 25.80 μm and ML: CTRL = 217.93 ± 21.45 μm vs. STZ = 325.72 ± 26.68 μm; for both, p < 0.05). Surprisingly, treatment with losartan (n=4) prevented the increase in colonic thickness observed in the STZ group (M = 684.34 ± 8.07 μm and ML = 206.63 ± 3.49 μm, p < 0.05), with no difference compared to the CTRL group (p > 0.05). Figure 16 ).

[0117] Based on the results obtained, STZ rats exhibited increased thickness of the muscle layer and mucosa, which was prevented in diabetic animals treated with losartan (STZ+LOS).

[0118] Typical signs of T1DM observed in rats include hyperglycemia, polydipsia, polyphagia, and weight loss.

[0119] Two weeks after STZ induction, the following can be observed:

[0120] - Increased structural differences and thickness between the colon and ileum in STZ rats;

[0121] - Treat with losartan to prevent muscle layer thickening.

[0122] These results suggest that intestinal remodeling in the ileum and colon of diabetic rats may be associated with AT1R activation.

[0123] Experiment 3 – Assessment of GI alterations in diabetic companion animals

[0124] The following criteria were used to assess whether diabetic companion animals also exhibited GI changes:

[0125] - Questionnaires were used to characterize the owner's perception of changes in digestion;

[0126] - Ultrasound (US) evaluation; and

[0127] - Histopathological evaluation.

[0128] In companion animals, diabetes mellitus (DM) is one of the most common metabolic diseases, occurring in 1 in 300 patients (0.21%–1.24% in cats and 0.34%–1.2% in dogs). It is estimated that 1 in 100 dogs reaching the age of 12 years will develop diabetes. Most cases of spontaneous diabetes occur in middle-aged dogs and older cats. In dogs, females are affected twice as frequently as males, but obese male cats appear to be more commonly affected than females. There are almost no reports of changes in the glycemic index (GI) in diabetic pets.

[0129] 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 motility disorders in critically ill dogs and cats, and thickened small intestine is associated with intestinal impaction in diabetic dogs. In a review of DM cases seen at Colorado State University Veterinary Teaching Hospital (CSU-YTH), approximately 38% of those dogs and 31% of those cats also had gastrointestinal (GI) disorders.

[0130] For this assessment, seven domestically kept, spayed diabetic cats (five males and two females) with no history of digestive system disease were studied, and they had the following characteristics:

[0131] Time from DM diagnosis: 3 days to 5 years

[0132] Age: 12.85 ± 1.5 years (range from 6 to 19 years)

[0133] Weight: 5.14 ± 0.90 kg (range from 2.1 to 9 kg)

[0134] Blood glucose level: 361 ± 62.11 mg / dL

[0135] All seven cats exhibited polyuria and polydipsia, with four showing polyphagia and three showing weight loss.

[0136] Owners of diabetic cats answered questionnaires to characterize their perceptions of digestive changes, and the following results have been obtained:

[0137] - Only 1 owner (14%) reported no change in their cat's GI health, while 86% of owners reported at least one change;

[0138] - In the group of cats that showed changes in GI, 1 cat (17%) had increased defecation frequency; 2 cats (33%) vomited at least twice a week; and 3 cats (50%) showed changes in excretion behavior, defecating outside the litter box or meowing while defecating.

[0139] Ultrasound evaluation was performed on seven domestically kept, spayed diabetic cats (five males and two females) with no history of digestive system disease. These cats had been diagnosed with DM for periods ranging from 1 month to 5 years. The entire GI tract was examined, and each section was evaluated three times (Table 4).

[0140] Table 4 – Ultrasound Evaluation (7 cats)

[0141]

[0142] RV – Reference Value

[0143] All cats exhibited the following gastric wall thickness measurements: folds = 5.40 ± 0.47 mm (reference, RV = 4 mm) and interfolds = 2.71 ± 0.07 mm (RV = 2 mm); duodenum = 3.15 ± 0.07 mm (RV = 2.5 mm); jejunum = 3.14 ± 0.15 mm (RV = 2.5 mm). On the other hand, the ileum and colon showed normal thickness in all cats: 3.07 ± 0.11 mm (RV = 3.2 mm) and 2.13 ± 0.18 mm (RV = 2.5 mm), respectively. Ultrasound images from those portions were obtained and are shown in... Figure 18 middle.

[0144] In addition, histopathological evaluation was performed on two diabetic cats that underwent necropsy. Inflammatory infiltration was observed in all sections penetrating the GI tract, and collagen deposits were identified in the muscle layer and between the mucosal crypts and the mucomuscularis mucosae (using Masson's trichrome staining). Figure 19 The histopathological evaluation showed: inflammatory infiltration (black squares); collagen deposition in the muscle layer and between the mucosal crypts and the muscularis mucosae (black dashed circles).

[0145] Experiment 3 – Final Conclusion

[0146] Based on the obtained data, the following conclusions can be drawn:

[0147] - Diabetic cats may experience symptoms similar to those observed in human diabetic patients;

[0148] - Diabetic cats may experience intestinal remodeling similar to that described in humans and laboratory animals;

[0149] - The gradual decrease in GI thickness in the distal direction is consistent with previous observations in diabetic laboratory animals, suggesting that a common mechanism may be involved;

[0150] - The gastrointestinal tract of diabetic cats exhibits inflammatory infiltration and fibrosis.

[0151] Overall conclusions of the experiment

[0152] These findings reveal that diabetic cats undergo intestinal remodeling similar to that described in humans and laboratory animals. Furthermore, the gradual decrease in GI thickness in the distal direction is consistent with previous observations in diabetic laboratory animals, suggesting a possible shared mechanism.

[0153] These data demonstrate a novel application of ACEIs or ARAs in the prevention and / or treatment of gastrointestinal changes observed in companion animals affected by diabetes.

[0154] Therefore, although these experiments were conducted in companion animals, they strongly suggest that people with T1DM or T2DM who experience GI changes will have similar results as obtained through this experiment, namely, treatment and / or prevention of GI changes when treated with ARA and ACEI.

[0155] These findings prove that:

[0156] - Significant gastrointestinal changes were observed in rat models of type 1 (STZ model) and type 2 (GK model) diabetes.

[0157] - Diabetic cats experience intestinal remodeling similar to that described in humans and laboratory animals;

[0158] Intestinal changes in the type 1 DM rat model are associated with AT1 receptor activation induced by angiotensin II, meaning that blocking these receptors with ARA (such as losartan) effectively prevents intestinal remodeling.

Claims

1. Angiotensin-converting enzyme inhibitors or angiotensin II receptor antagonists, which are used for the prevention and / or treatment of gastrointestinal changes associated with diabetes.

2. The angiotensin-converting enzyme inhibitor or angiotensin II receptor antagonist according to claim 1, for use in the prevention and / or treatment of gastrointestinal changes associated with type 1 diabetes.

3. The angiotensin-converting enzyme inhibitor or angiotensin II receptor antagonist according to claim 1, for use in the prevention and / or treatment of gastrointestinal changes associated with type II diabetes.

4. The angiotensin II receptor antagonist for the said use according to any one of claims 1-3, wherein, The angiotensin receptor antagonist is a type I or type II receptor antagonist.

5. The angiotensin II receptor antagonist for the said use according to any one of claims 1-3, wherein, The angiotensin type I receptor antagonist (ARA) mentioned is losartan, but is not limited to it.

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