Application of chenodeoxycholic acid in the preparation of drugs for canine acute pancreatitis

Abstract

This invention belongs to the field of veterinary pharmacy and relates to the application of chenodeoxycholic acid (CDCA) in the preparation of drugs for treating canine acute pancreatitis. This invention is the first to propose the application of CDCA in the preparation of drugs for treating canine acute pancreatitis, clarifying that it achieves therapeutic effects by reducing pancreatic CT values, alleviating pancreatic edema and necrosis, and improving pancreatic tissue pathological damage, providing a novel strategy and drug option for targeted treatment of canine acute pancreatitis.

Description

Technical Field

[0001] This invention belongs to the field of veterinary pharmacy, specifically the new pharmaceutical uses of chenodeoxycholic acid, particularly its application in the preparation of drugs for canine acute pancreatitis. Background Technology

[0002] Canine pancreatitis is a common digestive system disease in clinical practice, often accompanied by acinar cell necrosis, activation of the inflammatory cascade, and microcirculatory disturbances. It can progress to hemorrhagic necrotizing pancreatitis, leading to multiple organ failure, and its incidence is showing an increasing trend year by year. However, its specific pathogenesis and key etiologies have not been fully elucidated.

[0003] Currently, there is no specific treatment for this disease. Clinical treatment mainly relies on non-specific methods such as fluid support, analgesia, and nutritional management. Antibiotics, for example, are only used for cases with concurrent infections and are ineffective against aseptic inflammation; their overuse can easily lead to dysbiosis. Glucocorticoids have controversial anti-inflammatory effects, lack a unified treatment regimen, and carry the risk of immunosuppression. While these methods can temporarily stabilize the condition, they are insufficient to effectively curb the pancreatic digestive process and the ensuing systemic inflammatory response, resulting in a still high mortality rate in severe cases. Therefore, the development of novel drugs that can intervene at the pathological level is of significant clinical urgency.

[0004] Chenodeoxycholic acid (CDCA) is an endogenous primary bile acid synthesized by the liver from cholesterol as a substrate. It participates in fat digestion and absorption and is also an important signaling molecule, exerting anti-inflammatory, anti-necrosis, and metabolic regulatory effects by regulating nuclear receptors (such as FXR) and membrane receptors (such as TGR5). Currently, the clinical application of CDCA is focused on the dissolution of gallstones in humans. There are no patents or clinical application reports on the use of CDCA in the preparation of drugs for treating acute pancreatitis in dogs. Its efficacy, mechanism of action, and dosing regimen in canine pancreatitis models have not been systematically studied, indicating a significant gap in clinical translation. Summary of the Invention

[0005] The purpose of this invention is to overcome the lack of effective treatment drugs for canine acute pancreatitis in the prior art, and to provide the application of chenodeoxycholic acid (CDCA) in the preparation of drugs for canine acute pancreatitis.

[0006] The technical solution provided by this invention is as follows:

[0007] Application of chenodeoxycholic acid in the preparation of drugs for canine acute pancreatitis.

[0008] Furthermore, the dosage of the chenodeoxycholic acid is 3-6 mg / kg daily.

[0009] Furthermore, the administration time of the chenodeoxycholic acid is 2 to 4 days.

[0010] Furthermore, the drug is used to inhibit the abnormal increase in serum amylase and lipase induced by acute pancreatitis in dogs.

[0011] Furthermore, the drug is used to reduce pancreatic tissue enlargement induced by acute pancreatitis in dogs.

[0012] Furthermore, the drug is used to alleviate pancreatic perfusion defects and microcirculatory disturbances induced by acute pancreatitis in dogs.

[0013] Furthermore, the drug is used to reduce the extent of peripancreatic fat space inflammation induced by acute pancreatitis in dogs.

[0014] Furthermore, the drug is used to alleviate pancreatic parenchymal necrosis induced by acute pancreatitis in dogs.

[0015] Furthermore, the drug is used to reduce the high organ index induced by acute pancreatitis in dogs.

[0016] Furthermore, the drug is used to alleviate pancreatic tissue pathological damage induced by acute pancreatitis in dogs.

[0017] Beneficial effects

[0018] This invention, using non-targeted metabolomics and targeted bile acid metabolomics, revealed significant disruptions in the bile acid metabolism profile during the pathogenesis of pancreatitis in dogs, with particularly pronounced changes in the level of the primary bile acid chenodeoxycholic acid (CDCA), suggesting a close association with disease progression. Further research revealed that CDCA, as an endogenous agonist of the farnesoid X receptor (FXR), can inhibit the expression of various pro-inflammatory factors and exert cytoprotective effects by regulating key inflammatory signaling pathways such as NF-κB. These mechanisms are highly consistent with the theoretical framework for alleviating pathological damage in pancreatitis. Furthermore, our previous animal experiments observed that supplementing with exogenous CDCA in a mouse model of pancreatitis significantly reduced pancreatic tissue edema, necrosis, and inflammatory infiltration, and improved multiple biochemical and pathological indicators, preliminarily confirming its potential therapeutic effect.

[0019] This invention is the first to propose the application of CDCA in the preparation of drugs for treating canine acute pancreatitis, clarifying that it achieves therapeutic effects by reducing pancreatic CT values, alleviating pancreatic edema and necrosis, and improving pancreatic tissue pathological damage. This provides a novel strategy and drug option for targeted treatment of canine acute pancreatitis. Furthermore, a systematic evaluation of the therapeutic effect of CDCA was conducted in a canine acute pancreatitis model, aiming to provide clinical practice with a novel treatment strategy based on bile acid metabolism regulation, thereby filling a gap in the current veterinary medicine field in this area. Attached Figure Description

[0020] Figure 1The changes in serum amylase (AMY) and lipase (LPS) activities in each group of experimental dogs after modeling are shown. A represents serum amylase and B represents lipase.

[0021] Figure 2 The images show a comparison of pancreatic tissue from different groups of experimental dogs after modeling. A represents the macroscopic morphological differences of intact pancreatic tissue, B represents the comparison of hematoxylin-eosin (H&E) staining, C represents the comparison of enhanced scan images, D represents the comparison of ultrasound images, E represents the results of organ index analysis, F represents the pathological score, and G represents the CT value of the pancreas. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0023] Example 1

[0024] 1. Laboratory animals:

[0025] This project used a total of 12 Beagles, weighing 7-11 kg, aged 1-1.5 years, of any sex, provided by Lianyungang Rongyang Biotechnology Co., Ltd. They were routinely housed in the animal laboratory of the College of Veterinary Medicine, Yangzhou University. After blood routine and biochemical tests showed no abnormalities, they were used for subsequent experiments. The animal experimental procedures designed in this invention comply with the ethics of laboratory animal welfare and have been approved by the Ethics Committee of Yangzhou University (No. 202506001). After 7 days of acclimatization, the experimental animals were randomly divided into 3 groups (n=4): a healthy control group, an AP group, and a CDCA group.

[0026] 2. Instruments and equipment:

[0027] Routine surgical instruments, CT scanners (SinoVision Insitum CT 768 / 568), ultrasound scanners (Esaote MYLABTMX8VET), fully automated blood biochemistry analyzers (MT 8000), and fully automated blood cell analyzers (BC-760 CS) were all purchased from Mindray. Fully automated dehydrators (ASP 300S), fully automated embedding machines (EG1150H+C), and fully automated staining machines (ST5010) were all purchased from Leica, Germany. Disposable central venous catheters (dual-lumen 7Fr, Jiangsu Huaxing Medical Device Industry Co., Ltd.) were also used.

[0028] 3. Main medicines and reagents:

[0029] Chenodeoxycholic acid (HY-76847, MCE), physiological saline, propofol, isoflurane, hematoxylin stain, eosin stain, 4% paraformaldehyde, absorbable sutures, sodium heparin, blank capsules.

[0030] 4. Preoperative preparation:

[0031] Before the experiment began, all dogs in the AP and CDCA groups had a central jugular vein catheter placed, following the procedure outlined in T_CVMA 196—2024. This catheter was used for subsequent blood collection, intravenous fluid support, and enhanced CT scans.

[0032] 5. Disease model establishment:

[0033] The AP and CDCA groups established canine acute pancreatitis models using the established method of retrograde injection of sodium taurocholate into the accessory pancreatic duct. The procedure was as follows: An incision was made along the midline of the abdomen of the experimental dogs. The pancreas was located at the junction of the pylorus and duodenum, and the accessory pancreatic duct was identified. The lateral wall of the duodenum was incised, and the duodenal papillae were identified within the intestinal lumen. A portion of the soft tubing of a 22-gauge intravenous catheter was retained and connected to a syringe. The tubing was gently inserted into the opening of the accessory pancreatic duct, and a predetermined dose of sodium taurocholate solution (5 mg / kg) was rapidly injected. After injection, the accessory pancreatic duct was kept closed for approximately 5-10 minutes and then ligated with absorbable sutures. Approximately 5 minutes later, congestion and swelling of the pancreatic tissue were observed, indicating successful establishment of the AP model.

[0034] 6. Successful model validation and drug administration initiation:

[0035] Three hours after the modeling surgery was completed in each dog, venous blood was collected for testing. When serum amylase and lipase activities exceeded the normal range and ultrasound interpretation showed imaging characteristics consistent with acute pancreatitis, the model was considered successful, and medication was initiated for the dogs in the CDCA group.

[0036] 7. Drug Packaging Solution:

[0037] The dosage of CDCA is 3-6 mg / kg daily. The specific procedure is as follows: Calculate the total daily CDCA requirement for each dog based on its measured weight. Weigh the total amount precisely using a 0.01% analytical balance and divide the total daily dose equally into two blank capsules. The administration time is determined based on each dog's first administration time (i.e., after successful model validation): at the same time each day thereafter, administer both capsules at once using a dog / cat-specific medication dispenser to ensure accurate dosage. This treatment regimen involves once-daily administration for 3 consecutive days. The AP group and the healthy control group were given the same amount of blank capsules according to the same time logic.

[0038] 8. Supportive care during the trial:

[0039] During the 3-day observation period, all dogs were kept on a fast and abstain from water. To maintain vital signs, correct dehydration, and maintain fluid balance, intravenous fluid resuscitation was administered daily to each dog via a central jugular vein catheter. The total resuscitation volume was calculated based on the dog's daily maintenance fluid requirement (approximately 50-60 mL / kg) and the experimental dehydration status. In practice, each dog received a total of 500 mL of mixed maintenance fluid (0.9% saline and 5% glucose mixed in a 2:1 ratio) daily at an infusion rate of 10-15 mL / kg / h.

[0040] Example 2

[0041] Blood sample collection and serum biochemical marker testing:

[0042] To dynamically assess the process of pancreatic injury and repair, whole blood samples were collected via central jugular vein catheterization before modeling (0h) and at 3, 6, 12, 24, 36, 48, and 72h after modeling. Serum amylase (AMY) and lipase (LPS) activities were detected using a fully automated biochemical analyzer and accompanying reagent kits.

[0043] Dynamic monitoring of serum samples before and at 3, 6, 12, 24, 36, 48, and 72 hours after modeling revealed characteristic changes in the amylase and lipase activities of each experimental dog group.

[0044] Throughout the 72-hour observation period, serum amylase and lipase activities in the control group dogs remained stable without significant fluctuations, and their values ​​remained within the normal physiological range. In the AP group, the activities of both enzymes began to increase significantly 3 hours after modeling, reaching a peak at 12 hours, with peak levels significantly higher than the control group. Subsequently, enzyme activities decreased slowly, but remained above the normal physiological range until 72 hours. Enzyme activities in the CDCA group also increased in the early post-modeling period (3-6 hours), but the increase was significantly less than that in the AP model group at the same time point. Subsequently, their enzyme activities showed a continuous decreasing trend after 12 hours, significantly faster than in the AP model group. By 72 hours, serum amylase and lipase activities in the treatment group dogs had essentially recovered to the upper limit of the normal range for the control group (see...). Figure 1 A, B).

[0045] The above data indicate that CDCA treatment can significantly inhibit the abnormal increase in serum amylase and lipase induced by acute pancreatitis and accelerate their recovery to normal.

[0046] Example 3

[0047] Imaging examinations:

[0048] To objectively verify the therapeutic effect of CDCA morphologically, all experimental dogs underwent enhanced computed tomography (CT) and ultrasound examinations 72 hours after modeling. Enhanced CT was used to quantitatively assess the extent of pancreatic perfusion defects, edema, and the proportion of necrotic areas; ultrasound was used to observe and compare pancreatic size, echogenicity, and peripancreatic fluid accumulation in real time. By systematically comparing the differences in imaging characteristics between the AP group and the CDCA group, the ameliorative effect of CDCA on pancreatic histopathological changes was visually confirmed in a non-invasive manner.

[0049] Seventy-two hours after modeling, the pancreas in the control group was normal in shape and size, with uniform parenchymal density. Contrast-enhanced CT scans showed uniform and significant enhancement, with clear peripancreatic structures and no exudation or fat interstitial infiltration. In the AP group, the pancreas was significantly enlarged, with unevenly decreased parenchymal density; contrast-enhanced CT scans showed uneven, slightly hypo-enhanced enhancement, suggesting perfusion defects and microcirculatory disturbances; blurred, linear high-density shadows were visible in the peripancreatic and retroperitoneal fat spaces. In contrast, the pancreas in the CDCA group was less enlarged, and its contrast-enhanced CT scans showed relatively uniform enhancement without obvious non-enhancing necrosis areas; the blurred shadows in the peripancreatic fat spaces and the extent of inflammatory infiltration were also significantly reduced (see...). Figure 2 C, G).

[0050] In the control group, the pancreas had clear borders, uniform intermediate echogenicity, no pancreatic duct dilation, and normal echogenicity of the surrounding adipose tissue. In the AP group, the pancreas was enlarged, and its borders became blurred due to surrounding inflammation; the parenchymal echogenicity was diffusely reduced and uneven; the peripancreatic adipose tissue presented irregular, patchy hyperechoic areas, sometimes accompanied by posterior acoustic shadowing. In the CDCA group, the pancreatic echogenicity was improved compared to the model group, with reduced unevenness; in the peripancreatic region, the range and intensity of hyperechoic areas caused by fat saponification were significantly reduced and weakened, resulting in higher discernibility of the pancreatic contour (see...). Figure 2 D).

[0051] Comprehensive enhanced CT and ultrasound evaluation showed that CDCA can effectively improve pancreatic microcirculatory disturbances caused by acute pancreatitis, reduce pancreatic parenchymal necrosis and severe inflammatory infiltration of peripancreatic adipose tissue, and confirm its protective effect on pancreatic morphology and structure from an imaging perspective.

[0052] Example 4

[0053] Assessment of gross changes in pancreatic tissue

[0054] To preliminarily assess the ameliorative effect of CDCA on pancreatic tissue lesions from a macroscopic perspective, pancreatic tissue was harvested from each group of experimental dogs under deep anesthesia 72 hours after modeling. During harvesting, intact pancreatic tissue was placed on a background board with a standard ruler for photographic fixation to record its macroscopic morphology. Through direct observation, differences between the two groups of pancreas were compared in terms of size, color, texture, degree of edema, capsule tension, and the presence or absence of visible necrotic foci or hemorrhages.

[0055] The control group's pancreas was regular in shape, with a clear outline, smooth surface, and a uniform pale pink color; it was firm to the touch, and the lobular structure of the pancreas was clearly visible, with no edema, hemorrhage, or necrotic foci observed. The AP group's pancreas showed significant diffuse enlargement, irregular outline, and an uneven surface. The tissue color changed to dark red or purplish-brown, indicating widespread congestion and diffuse hemorrhage. Scattered or confluent bright red to dark red hemorrhage foci were visible on the surface and cut surface. Some areas showed grayish-yellow, hard, and brittle patchy necrotic areas, intertwined with residual degenerated tissue, easily crumbling to the touch. The lobular structure of the pancreas was destroyed and blurred due to severe edema, inflammatory exudation, and necrosis dissolution. The CDCA group's pancreas showed a clear trend of improvement. Although the pancreas was enlarged, its degree was significantly less than that of the model group. The dark red and purplish-brown color of the tissue was significantly lighter, and the number and extent of visible hemorrhage foci were greatly reduced. In terms of texture, the tissue was harder than the control group, but its toughness and density were somewhat restored compared to the model group. The basic outline of the pancreas and most of its lobular structures are discernible (see...) Figure 2 A). Further organ index analysis showed that the organ index in the CDCA group was higher than that in the control group but significantly lower than that in the AP group, objectively reflecting the reduction in edema (see...). Figure 2 E).

[0056] Example 5

[0057] Pathological examination:

[0058] To further objectively evaluate the protective effect of CDCA on pancreatic tissue at the microscopic level, the pancreatic tissue samples obtained above were immediately fixed in 4% paraformaldehyde neutral buffer, and then dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) according to standard procedures.

[0059] The stained slides were examined under an optical microscope by pathologists unaware of the grouping information. An assessment was conducted using a histopathological scoring system adapted to the Schmidt criteria and breed-specific characteristics, with semi-quantitative scoring of indicators such as acinar necrosis, edema, inflammation, and hemorrhage.

[0060] Pathological and histological observation results:

[0061] The control group showed normal pancreatic tissue morphology and structure, with clear lobular structures. Acinar cells were neatly arranged, morphologically intact, with abundant cytoplasm and centrally located nuclei. There was no interstitial edema, only a very small number of scattered lymphocytes, and no neutrophil infiltration, necrosis, or hemorrhage foci. Pathological sections of the AP group showed significant interstitial edema with widened interlobular spaces, diffuse infiltration of neutrophils and lymphocytes, and extensive coagulative necrosis of acinar cells (characterized by cell structure disintegration, nuclear pyknosis, or lysis), along with scattered hemorrhage foci. Compared with the AP group, the CDCA group showed significantly improved pancreatic tissue pathological damage. Interstitial edema was significantly reduced; inflammatory cell infiltration was significantly reduced, mostly focal rather than diffuse; the area of ​​acinar cell necrosis was greatly reduced, with most areas preserving acinar structure, and only localized reversible damage such as cell vacuolation was observed; hemorrhage foci were extremely rare (see [link to article]). Figure 2 B). The lesions were assessed using a recognized pathological scoring system (such as the Schmidt criteria). Semi-quantitative scoring showed that the total histopathological score of the CDCA group was significantly lower than that of the acute pancreatitis model group (see [link to relevant data]). Figure 2 F).

Claims

1. Application of chenodeoxycholic acid in the preparation of drugs for canine acute pancreatitis.

2. The application according to claim 1, characterized in that, The dosage of the chenodeoxycholic acid is 3-6 mg / kg per day.

3. The application according to claim 1, characterized in that, The administration period for the chenodeoxycholic acid is 2-4 days.

4. The application according to claim 1, characterized in that, The drug is used to inhibit the abnormal increase in serum amylase and lipase induced by acute pancreatitis in dogs.

5. The application according to claim 1, characterized in that, The drug is used to reduce pancreatic tissue swelling induced by acute pancreatitis in dogs.

6. The application according to claim 1, characterized in that, The drug is used to alleviate pancreatic perfusion defects and microcirculatory disturbances induced by acute pancreatitis in dogs.

7. The application according to claim 1, characterized in that, The drug is used to reduce the extent of peripancreatic fat space inflammation induced by acute pancreatitis in dogs.

8. The application according to claim 1, characterized in that, The drug is used to alleviate pancreatic parenchymal necrosis induced by acute pancreatitis in dogs.

9. The application according to claim 1, characterized in that, The drug is used to reduce high organ index induced by acute pancreatitis in dogs.

10. The application according to claim 1, characterized in that, The drug is used to reduce pancreatic tissue pathological damage induced by acute pancreatitis in dogs.

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