Application of flavin monooxygenase 3 inhibitor in preparation of medicine for preventing and / or treating acute pancreatitis

Abstract

The invention provides application of a flavin monooxygenase 3 inhibitor in preparation of a medicine for preventing and / or treating acute pancreatitis, and belongs to the technical field of medicines. The flavin monooxygenase 3 inhibitor is methimazole or a pharmaceutically acceptable salt, a prodrug or a derivative of methimazole. Experimental results show that after the methimazole is orally taken, the nidus area of animal pancreatitis is obviously reduced, the pathological score is obviously improved, the in-vivo inflammation level is obviously inhibited, and the blood amylase and lipase levels are obviously reduced; the levels of pancreatitis factors and chemotactic factors are reduced, and the oxidative stress index is reduced. In addition, by combining methimazole with the traditional Chinese medicine monomer for inhibiting the trimethylamine oxide level, the curative effect is better. Compared with the prior art, the application has the advantages that the core regulation effect of the trimethylamine oxide in the pathological process of the acute pancreatitis is disclosed for the first time, the limitation of the traditional anti-inflammatory or enzyme replacement therapy is broken through, and the acute pancreatitis is treated by inhibiting the trimethylamine oxide level by inhibiting the flavin monooxygenase 3.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to the application of a flavin monooxygenase 3 inhibitor or a combination of a flavin monooxygenase 3 inhibitor and a natural product monomer component for reducing the level of trimethylamine-N-oxide in the prevention and / or treatment of acute pancreatitis. BACKGROUND

[0002] Acute pancreatitis (AP) is an acute inflammatory state caused by abnormal activation of digestive enzymes in the pancreas. It is characterized by pancreatic tissue autodigestion, inflammation, hemorrhage and necrosis, and can progress to severe acute pancreatitis (SAP), marked by systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). Although the mortality rate has decreased over the past decade, AP is still a fatal disease with a mortality rate of 10% to as high as 30%. Common causes include cholelithiasis, alcohol abuse, binge eating, drug reactions and hyperlipidemia. Current clinical management mainly includes symptomatic treatment such as fasting, intravenous fluid replacement, anti-infection, anti-inflammatory and analgesia. However, supportive treatment, although alleviating symptoms to some extent, cannot fundamentally prevent the inflammatory storm in the progression of pancreatitis, especially the poor inhibition of proinflammatory factors, resulting in a high mortality rate of severe pancreatitis. Therefore, it is urgent to explore therapeutic drugs that can suppress the severity of pancreatitis. SUMMARY

[0003] In view of this, the present application aims to provide a new drug and composition for preventing and / or treating acute pancreatitis.

[0004] To solve the technical problems of the present application, the present application provides the following technical solutions:

[0005] The present application proposes the pathogenicity of bacterial metabolite trimethylamine oxide in pancreatitis, and inhibition of flavin monooxygenase activity can block the synthesis of trimethylamine oxide and reverse pancreatic tissue inflammation and oxidative stress.

[0006] The present application provides a flavin monooxygenase 3 inhibitor for use in the preparation of a drug for preventing and / or treating pancreatitis.

[0007] In the above technical solution, further, the flavin monooxygenase 3 inhibitor is methimazole as shown in structural formula (I) or a pharmaceutically acceptable salt, prodrug or derivative thereof,

[0008]

[0009] In the technical solution, further, the pharmaceutically acceptable salt of methimazole includes but is not limited to hydrochloride, sulfate, hydrobromide, hydroiodide, formate, acetate or oxalate; the prodrug or derivative of methimazole includes but is not limited to carbimazole or methimazole thio-beta-D-glucoside.

[0010] In the technical solution, further, the inhibitor reduces the serum concentration of oxidized trimethylamine by inhibiting the activity of liver flavin monooxygenase 3, thereby relieving the inflammatory response of pancreatic tissue and reducing the level of oxidative stress.

[0011] The application also provides a use of a composition containing a flavin monooxygenase 3 inhibitor in the preparation of a medicament for preventing and / or treating pancreatitis, wherein the composition comprises the flavin monooxygenase 3 inhibitor (methimazole or a pharmaceutically acceptable salt, prodrug or derivative thereof) and at least one natural product monomer component.

[0012] In the technical solution, further, the natural product monomer component is rhein. Preferably, the mass ratio of the flavin monooxygenase 3 inhibitor (methimazole or a pharmaceutically acceptable salt, prodrug or derivative thereof) to rhein is 1:20 to 5:20. More preferably, the mass ratio of the flavin monooxygenase 3 inhibitor to rhein is 3:20.

[0013] In the technical solution, further, the natural product monomer component includes but is not limited to other natural product components that can significantly reduce the level of oxidized trimethylamine, such as emodin, aloe emodin, berberine, baicalin, etc., or pharmaceutically acceptable salts or derivatives thereof, etc.

[0014] In the technical solution, further, the medicament or composition further comprises a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

[0015] In the technical solution, further, the acute pancreatitis is an acute inflammatory lesion of the pancreatic tissue region caused by cholelithiasis, alcohol, blood vessels, trauma, infection, etc., which is manifested by abnormal elevation of serum amylase and / or lipase concentration and persistent upper abdominal pain, and the abdominal imaging examination result shows that it is consistent with the imaging changes of pancreatitis. Preferably, the acute pancreatitis is moderate to severe acute pancreatitis.

[0016] In the technical solution, further, the moderate to severe acute pancreatitis, in addition to the typical characteristics of pancreatitis, also has peripheral tissue exudation, necrosis or systemic inflammatory response syndrome.

[0017] The inventors found through experimental exploration that the level of trimethylamine-N-oxide is significantly increased in acute pancreatitis patients and acute pancreatitis rats. Trimethylamine-N-oxide aggravates the pancreatic injury, the levels of serum amylase and lipase, the levels of inflammatory factors IL-lbeta, IL-6 and active oxygen in pancreatitis rats. It is proposed that trimethylamine-N-oxide is a key proinflammatory factor for the severity of pancreatitis.

[0018] Further, the inventors propose through mechanism research and experimental verification that inhibition of flavin monooxygenase can prevent and / or treat the severity of acute pancreatitis. Flavin monooxygenase 3 (FMO3) is a key metabolic enzyme expressed in the liver, and its main function is to catalyze the oxidation of trimethylamine (TMA) to trimethylamine-N-oxide (TMAO). Methimazole is a competitive substrate of flavin monooxygenase 3 and can inhibit the formation of trimethylamine-N-oxide. The oral flavin monooxygenase inhibitor provided in the present application can significantly inhibit the severity of acute pancreatitis by reducing the level of trimethylamine-N-oxide; when combined with natural medicines that reduce the level of trimethylamine-N-oxide, it can further reduce the levels of serum amylase, lipase, inflammatory factors TNF-alpha, IL-6, and the expression levels of chemokines CCL2 and CCL3 in pancreatitis model mice (inhibition rate is more than 95%), and the level of pancreatic inflammation returns to normal, achieving a significantly better treatment effect than expected.

[0019] The present application first proposes that trimethylamine-N-oxide significantly increases the lesion area of acute pancreatitis, significantly increases the pathological score, and significantly increases the levels of serum amylase, lipase, inflammatory factors and oxidative stress, which is an important mechanism for the severity of acute pancreatitis. Inhibition of flavin monooxygenase can prevent and / or treat the severity of acute pancreatitis. Flavin monooxygenase 3 competitive inhibitors and compositions containing the inhibitors can significantly reduce the lesion area of acute pancreatitis, significantly improve the pathological score, significantly reduce the levels of serum amylase, lipase, inflammatory factors, chemokines and oxidative stress, and suppress the severity of acute pancreatitis. Since the mechanism of acute pancreatitis severity has not been elucidated, the existing clinical treatment options have very limited effect. Therefore, the present technical solution is proposed. The oral flavin monooxygenase inhibitor provided can significantly inhibit the severity of acute pancreatitis by reducing the level of trimethylamine-N-oxide; in addition, when combined with natural medicines that reduce the level of trimethylamine-N-oxide, it can further reduce the levels of serum amylase, lipase, inflammatory factors TNF-alpha, IL-6, and the expression levels of chemokines CCL2 and CCL3 in pancreatitis model mice, and the inhibition of proinflammatory factors is more than 95%, which is much better than expected and better than existing Chinese and Western treatment options. The above results show that the flavin monooxygenase inhibitor and its composition have outstanding technical effects in the treatment / prevention of acute pancreatitis, especially moderate to severe acute pancreatitis.

[0020] Experimental results showed that serum trimethylamine oxide levels were significantly higher in patients with acute pancreatitis than in healthy individuals; serum trimethylamine oxide levels also increased significantly during the progression of acute pancreatitis; trimethylamine oxide is an important risk factor for inducing an increase in the area of ​​acute pancreatitis lesions, aggravated pathological scores, and elevated levels of inflammation in vivo. Oral administration of methimazole (a competitive substrate of flavin monooxygenase 3) significantly reduced the area of ​​pancreatitis lesions in animals, significantly improved pathological scores, significantly suppressed in vivo inflammation levels, and significantly decreased serum amylase and lipase levels; pancreatic inflammatory factors, chemokine levels, and oxidative stress indicators were also reduced. Furthermore, the combined use of methimazole with a traditional Chinese medicine monomer that inhibits trimethylamine oxide levels showed even better efficacy. Compared with previous technical solutions, its advantage lies in revealing for the first time the core regulatory role of trimethylamine oxide in the pathological process of acute pancreatitis, breaking through the limitations of traditional anti-inflammatory or enzyme replacement therapy. By inhibiting the level of trimethylamine oxide through the inhibition of flavin monooxygenase 3, including the use of methimazole alone and the combination of methimazole with specific Chinese herbal monomers such as rhein, the anti-inflammatory effect exceeds 95% at the optimized efficacy dose. The efficacy effect exceeded expectations at the animal level, achieving etiological treatment. Attached Figure Description

[0021] Figure 1 The results of serum trimethylamine oxide assay in patients with acute pancreatitis;

[0022] Figure 2 The results of trimethylamine oxide determination in rats with acute pancreatitis after treatment with trimethylamine oxide;

[0023] Figure 3 HE pathological section of pancreas in rats with acute pancreatitis after treatment with trimethylamine oxide;

[0024] Figure 4 The statistical results of pancreatic tissue pathological scores in rats with acute pancreatitis after treatment with trimethylamine oxide.

[0025] Figure 5 Serum amylase levels were measured in rats with an acute pancreatitis model after treatment with trimethylamine oxide.

[0026] Figure 6 Serum lipase levels were measured in rats with an acute pancreatitis model after treatment with trimethylamine oxide.

[0027] Figure 7 The results of serum inflammatory factor IL-lbeta measurement in rats with acute pancreatitis after treatment with trimethylamine oxide;

[0028] Figure 8 The results of serum IL-6 measurement in rats with acute pancreatitis after treatment with trimethylamine oxide;

[0029] Figure 9Serum ROS determination results for acute pancreatitis model rats after treatment with trimethylamine-N-oxide;

[0030] Figure 10 Pancreatic FMO3 mRNA levels for acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and inhibitor-containing composition;

[0031] Figure 11 Serum TMAO levels for acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and inhibitor-containing composition.

[0032] Figure 12 Pancreatic HE pathological section results for acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and inhibitor-containing composition;

[0033] Figure 13 Pancreatic tissue pathological score statistical results for acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and inhibitor-containing composition;

[0034] Figure 14 Serum amylase determination results for acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and inhibitor-containing composition;

[0035] Figure 15 Serum lipase determination results for acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and inhibitor-containing composition;

[0036] Figure 16 Pancreatic TNF-α mRNA levels for acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and inhibitor-containing composition;

[0037] Figure 17 Pancreatic IL-1β mRNA levels for acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and inhibitor-containing composition;

[0038] Figure 18 Pancreatic CCL2 mRNA levels for acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and inhibitor-containing composition;

[0039] Figure 19 Pancreatic CCL3 mRNA levels for acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and inhibitor-containing composition;

[0040] Figure 20 Pancreatic malondialdehyde levels for acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and inhibitor-containing composition. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present application.

[0042] Example 1. Detection of trimethylamine-N-oxide levels in patients with acute pancreatitis

[0043] 1. Participant recruitment and grouping

[0044] Twenty-four patients with acute pancreatitis and twenty-four healthy volunteers without chronic metabolic, gastrointestinal, and tumor diseases were recruited from the Department of General Surgery and the Physical Examination Center of the First Affiliated Hospital of Dalian Medical University. Serum samples were collected after admission (3000 rpm centrifugation for 10 min). The relative levels of trimethylamine-N-oxide were obtained by non-targeted metabolomics analysis of serum samples from all participants. The relevant experiments were approved by the Ethics Committee of the First Affiliated Hospital of Dalian Medical University, and the informed consent of the subjects was obtained (YJ-KS-KY-2019-93).

[0045] 2. Analysis method

[0046] Non-targeted metabolomics was used to analyze blood samples on an Ultimate 3000 ultra-high performance liquid chromatography coupled with Q-Exactive module quadrupole orbitrap mass spectrometer (Thermo Scientific, USA). An Acquity IM HSSC18 column (Waters Co., USA, 2.1 x 100 mm) was used, with a sample injection volume of 5 μL, and the extracts were separated by reverse phase chromatography in positive ion mode. The mobile phase was A (0.1% formic acid / water) and B (0.1% formic acid / acetonitrile), and the separation gradient was increased from 2% B to 98% B within 10 min. The chromatographic column was Acquity TMBEH C18 (Waters Co., USA, 1.7 μm, 2.1 x 100 mm) was used in negative mode. Mobile phase A was water and mobile phase B was water / acetonitrile (5:95, v / v, both containing ammonium bicarbonate (0.4 g / L)). The initial 2% B was ramped to 100% in 10 min. The next 5 min was used for washing and equilibrating the column. The flow rate was 0.4 mL / min, and the column temperature was 50 °C. In addition, the sheath gas flow was set to 45 arb and the auxiliary gas flow was set to 10 arb. The capillary temperature was 320 °C, and the S-Lens RF level was 55%. Metabolic peak alignment and extraction were performed using Compound Discoverer software (Thermo Scientific, USA). Structure annotation was performed according to the instructions of MSI 11, using our in-house database (iPhenome TM SMOL high-resolution MS / MS spectral library), NIST tandem MS / MS library (National Institute of Standards and Technology), mzCloud library (Thermo Scientific, USA), human metabolome database (HMDB), and Kyoto Encyclopedia of Genes and Genomes (KEGG). The peak area of the annotated metabolites was extracted using TraceFinder software (Thermo Scientific, San Jose, USA). The mass accuracy for metabolite identification or structure annotation was controlled within ±5 ppm, and the chemical formula was confirmed by the introduction of at least one isotope within 10 ppm of the isotope information and a fitting score of 70% based on the relative isotope abundance plot of the accurate mass. In addition, the structure annotation of the metabolites was strictly confirmed using the retention time information and high-resolution MS / MS spectral similarity.

[0047] Statistical data were analyzed using GraphPad Prism 6.0 software (GraphPad; CA, USA) and expressed as mean ± SEM. The difference between two groups was compared using T-test. P < 0.05, P < 0.01, or P < 0.001, the difference was statistically significant.

[0048] 3. Results

[0049] As Figure 1 As shown in Table 1, the serum level of trimethylamine-N-oxide in the pancreatitis model patients was significantly higher than that in the normal control group, suggesting that the level of trimethylamine-N-oxide in acute pancreatitis patients was significantly increased (P < 0.05), suggesting that trimethylamine-N-oxide may play a certain role in acute pancreatitis.

[0050] Table 1 Quantitative results of serum trimethylamine-N-oxide in acute pancreatitis model patients (relative value, n = 24)

[0051]

[0052]

[0053] Example 2. Proinflammatory effects of trimethylamine oxide in a sodium taurocholate-induced acute pancreatitis model in rats

[0054] Pancreatic HE pathological sections, serum amylase, serum lipase, serum inflammatory factors IL-6, IL-1β, ROS levels are important indicators for evaluating the severity of acute pancreatitis and the level of oxidative stress in vivo.

[0055] 1. Experimental animals, instruments and reagents

[0056] SD rats (8 weeks old) were purchased from Beijing Huafukang Biotechnology Co., Ltd. The animals were raised in a SPF level environment (21 ± 2℃, 12 hour light cycle), and were free to eat and drink during the experiment. Trimethylamine oxide was purchased from TCI Shanghai Chemical Industry Development Co., Ltd. 4% tissue cell fixative, hematoxylin-eosin staining solution and other pathological reagents were purchased from Beijing Solaybao Biotechnology Co., Ltd. Acetonitrile, methanol and mass spectrometry grade formic acid were purchased from Thermo Fisher Scientific. Ultra-pure water was taken from a Millipore purification system, and other chemical reagents were purchased from National Pharmaceutical Chemical Reagent Co., Ltd.

[0057] 2. Analysis method

[0058] Pancreatic tissue hematoxylin-eosin (HE) pathological staining: after the pancreatic head was fixed in 4% tissue cell fixative (4% paraformaldehyde) for 24 hours, the automatic dehydration machine was dehydrated for 16 hours, and then the paraffin embedding machine was routinely embedded. After cutting, the sections were flat-mounted on glass slides, dried, stained with hematoxylin-eosin, dehydrated and transparentized, and then mounted for microscopic examination. Olympus BX53 upright microscope was used for photographing and recording.

[0059] Acute pancreatitis pathological scoring criteria:

[0060] (1) Pancreatitis histopathological lesion score

[0061] Mild edema and mild inflammatory cell infiltration of pancreatic tissue, 1 point; pancreatic tissue with obvious necrosis and inflammatory cell infiltration, 2 points; pancreatic tissue with severe necrosis and inflammatory cell infiltration, 3 points.

[0062] (2) Pancreatic cell infiltration score

[0063] Normal glandular tissue, 0 points; mild inflammatory cell infiltration, only a small amount of inflammatory cell infiltration, 1 point; inflammatory cells, obvious inflammatory cell infiltration, 2 points; severe inflammatory cell infiltration, severe inflammatory cell infiltration, 3 points; very severe inflammatory cell infiltration, very severe inflammatory cell infiltration, 4 points.

[0064] (III) Pancreatic tissue necrosis score

[0065] No necrosis of pancreatic tissue, the structure of the tissue is complete, 0 points; mild necrosis of pancreatic tissue, only a small amount of pancreatic tissue necrosis, 1 point; moderate necrosis of pancreatic tissue, pancreatic tissue necrosis is more obvious; severe necrosis of pancreatic tissue, pancreatic tissue necrosis is more serious, 3 points; very severe necrosis of pancreatic tissue, very severe necrosis of pancreatic tissue, 4 points.

[0066] The pathological score of pancreatitis is based on the cumulative score of the above three indexes to evaluate the degree of tissue lesion, the degree of inflammatory cell infiltration and the degree of pancreatic tissue necrosis. The higher the score, the more severe the lesion of pancreatitis and the more serious the inflammatory reaction.

[0067] The determination of pancreatic inflammatory factors interleukin-6 (IL-6) and interleukin-1 beta (IL-1β) was determined by ELISA kit, which was purchased from Thermo Fisher Scientific Company. The specific method is described in the instruction manual.

[0068] Serum alpha-amylase (starch-iodine colorimetry), lipase (microplate method), and reactive oxygen species (ROS) were determined by test kits, which were purchased from Nanjing Jiancheng Biological Technology Co., Ltd. The specific method is described in the instruction manual.

[0069] High performance liquid chromatography-triple quadrupole tandem mass spectrometer (AB Sciex TM , 4500MD) was used for quantitative analysis of tissue samples. The separation of compounds used ACE Excel PFP C18 column (1.7 μm, 2.1 mm x 100 mm, Avantor, England). The temperature of the chromatographic column was maintained at 40℃, and the temperature of the automatic sampler was 4℃. The mobile phase was 0.01% formic acid-water solution (mobile phase A) and acetonitrile (mobile phase B), and the flow rate was 0.3 mL / min. The gradient elution program was set as: 0.01 min, 20% B; 1.50 min, 24% B; 3.00 min, 40% B; 7.00 min, 55% B; 7.5 min, 70% B; 8.00 min, 98% B; 9.50 min, 98% B, 10 min, 20% B. After detection equilibrium, 1.5 minutes. Multiple reaction monitoring was used for quantification, m / z 76.1→58.1 for monitoring of trimethylamine oxide; m / z 85.1→68.1 for monitoring of internal standard.

[0070] Statistical data were analyzed using GraphPad Prism 6.0 software (GraphPad; CA, USA) and expressed as mean ± SEM. Differences between groups were compared using one-way ANOVA. P < 0.05 or P < 0.01, the difference was statistically significant.

[0071] 3. Experimental design and animal grouping

[0072] Experimental design: 18 male SD rats were randomly divided into 3 groups, 6 in each group, respectively: control group, acute pancreatitis model group, trimethylamine oxide treatment group.

[0073] According to the method reported in the literature, the classic method of retrograde injection of sodium taurocholate into the pancreatic duct was used to establish the model of severe acute pancreatitis in rats. The rats in the trimethylamine oxide treatment group were given gavage of trimethylamine oxide 100 mg / kg 7 days in advance. The control group, acute pancreatitis group and trimethylamine group were fasted for 12 hours before operation. After successful inhalation anesthesia with isoflurane, the rats in the model group and trimethylamine treatment group were taken in the supine position, the head and limbs were fixed, and the skin was prepared for operation in the surgical site. Under sterile operation, the abdomen was incised in the middle of the upper abdomen, the surgical field was fully exposed, the duodenum was taken out and flattened, and the common bile duct was clamped with an artery clamp. A 1 ml sterile syringe was used to gently puncture the confluence of pancreatic duct, and 3.5% sodium taurocholate solution (0.1 ml / 100g body weight) was injected retrograde, with a speed of 0.1 ml / min. During and after injection, the left hand gently pressed the injection point to prevent the injection drug from flowing back into the intestinal tract. After observing the congestion and edema of the pancreas, the needle was withdrawn, and the artery clamp was opened after about 2 minutes. The pancreas was carefully replaced, and the abdomen was sutured layer by layer. The control group was opened in the same way, and the pancreas was gently pushed back into the abdominal cavity.

[0074] The experimental rats were anesthetized with isoflurane inhalation 24 hours after operation. The rats were taken in the supine position, and the head and limbs were fixed on the mouse plate, and the back was raised to fully expose the abdominal aorta. The surgical area was routinely disinfected, the abdomen was opened along the midline, the abdominal aorta was bluntly dissociated, and blood was collected with a medical blood collection tube. 4℃, 3500rpm, centrifugation for 10min, take the upper clear liquid, store at-80℃ for detection; The head of the pancreas was fixed in 4% paraformaldehyde solution for histopathological observation; The remaining pancreatic tissue was washed with normal saline and quickly placed in liquid nitrogen, then transferred to-80℃ refrigerator for storage.

[0075] 4. Results

[0076] As Figure 2, as shown in Table 2, the serum level of trimethylamine-N-oxide in the acute pancreatitis model rats was significantly higher than that in the normal control group, indicating that the level of trimethylamine-N-oxide in the acute pancreatitis animal model was significantly increased (P<0.05). After treatment with trimethylamine-N-oxide, the level of trimethylamine-N-oxide in rats was significantly higher than that in the acute pancreatitis group (P<0.05), indicating that the level of trimethylamine-N-oxide in the animal body increased after treatment.

[0077] As Figure 3 , Figure 4 and Table 3, after modeling by retrograde injection of sodium taurocholate into the pancreatic duct, the HE staining results of the acute pancreatitis model rat group showed that the pancreatic tissue appeared inflammatory cell infiltration, acinar cell edema, vacuolization and necrosis, and there was exudation. The statistical results of pancreatic tissue pathological score were significantly higher than those of the control group, indicating that the acute pancreatitis modeling was successful. After treatment with trimethylamine-N-oxide, the pathological condition of pancreatic tissue was more severe than that of the model group, and the statistical results of pancreatic tissue pathological score were significantly increased (P<0.05), indicating that trimethylamine-N-oxide had a significant aggravating effect on acute pancreatitis.

[0078] As Figure 5 , as shown in Table 4, the serum amylase determination results of the acute pancreatitis model rats were higher than those of the normal control group (P<0.01), indicating that the animal pancreatitis modeling was successful. After treatment with trimethylamine-N-oxide, the serum amylase determination results were further increased by about 1.8 times (P<0.05). It is proved that trimethylamine-N-oxide has pro-inflammatory effect.

[0079] As Figure 6 , as shown in Table 5, the serum lipase determination results of the acute pancreatitis model rats were higher than those of the normal control group, and the statistical results had significant difference (P<0.001), indicating that the animal pancreatitis modeling was successful. After treatment with trimethylamine-N-oxide, the serum lipase level of rats was further increased compared with the model group, increased by about 1.8 times (P<0.05), with significant difference.

[0080] As Figure 7 , 8 , as shown in Tables 6 and 7, the serum inflammatory factors IL-1beta and IL-6 determination results of the acute pancreatitis model rats were higher than those of the normal control group, and there was obvious inflammatory reaction in the rats. After treatment with trimethylamine-N-oxide, the determination results of pancreatic inflammatory factors IL-lbeta and IL-6 were increased, and the increase ratio was about 75.6% and 27.1% (P<0.001). The above results all showed that trimethylamine-N-oxide had obvious promoting effect on the inflammatory reaction in the progress stage of acute pancreatitis, and was one of the risk factors for severe acute pancreatitis.

[0081] As Figure 9, The results of the determination of the active oxygen in the pancreas of the acute pancreatitis model rats shown in Table 8 were higher than those of the normal control group (P<0.01). After treatment with trimethylamine oxide, the level of the active oxygen in the pancreas of the rats was increased compared with the pancreatitis model group, and the difference was statistically significant (P<0.05). It is shown that trimethylamine oxide can increase the level of oxidative stress in the pancreas and aggravate the oxidative stress injury of the pancreas.

[0082] Table 2 The results of the determination of the trimethylamine oxide in the serum of the acute pancreatitis model rats after treatment with trimethylamine oxide (μg / mL, n=6)

[0083]

[0084] * Excluded suspicious data from statistics.

[0085] Table 3 The results of the HE pathological section score of the pancreas of the acute pancreatitis model rats after treatment with trimethylamine oxide (n=6)

[0086]

[0087] Table 4 The results of the determination of the serum amylase of the acute pancreatitis model rats after treatment with trimethylamine oxide (U / L, n=6)

[0088]

[0089] Table 5 The results of the determination of the serum lipase of the acute pancreatitis model rats after treatment with trimethylamine oxide (U / L, n=6)

[0090]

[0091] Table 6 The results of the determination of the serum inflammatory factor IL-lbeta of the acute pancreatitis model rats after treatment with trimethylamine oxide (pg / mL, n=6)

[0092]

[0093]

[0094] Table 7 The results of the determination of the serum inflammatory factor IL-6 of the acute pancreatitis model rats after treatment with trimethylamine oxide (pg / mL, n=6)

[0095]

[0096] Table 8 The results of the determination of the serum active oxygen of the acute pancreatitis model rats after treatment with trimethylamine oxide (Fi, n=6)

[0097]

[0098] Example 3. Therapeutic effect of flavin-containing monooxygenase 3 competitive inhibitor in the combination of Bombesin and Lipopolysaccharide induced acute pancreatitis mouse model

[0099] 1. Experimental animals, instruments and reagents

[0100] C57 mice (8 weeks old) were purchased from Beijing Huafukang Biotechnology Co., Ltd. The animals were raised in a SPF level environment (21 ± 2℃, 12 hours light cycle), and were free to eat and drink during the experimental period. 4% tissue cell fixative, hematoxylin-eosin staining solution were purchased from Solabio Biotechnology Co., Ltd. Bombesin (98%), lipopolysaccharide (95%) were purchased from MCE Biochemical Reagent Co., Ltd. and Jinke Long Biological Technology Co., Ltd. respectively. Trizol lysis solution was purchased from Thermo Fisher Scientific. Methimazole, traditional Chinese medicine monomer rhein were purchased from Beijing Solabio Biotechnology Co., Ltd. Pathological section technology support was provided by Wuhan Saiver Biotechnology Co., Ltd.

[0101] 2. Analysis method

[0102] Pancreatic tissue hematoxylin-eosin (HE) pathological staining: After the head of the pancreas was fixed in 4% tissue cell fixative (4% paraformaldehyde) for 24 hours, it was dehydrated in an automatic dehydration machine for 16 hours, and then embedded in paraffin with a conventional embedding machine. After being cut into sections, it was flat-mounted on glass slides, dried, and then subjected to hematoxylin-eosin staining. After dehydration and transparency, it was mounted and observed under a microscope. Photographs were taken with an Olympus BX53 upright microscope and recorded.

[0103] Acute pancreatitis pathological scoring criteria, refer to Example 2.

[0104] The expression levels of pancreatic inflammation factors tumor necrosis factor alpha (TNF-a), interleukin-lbeta (IL-lβ), chemokine ligand 2 (CCL2), chemokine ligand 3 (CCL3), and flavin monooxygenase 3 (FMO3) were determined by RT-qPCR. Trizol lysis solution was added to the pancreatic tissue to fully lyse the sample, and the supernatant was placed in another EP tube. Chloroform reagent was added and vortexed until emulsified to a milky white color. After standing at room temperature for 5 minutes, centrifugation was performed at 12000g for 15 minutes (4°C), and the supernatant was mixed with an equal volume of isopropanol reagent and allowed to stand for 10 minutes. The supernatant was discarded after centrifugation, and 1 ml of 75% ethanol was added to the precipitate and mixed evenly. After washing the RNA precipitate, the liquid was discarded, and the cap was opened to dry at low temperature. After the ethanol evaporated, RNase-free water was added to dissolve the precipitate, and total RNA solution was obtained. A small amount of total RNA solution was added to RNase-free water for dilution, and the absorbance values at 260 nm, 280 nm, and 320 nm were determined. The RNA sample with OD260 / OD280 between 1.8 and 2.2 was used for subsequent experiments. According to the kit instructions, the total RNA solution was diluted to 1 μg / μl for reverse transcription reaction, and the obtained cDNA was stored at -20°C. All primers were designed and synthesized by Shengong Bioengineering (Shanghai) Co., Ltd. according to the qPCR primer design principles, and the specific primer sequences are shown in Table 1. qPCR reaction was performed according to the kit operation instructions, and the results were analyzed by the 2^(-ΔΔCt) method.

[0105] Table 9 Primer sequences

[0106]

[0107]

[0108] Serum α-amylase (starch-iodine colorimetry), lipase (microplate method), and malondialdehyde (MDA) were determined using test kits purchased from Nanjing Jianshen Biological Technology Co., Ltd., and the specific methods are described in the instruction manual.

[0109] A high-performance liquid chromatography-triple quadrupole mass spectrometer (AB SCiex TMThe quantification of the level of trimethylamine-N-oxide in the tissue samples was performed using a Shimadzu LCMS- 4500MD. The separation of the compounds was performed using an ACE Excel PFP C18 column (1.7 pm, 2.1 mm x 100 mm, Avantor, England). The temperature of the column was maintained at 40 °C and the temperature of the autosampler was 4 °C. The mobile phase was 0.01% formic acid in water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.3 mL / min. The gradient elution program was set as follows: 0.01 min, 20% B; 1.50 min, 24% B; 3.00 min, 40% B; 7.00 min, 55% B; 7.5 min, 70% B; 8.00 min, 98% B; 9.50 min, 98% B, 10 min, 20% B. After the detection equilibration, 1.5 minutes were allowed. The quantification was performed using multiple reaction monitoring, m / z 76.1→58.1 for trimethylamine-N-oxide; m / z 85.1→68.1 for the internal standard.

[0110] Statistical data were analyzed using GraphPad Prism 6.0 software (GraphPad; CA, USA) and expressed as mean ± SEM. Differences between groups were compared using one-way ANOVA. P < 0.05 or P < 0.01, the difference was statistically significant.

[0111] 3. Experimental design and sample processing method

[0112] Experimental design and grouping of animals: 30 female mice were randomly divided into 5 groups, including normal control group, methimazole group, acute pancreatitis model group, methimazole treatment group, and methimazole combined with traditional Chinese medicine monomer treatment group.

[0113] (1) Normal control group: oral physiological saline 0.3 mL / time / 12 h;

[0114] (2) Methimazole group: oral methimazole 15 mg / mL, 0.2 mL / time, 14 days;

[0115] (3) Acute pancreatitis model group: caerulein combined with lipopolysaccharide induction + oral physiological saline 0.2 mL / time / 12 h;

[0116] (4) Methimazole treatment group: oral methimazole 15 mg / kg, 0.2 mL / time, 14 days + caerulein combined with lipopolysaccharide induction;

[0117] (5) Methimazole combined with traditional Chinese medicine monomer treatment group: oral methimazole 15 mg / kg, 0.2 mL / time, 14 days + oral rhein 100 mg / kg, 0.2 mL / time, 7 days + caerulein combined with lipopolysaccharide induction.

[0118] Methimazole treatment group was given methimazole (TMZ, Sulebao, China) 15 mg / kg by gavage for 14 days before the establishment of acute pancreatitis model (AP). Methimazole combined with traditional Chinese medicine monomer treatment group was given methimazole 15 mg / kg by gavage for 14 days + rhein 100 mg / kg by gavage for 7 days before the establishment of AP model. The normal control group, methimazole group, acute pancreatitis model group, methimazole treatment group, and methimazole combined with traditional Chinese medicine monomer treatment group were established from 8-week-old mice. After one week of adaptive feeding with ordinary feed, the model group and drug treatment group were continuously modeled for 10 times by intraperitoneal injection of caerulein (100 μg / kg) every hour. One hour after the last modeling of caerulein, lipopolysaccharide (10 mg / kg) was injected intraperitoneally. The experiment ended 24 hours after the injection of lipopolysaccharide, and the fasting serum of the animals was taken. Six pancreas tissues of each group of animals were used for pathological section staining and statistics.

[0119] 4. Results

[0120] As Figure 10 shown in Table 10, after the induction of caerulein combined with lipopolysaccharide modeling, the expression level of flavin monooxygenase 3 in the acute pancreatitis model group of mice was significantly increased, which was consistent with the clinical detection results. The level of flavin monooxygenase 3 in the oral methimazole (inhibitor) treatment group was significantly lower than that in the acute pancreatitis model group, indicating that methimazole significantly inhibited the level of flavin monooxygenase 3 (P<0.001); in addition, the level of flavin monooxygenase 3 in the methimazole combined with traditional Chinese medicine monomer group (rhein combined with inhibitor-containing composition) was further reduced to close to the normal level, indicating that rhein may enhance the metabolic regulation effect by synergistically inhibiting the level of flavin monooxygenase 3 (P<0.001).

[0121] As Figure 11 shown in Table 11, the serum trimethylamine N-oxide level of the pancreatitis model mice was higher than that of the normal control group, indicating that the trimethylamine N-oxide level of the acute pancreatitis model was increased (P<0.05), confirming the abnormal activation of trimethylamine N-oxide in acute pancreatitis. After treatment with methimazole, the trimethylamine N-oxide level of the mice was significantly reduced (P<0.01), indicating that inhibition of flavin monooxygenase 3 can inhibit the level of trimethylamine N-oxide, and its mechanism is directly related to blocking the trimethylamine (TMA) oxidation metabolism pathway mediated by flavin monooxygenase 3. After treatment with methimazole combined with traditional Chinese medicine monomers, the trimethylamine N-oxide level of the mice was further reduced and the difference was statistically significant (P<0.001), indicating that the inhibitory effect of trimethylamine N-oxide level was more significant after treatment with methimazole combined with traditional Chinese medicine monomer rhein.

[0122] As Figure 12 , Figure 13, and as shown in Table 12, after the induction of the model by the combination of caerulein and lipopolysaccharide, the HE staining results of the pancreatitis model mice group showed that inflammatory cell infiltration, acinar cell edema, vacuolization and necrosis occurred in the pancreatic tissue, and there was exudation. The statistical results of the pathological score of the pancreatic tissue were significantly higher than those of the control group, indicating that the pancreatitis model was successfully established. After oral administration of methimazole to normal mice, no obvious pathological changes were observed in the pancreatic tissue, indicating that methimazole had no toxic side effects on the pancreas. After oral administration of methimazole to acute pancreatitis mice, the pathological condition of the pancreas was significantly improved; at the same time, the treatment effect of methimazole combined with the single body of traditional Chinese medicine was better. The pathological score results showed that reducing the level of oxidized trimethylamine by inhibiting flavin monooxygenase 3 had a significant therapeutic effect on acute pancreatitis, and the effect was more significant when combined with the single body of natural product. The pathological score of the pancreas was reduced by more than 50%. Due to the complex mechanism of severe pancreatitis, there are limited treatment targets and drugs that can significantly inhibit the deterioration of the disease, thus highlighting the advantages of the technical solution.

[0123] As Figure 14 , as shown in Table 13, the serum amylase determination results of the pancreatitis model mice were significantly higher than those of the normal control group, confirming that the pancreatitis model mice were successfully established. After treatment with methimazole or methimazole combined with the single body of traditional Chinese medicine rhein, the serum amylase determination results were all decreased (P<0.001). The amylase level of the methimazole treatment group was reduced by 22.62% compared with the acute pancreatitis model group, and the amylase level of the methimazole combined with the single body of traditional Chinese medicine was reduced by 24.84% compared with the methimazole treatment group, indicating that the more obvious the reduction of the level of oxidized trimethylamine by inhibiting the level of flavin monooxygenase 3, the more significant the therapeutic effect.

[0124] As Figure 15 , as shown in Table 14, the serum lipase determination results of the pancreatitis model mice were higher than those of the normal control group, confirming that the mice were successfully established. After treatment with methimazole or methimazole combined with the single body of traditional Chinese medicine rhein, the serum amylase determination results were all significantly reduced (P<0.001). The lipase levels of the methimazole treatment group and the methimazole combined with rhein treatment group were reduced by 48.31% and 80.14%, respectively, compared with the acute pancreatitis model group, indicating that the treatment targeting flavin monooxygenase 3 was positively correlated with the amylase therapeutic effect.

[0125] As Figures 16-19, the determination results of pancreatic inflammatory factors TNF-alpha, IL-lbeta, CCL2, CCL3 of the pancreatitis model mice were higher than those of the normal control group, indicating that there was an inflammatory response in the acute pancreatitis mice. After treatment with methimazole respectively, the determination results of pancreatic inflammatory factors TNF-alpha, IL-lbeta, CCL2, CCL3 were significantly reduced, and the inhibition rates were 87.34%, 86.99%, 86.19%, and 80.41% respectively (P<0.05, P<0.001, P<0.01, P<0.001). Unexpectedly, compared with the methimazole treatment group, the pancreatic inflammatory factors TNF-alpha, IL-lbeta, CCL2, CCL3 of the oral methimazole combined with traditional Chinese medicine monomer rhein treatment group were reduced by 90.88%, 93.80%, 91.63%, and 91.48% respectively. Due to the complexity of the inflammation progression mechanism, this scheme can significantly curb the inflammation storm level of the pancreatitis progression stage, highlighting the technical advantage. It is confirmed that the above inhibitors and compositions containing the inhibitors have the potential for the treatment of acute pancreatitis, especially severe acute pancreatitis.

[0126] Oxidative stress injury is an important inducement for pancreatic acinar necrosis and apoptosis. The level of malondialdehyde is one of the factors reflecting the level of oxidative stress in the body. For example Figure 20 , as shown in Table 19, the determination results of malondialdehyde in the pancreas of the pancreatitis model mice were higher than those of the normal control group, suggesting that the oxidative stress level of the pancreatitis mice was increased. After treatment with trimethylamine oxide, the level of malondialdehyde in the pancreas of the mice was significantly higher than that of the pancreatitis model group (P<0.05), indicating that trimethylamine oxide can increase the level of oxidative stress in the body and exacerbate pancreatic injury. Compared with the acute pancreatitis group, after treatment with methimazole and methimazole combined with traditional Chinese medicine monomer rhein respectively, the determination results of malondialdehyde in the pancreas were significantly reduced, and the inhibition rates were 71.35% and 81.29% respectively (P<0.01, P<0.001). It is indicated that the flavin-containing monooxygenase 3 inhibitor and the composition containing the inhibitor can reduce the oxidative stress injury of acute pancreatitis by inhibiting the level of trimethylamine oxide.

[0127] Table 10 Relative level of flavin-containing monooxygenase 3 mRNA in acute pancreatitis model mice treated with flavin-containing monooxygenase 3 competitive inhibitor and composition containing the inhibitor (n=6)

[0128]

[0129]

[0130] Table 11 Determination results of serum trimethylamine oxide of acute pancreatitis model mice treated with flavin-containing monooxygenase 3 competitive inhibitor and composition containing the inhibitor (μg / ml, n=6)

[0131]

[0132] Table 12 HE pathological section score of pancreas of acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and composition containing the inhibitor (n=6)

[0133]

[0134]

[0135] Table 13 Serum amylase determination results (U / L, n=6) of acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and composition containing the inhibitor

[0136]

[0137] Table 14 Serum lipase determination results (U / L, n=6) of acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and composition containing the inhibitor

[0138]

[0139]

[0140] Table 15 Relative level of pancreatic inflammatory factor TNF-alpha mRNA (n=6) of acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and composition containing the inhibitor

[0141] Table 16 Determination results of pancreatic inflammatory factor IL-1 beta mRNA (pg / mL, n=6) of acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and composition containing the inhibitor

[0142]

[0143]

[0144] Table 17 Relative level of pancreatic chemotactic factor CCL2 mRNA (n=6) of acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and composition containing the inhibitor

[0145]

[0146] Table 18 Relative level of pancreatic chemotactic factor CCL3 mRNA (n=6) of acute pancreatitis model mice after treatment with flavin monooxygenase 3 competitive inhibitor and composition containing the inhibitor

[0147]

[0148]

[0149] Table 19. Acute pancreatitis model mice pancreatic malondialdehyde (MDA) assay results after flavin monooxygenase 3 competitive inhibitor and inhibitor-containing composition treatment (nmol / mg prot, n=6)

[0150]

[0151] The above only is the preferred embodiment of the present application, it should be pointed out that, for the ordinary skilled person in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Use of a flavin monooxygenase 3 inhibitor in the preparation of a medicament for preventing and / or treating acute pancreatitis.

2. Use according to claim 1, characterized in that, The flavin monooxygenase 3 inhibitor is methimazole or a pharmaceutically acceptable salt, prodrug or derivative thereof of formula (I), wherein the pharmaceutically acceptable salt of methimazole includes hydrochloride, sulfate, hydrobromide, hydroiodide, formate, acetate or oxalate; the prodrug or derivative of methimazole includes carbimazole or methimazole thio-β-D-glucoside.

3. Use according to claim 2, characterized in that, The inhibitor alleviates the inflammatory response of pancreatic tissue and reduces the level of oxidative stress by inhibiting the activity of liver flavin monooxygenase 3 and reducing the concentration of serum oxidized trimethylamine.

4. Use according to claim 1, characterized in that, The composition further comprises at least one monomer component of natural product in addition to the flavin monooxygenase 3 inhibitor of any one of claims 1-3.

5. Use of a composition comprising a flavin-containing monooxygenase 3 inhibitor for the manufacture of a medicament for the prevention and / or treatment of pancreatitis, characterized in that, The monomer component of natural product is rhein; preferably, the mass ratio of flavin monooxygenase 3 inhibitor to rhein is 1:20 to 5:20; more preferably, the mass ratio of flavin monooxygenase 3 inhibitor to rhein is 3:

20.

6. Use according to claim 5, characterized in that, The monomer component of natural product, in addition to the rhein, further comprises other natural product components capable of significantly reducing the level of oxidized trimethylamine, such as emodin, aloe emodin, berberine, baicalin or a pharmaceutically acceptable salt or derivative thereof.

7. Use according to claim 6, characterized in that, The medicament or composition further comprises a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

8. Use according to any one of claims 1 to 7, characterized in that, The acute pancreatitis is an acute inflammatory lesion of pancreatic tissue caused by cholelithiasis, alcohol, blood vessels, trauma, infection factors, manifested as abnormal elevation of serum amylase and / or lipase and persistent upper abdominal pain, and imaging examination of the abdomen shows changes consistent with pancreatitis; preferably, the acute pancreatitis is moderate to severe acute pancreatitis.

9. Use according to any one of claims 1 to 7, characterized in that, In addition to the typical characteristics of pancreatitis, the moderate to severe acute pancreatitis further has exudation, necrosis of surrounding tissues or systemic inflammatory response syndrome.

10. Use according to claim 9, characterized in that, ​

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