Application of (-)-clavamide or a composition containing (-)-clavamide in the manufacture of a drug for treating intestinal-related diseases

Abstract

The present invention provides the use of (‑)‑clavamide or a composition containing (‑)‑clavamide in the manufacture of a medicament for treating intestinal-related diseases. The present invention also provides the use of ‑(‑)‑clavamide in the preparation of a medicament for inhibiting p‑STAT3 overexpression-related diseases. The use of (‑)‑clavamide can improve weight loss, increased DAI scores, shortened colon and histopathological damage in ulcerative colitis, reduce the secretion of inflammatory factors, reverse the reduction in the migration ability of intestinal epithelial cells in colitis, etc., with significant effects.

Description

[0001] Application of (-)-clavamide or a composition containing (-)-clavamide in the manufacture of a drug for treating intestinal-related diseases Technical Field

[0002] The present invention relates to application of (-)-clavamide or a composition containing (-)-clavamide in manufacturing medicines for treating intestinal-related diseases. Background Art

[0003] Intestinal-related diseases, such as inflammation, ulcers, etc., such as colitis and proctitis, are common digestive tract diseases, often manifested by symptoms such as abdominal pain, diarrhea, and bloody stools, which seriously affect people's lives.

[0004] Clausinamide is a compound extracted from the fruit of the plant chamomilla. It has four chiral centers in its chemical structure and a total of 16 chiral compounds. Among them, (-)-clavamide (as shown in Formula I below) is a known compound with potential anti-Alzheimer's disease effects, which can enhance the basic synaptic transmission and high-frequency stimulation-induced long-term potentiation (LTP) in anesthetized or freely moving rats, and increase the sprouting of hippocampal synapses and mossy fibers; in addition, (-)-clavamide can also increase the cytoplasmic free calcium concentration (Ca) in the cortical neurons of newborn rats. 2+ ). Currently, the research on (-)-clavamide is still mainly centered around its effects on Alzheimer's disease or the central nervous system.

[0005] Formula (I):

[0006] . Summary of the invention

[0007] In one aspect, the present application relates to the use of (-)-clavamide or a composition comprising (-)-clavamide in the manufacture of a medicament for treating intestinal-related diseases; intestinal-related diseases include inflammatory bowel disease and enteritis-related intestinal cancer, etc.

[0008] Preferably, the drug has a significant effect by improving colon damage and inflammatory cell infiltration, thereby achieving the purpose of treating intestinal-related diseases.

[0009] Application of (-)-clavamide or a composition containing (-)-clavamide in preparing a drug for inhibiting diseases related to p-STAT3 overexpression.

[0010] Preferably, the drug can inhibit the overexpression of p-STAT3 and achieve the effect of alleviating ulcerative colitis.

[0011] Preferably, the drug, calculated as (-)-clavamide, has a daily dosage of 35-45 mg / kg, once a day for 10 consecutive days.

[0012] Preferably, the purity of the (-)-clavamide is greater than 99%.

[0013] Preferably, the medicament further comprises one or more pharmaceutically acceptable carriers or excipients, non-pharmaceutically active compounds and / or inert substances.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] When a drug prepared by (-)-clavamide or a composition containing (-)-clavamide is used in the treatment of intestinal-related diseases, such as enteritis, colitis, and especially ulcerative colitis, it can significantly alleviate DSS-induced weight loss, symptoms of ulcerative colitis with increased DAI scores, reduce DSS-induced shortening of colon length and colon tissue pathological damage, reduce the levels of proinflammatory cytokines in the serum and colon tissue of colitis mice, and significantly reverse the reduction in the migration ability of colon epithelial cells caused by DSS; (-)-clavamide may alleviate ulcerative colitis by inhibiting the overexpression of p-STAT3. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 Animal treatment procedures and experimental processes.

[0018] Figure 2 Figure 2 shows the weight changes of the four groups of mice after the treatment procedure, compared with the normal control group. * p<0.05, ** p<0.01; compared with the model group, # p<0.05, ## p<0.01.

[0019] Figure 3 The figure shows the average DAI scores of the four groups of mice.

[0020] Figure 4 Comparison of colon appearance and colon length of four groups of mice, compared with the normal control group. ** p<0.01; compared with the model group, ## p<0.01.

[0021] Figure 5 H&E staining of colon tissue; the black arrow indicates damage to the tissue mucosal layer, loss of mucosal epithelial cells, and disappearance of local intestinal gland structures; the red arrow indicates infiltration of inflammatory cells.

[0022] Figure 6 The concentrations of IL-1β and TNF-α in serum were analyzed by enzyme-linked immunosorbent assay and compared with those in the normal control group. * p<0.05, ** p<0.01; compared with the model group, # p<0.05, ## p<0.01.

[0023] Figure 7 The concentrations of interleukin IL-6, IL-1β and TNF-α in the supernatant of colon tissue were analyzed by enzyme-linked immunosorbent assay and compared with those in the normal control group. * p<0.05, ** p<0.01; compared with the model group, # p<0.05, ## p<0.01.

[0024] Figure 8 These are images of immunohistochemical staining of p-STAT3 in colon tissue.

[0025] Fig. 9 Western blot analysis of p-STAT3 levels in colon tissues; compared with the normal control group, ** p<0.01; compared with the model group, # p<0.05.

[0026] Fig.10 For the cell scratch experiment in the co-culture system, compared with the normal control group, ** p<0.01; compared with the model group, ## p<0.01. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0028] definition

[0029] Unless otherwise indicated, all scientific and technological terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. All patents, applications, published applications and other publications cited herein are incorporated herein by reference in their entirety. If the definition set forth in this section is contrary to or inconsistent with the definition described in the patent, application or other publication incorporated herein by reference, the definition set forth in this section takes precedence over the definition incorporated herein by reference.

[0030] As used herein, references herein to "about" a value or parameter include (and describe) embodiments for that value or parameter itself. For example, descriptions referring to "about X" include descriptions of "X".

[0031] Unless otherwise expressly stated, "subject" as used herein refers to mammals, including but not limited to humans, cattle, primates, horses, dogs, cats, pigs and sheep. Therefore, the compositions and methods provided herein are useful in human medicine and veterinary settings, including for agricultural animals and domestic pets. The subject can be a person who has been diagnosed with or suspected of having a condition described herein, such as a gut-related disease (e.g., intestinal inflammation). The subject can be a person who exhibits one or more symptoms associated with a condition described herein, such as a gut-related disease (e.g., intestinal inflammation). The subject can be a person who is genetically or otherwise susceptible to or at risk for a condition described herein, such as a gut-related disease (e.g., intestinal inflammation).

[0032] As used herein, the term "treatment" is a method for obtaining a beneficial or desired result (including a clinical result). For the purposes of the compositions, methods, and uses provided herein, a beneficial or desired clinical result includes, but is not limited to, one or more of the following: reducing one or more symptoms caused by a condition, alleviating the extent of a condition, stabilizing a condition (e.g., preventing or delaying worsening of the condition), preventing or delaying the spread of a condition (e.g., metastasis), delaying or slowing the progression of a condition, improving the disease state, providing disease remission (partial or total disease), reducing the dose of one or more other agents required to treat a condition, enhancing the effect of another agent used to treat a condition, improving the quality of life of an individual with a condition, and / or prolonging survival. The compositions, methods, and uses described herein contemplate any one or more of these therapeutic aspects.

[0033] A "therapeutically effective amount" refers to an amount of a compound or salt thereof sufficient to produce a desired therapeutic result (e.g., a reduction in severity or duration, stabilization of severity, or elimination of one or more symptoms of a disease described herein). For therapeutic uses, beneficial or desired results include, for example, reduction in one or more symptoms (biochemical, histological, and / or behavioral) caused by the disease, including its complications and intermediate pathological phenotypes that occur during the development of the disease or condition, improving the quality of life of a subject suffering from the disease or condition, reducing the dose of other agents required to treat the disease or condition, enhancing the effect of another agent, delaying the progression of the disease or condition, and / or prolonging the survival of the patient.

[0034] As used herein, "pharmaceutically acceptable" or "pharmacologically acceptable" refers to a material that is not biologically or otherwise undesirable, for example, the material can be incorporated into a pharmaceutical composition administered to a patient without causing any significant adverse biological effect or interacting in a deleterious manner with any other component of the composition in which it is contained. A pharmaceutically acceptable carrier or excipient preferably meets the required standards of toxicology and manufacturing testing.

[0035] Throughout this application, "(-)-clavamide", "clavamide" and "clavamide" are used interchangeably unless expressly stated otherwise.

[0036] In the following examples of the present invention, the preparation method of (-)-clavamide is described in document DOI: 10.1002 / cjoc.201201187.

[0037] Example 1

[0038] 1. Grouping of experimental animals, model establishment and operation procedures

[0039] Female BALB / c mice were provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Jinan, China) (Animal Production License No.; SCXK(Lu)20190003), aged 6-8 weeks, weighing 18-20 g / mouse. Mice were raised in a clean animal room at a temperature of 22°C-26°C, a relative humidity of 45%-65%, and a light-dark cycle of 12 h / 12 ​​h to establish a normal circadian rhythm and maintain normal physiological activities of mice.

[0040] Forty BALB / c mice were randomly divided into four groups (n=10 / group): normal control group (blank), DSS model group, DSS+5-aminosalicylic acid group (5-ASA group) and DSS+(-)-clavamide group (clavamide group). Except for the normal control group, the mice in the other groups were given 4% (w / v) DSS (dextran sulfate sodium) solution in drinking water for 7 days, and then distilled water for 3 days, see Figure 1 .

[0041] Treatment Procedure:

[0042] Combination Figure 1 , from day 1 to day 10,

[0043] Mice in the 5-ASA group were intragastrically administered with 5-ASA at 50 mg / kg once a day;

[0044] Mice in the chrysinamide group were intragastrically administered with (-)-chrysinamide, with a purity of 99.8%, 40 mg / kg each time, once a day;

[0045] The mice in the normal control group and the DSS model group were gavaged with normal saline, 40 mg / kg each time, once a day.

[0046] All mice were weighed and killed on the 11th day, and the main organs (including heart, liver, spleen, lung, kidney, colon) and blood were collected. The heart, liver, spleen, lung, and kidney were weighed to calculate the organ coefficient; the collected serum was used to detect inflammatory factors using ELISA. The mouse colon tissue was fixed with 4% paraformaldehyde and then stained with H&E and immunohistochemistry (IHC). Part of the colon tissue was stored at -80°C for immunoblot analysis.

[0047] 2. Disease Activity Index (DAI) score

[0048] During the experiment, the weight of the mice was recorded and counted at the same time every day, and the activity, diet and drinking status, stool characteristics, and blood in the stool were observed. The disease activity index (DAI) was used to evaluate the colitis every day. The higher the score, the more severe the inflammation. DAI = weight loss score + stool characteristics score + fecal occult blood / bloody stool score, see Table 1.

[0049] Table 1 Disease Activity Index (DAI) scoring method

[0050]

[0051] Record the weight changes of mice during treatment and draw a trend graph. Figure 2 ; The line graph of DAI scores of the four groups of mice is shown in Figure 3 ; Combine Figure 2 It can be seen that compared with the untreated normal control group mice, the weight of the model group (DSS model group) mice was significantly reduced; and the mice treated with (-)-clavamide significantly alleviated the weight loss. Figure 3 It can be seen that the average DAI score of mice in the model group was higher than that in the normal control group, while the mice treated with (-)-clavamide alleviated the increase in DAI score caused by DSS, indicating that (-)-clavamide can alleviate the symptoms of DSS-induced ulcerative colitis.

[0052] 3. Colon length measurement

[0053] The colon lengths of the four groups of mice were statistically analyzed. Figure 4 , combined with Figure 4 It can be seen that compared with the normal control group, the colon of the model group mice was significantly shortened, and the colon of the mice using (-)-championamide (championamide group) was almost indistinguishable from the colon of the mice in the normal control group, indicating that (-)-championamide can reverse the DSS-induced colon shortening phenomenon (P<0.01).

[0054] 4. Organ coefficient calculation

[0055] After killing the mice, the heart, liver, spleen, lungs, and kidneys of the mice were taken out, the surface moisture was absorbed with filter paper, and then weighed to calculate the organ coefficient. Organ coefficient = organ wet weight / body weight × 100%. The results are shown in Table 2;

[0056] Table 2 Comparison of organ coefficients of mice in different groups (mean ± SD)

[0057]

[0058] Combined with the organ coefficients in Table 2, it can be seen that there is no significant difference in the organ coefficients of the important organs between the chrysinamide group and the normal control group, which also shows that (-)-chrysinamide is safe to use; in addition, compared with the normal control group, the spleen coefficient of the mice in the model group was significantly increased (P<0.05), and the use of (-)-chrysinamide can significantly reverse the increase in the spleen coefficient of mice induced by DSS (P<0.05).

[0059] 5. Hematoxylin-eosin staining (H&E staining)

[0060] The colon was removed, opened longitudinally, washed with saline, fixed with 4% paraformaldehyde, and embedded in paraffin. The embedded tissue was sliced ​​into 4 μm slices using a microtome, and then stained using the conventional H&E staining method. The pathological changes of the mouse colon tissue were observed under a microscope and photographed. The results are shown in Figure 5 ;

[0061] Combination Figure 5 Histopathological staining showed that DSS induced damage to the mucosal layer, loss of mucosal epithelial cells, disappearance of local intestinal gland structures, and obvious inflammatory cell infiltration; the use of (-)-clavamide could significantly improve the above colonic histopathological damage;

[0062] comprehensive Figure 1-Figure 5 The results showed that oral administration of (-)-clavamide could reduce colon inflammation and damage caused by dextran sodium sulfate (DSS).

[0063] 6. Enzyme-linked immunosorbent assay

[0064] ELISA kits were used to detect the secretion of IL-6, IL-1β, and TNF-α. Each experiment was performed three times, and the cytokine levels were expressed as pg ml −1 The secretion of IL-1β and TNF-α in serum was Figure 6 , the secretion of IL-6, IL-1β and TNF-α in colon tissue is shown in Figure 7 ;

[0065] Combination Figure 6 and Figure 7 It can be seen that the use of (-)-clavamide can reduce the levels of pro-inflammatory cytokines in the serum and colon of mice with dextran sulfate sodium (DSS)-induced colitis.

[0066] 7. Immunohistochemistry (IHC)

[0067] After the paraffin sections of colon tissue were dewaxed to water, conventional IHC was performed to evaluate the expression of p-STAT3 in colon tissue sections. Figure 8 .

[0068] 8. Western blot

[0069] The protein expression of p-STAT3 in colon tissue was detected. The results are shown in Fig. 9 .

[0070] It can be seen that the effect of (-)-clavamide of the present invention on intestinal-related diseases is related to the influence on colon p-STAT3 expression; that is, (-)-clavamide can be used to prepare drugs for treating diseases related to p-STAT3 expression; p-STAT3 (or pSTAT3) is phosphorylation signal transduction and transcription factor 3, and overexpression of pSTAT3 often causes some digestive tract, such as intestinal, especially colon-related diseases. For example, in patients with colitis, increased expression of p-STAT3 often occurs; and it can be seen from the experimental results of the present invention that (-)-clavamide can inhibit the overexpression of p-STAT3; therefore, (-)-clavamide or a composition containing (-)-clavamide can be used in the manufacture of drugs for downregulating overexpression of colon p-STAT3 in a subject.

[0071] Combination Figure 8 IHC and Fig. 9 The results of Western blotting showed that (-)-clavamide treatment inhibited the overexpression of p-stat3 induced by DSS.

[0072] 9. Cell scratch assay in co-culture system

[0073] In order to study the effect of (-)-clavamide on the repair of damaged intestinal epithelium, a co-culture system using mouse colon epithelial cells (MCECs) and mouse peritoneal macrophages (Mφs) was created to simulate the inflammatory microenvironment around the intestinal epithelium and simulate intestinal epithelial damage in UC by cell scratching. The construction method of the cell co-culture system is as follows:

[0074] The peritoneal Mφs of mice in the normal control group, model group, and clavamide group were collected and 2×10^ 6 The cells were inoculated at a density of 1.5 μm in the upper chamber of a 6-well Transwell chamber (membrane pore size: 0.4 μm) and incubated in 5% CO 2， Cultured in Dulbecco's modified Eagle's medium (DMEM) in a 37°C incubator. MCECs were placed in a 6-well culture plate and cultured in a 5% CO2, 37°C incubator until the cell density reached 80%. Cell scratching was then performed, and then the upper chamber of the Transwell chamber containing peritoneal Mφs was transferred to a 6-well culture plate and co-cultured with MCECs. Photos were taken and observed under a microscope at 0 hours and 24 hours after scratching. The cell migration rate was calculated, and the results are shown in Fig.10 ;

[0075] Fig.10 The AC scratch test shows the effect of mouse colon epithelial cells (MCECs) and peritoneal macrophages (Mφs) from different groups of mice on the migration ability of mouse colon epithelial cells (MCECs); Fig.10 A is a schematic diagram of the co-culture system of mouse peritoneal macrophages (Mφs) and mouse colon epithelial cells (MCECs); Fig.10 B is the migration rate of MCECs cells in scratch test, compared with the normal control group. ** p<0.01; compared with the model group, ## p<0.01; Fig.10 C is a cell scratch test of MCECs, and the cell photos at 0 hours and 24 hours after scratching; the experimental results show that compared with the normal control group, the presence of Mφs in the model group mice significantly reduced the cell migration ability of MCECs, while compared with the model group, the Mφs in the (-)-clavamide treated group mice of the present invention significantly reversed the reduction of MCECs cell migration ability. This shows that the (-)-clavamide of the present invention is helpful for the repair of damaged intestinal epithelium. ( Fig.10 AC).

[0076] Statistical analysis

[0077] All data were analyzed using GraphPad Prism software (GraphPad Software Inc.). All results are presented as mean ± SD of three replicates. One-way ANOVA was used to compare multiple groups of data, and independent sample t-test was used to compare two groups of data. P < 0.05 was considered statistically significant. Details of each statistical analysis are provided in the figure legends.

[0078] Although the present invention has been described in detail by reference to the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. Use of (-)-clavamide or a composition containing (-)-clavamide in the manufacture of a medicament for treating ulcerative colitis.

2. The use according to claim 1, characterized in that The drug can improve colon damage and inflammatory cell infiltration.

3. The use according to claim 1, characterized in that The drug can inhibit the overexpression of p-STAT3.

4. The use according to claim 1, characterized in that The drug, calculated as (-)-clavamide, has a daily dosage of 35-45 mg / kg, once a day for 10 consecutive days.

5. The use according to claim 4, characterized in that The purity of the (-)-clavamide is greater than 99%.

6. The use according to claim 5, characterized in that The medicament further comprises one or more pharmaceutically acceptable carriers.

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