CN110664791A

CN110664791A - Application of chloride channel inhibitor lanjiquinone in anti-diarrhea drugs

Info

Publication number: CN110664791A
Application number: CN201911071397.2A
Authority: CN
Inventors: 于波; 杨红; 朱小娟
Original assignee: Liaoning Normal University
Current assignee: Liaoning Normal University
Priority date: 2019-07-22
Filing date: 2019-11-05
Publication date: 2020-01-10

Abstract

The invention relates to application of a chloride ion channel inhibitor lanjiquinone in an anti-diarrhea drug. Intestinal canal Cl^‑The channel is used as a key channel for intestinal fluid secretion and becomes a potential diarrhea treatment target. The invention relates to Lanceoquinone p-intestinal Cl^‑Discovery of channel inhibitory activity, particularly therapeutic effects of oral lanugo quinone on bacterial and viral induced diarrhea. Studies have shown that lanugenon reversibly inhibits TMEM16A channel activity against CaCC and CFTR Cl^‑The channels also have an inhibitory effect. The gavage lanuginose can reduce intestinal juice secretion induced by STa and CT, and obviously improve diarrhea symptoms induced by cholera toxin and rotavirus. In addition, intestinal motility studies have shown thatThe snow quinone can obviously slow down intestinal peristalsis and inhibit contraction of intestinal smooth muscle, but has no obvious influence on contraction frequency. The lanugenone can be applied to medicines for treating cholera, traveler's diarrhea and rotavirus diarrhea, develops new pharmacological activity of the lanugenone, and provides a new choice for research and development of anti-diarrhea medicines.

Description

Application of chloride channel inhibitor lanjiquinone in anti-diarrhea drugs

Technical Field

The invention relates to the field of medicines, in particular to an inhibitory effect of lankeequinone on chloride ion channels and application thereof in treatment of bacterial and viral diarrhea.

Background

Diarrhea is a serious health-threatening disease, with high morbidity and mortality worldwide, particularly in children and the elderly. According to the incomplete statistics of 2015, about 577,000 children under 5 years of age and 502,000 elderly over 70 years of age die worldwide from secretory diarrhea. In developing countries, the enteropathogenic bacteria causing diarrhea are mainly vibrio cholerae and pathogenic escherichia coli, and the pathogenic viruses are mainly rotavirus and enteroinvasive virus.

The intestinal tract is an important digestive organ of the human body, and the main physiological functions relate to the physiological processes of substance absorption, intestinal fluid secretion, intestinal movement and the like. The flow of fluid between the intestine and the blood is mainly influenced by Na⁺、Cl^-、HCO₃ ^-And K⁺And driving of solutes such as glucose. Intestinal fluid secretion is mainly Cl passing through the basal and apical membranes^-Channel and transporter pairs Cl^-Is achieved by secretion. Cl involved in intestinal fluid secretion^-The channels are mainly cystic fibrosis transmembrane conductance regulator (CFTR) and calcium activated chloride channel (CaCC). Cholera toxin secreted by vibrio cholerae and enterotoxigenic escherichia coli and heat-resistant escherichia coli endotoxin can increase intracellular cAMP, cGMP and Ca²⁺And both of these molecules can activate CFTR activity. The bacteria also can increase the levels of various receptor agonists, neurotransmitters and neuropeptide receptors in body fluids, such as 5-hydroxytryptamine (5-HT), Vasoactive Intestinal Peptide (VIP) and galanin type I receptors, thereby activating intestinal cells to Cl^-Secretion of (5). Rotavirus infection can cause fluid secretion and changes in the epithelial structure of the intestinal tract, causing age-related secretory diarrhea. About 50 million infants die of rotavirus diarrhea every year in the world, and in China, infants hospitalized due to rotavirus infection account for about 40 percent of all infants hospitalized due to acute gastrointestinal diseases. Current studies indicate that the non-structural protein (NSP4) produced by rotavirus causes intracellular Ca by binding to the membrane receptor (integrin. alpha.1beta.2), or by binding to the receptor via glycerol peptide, or by stimulating the enteric nerve²⁺Increased concentration, Cl production by CaCC on the cell surface^-It is secreted to cause diarrhea. In addition to the liquid secretion mechanism, Cl^-The channels are also involved in the regulation of intestinal motility. TMEM16A, a known CaCC, has high expression in Interstitial Cajal Cells (ICC) in the intestinal tract. The ICC has the main function as a pacing point and is involved in the generation and conduction of gastrointestinal electrical slow waves, and constitutes a link between sensory nerves and smooth muscle cells. The normal contraction function of the intestinal muscles is mainly dependent on the coordination activity of the Enteric Nervous System (ENS) and ICC, and various digestive tract dyskinetic diseases are closely related to ICC abnormalities, such as: megacolon, constipation, abdominal pain and constipation which are frequently occurred in patients with long-term insulin-dependent diabetes mellitus. The study shows that TMEM16A protein has specific expression in ICC of all parts of gastrointestinal tracts of birds, non-human primates and humans, and the CaCC blocking agents niflumic acid and DIDS can weaken contraction frequency of stomach and small intestine in a dose-dependent mode and block slow wave generation. The selective inhibitor of TMEM16A can obviously weaken the contraction of smooth muscle of intestinal segment of mouse, while the activator of TMEM16A can enhance the contraction and restore the contraction force of small intestine after atropine inhibition. The lack of function of TMEM16A prevented the mouse gastrointestinal tract from forming slow waves, but did not affect the formation of the ICC network. The above results demonstrate the fundamental role of TMEM16A in the generation of slow waves in the gastrointestinal tract and in the regulation of smooth muscle contractility.

In view of Cl^-Channel activation may be a common mechanism for the development of multiple diarrheas, and therefore screening against different Cl's from combinatorial chemical small molecule libraries^-The channel inhibitors become hot spots of research on treatment and prevention of diarrhea, and a plurality of small molecule inhibitors belonging to different structural families are discovered at present, and part of inhibitors are applied to clinical disease treatment.

Disclosure of Invention

In order to make up for the blank of the prior art, the invention provides the application of a chloride channel inhibitor lanugo quinone (plumbagin) in the preparation of anti-diarrhea drugs and drugs for intestinal motility disorder diseases.

The invention has the following inventive concept: the natural small molecules have the characteristics of various structures, high bioactivity, relatively stable structure, high bioavailability, small toxic and side effects and the like, the inventor discovers the lanugenon from a natural small molecule library, and the lanugenon inhibits Cl at the apical membrane side of the FRT cell transfected with TMEM16A^-Current, which has TMEM16A inhibitory activity. The regulation activity of the lanugenone on CFTR and intestinal epithelial CaCC is firstly researched in vitro by using various means, the intestinal fluid secretion and intestinal power regulation of the lanugenone are systematically analyzed by using isolated mouse intestinal tissues, and finally the diarrhea resistance effect of the lanugenone is proved on various mouse diarrhea models, so that the lanugenone has higher application value in the aspect of clinical diarrhea resistance treatment.

The compound lancedrone has the following structure:

the experiment of the invention proves that the natural Cl^-Lancetone, a channel inhibitor, has inhibitory effects on CaCC and CFTR activity on HT-29 cells in colon cancer.

Lancequinone reduces intestinal fluid secretion induced by E.coli heat stable enterotoxin (STa) and Cholera Toxin (CT).

The diarrhea inhibiting rate of the lanugo quinone is more than 40% in a cholera toxin newborn mouse diarrhea model, and is 50% in a rotavirus-induced newborn mouse diarrhea model.

The lancedrone obviously inhibits the movement of the intestinal tract and increases the absorption time of intestinal tract liquid; inhibit intestinal smooth muscle contraction force, but has no obvious effect on contraction frequency.

Therefore, the lanugenone and the pharmaceutically acceptable salts thereof are prepared into oral preparations and used for preparing the medicines for resisting diarrhea and intestinal motility disorder-related diseases.

Such diseases or conditions include: treating intestinal motility disorder, diarrhea caused by cholera toxin or rotavirus diarrhea.

The invention discovers a novel pharmacological activity of the lanugenone, namely, by inhibiting Cl^-The channel activity can inhibit intestinal fluid secretion and intestinal peristalsis enhancement induced by intestinal pathogens. Lanceoquinone can be used as a lead compound for treating cholera toxin-induced diarrhea, traveler's diarrhea, rotavirus diarrhea and other types of secretory diarrhea.

Drawings

FIG. 1 short circuit current measurement of inhibition of TMEM16A by lancedrone;

FIG. 2 short circuit current measurement of the inhibitory effect of lankeequinone on the intestinal epithelium CaCC and CFTR;

FIG. 3 Lancequinone inhibits the secretion of intestinal fluids induced by STa and CT in isolated intestinal tracts;

FIG. 4 Lancequinone inhibits cholera toxin and rotavirus infection induced neonatal mouse diarrhea;

FIG. 5 shows the inhibitory effect of lancedrone on intestinal motility.

Detailed Description

In order to better explain the technical solution of the present invention, the following will describe the embodiments of the present invention in further detail with reference to examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The langjiquinone standard used in the present invention was purchased from Dalian Meilun Biotechnology Ltd. The specific information is as follows: the purity HPLC of the lanthaquinone is more than or equal to 98 percent; the molecular formula is as follows: C11H8O 3; molecular weight: 188.18.

example 1 determination of short-circuit Current of inhibitory Effect of Lanceoquinone on TMEM16A

Insert (1.12 cm) grown in Snapwell²Surface area) stably expressing TMEM16A were mounted in a using chamber. FRT cells were bathed in carbonate buffer (apical buffer containing 65mM NaCl, 65mM Na-gluconate, 2.7mM KCl, 1.5mM KH)₂PO₄，0.5mM MgCl₂，2mM CaCl₂10mM Hepes and 10mM glucose, pH 7.4; the substrate membrane side buffer solution contains: 130mM NaCl, 2.7mM KCl,1.5mMKH₂PO₄，0.5mM MgCl₂，2mM CaCl₂10mM Hepes and 10mM glucose, pH 7.4), after 30min, the basement membrane of FRT cells was permeabilized using 250. mu.g/mL amphotericin b. Short circuit current values were measured by VCC MC6 multi-channel voltage/current clamp and recorded using acquisition and analysis 2.3(Worldprecision Instruments, USA.) the results of the experiment are shown in FIG. 1, TMEM16A Selective activator T16A_inh-A01 was able to activate channel opening of TMEM16A, exhibiting Cl^-The current rise phenomenon, lancedaquinone (1-20 μ M), was able to suppress TMEM16A mediated Cl-current in a dose-dependent manner (FIG. 1A). The use of langevine (1 and 5. mu.M) for the basement membrane test had no inhibitory effect on Cl-current, and 20. mu.M gave a slight inhibition; while addition of langevine (1, 5 and 20. mu.M) on the apical membrane side completely suppressed Cl-current (FIG. 1B), demonstrating that langevine suppressed its TMEM16A channel activity by acting directly with TMEM 16A.

Example 2 short-circuit Current measurement of inhibitory Effect of lankeenone on CaCC and CFTR in intestinal epithelium

Insert (1.12 cm) grown in Snapwell²Surface area) of colon cancer HT-29 cells are mounted in a Ussing chamber. The same carbonate buffer (119mM NaCl, 0.6mM KH) was added to the apical and basement membrane chambers of HT-29 cells₂PO₄，2.4mM K₂HPO₄，1.2mM MgCl₂，1.2mM CaCl₂，21mMNaCO₃And 10 mmlucose, pH 7.4), short circuit current values were measured by VCC MC6 multi-channel voltage/current clamp and recorded using Acquireand analysis 2.3(world precision Instruments, usa). As shown in the figure, ATP can stimulate Ca release from cellular calcium stores via P2Y receptors on cell membranes²⁺And further activates the CaCC activity. It was determined that addition of 1-20. mu.M of lankeequinone inhibited ATP-induced Cl-current at the apical membrane of HT-29 cells (FIG. 2A). CPT-cAMP (a non-hydrolyzable, intracellular-permeable cAMP analog) added to extracellular fluid can penetrate the cell membrane into the cell, rapidly increase cAMP level and further activate CFTR to open, and the application of lanuginone to the apical membrane side can suppress the Cl-current mediated by CFTR (FIG. 2B).

Example 3 Lancequinone inhibits the secretion of intestinal fluids induced by STa and CT in isolated intestinal tracts

Male C57 mice (8-10 weeks old) were fasted and kept without water for 24h before the experiment, and at the time of the experiment, the mice were anesthetized by intraperitoneal injection of pentobarbital sodium (40mg/kg), and after small abdominal incisions, 4 closed mid-jejunums (each about 15mm) were separated with sutures. mu.L of physiological saline 100. mu.L containing STa (0.1. mu.g) or CT (1. mu.g) and 100. mu.L containing STa (0.1. mu.g) + lanugenone (20. mu.M) or CT (1. mu.g) + lanugenone (20. mu.M) were injected into the ligated intestinal sections, and the abdominal incision was sutured with a suture, and the mice were allowed to recover from anesthesia. After 4h, mice were sacrificed by intraperitoneal excess sodium pentobarbital (100mg/kg), sections of intestine were removed, intestinal fluid secretion was calculated as weight/length of intestine, data were expressed as mean ± SEs and statistical analysis of module differences was performed by Graphpad Prism 7 software, p < 0.05 represents significant differences, expressed as x. As shown in FIG. 3, in the established model of STa and CT-induced intestinal secretions, fluid accumulation in the sections of STa and CT treated intestines was significantly increased compared to the sections of saline treated intestines, while fluid accumulation in the sections of langevin injected intestines was significantly decreased, which was effective in comparison to the known CFTR inhibitor CFTR_inhThe effect of-172 is similar.

Example 4 Lanceoquinone inhibits cholera toxin and rotavirus infection-induced diarrhea in newborn mice

Neonatal C57 mice (3-4 days) gavage 3. mu.g CT or 25. mu.L (1.2X 10)⁷pfu/mL) monkey SA-11 rotavirus. In the CT-induced mouse diarrhea model, mice were gavaged with 0.4 μ g of lanugenone twice daily for 8h prior to gavage CT and two days after gavage CT. In the rotavirus-induced mouse diarrhea model, the treated mice were gavaged with lanuginone 1 time per day within 3 days after inoculation with rotavirus. The water content of the feces is measured by a watermark method, the concrete operation is that the abdomen of the mouse is slightly kneaded to promote the defecation of the mouse, and the feces is inserted into a small hole (the height is 1.5mm, the diameter is 1mm, and the volume of the feces is 1.17 mm) of a polydimethylsiloxane flat plate³) The cylindrical feces are pushed out by a metal push rod and placed on a cellulose acetate membrane, so that the feces are in contact with the membrane for 2min in a humid environment. Observing the watermark left on the film by the feces through a body type microscope and passing through a digital phaseImages were recorded and the water content of mouse feces was evaluated by measuring the area of the watermark using image J software. As shown in FIG. 4, the water content of feces of both gavage CT and rotavirus-inoculated mice was significantly increased compared to normal mice, especially on

days

1 and 2 after CT infection and

days

2 and 3 after rotavirus inoculation, whereas the water content of feces of mice treated with lanugenon by gavage administration was decreased to various degrees, and was most significantly decreased on

days

1 and 2 after CT infection and

days

2 and 3 after rotavirus inoculation.

Example 5 inhibitory Effect of Lanceoquinone on intestinal motility

C57 (8-10 weeks old) is fasted and fed without water supply for 24 hours before intestinal motility measurement, and then normal saline and E are fed in a stomach irrigation mode_act(TMEM16A activator), lanmoquinone or E_act+ lancedoquinone. After 15min, mice were gavaged with 0.2mL 10% charcoal powder (suspended in 5% acacia gum), and after 30min the mice were sacrificed and the whole intestine was isolated. The peristalsis index (%) — the distance moved by the activated charcoal/the distance from duodenum to cecum × 100. In the smooth muscle contraction experiment, the ileum was removed from the anesthetized mice and washed with cold bicarbonate solution to remove intestinal contents. The ileal segment (. about.10 mM) was connected to the tension sensor and equilibrated in bicarbonate solution for 60min, maintaining a static tension of 1mM, with buffer changes every 20 min. The tension was continuously monitored using tissue organ perfusion system DMT 750TOBS and analyzed using 750TOBS software (Denmark DMT Corp.). Fig. 5A shows that after gavage with lanugenon, the intestinal motility index of activated charcoal in adult mice was 50.1%, lower than that of the normal saline group (66.2%). Due to E_actGastrointestinal motility is promoted by activation of TMEM 16A-mediated rhythmic contractions of the intestine, thus inducing E_actA positive control was set. Intraperitoneal injection E_actAfter (50 μ M), the motility index rose to 83.2% and the effect was reversed by lancedoquinone to a motility index of 56.5% and close to normal physiological levels. Smooth muscle contraction experiments on isolated intestinal segments further validated the gut motor regulation mechanism of lanugenon. Normal adult mice contract in isolated ileum segment with coordinated contractility and spontaneous rhythmicity, and are treated with lancedoquinoneAfter 2min, the intestinal segment contractility decreased significantly. Furthermore, the rhythmicity and coordination of the intestinal segment was gradually lost after 3min of treatment (fig. 5B). Analysis of the tensile record showed that the contraction force was significantly lower for the section of lan-xuequin treated intestine compared to the normal section of intestine, but there was no significant difference in contraction frequency (fig. 5C).

Claims

1. An application of a chloride channel inhibitor lanugo quinone in preparing anti-diarrhea drugs is characterized in that the compound lanugo quinone shown in formula (I) is applied in preparing anti-bacterial diarrhea or viral diarrhea drugs;

2. the application of the chloride ion channel inhibitor lanjiquinone in the preparation of medicines for treating intestinal motility disorder is characterized in that the compound lanjiquinone shown in the formula (I) is applied in the preparation of medicines for treating intestinal motility disorder;

3. the use according to claim 1, wherein the diarrhea comprises cholera toxin-induced diarrhea or rotavirus diarrhea.

4. Use according to claim 1 or 2, characterized in that the compound and the pharmaceutically acceptable salts thereof are formulated as an oral preparation for the treatment of diarrhea and intestinal motility disorder-related diseases.