Application of Prevotella in the preparation of a drug for treating or adjuvantly treating hypertension

Abstract

The present invention relates to the use of Prevotella copri in preparing a drug for treating or assisting in treating hypertension. The present invention confirms for the first time the key role of Prevotella copri in treating hypertension and verifies it in a rat model. By increasing the number of Prevotella copri in the intestinal flora, blood pressure can be effectively lowered. In addition, Prevotella copri is a microorganism that normally exists in the intestine and does not produce additional side effects on the human body. It has an unparalleled drug safety advantage over similar drugs. The drug and pharmaceutical composition prepared by using Prevotella copri can be used to treat hypertension diseases including essential hypertension and salt-sensitive hypertension, and have important application value.

Description

Technical Field

[0001] Embodiments of the present invention generally relate to the field of biomedical technologies, and more specifically to the use of Prevotella in the preparation of a medicament for treating or adjuvantly treating hypertension. Background Art

[0002] In recent years, the incidence of hypertension has increased rapidly. Hypertension is a clinical syndrome mainly manifested by elevated systemic arterial pressure, and it is a systemic disease that can be accompanied by functional or organic changes in organs such as the heart, brain, blood vessels, and kidneys. Since there is a close causal relationship between the human blood pressure level and the incidence and mortality risks of cardiovascular diseases, preventing and controlling hypertension has become the core strategy to curb the development of cardiovascular diseases at present. Once diagnosed, all hypertensive patients are recommended to initiate drug treatment immediately while intervening in lifestyle.

[0003] According to the mechanism of action of drugs, currently, the drugs used for targeted treatment of hypertension are divided into five categories: angiotensin-converting enzyme inhibitors, diuretics, receptor blockers, calcium antagonists, and angiotensin receptor antagonists. However, in clinical applications, the effective rate of antihypertensive drugs is only 60% - 80%, but the incidence rate of side effects is as high as 15% - 20%. In addition, hypertension treatment also faces problems such as low treatment rate, low control rate, and poor compliance of patients. Therefore, it is extremely urgent to develop safer and more efficient antihypertensive drugs.

[0004] Although current research shows that the gut microbiota in the human body is closely related to the occurrence and development of diseases such as hypertension and diabetes, due to the complex types of microbiota and the incomplete understanding of the mechanism of action, the application of gut bacteria in the treatment of the above diseases is not extensive, and researchers mainly focus on diabetes related to the digestive system at present, while hypertension diseases are hardly involved. Summary of the Invention

[0005] In view of the above problems, the present invention provides the use of Prevotella copri in the preparation of a medicament for treating or adjuvantly treating hypertension, and further provides a medicament and a pharmaceutical composition for treating or adjuvantly treating hypertension.

[0006] According to the first aspect of the present invention, there is provided the use of Prevotella copri in the preparation of a medicament for treating or adjuvantly treating hypertension.

[0007] The Prevotella copri is a known gut colonizing bacterium, and all Prevotella copri mentioned in the present invention can be obtained through publicly available channels on the market.

[0008] In one embodiment, the Prevotella copri is viable Prevotella copri cells and / or the culture supernatant of Prevotella copri.

[0009] In one embodiment, the hypertensive disease is essential hypertension or salt-sensitive hypertension.

[0010] In one embodiment, the drug comprises a pharmaceutically effective dose of Prevotella copri and a pharmaceutically acceptable carrier.

[0011] The pharmaceutically acceptable carrier is one or more of skim milk, lactose, glucose, sucrose, sorbitol, mannose, trehalose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl paraben, propyl paraben, talc, magnesium stearate or mineral oil.

[0012] In one embodiment, the drug can be in any pharmaceutically feasible dosage form or forms, including but not limited to tablets, capsules, oral liquids or lyophilized powders.

[0013] Specifically, the drug can be in dosage forms such as capsules, soft capsules, orally disintegrating tablets, chewable tablets, buccal tablets, sublingual tablets, dripping pills, granules, infusion, powders, suspensions, sustained-release tablets and sustained-release capsules, solutions, suspensions, pills, etc. These dosage forms contain various excipients, disintegrants, binders, lubricants, sweeteners, flavoring agents, coloring agents, emulsifiers and diluents, etc.

[0014] According to a second aspect of the present invention, there is provided a drug for treating or adjuvantly treating hypertension, the drug comprising a pharmaceutically effective dose of Prevotella copri.

[0015] In one embodiment, the Prevotella copri is viable Prevotella copri cells and / or the culture supernatant of Prevotella copri.

[0016] According to a third aspect of the present invention, there is provided a pharmaceutical composition for treating or adjuvantly treating hypertension, the pharmaceutical composition comprising a pharmaceutically effective dose of Prevotella copri. The pharmaceutical composition further comprises other drugs or drug ingredients that can be used for treating or adjuvantly treating hypertension.

[0017] In one embodiment, the Prevotella copri is viable Prevotella copri bacteria and / or the culture supernatant of Prevotella copri.

[0018] Based on the above technical solution, the following beneficial effects are achieved:

[0019] 1. The present invention first confirms the key role of Prevotella copri in the treatment of hypertension diseases, which has been verified in various hypertensive rat models. By increasing the number of Prevotella copri in the intestinal flora, blood pressure can be effectively reduced. Moreover, since Prevotella copri itself is a microorganism that normally exists in the intestine and will not cause additional side effects to the human body, it has a drug safety advantage that cannot be compared with similar drugs. It is easy for patients to accept and has clinical promotion value, and is expected to solve the problems of poor treatment effect of hypertension, low patient compliance, and large drug side effects. In addition, Prevotella copri is simple to obtain and can be quickly put into production to obtain highly effective preparations.

[0020] 2. The present invention uses the key role of Prevotella copri in the treatment of hypertension diseases to prepare corresponding products such as drugs and pharmaceutical compositions. These products can be used to treat various hypertension diseases including essential hypertension and salt-sensitive hypertension, and have important application values.

[0021] It should be understood that the content described in this part is not intended to identify the key or important features of the embodiments of the present invention, nor is it used to limit the scope of the present invention. Other features of the present invention will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Combined with the drawings and referring to the following detailed description, the above and other features, advantages and aspects of the embodiments of the present invention will become more obvious. In the drawings, the same or similar reference numerals denote the same or similar elements.

[0023] Figure 1 Shows the relative abundances and differential strains of the top 15 strains in terms of relative abundance in the fecal samples of healthy people (Health) and newly diagnosed hypertensive patients (HTN) in Example 1 of the present invention; where:

[0024] A is a schematic diagram of the top 15 strains in terms of relative abundance in the Health group and the HTN group, and the vertical axis represents the relative abundance;

[0025] Figure B shows the comparison of the relative abundances of Prevotella copri in the Health group and the HTN group. The vertical axis represents the relative abundance, * p < 0.05;

[0026] Figure C shows the differential strain map between the Health group and the HTN group by LEfSe analysis.

[0027] Figure 2 It shows the effects of Prevotella copri in Example 3 of the present invention on the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of rats in a spontaneously hypertensive rat model (SHR); where:

[0028] Figure A is a schematic diagram of the change in SBP of SHR rats after adding Prevotella copri;

[0029] Figure B is a schematic diagram of the change in DBP of SHR rats after adding Prevotella copri;

[0030] SHR-control represents the control group, SHR-ABX represents the pseudo-sterile model group, SHR-ABX-PC represents the pseudo-sterile + PC group, and SHR-PC represents the PC group.

[0031] Figure 3 It shows the effects of Prevotella copri in Example 4 of the present invention on the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of mice in an AngII-induced hypertensive mouse model; where:

[0032] Figure A is a schematic diagram of the change in SBP of AngII-induced mice after adding Prevotella copri;

[0033] Figure B is a schematic diagram of the change in DBP of AngII-induced mice after adding Prevotella copri;

[0034] control represents the normal saline control group, model represents the AngII + solvent treatment group, PC represents the AngII + PC treatment group, ** p < 0.01, *** p < 0.001.

[0035] Figure 4 It shows the effects of Prevotella copri in Example 5 of the present invention on the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of rats in a high-salt-induced hypertensive rat model (HSD); where:

[0036] Figure showing the changes in SBP of HSD rats after adding Prevotella copri

[0037] Figure showing the changes in DBP of HSD rats after adding Prevotella copri

[0038] control represents the gavage solvent control group, PC represents the gavage Prevotella copri group, * p < 0.05, ** p < 0.01. Detailed implementation manners

[0039] The following describes exemplary embodiments of the present invention with reference to the accompanying drawings. Various details of the embodiments of the present invention are included to facilitate understanding, and they should be considered merely exemplary. Therefore, those of ordinary skill in the art should recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present invention. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0040] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. The test reagents used in the following embodiments are all conventional biochemical reagents without special instructions; the experimental methods are all conventional methods without special instructions.

[0041] The blood pressures of the healthy control groups involved in the embodiments are all normal, and the hypertensive rats and model rats all meet the diagnostic criteria.

[0042] Fecal specimens, reagent materials used in the embodiments and their sources:

[0043] 1. Specimens

[0044] The feces used in Example 1 were from healthy people and newly diagnosed hypertensive patients, and the feces used in Example 2 were from spontaneously hypertensive rats (SHR) and control rats (WKY).

[0045] 2. Main reagent materials.

[0046] Fecal microbial genomic DNA extraction kit (QIAamp® PowerFecal® Pro DNA); TB green (Takara); Emerald Amp PCR Master Mix (Takara); 7-week-old male SHR rats, Wistar-Kyoto (WKY) rats and Wistar rats (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.); 7-week-old c57 mice (Shanghai Slake Laboratory Animal Co., Ltd.); 8% NaCl feed, 0.4% NaCl feed (Shuyu Biotechnology (Shanghai) Co., Ltd.); pipette tips (specifications: 10μL, 100μL and 1mL), EP tubes (10mL, 100mL and 1mL) and eight-tube strips (0.2mL) (Axygen); sterile centrifuge tubes (15mL and 50mL), pipettes (5mL and 10mL); intelligent non-invasive blood pressure monitor for mice and rats (Beijing Ruanlong Biotechnology Co., Ltd.); Prevotella copri bacteria used for gavage (DSM 18205, Shangcheng Beina Chuanglian Biotechnology Co., Ltd.).

[0047] ABX: a suspension of 200 mg / mL vancomycin, 400 mg / mL ampicillin, 400 mg / mL neomycin, and 400 mg / mL metronidazole (antibiotics purchased from Sangon Biotechnology), administered intragastrically at a volume of 0.5 mL / kg.

[0048] The components of mGAM bacterial culture medium are as follows (per 1000 mL system): 15.0 g of pancreatic peptone, 10.0 g of tryptone, 3.0 g of soy peptone, 5.0 g of yeast extract powder, 2.0 g of beef powder, 13.5 g of digested serum powder, 1.2 g of beef liver extract powder, 3.0 g of glucose, 2.5 g of potassium dihydrogen phosphate, 3.0 g of sodium chloride, 0.3 g of soluble starch, 0.3 g of L-cysteine, 0.15 g of sodium thioglycolate, pH value 7.2.

[0049] Prevotella copri bacteria were used for pre-oral gavage treatment: Prevotella copri bacteria were stored in a freezing solution (glycerol:mGAM=1:9) at -80℃. When reviving, the Prevotella copri strain was completely thawed in the freezing solution in an anaerobic incubator, and then the bacterial solution was distributed to an appropriate amount of mGAM medium on a clean bench, gently mixed, and cultured in an anaerobic incubator for 24 h. When subculturing, 1 mL of the bacterial solution was added to 40 mL of fresh blank medium and mixed. Before gavage, the bacterial solution was centrifuged in a high-speed refrigerated centrifuge (4000 rpm, 10 min), the supernatant was removed, and the precipitate was resuspended in sterile PBS to prepare a bacterial suspension of 1010 CFU / mL. The diluted bacterial solution sample was divided into sterilized 1.5 mL centrifuge tubes, sealed with sealing strips, and placed on ice for standby and transportation.

[0050] ABX: a suspension of 200 mg / mL vancomycin, 400 mg / mL ampicillin, 400 mg / mL neomycin, and 400 mg / mL metronidazole (antibiotics purchased from Sangon Biotechnology), administered intragastrically at a volume of 0.5 mL / kg.

[0051] Example 1: Differences in intestinal flora between healthy people and patients with newly diagnosed hypertension

[0052] The samples in this example were collected from some hypertensive patients and healthy people who visited the Cardiovascular Hospital of the First Affiliated Hospital of Shantou University Medical College from September 2022 to September 2023. All study participants were over 18 years old. The hypertension identification standard is defined in accordance with the "Guidelines for the Prevention and Treatment of Hypertension in China (Revised Edition 2018)": systolic blood pressure (SBP) ≥ 140 mm Hg and (or) diastolic blood pressure (DBP) ≥ 90 mm Hg for three consecutive measurements of office blood pressure on different days.

[0053] After fasting overnight as instructed, the participants of the above study collected stool samples using sterile stool collection boxes, quickly placed them in ice boxes, and transported them to a -80°C environment as soon as possible for storage for subsequent experiments. The patients' basic information (name, gender, age, height, weight, contact information) and necessary clinical information reflecting liver and kidney function and blood lipids were collected through electronic medical records and follow-up.

[0054] The QIAamp® PowerFecal® Pro DNA extraction kit was used to extract microbial genomic DNA from stool samples according to the instructions.

[0055] Metagenomic testing was performed on stool DNA samples from healthy people (Health) and patients with newly diagnosed hypertension (HTN). The results are shown in Figure 1, compared with the healthy population group, the abundance of Prevotella copri decreased in the group of newly diagnosed hypertensive patients ( Figure 1 A), and a t-test found that the abundance of Prevotella copri increased significantly in the healthy population group ( Figure 1 B), and Lefse analysis showed that Prevotella copri was significantly enriched in the healthy population group ( Figure 1 C).

[0056] Example 2: Abundance difference of Prevotella in the intestines of spontaneously hypertensive rats (SHR) and control rats (WKY)

[0057] Seven-week-old spontaneously hypertensive rats (SHR) and WKY (Wistar Kyoto) rats as controls were housed in the Experimental Animal Department of Central South University. The experimental animals were housed in a SPF environment, maintaining a 12-hour day-night cycle, with the temperature maintained at 23 - 26 °C and the humidity maintained at 50 ± 10%. They were fed with standard experimental animal feed and the animals had access to water at any time.

[0058] After one week of adaptive feeding, animal fecal samples were collected using sterilized filter paper into sterile cryotubes and immediately frozen in liquid nitrogen, and then stored at -80 °C for subsequent experiments.

[0059] The fecal samples of spontaneously hypertensive rats SHR and control rats WKY were subjected to 16S rRNA gene sequencing, and the results showed obvious changes in the abundance levels of Prevotella in both. For example, compared with the WKY group, the relative abundance of Prevotellaceae_Ga6A1_group in the SHR group decreased by 3 times, the relative abundance of Prevotellaceae_NK3B31_group decreased by 4 times, and the relative abundance of Prevotella_9 decreased by 17 times. And through real-time quantitative PCR using specific primers, the results found that Prevotella copri decreased by more than 200 times in SHR rats compared with WKY.

[0060] Example 3: Effect of Prevotella copri on blood pressure in a spontaneously hypertensive rat model (SHR)

[0061] Seven-week-old spontaneously hypertensive rats (SHR) were housed in the Experimental Animal Department of Central South University. The experimental animals were housed in a SPF environment, maintaining a 12-hour day-night cycle, with the temperature maintained at 23 - 26 °C and the humidity maintained at 50 ± 10%. They were fed with standard experimental animal feed and the animals had access to water at any time.

[0062] Number and weigh the experimental animals, and randomly group them to ensure that the weights of the experimental animals in each group are comparable. In this example, SHR rats were divided into four groups of 10 rats each, namely the control group (SHR-control), the pseudo-sterile model group (SHR-ABX), the pseudo-sterile + PC group (SHR-ABX-PC), and the PC group (SHR-PC).

[0063] The pseudo-sterile model group was continuously gavaged with a combination of four antibiotics (ampicillin 20 mg / kg, vancomycin 10 mg / kg, neomycin sulfate 20 mg / kg, and metronidazole 20 mg / kg) for 5 days to try to knockdown the intestinal bacteria in the rats to simulate a sterile environment. Three days after stopping the drug, the pseudo-sterile + PC group and the PC group were continuously gavaged with the prepared PC bacterial solution once a day, and each rat was gavaged with 1 mL of diluted Prevotella copri bacterial solution each time. The control group and the pseudo-sterile model group were both gavaged with an equal volume of PBS for 5 weeks. During this process, the changes in the blood pressure of the rats were continuously recorded every week before and after giving the bacteria.

[0064] The tail cuff method was used to measure the blood pressure of the rats. After the rats were fixed by a special fixator, they were placed in the instrument and preheated at 39°C for 15 - 20 min to fully dilate the blood vessels in their tails for blood pressure measurement. The blood pressure was measured 3 times at intervals of 10 s. The starting point of the waveform was taken as the systolic blood pressure, and the highest point of the waveform was taken as the diastolic blood pressure. A relatively quiet environment was required during the measurement, and the blood pressure measurement was completed by the same person. All rats underwent blood pressure adaptation training before gavage treatment to reduce inaccurate blood pressure caused by animal stress.

[0065] See Figure 2 , and it was found that there was no difference in the change of SBP between the control group and the pseudo-sterile model group of rats. However, regardless of the normal flora state or the pseudo-sterile state after antibiotic treatment, after adding Prevotella copri bacteria, the SBP of the pseudo-sterile + PC group and the PC group decreased by about 20 mmHg ( Figure 2 A), and the decrease was significant; the DBP of the pseudo-sterile + PC group decreased slightly compared with the pseudo-sterile model group, and the DBP of the PC group decreased by about 20 mmHg compared with the control group ( Figure 2 B).

[0066] Example 4: Effect of Prevotella copri bacteria on blood pressure in a mouse model of angiotensin II (AngII)-induced hypertension

[0067] 7-week-old c57 mice were raised in the Experimental Animal Department of Central South University. The experimental animals were raised in a SPF environment, maintaining a 12-hour day-night cycle, with the temperature maintained at 23 - 26°C and the humidity maintained at 50 ± 10%. They were fed with standard experimental animal feed and the animals could drink water at any time.

[0068] One day before the operation, the implanted capsule osmotic minipump (RWD, 1002w) was placed in physiological saline and activated in an incubator at 37 °C for 24 hours. The body weight of the mice was weighed, and the total amount of Ang II required for the experiment was calculated (the volume of each micro-sustained release pump was 100 μl). The liquid preparation and pump filling were carried out in a sterile laminar flow hood, and the control group was perfused with an equal volume of physiological saline.

[0069] The mice were anesthetized with isoflurane using an anesthesia machine. After disinfection with 75% ethanol in the surgical area of the neck, a vertical incision about 1 cm long was made at the back of the neck of the mice. Hemostatic forceps were used to bluntly separate the skin and subcutaneous tissue, and the implanted capsule osmotic minipump was buried subcutaneously for Ang II (500 ng / kg / min) or physiological saline perfusion to establish an Ang II-induced hypertensive mouse model.

[0070] The model mice were numbered and randomly divided into groups to ensure that the body weights of the experimental animals in each group were comparable. In this example, the SHR rats were divided into three groups, with 10 rats in each group, namely the physiological saline control group (control), the Ang II + solvent treatment group (model), and the Ang II + PC treatment group (PC).

[0071] The three groups of mice were perfused with an equal volume of physiological saline or Ang II at regular intervals through the minipump. The Ang II + PC treatment group was gavaged with the prepared PC bacterial solution, once a day, 1 mL of diluted Prevotella copri bacterial solution per mouse each time. The Ang II + solvent treatment group was gavaged with an equal volume of PBS for two weeks. During this process, the blood pressure of the mice was measured on the 5th, 9th, and 13th days.

[0072] See Figure 3 , and it was found that after adding Prevotella copri, the blood pressure of the mice in the Ang II + PC treatment group was significantly lower than that in the Ang II + solvent treatment group. Among them, the SBP decreased by about 30 mmHg ( Figure 3 A), and the DBP decreased by about 20 mmHg ( Figure 3 B).

[0073] Example 5: Effect of Prevotella copri on blood pressure in a high-salt-induced hypertensive rat model

[0074] 7-week-old Wisar mice were housed in the Experimental Animal Department of Central South University. The experimental animals were kept in a SPF environment, with a 12-hour day-night cycle, the temperature maintained at 23 - 26 °C, the humidity maintained at 50 ± 10%, and they were fed with standard experimental animal feed and had free access to water.

[0075] Wistar rats were given 8% high-salt diet to develop a hypertension model. Then, the high-salt-induced hypertensive model rats were randomly divided into two groups: the control group (control) and the PC group (PC). The PC group was continuously gavaged with the prepared PC bacterial solution once a day, 1 mL of diluted Prevotella copri bacterial solution per rat each time, and the control group was gavaged with an equal volume of PBS for four weeks. During this process, the blood pressure changes of the rats were recorded continuously every week before and after bacteria administration.

[0076] See Figure 4 , and it was found that the addition of Prevotella copri could significantly reduce the blood pressure of high-salt-induced hypertensive rats, with the SBP decreasing by about 15 mmHg ( Figure 4 A), and the DBP decreasing by about 15 mmHg ( Figure 4 B).

[0077] The above results indicate that Prevotella copri plays a key role in the prevention and treatment of hypertension. It has been verified that the relative abundance of Prevotella copri is significantly decreased in hypertensive patients, in the primary hypertensive rat model (SHR), the AngII-induced hypertensive mouse model, and the high-salt-induced hypertensive rat model. After further intervention with Prevotella copri in the primary hypertensive rat model (SHR), the AngII-induced hypertensive mouse model, and the high-salt-induced hypertensive rat model, it was found that the blood pressure of the experimental subjects in each model was significantly reduced. In summary, Prevotella copri can reduce systolic blood pressure and diastolic blood pressure, with good therapeutic effects. Moreover, since it is a normal microorganism in the intestine and will not cause additional side effects to the human body, it has a drug safety advantage that cannot be compared with similar drugs.

[0078] In addition, the species diversity of the Prevotella genus is very high, and it is difficult to directly infer the characteristics of a specific Prevotella species to the rest of the Prevotella. At present, there is no research report on the relationship between Prevotella copri and hypertension. This invention clearly demonstrates for the first time the therapeutic effect of Prevotella copri on hypertension at the species level.

[0079] The embodiments of the present invention have been described above. The above description is exemplary and not exhaustive, and is also not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The choice of terms used herein is intended to best explain the principles of the embodiments, the practical application, or the improvement of the technology in the market, or to enable other ordinary technicians in the art to understand the embodiments disclosed herein.

Claims

1. Application of Prevotella in the preparation of a medicament for treating hypertension, wherein the strain number of the Prevotella is DSM 18205. Prevotella copri Prevotella copri The Prevotella Prevotella copri Prevotella copri has the strain number DSM 18205.

2. The Prevotella bacterium according to claim 1 Prevotella copri Use in the preparation of a medicament for treating hypertension, characterized in that The Prevotella bacterium Prevotella copri is the Prevotella bacterium Prevotella copri in live cell form.

3. The Prevotella bacterium according to claim 1 Prevotella copri Use in the preparation of a medicament for treating hypertension, characterized in that The hypertension is essential hypertension or salt-sensitive hypertension.

4. The Prevotella bacterium according to claim 1 Prevotella copri Use in the preparation of a medicament for treating hypertension, characterized in that The drug comprises a pharmaceutically effective dose of Prevotella bacteria Prevotella copri and a pharmaceutically acceptable carrier.

5. Use of the Prevotella bacterium according to claim 1 Prevotella copri in the preparation of a medicament for treating hypertension, characterized in that The dosage form of the drug is tablet, capsule, oral liquid or lyophilized powder.

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