Application of LMK235 in preparation of medicine for treating chronic urinary tract infection

Abstract

The invention discloses application of a deacetylase inhibitor LMK235 in preparation of a medicine for treating chronic urinary tract infection and a medicine preparation of the deacetylase inhibitor LMK235, and belongs to the field of biological medicine. The chronic urinary tract infection is induced by urethral pathogenic escherichia coli. Experiments show that the LMK235 effectively reduces the bacterial quantity in urine and the bacterial quantity in the bladder by remarkably reducing colonization of urethral pathogenic Escherichia coli in the bladder in a mouse body; the level of inflammatory cytokines IL-1b in the bladder is obviously reduced, so that the chronic bladder inflammation caused by the infection of the urethral pathogenic escherichia coli is improved. The application field of the deacetylase inhibitor LMK235 is widened, an experimental basis is provided for clinical application of the LMK235 in the aspect of preparing the urinary system infection treatment medicine, and the deacetylase inhibitor LMK235 has good application prospects and huge potential value and social significance.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of LMK235 in preparing a drug for treating chronic urinary tract infection and a drug preparation thereof. Background Art

[0002] Urinary tract infection is caused by various pathogens invading the urinary system, among which uropathogenic Escherichia coli (Uropathogenic Escherichia coli , UPEC) is the main pathogen. Based on the genetic, biological and behavioral factors of the host, women are more susceptible to this disease. There are three main results of host-microbe interactions in the urinary tract: a. Asymptomatic bacteriuria; b. Acute infection; c. Some patients are highly susceptible and develop severe acute infection, recurrent infection and chronic disease state. One of the strongest risk factors for urinary tract infection is a history of UTI, but why some people are more likely to develop and maintain UTI remains unclear. The development of long-term persistent infection is called chronic cystitis, that is, chronic urinary tract infection, characterized by persistent high titer bacteriuria, immunopathology and high bladder bacterial load. About 50% of women and 12% of men will have symptomatic infections in their lifetime, 20%-30% of women with UTI will experience recurrent infection (recurrent UTI, rUTI), and about 53% of women over 55 years old will have recurrences, and some people suffer 6 times or more per year. The recurrence rates of pulmonary and gastrointestinal bacterial infections are approximately 10% and 1.5%-12%, respectively, which shows that the recurrence rate of UTI is very high. Long-term unhealed infection can easily lead to renal fibrosis and chronic renal failure. Repeated medical treatment causes tremendous physical, mental and financial pressure on patients, and repeated antibiotic treatment will also destroy the normal flora structure in the body; from a social perspective, the "golden position" of antibiotics in treating UTI is gradually decreasing, and the significant increase in bacterial resistance has brought a heavy medical burden. The overall incidence of UTI has had a great impact at the population level, becoming one of the global public health issues and an issue that urgently needs to be addressed clinically.

[0003] Urinary tract infections are usually treated with antibiotics. However, UPEC strains can still be found in the urinary tract within weeks after antibiotic treatment, and multidrug-resistant strains are also increasing year by year, highlighting the importance of developing alternative treatment strategies. Current research focuses on UPEC virulence factors, developing vaccines against UPEC iron carriers, and some anti-adhesive agents targeting UPEC pili. For example, mannose and its derivatives (mannosides) have been shown to be receptor analogs of the adhesin FimH of type 1 pili, which can block the binding and colonization of UPEC in the bladder, thereby reducing the toxicity of UPEC, and targeting the globular tetrasaccharide of P pili as a non-antibiotic therapy for the treatment of UTI. However, there are relatively few studies on host targets for host-pathogen interactions, and there is a lack of in-depth research on epigenetic regulation mechanisms.

[0004] Histone deacetylase (HDAC) can regulate gene transcription and cell function by removing histone acetylation modification. HDAC can be divided into four categories according to structure and function. Class I HDACs include HDAC1~3 and HDAC8; Class II HDACs include Class IIa (HDAC4, HDAC5, HDAC7, HDAC9) and Class IIb (HDAC6 and HDAC10); Class III HDACs include SIRT1~7; and HDAC11 is the only member of Class IV. Inhibition of HDAC can restore histone acetylation levels and activate anti-inflammatory, apoptosis-inducing or differentiation-related genes. Histone deacetylase inhibitors (HDACi) are currently used clinically to treat various types of cancer. HDAC5 is a key enzyme that regulates gene expression through histone deacetylation. It removes acetyl groups on histone proteins and changes the accessibility of DNA to transcription factors, thereby affecting a variety of cellular processes including inflammation and immune response, making HDAC5 an important target for studying infectious diseases and inflammatory responses. Recent studies have shown that HDAC5 promotes inflammatory responses under the stimulation of specific pathogens. For example, HDAC5 enhances the inflammatory response of macrophages induced by Mycoplasma pneumoniae by activating the NF-kB signaling pathway. Such mechanisms highlight the central role of HDAC5 in mediating inflammatory responses in infectious diseases, enabling the immune system to effectively respond to pathogens. Therefore, inhibiting HDAC5 provides a new type of therapy for the treatment of infectious diseases. HDACi can enhance the host's ability to resist infection by regulating the expression of antimicrobial peptides (such as cathelicidin and defensins). This potential therapeutic significance shows that HDAC5 is an important target for the development of drugs against infection.

[0005] LMK235 is an HDACi with high selectivity for HDAC4 / 5, which can inhibit the activity of HDAC5, HDAC4, HDAC6, HDAC1, HDAC2, HDAC11 and HDAC8, with IC50 values ​​of 4.22 nM, 11.9 nM, 55.7 nM, 320 nM, 881 nM, 852 nM and 1278 nM, respectively. Studies have shown that LMK-235 can improve intestinal dysfunction in sepsis models by inhibiting the NF-kB pathway. In addition, LMK235 inhibits inflammation and fibrosis by inhibiting the lysine-specific demethylase 1-related pathway, thereby improving cardiac dysfunction after myocardial infarction. However, there is still no literature report on the use of LMK235 in the treatment of chronic urinary tract infections. Summary of the invention

[0006] The purpose of the present invention is to provide the use of the sirtuin inhibitor LMK235 in the preparation of a drug for treating chronic urinary tract infection and its pharmaceutical preparation, so as to provide important data for further developing drugs for urinary tract infection.

[0007] To achieve the above purpose, the present invention discloses the following technical contents: The invention discloses an application of a deacetylase inhibitor LMK235 in the preparation of a drug for treating chronic urinary tract infection, wherein the chronic urinary tract infection refers to a chronic bladder infection caused by urethra pathogenic Escherichia coli UPEC.

[0008] The specific application of LMK235 is as follows: (1) by significantly reducing the colonization of urethral pathogenic Escherichia coli in the bladder of mice, the bacterial count in urine and bladder was effectively reduced; (2) by significantly reducing the level of inflammatory cytokine IL-1b in the bladder, thereby improving chronic bladder inflammation caused by urethral pathogenic Escherichia coli infection.

[0009] Preferably, the dosage of LMK235 is 5 mg LMK235 / kg body weight.

[0010] This LMK235 was purchased from MCE (catalog number: HY-18998, CAS number: 1418033-25-6), molecular formula: C 15 H 22 N2O4, molecular weight: 294.35, chemical formula is as follows:

[0011] The present invention further discloses a pharmaceutical composition of LMK235 or a product containing LMK235 and the use of the composition in preparing a drug for treating chronic urinary tract infection. The product containing LMK235 mainly refers to tablets, granules, powders, suspensions, hard capsules, soft capsules or syrups prepared by adding pharmaceutically acceptable pharmaceutical excipients, wherein the tablets include: dispersible tablets, lozenges, chewable tablets or effervescent tablets.

[0012] The composition can be formulated into tablets, dispersible tablets, sugar-coated tablets, granules, dry powders, solutions or capsules. Lactose or starch can be used as carriers for preparing oral pharmaceutical compositions, and gelatin, sodium carboxymethylcellulose, methylcellulose, polyvinyl pyrrolidone, etc. are suitable binders. Starch or microcrystalline cellulose can be selected as disintegrants, often talcum powder, colloidal silica, glyceryl stearate, calcium or magnesium stearate, polyethylene glycol-4000, polyethylene glycol-6000, sodium metabisulfite, etc.; as suitable anti-adhesives and lubricants. For example, tablets can be prepared by pressing wet granules. The active ingredient is mixed with a carrier and optionally with a portion of a disintegrant additive, and the mixture is granulated with an aqueous solution, an alcoholic or an aqueous alcoholic solution of a binder in a suitable device, and the dried granules are then added with other disintegrants, lubricants and anti-adhesives to tablet the mixture.

[0013] Embodiments of the invention relate to methods of treating chronic urinary tract infections using LMK235.

[0014] The implementation method is as follows: 1) Select 6-8 week old SPF female C57BL / 6J mice, give them 5 mg / kg LMK235 intraperitoneally every other day for 2 weeks, monitor the weight, status and survival of the mice, and make it clear that the dose is non-toxic to the mice; 2) Select 6-8 week old SPF female C57BL / 6J mice, use UPEC CFT073 as the infection strain, and construct a chronic UTI model: give the mice 5 mg / kg LMK235 intraperitoneally one day before UPEC urethral infection and every other day for 2 weeks after infection, monitor the weight, status and survival of the mice, collect the urine of each group of mice, dilute and spread it, and count the urine bacteria; the mice were killed 14 days later, and the infection and inflammation of the bladder were evaluated by indicators such as the bladder bacteria count and the level of inflammatory factors in the bladder tissue.

[0015] The embodiments effectively treat chronic urinary tract infections caused by UPEC and avoid the disadvantages of traditional antibiotic-based treatment regimens. Thus, the current embodiments do not contribute to the generation of multi-drug resistant strains and do not adversely affect the microflora in the gastrointestinal tract. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Flow chart of the process of establishing a mouse model of chronic urinary tract infection with LMK235 intervention; Figure 2 This is a body weight comparison chart of normal mice treated with LMK235; Figure 3This is a comparison of the urine bacterial counts of mice with chronic UTI treated with LMK235 1 day after infection (1 dpi) and 14 days after infection (14 dpi); A is the urine bacterial count 1 day after infection, and B is the urine bacterial count 14 days after infection; Figure 4 This is a comparison chart of the bladder bacterial count in mice with chronic urinary tract infection caused by UPEC after LMK235 intervention; Figure 5 This is a comparison of the effects of LMK235 intervention on the level of inflammatory factor IL-1β in the bladder of mice with chronic urinary tract infection caused by UPEC. DETAILED DESCRIPTION

[0017] The present invention is described below through specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention. Unless otherwise specified, the LMK235 used in the present invention was purchased from MCE, and other reagents, methods and equipment are conventional reagents, methods and equipment in the art. The materials such as the kits used in the following examples are all commercially available.

[0018] Example 1: Construction of a mouse model of chronic urinary tract infection treated with LMK235 SPF female C57BL / 6J mice aged 6-8 weeks were selected, and UPEC CFT073 was used as the infection strain to construct a chronic UTI model. The experimental animals were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. The experimental animal production license number is SCXK (Su) 2018-0008; the experimental animal use license number is SYXK (Su) 2020-0048 (barrier environment).

[0019] like Figure 1 As shown, the mice were randomly divided into four groups: normal group (NC), LMK235 intervention group (LMK235), chronic urinary tract infection group (UTI) and LMK235 intervention UTI group (LMK235+UTI). One day before infection, the LMK235 group and LMK235+UTI group were intraperitoneally injected with 5 mg / kg of LMK235, and the NC group and UTI group were intraperitoneally injected with an equal volume of solvent. The infection operation was to select a 24G intravenous indwelling needle, insert it into the mouse urethra with a soft tube, and inoculate 1×10 8CFU / 50 mL of CFT073 bacterial solution, and mice were infected again 24 hours later (in the same way as the first time), which was conducive to the establishment of a chronic UTI model in mice. Chronic cystitis was defined as high titer (>10 4 CFU / mL) and high titers (>10 4 The mice were continuously observed, their body weights recorded, and the amount of bacteria in their urine monitored. 4 CFU / mL is used as an indicator of successful establishment of chronic UPEC infection. Comparison of the body weights of the NC group and the LMK235 group shows that the body weight of mice after intraperitoneal injection of LMK235 is stable, slightly increased, and in good condition, with no difference in body weight from the NC group, indicating that treatment with 5 mg / kg LMK235 (the dosage for synthetic humans is 0.55 mg / kg) has no toxic side effects on mice ( Figure 2 ). Mice were killed 2 weeks after infection, and urine, bladder tissue and other samples were taken for subsequent experiments.

[0020] Example 2: LMK235 treatment significantly reduces urine bacterial counts in chronic UTI mice and reduces the incidence of chronic UTI The urine of mice was collected at different time points after infection and graded dilution was performed, such as Figure 3 As shown in Figure 2, LMK235 treatment can reduce the pyuria caused by infection in mice (visual observation) and reduce the amount of urine bacteria. It can be seen that 1 day after infection, the amount of urine bacteria in the UTI group and the LMK235+UTI group was higher than 10 4 CFU / mL, but the urine bacterial count in the LMK235+UTI group was significantly lower than that in the UTI group ( Figure 3 A). As the infection time increased, the urine bacterial count of some mice gradually decreased within 2 weeks. On day 14 after infection, the urine bacterial count of 7 out of 9 mice in the UTI group was higher than 10 4 CFU / mL, while 5 out of 9 mice in the LMK235+UTI group had urine bacterial counts below 10 4 CFU / mL, did not progress to chronic UTI, and the urine bacterial count in the LMK235+UTI group was significantly lower than that in the UTI group ( Figure 3 B). The above results show that LMK235 significantly reduced the amount of urine bacteria in chronic UTI mice and reduced the occurrence of chronic UTI.

[0021] Example 3: LMK235 treatment significantly reduces UPEC colonization in the bladder of chronic UTI mice Mice were sacrificed 14 days after infection and bladder tissues were taken and weighed for homogenization. The homogenate was diluted in gradients and spread on solid plates. After overnight culture in a 37°C incubator, the number of colonies was counted to evaluate the bacterial load of bladder tissue in the UTI group and the LMK235-intervention UTI group. The results showed that compared with the injection of normal saline, treatment with 5 mg / kg LMK235 significantly reduced the colonization of UPEC in the bladder ( Figure 4 ), indicating that LMK235 can be used to treat chronic bladder infection caused by UPEC.

[0022] Example 4: LMK235 treatment significantly reduces the level of inflammatory factor IL-1β in the bladder of chronic UTI mice The enzyme-linked immunosorbent assay (ELISA) experiment was used to detect the level of inflammatory factor IL-1β in the bladder of chronic UTI mice: the mice were sacrificed 14 days after infection and the bladder tissue was taken. After weighing, it was homogenized with buffer and then centrifuged at 10000 rpm / min for 10 minutes to collect the supernatant. According to the manufacturer's instructions, the level of IL-1β in the tissue supernatant was detected using a mouse IL-1β ELISA kit (Invitrogen, ThermoFisher, USA), and the results were read using a 450nm microplate reader (Bio-RadiMARK, USA). Figure 5 As shown, the IL-1β level in the bladder tissue of the UTI group was significantly higher than that of the NC group, while the IL-1β level in the bladder tissue of the LMK235+UTI group was significantly lower than that of the UTI group. This indicates that LMK235 significantly reduces the level of inflammatory factor IL-1β in the bladder of chronic UTI mice, thereby improving the chronic bladder inflammation caused by UPEC infection.

[0023] Example 5: Tablets containing 35 mg of active ingredient per tablet are prepared as follows: Dosage / tablet LMK235: 35mg Microcrystalline cellulose: 55 mg Starch: 45mg Hydroxymethylcellulose: 4 mg Sodium carboxymethyl starch: 5 mg Magnesium stearate: 1 mg Talc: 1mg Take appropriate amounts of each ingredient, sieve the active ingredient, starch and cellulose, and mix them thoroughly; mix the hydroxymethylcellulose solution with the above powder, sieve to obtain wet granules, dry at 50-60°C, pre-sieve sodium carboxymethyl starch, magnesium stearate and talc, and then add them to the above granules for tableting.

[0024] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Use of the deacetylase inhibitor LMK235 or a product containing LMK235 in the preparation of a drug for treating chronic urinary tract infection.

2. The use according to claim 1, wherein the chronic urinary tract infection refers to: chronic bladder infection caused by urethral pathogenic Escherichia coli.

3. Use of the deacetylase inhibitor LMK235 or a product containing LMK235 in the preparation of drugs for protecting the health of the urinary system.

4. The use according to claim 1, characterized in that: The dosage of LMK235 is 5 mg / kg.

5. A pharmaceutical composition containing the sirtuin inhibitor LMK235 or a product containing LMK235 according to claim 1, characterized in that: Pharmaceutically acceptable excipients are added to prepare tablets, granules, powders, suspensions, hard capsules, soft capsules or syrups, wherein the tablets include dispersible tablets, lozenges, chewable tablets or effervescent tablets.

6. Use of the deacetylase inhibitor LMK235 or a pharmaceutical composition containing LMK235 according to claim 5 in the preparation of a drug for treating chronic urinary tract infection.

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