Use of lmk235 in the manufacture of a medicament for the treatment of chronic urinary tract infections

Abstract

The application discloses application of deacetylase inhibitor LMK235 in preparation of a medicine for treating chronic urinary tract infection and a medicine preparation thereof, and belongs to the field of biological medicines. The chronic urinary tract infection is induced by uropathogenic Escherichia coli. Experiments show that LMK235 can effectively reduce the amount of bacteria in urine and the amount of bacteria in the bladder by significantly reducing the colonization of uropathogenic Escherichia coli in the bladder in mice; and the chronic inflammation of the bladder caused by uropathogenic Escherichia coli infection can be improved by significantly reducing the level of inflammatory cytokine IL-1b in the bladder. The application widens the application field of the deacetylase inhibitor LMK235, provides an experimental basis for clinical application of LMK235 in preparation of a medicine for treating urinary system infection, and has a good application prospect, great potential value and social significance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of LMK235 in the preparation of drugs for treating chronic urinary tract infections and its pharmaceutical formulations. Background Technology

[0002] Urinary tract infections are caused by various pathogens invading the urinary system, among which urethral pathogenic Escherichia coli (Uropathogenic Escherichia coli) is one such pathogen. Escherichia coli Urinary tract infection (UTI) is the most common pathogen. Women are more susceptible to this disease due to host genetic, biological, and behavioral factors. Host-microbe interactions in the urinary tract produce three main outcomes: a. asymptomatic bacteriuria; b. acute infection; c. a high susceptibility in some patients to develop severe acute infection, recurrent infection, and a chronic disease state. One of the strongest risk factors for UTI is a history of UTI, but why some people are more likely to develop and maintain UTI remains unclear. The development of a long-term persistent infection is called chronic cystitis, or chronic urinary tract infection, characterized by persistent high-titer bacteriuria, immunopathology, and a high bladder bacterial load. Approximately 50% of women and 12% of men will experience a symptomatic infection in their lifetime. 20%–30% of women with UTI will experience recurrent UTI (rUTI), with approximately 53% of women over 55 experiencing recurrence, and some suffering six or more times per year. The recurrence rates for pulmonary and gastrointestinal bacterial infections are approximately 10% and 1.5%–12%, respectively, demonstrating the very high recurrence rate of UTI. Prolonged, untreated infections can easily lead to renal fibrosis and chronic renal failure. Repeated medical visits place enormous physical, psychological, and financial burdens on patients, and repeated antibiotic treatments can disrupt the body's normal flora. From a societal perspective, the "golden opportunity" for antibiotic treatment of UTIs is gradually diminishing, and the significant increase in bacterial resistance is creating a heavy medical burden. The overall incidence of UTIs has already had a significant impact on the population level, becoming a global public health issue and a problem that urgently needs to be addressed clinically.

[0003] Urinary tract infections (UTIs) are typically treated with antibiotics; however, UPEC strains can still be found in the urinary tract weeks after antibiotic treatment, and the number of multidrug-resistant strains is increasing annually, highlighting the importance of developing alternative treatment strategies. Current research focuses on the virulence factors of UPECs, developing vaccines targeting UPEC siderophores and anti-adhesion agents targeting UPEC pili. For example, mannose and its derivatives (mannosides) have been shown to be receptor analogs of the type 1 pili's adhesin FimH, blocking UPEC binding and colonization in the bladder, thereby attenuating UPEC virulence, and globular tetrasaccharides targeting P pili are being explored as non-antibiotic therapies for UTIs. However, research on host targets of host-pathogen interactions is relatively limited, and in-depth studies on epigenetic regulatory mechanisms are still lacking.

[0004] Histone deacetylases (HDACs) regulate gene transcription and cellular function by removing histone acetylation modifications. HDACs are classified into four classes based on their 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 Class IV member. Inhibition of HDACs can restore histone acetylation levels and activate genes related to anti-inflammation, apoptosis induction, or differentiation. 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. By removing acetyl groups from histone proteins, it alters DNA accessibility to transcription factors, thereby affecting various cellular processes, including inflammation and immune responses, making HDAC5 an important target for studying infectious diseases and inflammatory responses. Recent studies have shown that HDAC5 promotes inflammatory responses in response to specific pathogens. For example, HDAC5 enhances the Mycoplasma pneumoniae-induced macrophage inflammatory response by activating the NF-κB signaling pathway. These mechanisms highlight the central role of HDAC5 in mediating inflammatory responses in infectious diseases, enabling the immune system to respond effectively to pathogens. Therefore, inhibiting HDAC5 offers a novel therapeutic approach for infectious diseases. HDACi can enhance the host's anti-infective capabilities by regulating the expression of antimicrobial peptides such as cathelicidin and defenseins. This potential therapeutic implication suggests that HDAC5 is an important target for developing drugs targeting infections.

[0005] LMK235 is an HDACi with high selectivity for HDAC4 / 5, inhibiting 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 a sepsis model by inhibiting the NF-κB pathway. Furthermore, LMK235 can suppress inflammation and fibrosis by inhibiting the lysine-specific demethylase 1-related pathway, thereby improving post-myocardial infarction heart failure. However, there are no published reports on the therapeutic effects of LMK235 on chronic urinary tract infections. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the deacetylase inhibitor LMK235 in the preparation of drugs for treating chronic urinary tract infections and its pharmaceutical formulation, providing important data for the further development of drugs targeting urinary tract infections.

[0007] To achieve the above objectives, the present invention discloses the following technical contents:

[0008] The use of the deacetylase inhibitor LMK235 in the preparation of a drug for treating chronic urinary tract infections. The chronic urinary tract infection referred to here is a chronic bladder infection caused by urethral pathogenic Escherichia coli (UPEC).

[0009] The specific application of LMK235 is as follows: (1) by significantly reducing the colonization of urethropathogenic Escherichia coli in the bladder in mice, the amount of bacteria in urine and bladder is effectively reduced; (2) by significantly reducing the level of the inflammatory cytokine IL-1b in the bladder, the chronic inflammation of the bladder caused by urethropathogenic Escherichia coli infection is improved.

[0010] Preferably, the dosage of LMK235 is 5 mg LMK235 / kg body weight.

[0011] This LMK235 was purchased from MCE (Catalogue No.: HY-18998, CAS No.: 1418033-25-6), molecular formula: C 15 H 22 N₂O₄, molecular weight: 294.35, chemical formula as follows:

[0012] This invention further discloses pharmaceutical compositions containing LMK235 or LMK235-containing products, and the use of such compositions in the preparation of medicaments for treating chronic urinary tract infections. The LMK235-containing products mainly refer 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.

[0013] The composition can be formulated into tablets, dispersible tablets, sugar-coated tablets, granules, dry powders, solutions, or capsules. For the preparation of oral pharmaceutical compositions, lactose or starch can be used as a carrier, with gelatin, sodium carboxymethyl cellulose, methylcellulose, polyvinylpyrrolidone, etc., being suitable binders. Starch or microcrystalline cellulose can be used as disintegrants, and talc, colloidal silica gel, glyceryl stearate, calcium or magnesium stearate, polyethylene glycol-4000, polyethylene glycol-6000, sodium metabisulfite, etc., are commonly used as anti-adhesives and lubricants. For example, tablets can be prepared by compressing wet granules. The active ingredient is mixed with a carrier and, selectively, a disintegrant additive. This mixture, along with an aqueous, alcoholic, or aqueous-alcoholic solution of the binder, is granulated in a suitable device. The dried granules are then compressed into tablets with the addition of other disintegrants, lubricants, and anti-adhesives.

[0014] The embodiments described in this invention relate to a method for treating chronic urinary tract infections using LMK235.

[0015] The implementation method is as follows: 1) Select 6-8 week old SPF-grade female C57BL / 6J mice and administer 5 mg / kg of LMK235 via intraperitoneal injection every other day for 2 weeks. Monitor the weight, condition, and survival of the mice to confirm that this dose has no toxic effect on the mice; 2) Select 6-8 week old SPF-grade female C57BL / 6J mice and use UPEC CFT073 as the infecting strain to construct a chronic UTI model: Administer 5 mg / kg of LMK235 via intraperitoneal injection every other day for 1 day before and 2 weeks after UPEC transurethral infection of the mice. Monitor the weight, condition, and survival of the mice and collect urine from each group of mice. Dilute and spread the urine to count the bacterial count; 14 days later, sacrifice the mice and assess the infection and inflammation of the bladder using indicators such as bladder bacterial count and bladder tissue inflammatory factor levels.

[0016] This implementation method effectively treats chronic urinary tract infections caused by UPEC and avoids the drawbacks of traditional antibiotic-based treatments. Therefore, the current implementation method does not contribute to the development of multidrug-resistant strains or adversely affect the gut microbiota. Attached Figure Description

[0017] Figure 1 Flowchart of the process for establishing a mouse model of chronic urinary tract infection using LMK235 intervention;

[0018] Figure 2 A comparison of body weight in normal mice treated with LMK235;

[0019] Figure 3Comparison chart of the bacterial counts in the urine of mice with chronic UTI intervened by LMK235 at 1 day post-infection (1 dpi) and 14 days post-infection (14 dpi); where A shows the bacterial count in the urine at 1 dpi and B shows the bacterial count in the urine at 14 dpi.

[0020] Figure 4 Comparison chart of the bacterial counts in the bladder of mice with chronic urinary tract infection caused by UPEC intervened by LMK235.

[0021] Figure 5 Comparison chart of the level of inflammatory factor IL-1β in the bladder of mice with chronic urinary tract infection caused by UPEC intervened by LMK235. Detailed implementation manners

[0022] The present invention will be described below through specific implementation examples. Unless otherwise specified, the technical means used in the present invention are all methods well-known to those skilled in the art. In addition, the implementation examples should be understood as illustrative and not restrictive of the scope of the present invention. The essence and scope of the present invention are only defined by the claims. For those skilled in the art, without departing from the essence and scope of the present invention, various changes or modifications to the material components and dosages in these implementation examples also fall within the protection scope of the present invention. Unless otherwise specified, LMK235 used in the present invention is purchased from MCE Company, and other reagents, methods and equipment are all conventional reagents, methods and equipment in the technical field. The materials such as the kits used in the following examples are all commercially available.

[0023] Example 1: Construction of a mouse model of chronic urinary tract infection treated with LMK235

[0024] Select 6-8-week-old SPF-grade female C57BL / 6J mice, and use UPEC CFT073 as the infection strain to construct a chronic UTI model. The experimental animals are purchased from Jiangsu Jicui Yakang Biotech Co., Ltd. Experimental animal production license number: SCXK (Su) 2018-0008; Experimental animal use license number: SYXK (Su) 2020-0048 (barrier environment).

[0025] As Figure 1 shown, the mice are 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 the LMK235+UTI group were given an intraperitoneal injection of 5 mg / kg of LMK235, and the NC group and the UTI group were given an intraperitoneal injection of an equal volume of solvent. The infection operation was to select a 24G intravenous indwelling needle, insert a soft tube into the urethra of the mouse, and inoculate 1×10 8Mice were re-infected with CFU / 50 mL of CFT073 bacterial suspension after a 24-hour interval (using the same method as the first infection) to facilitate the establishment of a mouse model of chronic UTI. Chronic cystitis was defined as high titers (>10). 4 Persistent bacteriuria (CFU / mL) and high titers (>10 CFU / mL) 2-4 weeks after infection. 4 CFU / organ colonization of bladder tissue was achieved. Mice were continuously monitored for condition, weight, and urinary bacterial count; persistent bacteriuria >10- was considered normal. 4 CFU / mL was used as an indicator of successful chronic UPEC infection construction. Comparison of body weight between the NC and LMK235 groups showed that mice with stable body weight and slight weight gain after intraperitoneal injection of LMK235 were in good condition, with no difference in body weight compared to the NC group. This indicates that treatment with 5 mg / kg LMK235 (the synthetic human dose is 0.55 mg / kg) had no toxic side effects on mice. Figure 2 Mice were euthanized two weeks after infection, and urine, bladder tissue, and other samples were collected for subsequent experiments.

[0026] Example 2: LMK235 treatment significantly reduced urinary bacterial count in mice with chronic UTI and decreased the incidence of chronic UTI.

[0027] Urine samples were collected from mice at different time points after infection and serially diluted, such as... Figure 3 As shown, LMK235 treatment can alleviate infection-induced pyuria in mice (gross observation) and reduce urinary bacterial count. It can be seen that one day after infection, the urinary bacterial count in both the UTI group and the LMK235+UTI group was higher than 10. 4 CFU / mL, but the urinary 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 urinary bacterial count in some mice gradually decreased over a two-week period. At 14 days post-infection, 7 out of 9 mice in the UTI group had urinary bacterial counts higher than 10. 4 CFU / mL, while in the LMK235+UTI group, 5 out of 9 mice had urinary bacterial counts below 10 CFU / mL. 4 CFU / mL, did not progress to chronic UTI, and the urinary bacterial count in the LMK235+UTI group was significantly lower than that in the UTI group ( Figure 3 B). The above results indicate that LMK235 significantly reduced the amount of bacteria in the urine of mice with chronic UTI and reduced the incidence of chronic UTI.

[0028] Example 3: LMK235 treatment significantly reduced UPEC colonization in the bladder of mice with chronic UTI.

[0029] Fourteen days post-infection, mice were sacrificed and bladder tissue was collected, weighed, and homogenized. The homogenate was serially diluted and spread onto solid plates, incubated overnight at 37°C, and colony counts were performed to assess the bacterial load in the bladder tissue of the UTI group and the LMK235-treated UTI group. Results showed that compared with saline injection, treatment with 5 mg / kg LMK235 significantly reduced UPEC colonization in the bladder. Figure 4 This indicates that LMK235 can be used to treat chronic bladder infections caused by UPEC.

[0030] Example 4: LMK235 treatment significantly reduced the level of the inflammatory factor IL-1β in the bladder of mice with chronic UTI.

[0031] The level of IL-1β, an inflammatory factor in the bladder of mice with chronic UTI, was detected using an enzyme-linked immunosorbent assay (ELISA). Mice were sacrificed 14 days post-infection, and bladder tissue was harvested, weighed, homogenized with buffer, and then centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected. Following 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 450 nm 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 the inflammatory factor IL-1β in the bladder of mice with chronic UTI, thereby improving chronic bladder inflammation caused by UPEC infection.

[0032] Example 5: Tablets containing 35 mg of active ingredient per tablet were prepared as follows: Dosage / tablet

[0033] LMK235: 35mg

[0034] Microcrystalline cellulose: 55mg

[0035] Starch: 45mg

[0036] Hydroxymethylcellulose: 4mg

[0037] Sodium carboxymethyl starch: 5mg

[0038] Magnesium stearate: 1mg

[0039] Talc: 1mg

[0040] Take appropriate amounts of each ingredient, sieve the active ingredient, starch and cellulose, and mix them thoroughly; mix the hydroxymethyl cellulose solution with the powder above, sieve, and obtain wet granules. Dry them at 50-60℃. Pre-sieve the sodium carboxymethyl starch, magnesium stearate and talc powder, and then add them to the granules above for tableting.

[0041] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. The use of the deacetylase inhibitor LMK235 or products containing LMK235 in the preparation of drugs for the treatment of chronic urinary tract infections; wherein the chronic urinary tract infection refers to chronic bladder infection caused by pathogenic Escherichia coli of the urethra.

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