Berberine synergistic enhancement of antibacterial activity of su3327 and use thereof in preparation of anti-infective drugs

Abstract

The application provides a new use of berberine combined with a C-JUN N-terminal kinase inhibitor SU3327 in preparation of a medicine for resisting fungal and bacterial infections, and a use of a composition of SU3327 and berberine in preparation of a medicine for resisting fungal and bacterial infections with enhanced efficacy. The application not only proves that the berberine synergistically enhances the antibacterial activity of SU3327 through checkerboard minimum inhibitory concentration tests and in-vitro bacterial growth curves, but also proves the effectiveness of the in-vivo treatment of the combination of berberine and SU3327 on avian Candida albicans infections through clinical tests. The application provides a new use of berberine synergistically enhancing SU3327 in treatment of fungal and bacterial infections.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the field of antibacterial agents, and particularly to an antibacterial activity of SU3327 enhanced by berberine and its application in the preparation of antibacterial drugs in combination with SU3327. Background Technology

[0002] Candida albicans is an important opportunistic pathogenic fungus that is usually present in the upper digestive tract of pigeons. In normal individuals, it exists in small quantities and does not cause disease. However, when the body's immune function or general defense is weakened, or when the normal flora structure is imbalanced, it can transform into a pathogen, harming pigeons, especially squabs, which are particularly susceptible to infection. This disease is commonly known as thrush, also called fungal stomatitis, candidal stomatitis, sour crop disease, and candidiasis. Its most prominent characteristic is the formation of a yellowish-white, cheesy pseudomembrane on the mucous membranes of the esophagus and crop of affected pigeons. After peeling off the pseudomembrane, erosions and ulcers are visible. When squabs are infected with Candida albicans, they usually show no obvious symptoms before leaving the nest, but symptoms become apparent after leaving the nest. Their crops become noticeably dry and hard, they become lethargic, their feathers become ruffled, they are reluctant to move, their appetite decreases or even stops, they grow poorly, become emaciated, and they are prone to secondary infections such as trichomoniasis and bacterial infections. As reported in the literature, Candida albicans often leads to secondary or co-infection with Staphylococcus aureus (Van Dyck K, Viela F, Mathelié-Guinlet M, Demuyser L, Hauben E, Jabra-Rizk MA, Vande Velde G, Dufrêne YF, Krom BP, Van Dijck P. Adhesion of Staphylococcus aureus to Candida albicans During Co-Infection Promotes Bacterial Dissemination Through the Host Immune Response. Front Cell Infect Microbiol. 2021 Feb 2; 10:624839.), ultimately resulting in defective pigeons. Currently, there are no effective treatments for Candida albicans infection or co-infections caused by Candida albicans in veterinary clinics.

[0003] Berberine, also known as berberine alkaloid, was originally extracted from the traditional Chinese medicine Ligusticum chuanxiong. Currently, it can be extracted from various herbal plants or synthesized artificially. Its chemical structure is tetramethylpyrazine. It has been proven to have a variety of biological functions, including anti-inflammatory, antioxidant, antimicrobial, immunomodulatory, blood sugar control, and cardiovascular protection. Clinically, it is often used to treat diarrhea, diabetes, and cardiovascular diseases. Berberine has a good effect on the treatment of infectious diarrhea. Clinically, it is mainly used in the form of berberine hydrochloride or berberine sulfate. Meanwhile, some in vitro studies have found that berberine also has certain antibacterial activity, with weaker antibacterial activity against Gram-negative bacteria (MIC) ranging from 512 to 1024 μg / mL, and stronger antibacterial activity against Gram-positive bacteria (MIC usually between 128 and 512 μg / mL) (Xia Shuai, Ma Liyan, Xie Miaorong. Study on in vitro antibacterial activity of berberine hydrochloride against Staphylococcus aureus [J]. Journal of Clinical and Experimental Medicine, 2022, 21(07):673-678); In addition, berberine also has certain therapeutic effects on fungal infections, such as Studies have found that berberine has certain activity against Candida albicans, with a MIC generally between 64-128 μg / mL (Yong Jiangyan, Wang Hai, Huang Xiaoxue, et al. Effect of berberine hydrochloride combined with fluconazole on calcium homeostasis of drug-resistant Candida albicans [J]. Chinese Journal of Pathogenic Biology, 2020, 15(08):903-909), and the MIC range of activity against Cryptococcus neoformans is between 8-168 μg / mL (Xu Jialong, Song Haolei, Chen Xiaoqin, et al. Activity and mechanism of action of berberine against Cryptococcus neoformans [J]. Acta Microbiologica Sinica, 2023, 63(04):1541-1550.).

[0004] SU3327 (also known as halicin, CAS No.: 40045-50-9) is an N-terminal kinase inhibitor of C-JUN, the structure of which is shown below. Figure 1SU3327 is a potent, selective, and substrate-competitive JNK inhibitor with an IC50 of 0.7 μM. SU3327 also inhibits the protein-protein interaction between JNK and JIP with an IC50 of 239 nM. SU3327 has low activity against p38α and Akt kinases. Previous studies have identified SU3327 as a potential antidiabetic drug (De SK, Stebbins JL, Chen LH, Riel-Mehan M, Machleidt T, Dahl R, Yuan H, Emdadi A, Barile E, Chen V, Murphy R, Pellecchia M. Design, synthesis, and structure-activity relationship of substrate competitive, selective, and in vivo active triazole and thiadiazole inhibitors of the c-Jun N-terminal kinase. J Med Chem. 2009 Apr 9; 52(7):1943-52.). Another literature has reported that SU3327 exhibits certain antibacterial activity against common clinical pathogens such as Enterococcus, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus (Stokes JM, Yang K, Swanson K, Jin W, Cubillos-Ruiz A, Donghia NM, MacNair CR, French S, Carfrae LA, Bloom-Ackermann Z, Tran VM, Chiappino-Pepe A, Badran AH, Andrews IW, Chory EJ, Church GM, Brown ED, Jaakkola TS, Barzilay R, Collins JJ. A Deep Learning Approach to Antibiotic Discovery. Cell. 2020 Apr 16; 181(2):475-483.). Summary of the Invention

[0005] To address the problem of drug resistance in multidrug-resistant Gram-positive bacteria and the lack of effective drugs, this invention provides a combination of natural products with significant synergistic antibacterial effects.

[0006] The first aspect of the present invention discloses a composition for antibacterial and antifungal infection, specifically using SU3327 in combination with berberine, where the two are not simply additive in function, but achieve a synergistic antibacterial effect.

[0007] This invention demonstrates, through checkerboard minimum inhibitory concentration (MIC) tests and animal experiments, that the combination of SU3327 and berberine exhibits significant synergistic antibacterial and antifungal activity. The bacteria tested included Staphylococcus aureus and methicillin-resistant Staphylococcus aureus; the fungi tested included Candida albicans.

[0008] A second aspect of the present invention discloses a novel use of the above-described composition in the preparation of a medicament having antibacterial and antifungal efficacy.

[0009] Furthermore, the aforementioned drugs may be drugs for treating bacterial infections or drugs for treating fungal infections.

[0010] Furthermore, the berberine is berberine or a pharmaceutically acceptable salt thereof, specifically berberine hydrochloride or berberine sulfate.

[0011] Furthermore, the fungus is Candida albicans.

[0012] Furthermore, the bacteria are Gram-positive bacteria.

[0013] Furthermore, the Gram-positive bacteria include Staphylococcus aureus.

[0014] Further, the bacteria are Staphylococcus aureus; preferably, the Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus.

[0015] Furthermore, the fungus is Candida albicans.

[0016] The antibacterial or antifungal drugs provided by this invention include SU3327 and berberine.

[0017] Furthermore, the mass ratio of SU3327 to berberine is (1.25-10):(1.8-14.4).

[0018] The active ingredients in the above-mentioned drugs may be only SU3327 and berberine; they may also include other active ingredients, which can be determined by those skilled in the art based on their antibacterial effects.

[0019] The above-mentioned drugs can kill and / or inhibit bacteria and fungi;

[0020] The dosage form of the drug is one of the following: tablet, ointment, cream, capsule, sustained-release tablet, controlled-release tablet, oral liquid, syrup, injection, drop pill, or lyophilized powder for injection.

[0021] Furthermore, other antimicrobial products containing the above-mentioned antimicrobial composition are also within the scope of protection of this invention.

[0022] The dosage form of the above-mentioned antibacterial products may be any one of the following: cream, ointment, tablet, suspension, capsule, sustained-release tablet, controlled-release tablet, oral liquid, syrup, injection dosage form, drop pill, or lyophilized powder injection dosage form.

[0023] This invention opens up a new application for the combined use of SU3327 and berberine, namely, that the combination has significant synergistic antibacterial and antifungal activities, and has important clinical application value in the treatment of multidrug-resistant Gram-positive bacterial infections and fungal infections. Attached Figure Description

[0024] Figure 1 This invention provides the results of the checkerboard method for the combined antibacterial action of berberine and SU3327 against methicillin-resistant Staphylococcus aureus.

[0025] Figure 2 This invention provides the results of a checkerboard method for the synergistic antibacterial effect of berberine combined with SU3327 against Candida albicans ATCC12301.

[0026] Figure 3 Representative images from autopsy of spontaneously contracted Candida albicans infection in clinical settings. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] The berberine used in the following experiments was in the form of berberine hydrochloride, i.e., berberine hydrochloride, CAS No.: 633-65-8, molecular formula: C20H18ClNO4, molecular weight: 372.82, purchased from Aladdin Reagent Company, with a purity ≥98%. SU3327 was purchased from MCE Reagent Company, USA, with a purity ≥99%. A certain amount of berberine hydrochloride was weighed to prepare a stock solution with a concentration of 128 mg / mL. SU3327 was prepared using DMSO to prepare a stock solution with a concentration of 40 mg / mL. All prepared stock solutions were stored at -20 degrees Celsius.

[0030] The pathogens used in the following experiments:

[0031] Methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA33591) was provided by the National Center for Veterinary Drug Safety Evaluation (Beijing).

[0032] The standard cytotoxic strain of Candida albicans, ATCC12301, was purchased from Shanghai Fuxiang Biotechnology Co., Ltd.

[0033] Bacterial culture medium was used in the following experiments:

[0034] YPD medium: Weigh 20g glucose, 10g yeast extract, and 20g trypsin using an electronic balance, then add 800mL deionized water. Dissolve the solids completely on a magnetic stirrer, then bring the volume to 1L with deionized water and dispense into Erlenmeyer flasks. If preparing solid medium, add 2% agar as needed, and autoclave at 120℃ for 30 minutes. Then, store at room temperature for later use.

[0035] MHB broth culture medium was purchased from Beijing Luqiao Technology Co., Ltd., and the preparation method is as follows: weigh 25.0g into 1L of distilled water, heat to boiling until completely dissolved, autoclave at 121℃ for 15min, and set aside.

[0036] Liquid thioglycolate (FTG) composition (g / L): tryptone 15.0 g / L, yeast extract 5.0 g / L, sodium thioglycolate 0.5 g / L, glucose 5.0 g / L, sodium chloride 2.5 g / L, L-cysteine ​​0.5 g / L, resazurin 0.001 g / L, agar 0.75 g / L.

[0037] MHA culture medium was purchased from Beijing Luqiao Technology Co., Ltd., and the preparation method is as follows: weigh 38.0g into 1L of distilled water, heat to boiling until completely dissolved, autoclave at 121℃ for 15min, cool to 55℃ and pour into plates for later use.

[0038] Brain-Heart Infusion Culture Medium (BHI) was purchased from Beijing Luqiao Technology Co., Ltd., and the preparation method is as follows: Weigh 38.5g of this product, heat and stir to dissolve in 1000mL of distilled water, adjust the pH to 7.3, autoclave at 121℃ for 15 minutes, and set aside.

[0039] BHI solid culture medium was purchased from Beijing Luqiao Technology Co., Ltd., and the preparation method is as follows: Weigh 50.0g of this product into 1000mL of distilled water, heat to boiling until completely dissolved, autoclave at 121℃ for 20min, cool to 55℃ and pour into plates for later use.

[0040] Example 1: Evaluation of the antibacterial effect of the combined use of SU3327 and berberine

[0041] Combined drug index (FICI) determination of SU3327 and berberine hydrochloride against pathogens: The combined antibacterial effect of SU3327 and berberine hydrochloride against Staphylococcus aureus ATCC29213 and methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA33591) was determined using the checkerboard method.

[0042] Specifically, methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA33591) strain was cultured normally using MHB broth. The checkerboard method was performed as follows: SU3327 (as drug A) and berberine (as drug B) were serially diluted with MHB broth at maximum concentrations of 80 μg / mL and 128 μg / mL, respectively. 50 μL of MHB broth containing the two drugs at different concentrations was added along the horizontal and vertical axes of a 96-well microplate, respectively. Then, 50 μL of each pathogenic bacterial suspension was added to each well, resulting in a final bacterial count of 2 × 10⁶ bacteria per well. 5 CFU was incubated at 37℃ for 24 hours, and the results were observed. The minimum inhibitory concentrations (MICs) of the two drugs, used alone and in combination, were recorded, and the FICI values ​​(partial inhibitory concentration index) were calculated according to the following formula.

[0043] The results are as follows:

[0044] like Figure 1 As shown, for methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA33591), the MICs of SU3327 and berberine hydrochloride used alone were >20 μg / mL and >128 μg / mL, respectively. Based on the optimal ratio for synergistic effect calculated using the checkerboard method, the MICs of SU3327 and berberine hydrochloride decreased to 5 μg / mL and 64 μg / mL, respectively (i.e., the optimal ratio of SU3327 to berberine is 1:11.5), with a synergistic index (FICI) of 0.5, indicating a synergistic effect.

[0045] Example 2: Synergistic inhibitory effect of SU3327 and berberine on Candida albicans

[0046] The combination drug index (FICI) of SU3327 and berberine hydrochloride against pathogens was determined using the checkerboard method. The FICI value of the combined application of SU3327 and berberine hydrochloride against the standard accusation strain ATCC12301 of Candida albicans was determined. The specific procedure was as follows: The FICI between different drugs was determined using the checkerboard broth dilution method. Single fungal colonies were picked and cultured in Sabouraud dextrose broth at 27°C for 48 hours. The fungal turbidity was adjusted to 0.5 using a McFarland turbidimeter, and then diluted to 1.0 × 10³ CFUs / mL with Sabouraud dextrose medium. 100 μL of Sabouraud dextrose medium was added to each well of a 96-well U-shaped plate. The corresponding concentration of the drug was added to the first 10 wells of the eighth row, and serially diluted upwards to the second row. Other test drugs were added to the wells of the first column, and serially diluted from left to right to the ninth column. 100 μL of the diluted test bacterial solution was added to each of columns 1-10. Columns 11 and 12 show the negative and positive controls, respectively, containing only Sabouraud dextrose medium and the test bacterial culture. After incubating the 96-well plate at 27°C for 24 hours, the results were read. The lowest drug concentration that inhibits fungal growth and is visible to the naked eye was taken as the MIC value of the drug.

[0047] FICI = MIC(Drug A combined) / MIC(Drug A alone) + MIC(Drug B combined) / MIC(Drug B alone). Judgment criteria: FICI ≤ 0.5, synergistic effect; 0.5 < FICI ≤ 1, additive effect; 1 < FICI ≤ 2, irrelevant effect; FICI > 2, antagonistic effect. That is: FICI of SU3327 and berberine = MIC(berberine combined) / MIC(berberine alone) + MIC(SU3327 combined) / MIC(SU3327 alone).

[0048] The results are shown below:

[0049] Figure 2 As shown, the MICs of SU3327 and berberine hydrochloride alone against ATCC12301 were 40 μg / mL and 128 μg / mL, respectively. Calculations based on the optimal ratio for synergistic effect using the checkerboard method reduced the MICs of SU3327 and berberine hydrochloride to 2.5 μg / mL and 16 μg / mL, respectively (i.e., SU3327 and berberine). The synergistic index (FICI) was 0.1875. According to the checkerboard method results, the synergistic effect was observed in the range of SU3327 to berberine hydrochloride mass ratios of (1.25-10):(2-16), which is equivalent to a mass ratio of SU3327 to berberine hydrochloride of (1.25-10):(1.8-14.4). This indicates that the combined use of SU3327 and berberine has significant synergistic antibacterial activity against Candida albicans.

[0050] Example 3: Evaluation of the efficacy of SU3327 and berberine alone and in combination in the treatment of clinical Candida albicans infection in avian birds.

[0051] (1) Animal grouping and treatment

[0052] The squabs used in this experiment were provided by a pigeon farm in Jiangmen City, Guangdong Province. This experiment primarily used clinically ill squabs aged 25-28 days. The main clinical symptoms included ulcers and yellow cheesy material in the squabs' mouths, and varying degrees of white, ulcerative lesions on the crop, lungs, and other organs upon necropsy (see...). Figure 3 (Representative image of a diseased pigeon's necropsy).

[0053] Generally, pigeons weighing 450 grams meet the market standard. Pigeons weighing less than 300 grams usually have other diseases that lead to organ failure and cannot be used as experimental subjects. Therefore, according to the experimental requirements, the sick pigeons were screened to a certain extent, and finally 64 pigeons weighing 300-400 grams were selected. The experiment was divided into 4 groups, with 16 pigeons in each group.

[0054] Detailed grouping and administration are as follows:

[0055] Placebo control group: The corresponding solvent control was given, i.e., 1 mL of sodium carboxymethyl cellulose and 0.5%.

[0056] In the berberine hydrochloride combined with SU3327 treatment group: oral administration was carried out at a ratio of SU3327 to berberine hydrochloride of 1:6.4. The final dose of SU3327 was 3.12 mg / kg body weight, and the dose of berberine hydrochloride was 20 mg / kg body weight (equivalent to 18 mg / kg body weight of berberine).

[0057] Berberine-only group: Each pigeon was orally administered berberine hydrochloride solution at a dose of 20 mg / kg body weight, which is equivalent to 18 mg / kg body weight of berberine.

[0058] SU3327 monotherapy group: Each pigeon was orally administered SU3327 solvent at a dose of 3.12 mg / kg body weight.

[0059] The treatment cycle is 10 days, with medication administered once every two days, for a total of 5 doses.

[0060] (2) Evaluation criteria for treatment efficacy

[0061] (1) Sensory judgment

[0062] Observe the pigeon's mental state, changes in the color of the oral pseudomembrane, the fullness of the crop, and the degree of emaciation;

[0063] Observe the pigeon droppings.

[0064] Assessing the pigeon's recovery progress:

[0065] Full recovery: Normal feeding, full body shape restored, and meets market standards;

[0066] Semi-recovered: Feeding normally, but not fully recovered to full size, and not yet meeting market standards;

[0067] Emaciation in sick pigeons: empty crop, thin body, poor mental state.

[0068] For pigeons that have fully recovered, partially recovered, or become emaciated, one pigeon from each group was randomly selected for dissection, and the oral cavity, crop, and internal organs were observed.

[0069] (3) Test Results

[0070] The treatment effects are shown in Table 1. In the placebo control group, the pigeons' symptoms did not improve significantly, with 6 deaths and 0 recoveries. In the SU3327 treatment group, 1 pigeon died and 7 recovered, with a recovery rate of 43.75%. In the berberine-only treatment group, 8 pigeons recovered, with a recovery rate of 50%. In the SU3327 combined with berberine treatment group, 14 pigeons recovered, with a recovery rate of 87.5%. These results indicate that SU3327 combined with berberine treatment has significant advantages over either treatment alone, with a SU3327 to berberine mass ratio of 1:5.8.

[0071] Table 1. Evaluation of the efficacy of SU3327 combined with berberine in treating Candida albicans infection in pigeons.

[0072]

[0073] The embodiments described above are merely examples for clearly illustrating the present disclosure and are not intended to limit the implementation of the present disclosure. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this disclosure.

Claims

1. A composition for antibacterial and antifungal infection control, comprising berberine and SU3327, an N-terminal kinase inhibitor of C-JUN.

2. Use of the composition of claim 1 in the preparation of an medicament effective against methicillin-resistant Staphylococcus aureus and / or Candida albicans infections.

3. The use according to claim 2, characterized in that, The mass ratio of SU3327 to berberine is (1.25-10):(1.8-14.4) or 1:11.

5.

4. The use according to claim 2, characterized in that, The final animal treatment doses of SU3327 and berberine were 3.12 mg / kg and 18 mg / kg body weight, respectively, in a ratio of 1:5.

8.

5. The use according to claim 2, characterized in that, The dosage form of the drug is one of the following: cream, sustained-release tablet, controlled-release tablet, oral liquid, injection, pill, or lyophilized powder for injection.

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