Application of combined composition in preparation of medicine for resisting fungal and bacterial infection

Abstract

The invention relates to an application of a combined composition in preparation of a medicine for resisting fungal and bacterial infection. The active components of the combined composition are antibiotics and antibiotic adjuvants, the antibiotic adjuvant is tripterine or a pharmaceutically acceptable salt thereof. The invention creatively finds that the tripterine or the medicinal salt thereof can be combined with the antibiotics to have a very excellent effect in antifungal and bacterial infection, especially in antifungal infection, and the tripterine or the medicinal salt thereof can enhance the antibacterial effect, especially the antifungal effect, of the antibiotics; according to the present invention, the antibacterial sensitivity of the antibiotic is significantly increased, the generation of the drug resistance of fungi or bacteria is inhibited, the combination composition provides the new strategy for the efficient antibacterial effect as the antibiotic adjuvant-antibiotic drug platform, and the application dosage form and the application scene are diversified.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the application of a combination composition in the preparation of drugs for antifungal and antibacterial infections. Background Technology

[0002] Bacterial and fungal infections are a major cause of human death, and the discovery of antibiotics has significantly reduced mortality rates among patients with these infections. However, the overuse of antibiotics has led to the rapid development of multidrug-resistant bacteria, posing a long-term threat to human health and the sustainable production and development of food. The development of novel antibiotics is challenging and time-consuming, severely limiting the clinical treatment of drug-resistant infections. The arrival of the post-antibiotic era urges us to develop new treatment options to address the concerning situation of antibiotic resistance. The discovery of antibiotic adjuvants offers hope for addressing the obstacles in the development of new antibiotics and the inadequacy of existing drugs to combat drug-resistant infections. Antibiotic adjuvants, used in combination with antibiotics, enhance their efficacy, offering new hope for restoring the activity of existing antibiotics and inhibiting the development of resistance, while also providing an orthogonal strategy for the discovery of new antibiotics.

[0003] Tripterygium wilfordii, also known as yellow wax vine or water vine, possesses various medicinal properties, including dispelling wind and dampness, promoting blood circulation, reducing swelling and relieving pain, and anti-inflammatory and detoxifying effects. Celastrol (CST), also known as southern celestine, is a triterpenoid active ingredient in Tripterygium wilfordii extract, exhibiting diverse biological activities. Studies have found that celastrol possesses anti-tumor, anti-rheumatoid, and anti-Parkinson's disease effects. With ongoing research, the various medicinal effects of celastrol are being continuously developed and applied. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide an application of a combination composition in the preparation of a drug for treating fungal and bacterial infections.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides the use of a combination composition in the preparation of a medicament for treating fungal and bacterial infections, wherein the active component of the combination composition is an antibiotic and an antibiotic adjuvant; the antibiotic adjuvant is triptolide or a pharmaceutically acceptable salt thereof.

[0007] This invention creatively discovers that triptolide or its pharmaceutically acceptable salts can be used in combination with antibiotics to achieve excellent effects in antifungal and bacterial infections, especially in antifungal infections. In particular, triptolide or its pharmaceutically acceptable salts can enhance the antibacterial activity of antibiotics, especially their antifungal activity, increase the antibacterial sensitivity of antibiotics, and inhibit the development of fungal or bacterial resistance. This combination composition, as an antibiotic adjuvant-antibiotic drug platform, provides a new strategy for achieving highly efficient antibacterial activity, and its application formulations and application scenarios are very diverse.

[0008] The aforementioned "medicinal salts" include acid salts or basic salts;

[0009] The acid salts include hydrochloride, sulfate, phosphate, citrate, hydrobromide, acetate, benzoate, benzenesulfonate, tartrate, carbonate, citrate, gluconate, lactate, malate, methanesulfonate, stearate, valerate, or nitrate;

[0010] The basic salts include sodium salts, calcium salts, potassium salts, zinc salts, or meglumine salts.

[0011] Preferably, the antibiotic comprises any one or a combination of at least two of the following: amphotericin B, nystatin, miconazole, ketoconazole, fluconazole, itraconazole, voriconazole, posaconazole, flucytosine, terbinafine, caspofungin, micafungin, anisofungin, griseofulvin, penicillin, amoxicillin, ampicillin, cefadroxil, cephalexin, ceftazidime, imipenem, amikacin, gentamicin, doxycycline, tetracycline, minocycline, erythromycin, clarithromycin, azithromycin, sulfadiazine, ciprofloxacin, levofloxacin, metronidazole, clindamycin, or fosfomycin.

[0012] Preferably, the fungus includes any one or a combination of at least two of the following: Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus oryzae, Fusarium, Cryptococcus neoformans, Candida albicans, Mucor, Histoplasma capsulatum, Sarcodactylum, Coccidioides, Cryptococcus gutterus, or Paracoccidioides.

[0013] Preferably, the bacteria include any one or a combination of at least two of the following: Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Escherichia coli, Salmonella, Shigella, Streptococcus pneumoniae, Staphylococcus aureus, Mycobacterium tuberculosis, or Clostridium difficile.

[0014] Preferably, the molar ratio of the antibiotic to the antibiotic adjuvant is 1:(0.001-500), such as 1:0.001, 1:0.01, 1:0.05, 1:0.1, 1:0.5, 1:1, 1:5, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:100, 1:150, 1:200, 1:300, 1:400, 1:150, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0015] Preferably, the combination is a single compound preparation or a combination of an antibiotic adjuvant and a separate preparation of each antibiotic.

[0016] Preferably, when the combination is a combination of an antibiotic adjuvant and a single formulation of an antibiotic, the administration method can be simultaneous, cross-administration, or sequential administration.

[0017] Preferably, the formulation is any pharmaceutically acceptable dosage form.

[0018] Preferably, the dosage form of the preparation includes an oral dosage form, an injectable dosage form, or a topical dosage form.

[0019] Preferably, the administration route of the drug includes gastrointestinal administration, injection administration, transdermal administration, mucosal administration, inhalation administration, or local administration.

[0020] Preferably, the combination composition further contains pharmaceutically acceptable excipients.

[0021] Preferably, the excipients include any one or a combination of at least two of the following: carrier, diluent, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0022] In a second aspect, the present invention provides a combined pharmaceutical composition for treating fungal and bacterial infections, wherein the active component of the combined pharmaceutical composition is an antibiotic and an antibiotic adjuvant;

[0023] The antibiotic adjuvant is triptolide or its pharmaceutically acceptable salt;

[0024] The antibiotic is selected from any one or a combination of at least two of the following: amphotericin B, nystatin, miconazole, ketoconazole, fluconazole, itraconazole, voriconazole, posaconazole, flucytosine, terbinafine, caspofungin, micafungin, anisofungin, griseofulvin, penicillin, amoxicillin, ampicillin, cefadroxil, cephalexin, ceftazidime, imipenem, amikacin, gentamicin, doxycycline, tetracycline, minocycline, erythromycin, clarithromycin, azithromycin, sulfadiazine, ciprofloxacin, levofloxacin, metronidazole, clindamycin, or fosfomycin.

[0025] This invention utilizes triptolide or its pharmaceutically acceptable salts as antibiotic adjuvants. By combining it with multiple antibiotics, an antibiotic adjuvant-antibiotic drug platform is constructed. Leveraging the adjuvant effect of triptolide, the antibacterial activity, especially antifungal activity, of antibiotics is enhanced, reducing the occurrence of bacterial and fungal resistance. Compared to antibiotic therapy alone, the antibiotic adjuvant-antibiotic drug platform lowers the minimum effective therapeutic concentration of antibiotics, effectively broadening the therapeutic window and improving treatment safety. Furthermore, this antibiotic adjuvant-antibiotic drug platform can be formulated into various dosage forms to meet the needs of different treatment scenarios and antibiotics with varying physicochemical characteristics, while also satisfying clinical needs for oral, injectable, inhaled, or topical administration.

[0026] The aforementioned "medicinal salts" include acid salts or basic salts;

[0027] The acid salts include hydrochloride, sulfate, phosphate, citrate, hydrobromide, acetate, benzoate, benzenesulfonate, tartrate, carbonate, citrate, gluconate, lactate, malate, methanesulfonate, stearate, valerate, or nitrate;

[0028] The basic salts include sodium salts, calcium salts, potassium salts, zinc salts, or meglumine salts.

[0029] Preferably, the molar ratio of the antibiotic to the antibiotic adjuvant is 1:(0.001-500), such as 1:0.001, 1:0.01, 1:0.05, 1:0.1, 1:0.5, 1:1, 1:5, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:100, 1:150, 1:200, 1:300, 1:400, 1:150, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0030] Preferably, the fungus includes any one or a combination of at least two of the following: Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus oryzae, Fusarium, Cryptococcus neoformans, Candida albicans, Mucor, Histoplasma capsulatum, Sarcodactylum, Coccidioides, Cryptococcus gutterus, or Paracoccidioides.

[0031] Preferably, the bacteria include any one or a combination of at least two of the following: Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Escherichia coli, Salmonella, Shigella, Streptococcus pneumoniae, Staphylococcus aureus, Mycobacterium tuberculosis, or Clostridium difficile.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] This invention creatively discovers that triptolide or its pharmaceutically acceptable salts can be used in combination with antibiotics to have excellent effects in antifungal and bacterial infections, especially in antifungal infections. In particular, triptolide or its pharmaceutically acceptable salts can enhance the antibacterial effect of antibiotics, especially antifungal effect, increase the antibacterial sensitivity of antibiotics, and inhibit the occurrence of fungal or bacterial resistance.

[0034] This invention utilizes triptolide or its pharmaceutically acceptable salts as antibiotic adjuvants. By combining it with multiple antibiotics, an antibiotic adjuvant-antibiotic drug platform is constructed. Leveraging the adjuvant effect of triptolide, the antibacterial activity, especially antifungal activity, of antibiotics is enhanced, reducing the occurrence of bacterial and fungal resistance. Compared to antibiotic therapy alone, the antibiotic adjuvant-antibiotic drug platform lowers the minimum effective therapeutic concentration of antibiotics, effectively broadening the therapeutic window and improving treatment safety. Furthermore, this antibiotic adjuvant-antibiotic drug platform can be formulated into various dosage forms to meet the needs of different treatment scenarios and antibiotics with varying physicochemical characteristics, while also satisfying clinical needs for oral, injectable, inhaled, or topical administration. Attached Figure Description

[0035] Figure 1 The image shows the FICI results of the combined antibacterial experiment of triptolide with multiple antibiotics.

[0036] Figure 2 The image shows the FICI results of the combined antibacterial experiment of triptolide with multiple antibiotics.

[0037] Figure 3 The diagram shows the docking results of triptolide with CYP51 molecules;

[0038] Figure 4 The image shows the antimicrobial kinetic curves of the CST-Flu combination against Fusarium and Aspergillus fumigatus.

[0039] Figure 5 The image shows the antimicrobial kinetics curve of a combination of triptolide or its medicinal salt and fluconazole against Aspergillus fumigatus. Detailed Implementation

[0040] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0041] Example 1

[0042] Evaluation of the minimum inhibitory concentration (MIC) of a single drug:

[0043] This example describes the use of triptolide (CST) alone, as well as various antibiotics alone (including amphotericin B-AmB, nystatin-Nys, miconazole-Mnc, ketoconazole-Ket, fluconazole-Flu, itraconazole-Itr, voriconazole-Vor, posaconazole-Pos, flucytosine-Fly, terbinafine-Ter, caspofungin-Cas, micafungin-Mcf, anisofungin-Anf, griseofulvin-Gri, penicillin-Ben, amoxicillin-Amo, ampicillin-Amp, cefadroxil-Cfd, cephalexin-Cfa, ceftazidime-Cef, imipenem-Imi, amikacin-Ami, gentamicin-Gen, doxycycline-Dox, tetracycline-Tet, minocycline-Min, erythromycin-Ery, clavatin, etc.). The minimum inhibitory concentrations (MICs) of various fungi (including Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus oryzae, Fusarium, Cryptococcus neoformans, Candida albicans, Mucor, Histoplasma capsulatum, Sarcodactylus, Coccidioides, Cryptococcus gutterus, or Paracoccidioides) and bacteria (including Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Escherichia coli, Salmonella, Shigella, Streptococcus pneumoniae, Staphylococcus aureus, Mycobacterium tuberculosis, or Clostridium difficile) were determined using the following methods:

[0044] (1) Inoculate the fungi or bacteria to be tested onto various solid culture plates (see Table 1 for details) and incubate them in a 37°C incubator for 24 hours.

[0045] (2) Select single clones from solid culture medium and inoculate them into various liquid culture media (see Table 1 for details) and incubate them in an incubator at 37°C for 24 hours.

[0046] (3) Weigh out appropriate amounts of the antibiotic powder or tripterygium wilfordii to be tested, dissolve them thoroughly in sterile double-distilled water, filter through a 0.22 μm filter membrane, and prepare a stock solution (0.64 mg / mL) for later use.

[0047] (4) Dilute the above stock solution to the highest concentration of the drug to be tested (64 μg / mL) using liquid culture medium, and perform two-fold dilutions sequentially in a sterile 96-well plate in a biosafety cabinet:

[0048] Add 200 μL of the highest concentration of the drug to be tested to the first well (A1). Add 100 μL of liquid culture medium to wells A2 to A12. Then, aspirate 100 μL from well A1 and add it to well A2. Mix thoroughly and then aspirate 100 μL from well A2 and add it to well A3. Continue this serial dilution until well A12, ensuring that each well contains 100 μL of drug-containing liquid culture medium. Perform three replicates per well.

[0049] (5) Add 1 mL of liquid culture medium to a transparent plastic test tube and zero the turbidimeter. Then, pipette the freshly cultured liquid culture medium of the test strain and mix it well. Adjust the turbidity to 0.5 McFarland turbidity (MCF) and dilute it 20 times with liquid culture medium for later use.

[0050] (6) Take 10 μL of each diluted bacterial suspension and add it sequentially to the drug-containing liquid culture medium prepared in step (4) for each well. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD in each well. 600 The 96-well plate was incubated at 37°C for 16 hours. The OD values ​​in each well were monitored using a microplate reader. 600 No bacterial growth (during the test, OD) 600 The lowest drug concentration at which the value does not change significantly is the minimum inhibitory concentration (MIC) of the drug against this bacterium.

[0051] The results are shown in Tables 2-8.

[0052] Table 1

[0053]

[0054]

[0055] Example 2

[0056] Evaluation of the minimum inhibitory concentration (MIC) of the combination therapy:

[0057] This example compares triptolide (CST) with various antibiotics (including amphotericin B-AmB, nystatin-Nys, miconazole-Mnc, ketoconazole-Ket, fluconazole-Flu, itraconazole-Itr, voriconazole-Vor, posaconazole-Pos, flucytosine-Fly, terbinafine-Ter, caspofungin-Cas, micafungin-Mcf, anidoxime-Anf, griseofulvin-Gri, penicillin-Ben, amoxicillin-Amo, ampicillin-Amp, cefadroxil-Cfd, cephalexin-Cfa, ceftazidime-Cef, imipenem-Imi, amikacin-Ami, gentamicin-Gen, doxycycline-Dox, tetracycline-Tet, minocycline-Min, erythromycin-Ery, clarithromycin-...). The minimum inhibitory concentrations (MICs) of various fungi (including Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus oryzae, Fusarium, Cryptococcus neoformans, Candida albicans, Mucor, Histoplasma capsulatum, Sarcodactylus, Coccidioides, Cryptococcus gutterus or Paracoccidioides) and bacteria (including Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Escherichia coli, Salmonella, Shigella, Streptococcus pneumoniae, Staphylococcus aureus, Mycobacterium tuberculosis or Clostridium difficile) were determined using Cla, Azithromycin-Azi, Sulfadiazine-Sul, Ciprofloxacin-Cip, Levofloxacin-Lev, Metronidazole-Met, Clindamycin-Cli, Fosfomycin-Fos) as described below:

[0058] (1) Inoculate the fungi or bacteria to be tested onto various solid culture plates (see Table 1 for details) and incubate them in a 37°C incubator for 24 hours.

[0059] (2) Select single clones from solid culture medium and inoculate them into various liquid culture media (see Table 1 for details) and incubate them in an incubator at 37°C for 24 hours.

[0060] (3) Weigh out appropriate amounts of the antibiotic powder or tripterygium wilfordii to be tested, dissolve them thoroughly in sterile double-distilled water, filter through a 0.22 μm filter membrane, and prepare a stock solution (0.64 mg / mL) for later use.

[0061] (4) Prepare gradient stock solutions (128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL, 0.6 mL each) according to the gradient dilution method in Example 1 for later use.

[0062] (5) Take a sterile 96-well plate and perform gradient preparation of the composition in a biosafety cabinet as follows:

[0063] Group 1: 50 μL of 128 μg / mL antibiotic was added to each of wells A1 to A12. Then, 50 μL of triptolide at concentrations of 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, and 0.0625 μg / mL were added to each well. Each well was replicated in triptolides.

[0064] Group 2: Add 50 μL of 64 μg / mL antibiotic to each of wells B1 to B12. Then add 50 μL of triptolide in each of wells B1 to B12 in the same concentration gradient as shown in Group 1. Repeat each group in 3 parallel steps.

[0065] Group 3: Add 50 μL of 32 μg / mL antibiotic to each of wells C1 to C12, and then add 50 μL of triptolide in each of wells C1 to C12 in the same concentration gradient as shown in Group 1. Repeat each group in 3 parallel sets.

[0066] Group 4: Add 50 μL of 16 μg / mL antibiotic to each well from D1 to D12. Then add 50 μL of triptolide in each well from D1 to D12 in the same concentration gradient as shown in Group 1. Repeat each well in 3 parallel groups.

[0067] Group 5: Add 50 μL of 8 μg / mL antibiotic to each of wells E1 to E12. Then add 50 μL of triptolide in each of wells E1 to E12 in the same concentration gradient as shown in Group 1. Repeat each group in 3 parallel sets.

[0068] Group 6: Add 50 μL of 4 μg / mL antibiotic to each of wells F1 to F12. Then add 50 μL of triptolide in each of wells F1 to F12 in the same concentration gradient as shown in Group 1. Repeat each group in 3 parallel sets.

[0069] Group 7: Add 50 μL of 2 μg / mL antibiotic to each of wells G1 to G12. Then add 50 μL of triptolide in each of wells G1 to G12 in the same concentration gradient as shown in Group 1. Repeat each group in 3 parallel sets.

[0070] Group 8: Add 50 μL of 1 μg / mL antibiotic to each well from H1 to H12. Then add 50 μL of triptolide in each well from H1 to H12 in the same concentration gradient as shown in Group 1. Repeat each well in 3 parallel groups.

[0071] Group 9: Add 50 μL of 0.5 μg / mL antibiotic to each well I1 to I12. Then add 50 μL of triptolide in each well according to the concentration gradient shown in Group 1. Repeat each well 3 times.

[0072] Group 10: Add 50 μL of 0.25 μg / mL antibiotic to each well J1 to J12. Then add 50 μL of triptolide in each well according to the concentration gradient shown in Group 1. Repeat each well 3 times.

[0073] Group 11: Add 50 μL of 0.125 μg / mL antibiotic to each well K1 to K12. Then add 50 μL of triptolide in each well according to the concentration gradient shown in Group 1. Repeat each well in 3 parallel groups.

[0074] Group 12: Add 50 μL of 0.0625 μg / mL antibiotic to each well from L1 to L12. Then add 50 μL of triptolide in each well from L1 to L12 in the same concentration gradient as shown in Group 1. Repeat each well in 3 parallel groups.

[0075] (6) Add 1 mL of liquid culture medium to a transparent plastic test tube and zero the turbidimeter. Then, pipette the freshly cultured liquid culture medium of the test strain and mix it well. Adjust the turbidity to 0.5 McFarland turbidity (MCF) and dilute it 20 times with liquid culture medium for later use.

[0076] (7) Take 10 μL of each diluted bacterial suspension and add it sequentially to the drug-containing liquid culture medium prepared in step (5) for each well. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD in each well. 600 The 96-well plate was incubated at 37°C for 16 hours. The OD values ​​in each well were monitored using a microplate reader. 600 No bacterial growth (during monitoring period, OD) 600 The lowest drug concentration at which the value does not change significantly is the minimum inhibitory concentration (MIC) of the composition against this bacterium.

[0077] The results are shown in Tables 2-8 (where a(b) represents the concentration a of A and the concentration b of B in the AB composition).

[0078] Table 2

[0079]

[0080]

[0081] Table 3

[0082] Combination / Single Drug Aspergillus niger Aspergillus flavus Aspergillus oryzae Fusarium AmB-CST 1(4) 0.25(1) 0.5(8) 0.25(1) Nys-CST 1(4) 1(2) 2(1) 4(1) Mnc-CST 2(8) 2(1) - 4(1) Ket-CST 0.5(4) 0.125(1) 1(0.5) 0.5(0.5) Flu-CST 4(1) 0.5(1) 1(1) 0.25(0.25) Itr-CST 8(1) 0.5(2) 0.25(1) 4(1) Vor-CST 2(8) 0.0625(0.5) 0.125(2) 1(0.5) Pos-CST - 0.0625(0.25) 0.03125(8) 0.5(0.5) Fly-CST 1(4) 1(0.5) 8(4) 4(1) Ter-CST 0.0625(0.5) 4(0.5) 1(4) - Cas-CST 0.0625(0.5) 0.03215(0.0625) 1(2) 0.5(0.25) Mcf-CST 0.03125(1) 0.03125(1) 0.03125(0.125) 0.25(0.125) Anf-CST 0.03125(1) 0.03215(0.125) 0.0625(0.0625) 0.25(0.125) Gri-CST 16(8) - - 1(1) AmB 4 1 1 1 Nys 4 16 8 16 Mnc 8 8 - 32 Ket 2 0.5 64 64 Flu 16 2 4 4 Itr 16 2 0.5 64 Vor 4 0.25 0.5 8 Pos - 0.125 0.125 2 Fly 4 4 64 32 Ter 0.125 32 64 - Cas 1 0.0625 8 2 Mcf 0.125 0.125 0.0625 0.5 Anf 0.0625 0.0625 0.125 1 Gri 64 - - 32 CST 32 8 32 4

[0083] Table 4

[0084]

[0085]

[0086] Table 5

[0087]

[0088]

[0089] Table 6

[0090]

[0091]

[0092] Table 7

[0093]

[0094]

[0095] Table 8

[0096]

[0097]

[0098] From the data results of Table 2 - Table 8, it can be seen that celastrol improves the antibacterial activities of antibiotics against various fungi and various bacteria by being used in combination with various antibiotics. Compared with the antibiotics alone or celastrol alone, the composition significantly reduces the minimum inhibitory concentration (MIC) of antibiotics, effectively broadens the therapeutic window of the drug, and improves the safety of treatment.

[0099] Example 3

[0100] Calculation of the combined antibacterial index (FICI):

[0101] According to FICI = MIC(antibiotics in combination) / MIC(antibiotics alone) + MIC(celastrol in combination) / MIC(celastrol alone) for result determination: FICI ≤ 0.5 is judged as synergistic effect, 0.5 < FICI ≤ 1 is judged as additive effect, 1 < FICI ≤ 2 is judged as irrelevant effect, and FICI > 2 is judged as antagonistic effect. The results are as Figure 1 and Figure 2 shown.

[0102] From Figure 1 and Figure 2The results show that in the combination compositions involved in this invention, triptolide exhibits significant synergistic effects with most antibiotics, effectively inhibiting the growth of fungi and bacteria. The Flu-CST combination demonstrates excellent synergistic effects against Aspergillus fumigatus; 0.25 μg / mL of triptolide can reduce the MIC of fluconazole to 1 / 32 of its original value, showing significantly better antibacterial sensitization effects than antibiotic adjuvants found in the prior art. Furthermore, some results also indicate antagonistic effects between triptolide and some antibiotics, with minocycline showing antagonistic effects with triptolide in inhibiting the growth of Acinetobacter baumannii. This invention provides guidance for the combined use of triptolide with different antibiotics to treat various bacterial and fungal infections.

[0103] Example 4

[0104] Molecular docking results of triptolide with the antibacterial target CYP51:

[0105] Semi-flexible docking of the 3D structure of CYP51 protein (NCBI Gene ID: 13121) (PDBID: 6CR2) with CST (PubChem CID: 122724) molecule was studied using AutoDock 4.2.6 software. The docking box size was set to 100×100×100 points, with a spacing of [missing information]. Centered on the enzyme's active site, the results with the lowest binding energy and the highest number of hydrogen bonds were selected. Images were processed using Discovery Studio 2021 (Biovia, San Diego, CA, USA) software to display the intermolecular hydrogen bond and other binding mechanisms of small molecules and proteins in both two-dimensional and three-dimensional formats.

[0106] The docking results are as follows Figure 3 As shown, CST fits well into the active pocket of the CYP51 protein, with a binding energy of -12.23 kcal / mol. Specifically, CST forms strong intermolecular hydrogen bonds with the SER311 amino acid residues and exhibits π-σ and π-alkyl interactions with iron porphyrin, effectively promoting the binding of CST to the CYP51 protein. CYP51, as the most widely distributed member of the cytochrome P450 family, is a key enzyme in the synthesis of biosterols. Tripterygium wilfordii can specifically bind to the CYP51 protein, thereby inhibiting sterol synthesis in fungi and exerting antibacterial activity.

[0107] Example 5

[0108] This embodiment provides a combination composition prepared by physically mixing fluconazole and tripterygium wilfordii at a mass ratio of 4:1. The inhibitory effects of this combination composition on Fusarium and Aspergillus fumigatus were evaluated.

[0109] (1) Dilute the overnight cultured Aspergillus fumigatus and Fusarium oxysporum cultures with RPMI 1640 medium to a spore concentration of 1×10⁻⁶. 6 CFU / mL, for later use.

[0110] (2) Add 100 μL of diluted bacterial solution to each well of a sterile 96-well plate, and then add 100 μL of the combined solution to make the final total concentration of fluconazole 0.25, 0.5 and 1 μg / mL, respectively. PBS was set as a blank control group for each group.

[0111] (3) Subsequently, the sample was placed in an incubator and incubated statically at 37°C. The A value in each well was measured every 2 hours using a multi-functional microplate reader at a wavelength of 600 nm. The results were recorded continuously for 48 hours. Figure 4 As shown.

[0112] Depend on Figure 4 It was found that the combination therapy exhibited a concentration-dependent antibacterial effect. At a concentration of 1 μg / mL, it was able to effectively inhibit the growth of Fusarium and Aspergillus fumigatus.

[0113] Example 6

[0114] This embodiment provides several combination compositions, prepared by physically mixing fluconazole and triptolide or its pharmaceutical salts (triptolide sodium salt, triptolide phosphate) at a mass ratio of 4:1. The inhibitory effect of this combination composition on Aspergillus fumigatus was evaluated.

[0115] (1) Dilute the overnight cultured Aspergillus fumigatus culture with RPMI 1640 medium to a spore concentration of 1×10⁻⁶. 6 CFU / mL, for later use.

[0116] (2) Add 100 μL of diluted bacterial solution to each well of a sterile 96-well plate, and then add 100 μL of the combined solution of each group to prepare the final total concentration of fluconazole to 1 μg / mL. Set PBS as a blank control group.

[0117] (3) Subsequently, the sample was placed in an incubator and incubated statically at 37°C. The A value in each well was measured every 2 hours using a multi-functional microplate reader at a wavelength of 600 nm. The results were recorded continuously for 48 hours. Figure 5 As shown.

[0118] Depend on Figure 5 It is known that triptolide medicinal salt has the same antibacterial effect as triptolide. Both can effectively inhibit the growth of Aspergillus fumigatus at a concentration of 1 μg / mL.

[0119] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0120] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0121] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. The use of a combination composition in the preparation of a medicament for antifungal and antibacterial infections, characterized in that, The active components of the combined composition are an antibiotic and an antibiotic adjuvant; the antibiotic adjuvant is triptolide or its pharmaceutically acceptable salt.

2. The application according to claim 1, characterized in that, The antibiotics include any one or a combination of at least two of the following: amphotericin B, nystatin, miconazole, ketoconazole, fluconazole, itraconazole, voriconazole, posaconazole, flucytosine, terbinafine, caspofungin, micafungin, anisofungin, griseofulvin, penicillin, amoxicillin, ampicillin, cefadroxil, cephalexin, ceftazidime, imipenem, amikacin, gentamicin, doxycycline, tetracycline, minocycline, erythromycin, clarithromycin, azithromycin, sulfadiazine, ciprofloxacin, levofloxacin, metronidazole, clindamycin, or fosfomycin.

3. The application according to claim 1, characterized in that, The fungi include any one or a combination of at least two of the following: Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus oryzae, Fusarium, Cryptococcus neoformans, Candida albicans, Mucor, Histoplasma capsulatum, Cetacea, Coccidioides, Cryptococcus gutterus, or Paracoccidioides.

4. The application according to claim 1, characterized in that, The bacteria include any one or a combination of at least two of the following: Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Escherichia coli, Salmonella, Shigella, Streptococcus pneumoniae, Staphylococcus aureus, Mycobacterium tuberculosis, or Clostridium difficile.

5. The application according to claim 1, characterized in that, The molar ratio of the antibiotic to the antibiotic adjuvant is 1:(0.001-500).

6. The application according to claim 1, characterized in that, The combination is a single compound preparation or a combination of an antibiotic adjuvant and an individual antibiotic preparation; when it is a combination of an antibiotic adjuvant and an individual antibiotic preparation, it can be administered simultaneously, alternately, or sequentially. Preferably, the dosage form of the preparation includes an oral dosage form, an injectable dosage form, or a topical dosage form; Preferably, the administration route of the drug includes gastrointestinal administration, injection administration, transdermal administration, mucosal administration, inhalation administration, or local administration; Preferably, the combination composition further contains pharmaceutically acceptable excipients; Preferably, the excipients include any one or a combination of at least two of the following: carrier, diluent, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

7. A combined pharmaceutical composition for antifungal and antibacterial infections, characterized in that, The active components of the combined pharmaceutical composition are an antibiotic and an antibiotic adjuvant; The antibiotic adjuvant is triptolide or its pharmaceutically acceptable salt; The antibiotic is selected from any one or a combination of at least two of the following: amphotericin B, nystatin, miconazole, ketoconazole, fluconazole, itraconazole, voriconazole, posaconazole, flucytosine, terbinafine, caspofungin, micafungin, anisofungin, griseofulvin, penicillin, amoxicillin, ampicillin, cefadroxil, cephalexin, ceftazidime, imipenem, amikacin, gentamicin, doxycycline, tetracycline, minocycline, erythromycin, clarithromycin, azithromycin, sulfadiazine, ciprofloxacin, levofloxacin, metronidazole, clindamycin, or fosfomycin.

8. The combined pharmaceutical composition according to claim 7, characterized in that, The molar ratio of the antibiotic to the antibiotic adjuvant is 1:(0.001-500).

9. The combined pharmaceutical composition according to claim 7, characterized in that, The fungi include any one or a combination of at least two of the following: Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus oryzae, Fusarium, Cryptococcus neoformans, Candida albicans, Mucor, Histoplasma capsulatum, Cetacea, Coccidioides, Cryptococcus gutterus, or Paracoccidioides.

10. The combination pharmaceutical composition according to claim 7, characterized in that, The bacteria include any one or a combination of at least two of the following: Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Escherichia coli, Salmonella, Shigella, Streptococcus pneumoniae, Staphylococcus aureus, Mycobacterium tuberculosis, or Clostridium difficile.

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