A polyene antifungal drug composition and its application

Abstract

The present invention discloses a pharmaceutical composition for preventing and treating diseases caused by fungi, which comprises a polyene antifungal drug or a pharmaceutically acceptable salt thereof and vitamin D or an analog thereof. The mass ratio of the polyene antifungal drug to vitamin D is 600:1 to 5:1, preferably 250:1 to 25:1. The pharmaceutical composition described in the present application has excellent antifungal activity, and its active ingredients have good synergistic effects.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a pharmaceutical composition for preventing and treating diseases caused by fungi, which comprises a polyene antifungal drug or a pharmaceutically acceptable salt thereof and vitamin D or an analog thereof. Technical Background

[0002] Fungal infections can be divided into superficial fungal diseases and deep fungal diseases according to the parts of the human body invaded. The main pathogens of the former are dermatophytes, Malassezia and Candida, which affect more than 1 billion people each year; the main pathogens of the latter are conditionally pathogenic fungi represented by Candida, Aspergillus, Mucor and Cryptococcus.

[0003] Fungal infections are mainly treated with drugs, but the available drugs are very limited, mainly azoles (fluconazole, voriconazole, etc.), echinocandins (caspofungin, micafungin, etc.), and polyenes (amphotericin B). Among them, amphotericin B is an important drug for the treatment of deep fungal infections, with significant efficacy. It is one of the most broad-spectrum and least resistant drugs among the existing antifungal agents, especially for those who are resistant to azoles and echinocandins. It is often used as the last line of defense, but the high incidence of dose-dependent nephrotoxicity and infusion-related adverse reactions limits its clinical application. How to enhance its efficacy and reduce toxicity is one of the key tasks in the research and development of antifungal drugs.

[0004] At present, a variety of molecules that have been reported to enhance the efficacy of polyene amphotericin B and nystatin include small molecule compounds derived from plants such as pure polyphenol curcumin, berberine, allicin, acteoside, plumbagin, etc.; microbial extracts such as phialotide F, nectriatide, simpotentin; and marketed drugs such as rifapentine and fingolimod, etc. However, most of these molecules only enhance the efficacy of amphotericin B within a narrow concentration range in vitro, and their safety and efficacy in vivo are unknown, so they cannot be used directly in clinical practice.

[0005] In terms of attenuation, new lipid preparations of amphotericin B have been developed, including liposomal amphotericin B, amphotericin B lipid complexes, and amphotericin B colloidal dispersion bundles, which have reduced toxicity but are expensive, making them difficult to use, especially for patients in developing countries, where there are more patients with invasive fungal infections and higher mortality rates. Moreover, the attenuation of liposomal amphotericin B is achieved by reducing its renal distribution, but the kidney is often the organ with the highest bacterial load in invasive fungal infections, so it often requires a larger dose to achieve the same effect of clearing pathogens in the kidney.

[0006] It can be seen that the existing antifungal drugs have problems such as large toxic side effects, limited clinical use and lack of drug compositions. It is still necessary to continue to find safe and effective ways to enhance the efficacy and reduce the toxicity of amphotericin B to expand its scope of use and increase the survival rate of patients with deep fungal infections.

[0007] In view of this, the technical solution of the present invention is proposed. Summary of the invention

[0008] The purpose of the present invention is to provide an antifungal drug composition, which can effectively reduce the minimum inhibitory concentration of polyene antifungal drugs, increase their in vivo efficacy, and reduce their nephrotoxicity, thereby expanding their scope of application and improving the survival chance of patients with fungal infections.

[0009] Therefore, in a first aspect, the present invention provides an antifungal pharmaceutical composition comprising component A and component B, wherein component A is selected from polyene antifungal drugs or pharmaceutically acceptable salts thereof; and component B is selected from vitamin D or its analogs.

[0010] Preferably, the A component comprises amphotericin B or nystatin, and the B component is selected from vitamin D 2 , Vitamin D 3 , 25(OH)D, 1,25(OH) 2 D or idecalcitol.

[0011]

[0012] Preferably, the A component is amphotericin B and the B component is vitamin D 3 .

[0013] Preferably, the mass ratio of the polyene antifungal drug or a pharmaceutically acceptable salt thereof to vitamin D or an analog thereof is 600:1 to 5:1, preferably 250:1 to 25:1.

[0014] The pharmaceutical composition of the present invention further comprises one or more pharmaceutically acceptable carriers.

[0015] The pharmaceutically acceptable carrier of the present invention refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to humans or other vertebrates, and also includes natural or synthetic organic or inorganic ingredients, which are combined with active ingredients to facilitate application.

[0016] Specifically, the pharmaceutically acceptable carriers of the present invention include substances contained in the dosage form in addition to the active ingredient, including but not limited to liposomes, microcapsules, microspheres, micelles, nanoparticles, lipid complexes, colloidal dispersions, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, adhesives, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, flavoring agents or antioxidants in the pharmaceutical field.

[0017] Among them, the dosage form can be an injection, solution, tablet, capsule, lozenge, pill, lozenge, gel, syrup, suspension, tincture, liniment, spirit, powder, oil, paste, plaster, film, aerosol, lozenge, patch, nasal drops, ear drops, suppository, liposome-encapsulated or liposome-encapsulated lyophilized preparation of active compounds or lipid complexes in aqueous suspension.

[0018] The pharmaceutical composition provided by the present invention is in the form of a pharmaceutical composition comprising a polyene antifungal drug or a pharmaceutically acceptable salt thereof and vitamin D or an analog thereof; or in the form of a combination of the respective preparations of a polyene antifungal drug or a pharmaceutically acceptable salt thereof and vitamin D or an analog thereof (e.g., a medicine kit).

[0019] The present invention also provides a method for preparing such a pharmaceutical composition, which comprises placing the active ingredients of the pharmaceutical composition of the present invention in a pharmaceutically acceptable carrier in a certain proportion.

[0020] On the other hand, the present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of antifungal drugs.

[0021] Furthermore, the pharmaceutical composition is used to prevent and / or treat diseases caused by fungi.

[0022] Preferably, the fungus is selected from the genus Candida, Aspergillus, Histoplasma, Coccidioides, Mucor, Cryptococcus, Chromoblastomyces, Trichophyton or Microsporum.

[0023] Preferably, the fungus is selected from Candida albicans, Candida auris, Candida krusei, Aspergillus fumigatus, Histoplasma capsulatum, Pneumocystis, Mucor, Cryptococcus neoformans, Blastomyces chromogenes, Trichophyton rubrum or Microsporum canis.

[0024] Furthermore, the pharmaceutical composition of the present invention can be administered by injection, oral administration or topical administration depending on the dosage form.

[0025] The injection administration includes subcutaneous injection, intramuscular injection, intravenous injection and intravenous drip.

[0026] The local administration includes administration to the lungs by inhalation, administration to the local surface of the skin and mucous membranes, or administration to the tissues under the local skin and mucous membranes.

[0027] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects:

[0028] ① In vitro, vitamin D or its analogs can reduce the minimum inhibitory concentration of polyene antifungal drugs by 2-4 times;

[0029] ② In the body of wax moth, compared with the use of polyene drugs alone, the pharmaceutical composition can increase the survival rate of wax moth infected with fungi by 15%-40%;

[0030] ③ In mice infected with vulvovaginal Candida, the drug composition can reduce the vaginal bacterial load of mice by about 10% compared with the use of polyene drugs alone. 2 CFU / g;

[0031] ④ In mice with systemic fungal infection, compared with the use of polyene drugs alone, the drug combination can increase the survival rate of mice by 30%-40% when used orally + by injection or by injection, and reduce the bacterial load in the target organ kidney by about 10 2 CFU / g;

[0032] ⑤ In mice with systemic fungal infection, compared with the use of polyene drugs alone, the pharmaceutical composition reduced kidney damage in mice, as shown by a decrease of about 60% in creatinine and urea nitrogen levels;

[0033] ⑥ The mechanism by which the pharmaceutical composition increases the efficacy of polyene antifungal drugs is clear, which is mainly to increase the content of ergosterol, the target of polyene antifungal drugs in fungi, and enhance the binding ability of polyene antifungal drugs with ergosterol. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The present invention shows the survival rate of wax moth infected with Candida albicans treated with a pharmaceutical composition of vitamin D or its analogs and polyenes.

[0035] Figure 2 The present invention shows the survival rate of wax moth infected with Aspergillus fumigatus treated with a pharmaceutical composition of vitamin D or its analogs and polyenes.

[0036] Figure 3 The present invention shows the survival rate of wax moth infected with Cryptococcus neoformans treated with a pharmaceutical composition of vitamin D or its analogs and polyenes.

[0037] Figure 4 The invention shows the efficacy of a pharmaceutical composition of vitamin D or its analogs and amphotericin B in treating vaginal candida infection.

[0038] Figure 5Figure 3. Survival of mice treated with vitamin D or its analogs in combination with amphotericin B for systemic fungal infection.

[0039] Figure 6 Kidney bacterial load in mice treated with vitamin D or its analogs in combination with amphotericin B for systemic fungal infection.

[0040] Figure 7 This study shows the effects of vitamin D or its analogs combined with amphotericin B on plasma CR and BUN levels in mice with systemic fungal infection.

[0041] Figure 8 The present invention shows the survival rate of mice infected with systemic Candida albicans treated with a pharmaceutical combination of vitamin D and polyenes.

[0042] Fig. 9 The present invention shows the survival rate of mice infected with systemic Aspergillus fumigatus treated with a pharmaceutical combination of vitamin D and polyenes. Fig.10 The present invention shows the survival rate of mice infected with systemic Cryptococcus neoformans treated with a pharmaceutical combination of vitamin D and polyenes.

[0043] Fig.11 Non-targeted metabolomics results for the antifungal activity of a pharmaceutical combination of vitamin D and polyenes.

[0044] Fig.12 It indicates the expression of genes related to the ergosterol synthesis pathway in bacterial cells when vitamin D is combined with polyenes.

[0045] Fig.13 It indicates the level of ergosterol in bacterial cells when vitamin D is combined with polyenes.

[0046] Fig.14 This indicates the effect of vitamin D combined with polyenes on the binding of polyenes to ergosterol. DETAILED DESCRIPTION

[0047] The present invention is further described and explained below in conjunction with examples, but these examples are not intended to limit the scope of the present invention.

[0048] Example 1 Preparation of a pharmaceutical composition of a polyene antifungal drug and vitamin D or its analogs

[0049] (1) Storage solution preparation: Vitamin D is prepared using anhydrous ethanol 2 , Vitamin D 3 , 25(OH)D, 1,25(OH) 2 D. Idecalcitol stock solution (20 mg / ml), dimethyl sulfoxide (DMSO) was used to prepare amphotericin B and nystatin stock solutions (20 mg / ml).

[0050] (2) Composition preparation method 1: Vitamin D2 , Vitamin D 3 , 25(OH)D, 1,25(OH) 2 D, idecalciferol, amphotericin B, and nystatin were diluted 2-fold to a final concentration of 12.5 μg / ml to 1600 μg / ml. According to the chessboard method, a combination solution was prepared. For example, each concentration of vitamin D 3 Mix equal volumes of amphotericin B at different concentrations to obtain vitamin D 3 (6.25μg / ml)-amphotericin B (6.25μg / ml), vitamin D 3 (6.25μg / ml)-amphotericin B (12.5μg / ml), vitamin D 3 (6.25μg / ml)-Amphotericin B (25μg / ml) to Vitamin D 3 (6.25μg / ml)-amphotericin B (800μg / ml), to vitamin D 3 (800 μg / ml)-Amphotericin B (800 μg / ml).

[0051] (3) Composition preparation method 2: dilute amphotericin B and nystatin to 0.1 mg / ml to 1.2 mg / ml with 0.5% glucose solution, and add vitamin D 2 , Vitamin D 3 , 25(OH)D, 1,25(OH) 2 D. Idecalcitol is diluted to 2 μg / ml and 20 μg / ml. The two are combined in a 1:1 volume ratio, and the final concentrations are amphotericin B and nystatin (0.05 mg / ml to 0.06 mg / ml); vitamin D 2 , Vitamin D 3 , 25(OH)D, 1,25(OH) 2 D. Idecalcitol (1 μg / ml and 10 μg / ml). The final mass ratio of the polyene antifungal drug or its pharmaceutically acceptable salt to vitamin D or its analogue is 600:1-5:1, and further the mass ratio of the polyene antifungal drug or its pharmaceutically acceptable salt to vitamin D or its analogue is 250:1-25:1.

[0052] Example 2 Evaluation of in vitro antifungal activity of vitamin D or its analogs in combination with amphotericin B

[0053] (1) Strains and culture medium

[0054] Representative strains of Candida, Aspergillus, Histoplasma, Coccidioides, Mucor, Cryptococcus, Chromoblastomyces, Trichophyton or Microsporum were selected and numbered as follows: Candida albicans (SC5314, CA1-3), Candida auris (B11243, B11221), Candida krusei (CK1, CK2), Cryptococcus neoformans (ATCC 32609, CN2), Aspergillus fumigatus (ATCC1022, AF17, AF23), Mucor (NRRL3631), Pneumocystis (ATCC PRA-159), Histoplasma (G217B), Chromoblastomyces (CBS114402), Trichophyton rubrum (ATCC4438), Microsporum canis (ATCC36299). The strains were stored in YPD+glycerol medium at -80°C, subcultured on YPD agar plates, and activated in YPD liquid medium.

[0055] (2) Experimental methods

[0056] Vitamin D detection using the chessboard method 2 , Vitamin D 3 , 25(OH)D, 1,25(OH) 2 D. The minimum inhibitory concentration (MIC) of idecalcitol combined with amphotericin B against the above fungi 50 The strain was cultured in YEPD agar medium at 35°C for 24 h. After activation twice, the activated strain was picked up with an inoculation loop and diluted with RPMI 1640 culture medium. The cell count was performed using a cell counting plate to adjust the concentration of the bacterial suspension to the required working concentration (2.0-2.5)×10 3 CFU / ml, freshly prepared before use. Dilute the drug stock solution to 2 times the working concentration with RPMI 1640 liquid culture medium. Take 100μl of the diluted drug solution and add it to columns 2-11 of the 96-well plate at a time; column 1 is the growth control well (no drug is added, replaced by 100μl of RPMI 1640 culture medium), and column 12 is the blank control well (only 200μl of RPMI 1640 culture medium is added). Add 100μl of bacterial suspension to the working concentration in columns 1-11 of the 96-well plate. After incubating the plate with bacterial solution and drug at 35°C for 48h, use a microplate reader to detect the OD value at a wavelength of 490nm. The background reading is subtracted from each well and compared with the results of the drug-free bacterial suspension wells. The lowest drug concentration that inhibits 50% fungal growth is used as the final MIC. 50 Results. Fractional inhibitory concentration (FICI) was calculated, ≤0.5 for synergistic effect; 0.5-4 for no interaction; >4 for antagonism. The experiment was repeated 3 times.

[0057] (3) Experimental results

[0058] Vitamin D in the present invention 2, Vitamin D 3 , 25(OH)D, 1,25(OH) 2 D. The antifungal activity of idecalcitol and amphotericin B was determined by the above experimental methods. The results are shown in Tables 1-5. The synergistic effect of the above vitamin D and its analogs on amphotericin B is common in Candida, Aspergillus, Histoplasma, Coccidioides, Mucor, Cryptococcus, Chromosomyces, Trichophyton, and Microsporum. In the above fungi, vitamin D and its analogs can reduce the minimum inhibitory concentration of amphotericin B by 2-4 times.

[0059] Table 1 Vitamin D 2 In vitro antifungal MICs in combination with amphotericin B 50 (μg / ml)

[0060]

[0061] Table 2 Vitamin D 3 In vitro antifungal MICs in combination with amphotericin B 50 (μg / ml)

[0062]

[0063] Table 3 In vitro antifungal MICs of 25(OH)D combined with amphotericin B 50 (μg / ml)

[0064]

[0065] Table 41,25(OH) 2 In vitro antifungal MICs of D combined with amphotericin B 50 (μg / ml)

[0066]

[0067] Table 5 In vitro antifungal MICs of the combination of idecalcitol and amphotericin B 50 (μg / ml)

[0068]

[0069] Example 3 Evaluation of in vitro antifungal activity of vitamin D or its analogs in combination with nystatin

[0070] Vitamin D 3 Evaluation of the in vitro antifungal activity of the combination of vitamin D and nystatin using the interaction with nystatin as an example

[0071] (1) Experimental methods

[0072] The experimental method is as described in Example 2.

[0073] (2) Experimental results

[0074] Vitamin D in the present invention 3 As shown in Table 6, the synergistic effect of vitamin D on nystatin is common in Candida, Aspergillus, Histoplasma, Coccidioides, Mucor, Cryptococcus, and Blastomyces. In the above fungi, vitamin D and its analogs can reduce the minimum inhibitory concentration of amphotericin B by 2-4 times.

[0075] Table 6 Vitamin D 3 In vitro antifungal MICs in combination with nystatin 50 (μg / ml)

[0076]

[0077] Example 4: Efficacy of a pharmaceutical composition of vitamin D or its analogs and polyenes in treating fungal infection of wax moth

[0078] Taking the common clinical Candida albicans, Aspergillus fumigatus and Cryptococcus as examples, the synergistic activity of vitamin D or its analogs on polyene antifungal drugs was evaluated in the wax moth.

[0079] (1) Experimental methods

[0080] First, select the last instar wax moth larvae (about 3 cm in length and 0.5 g in weight), and try to select healthy larvae without black spots. Randomly divide them into groups, 20 in each group, and place them in a 10 cm diameter plate. Candida albicans SC5314, Aspergillus fumigatus AF17, and Cryptococcus neoformans ATCC 32609 were activated in YPD liquid culture medium until the end of logarithmic growth, and the cells were collected and washed twice with PBS. The bacterial solution concentration was adjusted to 1×10 8 CFU / ml. Take the wax moth and fix it with the abdomen facing up. Disinfect the injection site of the wax moth larva with alcohol. Hold the insect body and gently pierce the second leg of the lower right with a Hamilton microinjection needle. Inject 10μl of bacterial solution from the needle tip to the middle of the insect body. After the model is successfully established, place it on a clean paper towel and let it stand for 1 minute. Inject 10μl of drugs in the second leg of the lower left, respectively, vitamin D 2 , Vitamin D 3 , 25(OH)D, 1,25(OH) 2 D, idecalciferol, amphotericin B or a mixture of the above vitamin D or analogs with amphotericin B or nystatin, the concentration of vitamin D or analogs is 0.2-2μg / kg, and the concentration of amphotericin B is 0.5-1mg / kg. The infection control group was injected with the same volume of normal saline. The dead insect bodies were observed and removed regularly every day, and the survival time of each group of wax moths was recorded. The survival rate of wax moths was calculated after 3 days.

[0081] (3) Experimental results

[0082] The experimental results are as follows Figure 1-3 As shown. In the control group (WT), all wax moths infected with Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans died on the fourth day. 2 , Vitamin D 3 , 25(OH)D, 1,25(OH) 2 D. Idecalcitol has no significant effect on the survival rate of wax moth; injection of amphotericin B alone increases the survival rate of wax moth by 50%-70%; vitamin D 2 , Vitamin D 3 , 25(OH)D, 1,25(OH) 2 D. The combination of idecalcitol and amphotericin B or nystatin can increase the survival rate of G. mellonella to 100% (P<0.05).

[0083] Example 5: Efficacy of a pharmaceutical composition of vitamin D or its analogs and amphotericin B in the topical treatment of vaginal candidal infections

[0084] (1) Experimental methods

[0085] 18-20g female BALB / c mice were intraperitoneally injected with 100μl of estrogen (100μl of sesame oil containing 0.5mgβ-estradiol) and 100μl of cyclophosphamide (100mg / kg) three days before infection and every three days thereafter. On the day of infection, Candida albicans SC5314 activated overnight in liquid YPD was collected, washed twice with PBS, and the bacterial solution was adjusted to a concentration of 1×10 7 CFU / ml. The mice were anesthetized with chloral hydrate, and 10 μl of bacterial solution was injected into the vagina of each mouse. The mouse hips were raised and left to stand for half an hour. On the second day after infection, the corresponding groups (3 mice in each group) were given vitamin D 2 , Vitamin D 3 , 25(OH)D, 1,25(OH) 2 D, idecalcitol, amphotericin B or the above vitamin D or analogs and amphotericin B pharmaceutical composition (prepared according to the composition preparation method 2 described in Example 1), the concentration of vitamin D or analogs is 0.5 mg / ml, and the concentration of amphotericin B is 0.5-1 mg / kg, for 3 consecutive days. After administration, the mouse hips are kept elevated for half an hour. On the 4th day, the mouse vagina is irrigated with PBS, and the diluted PBS is smeared on the YPD plate and cultured for 48 hours before counting.

[0086] (2) Experimental results

[0087] like Figure 4 As shown, vitamin D alone 2 , Vitamin D3 , 25(OH)D, 1,25(OH) 2 D. Idecalcitol had no significant effect on the vaginal bacterial load in mice. Compared with amphotericin B alone, the pharmaceutical composition could reduce the vaginal bacterial load in mice by about 10 2 CFU / g(P<0.05).

[0088] Example 6 Efficacy and toxicity of vitamin D or its analogs combined with polyene drugs in the treatment of systemic fungal infection in mice

[0089] (1) Experimental methods

[0090] Candida albicans SC5314 was activated in YPD liquid medium until the end of logarithmic growth, the cells were collected, and washed twice with PBS. The concentration of the bacterial solution was adjusted to 2×10 6 CFU / ml. Female BALB / c mice weighing 18-22 g were randomly divided into 10 groups, NS (injected with saline), WT (injected with SC5314), WT+vitamin D 2 , WT+vitamin D 3 、WT+25(OH)D、WT+1,25(OH) 2 D, WT+idecalciferol, WT+amphotericin B or WT+the above vitamin D or analogs were used in combination with amphotericin B. The bacterial solution was injected into different mice via the tail vein, 100 μl per mouse (the amount of bacteria for modeling was 2×10 5 The dose of vitamin D or its analogues was 0.2-0.5 μg / kg, which was administered orally from 3 days before infection to the end of the experiment 30 days after infection; the dose of amphotericin B or nystatin was 0.5-1 mg / kg, which was injected intraperitoneally from 12 hours after infection for 7 consecutive days.

[0091] Survival rate: The survival status of mice in each group was observed and recorded every day. After 30 days of observation, the survival rate was calculated and the surviving mice were killed. The survival curve was drawn and statistically tested by Log-rank test.

[0092] Kidney bacterial load: On the 3rd and 7th days after modeling, the left kidney of each group of mice (3 mice / group) was taken, weighed and ground, and the tissue grinding solution was diluted in multiples and smeared on YPD plates. The number of colonies was counted after incubation at 30°C for 48 hours. The results were statistically analyzed using Student tTest.

[0093] Renal function: Blood was collected after the treatment and the levels of creatinine and urea nitrogen were measured using HITACHI 3100Automatic Analyzer.

[0094] (2) Experimental results

[0095] Survival rate Figure 5 As shown, within 5 days of systemic Candida albicans infection, all mice died. Oral administration of vitamin D alone 2 , Vitamin D 3 , 25(OH)D, 1,25(OH) 2 D. Idecalcitol had no significant effect on the survival rate of mice. Compared with amphotericin B alone, the combination of the above vitamin D or its analogs with amphotericin B could increase the survival rate of mice from 50%-60% to 90%-100% (P<0.05).

[0096] Kidney bacterial load Figure 6 As shown in the figure, the bacterial load could not be detected because the mice in the untreated group and the group treated with vitamin D or its analogs alone died within 7 days. On the 5th day of treatment, the combination of vitamin D or its analogs and amphotericin B could reduce the bacterial load in the target organ kidney of mice by 10% compared with amphotericin B alone. 2 CFU / g(P<0.05).

[0097] Renal function Figure 7 As shown, on the 5th day of treatment, the combination of the above-mentioned vitamin D or its analogs with amphotericin B can reduce the plasma creatinine and urea nitrogen levels of mice by about 60% compared with the use of amphotericin B alone.

[0098] Example 7 Efficacy of a pharmaceutical composition of vitamin D or its analogs and polyene drugs in treating systemic fungal infection in mice

[0099] (1) Experimental methods

[0100] Candida albicans SC5314, Aspergillus fumigatus AF17, and Cryptococcus neoformans ATCC 32609 were activated in YPD liquid medium until the end of logarithmic growth, and the cells were collected and washed twice with PBS. The concentration of the bacterial solution was adjusted to 2×10 6 CFU / ml. Female BALB / c mice weighing 18-22 g were randomly divided into 10 groups, including NS (injected with saline), WT (injected with SC5314 or AF17 or ATCC 32609), WT+vitamin D 2 , WT+vitamin D 3 、WT+25(OH)D、WT+1,25(OH) 2 D, WT+idecalciferol, WT+amphotericin B or WT+the above vitamin D or analogs were respectively combined with amphotericin B, and the drugs were prepared according to the composition preparation method 2 described in Example 1. The bacterial solution was injected into different mice through the tail vein, 100 μl per mouse (the amount of bacteria for modeling was 2×10 5All drugs were injected intraperitoneally 12 h after infection for 7 consecutive days. The survival status of mice in each group was observed and recorded every day. After 30 days of observation, the survival rate was calculated, and the survival curve was drawn and statistically tested by Log-rank test.

[0101] (2) Experimental results

[0102] The survival rates of mice infected with Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans were as follows: Figure 8 , Fig. 9 , Fig.10 As shown, vitamin D alone was injected intraperitoneally 2 , Vitamin D 3 , 25(OH)D, 1,25(OH) 2 D. Idecalcitol had no significant effect on the survival rate of mice. Compared with amphotericin B alone, the pharmaceutical combination could increase the survival rate of mice by 30%-40% (P<0.05).

[0103] Example 8 Mechanism by which vitamin D or its analogs increase the antifungal efficacy of polyene drugs

[0104] Vitamin D 3 Taking the combination with amphotericin B as an example, the mechanism by which vitamin D or its analogs increase the efficacy of polyene antifungal drugs was clarified through non-targeted metabolomics, RT-qPCR, HPLC, ITC and other methods.

[0105] (1) Experimental methods

[0106] a. Non-targeted metabolomics detection of changes in metabolites in bacterial cells after drug action: 70 mg of bacterial cells were collected, 500 μl of pre-cooled methanol was added, 500 μl of pre-cooled ultrapure water was added, vortexed for 30 seconds, 100 mg of glass beads were added, liquid nitrogen was frozen for 5 minutes, freeze-thawed at room temperature, grinded at 55 Hz for 2 minutes, 12000 rpm at 4°C for 10 minutes, and the supernatant was vacuum dried. 300 μl of 2-chlorophenylalanine (4 ppm) methanol aqueous solution (1:1, 4°C) was added for re-dissolution, filtered through a 0.22 μm membrane, and detected by LC-MS.

[0107] b. RT-qPCR detection of gene transcription levels in the ergosterol biosynthesis pathway: Phenol-chloroform and Trizol methods were used for RNA extraction of Candida albicans and tissues, respectively. Candida albicans treated with drugs for 6 hours were collected, washed twice with DEPC water, resuspended in 300 μl pre-cooled AE buffer, added with 25 μl 20% SDS, 300 μl phenol: chloroform (1:1), mixed at high speed for 20 seconds, transferred to a 65°C water bath for 5 minutes, placed on ice for 10 minutes, centrifuged, and the supernatant was aspirated. Repeat once. Add ETOH+NaAc and let stand at -20°C for 1 hour. Wash with 75% ethanol, dry, and ddH2 O was used to dissolve the RNA and detect the RNA concentration. After reverse transcription, RT-qPCR was performed using cDNA as a template and the SYBR primer Ex Taq system with primers for genes related to ergosterol, oxidative stress, and RAAS pathways. The 2 -ΔΔCt .

[0108] c. Detection of whole-cell ergosterol content by HPLC: Collect the bacteria incubated with drugs for 6 hours and wash twice with PBS. Add 3 ml of methanol and 1 mg of cholesterol as internal standard. Add glass beads for homogenization for 30 seconds, repeat 8-10 times, and then incubate at 320 rpm for 1 hour. Centrifuge, collect the supernatant, vacuum dry, and resuspend in 500 μl of methanol. Prepare ergosterol and cholesterol standards by reverse 250 mm × 4 mm C 18 The chromatographic column was separated at 30°C and eluted in 100% methanol at a flow rate of 1 ml / min. The signals at 282 nm and 210 nm were detected, the peak areas at 12.5 min and 15 min were recorded, and a standard curve was drawn. Under the same detection conditions, the corresponding peak areas in the whole cell and cell membrane ergosterol extract samples were detected, and quantified according to the internal standard and the standard curve.

[0109] d. Isothermal titration calorimetry to detect the affinity of amphotericin B for membrane-embedded ergosterol: prepare palmitoyloleoylphosphatidylcholine (POPC) stock solution (20 mg / ml) and ergosterol stock solution (4 mg / ml),

[0110] Store at -20°C in dry argon. The two solutions were heated to 25°C and added to 13×100 mm test tubes. 1.2 ml POPC and 350 μl ergosterol solution were added to one test tube, and only 1.2 ml POPC was added to the other test tube. The solvent was removed by nitrogen flow. The dried lipid film was stored in a high vacuum environment for at least 8 h before use. The lipid film was then hydrated with 1 ml K buffer (5 mM HEPES, pH 7.4) and vortexed vigorously for about 3 min to form multilamellar vesicles (MLVs). MLVs were passed through a 0.20 μm Millipore (Billerica, MA) polycarbonate filter 21 times in Avanti Polar Lipids to form large unilamellar vesicles (LUVs). The LUV suspension was diluted 10 times with K buffer and 10 μl of each bottle was added to three 7 ml vials. The solvent was removed by nitrogen flow. Set up a vial without lipids as a blank control, add 450 μl of 8.9MH 2 SO 4 The four samples were placed in an aluminum block at 225°C for 25 min, 23°C for 5 min, and 150 μl of 30% w / v H 2 O2 Aqueous solution, 225℃ aluminum block for 30min, then moved to 23℃ and cooled for 5min, 3.9ml water was added to each sample. 500μl 2.5% w / v ammonium molybdate was added, vortexed vigorously 5 times, 500μl 10% w / v ascorbic acid was added and rotated 5 times. The vial was sealed with a PTFE-lined cap, placed in an aluminum block at 100℃ for 7min, cooled at 23℃ for 15min, and analyzed by UV / visible spectroscopy. The method for determining total phosphorus was to measure the absorbance at 820nm and compare this value with the standard curve obtained by this method and standard phosphorus solutions of known concentration to determine the phosphorus concentration. Ergosterol content was determined by spectrophotometry. Three independent samples were prepared, and 50μl of LUV suspension was added to 450μl 2:18:9 hexane:isopropanol:water (v / v / v), and then vortexed vigorously for about 1min. The concentration of ergosterol in the solution was detected by UV / Vis spectroscopy (NanoDrop 2000) with an extinction coefficient of 10400 l / mol / cm and UVmax of 282 nm. The percentage of ergosterol was determined by comparing it with the concentration of phosphorus. Amphotericin B solution or amphotericin B and vitamin D were added to the sample cell. 3 The solution was mixed and LUV was added to the syringe. The injection method was continuous injection of LUV, with the first injection of 0.4μl, followed by 19 injections of 2μl increments, with an interval of 150s. The experiment was carried out at 25°C, and the data analysis and titration curve fitting used MicroCal Origin software assuming a single binding site mode to calculate the Kd value. In order to correct for the heat of dilution and mixing, the heat of the last injection of each run was subtracted from all the injection heats for that particular experiment. The total heat change during the experiment was calculated in this way, and the formula is as follows: Where i = number of injections, n = total number of injections, Δhi = heat of the ith injection, and Δhn = heat of the last injection.

[0111] (2) Experimental results

[0112] like Fig.11 As shown in the figure, compared with amphotericin B alone, the combination of vitamin D and amphotericin B significantly increased the level of steroid metabolites in fungal cells. Further detection of the gene transcription level of the final stage of ergosterol synthesis, a known target of amphotericin B, and the content of the final product ergosterol found that compared with amphotericin B alone, the combination of vitamin D and amphotericin B increased the transcription level of 8 / 13 genes ( Fig.12 ) and the content of ergosterol in the final product ( Fig.13 ). The results of further testing the binding ability of amphotericin B to the target ergosterol by titration exothermicity showed that when vitamin D was combined with amphotericin B, the exothermicity of the combination binding to ergosterol was more than 3 times higher than that of amphotericin B alone ( Fig.14 ), indicating that the above-mentioned pharmaceutical composition increases the antifungal effect of polyene drugs by increasing the ergosterol content in fungal cells and the combination of polyene drugs with ergosterol.

[0113] Finally, it is necessary to explain that the specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. Use of an antifungal drug composition in the preparation of an antifungal drug, characterized in that: The antifungal drug composition consists of component A and component B, wherein component A is selected from amphotericin B, component B is selected from 25(OH)D, and the fungus is selected from Cryptococcus neoformans ATCC32609 or Aspergillus fumigatus ATCC1022.

2. The use according to claim 1, characterized in that: The administration method of the pharmaceutical composition is selected from injection, oral administration or local administration.

3. Use of an antifungal drug composition in the preparation of an antifungal drug, characterized in that: The antifungal drug composition consists of component A and component B, wherein component A is selected from amphotericin B, component B is selected from 1,25(OH)2D, and the fungus is selected from Candida albicans SC5314, Cryptococcus neoformans ATCC32609 or Pneumocystis carinii ATCC PRA-159.

4. The use according to claim 3, characterized in that: The administration method of the pharmaceutical composition is selected from injection, oral administration or local administration.

5. Use of an antifungal drug composition in the preparation of an antifungal drug, characterized in that: The antifungal drug composition consists of component A and component B, wherein component A is selected from amphotericin B, component B is selected from idecalciferol, and the fungus is selected from Cryptococcus neoformans ATCC32609.

6. The use according to claim 5, characterized in that: The administration method of the pharmaceutical composition is selected from injection, oral administration or local administration.

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