An antifungal fatty acid inhibitor

Abstract

The application relates to the technical field of medicines, and relates to an antifungal fatty acid inhibitor, in particular to application of D-646 in preparation of an antifungal medicine; the fungus inhibited by the antifungal medicine does not include Candida glabrata. The application provides a new scheme for prevention and treatment of fungi.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technology, and in particular to an antifungal fatty acid inhibitor. Background Technology

[0002] It is estimated that 1.2 billion people worldwide are infected with fungi each year, resulting in more than 1.5 million deaths. Currently, there are four main classes of antifungal drugs used clinically: azoles, polyenes, echinocandins, and flucytosine. With the widespread use of antifungal drugs, pathogenic fungi are increasingly developing resistance to these drugs. Because fungi and human cells are both eukaryotic cells, the number of targets that can specifically act on fungal pathogens is relatively limited, making the development of new antifungal drugs or new targets quite difficult.

[0003] In view of this, this application is filed. Summary of the Invention

[0004] This application provides one or more embodiments of an antifungal fatty acid inhibitor, the technical solution of which includes:

[0005] One or more embodiments of this application provide the use of ND-646 in the preparation of antifungal drugs;

[0006] The fungi inhibited by the antifungal drug do not include Candida glabrata.

[0007] In some embodiments of this application, the Candida glabrata includes one or more of C. glabrata 0659, C. glabrata 0778, C. glabrata 1084, and C. glabrata 1167.

[0008] In some embodiments of this application, the fungi inhibited by the antifungal drug include fluconazole-resistant fungi.

[0009] In some embodiments of this application, the fungi inhibited by the antifungal drug include fluconazole-sensitive fungi.

[0010] In some embodiments of this application, the fungi inhibited by the antifungal drug include pathogenic fungi.

[0011] In some embodiments of this application, the pathogenic fungus includes one or more of Candida and Aspergillus.

[0012] In some embodiments of this application, the Candida includes one or more of Candida auris and Candida tropicalis.

[0013] In some embodiments of this application, the Aspergillus includes one or more of Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, and Aspergillus terreus.

[0014] One or more embodiments of this application also provide a pharmaceutical composition comprising ND-646 and voriconazole, wherein the ratio of ND-646 to voriconazole is (0.07–2.28) μg: (0.004–0.125) μg.

[0015] One or more embodiments of this application provide an antifungal agent comprising: the pharmaceutical composition thereof, with pharmaceutically acceptable excipients.

[0016] One or more embodiments of this application also provide a method for inhibiting fungi, the method comprising the following steps:

[0017] By contacting the target fungus with the antibacterial ingredient, the target fungus is inhibited;

[0018] The target fungi do not include Candida glabrata;

[0019] The antibacterial component is ND-646, the pharmaceutical composition, or the antifungal agent.

[0020] In some embodiments of this application, the Candida glabrata includes one or more of C. glabrata 0659, C. glabrata 0778, C. glabrata 1084, and C. glabrata 1167.

[0021] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Figure showing the results of a micro-broth dilution experiment;

[0024] Figure 2 The time-kill curves of ND-646 against Candida albicans (A) and Candida auris (B);

[0025] Figure 3 The chemical structure and drug susceptibility test results of ND-630 are shown in the figure.

[0026] Figure 4The chemical structure and drug susceptibility results of Acetyl-coA carboxylase-IN-1 are shown in the figure.

[0027] Figure 5 Figure showing the transformation of cells into hyphae in different drug treatment groups;

[0028] Figure 6 For in vivo anti-infection experiments, (A) animal experimental procedures, (B) photos of mouse models in different groups after the first treatment, (C) bacterial count of skin tissue in different treatment groups, and (D) histochemical staining of skin tissue in different treatment groups. Detailed Implementation

[0029] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0031] the term

[0032] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0033] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0034] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0035] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0036] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0037] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0038] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0039] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0040] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0041] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0042] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0043] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0044] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0045] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0046] A first aspect of this application provides the use of ND-646 in the preparation of antifungal drugs;

[0047] The fungi inhibited by the antifungal drug do not include Candida glabrata.

[0048] In some embodiments, the Candida glabrata includes one or more of C. glabrata 0659, C. glabrata 0778, C. glabrata 1084, and C. glabrata 1167.

[0049] In some embodiments, the fungi inhibited by the antifungal drug include fluconazole-resistant fungi.

[0050] In some embodiments, the fungi inhibited by the antifungal drug include fluconazole-sensitive fungi.

[0051] In some embodiments, the fungi inhibited by the antifungal drug include pathogenic fungi.

[0052] In some embodiments, the pathogenic fungus includes one or more of Candida and Aspergillus.

[0053] In some implementations, the Candida includes one or more of Candida auris and Candida tropicalis.

[0054] In some embodiments, the Aspergillus includes one or more of Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, and Aspergillus terreus.

[0055] A second aspect of this application provides a pharmaceutical composition comprising ND-646 and voriconazole, wherein the ratio of ND-646 to voriconazole is (0.07–2.28) μg : (0.004–0.125) μg. Optionally, the ratio of ND-646 to voriconazole is (0.07–0.11) μg : (0.004–0.008) μg. For example, the dosage ratios are 0.07 μg: 0.004 μg, 0.07 μg: 0.006 μg, 0.07 μg: 0.008 μg, 0.085 μg: 0.004 μg, 0.085 μg: 0.006 μg, 0.085 μg: 0.008 μg, 0.1 μg: 0.004 μg, 0.1 μg: 0.006 μg, 0.1 μg: 0.008 μg, 0.11 μg: 0.004 μg, 0.11 μg: 0.006 μg, and 0.11 μg: 0.008 μg. Further optionally, the ratio of ND-646 to voriconazole is 0.07 μg: 0.004 μg.

[0056] A third aspect of this application provides an antifungal agent comprising: the pharmaceutical composition thereof, with pharmaceutically acceptable excipients.

[0057] A fourth aspect of this application provides a method for inhibiting fungi, the method comprising the following steps:

[0058] By contacting the target fungus with the antibacterial ingredient, the target fungus is inhibited;

[0059] The target fungi do not include Candida glabrata;

[0060] The antibacterial component is ND-646, the pharmaceutical composition, or the antifungal agent.

[0061] In some embodiments, the Candida glabrata includes one or more of C. glabrata 0659, C. glabrata 0778, C. glabrata 1084, and C. glabrata 1167.

[0062] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0063] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0064] ND-646 is not cytotoxic. However, research on ND-646 has mainly focused on the oncology field and has not yet been applied to the antifungal field. This application investigated the growth inhibitory effects of ND-646 on various fungal strains, testing not only Candida species but also filamentous fungi; and found that ND-646 had a synergistic effect when used in combination with voriconazole. Results showed that ND-646 had antibacterial effects against various fungi, particularly Candida albicans (including sensitive and resistant strains) and Candida auris, with minimum inhibitory concentrations (MICs) ranging from 0.25 to 2 μmol / L. The drugs used in this application have undergone safety verification, and compared to some directly tested extract components, the pharmacokinetic and toxicological tests of the drugs used in the experiment are more complete. This application involves the following:

[0065] I. Drug Used: ND-646. Previous studies have shown that this drug can inhibit tumor growth by suppressing fatty acid synthesis in cells, and preliminary clinical trials have been conducted, demonstrating its safety for humans. However, there are no literature reports on its use for antifungal purposes. Therefore, this drug was used for fungal susceptibility testing in this application embodiment.

[0066] II. Strains used: Candida albicans, Candida glabrata, Candida tropicalis, Candida auris, and filamentous fungi (including Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, and Aspergillus terreus).

[0067] III. Drug Sensitivity Testing: To verify whether ND-646 has an inhibitory effect on Candida growth, a micro-broth dilution test was conducted according to the clinical reference method provided by the Clinical Laboratory Standards Institute (CLSI) to incubate the fungi for 24 hours (48 hours for filamentous fungi) to observe the antifungal effect of the drug.

[0068] IV. Efficacy Testing of New Drugs Combined with Antifungal Drugs: Drug combination therapy is a commonly used strategy to overcome the problem of fungal resistance. Due to their different mechanisms of action, combining two different drugs may produce different interactions, such as synergistic, additive, or antagonistic effects, which also determines the choice of drug combinations. This study explores the efficacy of this drug combined with commonly used antifungal drugs in clinical practice to explore new drug combination strategies.

[0069] V. Effects of the new drug on fungal virulence factors: The formation of hyphae is one of the manifestations of fungal virulence. By examining the differences in the transformation of fungal cells into hyphae under the action of the drug, the effects of ND-646 on fungal virulence factors can be determined.

[0070] VI. Anti-infective effects of the new drug in vivo: A mouse model of Candida infection of the skin was established. Different groups were treated with the carrier, fluconazole, amphotericin B, and ND-646, respectively. The antifungal effect of ND-646 in vivo was analyzed by statistical analysis of bacterial load and tissue staining of the skin tissue.

[0071] Specifically, the embodiments of this application include the following:

[0072] 1 Method

[0073] 1.1 Drug susceptibility testing of ND-646 against pathogenic fungi

[0074] (1) Drug concentration setting

[0075] For various Candida strains, drug susceptibility testing was first conducted using high concentration ranges (0μM, 1μM, 2μM, 4μM, 8μM, 16μM, 32μM, 64μM, 128μM). Then, drug susceptibility testing was conducted on strains with higher susceptibility, with drug concentrations set to 0μM, 0.06μM, 0.125μM, 0.25μM, 0.5μM, 1μM, and 2μM.

[0076] (2) Broth microdilution (BMD)

[0077] Add 100 μL of RPMI 1640 medium mixed with the above-mentioned gradient concentrations of ND-646 to a 96-well plate, and inoculate with 100 μL of bacterial suspension to a final density of 2.5 × 10⁻⁶. 3 CFU / mL ~5×10 3 CFU / mL, incubated in a 35℃ constant temperature incubator for 24h (48h for filamentous fungi), and the MIC results were interpreted.

[0078] 1.2 Efficacy test of ND-646 in combination with other antifungal drugs

[0079] The efficacy of ND-646 in combination with four commonly used clinical antifungal drugs was tested using the checkerboard method. Fluconazole (FLC), amphotericin B (AMB), voriconazole (VOR), and micafungin (MCF), the most commonly used drugs in clinical practice, were selected and used in combination with ND-646.

[0080] Fluconazole was administered at gradient concentrations of 0 μg / mL, 0.06 μg / mL, 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, while ND-646 was administered at concentrations of 0 μM, 0.06 μM, 0.125 μM, 0.25 μM, 0.5 μM, 1 μM, and 2 μM. Micro-broth dilution experiments were conducted by adding the drug in both longitudinal and transverse directions according to its concentration. 96-well plates inoculated with 100 μL of fungal suspension and 100 μL of drug-containing culture medium were incubated at 35°C for 24 h. The minimum inhibitory concentration (MIC) of ND-646 at a fluconazole concentration of 0%, the MIC of fluconazole at a ND-646 concentration of 0%, and the minimum inhibitory concentration (MIC) of each drug when used in combination were determined. The combined inhibitory index was calculated using the formula to assess the efficacy of the two drugs in combination.

[0081] Following the above procedure, the combined effects of ND-646 with amphotericin B (AMB), voriconazole (VOR), and micafungin (MCF) were tested.

[0082] 1.3 Effects of ND-646 on mycelial growth

[0083] Candida albicans ATCC 90028 cells that had reached the plateau phase were treated with different drugs (control group - fluconazole - amphotericin B - ND-646) and cultured at 37°C for 12-24 hours. Samples were collected and mycelial growth was observed under a microscope. The ratio of mycelia to cells was calculated to determine the differences in the effects of drugs on mycelial transformation.

[0084] 1.4 In vivo anti-infection experiment

[0085] A fungal abrasion model was used. Twelve female BALB / c mice, aged 6-8 weeks and weighing approximately 20 grams, were anesthetized, and their dorsal fur was shaved before creating the abrasion wound. The tissue was carefully scraped within a defined 8mm × 8mm area using a sterile 15-gauge scalpel. No bleeding occurred at the skin barrier in the scraped area. Following the abrasion, 20 μL of Candida albicans ATCC 90028 suspension (in 0.85% physiological saline, approximately 10 μL) was added. 9 CFU mL -1The inoculation solution was applied to the abrasion wound and allowed to air dry. Three hours after inoculation, the infected wound was treated locally. Mice were divided into four groups: a vector group (without antibiotics), a fluconazole group, amphotericin B group, and ND-646 group. Mice underwent two treatments: the first treatment was administered 3 hours after infection, and the second treatment was administered 24 hours after the first treatment. Twenty-four hours after the second treatment, the mice were euthanized. Wound tissue was harvested; one-third of the tissue was rinsed with YPD medium to collect fungal cells for colony counting, and two-thirds of the tissue was fixed in 10% neutral formalin solution for H&E / PAS staining and histological studies.

[0086] 2. Results

[0087] 2.1. Inhibitory effect on the growth of Candida albicans

[0088] Micro-broth dilution results demonstrated the inhibitory effect of the lipid-suppressing drug ND-646 on both fluconazole-resistant and fluconazole-sensitive Candida albicans strains. The fluconazole-resistant Candida albicans strains were C. albicans 23w and C. albicans 0368; the fluconazole-intermediate Candida albicans strains were C. albicans 23b and C. albicans 23z; and the fluconazole-sensitive Candida albicans strains were C. albicans 0165, C. albicans 1561, C. albicans 1658, C. albicans 1884, C. albicans 1973, C. albicans 1887, C. albicans 18113, C. albicans 23z 09031, C. albicans 23g, C. albicans ATCC 90028, C. albicans 0409, and C. albicans 0504.

[0089] Figure 1 The results of the micro-broth dilution experiment of ND-646 on 10 strains of Candida albicans are shown. It can be seen that the minimum inhibitory concentration range is 0.25μM to 2μM. Figure 1 In the middle, the left column from top to bottom consists of C. albicans 1973, C. albicans 0368, C. albicans 1887, C. albicans 18113, and C. albicans 0409; the right column from top to bottom consists of C. albicans 1561, C. albicans 0165, C. albicans 1884, C. albicans 1658, and C. albicans 0504.

[0090] In the experiment, the drug susceptibility of 16 Candida albicans strains to ND-646 was tested. The MIC results are shown in Table 1. ND-646 showed good growth inhibition effects on both strains sensitive to and resistant to other antifungal drugs.

[0091] Table 1. Antimicrobial susceptibility test results of ND-646 against Candida albicans

[0092]

[0093]

[0094] 2.2 Growth inhibitory effect on other Candida strains

[0095] In the experiment, the drug susceptibility of ND-646 to four non-albino Candida species (two Candida auris strains and two Candida tropicalis strains) was tested. The MIC results are shown in Table 2. ND-646 showed good growth inhibition effect on both Candida tropicalis and Candida auris.

[0096] Table 2. Antimicrobial susceptibility testing results of ND-646 against non-Candida albicans.

[0097] Strains MIC of ND-646 (μM) C.auris 23z 2 C.auris 211 1 C. tropicalalis 2365 2 C. tropicalalis 2581 8

[0098] 2.3 Growth-inhibiting effect on filamentous fungi

[0099] In this experiment, the susceptibility of ND-646 to 10 clinically common filamentous fungi (3 Aspergillus fumigatus, 3 Aspergillus niger, 2 Aspergillus flavus, and 2 Aspergillus terreus) was tested. The MIC results are shown in Table 3. ND-646 showed good growth inhibition against Aspergillus niger, Aspergillus flavus, and Aspergillus terreus, but the overall MIC value of filamentous fungi was higher than that of Candida species. The MIC value of Aspergillus fumigatus against ND-646 was higher than that of the other Aspergillus strains.

[0100] Table 3. Antimicrobial susceptibility test results of ND-646 against filamentous fungi

[0101] Strains MIC of ND-646 (μM) A. fumigatus 50 64 A. fumigatus 33 64 A. fumigatus 2 64 A.niger 23 8 A.niger 54 16 A.niger 13 16 A.flavus 20 4 A.flavus 53 8 A. terreus 28 16 A. terreus 30 16

[0102] 2.4ND-646's inhibitory effect on fungal growth

[0103] To investigate the mechanism of action of ND-646 on fungal growth, this application embodiment used gradient concentrations of the drug to culture Candida albicans and Candida auris for a long time, and measured the absorbance values ​​at different time points to measure the number of fungi. The culture conditions are as described in section 1.1.

[0104] Figure 2 The time-kill curves of ND-646 against Candida albicans (Figure A) and Candida auris (Figure B) are shown. Based on... Figure 2 The bacterial count curves of the untreated group and the low-drug-concentration group were consistently higher than those of the high-drug-concentration group, and the bacterial count decreased with increasing drug concentration. The time-varying curves showed that differences in bacterial count between the groups began to appear at the 8th hour, and this trend did not change significantly over time. This indicates that the drug's effect on fungal cells is concentration-dependent.

[0105] 2.5 Tests on the inhibitory effects of other ACC inhibitors on fungal growth

[0106] Antimicrobial susceptibility testing of other ACC inhibitors (see section 1.1 for details) revealed that inhibitors with different structures did not exhibit varying degrees of growth inhibition against the same strain.

[0107] To investigate whether all ACC inhibitors have fungal growth inhibitory effects, the homologue of ND-646, ND-630 (structural formula as shown), was used. Figure 3 Drug susceptibility testing of Candida albicans was performed (as shown in Figure A). Figure 3 The chemical structure and drug susceptibility results of ND-630 are shown below, where "1561" refers to "C. albicans 1561" and "1658" refers to "C. albicans 1658". Based on previous studies, ND-630 is a liver-specific fatty acid synthesis inhibitor. Strains C. albicans 1561 and C. albicans 1658 were selected. According to Table 1, the minimum inhibitory concentrations (MICs) of ND-646 against these two strains were 0.5 μM and 0.25 μM, respectively. Drug susceptibility testing was conducted (results are shown in Table 1). Figure 3 (Figure B) It was found that 32 μM ND-630 had no significant growth inhibitory effect on the two Candida albicans strains. Compared with ND-630, ND-646 had a lower antibacterial concentration and showed a better Candida growth inhibitory effect.

[0108] Drug susceptibility testing of Candida albicans was conducted using another ACC inhibitor, Acetyl-coA carboxylase-IN-1, which, according to previous studies, has antibacterial activity against Escherichia coli. Figure 4 The chemical structure and drug susceptibility results of Acetyl-coA carboxylase-IN-1 are shown. Strains C. albicans 1561 and C. albicans 1658 were selected. Referring to Table 1 above, the minimum inhibitory concentrations (MICs) of ND-646 against these two strains were 0.5 μM and 0.25 μM, respectively. Figure 4Figure B shows that the MICs of Acetyl-coA carboxylase-IN-1 against these two strains are 8 μM and 2 μM, respectively, which are higher than the MIC of ND-646.

[0109] Table 4. MIC (μM) of ND-646 and other ACC inhibitors

[0110] drug C. albicans 1561 C. albicans 1658 ND-646 0.5 0.25 ND-630 ＞32 ＞32 Acetyl-coA carboxylase-IN-1 8 2

[0111] 2.6 Combination test with existing antifungal drugs

[0112] The specific steps are as described in section 1.2, and the results are shown in the table below.

[0113] Table 5. MIC values ​​of ND-646 when used in combination with traditional antifungal drugs and when used alone (ATCC 90028 against Candida albicans).

[0114]

[0115]

[0116] In the table: FICI (fractional inhibitory concentration index), the combined antibacterial index; FICI = MIC A drug combination / MIC A drug monotherapy + MIC B drug combination / MIC B drug monotherapy. When FICI ≤ 0.5, it indicates a synergistic effect; 0.5 to < 1 indicates a partial synergistic effect; 1 indicates an additive effect; > 1 to < 4 indicates an irrelevant effect; ≥ 4 indicates an antagonistic effect.

[0117] Table 5 shows the results of the checkerboard method for detecting the combined use of ND-646 and traditional antifungal drugs. Through the calculation of the combined antifungal index FICI, it was found that ND-646 and voriconazole have a synergistic effect when used together.

[0118] 2.7 Effects on mycelial transformation

[0119] To investigate the effects of ND-646 on fungal virulence factors, this application's embodiments examined the drug's influence on the transformation of Candida albicans from cells to hyphae. Amphotericin B and fluconazole were used as positive controls, while untreated samples served as negative controls, as detailed in section 1.3.

[0120] The results are as follows Figure 5 As shown, Figure 5 The transformation of cells into hyphae in different drug treatment groups. Figure 5 It can be seen that, compared with the negative control group, the number of hyphae in the ND-646 treatment group was significantly reduced, which suggests that the drug may have inhibited hyphal growth.

[0121] 2.8 In vivo experiments to verify the antifungal effect of the drug

[0122] After studying the effects of ND-646 on fungal cell and hyphal growth in vitro, further validation is needed to determine how this drug acts on fungal cells in vivo. A mouse model of skin infection was constructed, treated with different drugs, and euthanized. A portion of the skin tissue was harvested for colony counting, and another portion was subjected to HE and PAS histochemical staining. Specific procedures are described in section 1.4, and the results are as follows. Figure 6 As shown, Figure 6 The diagram shows an in vivo anti-infection experiment. Figure A shows the animal experimental procedure; Figure B shows photographs of mouse models in different groups after the first treatment; Figure C shows the bacterial count in the skin tissue of different treatment groups; and Figure D shows the histochemical staining of skin tissue from different treatment groups. Figure 6 It can be seen that, compared with the control group, the number of fungal cells in the drug treatment group was significantly reduced, and the significance was even greater in the ND-646 treatment group. Figure 6 (See Figure C), which shows that ND-646 can also exert a good antifungal effect in vivo.

[0123] Overall, the embodiments of this application tested the inhibitory effect of ND-646 on fungal growth, demonstrating its antibacterial effect against a variety of common clinical pathogenic Aspergillus fungi. Furthermore, when used in combination with voriconazole, it exhibits a synergistic effect in inhibiting the growth of Candida albicans. The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described; however, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification.

[0124] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. Application of ND-646 in the preparation of antifungal drugs; The antifungal drugs inhibit Candida and Aspergillus, specifically Candida albicans 23w, 0368, 23b, 23z, 0165, 1561, 1658, 1884, 1973, 1887, 18113, 23z 09031, 23g, ATCC 90028, 0409, 0504, 23z, 211, 2365, and 2581.

2. The application according to claim 1, characterized in that, The Aspergillus species include one or more of Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, and Aspergillus terreus.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises ND-646 and voriconazole, wherein the mass ratio of ND-646 to voriconazole is 0.07:0.

004.

4. An antifungal agent, characterized in that, The antifungal agent comprises: the pharmaceutical composition of claim 3, and pharmaceutically acceptable excipients.

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