An antimicrobial peptide, its pharmaceutical composition, and its application

Abstract

This invention discloses an antimicrobial peptide, its pharmaceutical composition, and its applications. The invention optimizes the structure of the antimicrobial peptide through deuteration modification technology, significantly enhancing its targeted binding ability to β-1,3-D-glucan synthase. The affinity of this antimicrobial peptide for the aforementioned target is 9-27 times that of the control peptide. In vitro assays show that its minimum inhibitory concentration (MIC) against nine standard Candida strains is consistently 0.002 μg / mL, exhibiting significantly superior activity compared to fluconazole, anidoxurine, and the control peptide. It also exhibits extremely low cytotoxicity against L02 human normal hepatocytes. In in vivo experiments, in a neutropenic mouse model of Candida albicans infection, the antimicrobial peptide at all doses showed superior reduction in renal fungal load compared to anidoxurine and the control peptide. The antimicrobial peptide of this invention combines highly efficient anti-Candida activity, low cytotoxicity, and excellent in vivo efficacy, providing a safe and effective candidate drug for the treatment of drug-resistant candidiasis and possessing good clinical translational value.

Description

Technical Field

[0001] This invention relates to an antimicrobial peptide, its pharmaceutical composition, and its application, belonging to the field of biomedicine. Background Technology

[0002] Invasive candidiasis is the most prevalent and deadliest fungal infection worldwide, especially among immunocompromised individuals such as intensive care patients, cancer patients undergoing radiotherapy and chemotherapy, and organ transplant recipients, where infection rates and mortality rates continue to rise. Clinical data shows that more than 1.56 million new cases of invasive candidiasis occur globally each year, with bloodstream candidiasis having a mortality rate as high as 30% to 45%, making it one of the leading causes of hospital-acquired infection-related deaths.

[0003] With the irrational use of broad-spectrum antibiotics, the widespread use of immunosuppressants, and the routine use of invasive procedures such as central venous catheterization and mechanical ventilation, the problem of Candida resistance is becoming increasingly serious—the prevalence of multidrug-resistant strains such as fluconazole-resistant Candida glabrata and Candida auris has become a major global public health threat. Candida auris not only possesses extremely strong nosocomial transmission capabilities and is difficult to identify, but its bloodstream infection has a crude mortality rate of 30%–60%, and it is generally resistant to commonly used antifungal drugs, leading to a situation where clinical treatment is hampered by a lack of effective treatments.

[0004] In current clinical antifungal treatment systems, traditional drugs have many limitations that are difficult to overcome:

[0005] Triazole drugs (such as fluconazole): As a basic clinical drug, its target is fungal cytochrome P450 enzyme. The single target makes it easy to induce drug resistance. It has almost no inhibitory activity against fluconazole-resistant strains, and its tissue penetration is insufficient, which cannot meet the treatment needs of infections in special sites. Long-term use of high doses can also lead to liver damage.

[0006] Echinocandins (such as anifen): Although they target β-1,3-D-glucan synthase and are effective against some drug-resistant Candida, they still have problems such as limited target affinity and insufficient in vivo fungal clearance efficiency. Furthermore, adverse reactions may still occur in clinical applications, resulting in a narrow therapeutic window.

[0007] While existing peptide drug candidates possess the unique advantage of multi-target action and are less prone to inducing drug resistance, they generally suffer from bottlenecks such as weak antibacterial activity, insufficient target binding specificity, poor in vivo stability, and high cytotoxicity. To date, no highly effective and low-toxicity peptide drug targeting fluconazole-resistant Candida has been approved for marketing. Taking the following compound reported in published patent CN114616238A as an example (used as a control peptide in this article):

[0008]

[0009] Control peptide 1

[0010]

[0011] Control peptide 2

[0012]

[0013] Control peptide 3.

[0014] The aforementioned deuterated compounds exhibited dual drawbacks in practical applications: insufficient antifungal activity and high cytotoxicity. This directly hindered their further development and application in the clinical translation process of antimicrobial peptides. Therefore, based on the urgent needs of clinical treatment and the core technological goals of antimicrobial drug development, developing novel antimicrobial peptides that combine high antimicrobial activity, low toxicity, and high biocompatibility is of irreplaceable and crucial significance for overcoming the clinical treatment challenges caused by drug-resistant fungi and meeting the dual needs of clinical application and technology transfer.

[0015] Therefore, developing a novel antifungal drug with strong target affinity, significant activity against drug-resistant Candida, low cytotoxicity, and excellent in vivo efficacy has become an urgent need to solve the current clinical treatment dilemma. It has important clinical value and public health significance for reducing the mortality rate of invasive candidiasis and curbing the spread of drug-resistant strains. Summary of the Invention

[0016] Purpose of the invention: The purpose of this invention is to provide an antimicrobial peptide and its pharmaceutical composition, which aims to enhance the binding affinity of the parent nucleus to β-1,3-D-glucan synthase through deuteration structural modification, improve stability and water solubility, cover drug-resistant strains, solve the problems of frequent drug administration and drug resistance of existing drugs, and provide a highly efficient, long-acting and safe new treatment option for invasive fungal infections.

[0017] Technical solution: The antimicrobial peptide of the present invention has the following chemical structural formula:

[0018] ;

[0019] Wherein, the R basis is selected from , , , .

[0020] The pharmaceutical composition of the present invention contains the antimicrobial peptide or its pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug as an active ingredient, with or without pharmaceutically acceptable excipients.

[0021] Furthermore, the pharmaceutically acceptable excipients include one or more of the following: excipients, diluents, lubricants, flow aids, wetting agents, emulsifiers, and pH buffers.

[0022] Furthermore, the dosage forms of the pharmaceutical composition include injections, tablets, capsules, granules, oral liquids, sprays, nasal drops, eye drops, ear drops, suppositories, ointments, enteric-coated tablets, orally disintegrating tablets, oral instant films, creams, gels, patches, coatings, microcapsules, implants, syrups, effervescent tablets, sponges, lotions, liposome / nanoparticle dosage forms, inhaled powders, oral microcapsules, powders, chewable tablets, sustained-release tablets, suspensions, nebulizing solutions, and capsules.

[0023] Furthermore, injectable preparations include injection solutions, powder injections, infusion solutions, and sustained-release microsphere injections.

[0024] The application of the antimicrobial peptide or the pharmaceutical composition described in this invention in the preparation of microbial inhibitors.

[0025] Furthermore, the microorganism is a fungus.

[0026] Further, the fungi include one or more of the genera *Candida* and *Aspergillus*; wherein the *Candida* fungi include one or more of *Candida albicans*, *Candida glabrata*, *Candida krusei*, *Candida tropicalis*, *Candida auris*, *Candida lactea*, *Candida guinea*, *Candida parapsilosis*, and *Candida Portugueseia*; and the *Aspergillus* fungi include one or more of *Aspergillus fumigatus*, *Aspergillus flavus*, *Aspergillus lanceolata*, *Aspergillus alba*, *Aspergillus niger*, and *Aspergillus ochre*.

[0027] The use of the antimicrobial peptides or pharmaceutical compositions described in this invention in the preparation of drugs for the prevention and / or control of fungal infection-related diseases.

[0028] Further, the fungi include one or more of the genera *Candida* and *Aspergillus*; wherein the *Candida* fungi include one or more of *Candida albicans*, *Candida glabrata*, *Candida krusei*, *Candida tropicalis*, *Candida auris*, *Candida lactea*, *Candida guinea*, *Candida parapsilosis*, and *Candida Portugueseia*; and the *Aspergillus* fungi include one or more of *Aspergillus fumigatus*, *Aspergillus flavus*, *Aspergillus lanceolata*, *Aspergillus alba*, *Aspergillus niger*, and *Aspergillus ochre*.

[0029] Furthermore, the fungal infection-related diseases include one or more of the following: respiratory system infections, urinary system infections, musculoskeletal system infections, skin and soft tissue infections, systemic infections, circulatory system infections, digestive system infections, nervous system infections, endocrine system infections, and reproductive system infections.

[0030] The aforementioned applications include fungal infections affecting the respiratory system, such as pulmonary aspergillosis, pulmonary candidiasis, pulmonary cryptococcosis, and pulmonary mucormycosis; the urinary system, such as candidal urethritis, cystitis, pyelonephritis, and renal candidiasis; the musculoskeletal system, such as fungal arthritis and osteomyelitis; the skin and soft tissues, such as tinea corporis, tinea cruris, tinea pedis, onychomycosis, tinea versicolor, candidal intertrigo, cutaneous aspergillosis, cutaneous mucormycosis, and sporotrichosis; and systemic disseminated candidiasis, invasive aspergillosis, and malaria. Neifie basidium infection; fungal endocarditis and fungal sepsis of the circulatory system; oral candidiasis (thrush), esophageal candidiasis, intestinal candidiasis, and fungal hepatitis of the digestive system; cryptococcal meningitis, aspergillosis, and mucormycosis of the nervous system; candidal pancreatitis and thyroid fungal infection of the endocrine system; and vulvovaginal candidiasis (vaginal candidiasis) in women and candidal balanitis in men of the reproductive system.

[0031] Preferred "pharmaceuticalally acceptable excipients" are substances suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., substances with a reasonable benefit / risk ratio. They also include various excipients and diluents, and may contain liquids such as water, saline, glycerin, and ethanol, or auxiliary substances such as lubricants, glidants, wetting agents, emulsifiers, and pH buffers.

[0032] Preferably, the drug dosage form includes injections, oral preparations, or topical preparations, and the topical preparations include eye drops or lotions, wherein the dosage range of the antimicrobial peptide in the dosage form is: injections 0.001-1000 mg / kg; oral preparations 0.001-1000 mg / kg; topical preparations 1 / 10000-30% / vial; eye drops 1 / 10000-30% / vial; lotions 1 / 100000-20‰ / vial.

[0033] The core innovations of this invention are as follows:

[0034] This invention develops a novel class of antifungal and antimicrobial peptides through structural design and modification. Its core innovations achieve breakthroughs across three main dimensions: target binding specificity, antifungal activity, and safety, as detailed below:

[0035] 1. Innovative Target Binding: Ultra-high affinity targeting β-1,3-D-glucan synthase

[0036] The antimicrobial peptides of this invention achieve specific and efficient binding to the key fungal target β-1,3-D-glucan synthase through precise structural design: the dissociation constants of antimicrobial peptides 1-4 and β-1,3-D-glucan synthase were determined by microthermophoresis (MST). K dThe concentration is only 0.002~0.005 nM, which is far superior to the traditional antifungal drug anidoxuridine and control peptides 1-3; the control drug fluconazole has no binding activity to this enzyme (result shows "No"), further confirming the target specificity of the antimicrobial peptide of this invention. Its mechanism of action is completely different from that of fluconazole, which can effectively avoid the cross-resistance problem of fluconazole-resistant strains and provide a new mechanism of action for the treatment of drug-resistant Candida infections.

[0037] 2. Innovative antifungal activity: Highly and uniformly inhibits drug-resistant Candida, with significant in vitro and in vivo efficacy.

[0038] The antimicrobial peptides of this invention exhibit anti-Candida activity far exceeding that of traditional drugs in both in vitro and in vivo experiments, and also possess the core advantages of "high efficacy and strong uniformity":

[0039] Breakthrough in in vitro activity: Antimicrobial peptides 1-4 exhibited significantly higher antifungal activity against both Candida and Aspergillus fungi than commonly used clinical drugs. Against nine Candida strains, fluconazole showed no inhibitory effect on some strains (MIC > 64 μg / mL), anisulin's MIC was 0.25–0.5 μg / mL, and the control peptide 1-3's MIC was 0.015 μg / mL; however, antimicrobial peptides 1-4 had a MIC of 0.002 μg / mL against all strains, representing 7.5 times the activity of the control peptide 1-3. Against seven Aspergillus strains, fluconazole showed no effective inhibition (MEC > 64 μg / mL), anisulin's MEC was 0.25–0.5 μg / mL, and the control peptide 1-3's MEC was 0.015 μg / mL; the MEC of antimicrobial peptides 1-4 was as low as 0.002 μg / mL. In summary, antimicrobial peptides 1-4 possess both high efficiency and broad-spectrum antifungal activity, making them excellent candidates for clinical antifungal treatment (especially for infections caused by drug-resistant strains).

[0040] Excellent in vivo efficacy: This study validated the in vivo antifungal activity of antimicrobial peptides 1-4 using a mouse model of fluconazole-resistant Candida albicans infection with neutrophil reduction. Antimicrobial peptides 1-4 showed a significant advantage over the control peptide in reducing Candida albicans viral load in mouse kidneys: at a dose of 0.5 mg / kg, the viral load of the control peptide was log... 10 The bacterial load values ​​were 1.79–3.17, while the antimicrobial peptides were only 0.38–0.45, which was only 1 / 3 or even lower than the control peptides; at a dose of 1.5 mg / kg, the control peptides were 0.98–1.98, while the antimicrobial peptides were only 0.17–0.25, which was only 1 / 4 or even lower than the control peptides; at a dose of 4.5 mg / kg, the control peptides were 0.29–0.79, while the antimicrobial peptides were only 0.04–0.07, which was only 1 / 7 or even lower than the control peptides. Meanwhile, the 24-hour bacterial load log in the infected control group was... 10The viral load of anidoxime at all doses was as high as 4.91, and the viral load of anidoxime at all doses was also significantly higher than that of antimicrobial peptides, further demonstrating the potent in vivo antibacterial activity of antimicrobial peptides. Compared with the control drugs anidoxime and control peptides, antimicrobial peptides 1-4 showed more significant in vivo fungal clearance at the same dosage, providing a potential candidate for the treatment of drug-resistant Candida infections and possessing good clinical translational value.

[0041] 3. Safety Innovation: Highly advantageous with low nephrotoxicity, significantly broadening the therapeutic window.

[0042] This invention's antimicrobial peptides, through structural optimization, significantly reduce toxicity to normal host cells, solving the core challenge of "balancing efficacy and toxicity" in traditional antifungal drugs. Compared to the safety of fluconazole (relative ratio ≈ 0), the relative safety ratio of anidoxurine is approximately 10, and the relative ratios of control peptides 1-3 are approximately 18-20. Antimicrobial peptides 1-4 exhibit significantly superior safety, with relative ratios to fluconazole exceeding 180. Antimicrobial peptide 3, in particular, has a relative ratio approaching 200, representing a safety improvement of over 100 times compared to fluconazole and over 18 times compared to anidoxurine. This significantly reduces the risk of damage to normal cells, overcoming the clinical dilemma of fluconazole's "high toxicity" and surpassing the safety levels of anidoxurine and control peptides 1-3. This provides a solid foundation for the development of novel safe anti-infective agents and is expected to reduce adverse reactions caused by drug toxicity in clinical applications.

[0043] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) Strong target binding specificity, breaking the drug resistance dilemma and significantly broadening the scope of application of antifungal drugs. (2) Highly efficient and uniform antifungal activity, with excellent in vitro and in vivo efficacy. (3) Outstanding safety and a significantly broadened therapeutic window. (4) The antimicrobial peptide of the present invention also has advantages such as good biocompatibility, high stability, simple synthesis process, and controllable cost, and has shown excellent therapeutic effects in various infection models, possessing great potential for development into a new type of anti-infective drug. It is expected to be widely used in the prevention and treatment of various infections such as respiratory system, urinary system, and skin soft tissue, bringing breakthrough progress to the treatment of multidrug-resistant bacterial infections in clinical practice, and can be regarded as a safe, green, and highly efficient ideal antimicrobial agent to replace traditional antibiotics. Attached Figure Description

[0044] Figure 1 The HPLC chromatogram of antimicrobial peptide 1 is shown.

[0045] Figure 2 This is the mass spectrum of antimicrobial peptide 1;

[0046] Figure 3 The HPLC chromatogram of antimicrobial peptide 2;

[0047] Figure 4 This is the mass spectrum of antimicrobial peptide 2;

[0048] Figure 5 The HPLC chromatogram of antimicrobial peptide 3;

[0049] Figure 6 This is the mass spectrum of antimicrobial peptide 3;

[0050] Figure 7 The HPLC chromatogram of antimicrobial peptide 4;

[0051] Figure 8 This is the mass spectrum of antimicrobial peptide 4;

[0052] Figure 9 This is the result of an in vitro hepatotoxicity assay for the antimicrobial peptide. Detailed Implementation

[0053] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0054] The polypeptide compound in this embodiment of the invention, antimicrobial peptide 1, is:

[0055]

[0056] (I)

[0057] Antimicrobial peptide 2 is:

[0058]

[0059] (II)

[0060] Antimicrobial peptide 3 is:

[0061]

[0062] (III)

[0063] Antimicrobial peptide 4 is:

[0064]

[0065] (IV)

[0066] Example 1: Preparation method of antimicrobial peptides 1-4

[0067] 1. Preparation and purification of antimicrobial peptide 1

[0068] Under cold bath conditions, ethyl 2-(methylamino)-2-oxoacetate (0.41 g, 3.1 mmol, CAS No. 18522-95-7) was dissolved in 100 mL of dry tetrahydrofuran. Lithium aluminum deuteride (0.36 g, 8.7 mmol) was added in portions, and the mixture was stirred at this temperature for 3 h. The temperature was then slowly raised to 66 °C and refluxed for 12 h. The mixture was then cooled to 20–25 °C, and water was slowly added to quench the reflux. The mixture was filtered to obtain 0.23 g of a colorless liquid, namely 2-(methylamino)ethane-1,1,2,2-d4-1-ol, with a yield of 93.7%.

[0069] At room temperature, 0.23 g (2.9 mmol) of 2-(methylamino)ethane-1,1,2,2-d4-1-ol and iodomethane-d3 (0.91 g (6.4 mmol) were added to 10 mL of methanol, followed by sodium methoxide (0.33 g (6.1 mmol)). The mixture was stirred at room temperature for 24 h, and the solvent was removed under reduced pressure. The residue was recrystallized from ethanol to give 0.50 g of a white solid, namely 2-hydroxy-N-methyl-N,N-di(methyl-d3)ethylammonium-1,1,2,2-d4, with a yield of 71.5%.

[0070] At room temperature, 0.50 g (2.1 mmol) of 2-hydroxy-N-methyl-N,N-di(methyl-d3)ethylammonium-1,1,2,2-d4 iodide was dissolved in methanol, and 1.1 g (4.6 mmol) of silver oxide was added. The mixture was stirred at room temperature for 12 h, the solid was filtered off, the pH was adjusted to about 4 with 1 M hydrochloric acid, the solvent was evaporated under reduced pressure, ethanol was added, the insoluble matter was filtered off, and the mixture was concentrated under reduced pressure to obtain 0.3 g of 2-hydroxy-N-methyl-N,N-di(methyl-d3)ethylammonium-1,1,2,2-d4 chloride, with a yield of 95.4%.

[0071] Anidifene (12.5 mg, 0.01 mmol, purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number: T92381) was dissolved in anhydrous dimethyl sulfoxide (0.5 mL), and 2-hydroxy-N-methyl-N,N-di(methyl-d3)ethylammonium chloride-1,1,2,2-d4 (34.80 mg, 0.23 mmol) and a 1,4-dioxane solution containing 4 mol / L hydrochloric acid (2.5 μL, 0.01 mmol) were added. The reaction system was stirred at room temperature for 2 days, followed by heating at 40 °C for about 8 hours. After the reaction was completed, the system was diluted 10 times with a water-acetonitrile mixture (9:1, V / V), and the product was separated and purified by preparative high performance liquid chromatography (HPLC). The purified product was freeze-dried to obtain 5.4 mg of white solid product antimicrobial peptide 1.

[0072] The chromatographic column was a Waters Nova-Pak HR C18 (6 μm, 19 mm × 300 mm), and the column temperature was set to 40℃. The mobile phase system consisted of phase A (an aqueous solution containing 0.1% trifluoroacetic acid) and phase B (an acetonitrile solution containing 0.1% trifluoroacetic acid). A gradient elution program was used, with a flow rate controlled at 3 mL / min. The gradient conditions were that the proportion of mobile phase B linearly increased from 10% to 42% within 0–5 min, and further linearly increased from 42% to 50% within 5–25 min. The detection wavelength was set to 300 nm. Purity analysis results showed that the purity of antimicrobial peptide 1 reached 97.341% (see...). Figure 1 The corresponding HPLC retention time was 11.157 min; electrospray ionization positive ion mode LC / MS analysis showed that its mass-to-charge ratio (m / z) was 1237.5 [M+H]. + This confirms that the molecular structure of the product is consistent with that of the target compound.

[0073] 2. Preparation and purification of antimicrobial peptide 2

[0074] Under cold bath conditions, ethyl glycine (0.32 g, 3.1 mmol, CAS No. 459-73-4) was dissolved in 100 mL of dry tetrahydrofuran. Lithium aluminum deuteride (0.36 g, 8.7 mmol) was added in portions. The mixture was kept warm and stirred for 3 h, then slowly heated to 66 °C and refluxed for 12 h. The mixture was then cooled to 20–25 °C, and water was slowly added to quench the reflux. The mixture was filtered to obtain 0.18 g of colorless liquid, namely 2-aminoethane-1,1-d2-1-ol, with a yield of 92%.

[0075] At room temperature, 0.18 g (2.9 mmol) of 2-aminoethane-1,1-d2-1-ol and 1.4 g (9.5 mmol) of iodomethane-d3 were added to 10 mL of methanol, followed by the addition of sodium methoxide (0.33 g, 6.1 mmol). The mixture was stirred at room temperature for 24 h, and the solvent was removed under reduced pressure. The residue was recrystallized from ethanol to give 0.50 g of a white solid, namely 2-hydroxy-N,N,N-tris(methyl-d3)ethane-1-ammonium-2,2-d2 iodide, with a yield of 71.2%.

[0076] At room temperature, 0.50 g (2.1 mmol) of 2-hydroxy-N,N,N-tris(methyl-d3)ethane-1-ammonium-2,2-d2 iodide was dissolved in methanol, and 1.1 g (4.6 mmol) of silver oxide was added. The mixture was stirred at room temperature for 12 h, the solid was filtered off, the pH was adjusted to about 4 with 1 M hydrochloric acid, the solvent was evaporated under reduced pressure, ethanol was added, the insoluble matter was filtered off, and the mixture was concentrated under reduced pressure to obtain 0.3 g of 2-hydroxy-N,N,N-tris(methyl-d3)ethane-1-ammonium-2,2-d2 chloride, with a yield of 94.8%.

[0077] Anidifene (12.5 mg, 0.01 mmol) was dissolved in anhydrous dimethyl sulfoxide (0.5 mL), and 2-hydroxy-N,N,N-tris(methyl-d3)ethane-1-ammonium-2,2-d2 chloride (35.04 mg, 0.23 mmol) and a 1,4-dioxane solution containing 4 mol / L hydrochloric acid (2.5 μL, 0.01 mmol) were added. The reaction system was stirred at room temperature for 2 days, followed by heating at 40 °C for about 8 hours. After the reaction was completed, the system was diluted 10 times with a water-acetonitrile mixture (9:1, V / V), and purified by preparative high-performance liquid chromatography (HPLC). The purified product was freeze-dried to obtain 5.1 mg of antimicrobial peptide 2 with a purity of 95.994% (see [link to HPLC]). Figure 3 The corresponding HPLC retention time was 9.842 min; the mass-to-charge ratio of the antimicrobial peptide was 1238.5 [M+H] as determined by electrospray ionization positive ion mode LC / MS. + This confirms that its molecular structure is consistent with the target product.

[0078] 3. Preparation and purification of antimicrobial peptide 3

[0079] Under cold bath conditions, ethyl N,N-dimethylaminoacetate (0.42 g, 3.2 mmol, CAS No. 33229-89-9) was dissolved in 100 mL of dry tetrahydrofuran. Lithium aluminum deuteride (0.38 g, 9.0 mmol) was added in portions, and the mixture was stirred at this temperature for 3 h. The temperature was then slowly raised to 66 °C and refluxed for 12 h. The mixture was then cooled to 20–25 °C, and water was slowly added to quench the reflux. The mixture was filtered to obtain 0.27 g of a colorless liquid, namely 2-(dimethylamino)ethane-1,1-d2-ol, with a yield of 96%.

[0080] At room temperature, 0.28 g (3.1 mmol) of 2-(dimethylamino)ethane-1,1-d2-1-ol and 0.47 g (3.3 mmol) of iodomethane-d3 were added to 10 mL of methanol, followed by sodium methoxide (0.34 g, 6.3 mmol). The mixture was stirred at room temperature for 24 h, and the solvent was evaporated under reduced pressure. The residue was recrystallized from ethanol to give 0.51 g of a white solid, namely 2-hydroxy-N,N-dimethyl-N-(methyl-d3)ethyl-1-ammonium-2,2-d2, with a yield of 70%.

[0081] At room temperature, 0.51 g (2.2 mmol) of 2-hydroxy-N,N-dimethyl-N-(methyl-d3)ethyl-1-ammonium-2,2-d2 iodide was dissolved in methanol, and 1.11 g (4.8 mmol) of silver oxide was added. The mixture was stirred at room temperature for 12 h, the solid was filtered off, the pH was adjusted to about 4 with 1 M hydrochloric acid, the solvent was evaporated under reduced pressure, ethanol was added, the insoluble matter was filtered off, and the mixture was concentrated under reduced pressure to obtain 0.3 g of 2-hydroxy-N,N-dimethyl-N-(methyl-d3)ethyl-1-ammonium-2,2-d2 chloride, with a yield of 94.3%.

[0082] Anidifene (12.5 mg, 0.01 mmol) was dissolved in anhydrous dimethyl sulfoxide (0.5 mL), and 2-hydroxy-N,N-dimethyl-N-(methyl-d3)ethyl-1-ammonium-2,2-d2 chloride (33.51 mg, 0.23 mmol) and a 1,4-dioxane solution containing 4 mol / L hydrochloric acid (2.5 μL, 0.01 mmol) were added. The reaction system was stirred at room temperature for 2 days, followed by heating at 40 °C for about 8 hours. After the reaction was completed, the system was diluted 10 times with a water-acetonitrile mixture (9:1, V / V), and purified by preparative high-performance liquid chromatography (HPLC). The purified product was freeze-dried to obtain 5.6 mg of solid antimicrobial peptide 3 with a purity of 96.921% (see [link to HPLC]). Figure 5 The corresponding HPLC retention time was 5.983 min; the mass-to-charge ratio of the antimicrobial peptide was 1232.4 [M+H] as determined by electrospray ionization positive ion mode LC / MS. + This confirms that its molecular structure is consistent with the target product.

[0083] 4. Preparation and purification of antimicrobial peptide 4

[0084] At room temperature, 0.22 g (3.0 mmol, CAS No. 109-83-1) and iodomethane-d3 (0.93 g, 6.6 mmol) were added to 10 mL of methanol, followed by sodium methoxide (0.34 g, 6.3 mmol). The mixture was stirred at room temperature for 24 h, and the solvent was removed under reduced pressure. The residue was recrystallized from ethanol to give 0.50 g of a white solid, namely 2-hydroxy-N-methyl-N,N-di(methyl-d3)ethane-1-ammonium iodide, with a yield of 70.1%.

[0085] At room temperature, 0.51 g (2.1 mmol) of 2-hydroxy-N-methyl-N,N-di(methyl-d3)ethane-1-ammonium iodide was dissolved in methanol, and 1.1 g (4.7 mmol) of silver oxide was added. The mixture was stirred at room temperature for 12 h, the solid was filtered off, the pH was adjusted to about 4 with 1 M hydrochloric acid, the solvent was evaporated under reduced pressure, ethanol was added, the insoluble matter was filtered off, and the mixture was concentrated under reduced pressure to obtain 0.3 g of 2-hydroxy-N-methyl-N,N-di(methyl-d3)ethane-1-ammonium chloride, with a yield of 96.6%.

[0086] Anidifene (12.5 mg, 0.01 mmol) was dissolved in anhydrous dimethyl sulfoxide (0.5 mL), and 2-hydroxy-N-methyl-N,N-di(methyl-d3)ethane-1-ammonium chloride (33.86 mg, 0.23 mmol) and a 1,4-dioxane solution containing 4 mol / L hydrochloric acid (2.5 μL, 0.01 mmol) were added. The reaction system was stirred at room temperature for 2 days, followed by heating at 40 °C for about 8 hours. After the reaction was completed, the system was diluted 10 times with a water-acetonitrile mixture (9:1, V / V), and the product was separated and purified by preparative high-performance liquid chromatography (HPLC). The purified product was freeze-dried to obtain 5.3 mg of solid antimicrobial peptide 4 with a purity of 98.712% (see [link to product details]). Figure 7 The corresponding HPLC retention time was 5.892 min; the mass-to-charge ratio of the antimicrobial peptide was 1233.5 [M+H] as determined by electrospray ionization positive ion mode LC / MS. + This confirms that its molecular structure is consistent with the target product.

[0087] Combination Figure 1-4 The HPLC chromatograms show that the peak area of ​​each antimicrobial peptide exceeds 95% (97.341% for antimicrobial peptide 1, 95.994% for antimicrobial peptide 2, 96.921% for antimicrobial peptide 3, and 98.712% for antimicrobial peptide 4), indicating that the prepared antimicrobial peptides 1-4 have high purity. The peptides prepared above were identified and analyzed using an Agilent 1200 tandem with an AB SCIEX API3200, and the results are as follows. Figure 1-4 And as shown in Table 1:

[0088] Table 1. Basic chemical parameters, HPLC retention time, and mass-to-charge ratio (m / z) of antimicrobial peptides.

[0089] .

[0090] Example 2: Microthermophoresis (MST) to determine the interaction between antimicrobial peptides and β-1,3-D-glucan synthase

[0091] In this study, fluconazole (Shanghai Yuanye Biotechnology Co., Ltd., catalog number: B33743), non-deuterated anidoxurine, and a control peptide (CN114616238A) were used as control drugs. The control peptide was synthesized by Shanghai Jier Biochemical Co., Ltd. (purity >96%) and could be used without further purification. The binding affinity of antimicrobial peptides 1-4, fluconazole, anidoxurine, and the control peptide to β-1,3-D-glucan synthase (Sigma-Aldrich, catalog number 67138) was determined using micro-thermophoresis. The specific procedures are as follows:

[0092] Solution preparation: A series of dilution solutions of the antimicrobial peptide were prepared using PBS buffer (formulation: 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4, 0.05% Tween-20, pH 7.4). The concentration gradients were set as follows: 0.00025, 0.0005, 0.001, 0.002, 0.004, 0.008, 0.016, 0.032, 0.064, 0.128, 0.256, 0.512, 1.024, 2.048, 4.096, 8.192, and 10 nmol / L. The control drugs fluconazole, non-deuterated anidoxuridine, and control peptide were also prepared with the same concentration gradients using the same buffer solution. Sample mixing: The antimicrobial peptide solution / fluconazole solution / non-deuterated anidoxurine solution / control peptide solution of each concentration were thoroughly mixed with β-1,3-D-glucan synthase solution at a final concentration of 0.1 nM at a volume ratio of 1:1. The mixture was then injected into a standard glass capillary tube. Detection and analysis: Detection was performed using a NanoTemper Monolith NT.115 molecular interaction analyzer with the following parameters: LED power 50%, MST power 20%, on-time 30 seconds, off-time 5 seconds, and experimental temperature 37℃. Data were analyzed using NanoTemper Analysis software to obtain the dissociation constants of each test sample and enzyme. K d , nM).

[0093] As shown in Table 2, the affinity of antimicrobial peptides 1-4 for β-1,3-D-glucan synthase (expressed as dissociation constant) K d measure, K d The smaller the affinity, the stronger the binding affinity. It was significantly superior to the three control peptides: fluconazole, anifenazole, and the control peptide. Fluconazole showed no binding activity against this enzyme (result: "No"), while antimicrobial peptides 1-4... K d As low as 0.002~0.005 nM; Anifene K dIt is 0.791 nM; the control peptide is K d The concentration ranges from 0.043 to 0.053 nM, and the affinity of the antimicrobial peptides is 9 to 27 times that of the control. This demonstrates that the targeted binding ability of antimicrobial peptides 1-4 to β-1,3-D-glucan synthase is far superior to that of the control, providing a molecular basis for their potent antifungal activity.

[0094] Table 2. Affinity results of antimicrobial peptides 1-4, fluconazole, anidoxurine, and control peptides to β-1,3-D-glucan synthase.

[0095] .

[0096] Example 3: Assay of the antimicrobial activity of antimicrobial peptides against Candida albicans

[0097] This embodiment aims to evaluate the in vitro antifungal activity of antimicrobial peptides by determining the 24-hour minimum inhibitory concentrations (MICs, in μg / mL) of fluconazole, non-deuterated anidoxurine, control peptides 1-3, and antimicrobial peptides 1-4 against nine standard Candida species (Candida albicans ATCC 76615, Candida glabrata ATCC 90030, Candida krusei ATCC 14243, Candida tropicalis ATCC 90874, Candida auris ATCC MYA-5001, Candida lactis ATCC 66028, Candida guinea natans ATCC 6260, Candida parapneumoniae ATCC 22019, and Candida Portugueseia ATCC 42720). The strains used were... Cryopreservation with glycerol at 80℃ was performed. Before the experiment, the culture was subcultured at least twice on Sabouraud dextrose agar at 35℃ without antibiotics to restore activity and purity. MIC determination followed the CLSI microdilution method combined with an automated liquid handling system: five activated colonies with a diameter ≥1mm were picked and suspended in 5mL of sterile 0.85% physiological saline, vortexed for 15 seconds, and then adjusted to 0.5 McFarland turbidity using a spectrophotometer (530nm) (corresponding to a stock suspension concentration of 1×10⁻⁶). 6 ~5×10 6 (CFU / mL), then serially diluted 1:2000 in RPMI 1640 medium to prepare a final concentration of 5×10⁻⁶ CFU / mL. 3 ~2.5×10 3The inoculum suspension was prepared at CFU / mL; then the test drug was prepared into a 5.12 mg / mL stock solution, and serially diluted 1:1 in 17-well deep-plates to obtain drug gradients of 2560, 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.6, 0.3, 0.15, 0.075, and 0 μg / mL. 10 μL of each gradient was then transferred to a 96-well test plate containing 190 μL of RPMI 1640 medium, and 10 μL of the inoculum suspension was added to form a 200 μL well. A final concentration gradient of μL (drug diluted 20-fold) was prepared on the test plate to cover 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03, 0.015, 0.0075, 0.00375, 0.002, and 0 μg / mL. After sealing the test plate, it was incubated at 35°C for 24 hours, and the growth was observed using a microplate reader. The MIC was defined as the lowest drug concentration that could completely inhibit the visible growth of the strain.

[0098] The results are shown in Table 3. The in vitro antifungal activity of antimicrobial peptides 1-4 against nine standard strains of Candida albicans, Candida glabrata, and Candida krusei was significantly better than that of fluconazole, anidoxuridine, and the control peptide. The core advantages are as follows: the minimum inhibitory concentration (MIC) of fluconazole against most Candida strains, such as Candida albicans ATCC76615 and Candida glabrata ATCC90030, was >64. Only the MIC against Candida lactis ATCC66028, Candida guinea natans ATCC6260, and Candida davidii ATCC42720 was 32, indicating that most of the tested Candida strains showed a resistance phenotype to fluconazole. In contrast, the MIC of antimicrobial peptides 1-4 was only 0.002 μg / mL, which completely solved the problem of fluconazole resistance to Candida. Compared to anidoxurine, the activity is increased by more than 100 times, resulting in significantly enhanced efficacy. Anidoxurine's MIC against different strains ranges from 0.25 to 0.5 μg / mL, while the MIC of antimicrobial peptides 1-4 remains stable at 0.002 μg / mL, achieving complete inhibition at extremely low concentrations. Compared to the control peptide, the activity is further upgraded, and the inhibition threshold is further lowered. The MIC of control peptide 1-3 is 0.015 μg / mL, while the MIC of antimicrobial peptide 1-4 (0.002 μg / mL) is 7.5 times higher, further improving antifungal efficacy compared to the control peptide. Furthermore, antimicrobial peptides 1-4 exhibited consistently high inhibitory effects against nine different Candida species (including clinically high-risk strains of resistance), without specific resistance, providing a superior new drug candidate for clinical treatment of Candida infections (especially resistant strains).

[0099] Table 3. Minimum inhibitory concentration (MIC) of antimicrobial peptides against Candida albicans

[0100] .

[0101] Example 4: Determination of the anti-Aspergillus activity of antimicrobial peptides

[0102] This embodiment aims to evaluate the in vitro antifungal activity of the target antimicrobial peptides by determining the minimum effective concentrations (MEC, unit: μg / mL) of fluconazole, non-deuterated anidoxuron, control peptides 1-3, and antimicrobial peptides 1-4 against seven standard Aspergillus strains (Aspergillus fumigatus ATCC 204305, Aspergillus flavus ATCC 22546, Aspergillus lanceolata ATCC 10058, Aspergillus white mold ATCC 13686, Aspergillus niger ATCC 16888, Aspergillus ochraceus ATCC 96919, and Aspergillus fumigatus ATCC MYA-3626). The strains were preserved in -80℃ glycerol cryovials. Before the experiment, they were streaked onto antibiotic-free potato dextrose agar slant medium and continuously subcultured at 35℃ for 7 days to restore the sporulation capacity and culture purity of the strains. The spores on the slant culture medium were then thoroughly washed with 5 mL of sterile physiological saline. The spores were filtered through two layers of sterile gauze to remove hyphal fragments, and vortexed for 30 seconds to ensure uniform dispersion. Turbidity was measured using a 530 nm spectrophotometer and adjusted to 0.5 McFarland turbidity. Simultaneously, viable cell counts were performed using a hemocytometer to verify that the stock spore suspension concentration was 1 × 10⁻⁶. 6 ~5×10 6 CFU / mL; then serially diluted 1:500 with RPMI 1640 medium to prepare a final concentration of 2×10⁻⁶ CFU / mL. 3 ~1×10 4 Prepare an inoculum suspension of CFU / mL for later use. The test drug was prepared as a 10.24 mg / mL stock solution and serially diluted 1:1 in 17-well deep-plate columns to form a deep-plate concentration gradient: 5120, 2560, 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.6, 0.3, 0.15, 0 μg / mL. 10 μL of the deep-plate drug solution was transferred to a 96-well test plate containing 190 μL of RPMI 1640 medium, and 10 μL of inoculum suspension (drug diluted 20-fold) was added to form a final test plate concentration gradient: 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03, 0.015, 0.0075, 0.002, 0 μg / mL. μg / mL; after sealing the test plate, incubate at 35℃ for 48 hours; after incubation, observe the hyphal growth status of each well using a microplate observer, and the lowest drug concentration at which obvious hyphal morphological changes (such as reduced branching and shortened hyphae) can be observed.

[0103] The results are shown in Table 4. The minimum effective concentration (MEC) of antimicrobial peptides 1-4 against the seven standard Aspergillus strains showed an overwhelming advantage compared to fluconazole, anidoxuron, and control peptide 1-3: Fluconazole had an MEC > 64 μg / mL against all seven Aspergillus strains (no effective inhibition), while the MEC of antimicrobial peptide 1-4 was only 0.002 μg / mL, directly filling the antibacterial gap of fluconazole; the MEC of anidoxuron was 0.25–0.5 μg / mL, and the activity of antimicrobial peptide 1-4 was 125–250 times that of anidoxuron; the MEC of control peptide 1-3 was 0.015 μg / mL, and the activity of antimicrobial peptide 1-4 was also 7.5 times that of anidoxuron. This demonstrates that antimicrobial peptide 1-4 can effectively inhibit Aspergillus at extremely low concentrations, providing a better candidate for combating Aspergillus infection.

[0104] Table 4. Minimum Effective Concentration (MEC) of Antimicrobial Peptides against Aspergillus

[0105] .

[0106] Example 5: In vitro hepatotoxicity assay of antimicrobial peptides

[0107] This experiment used the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazole bromide (MTT) colorimetric method to evaluate hepatotoxicity. The specific procedure was as follows: First, L02 normal human hepatocytes were seeded in DMEM medium containing 10% fetal bovine serum and incubated at 37°C with 5% CO2 until the logarithmic growth phase. Then, they were incubated with 2.5 × 10⁻⁶ cells / mL. 3 Cells were seeded at a density of 1 / well in 96-well plates and pre-cultured for 24 hours until complete cell adhesion. Then, serially diluted test antimicrobial peptides 1-4, anifenazole, fluconazole, or control peptides 1-3 were added to the wells of adherent cells, with final concentration gradients of 0.0075, 0.015, 0.03, 0.06, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, and 256 μg / mL, and cultured in the dark for 48 hours. After drug treatment, the drug-containing medium was removed, and 110 μL of freshly prepared MTT working solution (composed of 10 μL of 5 mg / mL MTT-PBS solution and 100 μL of DMEM medium) was added to each well. The cells were incubated in the dark for 3 hours to induce formazan crystal formation. After discarding the MTT solution, 100 μL of a solution containing 40% N,N... was added to each well. A solution of dimethylformamide, 16% sodium dodecyl sulfate, and 2% glacial acetic acid was prepared and shaken for 10 minutes to fully dissolve the crystals. The absorbance of each well was measured at 570 nm using a microplate reader. Wells in untreated blank culture medium (defined as 100% cell viability) served as negative controls, and wells treated with fluconazole served as positive controls (defined as 0% cell viability). The half-maximal inhibitory concentration (IC50) was calculated using a nonlinear regression model.50 The in vitro hepatotoxicity of each test sample was assessed by measuring the concentration of the compound that reduced cell viability by 50% compared to the negative control.

[0108] This experiment used the MTT assay to evaluate the toxicity of the test samples to L02 normal human hepatocytes, with the "IC50" assay as the criterion. 50 The "ratio to fluconazole" characterizes safety (a higher ratio indicates lower cytotoxicity and better safety), and the results are as follows: Figure 9 As shown: using the safety profile of fluconazole as a baseline (relative ratio ≈ 0), the relative safety ratio of anidoxurine was approximately 10, and the relative ratios of control peptides 1-3 were approximately 18-20. However, the safety performance of antimicrobial peptides 1-4 was significantly better than the aforementioned control drugs, with relative safety ratios to fluconazole all exceeding 180, and the ratio for antimicrobial peptide 3 approaching 200. This result indicates that the hepatocellular safety of antimicrobial peptides 1-4 is more than 100 times better than fluconazole and more than 18 times better than anidoxurine, significantly reducing the risk of damage to normal cells. This characteristic not only effectively improves the toxicity issues existing in the clinical application of fluconazole but also demonstrates superior safety compared to anidoxurine and control peptides 1-3, providing crucial data support for the development of highly safe antifungal agents. It is expected to reduce drug-related adverse reactions in clinical treatment and improve patient tolerance.

[0109] Example 6: In vivo anti-Candida activity assay of antimicrobial peptides

[0110] In this embodiment, to evaluate the in vivo antifungal activity of injectable antimicrobial peptides 1-4 and control drugs anifenfenidine and 1-3, a mouse model of Candida albicans infection with neutropenia was constructed, and the drugs were administered via intraperitoneal injection. The colony-forming units (CFU) in kidney tissue were used as the core evaluation index to analyze the in vivo activity of the drugs. The strain used in the experiment was Candida albicans ATCC76615 (fluconazole-resistant strain). After being frozen at -80°C, the strain was rapidly thawed upon recovery and inoculated onto Sabouraud dextrose agar (SDA) plates, and activated by incubation at 35°C for 24 hours. Subsequently, the colonies were transferred to phosphate-buffered saline (PBS) and diluted to a final concentration of 3.8 × 10⁻⁶. 5CFU / mL was used as the inoculum for infection. Seven-week-old female CD-1 mice (weighing 15-30g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used as experimental animals, with 20 groups (n=5 per group): control group 2 hours after infection (kidney harvested 2 hours after infection, no drug administration), control group 24 hours after infection (kidney harvested 24 hours after infection, no drug administration), anidoxurine group (dose 0.5, 1.5, 4.5 mg / kg), control peptide 1-3 group (dose 0.5, 1.5, 4.5 mg / kg), and antimicrobial peptide 1-4 group (dose 0.5, 1.5, 4.5 mg / kg). Model establishment and drug administration procedure: Four days and one day before infection, mice were intraperitoneally injected with 150 mg / kg cyclophosphamide at 10 mL / kg body weight to establish a neutropenia model; subsequently, infection was completed by intravenous injection of 0.1 mL of the inoculum via the tail vein; two hours after infection, each drug administration group was intraperitoneally injected with the corresponding dose of the test compound at 10 mL / kg body weight, while the infection control group received no drug administration. Sample processing and detection: Mouse kidney tissue was aseptically collected 2 hours after infection (infection control group) and 24 hours after infection (other groups). 2 mL of sterile PBS was added to each mouse kidney, and the tissue was homogenized thoroughly using a Polytron 3100 tissue homogenizer. The homogenate was serially diluted, and 0.1 mL of each dilution was spread on SDA plates. After incubation at 35°C overnight, the number of colonies was counted, and the CFU value of each mouse kidney was calculated.

[0111] The results are shown in Table 5. Antimicrobial peptides 1-4 showed a significant advantage over the control peptide in reducing Candida albicans load in mouse kidneys: at a dose of 0.5 mg / kg, the bacterial load of the control peptide was log... 10 The bacterial load values ​​were 1.79–3.17, while the antimicrobial peptides were only 0.38–0.45, which is one-third or even lower than the control peptides. At a dose of 1.5 mg / kg, the control peptides were 0.98–1.98, while the antimicrobial peptides were only 0.17–0.25, which is one-quarter or even lower than the control peptides. At a dose of 4.5 mg / kg, the control peptides were 0.29–0.79, while the antimicrobial peptides were only 0.04–0.07, which is one-seventh or even lower than the control peptides. Meanwhile, the 24-hour bacterial load log in the infected control group was... 10 The viral load of anidoxime at all doses was as high as 4.91, and the viral load of anidoxime at all doses was also significantly higher than that of antimicrobial peptides, further demonstrating the potent in vivo antibacterial activity of antimicrobial peptides. Compared with the control drugs anidoxime and control peptides 1-3, antimicrobial peptides 1-4 showed more significant in vivo fungal clearance at the same dosage, providing a potential candidate for the treatment of drug-resistant Candida infections and possessing good clinical translational value.

[0112] Table 5. Antimicrobial peptides 1-4 effectively reduced the mean log of Candida albicans CFU in mouse kidneys. 10 value

[0113] .

[0114] In specific embodiments of the present invention, the Chinese meanings of the English abbreviations used in the application documents are shown in Table 6:

[0115] Table 6. English Abbreviations and Corresponding Chinese Names

[0116] .

Claims

1. An antimicrobial peptide, characterized in that, The chemical structural formula is as follows: ； Wherein, the R basis is selected from , , , .

2. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the antimicrobial peptide as described in claim 1 or a pharmaceutically acceptable salt thereof as the active ingredient, with or without pharmaceutically acceptable excipients.

3. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutically acceptable excipients include one or more of the following: excipients, diluents, lubricants, flow aids, wetting agents, emulsifiers, and pH buffers.

4. The pharmaceutical composition according to claim 2, characterized in that, The dosage forms of the pharmaceutical compositions include injections, capsules, granules, oral liquids, sprays, nasal drops, eye drops, ear drops, suppositories, ointments, enteric-coated tablets, orally disintegrating tablets, oral instant films, creams, gels, patches, coatings, microcapsules, implants, syrups, effervescent tablets, sponges, lotions, inhaled powders, oral microcapsules, powders, chewable tablets, sustained-release tablets, suspensions, and nebulized solutions.

5. The use of the antimicrobial peptide of claim 1 or the pharmaceutical composition of any one of claims 2-4 in the preparation of microbial inhibitors, characterized in that, The microorganism is a fungus; The fungi include one or more of the genera *Candida* and *Aspergillus*; wherein the *Candida* fungi include one or more of *Candida albicans*, *Candida glabrata*, *Candida krusei*, *Candida tropicalis*, *Candida auris*, *Candida lactea*, *Candida guinea*, *Candida parapsilosis*, and *Candida Portugueseia*; and the *Aspergillus* fungi include one or more of *Aspergillus fumigatus*, *Aspergillus flavus*, *Aspergillus lanceolata*, *Aspergillus alba*, *Aspergillus niger*, and *Aspergillus ochre*.

6. The use of the antimicrobial peptide of claim 1 or the pharmaceutical composition of any one of claims 2-4 in the preparation of a medicament for the prevention and / or control of fungal infections, characterized in that... The fungi include one or more of the genera *Candida* and *Aspergillus*; wherein the *Candida* fungi include one or more of *Candida albicans*, *Candida glabrata*, *Candida krusei*, *Candida tropicalis*, *Candida auris*, *Candida lactea*, *Candida guinea*, *Candida parapsilosis*, and *Candida Portugueseia*; and the *Aspergillus* fungi include one or more of *Aspergillus fumigatus*, *Aspergillus flavus*, *Aspergillus lanceolata*, *Aspergillus alba*, *Aspergillus niger*, and *Aspergillus ochre*.

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