Application of carbonyl cyanide-4-trifluoromethoxyphenylhydrazone in the prevention and treatment of Aspergillus fumigatus infection

Abstract

The present invention belongs to the technical field of aspergillosis treatment, and specifically discloses the use of carbonyl cyanide-4-trifluoromethoxyphenylhydrazone in the prevention and treatment of Aspergillus fumigatus infection, that is, using it as the sole active ingredient in the preparation of a drug for preventing or treating diseases caused by Aspergillus fumigatus infection, or for the preparation of an antibacterial and / or bactericidal product for Aspergillus fumigatus. The present invention first discovered that carbonyl cyanide-4-trifluoromethoxyphenylhydrazone has a significant inhibitory effect on both wild-type and drug-resistant strains of the human pathogenic fungus Aspergillus fumigatus, and can inhibit its colony growth, hyphal growth, and conidia formation, thereby providing a new therapeutic drug for Aspergillus fumigatus infection. At the same time, it solves the problem of antibiotic resistance that plagues the clinical treatment of Aspergillus fumigatus infection, and can circumvent the disadvantage that antibiotics are prone to drug resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of aspergillosis treatment, and particularly relates to application of carbonyl cyanide-4-trifluoromethoxyphenylhydrazone in the prevention and treatment of Aspergillus fumigatus infection. Background Art

[0002] Aspergillus fumigatus, a member of the genus Aspergillus, is a filamentous fungus that is opportunistically pathogenic to humans. It primarily infects the lungs, producing large numbers of conidia that are inhaled into the lungs. On average, a person inhales hundreds of conidia daily. When inhaled, these spores can cause lung infection. Aspergillus fumigatus colonies grow rapidly and are dark green, becoming darker in color as they age. Conidial heads are columnar, bluish-green when young, turning dark green to dark brown or coal-colored as they mature. Conidiophores are short, smooth, and often green.

[0003] Invasive aspergillosis includes pulmonary aspergillosis, aspergillosis rhinosinusitis, cerebral aspergillosis, and disseminated aspergillosis. Globally, approximately 200,000 cases of invasive aspergillosis occur annually, but the true number is far higher due to misdiagnosis. The lack of precise diagnostic and treatment strategies results in a mortality rate of 50%-100%.

[0004] Pulmonary aspergillosis (Ps) is a common fungal lung infection in clinical practice. The spectrum of this disease is vast, and its severity depends on the health of the host's immune system. While not a serious problem for individuals with normal immunity, invasive aspergillosis primarily affects immunocompromised patients, such as those with cancer, malignancies, chemotherapy, or organ transplants. The disease often manifests differently between neutropenic and non-neutropenic patients.

[0005] Clinically, the main drugs used to treat pulmonary aspergillosis include polyenes, echinocandins, and azoles. Polyenes, including amphotericin B (AmB), offer excellent efficacy but significant adverse reactions. Echinocandins, including caspofungin (CAS), have low oral availability and are expensive. Azoles, primarily voriconazole (VRC) and itraconazole (ITR), are first-line antifungal drugs with few adverse reactions, relative safety, and high bioavailability. The antifungal mechanism of action of azoles is through binding to lanosterol 14a demethylase (CYP51), a key enzyme in ergosterol synthesis, on the fungal cell membrane, inhibiting ergosterol synthesis, leading to the accumulation of toxic alcohols and exerting a bactericidal effect.

[0006] However, with the widespread use of triazoles, the incidence of Aspergillus fumigatus resistance has increased annually. For example, in the Netherlands, the five-year resistance rate caused by Aspergillus fumigatus increased from 0.79% (1996-2001) to 7.04% (2012-2016). Due to differences in detection and reporting in different countries or regions, the resistance rate of Aspergillus fumigatus to azoles is only 3.2% in some countries, but has reached 36.3% in others. In my country, the resistance rate of Aspergillus fumigatus to azoles is 1.4%-5.6%.

[0007] Carbonyl cyanide-4-trifluoromethoxyphenylhydrazone (FCCP) is a potent uncoupler of oxidative phosphorylation in mitochondria, disrupting ATP synthesis by transporting protons. In vitro, FCCP treatment can induce intracellular Ca 2+ FCCP rapidly increases the expression of mitochondrial proteins up to 2-fold, while also inhibiting protein synthesis. This inhibition of protein translation is associated with increased phosphorylation of eIF2α and increased double-stranded RNA-dependent protein kinase activity. FCCP also slightly reduces ATP and reactive oxygen species levels, increases the expression of mitochondrial genes such as Tfam and COXIV, induces quiescent morphological characteristics in mouse hematopoietic stem cells, and inhibits TGF-β signaling. In vivo, FCCP treatment significantly reduces mitochondrial membrane potential, ATP production, and the number of intracellular cell masses in blastocysts at the 8-cell stage in mouse embryos, but has no effect on blastocyst development. This disruption of embryonic mitochondrial function is accompanied by a decrease in birth weight in female offspring after implantation, a suppression that persists until weaning. Although FCCP-treated male mice exhibit impaired glucose tolerance, similar to female mice, their insulin sensitivity and adiposity remain unchanged between 4 and 14 weeks of age. Reduced mitochondrial function in pre-implanted embryos, coupled with reduced ATP output, impacts offspring phenotype.

[0008] Carbonyl cyanide-4-trifluoromethoxyphenylhydrazone (FCCP) has been reported to be used as an uncoupling agent in cell culture, including studies on its effects on thermogenic adipocytes, its effects on mouse mitochondrial β-oxidation, and its metabolic effects on mouse bone marrow-derived dendritic cells. FCCP can also affect various activities involving cellular calcium ions. It can also inhibit K + Background current and induced small inward current, decreased pH by 0.1 units, and induced intracellular Na + FCCP can stimulate Mg 2+ -ATPase activity, inhibiting β-amyloid protein production, and mimicking the effects of the selective glutamate receptor agonist N-methyl-D-aspartate (NMDA) on mitochondrial superoxide production.

[0009] The increasing number of drug-resistant strains of Aspergillus fumigatus poses a major challenge to the treatment of aspergillosis. Therefore, there is an urgent need to develop new drugs that can effectively treat Aspergillus fumigatus infection. Summary of the Invention

[0010] The main purpose of the present invention is to provide a use of carbonyl cyanide-4-trifluoromethoxyphenylhydrazone in the prevention and treatment of Aspergillus fumigatus infection, so as to solve the problem that the prior art lacks therapeutic drugs for Aspergillus fumigatus resistant bacteria infection.

[0011] The present invention unexpectedly discovered that carbonyl cyanide-4-trifluoromethoxyphenylhydrazone has an inhibitory effect on wild-type Aspergillus fumigatus strains and, in particular, clinically isolated drug-resistant strains of Aspergillus fumigatus, and its effect on the latter is significantly stronger than that of existing antibiotics. It may replace antibiotics and become a new drug for treating Aspergillus fumigatus, thus obtaining the present invention.

[0012] The present invention adopts the following technical solutions:

[0013] The present invention provides the use of carbonyl cyanide-4-trifluoromethoxyphenylhydrazone or a pharmaceutically acceptable salt thereof as the sole active ingredient in any of the following: (i) for preparing a medicament for preventing or treating a disease caused by Aspergillus fumigatus infection; (ii) for preparing an antibacterial and / or bactericidal product for Aspergillus fumigatus.

[0014] In some embodiments, the Aspergillus fumigatus is a wild-type Aspergillus fumigatus.

[0015] In some embodiments, the Aspergillus fumigatus is resistant to azole, echinocandin, or polyene antifungals, preferably resistant to azole antifungals, more preferably resistant to triazole antifungals, and most preferably resistant to itraconazole and / or voriconazole. Azoles include imidazoles and triazoles, with imidazoles including but not limited to ketoconazole, miconazole, econazole, or clotrimazole, and triazoles including but not limited to itraconazole, voriconazole, fluconazole, posaconazole, or lavuconazole; echinocandins include but not limited to caspofungin, micafungin, or anidulafungin; and polyenes include but not limited to amphotericin B or nystatin.

[0016] In some embodiments, diseases caused by Aspergillus fumigatus infection include but are not limited to pulmonary aspergillosis, aspergillosis rhinosinusitis, cerebral aspergillosis, or disseminated aspergillosis.

[0017] In some embodiments, the aforementioned drug is a drug that inhibits the growth of Aspergillus fumigatus colonies, inhibits the growth of Aspergillus fumigatus hyphae, or inhibits the production of Aspergillus fumigatus conidia.

[0018] In some embodiments, the aforementioned medicaments further include pharmaceutically acceptable excipients, such as fillers, wetting agents, binders, disintegrants, lubricants, solvents, emulsifiers, solubilizers, preservatives, pH adjusters, osmotic pressure regulators, suspending agents, plasticizers, thickeners, humectants, stabilizers, glidants, filter aids, defoamers, coating agents, surfactants, antioxidants, colorants, flavoring agents, or buffers. Exemplary fillers include, but are not limited to, starch, powdered sugar, compressible starch, lactose, microcrystalline cellulose, dextrin, mannitol, and inorganic salts. Wetting agents include, but are not limited to, water and ethanol. Binders include, but are not limited to, starch slurry, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, gelatin solution, sucrose solution, and aqueous polyvinyl pyrrolidone solution. Disintegrants include, but are not limited to, dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, citric acid, and sodium bicarbonate. Lubricants include, but are not limited to, magnesium stearate, micronized silica gel, talc, and polyethylene glycol. Solvents include, but are not limited to, water, ethanol, and glycerin. Emulsifiers include, but are not limited to, poloxamer, Tween 80, Span 80, sorbitan oleate, and distearoylphosphatidylcholine. Cosolvents include, but are not limited to, polyethylene glycol 400, polysorbates, and polyoxyethylene fatty acid esters. Preservatives include, but are not limited to, benzoic acid, sorbic acid, and ethanol.

[0019] In some embodiments, the dosage form of the aforementioned drugs includes any pharmaceutically acceptable dosage form, including but not limited to tablets, granules, powders, capsules, pills, solutions, emulsions, suspensions, injections, aerosols, pills or drops.

[0020] In some embodiments, to achieve the desired antibacterial effect, the drug may be administered by any known method, including but not limited to oral administration, injection, sublingual administration, or oral spray. The method and dosage may vary widely depending on the desired antibacterial effect, the nature and severity of the disease being treated, and the individual condition of the patient or animal. For example, the dosage may be set within the range of 5 to 25 μM.

[0021] The beneficial effects of the present invention are:

[0022] The present invention discovers for the first time that carbonyl cyanide-4-trifluoromethoxyphenylhydrazone has a significant inhibitory effect on both wild-type strains and resistant strains of the human pathogenic fungus Aspergillus fumigatus, and can inhibit its colony growth, hyphal growth and conidia formation, thereby providing a new therapeutic drug for Aspergillus fumigatus infection. At the same time, it solves the problem of antibiotic resistance that has plagued the clinical treatment of Aspergillus fumigatus infection and can circumvent the disadvantage that antibiotics are prone to drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1The results of the inhibitory concentration test of FCCP against wild-type Aspergillus fumigatus;

[0024] Figure 2 A is the result of the inhibitory concentration test of FCCP against the drug-resistant strain DZ-2 of Aspergillus fumigatus;

[0025] Figure 2 B is the result of the inhibitory concentration test of FCCP against the drug-resistant strain 12L-5 of Aspergillus fumigatus;

[0026] Figure 2 C is the experimental result of the inhibitory concentration of FCCP against the drug-resistant strain WXL6 of Aspergillus fumigatus;

[0027] Figure 3 A is the experimental result of the effect of FCCP concentration on the phenotype of Aspergillus fumigatus colonies;

[0028] Figure 3 B is the experimental results of the effect of FCCP concentration on the growth of Aspergillus fumigatus hyphae;

[0029] Figure 3 C is the experimental result of the effect of FCCP concentration on the conidia production of Aspergillus fumigatus;

[0030] Figure 4 The results of the experiment on the effects of azole drugs on the colony phenotypes of wild-type Aspergillus fumigatus and drug-resistant strains of Aspergillus fumigatus are shown in Figure 2. WT: wild-type Aspergillus fumigatus strain; 12L-5, DZ-2, WXL6: drug-resistant strains of Aspergillus fumigatus; ITZ: itraconazole; VCZ: voriconazole.

[0031] Figure 5 The experimental results show the effect of FCCP concentration on the colony phenotype of wild-type Aspergillus fumigatus strain and drug-resistant strain of Aspergillus fumigatus; WT: wild-type Aspergillus fumigatus strain; 12L-5, DZ-2, WXL6: drug-resistant strains of Aspergillus fumigatus;

[0032] Figure 6 These are the experimental results of the effect of FCCP concentration on the mitochondrial morphology of Aspergillus fumigatus. DETAILED DESCRIPTION

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] Experimental Materials

[0035] 1. Strains: The wild-type (WT) Aspergillus fumigatus strain used in this experiment was the clinical isolate A1163; strains 12L-5, DZ-2, and WXL6 were derived from clinical isolates resistant to azole drugs (itraconazole and voriconazole); strain ArgA-GFP is a laboratory-constructed strain of Aspergillus fumigatus mitochondrial protein ArgA labeled with a fluorescent protein.

[0036] 2. Culture medium: Minimal medium (MM) (1 L): 20× Salts 50 mL, 1000× Trace elements 1000 μL, Glucose 10 g, adjust pH to 6.5 with NaOH; solid culture medium requires the addition of 2% (w / v) agar. Autoclave at 115°C for 20 min.

[0037] 3. Dissolve FCCP in DMSO and mix thoroughly to make a 10 mM solution. Store at -20°C until use.

[0038] Example 1 MIC determination of FCCP against different Aspergillus fumigatus strains

[0039] The FCCP in the present invention was tested for its minimum inhibitory concentration (MIC) against wild-type Aspergillus fumigatus and its azole-resistant strains using the broth microdilution method in a 96-well plate according to the CLSIM60 protocol guidelines of the American Committee for Clinical Standards. Briefly, the same number of conidia (10 5 ) was diluted into RPMI 1640 medium containing FCCP gradient dilution solution, 200 μL was transferred to a 96-well plate, and cultured at 37°C for 48 hours, after which the colony formation was observed.

[0040] The results of the antibacterial concentration test of FCCP against wild-type Aspergillus fumigatus are as follows: Figure 1 As shown in FIG, the results of the antibacterial concentration determination of Aspergillus fumigatus resistant strain DZ-2, Aspergillus fumigatus resistant strain 12L-5, and Aspergillus fumigatus resistant strain WXL6 are respectively as shown in FIG. Figure 2 A. Figure 2 B. Figure 2 As shown in Figure C, the MIC of FCCP against wild-type Aspergillus fumigatus and various azole-resistant strains of Aspergillus fumigatus was 16 μg / mL. Using the same method as above, the MICs of the azole drug itraconazole (ITZ) against the aforementioned wild-type and resistant strains of Aspergillus fumigatus were 0.4 μg / mL and 20 μg / mL, respectively. Furthermore, further studies revealed that the inhibitory concentration of FCCP against the aforementioned wild-type and resistant strains of Aspergillus fumigatus was significantly lower than that against other Aspergillus species, such as Aspergillus flavus and Aspergillus niger.

[0041] Example 2 Experimental Study on the Effect of FCCP on Colony Phenotype, Mycelial Growth and Conidia Production of Aspergillus fumigatus

[0042] 1. Effects on Colony Phenotype

[0043] Different concentrations (5×10 6 , 5×10 5 , 5×10 4 2 μL of wild-type Aspergillus fumigatus (A. fumigatus) culture solution (100 μL / mL) was spotted onto MM solid plates containing different drugs. The plates were incubated at 37°C for 48 hours. The effects of the drugs on the colony phenotype were observed.

[0044] The experimental results of the effect of FCCP concentration on the phenotype of Aspergillus fumigatus colonies are as follows Figure 3 As shown in A. It can be seen that FCCP inhibits the growth of Aspergillus fumigatus colonies.

[0045] The experimental results of the effects of azole drugs on the colony phenotype of wild-type Aspergillus fumigatus and drug-resistant strains of Aspergillus fumigatus are as follows Figure 4 As shown in the figure, wild-type and azole-resistant bacteria (Cyp511A-12L-5, DZ-2, and Non-Cyp511A-Wxl6) were observed by spotting on plates containing 0.5 μg / mL itraconazole (ITZ) and 0.5 μg / mL voriconazole (VCZ). Wild-type colonies completely failed to grow, while azole-resistant bacteria (Cyp511A-12L-5, DZ-2, and Non-Cyp511A-Wxl6) continued to grow, unaffected by ITZ and VCZ. This demonstrates that 12L-5, DZ-2, and WXL6 are azole-resistant.

[0046] The experimental results of the effect of FCCP concentration on the colony phenotype of wild-type Aspergillus fumigatus strains and drug-resistant Aspergillus fumigatus strains are as follows: Figure 5 As shown in the figure, it can be seen that FCCP not only inhibits the growth of wild-type Aspergillus fumigatus, but also inhibits the growth of azole-resistant Aspergillus fumigatus.

[0047] 2. Effect on mycelial growth

[0048] To visualize the changes in hyphae, wild-type Aspergillus fumigatus strains were placed on coverslips in MM liquid medium containing different concentrations of FCCP and grown at 37°C for 11 hours. Images were captured using a laser confocal microscope (200×). Figure 3 As shown in B, the results showed that FCCP inhibited the growth of Aspergillus fumigatus hyphae.

[0049] 3. Effects on conidia production

[0050] From a concentration of 5×104 Take 100 μL of the wild-type Aspergillus fumigatus bacterial suspension at a concentration of 100 μL / mL and spread it on MM solid plates containing different concentrations of FCCP. Incubate at 37°C for 48 hours. Afterwards, collect the spores with sterile water and count them. Figure 3 As shown in C, the results showed that FCCP inhibited the conidia production of Aspergillus fumigatus.

[0051] Example 3 Effect of FCCP on the mitochondrial morphology of Aspergillus fumigatus

[0052] To observe changes in mitochondrial morphology, mitochondrial GFP-labeled bacteria (ArgA-GFP) were grown on coverslips in liquid MM medium containing different concentrations of FCCP at 37°C for 12 hours. Images were acquired using a laser confocal microscope (630×). Figure 6 As shown, the results showed that FCCP caused the mitochondrial fragmentation of Aspergillus fumigatus, thereby inhibiting the growth of Aspergillus fumigatus.

[0053] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. Use of carbonyl cyanide-4-trifluoromethoxyphenylhydrazone or a pharmaceutically acceptable salt thereof as the sole active ingredient in any of the following: (i) for the preparation of a medicament for preventing or treating a disease caused by infection with Aspergillus fumigatus; (ii) for preparing a bacteriostatic and / or bactericidal product for Aspergillus fumigatus.

2. The method according to claim 1, wherein the Aspergillus fumigatus is a wild-type Aspergillus fumigatus.

3. The use according to claim 1, wherein the Aspergillus fumigatus is resistant to azoles, echinocandins or polyene antifungals.

4. The use according to claim 3, wherein the Aspergillus fumigatus is resistant to azole antifungal drugs.

5. The use according to claim 4, wherein the Aspergillus fumigatus is resistant to triazole antifungal drugs.

6. The use according to claim 5, wherein the Aspergillus fumigatus is resistant to itraconazole and / or voriconazole.

7. The use according to claim 1, wherein the disease is selected from pulmonary aspergillosis, aspergillosis rhinosinusitis, cerebral aspergillosis or disseminated aspergillosis.

8. The use according to claim 1, wherein the drug is a drug for inhibiting the growth of Aspergillus fumigatus colonies.

9. The use according to claim 1, wherein the drug is a drug for inhibiting the growth of Aspergillus fumigatus hyphae.

10. The use according to claim 1, wherein the drug is a drug for inhibiting the production of conidia of Aspergillus fumigatus.

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