Application of a cyclodiene platinum complex in the preparation of drugs against drug-resistant fungi

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to the application of norbornadiene dichloroplatinum (II) and its composition with (1,5-cyclooctadiene) dichloroplatinum (II) in the preparation of anti-resistant fungal drugs. The present invention first found that norbornadiene dichloroplatinum (II) has strong antibacterial activity against wild-type and fluconazole-tolerant Candida albicans, and has low cytotoxicity, and the concentration required for 95% inhibition rate is 0.36 μM, which is better than the currently reported best metal compound (1,5-cyclooctadiene) dichloroplatinum (II), and can overcome the drug resistance of fungi. At the same time, the present invention also found that norbornadiene dichloroplatinum (II) and (1,5-cyclooctadiene) dichloroplatinum (II) have a synergistic antibacterial effect, and the composition formed by the two can further improve the inhibition rate of Candida albicans, and reduce the amount of drug used, providing a new treatment for Candida albicans infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a cyclodiene platinum (II) complex - norbornadiene dichloroplatinum (II) (English name: Dichloro (norbornadiene) platinum (II), CAS number: 12152-26-0) and a composition thereof with (1,5-cyclooctadiene) dichloroplatinum (II) in the preparation of drugs for resisting drug-resistant fungal infections. Background Art

[0002] In recent years, the incidence of invasive fungal infections has increased in people with low immunity, becoming an important cause of death [Wenjie Fang, Junqi Wu, Mingrong Cheng, Xinlin Zhu, Mingwei Du, Chang Chen, Kangkang Zhi and Weihua Pan. Diagnosis of invasive fungal infections challenges and recent developments [J]. Journal of Biomedical Science. 2023, 30: 42]. People with low immunity mainly include AIDS patients, diabetic patients, malignant tumor patients, patients undergoing radiotherapy and chemotherapy, and patients using immunosuppressants for a long time. According to reports in international authoritative journals [Kevin S Ikuta, Tomislav Mohsen Naghavi. Global incidence and mortality of severe fungal disease. Lancet Infect Dis, 2024, 24(5): e268】, the mortality rate of fungal infection in these people is as high as 40%, resulting in 3.75 million deaths worldwide each year, of which 90% are caused by fungal pathogens Candida albicans, Cryptococcus neoformans and Aspergillus. According to the 2023 edition of the Guidelines for Fungal Prevention and Control in my country, the main antifungal drugs used in clinical practice include polyenes (such as amphotericin B) and nitrogen azoles (such as fluconazole), especially fluconazole (Fluconazole, FLU), which has become the first choice for the prevention and treatment of infections caused by Candida albicans due to its safety, effectiveness and low price. However, due to the frequent use of fluconazole in clinical treatment, the number of cases of drug resistance has gradually increased, which has brought great challenges to the clinical treatment of fungal infections [Jing Ran, Hou Xin, Xiao Meng. Distribution characteristics of invasive yeast in intensive care units of member units of China's Invasive Fungal Resistance Monitoring Network and changes in sensitivity to triazole drugs. Chinese Journal of Infection and Chemotherapy, 2020, 20(2): 175-180; CDC Antibiotic Resistance Threats in the United States. https: / / www.cdc.gov / DrugResistance / Biggest-Threats]. Therefore, it is urgent to develop new antifungal drugs with unique mechanisms of action to solve the problem of drug resistance. However, there are currently less than 10 antifungal drugs in the pipeline worldwide, and all of them are organic small molecule drugs [Gintjee TJ, Donnelley MA, Thompson, GRAspiring Antifungals: Review of Current Antifungal Pipeline Developments. J. Fungi 2020, 6, 28].Therefore, the scientific community began to pay attention to the antifungal role of metal compounds [Frei, A.; Zuegg, J.; Elliott, AG; Baker, M.; Braese, S.; Brown, C.; Chen, F.; Dowson, CG; Dujardin, G.; Jung, N.; King, AP; Mansour, AM; Massi, M.; Moat, J.; Mohamed, HA; Renfrew, AK; Rutledge, PJ; Sadler, PJ; Todd, MH; Willans, CE; Wilson, JJ; Cooper, MA; Blaskovich, MAT, Metal Complexes as a Promising Source for New Antibiotics. Chem. Sci. 2020, 11, 2627-2639.].

[0003] Since the American scientist B. Rosenberg reported the anticancer activity of platinum complexes in 1969, people have successfully developed a number of platinum anticancer drugs with unique mechanisms of action, especially cisplatin, carboplatin and oxaliplatin, which have now become the first-line drugs for clinical chemotherapy of many malignant tumors [Johnstone, Timothy C., Suntharalingam, Kogularamanan, Lippard, Stephen J. The next generation of platinum drugs: Targeted Pt (II) agents, nanoparticle delivery, and Pt (IV) prodrugs. Chem. Rev. 2016, 116, 3436-3486.]. At the same time, people have also found that many metal compounds, especially platinum complexes, have good antimicrobial effects [Frei, A. Metal Complexes, an Untapped Source of Antibiotic Potential Antibiotics 2020, 9, 90.]. To this end, in 2022, Swiss scientist A. Frei and Australian scientist MATBlaskovich's team specifically summarized, analyzed and compared the reported metal complexes with antifungal effects [Metal Complexes as Antifungals From a Crowd-Sourced Compound Library to the First In Vivo Experiments. JACS Au 20222 (10), 2277-2294]. Based on the antifungal activity and preliminary toxicity of these complexes, they believe that (1,5-cyclooctadiene) dichloroplatinum (II) (Pt (1,5-COD) Cl2) is the most worthy of further research and development. Compared with polyenes and azoles used in clinical practice, Pt (1,5-COD) Cl2 has a different antibacterial mechanism and is also effective against drug-resistant fungi, but it still has the defects of low activity and relatively high toxicity, and it is necessary to further search for better antifungal platinum complexes. The chemical results of Pt (1,5-COD) Cl2 are shown in Formula I:

[0004] Summary of the invention

[0005] In view of the defects of Pt(1,5-COD)Cl2, the present invention provides a platinum complex with higher antifungal activity and lower toxicity - norbornadiene dichloroplatinum (II) for use in the preparation of drugs for the prevention of drug-resistant fungal infections. The present invention first discovered that norbornadiene dichloroplatinum (II) has high antifungal activity and is particularly effective against drug-resistant fungi, has low cytotoxicity, is significantly better than Pt(1,5-COD)Cl2, and has a synergistic antibacterial effect with Pt(1,5-COD)Cl2. Therefore, it has the prospect of being developed into a drug for the prevention of drug-resistant fungal infections.

[0006] The norbornadiene dichloroplatinum (II) described in the present invention is a cyclodiene platinum (II) complex, with the English name Dichloro (norbornadiene) platinum (II), the molecular formula abbreviated as Pt (NBD) Cl2, the chemical structure as shown in Formula II, and a unique stereo configuration.

[0007]

[0008] The norbornadiene dichloroplatinum (II) [Pt(NBD)Cl2] of the present invention can be synthesized by reacting commercially available platinum raw material K2PtCl4 with norbornadiene in a mixed system of acetic acid and water. Under optimal reaction conditions, the yield can reach more than 90%.

[0009] The Pt(NBD)Cl2 of the present invention has a high antibacterial activity against Candida albicans that tolerates the commonly used antifungal drug fluconazole FLU, showing a good dose-effect relationship, and the half inhibition concentration is MIC 50 The MIC of Pt(1,5-COD)Cl2 under the same test conditions is 0.10μM. 50 The MIC of Pt(NBD)Cl2 and Pt(1,5-COD)Cl2 is 0.18μM, and the inhibition rate of fluconazole against drug-resistant Candida albicans is less than 12% even if the concentration is increased to 0.84μM. For wild-type Candida albicans, the inhibition rate of 0.03μM FLU can reach more than 50%, while the ...%. 50 The results showed that the antibacterial activity of Pt(NBD)Cl2 was higher than that of Pt(1,5-COD)Cl2 and FLU for the drug-resistant white rosacea.

[0010] The concentration of Pt(NBD)Cl2 described in the present invention to achieve a 96% inhibition rate against drug-resistant Candida albicans is 0.36 μM, while at similar concentrations, the inhibition rate of Pt(1,5-COD)Cl2 (0.34 μM) is 87%, and the inhibition rate of FLU (0.40 μM) is 4.9%. These experimental data further illustrate that the activity of Pt(NBD)Cl2 against drug-resistant fungi is better than that of Pt(1,5-COD)Cl2 and FLU.

[0011] In order to evaluate the cytotoxicity of Pt(NBD)Cl2 described in the present invention, the inhibitory effect on the growth of human liver cancer strain HepG2 was tested by CCK8 standard method, and compared with cytotoxic anticancer drugs cisplatin, Pt(1,5-COD)Cl2 and FLU. The results showed that cisplatin has very strong cytotoxicity. When the concentration reaches 1.6μM, it can kill 20% of the cells and the cell survival rate is only 80%. At the same concentration, Pt(NBD)Cl2, Pt(1,5-COD)Cl2 and FLU have almost no cytotoxicity, and the cell survival rate is close to 100%. At the same time, from the inhibition rate of cells at various concentrations, there is also a trend that Pt(NBD)Cl2 has less toxicity.

[0012] The compound Pt(NBD)Cl2 described in the present invention has an inhibition rate of 96% against drug-resistant Candida albicans at 0.36 μM, and does not show cytotoxicity even when its concentration is increased to 1.6 μM, indicating that the use of Pt(NBD)Cl2 to treat drug-resistant fungal infections will be safe and effective.

[0013] The compound Pt(NBD)Cl2 described in the present invention is used in combination with Pt(1,5-COD)Cl2, and the antibacterial effect is significant, indicating that the two compounds have obvious synergistic effects. The antibacterial rate of 0.02μM Pt(NBD)Cl2+0.04μM Pt(1,5-COD)Cl2 against drug-resistant Candida albicans reaches 89%, while when they are used alone, the antibacterial rate of 0.02μM Pt(NBD)Cl2 is 5%, and the antibacterial rate of 0.04μM Pt(1,5-COD)Cl2 is 10%. The antibacterial rate of 0.04μM Pt(NBD)Cl2+0.02μM Pt(1,5-COD)Cl2 against drug-resistant Candida albicans reaches 96%, while when they are used alone, the antibacterial rate of 0.04μM Pt(NBD)Cl2 is 15%, and the antibacterial rate of 0.02μM Pt(1,5-COD)Cl2 is 9%. These experimental results also suggest that the antibacterial mechanism of Pt(NBD)Cl2 may be different from that of Pt(1,5-COD)Cl2, which deserves further study.

[0014] While carrying out the research of this project, the applicant of the present invention also synthesized and tested other cyclodiene platinum (II) complexes, including (1,3-cyclooctadiene) dichloroplatinum (II), (1,4-cyclohexadiene) dichloroplatinum (II), (1,3-cyclohexadiene) dichloroplatinum (II), and (cyclopentadiene) dichloroplatinum (II), but did not find that their antifungal activity was better than that of the compound Pt(NBD)Cl2 described in the present invention.

[0015] Therefore, on the one hand, the present invention provides an application of a complex Pt(NBD)Cl2 having an antibacterial effect superior to that of Pt(1,5-COD)Cl2 being developed internationally in the preparation of drugs against drug-resistant fungi. On the other hand, the present invention provides an application of a combination of Pt(NBD)Cl2 and Pt(1,5-COD)Cl2 having higher antibacterial activity in the preparation of drugs against drug-resistant fungi. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the concentration-inhibition rate curve of FLU against wild-type and drug-resistant Candida albicans;

[0017] Figure 2 The concentration-inhibition rate curve of FLU, Pt(1,5-COD)Cl2 and Pt(NBD)Cl2 against drug-resistant Candida albicans;

[0018] Figure 3 MIC of FLU, Pt(1,5-COD)Cl2 and Pt(NBD)Cl2 against drug-resistant Candida albicans 50 Result graph;

[0019] Figure 4 The graph shows the inhibition rate of FLU, Pt(1,5-COD)Cl2 and Pt(NBD)Cl2 against drug-resistant Candida albicans at similar concentrations;

[0020] Figure 5 The concentration-survival rate curve of DDP, FLU, Pt(1,5-COD)Cl2 and Pt(NBD)Cl2 on the growth of HepG2 cells, where A is the highest concentration of FLU in the antibacterial experiment, and B is the highest concentration of Pt(1,5-COD)Cl2 and Pt(NBD)Cl2 in the antibacterial experiment. DETAILED DESCRIPTION

[0021] Example 1

[0022] The present invention relates to the synthesis of the compound Pt(NBD)Cl2

[0023] Weigh 10g of potassium chloroplatinite (purchased from Guiyan Platinum Co., Ltd.), heat and dissolve it with 50mL of water, add 50mL of glacial acetic acid and 2mL of hydrochloric acid, and stir until the solution is clear. Slowly add 10mL of norbornadiene (purchased from AlfaAesar), reflux at 60°C for 2 hours, the red solution gradually fades and a light yellow precipitate is precipitated. After cooling and filtering, wash with water 3 times, ethanol 2 times, acetone 1 time, and then vacuum dry at 60°C for 2 hours to obtain 8.1g of light yellow solid product with a yield of 93%.

[0024] Structural characteristic parameters: <1> Elemental analysis: Measured C 23.4%, H 2.23% (calculated C 23.5%, H 2.24%); <2> Platinum content analysis: measured value Pt 54.4% (calculated value Pt 54.6%); <3> 1 HNMR (DMSO, ppm): δ H 5.51(m,4H,H-1,H-3,H-4,H-6),δ H 2.23(m,4H,H-2,H-5),δ H 2.59 (m, 2H, H-7); <4> 13 C NMR (DMSO, ppm):δ C 77.67 (C=C), 68.1 (CH), 48.1 (CH2). These parameters are consistent with the chemical structure of the present invention, and the chemical structure analysis of the present invention is shown in Formula III.

[0025]

[0026] Example 2

[0027] The activity of the compound Pt(NBD)Cl2 against drug-resistant fungi

[0028] Antibacterial activity was tested by conventional ELISA

[0029] 1. Sources of drugs and strains

[0030] Drugs: Pt(1,5-COD)Cl2 (purchased from Alfa Aesar), fluconazole (FLU) (purchased from MedChemExpress Biotechnology Company) and Pt(NBD)Cl2 (synthesized by the method in Example 1). All drugs were prepared into drug solutions of different concentrations using DMSO as a solvent. All drug solutions were prepared and used immediately.

[0031] Strains: Both wild-type and resistant Candida albicans were clinical isolates [Wen-Xuan, Wang, Xinxiang, et al. Cytochalasans from the Endophytic Fungus Xylaria cf. curta with Resistance Reversal Activity against Fluconazole-Resistant Candida albicans. [J]. Organic letters, 2019. DOI: 10.1021 / acs.orglett.9b00015.] [Wu Z, Wu QF, Yuan WL, et al. Arthrocolins Synergizing with Fluconazole Inhibit Fluconazole-Resistant Candida albicans by Increasing Riboflavin Metabolism and Causing Mitochondrial Dysfunction andAutophagy[J].MicrobiologySpectrum,2023,11(2).DOI:10.1128 / spectrum.04051-22.】, donated by the State Key Laboratory of Conservation and Utilization of Biological Resources of Yunnan Province, and revived before use.

[0032] 2. ELISA test for antibacterial activity

[0033] In the experimental group, 100 μl of Sabouraud dextrose liquid medium (SDB) and 100 μl of 5×10 5 CFU / ml drug-resistant Candida albicans liquid, and then use the multiple dilution method to add different concentrations of liquid to the 96-well plate in sequence. Add 100μl 5×10 5 CFU / mL of drug-resistant Candida albicans was recorded as the experimental group. 100 μl SDB and 100 μl 5×10 5 CFU / ml drug-resistant Candida albicans liquid (the difference between the control group and the experimental group is that no liquid is added), the blank group is added with 200μl SDB per well, and each treatment is set up 3 replicates. After each group was cultured in a biochemical incubator at 25℃ for 24 hours, the 96-well plate was taken out of the incubator and the absorbance (OD) was measured at a wavelength of 600nm using an enzyme reader. GSPSS28.0 software was used to calculate the MIC of each group 50 The inhibition rate was calculated using formula I.

[0034]

[0035] The antibacterial rates of FLU against wild-type and drug-resistant Candida albicans are shown in Table 1 and Figure 1 From Table 1 and Figure 1 The test data show that at a concentration of 0.84 μM, the inhibition rate of FLU on wild-type Candida albicans reached 85.66%, while the inhibition rate on the resistant strain was only 11.72%, indicating that the resistant Candida albicans selected in the experiment belongs to the (FLU) resistant type, and the MIC of Pt(1,5-COD)Cl2 and Pt(NBD)Cl2 on wild-type Candida albicans is 2.34%. 50 0.11 μM and 0.42 μM respectively.

[0036] Table 1 Inhibition rate of different concentrations of FLU, Pt(1,5-COD)Cl2 and Pt(NBD)Cl2 on wild-type Candida albicans

[0037]

[0038] The inhibition rates of different concentrations of FLU, Pt(1,5-COD)Cl2 and Pt(NBD)Cl2 on drug-resistant Candida albicans are shown in Table 2. From the data in Table 2, it can be seen that the three drugs all showed a good dose-effect relationship on drug-resistant Candida albicans. The curves between the concentrations of the three drugs and the inhibition rates were plotted ( Figure 2 ), and calculate the half inhibitory concentration MIC according to the curve 50 MIC of FLU, Pt(1,5-COD)Cl2 and Pt(NBD)Cl2 against drug-resistant Candida albicans 50 2.72 μM, 0.18 μM and 0.10 μM respectively (Table 3 and Figure 3 ), indicating that the drug Pt(NBD)Cl2 has the highest antibacterial activity.

[0039] The inhibition rates of FLU, Pt(1,5-COD)Cl2 and Pt(NBD)Cl2 against drug-resistant Candida albicans at similar concentrations are shown in Tables 4 and Figure 4 From Table 4 and Figure 4 The results show that the concentration of Pt(NBD)Cl2 required to achieve an inhibition rate of 96.75% is 0.36 μM (Table 4), while at similar concentrations, the inhibition rates of Pt(1,5-COD)Cl2 (0.34 μM) and FLU (0.42 μM) were 87.44% and 4.92%, respectively. These experimental data further demonstrate that the antibacterial activity of Pt(NBD)Cl2 is superior to that of Pt(1,5-COD)Cl2 and FLU.

[0040] Table 2 Inhibition rate of different concentrations of FLU, Pt(1,5-COD)Cl2 and Pt(NBD)Cl2 on drug-resistant Candida albicans

[0041]

[0042] Table 3 MIC of FLU, Pt(1,5-COD)Cl2 and Pt(NBD)Cl2 against drug-resistant Candida albicans 50

[0043]

[0044] Note: VS.FLU, **P<0.01, ***P<0.001 (One-WayANOVA)

[0045] Table 4 Inhibition rate of FLU, Pt(1,5-COD)Cl2 and Pt(NBDCl2 against drug-resistant Candida albicans at similar concentrations

[0046]

[0047] Note: VS.FLU, *P<0.05, **P<0.01, ***P<0.001 (One-WayANOVA)

[0048] Example 3

[0049] Cytotoxicity of Pt(NBD)Cl2

[0050] The human liver cancer cell line HepG2 [Wang Y, Yang XN, Zhu X, et al. Role of Metabolic Activation in Elemicin-Induced Cellular Toxicity [J]. Journal of Agricultural and Food Chemistry, 2019, 67 (29). DOI: 10.1021 / acs.jafc.9b02137.] (gift from Southwest Biodiversity Laboratory) was selected as the experimental cell, and the platinum cytotoxic drug cisplatin (DDP) (purchased from Shanghai Xianding Biotechnology Co., Ltd.) was used as the positive control group. The conventional CCK-8 method was used to determine the effect of the compound on cell growth. After the cells grew to the logarithmic phase, the cells were inoculated in a 96-well plate (5000 cells / well) and incubated at 37°C and 5% CO2 for 24 hours. When the cells attached to the bottom, the original culture medium was discarded. According to the grouping information, 100 μl of gradient diluted culture medium containing different concentrations of the test substance was added. After 48 hours, 10 μl of CCK-8 was added to each well and incubated for 1 hour at 37°C and 5% CO2 in the dark. The absorbance at 450 nm was measured by an ELISA instrument, and the survival rate of the drug on cell growth was calculated according to Formula II to determine the cytotoxicity.

[0051]

[0052] The results of the experiments on the survival rate of HepG2 cell growth by DDP, FLU, Pt(1,5-COD)Cl2 and Pt(NBD)Cl2 are shown in Tables 5 and Figure 5 From Table 5 and Figure 5 From the results, for the convenience of statistical analysis, the concentration is unified as molar concentration. Cisplatin, a platinum anticancer drug, can inhibit the growth of cancer cells and show obvious cytotoxicity. The inhibition rate of 1.6μM reaches 19.8%, while at the same concentration, the survival rate of FLU, Pt(1,5-COD)Cl2 and Pt(NBD)Cl2 is close to 99%, with almost no cytotoxicity. When the concentration rises to 8μM, the cell survival rate of the cisplatin group is only 9.4%, while the survival rates of the cells in the FLU, Pt(1,5-COD)Cl2 and Pt(NBD)Cl2 groups are 84.4, 88.3 and 92.9%, respectively. Judging from the survival rates at various concentrations, there is a trend that Pt(NBD)Cl2 has the least cytotoxicity.

[0053] Combined with the above antibacterial activity, it can be seen that the inhibition rate of 0.36μM Pt(NBD)Cl2 against drug-resistant Candida albicans reached 95%. In the cell survival experiment, 1.6μM Pt(NBD)Cl2 can still maintain a cell survival rate of more than 99%, indicating that Pt(NBD)Cl2 is safe and effective for antibacterial treatment, which is better than FLU and Pt(1,5-COD)Cl2.

[0054] Table 5 Effects of DDP, FLU, Pt(1,5-COD)Cl2 and Pt(NBD)Cl2 on the survival rate of HepG2 cells

[0055]

[0056] Example 4

[0057] Activity of Pt(NBD)Cl2 combined with Pt(1,5-COD)Cl2 against drug-resistant fungi

[0058] According to the antibacterial test method of Example 1, the antibacterial activity of the composition of Pt(NBD)Cl2 and Pt(1,5-COD)Cl2 at different concentration ratios against drug-resistant Candida albicans was tested, and the results are shown in Table 6. From the data in Table 6, it can be seen that the antibacterial activity of the composition of Pt(NBD)Cl2 and Pt(1,5-COD)Cl2 at different concentration ratios is significantly greater than that of single Pt(1,5-COD)Cl2 and single Pt(NBD)Cl2, showing a good antibacterial synergistic effect, which also suggests that the antibacterial mechanism of Pt(NBD)Cl2 may be different from that of Pt(1,5-COD)Cl2. Among them, the antibacterial effect of the combination of 0.02μM Pt(1,5-COD)Cl2+0.04μM Pt(NBD)Cl2 is the best, and the antibacterial rate reaches 96.16%. Such a low concentration achieves such a high antibacterial rate, suggesting that the composition of this concentration ratio is safer and more effective for antifungal treatment. The inventor is currently intensively studying the synergistic mechanism.

[0059] Table 6 Inhibition rate of Pt(NBD)Cl2 and Pt(1,5-COD)Cl2 combined with drug resistant Candida albicans

[0060]

[0061] Note: VS.FLU, *P<0.05, **P<0.01, ***P<0.001 (One-WayANOVA); VS.Pt(1,5-COD)Cl2,

[0062] #P<0.05, ##P<0.01, ###P<0.001 (One-WayANOVA)

[0063] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of a cyclodiene platinum (II) complex - norbornadiene dichloroplatinum (II) in the preparation of drugs for preventing and treating Candida albicans.

2. A pharmaceutical composition having Candida albicans inhibitory activity, characterized in that: Including norbornadiene dichloroplatinum(II) and (1,5-cyclooctadiene) dichloroplatinum(II); The molar mass ratio of the norbornadiene dichloroplatinum (II) to the (1,5-cyclooctadiene) dichloroplatinum (II) is 2:

1.

3. Use of the pharmaceutical composition according to claim 2 in the preparation of a medicament for preventing and / or treating Candida albicans infection.

4. The use according to claim 1 or 3, characterized in that: The Candida albicans includes wild-type Candida albicans and / or drug-resistant Candida albicans.

5. The application according to claim 4, characterized in that: The drug-resistant Candida albicans includes fluconazole.

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