Antifungal activity of liriodenine on clinical strains of Cryptococcus neoformans and Cryptococcus gattii species complexes

Adriele Dandara Levorato-Vinche Marcia de Souza Carvalho Melhem Lucas Xavier Bonfietti Iván de-la-Cruz-Chacón Carmen Sílvia Fernandes Boaro Alexandre Todorovic Fabro Gisela Ferreira Julhiany de Fátima da Silva Daniela Carvalho dos Santos Beatriz Aparecida Soares Pereira Camila Marçon Lariza Maza Lídia Raquel de Carvalho Rinaldo Poncio Mendes About the authors

Abstract

Background:

Cryptoccocal meningitis continues to present high incidence among AIDS patients. The treatment of choice is the synergistic combination of flucytosine (5-FC) with amphotericin B deoxycholate (AmBd) or its lipid formulations. However, 5-FC is unavailable in many countries and AmB demands hospitalization. The combination of AmB with the fungistatic fluconazole (FLC) or the use of high FLC daily doses alone became the choice. Nonetheless, sterilization of cerebrospinal fluid is delayed with FLC monotherapy, mainly with high fungal burden. These findings suggest the search for new antifungal compounds, such as liriodenine.

Methods:

Liriodenine antifungal activity was evaluated by three procedures: determining the minimum inhibitory concentration (MIC) on 30 strains of the Cryptococcus neoformans (C. neoformans) complex and 30 of the Cryptococcus gattii (C. gattii) complex, using EUCAST methodology and amphotericin B deoxycholate as control; performing the time-kill methodology in two strains of the C. neoformans complex and one of the C. gattii complex; and injury to cryptococcal cells, evaluated by transmission electron microscopy (TEM). Liriodenine absorption and safety at 0.75 and 1.50 mg.kg-1 doses were evaluated in BALB/c mice.

Results:

Liriodenine MICs ranged from 3.9 to 62.5 μg.mL-1 for both species complexes, with no differences between them. Time-kill methodology confirmed its concentration-dependent fungicidal effect, killing all the strains below the limit of detection (33 CFU.mL-1) at the highest liriodenine concentration (32-fold MIC), with predominant activity during the first 48 hours. Liriodenine induced severe Cryptococcus alterations - cytoplasm with intense rarefaction and/or degradation, injury of organelles, and presence of vacuoles. Liriodenine was better absorbed at lower doses, with no histopathological alterations on the digestive tract.

Conclusion:

The fungicidal activity confirmed by time-kill methodology, the intense Cryptococcus injury observed by TEM, the absorption after gavage administration, and the safety at the tested doses indicate that the liriodenine molecule is a promising drug lead for development of anticryptococcal agents.

Keywords:
Liriodenine; Yeast; Fungi; Fungal cell ultrastructure; Cryptococcus spp.; Time-kill methodology

Background

The increased incidence of fungal infections, mainly in immunosuppressed patients, and the emergence of resistant relevant isolates in many countries worldwide for all available compounds, except amphotericin B (AmB), demonstrate the importance of looking for new antifungal compounds, mainly those recommended for treatment of systemic diseases [11. Silva LN, Oliveira SSC, Magalhães LB, Andrade Neto VV, Torres-Santos EC, Carvalho MDC, Pereira MD, Branquinha MH, Santos ALS. Unmasking the Amphotericin B Resistance Mechanisms in Candida haemulonii Species Complex. ACS Infect Dis. 2020 May 8;6(5):1273-82., 22. Wiederhold NP. Antifungal resistance: current trends and future strategies to combat. Infect Drug Resist. 2017 Aug 29;10:249-59. ].

Cryptococcosis is a systemic mycosis caused by encapsulated and non-fermentative yeasts of the Cryptococcus neoformans (C. neoformans) and Cryptococcus gattii (C. gattii) species complexes [33. Kwon-Chung KJ, Fraser JA, Doering TL, Wang Z, Janbon G, Idnurm A, Bahn YS. Cryptococcus neoformans and Cryptococcus gattii, the etiologic agents of cryptococcosis. Cold Spring Harb Perspect Med. 2014 Jul 1;4(7):a019760.]. The lungs are the primary portal of entry and focus of this infection, but life-threatening meningitis is its main clinical manifestation, including in AIDS patients [44. Rajasingham R, Smith RM, Park BJ, Jarvis JN, Govender NP, Chiller TM, Denning DW, Loyse A, Boulware DR. Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis. Lancet Infect Dis. 2017 Aug;17(8):873-81., 55. Jarvis JN, Boulle A, Loyse A, Bicanic T, Rebe K, Williams A, Harrison TS, Meintjes G. High ongoing burden of cryptococcal disease in Africa despite antiretroviral roll out. AIDS. 2009 Jun 1;23(9):1182-3.].

Treatment of cryptococcal meningitis is carried out with the synergistic combination of flucytosine with AmBd or its lipid formulations. Nonetheless, flucytosine is unavailable in many countries, and AmB demands hospitalization, leading to the use of the combination AmB and fluconazole (FLC) for the initial treatment in several countries such as Brazil, or the use of higher doses of FLC as it is in many African countries [66. Perfect JR, Dismukes WE, Dromer F, Goldman DL, Graybill JR, Hamill RJ, Harrison TS, Larsen RA, Lortholary O, Nguyen MH, Pappas PG, Powderly WG, Singh N, Sobel JD, Sorrell TC. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of america. Clin Infect Dis. 2010 Feb 1;50(3):291-322.]. Since FLC is fungistatic and causes a slow clearance of the fungus, other regimens and/or combinations with immunomodulators have been evaluated [66. Perfect JR, Dismukes WE, Dromer F, Goldman DL, Graybill JR, Hamill RJ, Harrison TS, Larsen RA, Lortholary O, Nguyen MH, Pappas PG, Powderly WG, Singh N, Sobel JD, Sorrell TC. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of america. Clin Infect Dis. 2010 Feb 1;50(3):291-322.

7. Moretti ML, Resende MR, Lazéra MS, Colombo AL, Shikanai-Yasuda MA. Consenso em criptococose--2008 [Guidelines in cryptococcosis--2008]. Rev Soc Bras Med Trop. 2008 Sep-Oct;41(5):524-44.
-88. Armstrong-James D, Brown GD, Netea MG, Zelante T, Gresnigt MS, van de Veerdonk FL, Levitz SM. Immunotherapeutic approaches to treatment of fungal diseases. Lancet Infect Dis. 2017 Dec;17(12):e393-402.].

A number of plants produce molecules with antimicrobial activity, including several species of the Annonaceae family, which produce benzylisoquinoline alkaloids [99. Van Vuuren S, Holl D. Antimicrobial natural product research: A review from a South African perspective for the years 2009-2016. J Ethnopharmacol. 2017 Aug 17;208:236-52.]. One of the most common ones is liriodenine, an alkaloid aporphine type found in around 90 genera and 300 Annonaceae species [1010. González-Esquinca, Alma Rosa et al. Alkaloids and acetogenins in Annonaceae development: biological considerations. Rev Bras Frutic. 2014;36(spe 1):1-16., 1111. Lúcio ASSC, Silva Almeida JRG, Cunha EVL, Tavares JF, Barbosa Filho JM. “Chapter Five - Alkaloids of the Annonaceae: occurrence and a compilation of their biological activities,” In: H. J. Knölker (ed.). The Alkaloids: Chemistry and Biology, Cambridge, MA: Elsevier p. 233-409. 2015.] with activity on fungi, bacteria, and protozoa [1212. Hufford CD, Funderburk MJ, Morgan JM, Robertson LW. Two antimicrobial alkaloids from heartwood of Liriodendron tulipifera L. J Pharm Sci. 1975 May;64(5):789-92.

13. Hufford CD, Sharma AS, Oguntimein BO. Antibacterial and antifungal activity of liriodenine and related oxoaporphine alkaloids. J Pharm Sci. 1980 Oct;69(10):1180-3.

14. Graziose R, Rathinasabapathy T, Lategan C, Poulev A, Smith PJ, Grace M, Lila MA, Raskin I. Antiplasmodial activity of aporphine alkaloids and sesquiterpene lactones from Liriodendron tulipifera L. J Ethnopharmacol. 2011 Jan 7;133(1):26-30.

15. Costa EV, da Cruz PE, de Lourenço CC, de Souza Moraes VR, de Lima Nogueira PC, Salvador MJ. Antioxidant and antimicrobial activities of aporphinoids and other alkaloids from the bark of Annona salzmannii A. DC. (Annonaceae). Nat Prod Res. 2013;27(11):1002-6.

16. de-La-Cruz-Chacón I, González-Esquinca AR. Liriodenine alkaloid in Annona diversifolia during early development. Nat Prod Res. 2012;26(1):42-9.
-1717. de-La-Cruz-Chacón I, González-Esquinca AR, Guevara Fefer P, Jímenez Garcia LF. Liriodenine, early antimicrobial defence in Annona diversifolia. Z Naturforsch C J Biosci. 2011 Jul-Aug;66(7-8):377-84.].

Levorato-Vinche et al. [1818. Vinche ADL, de-la-Cruz-Chacón I, González-Esquinca AR, da Silva JF, Ferreira G, Dos Santos DC, Garces HG, de Oliveira DVM, Marçon C, Cavalcante RS, Mendes RP. Antifungal activity of liriodenine on agents of systemic mycoses, with emphasis on the genus Paracoccidioides. J Venom Anim Toxins Incl Trop Dis. 2020 Oct 28;26:e20200023. doi: 10.1590/1678-9199-JVATITD-2020-0023.
https://doi.org/10.1590/1678-9199-JVATIT...
] evaluated the in vitro antifungal activity of liriodenine on agents of systemic mycoses, and it showed a minimum inhibitory concentration (MIC) of 62.5 µg.mL-1 on most of the isolates. Moreover, its activity was fungicidal on susceptible isolates.

The present study aimed at evaluating liriodenine antifungal activity on isolates of both C. neoformans and C. gattii species complexes using the time-kill curve and the analysis of cell alterations by electron microscopy.

Methods

Antifungal compounds

AmBd (Sigma Chemical Company, St. Louis, MO, USA) and liriodenine were used for MIC testing. Moreover, a time-kill evaluation of liriodenine was performed.

Source and extraction of liriodenine

Liriodenine was obtained from Annona mucosa Jacq. planted at the geographical coordinates 22º59’27’’S and 48º28’22’’W in Rio Sul locality, Botucatu (São Paulo state, Brazil). The taxonomic sample was under reference voucher number 33185 of the BOTU Herbarium - São Paulo State University (UNESP), Botucatu (São Paulo state, Brazil). This species is neither threatened nor protected. Liriodenine is an oxoaporphine alkaloid, yellow needle, fluorescent, plain, with an oxo group in position 7, isolated from the root barks.

Liriodenine was extracted from root bark (1,000 g) in the Laboratory of Botany of Botucatu Biosciences Institute, São Paulo State University (UNESP), Brazil, according to a previously reported methodology [1919. De-la-Cruz-Chacón I, López-Fernández NY, Riley-Saldaña CA, Castro-Moreno M, González-Esquinca AR. Antifungal activity in vitro of Sapranthus microcarpus (Annonaceae) against phytopathogens. Acta Bot Mex. 2019;126: e1420.].

Stock solutions

Stock solutions of each agent were prepared using dimethyl sulfoxide (DMSO). Stock solutions of 1000 μg.mL-1 were separated into aliquots and stored at -70ºC until they were used. RPMI 1640 liquid medium (Sigma) buffered to a pH 7.0 with 0.165 M morpholinepropanesulfonic acid (Sigma) was used to obtain final tested agent concentrations. To demonstrate that DMSO did not affect the growth of the studied strains, fungal colonies were grown in the presence of final (1% vol/vol) DMSO concentration and compared to growth in DMSO-naïve conditions.

Microorganisms

Sixty strains, previously identified by molecular typing [2020. Meyer W, Castañeda A, Jackson S, Huynh M, Castañeda E, IberoAmerican Cryptococcal Study Group. Molecular typing of IberoAmerican Cryptococcus neoformans isolates. Emerg Infect Dis. 2003 Feb;9(2):189-95.], were evaluated in this study. Thirty of them belonged to the C. neoformans complex and the other 30 to the C. gattii complex. Regarding AFST-EUCAST recommendations, two quality control strains - Candida krusei (ATCC 6258) and Candida parapsilosis (ATCC 22019) - were also evaluated along with the tests. More than 99% of the QC results were within the acceptable limits. These isolates were kept frozen in 15% glycerol solutions at -20 °C at the Laboratory of Tropical Diseases, Section of Medical Mycology, Experimental Research Unit (Unipex), Botucatu Medical School, UNESP, until they were ready for use in the study. Before testing, each strain was plated on CHROMagar Candida® (Becton Dickinson, Franklin Lakes, NJ, USA) to ensure purity and viability.

Cryptococcal susceptibility testing

The in vitro susceptibility to liriodenine and to AmBd was evaluated by the broth microdilution method, according to the European Committee on Susceptibility Testing (EUCAST) [2121. Arendrup MC, Meletiadis J, Mounton JW, Lagrou K, Hamal P, Guinea J, the Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST). EUCAST definitive document E.DEF 7.3.1. Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts. Copenhagen: EUCAST/AFST; 2017, Jan. Available from: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Files/EUCAST_E_Def_7_3_1_Yeast_testing__definitive.pdf
https://www.eucast.org/fileadmin/src/med...
]. The antifungal epidemiologic cutoff values (ECOFFs) and clinical breakpoints were proposed according to the EUCAST E.Def 7.3 document (European Committee on Antimicrobial Susceptibility Testing), and by using colorimetric indicators [2222. Arendrup MC, Meletiadis J, Mouton JW, Lagrou K, Hamal P, Guinea J, Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST). EUCAST definitive document E.DEF 7.3.2. Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts. Copenhagen: EUCAST/AFST; 2020, Apr. Available from: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Files/EUCAST_E_Def_7.3.2_Yeast_testing_definitive_revised_2020.pdf.
https://www.eucast.org/fileadmin/src/med...
].

Serial dilutions of the stock solution using RPMI-1640 as diluent were performed, and 10 concentrations of liriodenine ranging from 0.97 to 500 μg.mL-1 were obtained. Volumes of 100 μL of each concentration were distributed in microplates with 96 wells. AmBd was included as a positive control in 10 concentrations from 0.03 to 16 μg.mL-1. The inocula of the fungal cells were adjusted to a final concentration of 1-5 × 105 cells/mL, and 100 μL were added to each well containing either liriodenine or AmBd, and to the control wells without antifungal compounds. The plates were incubated at 35 (C ± 2 ºC for 48 h, and then the reading was performed with a microdilution plate reader under 450 nm wavelength. The antifungal concentration that elicited an relative absorbance ≤ 50% or < 90% of that observed in the negative control well (without antifungal compound) was considered the MIC of liriodenine and AmBd, respectively. All the tests were carried out in triplicate [2121. Arendrup MC, Meletiadis J, Mounton JW, Lagrou K, Hamal P, Guinea J, the Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST). EUCAST definitive document E.DEF 7.3.1. Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts. Copenhagen: EUCAST/AFST; 2017, Jan. Available from: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Files/EUCAST_E_Def_7_3_1_Yeast_testing__definitive.pdf
https://www.eucast.org/fileadmin/src/med...
].

Time-kill studies with liriodenine

The time-kill studies were conducted with two strains (strain 32 and strain 57) of the C. neoformans complex and one (strain 17) of the C. gattii complex, according to the procedures of Klepser et al. [2323. Klepser ME, Ernst EJ, Lewis RE, Ernst ME, Pfaller MA. Influence of test conditions on antifungal time-kill curve results: proposal for standardized methods. Antimicrob Agents Chemother. 1998 May;42(5):1207-12.] and Silva et al. [2424. Silva DC, Martins MA, Szeszs MW, Bonfietti LX, Matos D, Melhem MS. Susceptibility to antifungal agents and genotypes of Brazilian clinical and environmental Cryptococcus gattii strains. Diagn Microbiol Infect Dis. 2012 Apr;72(4):332-9.]. Initially, the isolates were subcultured on potato dextrose agar plates. Individual colonies (≥ 1mm) from 48-h culture were suspended in 10-mL buffered RPMI 1640 with 2 % glucose and L-glutamine medium. Isolates were grown overnight with shaking at 35 °C. The initial inoculum was adjusted to 0.5 McFarland turbidity standard (106 CFU.mL-1). One milliliter of the adjusted fungal suspension was then added to either a 9-mL MOPS-buffered RPMI medium alone (control) or a solution of culture medium containing liriodenine. Liriodenine was tested in eight concentrations calculated as multiples of the MIC values (0.5×, 1×, 2×, 3×, 4×, 8×, 16× and 32× MIC). The test tubes were incubated at 35 (C ± 2 °C under agitation. Fungal growth was monitored over a time-course of 72 h (0, 2, 4, 6, 8, 12, 24, 48, 72 h). For every sampled time point, 0.5 mL of the tube content was removed, serially diluted 1:10 in sterile deionized water, and viable counts were determined in 30 μL plated on Sabouraud dextrose agar [2525. Klepser ME, Malone D, Lewis RE, Ernst EJ, Pfaller MA. Evaluation of voriconazole pharmacodynamics using time-kill methodology. Antimicrob Agents Chemother. 2000 Jul;44(7):1917-20.

26. Pappalardo MC, Szeszs MW, Martins MA, Baceti LB, Bonfietti LX, Purisco SU, Baez AA, Melhem MS. Susceptibility of clinical isolates of Cryptococcus neoformans to amphotericin B using time-kill methodology. Diagn Microbiol Infect Dis. 2009 Jun;64(2):146-51.
-2727. Nooney L, Matthews RC, Burnie JP. Evaluation of Mycograb, amphotericin B, caspofungin, and fluconazole in combination against Cryptococcus neoformans by checkerboard and time-kill methodologies. Diagn Microbiol Infect Dis. 2005 Jan;51(1):19-29.].

Analysis

The data were analyzed according to Klepser et al [2323. Klepser ME, Ernst EJ, Lewis RE, Ernst ME, Pfaller MA. Influence of test conditions on antifungal time-kill curve results: proposal for standardized methods. Antimicrob Agents Chemother. 1998 May;42(5):1207-12.]. Colony counting data, in log10 CFU per milliliter, from time-kill studies in triplicate, were averaged and plotted as function of time for each isolate. The rate and extent of liriodenine antifungal activity were assessed. Fungicidal activity was defined as a ≥ 99.9% reduction in the number of CFU per milliliter from the starting inoculum count, and fungistatic activity occurred when this decrease was < 99.9%.

Transmission electron microscopy

Transmission electron microscopy (TEM) was performed at the Center of Electron Microscopy of São Paulo State University (UNESP), Botucatu Biosciences Institute (São Paulo state, Brazil), regarding its previously reported specifications [1818. Vinche ADL, de-la-Cruz-Chacón I, González-Esquinca AR, da Silva JF, Ferreira G, Dos Santos DC, Garces HG, de Oliveira DVM, Marçon C, Cavalcante RS, Mendes RP. Antifungal activity of liriodenine on agents of systemic mycoses, with emphasis on the genus Paracoccidioides. J Venom Anim Toxins Incl Trop Dis. 2020 Oct 28;26:e20200023. doi: 10.1590/1678-9199-JVATITD-2020-0023.
https://doi.org/10.1590/1678-9199-JVATIT...
].

Two strains, numbered 21 (C. gattii) and 41 (C. neoformans), were treated with the respective liriodenine MICs in a 24-well plate. The final volumes of liriodenine and inoculum were adjusted to 1 mL, and Karnovsky’s fixative was added after 48-h incubation at 37 °C. Following this period, the material was removed from the fixative and washed three times for 5 min each in 0.1 M phosphate buffer, pH 7.3. The material was immersed in 0.1 M osmium tetroxide, pH 7.3, for 2 h. Next, the material was washed three times for 10 min each in distilled water and immersed in 0.5% uranyl acetate for approximately 2 h. After dehydration in an increasing acetone series, a mixture of Araldite® resin + 100% acetone (1:1) was added, and the material was left to stand at room temperature for 12 h. Pure resin was added for approximately one hour at 37ºC, and the material was embedded. Ultrathin (90 nm) sections were cut from the blocks and counterstained with uranyl acetate in 50% alcohol for 20 min, followed by counterstaining with lead citrate for 10 min. The sections were observed with a Tecnai Spirit transmission electron microscope (FEI Company).

Statistical analysis

The comparison between the MIC values of different species and the antifungal compounds was carried out using the Mann-Whitney test. The correlation of MIC between antifungal compounds was performed by the Spearman rank correlation coefficient. Statistical tests were performed by using SAS (SAS Institute, Cary, NC, USA). Significance was set up at p ≤ 0.05.

In vivo evaluation of absorption and toxicity

Animals

Four isogenic albino male BALB/c mice, five to seven weeks of age and 25 g average weight, were obtained from the husbandry of the Experimental Laboratory of Infectious and Parasitic Diseases of Botucatu Medical School, São Paulo State University (UNESP), Botucatu (São Paulo state, Brazil). The animals were kept in boxes containing two animals on average, with a bed of pressure-treated wood shavings, in an environment ranging 23-25 °C, with lighting controlled by electromechanical time switch, that is, 12 h with the light on and 12 h in the dark. They received filtered water and commercial mice diet ad libitum.

Liriodenine preparation and administration in uninfected mice

Liriodenine was dissolved in 4% DMSO. For the preparation of the doses 0.75 and 1.50 mg.kg-1, 1.5 and 3.0 mg of liriodenine, respectively, 0.240 mL of 4% DMSO were added. Liriodenine was given by gavage in a single dose at 12:00a.m. Each animal received 0.120 mL of the liriodenine-containing solution. The doses used were defined based on the results of the in vitro sensitivity test [1818. Vinche ADL, de-la-Cruz-Chacón I, González-Esquinca AR, da Silva JF, Ferreira G, Dos Santos DC, Garces HG, de Oliveira DVM, Marçon C, Cavalcante RS, Mendes RP. Antifungal activity of liriodenine on agents of systemic mycoses, with emphasis on the genus Paracoccidioides. J Venom Anim Toxins Incl Trop Dis. 2020 Oct 28;26:e20200023. doi: 10.1590/1678-9199-JVATITD-2020-0023.
https://doi.org/10.1590/1678-9199-JVATIT...
]. Two uninfected mice received 0.75 mg.kg-1 and other two 1.50 mg.kg-1 of liriodenine in a single dose.

Blood collection and euthanasia of the mice

The animals were submitted to blood collection and euthanasia six and 12 h after liriodenine administration. Blood samples were collected from each animal by cardiac puncture to determine the serum levels of liriodenine.

Mice were initially anesthetized and killed with 0.08 μL of a solution containing 24 μL ketamine, 32 μL xylosin, and 24 μL distilled water, which was administered intraperitoneally with the aid of a 1.0 mL disposable syringe. After sedation, the blood was collected by cardiac puncture using a 1.0 mL syringe and a 25 × 7-gauge needle. The blood was transferred to a sterile test tube without anticoagulants and, subsequently, centrifuged at 3.500 rpm in a Revan Cycle C I centrifuge.

Serum dosage of liriodenine

The serum samples from the mice were diluted in 15 mL deionized water to increase the volume of the working solution. In order to extract the fat present in the serum, the mixture was placed in a separatory funnel with 10 mL hexane, from which the aqueous phase was collected and then adjusted to 9.5 pH with saturated solution of Na2CO3. After that, it was placed in a separatory funnel with 10 mL chloroform, and the chloroform phase containing the liriodenine was then collected. For the identification and quantification of the liriodenine, the sample was kept to stand until the chloroform was completely evaporated, then dissolved in methanol and analyzed in High Performance Liquid Chromatography (HPLC), according to the specifications modified by de-La-Cruz-Chacón et al. [16, 17].

The samples were eluted in isocratic mode - 80% methanol (J. T. Baker, HPLC grade) and 20% ultrapure water - and the pH was adjusted to 3.0 with acetic acid. The flow rate of the mobile phase was 1.0 mL/min and the total time was 15 min, with 254 nm wavelength and 20 μL injection volume. The compartment of the chromatographic column oven was programmed to 30 °C. The equipment used was an HPLC (Thermo Scientific Dionex Ultimate 3000) equipped with a 0UV-VIS detector, an automatic injector with an oven and a four-channel pump with built-in degasser, and LUNA® 5 μm C18 (250 × 4.6 mm) column by Phenomenex [2828. Moreira DL, Leitão GG. Quantitative determination of liriodenine and moupinamide in five species of Mollinedia by high performance liquid chromatography. Phytochem Anal. 2001 Jul-Aug;12(4):223-5.].

The serum level of each sample was measured in triplicate, using the average of the concentrations found.

Histopathological evaluation

The histopathological examination of the intestines was performed in all animals. The intestines were collected and fixed in formalin for 48 h, then transferred to 70% alcohol and embedded in paraffin. Cuttings were performed, and afterwards, the slides were stained with hematoxylin and eosin (H&E).

Results

Minimum inhibitory concentrations (MICs)

The MIC of liriodenine ranged from 3.9 µg.mL-1 to 62.5 µg.mL-1 for C. neoformans as well as for C. gattii (Figure 1). MIC90 was 31.25 µg.mL-1 for both species. All the strains were inhibited to liriodenine and presented no differences in susceptibility regarding the species (p > 0.05). In addition, the low MICs of AmB did not differ between the species (p > 0.05). MIC90 was 0.125 µg.mL-1 for both species, and all strains showed high susceptibility to AmB.

Figure 1.
Minimum inhibitory concentrations (MICs) of liriodenine (LRD) and amphotericin B (AmB) for 30 strains of the Cryptococcus neoformans complex and 30 strains of the Cryptococcus gattii complex, determined by broth microdilution method. Results as median and interquartile intervals, as well as geometric mean. The Mann-Whitney U rank test. Comparison of MICs as to Cryptococcus complex, (A) LRD: C. neoformans: 15.62 [15.62; 31.25], C. gattii: 31.25 [15.62; 31.25], p = 0.30; (B) AmB: C. neoformans: 0.6 [0.06; 0.06], C. gattii: 0.06 [0.06; 0.13], p = 0.45.

Time-kill studies

Figures 2, 3 and 4 show time-kill curves for liriodenine using three strains exposed to four different agent concentrations and one untreated control of each strain. Liriodenine induced a fungicidal effect in all three strains, yet the onset of the fungicidal activity depended on the tested concentration and differed among the strains. All strains were killed to below the limit of detection (33 CFU.mL−1), at the highest liriodenine concentration (32-fold MIC). The strains experienced the most killing during the first 48 h at high antimicrobial concentrations. For strains 57 and 32, the fungicidal activity decreased at lower concentrations.

Figure 2.
Time-kill curves for lireodenine against strain 17 of Cryptococcus gattii complex. Eight doubling dilutions are plotted, the highest concentration corresponds to 32 × MIC as measured with the EUCAST microdilution method, and the blue line represents growth in absence of lireodenine. The compound was added at timepoint 0 and monitored until 72 h. The limit of detection in the assay was 33 CFU/mL. CFU: colony forming units; MIC: minimum inhibitory concentration.

Figure 3.
Time-kill curves for lireodenine against strain 57 of Cryptococcus neoformans complex. Eight doubling dilutions are plotted, the highest concentration corresponds to 32 × MIC as measured with the EUCAST microdilution method, and the blue line represents growth in absence of lireodenine. The compound was added at timepoint 0 and monitored until 72 h. The limit of detection in the assay was 33 CFU/mL. CFU: colony forming units; MIC: minimum inhibitory concentration.

Figure 4.
Time-kill curves for lireodenine against strain 32 of Cryptococcus neoformans complex. Eight doubling dilutions are plotted, the highest concentration corresponds to 32 × MIC as measured with the EUCAST microdilution method, and the blue line represents growth in absence of lireodenine. The compound was added at timepoint 0 and monitored until 72 h. The limit of detection in the assay was 33 CFU/mL. CFU: colony forming units; MIC: minimum inhibitory concentration.

Transmission electron microscopy

Transmission electron microscopy (TEM) of untreated cryptococcal cells revealed round shape with regular contours, uniformly thick walls, and a polysaccharide capsule. The organelles were preserved and intact, enabling the detection of free ribosomes, a discrete rough endoplasmic reticulum, multivesicular bodies, vacuoles of a heterogeneous material and various sizes, and a few lipid droplets. Ligaments between cells and their buds were also demonstrated (Figures 5A and 5B).

However, liriodenine-treated cryptococcal cells presented significantly altered morphology. In both species complexes, the cytoplasm showed intense rarefaction and/or degradation, injury of organelles, vacuoles, or other structures, suggesting death of the microorganism (Figures 5B, 5C, 5E and 5F).

Figure 5.
Transmission electron microscopy of Cryptococcus neoformans and Cryptococcus gattii species complexes. (A) Untreated Cryptococcus neoformans species complex cells. (B, C) Liriodenine-treated Cryptococcus neoformans species complex cells at a concentration of 31.25 µg.mL-1. (D) Untreated Cryptococcus gattii species complex cells. (E, F) Liriodenine-treated Cryptococcus gattii species complex cells at a concentration of 62.5 µg.mL-1. PM: plasma membrane; PC: polysaccharide capsule; MVB: multivesicular bodies; VA: vacuoles; LI: lipids; RER: rough endoplasmic reticulum; CR: cytoplasmic rarefaction; cell membrane irregularity.

In vivo evaluation of absorption and toxicity

Clinical observation and histopathological analysis

Prior to the administration of liriodenine, all animals had shiny hair, and had been calm eating regularly. Such conditions did not change with the administration of liriodenine.

Nevertheless, 12 h after the administration of liriodenine, the mice that had received the dose of 1.50 mg.kg-1 of body weight had abdominal distension; the laparotomy revealed the presence of large amounts of intestinal gas (Additional file 1). These findings were not observed in animals that received liriodenine at the dose of 0.75 mg.kg-1 of body weight.

The cross-sectional histological analyses of the intestines in all the animals, which were observed using light microscopy, showed no pathological alterations with either of the doses administered or in either of the two different moments of euthanasia (Figure 6).

Figure 6.
Intestinal histomorphology of BALB/c mice after administration of liriodenine by gavage. Note the absence of pathological changes at different doses (0.75 and 1.50 mg.kg-1) of liriodenine and different moments of euthanasia (6 and 12 h of administration) [H&E; 5XX].

Serum dosage of liriodenine

Liriodenine was detected in the serum of both mouse groups that had received it at the doses of 0.75 mg.kg-1 and 1.50 mg.kg-1 of body weight, confirming the absorption of this substance after administration by gavage (Figure 7).

Figure 7.
Serum levels of liriodenine were observed at 6 and 12 h after the administration by gavage of both doses of 0.75 and 1.50 mg.kg-1 of body weight. Data from one animal per dose at the time. The serum level is the average of three repetitions performed in each sample.

However, serum levels of liriodenine showed different behavior due to the time of administration, according to the dose used in the mice: the ones that had received 0.75 mg.kg-1 presented an increase in concentration over time, while the opposite was observed in animals to which 1.50 mg.kg-1 had been administered.

Discussion

In spite of the well-conducted studies on the treatment of cryptococcal meningitis, many questions still remain unanswered in the clinical practice, stimulating the investigation of new compounds, such as liriodenine [66. Perfect JR, Dismukes WE, Dromer F, Goldman DL, Graybill JR, Hamill RJ, Harrison TS, Larsen RA, Lortholary O, Nguyen MH, Pappas PG, Powderly WG, Singh N, Sobel JD, Sorrell TC. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of america. Clin Infect Dis. 2010 Feb 1;50(3):291-322.

7. Moretti ML, Resende MR, Lazéra MS, Colombo AL, Shikanai-Yasuda MA. Consenso em criptococose--2008 [Guidelines in cryptococcosis--2008]. Rev Soc Bras Med Trop. 2008 Sep-Oct;41(5):524-44.
-88. Armstrong-James D, Brown GD, Netea MG, Zelante T, Gresnigt MS, van de Veerdonk FL, Levitz SM. Immunotherapeutic approaches to treatment of fungal diseases. Lancet Infect Dis. 2017 Dec;17(12):e393-402., 2929. Lewis RE. Current concepts in antifungal pharmacology. Mayo Clin Proc. 2011 Aug;86(8):805-17., 3030. Brüggemann RJ, Alffenaar JW, Blijlevens NM, Billaud EM, Kosterink JG, Verweij PE, Burger DM. Clinical relevance of the pharmacokinetic interactions of azole antifungal drugs with other coadministered agents. Clin Infect Dis. 2009 May 15;48(10):1441-58.]. The choice of this compound was based on some previous information: (a) liriodenine is extracted from Annona mucosa, a plant available in the Brazilian region where our university is located; (b) liriodenine extraction was performed in this university; (c) its chemical composition is known, making it easier to have a possible future production and modification of the molecule; (d) the literature review revealed some antimicrobial activity; (e) our previous study showed its activity on some fungi, mainly from the Cryptococcus genus [1818. Vinche ADL, de-la-Cruz-Chacón I, González-Esquinca AR, da Silva JF, Ferreira G, Dos Santos DC, Garces HG, de Oliveira DVM, Marçon C, Cavalcante RS, Mendes RP. Antifungal activity of liriodenine on agents of systemic mycoses, with emphasis on the genus Paracoccidioides. J Venom Anim Toxins Incl Trop Dis. 2020 Oct 28;26:e20200023. doi: 10.1590/1678-9199-JVATITD-2020-0023.
https://doi.org/10.1590/1678-9199-JVATIT...
].

Antifungal susceptibility testing (AST) has become a powerful tool in the choice and management of treatment of many systemic fungal diseases. Therefore, the protocols used in AST should be very well standardized and validated. The ones used in this study were RPMI 1640 buffered to a pH 7.0 with MOPS, incubation of time-kill samples at 35 °C with agitation, effect on antifungal carryover and evaluation for at least 24 hours compliant to Klepser et al. [2323. Klepser ME, Ernst EJ, Lewis RE, Ernst ME, Pfaller MA. Influence of test conditions on antifungal time-kill curve results: proposal for standardized methods. Antimicrob Agents Chemother. 1998 May;42(5):1207-12.] standardization, being most of them confirmed by Zaragoza et al. [3131. Zaragoza O, Mesa-Arango AC, Gómez-López A, Bernal-Martínez L, Rodríguez-Tudela JL, Cuenca-Estrella M. Process analysis of variables for standardization of antifungal susceptibility testing of nonfermentative yeasts. Antimicrob Agents Chemother. 2011 Apr;55(4):1563-70.]. In addition, the correlations between AST and clinical response are poor with MIC, increase a little with minimum fungicidal concentration (MFC), and are much better with the time-kill methodology [3232. Rodero L, Córdoba S, Cahn P, Soria M, Lucarini M, Davel G, Kaufman S, Canteros C, Guelfand L. Timed-kill curves for Cryptococcus neoformans isolated from patients with AIDS. Med Mycol. 2000 Jun;38(3):201-7.], which was carried out in this study. As already described for the fungicidal drug AMB, isolates can show different profiles in the time-kill methodology that are impossible to identify only by MIC [2424. Silva DC, Martins MA, Szeszs MW, Bonfietti LX, Matos D, Melhem MS. Susceptibility to antifungal agents and genotypes of Brazilian clinical and environmental Cryptococcus gattii strains. Diagn Microbiol Infect Dis. 2012 Apr;72(4):332-9.

25. Klepser ME, Malone D, Lewis RE, Ernst EJ, Pfaller MA. Evaluation of voriconazole pharmacodynamics using time-kill methodology. Antimicrob Agents Chemother. 2000 Jul;44(7):1917-20.
-2626. Pappalardo MC, Szeszs MW, Martins MA, Baceti LB, Bonfietti LX, Purisco SU, Baez AA, Melhem MS. Susceptibility of clinical isolates of Cryptococcus neoformans to amphotericin B using time-kill methodology. Diagn Microbiol Infect Dis. 2009 Jun;64(2):146-51.]. Future studies on pharmacokinetics and pharmacodynamics associated with time-kill data should improve the knowledge on the in vivo activity of liriodenine.

The present study demonstrated fungicidal activity of liriodenine on fungi of the C. neoformans and C. gattii species complexes, showing MIC values ranging from 3.9 to 62.5 µg.mL-1. Such results are similar to those obtained by Cruz et al. [3333. Cruz KS, Lima ES, da Silva MJA, de Souza ES, Montoia A, Pohlit AM, de Souza JVB. Screening and Antifungal Activity of a β-Carboline Derivative against Cryptococcus neoformans and C. gattii. Int J Microbiol. 2019 Jan 22;2019:7157845.], who observed the activity of the synthetic 8-nitrohormone on eight strains of fungi from the Cryptococcus genus, with MICs of 40 µg.mL-1; however, the authors did not evaluate whether the compound presented a fungistatic or fungicidal effect.

The MICs of liriodenine observed in the present study were lower than those showed by the fatty acid methyl esters extracted from seeds of Annona cornifolia (> 500 µg.mL-1) [3434. Lima LA, Johann S, Cisalpino PS, Pimenta LP, Boaventura MA. In vitro antifungal activity of fatty acid methyl esters of the seeds of Annona cornifolia A.St.-Hil. (Annonaceae) against pathogenic fungus Paracoccidioides brasiliensis. Rev Soc Bras Med Trop. 2011 Nov-Dec;44(6):777-80.], and they were also lower when compared to the essential oil and its major components linalool and geraniol extracted from Ocimum basilicum var. Maria Bonita, whose MICs ranged from 156 to 2500 µg.mL-1 [3535. Cardoso NN, Alviano CS, Blank AF, Romanos MT, Fonseca BB, Rozental S, Rodrigues IA, Alviano DS. Synergism Effect of the Essential Oil from Ocimum basilicum var. Maria Bonita and Its Major Components with Fluconazole and Its Influence on Ergosterol Biosynthesis. Evid Based Complement Alternat Med. 2016;2016:5647182.]. Nevertheless, higher activity was found with maytenin and pristimerin, extracted from Maytenus ilicifolia (Celastraceae), when evaluated against five isolates of the Cryptococcus genus, showing MICs and MFCs ranging from 0.48 to 3.90 µg.mL-1 and from 0.97 to 7.81 µg.mL-1, respectively [3636. Gullo FP, Sardi JC, Santos VA, Sangalli-Leite F, Pitangui NS, Rossi SA, de Paula E Silva AC, Soares LA, Silva JF, Oliveira HC, Furlan M, Silva DH, Bolzani VS, Mendes-Giannini MJ, Fusco-Almeida AM. Antifungal activity of maytenin and pristimerin. Evid Based Complement Alternat Med. 2012;2012:340787.].

Punicalagin, a hydrosoluble tannin extracted from Lafoensia pacari A. St.-Hil (Lythraceae), presented a fungistatic effect on fungi of the C. neoformans complex, with MICs ranging from 0.5 to 4.0 µg.mL-1, which are values lower than those observed in the present study. However, its fungicidal activity was demonstrated at 256 µg.mL-1 [3737. Silva TC, Zara ALSA, Sá FADS, Bara MTF, Ávila RI, Costa CR, Valadares MC, Santos ASD, Freitas VAQ, Silva MDRR. Antifungal potential of punicalagin against Cryptococcus neoformans species complex. Rev Inst Med Trop Sao Paulo. 2018 Oct 22;60:e60.].

The previous findings with liriodenine based on MFC [1818. Vinche ADL, de-la-Cruz-Chacón I, González-Esquinca AR, da Silva JF, Ferreira G, Dos Santos DC, Garces HG, de Oliveira DVM, Marçon C, Cavalcante RS, Mendes RP. Antifungal activity of liriodenine on agents of systemic mycoses, with emphasis on the genus Paracoccidioides. J Venom Anim Toxins Incl Trop Dis. 2020 Oct 28;26:e20200023. doi: 10.1590/1678-9199-JVATITD-2020-0023.
https://doi.org/10.1590/1678-9199-JVATIT...
] were confirmed by the present study, in which the time-kill methodology was carried out. In addition, the intense morphological alterations demonstrated by TEM - suggestive of killed cryptococcal cells - seem to justify liriodenine fungicidal activity. To the best of our knowledge, this is the second study showing the effect of an antifungal compound on cryptococcal cells using TEM.

The mechanism of action of liriodenine has yet to be demonstrated. The results of this study could be taken into consideration, contributing to fill this gap. The in vitro and in vivo inhibition of the topoisomerase II enzyme of the DNA, alike the quinolones, suggest an interference in the RNA and protein synthesis [3838. Woo SH, Reynolds MC, Sun NJ, Cassady JM, Snapka RM. Inhibition of topoisomerase II by liriodenine. Biochem Pharmacol. 1997 Aug 15;54(4):467-73.]. Furthermore, melanin, produced by the action of phenol oxidase on L-DOPA [3939. Moore GS, Atkins RD. The fungicidal and fungistatic effects of an aqueous garlic extract on medically important yeast-like fungi. Mycologia. 1977 Mar-Apr;69(2):341-8., 4040. Fromtling RA, Bulmer GS. In vitro effect of aqueous extract of garlic (Allium sativum) on the growth and viability of Cryptococcus neoformans. Mycologia. 1978 Mar-Apr;70(2):397-405.], is a virulence factor for fungi of the Cryptococcus genus for protecting them from oxidative stress, phagocytosis, the action of antifungal compounds, as well as for modifying the host immune response [4141. Rosas AL, Casadevall A. Melanization affects susceptibility of Cryptococcus neoformans to heat and cold. FEMS Microbiol Lett. 1997 Aug 15;153(2):265-72.

42. Nosanchuk JD, Casadevall A. Impact of melanin on microbial virulence and clinical resistance to antimicrobial compounds. Antimicrob Agents Chemother. 2006 Nov;50(11):3519-28.

43. Wang Y, Casadevall A. Growth of Cryptococcus neoformans in presence of L-dopa decreases its susceptibility to amphotericin B. Antimicrob Agents Chemother. 1994 Nov;38(11):2648-50.

44. Van Duin D, Casadevall A, Nosanchuk JD. Melanization of Cryptococcus neoformans and Histoplasma capsulatum reduces their susceptibilities to amphotericin B and caspofungin. Antimicrob Agents Chemother. 2002 Nov;46(11):3394-400.

45. Mednick AJ, Nosanchuk JD, Casadevall A. Melanization of Cryptococcus neoformans affects lung inflammatory responses during cryptococcal infection. Infect Immun. 2005 Apr;73(4):2012-9.
-4646. Liu L, Tewari RP, Williamson PR. Laccase protects Cryptococcus neoformans from antifungal activity of alveolar macrophages. Infect Immun. 1999 Nov;67(11):6034-9.]. As liriodenine inhibits the melanin synthesis in PC-12 cells, this anti-melanin activity could play a role in its anticryptococcal activity [4747. Jin CM, Lee JJ, Yang YJ, Kim YM, Kim YK, Ryu SY, Lee MK. Liriodenine inhibits dopamine biosynthesis and L-DOPA-induced dopamine content in PC12 cells. Arch Pharm Res. 2007 Aug;30(8):984-90.]. Finally, liriodenine causes an imbalance in the iron cell homeostasis, leading to the accumulation of the mitochondrial iron, a decrease in the number of iron enzymes, and an increase in the oxidative stress, which causes fungal death [4848. Tripathi SK, Xu T, Feng Q, Avula B, Shi X, Pan X, Mask MM, Baerson SR, Jacob MR, Ravu RR, Khan SI, Li XC, Khan IA, Clark AM, Agarwal AK. Two plant-derived aporphinoid alkaloids exert their antifungal activity by disrupting mitochondrial iron-sulfur cluster biosynthesis. J Biol Chem. 2017 Oct 6;292(40):16578-93.].

Ultrastructural studies of antifungal compound actions on Cryptococcus cells are scarce [4949. Nosanchuk JD, Cleare W, Franzot SP, Casadevall A. Amphotericin B and fluconazole affect cellular charge, macrophage phagocytosis, and cellular morphology of Cryptococcus neoformans at subinhibitory concentrations. Antimicrob Agents Chemother. 1999 Feb;43(2):233-9.

50. Borges M. “Changes in fungal ultrastructure after itraconazole treatment.” In: R. A. Fromtling, editorRecent trends in discovery, development and evaluation of antifungal agents. (Barcelona: J.R. Prous Sience Publishers). p. 23-36. 1987.
-5151. Guerra CR, Ishida K, Nucci M, Rozental S. Terbinafine inhibits Cryptococcus neoformans growth and modulates fungal morphology. Mem Inst Oswaldo Cruz. 2012 Aug;107(5):582-90.]. Subinhibitory concentrations, from 0.125 to 0.5 of MIC either of AmB or of fluconazole, alter cell and capsule size, and cell shape at scanning electron microscopy [4949. Nosanchuk JD, Cleare W, Franzot SP, Casadevall A. Amphotericin B and fluconazole affect cellular charge, macrophage phagocytosis, and cellular morphology of Cryptococcus neoformans at subinhibitory concentrations. Antimicrob Agents Chemother. 1999 Feb;43(2):233-9.]. Terbinafine, a fungistatic compound, causes detachment of the cell membrane from the cell wall, which probably results in membrane impairment, as well as increased cytoplasmic vacuoles that could account for the lipid accumulation and mitochondrial swelling [5151. Guerra CR, Ishida K, Nucci M, Rozental S. Terbinafine inhibits Cryptococcus neoformans growth and modulates fungal morphology. Mem Inst Oswaldo Cruz. 2012 Aug;107(5):582-90.]. Some of these findings, including irregularity of cytoplasmic membranes and cytoplasmic vacuoles, are also observed with liriodenine, a fungicidal compound.

The evaluation of liriodenine serum levels after gavage administration suggests better absorption with the lower dose used, 0.75 mg.kg-1, which may be justified by the gas formation determined by the dose of 1.50 mg.kg-1. Thus, the abdominal distension due to gas formation could lead to a reduction in the absorption of liriodenine since the histopathological evaluation with the H&E staining of the intestinal fragments did not reveal any organic lesion. This result prompts the development of further studies on liriodenine or its derivatives as an anticryptococcal compound.

The potential weakness of this study is MIC90 higher than that observed with AmB. Such finding suggests the investigation of liriodenine derivatives with the same fungicidal activity but with lower MICs, which is ongoing. The best derivatives will progress into the determination of the mechanism of action, pharmacokinetic studies, and combination with other antifungal compounds.

Conclusions

The present in vitro studies of liriodenine on fungi of C. neoformans and C. gattii species complexes demonstrated intense structural alterations of the yeast cells and fungicidal activity when assessed by time-kill methodology. These results suggest that this molecule can be a promising prototype of anticryptococcal compounds. Investigations on modifications of the liriodenine molecule to improve its pharmacological properties and experimental cryptococcal infection are currently ongoing for evaluating the safety and efficacy of this antifungal compound and its derivatives.

Acknowledgments

We thank Rinaldo José Ortiz, from the University Library of Botucatu Campus, UNESP, for organizing the references.

References

  • 1. Silva LN, Oliveira SSC, Magalhães LB, Andrade Neto VV, Torres-Santos EC, Carvalho MDC, Pereira MD, Branquinha MH, Santos ALS. Unmasking the Amphotericin B Resistance Mechanisms in Candida haemulonii Species Complex. ACS Infect Dis. 2020 May 8;6(5):1273-82.
  • 2. Wiederhold NP. Antifungal resistance: current trends and future strategies to combat. Infect Drug Resist. 2017 Aug 29;10:249-59.
  • 3. Kwon-Chung KJ, Fraser JA, Doering TL, Wang Z, Janbon G, Idnurm A, Bahn YS. Cryptococcus neoformans and Cryptococcus gattii, the etiologic agents of cryptococcosis. Cold Spring Harb Perspect Med. 2014 Jul 1;4(7):a019760.
  • 4. Rajasingham R, Smith RM, Park BJ, Jarvis JN, Govender NP, Chiller TM, Denning DW, Loyse A, Boulware DR. Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis. Lancet Infect Dis. 2017 Aug;17(8):873-81.
  • 5. Jarvis JN, Boulle A, Loyse A, Bicanic T, Rebe K, Williams A, Harrison TS, Meintjes G. High ongoing burden of cryptococcal disease in Africa despite antiretroviral roll out. AIDS. 2009 Jun 1;23(9):1182-3.
  • 6. Perfect JR, Dismukes WE, Dromer F, Goldman DL, Graybill JR, Hamill RJ, Harrison TS, Larsen RA, Lortholary O, Nguyen MH, Pappas PG, Powderly WG, Singh N, Sobel JD, Sorrell TC. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of america. Clin Infect Dis. 2010 Feb 1;50(3):291-322.
  • 7. Moretti ML, Resende MR, Lazéra MS, Colombo AL, Shikanai-Yasuda MA. Consenso em criptococose--2008 [Guidelines in cryptococcosis--2008]. Rev Soc Bras Med Trop. 2008 Sep-Oct;41(5):524-44.
  • 8. Armstrong-James D, Brown GD, Netea MG, Zelante T, Gresnigt MS, van de Veerdonk FL, Levitz SM. Immunotherapeutic approaches to treatment of fungal diseases. Lancet Infect Dis. 2017 Dec;17(12):e393-402.
  • 9. Van Vuuren S, Holl D. Antimicrobial natural product research: A review from a South African perspective for the years 2009-2016. J Ethnopharmacol. 2017 Aug 17;208:236-52.
  • 10. González-Esquinca, Alma Rosa et al. Alkaloids and acetogenins in Annonaceae development: biological considerations. Rev Bras Frutic. 2014;36(spe 1):1-16.
  • 11. Lúcio ASSC, Silva Almeida JRG, Cunha EVL, Tavares JF, Barbosa Filho JM. “Chapter Five - Alkaloids of the Annonaceae: occurrence and a compilation of their biological activities,” In: H. J. Knölker (ed.). The Alkaloids: Chemistry and Biology, Cambridge, MA: Elsevier p. 233-409. 2015.
  • 12. Hufford CD, Funderburk MJ, Morgan JM, Robertson LW. Two antimicrobial alkaloids from heartwood of Liriodendron tulipifera L. J Pharm Sci. 1975 May;64(5):789-92.
  • 13. Hufford CD, Sharma AS, Oguntimein BO. Antibacterial and antifungal activity of liriodenine and related oxoaporphine alkaloids. J Pharm Sci. 1980 Oct;69(10):1180-3.
  • 14. Graziose R, Rathinasabapathy T, Lategan C, Poulev A, Smith PJ, Grace M, Lila MA, Raskin I. Antiplasmodial activity of aporphine alkaloids and sesquiterpene lactones from Liriodendron tulipifera L. J Ethnopharmacol. 2011 Jan 7;133(1):26-30.
  • 15. Costa EV, da Cruz PE, de Lourenço CC, de Souza Moraes VR, de Lima Nogueira PC, Salvador MJ. Antioxidant and antimicrobial activities of aporphinoids and other alkaloids from the bark of Annona salzmannii A. DC. (Annonaceae). Nat Prod Res. 2013;27(11):1002-6.
  • 16. de-La-Cruz-Chacón I, González-Esquinca AR. Liriodenine alkaloid in Annona diversifolia during early development. Nat Prod Res. 2012;26(1):42-9.
  • 17. de-La-Cruz-Chacón I, González-Esquinca AR, Guevara Fefer P, Jímenez Garcia LF. Liriodenine, early antimicrobial defence in Annona diversifolia. Z Naturforsch C J Biosci. 2011 Jul-Aug;66(7-8):377-84.
  • 18. Vinche ADL, de-la-Cruz-Chacón I, González-Esquinca AR, da Silva JF, Ferreira G, Dos Santos DC, Garces HG, de Oliveira DVM, Marçon C, Cavalcante RS, Mendes RP. Antifungal activity of liriodenine on agents of systemic mycoses, with emphasis on the genus Paracoccidioides J Venom Anim Toxins Incl Trop Dis. 2020 Oct 28;26:e20200023. doi: 10.1590/1678-9199-JVATITD-2020-0023.
    » https://doi.org/10.1590/1678-9199-JVATITD-2020-0023
  • 19. De-la-Cruz-Chacón I, López-Fernández NY, Riley-Saldaña CA, Castro-Moreno M, González-Esquinca AR. Antifungal activity in vitro of Sapranthus microcarpus (Annonaceae) against phytopathogens. Acta Bot Mex. 2019;126: e1420.
  • 20. Meyer W, Castañeda A, Jackson S, Huynh M, Castañeda E, IberoAmerican Cryptococcal Study Group. Molecular typing of IberoAmerican Cryptococcus neoformans isolates. Emerg Infect Dis. 2003 Feb;9(2):189-95.
  • 21. Arendrup MC, Meletiadis J, Mounton JW, Lagrou K, Hamal P, Guinea J, the Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST). EUCAST definitive document E.DEF 7.3.1. Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts. Copenhagen: EUCAST/AFST; 2017, Jan. Available from: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Files/EUCAST_E_Def_7_3_1_Yeast_testing__definitive.pdf
    » https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Files/EUCAST_E_Def_7_3_1_Yeast_testing__definitive.pdf
  • 22. Arendrup MC, Meletiadis J, Mouton JW, Lagrou K, Hamal P, Guinea J, Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST). EUCAST definitive document E.DEF 7.3.2. Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts. Copenhagen: EUCAST/AFST; 2020, Apr. Available from: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Files/EUCAST_E_Def_7.3.2_Yeast_testing_definitive_revised_2020.pdf
    » https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Files/EUCAST_E_Def_7.3.2_Yeast_testing_definitive_revised_2020.pdf
  • 23. Klepser ME, Ernst EJ, Lewis RE, Ernst ME, Pfaller MA. Influence of test conditions on antifungal time-kill curve results: proposal for standardized methods. Antimicrob Agents Chemother. 1998 May;42(5):1207-12.
  • 24. Silva DC, Martins MA, Szeszs MW, Bonfietti LX, Matos D, Melhem MS. Susceptibility to antifungal agents and genotypes of Brazilian clinical and environmental Cryptococcus gattii strains. Diagn Microbiol Infect Dis. 2012 Apr;72(4):332-9.
  • 25. Klepser ME, Malone D, Lewis RE, Ernst EJ, Pfaller MA. Evaluation of voriconazole pharmacodynamics using time-kill methodology. Antimicrob Agents Chemother. 2000 Jul;44(7):1917-20.
  • 26. Pappalardo MC, Szeszs MW, Martins MA, Baceti LB, Bonfietti LX, Purisco SU, Baez AA, Melhem MS. Susceptibility of clinical isolates of Cryptococcus neoformans to amphotericin B using time-kill methodology. Diagn Microbiol Infect Dis. 2009 Jun;64(2):146-51.
  • 27. Nooney L, Matthews RC, Burnie JP. Evaluation of Mycograb, amphotericin B, caspofungin, and fluconazole in combination against Cryptococcus neoformans by checkerboard and time-kill methodologies. Diagn Microbiol Infect Dis. 2005 Jan;51(1):19-29.
  • 28. Moreira DL, Leitão GG. Quantitative determination of liriodenine and moupinamide in five species of Mollinedia by high performance liquid chromatography. Phytochem Anal. 2001 Jul-Aug;12(4):223-5.
  • 29. Lewis RE. Current concepts in antifungal pharmacology. Mayo Clin Proc. 2011 Aug;86(8):805-17.
  • 30. Brüggemann RJ, Alffenaar JW, Blijlevens NM, Billaud EM, Kosterink JG, Verweij PE, Burger DM. Clinical relevance of the pharmacokinetic interactions of azole antifungal drugs with other coadministered agents. Clin Infect Dis. 2009 May 15;48(10):1441-58.
  • 31. Zaragoza O, Mesa-Arango AC, Gómez-López A, Bernal-Martínez L, Rodríguez-Tudela JL, Cuenca-Estrella M. Process analysis of variables for standardization of antifungal susceptibility testing of nonfermentative yeasts. Antimicrob Agents Chemother. 2011 Apr;55(4):1563-70.
  • 32. Rodero L, Córdoba S, Cahn P, Soria M, Lucarini M, Davel G, Kaufman S, Canteros C, Guelfand L. Timed-kill curves for Cryptococcus neoformans isolated from patients with AIDS. Med Mycol. 2000 Jun;38(3):201-7.
  • 33. Cruz KS, Lima ES, da Silva MJA, de Souza ES, Montoia A, Pohlit AM, de Souza JVB. Screening and Antifungal Activity of a β-Carboline Derivative against Cryptococcus neoformans and C. gattii Int J Microbiol. 2019 Jan 22;2019:7157845.
  • 34. Lima LA, Johann S, Cisalpino PS, Pimenta LP, Boaventura MA. In vitro antifungal activity of fatty acid methyl esters of the seeds of Annona cornifolia A.St.-Hil. (Annonaceae) against pathogenic fungus Paracoccidioides brasiliensis Rev Soc Bras Med Trop. 2011 Nov-Dec;44(6):777-80.
  • 35. Cardoso NN, Alviano CS, Blank AF, Romanos MT, Fonseca BB, Rozental S, Rodrigues IA, Alviano DS. Synergism Effect of the Essential Oil from Ocimum basilicum var. Maria Bonita and Its Major Components with Fluconazole and Its Influence on Ergosterol Biosynthesis. Evid Based Complement Alternat Med. 2016;2016:5647182.
  • 36. Gullo FP, Sardi JC, Santos VA, Sangalli-Leite F, Pitangui NS, Rossi SA, de Paula E Silva AC, Soares LA, Silva JF, Oliveira HC, Furlan M, Silva DH, Bolzani VS, Mendes-Giannini MJ, Fusco-Almeida AM. Antifungal activity of maytenin and pristimerin. Evid Based Complement Alternat Med. 2012;2012:340787.
  • 37. Silva TC, Zara ALSA, Sá FADS, Bara MTF, Ávila RI, Costa CR, Valadares MC, Santos ASD, Freitas VAQ, Silva MDRR. Antifungal potential of punicalagin against Cryptococcus neoformans species complex. Rev Inst Med Trop Sao Paulo. 2018 Oct 22;60:e60.
  • 38. Woo SH, Reynolds MC, Sun NJ, Cassady JM, Snapka RM. Inhibition of topoisomerase II by liriodenine. Biochem Pharmacol. 1997 Aug 15;54(4):467-73.
  • 39. Moore GS, Atkins RD. The fungicidal and fungistatic effects of an aqueous garlic extract on medically important yeast-like fungi. Mycologia. 1977 Mar-Apr;69(2):341-8.
  • 40. Fromtling RA, Bulmer GS. In vitro effect of aqueous extract of garlic (Allium sativum) on the growth and viability of Cryptococcus neoformans Mycologia. 1978 Mar-Apr;70(2):397-405.
  • 41. Rosas AL, Casadevall A. Melanization affects susceptibility of Cryptococcus neoformans to heat and cold. FEMS Microbiol Lett. 1997 Aug 15;153(2):265-72.
  • 42. Nosanchuk JD, Casadevall A. Impact of melanin on microbial virulence and clinical resistance to antimicrobial compounds. Antimicrob Agents Chemother. 2006 Nov;50(11):3519-28.
  • 43. Wang Y, Casadevall A. Growth of Cryptococcus neoformans in presence of L-dopa decreases its susceptibility to amphotericin B. Antimicrob Agents Chemother. 1994 Nov;38(11):2648-50.
  • 44. Van Duin D, Casadevall A, Nosanchuk JD. Melanization of Cryptococcus neoformans and Histoplasma capsulatum reduces their susceptibilities to amphotericin B and caspofungin. Antimicrob Agents Chemother. 2002 Nov;46(11):3394-400.
  • 45. Mednick AJ, Nosanchuk JD, Casadevall A. Melanization of Cryptococcus neoformans affects lung inflammatory responses during cryptococcal infection. Infect Immun. 2005 Apr;73(4):2012-9.
  • 46. Liu L, Tewari RP, Williamson PR. Laccase protects Cryptococcus neoformans from antifungal activity of alveolar macrophages. Infect Immun. 1999 Nov;67(11):6034-9.
  • 47. Jin CM, Lee JJ, Yang YJ, Kim YM, Kim YK, Ryu SY, Lee MK. Liriodenine inhibits dopamine biosynthesis and L-DOPA-induced dopamine content in PC12 cells. Arch Pharm Res. 2007 Aug;30(8):984-90.
  • 48. Tripathi SK, Xu T, Feng Q, Avula B, Shi X, Pan X, Mask MM, Baerson SR, Jacob MR, Ravu RR, Khan SI, Li XC, Khan IA, Clark AM, Agarwal AK. Two plant-derived aporphinoid alkaloids exert their antifungal activity by disrupting mitochondrial iron-sulfur cluster biosynthesis. J Biol Chem. 2017 Oct 6;292(40):16578-93.
  • 49. Nosanchuk JD, Cleare W, Franzot SP, Casadevall A. Amphotericin B and fluconazole affect cellular charge, macrophage phagocytosis, and cellular morphology of Cryptococcus neoformans at subinhibitory concentrations. Antimicrob Agents Chemother. 1999 Feb;43(2):233-9.
  • 50. Borges M. “Changes in fungal ultrastructure after itraconazole treatment.” In: R. A. Fromtling, editorRecent trends in discovery, development and evaluation of antifungal agents. (Barcelona: J.R. Prous Sience Publishers). p. 23-36. 1987.
  • 51. Guerra CR, Ishida K, Nucci M, Rozental S. Terbinafine inhibits Cryptococcus neoformans growth and modulates fungal morphology. Mem Inst Oswaldo Cruz. 2012 Aug;107(5):582-90.

  • Availability of data and materials

    All date generated or analyzed during the study are included in this article.
  • Funding

    ADLV was the recipient of a doctoral scholarship from the Coordination for the Improvement of Higher Education Personnel (CAPES).
  • Ethics approval

    The study was submitted to and approved by the Research Ethics Committee (protocol number 2.257.787) as well as by the Ethics Committee on Animal Research (protocol 127/2017), both from Botucatu Medical School (FMB) of São Paulo State University (UNESP).
  • Consent for publication

    Not applicable.

Publication Dates

  • Publication in this collection
    05 Sept 2022
  • Date of issue
    2022

History

  • Received
    08 Feb 2022
  • Accepted
    27 July 2022
Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP/UNESP) Av. Universitária, 3780, Fazenda Lageado, Botucatu, SP, CEP 18610-034, Brasil, Tel.: +55 14 3880-7693 - Botucatu - SP - Brazil
E-mail: editorial.jvatitd@unesp.br