Astemizole and an Analogue Promote Fungicidal Activity of Fluconazole
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Medical Mycology March 2010, 48, 255–262 Astemizole and an analogue promote fungicidal activity of fl uconazole against Cryptococcus neoformans var. grubii and Cryptococcus gattii KIEM VU & ANGIE GELLI Department of Pharmacology, School of Medicine, University of California, Genome and Biomedical Sciences Facility, Davis, California, USA Downloaded from https://academic.oup.com/mmy/article/48/2/255/1013134 by guest on 27 September 2021 Cryptococcus neoformans is the leading cause of fungal meningitis, a life-threatening infection that occurs predominately in immuocompromised patients. Current drug therapies are limited to amphotericin B, fl ucytosine and the azoles since the echinocandins have no demonstrated activity against yeast like pathogens. Fluconazole, a drug belonging to the azole class and often the only available antifungal in the developing world, is fungistatic and therefore not effective in clearing cryptococcal infections in immunosuppressed individuals. Here we report that astemizole and a closely related analog (A2) promoted in vitro fungicidal activity of fl uconazole against Cryptococcus neoformans var. grubii and Cryptococcus gattii . Astemizole, a second-generation antihistamine drug used as an H1 antagonist, has also been found to have antimalarial activity. Disk diffusion assays and MIC and MFC analysis confi rmed that the inhibitory concentrations of these drug combinations were fungicidal. When tested in vivo , astemizole or A2 in combination with fl uconazole signifi cantly improved the survival of Galleria mellonella (wax moth caterpillar) that had been previously challenged with C. neoformans but not when caterpillars were challenged with a fl uconazole-resistant strain. The fi ndings reported here suggest that fungicidal combinations between azoles and other existing drugs may represent an alternative strategy for improving treatments for fungal infections. Keywords fl uconazole, astemizole, Cryptococcus neoformans, fungicidal, drug Introduction meningitis is a grave infection and even when treated with antifungals it causes high morbidity and mortality in The incidence of opportunistic invasive fungal infections immunocompromised patients and in seemingly normal in immunocompromised individuals continues to be a individuals [ 5 ]. Lifelong suppressive therapy is usually serious health issue [ 1 – 3 ]. Advancements in the treatment required in AIDS patients suffering from cryptococcosis of malignancies, autoimmune disease and organ transplan- because these infections are commonly not cleared in these tation have resulted in a cohort of patients who are highly individuals [ 6 ]. susceptible to life-threatening fungal infections. Candida Fluconazole is a commonly used drug from the azole species represent the fourth-most common cause of blood- class of antifungals [ 7 ,8 ]. It is often the drug of choice for stream infections [ 4 ] and Cryptococcus neoformans var. long-term maintenance therapy in AIDS patients because grubii and Cryptococcus gatti are the leading cause of fun- it can be administered orally, lacks signifi cant side effects gal infections of the nervous system [ 2 ,3 ,5 ]. Cryptoccocal and penetrates the central nervous system [ 6, 9 ]. Azoles tar- get lanosterol 14α-demethylase – an essential enzyme in Received 12 January 2009; Received in Final version form 28 April 2009; the biosynthesis of ergosterol [ 7 ,8 ]. A signifi cant downside Accepted 1 June 2009 of fl uconazole and other azoles is that they are fungistatic Correspondence: A. Gelli, Genome and Biomedical Sciences Facility, Room 3503, Department of Pharmacology, School of Medicine, University rather than fungicidal and as a result cryptococcal infec- of California, 451 Health Sciences Drive, Davis, CA 95616, USA. tions will persist in the absence of a functional immune Tel: ϩ1 530 754 6446; fax: ϩ1 530 754 7710; E-mail: [email protected] system. The use of fl uconazole for maintenance therapy © 2010 ISHAM DOI: 10.3109/13693780903081968 256 Vu & Gelli in immunocompromised patients has been associated with cascontent/registry/index.html ) and purchased from Ana- the development of fl uconazole-resistance in cryptococcal logix Inc (Burlington, WI). All drugs were dissolved in infections [ 9 ]. Given their low toxicity and good infi ltration dimethylsulfoxide to achieve the following concentrations; into the nervous system, azoles would be ideal drugs if they fl uconazole 4 mg/ml, astemizole 4 mg/ml, A1 10 mg/ml, were fungicidal instead. Interestingly, it has been shown A2 10 mg/ml and A3 10 mg/ml. that azoles become fungicidal against Candida spp. when combined with inhibitors of calcineurin [ 10 ,11 ]. Disk diffusion halo assays In this study we found that astemizole and a closely related analog (A2) promoted in vitro fungicidal activity of Strains were inoculated into liquid medium and grown fl uconazole against Cryptococcus neoformans var. grubii overnight at 30°C. Approximately 2 10 7 cells were inocu- and Cryptococcus gattii . Astemizole is a second-generation lated into 8 ml of top agar pre-warmed at 42°C and subse- Downloaded from https://academic.oup.com/mmy/article/48/2/255/1013134 by guest on 27 September 2021 antihistamine drug that is used as an H1 antagonist and it also quently spread onto YPD plates. All drugs and the solvent possesses antimalarial activity [ 12, 13 ]. The MFCs observed control were applied to 6-mm BBL disks (Becton Dickinson). were similar to the MIC analysis and the results of the These disks were placed on the solidifi ed top agar surface MFCs confi rmed that the inhibitory concentrations of these of the YPD plates as indicated and the strains were grown drug combinations were fungicidal against C. neoformans at 30°C for 48 h. All disk diffusion assays were performed var. grubii and C. gattii . Disk diffusion assays were con- as explained above. Microscope observation at 2.5 ϫ sistent with the MIC analysis. The combination of drugs magnifi cation was used to examine whether there was an also signifi cantly improved the survival of the wax moth enhancement of halo clearing that resulted from the com- caterpillar, Galleria mellonella, but not when caterpillars bination of drugs versus drug alone [ 10 ]. where challenged with a fl uconazole-resistant strain of C. neoformans var. grubii . This system was demonstrated to Antifungal drug activity testing by CLSI/NCCLS criteria function as a useful model to examine the in vivo effi cacy of antifungal agents in larvae of G. mellonella previously Checkerboard titrations were performed in order to assess challenged with C. neoformans var. grubii [ 14, 15 ]. Collec- drug interactions according to the Clinical and Laboratory tively, these observations support the notion that fungicidal Standards Institute (CLSI) (previously known as NCCLS – combinations between azoles and other existing drugs National Committee for Clinical Laboratory Standards may lead to improvements in treatment regimes for fungal [ 16 ]. In vitro testing was carried out in RPMI 1640 medium infections. containing L-glutamine, without sodium bicarbonate and buffered to pH 7.0 with MOPS in 96-well plates (96- well cell culture cluster, fl at-bottom, Costar). Inocula of Materials and methods C. neoformans (100 μl) were prepared in accordance with the CLSI standard (M27-A2) [ 16 ], added to the 96-well Strains, media and chemicals plates and incubated for 48 h at 35°C without shaking. The strains used in this study were as follows; Cryptococcus Readings were taken by measuring the optical density neoformans var. grubii (H99, ATCC 208821, Rockville (OD600 ) with a microplate reader. The MIC of drugs alone MD) , Cryptococcus gattii (NIH444), and strains T1-5796 or in combination was defi ned as the lowest drug con- and 89-6105797 (both of which represent fl uconazole- centration in a well at which 100% reduction in optical resistant C. neoformans var. grubii isolates). The latter density was observed compared to the no-drug control three strains were generously provided by J. Heitman well. Fractional inhibitory concentrations (FICs) and FIC (Duke University). All test isolates were grown on YPD indices ( FIC) were determined as previously described (Yeast extract, Peptone, Dextrose (2% vol/vol). The top [ 4 ]. The FIC index is defi ned as the sum of the FICs for agar used in the disk diffusion assays was 0.7% Bactor each of the drugs and the FICs are defi ned as the MIC Agar (Difco) in water. Fluconazole and astemizole were of each drug when used in combination divided by the purchased from Sigma-Aldrich (St. Louis, MO). The three MIC of the drug when used alone [ 4 ]. Drug interactions astemizole analogues, i.e., (A1, 4-Piperidinamine, N-[1- were based on ⌺ FIC indices and classifi ed as synergistic [(4-fluorophenyl)methyl]-1H-benzimidazol-2-yl]-1-[(4- ( FIC Ͻ 0.5), indifferent ( FIC ϭ 1 through 4), or methoxyphenyl)acetyl];, A2, 1H-Benzimidazole-2-amine, antagonistic (⌺ FIC Ͼ 4) [ 4 ]. Minimum fungicidal concen- 1-[2-(4-methoxyphenyl)ethyl]-N-[1-[2-(4-methoxyphenyl) trations (MFCs) were determined by plating 100 μl from ethyl]-4-piperidinyl] and A3, 1H-Benzimidazole-2-amine, each well with growth inhibition onto YPD plates that were N-[(4-methoxyphenyl)methyl]-1-[2-(1-piperidinyl)ethyl] then incubated at 30°C for 72 h. The lowest concentration were identifi ed through the Chemical Abstracts Service that yielded three or fewer colonies was recorded as the (CAS) registry website ( http://www.cas.org/expertise/ MFC [ 16 ]. © 2010 ISHAM, Medical Mycology, 48, 255–262 Fungicidal activity of fl uconazole 257 Galleria mellonella killing assays San Diego, CA). Estimation of differences in survival (log rank and Wilcoxon tests) was analyzed by the Kaplan- The G. mellonella killing assays were carried out as Meier method using GraphPad Prism software. A P value previously described [ 2,3 ]. Briefl y, G . mellonella wax of Ͻ0.05 was considered signifi cant. Each experiment was moth caterpillars (or larvae) (Vanderhorst, Inc., St. Marys, repeated three times and the data shown here are from a Ohio) in the fi nal instar stage were housed in the dark and representative experiment. used within 7 days from the day of shipment.