Synergistic Anti-candidal Activity of Tetrandrine on Ketoconazole: an Experimental Study

Abstract

With the widespread use of azoles, drug resistant Candida albicans strains are increasing. The study examined the synergism of tetrandrine (TET) on ketoconazole (KCZ) candidacidal activity. The protocol M27-A2 of the Clinical and Laboratory Standards Institute (CLSI) was adopted and the minimum inhibitory concentrations (MICs) were determined for KCZ alone and in combination with a TET level that was noncytotoxic for C. albicans strains CA-1 through CA-17, with no CA-10. Colony counting techniques were used to construct time-kill curves. CA-15 was used to build the mouse candidal vaginitis model. After randomization, drugs were administered vaginally once daily from days 3–10 (both KCZ and TET were 26 mg/kg/day and 13 mg/kg/day, respectively, administered in different combinations). Mouse vaginal lavage fluid was obtained at days 2, 6, and 11 after inoculation for fungal load analysis, and vaginal tissue was obtained for pathological examination. MICs of KCZ alone and combined with 30 µg/mL TET for the C. albicans strains were 1–32 µg/mL and 0.0038–0.2500 µg/mL, respectively (t = 24.624, p = 0.000). Time-kill curves showed that at 48 h the viable cell counts of strains treated with KCZ + TET were at least 2 log₁₀ CFU/mL lower compared to strains treated with corresponding doses of KCZ (p = 0.000). At day 6, the fungal load in the KCZ 26 mg/kg/day + TET 26 mg/kg/day mice was significantly lower than the KCZ 26 mg/kg/day mice (1.17 ± 1.17 × 10⁴ CFU/mL and 9.33 ± 3.08 × 10⁴ CFU/mL, respectively, p = 0.000). Mucosal and submucosal fungal clearances were complete and vaginal mucosal edema was slight with minimal inflammatory cell infiltration. We conclude that noncytotoxic doses of TET synergistically enhance KCZ candidacidal activity in vitro and in vivo.

Key words

synergism - tetrandrine - ketoconazole - Candida albicans - antifungal susceptibility testing

References

• 1 Almirante B, Rodríguez D, Park B J, Cuenca-Estrella M, Planes A M, Almela M, Mensa J, Sanchez F, Ayats J, Gimenez M, Saballs P, Fridkin S K, Morgan J, Rodriguez-Tudela J L, Warnock D W, Pahissa A. Epidemiology and predictors of mortality in cases of Candida bloodstream infection: results from population-based surveillance, Barcelona, Spain, from 2002 to 2003.  J Clin Microbiol. 2005;  43 1829-1835
• 2 Chattopadhyay A, Gray L R, Patton L L, Caplan D J, Slade G D, Tien H C, Shugars D C. Salivary secretory leukocyte protease inhibitor and oral candidiasis in human immunodeficiency virus type 1-infected persons.  Infect Immun. 2004;  72 1956-1963
• 3 Beck-Sagué C M, Jarvis W R. The National Nosocomial Infection Surveillance System. Secular trends in the epidemiology of nosocomial fungal infections in the United States, 1980–1990.  J Infect Dis. 1993;  167 1247-1251
• 4 Rex J H, Bennett J E, Sugar A M, Pappas P G, van der Horst C M, Edwards Jr J E, Washburn R G, Scheld W M, Karchmer A W, Dine A P, Levenstein M J, Douglas Webb C. The Candidemia Study Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group. A randomized trial comparing fluconazole with amphotericin B for the treatment of candidemia in patients without neutropenia.  N Engl J Med. 1994;  331 1325-1330
• 5 Edwards Jr J E, Bodey G P, Bowden R A, Büchner T, de Pauw B E, Filler S G, Ghannoum M A, Glauser M, Herbrecht R, Kauffman C A, Kohno S, Martino P, Meunier F, Mori T, Pfaller M A, Rex J H, Rogers T R, Rubin R H, Solomkin J, Viscoli C, Walsh T J, White M. International conference for the development of a consensus on the management and prevention of severe candidal infections.  Clin Infect Dis. 1997;  25 43-59
• 6 Hazen K C, Baron E J, Lopes Colombo A, Girmenia C, Sanchez-Sousa A, del Palacio A, de Bedout C, Gibbs D L. Comparison of the susceptibilities of Candida spp. to fluconazole and voriconazole in a 4-year global evaluation using disk diffusion.  J Clin Microbiol. 2003;  41 5623-5632
• 7 Li F, Zhang H. In vitro study of the synergistic effect of tetrandrine and fluconazole against Candida albicans.  Chin J Dermatol. 2006;  39 454-456
• 8 Wang K, Zhang H, Jiang H, Shi J, Gao A, Ho H, Chao H. In vitro study on tetrandrine as a synergist to fluconazole in murine model of vaginal candidiasis.  Chin J Zoonoses. 2007;  23 474-478
• 9 White T C, Pfaller M A, Rinaldi M G, Smith J, Redding S W. Stable azole drug resistance associated with a substrain of Candida albicans from an HIV-infected patient.  Oral Dis. 1997;  3 S102-S109
• 10 National Committee for Clinical Laboratory Standards .Reference method for broth dilution antifungal susceptibility testing of yeasts: approved standard [S]. NCCLS document M27-A. Wayne, Pennsylvania; NCCLS 1997
• 11 Marchetti O, Moreillon P, Entenza J M, Vouillamoz J, Glauser M P, Bille J, Sanglard D. Fungicidal synergism of fluconazole and cyclosporine in Candida albicans is not dependent on multidrug efflux transporters encoded by the CDR1, CDR2, CaMDR1, and FLU1 genes.  Antimicrob Agents Chemother. 2003;  47 1565-1570
• 12 Mukherjee P K, Sheehan D J, Hitchcock C A, Ghannoum M A. Combination treatment of invasive fungal infections.  Clin Microbiol Rev. 2005;  18 163-194
• 13 Roling E E, Klepser M E, Wasson A, Lewis R E, Ernst E J, Pfaller M A. Antifungal activities of fluconazole, caspofungin (MK0991), and anidulafungin (LY 303366) alone and in combination against Candida spp. and Crytococcus neoformans via time-kill methods.  Diagn Microbiol Infect Dis. 2002;  43 13-17
• 14 Chen Z, Kong X. Study of Candida albicans vaginitis model in Kunming mice.  J Huazhong Univ Sci Technolog Med Sci. 2007;  27 307-310
• 15 Fidel Jr P L, Cutright J L, Tait L, Sobel J D. A murine model of Candida glabrata vaginitis.  J Infect Dis. 1996;  173 425-431
• 16 Lopez-Medrano R. Grupo de Microbiologos de la Sociedad Castellano-Leonesa de Microbilogia . Invasive aspergillosis in Castilla y Leon and Cantabria: years 1998 and 1999.  Rev Iberoam Micol. 2001;  18 70-75
• 17 Wroblewsla M M, Swoboda-Kopec E, Rokosz A, Krawczyk E, Marchel H, Luczak M. Epidermiology of clinical isolates of Candida albicans and their susceptibility to triazoles.  Int J Antimicrob Agents. 2002;  20 472-475
• 18 Cernicka J, Subik J. Resistance mechanisms in fluconazole-resistant Candida albicans isolates from vaginal candidiasis.  Int J Antimicrob Agents. 2006;  27 403-408
• 19 Canuto M M, Rodero F G. Antifungal drug resistance to azoles and polyenes.  Lancet Infect Dis. 2002;  2 550-563
• 20 Hossain M A, Reyes G H, Long L A, Mukherjee P K, Ghannoum M A. Efficacy of caspofungin combined with amphotericin B against azole-resistant Candida albicans.  J Antimicrob Chemother. 2003;  51 1427-1429
• 21 Gil-Lamaignere C, Muller F M C. Differential effects of the combination of caspofungin and terbinafine against Candida albicans, Candida dubliniensis and Candida kefyr.  Int J Antimicrob Agents. 2004;  23 520-523
• 22 Gao A, Zhang H, Mo H, Jiang H, Wang K, Chao H. Molecular mechanism of the synergy between tetrandrine and fluconazole against Candida albicans.  Chin J Dermatol. 2008;  41 97-100
• 23 Zhang H, Gao A, Li F, Zhang G, Ho H, Liao W. Mechanism of action of tetrandrine, a natural inhibitor of Candida albicans drug efflux pumps.  Yakugaku Zasshi. 2009;  129 623-630
• 24 Fu L, Liang Y, Deng L, Ding Y, Chen L, Ye Y, Yang X, Pan Q. Characterization of tetrandrine, a potent inhibitor of P-glycoprotein-mediated multidrug resistance.  Cancer Chemother Pharmacol. 2004;  53 349-356
• 25 Wang F P, Wang L, Yang J S, Nomura M, Miyamoto K I. Reversal of P-glycoprotein-dependent resistance to vinblastine by newly synthesized bisbenzylisoquinoline alkaloids in mouse leukemia P388 cells.  Biol Pharm Bull. 2005;  28 1979-1982
• 26 Wang G, Lemos J R. Tetrandrine: a new ligand to block voltage-dependent Ca2+ and Ca(+)-activated K+ channels.  Life Sci. 1995;  56 295-306
• 27 Liu L F, Chen N M, Cai G P, Li Z L, Yang J G, Li Y R. Studies on the effect of tetrandrine on microtubules. I. Biochemical observation and electron microscopy.  Ecotoxicol Environ Saf. 1988;  15 142-148
• 28 Li F, Lu B, Chen W. Evaluation of the safety of tetrandrine loaded sustained release microcapsules for lung targeting.  Chin J Mod Appl Pharm. 2002;  19 478-480