High-Dose Ascorbate with Low-Dose Amphotericin B Attenuates Severity of Disease in a Model of the Reappearance of Candidemia During Sepsis in the Mouse

Am J Physiol Regul Integr Comp Physiol 309: R223–R234, 2015. First published May 20, 2015; doi:10.1152/ajpregu.00238.2014. High-dose ascorbate with low-dose amphotericin B attenuates severity of disease in a model of the reappearance of candidemia during sepsis in the mouse Asada Leelahavanichkul,1,2 Poorichaya Somparn,3 Tanabodee Bootprapan,1 Hongbin Tu,4 Pattarin Tangtanatakul,1 Ratchanok Nuengjumnong,2 Navaporn Worasilchai,1 Khajohn Tiranathanagul,5 Somchai Eiam-ong, Mark Levine,4 Ariya Chinampon,1 and Nattachai Srisawat5 1Department of Microbiology, Chulalongkorn University, Bangkok, Thailand; 2Center of Excellence in Immunology and Immune-Mediated Diseases, Department of Microbiology, Faculty of Medicine and STAR on Craniofacial and Skeletal Disorders, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand; 3Research Affairs, Department of Medicine, Chulalongkorn University, Bangkok, Thailand; 4National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland; and 5Nephrology Unit, Department of Medicine, Chulalongkorn University, Bangkok, Thailand Submitted 16 June 2014; accepted in final form 5 May 2015 Leelahavanichkul A, Somparn P, Bootprapan T, Tu H, Tang- immune response in sepsis may present as a hyperimmune tanatakul P, Nuengjumnong R, Worasilchai N, Tiranathanagul characteristic with a rapid natural course of disease and/or an K, Eiam-Ong S, Levine M, Chinampon A, Srisawat N. immunosuppression phase responsible for nosocomial infec- High-dose ascorbate with low-dose amphotericin B attenuates tion (55, 62). Candidiasis, especially resulting from Candida severity of disease in a model of the reappearance of candidemia albicans, is one of the leading worldwide nosocomial infec- during sepsis in the mouse. Am J Physiol Regul Integr Comp Physiol 309: R223–R234, 2015. First published May 20, 2015; tions occurring after the onset of sepsis (30, 49). The mortality doi:10.1152/ajpregu.00238.2014.—Amphotericin B (Ampho B) is rate of patients with postsepsis systemic candidiasis is more a fungicidal drug that causes cell wall injury. Pharmacological than 50%, with more than 25% of cases of candidiasis occur- ascorbate induces the extracellular prooxidants, which might enter ring in patients who had developed sepsis (17, 22). Although the Ampho B-induced cell wall porosity and act synergistically. the incidences of infection from other fungal species are We tested low-dose Ampho B with a short course of pharmaco- increasing in patients in the intensive care unit, C. albicans still logical ascorbate using a mouse model of sepsis preconditioned remains the leading cause of nosocomial fungal infection (22, with an injection of Candida albicans 6 h prior to cecal ligation 48, 51). The increasing prevalence of nosocomial fungal in- and puncture (CLP). In this model, candidemia reappeared as early fection in cases of sepsis may be due to the growing use of as 6 h after CLP with a predictably high mortality rate. This broad-spectrum antibiotics coupled with the advancement of characteristic mimics sepsis in the phase of immunosuppression in patients. Using the model, at 12- and 18-h post-CLP, we admin- intensive care supports (8, 29, 37, 43, 59). istered isotonic (pH neutralized) pharmacological ascorbate intra- Amphotericin B (Ampho B) is a traditional broad-spectrum, venously with low-dose Ampho B or sodium deoxycholate, vehi- dose-dependent fungicidal drug, which is a pore-forming agent cle-controlled, administered IP. The survival rate of low-dose that acts though ergosterol inhibition. Ampho B, unfortunately, Ampho B plus ascorbate was 53%, compared with Ͻ11% for has substantial dose-dependent nephrotoxicity, and liposomal low-dose Ampho B or high-dose Ampho B alone. In addition, a amphotericin B is expensive (11). If Ampho B can be used at beneficial effect was demonstrated in terms of kidney damage, a lower dose in combination with another agent, perhaps liver injury, spleen histopathology, and serum markers at 24 h after nephrotoxicity and cost will be reduced. When given intrave- CLP. Kidney injury was less severe in low-dose Ampho B plus nously as a pharmacological agent, ascorbate (ascorbic acid or ascorbate combination therapy due to less severe sepsis. Moreover, vitamin C) demonstrates antineoplastic actions via the gener- ascorbate enhanced the effectiveness of phagocytosis against C. albicans in human phagocytic cells. Taken together, the data ation of extracellular hydrogen peroxide and downstream re- indicate that the new mouse model simulates sepsis-induced im- active oxygen species (ROS) (5–7, 37, 38). ROS formed in the munosuppression and that the combination of pharmacological presence of pharmacological ascorbate are less toxic to the host ascorbate with an antifungal drug is a potentially effective treat- tissue, but are very toxic to cancer cells in vitro and in vivo. We ment that may reduce nephrotoxicity, and perhaps also increase hypothesized that extracellular hydrogen peroxide generated fungicidal activity in patients with systemic candidiasis caused by by pharmacological ascorbate could enter the fungal cell wall, Candida albicans. which is injured or made vulnerable by the fungicidal pore- Candida albicans; ascorbate; sepsis; amphotericin B forming agent and enhanced antifungal therapeutic effectiveness (4–7). It should be noted that lower doses of ascorbate, given as a drug, may attenuate bacterial sepsis via additional SEPSIS IS A SERIOUS, GENERALIZED immune response to systemic mechanisms (1, 58, 63–65, 67). If ascorbate demonstrates infection, independent of the infecting organisms (42). The actions that support antibacterial and antifungal activities, then ascorbate in combination with Ampho B may be an effective combined therapy. Address for reprint requests and other correspondence: A. Leelahavanich- kul, Immunology Unit, Dept. of Microbiology, Chulalongkorn Univ., Bangkok To study ascorbate in combination with antifungal agents, an 10330, Thailand. animal model system is necessary. The available model for http://www.ajpregu.org R223 Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (079.016.061.055) on May 28, 2018. Copyright © 2015 American Physiological Society. All rights reserved. R224 HIGH-DOSE ASCORBATE Table 1. The minimal inhibitory concentration that BioTek, Shoreline, WA) by the optical density at 630 nm at 0.45 (OD demonstrated antifungal resistance to the Candida albicans 630 nm at 0.45). The procedure for LD50 was described in detail in the used in this study animal section below. For the germ tube test, 300 blastospores of C. albicans, as determined by hemocytometer, were placed in 50 ␮lof Drug MIC, ng/ml serum from a septic mouse (24 h after CLP) or serum from a sham mouse, incubated at 37°C for 3 h, then counted by hemocytometer. Amphotericin B 125 Fluconazole 125 The percentage of the ratio of blastospores with germ tube formation Itraconazole 31.3 to total blastospores was calculated. Ketonazole 31.3 Posaconazole 31.3 Animal and Animal Models Voriconazole 31.3 Anidulafungin 15 The U.S. National Institutes of Health (NIH) criteria and protocols Caspofungin 15 for the use and treatment of laboratory animals were followed (NIH MIC, minimal inhibitory concentration. publication protocols no. 85-23, revised 1985). Male, 8–10-wk old BalB/C mice (National Laboratory Animal Center, Nakornpathom, Thailand) were used. The animal protocols were approved by the Candida infection, characterized by Candida injection after Institutional Animal Care and Use Committee of the Faculty of sepsis (9), may not reflect Candida infections in humans, Medicine, Chulalongkorn University, Bangkok, Thailand. All proce- which mostly resulted from endogenous sources (50). As a dures were performed under isoflurane anesthesia. result, we developed a new sepsis-induced candidemia reap- Candida albicans injection model. Resuspended C. albicans in normal saline solution (NSS) adjusted into different doses of optical pearance model that is characterized by the reactivation of an density at 630 nm in the same volume of each preparation (100 ␮l) exogenous Candida administration prior to the onset of sepsis. was injected through the tail vein. The LD50 was extrapolated from We used this new system to test Ampho B administered alone the correlation equation of the graph of the 7-day mortality of mice, and in combination with pharmacological ascorbate. which was injected with C. albicans in different OD 630-nm doses (0.8, 0.6, 0.4, 0.2, and 0.1). The Candida dose with 50% mortality rate METHODS was calculated and identified as LD50. C. albicans Preparation, Characterization, and Germ Tube Test Cecal ligation and puncture sepsis model. The cecal ligation and puncture (CLP) procedure was described in detail previously (34), C. albicans was isolated from blood samples (Mycology Unit, King with some modifications. Briefly, the cecum was ligated at 12 mm Chulalongkorn Memorial Hospital), identified by morphology to- from the cecal tip and punctured twice with a 23-gauge needle, gether with the API 20C AUX yeast identification kit (Biomuriex, leaving a small amount of fecal material exposed. The abdominal Lyon, France), and stored in Sabouraud dextrose agar (SDA) (Thermo incision was closed in two layers with 6-0 nylon sutures. Antibiotic Scientific, Hampshire, UK) at Ϫ80°C. Before conducting all of the (imipenem/cilastatin 7 mg/kg in 0.3 ml NSS) was administered experiments, C. albicans was subcultured

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