Karl V. Clemons

Itraconazole, a Commonly Used Antifungal that Inhibits Hedgehog Pathway Activity and Cancer Growth

In a screen of drugs previously tested in humans we identified itraconazole, a systemic antifungal, as a potent antagonist of the Hedgehog (Hh) signaling pathway that acts by a mechanism distinct from its inhibitory effect on fungal sterol biosynthesis. Systemically administered itraconazole, like other Hh pathway antagonists, can suppress Hh pathway activity and the growth of medulloblastoma in a mouse allograft model and does so at serum levels comparable to those in patients undergoing antifungal therapy. Mechanistically, itraconazole appears to act on the essential Hh pathway component Smoothened (SMO) by a mechanism distinct from that of cyclopamine and other known SMO antagonists, and prevents the ciliary accumulation of SMO normally caused by Hh stimulation.

Candida parapsilosis: a review of its epidemiology, pathogenesis, clinical aspects, typing and antimicrobial susceptibility

The Candida parapsilosis family has emerged as a major opportunistic and nosocomial pathogen. It causes multifaceted pathology in immuno-compromised and normal hosts, notably low birth weight neonates. Its emergence may relate to an ability to colonize the skin, proliferate in glucose-containing solutions, and adhere to plastic. When clusters appear, determination of genetic relatedness among strains and identification of a common source are important. Its virulence appears associated with a capacity to produce biofilm and production of phospholipase and aspartyl protease. Further investigations of the host-pathogen interactions are needed. This review summarizes basic science, clinical and experimental information about C. parapsilosis.

Comparative efficacies of conventional amphotericin b, liposomal amphotericin B (AmBisome), caspofungin, micafungin, and voriconazole alone and in combination against experimental murine central nervous system aspergillosis.

ABSTRACT Central nervous system (CNS) aspergillosis is a severe disease that responds poorly to current therapies. The current studies examined the efficacies of several antifungal agents alone or in combination with a murine model of CNS aspergillosis. Immunosuppressed mice were infected intracerebrally with Aspergillus fumigatus and treated with an amphotericin B preparation, an echinocandin, or voriconazole (VCZ) given alone or in combination. Monotherapy studies showed that micafungin (MICA), caspofungin (CAS), VCZ, conventional amphotericin B (AMB), Abelcet (ABLC) (a lipid-carried AMB formulation; Enzon Pharmaceuticals, Inc.), and AmBisome (AmBi) (liposomal AMB; Gilead Sciences, Inc.) were efficacious. However, doses of AmBi above 15 mg/kg of body weight showed reduced efficacy. Neither MICA nor CAS showed dose responsiveness at the doses tested (1, 5, or 10 mg/kg). Only the 40-mg/kg dose of VCZ was effective. AmBi and ABLC showed dose responsiveness, with 10-mg/kg doses causing a significant reduction in fungal burden; they had equivalent activities at the 10-mg/kg dose. Suboptimal dosages of AmBi in combination with MICA, CAS, or VCZ were effective in prolonging survival. However, significantly enhanced activity was demonstrated only with AmBi and VCZ in combination. AmBi in combination with MICA or CAS showed a trend toward enhanced activity, but the combination was not significantly superior to monotherapy. The use of AmBi with CAS or VCZ at optimal doses did not improve efficacy. Cure was not attained with any dosage combinations. These results indicate that AmBi in combination with VCZ may be superior for treatment of CNS aspergillosis; combinations of AmBi and MICA or CAS were not antagonistic and may have a slight benefit.

Cytokine treatment of central nervous system infection: efficacy of interleukin-12 alone and synergy with conventional antifungal therapy in experimental cryptococcosis.

Cell-mediated immune responses appear to be critical in the outcome of cryptococcosis. Interleukin-12 (IL-12) was studied for its potential use as a therapeutic agent because of its stimulation of natural killer cells and gamma interferon production by stimulated T cells and natural killer cells. Gamma interferon-activated macrophages are important in host resistance against cryptococcosis. In two separate studies, male BALB/c mice were infected intravenously with Cryptococcus neoformans. In the first study, mice received either no treatment, 5.0 mg of fluconazole alone per kg of body weight per day (by gavage twice daily), or IL-12 subcutaneously at 0.01, 0.1, or 1.0 microgram/day once daily (low-dose study) alone or in combination with 5.0 mg of fluconazole per kg/day. In a second study (high dose), the dosages of IL-12 used were 1.0, 2.5, or 5.0 micrograms/day. Therapy was given for 10 consecutive days, and the number of CFU of C. neoformans remaining in various organs was quantitated 1 or 2 days after administration of the last dose. In the low-dose study, IL-12 at 0.1 or 1.0 microgram reduced the level of brain infection by approximately 10-fold (P < 0.05) and IL-12 at 1.0 or 0.1 microgram/day enhanced the efficacy of fluconazole. In liver, both the efficacy of IL-12 alone (0.01 or 0.1 microgram; P < 0.05) and enhancement of the efficacy of fluconazole (P < 0.05) were seen. No efficacy of IL-12 was seen in spleens or lungs, although spleen weights increased fourfold in mice given 1.0 microgram of IL-12 per day. In the high-dose study, all IL-12 doses alone again reduced the levels of brain infection (5- to 8-fold; P < 0.05) when the two were given in combination. No overt toxicities were observed at any dose, and overall, 1.0 microgram of IL-12 per day was found to be the optimal dosage for reducing infection in the brain. To our knowledge, this is the first demonstration of the efficacy of cytokine therapy in systemic and particularly brain infections with C. neoformans. The stimulation of cell-mediated immunity represents a new approach to therapy and can enhance suboptimal antimicrobial chemotherapy. IL-12 should be considered for further study and for clinical trials. These studies suggest that other opportunistic central nervous system pathogens should also be investigated. Images

Candida parapsilosis fungemia in neonates: genotyping results suggest healthcare workers hands as source,and review of published studies

An outbreak of Candida parapsilosis fungemia involving 17 neonatal intensive care unit (NICU) patients was studied. There were 14 blood culture and nine colonizing isolates from other sites available. The hands of NICU healthcare workers (HCW) yielded eight isolates. Screening of the isolates by random amplified polymorphic DNA (RAPD) method showed only three profiles. Typing by restriction fragment length polymorphism (RFLP) revealed all blood isolates were RFLP subtype VII-1. Among the nine infant colonizing isolates, there were four different RFLP subtypes; four of the isolates were subtype VII-1. Seven of the eight isolates from HCW were RFLP subtype VII-1. The majority of infant colonizers were not found in the blood, suggesting a possible direct spread of the epidemic subtype VII-1 strain from HCW hands to infant blood. The source of the infant colonizing strains is unclear, but non-VII-1 strains may be largely of maternal origin and VII-1 strains from HCW. These findings reinforce prior studies that have implicated HCW hands as the source of nosocomial, including neonatal, fungemia.

Assessment of the Paradoxical Effect of Caspofungin in Therapy of Candidiasis

ABSTRACT Paradoxical growth of some Candida albicans isolates in the presence of caspofungin (CAS) in vitro has been demonstrated previously. We sought to determine whether a similar phenomenon occurred in vivo. A systemic model of candidiasis was studied in CD-1 mice by intravenous inoculation of different isolates of C. albicans. Infected animals were treated with CAS at various dosages (0.01 to 20 mg/kg) and CFU remaining in the kidneys determined. Four clinical isolates that showed paradoxical growth in vitro and one that did not were tested. Recovery of CFU from the kidneys showed that dosages of CAS at 0.1 mg/kg and above were efficacious in the reduction of C. albicans, but were not curative. Against isolates that show paradoxical growth in vitro, CAS was efficacious, but lacked dose responsiveness above 0.5 mg/kg against three of the four. One isolate, 95-68, showed paradoxical growth in vivo with significantly higher CFU recovered from mice given CAS at 20 mg/kg than those given CAS at 5 mg/kg, but the effect was not reproducible in a subsequent experiment. When CAS was given prophylactically and therapeutically, improved efficacy and cure rate were observed. Overall, these data indicate that CAS is highly efficacious against systemic murine candidiasis and a paradoxical effect was not reproducibly demonstrated in vivo.

Efficacy of micafungin alone or in combination against systemic murine aspergillosis.

ABSTRACT We tested the efficacy of micafungin (FK) alone or in combination with other antifungals against systemic murine aspergillosis. FK alone at 10 mg/kg of body weight/dose prolonged survival (P = 0.01) and reduced CFU in the brain and kidney. Combination therapy that used suboptimal FK with amphotericin B or itraconazole prolonged survival. Although no survivors were free of infection, no antagonism was seen. Nikkomycin Z with FK showed significantly greater potency (P < 0.01) than either alone.

Role of IL-10 in invasive aspergillosis : increased resistance of IL-10 gene knockout mice to lethal systemic aspergillosis

IL‐10 is associated with a Th2 response, down‐regulation of a Th1 response and macrophage activation. We assessed the role of IL‐10 during systemic infection with Aspergillus fumigatus. Systemic aspergillosis was established in female C56Bl/6 IL‐10−/– (KO) and wild‐type (WT) C57Bl/6 mice by i.v. administration of 1 × 105−6 × 105 conidia of A. fumigatus. In two experiments, KO survived longer than did WT (P < 0·001). Determination of fungal burdens in the kidneys and brain showed that KO carried significantly lower burdens in both organs than did WT on day 3 (P < 0·001). Semiquantitative histological analyses showed fewer inflammatory foci/mm2 in brain and kidneys of KO than WT (P < 0·03 and < 0·001, respectively) and that extent of infection and associated tissue injury were greater in WT. Although beneficial in some bacterial infections, exogenous IL‐10 has been shown deleterious in models of fungal infection. Our data indicate IL‐10 is deleterious during systemic aspergillosis infection, increasing the host susceptibility to lethal infection. We speculate this might be related to greater Th2 or lesser Th1 responses, or down‐regulation of macrophage responses, in WT compared with KO.

Discrepancies in bioassay and chromatography determinations explained by metabolism of itraconazole to hydroxyitraconazole: studies of interpatient variations in concentrations.

Pharmacologic studies of itraconazole (IZ), a triazole antifungal, indicated unexplained differences between bioassay and chromatographic determinations and large variations in steady-state blood concentrations. We show that concentrations of a hydroxylated metabolite, hydroxyitraconazole (HIZ), are approximately twofold higher than IZ over a range of concentrations. Though HIZ and IZ appear equipotent against selected pathogens, HIZ is two to three times more active against a commonly used bioassay fungus but minimally affects IZ activity. Hence, HIZ probably contributes importantly to the therapeutic activity attributed to IZ and contributes approximately four to six times the activity of IZ in bioassays, explaining discrepancies observed between assay methods.

The contribution of animal models of aspergillosis to understanding pathogenesis, therapy and virulence

Animal models of aspergillosis have been used extensively to study various aspects of pathogenesis, innate and acquired host-response, disease transmission and therapy. Several different animal models of aspergillosis have been developed. Because aspergillosis is an important pulmonary disease in birds, avian models have been used successfully to study preventative vaccines. Studies done to emulate human disease have relied on models using common laboratory animal species. Guinea pig models have primarily been used in therapy studies of invasive pulmonary aspergillosis (IPA). Rabbits have been used to study IPA and systemic disease, as well as fungal keratitis. Rodent, particularly mouse, models of aspergillosis predominate as the choice for most investigators. The availability of genetically defined strains of mice, immunological reagents, cost and ease of handling are factors. Both normal and immunosuppressed animals are used routinely. These models have been used to determine efficacy of experimental therapeutics, comparative virulence of different isolates of Aspergillus, genes involved in virulence, and susceptibility to infection with Aspergillus. Mice with genetic immunological deficiency and cytokine gene-specific knockout mice facilitate studies of the roles cells, and cytokines and chemokines, play in host-resistance to Aspergillus. Overall, these models have been critical to the advancement of therapy, and our current understanding of pathogenesis and host-resistance.