Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by recurrent life-threatening bacterial and fungal infections. We characterized the effects of single and combination antifungal therapy on survival, histopathology, and laboratory markers of fungal burden in experimental aspergillosis in the p47 phox؊/؊ knockout mouse model of CGD. CGD mice were highly susceptible to intratracheal Aspergillus fumigatus challenge, whereas wild-type mice were resistant. CGD mice were challenged intratracheally with a lethal inoculum ( Invasive aspergillosis is a major cause of morbidity and mortality in highly immunocompromised persons. Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase complex in which phagocytes are defective in generating the reactive oxidant superoxide anion and its metabolites, hydrogen peroxide, hydroxyl anion, and hypohalous acid. Activation of preformed granular proteases is likely to be principally responsible for NADPH oxidase-mediated destruction of pathogens (25, 31). As a result of the defect in this key host defense pathway, CGD patients suffer from recurrent life-threatening bacterial and fungal infections. CGD patients are susceptible to a broad spectrum of opportunistic filamentous fungi, but Aspergillus infection is by far the most common. Invasive aspergillosis is the most important cause of mortality in CGD (10,(26)(27)(28). Genetically engineered CGD mice (Xlinked and p47 phoxϪ/Ϫ ) recapitulate the human disease and are highly susceptible to Aspergillus infection (5, 9, 24). p47phoxϪ/Ϫ mice were used in experimental pulmonary aspergillosis to evaluate single and combination antifungal regimens on the basis of survival, histopathology, and fungal burden in lungs. The following four treatment groups were evaluated: (i) vehicle, (ii) amphotericin B, (iii) micafungin, and (iv) amphotericin B plus micafungin. The combination of amphotericin B and micafungin was more effective than either agent alone in prolonging survival after Aspergillus fumigatus challenge. Our study also demonstrated unique features of the CGD mouse model that are distinct from other immunocompromised animal models of experimental aspergillosis.(Material in this paper was presented in abstract form at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., October 2004 [abstr. M-232].) MATERIALS AND METHODS Mice. Mice with a targeted disruption of the p47phox gene have a defective NADPH oxidase, rendering phagocytes incapable of generating measurable superoxide (12). CGD mice were derived from C57BL/6 and 129 intercrosses, and mice used were backcrossed either 5 (N5) or 14 (N14) generations in the C57BL/6 background. In all experiments, treatment groups were matched with respect to age (16 to 24 weeks) and generation of backcrossing. Mice were bred

A flexible approach to response surface modeling for the study of the joint action of three active anticancer agents is used to model a complex pattern of synergism, additivity and antagonism in an in vitro cell growth assay. The method for determining a useful nonlinear response surface model depends upon a series of steps using appropriate scaling of drug concentrations and effects, raw data modeling, and hierarchical parameter modeling. The method is applied to a very large in vitro study of the combined effect of Trimetrexate (TMQ), LY309887 (LY), and Tomudex (TDX) on inhibition of cancer cell growth. The base model employed for modeling dose-response effect is the four parameter Hill equation [1]. In the hierarchical aspect of the final model, the base Hill model is treated as a function of the total amount of the three drug mixture and the Hill parameters, background B, dose for 50% effect D50, and slope m, are understood as functions of the three drug fractions. The parameters are modeled using the canonical mixture polynomials from the mixture experiment methodologies introduced by Scheff [2]. We label the model generated a Nonlinear Mixture Amount model with control observations, or zero amounts, an "NLMAZ" model. This modeling paradigm provides for the first time an effective statistical approach to modeling complex patterns of local synergism, additivity, and antagonism in the same data set, the possibility of including additional experimental components beyond those in the mixture, and the capability of modeling three or more drugs.

Response surface methods for the study of multiple-agent interaction allow one to model all of the information present in full concentration-effect data sets and to visualize and quantify local regions of synergy, additivity, and antagonism. In randomized wells of 96-well plates, Aspergillus fumigatus was exposed to various combinations of amphotericin B, micafungin, and nikkomycin Z. The experimental design was comprised of 91 different fixed-ratio mixtures, all performed in quintuplicate. After 24 h of drug exposure, drug effect on fungal viability was assessed using the tetrazolium salt 2,3-bis {2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium-hydroxide} (XTT) assay. First, we modeled each fixed-ratio combination alone using the four-parameter Hill concentration-effect model. Then, we modeled each parameter, including the 50% inhibitory concentration (IC 50 ) effect, versus the proportion of each agent using constrained polynomials. Finally, we modeled the three-agent response surface overall. The overall four-dimensional response surface was complex, but it can be explained in detail both analytically and graphically. The grand model that fit the best included complex polynomial equations for the slope parameter m and the combination index (equivalent to the IC 50 for a fixed-ratio concentration, but with concentrations normalized by the respective IC 50 s of the drugs alone). There was a large region of synergy, mostly at the nikkomycin Z/micafungin edge of the ternary plots for equal normalized proportions of each drug and extending into the center of the plots. Applying this response surface method to a huge data set for a three-antifungal-agent combination is novel. This new paradigm has the potential to significantly advance the field of combination antifungal pharmacology.Invasive aspergillosis is a major cause of morbidity and mortality in highly immunocompromised persons (15). Risk factors for invasive aspergillosis include prolonged neutropenia, hematopoietic stem cell transplantation, solid organ transplantation, advanced AIDS, and chronic granulomatous disease (22). The expansion of the number of antifungal drugs with different modes of action that are used in the clinic and that are at preclinical stages of development has generated significant interest in combination antifungal therapy for invasive aspergillosis (4,11,13,14,25). The recent sequencing of the Aspergillus fumigatus genome will provide additional tools for the identification of novel targets for drug development (19). These important advances create a greater need to model effectively the combination of antifungal agents in vitro and in vivo.In the past, various approaches to the assessment of synergy, additivity, and antagonism have been developed. They include older graphical isobologram methods and newer statistical response surface methods. The older methods are mostly limited to two-agent interactions; however, several statistical response surface methods can be used for three-agent and higher order combinations. R...

Preclinical studies at Roswell Park Cancer Institute by Minderman, Cao, and Rustum (unpublished results) showed that a combination of SN-38 and 5-FU against HCT-8 human colon carcinoma cells in vitro was synergistic, with the best interaction occurring when the drugs were added sequentially, SN-38 first. Their in vivo studies using HCT-8 tumor xenografts implanted s.c. in nude athymic mice demonstrated superior efficacy for a sequential i.v. administration of CPT-11, 24 hr before 5-FU. On the basis of these studies, our group has begun to evaluate effects of RFS2000 (9-nitro-20(S)-camptothecin) (9-NC) in combination with a series of other antitumor agents. Using a panel of human tumor cell lines including A121 ovarian cancer, HCT-8 colon cancer, H-460 NSCLC, HT-1080 fibrosarcoma, and MCF7 mammary cancer, we found that a 2-hr exposure to 9-NC resulted in ID 50 values of <1.0 M, whereas continuous exposure to drug resulted in ID 50 values of <1.0 nM. Tumor growth inhibitory activities of 5-FU, gemcitabine, and paclitaxel were determined for comparison. Combinations of these agents were evaluated with 9-NC using the human HCT-8 colon tumor cell line. Concurrent and sequential combinations of 9-NC with 5-FU had some regions of the concentration-effect surface with local synergy and some with local antagonism. However, sequential combination of 9NC or SN-38 followed by 5-FU, 24 hr later appeared to be highly synergistic at high dose-effect levels (i.e., ID 90 ), suggesting that sequential drug administration may be more efficacious at high effect level and that the order of drug addition is very important. Overall, our results were similar to that found earlier by Rustum's group with CPT11 (or SN-38) and 5-FU, suggesting that sequential combination of 9-NC (or other camptothecin analogues) followed by 5-FU has potential for the treatment of cancer in man.