INTRODUCTION

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Among neutropenic patients with hematological malignancy, infections remain a major problem. Nowadays, the majority of bacterial infections can be treated or prevented successfully by using broad-spectrum antibiotics. Fungal infections, on the other hand, remain a common cause of morbidity and mortality in patients at risk of neutropenia (1). Moreover, during the past two decades, the incidence of fungal infections in this patient population has increased dramatically, the major pathogens being Candida spp. and Aspergillus spp.

Systemic fungal infections remain the major cause of death in neutropenic patients with hematological malignancy, and optimal management of these opportunistic infections is a major challenge for clinicians. Among the measures that can be taken to prevent fungal infections, environmental and hygienic control and elimination of well-known sources of infection are important.

Early studies of the prophylactic use of oral drugs like amphotericin B, nystatin, clotrimazole, miconazole, and ketoconazole yielded unsatisfactory results because of poor absorption, a narrow spectrum, poor compliance, and toxicity (4; E. J. Bow, M. Laverdiere, N. Lussier, C. Rotstein, and S. Ioannou, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. LM-88, p. 381, 1997). Neutropenic patients with persistent fever refractory to antibiotic treatment are often treated empirically with intravenous amphotericin B, despite its toxicity. Prophylactic treatment with fluconazole, an azole antifungal agent (15), is widely used nowadays. However, fluconazole, either oral or intravenous, offers no protection against Aspergillus spp., and several pathogenic Candida spp. are either resistant (C. krusei) or less sensitive (C. glabrata) to this compound (8, 17, 18). Intranasal administration of amphotericin B did not show a significant benefit over placebo as prophylaxis against aspergillosis, and although the liposomal formulations of amphotericin B seem to be safer, the clinical efficacy has not yet been proven, and few data are available with regard to prophylaxis (14). Moreover, prophylactic treatment with these liposomal formulations is expensive, thereby limiting its potential as a commonly used prophylactic agent (6). Itraconazole is a broad-spectrum triazole antifungal agent currently available as a capsule formulation for the treatment of systemic mycoses such as aspergillosis, candidosis, cryptococcosis, histoplasmosis, and several endemic mycoses; it has demonstrated clinical activity against Aspergillus infections (3). The newer oral solution formulation (10% itraconazole in a 40% hydroxypropyl--cyclodextrin [HDCD] solution) has overcome the limitations of the capsule formulation in that both adequate plasma drug concentrations are reached in the neutropenic host (12, 13) and it can be administered with or without food (2, 16). The prophylactic use of itraconazole oral solution offers an alternative at least as effective as fluconazole (10). Treatment with this cyclodextrin-containing solution is well tolerated (11).

This trial was initiated to compare the efficacy of itraconazole oral solution with nonabsorbable oral amphotericin B in the prevention of systemic and superficial fungal infections in patients with hematological malignancy and profound neutropenia. Furthermore, the frequency of and the time to initiation of intravenous systemic antifungals (e.g., amphotericin B) were compared.

	MATERIALS AND METHODS

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Patients. Male or female patients of at least 18 years of age, with a life expectancy of at least 14 days, and who had one of the following underlying conditions were eligible for this trial: acute leukemia scheduled for remission or induction or consolidation or reinduction chemotherapy; autologous bone marrow transplantation; myelodysplastic syndrome and scheduled to receive leukemia-like cytostatic therapy; chemotherapy for the blast crisis of chronic myelogenous leukemia; or lymphoma or myeloma and undergoing aggressive chemotherapy (e.g., platinum-based salvage regimen). In addition, their neutrophil count was expected to be <500/µl for at least 14 days, and they were not to have signs or symptoms of fungal infection (fungal colonization was allowed). Informed consent was obtained from each patient or from the patient's relative, guardian, or representative. Patients were excluded from the trial if they had been diagnosed with a proven or suspected deep fungal infection (including all cases without mycological sampling) during previous episodes of neutropenia, presented with a chest X ray suggestive of fungal infection, or with a fever of unknown origin (>38.5°C, rectal temperature), had received systemic antifungal therapy within 2 weeks before trial entry or topical intraoral antifungal therapy within 1 week before trial entry, were receiving terfenadine, astemizole, phenytoin, phenobarbital, rifampin, warfarin, loratadine, oral midazolam, triazolam, or cisapride or had received these medications in the 2-week period prior to trial entry or were expected to receive granulocyte-macrophage-colony-stimulating factor (GM-CSF), M-CSF, interleukin 3 (IL-3) or other growth factors (except for G-CSF, which was allowed). Women who were known to be pregnant or breast feeding, or who were of childbearing potential and not practicing adequate birth control were also excluded, as were patients who were unable to take oral medication, had liver disease defined as liver enzyme levels (alanine aminotransferase [ALAT] or aspartate aminotransferase [ASAT]) 4 times the upper normal limit at trial entry, were receiving investigational drugs other than anticancer regimens concurrently or within 1 month prior to trial entry, had previously shown hypersensitivity to azole antifungals or to oral amphotericin B, were known to be human immunodeficiency virus positive, had profuse diarrhea on presentation, or were entered into the trial previously.

Treatments. Patients were, on the basis of a computer-generated randomization list, grouped in blocks of four, assigned to receive double-blind, double-placebo treatment with one of the two active trial substances. Because of the double-blind, double-placebo nature of the trial, patients received both the solution and the capsule regimen: they received either active itraconazole solution together with placebo capsules or active amphotericin B capsules together with placebo solution. Prophylaxis was started on the first day of chemotherapy. Itraconazole oral solution (2.5 mg/kg of body weight twice a day) was provided as 100-ml bottles containing 100 mg of itraconazole per 10 ml of HPCD solution or HPCD-identical placebo. Oral amphotericin B (500 mg four times a day) was administered as 250-mg (Fungizone) capsules or identical placebo capsules. The trial medications were preferably to be taken without a meal. The oral solution was to remain in contact with the oral mucosa for at least 10 s and then was swallowed; it was not to be diluted.

Effectiveness assessments. The leukocyte count and the neutrophil count were recorded at baseline and during prophylaxis, daily, or at least three times weekly. All changes in urinary and intravenous catheters were to be recorded. Removed catheters were examined for the presence of yeast or molds in case of a fungal trial end point. Identification of the isolated yeast or mold was always to be done up to the species level. A chest X ray was to be carried out at baseline and weekly thereafter during prophylaxis. Signs and symptoms potentially attributable to superficial or deep fungal infection were recorded. Both during and after prophylaxis, additional tests (biopsy, computed tomography [CT] scan, chest X ray, and bronchoalveolar lavage [BAL]) were to be carried out whenever possible and acceptable in terms of the clinical condition of the patient. In addition, BAL was to be carried out in case of pulmonary infiltrates. In the case of the CT scan, high-resolution scanning was to be utilized.

Trial end point. Prophylactic treatment was stopped at the end of the neutropenic period, when a fungal infection trial end point was reached earlier, or when the investigator considered it, for safety reasons, in the best interest of the patient that or he/she be withdrawn. The end of neutropenia was defined as at least one neutrophil count higher than 500/µl. The fungal infection trial end point was classified according to one of the following criteria.

Assessments by the independent Expert Committee. The Expert Committee classification was a reclassification of the investigator's postprophylaxis end point. The Expert Committee, which was composed of three members with specific expertise on fungal infections during neutropenia and not participating into the trial, was provided with copies of all test results used to substantiate a diagnosis, the original CT scan or X rays providing evidence of fungal infection, as well as data on autopsy or immediate postmortem biopsies in order to enable them to reevaluate the classification of all trial end points prior to analysis. This final overall assessment was carried out blinded for the prophylactic trial medication.

Plasma drug concentrations. A 5-ml blood sample was taken immediately before the morning intake once weekly during prophylaxis and at the end of prophylaxis for the measurement of plasma itraconazole and hydroxyitraconazole concentrations. Date and hour of sampling and of the intake of study medication in the 24-h period before sampling were recorded. Concentrations of itraconazole and hydroxyitraconazole in plasma were determined by high-performance liquid chromatography with UV detection. The quantification limit of the assay was 10 ng/ml.

Safety assessments. The date of onset and duration of any adverse event that had occurred during the trial period (including any intercurrent disease) were noted, as well as their intensity, frequency, action taken, presumed drug relatedness, and outcome. The occurrence of serious adverse events was documented. Laboratory parameters (potassium, sodium, calcium, ALAT, ASAT, alkaline phosphatase, -glutamyltransferase, total cholesterol, creatinine, urea, and total bilirubin) were determined at selection and on a twice-weekly basis during the trial.

Statistical analysis. The statistical analysis of all data was performed by the Janssen Research Foundation. The primary population was the intent-to-treat population, which included all randomized patients with at least one drug administration. The subgroup of patients with neutropenia of <0.5 × 10⁹/liter for at least 14 days (i.e., high-risk patients for fungal infection) was also analyzed. The comparability among the trial groups was evaluated relative to demographic and baseline variables.

	RESULTS

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Investigators from 49 centers in seven different countries (Austria, Belgium, France, Greece, The Netherlands, Portugal, and Spain) recruited 557 patients (from July 1994 to April 1997) who were randomized to treatment: 281 received itraconazole, and 276 were given amphotericin B (intent-to-treat population).

There were no significant intergroup differences in baseline characteristics; data on sex, age, underlying disease, therapy of the underlying disease, duration of neutropenia, and duration of treatment are summarized in Table 1. The median (minimum to maximum) durations of neutropenia were 18 (0 to 84) days in itraconazole-treated patients and 20 (0 to 88) days in amphotericin B-treated patients. In line with these results, the median durations of treatment were 19 (1 to 56) days in the itraconazole group and 18 (2 to 56) days in the amphotericin B group. The main underlying pathology was leukemia (71% of the patients in each group).

Invasive aspergillosis. Invasive aspergillosis was documented in five (1.8%) itraconazole-treated patients and in nine (3.3%) amphotericin B-treated patients (P = 0.264) (Table 2); of these, only one patient in the itraconazole group compared with four patients in the amphotericin B group died (Table 3). The pathogens that were identified were Aspergillus fumigatus in all cases, except for one in the amphotericin B group (Aspergillus terreus) (Table 3).

Other fungal infections. Apart from invasive aspergillosis, other proven deep fungal infections were noted in three (1.1%) patients in the itraconazole group and in four (1.4%) patients in the amphotericin B group (Table 2; P is not significant); of these, none of the itraconazole patients and one amphotericin B patient died (Table 3). In the amphotericin B group, all four fungal infections were documented in the blood. (Candida krusei, Candida parapsilosis, Geotrichium spp., and Fusarium nygamai were each noted in one patient.) In the itraconazole group, one patient had a Candida albicans infection of the blood, one had a skin biopsy positive for Cryptococcus neoformans (maculopapular eruptions), and one had a positive culture for Candida glabrata (pus and an abscess in the right iliac fossa).

Initiation of systemic antifungal treatment. During the trial, systemic antifungal treatments (intravenous amphotericin B [any formulation], fluconazole, and voriconazole) were initiated in 114 (41%) itraconazole-treated patients and in 132 (48%) oral amphotericin B-treated patients. The Kaplan-Meier analysis of time to an event showed that, in the first quartile of patients, the times to first administration of systemic antifungals were 18 days in the itraconazole group and 15 days in the amphotericin B group; this intergroup difference tended to be significant (P = 0.055). In the analysis of high-risk patients, these times were 18 and 14 days, respectively; this intergroup difference was statistically significant (P = 0.029) (Fig. 1).

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Time to use of systemic antifungals for eligible patient analysis (neutrophil count of <0.5 × 109/liter for at least 14 days).

Survival. The overall mortalities were 6% (n = 18) in the itraconazole group and 8% (n = 23) in the oral amphotericin B group (P = 0.384). Of those, five patients died with a proven deep fungal infection in the oral amphotericin B group versus only one in the itraconazole group.

Plasma itraconazole concentrations. Mean concentrations of itraconazole in plasma were 512 ng/ml after 1 week of treatment and increased further to 764, 1,028, and 1,253 ng/ml after 2, 3, and 4 weeks of treatment, respectively (Table 4). The efficacy levels were achieved shortly after initiation of treatment. From 1 week after the start of treatment onwards, about 80% of the patients had predose concentrations of itraconazole in plasma greater than 250 ng/ml, which is considered to be the minimum level required for efficacy. After 4 weeks of treatment, these levels were attained in 96% of the patients (Table 4).

Subanalysis of high-risk patients. As outlined in the protocol, we analyzed the subgroup of all patients with neutropenia of <0.5 × 10⁹/liter for at least 14 days (201 itraconazole patients and 186 amphotericin B patients). Due to the combination of depth and duration of neutropenia, this group is considered to be a high-risk population for fungal infection.

Safety. Adverse events were reported in 222 (79%) itraconazole-treated patients and in 205 (74%) amphotericin B-treated patients. The most frequently reported adverse events were of gastrointestinal origin (Fig. 2). Rash (17 and 13%, respectively) and hypokalemia (11% for itraconazole and 7% for amphotericin B) were the other most frequently reported adverse events. The incidences of adverse events were similar in the two groups.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Most frequently reported adverse events for the patients in this study treated with itraconazole (Itr) or amphotericin B (Ampho B).

	DISCUSSION

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The current trial was set up to establish the potential role of the broad-spectrum azole antifungal itraconazole in preventing fungal infections in patients with a hematological malignancy including autologous bone marrow transplant patients expected to be neutropenic for at least 14 days. Attitudes towards antifungal prophylaxis are different between countries and between centers. As a remnant of the gastrointestinal decontamination approach, the still widely used oral nonabsorbable agent amphotericin B was selected as the comparative agent.

Collection of adequately documented fungal end point data has been recognized as a serious difficulty for prophylactic evaluation, since confirmation is often obtained only after initiation of intravenous amphotericin B or at autopsy. Therefore, the 4-week postprophylaxis visit was considered essential to collect additional postprophylaxis evidence of suspected and deep fungal infection. An overall and blinded assessment by the Expert Committee harmonized the interpretation for a limited number of difficult cases.

The incidences of aspergillosis (1.8%) and proven deep fungal infection (2.8%, including invasive aspergillosis) observed in the itraconazole recipients were very similar to those observed previously. In two large prophylactic trials with itraconazole in neutropenic patients (9, 10), Aspergillus infection has been reported in 0 and 2% and proven deep fungal infections have been reported in 0.5 and 2.5% of the itraconazole-treated patients, respectively. In the present and both previous trials combined, a clinically relevant reduction in the number of cases of deep fungal infection including Aspergillus infection was obtained in favor of itraconazole oral solution compared with the use of oral amphotericin B, placebo, and fluconazole (14 versus 28 cases, respectively). Related to this observation is the number of patients dying with proven deep fungal infections, mostly aspergillosis: 2 in the itraconazole arms versus 14 in the comparative arms (oral amphotericin B, n = 5; placebo, n = 5; and fluconazole, n = 4). Based on the blinded interim analysis, it became evident that the required number of documented cases of aspergillosis in the present trial could not be obtained by recruiting all patients as stipulated in the sample size calculation. Statistical conclusions on very small incidences are difficult to interpret. The majority of the patient population in this trial mainly consisted of neutropenic patients with a lower risk for mold infections. The risk for aspergillosis is higher for patients undergoing allogeneic bone marrow transplantation, for patients with acute myeloid leukemia in second relapse, or patients with graft-versus-host disease treated with high-dose steroids. Only a few high-risk patients were included in this study.

All cases of invasive aspergillosis were identified in France, except for one in the itraconazole group, which was observed in The Netherlands. An explanation for the difference could be that the incidence of aspergillosis varies strongly between different hospitals, even in the same country. Furthermore, 46% of the total patient sample was recruited in France (129 of 281 patients in the itraconazole group and 125 of 276 patients in the oral amphotericin B group).

As expected from the trial comparison versus amphotericin B capsules, a statistically significant (P = 0.004) higher incidence of superficial candidosis, mainly oropharyngeal candidosis, was documented. Also, for itraconazole oral solution, which is effective in the treatment of oral and esophageal candidosis (5), this effect is not surprising. The clinical relevance is, however, less clear. A significant relationship with deep candidosis could not be established in this trial. The administration of empiric or therapeutic intravenous amphotericin B is preferably to be avoided due to the potential of severe renal toxicity and other adverse experience. In the itraconazole oral solution arm of the trial, the instantaneous risk of administration of intravenous amphotericin B was 33% lower than that in the oral amphotericin B arm. This difference was marginally statistically significant (P = 0.052) and is considered to be of clinical relevance. More than in therapeutic trials, the safety evaluation of a proposed drug for prophylaxis is very important. Interpreting safety aspects of one component of a multidrug regimen in highly immunocompromised patients is difficult. Even though HPCD was also used in the placebo formulation, the double-blind, double-placebo design of the trial and the standard methodology to evaluate adverse events and to classify biochemical parameters have been instrumental in this assessment.

In general, treatment with the itraconazole oral solution is well tolerated (11). Any diarrhea induced by treatment with itraconazole oral solution is most likely due to the HPCD carrier, which is known to induce soft stools and diarrhea from a daily dose of 16 g onwards. This dose was administered in the placebo as well as in the active formulation. However, nausea and vomiting, which both belong to the adverse event profile of both itraconazole capsules and oral solution, did occur to similar extents in both groups. In the population studied, the overall reporting of gastrointestinal adverse events mirrors the clinical day-by-day observations which occur independent of a clinical trial setting. Other adverse events for itraconazole are rashes, transient increases in liver function tests, and hypokalemia, but again, no relevant differences were observed.

Biochemistry abnormalities are inherent in the patient population studied. All observed increases and decreases were similar in both groups, pointing at the background laboratory abnormalities in this population. The hypokalemia confirmed findings in the adverse event analysis.

In conclusion, prophylactic administration of itraconazole oral solution at 5 mg/kg daily in patients with hematological malignancies and neutropenia significantly reduced the incidence of superficial fungal infections. The number of systemic fungal infections, the number of deaths due to proven deep fungal infections, the incidence of administration of systemic antifungals, and the time prior to this administration were influenced positively by the administration of itraconazole oral solution.