INTRODUCTION

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Cancer treatment and bone marrow transplantation/stem cell transplantation (BMT/SCT) regimens involve intensive cytotoxic chemotherapy, which often leads to prolonged myelosuppression and increased risk for opportunistic mycoses (5, 30, 31, 33, 39). Graft versus host disease (GVHD) following BMT/SCT may require immunosuppression that also increases the risk of invasive fungal infections. Despite empirical use of amphotericin B and the prophylactic use of antifungal triazoles, invasive fungal infections due to resistant Candida spp., Aspergillus spp., Fusarium spp., and zygomycetes, as well as other fungal pathogens, continue to emerge (6, 10, 11, 40). Infections due to these organisms may be unresponsive even to maximal tolerated doses of conventional deoxycholate amphotericin B (DAMB; 1.0 to 1.5 mg/kg of body weight/day). Moreover, when responses do occur, treatment may be accompanied by frequent dose-limiting nephrotoxicity (14, 43).

Liposomal amphotericin B (L-AMB; AmBisome) was found in preclinical studies to be as effective or more effective but less nephrotoxic than conventional amphotericin B in the treatment of experimental invasive pulmonary aspergillosis (13, 15, 37). Consistent with these findings are the results of four open label clinical trials which also demonstrated antifungal efficacy in immunocompromised patients (28, 29, 35) and a randomized trial in patients with invasive fungal infections complicating hematologic malignancies that found superior efficacy and safety (23). Toxicity has been minimal despite much higher dosages of amphotericin B being delivered than can be safely administered when given as DAMB. The reduced toxicity of this formulation thus, allows the administration of much higher doses of amphotericin B than that of the deoxycholate product (13, 18, 19, 27, 44).

Preclinical and clinical data demonstrate a dose-response relationship of the class of amphotericin B compounds against invasive fungal infections (4, 13, 17, 41, 42). An earlier study found that L-AMB had no dose-limiting toxicity between 1.0 and 7.5 mg/kg/day (45). As invasive fungal infections in immunocompromised patients may be unresponsive to approved dosages of L-AMB (3 to 5 mg/kg/day), clinicians will often increase the dosage of L-AMB to 7.5 mg/kg/day.

Little is known, however, about the safety, tolerance, and plasma pharmacokinetics of amphotericin B at these higher dosages of L-AMB. Moreover, the maximum tolerated dose (MTD) of L-AMB also is unknown. We therefore investigated the safety, tolerance, plasma pharmacokinetics, and MTD of L-AMB in an open label, multicenter, and sequential dose escalation study of the treatment of invasive fungal infections.

	MATERIALS AND METHODS

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Study design. This study was designed as an open-label, multicenter, sequential dose escalation trial for the assessment of the safety, tolerance, plasma pharmacokinetics, and MTD of intravenous L-AMB. L-AMB was administered at four high dosage levels in a population of immunocompromised adults requiring antifungal therapy for invasive fungal infections. The study drug consisted of intravenous L-AMB (AmBisome; Fujisawa Healthcare, Inc. USA). Doses of L-AMB were calculated and expressed as the amount of amphotericin B administered. For example, a dose of 50 mg of L-AMB refers to the administration of an amount of L-AMB that contains 50 mg of amphotericin B. Patients were eligible for study if (i) they were between the ages of 18 and 80 years and were undergoing bone marrow transplantation, were receiving active chemotherapy for neoplastic disease or immunosuppressive therapy for solid organ transplantation or aplastic anemia, or were infected with human immunodeficiency virus (HIV) and (ii) they had evidence of an invasive mycosis due to a filamentous fungus.

Monitoring of safety and tolerance. In order to monitor the safety and tolerance of L-AMB infusions, patients were closely observed for side effects during the administration of study drug. A previously validated bedside data extraction sheet was utilized by the nursing staff to record serial vital signs during and after infusion, as well as signs and symptoms of infusion-related toxicity (41, 45, 50). This data extraction sheet then became a source document for reporting infusion-related toxicity. Pulse and blood pressure were monitored immediately before, at 15 and 30 min, and at the end of the infusion. Between doses, temperature and vital signs were recorded every 4 h during waking hours. Signs, symptoms, and reported side effects associated with drug infusion or occurring at any time during the study period were documented and assessed for a relationship to the study drug.

Pharmacokinetic sampling. Pharmacokinetic sampling was performed on days 1 and 7 and on the last day of study drug. Two-milliliter venous blood samples were drawn at each time point. The blood was centrifuged and the plasma fraction was stored at 70°C until analysis. Samples were stored for a median of 4 months before analysis. First dose and day 7 pharmacokinetic sample collection times were as follows: prior to dosing, at the end of 60 min of infusion, at 65, 75, 90, and 120 min, and then at 3, 4, 6, 8, 12, 18, and 24 h postinfusion. Daily trough samples (immediately prior to the next dose) were subsequently obtained on days 3, 4, 5, and 6. Last-dose pharmacokinetic sample time points were then obtained prior to dose, at the end of 60 min of infusion, at 65, 75, 90, and 120 min, and then at 3, 4, 6, 8, 12, 18, and 24 h postinfusion, followed by wash-out samples 3, 7, and 14 days after the last dose of L-AMB.

Analytical methods for L-AMB assay. Concentrations of amphotericin B were determined by a validated high-performance liquid chromatography assay (3). Following methanol deproteinization, amphotericin B and the internal standard, 3-nitrophenol, were separated by reversed-phase chromatography and detected by UV absorbance at 406 nm. The high-performance liquid chromatography system consisted of a Bio-Rad autosampler (Bio-Rad, Hercules, Calif.), Spectra-Physics Model 250 pump (Thermo Separations, San Jose, Calif.), Waters Model 441 UV-VIS detector (Waters Corp, Milford, Mass.), column heater, Supelcosil ABZ + Plus column (3-µm-diameter particle size, 150 by 4.6 mm [inner diameter]; Supelco, Bellefonte, Pa.), and a Keystone CL8 guard column (Western Analytical, Murrieta, Calif.). Data were collected and integrated on a VG Multichrom Data System for VAX/VMS. The lower limit of quantitation of the assay was 0.1 µg/ml. Two overlapping standard curves were used: 0.05 to 20 µg/ml and 0.5 to 200 µg/ml. The unweighted correlation coefficient was 0.998 for both curves, with an interday and intraday coefficient of variation of 1.8 to 11.2% and 6.9 to 10.1%, respectively.

Pharmacokinetic calculations. The pharmacokinetic profile of amphotericin B following L-AMB administration was determined by noncompartmental analysis. The terminal elimination half-life (t_1/2) was obtained from plasma data in the postdistribution phase. The elimination rate constant k was defined as 0.693/t_1/2. The area under the concentration-time curve from 0 to 24 h (AUC_0-24) and area under the first moment of the concentration-time curve from 0 to 24 h (AUMC_0-24) were calculated using the linear trapezoidal method. The AUC_0- was determined as follows: AUC_0-24 + AUC_24-, with AUC_24- extrapolated from C_t/k, where C_t was the last measured concentration. The AUMC_0- was calculated similarly, with AUMC_24- extrapolated from (C_t × t)/k + C_t/k². The mean residence time (MRT) was calculated as AUMC_inf/AUC_inf. Total body clearance (CL) was calculated as dose/AUC_0-. The volume of distribution (V) was calculated as follows: V = CL/k. The volume of distribution at steady state (V_ss) was calculated as follows: V_ss = (dose)(AUMC_0-)/(AUC_0-)²  (dose)(infusion time)/2 × AUC_0-.

1

(1+2)

3

4

Monitoring and assessment of efficacy. Efficacy was assessed by clinical response, radiological response, mycological response, and survival. Serial blood cultures, urine cultures, and chest radiographs were performed for all patients. Blood was cultured using lysis centrifugation (Wampole) and BacTAlert (Organon Teknika) systems. Serial chest CT scans were performed for monitoring therapeutic response of invasive fungal infections of the respiratory tract and sinuses.

Statistical analysis. All patients who received at least one dose of study drug (modified intent to treat population) were included in the safety and efficacy analyses. A separate analysis of response was assessed for patients who received at least 7 days of therapy. This MTD study was not powered to detect differences in efficacy across dosage groups. Comparisons of the mean pharmacokinetic values between the first versus last doses and between different dose levels of L-AMB were performed using two-tailed unpaired Student's t test. A P value of 0.05 was considered to be statistically significant.

	RESULTS

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Study patient population. A total of 44 patients enrolled in the study and received at least three doses of L-AMB (Table 1). These patients had a mean age of 43.2 years (range, 19 to 70 years). Thirty-five patients were male and nine were female. A total of 29 (66%) patients underwent BMT/SCT; 20 received allogeneic and 9 received autologous transplants. Nine (21%) had graft versus host disease (GVHD). The remaining 15 patients received antineoplastic chemotherapy, underwent solid organ transplantation, or had aplastic anemia or HIV infection. Consistent with the seriously ill condition of many of the patients enrolled into this study, approximately one-half of all patients had baseline elevation of serum creatinine, AST, ALT, or bilirubin. Proven fungal infections were documented for 21 patients, probable fungal infection was documented for 10, and possible fungal infection was documented for 13. The type of invasive fungal infection and the sites of these infections are summarized in Table 2. Invasive pulmonary aspergillosis was the predominant cause of infection, followed by three cases of zygomycosis and two cases of fusariosis. Patients may have had more than one site of infection.

Duration of administration of L-AMB. The number of infusions of L-AMB ranged from 1 to 83, with an actual cumulative dose ranging from 13.6 to 972.1 mg/kg (Table 3). The duration of the day 1 infusion ranged from 1 to 2.6 h. All patients stopped therapy within the protocol-specified maximum of 100 days. The median day for administration of the last study dose ranged from day 9 (15-mg/kg group) to day 24 (7.5-mg/kg group). A total of 9 (20%) patients discontinued taking the study drug due to an adverse event. These included elevated serum creatinine, renal failure, acute renal failure, pancreatitis, hyperbilirubinemia, hypotension associated with infusion, relapse of primary malignancy, cardiorespiratory failure, and multisystem organ failure; the latter three adverse events were attributed to underlying diseases and not to the study drug. Discontinuation was unrelated to dosage group, occurring in 38% of patients in the 7.5-mg/kg group, 10% in the 10-mg/kg group, 14% in the 12.5-mg/kg group, and 21% in the 15-mg/kg group.

Infusion-related reactions. Safety and tolerance were monitored prospectively with particular attention to infusion-related reactions and to nephrotoxicity. Premedications were not administered per protocol for infusion-related reactions prior to the day 1 study drug infusion. Any adverse events with onset during or within 1 h of completion of the study drug infusion were recorded as infusion-related reactions.

Laboratory parameters. The frequency of abnormal values for laboratory parameters of renal and hepatic function is presented in Table 4. One-half (22 of 44 [50%]) of patients had serum creatinine values that were 1.5 times greater than the baseline and >1.2 mg/dl at some time during the study. Fourteen (32%) had a value 2 times greater than the baseline. Excluding one patient, peak creatinine levels in serum ranged from 0.9 to 5.8 mg/dl during the study. The one patient who was HIV positive with sinus zygomycosis due to Rhizopus sp. was enrolled in the 12.5-mg/kg dosage cohort and received L-AMB over the course of 99 days. The level of creatinine in serum ranged between 1.4 and 2.3 mg/dl for 94 days. However, the creatinine level in serum increased abruptly to 6.4 mg/dl in one patient on day 100 and L-AMB was discontinued. No patients required hemodialysis.

Overall adverse events. All patients experienced at least one adverse event. The more common adverse events included fever (21 out of 44 [48%]), increased creatinine level (20 out of 44 [46%]), hypokalemia (17 out of 44 [39%]), chills (21 out of 44 [32%]), and abdominal pain (11 out of 44 [25%]). No obvious dose relationship with respect to the overall frequency of adverse events was observed.

Pharmacokinetics. The pharmacokinetic profile of L-AMB administered at dosages of 7.5, 10, 12.5, or 15 mg/kg/day as a 2-h infusion is summarized in Fig. 1 and the noncompartmental parameters in Table 5. The interpatient concentration-time data were highly variable within a dosage group. The mean (± standard deviation [SD]) AUC₂₄s after the initial dose were 692 ± 834, 1,062 ± 971, 860 ± 390, and 554 ± 174 for the respective ascending doses.

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Plasma concentration-time profiles of L-AMB at doses of 7.5 mg/kg (), 10.0 mg/kg (), 12.5 mg/kg (), and 15.0 mg/kg () on day 1 (left panel), day 7 (middle panel), and last day (right panel) of infusion.

Overall success. End-of-treatment response rates for all patients are summarized by dosage group in Table 6. Approximately one-half of the patients (23 out of 44 [52%]) had a response (complete or partial) or stabilization of infection. A somewhat greater response and stabilization rate (20 out of 31 [64%]) was observed in patients administered at least 7 doses of study drug (efficacy evaluable population). Response rates were higher in the first three dosage groups. The 15-mg/kg dosage group consisted of more patients who were either nonevaluable or had higher failure rates due to a relatively high number of patients with advanced disease at study entry. Response rates for patients with baseline definite or probable infection were similar to those of the overall response rate.

	DISCUSSION

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This study of the safety, tolerance, and plasma pharmacokinetics of L-AMB found no demonstrable dose-limiting nephrotoxicity or infusion-related toxicity over a dosage range from 7.5 to 15.0 mg/kg/day. Hypokalemia developed more frequently at dosages >10 mg/kg/day than at dosages of 10 mg/kg/day. Overall infusion-related toxicity was relatively low; however, an idiosyncratic non-dose-dependent syndrome of acute dyspnea and chest pain was observed in three patients. This could be ameliorated by diphenhydramine administration. There was a distinctly nonlinear profile of the plasma pharmacokinetics of L-AMB over the dosage range of 7.5 to 15.0 mg/kg/day. Furthermore, C_max and AUC achieved an upper limit at 10 mg/kg/day. Further dosage increases did not result in an increase in C_max or AUC. Although this MTD study was not statistically powered for assessment of dose-dependent efficacy, L-AMB was effective in the treatment of severe infection in many of these critically ill patients.

The nephrotoxicity of amphotericin B may be understood in terms of its effects on glomerular and tubular functions. As evidenced by serial creatinine concentrations in serum, there was remarkably little glomerular nephrotoxicity at relatively high dosages from 7.5 to 15.0 mg/kg/day. This preservation of glomerular function by lipid formulations of amphotericin B is related to several possible mechanisms. Lipid formulations of amphotericin B have a high affinity for binding to high-density lipoproteins (HDL) (46-49). Binding to HDL promotes uptake in the reticuloendothelial system (RES), which has a relatively high level of expression of HDL receptors. By comparison, conventional deoxycholate amphotericin B has a higher affinity for low-density lipoproteins (LDL). Receptors for LDL are more highly expressed on glomerular endothelial cells and may contribute to the relatively high concentration of conventional amphotericin B in kidneys in comparison to those of L-AMB (22, 37). The preferential uptake of L-AMB by hepatic and splenic macrophages and correspondingly reduced concentration in the kidney further contributed to reduced nephrotoxicity and an improved therapeutic index in comparison to deoxycholate amphotericin B. Another potential mechanism contributing to decreased nephrotoxicity of lipid formulations of amphotericin B is the preferential selected diffusion of the amphotericin B molecule to fungal cell membranes versus mammalian cell membranes (1, 2, 26). The release of amphotericin B molecules from the lipid vehicle via fungal phospholipases onto the fungal cell membrane may further enhance the host-pathogen specificity of lipid formulations of amphotericin B (32).

Although there was no evidence of dose-dependent glomerular dysfunction in this study of high-dose L-AMB, there was a significant difference in the frequency of hypokalemia in patients receiving higher dosages of L-AMB than in those receiving lower dosages. These findings suggest that the renal tubular epithelium may be more sensitive to high dosages of L-AMB then cells mediating glomerular filtration. Hypokalemia developed in patients who were already undergoing close monitoring and routine supplementation of potassium. Thus, the use of high-dose L-AMB at dosages greater than 10 mg/kg/day warrants a particularly meticulous approach to monitoring of potassium levels in serum and may require preemptive administration of higher daily potassium supplementation than would routinely be administered without lipid formulation of amphotericin B.

Infusion-related toxicity was prospectively monitored using a bedside data acquisition sheet. Of the 815 infusions administered, a total of 133 (16.3%) was associated with infusion-related reactions. The relative infrequency of infusion-related reactions is consistent with earlier observations of L-AMB (41, 45, 50). However, this current study also found the development of an idiosyncratic reaction associated with severe substernal chest discomfort, dyspnea, and flank and/or abdominal pain. Developing within the first 1 to 2 min of infusion of L-AMB, these events were not dependent upon dosage or rate of infusion. This acute infusion-related toxicity of L-AMB was managed by the discontinuation of the infusion and the intravenous administration of 25 to 50 mg of diphenhydramine. The symptom complex of dyspnea and flank and abdominal pain resolved rapidly with these interventions. Following resolution of these symptoms, the infusion was resumed with no subsequent infusion-related toxicity. Subsequent infusions were preceded by the administration of diphenhydramine as 25 to 50 mg of intravenous fluid for prevention of infusion-related toxicity. This syndrome, which has been observed previously with L-AMB and other lipid formulations of amphotericin B (24), was recently reviewed by Johnson et al. (20). The mechanism of this infusion-related toxicity is not well understood but may be mediated by histamine. Given the small volume of compound that is infused, the absolute amount of liposomal amphotericin B does not appear to contribute to this toxicity. Instead, the reaction appears to be idiosyncratic and perhaps related to several interactions between the lipid material and host factors. Unlike the reactions of conventional amphotericin B that are mediated by tumor necrosis factor alpha (TNF-) (7, 25), this distinctive syndrome of L-AMB appears to be more histamine mediated. Szebeni et al. report that some liposomes may activate complement and release C5a, resulting in a liposome-induced pulmonary hypertension with a complement-induced pseudoallergic reaction (36).

Our present study and other reports underscore the importance of being vigilant of this idiosyncratic acute liposome-infusion-related syndrome. Nonetheless, this study found that these infusion-related reactions of L-AMB are not dose dependent and that the MTD is 15 mg/kg/day. While the higher frequency of infusion-related toxicity could be related to higher peak levels in the plasma of the 7.5- and 10.0-mg/kg cohorts, only a small fraction of the total dosage was infused by the time the idiosyncratic symptoms began.

The plasma pharmacokinetics of L-AMB over the range of dosages from 7.5 to 15 mg/kg/day was clearly nonlinear. The C_max and AUC appeared to reach maximum levels at 10 mg/kg/day. This nonlinear disposition of L-AMB suggests that new clearance mechanisms are induced or activated at levels exceeding 10 mg/kg/day. Amphotericin B is eliminated from the circulation by the RES, biliary tract, and urinary tract (21, 38). Elimination of L-AMB from the circulation also is dependent on the RES (2, 22, 34, 38). Higher concentrations of L-AMB may induce a concentration-driven clearance mechanism for amphotericin B, as observed for the renal elimination of carprofen (8) and reticuloendothelial elimination of hemoglobin (9) or amphotericin B lipid complex (44). These clearance mechanisms may be related to enhanced uptake by the RES, perhaps mediated by low-affinity receptors for lipids that permit clearance of these liposomes at relatively high plasma concentrations. Such enhanced uptake by the RES would explain high concentrations of amphotericin B in the liver, spleen, and bone marrow. The decreasing variability of pharmacokinetic parameters at the higher dosage range also may be related to the induction of a clearance mechanism (e.g., low-affinity reticuloendothelial receptors) that may enhance a more uniform interpatient clearance. That patients in the two higher dosage groups had greater mortality may also have contributed to the dosage dependency in the model.

The therapeutic implications of a nonlinear dosage-dependent distribution are important for treatment of fungal infections in non-RES tissues. For example, this study of the plasma pharmacokinetics of high-dosage L-AMB suggests that 10 mg/kg would be the upper dosage limit for treatment of fungal infections of the central nervous system (CNS). Based upon earlier studies in the treatment of experimental Candida meningoencephalitis, C_max and AUC were the principal pharmacodynamic determinants of antifungal efficacy of amphotericin B and its lipid formulation (16). There was a direct relationship between peak concentrations in plasma, AUCs, and cerebral tissue concentrations, which in turn correlated directly with greater antifungal efficacy. The greatest antifungal efficacy in the CNS was observed with deoxycholate amphotericin B and L-AMB, which also exhibited the highest peak plasma concentrations, AUCs, and cerebral tissue concentrations. As a logical extension of these findings, the higher dosages of L-AMB beyond 10 mg/kg would be unlikely to impact on fungal infection of the central nervous system, if CNS penetration is based on C_max and AUC are not increased with a corresponding increase in dosage. By comparison, an infection of RES tissues, such as the liver and spleen, as in chronic disseminated candidiasis may benefit from dosages exceeding 10 mg/kg/day. Liposomal amphotericin at such high dosages would likely be deposited in RES tissues.

The implications for such nonlinear plasma pharmacokinetics on treatment of fungal infections of the lungs remain to be determined. Earlier work demonstrated a dosage-dependent relationship between the dosage of L-AMB and parameters of efficacy (13). Such dosage-dependent relationships for liposomal formulations of amphotericin B also exist for other lipid formulations (4, 42). Further experimental investigation of antifungal pharmacodynamics of these higher dosages is clearly warranted.

This study was designed for determination of the MTD in patients with invasive fungal infections and not the optimal therapeutic dosage for treatment of these mycoses. A study designed to assess the optimal therapeutic dosage would require approximately 200 patients per dosage group in order to be sufficiently powered. Comparison of the overall response rate of 46% in this study with those of other clinical trials with L-AMB must be undertaken cautiously. Differences in underlying immunosuppression, patterns of infection, and etiologic organisms preclude statistical comparisons across studies. This problem is exemplified with two studies demonstrating response rates ranging from 39% (12) to 56% (11) for invasive aspergillosis. A case-matched study minimally and a randomized trial ideally permits sufficient certainty for comparison of responses in these complex infections. Nonetheless, one can infer that L-AMB is effective in producing complete and partial responses in approximately 40% of seriously ill patients with invasive fungal infections due to Aspergillus species and other molds. Whether these higher dosages of 7.5 to 15 mg/kg/day are more efficacious then the Food and Drug Administration-approved dosage of 5 mg/kg/day remains to be determined through carefully designed comparative trials. In the absence of such clinical trials, however, a pragmatic approach for patients who are critically ill with invasive fungal infections progressing through L-AMB at 5 mg/kg/day would be to increase the dosage of L-AMB to 7.5 or 10 mg/kg/day. Whether exceeding 10 mg/kg/day for an individual patient is therapeutically beneficial may depend upon the site of infection. For infection progressing in the CNS, it is not clear that further increment of the dosage beyond 10 mg/kg/day would have benefits. Instead, using an alternative agent such as an investigative antifungal triazole or using a combination of L-AMB with an echinocandin would be potential alternative therapeutic approaches for such patients. Certainly, for patients whose invasive fungal infections are progressing in the lungs, liver, spleen, or other non-CNS site while receiving 10 mg/kg/day of L-AMB, raising the dosage to 12.5 or 15.0 mg/kg/day is a rational therapeutic alternative.