Minimum concentration of Amphotericin B in serum according to the formulation, dose, and daily or prolonged intermittent therapeutic regimen

Abstract INTRODUCTION: The therapeutic efficacy of daily amphotericin B infusion is related to its maximum concentration in blood; however, trough levels may be useful in intermittent regimens of this antifungal drug. METHODS : High performance liquid chromatography (HPLC) was used to determine the minimum concentration (Cmin) of amphotericin B in the serum of patients receiving deoxycholate (D-Amph) or liposomal amphotericin B (L-AmB) for the treatment of cryptococcal meningitis (n=28), histoplasmosis (n=8), paracoccidioidomycosis (n=1), and leishmaniasis (n=1). RESULTS: Daily use of D-Amph 30 to 50 mg or L-AmB 50 mg resulted in a similar Cmin, but a significant increase ocurred with L-AmB 100 mg/day. The geometric mean Cmin tended to decrease with a reduction in the dose and frequency of intermittent L-AmB infusions: 357 ng/mL (100 mg 4 to 5 times/week) > 263 ng/mL (50 mg 4 to 5 times/week) > 227 ng/mL (50 mg 1 to 3 times/week). The impact on Cmin was variable in patients whose dose or therapeutic scheme was changed, especially when administered the intermittent infusion of amphotericin B. The mean Cmin for each L-AmB schedule of intermittent therapy was equal or higher than the minimum inhibitory concentration of amphotericin B against Cryptococcus isolates from 10/12 patients. The Cmin of amphotericin B in patients with cryptococcal meningitis was comparable between those that survived or died. CONCLUSIONS: By evaluating the Cmin of amphotericin B, we demonstrated the therapeutic potential of its intermittent use including in the consolidation phase of neurocryptococcosis treatment, despite the great variability in serum levels among patients.

Determination of amphotericin B in PLA-PEG blend nanoparticles by HPLC-PDA

In this work, we developed and validated an effective reversed-phase HPLC method with photodiode array (PDA) detection for the quantitative analysis of amphotericin B (AmB) in poly(lactide)-poly(ethylene glycol) (PLA-PEG) blend nanoparticles. Chromatographic runs were performed on a reverse phase C18 column using a mobile phase comprising a 9% acetic acid and acetonitrile mixture (40:60, v/v) under isocratic elution with a flow rate of 1 mL/min. AmB was detected at a wavelength of 408 nm. The validation process was performed considering the selectivity, linearity, precision, accuracy, robustness, limit of detection (LOD) and limit of quantitation (LOQ) of the method. A concentration range of 1-20 µg/mL was used to construct a linear calibration curve. The LOQ and LOD were 55 and 18 ng/mL, respectively. The mean recovery of AmB from the samples was 99.92% (relative standard deviation (RSD) = 0.34%, n=9), and the method was robust for changes in the flow rate of the mobile phase (maximum RSD=4.82%). The intra- and inter-assay coefficients of variation were less than 0.59%. The method was successfully used to determine the entrapment efficiency of AmB in PLA-PEG blend nanoparticles.

Neste trabalho desenvolveu-se e validou-se um efetivo método por cromatografia líquida de alta eficiência (CLAE) em fase reversa com detecção por fotodiodos para a análise quantitativa de anfotericina B (AmB) em nanopartículas compostas por blendas de poli(ácido lático)-polietilenoglicol (PLA-PEG). Corridas cromatográficas foram realizadas sob coluna C18 de fase reversa com fase móvel consistindo de ácido acético 9% e acetonitrila (40:60, v/v), em eluição isocrática com fluxo de 1 mL/min. A AmB foi detectada no comprimento de onda de 408 nm. O processo de validação foi realizado considerando a seletividade, linearidade, precisão, exatidão, robustez, limite de detecção (LD) e limite de quantificação (LQ) do método. Uma faixa de concentração entre 1-20 µg/mL foi usada para obter a curva-padrão linear. Os valores de LD e LQ foram 55 e 18 ng/mL, respectivamente. A recuperação média da AmB a partir das amostras foi de 99,92% (desvio padrão relativo = 0,34%, n=9) e o método foi robusto, considerando alterações no fluxo da fase móvel (desvio padrão relativo máximo=4,82%). Os coeficientes de variação intra e inter dia foram inferiores a 0,59%. O método foi utilizado com sucesso para a determinação da eficiência de encapsulação da AmB em nanopartículas de PLA-PEG.

Leishmaniasis cutánea y anfotericina B liposomal: Reporte de caso / Cutaneous leishmaniasis and liposomal amphotericin B: Case report

La leishmaniasis cutánea es una zoonosis producida por diferentes especies del parásito del género Leishmania . Existen 2 tipos de leishmaniasis, la que se conoce como del nuevo mundo y la del viejo mundo, que son causadas por diferentes especies. La forma de leishmaniasis más común en nuestro medio es la cutánea, rara vez con compromiso sistémico aun en pacientes HIV positivos. El tratamiento consiste en Glucantime, alternativamente miltefosine, los cuales tienen una toxicidad signifi cativa y pueden fallar en un número sustancial de casos. La opción de tratamiento para estos pacientes es la anfotericina B, de las cuales la forma liposomal tiene menor toxicidad renal pero un alto costo y difi cultades para su disponibilidad. Se reporta el caso de un paciente con enfermedad renal crónica y leishmaniasis cutánea, en quien estaba contraindicado el Glucantime y presentó falla terapéutica con miltefosine. Recibió tratamiento con anfotericina B liposomal en una dosis única de 15 mg/kg con adecuada tolerancia, deterioro no signifi cativo de la función renal y resolución de las lesiones cutáneas hasta los 30 y 60 días de seguimiento.

Cutaneous leishmaniasis is a zoonosis caused by various species of Leishmania parasite. There are two types, known as New World and Old World leishmaniasis, which are caused by different species. The most common form of leishmaniasis in our area is mucocutaneous, which rarely has systemic involvement, even in HIV-positive patients. Glucantime is the first choice of therapy, or alternatively miltefosine, both of which have significant toxicity and can fail in a substantial number of cases. The alternative choice of treatment for these patients is amphotericin B, from which the liposomal form has less renal toxicity but a high cost and low availability. We report the case of a patient with chronic renal disease and cutaneous leishmaniasis, in whom Glucantime was contraindicated and therapeutic failure with miltefosine occurred. He was treated with liposomal amphotericin B in a single dose of 15mg/kg with adequate tolerance, no significant deterioration of renal function and resolution of skin lesions after 30 and 60 days of follow up.

Changing strategies for treatment of systemic mycoses

There have been a number of changes in strategies in antifungal therapy in the past few years. AIDS related mycoses hade decreased, and the increse of fluconazole resistant Candida albicans may be slowing because fewer severely immune depressed patients require constant fluconazole suppression. Candida species continue to be relatively commun blood culture isolates. About half of these are C.albicans and half non-albicans species. In recent years, we have moved from the use of amphotericin B to fluconazole for initial treatment of candidemia. We have seen fluconazole resistent isolates emerge, primarily C.glabrata and a few C.krusei, but also C.albicans. It is unclear whether the increasing use of fluconazole in intensive care units will worsen this problem. There appears to be no advantage for the lipid formulations of amphotericin B, though they are useful to reduce or prevent renal toxicity. In the United States and Europe, prevention and treatment of aspergillosis have become increasingly important. There are increasing data suggesting that lipid formulations are more effective for both treatment and prevention of invasive disease in the most vulnerable patients with this infection. Renal toxicity is reduced but not avoided by use of the lipid formulations of amphotericin B. For those patients with less acutely progressing disease, the triazoles may be effective options. It is unclear at present whether itraconazole, voriconazole, or posaconazole will be the most favored drug. One promising new class, now in clinical trials, is the echinocandin group. Other agents, such as the sordarins, the chitin synthase inhibitors, and topoisomerase inhibitors, have promise but are much earlier in development. Unfortunately, we still have >50 (percent) treatment failure with acute invasive aspergillosis, and 20 (percent)-30 (percent) failures with candidemia. Now that we have multiple classes of antifungal drugs available, and others in preclinical trials, it would be advantageous to begin more active exploration of conbination therapy with antifungals and with combined immune modulators and antifungals.

Antifungal susceptibility testing: Progress and future developments

The establishment of a standardized broth reference method for antifungal susceptibility testing of yeastshas opened the door to a number of interesting and useful developments. The adaptation of the reference macrodilution method to a microdilution method has significantly increased the clinical utility of antifungal susceptibility testing, and both methods are now included in the NCCLS document M27-A. The publication of quality control limits for five antifungal agents, coupled with the estabilisment of interpretive MIC breakpoints for three agents, provides useful parameters to survey clinical isolates of Candida and other yeast species. Adaptations of the M27 microdilution method for testing molds has also proved feasible. These developments have made it possible for a number of recent studies designed to expand the capabilities of laboratories to perform antifungal susceptibility testing and to enhance our understanging of trends in antifungal susceptibility. The availability of reference methods also provides a useful touchstone for the development of commercial products that promise to be more user friendly and to further improve test standartization. Products in varying stages of development include two colorimetric microdilution methods (Sensititre and KPI) and the Etest stable gradient MIC method. Preliminary data indicate that these methods are viable alternatives to the reference method for testing of yeasts. Furthermore, Etest may also prove useful for testing molds. Future expectations for antifungal susceptibility testing includes improved ability to detect amphotericin B resistence, development of an NCCLS document for susceptibility testing of molds, and application of these methods for testing dematophytes. Incorporation of antifungal susceptibility testing methods into the clinical trials of new antifungal agents will facilitate the establishment of clinical correlates and further enhance the clinical utility of antifungal susceptibility testing.

Efficacy and tolerabiliy of loposomal amphotericin B (Ambisome) in the treatment of visceral leishmaniasis in Brazil

Thirty-two patients were enrolled in an open-label, dose/schedule ranging clinical trial to evaluate the efficacy and tolerability of loposomal amphotericin B (Ambisome) in the treatment of visceral leishmaniasis. All the patients received a dose of 2mg/kg daily for the first 4 days, followed by a single repeat dose of 2mg/kg at day 10 in 4 patients (total dose 10mg/kg); repeat doses on days 5, 6, and 10 in 13 patients (total dose 14/kg); or daily doses were continued on days 5 through 10 in 15 patients (total dose 20mg/kg). Patients had a mean age of 9 years, ranging between 3 and 26 years. Their mean weight was 25.9kg, ranging between 9.5kg and 75kg. All patients had splenomegaly,31/32 hepatomegaly, and 20 patients tested had leishmania documented on splenic aspirate. Six of the 32 patients were treated after relapse following antimony therapy. The duration of illness prior to therapy was a mean of 2 months, ranging between 2 weeks and 23 months. During and after treatment, there were significant reductions in liver and spleen size, and significant increases in body weight, hemoglobin levels and white blood cell counts. All patients showed initial cure at the 1 mouth follow-up. Seven patients relapsed between 2 and 6 months after the start of treatment. There was no dose relationship to the occurrence of relapse. The relapse rate in children 5 years of age or less was 7/15 (47 percent). Associated causes of relapse were refractory disease (i.e., previous relapses) in 2, severe malnutrition in 1, and concurrent disease (meningococcal meningitis) in 1. In the other 2 cases, no associated event was observed except young age (ages 3 and 5 years). One relapsed patient was treated successfully with 14 days of lipid amphotericin B, and the others were cured by use of antimony for 20 to 30 days. There were no dose related adverse events. The most common event was fever which occurred in 13/32 patients (41 percent); 3/4 patients in the 10mg dose group, 7/13 in the 14mg dose group, and 3/15 in the 20mg dose group. Three patients had cardiac arrhythmia, one also with myocarditis diagnosed 2 weeks after therapy was discontinued. One patient developed hepatitis after dose 3 and the drug was discontinued. We concluded that liposomal amphotericin B is effective in a daily dose of 2mk/kg given for 5-10 doses as an initial cure, but that relapse occurs in young children, particularly those with documented treatment resistant disease or concurrent malnutrition...

Pruebas de sensibilidad in vitro para levaduras: evaluación de un micrométodo / In vitro susceptibility tests for yeasts: evaluation of a micromethod

Se evaluó un micrométodo para la realización de pruebas de sensibilidad de levaduras frente a antifúngicos, basado en el macrométodo en medio líquido estandarizado por el National Committee for Clinical Laboratory Standards (NCCLS) Subcommittee on Antifungal Susceptibility Testing. En este trabajo se compararon ambos métodos utilizando 6 cepas de referencia de diferente sensibilidad a los siguientes antifúngicos, anfotericina B (AMB), flucitosina (5FC), fluconazol (FCZ), itraconazol (ITZ), ketoconazol (KTZ) y miconazol (MCZ). Se observaron variaciones de sólo 1 ó 2 diluciones entre los resultados de las concentraciones inhibitorias mínimas (CIM) obtenidas con las dos técnicas. Asimismo, se compararon las lecturas visuales de CIM por micrométodo con las mediciones turbidimétricas del crecimiento en distintas concentraciones de antifúngicos frente a 47 aislamientos de Candida albicans. Existió una correlación significativa (p<0.001) entre CIM visual y la inhibición del 80 por ciento de crecimiento determinada por turbidimetría con AMB, 5FC, FCZ, ITZ y MCZ; en cambio no hubo correlación alguna para KTZ (p=1.00)