RESUMO
By selecting a unique combination of lipids and amphotericin B, the liposome composition for AmBisome® (L-AmBis) has been optimized resulting in a formulation that is minimally toxic, targets to fungal cell walls, and distributes into and remains for days to weeks in various host tissues at drug levels above the MIC for many fungi. Procedures have been standardized to ensure that large scale production of the drug retains the drug's low toxicity profile, favorable pharmacokinetics and antifungal efficacy. Tissue accumulation and clearance with single or multiple intravenous administration is similar in uninfected and infected animal species, with tissue accumulation being dose-dependent and the liver and spleen retaining the most drug. The efficacy in animals appears to be correlated with drug tissue levels although the amount needed in a given organ varies depending upon the type of infection. The long-term tissue retention of bioactive L-AmBis in different organs suggests that for some indications, prophylactic and intermittent drug dosing would be efficacious reducing the cost and possible toxic side-effects. In addition, preliminary preclinical studies using non-intravenous routes of delivery, such as aerosolized L-AmBis, catheter lock therapy, and intravitreal administration, suggest that alternative routes could possibly provide additional therapeutic applications for this antifungal drug.
Assuntos
Anfotericina B/administração & dosagem , Anfotericina B/farmacologia , Antifúngicos/administração & dosagem , Antifúngicos/farmacologia , Micoses/tratamento farmacológico , Anfotericina B/efeitos adversos , Anfotericina B/farmacocinética , Animais , Antifúngicos/efeitos adversos , Antifúngicos/farmacocinética , Química Farmacêutica , Relação Dose-Resposta a Droga , Liberação Controlada de Fármacos , Humanos , Lipossomos , Fígado/metabolismo , Baço/metabolismo , Distribuição TecidualRESUMO
Given the interest in the ectodomain of the matrix 2 (M2e) channel protein as a target for development of a universal influenza vaccine, we examined the role of the antigen configuration of M2e in generating a protective immune response. A series of M2e mutations and a truncated M2e segment were prepared as a means of controlling the formation of monomer, dimer, and higher order multimeric forms of M2e. Each of these M2e peptides was incorporated into a liposome-based vaccine technology platform previously shown to stimulate a protective response to influenza A infection using M2e as a mixture of monomers, dimers and multimers (L-M2e1-HD/MPL). Our results using these modified forms of M2e produced 90-100% survival following lethal challenge with H1N1 (A/PR/8/34) in both inbred BALB/c and outbred Swiss Webster mice vaccinated with a truncated monomeric form of the M2 protein, M2e1-15 in liposomes. These observations show that a tetrameric configuration is not required to elicit significant protection when the M2e antigen is formulated in immunogenic liposomes and further, that the first 15 amino acids of M2e likely play a primary role in providing the protective immune response.
Assuntos
Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas da Matriz Viral/imunologia , Animais , Feminino , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/química , Lipossomos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Infecções por Orthomyxoviridae/imunologia , Multimerização Proteica , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genéticaRESUMO
Unilamellar liposomes that retain their contents in the systemic circulation can alter the pharmacokinetics of anticancer agents in favorable ways. It has long been recognized that certain liposome compositions may increase the local drug concentration substantially above that achievable with a free drug. We report here that liposomes can alter the in vivo disposition of an entrapped drug not only on a macroscopic but also on a microscopic scale. We show through in vitro studies that intact liposomes composed of distearoylphosphatidylcholine and cholesterol and containing daunorubicin (DaunoXome) are taken up into P1798 tumor cells. These liposomes produce an enhanced cytotoxicity relative to the free drug for incubation times longer than about 8 h. For in vivo studies, we developed and used a noninvasive fluorescence imaging technique to follow the accumulation of liposomal daunorubicin within murine tumors. With this method, we show that the maximum concentration of the available liposomal drug in tumors exceeds that of the free drug, and additionally, liposomal daunorubicin persists at high levels for several days. Total liposome-delivered drug fluorescence from whole tumor extracts peaks at about 8 h. In comparison, the fluorescence intensity of daunorubicin demonstrate persistent high levels of daunorubicin fluorescence within cells and throughout the tumor masses. Free daunorubicin, in contrast, transiently achieves modest levels of fluorescence and rapidly drops to background within a few h. These results indicate distinct mechanisms for the localization of free and liposomal daunorubicin, suggesting that liposmal daunorubicin can provide sustained intracellular levels of the drug within the tumor.
Assuntos
Daunorrubicina/farmacocinética , Animais , Sobrevivência Celular/efeitos dos fármacos , Daunorrubicina/administração & dosagem , Preparações de Ação Retardada , Portadores de Fármacos , Feminino , Lipossomos , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/metabolismo , Linfoma não Hodgkin/patologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Células Tumorais CultivadasRESUMO
AmBisome is a lyophilized preparation of liposomal amphotericin B. The acute intravenous toxicity of AmBisome was evaluated in mice and rats, and the LD50S were found to be greater than 175 and 50 mg/kg, respectively. The corresponding LD50S for conventional amphotericin B were approximately 2.3 and 1.6 mg/kg for mice and rats, respectively. The multiple dose toxicity test confirmed that AmBisome was well tolerated by both species. There were no deaths observed among mice receiving 25 or 50 mg/kg AmBisome for 14 days, and only two deaths among mice receiving 75 mg/kg AmBisome. One rat died in the group receiving 25 mg/kg AmBisome for 30 days. However, five of ten and nine of ten rats died in the groups treated with 50 and 75 mg/kg AmBisome, respectively. Hepatotoxicity was evident by elevation in serum liver enzyme levels for these groups. Initial pharmacokinetic evaluations demonstrated that peak plasma concentrations of 87 and 118 mg/kg, respectively, were attained in mice and rats after injection with 5 mg/kg AmBisome. Terminal plasma half-lives of 3.36 and 7.56 h were calculated for mice and rats, respectively. Tissue accumulations of amphotericin B resulting from multiple dose intravenous administration of either conventional amphotericin B or AmBisome were determined. At equivalent doses of 1 mg/kg, AmBisome treatment resulted in higher liver and spleen uptake of drug, but lower kidney and lung uptake than amphotericin B. At 5 mg/kg, AmBisome treatment resulted in concentrations of drug in the kidney and lungs that were comparable to corresponding tissue levels observed in the group treated with 1 mg/kg conventional amphotericin B.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Anfotericina B/administração & dosagem , Anfotericina B/farmacologia , Anfotericina B/toxicidade , Animais , Colesterol , Portadores de Fármacos , Feminino , Liofilização , Dose Letal Mediana , Lipossomos , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilcolinas , Ratos , Ratos EndogâmicosRESUMO
The antifungal efficacy and cellular toxicity of AmBisome(R) and non-liposomal amphotericin B were compared in cultured epidermal Langerhans cells infected with Candida glabrata. Uptake of the yeast was determined by light and electron microscopy, and viability was assessed by plating dilutions of lysates from yeast-infected Langerhans cells and counting colony forming units. The Candida-infected Langerhans cells were incubated for 6, 24 or 48 h with 12.5 micro ml-1 of AmBisome or non-liposomal amphotericin B, non-drug-containing liposomes or media. Intracellular C. glabrata incubated with media or non-drug-containing liposomes showed a 2 log increase in cfu, and microscopic examination revealed budding yeast within the Langerhans cells. Both liposomal and non-liposomal amphotericin B treatment reduced intracellular growth of C. glabrata by 5 logs over 48 h of incubation. A morphometric analysis of cell ultrastructure demonstrated that AmBisome-treated Langerhans cells retained their cell architecture, but Langerhans cells treated with non-liposomal amphotericin B were characterized by the absence of intact organelles, disrupted non-granular cytoplasm and the presence of many large vacuoles. In conclusion, AmBisome was significantly less toxic for epidermal Langerhans cells than amphotericin B, but demonstrated comparable antifungal efficacy. After 48 h of drug exposure, both forms of amphotericin B effectively inhibited intracellular growth of C. glabrata, but only AmBisome did not damage the Langerhans cells.
Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Células de Langerhans/microbiologia , Animais , Animais Recém-Nascidos , Candida/fisiologia , Células Cultivadas , Células de Langerhans/efeitos dos fármacos , Células de Langerhans/ultraestrutura , Lipossomos , CamundongosRESUMO
This investigation examined the therapeutic efficacy of AmBisome, a unilamellar (55-75 nm) liposome amphotericin B preparation with a murine LD50 by the intravenous route of greater than 175 mg/kg amphotericin B. Both fungal burden and survival were used to evaluate the drug's efficacy against murine candidosis and cryptococcosis. Single and multiple dose intravenous treatment with AmBisome (2.5, 5.0 and 10.0 mg/kg) reduced the colony forming units/mg kidney in candida-infected mice by 99% and improved survival by at least 40% relative to untreated control mice. Repeated intravenous dosing of candida-infected mice with equivalent amounts (0.75 mg/kg) of conventional amphotericin B (Fungizone) or AmBisome showed comparable reduction of yeasts in the kidneys. When mice were infected systemically with Cryptococcus neoformans, all but one of the 30 mice given AmBisome (5.0, 7.5 or 10.0 mg/kg) survived until the experiment was terminated 35 days after infection. Liver and spleen cultures from AmBisome-treated mice were negative for fungal growth. All the mice given conventional amphotericin B intraperitoneally at 4.5 mg/kg survived and cleared the infection from the livers although some of the mice had infected spleens. The percentage of cultured brains free of cryptococcus was 89% following treatment with 10.0 mg/kg AmBisome, and 80% with 4.5 mg/kg conventional drug. These preclinical studies of systemic candidosis and cryptococcosis demonstrate comparable efficacy of AmBisome and conventional amphotericin B at low doses and improved efficacy with AmBisome at doses higher than can be safely administered of the conventional drug.