Determination of free and liposomal Amphotericin B in human plasma by liquid chromatography–mass spectroscopy with solid phase extraction and protein precipitation techniques

Amphotericin B is available in various drug delivery systems such as cholesteryl sulfate complex, as lipid complex, and as liposomal formulation. The separation and measurement of free drug (drug which is not bound with liposomal lipids) and liposomal drug (drug which is entrapped in liposomes) in the human plasma after injection of liposomal Amphotericin B is of prime importance due to toxicity concerns. A robust, specific and sensitive method has been developed to effectively separate and then quantify the free drug and liposomal drug, present in human plasma. This method utilizes solid phase extraction Oasis HLB cartridges, which retains the free drug and the liposomal Amphotericin B was eluted from the cartridge in first step. The eluted liposomal Amphotericin B was then extracted from lipids by protein precipitation method using 2% dimethylsulfoxide (DMSO) in acetonitrile. After separation and extraction, the quantification of free and liposomal fractions of Amphotericin B was performed by HPLC–MS–MS technique. The chromatographic separation was performed using Chromolith Performance RP 18e column. The mobile phase composed of 5 mM ammonium acetate, methanol and acetonitrile and a gradient elution program was used. The calibration curves were found to be linear for free Amphotericin B (0.25–15.0 μg/ml) and liposomal Amphotericin B (1.0–100.0 μg/ml). The recovery was about 96% for free Amphotericin B and about 92% for liposomal Amphotericin B. Recoveries were consistent over the linearity ranges defined. The intra-batch and inter-batch accuracy and precision fulfilled the international requirements. The stability of free and liposomal Amphotericin B was assessed under different storage conditions.     

Liposomal amphotericin-B in the control of experimental aspergillosis in mice: Part I--Relative therapeutic efficacy of free and liposomal amphotericin-B

An animal model system for aspergillosis in BALB/c mice has been developed to evaluate the therapeutic efficacy of liposomal Amphotericin-B (Amp-B) and commercial Amp-B (Fungizone). Amp-B was intercalated into liposomes composed of egg phosphatidylcholine, phosphatidylethanolamine and cholesterol in a molar ratio of 6:1:3. A single dose (0.5 mg/kg body wt) of Amp-B both alone as well as in liposomal preparation was injected (i.v.) into animals infected with Aspergillus fumigatus. An increase in survival rate of animals and decrease in fungal count in lung, the most affected organ, were observed with liposomal formulation. Inclusion of Amp-B into liposomes also reduced the toxicity of the drug. Tissue distribution analysis of Amp-B by HPLC showed an increase in concentration of the drug in lung for both free and liposomal Amp-B in infected animals as compared to normal. Use of liposomal Amp-B increased the concentration of the drug in the disease affected organs such as lung, spleen. A longer persistence of the drug in the infected organs was also observed. The results suggest that inclusion of Amp-B in liposomes decreases its toxicity and improves therapeutic efficacy which at least in part could be due to more deposition and longer persistence of the drug in infected tissues.     

Optimizing efficacy of Amphotericin B through nanomodification

The polyene antibiotic Amphotericin B (AMB) is one of the first therapeutic agents to be marketed commercially as nanosized formulations in which the drug is associated with lipids as liposomes or complexes. In this way, its renal toxicity is reduced and its therapeutic index improved. This review summarizes the particular properties of AMB which justify this type of formulation and the early work leading up to their development. The clinical results obtained in the treatment of fungal infections are reviewed and their activity against leishmaniasis is also evoked. Some newer formulations of AMB, based on both lipids and polymers are described. In particular, their potential by the oral and pulmonary routes are discussed. Finally, the development of targeted systems to deliver the drug to specific cells and tissues is considered.     

Evaluation of antifungal pharmacodynamic characteristics of AmBisome against Candida albicans

A liposomal formulation of Amphotericin B (AmBisome), with small unilamellar vesicles containing amphotericin B, shows characteristic pharmacokinetics as liposomes, and in consequence, has different pharmacological activity and toxicity from amphotericin B deoxycholate (Fungizone). In this study, we evaluated the antifungal pharmacodynamic characteristics of AmBisome against Candida albicans using the in vitro time-kill method and murine systemic infection model. A time-kill study indicated that the in vitro fungicidal activities of AmBisome and Fungizone against C. albicans ATCC 90029 increased with increasing drug concentration. For in vivo experiments, leucopenic mice were infected intravenously with the isolate 4 hr prior to the start of therapy. The infected mice were treated for 24 hr with twelve dosing regimens of AmBisome administered at 8-, 12-, 24-hr dosing intervals. Correlation analysis between the fungal burden in the kidney after 24 hr of therapy and each pharmacokinetic/pharmacodynamic parameter showed that the peak level/MIC ratio was the best predictive parameter of the in vivo outcome of AmBisome. These results suggest that AmBisome, as well as Fungizone, has concentration-dependent antifungal activity. Furthermore, since AmBisome can safely achieve higher concentrations in serum than Fungizone, AmBisome is thought to have superior potency to Fungizone against fungal infections.     

Effects of the detergent sucrose monolaurate on binding of amphotericin B to sterols and its toxicity for cells

Amphotericin B (AmB) is a potent antifungal agent used to treat patients with systemic mycoses. The cytotoxicity of AmB is related to its binding to membrane sterols and its clinical usefulness is based on its greater affinity to ergosterol, the fungal sterol, compared to the mammalian cell sterol, cholesterol (1-3). Here we report that sucrose monolaurate (L.S.) decreased the binding of AmB to cholesterol without interfering with its binding to ergosterol. Furthermore, the toxicity of AmB for mouse erythrocytes (RBC) and cultured mouse fibroblasts, L-929, cells was significantly decreased by low concentrations of L.S., whereas under the same conditions, its toxicity for Candida albicans was unaffected. We observed a very good correlation between the spectroscopic and cell studies. The results reported here on the effects of L.S. on the selectivity of AmB toxicity for fungal cells compared to animal cells and the relative nontoxic nature of sugar esters suggest a potential for compounds of this type to enhance the therapeutic index of AmB.     

Invasive maxilar sinusitis by Rhizopus oryzae

We herein present a diabetic with non Hodgkin lymphoma patient that had been treated with steroids and developed fungal invasive sinusitis. The patient had intensive facial pain that did not respond to antibiotics and on clinical inspection had a necrotic lesion on right nasal area. A smear and biopsy tissue showed broad non septate hyphae and on cultures Rhizopus oryzae was isolated. There was an unfavorable outcome, and the patient died even though liposomal Amphotericin B was administered and surgical treatment was performed.     

Effect of cisplatin containing liposomes formulated by unsaturated chain-containing lipids on gynecological tumor cells

Gynecological tumors are major therapeutic areas of platinum-based anticancer drugs. Here, we report the characterization and in vitro biological assays of cisplatin-containing Egg L-α-phosphatidylcholine liposomes with different amounts of cholesterol. Dynamic light scattering estimated sizes of all obtained liposomes in the 100?nm range that are suitable for in vivo use. On the basis of these data and of the drug loading values, the best formulation has been selected. Stability and drug release properties of platinum-containing liposomes have been verified in serum. The growth inhibitory effects of both liposomal and free drug in a panel of ovarian and breast human cancer cell lines, characterized by a different drug sensitivity, give comparable or better results with respect to free cisplatin drug.     

Liposomal hamycin in the control of experimental aspergillosis in mice: relative toxicity, therapeutic efficacy and tissue distribution of free and liposomal hamycin.

Therapeutic efficacy of liposomal Hamycin has been evaluated in an animal model system for aspergillosis in Balb/c mice. Hamycin was intercalated into soya phosphatidyl choline (SPC), SPC: choline (1:1, vol./vol.) and DMPC liposomes. A single dose of either 0.1 mg/kg, 0.25 mg/kg or 0.5 mg/kg of liposomal Hamycin and 0.1 mg/kg of free Hamycin was injected (i.v.) into animals infected with Aspergillus fumigatus. An increase in the survival rate of animals along with decrease in fungal count in various organs was observed with liposomal administration. Incorporation of cholesterol into liposomes decreased the in vivo toxicity of Hamycin in a dose dependent manner. However, antifungal activity both in the presence and absence of cholesterol showed marked variation as compared to that of non-aromatic polyenes, e.g. amphotericin B. Analysis of Hamycin distribution by HPLC in various tissues revealed higher blood concentration of this drug, when given in free form, compared to its liposomised form. These studies suggest that liposomal Hamycin is more effective than free Hamycin in controlling the experimental Aspergillosis.     

Mechanism of Action of Amphotericin B at the Cellular Level. Its Modulation by Delivery Systems

Abstract

Amphotericin B (AmB) is the drug of choice for the treatment of systemic fungal infections, but its use is hampered by its severe side-effects. A better understanding of its mechanisms of action is needed to develop new AmB formulations with an optimal selectivity between fungal and mammalian cells. Interactions between AmB and cells depend on the concentration of the drug. Stimulatory effects, modulation of the activity of immunocompetent cells and inhibition of yeast adherence are early events that precede the actual cellular toxicity. If membrane permeability alterations are considered to be the first toxic step, cell death results not only from osmotic imbalances, but also from additional mechanisms, such as lipid peroxidation, inhibition of membrane enzymes and blockade of endocytosis. The selectivity between fungal and mammalian cells takes its origin from the difference in the nature of the membrane sterol: ergosterol in fungi, cholesterol in mammalian cells. Transmembrane pores result from different mechanisms according to the sterol: ergosterol-AmB complexes are formed from monomelic AmB in solution, which is the only form present in aqueous medium at low AmB concentrations, whereas pores in the cholesterol containing membrane result from the adsorption onto the membrane surface of aqueous self-associated AmB, that appears in medium when AmB concentration increases. The liposomes seem to sequester AmB in a manner which makes it unavailable for mammalian cells, but maintains its access to fungal cells. The transfer of AmB by progressive diffusion of free AmB through the aqueous phase could explain the enhancement of the therapeutic index of the drug by liposomes, since the induction of pore formation needs a higher threshold of drug for host cell than for fungal cell membranes. The closed structure of the vehicle is not required to enhance the selectivity of the drug: esters of sucrose or high concentration of sodium deoxycholate afford a protective effect as well. Macrophages, after phagocytosis of liposomal AmB, may be considered as a reservoir of AmB, from which the drug is progressively released. Finally, the strong binding of AmB to the delivery system reduces the amount of drug bound to serum components and thus the endocytosis of AmB through the LDL receptor, resulting in lower toxicity.     

In vitro cytotoxic study of immunoliposomal doxorubicin targeted to human CD34(+) leukemic cells

The expression of CD34 antigen in acute myelogenous leukemias is considered an unfavourable prognosis marker for response to anticancer drugs and duration of remission. This study investigated the applicability of long-circulating immunoliposomes loaded with doxorubicin targeted to CD34 antigen present on MDR(+) human myelogenous leukemia KG-1a cell line. Immunoliposomal doxorubicin showed a higher cytotoxicity against KG-1a cells than non-targeted liposomal doxorubicin, but it did not improve over that of free drug. Although no reversal of doxorubicin resistance was found to occur through its liposomal encapsulation, a therapeutic benefit can be obtained by the selective cytotoxicity observed. Endocytosis studies demonstrated that, after binding to CD34 antigen, the immunoliposomes are not internalized by the KG-1a cells and so the cytotoxic effect might be due to drug released into the space near the cell membrane. Thus, immunotargeting of liposomal doxorubicin to CD34(+) leukemic cells may only provide an ex vivo strategy for site-selective CD34(+) leukemia cell killing.     

Human recombinant antibody to HSP90: a natural partner in combination therapy

Recent years have seen the development of the concept of combination therapy for treating severe fungal sepsis. The advantages of this approach are a potential improvement in patient survival and a reduction in the chance of resistance developing to each of the single agents. The disadvantage is that combining drugs may increase the chance of toxicity. Mycograb is a genetically recombinant antibody against fungal heat shock protein 90 (hsp90) which is poised to become the mainstay of combination therapy. This paper presents data on how hsp90 is important to fungi and what role it might play in human disease with possible interactions with interleukin 6 and nitric oxide. There is discussion of preclinical data demonstrating synergy in vitro between Mycograb and amphotericin B and caspofungin. The progress of Mycograb through a Phase II pharmacokinetic study when used in escalating doses with a liposomal amphotericin B preparation has also been reviewed. The concepts behind a Phase II pivotal study, where Mycograb or a placebo was given in combination with a liposomal amphotericin B drug for five days for the treatment of disseminated candidiasis are discussed.     

几种新型抗真菌药物简介

脂质型二性霉素B,新型唑类药物如伏立康唑,以及作用于真菌细胞壁的药物如卡泊芬净和米卡芬净的问世,反映了抗真菌药物研究向高效、广谱、低毒的方向发展的一个特点,无疑为各种类型真菌感染的药物治疗提供了新型的有力的手段;与传统的抗真菌药物如脱氧胆酸二性霉素B和氟康唑等相比,这些药物临床疗效好,毒副作用低,对于系统性真菌感染的防治具有重大意义,文中就这方面的信息做了简介。     

Anfotericina B liposomal: farmacología clínica,farmacocinética y farmacodinamia

Liposomal amphotericin B is a lipid formulation of the antifungal drug amphotericin B with some distinguishing characteristics in its pharmacological behavior that entail some clinical differences of great interest. The significant improvement in the systemic and renal tolerability is one of them. This fact is related to the great stability of the liposome, promoted by its negative charge, the presence of cholesterol and the remarkable thermo-stability of the remaining lipids that compose it. In this situation, amphotericin B seems to be released from the liposome not spontaneously but when the liposome binds to the ergosterol in the fungal cell membrane. For this reason, there is almost no free amphotericin B in plasma or tissues, although it seems that its availability is greater when there is fungal infection. As a consequence, when the pharmacokinetic behavior is studied, the concentration and availability of liposomal amphotericin B are very high, and its volume of distribution is reduced in comparison with the other formulations.     

The effects of amphotericin B on pure and ergosterol- or cholesterol-containing dipalmitoylphosphatidylcholine bilayers as viewed by 2H NMR

Amphotericin B (AmB) is a widely used polyene antibiotic to treat systemic fungal infections. This drug is known to be lethal to fungal cells but it has also side effect toxicity on mammalian cells. The mechanism of action of AmB is thought to be related to the difference of the main sterol present in the mammalian and the fungal cells, namely cholesterol and ergosterol, respectively. The effect of AmB has been investigated on pure dipalmitoylphosphatidylcholine (DPPC) and on cholesterol- and ergosterol-containing DPPC bilayers by 2H NMR spectroscopy. The 2H NMR results first confirm that AmB forms a complex with sterol-free DPPC bilayers, the interaction causing the structurization of the lipids and the increase of the gel-to-lamellar fluid DPPC phase transition temperature with increasing concentration of the antibiotic. The results also show that the effects of AmB on cholesterol- and ergosterol-containing DPPC bilayers are remarkably different. On one hand, the drug causes an increase of the orientational order of the lipid acyl chains in cholesterol-containing membranes, mostly in high cholesterol content membranes. On the other hand, the addition of AmB disorders the DPPC acyl chains when ergosterol is present. This is thought to be due to the direct complexation of the ergosterol by AmB, causing the sterol ordering effect to be weaker on the lipids.     

Cryptococcal therapies and drug targets: the old,the new and the promising

Half a century after the introduction of Amphotericin B the management of cryptococcosis remains unsatisfactory. The disease, caused primarily by the two fungal species Cryptococcus neoformans and Cryptococcus gattii, remains responsible for considerable morbidity and mortality despite standard medical care. Current therapeutic options are limited to Amphotericin B, azoles and 5‐flucytosine. However, this organism has numerous well‐characterized virulence mechanisms that are amenable to pharmacological interference and are thus potential therapeutic targets. Here, we discuss existing approved antifungal drugs, resistance mechanisms to these drugs and non‐standard antifungal drugs that have potential in treatment of cryptococcosis, including immunomodulatory strategies that synergize with antifungal drugs, such as cytokine administration or monoclonal antibodies. Finally, we summarize attempts to target well‐described virulence factors of Cryptococcus, the capsule or fungal melanin. This review emphasizes the pressing need for new therapeutic alternatives for cryptococcosis.     

Candida albicans Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses

Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-β-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen Candida albicans by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes Candida albicans cell wall-associated β-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to Candida albicans but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to Candida albicans in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.     

Biosynthesis of amphotericin derivatives lacking exocyclic carboxyl groups

Amphotericin B is a medically important antifungal antibiotic that is also active against human immunodeficiency virus, Leishmania parasites, and prion diseases. The therapeutic use of amphotericin B is restricted by severe side effects that can be moderated by liposomal formulation or structural alteration. Chemical modification has shown that suppression of charge on the exocyclic carboxyl group of amphotericin B substantially reduces toxicity. We report targeted deletions of the amphN cytochrome P450 gene from the chromosome of the amphotericin-producing bacterium Streptomyces nodosus. The mutant strains produced amphotericin analogues in which methyl groups replace the exocyclic carboxyl groups. These compounds retained antifungal activity and had reduced hemolytic activity.     

Targeted delivery of indinavir to HIV-1 primary reservoirs with immunoliposomes

The tissue distribution of indinavir, free or incorporated into sterically stabilized anti-HLA-DR immunoliposomes, has been evaluated after a single subcutaneous injection to C3H mice. Administration of free indinavir resulted in low drug levels in lymphoid organs. In contrast, sterically stabilized anti-HLA-DR immunoliposomes were very efficient in delivering high concentrations of indinavir to lymphoid tissues for at least 15 days post-injection increasing by up to 126 times the drug accumulation in lymph nodes. The efficacy of free and immunoliposomal indinavir has been evaluated in vitro. Results showed that immunoliposomal indinavir was as efficient as the free agent to inhibit HIV-1 replication in cultured cells. The toxicity and immunogenicity of repeated administrations of liposomal formulations have also been investigated in rodents. No significant differences in the levels of hepatic enzymes of mice treated with free or liposomal indinavir were observed when compared to baseline and control untreated mice. Furthermore, histopathological studies revealed no significant damage to liver and spleen when compared to the control group. Liposomes bearing Fab′ fragments were 2.3-fold less immunogenic than liposomes bearing the entire IgG. Incorporation of antiviral agents into sterically stabilized immunoliposomes could represent a novel therapeutic strategy to target specifically HIV reservoirs and treat more efficiently this retroviral infection.     

Targeted delivery of indinavir to HIV-1 primary reservoirs with immunoliposomes.

The tissue distribution of indinavir, free or incorporated into sterically stabilized anti-HLA-DR immunoliposomes, has been evaluated after a single subcutaneous injection to C3H mice. Administration of free indinavir resulted in low drug levels in lymphoid organs. In contrast, sterically stabilized anti-HLA-DR immunoliposomes were very efficient in delivering high concentrations of indinavir to lymphoid tissues for at least 15 days post-injection increasing by up to 126 times the drug accumulation in lymph nodes. The efficacy of free and immunoliposomal indinavir has been evaluated in vitro. Results showed that immunoliposomal indinavir was as efficient as the free agent to inhibit HIV-1 replication in cultured cells. The toxicity and immunogenicity of repeated administrations of liposomal formulations have also been investigated in rodents. No significant differences in the levels of hepatic enzymes of mice treated with free or liposomal indinavir were observed when compared to baseline and control untreated mice. Furthermore, histopathological studies revealed no significant damage to liver and spleen when compared to the control group. Liposomes bearing Fab' fragments were 2.3-fold less immunogenic than liposomes bearing the entire IgG. Incorporation of antiviral agents into sterically stabilized immunoliposomes could represent a novel therapeutic strategy to target specifically HIV reservoirs and treat more efficiently this retroviral infection.     

Preparation, relative toxicity, chemotherapeutic activity, and pharmacokinetics of liposomal SJA-95: a new polyene macrolide antibiotic

The research work was designed to compare the relative toxicity, chemotherapeutic activity, and pharmacokinetic parameters of liposomal incorporated SJA-95 with that of free SJA-95, with an objective to reduce toxicity and improve therapeutic activity in vivo. Liposomal-incorporated SJA-95 (Lip SJA-95), prepared using the proliposome method, was found to exhibit a higher LD(50) value in mice, and the relative toxicity was about 2.5 times lower than that of the free drug. Lip SJA-95 treatment in experimental mice model of Candidiasis showed increased survival and reduced fungal loads in various organs. The pharmacokinetic profile of the free and liposomal drug was evaluated by administration of free and Lip SJA-95 intravenously to healthy albino rabbits in a crossover fashion. Lip SJA-95 showed an initial fall in plasma levels and longer half-life. The improved microbial clearance following treatment with Lip SJA-95 could be attributed partly to an increased tissue uptake, which was reflected in a marked increase in volume of distribution (V(d)) and longer half-life (T(1/2)). The present results clearly indicated that Lip SJA-95 treatment led to prolonged survival time, effective microbiological clearance, and reduced toxicity in the mice model of Candidiasis.