Lipid-based formulations of amphotericin B

Amphotericin B remains the drug of choice for the treatment of invasive fungal infections and visceral leishmaniasis. However, both the dose-dependent nephrotoxicity and the low response rates (10-80%) associated with amphotericin B limit its clinical use. The first marketed formulation of amphotericin B with deoxycholate, Fungizone, remains the "gold standard" in spite of its renal toxicity. Several investigations have been made to reduce the nephrotoxicity of amphotericin B by formulation strategies. Lipid-based formulations of amphotericin B were found to reduce toxicity and to increase tolerance and therapeutic efficacy. Three lipid formulations are now available in most countries: liposomal amphotericin B (AmBisome), amphotericin B lipid complex and amphotericin B colloidal dispersion. Amphotericin B colloidal dispersion was less nephrotoxic, but immediate reactions to this formulation were as frequent and severe as those to amphotericin B. Amphotericin B lipid complex appeared to be as effective as amphotericin B, with improved general and renal tolerability. Several comparative studies have confirmed that AmBisome has similar or superior efficacy relative to amphotericin B in various fungal infections, in visceral leishmaniasis and also in the empirical treatment of febrile neutropenia. Renal and general tolerability is excellent. A significant drawback to the newer, less toxic, commercial lipid-based formulations is their cost. There is a need to develop more affordable lipid-based formulations of amphotericin B.     

Toxicity of amphotericin B to analbuminemic rats

Nagase analbuminemic rats (NAR), a strain derived from Sprague Dawley rats (SDR), are suitable to determine the in vivo effect of hyperlipidemia and analbuminemia on the toxicity of the antifungal agent amphotericin B (AB). Cholesterol content was increased in all plasma lipoprotein fractions when SDR serum was compared with NAR serum. Incubation of AB with plasma from animals of both strains and further isolation of plasma lipoprotein by ultracentrifugation, showed that, while in SDR approximately 40% of the injected AB was in the lipoprotein fraction, in NAR almost all (approximately 80%) AB was associated with lipoproteins, especially in the low density lipoprotein fraction. AB spectrophotometry showed differences in the extent of AB aggregation between SDR and NAR plasma fractions. The lack of albumin results in aggregation of the AB present in the infranatant fraction. In vivo, a strong reduction in lethality was observed for NAR when compared to SDR (LD 50% of 6.4 and 1.9 mg/kg respectively). The preferential distribution of AB in the plasma lipoprotein fraction seems to affect pharmacological parameters, with a consequent reduction of toxicity.     

Clinical formulations of amphotericin B]

The clinical use of amphotericin B is impaired by its poor water solubility and by the severity of its side effects. Several amphotericin B formulations have already been prepared in an attempt to overcome these disadvantages. The following methods have been proposed to solubilize amphotericin B in water: the complexation of amphotericin B with metallic ions, sodium tetraborate or gamma cyclodextrin or the synthesis of semi-synthetic derivatives. Another approach was to use a carrier (liposomes, lipoproteins, emulsions or surfactants) to target amphotericin B. This paper summarizes and criticizes these formulations.     

The lipid formulations of amphotericin B

Amphotericin B spectrum covers most of the fungal pathogens involved in human diseases. Its use is limited by infusion-related effects and nephrotoxicity. As a result of strong lipophilic properties, encapsulation in liposomes or binding to lipid complexes led to the development of lipid formulations in an attempt to increase both efficacy and safety. Three lipid formulations of amphotericin B are commercially available: a liposomal preparation, a lipid complex and a colloidal dispersion. They differ in their lipid composition, shape, pharmacokinetic behaviour and clinical effects. The nephrotoxicity of these formulations is significantly decreased compared to their parent compound. Infusion-related events are lowest with liposomal amphotericin B. Increased efficacy of the lipid formulations over conventional amphotericin B, however, still has to be demonstrated. These formulations are mainly indicated for the treatment of documented fungal infections in patients failing conventional amphotericin B or with renal impairment. Liposomal amphotericin B is also indicated for empirical therapy of suspected fungal infections in febrile neutropenic patients giving this compound an advantage over the two other formulations. Lipid formulations of amphotericin B are extremely expensive. Whether the increase in cost translates into a long-term benefit for the patient is still unknown.     

Lipid-based amphotericin B formulations: from animals to man

Amphotericin B has been the mainstay of systemic antifungal therapy for over 30 years, despite its serious side-effects, and, although numerous alternative antifungal agents have been developed, none to date has matched the efficacy of amphotericin B. However, modern drug delivery technology has improved the safety of amphotericin B by incorporating it into lipid-based delivery systems, including liposomes. Three such formulations, based on the natural affinity of amphotericin B for lipids, are currently marketed. All increase the therapeutic index of amphotericin B, thereby allowing more aggressive treatment than is possible with the conventional product. However, they differ in structure, side-effect profiles and evidence of proven efficacy as discussed in this review.     

Stability and transdermal absorption of topical amphotericin B liposome formulations

The aim of this study was to characterize the stability and transdermal absorption of amphotericin B (AmB: 0.05 mg/mg lipid) in hydrogenated soya phosphatidylcholine/cholesterol/charged lipid {dicetyl phosphate (−) or stearylamine (+)} liposomes at molar ratios of 1:1:0, 7:2:0, 7:2:1(−) and 7:2:1(+). The AmB contents in liposomes were determined by HPLC with UV detection at 382 nm. Stabilities of AmB in liposome formulations were compared with those in solution and powder forms, during storage at 4, 30 and 45 °C for 90 days. Absorption studies of AmB across the rat skin were conducted, using vertical Franz diffusion cells at 37 °C for 24 h. The slowest degradation was observed in the positive liposome (7:2:1(+)AmB), with shelf life of 1 year (30 °C). In comparison, the shelf lives of AmB in solution and powder were 4 and 14 days, respectively. AmB in positive liposomes seemed to demonstrate the highest flux in stratum corneum (58 ng/cm²/h), while the highest flux in viable epidermis (23 ng/cm²/h) was observed in negative liposomes. AmB entrapped in charged liposomes showed sustained skin absorption. The positively charged liposome might be the best formulation for AmB, due to its higher stability than other formulations.     

Studies of the effects of antifungal cationic derivatives of amphotericin B on human erythrocytes

The novel group of amphotericin B (AmB) cationic derivatives has been examined in terms of relationship between self-association and selective toxicity. In all determinations AmB has been used as reference compound. In vitro toxic effects of the compounds on human erythrocytes were determined by measuring leakage of intracellular potassium ions and hemolysis. Antifungal effects were determined as MIC and intracellular potassium loss. The compounds self-association was followed by UV-Vis spectroscopy. The results suggested that: i) unlike AmB the monomer/self-associated species ratio is not an essential in governing the selective toxicity of the derivatives studied; ii) the presence of a bulky substituent in the AmB molecule, preferably located at the amino group of mycosamine moiety is the structural factor essential for the selective toxicity improvement.     

New formulations and derivatives of amphotericin B for treatment of leishmaniasis

The clinical treatment of leishmaniasis is based on a limited number of drugs, which are associated with adverse effects and have already induced resistance. Amphotericin B (AmB), a polyene antibiotic produced by Streptomyces sp, is the only anti-leishmanial drug which has not induced clinical resistance since its discovery in 1956. The limiting factor in the use of AmB is its toxic effects, mainly nephrotoxicity. The maximal dose of AmB for human use is 1.5 mg/kg which sometimes is not sufficient for cure. The mode of action of AmB is associated with its toxicity: it selectively binds to parasite membrane ergosterol but also, to a lesser extent, to human cholesterol. Apart from this mechanism, AmB has immunomodulatory effects, some of them are deleterious. Reduction of the toxic effects by using lipid formulations allows the infusion of higher doses of AmB. Unfortunately, these formulations are relatively expensive and therefore out of reach for patients in need, in the endemic areas. All the existing formulations are given parenterally, which has obvious disadvantages; most important is the need for hospitalization or multiple visits in the clinic. The current efforts to improve AmB are directed at the production of AmB aggregates in liquid solutions, encapsulation with lipid components, and solubilization by binding to soluble polymers. The expected improved treatment resulting from use of the new formulations is based on better pharmacokinetics, reduced toxicity originating from slow release, targeting to the infected organ and an altered pattern of immune responses (related to AmB). Of particular importance are the attempts to produce derivatives for oral treatment, which will decrease costs of hospitalization and improve applicability for children and the elderly population.     

Liposomal and lipid formulations of amphotericin B. Clinical pharmacokinetics.

Amphotericin B remains a very important drug for the treatment of fungal infections despite its toxicity. Encapsulation of amphotericin B into liposomes appears to reduce the toxic effects and to improve the clinical efficacy, allowing higher dosages to be given. The exact mechanism behind the reduced toxicity is not yet known. Amphotericin B is widely distributed after intravenous administration as the deoxycholate solubilisate. The highest concentrations are found in the liver, spleen and kidney. Protein binding and binding to the tissues is very high. The fate of the drug in the body is not known in detail. Renal and biliary excretion are both low and no metabolites have been identified. The drug is still detectable in the liver, spleen and kidney for as long as 1 year after stopping therapy. The pharmacokinetics of the different liposomal amphotericin B or lipid complexes of amphotericin B, which were recently developed, are quite diverse. A number of these preparations, such as amphotericin B lipid complex (ABLC), 'AmBisome' and amphotericin B colloidal dispersion (ABCD) are in clinical development. Their pharmacokinetics depend to a large extent on the composition and particle size of the liposomes or lipid complexes. Relatively large structures such as ABLC are rapidly taken up by the mononuclear phagocyte system, whereas smaller liposomes remain in the circulation for prolonged periods. In all studies only the total amphotericin B (both free and liposome- or lipid-associated) concentrations were determined. There is a need for studies correlating clinical efficacy and tolerability of liposomal amphotericin B with the pharmacokinetic properties of these formulations.     

Toxicity of amphotericin B emulsion to cultured canine kidney cell monolayers.

The effect of amphotericin B in an emulsion formulation on the integrity of monolayers of kidney cells has been studied. Whereas a conventional solubilized amphotericin formulation (Fungizone, Squibb) caused a loss in monolayer integrity at concentrations above 1 microgram mL-1, the emulsion formulation had no measurable effect on confluence at amphotericin concentrations up to 100 micrograms mL-1. The emulsion retained a comparable antifungal activity to that of Fungizone against Saccharomyces cerevisiae in suspension culture. These results parallel the observed erythrocyte lysis data obtained previously using amphotericin B emulsions, and suggest that the emulsion formulation may have a lower toxicity and improved therapeutic potential over existing formulations.     

Variability in polyene content and cellular toxicity among deoxycholate amphotericin B formulations

STUDY OBJECTIVE: To evaluate the toxicity of amphotericin B deoxycholate formulations. DESIGN: In vitro experiment. SETTING: University research center. MATERIAL: Human mononuclear THP-1 cells. INTERVENTION: The human mononuclear cells were exposed in vitro for 2 hours to the following deoxycholate formulations of amphotericin B, in 2.5- and 5-micro/ml concentrations: Apothecon, Pharmacia, Sigma, Gensia, Pharma-Tek, and VHA. MEASUREMENTS AND MAIN RESULTS: Toxicity of the amphotericin B formulations were assessed by measuring interleukin (IL)-1beta expression in an in vitro model. Amphotericin B content was measured by enzyme-linked immunosorbent assay (ELISA), and amphotericin A and B contents were assessed by spectrophotometry. Endotoxin contamination was evaluated in all reagents. Expression of IL-1beta from Sigma, Pharmacia, and Pharma-Tek formulations was increased approximately 250%, 50%, and 25%, respectively, compared with amphotericin A. Amphotericin B content of Sigma, Pharmacia, Pharma-Tek, and Gensia formulations, as measured by ELISA, was increased approximately 450%, 200%, 200%, and 100%, respectively, compared with Apothecon. This variation could not be explained by differences in amphotericin A or B content as measured by spectrophotometry. CONCLUSION: Amphotericin B is obtained from a fermentation plant and manufactured as a pharmaceutical at different facilities. Both previous clinical observations and the current in vitro evaluation revealed significant differences among the formulations. Likely, other polyenes or pyrogenic toxins in differing amounts are in these formulations, thus explaining the variability in toxicity observed among the formulations.     

Solubility and stability of amphotericin B in human serum

The solubility and stability of amphotericin B in human serum in vitro was assessed using a sensitive high performance liquid chromatographic technique. The solubility of amphotericin B at concentrations ranging from 0.5 micrograms/ml to 10 micrograms/ml in human serum at pH 7.4, at 37 degrees C, and in 5% CO2 (simulated in vivo conditions) showed no significant difference compared with the solubility at 23 degrees C in room air after a 1 h incubation period. Dissolution of the colloidal suspension at a concentration of 2 micrograms/ml reached 81% in 3 min with a small increase in solubility by 60 min. Amphotericin B was very stable (less than 5% loss) in human serum at -20 degrees C for up to 14 days with only a 10% loss of drug at 6 months. In dimethylsulfoxide, however, losses at 6 months were greater than 60%. We conclude that amphotericin B solubilizes, but not completely, from a colloidal form in human serum under simulated in vivo conditions at 37 degrees C and at 23 degrees C in room air over 1 hour. Additionally, amphotericin B is stable in frozen serum for 6 months.     

Assessment of novel oral lipid-based formulations of amphotericin B using an in vitro lipolysis model

The purpose of this study was to investigate the intraluminal processing of novel oral lipid-based formulations of amphotericin B using an in vitro lipolysis model. Amphotericin B (AmB) was formulated in three lipid-based formulations consisting of different lipid components: iCo-009, iCo-010 and iCo-011. Various lipid loads (0.25, 0.5, 1 and 2 g) were digested using the lipolysis model to assess AmB distribution among the lipolysis phases. The duration of lipolysis was comparable among the three formulations except for 2 g load of iCo-009 which had a significantly longer lipolysis than iCo-010 and iCo-011. The lipid components of iCo-009 experienced lower extent of lipolysis as compared to other formulations. Amphotericin B concentration in the aqueous phases was the highest with iCo-010 which also had the lowest sediment recovery. Amphotericin B levels in the undigested lipid layers were comparable between iCo-009 and iCo-010 and were higher than with iCo-011. Given the observation that iCo-010 had the highest aqueous micellar solubilization and the lowest sediment recovery of AmB among the tested formulations, these results could potentially be used to interpret and predict the in vivo performance of AmB- SEDDS formulations in future studies.     

高效液相色谱法测定人血浆中两性霉素B浓度

目的　测定人血浆中两性霉素B浓度。方法　色谱柱为SupelcosilLC 18 DB( 4 .5mm× 2 5 0mm ,5 μm) ,流动相为0 .0 2 5mol/L乙二胺四乙酸二钠 (EDTA 2Na) 乙腈 ( 5 0∶5 0 ) ,柱温 :2 5℃ ,流速 1.0mL/min ,检测波长 4 0 5nm。结果　人血浆中两性霉素B浓度在 0 .0 5～ 4 .0 μg/mL范围内线性关系良好 ,回归方程为 :C =5 .0 4× 10 -6A + 0 .0 7(r =0 .9994 )。高、中、低 3个浓度平均回收率为 ( 98.4± 6 .9) % ;日内RSD≤ 3.5 % ,日间RSD≤ 7.6 %。结论　本法灵敏、快速、重现性好 ,分析时间短 ,可用于临床药代动力学研究及血药浓度监测     

Stability studies with amphotericin B and amphotericin B methyl ester.

In solid form, amphotericin B and amphotericin B methyl ester free base exhibit similar stability. Acid salts of the methyl ester derivative stored under identical conditions are less stable. In solution, amphotericin B is generally more stable than its methy ester salts. However, when pH is adjusted to 6.0 and storage temperature held at 5 degrees C the methyl ester salts reflect the stability exhibited by the parent compound, amphotericin B.     

In vitro effect of new formulations of amphotericin B on amastigote and promastigote forms of Leishmania infantum

The in vitro antileishmanial activities of various new amphotericin B (AMB) formulations were investigated, including microspheres of hydrophilic albumin with three AMB aggregation forms (monomeric, dimeric and multiaggregate) and the polymers of polylactic-co-glycolic acid, Resomer RG502 and RG503 with the multiaggregate AMB form. This in vitro study was performed on the extracellular promastigote form and the intracellular amastigote form of a canine strain of Leishmania infantum (UCM 20) using the infected J774 murine macrophage-like cell line. Albumin-encapsulated forms did not show any toxicity for murine cells and had lower median effective concentration (EC50) values (ca. 0.003 microg/mL) for L. infantum amastigotes than free formulations (0.03 microg/mL). In addition, the aggregation state of AMB had a notable effect on the antileishmanial activity of the drug. Results obtained in vitro point towards interest in monomeric AMB encapsulated in microspheres in the chemotherapeutic control of leishmaniasis.     

浊度法测定两性霉素B及其脂质体的效价

目的建立浊度法测定两性霉素B及其脂质体效价的方法。方法啤酒酵母为实验菌,加菌量2%～3.3%(V/V),(37±1)℃培养6～8h测定。结果抗生素线性浓度为0.04～0.10μg/ml,一剂量法、二剂量法的平均回收率分别为103.5%(n=5)、106.6%(n=2),日内RSD分别为1.64%、0.38%,日间RSD分别为2.79%(n=8)、0.87%(n=3)。结论本方法灵敏,快速,可作为测定两性霉素B及其脂质体的效价的方法。     

Buffer capacity and precipitation control of pH solubilized phenytoin formulations.

The main objective of this investigation is to develop a phenytoin (DPH) intravenous formulation that does not precipitate upon dilution. The effect of the buffer capacity at pH 12 of several DPH formulations on the extent and lag-time of DPH precipitation upon dilution with Sorensen's phosphate buffer (SPB) is evaluated. DPH precipitation was evaluated by means of static and dynamic in vitro dilution methods. It is shown that an increase in the formulation buffer capacity decreases substantially the extent of DPH precipitation and increases the lag-time for precipitation. In addition, a comparison between static and dynamic in vitro methods to measure precipitation is presented.     

Potential role of aerosolized amphotericin B formulations in the prevention and adjunctive treatment of invasive fungal infections

The incidence of invasive fungal infections (IFIs) continues to increase, largely due to the steady rise in the number of at-risk patients and the increased use of aggressive immunosuppressant agents. Many available treatments are often limited by concerns about efficacy, safety, drug interactions, and/or cost. Owing to the poor treatment outcomes of immunosuppressed patients with IFIs, new preventative and treatment strategies are being investigated. Among these are the aerosolized formulations of amphotericin B. Published experience with the use of aerosolized amphotericin B deoxycholate (AmBd) in the prevention of IFIs has raised concerns regarding challenges in drug administration and tolerability. However, evolving data regarding administration of lipid-based formulations of amphotericin B indicate potential advantages over AmBd in the prevention and adjunctive treatment of IFIs.