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.     

Lipid-based amphotericin B for the treatment of fungal infections

The frequency of life-threatening fungal infections has increased dramatically over the past few decades. For more than 30 years amphotericin B has been the standard treatment for systemic and deep-seated fungal infections, primarily because of its broad spectrum of activity. Its usefulness is limited by a relatively high frequency of significant adverse events including infusion-related reactions and nephrotoxicity. In an effort to overcome these side effects, a number of lipid-based formulations were developed, each with its own composition and pharmacokinetic behavior. The clinical significance of these differences is unknown. Available clinical data suggest the formulations have a reduced propensity for causing nephrotoxicity. However, considering limited efficacy data, they should be reserved as second-line therapy for patients who cannot tolerate or fail an adequate trial of conventional amphotericin B or cannot benefit from other antifungal agents.     

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: a review of the last 10 years of use

The last decade has been remarkable for the dramatic increase in the prevalence of serious fungal infections in patients with haematological disorders and neutropenic cancer patients. The mortality rate of deep-seated infection has been in excess of 90% and there is no doubt that this is one of the greatest challenges currently facing haematologists and oncologists. The development of the lipid-based drugs - liposomal amphotericin (AmBisome(R)), amphotericin B lipid complex, ABLC (Abelcet(R)), amphotericin B colloidal dispersion, Amphocil (ABCD(R)), has meant that doses of amphotericin B can be safely escalated for the first time whilst the problems of nephrotoxicity, infusion related reactions (including chills, rigors, fevers and hypoxia) can be reduced. These toxicities are variably reduced with AmBisome more than Abelcet and more than Amphocil and there is little information from randomised trials other than for AmBisome. AmBisome used in the setting of persistent fever and neutropenia not responding after 3-4 days of intravenous antibiotics, is associated with less breakthrough systemic fungal infections. There is also much less need for premedication, including steroids, compared with amphotericin B and Abelcet. The use of intermittent doses of Ambisome given prophylactically is now being explored. A new and exciting era of antifungal therapy is opening up with new compounds, such as itraconazole voriconazole, posaconazole and echinocandins, being investigated and for the first time, we also have options for combination therapy and prophylaxis.     

Lipid-based formulations for intestinal lymphatic delivery

The current state of the art of intestinal lymphatic transport is given by reviewing the more recent publications, which have utilized lipid-based vehicles. The results published often show variable trends depending on, the design of the vehicle, the components used, the physicochemical properties of the drug, the animal model and experimental techniques, these variables often make direct comparisons difficult. Traditionally intestinal lymphatic delivery has been expressed as a percentage of the dose transported in the lymph. Using this parameter results obtained to date, with lipid-based vehicles, are somewhat disappointing maximising at approximately 20–30%, for highly lipophilic compounds including DDT and halofantrine (Hf). Recent data, monitoring Hf, in a fed versus fasted dog study, have shown that a higher degree of lymphatic transport is possible (>50% dose) in the postprandial state, this study should result in stimulating renewed interest in the potential of achieving significant levels of lymphatic targeting. Although some relevant features controlling lymphatic transport have been identified over the years a deeper appreciation of all the mechanisms, which is vital for therapeutic exploitation of lymphatic transport, is still unrealized. This review analyses the success and limitations of a formulation approach using lipid-based vehicles and highlights potential areas for further research.     

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.     

Toxicity of solubilized and colloidal amphotericin B formulations to human erythrocytes

The toxicity of a number of solubilized or colloidal amphotericin B formulations to human erythrocytes has been studied in-vitro. All the solubilized formulations studied, using poloxamer F127 or L92, or sodium deoxycholate as solubilizing agents, showed similar toxicity, erythrocyte lysis being greater than 90% for amphotericin B concentrations between 4 to 8 micrograms mL-1. Emulsion formulations stabilized by poloxamers showed reduced toxicity, while those stabilized by egg lecithin showed less than 5% erythrocyte lysis up to an amphotericin B concentration of 200 micrograms mL-1. The mechanisms of the differential toxicity is considered to be due to the differences in the equilibrium concentration of free amphotericin B in the aqueous phase.     

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.     

Lipid-based formulations for oral administration of poorly water-soluble drugs

Lipid-based drug delivery systems have shown great potentials in oral delivery of poorly water-soluble drugs, primarily for lipophilic drugs, with several successfully marketed products. Pre-dissolving drugs in lipids, surfactants, or mixtures of lipids and surfactants omits the dissolving/dissolution step, which is a potential rate limiting factor for oral absorption of poorly water-soluble drugs. Lipids not only vary in structures and physiochemical properties, but also in their digestibility and absorption pathway; therefore selection of lipid excipients and dosage form has a pronounced effect on the biopharmaceutical aspects of drug absorption and distribution both in vitro and in vivo. The aim of this review is to provide an overview of the different lipid-based dosage forms from a biopharmaceutical point of view and to describe effects of lipid dosage forms and lipid excipients on drug solubility, absorption and distribution.     

Lipid-based nanoparticle formulations for small molecules and RNA drugs

ABSTRACT

Introduction: Liposomes and lipid-based nanoparticles (LNPs) effectively deliver cargo molecules to specific tissues, cells, and cellular compartments. Patients benefit from these nanoparticle formulations by altered pharmacokinetic properties, higher efficacy, or reduced side effects. While liposomes are an established delivery option for small molecules, Onpattro® (Sanofi Genzyme, Cambridge, MA) is the first commercially available LNP formulation of a small interfering ribonucleic acid (siRNA).

Areas covered: This review article summarizes key features of liposomal formulations for small molecule drugs and LNP formulations for RNA therapeutics. We describe liposomal formulations that are commercially available or in late-stage clinical development and the most promising LNP formulations for ASOs, siRNAs, saRNA, and mRNA therapeutics.

Expert opinion: Similar to liposomes, LNPs for RNA therapeutics have matured but still possess a niche application status. RNA therapeutics, however, bear an immense hope for difficult to treat diseases and fuel the imagination for further applications of RNA drugs. LNPs face similar challenges as liposomes including limitations in biodistribution, the risk to provoke immune responses, and other toxicities. However, since properties of RNA molecules within the same group are very similar, the entire class of therapeutic molecules would benefit from improvements in a few key parameters of the delivery technology.     

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.     

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.     

Lipid-based oral multiparticulate formulations - advantages, technological advances and industrial applications

INTRODUCTION: Lipid-based formulations have emerged as potential dosage forms to harvest effectively the therapeutic benefits of existing lipophilic molecules and new chemical entities. Compared with existing excipients, lipids by virtue of their unique physicochemical properties and resemblance to in vivo components demonstrate the extra advantage of improving the bioavailability of lipophilic and highly metabolizable drugs. Moreover, if used as the major excipient, lipids can reduce the required dose and even the toxicity of drugs with poor aqueous solubility. AREAS COVERED: This review deals with the importance of multiparticulate systems in drug delivery, the therapeutic and manufacturing advantages of lipids as excipients, and the technological advances made so far in utilizing lipids for multiparticulate dosage forms, with emphasis on their application and success on an industrial scale. Lipids are being widely formulated into different multiparticulate dosage forms using innovative modifications of conventional equipment with relative ease, using methods such as melt-granulation, adsorption on solid support, spray-cooling, melt-extrusion/spheronization, freeze-pelletization, pastillation, and so on. EXPERT OPINION: There is still a need to design new simple dosage forms and to upgrade existing manufacturing technology, in order to open up new avenues in drug delivery, for preparing patent non-infringing formulations of existing drug products, and to help patients receive treatment at an affordable cost.     

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.     

A comparison of the activities of three amphotericin B lipid formulations against experimental visceral and cutaneous leishmaniasis

The polyene antibiotic, amphotericin B, the gold standard for systemic fungal infections is also a recommended second line treatment for visceral, cutaneous and mucocutaneous leishmaniasis. Acute toxicity has limited the use of amphotericin B but less toxic lipid formulations, AmBisome®, Amphocil™ and Abelcet®, have shown potential for the treatment of clinical visceral and mucocutaneous leishmaniasis. This study compares the in vitro and in vivo anti-leishmanial activity of Fungizone and the three lipid formulations. AmBisome and Amphocil were more active (ED₅₀ values 0.3 and 0.7mg/kg, respectively) than Abelcet (ED₅₀ 2.7mg/kg) against L. donovani in a mouse model. Against L. major in vivo, AmBisome at a dose of 25mg/kg was the most successful at reducing lesion size, with Amphocil also showing activity while Abelcet was inactive. In the L. donovani — peritoneal macrophage (PEM) model Fungizone and Amphocil were significantly more active (ED₅₀ values 0.013 and 0.02 μg/ml, respectively) than AmBisome and Abelcet (ED₅₀ values 1.5 and 2.6 μg/ml). This trend was similar in the L. major — PEM model (Fungizone>Amphocil>AmBisome>Abelcet). THP-1 macrophages infected with L. donovani amastigotes showed a different profile with Amphocil=Abelcet>AmBisome>Fungizone. Differences could be due to the interaction of the formulations with the biological milieu and uptake into different cell types.     

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.