INTRODUCTION

The development of effective, safe and low-cost drug and delivery systems has been a key goal of the pharmaceutical research and industry for decades. Recently, the use of combinatory chemistry and high-throughput screening have provided an increasing number of drug candidates with poor aqueous solubility [1]. Low solubility in water represents an important factor limiting the drug dissolution rate. Consequently, absorption and bioavailability after oral administration of these drugs is also unsatisfactory, thus reducing the therapeutic efficacy and safety of these poorly water-soluble molecules [2].

Amphotericin B (AmB) is a polyene antibiotic, first isolated from Streptomyces nodosus in 1945 [3]. The molecule consists of an elongated circular structure presenting hydrophilic polyhydroxyl and hydrophobic polyene domains. As a consequence of having both apolar and polar sides to its lactone ring combined with the presence of ionizable carboxyl and amine groups, AmB molecule presents both amphoteric and amphiphilic behavior (Figure 1). Hence, AmB is poorly soluble in aqueous solvents and in many organic solvents. At a pH below 2 or above 11, AmB is water-soluble, although it is unstable [4]. As a result of its very low solubility and membrane permeability, AmB is characterized as a type IV drug by the Biopharmaceutical Classification System [5, 6].

Figure 1. Chemical structure of amphotericin B [7].

The amphipathic nature of AmB causes it to self-associate and aggregate according to its concentration in water [8, 9]. Thus, only monomers are formed at concentrations of 5 x 10^-

8 M and below while from 5 x 10^-5 M to higher concentrations, only aggregates are found. The two forms co-exist between 5 x 10^-6 M and 5 x 10^-7 M [3] (Figure 2). Interestingly, depending on its concentration, AmB in water may form a mixture of water-soluble monomers and oligomers with insoluble aggregates. The AmB aggregate forms are usually described as

“water-soluble oligomers” and “water-insoluble aggregates” [9]. These different aggregate forms of AmB have been shown to be directly related to its toxicity. Water-soluble oligomers are defined as the most toxic form of the drug while larger water-insoluble aggregates are believed to be less toxic [9].

Figure 2. Absorption spectra of aqueous solutions of AmB-DOC at different concentrations at 25ºC. Peaks at 363, 385 and 408 nm are due to the monomeric form while the peak at 327 nm indicate the presence of aggregates of AmB [10].

AmB provokes both acute and chronic toxic side effects. The acute toxicity of AmB is infusion-related and caused by the production of proinflammatory cytokines by innate immune cells. As a microbial product, it is believed that AmB can stimulate mammalian immune cells via toll-like receptors [11]. Nausea, vomiting, rigors, fever, hypertension/hypotension, and hypoxia are observed as the main symptoms of acute toxicity of AmB deoxycholate, the “conventional” formulation [12]. Among these side effects, hyperkalemia is considered the most life-threatening consequence due to the potential for

developing fatal cardiac arrhythmias because of the leakage of potassium (K⁺) from the intracellular compartment [12]. Among the chronic toxicities caused by therapy with AmB, distal renal tubular acidosis with hypomagnesaemia and hypokalemia is the most important side effect [13]. Shigemi and coworkers have evaluated retrospectively the frequency of anemia, thrombocytopenia, nephrotoxicity, hepatotoxicity and hypokalemia induced by the administration of liposomal formulations of AmB (L-AmB) [14]. The relationship between the daily dose of L-AmB and these side effects was also investigated. They observed that both anemia and thrombocytopenia occurred in a dose-dependent manner [14]. Nephrotoxicity was seen to be associated with a greater cumulative dose of AmB and concomitant administration of other nephrotoxic drugs, such as cyclosporine and streptomycin [14]. However, the study was not powerful enough to determine the influence of AmB on the hepatotoxicity and hypokalemia.

For over 50 years, AmB deoxycholate (AmB-DOC), the conventional colloidal dispersion marketed as Fungizone^®, has been the treatment of choice for fungal infections, despite significant adverse effects, notably severe nephrotoxicity. As well as AmB-DOC, several other formulations incorporating amphiphiles have been developed in order to improve the drug solubility and bioavailability and decrease its toxicity. In the last 15 years, several new formulations have emerged. For example, in AmBisome^® the drug is incorporated into small unilamellar liposomes to overcome its toxic effects. Colloidal drug delivery systems have incited great interest for decades due to their multiple advantages for the administration, stability and efficacy of active molecules. These drug carriers are able to provide improved biodistribution and bioavailability, reduced toxicity and better selectivity to both hydrophilic and hydrophobic molecules.

Different approaches have been adopted with a view towards decreasing the toxicity of AmB, such as its complexation with other agents such as surfactants, cyclodextrins, lipids, polymers and carbon nanotubes [15-25]. The aim of this article is to provide an overview of publications from the last ten years, which have reported relevant findings in the pharmaceutical technology field, especially on the development of delivery systems for AmB showing promising results. For this purpose, a systematic literature survey was performed on the database compiled by ISI Web of Knowledge and National Center for Biotechnology Information and published between 2002 and 2012 using “nanotechnology”, “amphotericin B”, “colloidal drug delivery system”, “microemulsion”, “nanoemulsion”, “emulsion”

“micelles”, “amphiphile”, liposomes”, “nanoparticles”, and “carbon nanotubes” as search

terms. The quantitative results are shown in Figure 3.

Figure 3. Timeline of publications on AmB formulations, such as amphiphile-based systems, liposomes, lipid-based systems, emulsions, microemulsions and nanoemulsions, nanoparticles and carbon nanotubes, indexed in Web of Science from 2002 until 2012.