Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy.

Tessa Rui Min Tan,Kong Meng Hoi,Say Kong Ng,Peiqing Zhang,Marco Rito-Palomares

doi:10.1371/journal.pone.0152112

Abstract

Amphotericin B (AMB) is a highly hydrophobic antifungal, whose use is limited by its toxicity and poor solubility. To improve its solubility, AMB was reacted with a functionalized polyethylene glycol (PEG), yielding soluble complex AmB-PEG formulations that theoretically comprise of chemically conjugated AMB-PEG and free AMB that is physically associated with the conjugate. Reverse-phase chromatography and size exclusion chromatography methods using HPLC were developed to separate conjugated AMB-PEG and free AmB, enabling the further characterization of these formulations. Using HPLC and dynamic light scattering analyses, it was observed that the AMB-PEG 2 formulation, having a higher molar ratio of 2 AMB: 1 PEG, possesses more free AMB and has relatively larger particle diameters compared to the AMB-PEG 1 formulation, that consists of 1 AMB: 1 PEG. The identity of the conjugate was also verified using mass spectrometry. AMB-PEG 2 demonstrates improved antifungal efficacy relative to AMB-PEG 1, without a concurrent increase in in vitro toxicity to mammalian cells, implying that the additional loading of free AMB in the AMB-PEG formulation can potentially increase its therapeutic index. Compared to unconjugated AMB, AMB-PEG formulations are less toxic to mammalian cells in vitro, even though their MIC50 values are comparatively higher in a variety of fungal strains tested. Our in vitro results suggest that AMB-PEG 2 formulations are two times less toxic than unconjugated AMB with antifungal efficacy on Candida albicans and Cryptococcus neoformans.

Highlights

Amphotericin B (AMB), a polyene antifungal agent, is frequently used to treat systemic fungal infections
To evaluate whether additional AMB can be loaded on AMB-polyethylene glycol (PEG) conjugates formed through the reaction with MS (PEG)4, AMB-PEG formulations reacted at increasing AMB:PEG molar ratios of 1:1, 2:1, 4:1 and 10:1, were diluted in PBS-EDTA (Fig 1B)
Given the low solubility of unreacted AMB, this suggests that free AMB in the 2:1 formulation may be associated with the soluble AMB-PEG conjugate thereby increasing its aqueous solubility, and this association may be saturated at an AMB:PEG molar ratio between 2:1 and 4:1

Summary

Introduction

Amphotericin B (AMB), a polyene antifungal agent, is frequently used to treat systemic fungal infections. Such infections are often opportunistic, and cause significant morbidity and mortality among immunocompromised individuals [1]. Crystalline AMB is highly hydrophobic, virtually insoluble in water and only sparingly soluble in many organic solvents, making drug delivery challenging [5]. It has a low therapeutic index, and limitations of intravenous AMB therapy include severe side-effects [6] and poor aqueous solubility, which can potentially reduce its efficacy in certain fungal lesions [7]. To the kidneys, is drug-mediated and is a chronic side-effect of AMB treatment [9]

Methods

Results

Conclusion