Critical process parameters in manufacturing of liposomal formulations of amphotericin B
Graphical abstract
Introduction
Amphotericin B (AMB) is a mainstay antibiotic for treatment of fungal infections. The drug, commercially available in several different formulations, is frequently administered to patients with immunodeficiency, including those suffering from AIDS or undergoing chemotherapy (Barratt and Bretagne, 2007, Rodrigues and Henriques, 2017, Tonin et al., 2017, Torrado et al., 2008). Despite the wide clinical use of this drug, duration of therapy and doses of AMB are limited by its high toxicity, which appears to be least pronounced in its liposomal formulation marketed as AmBisome (Barratt and Bretagne, 2007, Falci et al., 2015, Groll et al., 2000, Hamill, 2013, Kwong et al., 2001, Mishra et al., 2013, Serrano et al., 2013, Silver and Rostas, 2018, Torrado et al., 2008).
The drug’s efficacy against fungal cells and associated toxicity to blood cells stems from the amphiphilic nature of the AMB molecule (Fig. 1) (Gruszecki et al., 2003). Hydrophobic and hydrophilic functional groups of AMB induce self-association (aggregation), causing formations of oligomeric structures (Bartlett et al., 2004, Bolard et al., 2009, Castanho et al., 1992). When these oligomers are embedded in bio membranes, hydrophobic regions are presumably oriented towards the membrane’s lipid core, forming pores lined by hydrophilic regions. Electrolyte leakage through membrane pores leads then to cell death (Barratt and Bretagne, 2007, Bartlett et al., 2004, Stoodley et al., 2007). Additional complexity may originate from the interactions of AMB with negatively charged phospholipids and sterols. The therapeutic window of AMB is thus determined by differences in the binding to the predominant sterols of fungal (ergosterol) vs. humans (cholesterol) cells (Liu et al., 2017, Mishra et al., 2013, Rodrigues and Henriques, 2017, Serrano et al., 2013, Silver and Rostas, 2018).
Fine features of the aggregates presented in drug products have a strong impact on toxicity towards cholesterol-containing cells (Liu et al., 2017). Therefore, the drug’s toxicity is highly related to the formulation ingredients and preparation processes. Reducing the toxicity of AMB will widen its therapeutic window, allowing increased doses with reduced side effects (Takemoto et al., 2004).
AMB is very poorly soluble in both aqueous media (Barratt and Bretagne, 2007, Hamill, 2013, Liu et al., 2017, Rodrigues and Henriques, 2017, Wijnant et al., 2018) and highly hydrophobic milieu (Torrado et al., 2008) (such as the core of lipid membrane bilayers). Therefore, the monomeric form of AMB is typically present in negligible concentrations and appears to be a minor contributor to toxicity. An increase in solubility and reduction of toxicity were both achieved by conversion of homo-aggregates (i.e. aggregates or oligomers of pure AMB) to hetero-aggregates containing detergents or lipids. Such structures were found to be less likely to form pores in cholesterol-containing membranes (Barratt and Bretagne, 2007, Bartlett et al., 2004, Kwong et al., 2001, Petit et al., 1999).
Fungizone is a conventional AMB drug product formed from AMB-deoxycholate (AMB-DOC) hetero-aggregated structures, a historically first formulation in clinical use (Kwong et al., 2001, Mishra et al., 2013, Rodrigues and Henriques, 2017, Takemoto et al., 2004). At low drug-to-detergent ratios, AMB is encapsulated in classical DOC micelles (which have an aggregation number near 10). Fungizone preparation, which has a higher drug-to-detergent ratio, results in the formation of different phases that maintain the solubility of the super-aggregate.
Fungizone offers a common reference point for experimental AMB formulations (Bartlett et al., 2004, Kwong et al., 2001, van Etten et al., 2000). An improvement in Fungizone’s toxicity was achieved with heat treatment, causing the formation of super-aggregated structures (Bartlett et al., 2004, Kwong et al., 2001, Petit et al., 1999). While less toxic, heat-treated Fungizone was equally effective in treating fungal infections in animal models as untreated Fungizone (Bartlett et al., 2004).
Additional reductions in toxicity were demonstrated in formulations where AMB was entrapped in the hydrophobic core of phospholipid vesicle (liposomal) membranes (Mishra et al., 2013, Takemoto et al., 2004, Tonin et al., 2017). These structures alter the pharmacodynamics of the drug, resulting in lower AMB levels in the kidney and lungs compared to Fungizone (Torrado et al., 2008). Liposomal AMB formulations are thus less toxic than Fungizone and more effective in treating fungal infections allowing for higher exposure and longer duration of the therapy (Bartlett et al., 2004, Bolard et al., 2009, Kwong et al., 2001, Leenders et al., 1996, Mishra et al., 2013, van Etten et al., 2000, Wijnant et al., 2018).
AmBisome is currently considered as a standard liposome-based commercial AMB formulation (Serrano et al., 2013, Wijnant et al., 2018). The solubilization of AMB in AmBisome (supporting ∼4 mg AMB per mL) and the safety profile of liposomal formulations make the drug convenient for parenteral administration (“AmBisome, 2012, Liu et al., 2017). The pharmacodynamics and toxicological benefits of AmBisome (attributed to lipid entrapment) are commonly considered to outweigh higher production costs (Bartlett et al., 2004, Kwong et al., 2001, Torrado et al., 2008).
This study evaluated the key steps in the complex liposomal AMB formulation process and their impact on the final product’s critical quality attributes (CQAs). These test formulations were compared to CQAs of AmBisome, an approved and commercially available drug product. The task in hands was complicated by the fact that there are four separate steps in the manufacturing process (Proffitt et al., 1999, Adler et al. 1999,), each involving multiple (4–8) physico-chemical variables, and possible different equipment choice and settings. Furthermore, the water-insoluble nature of the API has been associated with serious difficulties in analyzing the API status in-situ and required development of the additional analytical assays.
Section snippets
Materials
The AmBisome standard was prepared from reconstituted AmBisome (Prod. Code: 305130) Gilead Biosciences Inc. (San Dimas, CA). The following chemicals were used during Step 1 of the manufacturing process: Amphotericin B (AMB, API, Drug Substance (DS)) Alfa Aesar (Haverhill, MA) and BOC Sciences (Shirley, NY); Chloroform (>99.8%, Cat: 40974) Alfa Aesar (Haverhill, MA), Cholesterol (>99%, Cat: C-8667) Sigma (St. Louis, MO), Di-Stearoyl-Phosphatidyl-Glycerol (DSPG, sodium salt, Cat: 5600400), Lipoid
Results
The first step of the manufacturing protocol allows the API to be solubilized in a methanol–chloroform mixture to form optically clear solutions at very high concentration (20–40 mg/mL), nearly two orders of magnitude above the solubility of pure AMB in methanol, through the formation of soluble drug-lipid complexes. The second step of the formulation is aimed to remove the organic solvent by spray drying. Spray-dried solid material, which is ∼12% w:w API, is then hydrated in succinate-buffered
Discussion
Most of the reported extensive physico-chemical and manufacturing studies related to the approved liposomal drugs deal with doxorubicin-containing Doxil (Gaspani and Milani, 2013), which is now available as both branded and generic product (the first FDA approved generic liposomal drug product). To underscore the importance of the current study, it is necessary to highlight the differences between Doxil and products involving AMB. The first major difference relates to the luminal location of
Conclusions
Critical process parameters in the manufacturing process of an Amphotericin B liposomal formulation were investigated, and the corresponding critical quality attributes have been established. This process was based on the acid-aided formation of drug-lipid complexes in a methanol-chloroform mixture (Step 1) followed by spray drying (Step 2), particle sizing by microfluidization (Step 3), and lyophilization (Step 4). An exceedingly low aqueous solubility of the API and propensity to aggregation
Conflict of interest
None declared.
Acknowledgements
This work was supported by the FDA’s BAA grant award to Neo-Advent Technologies, LLC (Littleton, MA), Contract HHSF223201610093C (PI Dr. Alex Nivorozhkin). We are grateful to Shirley Rodriguez-Morales for her devoted technical assistance in the experimental part of this project.
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