A client requested help in the development of a liposomal platform to meet a new clinical need for an antifungal API. The formulation goals included antifungal activity at low doses, acceptable shelf stability at room temperature or cold storage (2-8 °C), low toxicity, a highly scalable manufacturing process, liposome size suitable for sterile filtration of final product and high encapsulation efficiency of the API-loaded liposomes. To fit within these parameters, we developed a liposome library using combinations of phospholipids and cationic lipids (up to 32), as well as multiple lipid-to-API ratios for analysis of efficacy (Minimum Inhibitory Concentration – MIC and Minimum Toxic Concentration – MTC) at the client’s CRO.
The use of cationic lipids in liposomal formulations can be a double-edged sword. Cationic lipids can improve delivery and uptake of liposomes and increase liposome stability through electrostatic stabilization.On the other side of the sword, however, many cationic lipids can increase toxicity of the liposomes. In these studies, we tested multiple cationic lipids compared these liposomes with cationic components against liposomes composed entirely of phospholipids. This approach allowed us to identify lipid compositions that resulted in both low toxicity to human cells and high antifungal activity for the drug products. Moreover, in this library of liposomal formulations, the API encapsulation for the majority of lipid combinations was high but we did observe that lipid composition influenced the rate at which the API degradation occurred in various formulations. Through this observation, we were able to narrow our selection of candidates for further study as development continued.
We were able to identify three formulation candidates from the library that were both effective against many fungal species (as indicated by low MIC values). In all three final candidates, the MIC values were less than or equal to those of the control formulation. In two isolated fungal species, low concentration inhibition was observed. However, the species as a whole appeared resistant to this API in the control formulation (>64-fold difference in concentration).
Assessing toxicity in human cells for these formulations revealed that at higher concentrations (~125 µg/mL of API) the lactate dehydrogenase (LDH) release measured was 1/3 the release observed in the control formulation. This shows how these formulations are likely to be less toxic than the control formulation. Additionally, we moved candidates from our initial screening library of small volume bench-scale formulations to larger-scale pilot processes. These processes were capable of producing liters of material without observing negative impacts on particle characteristics or other quality criteria.
Scaling liposomal mixing processes can be challenging, but in this case we demonstrated that our approach to a solvent injection process resulted in a continuous mixing that performed optimally at 5X scale with potential for further scale-up.
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