In Vitro potassium release assay for liposomal formulations of Amphotericin B – a requirement for Bio-IND applications

Ritika Uppal Mukherjee, PhD, Section Head, DMPK, ritika.u@advinus.com; Bhavesh Patel, PhD, Head of Department, DMPK, bhavesh.patel@advinus.com

Amphotericin B remains the standard of care for life-threatening fungal infections. However, its use is limited due to its severe acute and chronic toxicity. Amphotericin B forms pores in sterol-containing membranes, resulting in leakage of monovalent ions such as potassium and other cell constituents. This is the primary mechanism of action for its anti-fungal activity, as well as toxicity.

Liposomal formulations where Amphotericin B is embedded in the phospholipid layer have been developed to improve this safety profile. These formulations achieve slow release of Amphotericin B and reduce toxicity while retaining potency. During the COVID-19 pandemic, Amphotericin B liposomal formulation was the single line of treatment for patients suffering from Mucormycosis.

Due to high demand, generic companies are investing in developing similar products. To prove bioequivalence of such products, one of the studies that needs to be performed (based on the draft Office of Generic Drugs guidance) is the in vitro potassium release assay.

Eurofins Advinus has:

• Established this assay to compare the potassium released from red blood cells upon incubation with reference (RLD) and test formulations to support Bio-IND applications.

• Measured potassium concentration using Inductively Coupled Plasma Mass Spectrometry (ICP-MS).

• Validated the in vitro potassium release assay and bioanalytical method in RBCs from human and preclinical species.

The in vitro assay can be used during screening or batch release of exhibit batches of generic Amphotericin B formulations. The assay developed is a high throughput assay performed in 96-well plates and replaces traditional methods of potassium analysis with more sensitive ICP-MS. The concentration of formulation that causes 50% release of potassium is considered as K50 and is a measure of relative toxicity. This in vitro assay substitutes the need for in vivo studies to prove similarity of the test formulations to the innovator product. For more information, visit: www.advinus.com