Formulation strategies to help de-risking drug development

Amphotericin B is a well-known and mature antifungal drug which is used in the treatment of bloodborne parasitic and fungal infections. However, its use has been limited by dose-dependent kidney toxicity. Oral formulation approaches have focused on the dual problem of solubility and permeability of Amphotericin B (AmphB), which is poorly water soluble, amphoteric and has extremely low oral bioavailability.

In this article, we would like to highlight some of the key features in the history of AmphB and the attempts to reduce its toxicity through formulation strategies. Recently, an analogue compound with potential reduced toxicity has been discovered. This new analogue could be considered as a new lead, and may enter a drug development program. As such, this compound could benefit from pre-formulation studies for further drug development. We will therefore examine the overall approach that need to be implemented to aim for an efficient drug developability.

INTRODUCTION – THE HISTORY OF AMPHOTERICIN B

Since the 60s, the antifungal drug AmphB is known for its efficacy but is also responsible for severe renal toxicity, as its main critical side-effect. Recently, A. Maji et al. have published some outstanding results regarding the mechanism of action and the impact of AmphB¹ chemical modifications on toxicity activity. Although clinical trials are needed to confirm the effectiveness in humans, their published results emphasize the importance of in-depth (bio)physical and chemical characterization of Active Pharmaceutical Ingredients (APIs) in order to select and/or improve the therapeutic benefit of drug substance. AmphB is a critical antifungal drug, used for a wide range of systemic fungal infections. However, due to its extensive side effects and especially its renal toxicity, AmphB is often reserved for severe infections.

AmphB is a natural product that was first isolated from Streptomyces nodosus in 1955, and medically used in 19582. Since then, it has been recognized as a highly effective drug with low incidence of drug resistance in the treated pathogens. AmphB is on the World Health Organization’s List of Essential Medicines³.

CHEMISTRY – BIOSYNTHESIS

The complete stereochemical structure was determined in 1970 by an X-ray structure of a N-iodoacetyl derivative. The first synthesis of this polyene macrolide compound within its enantiomeric form was achieved in 1987 by K. C. Nicolaou⁴.

Amphotericin B, a polyene macrolide compound, is made up of sixteen ‘C2’ acetate and three ‘C3’ propionate units. The growing chain is constructed by a series of Claisen reactions. After cyclisation, the macrolactone core undergoes further modification by hydroxylation, methylation and glycosylation.

Due to the polyfunctional structure of this polyene, there are multiple possibilities to investigate the impact of chemical groups involved in the therapeutic benefits and toxicity effect. It can also contribute to the understanding of the mechanism of action.

BIO-PHYSICAL-CHEMICAL CHARACTERIZATION OF AMPHOTERICIN B

With the urgent need to develop new drugs for treating fungal infections, the multi-disciplinary work of A. Maji et al.¹ concentrates on understanding the mechanism of action and toxicity at the molecular level of AmphB.

Based on biophysical characterization and within a modular synthesis approach, the authors showed that AmphB mainly kills fungi by forming large spongelike aggregates that remove ergosterol from the cell membrane, a compound abundantly present in fungi cells. Mammalian and fungal membranes both contain sterols. More specifically, ergosterol, a fungal sterol, is more sensitive to AmphB than cholesterol, the common mammalian sterol. Bacteria are not affected as their cell membranes do not usually contain sterols. The removal and aggregation of ergosterol from the fungi cell membranes cause membrane disruption and cell death.

TUNING CHEMISTRY MODIFICATIONS VERSUS BIOLOGICAL ACTIVITY

To demonstrate how structural changes may affect the drug’s biological activity, the Researchers have synthesized several analogues of AmphB by varying specific functionalities of the molecule.

Chemical biology studies using these analogues in human kidney cells and in mice confirmed that renal toxicity is driven mainly by the binding of AmphB to cholesterol in kidney cell membranes.

By generating high resolution structures of AmphB with and without bound sterol molecules, comparative analysis of these structures revealed that AmphB’s toxicity is driven by its binding to cholesterol in kidney cell membranes.

By modifying the drug’s structure, Maji et al. created a renal-sparing analogue, AM-2-19, which maintains potent fungicidal activity while evading resistance. The findings suggest a potential general approach for mitigating renal toxicity in polyene macrolide antifungals. Clinical trials will be needed to confirm the effectiveness of this analogue in humans. The study emphasizes the untapped potential for rational development of small-molecule therapeutics.

Thanks to high-resolution structures of the drug guided by a rational approach, the researchers are paving the way to a new class of antifungals with lower renal toxicity. Ultimately, their work based on understanding the impact of chemical structure of AmphB enabled to optimize its selective action on ergosterol and reduce renal toxicity.

FORMULATION STUDIES

AmphB is a yellow solid product that can be found in both neutral and zwitterionic forms, possessing a polar and apolar side of the lactone ring, polyene chain and ionizable carboxyl and amine groups, which provide amphoteric properties to the molecule. These physicochemical properties are the reason why AmphB is poorly soluble in aqueous media, as well as several organic solvents, leading, in some cases, to self-aggregation. The aggregation state can modify its activity and pharmacokinetic properties.

These features, along with its high molecular weight of 924 Da and negligible gastrointestinal or blood–brain barrier permeability do not make AmphB a typical druggable molecule, placing it in Biopharmaceutical Classification System⁵ (BCS) Class IV.

The pharmacokinetics, toxicity and activity are clearly dependent on the type of AmphB formulation. Several drug delivery strategies have been investigated, the most extensively studied system is encapsulation into liposomes⁶ for parenteral administration. Moreover, significant efforts have been made to develop lipidbased delivery system for oral formulation, such as solid lipid nanoparticles and self-emulsifying systems, to overcome solubility problems and enhance bioavailability. Polymer-based formulations have also been investigated, such as ethylcellulose-based nanoparticles⁷. New drug delivery systems such as liposomes, nanospheres and microspheres can result in higher concentrations of AmphB in the liver and spleen, but lower concentrations in kidney and lungs, so decreasing its toxicity⁸.

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