Solid and solution properties of the antimicrobial agent clofazimine

Abstract

One strategy to combat antimicrobial resistant, a looming global threat, is to repurpose existing drug substances to fill the antibiotic discovery void until new treatments become available. Clofazimine is a hydrophobic antimicrobial agent which shows promise in vitro against most Gram-positive bacteria, including multidrug-resistant strains of Mycobacterium tuberculosis, Clostridium difficile and Staphylococcus aureus. However, the clinical use of clofazimine is hampered by low solubility, which results in a poor correlation between in vitro antimicrobial activity and in vivo success. The aim of this project was to study the solid and solution properties of clofazimine and optimise these properties to formulate the drug into a more effective antimicrobial agent. In the first phase of this project, a systematic characterisation of the solution and solid states properties of the two known polymorphs of clofazimine was carried out and this study led to the discovery of two novel polymorphs, one of which was the most thermodynamically stable under ambient conditions. None of these polymorphic forms of clofazimine displayed any detectable water solubility. The large hydrophobic skeleton of CFZ made solvation in water challenging, and it was established that only CFZ in the protonated state could improve water solubility. Thus, the use of pharmaceutical salts of clofazimine as supersaturating drug delivery systems was investigated. By screening potential salt forms in biorelevant dissolution media, the optimal salt form could be identified. This study resulted in the identification of several novel salts of CFZ, three of which displayed improved solution behaviour in biorelevant media compared to the existing commercial formulation, adequate long-term stability, and better in vitro antimicrobial activity compared to the free base. Despite the improvements in solution behaviour obtained from the new salt forms, the protonated clofazimine species obtained from their dissociation in biorelevant media exhibited poor solution stability and rapidly precipitated from solution following administration. This was found to be due to the common ion effect at low pH and deprotonation at higher pH. During the development of the salt screening protocol, certain amphipathic elements of the biorelevant media were noted to have a significant influence on the solution concentration of clofazimine. Thus, the effects of the various components of the biorelevant media on the solution behaviour of clofazimine were studied. Here, it was observed that the presence of bile acids, phospholipid and the digestive protein pepsin could increase the solution concentration of clofazimine. The presence of bile acids, phospholipid and pepsin also increased the induction time of clofazimine hydrochloride in low pH media, thus affecting the crystallisation kinetics of the salt. It was discovered that the digestive enzyme pepsin has a strong affinity for clofazimine molecules, which could overcome the common ion effect in the low pH gastric system and solubilise the API in the higher pH intestinal media. Pepsin was shown to solubilise clofazimine under gastric conditions and deliver the drug to the lower intestinal system, the site of C. difficile infections. The effectiveness of this enzyme-mediated drug delivery system was demonstrated in vitro in a dynamic dissolution system as well as in bioactivity assays against C. difficile