An Expandable Mechanopharmaceutical Device (1): Measuring the Cargo Capacity of Macrophages in a Living Organism

Abstract

Purpose: Clofazimine (CFZ) is an FDA-approved, poorly soluble small molecule drug that precipitates as crystal-like drug inclusions (CLDIs) which accumulate in acidic cytoplasmic organelles of macrophages. In this study, we considered CLDIs as an expandable mechanopharmaceutical device, to study how macrophages respond to an increasingly massive load of endophagolysosomal cargo. Methods: First, we experimentally tested how the accumulation of CFZ in CLDIs impacted different immune cell subpopulations of different organs. Second, to further investigate the mechanism of CLDI formation, we asked whether specific accumulation of CFZ hydrochloride crystals in lysosomes could be explained as a passive, thermodynamic equilibrium phenomenon. A cellular pharmacokinetic model was constructed, simulating CFZ accumulation driven by pH-dependent ion trapping of the protonated drug in the acidic lysosomes, followed by the precipitation of CFZ hydrochloride salt via a common ion effect caused by high chloride concentrations. Results: While lower loads of CFZ were mostly accommodated in lung macrophages, increased CFZ loading was accompanied by organ-specific changes in macrophage numbers, size and intracellular membrane architecture, maximizing the cargo storage capabilities. With increasing loads, the total cargo mass and concentrations of CFZ in different organs diverged, while that of individual macrophages converged. The simulation results support the notion that the proton and chloride ion concentrations of macrophage lysosomes are sufficient to drive the massive, cell type-selective accumulation and growth of CFZ hydrochloride biocrystals. Conclusion: CLDIs effectively function as an expandable mechanopharmaceutical device, revealing the coordinated response of the macrophage population to an increasingly massive, whole-organism endophagolysosomal cargo load.

Quantitative Analysis of the Phase Transition Mechanism Underpinning the Systemic Self-Assembly of a Mechanopharmaceutical Device

“…For the most part, the serum concentrations of CFZ were largely determined independently from parameters governing the soluble-to-insoluble phase transition of CFZ and its accumulation as intracellular CLDI precipitates within macrophages. The increase in the half-life of CFZ was expectedly coupled to the expansion of the drug volume of distribution, which can be explained by the thermodynamic and cellular mechanisms responsible for the precipitation of the drug within macrophages [10,12,16]. The modeling approach presented here suggests that the actual concentrations of the drug in the blood are minimally affected by the mechanisms governing the phase transition of the drug throughout the dosing period.…”

An Adaptive Biosystems Engineering Approach towards Modeling the Soluble-to-Insoluble Phase Transition of Clofazimine

“…The accumulation of CFZ in macrophage lysosomes has been shown to occur by thermodynamically favorable conditions that lead to the precipitation of drugs by pH-dependent ion trapping. The protonated species of the weak base in the lysosome of the macrophage, together with the formation of the very insoluble hydrochloride (CFZ-HCl) salt, form a highly stable crystal upon interaction of the protonated drug with chloride [6][7][8]. In macrophage lysosomes, the hydrochloride crystals are found within membrane-associated complexes referred to as crystal-like drug inclusions (CLDIs).…”

“…Although it has been used clinically for many decades [10,11], CFZ pharmacokinetics are characterized by an increasing, dose-dependent half-life and high interindividual variability [12], which complicates pharmacokinetics analysis. In an experimental mouse model used to analyze the mechanisms underlying the complex pharmacokinetics of CFZ, the dynamic half-life of CFZ was associated with the formation and accumulation of hydrochloride salt as CLDIs grew and expanded within macrophage lysosomes [6,13]. This increasing accumulation was accompanied by changes in the distribution of the drug within the organism, as well as the formation of local drug depots that remained long after treatment stopped [6].…”

“…In an experimental mouse model used to analyze the mechanisms underlying the complex pharmacokinetics of CFZ, the dynamic half-life of CFZ was associated with the formation and accumulation of hydrochloride salt as CLDIs grew and expanded within macrophage lysosomes [6,13]. This increasing accumulation was accompanied by changes in the distribution of the drug within the organism, as well as the formation of local drug depots that remained long after treatment stopped [6]. While the soluble phase of CFZ is the pharmacologically active moiety that can freely exert antibacterial or antiviral activity [1,2,10], the CLDI drug depot could be beneficial since it concentrates in macrophages, which are directly involved in combating infection.…”

“…While the soluble phase of CFZ is the pharmacologically active moiety that can freely exert antibacterial or antiviral activity [1,2,10], the CLDI drug depot could be beneficial since it concentrates in macrophages, which are directly involved in combating infection. Crystal-like drug inclusions have also been shown to increase with repeated dosing in both mice and humans [6]. Due to the high concentration of macrophages in the spleen, this organ sequesters CFZ at 10 to 100 times the concentration of other tissues [6,14].…”