Biophysics Seminar: Josh Karslake and Tehetina Woldemichael, Post-Doctoral Students

Tehetina Woldemichael

"Exploring Drug Bioaccumulation and Stabilization with respect to Endolysosomal Ion Homeostasis Using a Systems-based Mathematical Modeling Approach"
Even though most of the FDA approved drugs currently out in the market as well as in the process of development are weakly basic drugs, the bioaccumulation and stabilization of these drugs remains to be a topic that has not been well understood. For this purpose, we use a model drug, Clofazimine (CFZ), which is an FDA approved, weakly basic and poorly soluble drug that has been used worldwide to treat patients with leprosy and tuberculosis diseases, curing over 14 million people in the last twenty years, to investigate its bioaccumulation and stabilization properties. During prolonged oral administration, CFZ accumulates in macrophages of humans and mice as Crystal Like Drug Inclusions (CLDIs), which have been chemically characterized to be composed of hydrochloride salts of CFZ (CFZ-HCl) crystals. However, the mechanism by which the formation and stabilization of these insoluble complexes in cells is not known. Thus, due to sufficient proton and chloride levels in endolysosomes, we hypothesize that the drug accumulation and stabilization processes are occurring inside intracellular compartments of likely endolysosomal origin, in macrophages of humans as well as mice. To test our hypothesis, we adapt a systems-based mathematical lysosomal ion regulation model which consists of lysosomal membrane proteins, such as, the Vacuolar ATPase (V-ATPase), Cl-/H+ antiporter known as CLC7, and membrane proton leak in order to investigate the key lysosomal parameters that play essential role in the physiological, dose-dependent CFZ-HCl crystal bioaccumulation. Furthermore, we examine the stabilization of the free base (CFZ) versus salt form (CFZ-HCl) of the drug by mathematically fitting an in vitro pH-dependent solubility data of CFZ-HCl crystals obtained at pH ranging between extracellular, cellular and subcellular pH values, and determining the solubility properties dictated by the apparent pKa, intrinsic free base and salt solubility, pHmax, and Ksp values. Collectively, our computational results as well as the CFZ-HCl solubility parameters in relation to the ion contents and pH of the microenvironment suggest that the physiological and preferential accumulation and stabilization mechanisms of CFZ-HCl crystals in macrophages, more specifically in macrophage lysosomes, are primarily determined by the lysosomal V-ATPase.

Jason Karslake
"Population Density Modulates Drug Inhibition and Treatment Outcomes for Bacterial Infections"

The inoculum effect (IE) is an increase in the minimum inhibitory concentration (MIC) of an antibiotic as a function of the initial size of a microbial population. The IE has been observed in a wide range of bacteria, implying that antibiotic efficacy may depend on population density. Such density dependence could have dramatic effects on bacterial population dynamics and potential treatment strategies, but explicit measures of per capita growth as a function of density are generally not available. Instead, the IE measures MIC as a function of initial population size, and population density changes by many orders of magnitude on the timescale of the experiment. Therefore, the functional relationship between population density and antibiotic inhibition is generally not known, leaving many questions about the impact of the IE on different treatment strategies unanswered. To address these questions, here we directly measured real-time per capita growth of Enterococcus faecalis populations exposed to antibiotic at fixed population densities using multiplexed computer-automated culture devices. We show that density-dependent growth inhibition is pervasive for commonly used antibiotics, with some drugs showing increased inhibition and others decreased inhibition at high densities. For several drugs, the density dependence is mediated by changes in extracellular pH, a community-level phenomenon not previously linked with the IE. Using a simple mathematical model, we demonstrate how this density dependence can modulate population dynamics in constant drug environments. Then, we illustrate how time-dependent dosing strategies can mitigate the negative effects of density-dependence. Finally, we show that these density effects lead to bistable treatment outcomes for a wide range of antibiotic concentrations in a pharmacological model of antibiotic treatment. As a result, infections exceeding a critical density often survive otherwise effective treatments.