Database screening as a strategy to identify endogenous candidate metabolites to probe and assess mitochondrial drug toxicity.

Adverse drug reactions (ADRs) are considered an inherent risk of medication use, and some ADRs have been associated with off-target drug interactions with mitochondria. Metabolites that reflect mitochondrial function may help identify patients at risk of mitochondrial toxicity. We employed a database strategy to identify candidate mitochondrial metabolites that could be clinically useful to identify individuals at increased risk of mitochondrial-related ADRs. This led to L-carnitine being identified as the candidate mitochondrial metabolite. L-carnitine, its acetylated metabolite, acetylcarnitine and other acylcarnitines are mitochondrial biomarkers used to detect inborn errors of metabolism. We hypothesized that changes in L-carnitine disposition, induced by a "challenge test" of intravenous L-carnitine, could identify mitochondrial-related ADRs by provoking variation in L-carnitine and/or acetylcarnitine blood levels. To test this hypothesis, we induced mitochondrial drug toxicity with clofazimine (CFZ) in a mouse model. Following CFZ treatment, mice received an L-carnitine "challenge test". CFZ-induced changes in weight were consistent with previous work and reflect CFZ-induced catabolism. L-carnitine induced differences in whole blood acetylcarnitine concentrations in a manner that was dependent on CFZ treatment. This supports the usefulness of a database strategy for the discovery of candidate metabolite biomarkers of drug toxicity and substantiates the potential of the L-carnitine "challenge test" as a "probe" to identify drug-related toxicological manifestations.

Quantitative Raman chemical imaging of intracellular drug-membrane aggregates and small molecule drug precipitates in cytoplasmic organelles.

Raman confocal microscopes have been used to visualize the distribution of small molecule drugs within different subcellular compartments. This visualization allows the discovery, characterization, and detailed analysis of the molecular transport phenomena underpinning the Volume of Distribution - a key parameter governing the systemic pharmacokinetics of small molecule drugs. In the specific case of lipophilic small molecules with large Volumes of Distribution, chemical imaging studies using Raman confocal microscopes have revealed how weakly basic, poorly soluble drug molecules can accumulate inside cells by forming stable, supramolecular complexes in association with cytoplasmic membranes or by precipitating out within organelles. To study the self-assembly and function of the resulting intracellular drug inclusions, Raman chemical imaging methods have been developed to measure and map the mass, concentration, and ionization state of drug molecules at a microscopic, subcellular level. Beyond the field of drug delivery, Raman chemical imaging techniques relevant to the study of microscopic drug precipitates and drug-lipid complexes which form inside cells are also being developed by researchers with seemingly unrelated scientific interests. Highlighting advances in data acquisition, calibration methods, and computational data management and analysis tools, this review will cover a decade of technological developments that enable the conversion of spectral signals obtained from Raman confocal microscopes into new discoveries and information about previously unknown, concentrative drug transport pathways driven by soluble-to-insoluble phase transitions occurring within the cytoplasmic organelles of eukaryotic cells.

Distinguishing the Concentration- vs. Bioaccumulation-Dependent Immunological and Metabolic Effects of Clofazimine.

The antimycobacterial drug clofazimine (CFZ) is used as a single agent at high doses, to suppress the exaggerated inflammation associated with leprosy. Paradoxically, increasing doses of CFZ leads to bioaccumulation of CFZ in the spleen and other organs under physiologically relevant dosing regimens, without accompanying dose-dependent elevation in the concentrations of the circulating drug in the blood. In long-term oral dosing regimens, CFZ induces immunological and metabolic changes resulting in splenomegaly, while the mass of other organs decreases or remains unchanged. As an organ that extensively sequesters CFZ as insoluble drug precipitates, the spleen likely influences drug-induced inflammatory signaling. To probe the role of systemic drug concentrations vs. drug bioaccumulation in the spleen, healthy mice were treated with six different dosing regimens. A subgroup of these mice underwent surgical splenectomies prior to drug treatment to assess the bioaccumulation-dependent changes in immune system signaling and immune-system-mediated drug distribution. Under increasing drug loading, the spleen was observed to grow up to six times in size, sequestering over 10% of the total drug load. Interestingly, when the spleen was removed prior to CFZ administration, drug distribution in the rest of the organism was unaffected. However, there were profound cytokine elevations in the serum of asplenic CFZ-treated mice, indicating that the spleen is primarily involved in suppressing the inflammatory signaling mechanisms that are upregulated during CFZ bioaccumulation. Thus, beyond its role in drug sequestration, the spleen actively modulates the systemic effect of CFZ on the immune system, without impacting its blood concentrations or distribution to the rest of the organism.

Clofazimine-Mediated, Age-Related Changes in Skeletal Muscle Mitochondrial Metabolites.

Mitochondrial health declines with age, and older patients can demonstrate dysfunction in mitochondrial-rich tissues, such as cardiac and skeletal muscle. Aged mitochondria may make older adults more susceptible to adverse drug reactions (ADRs). We assessed mitochondrial metabolic function by measuring two metabolites, l-carnitine and acetylcarnitine, to determine their effectiveness as candidate clinical biomarkers for age-related, drug-induced alterations in mitochondrial metabolism. To study age- and medication-related changes in mitochondrial metabolism, we administered the FDA-approved mitochondriotropic drug, clofazimine (CFZ), or vehicle for 8 weeks to young (4-week-old) and old (61-week-old) male C57BL/6J mice. At the end of treatment, whole blood and cardiac and skeletal muscle were analyzed for l-carnitine, acetylcarnitine, and CFZ levels; muscle function was measured via a treadmill test. No differences were found in blood or cardiac carnitine levels of CFZ-treated mice, but CFZ-treated mice displayed lost body mass and alterations in endurance and levels of skeletal muscle mitochondrial metabolites. These findings demonstrate the age-related susceptibility of the skeletal muscle to mitochondria drug toxicity. Since drug-induced alterations in mitochondrial metabolism in skeletal muscle were not reflected in the blood by l-carnitine or acetylcarnitine levels, drug-induced catabolism and changes in muscle function appear more relevant to stratifying individuals at increased risk for ADRs.

Molecular design of a pathogen activated, self-assembling mechanopharmaceutical device.

Weakly basic small molecule drugs like clofazimine can be used as building blocks for endowing cells with unnatural structural and functional elements. Here, we describe how clofazimine represents a first-in-class mechanopharmaceutical device, serving to construct inert, inactive and stimulus responsive drug depots within the endophagolysosomal compartment of cells of living organisms. Upon oral administration, clofazimine molecules self-assemble into stable, membrane-bound, crystal-like drug inclusions (CLDI) that accumulate within macrophages to form a "smart" biocompatible, pathogen activatable mechanopharmaceutical device. Upon perturbation of the mechanism maintaining pH and ion homeostasis of these CLDIs, the inert encapsulated drug precipitates are destabilized, releasing bioactive drug molecules into the cell and its surrounding. The resulting increase in clofazimine solubility activates this broad-spectrum antimicrobial, antiparasitic, antiviral or cytotoxic agent within the infected macrophage. We present a general, molecular design strategy for using clofazimine and other small molecule building blocks for the cytoplasmic construction of mechanopharmaceutical devices, aimed at rapid deployment during infectious disease outbreaks, for the purpose of pandemic prevention.

Food effects on the formulation, dosing, and administration of cannabidiol (CBD) in humans: A systematic review of clinical studies.

Cannabidiol (CBD), a non-psychotropic phytocannabinoid from the Cannabis plant, is increasingly being pursued as a treatment for differing ailments. The bioavailability and pharmacokinetics of CBD are not well understood, and proper dosing schemes have not been adequately developed for its clinical use. CBD is a lipophilic molecule and exhibits low water solubility, so its formulation expectedly impacts its gastrointestinal absorption and subsequent blood plasma concentrations. In this review article, the food effects on CBD pharmacokinetics were analyzed. Clinical trials focusing on the performance of Epidiolex, the FDA-approved CBD formulation, were found in several databases and systematically analyzed in terms of administration method, dosing schedules, and patient characteristics. 44 data sets from clinical trials were found to be useful in the quantitative analysis. Following the normalization of all the pharmacokinetic data sets by dose and patient weight, CBD exhibited a much greater bioavailability in fed patients. For Epidiolex, administration in the fed state led to lower interindividual variability and more predictable pharmacokinetics. Considering all the different oral formulations of CBD, further analysis points to the main excipient of oral CBD formulations (refined sesame seed oil) as a major contributor to the dose-dependent variations in CBD pharmacokinetics, especially affecting the fasted state. We discuss the implications of these results on the downstream pharmacodynamics of endocannabinoid receptor modulation and its broad physiological implications.

L-Carnitine and Acylcarnitines: Mitochondrial Biomarkers for Precision Medicine.

Biomarker discovery and implementation are at the forefront of the precision medicine movement. Modern advances in the field of metabolomics afford the opportunity to readily identify new metabolite biomarkers across a wide array of disciplines. Many of the metabolites are derived from or directly reflective of mitochondrial metabolism. L-carnitine and acylcarnitines are established mitochondrial biomarkers used to screen neonates for a series of genetic disorders affecting fatty acid oxidation, known as the inborn errors of metabolism. However, L-carnitine and acylcarnitines are not routinely measured beyond this screening, despite the growing evidence that shows their clinical utility outside of these disorders. Measurements of the carnitine pool have been used to identify the disease and prognosticate mortality among disorders such as diabetes, sepsis, cancer, and heart failure, as well as identify subjects experiencing adverse drug reactions from various medications like valproic acid, clofazimine, zidovudine, cisplatin, propofol, and cyclosporine. The aim of this review is to collect and interpret the literature evidence supporting the clinical biomarker application of L-carnitine and acylcarnitines. Further study of these metabolites could ultimately provide mechanistic insights that guide therapeutic decisions and elucidate new pharmacologic targets.

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

Clofazimine (CFZ) is a poorly soluble, weakly basic, small molecule antibiotic clinically used to treat leprosy and is now in clinical trials as a treatment for multidrug resistant tuberculosis and COVID-19. CFZ exhibits complex, context-dependent pharmacokinetics that are characterized by an increasing half-life in long term treatment regimens. The systemic pharmacokinetics of CFZ have been previously represented by a nonlinear, 2-compartment model incorporating an expanding volume of distribution. This expansion reflects the soluble-to-insoluble phase transition that the drug undergoes as it precipitates out and accumulates within macrophages disseminated throughout the organism. Using mice as a model organism, we studied the mechanistic underpinnings of this increasing half-life and how the systemic pharmacokinetics of CFZ are altered with continued dosing. To this end, M. tuberculosis infection status and multiple dosing schemes were studied alongside a parameter sensitivity analysis (PSA) to further understanding of systemic drug distribution. Parameter values governing the sigmoidal expansion function that captures the phase transition were methodically varied, and in turn, the systemic concentrations of the drug were calculated and compared to the experimentally measured concentrations of drug in serum and spleen. The resulting amounts of drug sequestered were dependent on the total mass of CFZ administered and the duration of drug loading. This phenomenon can be captured by altering three different parameters of an expansion function corresponding to key biological determinants responsible for the precipitation and the accumulation of the insoluble drug mass in macrophages. Through this analysis of the context dependent pharmacokinetics of CFZ, a predictive framework for projecting the systemic distribution and self-assembly of precipitated drug complexes as intracellular mechanopharmaceutical devices of this and other drugs exhibiting similarly complex pharmacokinetics can be constructed.

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

Clofazimine (CFZ) is a weakly basic, small-molecule antibiotic used for the treatment of mycobacterial infections including leprosy and multidrug-resistant tuberculosis. Upon prolonged oral administration, CFZ precipitates and accumulates within macrophages throughout the host. To model the pharmacokinetics of CFZ, the volume of distribution (Vd) was considered as a varying parameter that increases with continuous drug loading. Fitting the time-dependent change in drug mass and concentration data obtained from CFZ-treated mice, we performed a quantitative analysis of the systemic disposition of the drug over a 20-week treatment period. The pharmacokinetics data were fitted using various classical compartmental models sampling serum and spleen concentration data into separate matrices. The models were constructed in NONMEM together with linear and nonlinear sigmoidal expansion functions to the spleen compartment to capture the phase transition in Vd. The different modeling approaches were compared by Akaike information criteria, observed and predicted concentration correlations, and graphically. Using the composite analysis of the modeling predictions, adaptive fractional CFZ sequestration, Vd and half-life were evaluated. When compared to standard compartmental models, an adaptive Vd model yielded a more accurate data fit of the drug concentrations in both the serum and spleen. Including a nonlinear sigmoidal equation into compartmental models captures the phase transition of drugs such as CFZ, greatly improving the prediction of population pharmacokinetics and yielding further insight into the mechanisms of drug disposition.

Photoacoustic imaging of clofazimine hydrochloride nanoparticle accumulation in cancerous vs normal prostates.

Prostate cancer was the most common form and had the second highest death rate of male cancer in the United States in 2015. Current diagnosis techniques, such as prostate-specific antigen tests, transrectal ultrasound scans, and biopsies, are often inconclusive, and in the latter case, invasive. Here, we explore the use of clofazimine hydrochloride nanoparticles (CFZ-HCl NPs), a repurposed formulation from an FDA-approved antimycobacterial agent, as a photoacoustic contrast agent for the evaluation of prostate cancer due to its macrophage-targeting capabilities and high optical absorbance at 495 nm. Using a transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model, our results indicate a preferential accumulation of intravenously injected CFZ-HCl NPs in cancerous prostates over normal prostates. Differences in accumulation of CFZ-HCl NPs between cancerous and normal prostates were determined using a two-wavelength unmixing technique via ex vivo photoacoustic imaging. Thus, intravenous injection of CFZ-HCl NPs leads to differences in the interactions of the particles with cancerous vs normal prostates, while allowing for photoacoustic detection and analysis of prostate cancer. These findings could lead to the development of a new noninvasive technique for the detection and monitoring of prostate cancer progression in an animal model that can potentially be translated to human patients.

Inkjet-printed micro-calibration standards for ultraquantitative Raman spectral cytometry.

Herein we report the development of a cytometric analysis platform for measuring the contents of individual cells in absolute (picogram) scales; this study represents the first report of Raman-based quantitation of the absolute mass - or the total amount - of multiple endogenous biomolecules within single-cells. To enable ultraquantitative calibration, we engineered single-cell-sized micro-calibration standards of known composition by inkjet-printer deposition of biomolecular components in microarrays across the surface of silicon chips. We demonstrate clinical feasibility by characterizing the compositional phenotype of human skin fibroblast and porcine alveolar macrophage cell populations in the respective contexts of Niemann-Pick disease and drug-induced phospholipidosis: two types of lipid storage disorders. We envision this microanalytical platform as the foundation for many future biomedical applications, ranging from diagnostic assays to pathological analysis to advanced pharmaco/toxicokinetic research studies.

Acoustic tweezing cytometry for mechanical phenotyping of macrophages and mechanopharmaceutical cytotripsy.

Macrophages are immune cells responsible for tissue debridement and fighting infection. Clofazimine, an FDA-approved antibiotic, accumulates and precipitates as rod-shaped, crystal-like drug inclusions within macrophage lysosomes. Drug treatment as well as pathophysiological states could induce changes in macrophage mechanical property which in turn impact their phenotype and function. Here we report the use of acoustic tweezing cytometry as a new approach for in situ mechanical phenotyping of macrophages and for targeted macrophage cytotripsy. Acoustic tweezing cytometry applies ultrasound pulses to exert controlled forces to individual cells via integrin-bound microbubbles, enabling a creep test for measuring cellular mechanical property or inducing irreversible changes to the cells. Our results revealed that macrophages with crystal-like drug inclusions became significantly softer with higher cell compliance, and behaved more elastic with faster creep and recovery time constants. On the contrary, phagocytosis of solid polyethylene microbeads or treatment with soluble clofazimine rendered macrophages stiffer. Most notably, application of ultrasound pulses of longer duration and higher amplitude in ATC actuated the integrin-bound microbubbles to mobilize the crystal-like drug inclusions inside macrophages, turning the rod-shaped drug inclusions into intracellular microblender that effectively destructed the cells. This phenomenon of acoustic mechanopharmaceutical cytotripsy may be exploited for ultrasound activated, macrophage-directed drug release and delivery.

Synthesis and Characterization of a Biomimetic Formulation of Clofazimine Hydrochloride Microcrystals for Parenteral Administration.

Clofazimine (CFZ) is a broad spectrum antimycobacterial agent recommended by the World Health Organization as a first line treatment for leprosy and second line treatment for multidrug resistant tuberculosis. Oral administration of CFZ leads to a red skin pigmentation side effect. Since CFZ is a weakly basic, red phenazine dye, the skin pigmentation side effect results from lipophilic partitioning of the circulating, free base (neutral) form of CFZ into the skin. Here, we developed a stable and biocompatible formulation of CFZ-HCl microcrystals that mimics the predominant form of the drug that bioaccumulates in macrophages, following long term oral CFZ administration. In mice, intravenous injection of these biomimetic CFZ-HCl microcrystals led to visible drug accumulation in macrophages of the reticuloendothelial system with minimal skin accumulation or pigmentation. In fact, no skin pigmentation was observed when the total amount of CFZ-HCl administered was equivalent to the total oral dose leading to maximal skin pigmentation. Thus, parenteral (injected or inhaled) biomimetic formulations of CFZ-HCl could be instrumental to avoid the pigmentation side effect of oral CFZ therapy.

An Expandable Mechanopharmaceutical Device (2): Drug Induced Granulomas Maximize the Cargo Sequestering Capacity of Macrophages in the Liver.

PURPOSE: Drug-induced liver injuries (DILI) comprise a significant proportion of adverse drug reactions leading to hospitalizations and death. One frequent DILI is granulomatous inflammation from exposure to harmful metabolites that activate inflammatory pathways of immune cells of the liver, which may act as a barrier to isolate the irritating stimulus and limit tissue damage. METHODS: Paralleling the accumulation of CFZ precipitates in the liver, granulomatous inflammation was studied to gain insight into its effect on liver structure and function. A structural analog that does not precipitate within macrophages was also studied using micro-analytical approaches. Depleting macrophages was used to inhibit granuloma formation and assess its effect on drug bioaccumulation and toxicity. RESULTS: Granuloma-associated macrophages showed a distinct phenotype, differentiating them from non-granuloma macrophages. Granulomas were induced by insoluble CFZ cargo, but not by the more soluble analog, pointing to precipitation being a factor driving granulomatous inflammation. Granuloma-associated macrophages showed increased activation of lysosomal master-regulator transcription factor EB (TFEB). Inhibiting granuloma formation increased hepatic necrosis and systemic toxicity in CFZ-treated animals. CONCLUSIONS: Granuloma-associated macrophages are a specialized cell population equipped to actively sequester and stabilize cytotoxic chemotherapeutic agents. Thus, drug-induced granulomas may function as drug sequestering "organoids" -an induced, specialized sub-compartment- to limit tissue damage.

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

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.

An Expandable Mechanopharmaceutical Device (3): a Versatile Raman Spectral Cytometry Approach to Study the Drug Cargo Capacity of Individual Macrophages.

PURPOSE: To improve cytometric phenotyping abilities and better understand cell populations with high interindividual variability, a novel Raman-based microanalysis was developed to characterize macrophages on the basis of chemical composition, specifically to measure and characterize intracellular drug distribution and phase separation in relation to endogenous cellular biomolecules. METHODS: The microanalysis was developed for the commercially-available WiTec alpha300R confocal Raman microscope. Alveolar macrophages were isolated and incubated in the presence of pharmaceutical compounds nilotinib, chloroquine, or etravirine. A Raman data processing algorithm was specifically developed to acquire the Raman signals emitted from single-cells and calculate the signal contributions from each of the major molecular components present in cell samples. RESULTS: Our methodology enabled analysis of the most abundant biochemicals present in typical eukaryotic cells and clearly identified "foamy" lipid-laden macrophages throughout cell populations, indicating feasibility for cellular lipid content analysis in the context of different diseases. Single-cell imaging revealed differences in intracellular distribution behavior for each drug; nilotinib underwent phase separation and self-aggregation while chloroquine and etravirine accumulated primarily via lipid partitioning. CONCLUSIONS: This methodology establishes a versatile cytometric analysis of drug cargo loading in macrophages requiring small numbers of cells with foreseeable applications in toxicology, disease pathology, and drug discovery.

Reverse Engineering the Intracellular Self-Assembly of a Functional Mechanopharmaceutical Device.

Weakly basic, poorly soluble chemical agents could be exploited as building blocks for constructing sophisticated molecular devices inside the cells of living organisms. Here, using experimental and computational approaches, we probed the relationship between the biological mechanisms mediating lysosomal ion homeostasis and the self-assembly of a weakly basic small molecule building block (clofazimine) into a functional, mechanopharmaceutical device (intracellular Crystal-Like Drug Inclusions - "CLDIs") in macrophage lysosomes. Physicochemical considerations indicate that the intralysosomal stabilization of the self-assembled mechanopharmaceutical device depends on the pHmax of the weakly basic building block and its affinity for chloride, both of which are consistent with the pH and chloride content of a physiological lysosomal microenvironment. Most importantly, in vitro and in silico studies revealed that high expression levels of the vacuolar ATPase (V-ATPase), irrespective of the expression levels of chloride channels, are necessary and sufficient to explain the cell-type dependent formation, stabilization, and biocompatibility of the self-assembled mechanopharmaceutical device within macrophages.

The Physicochemical Basis of Clofazimine-Induced Skin Pigmentation.

Clofazimine is a weakly basic, Food and Drug Administration-approved antibiotic recommended by the World Health Organization to treat leprosy and multi-drug-resistant tuberculosis. Upon prolonged treatment, clofazimine extensively bioaccumulates and precipitates throughout the organism, forming crystal-like drug inclusions (CLDIs). Due to the drug's red color, it is widely believed that clofazimine bioaccumulation results in skin pigmentation, its most common side effect. To test whether clofazimine-induced skin pigmentation is due to CLDI formation, we synthesized a closely related clofazimine analog that does not precipitate under physiological pH and chloride conditions that are required for CLDI formation. Despite the absence of detectable CLDIs in mice, administration of this analog still led to significant skin pigmentation. In clofazimine-treated mice, skin cryosections revealed no evidence of CLDIs when analyzed with a microscopic imaging system specifically designed for detecting clofazimine aggregates. Rather, the reflectance spectra of the skin revealed a signal corresponding to the soluble, free base form of the drug. Consistent with the low concentrations of clofazimine in the skin, these results suggest that clofazimine-induced skin pigmentation is not due to clofazimine precipitation and CLDI formation, but rather to the partitioning of the circulating, free base form of the drug into subcutaneous fat.

The physiological determinants of drug-induced lysosomal stress resistance.

Many weakly basic, lipophilic drugs accumulate in lysosomes and exert complex, pleiotropic effects on organelle structure and function. Thus, modeling how perturbations of lysosomal physiology affect the maintenance of lysosomal ion homeostasis is necessary to elucidate the key factors which determine the toxicological effects of lysosomotropic agents, in a cell-type dependent manner. Accordingly, a physiologically-based mathematical modeling and simulation approach was used to explore the dynamic, multi-parameter phenomenon of lysosomal stress. With this approach, parameters that are either directly involved in lysosomal ion transportation or lysosomal morphology were transiently altered to investigate their downstream effects on lysosomal physiology reflected by the changes they induce in lysosomal pH, chloride, and membrane potential. In addition, combinations of parameters were simultaneously altered to assess which parameter was most critical for recovery of normal lysosomal physiology. Lastly, to explore the relationship between organelle morphology and induced stress, we investigated the effects of parameters controlling organelle geometry on the restoration of normal lysosomal physiology following a transient perturbation. Collectively, our results indicate a key, interdependent role of V-ATPase number and membrane proton permeability in lysosomal stress tolerance. This suggests that the cell-type dependent regulation of V-ATPase subunit expression and turnover, together with the proton permeability properties of the lysosomal membrane, is critical to understand the differential sensitivity or resistance of different cell types to the toxic effects of lysosomotropic drugs.

Detecting ordered small molecule drug aggregates in live macrophages: a multi-parameter microscope image data acquisition and analysis strategy.

Following prolonged administration, certain orally bioavailable but poorly soluble small molecule drugs are prone to precipitate out and form crystal-like drug inclusions (CLDIs) within the cells of living organisms. In this research, we present a quantitative multi-parameter imaging platform for measuring the fluorescence and polarization diattenuation signals of cells harboring intracellular CLDIs. To validate the imaging system, the FDA-approved drug clofazimine (CFZ) was used as a model compound. Our results demonstrated that a quantitative multi-parameter microscopy image analysis platform can be used to study drug sequestering macrophages, and to detect the formation of ordered molecular aggregates formed by poorly soluble small molecule drugs in animals.