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.

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.

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.

A Family of Nonribosomal Peptides Modulate Collective Behavior in Pseudovibrio Bacteria Isolated from Marine Sponges*.

Although swarming motility and biofilms are opposed collective behaviors, both contribute to bacterial survival and host colonization. Pseudovibrio bacteria have attracted attention because they are part of the microbiome of healthy marine sponges. Two-thirds of Pseudovibrio genomes contain a member of a nonribosomal peptide synthetase-polyketide synthase gene cluster family, which is also found sporadically in Pseudomonas pathogens of insects and plants. After developing reverse genetics for Pseudovibrio, we isolated heptapeptides with an ureido linkage and related nonadepsipeptides we termed pseudovibriamides A and B, respectively. A combination of genetics and imaging mass spectrometry experiments showed heptapetides were excreted, promoting motility and reducing biofilm formation. In contrast to lipopeptides widely known to affect motility/biofilms, pseudovibriamides are not surfactants. Our results expand current knowledge on metabolites mediating bacterial collective behavior.

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.