Multiscale Biodistribution Analysis of Lipophilic, Poorly Soluble Drugs.

Clofazimine is a poorly soluble drug that accumulates as solid deposits in the body during prolonged oral administration. At the outset, we hypothesized that clofazimine accumulated intracellularly by a passive and spontaneous crystallization, and in various levels of experimental set-ups, from a tissue culture to mouse models. We found that clofazimine readily formed amorphous inclusions in complexes with intracellular membranes in MDCK cells, while different types of inclusions were found in the tissue macrophages of clofazimine-diet fed mice. Most of the inclusions in vivo appeared as vibrant red, birefringent, 10 – 20 µm length crystal-like structures; however, their physicochemical and morphological characteristics were inconsistent with those of pure clofazimine crystals. Most remarkably, among the inclusions from macrophages, we discovered a new cytoplasmic structure delimited by double membranes with internal supramolecular organizations resembling stacks of lipidic lamellae. Upon prolonged dosing, the intact clofazimine was redistributed from adipose tissues to the lymphatic organs paralleled by anti-inflammatory responses such as splenomegaly, liver microgranulomas, and an expansion of macrophage populations. In conclusion, instead of passive intracellular crystallization hypothesis, I propose that clofazimine accumulates in vivo by active sequestration in the immune system. By constructing intracellular crystal- and organelle-like “polyhedrosomes”, the macrophages can impact the clofazimine’s systemic pharmacokinetics and biodistribution, from micro to macro scale.Ph.D.Pharmaceutical SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91404/1/jsbaik_1.pd

Melatonin receptor 1 B polymorphisms associated with the risk of gestational diabetes mellitus

<p>Abstract</p> <p>Backgrounds</p> <p>Two SNPs in <it>melatonin receptor 1B </it>gene, <it>rs10830963 </it>and <it>rs1387153 </it>showed significant associations with fasting plasma glucose levels and the risk of Type 2 Diabetes Mellitus (T2DM) in previous studies. Since T2DM and gestational diabetes mellitus (GDM) share similar characteristics, we suspected that the two genetic polymorphisms in <it>MTNR1B </it>may be associated with GDM, and conducted association studies between the polymorphisms and the disease. Furthermore, we also examined genetic effects of the two polymorphisms with various diabetes-related phenotypes.</p> <p>Methods</p> <p>A total of 1,918 subjects (928 GDM patients and 990 controls) were used for the study. Two <it>MTNR1B </it>polymorphisms were genotyped using TaqMan assay. The allele distributions of SNPs were evaluated by <it>x</it>²models calculating odds ratios (ORs), 95% confidence intervals (CIs), and corresponding <it>P </it>values. Multiple regressions were used for association analyses of GDM-related traits. Finally, conditional analyses were also performed.</p> <p>Results</p> <p>We found significant associations between the two genetic variants and GDM, <it>rs10830963</it>, with a corrected <it>P </it>value of 0.0001, and <it>rs1387153</it>, with the corrected <it>P </it>value of 0.0008. In addition, we also found that the two SNPs were associated with various phenotypes such as homeostasis model assessment of beta-cell function and fasting glucose levels. Further conditional analyses results suggested that <it>rs10830963 </it>might be more likely functional in case/control analysis, although not clear in GDM-related phenotype analyses.</p> <p>Conclusion</p> <p>There have been studies that found associations between genetic variants of other genes and GDM, this is the first study that found significant associations between SNPs of <it>MTNR1B </it>and GDM. The genetic effects of two SNPs identified in this study would be helpful in understanding the insight of GDM and other diabetes-related disorders.</p

Clofazimine inclusions formed in macrophage-like cells <i>in vivo</i>.

<p>(<b>A</b>) Mice fed with clofazimine (above) showed reddish pigmentation visible in the ear, tail, and skin when compared to mice treated with vehicle only (below). (<b>B</b>) Weight gains in mice fed with and without clofazimine were comparable (N = 40, ▪, vehicle; ○, treated; *, P<0.01, end-point T-test). (<b>C</b>) Biochemical analysis of various organs revealed differences in the accumulation and retention of clofazimine after wash out (*, P<0.01, N = 5 per group, ANOVA). (<b>D</b>) Ruby red inclusions appeared in frozen sections of spleen, lung and liver, but not in kidneys of 8 wk supplemented diet. H, hepatocyte; V, blood vessel; M, microgranulomas. (<b>E</b>) Intracellular inclusions were extracted in perfusion-fixed liver upon ethanol-dehydration and staining with toluidine blue. Arrows indicate needle-like cavities remaining after extraction. (<b>F</b>) Histological sections revealed cellular changes in liver of mice fed with clofazimine. H&E staining, F4/80 macrophage specific marker, Masson's trichrome staining (MTS, collagen fibers), von Willebrand factor (vWF, endothelium) and alpha smooth muscle actin (αSMA). K, Kupffer cells. Scale bar  = 10 µm unless otherwise indicated.</p

Macrophages containing intracellular CLDIs were collected, plated and studied <i>in vitro</i>.

<p>(<b>A</b>) Bone marrow macrophage (BMM) and thioglycollate elicited peritoneal macrophages (PM) were obtained from mice fed with clofazimine, attached and spread on tissue culture plastic, and were stained with Hoechst 33342 to show nuclei. (<b>B</b>) Peritoneal macrophages with CLDIs migrated away from large clusters when plated on tissue culture dishes. (<b>C</b>) Illuminating peritoneal macrophages with blue (490 nm) light triggers clofazimine release (observed in TRITC channel) from CLDIs. (<b>D</b>) Once removed from cells, extracellular CLDIs grew in size and became irregular in morphology, unlike intracellular CLDIs. Red blood cells (d = 8 µm) in the background serve as size markers, for reference. (<b>E</b>) CLDIs inside bone marrow-derived cells in suspension, stained with Trypan Blue. Scale bars  = 10 µm unless otherwise indicated.</p

Deep-etch freeze-fracture electron microscopy of isolated CLDIs.

<p>(<b>A</b>) Pure isolated CLDIs stood out from surrounding ice and cytosolic debris based on their elongated polyhedral shape and internal layered structure. (<b>B</b>) Isolated CLDIs clearly lacked the outer double membrane covering. (<b>C</b>) Biochemically-isolated CLDI often showed outer layers of material that appeared to be peeling off from the structure.</p