Multiscale Distribution and Bioaccumulation Analysis of Clofazimine

Multiscale Distribution and Bioaccumulation Analysis of ClofazimineReveals a Massive Immune System-Mediated Xenobiotic SequestrationResponse

Jason Baik,a* Kathleen A. Stringer,b Gerta Mane,b Gus R. Rosaniaa

Department of Pharmaceutical Sciencesa and Department of Clinical, Social, and Administrative Sciences,b University of Michigan College of Pharmacy, Ann Arbor,Michigan, USA

Chronic exposure to some well-absorbed but slowly eliminated xenobiotics can lead to their bioaccumulation in living organ-isms. Here, we studied the bioaccumulation and distribution of clofazimine, a riminophenazine antibiotic used to treat myco-bacterial infection. Using mice as a model organism, we performed a multiscale, quantitative analysis to reveal the sites of clo-fazimine bioaccumulation during chronic, long-term exposure. Remarkably, between 3 and 8 weeks of dietary administration,clofazimine massively redistributed from adipose tissue to liver and spleen. During this time, clofazimine concentration in fatand serum significantly decreased, while the mass of clofazimine in spleen and liver increased by >10-fold. These changes wereparalleled by the accumulation of clofazimine in the resident macrophages of the lymphatic organs, with as much as 90% of theclofazimine mass in spleen sequestered in intracellular crystal-like drug inclusions (CLDIs). The amount of clofazimine associ-ated with CLDIs of liver and spleen macrophages disproportionately increased and ultimately accounted for a major fraction ofthe total clofazimine in the host. After treatment was discontinued, clofazimine was retained in spleen while its concentrationsdecreased in blood and other organs. Immunologically, clofazimine bioaccumulation induced a local, monocyte-specific upregu-lation of various chemokines and receptors. However, interleukin-1 receptor antagonist was also upregulated, and the acute-phase response pathways and oxidant capacity decreased or remained unchanged, marking a concomitant activation of an anti-inflammatory response. These experiments indicate an inducible, immune system-dependent, xenobiotic sequestration responseaffecting the atypical pharmacokinetics of a small molecule chemotherapeutic agent.

Clofazimine is an orally bioavailable, poorly soluble, highly li-pophilic (logP 7) antibiotic drug with a very long pharma-cokinetic half-life (16). It was first marketed in 1969 as Lam-prene, and in 1983 it was recommended by the World HealthOrganization as one of the components of multidrug therapyagainst leprosy (7). Subsequently, in 1986, it was approved by theU.S. Food and Drug Administration (FDA; http://www.accessdata.fda.gov/scripts/cder/drugsatfda/). During prolonged oral dosing,clofazimine is widely distributed and accumulates throughout theorganism and is eliminated very slowly by renal and hepatic routes(8). Clofazimine is deep red in color, so its accumulation in skinand other organs leads to a visible dark purple pigmentation (9).Although the human and animal pharmacology and pharmacoki-netics of clofazimine have been quantitatively studied duringshort-term treatment (1, 5), there is little quantitative, mechanis-tic information about the distribution of clofazimine duringchronic exposure or about the associated physiological responsesof the organism to clofazimine bioaccumulation. Like many otherlipophilic small molecule drugs, clofazimines long half-life andatypical pharmacokinetic properties have been ascribed to itshighly hydrophobic character leading to lipophilic partitioninginto adipose tissue.

Although clofazimine is clinically useful in the treatment ofMycobacterium leprae, new generations of clofazimine derivativesare being sought as drug candidates to treat multidrug-resistantstrains of Mycobacterium tuberculosis (10). These drug candidatescould benefit from decreased bioaccumulation and improvedpharmacokinetic properties. Clofazimine is also interesting as aprobe to study the physiological effects of long-term exposure tonatural product-derived, orally bioavailable, bioaccumulating

small molecule xenobiotics (11). Qualitatively (1214), clofazi-mine is known to form crystal-like drug inclusions (CLDIs) insidecells of the mononuclear phagocyte system. However, the extentto which clofazimine accumulates in these intracellular inclusionshas not been quantitatively established (12, 15, 16). Because it is alipophilic, weakly basic molecule (17), clofazimine is likely to ac-cumulate inside acidic organelles by a pH-dependent ion trappingmechanism (18). However, the calculated logD of clofazimineranges from 5 to 7 in physiological pH (see Fig. S1 in thesupplemental material), suggesting that a large fraction of thecompound can also partition into organelle membranes. In a kid-ney-derived epithelial cell line, clofazimine accumulated in drug-induced, autophagosome-like drug-membrane aggregates (19).These drug-membrane aggregates resembled the multilamellarbodies that form inside cells exposed to phospholipidosis-induc-ing, lysosomotropic amphiphilic cations (20, 21). Consistent with

Received 21 August 2012 Returned for modification 6 October 2012Accepted 8 December 2012

Published ahead of print 21 December 2012

Address correspondence to Gus R. Rosania, [email protected].

* Present address: Jason Baik, Department of Bioengineering and TherapeuticSciences, University of CaliforniaSan Francisco, San Francisco, California, USA.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.01731-12.

Copyright 2013, American Society for Microbiology. All Rights Reserved.

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lysosomal accumulation, clofazimine treatment also affects theactivity of lysosomal enzymes, both in vitro (22) and in vivo (23).

In mammals, the cells of the immune system are considered toplay a minor role in the disposition of xenobiotics and the sys-temic pharmacokinetics of small molecule drugs. Accordingly, wehypothesized that clofazimine sequestration in macrophages wasonly of minor consequence to the overall bioaccumulation anddistribution of clofazimine in vivo. To test this hypothesis, wemeasured the impact of this sequestration mechanism on the totalbioaccumulation and distribution of clofazimine. Taking advan-tage of its deep red color, low metabolic rate, and slow clearance(4, 24), we performed a multiscale biodistribution analysis of clo-fazimine, measuring its bioaccumulation in all of the major or-gans, adipose tissue, and serum, combined with microscopic andbiochemical analysis of CLDIs found in macrophages of the liverand spleen. Unexpectedly, the massive bioaccumulation of clo-fazimine in cells of the immune system accounted for dramaticchanges in the distribution of clofazimine between 3 and 8 weeksof treatment. Follow-up proteomic analysis revealed local changesin the levels of key cytokines, chemokines, and immunologicalsignaling molecules at the sites of clofazimine bioaccumulation,which were most consistent with a general anti-inflammatory re-sponse. These results suggest a candidate, macrophage-dependentxenobiotic sequestration response affecting the distribution, bio-accumulation, and other side effects of clofazimine duringchronic, long-term exposure.

MATERIALS AND METHODSAnimal experiments. Mice (4 week old, male BALB/c) were purchasedfrom the Jackson Laboratory (Bar Harbor, ME) and acclimatized for 2weeks in a specific-pathogen-free animal facility. All animal care was pro-vided by the University of Michigans Unit for Laboratory Animal Medi-cine (ULAM), and the experimental protocol was approved by the Com-mittee on Use and Care of Animals. Clofazimine (C8895; Sigma-Aldrich,St. Louis, MO) was dissolved in sesame oil (Roland, China, or Shirakiku,Japan) to achieve a concentration of 3 mg/ml, which was mixed withPowdered Lab Diet 5001 (PMI International, Inc., St. Louis, MO) to pro-duce a 0.03% drug to powdered feed mix. A corresponding amount ofsesame oil was mixed with chow for vehicle treatment (control). On av-erage, food consumption for a 25-g mouse was 3 g per day, resulting in10 mg of bioavailable drug/kg per day (25).

Biochemical analysis of clofazimine in tissues. Drug tissue contentswere analyzed by liquid chromatography-mass spectrometry (LC-MS)and spectrophotometrically, as previously described (1214). In brief, af-ter feeding either clofazimine- or vehicle-supplemented food, mice wereeuthanized by CO2, and blood and organs were harvested. Tissue (0.05 to0.1 g/ml of water) was homogenized, and 100 l of sample was mixed withthe same volume of 5 N NaOH and then extracted with 300 l of dichlo-romethane twice (6). After centrifugation (2,000 g, 10 min) to collectthe dichloromethane layer, the solvent was evaporated (40C) and recon-stituted in methanol for absorbance measurement (490 nm; Synergy-2plate reader; Biotek Instruments, Winooski, VT). Clofazimine concentra-tion was calculated from the standard curve generated by adding a knownamount of drug solution to a tissue extract from a vehicle-only treatedsample. The average extraction yield of clofazimine was 60 to 80% for allorgans except for abdominal fat, which yielded nearly a 100% recovery,and all contents were compensated for by the recovery yield. The accu-mulated drug mass was determined by converting clofazimine concentra-tions to mass by multiplying either the measured organ weight (duode-num, 0.33 0.3 g; jejunum plus ileum, 1.3 0.06 g [see Results for spleenand kidney weights]) or the reported organ weight (liver, 1.2 g; fat, 16.6%of body weight, which were 25.2 1.8 g at week 3 and 27.9 1.8 g at week8 [Jackson Laboratory, http://phenome.jax.org/db]). The total drug

amounts available in the body for week 3 and 8 were estimated by multi-plying absorption rate with cumulative weekly average body weight pre-sented in Fig. 1A. The excreted plus unaccounted drug fraction in theorganism was calculated by subtracting the measured organ contentsfrom the total drug amount. LC-MS was used to confirm that the mea-sured drug in tissue was metabolically intact (13).

Determination of clofazimine concentration in serum. Blood wascollected in microtainer serum separator tubes (catalog no. 3659656; Bec-ton Dickinson, Franklin lakes, NJ) and were allowed to clot at room tem-perature and centrifuged (7,000 g, 5 min). The resulting supernatantserum was submitted to the University of Michigans Nutrition and Obe-sity Research Center for LC-MS analysis. Samples (20 l) were extractedwith acetonitrile (60 l, 90% extraction efficiency) for 10 min at 4C withintermittent vortexing. After centrifugation (15,000 rpm, 4C), the super-natant was injected into Agilent 1200 RRLC coupled to 6410 Triple QuadLC-MS equipped with an Xbridge C18 column (2.5 m, 2.1 mm [innerdiameter] by 100 mm; Waters). Mobile phase A was 5 mM ammoniumacetate, adjusted to pH 9.9 with ammonium hydroxide, and mobile phaseB was acetonitrile. The flow rate was 0.35 ml/min, with a linear gradientfrom 50 to 100% phase B over 1.5 min, followed by holding at 100% for1.5 min, a return to 50% phase B, and then re-equilibration for 2.5 min.The mass spectrometer source conditions were set as follows: 325C, gasflow at 10 liters/min, nebulizer at 40 lb/in2, capillary at 4,000 V, andpositive ion mode. The MS acquisition parameters were as follows: MRMmode, transition 1 set at 473.1 to 1:431.1, a dwell time of 400 ms, frag-mentor set at 180, a collision energy of 40; transition 2 set at 473.1 to 429.1,a dwell time of 100 ms, fragmentor set at 180, and a collision energy of 40.A standard curve was generated using serum from a vehicle-only treatedmouse mixed with clofazimine stock solution from dimethyl sulfoxide,resulting in 10 different clofazimine concentrations between 0 and 30 M.The peak area was quantified using MassHunter Quantitative Analysissoftware, vB.04.00.

Isolation of CLDIs. Crystal-like drug inclusion (CLDI) purificationwas performed as previously described (13). In brief, organ homogenatesfrom two mice treated with clofazimine for 8 weeks were sonicated andthen centrifuged (100 g, 1 min) to remove debris. The resulting super-natants were mixed with 0.125% trypsin-EDTA solution (Gibco) and in-cubated at 37C for 1 h, followed by another centrifugation (100 g, 1min). The supernatants were centrifuged (21,000 g, 1 min), and thepelleted CLDIs were resuspended in water for analysis. For assaying pro-tein content of the samples, equal volumes of 5% sodium dodecyl sulfatesolution and sample were mixed, and the protein content was measuredby bicinchoninic acid (BCA) assay (Pierce 23227; Thermo Scientific).

Sample preparation for microscopy. Following the postmortem col-lection of blood samples, some euthanized mice were perfused via the leftventricle with Sorensens buffer (0.1 M), followed by Karnovskys fixative(3% paraformaldehyde, 2.5% glutaraldehyde, 0.1 M Sorensens buffer[pH 7.4]), until the liver was visibly clear of blood. Immediately afterperfusion, the organs were removed in preparation for either paraffinembedding or transmission electron microscopy (TEM).

For immunohistochemical staining, paraffin embedding of the perfu-sion-fixed organs and heat-mediated antigen retrieval were carried out inthe histology lab at the Pathology Cores of Animal Research (PCAR),ULAM, at the University of Michigan. Routine hematoxylin and eosin(H&E) and Massons trichrome staining (MTS), as well as immunohisto-chemistry of F4/80 (1/100 dilution, ab6640; Abcam), -smooth muscleactin (-SMA; 1/200, ab5694; Abcam), vWF (1/500, ab7356; Millipore),CD21 (1/200, ab75985; Abcam), and CD3T (1/300, RM-9107S; ThermoScientific) were carried out using horseradish peroxidase and intelliPATHFLX DAB chromogen (IPK5010; Biocare Medical, Concord, CA).

For TEM, organs were submerged in fixative, and diced into pieces(1 mm). The minced organs were preserved in a glass vial with fixativeand stored at 4C. After three rinses with Sorensens buffer (0.1 M), dicedtissues were stained with 1% osmium tetroxide in Sorensens buffer andwashed three times in Sorensens buffer. Dehydration was carried out with

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a graded ethanol-water series (50, 70, and 90% and two changes of 100%)for 15 min each. After transition through three changes of propyleneoxide, the tissues were infiltrated with Epon resin (Electron MicroscopySciences) and then polymerized at 60C for 24 h. Next, the blocks weresectioned (70 nm) using a ultramicrotome and mounted on a copper EMgrid (Electron Microscopy Sciences), which was post-stained with uranylacetate and lead citrate before imaging.

Cryosectioning was carried out using a Leica 3050S cryostat. Sampleswere sectioned to 10 m and mounted onto glass slide with a drop ofglycerol and cover glass. In preparation for cryosectioning, the organswere not perfused with fixative but instead were removed, immediatelysubmerged at an optimal cutting temperature (Tissue-Tek catalog no.4583; Sakura), and frozen (80C).

Transmitted light, polarization, and fluorescence microscopy. AnOlympus 51 upright epifluorescence/polarization microscopeequipped with 100 objective lens (1.40 NA, PlanApo oil emersion),cross polarizers, and a DP-70 color camera was used. For fluorescence, aU-MWIBA3 eGFP filter cube for the green channel and a U-MWG2 (rho-damine) filter cube for the red channel were used. Images were acquiredusing a DP controller 3.1.1.267 under the same exposure settings. Fordisplay purposes, the image brightness, contrast, and color balance wasadjusted using Microsoft PowerPoint and Adobe Photoshop. For controland experimental comparisons within the same figure, settings were ad-justed to the same.

TEM. Images were acquired using a Philips CM-100 TEM and digitallyrecorded using a Hamamatsu ORCA-HR camera system operated by Ad-vanced Microscopy Techniques software (Danvers, MA).

Measurement of antioxidant responses. To determine whether clo-fazimine administration induced oxidant stress, we measured manganesesuperoxide dismutase (MnSOD) in organs obtained from mice postmor-tem (26) that were then immediately frozen at 80C after harvesting. Inpreparation for immunoblotting, organ samples were thawed on ice andhomogenized as previously described (26). Just prior to assay, the proteinconcentration of homogenates was determined by using a BCA assay.

Homogenate samples (50 g of total protein) were subjected to gra-dient 4 to 20% SDS-PAGE and transferred to polyvinylidene difluoride.The membranes were blocked in 5% milk in phosphate-buffered saline(PBS)Tween buffer (1 PBS, 0.1% Tween) overnight (4C). The mem-branes were then cut into two sections according to the molecular weightsof MnSOD and actin. Each respective membrane was incubated with anantibody specific for MnSOD (1:5,000 dilution, rabbit anti-MnSOD an-tibody; Upstate Biotechnology, Charlottesville, VA) or actin (1:1,000,000dilution, mouse anti-actin; Sigma) for 1 h at room temperature. Afterbeing washed, the membranes were incubated with secondary antibodyconjugated to horseradish peroxidase (Santa Cruz Biotechnology, SantaCruz, CA) for 1 h at room temperature and washed again, and enhancedchemiluminescence was detected using an ECL Plus reagent kit (GEHealthcare Life Sciences, Piscataway, NJ) exposed to radiograph film(Fuji, Stamford, CT). Scanned protein bands were quantified by densi-tometry with ImageJ software (ImageJ 1.44b; National Institutes of Health[http://rsb.info.nih.gov/ij]). The ratio of the density of the MnSOD pro-tein band to the density of the corresponding actin protein band wasdetermined for each sample.

To further investigate the redox status of clofazimine-treated mouseorgans, we used a commercial antioxidant assay kit (CS0790; Sigma-Al-drich) that measures the formation of a ferryl myoglobin radical frommetmyoglobin and hydrogen peroxide, ABTS [2,2=azinobis(3-ethylbenz-thiazolinesulfonic acid)], to produce a radical cation. This cation is greenin color and can be spectrophotometrically measured at 405 nm. When

FIG 1 Changes in pigmentation were observed in clofazimine-treated mice. (A)Change in the body weight of mice over an 8-week clofazimine treatment period,followed by an 8-week washout period by feeding a drug-free, regular diet. Ani-mals in both groups gained weight over the 16 week study period (*, P 0.05). (B)Skin pigmentation was apparent at 8 weeks of treatment (right) compared to themouse treated with vehicle-only diet (left). (C) Significant pigmentation was ob-served in the lungs, liver, spleen, jejunum/ileum, and mesenteric lymph node(mLN). Pale orange pigmentation was observed in the hypodermis of the skin,abdominal fat, omental fat attached to mLN, and tips of the femurs. The color ofthe heart, brain, pancreas, kidney, and duodenum remained unchanged. (D) Eightweeks of drug treatment induced splenomegaly, which further increased during

washout period (WO) compared to the controls (drug-free diet) or 3 weeks oftreatment. In contrast, there was no mass change in the kidney. The data arethe means the SD of four to six animals per group. *, P 0.05; ***, P 0.001, ANOVA.

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antioxidants are present the production of the radical cation is suppressedin a concentration-dependent manner and the color intensity propor-tionally declines. The kit utilizes Trolox, a water-soluble vitamin E analog,as a standard or positive control antioxidant. We used the assay kit tomeasure the total oxidant content of the samples by titrating out theoxidizing capacity of each tissue lysate with increasing amounts of Trolox.All samples were adjusted to 20 g of protein at the starting point of eachtitration. All other aspects of the assay were conducted in accordance withthe manufacturers instructions.

Measurement of cytokine-chemokine responses. Changes in cyto-kine and chemokines in response to clofazimine treatment were measuredusing a cytokine array assay (R&D Systems mouse cytokine array, panel A,catalog no. ARY006). This assay, similar to the enzyme-linked immu-nosorbent assay, simultaneously measures the relative levels of 40 differ-ent cytokines and chemokines by utilizing selected capture antibodiesspotted in duplicate on nitrocellulose membranes. Tissue homogenateswere prepared according to the manufacturers instructions, and 300 gof lysate protein of the liver, spleen, brain, and lung samples from clofazi-mine-treated or control, vehicle-treated mice was added to a cocktail ofbiotinylated detection antibodies provided in the kit. The samples fromeach organ and the respective negative control samples were simultane-ously assayed. After the detection of antibody complexes by streptavidin-horseradish peroxidase chemiluminescence using radiographic film, thepixel density of each cytokine signal was quantified by densitometry usingImageJ. The data were normalized by dividing the density value of eachcytokine by the mean density value of the positive controls from the cor-responding membrane. The mean densities of each cytokine for each or-gan from vehicle-treated and clofazimine-treated mice were determinedand statistically compared.

Data analysis. For weight and drug concentration/mass analyses,three to six mice per each group were weighed weekly and, by the time oftheir euthanasia, individual organs were isolated and washed three timesin cold DPBS, surgically removing other connective tissues. Afterward,they were gently tab dried on gauze for weight measurements, which wereaveraged for each group. For intestines, the lumen were flushed withDPBS by injecting 5 to 10 ml of DPBS to remove the unabsorbed contents.All of the data points presented here are means the standard deviations(SD), from three to six organs. Statistical analyses were performed byanalysis of variance (ANOVA) with Tukeys HSD using the R softwarepackage.

For biochemical assays, the mean MnSOD/actin ratio, the mean ab-sorbance for each Trolox concentration, and the mean normalized densityvalue the SD of each cytokine from each organs were compared byunpaired Student t test with Welchs correction for differences in vari-ances when applicable. The resulting P values from the analysis of thecytokine data were tested using InStat (GraphPad Software, La Jolla, CA)and corrected for multiple comparisons by calculating the false discoveryrate (FDR) (27, 28).

RESULTSProlonged clofazimine administration led to visible changes inmouse organs. Based on the amount of food consumed per dayand clofazimines bioavailability (25), we estimated an intake of 10mg of bioavailable drug/kg per day, consistent with previous stud-ies (12). Prolonged administration (8 weeks) of this regimen andthe vehicle (sesame oil) mixed diet was well tolerated. Duringtreatment, body weight increased and continued to increase afterthe discontinuation of treatment (Fig. 1A). Red pigmentation ofthe skin was visible after the first week of clofazimine treatment(Fig. 1B). Dissection revealed visible pigmentation of the internalorgans (Fig. 1C). The lungs, pericardium, diaphragm, and chestcavity displayed dark patches, while the liver and spleen were uni-formly dark purple or black. The color of other organs was notnoticeably altered. The ileum appeared dark purple to black in

color after the first 3 weeks of treatment. Beyond the first 3 weeksof treatment, pigmentation progressed in a distal to proximal di-rection along the length of the intestine, up to the jejunal segment.Visual inspection of isolated jejunal and ileal sections revealedpatches of pigmentation on the outer walls of the intestine(Fig. 1C). This contrasted with the minimal changes in pig-mentation of duodenum and stomach (Fig. 1C).

The mesenteric lymph node and all other inspected lymphnodes (superficial cervicals, deep cervicals, mediastinal, axillary,brachial, thymus, pancreatic sheet, linguinal, lumbar, sciatic, andcaudal) appeared black in color. Omental fat and abdominal fatwere bright orange in color from the beginning of the first weekand remained orange in color through the 8-week treatment pe-riod (Fig. 1C). The femurs of clofazimine-treated mice appearedgray compared to the controls. The brain, spinal cord, sciaticnerve, pancreas, and kidney did not show any obvious changes inpigmentation.

In clofazimine-treated mice, the size of the mesenteric lymphnodes and the spleen increased significantly compared to controlmice. The spleen enlarged in mass by 3-fold (Fig. 1C and D), butthis was only apparent after 3 weeks of treatment. Of the internalcontrols, the mass of the kidneys (and other major organs) wascomparatively unaffected (Fig. 1D). The spleens mass continuedto increase following the discontinuation of clofazimine adminis-tration (Fig. 1D). During an 8-week drug washout phase, spleenmass increased by 20%, although the total body mass increasedonly by 3.5%, and the kidney mass remained constant. Thissplenomegaly phenotype suggested an active biological responseaccompanying local clofazimine bioaccumulation in this organ.

Interestingly, organ-specific differences in pigmentation re-flected local clofazimine bioaccumulation and CLDI formation.Lung parenchyma of mice treated with clofazimine for 8 weeks(8-week-clofazimine-treated mice) revealed heterogeneous distri-bution of red colored drug deposits with two distinctive subcellu-lar distribution patterns: scattered, small punctate of autophago-some-like drug inclusions (19) and prism-shaped CLDIs (13)(Fig. 2A). Although autophagosome-like drug inclusions were notmore than 2 m in diameter, CLDIs ranged from 5 to 20 m inlength. CLDIs were birefringent when examined using polarizedlight microscopy (Fig. 2B). In the kidneys, the distribution of clo-fazimine inclusions varied in different regions of the organ. Thekidney medulla (Fig. 2C) showed a pale pink pigmentation, whilethe cortex (Fig. 2D) exhibited CLDIs formed around the glomer-ulus and blood vessels (V). In the small intestine, CLDIs wereobserved only in the lamina propria of villi in the jejunum andileum (Fig. 2E). Enterocytes did not show any visible pigmenta-tion. The mesenteric lymph nodes were filled with large numbersof CLDIs (Fig. 2F) present at the periphery of germinal centers,but lymphocytes appeared to be devoid of drug inclusions. A sim-ilar pattern was observed in the spleen after 3 weeks (Fig. 2G) and8 weeks (Fig. 2H) of treatment, with CLDIs increasingly accumu-lating in macrophage-like cells of the marginal zone surroundingthe germinal centers, with fewer CLDIs found in the macrophagesof the red pulp. In the liver, CLDI distribution changed from ascattered pattern after 3 weeks of treatment (Fig. 2I) to a morelocalized pattern with CLDIs present mostly in the periphery ofblood vessels after 8 weeks of treatment (Fig. 2J).

Clofazimine mass corresponded to visual differences in pig-mentation and CLDI accumulation. Drug mass was assessed tostudy the changes in clofazimine bioaccumulation and distribu-




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tion during the course of treatment (Fig. 3). To calculate drugmass, we first measured drug concentration by dividing theamount of clofazimine extracted from tissue samples obtainedfrom the different organs by the weight of wet tissue. This analysiswas performed after 3 and 8 weeks of treatment and then againafter an 8-week washout phase (Fig. 3A). After 8 weeks of treat-ment, clofazimine concentration in the spleen, liver, and smallintestine (jejunum and ileum) increased by at least 1,500%, rela-tive to the measured concentration at the end of 3 weeks. Duringthe same period, the clofazimine concentration in adipose tissuesurprisingly decreased by 38%. This decrease corresponded to agradual, but massive redistribution of clofazimine from adiposetissue to spleen or liver after 3 weeks of treatment. Remarkably,although clofazimine concentration decreased in the blood andother organs consistent with its gradual clearance (1), clofazimineconcentration in spleen remained largely constant during thewashout phase.

After 8 weeks of treatment, the spleen, liver, jejunum, ileum,and fat contained 13 mg of clofazimine (Fig. 3B; see also Mate-rials and Methods). Although the clofazimine amount per weightmeasured in the liver was lower than that of the spleen (Fig. 3A),its greater weight and volume of the liver made it the largest stor-age compartment of clofazimine, containing up to 5.8 mg (39% oftotal drug available) after 8 weeks of treatment (Fig. 3B). Based ona mouse body fat of 16%, at week 8 there were 1.3 mg of clofazi-mine in fat (9% of total), which was about one-third of the mass infat at week 3 (3.1 mg, Fig. 3B). The clofazimine content in thejejunum and ileum (Fig. 3A) increased during treatment, in con-trast to the clofazimine content of the duodenum (Fig. 3C) or thelarge intestine (data not shown), which remained low. The com-bined average drug mass in the jejunum and ileum after 8 weeks oftreatment was 3 mg (Fig. 3B). In the lung, the amount of clofazi-mine peaked at 8 weeks and declined rapidly by 25% on averageduring the washout phase (Fig. 3C). Clofazimine mass in the du-odenum and kidney remained undetectable during the course ofthe experiment (Fig. 3C).

Interestingly, at 8 weeks, the mass of clofazimine in the liver,spleen, and intestine (jejunum and ileum) corresponded to 81%of the total drug absorbed during the 8-week treatment period.Remarkably, serum clofazimine concentrations were lower after 8weeks of treatment compared to those after 3 weeks of treatment(Fig. 3D), which corresponded to a shift in clofazimine mass bal-ance from the adipose tissue to the liver, spleen, and small intes-tines (Fig. 3E).

Clofazimine mass associated with CLDIs in spleen and liverexceeded the mass present in fat. To determine the fraction oftotal drug mass present in CLDIs, CLDIs were isolated from tissuehomogenates after sonication and differential centrifugation. Thisresulted in the removal of 95% of protein and a 10-fold en-richment in CLDIs. In the spleen, up to 91% of the total clofazi-mine mass in this organ was found in association with the isolatedCLDIs. The mean combined clofazimine mass present in the iso-lated CLDIs from the spleen and liver was 50% 38% of the totalmass found in these two organs. CLDI isolation and fractionationanalysis was restricted to the liver and spleen, because other organscontained more connective tissue which made it difficult to isolateCLDIs. In addition, since the total mass of clofazimine in liver andspleen amounted to 60% of the total mass of absorbed clofazimineat 8 weeks, 30 to 40% of the total consumed clofazimine mass wasfound in CLDIs isolated from these organs. Using LC-MS, we

FIG 2 Dark purple and brown pigmentation was associated with CLDI for-mation in organs of drug-treated mice. (A) Eight-week-clofazimine-treatedlung parenchyma showed distinct, vibrant red drug inclusions. (B) CLDIs werebirefringent under polarized light, whereas smaller, autophagosome-like druginclusions were not. (C) Kidney medulla of 8-week-clofazimine-treated ani-mals failed to exhibit evidence of CLDIs. (D) Kidney cortices of 8-week-clo-fazimine-treated animals showed CLDIs localized to the glomeruli (GM). (E)Jejunal/ileal sections after 8 weeks of clofazimine treatment showed CLDIslocalized at the lamina propria of intestinal villus, with no evidence of clofazi-mine accumulation in the enterocytes. (F) At 8 weeks of clofazimine treatment,the mesenteric lymph node showed prominent and heterogeneous CLDI dis-tribution in the periphery of the germinal centers (GC). (G and H) Spleensfrom clofazimine-treated mice showed extensive CLDI accumulation. (I and J)Liver exhibited scattered CLDIs after a 3-week clofazimine treatment period;liver exhibited localized clusters of CLDIs surrounding blood vessels (V) afteran 8-week treatment period. Scale bars, 20 m.

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confirmed that the molecular weight and retention time of clofazi-mine in liver and spleen corresponded to the pure compound; thisis consistent with prior reports that showed clofazimine containedin tissues remains metabolically intact (12, 29). Because the CLDI

isolation procedure is 100% efficient and the intestine and lungcontained 21% of the bodily clofazimine mass, the percentage oftotal clofazimine mass in the host associated with CLDIs may wellexceed 50%.

FIG 3 CLDI formation reflected variations of clofazimine content in various organs. (A) Concentration of clofazimine (CFZ) per gram of the spleen, liver,intestinal segments from the jejunum to ileum, and fat. (B) Total clofazimine (CFZ) mass in these organs were obtained by multiplying total weight of therespective organs. (C) The lungs accumulated clofazimine between 3 and 8 weeks, which were reduced during the washout phase (WO). The kidney andduodenum showed no difference between the treatments. (D) The clofazimine serum concentration, which peaked at an earlier point of treatment, decreasedduring further treatment and washout phase (*, P 0.05; **, P 0.01; ***, P 0.001; n 4 to 6 by ANOVA). (E) The total mass and relative fractions ofclofazimine in adipose tissue and various other organs changed dramatically between 3 and 8 weeks of treatment. The pie charts illustrate the mean relativeamounts of clofazimine in the different tissues, based on the total mass of clofazimine ingested at 3 and 8 weeks of treatment (n 4 to 6 mice).




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Changes in clofazimine serum concentration over time paral-leled its concentration in fat tissue (Fig. 3D), as expected based onpartitioning of clofazimine between serum and fat. The measuredserum concentrations were within a comparable range to previ-ously reported values, 2.6 to 7 M in animals and humans (1, 24).Although changes of clofazimine concentration in fat (Fig. 3A)paralleled concentration changes in serum (Fig. 3D), clofazimineconcentration and mass in the liver, spleen, and small intestineincreased over time and did not parallel the clofazimine concen-tration in serum. At 3 weeks, the ratio of total clofazimine mass infat relative to the liver and spleen was 9.4 4.5 (n 4). Never-theless, at 8 weeks, this ratio dramatically decreased to 0.15 0.03(n 4), indicating a major shift in clofazimine distribution. Basedon the amount of clofazimine present in CLDIs, the mass of clo-fazimine in CLDIs at 8 weeks was greater than the amount ofclofazimine in fat. We reasoned that clofazimine became seques-tered in CLDIs as a result of a sequestration mechanism that fa-vored increased partitioning of clofazimine from serum into liver,spleen, lungs, and intestines.

CLDIs accumulated in clusters of macrophage-like cells thatformed around the blood vessels of liver and spleen. In the lungs,only parenchymal macrophages showed evidence of CLDIs, ap-parent as polyhedral, membrane-bound intracellular cavities vis-ible by TEM (Fig. 4A). Morphologically, macrophages were iden-tified based on the shape of the nucleus, the prevalence oflysosomes and heterolysosomes in the cytoplasm, the presence ofabundant cytoplasm without rough endoplasmic reticulum, andmany surface pseudopodia. In the kidneys (Fig. 4B), peritubularmacrophages were found to contain CLDIs, while neighboringtubular cells, consisting of the epithelium of renal tubules, con-tained only autophagosome-like drug inclusions similar to thosereported to form in vitro using MDCK (Madin-Darby canine kid-ney) cell cultures (19). After 4 and 8 weeks of clofazimine treat-ment, a significant number of CLDI-containing macrophageswere evident in the jejunal submucosa (Fig. 4C) and spleen(Fig. 4D). In the liver, 4 weeks of treatment resulted in the pres-ence of CLDIs in macrophages (Fig. 4E) but not in hepatocytes.After 8 weeks of treatment there was an increase in the numberand size of the liver CLDIs (Fig. 4F), mostly in clusters of macro-phages that had formed adjacent to blood vessels. The clustersresembled microgranulomas, an inflammatory structure that usu-ally forms in response to localized bacterial or parasitic infections.

To confirm that the clofazimine sequestrating cells were mac-rophages, immunohistochemical analysis of samples from clofazi-mine-treated animals was performed using the macrophage-spe-cific anti-F4/80 antibody (Fig. 5). Microscopically, the amount ofF4/80 staining after 6 weeks of clofazimine treatment was greaterthan control, vehicle-treated mice, suggesting increased numbersof macrophages. Massons trichrome staining (MTS) revealed thepresence of fibrotic tissue associated with the F4/80-positive cellclusters. In contrast to anti-F4/80 staining, antibodies against-smooth muscle actin (SMA) or an endothelial cell marker (vonWillebrand factor [vWF]) did not show any changes in stainingpattern, yielding no evidence that smooth muscle or endothelialcells were involved in CLDI formation (see Fig. S2 in the supple-mental material). From the H&E-stained tissue samples, F4/80-positive cell clusters resembled microgranulomas (MG; see Fig.S2, arrow, in the supplemental material) formed by macrophagesfilled with empty intracellular cavities where CLDIs resided priorto being removed by the immunohistochemical staining process.

After the 8-week washout period, microgranulomas appeared tobe localized mostly around the blood vessels (30). The increase inperivascular microgranulomas between 3 and 8 weeks suggestsmonocyte recruitment from the circulation (31).

In the spleen, immunohistochemical analysis of tissue samplesobtained from clofazimine-treated animals revealed CLDI forma-tion associated with macrophages at the periphery of germinalcenters. The CLDIs were not present in lymphocytes which are theprevalent cell population in the germinal centers. Instead, CLDIswere localized to cells of the marginal zone of the germinal centers,adjacent to the red pulp where the erythrocytes are filtered (Fig. 6).In spleens from control, vehicle-treated mice, there was a cleardistinction between the F4/80-positive macrophages, CD21-pos-itive follicular dendritic cells, and CD3-positive early T cells (seeFig. S3 in the supplemental material). In 6-week-clofazimine-treated mouse spleens, immunohistochemical staining showed asimilar histological organization. However, intracellular CLDIcavities were mostly present in a subpopulation of F4/80-positivemacrophages at the marginal zones, in the periphery of the germi-nal centers. The intensity of F4/80 (or CD21) staining in these cellswas not as prominent as that of the macrophages or dendritic cellsof the red pulp.

In other tissues there were no obvious histological changesaccompanying CLDI formation (see Fig. S4 in the supplementalmaterial). In the intestine (see Fig. S4A), the staining of F4/80-positive cells was comparable between the treated and controlsamples. CD21 (follicular dendritic cell marker), and CD3 (early Tcell marker) staining did not show any significant differences be-tween the samples. In the kidneys, the cortex section showed anincreased level of macrophage specific F4/80 staining in the glo-merular region (see Fig. S4B, triangle, in the supplemental mate-rial) in proximity to blood vessels that stained positive for -SMAand vWF.

Clofazimine bioaccumulation was associated with decreasedlocal oxidant levels and an upregulated IL-1RA anti-inflamma-tory response. To determine whether clofazimine bioaccumula-tion was linked to pro-oxidant stress or activation of inflamma-tory response pathways, we assayed the levels of the inducibleantioxidant response protein MnSOD, measured the total antiox-idant capacity of various tissues, and profiled key chemokine andcytokine signaling molecules. We analyzed the major organs ex-hibiting the greatest amount of local clofazimine accumulation(spleen, liver, and lung), as well as a control organ showing anundetectable amount of clofazimine accumulation (brain). InWestern blots, MnSOD protein levels did not change in responseto clofazimine treatment (see Fig. S5A to D in the supplementalmaterial) except in the spleen (see Fig. S5C), where MnSOD ex-pression was slightly increased. The amount of MnSOD to totalprotein content remained low. Consistent with this observation,the absolute oxidant state of different organs was not altered byclofazimine treatment (Fig. 7, 0 mM Trolox). However, the totaloxidant capacity of the lungs (Fig. 7B) and spleen (Fig. 7C) weremore readily titrated by the addition of exogenous antioxidant(Trolox) relative to the liver (Fig. 7D) and brain (Fig. 7A). There-fore, in the lungs and spleen the local clofazimine bioaccumula-tion was associated with a reduction in oxidant capacity.

By measuring changes in the levels on immune signaling mol-ecules using a cytokine array, clofazimine bioaccumulation wasassociated with a remarkable phenomenon: a lymphatic organ-specific upregulation of the soluble anti-inflammatory protein in-

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terleukin-1 receptor antagonist (IL-1RA) (Table 1). IL-1RA is apotent, systemic anti-inflammatory protein (3235), and its in-duction appeared to be specifically associated with local clofazi-mine accumulation in the lung, liver, and spleen. In the brain, an

organ that did not exhibit clofazimine accumulation, IL-1RA wasdownregulated, as were many other monocyte chemoattractantproteins and a large number of other acute inflammatory responsemediators. Spleen samples from clofazimine-treated mice also ex-

FIG 4 TEM images of affected organs showed empty CLDI cavities present in the cytoplasm of macrophage-like cells. These cavities correspond to empty spacesthat are left behind after CLDI extraction by the sample preparation process. (A) Lung parenchyma macrophages (M) with cytoplasmic inclusion bodies after 4weeks of clofazimine treatment. Although autophagosome-like drug inclusions were preserved as dark osmiophilic bodies, CLDIs are shown as polyhedral inshape, empty cavities as they were washed out during sample preparation. (B) Eight-week-clofazimine-treated kidney tubular region with numerous renaltubular cells (T). In the peritubular region, a group of macrophages (M) appeared with CLDI cavities in the cytoplasm. (C) Four-week-clofazimine-treated micejejunal submucosa shows a number of macrophages with both autophagosome-like drug inclusions and CLDIs in the cytoplasm. (D) Eight-week-clofazimine-treated spleen showed significant amounts of CLDI cavities as if bundled together, presumably associated with macrophages. Lymphocytes (L), identified assmaller cells in size and also small cytoplasmic space around nuclei, lacked CLDI cavities. (E) Four-week-clofazimine-treated liver showed large numbers ofKupffer cells (M) with empty CLDI cavities surrounded by hepatocytes (H), identified by their round nuclei and granular vesicles containing glycogen and lipiddroplets. (F) Eight-week-clofazimine-treated liver developed microgranulomas around the blood vessels (V). Fibrocytes (F) along the edge of the vessels wereseparated from endothelium and macrophages at the core of the microgranulomas. The empty CLDI cavities were exclusively present in macrophages at the coreof the microgranulomas. Scale bars, 5 m.




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hibited indications of an IL-1RA-related anti-inflammatory re-sponse, evidenced by the significant downregulation of the T-cellchemokines CCL5 (36, 37) and CXCL9, as well as general down-regulation of many other cytokines and chemokines (see Table S1in the supplemental material). In the lungs, clofazimine bioaccu-mulation was also associated with increased IL-1RA levels (Table1), concomitant with the downregulation of CCL4 (38), CCL17(39), and TNF- (40) (Table 1), which are proinflammatory sig-naling mediators secreted by activated macrophages. The lungsfrom clofazimine-treated mice also had higher levels of chemo-kines involved in macrophage recruitment, including CCL2 (36,41) and CXCL1 (42, 43), the response-amplifying, growth-stimu-latory monocyte receptor, TREM-1 (44, 45), and the monocyteretention-promoting metalloprotease inhibitor, TIMP-1. Themajority of other soluble chemokines, cytokines, and receptormolecules assayed appeared to be decreased or unaffected by clo-fazimine treatment (see Table S1 in the supplemental material).Of the few proteins upregulated in treated samples, less than halfincreased to statistically significant levels. In addition to IL-1RA,only the macrophage recruitment signal-potentiating TREM-1

receptor (44, 45) was consistently increased in most clofazimine-treated samples (see Table S1 in the supplemental material).

DISCUSSION

In the present study, we quantitatively analyzed how the distribu-tion of clofazimine changed between 3 and 8 weeks of administra-tion and during an 8-week posttreatment washout period.Changes in clofazimine content and distribution occurred duringprolonged clofazimine treatment and correlated with major struc-tural and functional changes in the immune system. Our resultsprovide evidence that an inducible xenobiotic sequestration re-sponse mediated by a subpopulation of cells of the immune sys-tem is profoundly impacting the distribution and bioaccumula-tion of clofazimine. This is consistent with human autopsy reports(5, 15, 46) indicating the presence of clofazimine crystals in lym-phatic tissues. Furthermore, our results suggest that these crys-tals are not accidental or haphazard. In mice, they were present ina site-specific subpopulation of macrophages in the spleen, andlinked to an active, immune system-mediated, intracellular xeno-

FIG 5 Immunohistochemical analyses revealed liver microgranulomas containing CLDIs. Regions around the blood vessels (central vein [CV]) visualized byH&E staining underwent a structural transformation during clofazimine treatment (6 wk CFZ) as well as after clofazimine discontinuation (washout). The highermagnification showed microgranulomas (MG, arrows) and indicated that they were actually comprised of clusters of macrophages with a significant number ofCLDI cavities in the cytoplasm. Distribution of F4/80 staining shows the scattered distribution of Kupffer cells that aggregated in small clusters which were evenlydistributed throughout the section. A few larger microgranulomas (triangles) with some fibrosis (revealed by MTS) were also evident in the periphery of bloodvessels (SMA and anti-vWF immunohistochemistry, see Fig. S2 in the supplemental material). Intracellular cavities corresponded to the empty spaces that wereleft behind after CLDI extraction by the sample preparation process.

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biotic sequestration response associated with spleen enlargementand microgranuloma formation in the liver.

Our multiscale distribution and bioaccumulation analysis alsobrings into focus the long-standing pharmacokinetic assumptionthat highly lipophilic compounds stably partition from the seruminto adipose tissue. Although this assumption may be true duringshort-term clofazimine treatment (3 weeks), it was certainly nottrue after a long-term, 8-week treatment. Like other lipophilicmolecules, clofazimine partitioned into adipose tissues during ashort-term exposure period (8). When clofazimine absorptionand distribution was monitored in mice for either 1 or 5 days aftera daily dose of 40 mg/kg, the drug concentration in the lungs,spleen, and liver peaked at 6 h after each dose, followed by a sharpdecrease, and then remained at minimal level until another dosewas given. The drug content in fat increased continuously overeach 24-h period, which persisted with each daily treatmentthroughout the first 5 days of administration (8). Nevertheless,between 3 and 8 weeks of continuous exposure, we observed clo-fazimine dramatically redistributed from adipose tissue to liver,spleen, gut, and lungs.

Interestingly, CLDIs were never observed outside macro-phages and were always localized to the cells cytoplasm. Macro-phages are phagocytic cells, and they are the only cell type knownto possess a size-fractionating endolysosomal system (47), whichmay explain why CLDIs are found exclusively in these cells. Tox-icologically, one may have expected evidence of necrosis and thepresence of extracellular crystals at sites of microgranulomas for-mation. However, neither necrosis nor extracellular crystals wereobserved. CLDIs were homogeneous in size and shape, suggestingthat their distribution and morphology is under active cellularcontrol. Supporting the novelty and significance of these results,we also found that clofazimine formed a unique, liquid crystal-and organelle-like supramolecular organization inside macro-phages (13).

FIG 7 Clofazimine-induced CLDI formation reduced organ oxidant capacity.Oxidizing activity present in each homogenate led to an increase in absorbance (yaxis), which decreased in the presence of increasing concentration of an addedantioxidant (Trolox). (A) At all tested Trolox concentrations (x axis), the brain, anorgan that lacked clofazimine CLDI formation, exhibited similar oxidant titrationcurves in both vehicle- and clofazimine-treated mice. (B and C) However, in thelung (B) and spleen (C), organs in which CLDIs are particularly prominent, clo-fazimine-treated mice showed significantly (*, P 0.05) less absorbance thanvehicle-fed mice, at corresponding antioxidant (Trolox) concentration, indicatingthat the oxidizing capacity of clofazimine-treated animals can be more readilytitrated down by exogenous antioxidant. This reduction in the oxidizing capacitywas less apparent in the liver of clofazimine-treated mice compared to the liver ofvehicle-fed mice (D). n 3 per group, mean SD. *, P 0.05.

FIG 6 Immunohistochemical analysis of spleen revealed macrophage clusters exhibiting CLDI cavities in specific regions of the organ. CLDI cavities correspond toempty spaces that are left behind after CLDI extraction by the sample preparation process. In contrast to the control animals (6 wk Oil), the spleens of clofazimine-treatedanimals (6 wk CFZ) exhibited macrophages containing CLDI cavities accumulating at the periphery of the germinal centers (GC), which were rich in lymphocytes andlymphoblasts. Within this marginal zone region (arrow, magnified on the GC periphery panel), clusters of cells with CLDI cavities (triangle) were apparent.




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The development of splenomegaly upon prolonged clofazi-mine treatment indicated an active response mechanism affectingthe disposition of clofazimine (48). In addition, because there aredifferent kinds of resident tissue macrophages in the spleen (4951), it is possible that only a specialized subpopulation of macro-phages sequestered clofazimine. At 8 weeks of treatment, up to 1%of the mass of the spleen was comprised of clofazimine, with up to91% of the total clofazimine mass being present in association

with CLDIs. Since the major elimination route for this metaboli-cally stable drug is biliary clearance followed by fecal excretion (4,5), we reasoned that the decrease in clofazimine content in liverduring the washout period may result from direct eliminationthrough the bile once treatment is discontinued. Remarkably, thespleen continued to retain clofazimine even after the plasma con-centrations of clofazimine had significantly dropped.

In parallel to the observed, immune system-mediated, macro-

TABLE 1 Clofazimine-induced changes in cytokine and chemokine levels in the lungs, spleen, liver, and braina

Protein Change (%) Vehicle Clofazimine P FDR (%) Vehicle Clofazimine

Lungs

CCL2 334 0.22 0.10 0.94 0.12 0.0001 0.20

CXCL1 180 0.16 0.05 0.44 0.11 0.0002 0.20

TREM-1 168 0.13 0.05 0.35 0.08 0.0002 0.20

IL-1RA 100 0.81 0.04 1.62 0.05 0.0001 0.20

TIMP-1 81 0.31 0.05 0.56 0.08 0.0001 0.20

IL-4 36 0.13 0.03 0.08 0.02 0.0040 2.46

CCL17 47 0.13 0.03 0.07 0.03 0.0085 4.25

TNF- 47 0.13 0.03 0.07 0.03 0.0105 4.67

CCL4 61 0.12 0.04 0.04 0.01 0.0016 1.07

Spleen

IL-1RA 90 0.59 0.15 1.12 0.20 0.0004 0.53

CCL5 47 0.78 0.11 0.42 0.10 0.0001 0.40

CXCL9 88 0.68 0.10 0.08 0.03 0.0001 0.40

Liver

IL-1RA 315 0.27 0.20 1.12 0.10 0.0001 0.40

Brain

CCL4 33 0.19 0.03 0.13 0.01 0.0003 0.60

IL-7 36 0.14 0.02 0.09 0.04 0.0170 4.86

GM-CSF 40 0.24 0.05 0.15 0.01 0.0071 2.58

TNF- 41 0.20 0.03 0.12 0.03 0.0010 0.80

IL-1 42 0.26 0.04 0.15 0.04 0.0008 0.80

CXCL9 44 0.23 0.05 0.13 0.04 0.0029 1.66

CXCL13 47 0.15 0.03 0.08 0.02 0.0007 0.80

CXCL10 50 0.10 0.02 0.05 0.02 0.0028 1.66

IL-13 51 0.08 0.02 0.04 0.02 0.0050 2.00

GCSF 52 0.06 0.02 0.03 0.01 0.0030 1.66

CCL11 58 0.09 0.01 0.04 0.01 0.0117 3.90

IL-1 67 0.09 0.02 0.03 0.01 0.0001 0.40

IL-1RA 83 0.35 0.14 0.06 0.02 0.0045 1.96

a Clofazimine accumulation was associated with significant, site-specific changes in immune signaling molecules. The levels of 40 different key cytokines and chemokines wereanalyzed in lungs, spleen, liver, and brain samples of clofazimine- and vehicle-treated mice. Most acute-phase chemokines and cytokines did not show significant changes or weredownregulated in animals exposed to clofazimine (see Table S1 in the supplemental material). The unitless values in the table correspond to the pixel densities of the spots for eachprotein, averaged over all six vehicle- or clofazimine-treated spots, normalized by the averaged, measured pixel densities of the calibration, positive control spots included in eacharray. The data show the percent change in each respective cytokine between clofazimine- and vehicle-treated mice that, when compared, resulted in a P value of 0.05 and an falsediscovery rate (FDR) of 5%. The original spots from the arrays are shown in the last six columns of the table (each protein was probed in duplicate, for each vehicle- orclofazimine-treated sample). Data are means ( the standard deviations) of the signal intensities for each analyte from three mice/group. IL-1RA (indicated in boldface) wassignificantly upregulated in all organs in which clofazimine bioaccumulated.

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phage dependent, active xenobiotic sequestration response asso-ciated with the intracellular accumulation of clofazimine, the up-regulation of IL-1RA (34, 35, 52) could explain the potent anti-inflammatory effects of clofazimine reported in clinical studies (3,4). IL-1RA inhibits the binding of soluble IL-1 and IL-1 to theproinflammatory interleukin-1 (IL-1) receptor (34, 35, 52), lead-ing to a pronounced, systemic anti-inflammatory activity. In hu-mans, genetic mutations that lead to nonfunctional IL-1RA resultin a generalized auto-inflammatory disease affecting bones, jointsand skin from birth (53), and recombinant IL-1RA is an FDA-approved treatment for rheumatoid arthritis (33). Although IL-1RA is present at high basal levels and its mutation is known tohave major consequences for immune regulation (35, 53), this isthe first report linking increased IL-1RA to a specific, xenobioticaccumulation response pathway.

The complete lack of extracellular clofazimine crystals, thehighly controlled intracellular distribution of CLDIs (13), the ab-sence of obvious toxicological manifestations together with theincreased levels of anti-inflammatory IL-1RA suggest a protective,coordinated biological response. In vitro, clofazimine is cytotoxicand has been shown to induce the production of superoxide anionin rat peritoneal macrophages and human neutrophils ex vivo (54,55). In vitro, clofazimine can induce apoptosis via caspase activa-tion (56). Nevertheless, there were no obvious toxicological man-ifestations in vivo, and primary macrophages isolated from clo-fazimine-treated mice were viable and mobile, although theycontained many drug inclusions (13). In clofazimine-treatedmice, the amounts of inducible, anti-oxidant MnSOD did notincrease, which is consistent with an absence of oxidant stress.Furthermore, clofazimine increased the levels of the anti-inflam-matory IL-1RA signaling protein and decreased the levels of manyother proinflammatory cytokines and chemokines, while the ox-idizing capacity of the primary sites of clofazimine bioaccumula-tion decreased or remained unchanged. Collectively, these resultsdemonstrate that, in vivo, clofazimine induces a protective, mac-rophage-mediated xenobiotic sequestration response.

In conclusion, our results provide insights into the bioaccu-mulation-related side effects of clofazimine, unrelated to thedrugs primary mechanism of action. The extraordinarily longhalf-life, atypical pharmacokinetics and extensive bioaccumu-lation of clofazimine are not simply a consequence of lipophilicpartitioning into body fat. To our knowledge, this is the firststudy to implicate an immune-mediated drug sequestrationmechanism in a mammalian organism. Certainly, our observa-tions prompt many more questions that lie beyond the scope ofthis multiscale biodistribution study. In future experiments,we will address the exact role of the immune system on thebioaccumulation of clofazimine by taking advantage of genet-ically mutant mice lacking IL-1RA as well as the other chemo-kine genes involved in immune signaling. We also envisionanalyzing the bioaccumulation and distribution of differentclofazimine derivatives as well as other lipophilic compoundsto address whether this phenomenon represents a unique, id-iosyncratic side effect of clofazimine, or a more general xeno-biotic sequestration response. Most importantly, the experi-mental approach and results presented here offer a uniquelydifferent perspective into some of the chemotherapeutic prop-erties of clofazimine and next-generation clofazimine deriva-tives.

ACKNOWLEDGMENTS

The project was supported by National Institutes of Health (NIH) grantsGM007767 (J.B.), R01GM078200 (G.R.R.), and R15HD065594 (K.A.S).This study utilized Core Services supported by NIH grant DK089503 tothe University of Michigan. J.B. was also supported by a fellowship fromthe American Foundation for Pharmaceutical Education.

We thank Dorothy Sorenson (MIL, University of Michigan), PaulaArrowsmith (PCAR, University of Michigan), and Gerald Hish (ULAM,University of Michigan) for technical support. We thank Charles Burant,Nair Rodriguez-Hornedo, and David E. Smith for insightful comments.

The contents of this report are solely the responsibility of the authorsand do not necessarily represent the official views of the NIGMS, NICHD,or NIH.

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Multiscale Distribution and Bioaccumulation Analysis of Clofazimine Reveals a Massive Immune System-Mediated Xenobiotic Sequestration ResponseMATERIALS AND METHODSAnimal experiments.Biochemical analysis of clofazimine in tissues.Determination of clofazimine concentration in serum.Isolation of CLDIs.Sample preparation for microscopy.Transmitted light, polarization, and fluorescence microscopy.TEM.Measurement of antioxidant responses.Measurement of cytokine-chemokine responses.Data analysis.

RESULTSProlonged clofazimine administration led to visible changes in mouse organs.Clofazimine mass corresponded to visual differences in pigmentation and CLDI accumulation.Clofazimine mass associated with CLDIs in spleen and liver exceeded the mass present in fat.CLDIs accumulated in clusters of macrophage-like cells that formed around the blood vessels of liver and spleen.Clofazimine bioaccumulation was associated with decreased local oxidant levels and an upregulated IL-1RA anti-inflammatory response.

DISCUSSIONACKNOWLEDGMENTSREFERENCES

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Multiscale Distribution and Bioaccumulation Analysis of Clofazimine