PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of aacPermissionsJournals.ASM.orgJournalAAC ArticleJournal InfoAuthorsReviewers
 
Antimicrob Agents Chemother. Mar 2013; 57(3): 1218–1230.
PMCID: PMC3591914
Multiscale Distribution and Bioaccumulation Analysis of Clofazimine Reveals a Massive Immune System-Mediated Xenobiotic Sequestration Response
Jason Baik,a* Kathleen A. Stringer,b Gerta Mane,b and Gus R. Rosaniacorresponding authora
aDepartment of Pharmaceutical Sciences
bDepartment of Clinical, Social, and Administrative Sciences, University of Michigan College of Pharmacy, Ann Arbor, Michigan, USA
corresponding authorCorresponding author.
Address correspondence to Gus R. Rosania, grosania/at/umich.edu.
*Present address: Jason Baik, Department of Bioengineering and Therapeutic Sciences, University of California—San Francisco, San Francisco, California, USA.
Received August 21, 2012; Revisions requested October 6, 2012; Accepted December 8, 2012.
Chronic exposure to some well-absorbed but slowly eliminated xenobiotics can lead to their bioaccumulation in living organisms. Here, we studied the bioaccumulation and distribution of clofazimine, a riminophenazine antibiotic used to treat mycobacterial infection. Using mice as a model organism, we performed a multiscale, quantitative analysis to reveal the sites of clofazimine 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 fat and serum significantly decreased, while the mass of clofazimine in spleen and liver increased by >10-fold. These changes were paralleled by the accumulation of clofazimine in the resident macrophages of the lymphatic organs, with as much as 90% of the clofazimine mass in spleen sequestered in intracellular crystal-like drug inclusions (CLDIs). The amount of clofazimine associated with CLDIs of liver and spleen macrophages disproportionately increased and ultimately accounted for a major fraction of the total clofazimine in the host. After treatment was discontinued, clofazimine was retained in spleen while its concentrations decreased in blood and other organs. Immunologically, clofazimine bioaccumulation induced a local, monocyte-specific upregulation 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 response affecting the atypical pharmacokinetics of a small molecule chemotherapeutic agent.
Clofazimine is an orally bioavailable, poorly soluble, highly lipophilic (logP > 7) antibiotic drug with a very long pharmacokinetic half-life (16). It was first marketed in 1969 as “Lamprene,” and in 1983 it was recommended by the World Health Organization as one of the components of multidrug therapy against leprosy (7). Subsequently, in 1986, it was approved by the U.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 the organism and is eliminated very slowly by renal and hepatic routes (8). Clofazimine is deep red in color, so its accumulation in skin and other organs leads to a visible dark purple pigmentation (9). Although the human and animal pharmacology and pharmacokinetics of clofazimine have been quantitatively studied during short-term treatment (1, 5), there is little quantitative, mechanistic information about the distribution of clofazimine during chronic exposure or about the associated physiological responses of the organism to clofazimine bioaccumulation. Like many other lipophilic small molecule drugs, clofazimine's long half-life and atypical pharmacokinetic properties have been ascribed to its highly hydrophobic character leading to lipophilic partitioning into adipose tissue.
Although clofazimine is clinically useful in the treatment of Mycobacterium leprae, new generations of clofazimine derivatives are being sought as drug candidates to treat multidrug-resistant strains of Mycobacterium tuberculosis (10). These drug candidates could benefit from decreased bioaccumulation and improved pharmacokinetic properties. Clofazimine is also interesting as a probe to study the physiological effects of long-term exposure to natural product-derived, orally bioavailable, bioaccumulating small molecule xenobiotics (11). Qualitatively (1214), clofazimine is known to form crystal-like drug inclusions (CLDIs) inside cells of the mononuclear phagocyte system. However, the extent to which clofazimine accumulates in these intracellular inclusions has not been quantitatively established (12, 15, 16). Because it is a lipophilic, weakly basic molecule (17), clofazimine is likely to accumulate inside acidic organelles by a pH-dependent ion trapping mechanism (18). However, the calculated logD of clofazimine ranges from ~5 to ~7 in physiological pH (see Fig. S1 in the supplemental material), suggesting that a large fraction of the compound can also partition into organelle membranes. In a kidney-derived epithelial cell line, clofazimine accumulated in drug-induced, autophagosome-like drug-membrane aggregates (19). These drug-membrane aggregates resembled the multilamellar bodies that form inside cells exposed to phospholipidosis-inducing, lysosomotropic amphiphilic cations (20, 21). Consistent with lysosomal accumulation, clofazimine treatment also affects the activity of lysosomal enzymes, both in vitro (22) and in vivo (23).
In mammals, the cells of the immune system are considered to play a minor role in the disposition of xenobiotics and the systemic pharmacokinetics of small molecule drugs. Accordingly, we hypothesized that clofazimine sequestration in macrophages was only of minor consequence to the overall bioaccumulation and distribution of clofazimine in vivo. To test this hypothesis, we measured the impact of this sequestration mechanism on the total bioaccumulation and distribution of clofazimine. Taking advantage of its deep red color, low metabolic rate, and slow clearance (4, 24), we performed a multiscale biodistribution analysis of clofazimine, measuring its bioaccumulation in all of the major organs, adipose tissue, and serum, combined with microscopic and biochemical analysis of CLDIs found in macrophages of the liver and spleen. Unexpectedly, the massive bioaccumulation of clofazimine in cells of the immune system accounted for dramatic changes in the distribution of clofazimine between 3 and 8 weeks of treatment. Follow-up proteomic analysis revealed local changes in the levels of key cytokines, chemokines, and immunological signaling molecules at the sites of clofazimine bioaccumulation, which were most consistent with a general anti-inflammatory response. These results suggest a candidate, macrophage-dependent xenobiotic sequestration response affecting the distribution, bioaccumulation, and other side effects of clofazimine during chronic, long-term exposure.
Animal experiments.
Mice (4 week old, male BALB/c) were purchased from the Jackson Laboratory (Bar Harbor, ME) and acclimatized for 2 weeks in a specific-pathogen-free animal facility. All animal care was provided by the University of Michigan's Unit for Laboratory Animal Medicine (ULAM), and the experimental protocol was approved by the Committee 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 with Powdered Lab Diet 5001 (PMI International, Inc., St. Louis, MO) to produce a 0.03% drug to powdered feed mix. A corresponding amount of sesame oil was mixed with chow for vehicle treatment (control). On average, food consumption for a 25-g mouse was ~3 g per day, resulting in 10 mg of bioavailable drug/kg per day (25).
Biochemical analysis of clofazimine in tissues.
Drug tissue contents were analyzed by liquid chromatography-mass spectrometry (LC-MS) and spectrophotometrically, as previously described (1214). In brief, after feeding either clofazimine- or vehicle-supplemented food, mice were euthanized by CO2, and blood and organs were harvested. Tissue (0.05 to 0.1 g/ml of water) was homogenized, and 100 μl of sample was mixed with the same volume of 5 N NaOH and then extracted with 300 μl of dichloromethane twice (6). After centrifugation (2,000 × g, 10 min) to collect the dichloromethane layer, the solvent was evaporated (40°C) and reconstituted in methanol for absorbance measurement (490 nm; Synergy-2 plate reader; Biotek Instruments, Winooski, VT). Clofazimine concentration was calculated from the standard curve generated by adding a known amount of drug solution to a tissue extract from a vehicle-only treated sample. The average extraction yield of clofazimine was 60 to 80% for all organs except for abdominal fat, which yielded nearly a 100% recovery, and all contents were compensated for by the recovery yield. The accumulated drug mass was determined by converting clofazimine concentrations to mass by multiplying either the measured organ weight (duodenum, 0.33 ± 0.3 g; jejunum plus ileum, 1.3 ± 0.06 g [see Results for spleen and 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 week 8 [Jackson Laboratory, http://phenome.jax.org/db]). The total drug amounts available in the body for week 3 and 8 were estimated by multiplying absorption rate with cumulative weekly average body weight presented in Fig. 1A. The excreted plus unaccounted drug fraction in the organism was calculated by subtracting the measured organ contents from the total drug amount. LC-MS was used to confirm that the measured drug in tissue was metabolically intact (13).
Fig 1
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. Animals in both groups gained (more ...)
Determination of clofazimine concentration in serum.
Blood was collected in microtainer serum separator tubes (catalog no. 3659656; Becton Dickinson, Franklin lakes, NJ) and were allowed to clot at room temperature and centrifuged (7,000 × g, 5 min). The resulting supernatant serum was submitted to the University of Michigan's Nutrition and Obesity Research Center for LC-MS analysis. Samples (20 μl) were extracted with acetonitrile (60 μl, 90% extraction efficiency) for 10 min at 4°C with intermittent vortexing. After centrifugation (15,000 rpm, 4°C), the supernatant was injected into Agilent 1200 RRLC coupled to 6410 Triple Quad LC-MS equipped with an Xbridge C18 column (2.5 μm, 2.1 mm [inner diameter] by 100 mm; Waters). Mobile phase A was 5 mM ammonium acetate, adjusted to pH 9.9 with ammonium hydroxide, and mobile phase B was acetonitrile. The flow rate was 0.35 ml/min, with a linear gradient from 50 to 100% phase B over 1.5 min, followed by holding at 100% for 1.5 min, a return to 50% phase B, and then re-equilibration for 2.5 min. The mass spectrometer source conditions were set as follows: 325°C, gas flow at 10 liters/min, nebulizer at 40 lb/in2, capillary at 4,000 V, and positive ion mode. The MS acquisition parameters were as follows: MRM mode, transition 1 set at 473.1 to 1:431.1, a dwell time of 400 ms, fragmentor 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 treated mouse mixed with clofazimine stock solution from dimethyl sulfoxide, resulting in 10 different clofazimine concentrations between 0 and 30 μM. The peak area was quantified using MassHunter Quantitative Analysis software, vB.04.00.
Isolation of CLDIs.
Crystal-like drug inclusion (CLDI) purification was performed as previously described (13). In brief, organ homogenates from two mice treated with clofazimine for 8 weeks were sonicated and then centrifuged (100 × g, 1 min) to remove debris. The resulting supernatants were mixed with 0.125% trypsin-EDTA solution (Gibco) and incubated at 37°C for 1 h, followed by another centrifugation (100 × g, 1 min). The supernatants were centrifuged (21,000 × g, 1 min), and the pelleted CLDIs were resuspended in water for analysis. For assaying protein content of the samples, equal volumes of 5% sodium dodecyl sulfate solution and sample were mixed, and the protein content was measured by bicinchoninic acid (BCA) assay (Pierce 23227; Thermo Scientific).
Sample preparation for microscopy.
Following the postmortem collection of blood samples, some euthanized mice were perfused via the left ventricle with Sorensen's buffer (0.1 M), followed by Karnovsky's fixative (3% paraformaldehyde, 2.5% glutaraldehyde, 0.1 M Sorensen's buffer [pH 7.4]), until the liver was visibly clear of blood. Immediately after perfusion, the organs were removed in preparation for either paraffin embedding or transmission electron microscopy (TEM).
For immunohistochemical staining, paraffin embedding of the perfusion-fixed organs and heat-mediated antigen retrieval were carried out in the histology lab at the Pathology Cores of Animal Research (PCAR), ULAM, at the University of Michigan. Routine hematoxylin and eosin (H&E) and Masson's trichrome staining (MTS), as well as immunohistochemistry of F4/80 (1/100 dilution, ab6640; Abcam), α-smooth muscle actin (α-SMA; 1/200, ab5694; Abcam), vWF (1/500, ab7356; Millipore), CD21 (1/200, ab75985; Abcam), and CD3T (1/300, RM-9107S; Thermo Scientific) were carried out using horseradish peroxidase and intelliPATH FLX 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 fixative and stored at 4°C. After three rinses with Sorensen's buffer (0.1 M), diced tissues were stained with 1% osmium tetroxide in Sorensen's buffer and washed three times in Sorensen's buffer. Dehydration was carried out with a graded ethanol-water series (50, 70, and 90% and two changes of 100%) for 15 min each. After transition through three changes of propylene oxide, the tissues were infiltrated with Epon resin (Electron Microscopy Sciences) and then polymerized at 60°C for 24 h. Next, the blocks were sectioned (70 nm) using a ultramicrotome and mounted on a copper EM grid (Electron Microscopy Sciences), which was post-stained with uranyl acetate and lead citrate before imaging.
Cryosectioning was carried out using a Leica 3050S cryostat. Samples were sectioned to 10 μm and mounted onto glass slide with a drop of glycerol and cover glass. In preparation for cryosectioning, the organs were not perfused with fixative but instead were removed, immediately submerged at an optimal cutting temperature (Tissue-Tek catalog no. 4583; Sakura), and frozen (−80°C).
Transmitted light, polarization, and fluorescence microscopy.
An Olympus ×51 upright epifluorescence/polarization microscope equipped with ×100 objective lens (1.40 NA, PlanApo oil emersion), cross polarizers, and a DP-70 color camera was used. For fluorescence, a U-MWIBA3 eGFP filter cube for the green channel and a U-MWG2 (rhodamine) filter cube for the red channel were used. Images were acquired using a DP controller 3.1.1.267 under the same exposure settings. For display purposes, the image brightness, contrast, and color balance was adjusted using Microsoft PowerPoint and Adobe Photoshop. For control and experimental comparisons within the same figure, settings were adjusted to the same.
TEM.
Images were acquired using a Philips CM-100 TEM and digitally recorded using a Hamamatsu ORCA-HR camera system operated by Advanced Microscopy Techniques software (Danvers, MA).
Measurement of antioxidant responses.
To determine whether clofazimine administration induced oxidant stress, we measured manganese superoxide dismutase (MnSOD) in organs obtained from mice postmortem (26) that were then immediately frozen at −80°C after harvesting. In preparation for immunoblotting, organ samples were thawed on ice and homogenized as previously described (26). Just prior to assay, the protein concentration of homogenates was determined by using a BCA assay.
Homogenate samples (50 μg of total protein) were subjected to gradient 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 (4°C). The membranes were then cut into two sections according to the molecular weights of MnSOD and actin. Each respective membrane was incubated with an antibody specific for MnSOD (1:5,000 dilution, rabbit anti-MnSOD antibody; Upstate Biotechnology, Charlottesville, VA) or actin (1:1,000,000 dilution, mouse anti-actin; Sigma) for 1 h at room temperature. After being washed, the membranes were incubated with secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature and washed again, and enhanced chemiluminescence was detected using an ECL Plus reagent kit (GE Healthcare Life Sciences, Piscataway, NJ) exposed to radiograph film (Fuji, Stamford, CT). Scanned protein bands were quantified by densitometry with ImageJ software (ImageJ 1.44b; National Institutes of Health [http://rsb.info.nih.gov/ij]). The ratio of the density of the MnSOD protein band to the density of the corresponding actin protein band was determined for each sample.
To further investigate the redox status of clofazimine-treated mouse organs, we used a commercial antioxidant assay kit (CS0790; Sigma-Aldrich) that measures the formation of a ferryl myoglobin radical from metmyoglobin and hydrogen peroxide, ABTS [2,2′azinobis(3-ethylbenzthiazolinesulfonic acid)], to produce a radical cation. This cation is green in color and can be spectrophotometrically measured at 405 nm. When antioxidants are present the production of the radical cation is suppressed in a concentration-dependent manner and the color intensity proportionally declines. The kit utilizes Trolox, a water-soluble vitamin E analog, as a standard or positive control antioxidant. We used the assay kit to measure the total oxidant content of the samples by titrating out the oxidizing capacity of each tissue lysate with increasing amounts of Trolox. All samples were adjusted to 20 μg of protein at the starting point of each titration. All other aspects of the assay were conducted in accordance with the manufacturer's instructions.
Measurement of cytokine-chemokine responses.
Changes in cytokine and chemokines in response to clofazimine treatment were measured using a cytokine array assay (R&D Systems mouse cytokine array, panel A, catalog no. ARY006). This assay, similar to the enzyme-linked immunosorbent assay, simultaneously measures the relative levels of 40 different cytokines and chemokines by utilizing selected capture antibodies spotted in duplicate on nitrocellulose membranes. Tissue homogenates were prepared according to the manufacturer's instructions, and 300 μg of lysate protein of the liver, spleen, brain, and lung samples from clofazimine-treated or control, vehicle-treated mice was added to a cocktail of biotinylated detection antibodies provided in the kit. The samples from each organ and the respective negative control samples were simultaneously assayed. After the detection of antibody complexes by streptavidin-horseradish peroxidase chemiluminescence using radiographic film, the pixel density of each cytokine signal was quantified by densitometry using ImageJ. The data were normalized by dividing the density value of each cytokine by the mean density value of the positive controls from the corresponding membrane. The mean densities of each cytokine for each organ from vehicle-treated and clofazimine-treated mice were determined and 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 of their euthanasia, individual organs were isolated and washed three times in cold DPBS, surgically removing other connective tissues. Afterward, they were gently tab dried on gauze for weight measurements, which were averaged for each group. For intestines, the lumen were flushed with DPBS 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 by analysis of variance (ANOVA) with Tukey's HSD using the R software package.
For biochemical assays, the mean MnSOD/actin ratio, the mean absorbance for each Trolox concentration, and the mean normalized density value ± the SD of each cytokine from each organs were compared by unpaired Student t test with Welch's correction for differences in variances when applicable. The resulting P values from the analysis of the cytokine data were tested using InStat (GraphPad Software, La Jolla, CA) and corrected for multiple comparisons by calculating the false discovery rate (FDR) (27, 28).
Prolonged clofazimine administration led to visible changes in mouse organs.
Based on the amount of food consumed per day and clofazimine's bioavailability (25), we estimated an intake of 10 mg of bioavailable drug/kg per day, consistent with previous studies (12). Prolonged administration (8 weeks) of this regimen and the vehicle (sesame oil) mixed diet was well tolerated. During treatment, body weight increased and continued to increase after the discontinuation of treatment (Fig. 1A). Red pigmentation of the skin was visible after the first week of clofazimine treatment (Fig. 1B). Dissection revealed visible pigmentation of the internal organs (Fig. 1C). The lungs, pericardium, diaphragm, and chest cavity displayed dark patches, while the liver and spleen were uniformly dark purple or black. The color of other organs was not noticeably altered. The ileum appeared dark purple to black in color after the first 3 weeks of treatment. Beyond the first 3 weeks of treatment, pigmentation progressed in a distal to proximal direction along the length of the intestine, up to the jejunal segment. Visual inspection of isolated jejunal and ileal sections revealed patches of pigmentation on the outer walls of the intestine (Fig. 1C). This contrasted with the minimal changes in pigmentation of duodenum and stomach (Fig. 1C).
The mesenteric lymph node and all other inspected lymph nodes (superficial cervicals, deep cervicals, mediastinal, axillary, brachial, thymus, pancreatic sheet, linguinal, lumbar, sciatic, and caudal) appeared black in color. Omental fat and abdominal fat were bright orange in color from the beginning of the first week and remained orange in color through the 8-week treatment period (Fig. 1C). The femurs of clofazimine-treated mice appeared gray compared to the controls. The brain, spinal cord, sciatic nerve, pancreas, and kidney did not show any obvious changes in pigmentation.
In clofazimine-treated mice, the size of the mesenteric lymph nodes and the spleen increased significantly compared to control mice. The spleen enlarged in mass by >3-fold (Fig. 1C and andD),D), but this was only apparent after 3 weeks of treatment. Of the internal controls, the mass of the kidneys (and other major organs) was comparatively unaffected (Fig. 1D). The spleen's mass continued to increase following the discontinuation of clofazimine administration (Fig. 1D). During an 8-week drug washout phase, spleen mass increased by 20%, although the total body mass increased only by 3.5%, and the kidney mass remained constant. This splenomegaly phenotype suggested an active biological response accompanying local clofazimine bioaccumulation in this organ.
Interestingly, organ-specific differences in pigmentation reflected local clofazimine bioaccumulation and CLDI formation. Lung parenchyma of mice treated with clofazimine for 8 weeks (8-week-clofazimine-treated mice) revealed heterogeneous distribution of red colored drug deposits with two distinctive subcellular distribution patterns: scattered, small punctate of autophagosome-like drug inclusions (19) and prism-shaped CLDIs (13) (Fig. 2A). Although autophagosome-like drug inclusions were not more than 2 μm in diameter, CLDIs ranged from 5 to 20 μm in length. CLDIs were birefringent when examined using polarized light microscopy (Fig. 2B). In the kidneys, the distribution of clofazimine inclusions varied in different regions of the organ. The kidney medulla (Fig. 2C) showed a pale pink pigmentation, while the cortex (Fig. 2D) exhibited CLDIs formed around the glomerulus and blood vessels (V). In the small intestine, CLDIs were observed only in the lamina propria of villi in the jejunum and ileum (Fig. 2E). Enterocytes did not show any visible pigmentation. The mesenteric lymph nodes were filled with large numbers of CLDIs (Fig. 2F) present at the periphery of germinal centers, but lymphocytes appeared to be devoid of drug inclusions. A similar pattern was observed in the spleen after 3 weeks (Fig. 2G) and 8 weeks (Fig. 2H) of treatment, with CLDIs increasingly accumulating in macrophage-like cells of the marginal zone surrounding the germinal centers, with fewer CLDIs found in the macrophages of the red pulp. In the liver, CLDI distribution changed from a scattered pattern after 3 weeks of treatment (Fig. 2I) to a more localized pattern with CLDIs present mostly in the periphery of blood vessels after 8 weeks of treatment (Fig. 2J).
Fig 2
Fig 2
Dark purple and brown pigmentation was associated with CLDI formation in organs of drug-treated mice. (A) Eight-week-clofazimine-treated lung parenchyma showed distinct, vibrant red drug inclusions. (B) CLDIs were birefringent under polarized light, whereas (more ...)
Clofazimine mass corresponded to visual differences in pigmentation and CLDI accumulation.
Drug mass was assessed to study the changes in clofazimine bioaccumulation and distribution during the course of treatment (Fig. 3). To calculate drug mass, we first measured drug concentration by dividing the amount of clofazimine extracted from tissue samples obtained from the different organs by the weight of wet tissue. This analysis was performed after 3 and 8 weeks of treatment and then again after an 8-week washout phase (Fig. 3A). After 8 weeks of treatment, clofazimine concentration in the spleen, liver, and small intestine (jejunum and ileum) increased by at least 1,500%, relative to the measured concentration at the end of 3 weeks. During the same period, the clofazimine concentration in adipose tissue surprisingly decreased by 38%. This decrease corresponded to a gradual, but massive redistribution of clofazimine from adipose tissue to spleen or liver after 3 weeks of treatment. Remarkably, although clofazimine concentration decreased in the blood and other organs consistent with its gradual clearance (1), clofazimine concentration in spleen remained largely constant during the washout phase.
Fig 3
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 (more ...)
After 8 weeks of treatment, the spleen, liver, jejunum, ileum, and fat contained ~13 mg of clofazimine (Fig. 3B; see also Materials and Methods). Although the clofazimine amount per weight measured in the liver was lower than that of the spleen (Fig. 3A), its greater weight and volume of the liver made it the largest storage compartment of clofazimine, containing up to 5.8 mg (39% of total drug available) after 8 weeks of treatment (Fig. 3B). Based on a mouse body fat of 16%, at week 8 there were 1.3 mg of clofazimine in fat (9% of total), which was about one-third of the mass in fat at week 3 (3.1 mg, Fig. 3B). The clofazimine content in the jejunum and ileum (Fig. 3A) increased during treatment, in contrast to the clofazimine content of the duodenum (Fig. 3C) or the large intestine (data not shown), which remained low. The combined average drug mass in the jejunum and ileum after 8 weeks of treatment was 3 mg (Fig. 3B). In the lung, the amount of clofazimine peaked at 8 weeks and declined rapidly by 25% on average during the washout phase (Fig. 3C). Clofazimine mass in the duodenum and kidney remained undetectable during the course of the 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 8 weeks of treatment compared to those after 3 weeks of treatment (Fig. 3D), which corresponded to a shift in clofazimine mass balance from the adipose tissue to the liver, spleen, and small intestines (Fig. 3E).
Clofazimine mass associated with CLDIs in spleen and liver exceeded the mass present in fat.
To determine the fraction of total drug mass present in CLDIs, CLDIs were isolated from tissue homogenates after sonication and differential centrifugation. This resulted in the removal of >95% of protein and a >10-fold enrichment in CLDIs. In the spleen, up to 91% of the total clofazimine mass in this organ was found in association with the isolated CLDIs. The mean combined clofazimine mass present in the isolated CLDIs from the spleen and liver was 50% ± 38% of the total mass found in these two organs. CLDI isolation and fractionation analysis was restricted to the liver and spleen, because other organs contained more connective tissue which made it difficult to isolate CLDIs. In addition, since the total mass of clofazimine in liver and spleen amounted to 60% of the total mass of absorbed clofazimine at 8 weeks, 30 to 40% of the total consumed clofazimine mass was found in CLDIs isolated from these organs. Using LC-MS, we confirmed that the molecular weight and retention time of clofazimine in liver and spleen corresponded to the pure compound; this is consistent with prior reports that showed clofazimine contained in tissues remains metabolically intact (12, 29). Because the CLDI isolation procedure is <100% efficient and the intestine and lung contained >21% of the bodily clofazimine mass, the percentage of total clofazimine mass in the host associated with CLDIs may well exceed 50%.
Changes in clofazimine serum concentration over time paralleled its concentration in fat tissue (Fig. 3D), as expected based on partitioning of clofazimine between serum and fat. The measured serum concentrations were within a comparable range to previously reported values, 2.6 to 7 μM in animals and humans (1, 24). Although changes of clofazimine concentration in fat (Fig. 3A) paralleled concentration changes in serum (Fig. 3D), clofazimine concentration and mass in the liver, spleen, and small intestine increased over time and did not parallel the clofazimine concentration in serum. At 3 weeks, the ratio of total clofazimine mass in fat relative to the liver and spleen was 9.4 ± 4.5 (n = 4). Nevertheless, at 8 weeks, this ratio dramatically decreased to 0.15 ± 0.03 (n = 4), indicating a major shift in clofazimine distribution. Based on the amount of clofazimine present in CLDIs, the mass of clofazimine in CLDIs at 8 weeks was greater than the amount of clofazimine in fat. We reasoned that clofazimine became sequestered in CLDIs as a result of a sequestration mechanism that favored increased partitioning of clofazimine from serum into liver, spleen, lungs, and intestines.
CLDIs accumulated in clusters of macrophage-like cells that formed around the blood vessels of liver and spleen.
In the lungs, only parenchymal macrophages showed evidence of CLDIs, apparent as polyhedral, membrane-bound intracellular cavities visible by TEM (Fig. 4A). Morphologically, macrophages were identified based on the shape of the nucleus, the prevalence of lysosomes and heterolysosomes in the cytoplasm, the presence of abundant cytoplasm without rough endoplasmic reticulum, and many surface pseudopodia. In the kidneys (Fig. 4B), peritubular macrophages were found to contain CLDIs, while neighboring tubular cells, consisting of the epithelium of renal tubules, contained only autophagosome-like drug inclusions similar to those reported to form in vitro using MDCK (Madin-Darby canine kidney) cell cultures (19). After 4 and 8 weeks of clofazimine treatment, a significant number of CLDI-containing macrophages were evident in the jejunal submucosa (Fig. 4C) and spleen (Fig. 4D). In the liver, 4 weeks of treatment resulted in the presence of CLDIs in macrophages (Fig. 4E) but not in hepatocytes. After 8 weeks of treatment there was an increase in the number and size of the liver CLDIs (Fig. 4F), mostly in clusters of macrophages that had formed adjacent to blood vessels. The clusters resembled microgranulomas, an inflammatory structure that usually forms in response to localized bacterial or parasitic infections.
Fig 4
Fig 4
TEM images of affected organs showed empty CLDI cavities present in the cytoplasm of macrophage-like cells. These cavities correspond to empty spaces that are left behind after CLDI extraction by the sample preparation process. (A) Lung parenchyma macrophages (more ...)
To confirm that the clofazimine sequestrating cells were macrophages, immunohistochemical analysis of samples from clofazimine-treated animals was performed using the macrophage-specific anti-F4/80 antibody (Fig. 5). Microscopically, the amount of F4/80 staining after 6 weeks of clofazimine treatment was greater than control, vehicle-treated mice, suggesting increased numbers of macrophages. Masson's trichrome staining (MTS) revealed the presence of fibrotic tissue associated with the F4/80-positive cell clusters. In contrast to anti-F4/80 staining, antibodies against α-smooth muscle actin (SMA) or an endothelial cell marker (von Willebrand factor [vWF]) did not show any changes in staining pattern, yielding no evidence that smooth muscle or endothelial cells were involved in CLDI formation (see Fig. S2 in the supplemental 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 macrophages filled with empty intracellular cavities where CLDIs resided prior to being removed by the immunohistochemical staining process. After the 8-week washout period, microgranulomas appeared to be localized mostly around the blood vessels (30). The increase in perivascular microgranulomas between 3 and 8 weeks suggests monocyte recruitment from the circulation (31).
Fig 5
Fig 5
Immunohistochemical analyses revealed liver microgranulomas containing CLDIs. Regions around the blood vessels (central vein [CV]) visualized by H&E staining underwent a structural transformation during clofazimine treatment (6 wk CFZ) as well (more ...)
In the spleen, immunohistochemical analysis of tissue samples obtained from clofazimine-treated animals revealed CLDI formation associated with macrophages at the periphery of germinal centers. The CLDIs were not present in lymphocytes which are the prevalent cell population in the germinal centers. Instead, CLDIs were 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 clear distinction between the F4/80-positive macrophages, CD21-positive follicular dendritic cells, and CD3-positive early T cells (see Fig. S3 in the supplemental material). In 6-week-clofazimine-treated mouse spleens, immunohistochemical staining showed a similar histological organization. However, intracellular CLDI cavities were mostly present in a subpopulation of F4/80-positive macrophages at the marginal zones, in the periphery of the germinal centers. The intensity of F4/80 (or CD21) staining in these cells was not as prominent as that of the macrophages or dendritic cells of the red pulp.
Fig 6
Fig 6
Immunohistochemical analysis of spleen revealed macrophage clusters exhibiting CLDI cavities in specific regions of the organ. CLDI cavities correspond to empty spaces that are left behind after CLDI extraction by the sample preparation process. In contrast (more ...)
In other tissues there were no obvious histological changes accompanying CLDI formation (see Fig. S4 in the supplemental material). In the intestine (see Fig. S4A), the staining of F4/80-positive cells was comparable between the treated and control samples. CD21 (follicular dendritic cell marker), and CD3 (early T cell marker) staining did not show any significant differences between the samples. In the kidneys, the cortex section showed an increased level of macrophage specific F4/80 staining in the glomerular region (see Fig. S4B, triangle, in the supplemental material) in proximity to blood vessels that stained positive for α-SMA and vWF.
Clofazimine bioaccumulation was associated with decreased local oxidant levels and an upregulated IL-1RA anti-inflammatory response.
To determine whether clofazimine bioaccumulation was linked to pro-oxidant stress or activation of inflammatory response pathways, we assayed the levels of the inducible antioxidant response protein MnSOD, measured the total antioxidant capacity of various tissues, and profiled key chemokine and cytokine signaling molecules. We analyzed the major organs exhibiting the greatest amount of local clofazimine accumulation (spleen, liver, and lung), as well as a control organ showing an undetectable amount of clofazimine accumulation (brain). In Western blots, MnSOD protein levels did not change in response to clofazimine treatment (see Fig. S5A to D in the supplemental material) except in the spleen (see Fig. S5C), where MnSOD expression was slightly increased. The amount of MnSOD to total protein content remained low. Consistent with this observation, the absolute oxidant state of different organs was not altered by clofazimine treatment (Fig. 7, 0 mM Trolox). However, the total oxidant capacity of the lungs (Fig. 7B) and spleen (Fig. 7C) were more readily titrated by the addition of exogenous antioxidant (Trolox) relative to the liver (Fig. 7D) and brain (Fig. 7A). Therefore, in the lungs and spleen the local clofazimine bioaccumulation was associated with a reduction in oxidant capacity.
Fig 7
Fig 7
Clofazimine-induced CLDI formation reduced organ oxidant capacity. Oxidizing activity present in each homogenate led to an increase in absorbance (y axis), which decreased in the presence of increasing concentration of an added antioxidant (Trolox). (A) (more ...)
By measuring changes in the levels on immune signaling molecules using a cytokine array, clofazimine bioaccumulation was associated with a remarkable phenomenon: a lymphatic organ-specific upregulation of the soluble anti-inflammatory protein interleukin-1 receptor antagonist (IL-1RA) (Table 1). IL-1RA is a potent, systemic anti-inflammatory protein (3235), and its induction appeared to be specifically associated with local clofazimine accumulation in the lung, liver, and spleen. In the brain, an organ that did not exhibit clofazimine accumulation, IL-1RA was downregulated, as were many other monocyte chemoattractant proteins and a large number of other acute inflammatory response mediators. Spleen samples from clofazimine-treated mice also exhibited indications of an IL-1RA-related anti-inflammatory response, evidenced by the significant downregulation of the T-cell chemokines CCL5 (36, 37) and CXCL9, as well as general downregulation of many other cytokines and chemokines (see Table S1 in the supplemental material). In the lungs, clofazimine bioaccumulation was also associated with increased IL-1RA levels (Table 1), concomitant with the downregulation of CCL4 (38), CCL17 (39), and TNF-α (40) (Table 1), which are proinflammatory signaling mediators secreted by activated macrophages. The lungs from clofazimine-treated mice also had higher levels of chemokines involved in macrophage recruitment, including CCL2 (36, 41) and CXCL1 (42, 43), the response-amplifying, growth-stimulatory monocyte receptor, TREM-1 (44, 45), and the monocyte retention-promoting metalloprotease inhibitor, TIMP-1. The majority of other soluble chemokines, cytokines, and receptor molecules assayed appeared to be decreased or unaffected by clofazimine treatment (see Table S1 in the supplemental material). Of the few proteins upregulated in treated samples, less than half increased 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).
Table 1
Table 1
Clofazimine-induced changes in cytokine and chemokine levels in the lungs, spleen, liver, and braina
In the present study, we quantitatively analyzed how the distribution of clofazimine changed between 3 and 8 weeks of administration and during an 8-week posttreatment washout period. Changes in clofazimine content and distribution occurred during prolonged clofazimine treatment and correlated with major structural and functional changes in the immune system. Our results provide evidence that an inducible xenobiotic sequestration response mediated by a subpopulation of cells of the immune system is profoundly impacting the distribution and bioaccumulation of clofazimine. This is consistent with human autopsy reports (5, 15, 46) indicating the presence of clofazimine crystals in lymphatic tissues. Furthermore, our results suggest that these “crystals” are not accidental or haphazard. In mice, they were present in a site-specific subpopulation of macrophages in the spleen, and linked to an active, immune system-mediated, intracellular xenobiotic sequestration response associated with spleen enlargement and microgranuloma formation in the liver.
Our multiscale distribution and bioaccumulation analysis also brings into focus the long-standing pharmacokinetic assumption that highly lipophilic compounds stably partition from the serum into adipose tissue. Although this assumption may be true during short-term clofazimine treatment (<3 weeks), it was certainly not true after a long-term, 8-week treatment. Like other lipophilic molecules, clofazimine partitioned into adipose tissues during a short-term exposure period (8). When clofazimine absorption and distribution was monitored in mice for either 1 or 5 days after a 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 sharp decrease, and then remained at minimal level until another dose was given. The drug content in fat increased continuously over each 24-h period, which persisted with each daily treatment throughout the first 5 days of administration (8). Nevertheless, between 3 and 8 weeks of continuous exposure, we observed clofazimine dramatically redistributed from adipose tissue to liver, spleen, gut, and lungs.
Interestingly, CLDIs were never observed outside macrophages and were always localized to the cells' cytoplasm. Macrophages are phagocytic cells, and they are the only cell type known to possess a size-fractionating endolysosomal system (47), which may explain why CLDIs are found exclusively in these cells. Toxicologically, one may have expected evidence of necrosis and the presence of extracellular crystals at sites of microgranulomas formation. However, neither necrosis nor extracellular crystals were observed. CLDIs were homogeneous in size and shape, suggesting that their distribution and morphology is under active cellular control. Supporting the novelty and significance of these results, we also found that clofazimine formed a unique, liquid crystal- and organelle-like supramolecular organization inside macrophages (13).
The development of splenomegaly upon prolonged clofazimine treatment indicated an active response mechanism affecting the disposition of clofazimine (48). In addition, because there are different kinds of resident tissue macrophages in the spleen (4951), it is possible that only a specialized subpopulation of macrophages sequestered clofazimine. At 8 weeks of treatment, up to 1% of the mass of the spleen was comprised of clofazimine, with up to 91% of the total clofazimine mass being present in association with CLDIs. Since the major elimination route for this metabolically stable drug is biliary clearance followed by fecal excretion (4, 5), we reasoned that the decrease in clofazimine content in liver during the washout period may result from direct elimination through the bile once treatment is discontinued. Remarkably, the spleen continued to retain clofazimine even after the plasma concentrations of clofazimine had significantly dropped.
In parallel to the observed, immune system-mediated, macrophage dependent, active xenobiotic sequestration response associated with the intracellular accumulation of clofazimine, the upregulation 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-1α and IL-1β to the proinflammatory interleukin-1 (IL-1) receptor (34, 35, 52), leading to a pronounced, systemic anti-inflammatory activity. In humans, genetic mutations that lead to nonfunctional IL-1RA result in a generalized auto-inflammatory disease affecting bones, joints and 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 to have major consequences for immune regulation (35, 53), this is the first report linking increased IL-1RA to a specific, xenobiotic accumulation response pathway.
The complete lack of extracellular clofazimine crystals, the highly controlled intracellular distribution of CLDIs (13), the absence of obvious toxicological manifestations together with the increased levels of anti-inflammatory IL-1RA suggest a protective, coordinated biological response. In vitro, clofazimine is cytotoxic and has been shown to induce the production of superoxide anion in rat peritoneal macrophages and human neutrophils ex vivo (54, 55). In vitro, clofazimine can induce apoptosis via caspase activation (56). Nevertheless, there were no obvious toxicological manifestations in vivo, and primary macrophages isolated from clofazimine-treated mice were viable and mobile, although they contained many drug inclusions (13). In clofazimine-treated mice, the amounts of inducible, anti-oxidant MnSOD did not increase, which is consistent with an absence of oxidant stress. Furthermore, clofazimine increased the levels of the anti-inflammatory IL-1RA signaling protein and decreased the levels of many other proinflammatory cytokines and chemokines, while the oxidizing capacity of the primary sites of clofazimine bioaccumulation decreased or remained unchanged. Collectively, these results demonstrate that, in vivo, clofazimine induces a protective, macrophage-mediated xenobiotic sequestration response.
In conclusion, our results provide insights into the bioaccumulation-related side effects of clofazimine, unrelated to the drug's primary mechanism of action. The extraordinarily long half-life, atypical pharmacokinetics and extensive bioaccumulation of clofazimine are not simply a consequence of lipophilic partitioning into body fat. To our knowledge, this is the first study to implicate an immune-mediated drug sequestration mechanism in a mammalian organism. Certainly, our observations prompt many more questions that lie beyond the scope of this multiscale biodistribution study. In future experiments, we will address the exact role of the immune system on the bioaccumulation of clofazimine by taking advantage of genetically mutant mice lacking IL-1RA as well as the other chemokine genes involved in immune signaling. We also envision analyzing the bioaccumulation and distribution of different clofazimine derivatives as well as other lipophilic compounds to address whether this phenomenon represents a unique, idiosyncratic side effect of clofazimine, or a more general xenobiotic sequestration response. Most importantly, the experimental approach and results presented here offer a uniquely different perspective into some of the chemotherapeutic properties of clofazimine and next-generation clofazimine derivatives.
Supplementary Material
Supplemental material
ACKNOWLEDGMENTS
The project was supported by National Institutes of Health (NIH) grants GM007767 (J.B.), R01GM078200 (G.R.R.), and R15HD065594 (K.A.S). This study utilized Core Services supported by NIH grant DK089503 to the University of Michigan. J.B. was also supported by a fellowship from the American Foundation for Pharmaceutical Education.
We thank Dorothy Sorenson (MIL, University of Michigan), Paula Arrowsmith (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 authors and do not necessarily represent the official views of the NIGMS, NICHD, or NIH.
Footnotes
Published ahead of print 21 December 2012
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.01731-12.
1. Banerjee DK, Ellard GA, Gammon PT, Waters MF. 1974. Some observations on the pharmacology of clofazimine (B663). Am. J. Trop. Med. Hyg. 23:1110–1115. [PubMed]
2. Queiroz RHC, de Souza AM, Sampaio SV, Melchior E. 2002. Biochemical and hematological side effects of clofazimine in leprosy patients. Pharmacol. Res. 46:191–194. [PubMed]
3. Cholo MC, Steel HC, Fourie PB, Germishuizen WA, Anderson R. 2011. Clofazimine: current status and future prospects. J. Antimicrob. Chemother. 67:290–298. [PubMed]
4. O'Connor R, O'Sullivan JF, O'Kennedy R. 1995. The pharmacology, metabolism, and chemistry of clofazimine. Drug Metab. Rev. 27:591–614. [PubMed]
5. Mansfield RE. 1974. Tissue concentrations of clofazimine (B663) in man. Am. J. Trop. Med. Hyg. 23:1116–1119. [PubMed]
6. Venkatesan K, Deo N, Gupta UD. 2007. Tissue distribution and deposition of clofazimine in mice following oral administration with or without isoniazid. Arzneimittelforschung 57:472–474. [PubMed]
7. Greenwood D. 2008. Antimicrobial drugs: chronicle of a twentieth century medical triumph. Oxford University Press, Oxford, England.
8. Barry VC, Buggle K, Byrne J, Conalty ML, Winder F. 1960. Absorption, distribution and retention of the riminocompounds in the experimental animal. Ir. J. Med. Sci. 416:345–352. [PubMed]
9. McDougall AC, Horsfall WR, Hede JE, Chaplin AJ. 1980. Splenic infarction and tissue accumulation of crystals associated with the use of clofazimine (Lamprene; B663) in the treatment of pyoderma gangrenosum. Br. J. Dermatol. 102:227–230. [PubMed]
10. Dey T, Brigden G, Cox H, Shubber Z, Cooke G, Ford N. 2012. Outcomes of clofazimine for the treatment of drug-resistant tuberculosis: a systematic review and meta-analysis. J. Antimicrob. Chemother. doi:10.1093/jac/dks389. [PubMed]
11. Levy L, Randall H. 1970. A study of skin pigmentation by clofazimine. Int. J. Lepr. Other Mycobact. Dis. 38:404–416. [PubMed]
12. Aplin RT, McDougall AC. 1975. Identification of crystals of the rimino-phenazine compound B663 (Lamprene: clofazimine) in mouse spleen macrophages by thin layer chromatography and mass spectrum analysis. Experientia 31:468–469. [PubMed]
13. Baik J, Rosania GR. 2012. Macrophages sequester clofazimine in an intracellular liquid crystal-like supramolecular organization. PLoS One 7:e47494 doi:10.1371/journal.pone.0047494. [PMC free article] [PubMed]
14. McDougall AC. 1974. Electron microscope studies of the antileprosy drug B663 (clofazimine; Lamprene). Int. J. Lepr. Other Mycobact. Dis. 42:1–12. [PubMed]
15. Desikan KV, Ramanujam K, Ramu G, Balakrishnan S. 1975. Autopsy findings in a case of lepromatous leprosy treated with clofazimine. Lepr. Rev. 46:181–189. [PubMed]
16. Sukpanichnant S, Hargrove NS, Kachintorn U, Manatsathit S, Chanchairujira T, Siritanaratkul N, Akaraviputh T, Thakerngpol K. 2000. Clofazimine-induced crystal-storing histiocytosis producing chronic abdominal pain in a leprosy patient. Am. J. Surg. Pathol. 24:129–135. [PubMed]
17. Wan H, Holmen AG, Wang Y, Lindberg W, Englund M, Nagard MB, Thompson RA. 2003. High-throughput screening of pKa values of pharmaceuticals by pressure-assisted capillary electrophoresis and mass spectrometry. Rapid Commun. Mass Spectrom. 17:2639–2648. [PubMed]
18. de Duve C, de Barsy T, Poole B, Trouet A, Tulkens P, Van Hoof F. 1974. Commentary: lysosomotropic agents. Biochem. Pharmacol. 23:2495–2531. [PubMed]
19. Baik J, Rosania GR. 2011. Molecular imaging of intracellular drug-membrane aggregate formation. Mol. Pharm. 8:1742–1749. [PMC free article] [PubMed]
20. Alakoskela JM, Vitovic P, Kinnunen PK. 2009. Screening for the drug-phospholipid interaction: correlation to phospholipidosis. ChemMedChem 4:1224–1251. [PubMed]
21. Reasor MJ, Hastings KL, Ulrich RG. 2006. Drug-induced phospholipidosis: issues and future directions. Expert Opin. Drug Safety 5:567–583. [PubMed]
22. Sarracent J, Finlay CM. 1982. The action of Clofazimine on the level of lysosomal enzymes of cultured macrophages. Clin. Exp. Immunol. 48:261–267. [PubMed]
23. Sarracent J, Finlay CM. 1984. In vivo effect of clofazimine in the lysosomal enzyme level and immune complex phagocytosis of mouse peritoneal macrophages. Int. J. Lepr. Other Mycobact. Dis. 52:154–158. [PubMed]
24. Levy L. 1974. Pharmacologic studies of clofazimine. Am. J. Trop. Med. Hyg. 23:1097–1109. [PubMed]
25. Wishart DS, Knox C, Guo AC, Cheng D, Shrivastava S, Tzur D, Gautam B, Hassanali M. 2008. DrugBank: a knowledgebase for drugs, drug actions and drug targets. Nucleic Acids Res. 36:D901–D906. [PMC free article] [PubMed]
26. Stringer KA, Freed BM, Dunn JS, Sayers S, Gustafson DL, Flores SC. 2004. Particulate phase cigarette smoke increases MnSOD, NQO1, and CINC-1 in rat lungs. Free Radic. Biol. Med. 37:1527–1533. [PubMed]
27. Storey JD, Tibshirani R. 2003. Statistical methods for identifying differentially expressed genes in DNA microarrays. Methods Mol. Biol. 224:149–157. [PubMed]
28. Storey JD, Tibshirani R. 2003. Statistical significance for genomewide studies. Proc. Natl. Acad. Sci. U. S. A. 100:9440–9445. [PubMed]
29. Barry VC, Belton JG, Conalty ML, Denneny JM, Edward DW, O'Sullivan JF, Twomey D, Winder F. 1957. A new series of phenazines (rimino-compounds) with high antituberculosis activity. Nature 179:1013–1015. [PubMed]
30. Rhodin JA. 1975. An atlas of histology, vol 1 Oxford University Press, New York, NY.
31. Jenkins SJ, Ruckerl D, Cook PC, Jones LH, Finkelman FD, van Rooijen N, MacDonald AS, Allen JE. 2011. Local macrophage proliferation, rather than recruitment from the blood, is a signature of TH2 inflammation. Science 332:1284–1288. [PMC free article] [PubMed]
32. Schreuder H, Tardif C, Trump-Kallmeyer S, Soffientini A, Sarubbi E, Akeson A, Bowlin T, Yanofsky S, Barrett RW. 1997. A new cytokine-receptor binding mode revealed by the crystal structure of the IL-1 receptor with an antagonist. Nature 386:194–200. [PubMed]
33. Schiff MH. 2000. Role of interleukin 1 and interleukin 1 receptor antagonist in the mediation of rheumatoid arthritis. Ann. Rheum. Dis. 59(Suppl 1):i103–i108. [PMC free article] [PubMed]
34. Hannum CH, Wilcox CJ, Arend WP, Joslin FG, Dripps DJ, Heimdal PL, Armes LG, Sommer A, Eisenberg SP, Thompson RC. 1990. Interleukin-1 receptor antagonist activity of a human interleukin-1 inhibitor. Nature 343:336–340. [PubMed]
35. Arend WP, Malyak M, Guthridge CJ, Gabay C. 1998. Interleukin-1 receptor antagonist: role in biology. Annu. Rev. Immunol. 16:27–55. [PubMed]
36. Xu LL, Warren MK, Rose WL, Gong W, Wang JM. 1996. Human recombinant monocyte chemotactic protein and other C-C chemokines bind and induce directional migration of dendritic cells in vitro. J. Leukoc. Biol. 60:365–371. [PubMed]
37. Oppenheim JJ, Zachariae CO, Mukaida N, Matsushima K. 1991. Properties of the novel proinflammatory supergene “intercrine” cytokine family. Annu. Rev. Immunol. 9:617–648. [PubMed]
38. Bystry RS, Aluvihare V, Welch KA, Kallikourdis M, Betz AG. 2001. B cells and professional APCs recruit regulatory T cells via CCL4. Nat. Immunol. 2:1126–1132. [PubMed]
39. Romagnani S. 2002. Cytokines and chemoattractants in allergic inflammation. Mol. Immunol. 38:881–885. [PubMed]
40. Beutler B, Cerami A. 1989. The biology of cachectin/TNF–a primary mediator of the host response. Annu. Rev. Immunol. 7:625–655. [PubMed]
41. Kuziel WA, Morgan SJ, Dawson TC, Griffin S, Smithies O, Ley K, Maeda N. 1997. Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2. Proc. Natl. Acad. Sci. U. S. A. 94:12053–12058. [PubMed]
42. Devalaraja RM, Nanney LB, Du J, Qian Q, Yu Y, Devalaraja MN, Richmond A. 2000. Delayed wound healing in CXCR2 knockout mice. J. Invest. Dermatol. 115:234–244. [PMC free article] [PubMed]
43. Smith DF, Galkina E, Ley K, Huo Y. 2005. GRO family chemokines are specialized for monocyte arrest from flow. Am. J. Physiol. Heart Circ. Physiol. 289:H1976–H1984. [PubMed]
44. Colonna M, Facchetti F. 2003. TREM-1 (triggering receptor expressed on myeloid cells): a new player in acute inflammatory responses. J. Infect. Dis. 187(Suppl 2):S397–S401. [PubMed]
45. Ford JW, McVicar DW. 2009. TREM and TREM-like receptors in inflammation and disease. Curr. Opin. Immunol. 21:38–46. [PMC free article] [PubMed]
46. Balakrishnan S, Desikan KV, Ramu G. 1976. Quantitative estimation of clofazimine in tissue. Lepr. India 48:732–738. [PubMed]
47. Berthiaume EP, Medina C, Swanson JA. 1995. Molecular size-fractionation during endocytosis in macrophages. J. Cell Biol. 129:989–998. [PMC free article] [PubMed]
48. Griffis LC, Twerdok LE, Francke-Carroll S, Biles RW, Schroeder RE, Bolte H, Faust H, Hall WC, Rojko J. 2010. Comparative 90-day dietary study of paraffin wax in Fischer-344 and Sprague-Dawley rats. Food Chem. Toxicol. 48:363–372. [PubMed]
49. Backer R, Schwandt T, Greuter M, Oosting M, Jungerkes F, Tuting T, Boon L, O'Toole T, Kraal G, Limmer A, den Haan JM. 2010. Effective collaboration between marginal metallophilic macrophages and CD8+ dendritic cells in the generation of cytotoxic T cells. Proc. Natl. Acad. Sci. U. S. A. 107:216–221. [PubMed]
50. Leenen PJ, Radosevic K, Voerman JS, Salomon B, van Rooijen N, Klatzmann D, van Ewijk W. 1998. Heterogeneity of mouse spleen dendritic cells: in vivo phagocytic activity, expression of macrophage markers, and subpopulation turnover. J. Immunol. 160:2166–2173. [PubMed]
51. Aichele P, Zinke J, Grode L, Schwendener RA, Kaufmann SH, Seiler P. 2003. Macrophages of the splenic marginal zone are essential for trapping of blood-borne particulate antigen but dispensable for induction of specific T cell responses. J. Immunol. 171:1148–1155. [PubMed]
52. Dinarello CA. 2000. The role of the interleukin-1-receptor antagonist in blocking inflammation mediated by interleukin-1. N. Engl. J. Med. 343:732–734. [PubMed]
53. Aksentijevich I, Masters SL, Ferguson PJ, Dancey P, Frenkel J, van Royen-Kerkhoff A, Laxer R, Tedgard U, Cowen EW, Pham TH, Booty M, Estes JD, Sandler NG, Plass N, Stone DL, Turner ML, Hill S, Butman JA, Schneider R, Babyn P, El-Shanti HI, Pope E, Barron K, Bing X, Laurence A, Lee CC, Chapelle D, Clarke GI, Ohson K, Nicholson M, Gadina M, Yang B, Korman BD, Gregersen PK, van Hagen PM, Hak AE, Huizing M, Rahman P, Douek DC, Remmers EF, Kastner DL, Goldbach-Mansky R. 2009. An autoinflammatory disease with deficiency of the interleukin-1-receptor antagonist. N. Engl. J. Med. 360:2426–2437. [PMC free article] [PubMed]
54. Sahu A, Saha K, Banerjee NR, Sehgal VN, Jagga CR. 1991. Effect of anti-leprosy drugs on superoxide anion production by rat peritoneal macrophage with special reference to light exposed clofazimine. Int. J. Immunopharmacol. 13:419–428. [PubMed]
55. Van Rensburg CE, Van Staden AM, Anderson R. 1993. The riminophenazine agents clofazimine and B669 inhibit the proliferation of cancer cell lines in vitro by phospholipase A2-mediated oxidative and nonoxidative mechanisms. Cancer Res. 53:318–323. [PubMed]
56. Fukutomi Y, Maeda Y, Makino M. 2011. Apoptosis-inducing activity of clofazimine in macrophages. Antimicrob. Agents Chemother. 55:4000–4005. [PMC free article] [PubMed]
Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of
American Society for Microbiology (ASM)