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
) 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 (49
), 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
), 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
) could explain the potent anti-inflammatory effects of clofazimine reported in clinical studies (3
). IL-1RA inhibits the binding of soluble IL-1α and IL-1β to the proinflammatory interleukin-1 (IL-1) receptor (34
), 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
), 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
). 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.