Multiscale Distribution and Bioaccumulation Analysis of Clofazimine Reveals a Massive Immune System-Mediated Xenobiotic Sequestration Response

Abstract

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 acutephase response pathways and oxidant capacity decreased or remained unchanged, marking a concomitant activation of an antiinflammatory response. These experiments indicate an inducible, immune system-dependent, xenobiotic sequestration response affecting the atypical pharmacokinetics of a small molecule chemotherapeutic agent.

Clofazimine Biocrystal Accumulation in Macrophages Upregulates Interleukin 1 Receptor Antagonist Production To Induce a Systemic Anti-Inflammatory State

“…Such targeted accumulation of CFZ biocrystals was also detected in other organs such as CD68 ϩ alveolar macrophages (see Fig. S1 in the supplemental material), and F4/80 ϩ splenic macrophages (8,31). CFZ biocrystal formation in the liver downregulates Casp 1/IL-1␤ processing while enhancing IL-1RA expression.…”

“…Clofazimine (CFZ; Sigma-Aldrich, catalog no. C8895) was prepared in sesame oil (Shirakiku, Japan, or Roland, China) and Powdered Lab Diet 5001 (PMI International, Inc., St. Louis, MO) and orally administered to wild-type (WT) C57BL/6 mice or IL-1RA knockout mice (4 to 5 weeks old; Jackson Laboratory, Bar Harbor, ME) for up to 8 weeks ad libitum as previously described (8,24). Control mice were fed with the same diet without CFZ.…”

“…In previous studies, it has been shown that following prolonged oral administration of CFZ, the drug massively bioaccumulates as intracellular biocrystals in resident tissue macrophages (8)(9)(10)(11)(12). Therefore, it is natural to assume that this bioaccumulation phenomenon contributes to the drug's toxicity, whereas the soluble form of CFZ, which circulates and partitions to and from the different organs, is responsible for CFZ's mechanism of action.…”

Pharmacokinetics and Pharmacodynamics of Clofazimine in a Mouse Model of Tuberculosis

“…weeks of administration of approximately 10 mg/kg/day of clofazimine in the diet to uninfected BALB/c mice resulted in extremely high accumulation in the spleen, although they found much higher accumulation in their study (more than 10 mg/g spleen tissue), and that approximately 90% of the drug was sequestered in intracellular crystal-like drug inclusions (25). Interestingly, in our study, when clofazimine was administered at 25 mg/kg/day to infected mice, the concentration of the drug in the lungs and spleen stabilized at approximately 100 g/g tissue after 4 to 8 weeks of administration (Fig.…”

Detecting ordered small molecule drug aggregates in live macrophages: a multi-parameter microscope image data acquisition and analysis strategy

“…Clofazimine (CFZ) is a bright red phenazine drug that accumulates extensively in tissues throughout the organism, leading to the formation of deep red, crystal-like drug inclusions in vivo [27]. Following eight weeks of CFZ treatment, CLDIs were isolated from the spleen of a mouse and imaged with the polarization and fluorescence imaging system.…”