Chemical Analysis of Drug Biocrystals: A Role for Counterion Transport Pathways in Intracellular Drug Disposition

Keswani, Rahul K.; Baik, Jason; Yeomans, Larisa; Hitzman, Chuck; Johnson, Allison M.; Pawate, Ashtamurthy S.; Kenis, Paul J. A.; Rodrı́guez-Hornedo, Naı́r; Stringer, Kathleen A.; Rosania, Gus R.

doi:10.1021/acs.molpharmaceut.5b00032

Cited by 40 publications

(69 citation statements)

References 44 publications

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“…Our results demonstrated that CLDIs are comprised of subdomains of pure CFZ-hydrochloride crystals [34], bounded by multilamellar membrane structure of biological origin [18,19]. Consistent with these previous results, quantitative polarization microscopy revealed the manner in which microscopic domains of ordered CFZ molecules within CLDIs of live macrophages obtained from CFZ-treated mice differentially interact with polarized light.…”

Section: Discussionsupporting

confidence: 90%

“…Previously, we have characterized the molecular organization and chemical composition of CLDIs following biochemical isolation, using nuclear magnetic resonance, powder X-ray diffraction, transmitted electron microscopy, freeze fracture electron microscopy, liquid chromatography and mass spectrometry [18,19,34]. Our results demonstrated that CLDIs are comprised of subdomains of pure CFZ-hydrochloride crystals [34], bounded by multilamellar membrane structure of biological origin [18,19].…”

Section: Discussionmentioning

confidence: 75%

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

Rzeczycki

Yoon

Keswani

et al. 2017

Biomed. Opt. Express

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Abstract:Following prolonged administration, certain orally bioavailable but poorly soluble small molecule drugs are prone to precipitate out and form crystal-like drug inclusions (CLDIs) within the cells of living organisms. In this research, we present a quantitative multiparameter imaging platform for measuring the fluorescence and polarization diattenuation signals of cells harboring intracellular CLDIs. To validate the imaging system, the FDAapproved drug clofazimine (CFZ) was used as a model compound. Our results demonstrated that a quantitative multi-parameter microscopy image analysis platform can be used to study drug sequestering macrophages, and to detect the formation of ordered molecular aggregates formed by poorly soluble small molecule drugs in animals.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 75%

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

Rzeczycki

Yoon

Keswani

et al. 2017

Biomed. Opt. Express

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“…Many of these microorganisms tend to infect and reside within macrophages. Interestingly, chemical analysis of CFZ biocrystals has revealed that they are composed of protonated hydrochloride salt of CFZ (CFZ-HCl) within membrane-bound intracellular compartments (9). Therefore, the bioaccumulation of CFZ biocrystals in macrophages could serve to maximize the therapeutic efficacy of CFZ without significant side effects.…”

Section: Figmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Yoon

Keswani

Sud

et al. 2016

Antimicrob Agents Chemother

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Clofazimine (CFZ) is a poorly soluble antibiotic and anti-inflammatory drug indicated for the treatment of leprosy. In spite of its therapeutic value, CFZ therapy is accompanied by the formation of drug biocrystals that accumulate within resident tissue macrophages, without obvious toxicological manifestations. Therefore, to specifically elucidate the off-target consequences of drug bioaccumulation in macrophages, we compared the level of inflammasome activation in CFZ-accumulating organs (spleen, liver and lung) in mice after 2 and 8 weeks of CFZ treatment when the drug exists in soluble and insoluble (biocrystalline) forms, respectively. Surprisingly, the results showed a drastic reduction in caspase 1 and interleukin-1␤ (IL-1␤) cleavage in the livers of mice treated with CFZ for 8 weeks (8-week-CFZ-treated mice) compared to 2-week-CFZ-treated and control mice, which was accompanied by a 3-fold increase in hepatic IL-1 receptor antagonist (IL-1RA) production and a 21-fold increase in serum IL-1RA levels. In the lung and spleen, IL-1␤ cleavage and tumor necrosis factor alpha expression were unaffected by soluble or biocrystal CFZ forms. Functionally, there was a drastic reduction of carrageenan-and lipopolysaccharide-induced inflammation in the footpads and lungs, respectively, of 8-week-CFZ-treated mice. This immunomodulatory activity of CFZ biocrystal accumulation was attributable to the upregulation of IL-1RA, since CFZ accumulation had minimal effect in IL-1RA knockout mice or 2-week-CFZ-treated mice. In conclusion, CFZ accumulation and biocrystal formation in resident tissue macrophages profoundly altered the host's immune system and prompted an IL-1RA-dependent, systemic anti-inflammatory response.

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“…Clofazimine (CFZ) is a model xenobiotic - a weakly basic, FDA approved drug for the treatment of leprosy (1) that accumulates in macrophages, forming crystal-like-drug-inclusions (CLDIs) (2–7). Following biochemical isolation it is possible to analyze the composition of CLDIs, and we have elucidated that CLDIs are comprised of domains of pure CFZ hydrochloride crystals (CFZ-HCl), bound and separated by membranes likely of cellular origin (8). …”

Section: Introductionmentioning

confidence: 99%

A far‐red fluorescent probe for flow cytometry and image‐based functional studies of xenobiotic sequestering macrophages

et al. 2015

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Clofazimine (CFZ) is an optically active, red-colored chemotherapeutic agent that is FDA – approved for the treatment of leprosy and is on the World Health Organization's list of essential medications. Interestingly, CFZ massively accumulates in macrophages where it forms crystal–like–drug inclusions (CLDIs) following oral administration of the drug in animals and humans. Analysis of the fluorescence spectra of CLDIs formed by resident tissue macrophages revealed that CFZ, when accumulated as CLDIs, undergoes a red shift in fluorescence excitation (from Ex:540-570 nm to Ex:560-600 nm) and emission (Em: 560-580 nm to Em: 640-700 nm) signal relative to the soluble and free-base crystal forms of CFZ. Using epifluorescence microscopy, CLDI(+) cells could be identified, relative to CLDI(−) cells, based on a >3-fold increment in mean fluorescence signal at excitation 640 nm and emission at 670 nm. Similarly, CLDI(+) cells could be identified by flow cytometry, based on a >100-fold increment in mean fluorescence signal using excitation lasers at 640 nm and emission detectors >600 nm. CLDI's fluorescence excitation and emission was orthogonal to that of cell viability dyes such as propidium iodide and DAPI, cellular staining dyes such as Hoechst 33342 (nucleus) and FM 1-43 (plasma membrane), as well as many other fluorescently-tagged antibodies used for immunophenotyping analyses. In vivo, >85% of CLDI(+) cells in the peritoneal exudate were F4/80(+) macrophages and >97% of CLDI(+) cells in the alveolar exudate were CD11c(+). Most importantly, the viability of cells was minimally affected by the presence of CLDIs. Accordingly, these results establish that CFZ fluorescence in CLDIs is suitable for quantitative flow cytometric phenotyping analysis and functional studies of xenobiotic sequestering macrophages.

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Chemical Analysis of Drug Biocrystals: A Role for Counterion Transport Pathways in Intracellular Drug Disposition

Cited by 40 publications

References 44 publications

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

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

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

A far‐red fluorescent probe for flow cytometry and image‐based functional studies of xenobiotic sequestering macrophages

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