Newly Published Reports from the Rosania Research Group Describe Macrophages and their Drug-Sequestering Habits
The Rosania Research Group at the College of Pharmacy has published four reports detailing the pharmacological, immunological, and optical analyses on macrophage-targeted drug sequestration. Working with a molecule, clofazimine, which is an FDA-approved drug that is included on the list of World Health Organization’s essential medicines, these reports point to multiple biochemical and signaling mechanisms that are a result of intracellular drug aggregation and crystallization.
Clofazimine (CFZ) massively accumulates in macrophages, forming insoluble, intracellular crystal-like drug inclusions (CLDIs) or Biocrystals during long-term oral dosing. Detailed chemical analyses led by Dr. Rahul Keswani, a Research Fellow in the Rosania Group, point to a concentrative, chloride transport pathway acting to drive biocrystal formation and retention. Essentially, the lab has established that CLDIs are hydrochloride salt crystals of the drug enveloped within a biological membranous domain. Learn more in the July issue of Molecular Pharmaceutics.
“Understanding the mechanism by which biocrystals form can offer insight into the manner in which macrophages impact the bioaccumulation and distribution of CFZ, and potentially many other poorly soluble compounds that can exist in the organism in both soluble and insoluble forms,” said Dr. Keswani. “These mechanisms are not only relevant to the disposition of CFZ or other phenazine compounds, but may also lend insight into the metabolism and clearance of other drugs, nutrients, toxicants, and potentially, endogenous compounds that accumulate in macrophage lysosomes during the normal aging process or in diseased states. Our work further emphasizes the significant role that chloride channels play in xenobiotic metabolism, disposition and pharmacology.”
“For poorly soluble compounds that can exist both as soluble and insoluble forms within the cells of an organism, the mechanisms controlling the bioaccumulation and distribution of soluble and insoluble forms of these agents in the different cells and organs of the body are not known,” said Dr. Gus Rosania, Professor of Pharmaceutical Sciences. “Many FDA-approved drugs, for example, CFZ, amiodarone, azithromycin, chloroquine, and gefitinib fall within the class of poorly soluble compounds that are actively sequestered in macrophages. Elucidating the mechanisms affecting the solubility and accumulation of these drugs inside cells is relevant to understanding drug toxicity, disposition and eventual therapeutic efficacy.”
From a biomaterials and nanotechnology perspective, the presence of biocrystals within intracellular domains clearly illustrates how it is possible to endow cells with artificial structural and functional elements, using self-assembling, orally bioavailable small molecule building blocks. Biocrystals possess nanoscale structural features, with properties resembling liquid crystals. The many interesting chemical, physical, and biological properties of these biocrystals could serve as a starting point for developing new kinds of CFZ-based solid-state nano-derivatives with potential applications in diagnostics, drug delivery and therapeutics. Such analyses are possible via polarization light microscopy techniques that can help to identify such uniformly assembled structures, especially within biological systems. To establish this, Dr. Kyoung Ah Min, a former Research Fellow in the Rosania Group, led an investigation of more than twenty CFZ-based derivatives. By relating the extent of bioaccumulation to the measured optical properties of the intracellular inclusions formed by different phenazine derivatives, the group determined that certain chemical modifications at the R-imino group promoted the self-assembly of phenazines, specifically in macrophages. All compounds tended to preferentially accumulate in macrophages relative to epithelial cells, regardless of their chemical structure or physicochemical properties. “With our work on derivatives, we have demonstrated our ability to detect and quantify the distribution of small molecules inside cells and monitor the formation of ordered, insoluble complexes with use of polarized light microscopy,” continued Dr. Rosania. “This was possible by illuminating the cells with monochromated light at specific wavelengths and tuning the anisotropy signals to specifically detect the organization of the exogenous molecules.”
This work was ably supported through collaborations with Dr. Rudolph Oldenburg of the Marine Biological Laboratory, Woods Hole, MA and Dr. Scott Larsen of the Vahlteich Medicinal Chemistry Core at the College of Pharmacy. Article in press - Advanced Science.
Interestingly, these biocrystals have also been documented in humans following autopsies and toxicological case studies of CFZ-treated patients and while the drug is FDA-approved for clinical use, there have been no scientific studies examining the toxicological and immunological properties of the biocrystals, or any other biologically-derived insoluble drug complexes. While the formation of insoluble drug precipitates or crystals inside cells could be readily dismissed as an unwanted side effect, the work conducted by the Rosania Research Group proves that biocrystal formation is intrinsically linked to the drug’s macrophage targeting mechanism, as well as its downstream pharmacological and immunological effects. To monitor the macrophage-targeted accumulation effects of the drug, Dr. Keswani led another study to report the far-red shift in the fluorescence of the drug contained within these biocrystals that can be tracked with >99% precision with a cytometry and microscopy imaging approach. “By identifying the specific fluorescence of drug aggregates and crystals within cells, we see this as potentially driving research toward unchartered territory in therapeutic models,” continues Dr. Keswani. “Endogenous signals from drugs within cells can be tracked, thereby also tracking the cell itself. There is tremendous potential for harnessing this ability in the personalized medicine approaches and imaging modalities.”.Article in press, Cytometry A.
Finally, in a fourth paper, Dr. Gi Sang Yoon, Research Fellow in the Rosania Research Group, demonstrated the unique immunomodulatory effects of the drug biocrystals on toll-like receptor (TLR)-dependent down regulation of the inflammatory cytokine, tumor necrosis factor (TNF)-a and the up regulation of the anti-inflammatory cytokine, interleukin (IL)-1 receptor antagonist (RA). These findings implicate the existence of anti-inflammatory signaling pathways stemming from the formation and intracellular sequestration of the biocrystals in macrophages. Perhaps, most significantly, biocrystals undergo active phagocytosis without causing cytotoxicity and can effectively modulate cell surface TLR expression and function. In this context, the formulation of CFZ or other drugs as insoluble, biocrystal-like complexes may be readily justifiable as a novel, macrophage-targeted therapeutic approach for treating inflammatory diseases. To view the full article, see the July issue of Molecular Pharmaceutics.
“Through our work, we have established the ability of biocrystals to alter innate immune signaling in macrophages following phagocytosis in vitro,” said Dr. Yoon. “Therefore, we decided to compare the in vitro biocrystal-induced changes in TLR-dependent pro- and anti-inflammatory signaling pathways, in relation to those of freely soluble CFZ. Considering both the amount of CFZ added to the cells as well as the cell-associated CFZ content allowed equitable comparison of intracellular biocrystals with those of freely soluble CFZ. Following phagocytosis of biocrystals, we analyzed the associated changes in cell viability, mitochondrial integrity, and inflammatory signaling pathways downstream of TLRs which play a critical role in inflammation, innate immune response as well as in the initiation of adaptive immune responses.”
Dr. Kathleen Stringer, a Professor of Clinical and Translational Pharmacy and a member of the Rosania Research Group, added, “These drug biocrystals can function as intracellular drug depots, which could be exploited to influence macrophage-dependent immune signaling functions. This mechanism may explain, at least in part, why CFZ has been found to have clinical anti-inflammatory activity and has been used for the treatment of a variety of inflammatory disorders.”
The group is now focused on performing computational and systems biology analyses, and formulation chemistry studies coupled with the use of anti-inflammatory animal models to further the development of targeted crystalline therapeutics.