In Vivo Imaging of Antileukemic Drug Asparaginase Reveals a Rapid Macrophage-Mediated Clearance from the Bone Marrow

Abstract

The antileukemic drug asparaginase, a key component in the treatment of acute lymphoblastic leukemia, acts by depleting asparagine from the blood. However, little is known about its pharmacokinetics, and mechanisms of therapy resistance are poorly understood. Here, we explored the in vivo biodistribution of radiolabeled asparaginase, using a combination of imaging and biochemical techniques, and provide evidence for tissue-specific clearance mechanisms, which could reduce the effectiveness of the drug at these specific sites. Methods: In vivo localization of 111 In-labeled Escherichia coli asparaginase was performed in C57BL/6 mice by both small-animal SPECT/CT and ex vivo biodistribution studies. Mice were treated with liposomal clodronate to investigate the effect of macrophage depletion on tracer localization and drug clearance in vivo. Moreover, macrophage cell line models RAW264.7 and THP-1, as well as knockout mice, were used to identify the cellular and molecular components controlling asparaginase pharmacokinetics. Results: In vivo imaging and biodistribution studies showed a rapid accumulation of asparaginase in macrophage-rich tissues such as the liver, spleen, and in particular bone marrow. Clodronate-mediated depletion of phagocytic cells markedly prolonged the serum half-life of asparaginase in vivo and decreased drug uptake in these macrophage-rich organs. Immunohistochemistry and in vitro binding assays confirmed the involvement of macrophagelike cells in the uptake of asparaginase. We identified the activity of the lysosomal protease cathepsin B in macrophages as a rate-limiting factor in degrading asparaginase both in vitro and in vivo. Conclusion: We showed that asparaginase is rapidly cleared from the serum by liver-, spleen-, and bone marrow-resident phagocytic cells. As a consequence of this efficient uptake and protease-mediated degradation, particularly bone marrow-resident macrophages may provide a protective niche to leukemic cells.

Hypersensitivity reactions to asparaginase in mice are mediated by anti-asparaginase IgE and IgG and the immunoglobulin receptors FcεRI and FcγRIII

“…Therefore, as expected, staining peripheral blood cells from naïve mice with fluorochrome-labeled ASNase (Figure 1A) revealed no ASNase positive neutrophils, T cells, basophils, or B cells. Yet, consistent with the perceived mechanism of ASNase clearance involving macrophages of the reticuloendothelial system (RES), 25 a small percentage of macrophages/monocytes were ASNase positive (Figure 1A). Mice were given 100 mg of fluorochrome-labeled ASNase IV to verify its uptake by macrophages/monocytes.…”

Asparaginase-Phage P22 Nanoreactors: Toward a Biobetter Development for Acute Lymphoblastic Leukemia Treatment

“…In patients with ALL, ASNase is often administrated by intravenous infusion, where it is exposed to proteases that are necessary for hemostasis, fibrinolysis, and tissue repair [ 54 ]. ASNase is mostly inactivated by proteases such as cathepsin B and asparagine endopeptidase, expressed by leukemia cells and macrophages [ 55 , 56 , 57 , 58 ]. Therefore, the stability of non-PEGylated and PEGylated ASNase-P22 nanoreactors were evaluated in the presence of HBS at 37 °C and different times of incubation ( Figure 7 ).…”

Low Bioavailability and High Immunogenicity of a New Brand of E. coli l-Asparaginase with Active Host Contaminating Proteins

“…It reaches plasma peak levels within 2 h after infusion, with accumulation in liver, spleen and bone marrow. Over time, l -asparaginase levels decrease in the circulation, remain constant in bone marrow and increase in spleen and liver – the residence of most phagocytic cells of the reticuloendothelial system ( van der Meer et al, 2017 ). l -Asparaginase degradation occurs possibly by means of plasmatic and intracellular proteases, such as lysosomal cysteine proteases B cathepsin and asparaginyl endopeptidase, which are expressed in macrophages ( Patel et al, 2009 ; van der Meer et al, 2017 ).…”

“…Over time, l -asparaginase levels decrease in the circulation, remain constant in bone marrow and increase in spleen and liver – the residence of most phagocytic cells of the reticuloendothelial system ( van der Meer et al, 2017 ). l -Asparaginase degradation occurs possibly by means of plasmatic and intracellular proteases, such as lysosomal cysteine proteases B cathepsin and asparaginyl endopeptidase, which are expressed in macrophages ( Patel et al, 2009 ; van der Meer et al, 2017 ). Macrophage depletion using clodronate prolongs the l -asparaginase half-life significantly, revealing the central role of these cells in the pharmacokinetics of this drug ( van der Meer et al, 2017 ).…”

“…l -Asparaginase degradation occurs possibly by means of plasmatic and intracellular proteases, such as lysosomal cysteine proteases B cathepsin and asparaginyl endopeptidase, which are expressed in macrophages ( Patel et al, 2009 ; van der Meer et al, 2017 ). Macrophage depletion using clodronate prolongs the l -asparaginase half-life significantly, revealing the central role of these cells in the pharmacokinetics of this drug ( van der Meer et al, 2017 ). The adjuvant effect of impurities that is likely to underlie the higher anti-asparaginase antibody titres observed in animals injected with Leuginase® may also be operative in the activation of the innate immune cells of the reticuloendothelial system, therefore contributing to faster l -asparaginase elimination.…”