ALBERT

Description: Asparaginase (ASNase) is one of the cornerstones of the multi-drug treatment protocol that is used to treat acute lymphoblastic leukemia (ALL) in pediatric and adult patients. Despite the fact that ASNase has been used in ALL treatment protocols for decades, little is known about the biodistribution and the mechanism of ASNase turnover in vivo. A large inter-individual variation in ASNase pharmacokinetics is observed in patients. While elevated ASNase levels are associated with an increase in adverse events, underexposure, frequently caused by antibody mediated clearance, seriously reduces therapeutic efficacy. To date, it is not possible to predict pharmacokinetics of ASNase in individual patients and therefore current therapeutic protocols are supported by frequent monitoring of ASNase levels and adjustments of administration schemes. We used an in vivo imaging approach to study ASNase biodistribution and pharmacodynamics in a mouse model and provide in vitro and in vivo evidence that identifies the endo-lysosomal protease Cathepsin B in macrophages as a critical component of ASNase degradation. Results/Discussion Mice were injected with 111Indium-labeled ASNase and biodistribution was monitored by quantitative microSPECT/CT scans and ex vivo analysis of organs using a gamma counter. Over time, ASNase accumulated in the liver and particularly the spleen and the bone marrow. We hypothesized that macrophages in these organs, efficiently take up the ASNase, thereby rapidly clearing the active enzyme from the blood. Immunohistochemical analysis confirmed the presence of ASNase in cells positive for the murine macrophage marker F4/80. To confirm the importance of macrophage populations in ASNase clearance, we depleted mice from phagocytic cells by injection of clodronate liposomes, and studied ASNase biodistribution and kinetics. Indeed, clodronate pretreatment significantly diminished the accumulation of ASNase in the liver, spleen and the bone marrow while doubling the circulatory half-life of serum ASNase activity. We conclude from these experiments that macrophages determine the pharmacokinetics of asparaginase, which raises the question whether rapid clearance of the drug by bone marrow resident macrophages will negatively affect the depletion of asparagine in the bone marrow niche. We previously linked a germline mutation in the gene encoding endosomal protease Cathepsin B to strongly diminished asparaginase degradation in a pediatric ALL patient. To connect the macrophage mediated clearance to the proposed role of Cathepsin B in ASNase degradation, we studied the contribution of this protease in macrophage-mediated degradation of asparaginase. We used cell lines to show that Cathepsin B expression is induced during differentiation from monocytes towards macrophages. This is consistent with our finding that macrophages, but not monocytes, are capable of degrading ASNase. Furthermore, we used both chemical inhibition and RNAi mediated knockdown of Cathepsin B to show that this protease is required for ASNase degradation in these macrophages. Finally, by comparing Cathepsin B knockout mice with wildtype littermates, we demonstrated that loss of Cathepsin B activity significantly delayed clearance of serum asparaginase, consistent with a prominent role for this lysosomal protease in ASNase turnover. In conclusion, by using in vivo imaging we showed that asparaginase is efficiently cleared from the circulation by macrophages. In particular, bone marrow resident macrophages may provide a protective environment for leukemic cells by effectively removing the therapeutic protein from the bone marrow niche. However, both the prominent role of macrophages and the importance of the lysosomal protease Cathepsin B in asparaginase clearance, may allow the rational design of a next generation asparaginase. Disclosures Metselaar: Enceladus Pharmaceuticals: Employment, Equity Ownership.

Description: Various pathologic conditions, such as hemorrhage, hemolysis and cell injury, are characterized by the release of large amounts of heme. Recently, it was demonstrated that heme oxygenase (HO), the heme-degrading enzyme, and heme are able to modulate adhesion molecule expression in vitro. In the present study, the effects of heme and HO on inflammation in mice were analyzed by monitoring the biodistribution of radiolabeled liposomes and leukocytes in conjunction with immunohistochemistry. Small liposomes accumulate in inflamed tissues by diffusion because of locally enhanced vascular permeability, whereas leukocytes actively migrate into inflammatory areas through specific adhesive interactions with the endothelium and chemotaxis. Exposure to heme resulted in a dramatic increase in liposome accumulation in the pancreas, but also intestines, liver, and spleen exhibited significantly increased vascular permeability. Similarly, intravenously administered heme caused an enhanced influx of radiolabeled leukocytes into these organs. Immunohistochemical analysis showed differential up-regulation of the adhesion molecules ICAM-1, P-selectin, and fibronectin in liver and pancreas in heme-treated animals. Heme-induced adhesive properties were accompanied by a massive influx of granulocytes into these inflamed tissues, suggesting an important contribution to the pathogenesis of inflammatory processes. Moreover, inhibition of HO activity exacerbated heme-induced granulocyte infiltration. Here it is demonstrated for the first time that heme induces increased vascular permeability, adhesion molecule expression, and leukocyte recruitment in vivo, whereas HO antagonizes heme-induced inflammation possibly through the down-modulation of adhesion molecules.

Description: Asparaginase (ASNase) is one of the cornerstones of the multi-drug treatment protocol that is used to treat acute lymphoblastic leukemia (ALL) in pediatric and adult patients. Recent studies monitoring ASNase kinetics in patients provide evidence of a large inter-patient variability of serum ASNase concentrations and call attention to the negative effects of ASNase underexposure on treatment response and relapse risk. Despite the fact that ASNase has been used in ALL treatment protocols for decades, little is known about the biodistribution and the mechanism of ASNase turnover in patients. We used in vivo imaging to study the distribution and pharmacodynamics of ASNase in a mouse model. We injected mice with 3,000 International Units (I.U.)/kg ASNase, which was labeled with 20-25 MBq Indium-111 (In-111) and acquired micro-SPECT/CT images up 18 hours post injection. At this timepoint, serum ASNase activity has dropped to levels close to the detection limits. In addition to the expected uptake in the liver, SPECT/CT imaging revealed a rapid, strong and specific accumulation of radiolabeled ASNase in the bone marrow and spleen (Figure 1). Accumulation in these organs was confirmed by quantitative measurement of radiolabeled ASNase in the dissected organs (Figure 2). We hypothesized that macrophages which are present in high numbers in these organs, efficiently phagocytose the ASNase, thereby rapidly clearing the active enzyme from the blood. Autoradiography of spleen sections indeed showed high uptake of radiolabeled ASNase in the macrophage-rich red pulp of the spleen. Immunohistochemical stainings confirmed the presence of ASNase in cells positive for the murine macrophage marker F4/80. To provide additional evidence for the potential role of macrophages in the turnover of ASNase, we pretreated mice with a single injection of clodronate liposomes, which almost completely depletes the relevant organs from phagocytic cells. This pretreatment diminished the accumulation of ASNase in the liver, spleen and the bone marrow (Figure 2). Consistent with this notion, we found that clodronate pretreatment more than doubles the circulatory half-life of serum ASNase activity. We conclude from these experiments that ASNase is rapidly cleared from the serum by phagocytic cells. In particular, the efficient uptake of ASNase by spleen and bone marrow resident macrophages may protect leukemic cells from the nutrient depriving action of this drug and could thereby compromise therapeutic efficacy. Figure 1: SPECT/CT image of Asparaginase uptake Figure 1:. SPECT/CT image of Asparaginase uptake Lateral (A) and ventral (B) 3-dimensional volume projections of fused SPECT/CT scans of mice injected with 111Indium-labeled asparaginase (pseudocolor images with red being least intense and yellow most intense), 18 hours post injection. Numbers indicate relevant organs: 1 sternum, 2 liver, 3 spleen, 4 spine, 5, pelvis, 6 femur, 7 tibia. Figure 2: Biodistribution of Asparaginase in control and clodronate pretreated mice. Figure 2:. Biodistribution of Asparaginase in control and clodronate pretreated mice. Asparaginase uptake is depicted as percentage of the injected dose per gram of tissue (%ID/g) at 19 hours after injection in control (empty liposomes) and clodronate pretreated mice. Results are mean + standard deviation (n=5 for each group). 2-tailed t-test was used to test for significance: * p