Imaging of enzyme replacement therapy using PET

Abstract

Direct enzyme replacement therapy (ERT) has been introduced as a means to treat a number of rare, complex genetic conditions associated with lysosomal dysfunction. Gaucher disease was the first for which this therapy was applied and remains the prototypical example. Although ERT using recombinant lysosomal enzymes has been shown to be effective in altering the clinical course of Gaucher disease, Fabry disease, Hurler syndrome, Hunter syndrome, Maroteaux-Lamy syndrome, and Pompe disease, the recalcitrance of certain disease manifestations underscores important unanswered questions related to dosing regimes, tissue half-life of the recombinant enzyme and the ability of intravenously administered enzyme to reach critical sites of known disease pathology. We have developed an innovative method for tagging acid beta-glucocerebrosidase (GCase), the recombinant enzyme formulated in Cerezyme(R) used to treat Gaucher disease, using an (18)F-labeled substrate analogue that becomes trapped within the active site of the enzyme. Using micro-PET we show that the tissue distribution of injected enzyme can be imaged in a murine model and that the PET data correlate with tissue (18)F counts. Further we show that PET imaging readily monitors pharmacokinetic changes effected by receptor blocking. The ability to (18)F-label GCase to monitor the enzyme distribution and tissue half-life in vivo by PET provides a powerful research tool with an immediate clinical application to Gaucher disease and a clear path for application to other ERTs.

Fig. 1.

The biochemical reaction catalyzed by GCase and the mechanism of catalysis A) Reaction catalyzed by GCase during glycosphingolipid metabolism B) Double displacement mechanism used by GCase when X = OH and OR = ceramide. When the 2-OH is replaced by F and the Ceramide replaced by 2, 4-dinitrophenol, these substrate analogues form a long-lived covalent glycosyl-enzyme intermediate.

Fig. 2.

Macrophage uptake of intravenously administered recombinant GCase (structure used in this figure taken from Dvir et al. (21)). The recombinant GCase in Cerezyme, produced from Chinese hamster ovary cells, is modified to ensure that N-linked glycans contain terminal mannose residues thus conferring high affinity for the mannose receptor (MR) resident on the macrophage and other cell types. Internalization of the enzyme occurs through endocytosis of the MR receptor/GCase complex. Early endosomes containing GCase mature into late endosomes while receptor sorting leads to the recycling of the MR receptor back to the cellular membrane. Late endosomal and lysosomal fusion delivers the therapeutic enzyme to the lysosome which subsequently restores GlcCer to normal levels.

Fig. 3.

Radiochemical synthesis of the glycosidase labeling agent β-DNP-[¹⁸F]-FDG and active-site labeling of GCase.

Fig. 4.

Uptake of ¹⁸F-Glc-GCase in various organs. A) Biodistribution of ¹⁸F-Glc-GCase in various tissues at 2 h postinjection in mice. Mice were killed and tissue radioactivity was measured by γ counting (n = 4 for all organs except the gall bladder where n = 2) Bars represent the average tissue uptake plus/minus standard deviation (expressed as % injected dose/g of tissue). B) Comparison of the biodistribution of ¹⁸F-Glc-GCase as determined by PET (blue bars expressed % injected dose/mL) and by postmortem tissue γ counting (purple bars expressed % injected dose/g) 2 h postinjection. C) Liver uptake of ¹⁸F-Glc-GCase determined by PET in an untreated (green triangles) and a mannan-treated mouse (blue diamonds). The red square represents the postmortem tissue activity counts of the mannan-treated mouse 2.5 h postinjection D) Bar graphs comparing the rate of liver uptake for untreated mice vs mice receiving yeast mannan (15 mg/kg) prior to injection of ¹⁸F-Glc-GCase (n = 2 in each instance). The rates of uptake were determined by calculating the slope of the linear portion of the liver uptake curves over the first 15 min postinjection.

Fig. 5.

PET images obtained by injection of ¹⁸F-Glc-GCase into mice. Images are as follows: A) Coronal slice highlighting the accumulated uptake of tracer in spleen, kidneys, and liver over 2 h acquisition time B) Sagittal slice of the image shown in A) but highlighting the liver, bladder, and spine C) For comparison purposes the accumulated tracer uptake into organs of an untreated mouse summed over the first 20 min postinjection D) Accumulated tracer uptake into the organs of a mouse treated with mannan summed over the first 20 min postinjection