Distribution of chemically modified rhSulfamidase to CNS monitored by brain microdialysis and repeated CSF sampling after intravenous administration in rat

Abstract

Mucopolysaccharidosis type IIIA (MPS IIIA) is an autosomal recessive disorder caused by absence or deficiency of the lysosomal enzyme sulfamidase. Glycans of recombinant human sulfamidase were chemically modified to generate CM-rhSulfamidase with retained stability and catalytic activity, but with significantly reduced uptake in peripheral tissues. For the enzyme to modify the neuropathology of MPS IIIA disease, it has to reach the lysosomes of cells in the brain, hence pass the blood brain barrier (BBB). To monitor BBB passage of CM-rhSulfamidase in a time dependent manner, concentration in brain interstitial fluid was explored using push-pull microdialysis combined with CSF and serum collection. Push-pull microdialysis is a sampling technique facilitating continuous measurement of larger molecules (e.g. proteins), here applied in awake, freely-moving Sprague Dawley rats. Having established membrane recovery, surgery was performed on rats to implant guide cannulas for the microdialysis probes using a stereotaxic frame to pin-point exact location in the prefrontal cortex, and cannulas for repeated CSF and serum sampling in the cisterna magna and jugular vein, respectively. After the animals fully recovered, CM-rhSulfamidase was dosed intravenously and brain interstitial fluid was sampled by microdialysis, CSF and serum via their respective cannulas. Concentrations of CM-rhSulfamidase were determined by an immunoassay and data were subjected to quantitative pharmacokinetic analysis. While maximum serum concentration was observed shortly after administration, a delay was observed of CM-rhSulfamidase entering CSF and brain interstitial fluid. Maximum CSF and brain interstitial fluid concentrations differed somewhat between rats, but after reaching the maximum level reduction in concentration occurred at a similar rate between individual rats. Mean CM-rhSulfamidase concentrations versus time relationships were similar in brain interstitial fluid and CSF. Brain exposure was confirmed by determination of brain homogenate concentrations. In conclusion, CM-rhSulfamidase bio-distribution to CNS was quantified using a combined push-pull brain microdialysis, serum and CSF approach.