The brain cortical uptake of the HIRMAbCSGSH fusion protein is 0

The brain cortical uptake of the HIRMAbCSGSH fusion protein is 0.81 0.07% ID/100 g brain (Table 3). SGSH Western blot, the primary antibody was a rabbit antihuman SGSH antibody (Abcam, Cambridge, MA). The secondary antibody was a biotinylated horse antigoat IgG or biotinylated goat antirabbit IgG antibody (Vector Laboratories). The purity of the HIRMAbCSGSH fusion protein was verified by reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as previously described.9 The molecular weight (MW) standards were obtained from Thermo Fisher Scientific, Inc. (Rockford, IL), and Biorad Laboratories, Inc. (Hercules, CA). Samples tested in the Western blotting include the protein A purified HIRMAbCSGSH fusion protein, the protein A purified HIRMAb, and a recombinant fusion protein of amino terminal glutathione + = 4). * 0.01 difference from control. The HIRMAbCSGSH fusion CTMP protein was radiolabeled with the [125I]-BoltonCHunter reagent to a specific activity of 3.7 Ci/g and a TCA precipitation of 97%. The [125I]-HIRMAbCSGSH fusion protein (1200 Ci, 324 g) was injected IV in a male Rhesus monkey. The time course NPI-2358 (Plinabulin) of TCA precipitable [125I]-HIRMAbCSGSH fusion protein is usually shown in Physique ?Physique8.8. The percent of total plasma radioactivity that was precipitable by TCA was 96 1%, 95 1%, 94 1%, 89 1%, 84 2%, 79 1%, and 72 2%, respectively, at 2, 5, 15, 30, 60, 90, and 140 min after IV injection. A 2-exponential equation was fit to the plasma profile NPI-2358 (Plinabulin) of TCA-precipitable fusion protein (Experimental Section) to yield the pharmacokinetic (PK) parameters shown in Table 1. The [125I]-HIRMAbCSGSH fusion protein is rapidly cleared from plasma with a mean residence time of 62 4 min, a systemic volume of distribution (Vss) that is 2.5-fold greater the central compartment volume (Vc), and a high rate of systemic clearance, 1.11 0.03 mL/min/kg (Table 1). Open in a separate window Physique 8 Plasma TCA-precipitable [125I]-HIRMAbCSGSH fusion protein concentration, ng/mL, in the adult Rhesus monkey, is usually plotted vs time over a 140 min period after a single IV injection of 19 g/kg the fusion protein. Table 1 Pharmacokinetic Parameters of the HIRMAbCSGSH Fusion Proteina thead th style=”border:none;” align=”center” rowspan=”1″ colspan=”1″ parameter /th th style=”border:none;” align=”center” rowspan=”1″ colspan=”1″ models /th th style=”border:none;” align=”center” rowspan=”1″ colspan=”1″ value /th /thead em NPI-2358 (Plinabulin) T /em 1/21min5.5??0.8 em T /em 1/22min55??4MRTmin62??4VcmL/kg28??2VssmL/kg69??4AUCssgmin/mL16.8??0.5CLmL/min/kg1.11??0.03 Open in a separate window aParameters computed from the plasma profile in Figure ?Physique8.8. em T /em 1/21 and em T /em 1/22 are the half-times of plasma clearance for the first phase () and second phase () phases. The volume of distribution (VD) of the HIRMAbCSGSH fusion protein in total brain homogenate at 140 min after injection is usually high, 782 36 L/g, compared to the brain VD of a nonspecific human IgG1 NPI-2358 (Plinabulin) isotype control antibody, 20 6 L/g (Table 2). The brain VD of the IgG1 isotype control antibody represents the brain uptake of a molecule that is sequestered within the blood volume of brain, and which does not cross the BBB, as described previously.9 The VD of the HIRMAbCSGSH fusion protein in the postvascular supernatant, 666 71 L/g, is greater than the VD of the HIRMAbCSGSH fusion protein in the vascular pellet of brain, 24 17 L/g (Table 2), which indicates that NPI-2358 (Plinabulin) the majority of the HIRMAbCSGSH fusion protein has traversed the BBB and penetrated the brain parenchyma. The radioactivity in the postvascular supernatant represents intact HIRMAbCSGSH fusion.