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The novel antioxidants U-78517F and U-74006F, or lazaroids, are highly lipophilic organic molecules with poor brain uptake. To understand this paradoxical behavior better, continuous monolayers of Madin-Darby canine kidney (MDCK) epithelial cells with distinct apical (AP) and basolateral (BL) plasma membrane domains grown on polycarbonate membrane filters and plastic were used to examine the mechanism of transcellular diffusion. Independent kinetic experiments were used to quantify AP to BL flux, efflux from the AP and BL membranes and AP membrane partitioning as functions of bovine serum albumin (BSA) concentration. Fluxes were appropriately reduced to permeability coefficients (Pe) for the membrane, aqueous boundary layer (ABL) and filter, BSA-drug binding constants, and effective (Ke) and intrinsic (Kintr) membrane partition coefficients in the absence of metabolism. Both Pe and Ke decreased exponentially with increased BSA concentration and a concomitant decrease in free drug concentration. Uptake was ABL-controlled under the conditions used and its Pe was 1,000-fold faster than that for efflux due to a large Kintr. Therefore, diffusion across the cellular barrier was limited kinetically by the equilibrium between protein-bound drug and free drug partitioned into the cell membrane and the rate-limiting desorption of drug from the cell membrane into the aqueous receiver. This suggests that brain uptake of these lipophilic antioxidants is limited by interactions with plasma proteins and, possibly, by unfavorable partitioning from the endothelium into the underlying tissue. The present biophysical kinetic model is proposed as generally useful in studying the penetrative ability of other membrane interacting molecules.

ObjectivesSurvival Motor Neuron (SMN) protein levels may become key pharmacodynamic (PD) markers in spinal muscular atrophy (SMA) clinical trials. SMN protein in peripheral blood mononuclear cells (PBMCs) can be quantified for trials using an enzyme-linked immunosorbent assay (ELISA). We developed protocols to collect, process, store and analyze these samples in a standardized manner for SMA clinical studies, and to understand the impact of age and intraindividual variability over time on PBMC SMN signal.MethodsSeveral variables affecting SMN protein signal were evaluated using an ELISA. Samples were from healthy adults, adult with respiratory infections, SMA patients, and adult SMA carriers.ResultsDelaying PBMCs processing by 45 min, 2 hr or 24 hr after collection or isolation allows sensitive detection of SMN levels and high cell viability (>90%). SMN levels from PBMCs isolated by EDTA tubes/Lymphoprep gradient are stable with processing delays and have greater signal compared to CPT-collected samples. SMN signal in healthy individuals varies up to 8x when collected at intervals up to 1 month. SMN signals from individuals with respiratory infections show 3–5x changes, driven largely by the CD14 fraction. SMN signal in PBMC frozen lysates are relatively stable for up to 6 months. Cross-sectional analysis of PBMCs from SMA patients and carriers suggest SMN protein levels decline with age.ConclusionsThe sources of SMN signal variability in PBMCs need to be considered in the design and of SMA clinical trials, and interpreted in light of recent medical history. Improved normalization to DNA or PBMC subcellular fractions may mitigate signal variability and should be explored in SMA patients.

Scanning laser microscopy (SLM) was used to develop an assay to visualize the generation of intra‐cellular reactive oxygen species (ROS) and to evaluate the effect of the lipophilic antioxidant U‐87,663 on ROS formation. Cultured N18 neuroglioma cells were challenged by extracellular addition of cumene hydroperoxide, and subsequent intracellular generation of ROS was characterized by measuring the fluorescence intensity of the ROS indicator 2′,7′‐dichlorofluorescein (DCF). The kinetics of the reaction between ROS and the indicator DCF, or the antioxidant U‐87,663, can be most accurately assessed if results from individual cell clusters are analyzed independently. It is possible and necessary to account for these experimental and analytical properties in order to characterize the properties of the antioxidant activity precisely. We determined that the temporal increase in DCF fluorescence was consistent with the reaction of DCF with free radicals generated from cumene hydroperoxide, as was the loss of fluorescence from U‐87,663. The rate constants for the free radical reactions revealed that ROS reaction with DCF is 10 times faster than with U‐87,663. These differences in reaction rates combined with differences in the cellular distribution of the ROS indicator DCF, the antioxidant U‐87,663, and the bulk of the ROS prevented detection of any protection U‐87,663 may offer. © 1996 Wiley‐Liss, Inc.

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