Bivalent engagement of endothelial surface antigens is critical to prolonged surface targeting and protein delivery in vivo.

Targeted drug delivery to the endothelium has the potential to generate localized therapeutic effects at the blood-tissue interface. For some therapeutic cargoes, it is essential to maintain contact with the bloodstream to exert protective effects. The pharmacokinetics (PK) of endothelial surface-targeted affinity ligands and biotherapeutic cargo remain a largely unexplored area, despite obvious translational implications for this strategy. To bridge this gap, we site-specifically radiolabeled mono- (scFv) and bivalent (mAb) affinity ligands specific for the endothelial cell adhesion molecules, PECAM-1 (CD31) and ICAM-1 (CD54). Radiotracing revealed similar lung biodistribution at 30 minutes post-injection (79.3% ± 4.2% vs 80.4% ± 10.6% ID/g for αICAM and 58.9% ± 3.6% ID/g vs. 47.7% ± 5.8% ID/g for αPECAM mAb vs. scFv), but marked differences in organ residence time, with antibodies demonstrating an order of magnitude greater area under the lung concentration vs. time curve (AUCinf 1698 ± 352 vs. 53.3 ± 7.9 ID/g*hrs for αICAM and 1023 ± 507 vs. 114 ± 37 ID/g*hrs for αPECAM mAb vs scFv). A physiologically based pharmacokinetic model, fit to and validated using these data, indicated contributions from both superior binding characteristics and prolonged circulation time supporting multiple binding-detachment cycles. We tested the ability of each affinity ligand to deliver a prototypical surface cargo, thrombomodulin (TM), using one-to-one protein conjugates. Bivalent mAb-TM was superior to monovalent scFv-TM in both pulmonary targeting and lung residence time (AUCinf 141 ± 3.2 vs 12.4 ± 4.2 ID/g*hrs for ICAM and 188 ± 90 vs 34.7 ± 19.9 ID/g*hrs for PECAM), despite having similar blood PK, indicating that binding strength is more important parameter than the kinetics of binding. To maximize bivalent target engagement, we synthesized an oriented, end-to-end anti-ICAM mAb-TM conjugate and found that this therapeutic had the best lung residence time (AUCinf 253 ± 18 ID/g*hrs) of all TM modalities. These observations have implications not only for the delivery of TM, but also potentially all therapeutics targeted to the endothelial surface.

Established antimicrobial susceptibility testing methods with a new twist--points to consider and a glimpse of the future.

The developments seen in these systems allow speculation about future trends in antimicrobial susceptibility testing methods. Microbiology system manufacturers seem to be heeding the call of all industry, for greater automation, enhanced data management capabilities and increased flexibility (see Table 2 below). [table: see text] Cost seems to be less of an issue. This may be due to the decrease in the availability of medical technologists and the need to find systems with better throughput and increased productivity. Increased automation, data management capabilities, and walkaway technology may justify the additional cost of some of these systems. The computer software package provided with these systems is becoming increasingly important with the focus on quality assurance and utilization. Computer generated data analysis gives the microbiologist the tools to educate physicians through the use of selective reporting functions, antibiograms, cost analysis and drug effectiveness comparisons. Each of the four systems is unique and will probably find a niche among the various markets that exist in the United States, European and other specialized markets. The lack of automation in the ALAMAR system may be its selling point in those areas where automation is not affordable, but new ways are being sought for ease of interpretation of results. BIOMIC and CATHRA systems may be more beneficial to those microbiologists who do not want to stop doing traditional Bauer-Kirby or agar dilution methods, but require computer enhancements. ALADIN, may fill a niche to which other walkaway systems have not adapted, but because of its expense, will face more demands than the other three systems covered in this review.