Exploiting transferrin receptor for delivering drugs across the blood-brain barrier.

Delivery of large molecule drugs across the blood brain barrier is increasingly being seen as an achievable goal. Several technologies have been described where following peripheral administration the molecules can be detected in the brain. Foremost amongst these technologies are antibodies against the transferrin receptor. Following a burst of publications in the very early twenty first century, excitement seemed to wane as contrary data started to emerge. Over the last few years antibodies against transferrin receptor have again started to raise hopes of successful drug delivery to the central nervous system, as protein engineering techniques have allowed a more detailed understanding of the antibody properties necessary for successful transport across the blood brain barrier.

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier.

BACKGROUND: Modelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies. METHODS: Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting. RESULTS: Using a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS endothelial cell types can be obtained, and these can be passaged onto large numbers of cell culture inserts for in vitro permeability studies. The passaged brain and spinal cord endothelial cells are pure and express endothelial markers, tight junction proteins and intracellular transport machinery. Further, both models exhibit tight, functional barrier characteristics that are discriminating against large and small molecules in permeability assays and show functional expression of the pharmaceutically important P-gp efflux transporter. CONCLUSIONS: Our techniques allow the provision of high yields of robust sister cultures of endothelial cells that accurately model the blood-CNS barriers in vitro. These models are ideally suited for use in studying the biology of the blood-brain barrier and blood-spinal cord barrier in vitro and for pre-clinical drug discovery.

Isolation of blood-brain barrier-crossing antibodies from a phage display library by competitive elution and their ability to penetrate the central nervous system.

The blood-brain barrier (BBB) is a formidable obstacle for delivery of biologic therapeutics to central nervous system (CNS) targets. Whilst the BBB prevents passage of the vast majority of molecules, it also selectively transports a wide variety of molecules required to maintain brain homeostasis. Receptor-mediated transcytosis is one example of a macromolecule transport system that is employed by cells of the BBB to supply essential proteins to the brain and which can be utilized to deliver biologic payloads, such as antibodies, across the BBB. In this study, we performed phage display selections on the mouse brain endothelial cell line, bEND.3, to enrich for antibody single-chain variable fragments (scFvs) that could compete for binding with a known BBB-crossing antibody fragment, FC5. A number of these scFvs were converted to IgGs and characterized for their ability to bind to mouse, rat and human brain endothelial cells, and subsequent ability to transport across the BBB. We demonstrated that these newly identified BBB-targeting IgGs had increased brain exposure when delivered peripherally in mice and were also able to transport a biologically active molecule, interleukin-1 receptor antagonist (IL-1RA), into the CNS. The antagonism of the interleukin-1 system within the CNS can result in the relief of neuropathic pain. We demonstrated that the BBB-targeting IgGs were able to elicit an analgesic response in a mouse model of nerve ligation-induced hypersensitivity when fused to IL-1RA.

Increased F-18 FDG intestinal uptake in diabetic patients on metformin: a matched case-control analysis.

PURPOSE: A matched case-control study was performed to assess the relationship between metformin use and the degree of F-18 fluorodeoxyglucose (FDG) bowel activity in diabetic patients. MATERIALS AND METHODS: Seventy-seven diabetic patients referred to our department for a positron emission tomography/computed tomography study, including 45 on metformin, were compared with nondiabetic controls matched for sex, age, and body mass index. Positron emission tomography studies were obtained in a standard manner and reviewed in a blinded fashion. F-18 FDG uptake in the GI tract was evaluated quantitatively using maximal standardized uptake values and visually using a previously published semiquantitative scale. RESULTS: F-18 FDG uptake in small and large bowel was significantly increased in metformin patients compared with nondiabetic controls both visually and quantitatively (all P < 0.0001), as well as compared with nonmetformin patients with diabetes. Control sites (liver, fat, muscle) showed similar uptake. Multiple regression analysis confirmed that metformin was the variable most strongly associated with bowel uptake. CONCLUSION: Physiologic accumulation of F-18 FDG in bowel is increased in diabetic patients maintained on metformin.