Thomas J. Abbruscato

Protein expression of brain endothelial cell E-cadherin after hypoxia/aglycemia: influence of astrocyte contact

The blood-brain barrier (BBB) plays a crucial role in protecting the central nervous system (CNS) from any changes in homeostasis brought about by pathological conditions. Cerebrovascular permeability is an important factor in the development of cerebral edema following stroke [M. Plateel, E. Teissier, R. Cecchelli, Hypoxia, dramatically increases the nonspecific transport of blood-borne proteins to the brain. J. Neurochem. 68 (1997) 874-877] and any changes in its function can have detrimental neurological consequences. Recently, research has shown that an in vitro model of the BBB is sensitive to short exposures of hypoxia/aglycemia and that changes in endothelial cell calcium flux may be responsible for structural and functional variations in the BBB during ischemic stress [T.J. Abbruscato, T.P. Davis, Combination of hypoxia/aglycemia compromises in vitro BBB. J. Pharmacol. Exp. Ther. 289 (1999) 668-675]. Present experiments investigated bovine brain microvessel endothelial cell (BBMEC) expression of a Ca(2+)-dependent cell-cell adhesion molecule, E-cadherin, which has been shown to be important for blood-brain barrier function [D. Pal, K.L. Audus, T.J. Siahaan, Modulation of cellular adhesion in bovine brain microvessel endothelial cells by a decapeptide. Brain Research 747 (1997) 103-113]. Since it is believed that astrocyte-endothelial cell interaction is crucial for maintenance of in vivo BBB characteristics, we have attempted to optimize our isolation and culturing techniques to produce a reliable, in vitro model of the BBB that is suitable to study pathological conditions. Immunofluoresence experiments showed positive staining for E-cadherin, yet failed to show any change in cellular distribution of E-cadherin upon hypoxic/aglycemic exposure. In addition, culturing BBMECs with C6 conditioned medium (CM) had no effect on the localization of E-cadherin. Western blotting experiments showed that BBMECs express E-cadherin and this protein is decreased in a time dependent manner after various hypoxic/aglycemic exposures when endothelial cells are cultured alone or with C6 astrogliomas grown on a separate culture surface. When C6 astrocytes are grown directly opposed to endothelial cells, with a porous membrane between, we observed a slight attenuation in the decreased BBMEC expression of E-Cadherin after hypoxia/aglycemia exposure. This work has shown that the mammalian brain endothelial/astrocyte co-culture system is a useful model for studies of pathological conditions where BBB characteristics are maintained.

The effect of halogenation on blood–brain barrier permeability of a novel peptide drug☆

The utility of a drug depends on its ability to reach appropriate receptors at the target tissue and remain metabolically stable to produce the desired effect. To improve central nervous system entry of the opioid analgesic [D-Pen2, L-Pen5, Phe6] Enkephalin (DPLPE-Phe), our research group synthesized analogs that had chloro, bromo, fluoro, and iodo halogens on the para positions of the phenylalanine-4 residue. This study reports on investigation of the effect of halogenation on stability, lipophilicity, and in vitro blood-brain barrier permeability of a novel enkephalin analog DPLPE-Phe. The stability of each halogenated DPLPE-Phe analog as well as the amidated and nonamidated parent peptide was tested in plasma and brain. All peptides tested had a half-time disappearance >300 min except for DPLPE-Phe-NH2, which was found to have a half-life of 30 min in plasma. Octanol/saline distribution studies indicated addition of halogens to DPLPE-Phe-OH significantly increased lipophilicity except for p-[F-Phe4]DPLPE-Phe-OH. p-[Cl-Phe4]DPLPE-Phe-OH exhibited the most pronounced increase in lipophilicity. Para-bromo and para-chloro halogen additions significantly enhanced in vitro blood-brain barrier permeability, providing evidence for improved delivery to the central nervous system.

Bioavailability of Ziconotide in brain: influx from blood, stability, and diffusion.

Ziconotide is a selective peptide antagonist of the N-type calcium channel currently in clinical trials for analgesia. Ziconotide reached a maximal brain concentration of between 0.003 and 0.006% of the injected material per gram of tissue at 3-20 min after i.v. injection, and this decayed to below 0.001%/g after 2 h. The structurally distinct conopeptide SNX-185 (synthetic TVIA) was considerably more persistent in brain after i.v. administration, with 0.0035% of the injected material present at 2-4 h after i.v. injection, and 0.0015% present at 24 h. Similar results (i.e. greater persistence of SNX-185) were obtained when the peptides were perfused through in vivo dialysis probes implanted into the hippocampus. Image analysis and serial sectioning showed that diffusion of Ziconotide in the extracellular fluid around the dialysis probe was minimal, with the peptide located within 1 mm of the probe after 2 h. In vitro diffusion through cultured bovine brain microvessel endothelial cells (BBMEC) verified that a close structural analog of Ziconotide (SNX-194) passed through this blood-brain barrier (BBB) model as expected for peptides of similar physical properties (permeability coefficient of 6.5 x 10(-4) cm/g). Passage from blood to brain was also verified by in situ perfusion through the carotid artery. A statistically greater amount of radioactivity was found to cross the BBB after perfusion of radioiodinated Ziconotide compared to [14C]inulin. Capillary depletion experiments and HPLC analysis defined the brain location and stability.

The Effect of Glycosylation on the Uptake of an Enkephalin Analogue into the Central Nervous System

In contrast to unglycosylated controls, glycosylated [D-Cys2,5]enkephalin-ser-gly (glycosylated DCDCE-ser-gly) elicits analgesia after intraperitoneal administration. This was postulated to be due to the presence of the glucose moiety allowing the analogue to cross the BBB via the glucose carrier. To test this hypothesis, the present study investigated the biological stability and the CNS uptake ofunglycosylated and glycosylated DCDCE-ser-gly. Interestingly, the metabolic half-lives and ability to cross the in vitro BBB was found to be similar for both analogues. In situ brain perfusion indicated that the brain uptake of glycosylated DCDCE-ser-gly was greater than that for the vascular marker, [14C]sucrose, but similar to the CSF uptake of the peptide. CNS uptake of glycosylated DCDCE-ser-gly was not affected by replacing D- with L- glucose, nor with the addition of 10 µM unlabelled glycosylated DCDCE-ser-gly. In summary, the difference in analgesic response of glycosylated compared to unglycosylated DCDCE-ser-gly, is not related to either differing metabolic profiles, nor the ability of the glycosylated analogue to use the glucose carrier to enter the CNS. However, this study does not eliminate the involvement of a different low affinity, saturable uptake system taking the glycosylated, but not the unglycosylated form.