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The indolo[2,1 -b]quinazoline alkaloid tryptanthrin was previously identified as a potent anti-inflammatory compound with a unique pharmacological profile. It is a potent inhibitor of cyclooxy-genase-2, 5-lipooxygenase-catalyzed leukotriene synthesis, and nitric oxide production catalyzed by the inducible nitric oxide synthase. To characterize the pharmacokinetic properties of tryptanthrin, we performed a pilot in vivo study in male Sprague-Dawley rats (2mg/kg bw i.v.). Moreover, the ability of tryptanthrin to cross the blood-brain barrier was evaluated in three in vitro human and animal blood-brain barrier models. Bioanalytical UPLC-MS/MS methods used were validated according to current international guidelines. A half-life of 40.63 ± 6.66 min and a clearance of 1.00 ± 0.36 L/h/kg were found in the in vivo pharmacokinetic study. In vitro data obtained with the two primary animal blood-brain barrier models showed a good correlation with an immortalized human monoculture blood-brain barrier model (hBMEC cell line), and were indicative of a high blood-brain barrier permeation potential of tryptanthrin. These findings were corroborated by the in silico prediction of blood-brain barrier penetration. P-glycoprotein interaction of tryptanthrin was assessed by calculation of the efflux ratio in bidirectional permeability assays. An efflux ratio below 2 indicated that tryptanthrin is not subjected to active efflux. [ABSTRACT FROM AUTHOR]

Desmoteplase, a recombinant form of the plasminogen activator DSPAα1 from Desmodus rotundus, may offer improved clinical benefits for acute ischemic stroke treatment over the current therapy, recombinant tissue plasminogen activator (rtPA). Accumulating evidence suggests that clinical use of rtPA could be limited by unfavorable properties, including its ability to cross the blood–brain barrier (BBB), thus potentially adding to the pro-excitotoxic effect of endogenous tPA in cerebral parenchyma. Here, to investigate whether desmoteplase may display a safer profile than the structurally-related tPA, both agents were compared for their ability to cross the BBB and promote neurotoxicity.

Purpose.Studies were conducted to evaluate whether the use of an in vitromodel of the blood-brain barrier (BBB) resulted in more accurate predictions of the in vivotransport of compounds compared to the use of a human intestinal cell line (Caco-2). Methods.The in vitroBBB model employs bovine brain capillary endothelial cells co-cultured with primary rat astrocytes. The Caco-2 cells originate from a human colorectal carcinoma. The rat was used as experimental animal for the in vivostudies. Results.Strong correlations (r = 0.93-0.95) were found between the results generated by the in vitromodel of the BBB and two different methodologies to measure the permeability across the BBB in vivo. In contrast, a poor correlation (r = 0.68) was obtained between Caco-2 cell data and in vivoBBB transport. A relatively poor correlation (r = 0.74) was also found between the two in vitromodels. Conclusion.The present study illustrates the limitations of the Caco-2 model to predict BBB permeability of compounds in vivo. The results emphasize the fact that the BBB and the intestinal mucosa are two fundamentally different biologic barriers, and to be able to make accurate predictions about the in vivoCNS penetration of potential drug candidates, it is important that the in vitromodel possesses the main characteristics of the in vivoBBB.

Purpose. To investigate the mechanism underlying the entry of the analgesic peptide dalargin into brain using biodegradable polybutylcyanoacrylate (PBCA) nanoparticles (NP) overcoated with polysorbate 80. Methods. The investigations were carried out with PBCA NP and with non biodegradable polystyrene (PS) NP (200 nm diameter). Dalargin adsorption was assessed by HPLC. Its entry into the CNS in mice was evaluated using the tail-flick procedure. Locomotor activity measurements were performed to compare NP toxicities. BBB permeabilization by PBCA NP was studied in vitrousing a coculture of bovine brain capillary endothelial cells and rat astrocytes. Results. Dalargin loading was 11.7 µg/mg on PBCA NP and 16.5µg/ mg on PS NP. Adding polysorbate 80 to NP led to a complete desorption. Nevertheless, dalargin associated with PBCA NP and polysorbate 80 induced a potent and prolonged analgesia, which could not be obtained using PS NP in place of PBCA NP. Locomotor activity dramatically decreased in mice dosed with PBCA NP, but not with PS NP. PBCA NP also caused occasional mortality. In vitro, PBCA NP (10 µg/ml) induced a permeabilization of the BBB model. Conclusions. A non specific permeabilization of the BBB, probably related to the toxicity of the carrier, may account for the CNS penetration of dalargin associated with PBCA NP and polysorbate 80.

Lactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation; it accumulates in the brain during neurodegenerative disorders. Before determining Lf function in brain tissue, we investigated its origin and demonstrate here that it crosses the blood-brain barrier. An in vitromodel of the blood-brain barrier was used to examine the mechanism of Lf transport to the brain. We report that differentiated bovine brain capillary endothelial cells exhibited specific high (Kd= 37.5 nm; n= 90,000/cell) and low (Kd= 2 μm;n= 900,000 sites/cell) affinity binding sites. Only the latter were present on nondifferentiated cells. The surface-bound Lf was internalized only by the differentiated cell population leading to the conclusion that Lf receptors were acquired during cell differentiation. A specific unidirectional transport then occurred via a receptor-mediated process with no apparent intraendothelial degradation. We further report that iron may cross the bovine brain capillary endothelial cells as a complex with Lf. Finally, we show that the low density lipoprotein receptor-related protein might be involved in this process because its specific antagonist, the receptor-associated protein, inhibits 70% of Lf transport.

Purpose. The objective of this work was to assess, in vitro, the passage of P-glycoprotein dependent drugs across brain capillary endothelial cells, when these drugs are associated with a reversing agent. Methods. An in vitromodel of the blood-brain barrier consisting of a coculture of brain capillary endothelial cells and astrocytes was used. Results. We demonstrate that P-glycoprotein expression is upregulated by the presence of astrocytes. Uptake in the cells and transport across endothelial cell monolayers of vincristine, cyclosporin A and doxorubicin were studied. Using S9788 or verapamil as reversing agents, we found an increase in vincristine transport across the endothelial cell monolayers. On the other hand, the association of S9788 or verapamil with cyclosporin A failed to increase the transport of this drug. An increase in the transport of doxorubicin from luminal to abluminal compartment was also observed, due to endothelial cell monolayer breakdown. Conclusions. Using this model, it is possible to predict the passage of a P-glycoprotein dependent drug to the brain or its sequestration in brain capillary endothelial cells when this drug is associated with a reversing agent, or its toxicity on the blood-brain barrier integrity.

Acylated proteins play a crucial role in cellphysiology because of their increased interaction withmembranes. Their isolation is difficult as aconsequence of their low cellular concentration andtheir chemical preparation is problematic due tosolubility problems. Through the use of reversedmicelles, we produced tens of milligrams of acylatedribonucleases A, chosen as a model, purified them bysemi-preparative high performance liquidchromatography (HPLC) and characterized them by analyticalHPLC, capillary electrophoresis, mass spectrometry, peptide mapping, Edman degradation and enzyme activity. We nextscrutinized the interaction with an in vitro blood–brainbarrier model and demonstrated that palmitoylated andstearoylated ribonucleases A are transported from onecompartment to the other across the cellular monolayer,in contrast to the native enzyme.

The mechanism of translocation of UDP-GlcNAc, UDP-Gal and UDP-Glc into intracellular vesicles has been studied using thymocytes whose plasma membranes have been permeabilized with isotonic ammonium chloride. It has been previously shown that the intracellular vesicles have specific carriers for UDP-GlcNAc and UDP-Gal. We now report that the translocation of these two sugar nucleotides occurs via UDP-GlcNAc/UDP and UDP-Gal/UDP antiports. The entry of UDP-GlcNAc or UDP-Gal into vesicles was specifically dependent on the exit of UDP from these vesicles. In contrast, no antiport mechanism has been recovered with UDP-Glc for which no transport and accumulation into intracellular vesicles were observed.

In contrast to the endothelial cells in large vessels where LDL receptors are downregulated, brain capillary endothelial cells in vivo express an LDL receptor. Using a cell culture model of the blood-brain barrier consisting of a coculture of brain capillary endothelial cells and astrocytes, we observed that the capacity of endothelial cells to bind LDL is enhanced threefold when cocultured with astrocytes. We next investigated the ability of astrocytes to modulate endothelial cell LDL receptor expression. We have shown that the lipid requirement of astrocytes increases the expression of endothelial cell LDL receptors. Experiments with dialysis membranes of different pore size showed that this effect is mediated by a soluble factor(s) with relative molecular mass somewhere between 3,500 and 14,000. Substituting astrocytes with smooth muscle cells or brain endothelium with endothelium from the aorta or the adrenal cortex did not enhance the luminal LDL receptor expression on endothelial cells, demonstrating the specificity of the interactions. This factor(s) is exclusively secreted by astrocytes cocultured with brain capillary endothelial cells, but it also upregulates the LDL receptor on other cell types. This study confirms the notion that the final fine tuning of cell differentiation is under local control.

Incubation of mouse thymocytes with 10µM monensin for 1 hour induces morphological alterations characterized by the extensive dilatation and vacuolization of the Golgi complex. This effect is used to study the transport and utilization of labelled sugar nucleotides into intracellular vesicles by using thymocytes whose plasma membrane has been permeabilized by ammonium chloride treatment. It is demonstrated that monensin stimulates the incorporation of labelled sialyl, fucosyl, galactosyl, and N-acetylglucosaminyl residues. This enhanced incorporation is not due to a direct effect of monensin on glycosyltransferase activities themselves but is a consequence of a higher entry and accumulation of labelled sugar nucleotides in the dilated vesicles.

Endothelial cells and pericytes are closely associated in brain capillaries. Together with astrocytic foot processes, they form the blood–brain barrier. Capillaries were isolated from bovine brain cortex. Pure populations of endothelial cells and pericytes were isolated and cultured in vitro. Polarized monolayers of endothelial cells preferentially secreted immunoreactive endothelin-1 (Et-1) at their abluminal (brain-facing) membrane. They did not express receptors for Et-1. Pericytes expressed BQ-123-sensitive ETAreceptors for endothelins as evidenced by 125I-Et-1 binding experiments. These receptors were coupled to phospholipase C as demonstrated by intracellular calcium measurements using indo-1-loaded cells. Addition of Et-1 to pericytes induced marked changes in the cell morphology that were associated with a reorganization of F-actin and intermediate filaments. It is concluded that Et-1 is a paracrine mediator at the bovine blood–brain barrier and that capillary pericytes are target cells for endothelium-derived Et-1.

Stereocontrolled syntheses of immunologically relevant heptopyranose disaccharides were achieved in excellent yields by the trimethylsilyl triflate promoted glycosylation of the donor 1,3,4,6-tetra-O-acetyl-2-N-allyloxycarbonyl-2-amino-2-deoxy-β-d-glucopyranose with the adequate acceptors.