Effects of arsenolipids on in vitro blood-brain barrier model.

Arsenic-containing hydrocarbons (AsHCs), a subgroup of arsenolipids (AsLs) occurring in fish and edible algae, possess a substantial neurotoxic potential in fully differentiated human brain cells. Previous in vivo studies indicating that AsHCs cross the blood-brain barrier of the fruit fly Drosophila melanogaster raised the question whether AsLs could also cross the vertebrate blood-brain barrier (BBB). In the present study, we investigated the impact of several representatives of AsLs (AsHC 332, AsHC 360, AsHC 444, and two arsenic-containing fatty acids, AsFA 362 and AsFA 388) as well as of their metabolites (thio/oxo-dimethylpropionic acid, dimethylarsinic acid) on porcine brain capillary endothelial cells (PBCECs, in vitro model for the blood-brain barrier). AsHCs exerted the strongest cytotoxic effects of all investigated arsenicals as they were up to fivefold more potent than the toxic reference species arsenite (iAsIII). In our in vitro BBB-model, we observed a slight transfer of AsHC 332 across the BBB after 6 h at concentrations that do not affect the barrier integrity. Furthermore, incubation with AsHCs for 72 h led to a disruption of the barrier at sub-cytotoxic concentrations. The subsequent immunocytochemical staining of three tight junction proteins revealed a significant impact on the cell membrane. Because AsHCs enhance the permeability of the in vitro blood-brain barrier, a similar behavior in an in vivo system cannot be excluded. Consequently, AsHCs might facilitate the transfer of accompanying foodborne toxicants into the brain.

The effects of gold nanoparticles functionalized with ß-amyloid specific peptides on an in vitro model of blood-brain barrier.

We studied the effect of gold nanoparticle (AuNP) size, surface charge, concentration and morphology on the integrity of the blood-brain barrier (BBB) in a well-established in vitro model set-up. We focused on the effect of peptide functionalized hollow gold nanospheres and gold nanorods, which selectively bind to amyloidogenic ß-amyloid structures. These AuNP conjugates have already been successfully tested as photothermal absorbers for potential application in Alzheimer's disease (AD) therapy in an in vitro set-up, but may exhibit a low passage through the BBB due to their overall negative charge. Our results show that: (i) small (1.4 nm) AuNPs strongly affects the BBB integrity, (ii) negative surface charge impedes BBB passage, and (iii) this charge effect caused by the peptide is compensated by covalent coupling to a polyethylene glycol ligand stabilizing the particles in diluted manner.

Intestinal formation of trans-crocetin from saffron extract (Crocus sativus L.) and in vitro permeation through intestinal and blood brain barrier.

AIMS: Extracts of saffron (Crocus sativus L.) have traditionally been used against depressions. Recent preclinical and clinical investigations have rationalized this traditional use. Trans-crocetin, a saffron metabolite originating from the crocin apocarotenoids, has been shown to exert strong NMDA receptor affinity and is thought to be responsible for the CNS activity of saffron. Pharmacokinetic properties of the main constituents from saffron have only been described to a limited extent. Therefore the present in vitro study aimed to determine if crocin-1 and trans-crocetin are able to pass the intestinal barrier and to penetrate the blood brain barrier (BBB). Additionally, the intestinal conversion of glycosylated crocins to the lipophilic crocetin had to be investigated. Experiments with Caco-2 cells and two different porcine BBB systems were conducted. Further on, potential intestinal metabolism of saffron extract was investigated by ex vivo experiments with murine intestine. METHODOLOGY: In vitro Caco-2 monolayer cell culture was used for investigation of intestinal permeation of crocin-1 and trans-crocetin. In vitro models of porcine brain capillary endothelial cells (BCEC) and blood cerebrospinal fluid barrier (BCSFB) were used for monitoring permeation characteristics of trans-crocetin through the blood brain barrier (BBB). Intestine tissue and feces homogenates from mice served for metabolism experiments. RESULTS: Crocin-1, even at high concentrations (1000 µM) does not penetrate Caco-2 monolayers in relevant amounts. In contrast, trans-crocetin permeates in a concentration-independent manner (10-114 µM) the intestinal barrier by transcellular passage with about 32% of the substrate being transported within 2 h and a permeation coefficient of Papp 25.7 × 10(-)(6) ± 6.23 × 10(-)(6) cm/s. Trans-crocetin serves as substrate for pGP efflux pump. Trans-crocetin permeates BBB with a slow but constant velocity over a 29 h period (BCEC system: Papp 1.48 × 10(-)(6) ± 0.12 × 10(-)(6) cm/s; BCSFB system Papp 3.85 × 10(-)(6) ± 0.21 × 10(-)(6) cm/s). Conversion of glycosylated crocins from saffron extract to trans-crocetin occurs mainly by intestinal cells, rather than by microbiological fermentation in the colon. CONCLUSION: The here described in vitro studies have shown that crocins from saffron are probably not bioavailable in the systemic compartment after oral application. On the other side the investigations clearly have pointed out that crocins get hydrolyzed in the intestine to the deglycosylated trans-crocetin, which subsequently is absorbed by passive transcellular diffusion to a high extend and within a short time interval over the intestinal barrier. Crocetin will penetrate in a quite slow process the blood brain barrier to reach the CNS. The intestinal deglycosylation of different crocins in the intestine is mainly due to enzymatic processes in the epithelial cells and only to a very minor extent due to deglycosylation by the fecal microbiome. On the other side the fecal bacteria degrade the apocarotenoid backbone to smaller alkyl units, which do not show any more the typical UV absorbance of crocins. As previous studies have shown strong NMDA receptor affinity and channel opening activity of trans-crocetin the use of saffron for CNS disorders seems to be justified from the pharmacokinetic and pharmacodynamic background.

Predicting blood-brain barrier permeability of drugs: evaluation of different in vitro assays.

The ability of a drug to penetrate the blood-brain barrier (BBB) is essential for its use in the pharmaceutical treatment of CNS disorders. Five different in vitro methods to predict BBB permeability of drugs were compared and evaluated in the present study. All assays were performed with a consistent set of seven compounds and in the same physiological buffer to provide a basis for direct comparison of the results. Octanol-buffer and liposome-buffer partition coefficients were most conveniently obtained but failed to predict BBB permeability for certain drugs. The incorporation of drugs into lipid monolayers at the air-buffer interface was found to be a poor predictor of BBB permeability and was furthermore not considered suitable for screening due to the demanding experimental requirements. Permeability studies using Caco-2 cell monolayers provided a good correlation to an in vitro model of the BBB, which was based on primary cultured porcine brain capillary endothelial cells (PBCEC). However, differences in drug permeability between the intestine and brain derived cells were detected, limiting the advantages of the easy handling of the Caco-2 cell line compared to the more time-consuming primary culture of the BCEC.

Maltose phosphorylase from Lactobacillus brevis: purification, characterization, and application in a biosensor for ortho-phosphate.

With the goal to obtain maltose phosphorylase as a tool to determine ortho-phosphate, the enzyme from Lactobacillus brevis was purified to 98% by an expeditious FPLC-aided procedure which included anion exchange chromatography, gel filtration, and hydroxyapatite chromatography. The native maltose phosphorylase had a molecular mass of 196 kDa and consisted of two 88 kDa subunits. In isoelectric focusing two isoforms with pI values of 4.2 and 4.6 were observed. Maximum enzyme activity was obtained at 36 degrees C and pH 6.5 and was independent of pyridoxal 5'-phosphate. The apparent K(m) values with maltose and phosphate as substrates were 0.9 mmol l-1 and 1.8 mmol l-1, respectively. Maltose phosphorylase could be stored in 10 mM phosphate buffer pH 6.5 at 4 degrees C with a loss of activity of only 7% up to 6 months. The stability of the enzyme at high temperatures was enhanced significantly using additives like phosphate, citrate, and imidazole. The purified maltose phosphorylase was used as key enzyme in a phosphate sensor consisting of maltose phosphorylase and glucose oxidase. A detection limit of 0.1 microM phosphate was observed and the sensor response was linear in the range between 0.5 and 10 microM.