The use of 4-Hexylresorcinol as antibiotic adjuvant.

Ever decreasing efficiency of antibiotic treatment due to growing antibiotic resistance of pathogenic bacteria is a critical issue in clinical practice. The two generally accepted major approaches to this problem are the search for new antibiotics and the development of antibiotic adjuvants to enhance the antimicrobial activity of known compounds. It was therefore the aim of the present study to test whether alkylresorcinols, a class of phenolic lipids, can be used as adjuvants to potentiate the effect of various classes of antibiotics. Alkylresorcinols were combined with 12 clinically used antibiotics. Growth-inhibiting activity against a broad range of pro- and eukaryotic microorganisms was determined. Test organisms did comprise 10 bacterial and 2 fungal collection strains, including E. coli and S. aureus, and clinical isolates of K. pneumoniae. The highest adjuvant activity was observed in the case of 4-hexylresorcinol (4-HR), a natural compound found in plants with antimicrobial activity. 50% of the minimal inhibitory concentration (MIC) of 4-HR caused an up to 50-fold decrease in the MIC of antibiotics of various classes. Application of 4-HR as an adjuvant revealed its efficiency against germination of bacterial dormant forms (spores) and prevented formation of antibiotic-tolerant persister cells. Using an in vivo mouse model of K. pneumoniae-induced sepsis, we could demonstrate that the combination of 4-HR and polymyxin was highly effective. 75% of animals were free of infection after treatment as compared to none of the animals receiving the antibiotic alone. We conclude that alkylresorcinols such as 4-HR can be used as an adjuvant to increase the efficiency of several known antibiotics. We suggest that by this approach the risk for development of genetically determined antibiotic resistance can be minimized due to the multimodal mode of action of 4-HR.

Mechanistic insights into effect of surfactants on oral bioavailability of amorphous solid dispersions.

Drug delivery of poorly soluble drugs in form amorphous solid dispersions (ASDs) is an appealing method to increase in vivo bioavailability. For rational formulation design, a mechanistic understanding of the impact of surfactants on the performance of ASD-based formulations is therefore of importance. In this study, we used hot-melt extrusion to prepare ASDs composed of the model drug substance efavirenz with hydroxypropyl methylcellulose phthalate (HPMCP) as the base polymer, and surfactants. Molecular dynamics simulations and in vitro dissolution studies were used to investigate formation and drug release from polymer vesicles, and their ability to maintain a supersaturation state as a function of surfactant composition. It was possible to identify main factors regulating particle formation and to modify dissolution profiles with different excipient compositions. Animal studies in the rat, in combination with physiologically based pharmacokinetic modeling, demonstrated enhanced drug absorption from formed vesicles. The surfactant composition in the ASD had a direct influence on the morphology of these vesicles, as well as kinetics of drug release, and, therefore, the oral bioavailability. ASDs, prepared by hot-melt extrusion method, were optimized for dissolution and adsorption rates increase. Our findings contribute to a better understanding of dissolution behavior of ASDs with respect to the function of surfactants, aiming to facilitate a rational formulation development and an accelerated transition from in vitro systems to in vivo applications.

Insights into possibilities for grouping and read-across for nanomaterials in EU chemicals legislation.

This paper presents a comprehensive review of European Union (EU) legislation addressing the safety of chemical substances, and possibilities within each piece of legislation for applying grouping and read-across approaches for the assessment of nanomaterials (NMs). Hence, this review considers both the overarching regulation of chemical substances under REACH (Regulation (EC) No 1907/2006 on registration, evaluation, authorization, and restriction of chemicals) and CLP (Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures) and the sector-specific pieces of legislation for cosmetic, plant protection and biocidal products, and legislation addressing food, novel food, and food contact materials. The relevant supporting documents (e.g. guidance documents) regarding each piece of legislation were identified and reviewed, considering the relevant technical and scientific literature. Prospective regulatory needs for implementing grouping in the assessment of NMs were identified, and the question whether each particular piece of legislation permits the use of grouping and read-across to address information gaps was answered.

A combined mathematical model linking the formation of amorphous solid dispersions with hot-melt-extrusion process parameters.

Hot-melt extrusion allows for the continuous production of amorphous solid dispersions, which are used to enhance bioavailability of poorly soluble drugs in pharmaceutical drug delivery. To facilitate formulation and extrusion process development, we propose a mathematical model describing the formation of amorphous solid dispersions in the context of this process. The model is based on the calculation of two key process values: (1) time to dissolution of solid drug particles in molten polymer during extrusion and (2) mean residence of material in the extruder. We suggest that their linking allows for rational process design. Experimental data support the validity of our model for both key process values as well as the overall process. This modeling approach allows for fast and cost-effective formulation and extrusion process development as well as feasibility estimations in early stages of drug development.

Biomimetic artificial organelles with in vitro and in vivo activity triggered by reduction in microenvironment.

Despite tremendous efforts to develop stimuli-responsive enzyme delivery systems, their efficacy has been mostly limited to in vitro applications. Here we introduce, by using an approach of combining biomolecules with artificial compartments, a biomimetic strategy to create artificial organelles (AOs) as cellular implants, with endogenous stimuli-triggered enzymatic activity. AOs are produced by inserting protein gates in the membrane of polymersomes containing horseradish peroxidase enzymes selected as a model for natures own enzymes involved in the redox homoeostasis. The inserted protein gates are engineered by attaching molecular caps to genetically modified channel porins in order to induce redox-responsive control of the molecular flow through the membrane. AOs preserve their structure and are activated by intracellular glutathione levels in vitro. Importantly, our biomimetic AOs are functional in vivo in zebrafish embryos, which demonstrates the feasibility of using AOs as cellular implants in living organisms. This opens new perspectives for patient-oriented protein therapy.

Functionalized calcium carbonate microparticles for the delivery of proteins.

The recently introduced functionalized calcium carbonate (FCC), a porous microparticle with a nano-structured, lamellar surface, shows promising properties in the field of oral drug delivery. In this work, FCC was loaded with biomolecules e.g. lysozyme (Lys) and bovine serum albumin (BSA) in order to investigate its suitability to deliver protein based drugs. Loading efficiency for our model proteins was >90% and enzyme activity was preserved as demonstrated by Michaelis-Menten enzyme kinetic experiments. Circular dichroism analysis confirmed, that neither the structure of both model substances, nor the activity of Lys was affected by the loading process or the interaction with the surface of FCC. Electron microscopy (SEM) and mercury porosimetry were indicative of protein deposition on the particle surface as well as within the particle pores. Release properties were investigated in a customized flow cell, which simulates the conditions in the oral cavity. Depending on the isoelectric point of the investigated proteins, complete release was obtained within 1.5h. This work shows, that FCC is a suitable pharmaceutical excipient for delivery of proteins.

Selective stimulation of the JAK/STAT signaling pathway by silica nanoparticles in human endothelial cells.

Silica nanoparticles (SiNP) are frequently used in pharmaceutical formulations. Intravenously administered, these particles are in close contact with the vascular endothelium. However, preliminary safety assessments of these novel excipients have indicated that SiNP are potentially cytotoxic and can trigger inflammation. In order to elucidate mechanisms of SiNP mediated inflammation, cerebral microvascular endothelial cells and primary umbilical endothelial cells were incubated with SiNP at doses between 10ng/ml and 250µg/ml. Two types of 110nm SiNP with different surface charge were synthesized and characterized. Uptake, cell viability, apoptosis, necrosis, oxidative stress, as well as interferences with both JAK/STAT and NF-κB pathways were studied. SiNP uptake leads to a cell viability decrease and promotes generation of reactive oxygen species (ROS) in a time- and dose-dependent manner. Furthermore, SiNP are able to trigger the activation of the STAT1 pathway. In contrast, no significant activation of STAT3, STAT6 or NF-κB could be detected. Additionally, modulation of the major histocompatibility complex (MHC) class I proteins was observed for cationic SiNP at low doses. Our results show the potential of SiNP to trigger selective activation of inflammatory signaling pathways in endothelial cells and thereby contribute to a better understanding of the toxicological profile of SiNP.

New Processes for Freeze-Drying in Dual-Chamber Systems.

Dual-chamber systems can offer self-administration and home care use for lyophilized biologics. Only a few products have been launched in dual-chamber systems so far-presumably due to dual-chamber systems' complex and costly drug product manufacturing process. Within this paper, two improved processes (both based on tray filling technology) for freeze-drying pharmaceuticals in dual-chamber systems are described. Challenges with regards to heat transfer were tackled by (1) performing the freeze-drying step in a needle-down orientation in combination with an aluminum block, or (2) freeze-drying the drug product "externally" in a metal cartridge with subsequent filling of the lyophilized cake into the dual-chamber system. Metal-mediated heat transfer was shown to be efficient in both cases and batch (unit-to-unit) homogeneity with regards to sublimation rate was increased. It was difficult to influence ice crystal size using different methods when in use with an aluminum block due to its heat capacity. Using such a metal carrier implies a large heat capacity leading to relatively small ice crystals. Compared to the established process, drying times were reduced by half using the new processes. The drying time was, however, longer for syringes compared to vials due to the syringe design (long and slim). The differences in drying times were less pronounced for aggressive drying cycles. The proposed processes may help to considerably decrease investment costs into dual-chamber system fill-finish equipment. LAY ABSTRACT: Dual-chamber syringes offer self-administration and home care use for freeze-dried pharmaceuticals. Only a few products have been launched in dual-chamber syringes so far-presumably due to their complex and costly drug product manufacturing process. In this paper two improved processes for freeze-drying pharmaceuticals in dual-chamber syringes are described. The major challenge of freeze-drying is to transfer heat through a vacuum. The proposed processes cope with this challenge by (1) freeze-drying the drug product in the syringe in an orientation in which the product is closest to the heat source, or (2) freeze-drying the drug product outside the syringe in a metal tube. The latter requires filling the freeze-dried product subsequently into the dual-chamber syringe. Both processes were very efficient and promised to achieve similar freeze-drying conditions for all dual-chamber syringes within one production run. The proposed processes may help to considerably decrease investment costs into dual-chamber syringe fill-finish equipment.

Pharmacological characterization of designer cathinones in vitro.

BACKGROUND AND PURPOSE: Designer ß-keto amphetamines (e.g. cathinones, 'bath salts' and 'research chemicals') have become popular recreational drugs, but their pharmacology is poorly characterized. EXPERIMENTAL APPROACH: We determined the potencies of cathinones to inhibit DA, NA and 5-HT transport into transporter-transfected HEK 293 cells, DA and 5-HT efflux from monoamine-preloaded cells, and monoamine receptor binding affinity. KEY RESULTS: Mephedrone, methylone, ethylone, butylone and naphyrone acted as non-selective monoamine uptake inhibitors, similar to cocaine. Mephedrone, methylone, ethylone and butylone also induced the release of 5-HT, similar to 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) and other entactogens. Cathinone, methcathinone and flephedrone, similar to amphetamine and methamphetamine, acted as preferential DA and NA uptake inhibitors and induced the release of DA. Pyrovalerone and 3,4-methylenedioxypyrovalerone (MDPV) were highly potent and selective DA and NA transporter inhibitors but unlike amphetamines did not evoke the release of monoamines. The non-ß-keto amphetamines are trace amine-associated receptor 1 ligands, whereas the cathinones are not. All the cathinones showed high blood-brain barrier permeability in an in vitro model; mephedrone and MDPV exhibited particularly high permeability. CONCLUSIONS AND IMPLICATIONS: Cathinones have considerable pharmacological differences that form the basis of their suggested classification into three groups. The predominant action of all cathinones on the DA transporter is probably associated with a considerable risk of addiction.

The norepinephrine transporter inhibitor reboxetine reduces stimulant effects of MDMA ("ecstasy") in humans.

This study assessed the pharmacodynamic and pharmacokinetic effects of the interaction between the selective norepinephrine (NE) transporter inhibitor reboxetine and 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") in 16 healthy subjects. The study used a double-blind, placebo-controlled crossover design. Reboxetine reduced the effects of MDMA including elevations in plasma levels of NE, increases in blood pressure and heart rate, subjective drug high, stimulation, and emotional excitation. These effects were evident despite an increase in the concentrations of MDMA and its active metabolite 3,4-methylenedioxyamphetamine (MDA) in plasma. The results demonstrate that transporter-mediated NE release has a critical role in the cardiovascular and stimulant-like effects of MDMA in humans.

Report from the EPAA workshop: in vitro ADME in safety testing used by EPAA industry sectors.

There are now numerous in vitro and in silico ADME alternatives to in vivo assays but how do different industries incorporate them into their decision tree approaches for risk assessment, bearing in mind that the chemicals tested are intended for widely varying purposes? The extent of the use of animal tests is mainly driven by regulations or by the lack of a suitable in vitro model. Therefore, what considerations are needed for alternative models and how can they be improved so that they can be used as part of the risk assessment process? To address these issues, the European Partnership for Alternative Approaches to Animal Testing (EPAA) working group on prioritization, promotion and implementation of the 3Rs research held a workshop in November, 2008 in Duesseldorf, Germany. Participants included different industry sectors such as pharmaceuticals, cosmetics, industrial- and agro-chemicals. This report describes the outcome of the discussions and recommendations (a) to reduce the number of animals used for determining the ADME properties of chemicals and (b) for considerations and actions regarding in vitro and in silico assays. These included: standardisation and promotion of in vitro assays so that they may become accepted by regulators; increased availability of industry in vivo kinetic data for a central database to increase the power of in silico predictions; expansion of the applicability domains of in vitro and in silico tools (which are not necessarily more applicable or even exclusive to one particular sector) and continued collaborations between regulators, academia and industry. A recommended immediate course of action was to establish an expert panel of users, developers and regulators to define the testing scope of models for different chemical classes. It was agreed by all participants that improvement and harmonization of alternative approaches is needed for all sectors and this will most effectively be achieved by stakeholders from different sectors sharing data.

Discovery of potent and selective histamine H3 receptor inverse agonists based on the 3,4-dihydro-2H-pyrazino[1,2-a]indol-1-one scaffold.

A novel series of potent histamine H(3) receptor inverse agonists based on the 3,4-dihydro-2H-pyrazino[1,2-a]indol-1-one scaffold has been discovered. Several compounds display high selectivity over other histamine receptor subtypes and have favorable physicochemical properties, low potential for CYP450 enzyme inhibition and high metabolic stability in microsomal preparations. (R)-2-Cyclopropylmethyl-8-(1-isopropyl-piperidin-4-yloxy)-3-methyl-3,4-dihydro-2H-pyrazino[1,2-a]indol-1-one (8t) showed good in vivo efficacy after per os application in an acute rat dipsogenia model of water intake.

Characterization of (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one as a positive allosteric modulator of GABAB receptors.

BACKGROUND AND PURPOSE: As baclofen is active in patients with anxiety disorders, GABAB receptors have been implicated in the modulation of anxiety. To avoid the side effects of baclofen, allosteric enhancers of GABAB receptors have been studied to provide an alternative therapeutic avenue for modulation of GABAB receptors. The aim of this study was to characterize derivatives of (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (rac-BHFF) as enhancers of GABAB receptors. EXPERIMENTAL APPROACH: Enhancing properties of rac-BHFF were assessed in the Chinese hamster ovary (CHO)-Galpha16-hGABA(B1a,2a) cells by Fluorometric Imaging Plate Reader and GTPgamma[35S]-binding assays, and in rat hippocampal slices by population spike (PS) recordings. In vivo activities of rac-BHFF were assessed using the loss of righting reflex (LRR) and stress-induced hyperthermia (SIH) models. KEY RESULTS: In GTPgamma[35S]-binding assays, 0.3 microM rac-BHFF or its pure enantiomer (+)-BHFF shifted the GABA concentration-response curve to the left, an effect that resulted in a large increase in both GABA potency (by 15.3- and 87.3-fold) and efficacy (149% and 181%), respectively. In hippocampal slices, rac-BHFF enhanced baclofen-induced inhibition of PS of CA1 pyramidal cells. In an in vivo mechanism-based model in mice, rac-BHFF increased dose-dependently the LRR induced by baclofen with a minimum effective dose of 3 mg kg(-1) p.o. rac-BHFF (100 mg kg(-1) p.o.) tested alone had no effect on LRR nor on spontaneous locomotor activity, but exhibited anxiolytic-like activity in the SIH model in mice. CONCLUSIONS AND IMPLICATIONS: rac-BHFF derivatives may serve as valuable pharmacological tools to elucidate the pathophysiological roles played by GABAB receptors in the central and peripheral nervous systems.

Evaluation of the NR2B-selective NMDA receptor antagonist Ro 63-1908 on rodent behaviour: evidence for an involvement of NR2B NMDA receptors in response inhibition.

We have characterised the effects of the recently described NMDA NR2B subtype selective antagonist, Ro 63-1908, on spontaneous behaviour and in tasks sensitive to non-selective NMDA antagonists. In both rats and wild type mice, Ro 63-1908 (1-30mg/kg sc) produced a mild increase in motor activity of lesser magnitude than that elicited by dizocilpine. No signs of overt PCP-like stereotypy were seen in either species at equivalent doses. PPI was also unaffected. However, in mice lacking the NR2A subunit, Ro 63-1908 (3-30mg/kg) produced a profound hyperactivity of similar magnitude to dizocilpine but few other 'PCP-like' behaviours. In rats, Ro 63-1908 (1-10mg/kg) did not affect Morris water maze or delayed matching performance. In a 5-choice serial reaction time task, requiring rats to respond to a visual stimulus presented after a fixed time interval, Ro 63-1908 (0.3-3mg/kg) produced a dramatic increase in premature responses - accuracy was relatively unaffected. Finally in a DRL24 task, Ro 63-1908 (0.3-3mg/kg) reduced inter-response time, increased response rate, and consequently reduced efficiency. We conclude that the improved profile of Ro 63-1908 compared to NMDA channel blockers is due to both its selectivity for the NR2B vs. NR2A subunit containing receptors and its activity-dependent mechanism of action. However, in the 5-CSRT and DRL24 tasks, Ro 63-1908 produced behaviours suggestive of impaired response inhibition, implicating a critical role of NMDA NR2B transmission in this process.

Uptake of cationzied albumin coupled liposomes by cultured porcine brain microvessel endothelial cells and intact brain capillaries.

The suitability of protein-coupled liposomes as drug carriers for brain specific targeting was investigated using albumin (BSA) and cationized albumin (CBSA), respectively, as model proteins. Liposomes coated with polyethylene glycol (sterically stabilized, PEG-liposomes) were prepared from phosphatidylcholine, cholesterol, and a PEG-derivatized phospholipid and covalently coupled to thiolated BSA or CBSA. Liposomes were loaded with carboxy-fluorescein and rhodamine-labeled dipalmitoyl-phosphatidylethanolamine as hydrophilic and lipophilic marker compounds, respectively. The interaction of these constructs with monolayers of porcine brain capillary endothelial cells (BCEC) and freshly isolated porcine brain capillaries was studied by means of fluorescence assays and confocal laser scanning fluorescence microscopy (CLSFM). In contrast to BSA, CBSA was rapidly taken up by cultured BCECs. BSA-coupled liposomes did not interact with endothelial cells, whereas CBSA-coupled liposomes bound to cellular surfaces and exhibited time dependently a high intracellular accumulation. CBSA-conjugated liposomes were also taken up by intact brain capillaries. Cellular uptake could be inhibited by free cationized albumin, phenylarsineoxide, nocodazole, and filipin, but not by dansylcadaverine, suggesting a caveolae-mediated incorporation process. Immunostaining demonstrated a high expression of caveolin in the capillary endothelium. In conclusion, liposomes coupled to CBSA are taken up into brain endothelium via an endocytotic pathway and may therefore be a suitable carrier for drug delivery to the brain.

Evidence that nicotinic alpha(7) receptors are not involved in the hyperlocomotor and rewarding effects of nicotine.

Neuronal nicotinic receptors are comprised of combinations of alpha(2-9) and beta(2-4) subunits arranged to form a pentameric receptor. Currently, the principal central nervous system (CNS) subtypes are believed to be alpha(4)beta(2) and a homomeric alpha(7) receptor, although other combinations almost certainly exist. The identity of the nicotinic receptor subtype(s) involved in the rewarding effects of nicotine are unknown. In the present study, using some recently described subtype selective nicotinic agonists and antagonists, we investigated the role of the alpha(7) nicotinic receptor in the mediation of nicotine-induced hyperactivity and self-administration in rats. The alpha(7) receptor agonists AR-R 17779 and DMAC failed to stimulate locomotor activity in both nicotine-nontolerant and -sensitized rats. In contrast, nicotine and the putative alpha(4)beta(2) subtype selective agonist SIB1765F increased activity in both experimental conditions. In nicotine-sensitized rats, the high affinity (including the alpha(4)beta(2) subtype) nicotinic antagonist dihydro-beta-erythroidine (DHbetaE), but not the selective alpha(7) antagonist methyllycaconitine (MLA), antagonized a nicotine-induced hyperactivity. Similarly, DHbetaE, but not MLA, pretreatment reduced nicotine self-administration. Electrophysiology experiments using Xenopus oocytes expressing the human alpha(7) receptor confirmed AR-R 17779 and DMAC to be potent agonists at this site, and further studies demonstrated the ability of systemically administered AR-R 17779 to penetrate into the CNS. Taken together, these results indicate a negligible role of alpha(7) receptors in nicotine-induced hyperlocomotion and reward in the rat, and support the view for an involvement of a member from the high-affinity nicotinic receptor subclass, possibly alpha(4)beta(2). Issues such as drug potency, CNS penetration, and desensitization of the alpha(7) receptor are discussed.

Endocytosis and transcytosis of an immunoliposome-based brain drug delivery system.

Immunoliposomes conjugated with the OX26 monoclonal antibody to the rat transferrin receptor can be used for brain delivery of small molecules. In the present study the uptake of OX26-immunoliposomes by target cells as well as their transcytosis across the blood-brain barrier was investigated. Microscopy of RG2 rat glioma cells incubated with fluorescence labeled OX26-immunoliposomes revealed intracellular co-localization of liposomal cargo, the liposomal membrane bilayer and the OX26 monoclonal antibody. The distinct particulate staining pattern was indicative for accumulation of OX26-immunoliposomes within endosomal or lysosomal compartments. Prolonged incubations demonstrated endosomal release of the liposomal cargo propidium iodide to the cytoplasm. A maximum of 50% of propidium iodide was released from the endosomal compartment after 24 hours of incubation. Transcytosis was studied using an in vitro model of the blood-brain barrier consisting of immortalized RBE4 rat brain endothelial cells. OX26-immunoliposomes did permeate across the RBE4 cell monolayer and showed a permeability coefficient of P(app) = 1.6 x 10(-5) ml/s. Transport was inhibited at low temperature, by competition with free OX26 or by exchanging the OX26 monoclonal antibody for an unspecific isotype antibody. Transcytosis of OX26-immunolipsomes was confirmed in vivo by the brain perfusion and capillary depletion technique. OX26-immunoliposomes were detected within the post-vascular compartment of brain parenchyma (PS product = 2.4 microl/g/min.) and were not associated with the brain microvasculature.

Modulation of multidrug resistance protein expression in porcine brain capillary endothelial cells in vitro.

Multidrug resistance-associated protein (MRP) is a transport system that is involved in the elimination of xenobiotics and biologically active endogenous substrates. Recently, the presence of MRP has been demonstrated in cultured brain capillary endothelial cells (BCECs). The time-dependent, functional expression of MRP in porcine BCECs was investigated to assess the value of this cell culture model for drug transport at the blood-brain barrier. Western blot analysis was used to investigate MRP expression in freshly isolated porcine BCECs and compared to MRP expression at days 8 and 10 in culture. Subcellular localization of MRP was investigated by immunocytochemistry with an MRP-specific monoclonal antibody, MRPr1. Functional activity of MRP was assessed by efflux studies with the fluorescent MRP substrate glutathione-methylfluorescein (GS-MF). No significant MRP expression was detected in freshly isolated endothelial cells. However, MRP expression is up-regulated in cell culture in a time-dependent manner. Immunostaining revealed predominantly perinuclear and, to a lesser degree, plasma membrane localization of MRP. At 10 degrees C GS-MF efflux was significantly decreased, indicating the involvement of an energy-dependent transport system. Efflux of GS-MF was apparently inhibited by MK571, a specific inhibitor for MRP. Porcine BCECs demonstrate up-regulation of functional MRP expression during culture, as observed in human tissue, and therefore might serve as a useful in vitro system for studying MRP-mediated blood-brain barrier transport.

Characterization of transferrin receptor in an immortalized cell line of rat brain endothelial cells, RBE4.

The content and distribution of transferrin receptors in an immortalized cell line, RBE4, derived from rat cerebral capillary endothelial cells was investigated using the monoclonal antibody MRC OX-26 (OX-26 mAb) specific for the rat transferrin receptor. An ELISA assay was developed with which the OX-26 mAb can be determined quantiatively. The detection limit of the assay was 10 pg or 0.07 fmol of murine antibody. With this technique accurate measurement of native antibody is now possible without the need for isotope labeling (iodination). Immunostaining of confluent monolayers of RBE4 cells using an antibody directed against the tight junction associated protein ZO-1 was indicative for structural intactness of RBE4 cell monolayers. OX-26 immunostaining demonstrated localization of the transferrin receptor at the plasma membrane and/or in the cytosol. Binding studies showed saturation of OX-26 mAb binding. The antibody binding analysis gave a dissociation constant (KD) of 17.1 +/- 1.2 nmol/l. The total amount of transferrin receptors present per cell was 70,800 +/- 17,000. Our results indicate that receptor binding of OX-26 mAb can be studied using an in vitro cell culture model of rat brain mircrovessel endothelium in conjunction with an ELISA technique for detection of native antibody. This approach will be used to investigate mechanisms of transendothelial transport of OX-26 in vitro.

HIV protease inhibitor ritonavir: a more potent inhibitor of P-glycoprotein than the cyclosporine analog SDZ PSC 833.

The effect of P-glycoprotein inhibition on the uptake of the HIV type 1 protease inhibitor saquinavir into brain capillary endothelial cells was studied using porcine primary brain capillary endothelial cell monolayers as an in vitro test system. As confirmed by polymerase chain reaction and Western blot analysis, this system functionally expressed class I P-glycoprotein (pgp1A). P-Glycoprotein isoforms pgp1B or pgp1D could not be detected. The uptake of saquinavir into endothelial cells could be described as the result of a diffusional term of uptake and an oppositely directed saturable extrusion process. Net uptake of saquinavir into cultured brain endothelial cells could be increased significantly up to 2-fold by SDZ PSC 833 in a dose-dependent manner, with an IC(50) of 1.13 microM. In addition, the HIV protease inhibitor ritonavir inhibited p-glycoprotein-mediated extrusion of saquinavir with an IC(50) of 0.2 microM, indicating a high affinity of ritonavir for p-glycoprotein. In conclusion, we showed that the HIV protease inhibitor ritonavir is a more potent inhibitor of P-glycoprotein than the multidrug resistance (MDR)-reversing agent SDZ PSC 833. The inclusion of this drug in combination regimens may greatly facilitate brain uptake of HIV protease inhibitors, which is especially important in patients suffering from AIDS dementia complex.