Investigation of the high partition of YM992, a novel antidepressant,in rat brain - in vitro and in vivo evidence for the high binding in brain and the high permeability at the BBB

Brain extracellular fluid (ECF) concentration of YM992, a novel antidepressant, was determined using brain microdialysis to investigate the high partition of this drug to the brain after systemic administration to rats. Plasma, cerebrospinal fluid (CSF), ECF and brain concentrations were determined at the steady-state after intravenous infusion to rats. The concentration ratio of brain to plasma at the total concentration base was 71.3, while those of ECF to plasma and CSF to plasma at the free concentration base were comparable. The distribution volume in brain was 375 ml/g brain and in vitro binding of YM992 to rat brain was 98.1-98.5%, suggesting a high binding in the brain. The carotid artery injection study showed that the brain uptake index of YM992 was 141%, furthermore, the uptake clearance into brain after i.v. dosing to rats was 0.6 ml/min/g brain, indicating a high permeability at the blood-brain barrier (BBB). These findings suggest that the high partition of YM992 to rat brain is attributed to its high level of binding in the brain as well as its high permeability at the BBB.     

Involvement of an influx transporter in the blood–brain barrier transport of naloxone

Naloxone, a potent and specific opioid antagonist, has been shown in previous studies to have an influx clearance across the rat blood–brain barrier (BBB) two times greater than the efflux clearance. The purpose of the present study was to characterize the influx transport of naloxone across the rat BBB using the brain uptake index (BUI) method. The initial uptake rate of [³H]naloxone exhibited saturability in a concentration‐dependent manner (concentration range 0.5 µM to 15 mM ) in the presence of unlabeled naloxone. These results indicate that both passive diffusion and a carrier‐mediated transport mechanism are operating. The in vivo kinetic parameters were estimated as follows: the Michaelis constant, K_t, was 2.99±0.71 mM ; the maximum uptake rate, J_max, was 0.477±0.083 µmol/min/g brain; and the nonsaturable first‐order rate constant, K_d, was 0.160±0.044 ml/min/g brain. The uptake of [³H]naloxone by the rat brain increased as the pH of the injected solution was increased from 5.5 to 8.5 and was strongly inhibited by cationic H₁‐antagonists such as pyrilamine and diphenhydramine and cationic drugs such as lidocaine and propranolol. In contrast, the BBB transport of [³H]naloxone was not affected by any typical substrates for organic cation transport systems such as tetraethylammonium, ergothioneine or L ‐carnitine or substrates for organic anion transport systems such as p‐aminohippuric acid, benzylpenicillin or pravastatin. The present results suggest that a pH‐dependent and saturable influx transport system that is a selective transporter for cationic H₁‐antagonists is involved in the BBB transport of naloxone in the rat. Copyright © 2010 John Wiley & Sons, Ltd.     

A cholecystokinin-1 receptor agonist (CCK-8) mediates increased permeability of brain barriers to leptin

BACKGROUND AND PURPOSE: Leptin regulates energy expenditure and body weight by acting both on the hypothalamus and on peripheral targets. Central actions of leptin are enhanced by cholecystokinin (CCK). The interaction between leptin and CCK makes physiological sense, as rats lacking CCK1 receptors are resistant to peripheral leptin but not to leptin directly infused into the brain. We have recently reported that CCK enhances leptin effects by increasing the entry of leptin into the CNS. The aim of this work was to further characterize the effect of CCK (10 microg kg(-1)) on leptin kinetics as well as the CCK receptor subtype involved in the interaction between CCK and leptin. EXPERIMENTAL APPROACH: Experiments were carried out both in free-feeding and in fasted rats receiving a single dose of leptin (100 microg kg(-1); i.p.). Parameters analysed over the next 6 h were plasma and cerebrospinal fluid concentrations of leptin. KEY RESULTS: We observed that CCK-8 depressed the increase in plasma leptin that followed the i.p. injection and simultaneously increased leptin concentration in the cerebrospinal fluid from 92+/-25 to 230+/-24 pg mL(-1) (P<0.05). The effect of CCK-8 was totally prevented by the CCK1 receptor antagonist, SR-27,897 (0.3 mg kg(-1), s.c.), but not by the CCK2 receptor antagonist, L-365,260 (1 mg kg(-1)). CONCLUSIONS AND IMPLICATIONS: These results show that CCK plays a role in regulating the access of leptin to the brain and suggest that CCK analogues, acting on CCK1 receptors, might be useful drugs in improving leptin actions within the brain.     

Brain inducible nitric oxide synthase is involved in interleukin-1beta-induced activation of the central sympathetic outflow in rats

Nitric oxide (NO) has been recognized as a neurotransmitter or a neuromodulator in the central nervous system. Brain NO is mainly generated either by neuronal NO synthase (NOS) or by inducible NOS. Previously we reported that central NO is involved in the elevation of plasma noradrenaline levels induced by intracerebroventricularly (i.c.v.) administered interleukin-1beta in rats [Eur. J. Phamacol. 317 (1996) 61]. In the present study, therefore, we tried to characterize which type of NOS isoforms is involved in the cytokine-induced responses using selective inhibitors of each NOS isoform in urethane-anesthetized rats. I.c.v. administered interleukin-1beta (100 ng/animal) elevated plasma levels of noradrenaline but not adrenaline. The cytokine-induced elevation of plasma noradrenaline levels was attenuated by cycloheximide, an inhibitor of protein synthesis, in a dose-dependent manner (10 and 20 microg/animal, i.c.v.). S-ethylisothiourea (0.1 and 0.5 microg/animal, i.c.v.), an inhibitor of inducible NOS, dose-dependently reduced the cytokine-induced elevation of plasma noradrenaline levels, while 7-nitroindazole (5 and 10 microg/animal, i.c.v.), an inhibitor of neuronal NOS, had no effect. These results suggest the involvement of brain inducible NOS in the interleukin-1beta-induced activation of the central sympathetic outflow in rats.     

In Vivo Transport of a Dynorphin-like Analgesic Peptide,E-2078, Through the Blood–Brain Barrier: An Application of Brain Microdialysis

In vivo transport through the blood–brain barrier (BBB) has been demonstrated for a dynorphin-like analgesic peptide, CH₃-[¹²⁵I]Tyr-Gly-Gly-Phe-Leu-Arg-CH₃Arg-D-Leu-NHC₂H₅ ([¹²⁵I]E-2078). A remarkable time-dependent increase in the distribution volume of [¹²⁵I]E-2078 in the brain parenchyma separated from blood vessels and capillaries was observed during a brain perfusion. The distribution volume of [¹²⁵I]E-2078 in the brain parenchyma after 20 min of perfusion was 2.18 ± 0.09 µl/g brain (mean ± SE) and was significantly greater than the distribution volume of [³H]inulin (0.994 ± 0.138 (µl/g brain), providing in vivo evidence for the penetration of [¹²⁵I]E-2078 into the brain parenchyma. Brain microdialysis was carried out to collect directly the brain interstitial fluid (ISF) during the brain perfusion of [¹²⁵I]E-2078. No metabolite of [¹²⁵I]E-2078 in the brain ISF was found by high-performance liquid chromatographic analysis of the brain dialysate. The concentrations of [¹²⁵I]E-2078 and [¹⁴C]sucrose in the brain ISF were estimated based on an in vitro evaluation of dialysis clearance. The concentration ratio of [¹²⁵I]E-2078 between the brain ISF and the brain perfusate was determined to be 2.92 × 10^–l ± 0.50 × 10^–l and was approximately 100 times higher than that of [¹⁴C]sucrose (2.71 × 10^–3 ± 1.43 × 10^–3), demonstrating transport of [¹²⁵I]E-2078 through the BBB in vivo. On the other hand, no remarkable difference in the cerebrospinal fluid (CSF)-to-perfusate concentration ratios of [¹²⁵I]E-2078 and [¹⁴C]sucrose was observed, indicating little contribution of the blood–CSF barrier (BCSF barrier) transport to the penetration of [¹²⁵I]E-2078 into the brain.     

Different effects of dexamethasone and the nitric oxide synthase inhibitor L-NAME on caerulein-induced rat acute pancreatitis, depending on the severity

Effects of dexamethasone and N^G-nitro-L-arginine methyl ester (L-NAME), the nitric oxide (NO) synthase inhibitor, on caerulein-induced acute pancreatitis were examined in rats. Acute pancreatitis was induced by caerulein (20 μg/kg, s.c.) given repeatedly 2 or 4 times every hour, and serum amylase levels, pancreas weight and myeloperoxidase (MPO) activity were measured 6 h after the first injection of caerulein. Dexamethasone (3 mg/kg) and L-NAME (30 mg/kg) were administered p.o. 30 min before the first injection of caerulein. Caerulein caused moderate or severe pancreatitis, depending on the times of injections, resulting in different degrees of increase in serum amylase levels and pancreas weight, and the marked elevation of MPO activity was observed only after injections of caerulein given 4 times per hour. Both dexamethasone and L-NAME suppressed the severity of pancreatits, yet the effect of L-NAME as compared with dexamethasone was more potent against mild pancreatitis but less potent against severe pancreatitis. These results suggest that caerulein-induced acute pancreatitis shows different responsiveness to L-NAME and dexamethasone, depending on the severity; the former is more effective against pancreatitis with less inflammation, while the latter is more effective against pancreatitis with severe inflammation. It is assumed that endogenous NO may be involved in oedema formation as the early event in the development of acute pancreatitis.     

慢性心力衰竭患者血清嗜铬粒蛋白A、脑钠肽水平及左室质量指数相关性研究

目的观察慢性心力衰竭(CHF)患者外周血血清嗜铬粒蛋白A(CgA)水平的变化,探讨CgA的价值和临床意义。方法测定不同程度CHF患者(CHF组,49例)血清CgA、脑钠肽(BNP)水平及左室质量指数(LVM I),与12例正常健康人(对照组)进行对照,分析CHF组血清CgA与BNP水平及LVM I的相关性。结果 CHF组血清CgA、BNP水平及LVM I均显著高于对照组(P<0.01),且随着心力衰竭程度的加重而增加;CgA与BNP、LVM I分别具有良好的相关性(r=0.88,P<0.01;r=0.61,P<0.01)。结论心力衰竭患者CgA水平分别与BNP水平、LVM、INY-HA功能分级呈明显正相关,检测CgA水平有助于判断CHF病情的严重程度。     

A novel antioxidant,octyl caffeate,suppression of LPS/IFN-gamma-induced inducible nitric oxide synthase gene expression in rat aortic smooth muscle cells

In the present study, we investigated the effects and mechanisms of a novel potent antioxidant, octyl caffeate, on the induction of iNOS expression by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) in cultured primary rat aortic smooth muscle cells (RASMCs) in vitro and LPS-induced hypotension in vivo. Octyl caffeate (0.1-1.0 microM) exerted a concentration-dependent inhibition of iron-catalyzed lipid peroxidation in rat brain homogenates. Furthermore, octyl caffeate (20, 50, and 100 microM) concentration-dependently diminished the initial rate of superoxide-induced NBT reduction and the enzymatic activity of xanthine oxidase. It also concentration-dependently (1-50 microM) inhibited the NO production, iNOS protein and messenger RNA expressions upon stimulation by LPS (100 microg/mL)/IFN-gamma (100U/mL) in RASMCs. In addition, we found that octyl caffeate did not significantly affect IkappaBalpha degradation stimulated by LPS/IFN-gamma in RASMCs. On the other hand, octyl caffeate (10 and 50 microM) significantly suppressed activation of c-Jun-N-terminal kinase and extracellular signal-regulated kinase. Moreover, octyl caffeate (10mg/kg, i.v.) significantly inhibited the fall in mean arterial pressure stimulated by LPS (7.5mg/kg) in rats. In conclusion, we demonstrate that a novel potent antioxidant, octyl caffeate, significantly ameliorates circulatory failure of endotoxemia in vivo by a mechanism involving suppression of iNOS expression through inactivation of mitogen-activated protein kinases in RASMCs.     

Transport of Desglycinamide-Arginine Vasopressin Across the Blood–Brain Barrier in Rats as Evaluated by the Unit Impulse Response Methodology

The pharmacokinetic characteristics of desglycinamide-arginine vasopressin (DGAVP) with respect to its transport across the blood–brain barrier (BBB) were studied with the use of serial CSF sampling in an individual animal and the unit impulse response methodology. Transport rate is determined as BBB clearance, the volume of plasma per unit time cleared of the peptide by BBB transport, and the extent of transport as the percentage of the administered dose transported into the central nervous system. Plasma kinetics of DGAVP were shown to be linear within the dose range studied (50–150 µg), plasma mean residence time (MRT) being 18 ± 4 min (mean ± SE; n = 9). Elimination of DGAVP from CSF after icv administration was linear, with an MRT of 10 ± 1 min (n = 9). After iv administration of 100 µg DGAVP, CSF concentrations were detectable for 90 min. Transport from plasma to the central nervous system was linear. The BBB transport clearance value was 1.0 ± 0.3 µl/min, and 0.026 ± 0.007% of the administered dose was transported into the central nervous system. Results demonstrate that, within the concentration range studied, DGAVP is transported across the BBB by passive diffusion, although to a very low extent.     

Absorptive-Mediated Endocytosis of an Adrenocorticotropic Hormone (ACTH) Analogue,Ebiratide, into the Blood–Brain Barrier: Studies with Monolayers of Primary Cultured Bovine Brain Capillary Endothelial Cells

The internalization of a neuromodulatory adrenocorticotropic hormone (ACTH) analogue, [¹²⁵I]ebiratide (H-Met(O₂)-Glu[¹²⁵I]His-Phe-D-Lys-Phe-NH(CH₂)₈NH₂), was examined in cultured mono-layers of bovine brain capillary endothelial cells (BCEC). HPLC analysis of the incubation solution showed that [¹²⁵I]ebiratide was not metabolized during the incubation with BCEC. The acid-resistant binding of [¹²⁵I]ebiratide to BCEC increased with time for 120 min and showed a significant dependence on temperature and medium osmolarity. Pretreatment of BCEC with dansylcadaverine or phenylarsine oxide, endocytosis inhibitors, and 2,4-dinitrophenol, a metabolic inhibitor, decreased significantly the acid-resistant binding of [¹²⁵I]ebiratide. The acid-resistant binding of [¹²⁵I]ebiratide was saturable in the presence of unlabeled ebiratide (100 nM–1 mM). The maximal internalization capacity (B _max) at 30 min was 7.96 ± 3.27 µmol/mg of protein with a half-saturation constant (K _d) of 15.9 ± 6.4 µM. The acid-resistant binding was inhibited by basic peptides such as poly-L-lysine, protamine, histone, and ACTH but was not inhibited by poly-L-glutamic acid, insulin, or transferrin. These results confirmed that ebiratide is transported through the blood-brain barrier via an absorptive-mediated endocytosis.     

A comparative study on the in vitro cytotoxic responses of two mammalian cell types to fullerenes,carbon nanotubes and iron oxide nanoparticles

The present study was designed to evaluate and compare the time- and dose-dependent cellular response of human periodontal ligament fibroblasts (hPDLFs), and mouse dermal fibroblasts (mDFs) to three different types of nanoparticles (NPs); fullerenes (C₆₀), single walled carbon nanotubes (SWCNTs) and iron (II,III) oxide (Fe₃O₄) nanoparticles via in vitro toxicity methods, and impedance based biosensor system. NPs were characterized according to their morphology, structure, surface area, particle size distribution and zeta potential by using transmission electron microscopy, X-ray diffraction, Brunauer–Emmett–Teller, dynamic light scattering and zeta sizer analyses. The Mössbauer spectroscopy was used in order to magnetically characterize the Fe₃O₄ NPs. The hPDLFs and mDFs were exposed to different concentrations of the NPs (0.1, 1, 10, 50 and 100?μg/mL) for predetermined time intervals (6, 24 and 48?h) under controlled conditions. Subsequently, NP exposed cells were tested for viability, membrane leakage and generation of intracellular reactive oxygen species. Additional to in vitro cytotoxicity assays, the cellular responses to selected NPs were determined in real time using an impedance based biosensor system. Taken together, information obtained from all experiments suggests that toxicity of the selected NPs is cell type, concentration and time dependent.     

A critical review of the data related to the safety of quercetin and lack of evidence of in vivo toxicity, including lack of genotoxic/carcinogenic properties.

Quercetin is a naturally-occurring flavonol (a member of the flavonoid family of compounds) that has a long history of consumption as part of the normal human diet. Because a number of biological properties of quercetin may be beneficial to human health, interest in the addition of this flavonol to various traditional food products has been increasing. Prior to the use of quercetin in food applications that would increase intake beyond that from naturally-occurring levels of the flavonol in the typical Western diet, its safety needs to be established or confirmed. This review provides a critical examination of the scientific literature associated with the safety of quercetin. Results of numerous genotoxicity and mutagenicity, short- and long-term animal, and human studies are reviewed in the context of quercetin exposure in vivo. To reconcile results of in vitro studies, which consistently demonstrated quercetin-related mutagenicity to the absence of carcinogenicity in vivo, the mechanisms that lead to the apparent in vitro mutagenicity, and those that ensure absence of quercetin toxicity in vivo are discussed. The weight of the available evidence supports the safety of quercetin for addition to food.     

Several transport systems contribute to the intestinal uptake of Paraquat,modulating its cytotoxic effects

Paraquat (PQ) is an extremely toxic herbicide upon oral ingestion that lacks a specific antidote. In case of intoxication, treatment primarily relies on limiting its intestinal absorption. In this study, we elucidate the intestinal transport mechanisms of PQ uptake using Caco-2 cells as a model of the human intestinal epithelium. The cells were incubated with a wide range of PQ concentrations (0–5000 μM) for 24 h with or without simultaneous exposure to different transporters substrates/inhibitors including, choline or hemicolinium-3 (for choline carrier-mediated transport system inhibition) and putrescine, trifluoperazine, valine, lysine, arginine or N-ethylmaleimide (for basic amino acid transport systems inhibition). PQ cytotoxicity was evaluated by the MTT reduction assay and correlated with PQ intracellular levels quantified by gas chromatography-ion trap-mass spectrometry (GC–IT/MS). Potential interactions of PQ with the substrates/inhibitors of the transport systems were investigated and discarded by infrared spectroscopy.     

The effect of drug loading and multiple administration on the protein corona formation and brain delivery property of PEG-PLA nanoparticles

The presence of protein corona on the surface of nanoparticles modulates their physiological interactions such as cellular association and targeting property. It has been shown that α-mangostin (αM)-loaded poly(ethylene glycol)-poly(l-lactide) (PEG-PLA) nanoparticles (NP-αM) specifically increased low density lipoprotein receptor (LDLR) expression in microglia and improved clearance of amyloid beta (Aβ) after multiple administration. However, how do the nanoparticles cross the blood?brain barrier and access microglia remain unknown. Here, we studied the brain delivery property of PEG-PLA nanoparticles under different conditions, finding that the nanoparticles exhibited higher brain transport efficiency and microglia uptake efficiency after αM loading and multiple administration. To reveal the mechanism, we performed proteomic analysis to characterize the composition of protein corona formed under various conditions, finding that both drug loading and multiple dosing affect the composition of protein corona and subsequently influence the cellular uptake of nanoparticles in b.End3 and BV-2 cells. Complement proteins, immunoglobulins, RAB5A and CD36 were found to be enriched in the corona and associated with the process of nanoparticles uptake. Collectively, we bring a mechanistic understanding about the modulator role of protein corona on targeted drug delivery, and provide theoretical basis for engineering brain or microglia-specific targeted delivery system.