Preparation andin vivo evaluation of huperzine A-loaded PLGA microspheres

Huperzine A-loaded microspheres composed of poly(D,L-lactide-co-glycolide) were prepared by an ONV emulsion solvent evaporation method. The characterization of the microspheres such as drug loading, size, shape and release profile was described. The in vitro release in the initial 7 days was nearly linear with 10% released per day. Thereafter drug release rate became slow gradually and about 90% drug released at day 21. The in vitro release rate determined by dialysis bag method had a good correlation with the in vivo release rate. Huperzine A aqueous solution was intramuscularly injected (i.m.) at 0.4 mg/kg and microspheres were intramuscularly injected at 8.4 mg eq huperzine A/kg in rats. The maxium plasma concentration (Cmax ) after i.m. microspheres was only 32% of that after i.m. solution. Drug in plasma could be detected until day 14 and about 5% of administered dose was residued at the injection site at day 14. The relative bioavailability of huperzine A microspheres over a period of 14 days was 94.7%. Inhibition of acyecholinesterase activity (AchE) in rat's cortex, hippocampus and striatum could sustain for about 14 days. In conclusion, huperzine A-loaded microspheres possessed a prolonged and complete drug release with significant inhibition of AchE for 2 weeks in rats.     

In vivo genotoxicity study of titanium dioxide nanoparticles using comet assay following intratracheal instillation in rats

Titanium dioxide (TiO₂) is widely used as a white pigment in paints, plastics, inks, paper, creams, cosmetics, drugs and foods. In the present study, the genotoxicity of anatase TiO₂ nanoparticles was evaluated in vivo using the comet assay after a single or repeated intratracheal instillation in rats. The nanoparticles were instilled intratracheally at a dosage of 1.0 or 5.0 mg/kg body weight (single instillation group) and 0.2 or 1.0 mg/kg body weight once a week for 5 weeks (repeated instillation group) into male Sprague–Dawley rats. A positive control, ethyl methanesulfonate (EMS) at 500 mg/kg, was administered orally 3 h prior to dissection. Histopathologically, macrophages and neutrophils were detected in the alveolus of the lung in the 1.0 and 5.0 mg/kg TiO₂ groups. In the comet assay, there was no increase in % tail DNA in any of the TiO₂ groups. In the EMS group, there was a significant increase in % tail DNA compared with the negative control group. TiO₂ nanoparticles in the anatase crystal phase are not genotoxic following intratracheal instillation in rats.     

Genotoxicity of organic pollutants in source of drinking water on microalga Euglena gracilis

The potential toxicities of organic pollutants in the drinking water source at Meiliang Bay of Lake Taihu were investigated by comet assay and antioxidant enzyme approach on microalgae Euglena gracilis. The organic extracts of the water samples could induce DNA damage on microalgae cells. Statistically significant differences (P < 0.05) were observed at groups of 0.3×, 3× and 10× concentrations compared with the control and a solvent control (DMSO). The organic extracts also affected antioxidant enzyme activity and induced lipid peroxidation in the microalga. In the high dose group, there was an obvious increase in SOD content (P < 0.05). The results suggest that the concentrated organics from water sample extracts have adversary effects on E. gracilis and could possibly damage the ecosystem.     

In vivo antitumor efficacy of CW252053, a folate-based thymidylate synthase inhibitor

Previous studies have demonstrated that CW252053, a quinazoline antifolate, exhibits potent inhibitory activity against thymidylate synthase (TS) as well as cytotoxic activity against tumor cell lines in vitro. In this study, we evaluated the in vivo antitumor efficacy of CW252053 in the mouse tumor model. Female B6D2F1 mice were injected with LY3.7.2C TK-/- (thymidine kinase deficient mouse lymphoma) cells into the gastrocnemius muscle. Then, CW252053 was administered twice daily by intraperitoneal injection for 10 days, and tumor growth was monitored daily by leg diameter measurement. All animals in the vehicle, 5-FU, and low dose (30 mg/kg) CW252053 treated groups died between days 12 and 23 because of the tumor burden. In contrast, dosing with 60 mg/kg of CW252053 produced a cure rate against tumor growth of 37.5% and a survival rate of 50%. Even more significantly, a higher dose of CW252053 (120 mg/kg) elicited both a 100% cure rate and a 100% survival rate at the termination of the study, confirming that this compound has very potent in vivo antitumor activity against tumor growth. During the experimental period of this study no signs of toxicity were observed even at the high CW252053 dosage rate of 120 mg/kg.     

In vitro andin vivo antifungal activities of 6-[(N-4-bromophenyl) amino]-7-chloro-5,8-quinolinediones

Antifungal activities of 6-[(N-4-bromophenyl)amino]-7-chloro-5,8-quinolinedione (RCK7) were tested. The MIC values of RCK7 were determined for antifungal suceptibility,in vitro againstAspergillus niger, Cryptococcus neoformans andTrichophyton mentagrophyte by standard agar streak method.In vitro, RCK7 showed more potent antifungal activity than fluconazole and ketoconazole. Also, RCK7 was tested forin vivo antifungal activity in the treatment of systemic infection withCandida albicans in normal mice. The therapeutic potential of RCK7 had been assessed by evaluating their survival rate against systemic infections compared with that of ketoconazole. ED₅₀ of intraperitoneally administered RCK7 was 2.05±0.30 mg/kg but that of ketoconazole was 8.00±0.73 mg/kg, respectively. When RCK7 was administered intravenously at the ED₅₀ (2.05 mg/kg), the colony counts ofCandida albicans in the liver after 7 days and 14 days were reduced as likely as ketoconazole at the ED₅₀ (8.00 mg/kg), and the better survival rates than ketoconazole’s were achieved after 14 days. The results suggest that RCK7 may be a potent antifungal agent.     

饮水铅暴露对大鼠脑组织 APE1 表达的影响及与氧化应激的关系研究

目的 探讨饮水铅暴露对大鼠大脑皮质、 小脑、 海马组织中脱嘌呤脱嘧啶核酸内切酶 1（APE1）表达的影响及其与氧化应激的关系。方法 40 只刚断乳雄性 SD 大鼠按体质量随机区组法均分为 5 组, 对照组自由饮用去离子水, 4 个铅暴露组分别饮用 100、 200、 400 和 800 mg/L 醋酸铅溶液, 连续染毒 60 d 后, 取大脑皮质、 小脑和海马组织, 测定各组的超氧化物歧化酶 （SOD） 活力、 过氧化氢 （H2O2） 水平和丙二醛 （MDA） 含量, 蛋白印迹法检测 APE1 蛋白在各组织中的表达。结果 铅暴露后, 大脑皮质、 小脑、 海马中 APE1 蛋白表达水平均低于对照组, 且随染铅剂量的升高呈逐渐下降趋势（P＜0.05）; 随着染铅剂量的升高, 大脑皮质、 小脑和海马中的 SOD 活力基本呈下降趋势; 而 H2O2及 MDA 含量随染铅剂量的升高基本呈逐渐升高趋势, 大脑皮质、 小脑和海马组织的 APE1 蛋白表达水平与其 SOD 活力呈正相关（r 分别为 0.619、 0.380、 0.375, P < 0.05） ,而与 H2O2水平和 MDA 含量呈负相关（r 分别为-0.472、 -0.535、 -0.436, -0.514、 -0.486、 -0.316, P < 0.05）。结论 饮水铅暴露可导致大鼠脑组织 APE1 蛋白表达水平改变,且此种改变与铅所致的氧化应激有关。     

In vitro and in vivo evaluation of the effects of piperine on P-gp function and expression

Piperine, a major component of black pepper, is used as spice and nutrient enhancer. The purpose of the present study was to evaluate the effects of acute and prolonged piperine exposure on cellular P-gp expression and function in vitro and in vivo. Piperine at concentrations ranging from 10 to 100 microM, determined by MTT assay to be non-cytotoxic, was observed to inhibit P-gp mediated efflux transport of [(3)H]-digoxin across L-MDR1 and Caco-2 cell monolayers. The acute inhibitory effect was dependent on piperine concentration, with abolishment of [(3)H]-digoxin polarized transport attained at 50 microM of piperine. In contrast, prolonged (48 and 72 h) co-incubation of Caco-2 cell monolayers with piperine (50 and 100 microM) increased P-gp activity through an up-regulation of cellular P-gp protein and MDR1 mRNA levels. The up-regulated protein was functionally active, as demonstrated by a higher degree of [(3)H]-digoxin efflux across the cell monolayers, but the induction was readily reversed by the removal of the spice from the culture medium. Peroral administration of piperine at the dose of 112 microg/kg body weight/day to male Wistar rats for 14 consecutive days also led to increased intestinal P-gp levels. However, there was a concomitant reduction in the rodent liver P-gp although the kidney P-gp level was unaffected. Our data suggest that caution should be exercised when piperine is to be co-administered with drugs that are P-gp substrates, particularly for patients whose diet relies heavily on pepper.     

Establishing a total allowable concentration of o-toluidine in drinking water incorporating early lifestage exposure and susceptibility

o-Toluidine is a monocyclic aromatic amine present in the formulation of some materials that contact drinking water. NSF/ANSI 61 Annex A (2011) and US EPA (2005a) risk assessment guidelines were used to determine an acceptable drinking water level. Occupational exposure to o-toluidine is associated with an increased risk of bladder cancer but human disease rates could not be used to establish risk values due to inadequate exposure data and coexposures in the epidemiology cohorts. Chronic dietary exposure to o-toluidine hydrochloride was associated with benign and malignant tumors in both sexes of F344 rats and B6C3F1 mice. o-Toluidine is genotoxic in vitro and in vivo. A 10⁻⁵ cancer risk level was extrapolated from the human equivalent BMDL₁₀ of 13 mg/kg-day for the combined incidence of papillomas and carcinomas of the bladder transitional epithelium in female rats. Considering varying susceptibility to tumor development at different life stages, the unit risk was modified to incorporate potency adjustments for early-life exposures. A framework for lifestage adjustment is presented that makes assumptions evident. For this assessment, the lifetime unit risk derived was ∼2-fold greater than the unadjusted adult lifetime unit risk, and the resulting Total Allowable Concentration in drinking water is 20 μg/L.     

The effects of lipophilic substances on the shape of erythrocytes demonstrated by a new in vitro-method

Low aqueous solubility of lipophilic agents, such as free fatty acids, hampers proper in vitro demonstration of biological effects, yielding an ambiguous in vitro–in vivo correlation. We have therefore developed a method for evaluating the acute effects of lipophilic substances on the shape of erythrocytes and estimated EC₅₀ and Hill coefficient according to the sigmoidal E_max model.The test substance dissolved in medium-chain triglyceride is coated on a polycarbonate slide which serves as a cover sheet of a Bürker chamber. Freshly collected finger-tip blood is diluted with autologous EDTA–plasma and introduced into the chamber. After 10 min at 37 °C, the cells are photographed under microscope and the fractions of normal and defect cells are evaluated. No staining is needed and the cells are kept viable during the test period.With increasing chain length, fatty acids, aliphatic amines and alcohols all increased the fraction of defect erythrocytes in a concentration-dependent manner. The results indicate that several fatty acids are very potent in their acute actions on erythrocytes, and that this effect is due to chain length rather than conformation.

Conclusion

The technique offers a screening method for testing the harmful effects of small amounts of lipophilic substances on erythrocytes.     

Effects of the Yangtze River source of drinking water on metabolites of Mus musculus

The effects of the Yangtze River source of drinking water on metabolites of mouse (Mus musculus) were implemented to observe the environmental health issue of the water by use of the ¹H nuclear magnetic resonance (NMR) spectroscopy-based metabonomics. All the sampled mice were treated for 90 days. There were 20 organic pollutants discovered in the water with total concentration of 9.41 μg/l. The NMR spectra for the sampled mice were different at δ2.06, δ2.24 and δ5.22. The concentrations of alanine, glycoprotein, acetone and trimethylamine-N-oxide in the source water group mice were decreased but that of creatinine and glucose were increased, which indicated that hepatotoxicity and kidney dysfunction occurred. There were six parameters for the source water group mice were different or extremely different from that of the control group. And these metabolites are responsible for separation of the data along PC1 and PC2 which may be used as biomarkers to indicate the source water pollution. The results indicate that ¹H NMR-based metabonomic approach is a useful technique to test toxicity of xenobiotics on metabolites for observation of the environmental health issue of source water.     

基于生物可给性和靶器官毒性剂量法的乌梢蛇中铅和砷联合暴露评估

目的 通过考察乌梢蛇Zaocys dhumnades（Cantor）中铅（Pb）和砷（As）的生物可给性,并探索靶器官毒性剂量（TTD）法在评估中药中重金属联合暴露风险中的应用,为限量标准的制定提供参考。方法 通过胃-肠两步模拟消化（in vitro PBET）联合电感耦合等离子体质谱（ICP-MS）法对乌梢蛇中Pb和As的生物可给性进行考察,根据其生物可给性计算其日暴露量。采用危害指数（HI）法对于Pb和As联合暴露产生的健康风险进行初步筛查;进一步针对不同毒理学终点,采用TTD法对Pb和As的累积风险进行更加精确的评估。结果 8批乌梢蛇中Pb和As的合格率为100%。HI法的初步评估结果表明,所有批次乌梢蛇中Pb和As的HI值均＜1。TTD法评估结果表明,作用终点心血管系统、神经系统、肾脏、血液和睾丸,所有批次乌梢蛇的HI值均＜1,健康风险可接受。结论 基于生物可给性,探索乌梢蛇中Pb和As的累积风险评估方法,为中药外源性有害残留物风险评估的方法开发提供新的思路,为制定更加科学的限量标准提供技术支撑。     

5-氟尿嘧啶-聚α,β(2-羟乙基)-DL-天冬酰胺的合成及体内释放的研究

以5-氟尿嘧啶为模型药物,将药物以共价键的形式键合于生物降解型高分子材料聚-α,β(2-羟乙基)-DL-天冬酰胺上制成高分子载体药物,药物接入率达37.1%(w/w)。用红外和差热分析法对载体药物进行了表征。以纯种大白兔为实验动物,把载体药物制成混悬型和棒状型两种剂型,进行药物体内释放实验。结果表明:以棒状型药物给药在一定程度上可以降低释药初期的“爆释”现象,为进一步临床应用提供了重要依据。     

In vitro angiogenic activity ofAloe vera gel on calf pulmonary artery endothelial (CPAE) cells

Angiogenic activity ofAloe vera gel was investigated byin vitro assay. We obtained the most active fraction from dichloromethane extract ofAloe vera gel by partitioning between hexane and 90% aqueous methanol. The most active fraction (F3) increased the proliferation of calf pulmonary artery endothelial (CPAE) cells. In addition, F3 fraction induced CPAE cells to invade type I collagen gel and form capillary-like tube throughin vitro angiogenesis assay, and increased the invasion of CPAE cells into matrigel throughin vitro invasion assay. Furthermore, the effect on the mRNA expression of proteolytic enzymes which are key participants in the regulation of extracellular matrix degradation was investigated by northern blot analysis. F3 fraction enhanced mRNA expression of urokinase-type plasminogen activator (u-PA), matrix metalloproteinase-2 (MMP-2), and membrane-type MMP (MT-MMP) in CPAE cells whereas the expression of plasminogen activator inhibitor-1 (PAL-1) mRNA was not changed.     

In vitro and in vivo anti-hepatitis B virus activities of a plant extract from Geranium carolinianum L

Natural products provide a large reservoir of potentially active agents with anti-hepatitis B virus (HBV) activity. We examined the effect of the polyphenolic extract from Geranium carolinianum L. (PPGC) on HBV replication both in vitro and in vivo. In the human HBV-transfected liver cell line HepG(2) 2.2.15, PPGC effectively suppressed the secretion of the HBV antigens in a dose-dependent manner with IC(50) values of 46.85 microg/ml for HBsAg and 65.60 microg/ml for HBeAg at day 9. Consistent with the HBV antigen reduction, PPGC (100 microg/ml) also reduced HBV DNA level by 35.9%. In the duck hepatitis B virus (DHBV) infected ducks, after PPGC was dosed intragastricly (i.g.) once a day for 10 days, the plasma DHBV DNA level was reduced, with an ED(50) value of 47.54 mg/kg. In addition, Southern blot analysis confirmed the in vivo anti-HBV effect of PPGC in ducks and PPGC also reduced the plasma and the liver DHBV DNA level in a dose-dependent manner. Furthermore, significant improvement of the liver was observed after PPGC treatment, as evaluated by the histopathological analysis.     

双(α-呋喃甲酸)氧钒对糖尿病大鼠血糖的调节作用

目的研究双(α-呋喃甲酸)氧钒(VO-FA)对正常大鼠及链脲佐霉素(STZ)性糖尿病大鼠的降糖作用。方法以STZ诱导大鼠糖尿病模型。观察VO-FA灌胃给药后，对血糖、糖化血红蛋白、糖原和血清胰岛素等的影响。结果VO-FA ig两周后，剂量依赖性地降低STZ性糖尿病动物的血糖水平，但对正常动物的血糖无影响。VO-FA 56.8 mg·kg^-1可降低正常动物血清胰岛素水平，加倍剂量尚能降低STZ性糖尿病大鼠的血清胰岛素水平。此外，VO-FA 能降低糖尿病大鼠的糖化血红蛋白，明显改善糖耐量，增加肌糖原和肝糖原的含量，并呈现出量效关系，但对正常动物则没有影响。结论VO-FA可以改善糖尿病动物的糖代谢，而不影响正常动物的血糖水平。     

Study on dissolution and absorption of four dosage forms of isosorbide mononitrate: Level A in vitro–in vivo correlation

The objective of the present study was to develop a novel in vitro system to simulate the process of dissolution and permeation of oral solid dosage forms in vivo, and to establish a correlation between in vitro permeation and in vivo absorption that could predict the bioavailability (BA) and bioequivalence (BE) of congeneric products. The in vitro dissolution and absorption kinetics of four dosage forms of isosorbide mononitrate (ISMN) were evaluated by the USP basket/paddle system and drug dissolution/absorption simulating system (DDASS). The corresponding pharmacokinetic study was performed in beagle dogs. A comparative study was carried out between the classical and the novel method to estimate the effectiveness of the modified DDASS in simulating the course of dissolution and absorption in vivo. Indeed, the correlation coefficients of in vitro dissolution and in vivo absorption obtained from DDASS and dogs were higher. Moreover, a higher level A in vitro–in vivo correlation (IVIVC) between DDASS permeation and dog absorption was established, with correlation coefficients of 0.9968, 0.9872, 0.9921, and 0.9728. The DDASS method was more accurate at modeling the process of dissolution and absorption in vivo for both immediate-release (IR) and sustained-release (SR) dosage forms of ISMN.     

Effect of chronic exposure to aluminium on isoform expression and activity of rat (Na+/K+)ATPase.

The ability of aluminum to inhibit the (Na(+)/K(+))ATPase activity has been observed by several investigators. The (Na(+)/K(+))ATPase is characterized by a complex molecular heterogeneity that results from the expression and differential association of multiple isoforms of both catalytic (alpha) and regulatory (beta) subunits. For instance, three main alpha (alpha(1), alpha(2) and alpha(3)) and three beta (beta(1), beta(2) and beta(3)) subunit isoforms exist in vertebrate nervous tissue, whereas only alpha(1) and beta(1) have been identified in kidney. However, no studies have focused on determining the change in (Na(+)/K(+))ATPase isoforms caused by chronic exposure to aluminum and its relation with aluminum toxicity. In this study, adult male Wistar rats were submitted to chronic dietary AlCl(3) exposure (0.03 g/day of AlCl(3) for 4 months), and the activity and protein expression of (Na(+)/K(+))ATPase isozymes were studied in brain cortex synaptosomes and in kidney homogenates. The intracellular levels of adenine nucleotides, plasma membrane integrity, and aluminum accumulation were also studied in brain synaptosomes. Aluminum accumulation upon chronic dietary AlCl(3) administration significantly decreased the (Na(+)/K(+))ATPase activity measured in the presence of nonlimiting Mg-ATP concentrations, without compromising protein expression of alpha-subunit isoforms in brain and kidney. Aluminum-induced synaptosomal (Na(+)/K(+))ATPase inhibition was due to a reduction in the activity of isozymes containing alpha(1)-alpha(2) and alpha(3)-subunits. The onset of enzyme inhibition was accompanied by a decrease of the (Na(+)/K(+))ATPase sensitivity to submicromolar concentrations of ouabain, and it preceded major damage in plasma membrane integrity and energy supply, as revealed by the analysis of lactate dehydrogenase leakage and endogenous adenine nucleotides. The data suggest that, during chronic dietary exposure to AlCl(3), brain (Na(+)/K(+))ATPase activity drops, even if no significant alterations of catalytic subunit protein expression, cellular energy depletion, and changes in cell membrane integrity are observed. Implications regarding underlying mechanisms of aluminum neurotoxicity are discussed.     

Effects of alpha(1)-adrenoceptors and muscarinic cholinoceptors on water intake in rats

The present study investigated the effects of alpha(1)-adrenoceptors and muscarinic cholinoceptors on water intake in adult male rats. Intracerebroventricular (i.c.v.) injections were carried out in all experiments after 24-h deprivation of water. After deprivation, the volume of consumed water was measured for 1 h. Administration of pilocarpine, a muscarinic cholinoceptor agonist (0.5-1 microg/rat), and prazosin, the alpha(1)-adrenoceptors antagonist (2 microg/rat), increased, while scopolamine, a muscarinic cholinoceptor antagonist (5-10 microg/rat), and phenylephrine, an alpha(1)-adrenoceptor agonist (30 microg/rat), decreased water intake in rats. The activation of muscarinic cholinoceptors by pilocarpine attenuated the inhibitory effect induced by phenylephrine. Blockade of muscarinic cholinoceptors did not change the phenylephrine-induced response. Pretreatment with prazosin decreased the pilocarpine-induced response. However, pharmacological blockade of muscarinic cholinoceptors by scopolamine decreased the prazosin-induced effect on water intake. It is concluded that muscarinic cholinoceptors and alpha(1)-adrenoceptors may interact on water intake.     

In vivo toxicity studies of europium hydroxide nanorods in mice

Lanthanide nanoparticles and nanorods have been widely used for diagnostic and therapeutic applications in biomedical nanotechnology due to their fluorescence and pro-angiogenic properties to endothelial cells, respectively. Recently, we have demonstrated that europium (III) hydroxide [Eu^III(OH)₃] nanorods, synthesized by the microwave technique and characterized by several physico-chemical techniques, can be used as pro-angiogenic agents which introduce future therapeutic treatment strategies for severe ischemic heart/limb disease, and peripheral ischemic disease. The toxicity of these inorganic nanorods to endothelial cells was supported by several in vitro assays. To determine the in vivo toxicity, these nanorods were administered to mice through intraperitoneal injection (IP) everyday over a period of seven days in a dose dependent (1.25 to 125 mg kg^− 1 day^− 1) and time dependent manner (8–60 days). Bio-distribution of europium elements in different organs was analyzed by inductively coupled plasma mass spectrometry (ICPMS). Short-term (S-T) and long-term (L-T) toxicity studies (mice euthanized on days 8 and 60 for S-T and L-T, respectively) show normal blood hematology and serum clinical chemistry with the exception of a slight elevation of liver enzymes. Histological examination of nanorod-treated vital organs (liver, kidney, spleen and lungs) showed no or only mild histological changes that indicate mild toxicity at the higher dose of nanorods.     

Influence of Surface-Modifying Surfactants on the Pharmacokinetic Behavior of 14C-Poly (Methylmethacrylate) Nanoparticles in Experimental Tumor Models

Purpose. The aim of this study was to investigate the different pharmacokinetic behavior of surface-modified poly(methylmethacrylate) (PMMA) nanoparticles. Methods. The particles were ¹⁴C-labeled and coated with polysorbate 80, poloxamer 407, and poloxamine 908. Plain particles served as control particles. In vivo studies were performed in three tumor models differing in growth, localization, and origin. Particle suspensions were administered via the tail vein, and at given time animals were killed and organs were dissected for determination of PMMA concentration. Results. For the PMMA nanoparticles coated with poloxamer 407 or poloxamine 908, high and long-lasting concentrations were observed in the melanoma and at a lower level in the breast cancer model. In an intracerebrally growing glioma xenograft, the lowest concentrations that did not differ between the tumor-loaded and tumor-free hemispheres were measured. Organ distribution of the four investigated batches differed significantly. For instance, poloxamer 407- and poloxamine 908-coated particles circulated over a longer period of time in the blood, leading additionally to a higher tumor accumulation. In contrast, plain and polysorbate 80-coated particles accumulated mainly in the liver. The strong expression of vascular endothelial growth factor and Flk-1 in the melanoma correlated with high concentrations of PMMA in this tumor. Conclusion. The degree of accumulation of PMMA nanoparticles in tumors depended on the particle surface properties and the specific growth differences of tumors.