In vitro metabolism and covalent binding of ethylbenzene to microsomal protein as a possible mechanism of ethylbenzene-induced mouse lung tumorigenesis

This study was conducted to determine species differences in covalent binding of the reactive metabolites of ethylbenzene (EB) formed in the liver and lung microsomes of mouse, rat and human in the presence of NADPH. These data further the understanding of the mechanism by which EB causes mouse specific lung toxicity and a follow-up to our earlier report of the selective elevation, although minor, of the ring-oxidized reactive metabolites in mouse lung microsomes (Saghir et al., 2009). Binding assays were also conducted with or without 5-phenyl-1-pentyne (5P1P), an inhibitor of CYP 2F2, and diethyldithiocarbamate (DDTC), an inhibitor of CYP 2E1 to evaluate their role in the formation of the related reactive metabolites. Liver and lung microsomes were incubated with ¹⁴C-EB (0.22 mM) in the presence of 1 mM NADPH under physiological conditions for 60 min. In lung microsomes, binding activity was in the order of mouse (812.4 ± 102.2 pmol/mg protein) >> rat (57.0 ± 3.2 pmol/mg protein). Human lung microsomes had little binding activity (15.7 ± 1.4 pmol/mg protein), which was comparable to the no-NADPH control (9.9–16.7 pmol/mg protein). In liver microsomes, mouse had the highest activity (469.0 ± 38.5 pmol/mg protein) followed by rat (148.3 ± 14.7 pmol/mg protein) and human (89.8 ± 3.0 pmol/mg protein). Presence of 5P1P or DDTC decreased binding across species and tissues. However, much higher inhibition was observed in mouse (86% [DDTC] and 89% [5P1P]) than rat (56% [DDTC] and 59% [5P1P]) lung microsomes. DDTC showed ∼2-fold higher inhibition of binding in mouse and human liver microsomes than 5P1P (mouse = 85% vs. 40%; human = 59% vs. 36%). Inhibition in binding by DDTC was much higher (10-fold) than 5P1P (72% vs. 7%) in rat liver microsomes. These results show species, tissue and enzyme differences in the formation of reactive metabolites of EB. In rat and mouse lung microsomes, both CYP2E1 and CYP2F2 appear to contribute in the formation of reactive metabolites of EB. In contrast, CYP2E1 appears to be the primary CYP isozyme responsible for the reactive metabolites of EB in the liver.     

CYP2B6 haplotype and biological factors responsible for hepatotoxicity in HIV-infected patients receiving efavirenz-based antiretroviral therapy

Data on the pharmacogenetic markers of CYP2B6 and biological factors associated with hepatotoxicity in HIV-infected patients receiving an efavirenz-based antiretroviral therapy (ART) regimen are very limited. A total of 134 HIV-infected Thai adults were prospectively enrolled to receive a once-daily regimen of efavirenz 600 mg/tenofovir/lamivudine. Seven single nucleotide polymorphisms (SNPs) within CYP2B6 were genotyped using real-time PCR. At 12 weeks after ART, plasma efavirenz concentrations at 12 h after dosing were measured. The mean ± standard deviation patient age was 37 ± 8 years, and 77.6% were male. The median (IQR) CD4 count was 43 cells/mm³ (17–105 cells/mm³). Eighteen patients (13.4%) had positive anti-HCV and 5 patients (3.7%) had positive HBsAg. The frequencies of heterozygous/homozygous mutants of each SNP were 64C>T (11%), 499C>G (0%), 516G>T (55%), 785A>G (63%), 1375A>G (0%), 1459C>T (3%) and 21563C>T (62%). The three most frequent haplotypes identified included *1/*6 (40.3%), *1/*1 (34.3%) and *6/*6 (8.2%). The median (IQR) plasma efavirenz concentration was 2.3 mg/L (1.4–3.7 mg/L). At 24 weeks, median (IQR) serum ALP was 98 mg/dL (73–133 mg/dL) and direct bilirubin was 0.11 mg/dL (0.10–0.19 mg/dL). The proportion of grade 1 and grade 2 elevated serum ALP was 12.7% and 1.5%, respectively. By multivariate analysis, factors associated with high ALP, total bilirubin and direct bilirubin included CYP2B6 haplotype *6/*6, high serum ALP at Week 0 and positive anti-HCV (all P < 0.05). In summary, HIV-infected patients with the pharmacogenetic marker ‘CYP2B6 haplotype *6/*6’ may have increased susceptibility to hepatotoxicity with efavirenz-based ART.     

Intracellular disposition of arabinogalactan and asialofetuin in HepG2 cells

The binding and uptake of arabinogalactan and asialofetuin in HepG2 cells was kinetically characterized using ^I25I-labeled ligands. The number of binding sites (n) and the association constant (K) of arabinogalactan was 1.9 × 10⁵ ± 1.2 × 10⁵ sites/cell and 5.0 × 10⁶ ± 3.9 × 10⁶ M^? 1, respectively, whereas the n and K_a of asialofetuin was 2.7 × 10⁵ ± 1.1 × 10⁵ sites/cell and 1.1 × 10⁷ ± 0.7 × 10⁷ M^? 1, respectively. These results suggest that the binding capacity of HepG2 cells for arabinogalactan is lower than that for asialofetuin. Moreover, the amount of arabinogalactan uptake by HepG2 cells was lower than that of asialofetuin. Thus, asialofetuin was preferentially bound and internalized by hepatoma cells compared to arabinogalactan.     

The prototypic pharmacogenetic drug debrisoquine is a substrate of the genetically polymorphic organic cation transporter OCT1

Debrisoquine is a probe drug for in vivo phenotyping of human CYP2D6 metabolic activity. However, debrisoquine is positively charged under physiological conditions and it is unclear how it enters the hepatocytes to undergo CYP2D6 metabolism. We analysed whether debrisoquine is a substrate of the hepatic organic cation transporter OCT1 and whether drug–drug interactions at OCT1, or polymorphisms in OCT1 gene, affect debrisoquine uptake.Debrisoquine showed low carrier-independent membrane permeability (P_e of 0.01 × 10^?6 cm/s in artificial PAMPA membranes) and strongly inhibited the uptake of the model OCT1 substrate MPP⁺ (IC₅₀ of 6.2 ± 0.8 μM). Debrisoquine uptake was significantly increased in HEK293 cells overexpressing OCT1 compared to control cells. The OCT1-mediated uptake of debrisoquine followed Michaelis–Menten kinetics (K_M of 5.9 ± 1.5 μM and V_max of 41.9 ± 4.5 pmol/min/mg protein) and was inhibited by known OCT1 inhibitors and by commonly used drugs. OCT1-mediated debrisoquine uptake was reduced or missing in cells expressing loss-of-function OCT1 isoforms. Deletion of Met420 or substitution of Arg61Cys or Gly401Ser reduced V_max by 48, 63 and 91%, respectively, but did not affect the K_M. The OCT1 isoforms carrying Cys88Arg or Gly465Arg substitutions completely lacked OCT1-mediated debrisoquine uptake.In conclusion, debrisoquine is a substrate of OCT1 and has the potential to be used as a phenotyping marker for OCT1 activity. Moreover, variations in debrisoquine metabolic phenotypes and their associations with diseases may be due not only to genetic variations CYP2D6, but also in OCT1.     

Activation of nicotinamide N-methyltrasferase and increased formation of 1-methylnicotinamide (MNA) in atherosclerosis

Nicotinamide N-methyltrasferase (NMMT) catalyzes the conversion of nicotinamide (NA) to 1-methylnicotinamide (MNA). Recent studies have reported that exogenous MNAexerts anti-thrombotic and anti-inflammatory activity, suggesting that endogenous NMMT-derived MNA may play a biological role in the cardiovascular system.In the present study, we assayed changes in hepatic NNMTactivity and MNAplasma levels along the progression of atherosclerosis in apoE/LDLR^?/? mice, as compared to age-matched wild-type mice. Atherosclerosis progression in apoE/LDLR^?/? mice was quantified in aortic root, while hepatic NNMT activity and MNAplasma concentrations were concomitantly measured in 2-, 3-, 4-, and 6-month-old mice.In apoE/LDLR^?/? mice, atherosclerotic plaques developed in the aortic roots beginning at the age of 3 months and gradually increased in size, macrophage content, and inflammation intensity over time, as detected by Oil-Red O staining, CD68 immunostaining, and in situ zymography (MMP2/MMP9 activity). Hepatic NNMT activity was upregulated approximately two-fold in apoE/LDLR^?/? mice by the age of 2 months, as compared to wild-type mice (1.03 ± 0.14 vs. 0.64 ± 0.23 pmol/min/mg, respectively). MNAplasma concentrations were also elevated approximately two-fold (0.30 ± 0.13 vs. 0.17 ± 0.04 μmol/l, respectively). As atherosclerosis progressed, hepatic NMMTactivity and MNAplasma concentrations increased five-fold in 6-month-old apoE/LDLR^?/? mice at the stage of advanced atherosclerotic plaques (NMMT activity: 2.29 ± 0.34 pmol/min/mg, MNA concentration: 1.083 ± 0.33 μmol/l).In summary, the present study demonstrated that the progression of vascular inflammation and atherosclerosis was associated with the upregulation of hepatic NNMT activity and subsequent increase in endogenous MNAplasma levels. Given the anti-thrombotic and anti-inflammatory properties of exogenous MNA, robust activation of an endogenous NA-MNApathway in atherosclerosis may play an important compensatory role.     

The influence of CYP2A6 polymorphisms and cadmium on nicotine metabolism in Thai population

We investigated the influence of genetic, cadmium exposure and smoking status, on cytochrome P450-mediated nicotine metabolism (CYP2A6) in 182 Thai subjects after receiving 2 mg of nicotine gum chewing for 30 min. The urinary excretion of cotinine was normally distributed over a 2 h period (logarithmically transformed). Individuals with urinary cotinine levels in the ranges of 0.01–0.21, and 0.52–94.99 μg/2 h were categorized as poor metabolizes (PMs: 6.5%), and extensive metabolizers (EMs: 93.5%), respectively. The majority of EMs (45%) carried homozygous wild-type genotypes (CYP2A6*1A/*1A, CYP2A6*1A/*1B and CYP2A6*1B/*1B), whereas only 1% of PMs carried these genotypes. Markedly higher frequencies of EMs were also observed in all heterozygous defective genotypes including the null genotype (*4C/*4C; 1 subject).A weak but significant positive correlation was observed between total amounts of urinary cadmium excretion and total cotinine excretion over 2 h. Our study shows generally good agreement between CYP2A6 genotypes and phenotypes. Smokers accumulated about 3–4-fold higher mean total amounts of 2-h urinary cadmium excretion (127.5 ± 218.2 ng/2 h) than that of non-smokers (40.5 ± 78.4 ng/2 h). Among the smokers (n = 16), homologous wild-type genotype *1/*1 was significantly the predominant genotype (6/16) compared with other defective allele including *4C/*4C. In addition, 2 h urinary excretion of cotinine in smokers of all genotypes was significantly higher than non-smokers. The proportion of smokers who smoked more than 5 cigarettes/day was significantly higher in EMs in all CYP2A6 genotypes (n = 14) than in PMs (n = 0).     

Evaluation of cytochrome P450 1 (CYP1) and N-acetyltransferase 1 (NAT1) activities in HaCaT cells: Implications for the development of in vitro techniques for predictive testing of contact sensitizers

Xenobiotic metabolizing enzymes like cytochrome P450s and N-acetyltransferase are expressed in keratinocytes and professional antigen-presenting cells. Thus, biotransformation of chemicals applied to the skin can be relevant for their potential to cause skin toxicity and immune responses like allergic contact dermatitis. Considering the keratinocyte cell line HaCaT as a relevant in vitro tool for epidermal biotransformation, we specifically investigated CYP1 (EROD) and N-acetyltransferase 1 (NAT1) activities of three different HaCaT shipments and human primary keratinocytes (NHEK). Solvent treated HaCaT showed EROD levels near the detection limit (0.047 pmol/mg/min), primary keratinocytes (n = 4) were in a range between 0 and 0.76 pmol/mg/min. B[a]P (1 μM) induced EROD activities of 19.0 ± 0.9 pmol/mg/min (n = 11) in HaCaT and 5.8 ± 0.5 pmol/mg/min (n = 4) in NHEK. N-acetylation activities for para-aminobenzoic acid (PABA) were in average 3.4-fold higher in HaCaT compared to NHEK (8 ± 0.5 nmol/mg/min) and varied between the HaCaT shipments (range 12.0–44.5 nmol/mg/min). This was in good agreement with NAT1 promoter P1 dependent mRNA level and N-acetylation of the contact allergen para-phenylenediamine (PPD) under typical cell-based assay conditions. We conclude that HaCaT represent a suitable in vitro model for studying the qualitative contribution of epidermal phase1/phase2 metabolism to toxicological endpoints such as skin sensitization.     

Covalent inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by the carbamates JZL184 and URB597

Carboxylesterase type 1 (CES1) and CES2 are serine hydrolases located in the liver and small intestine. CES1 and CES2 actively participate in the metabolism of several pharmaceuticals. Recently, carbamate compounds were developed to inhibit members of the serine hydrolase family via covalent modification of the active site serine. URB597 and JZL184 inhibit fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively; however, carboxylesterases in liver have been identified as a major off-target. We report the kinetic rate constants for inhibition of human recombinant CES1 and CES2 by URB597 and JZL184. Bimolecular rate constants (k_inact/K_i) for inhibition of CES1 by JZL184 and URB597 were similar [3.9 (±0.2) × 10³ M^?1 s^?1 and 4.5 (±1.3) × 10³ M^?1 s^?1, respectively]. However, k_inact/K_i for inhibition of CES2 by JZL184 and URB597 were significantly different [2.3 (±1.3) × 10² M^?1 s^?1 and 3.9 (±1.0) × 10³ M^?1 s^?1, respectively]. Rates of inhibition of CES1 and CES2 by URB597 were similar; however, CES1 and MAGL were more potently inhibited by JZL184 than CES2. We also determined kinetic constants for spontaneous reactivation of CES1 carbamoylated by either JZL184 or URB597 and CES1 diethylphosphorylated by paraoxon. The reactivation rate was significantly slower (4.5×) for CES1 inhibited by JZL184 than CES1 inhibited by URB597. Half-life of reactivation for CES1 carbamoylated by JZL184 was 49 ± 15 h, which is faster than carboxylesterase turnover in HepG2 cells. Together, the results define the kinetics of inhibition for a class of drugs that target hydrolytic enzymes involved in drug and lipid metabolism.     

Binding of new aminopropan-2-ol compounds to bovine serum albumin, α1 acid glycoprotein and rat serum using equilibrium dialysis and LC/MS/MS

BackgroundThe binding of three new aminopropan-2-ol compounds briefly called 2F109, ANBL and TWo8 with potential cardiovascular activity to bovine serum albumin (BSA), α₁-acid glycoprotein (AGP) and to rat serum was studied. The chemical structures of these compounds are related to carvedilol. They possess an antiarrhythmic and hypotensive activity, and β- and α-adrenolytic mechanism of action. All analogues are weak bases with pKa values 8.65,8.85 and 8.26 for 2F109, ANBL and TWo8, respectively, and they possess lipophilic character (log P > 1.9584).MethodsThe extent of protein binding was determined using equilibrium dialysis in the range 2.5 – 900 μM, and 2.5 – 300 μM for binding of investigated compounds to BSA and AGP, respectively, and the quantitative measurement was done by LC/ESI-MS/MS assay.ResultsThe studied compounds bound to a single class of binding sites on BSA which was characterized by low affinity (K_d for 2F109 = 8.49 × 10^–5 M, for ANBL = 1.92 × 10^–5 M, and for TWo8 = 1.71 × 10^–5 M) and low capacity(n = 0.53 for 2F109,0.132 for ANBL and 0.13 for TWo8). The binding of 2F109, ANBL and TWo8 to AGP revealed one class of binding sites, with moderate affinity (K_d for 2F109 = 4.67 × 10^–6 M, for ANBL = 3.48 × 10^–5 M, and for TWo8 = 1.13 × 10^–5 M) and higher capacity (n = 2.21 for 2F109, 2.76 for ANBL and 2.28 for TWo8).ConclusionThe obtained data indicate that 2F109, ANBL and TWo8 moderately bind to BSA (34.2 – 71.2%) with low capacity (K_a = 6.21 × 10³–7.61 × 10³ M^–1)and strongly bind to AGP(71.5–85.5%)with moderate affinity (K_a = 7.94 × 104⁴.73 ×10⁵ M^–1).     

Activity of the enzymes of the antioxidative system in cadmium-treated Oxya chinensis (Orthoptera Acridoidae)

One purpose in this research was to determine the toxic effects of Cd on antioxidant enzymes of Oxya chinensis (Orthoptera: Acridoidae). Changes in the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (GPx) were measured in O. chinensis insects injected with Cd²⁺. Fifth-nymphs of O. chinensis insects were injected with Cd²⁺ at different concentrations (0, 0.55 × 10⁻⁴, 1.10 × 10⁻⁴, 1.65 × 10⁻⁴, 2.20 × 10⁻⁴, and 2.75 × 10⁻⁴ g g⁻¹). An increase in SOD activity in O. chinensis was observed at 1.10 × 10⁻⁴ to 2.75 × 10⁻⁴ g g⁻¹ Cd²⁺. The SOD activity was lower at 2.20 × 10⁻⁴and 2.75 × 10⁻⁴ g g⁻¹ than that at 1.10 × 10⁻⁴ and 1.65 × 10⁻⁴ g g⁻¹. It appears that SOD had a positive protective effect at low Cd²⁺ concentrations, and that this effect disappeared at high Cd²⁺ concentrations. CAT activity was accelerated to varying degrees at 1.10 × 10⁻⁴ to 2.75 × 10⁻⁴ g g⁻¹ for males and at 1.10 × 10⁻⁴, 2.20 × 10⁻⁴, and 2.75 × 10⁻⁴g g⁻¹ for females. CAT showed a strong detoxification effect with all treatments. GPx activity decreased with increasing Cd²⁺ concentration with all treatments for males and at 2.20 × 10⁻⁴ and 2.65 × 10⁻⁴g g⁻¹ for females. We showed that GPx activity had a weak detoxification function with all treatments for males and at high Cd²⁺ for females. Thus, CAT had a strong detoxification effect, whereas SOD had a medium and GPx had a weak detoxification effect. Among the three enzymes, CAT played an important role in the damaging mechanisms of reactive oxygen species in O. chinensis insects. Alterations of the antioxidant enzyme level under environmental stresses are suggested as indicators of biotic and abiotic stress.     

Pyrethroid insecticide exposure and semen quality of young Japanese men

The present study aimed at assessing the relationship between exposure to pyrethroid insecticides and semen quality in 323 university students recruited in a population-based manner in Metropolitan Tokyo. Urinary concentrations of pyrethroid insecticide metabolite, 3-phenoxybenzoic acid (3-PBA), were measured by LC/MS/MS and semen parameters were measured by following internationally harmonized protocols. Median urinary 3-PBA concentration was 0.641 ng/mL (specific gravity-adjusted, n = 322). Median values of semen volume, sperm concentration, motility, total number of sperm, and total number of motile sperm were 2.5 mL, 56 × 10⁶/mL, 61%, 141 × 10⁶, and 82 × 10⁶, respectively. Urinary concentration of 3-PBA was not selected as significant in multiple regression models indicating, in contrast to previous findings, that environmental exposure to pyrethroid insecticides did not affect semen quality. This inconsistency may be related to exposure to different pyrethroid insecticides and/or levels of exposure as well as to survey design (hospital- vs population-based subject recruitment).     

Menin regulates spinal glutamate-GABA balance through GAD65 contributing to neuropathic pain

BackgroundOur previous work found that tumor suppressor menin potentiates spinal synaptic plasticity in the context of peripheral nerve injury-induced neuropathic hypersensitivity, but the underlying molecular mechanisms are not clear. We hereby assessed the role of menin in regulating the spinal balance between glutamate and GABA and its contribution to the pathological condition of nerve injury-induced hypersensitivity.MethodsIn spared nerve injury induced C57BL/6 mice, mechanical withdrawal threshold was measured with von Frey filaments after intrathecal administration of small interfering RNA (siRNA) of MEN1 or/and subcutaneous histone deacetylase (HDAC) inhibitors to control the level of glutamic acid decarboxylase 65 (GAD65). Immunoblotting and high-performance liquid chromatography were used to detect the level of protein expression and spinal glutamate and GABA, respectively.ResultsGenetic knockdown of spinal menin alleviated nerve injury evoked mechanical hypersensitivity, which was strongly associated with the alteration of the spinal level of GAD65 that resulted in an imbalance of glutamate/GABA ratio from the baseline ratio of 5.8 ± 0.9 (×10⁻⁴) to the peak value of 58.6 ± 11.8 (×10⁻⁴) at the day 14 after SNI (p < 0.001), which was reversed by MEN1 siRNA to 14.7 ± 2.1 (×10⁻⁴) at the day 14 after nerve injury (p < 0.01). In further, selective inhibitors of HDACs considerably reversed the ratio of spinal glutamate and GABA, and also alleviated the mechanical withdrawal threshold markedly.ConclusionOur findings provide mechanistic insight into the contribution of the upregulated spinal menin to peripheral nerve injury induced neuropathic hypersensitivity by regulating glutamate-GABA balance through deactivating GAD65.     

On the Mechanism and Dynamics of Uptake and Permeation of Polyether-Copolyester Dendrimers Across an In Vitro Blood–Brain Barrier Model

Dendrimers have emerged as a promising drug delivery system due to their well defined size, tailorability, and multifunctional nature. However, their application in brain delivery is relatively a new area of research. The present study was aimed at evaluating the uptake and permeation of polyether-copolyester (PEPE) dendrimers across the blood–brain barrier model and exploring the underlying mechanisms. Saturation was observed in the uptake of rhodamine B labeled PEPE dendrimers by brain vascular endothelial (bEnd.3) cells at high concentrations. Clathrin and caveolin inhibitors produced partial inhibition of the dendrimer uptake, signifying contribution of both pathways in the uptake process. PEPE dendrimers were able to cross in vitro BBB model in high amounts with P_app of 19.7 ± 1.9 × 10^?6 cm/s and 38.6 ± 4.1 × 10^?6 cm/s for den-1-(G2)-400 and den-2-(G2)-400, respectively; and only 11–14% reduction in transendothelial electrical resistance during initial 4 h. The results of this study suggest that architecture of dendrimers plays a major role not only in influencing the extent and mechanism of uptake by bEnd.3 cells but also permeation across the BBB model. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3748–3760, 2009     

Determination of genotoxicity by the Comet assay applied to murine precision-cut lung slices

Precision-cut lung slices (PCLSs) are an organotypic lung model that is widely used in pharmacological, physiological, and toxicological studies. Genotoxicity testing, as a pivotal part of early risk assessment, is currently established in vivo in various organs including lung, brain, or liver, and in vitro in cell lines or primary cells. The aim of the present study was to provide the three-dimensional organ culture PCLS as a new ex vivo model for determination of genotoxicity using the Comet assay.Murine PCLS were exposed to increasing concentrations of ethyl methane sulfonate ‘EMS’ (0.03–0.4%) and formalin (0.5–5 mM). Tissue was subsequently dissociated, and DNA single-strand breaks were quantified using the Comet assay. Number of viable dissociated lung cells was between 4 × 10⁵ and 6.7 × 10⁵ cells/slice. Even treatment with EMS did not induce toxicity compared to untreated tissue control. As expected, DNA single-strand breaks were increased dose-dependently and significantly after exposure to EMS. Here, tail length rose from 24 μm to 75 μm. In contrast, formalin resulted in a significant induction of DNA cross-links.The effects induced by EMS and formalin demonstrate the usefulness of PCLS as a new ex vivo lung model for genotoxicity testing in the early risk assessment of airborne substances in the future.     

Citrus peel polymethoxylated flavones extract modulates liver and heart function parameters in diet induced hypercholesterolemic rats

The primary aim of this study was to investigate the effects of Ortanique peel polymethoxylated flavones extract (PMF^ort) on organ function parameters in the serum of hypercholesterolemic and normal rats. Thirty Sprague–Dawley rats were fed high cholesterol diets supplemented with 1.5% PMF^ort and niacin respectively for 49 days. Hypercholesterolemic rats fed PMF^ort had significant reductions in the activities of aspartate aminotransferase and alkaline phosphatase (69.12 ± 3.34 and 87.22 ± 8.42 U/L respectively) compared to the untreated hypercholesterolemic group (118.61 ± 4.85 and 132.62 ± 10.62 U/L respectively, p < 0.05). Supplementation of the diet with niacin or PMF^ort resulted in no significant differences in the serum levels of creatinine or urea in any of the groups. Total bilirubin was highest in the untreated hypercholesterolemic group. Supplementation of the diets of hypercholesterolemic rats with PMF^ort resulted in significant reductions in the activities of serum creatine kinase and lactate dehydrogenase (119.3 ± 25.3; 222.5 ± 50.3 U/L, p < 0.05) respectively relative to the untreated hypercholesterolemic group (257.2 ± 48.3; 648.8 ± 103 U/L, p < 0.05). The results would suggest that PMF^ort modulates hypercholesterolemia-associated organ injury in rats. PMF^ort could therefore be a suitable candidate for prophylactic and therapeutic treatment of hypercholesterolemia-associated organ injury.     

Therapeutic effect of Halofuginone on ITP mice by regulating the differentiation of Th cell subsets

The aim of the present study was to investigate the therapeutic effect of halofuginone (HF) in the treatment of idiopathic thrombocytopenic purpura (ITP) and explore the underlying mechanism. Sixty ITP mice were divided into four groups including control group, low dose group (25 mg/kg HF), medium dose group (50 mg/kg HF), and high dose group (100 mg/kg HF). Corresponding dose of HF was administrated by gavage daily in HF groups for 7 days, and the same volume of saline was given in control group. Platelet counts were 28.87 ± 3.91 × 10⁹/L, 57.13 ± 2.75 × 10⁹/L, 86.73 ± 3.06 × 10⁹/L and 89.73 ± 2.84 × 10⁹/L in control group, low dose group, medium dose group, and high dose group respectively, on day 7 after intragastrically administration of HF or saline. Compared with control group, three HF groups showed significantly increased levels of INF-γ and IL-2 (all P < 0.05), and significantly decreased concentrations of IL-4 and IL-10(all P < 0.05). The expression of T-bet mRNA increased and the expression of GATA-3 mRNA decreased (all P < 0.05) in ITP mice after intragastric administration with different dose of HF. HF significantly recovered peripheral platelet counts in ITP mice through promoting Th1 cell differentiation and attenuating Th2 differentiation in ITP mice.     

In vitro screening of reversible and time-dependent inhibition on CYP3A by TM208 and TM209 in rat liver microsomes

TM208 and TM209, dithiocarbamate derivatives with potential anti-cancer effects, were evaluated in reversible and time-dependent cytochrome P450 (CYP) 3A inhibition assays in rat liver microsomes using testosterone as probe substrate. Both compounds were found to be weak reversible inhibitors and moderate mechanism-based inhibitors of rat CYP3A. For reversible inhibition on rat CYP3A, the K_i values of competitive inhibition model were 12.10±1.75 and 13.94±1.31 μM, respectively. For time-dependent inhibition, the inactivation constants (K_l) were 31.93±12.64 and 32.91±15.58 μM, respectively, and the maximum inactivation rates (k_inact) were 0.03497±0.0069 and 0.07259±0.0172 min^?1 respectively. These findings would provide useful in vitro information for future in vivo DDI studies on TM208 or TM209.     

Development and statistical optimization of nefopam hydrochloride loaded nanospheres for neuropathic pain using Box–Behnken design

Nefopam hydrochloride (NFH) is a non-opioid centrally acting analgesic drug used to treat chronic condition such as neuropathic pain. In current research, sustained release nefopam hydrochloride loaded nanospheres (NFH-NS) were auspiciously synthesized using binary mixture of eudragit RL 100 and RS 100 with sorbitan monooleate as surfactant by quasi solvent diffusion technique and optimized by 3⁵ Box–Behnken designs to evaluate the effects of process and formulation variables. Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetric (DSC) and X-ray diffraction (XRD) affirmed absence of drug–polymer incompatibility and confirmed formation of nanospheres. Desirability function scrutinized by design-expert software for optimized formulation was 0.920. Optimized batch of NFH-NS had mean particle size 328.36 nm ± 2.23, % entrapment efficiency (% EE) 84.97 ± 1.23, % process yield 83.60 ± 1.31 and % drug loading (% DL) 21.41 ± 0.89. Dynamic light scattering (DLS), zeta potential analysis and scanning electron microscopy (SEM) validated size, charge and shape of nanospheres, respectively. In-vitro drug release study revealed biphasic release pattern from optimized nanospheres. Korsmeyer Peppas found excellent kinetics model with release exponent less than 0.45. Chronic constricted injury (CCI) model of optimized NFH-NS in Wistar rats produced significant difference in neuropathic pain behavior (p < 0.05) as compared to free NFH over 10 h indicating sustained action. Long term and accelerated stability testing of optimized NFH-NS revealed degradation rate constant 1.695 × 10⁻⁴ and shelf-life 621 days at 25 ± 2 °C/60% ± 5% RH.     

Hepatic Metabolism and Biliary Excretion of Valerenic Acid in Isolated Perfused Rat Livers: Role of Mrp2 (Abcc2)

The study was designed to investigate the hepatic metabolism and transport system of valerenic acid, a main active constituent of valerian, in isolated perfused livers from Wistar and Mrp2-deficient TR^? rats. After administration of 20 µM valerenic acid, the formation of seven valerenic acid glucuronides (M1–M7), namely two glucuronides of valerenic acid (M6, M7), four glucuronides of hydroxylated valerenic acid (M1, M3, M4, M5), and one glucuronide of hydroxylated dehydro-valerenic acid (M2) in bile and perfusate was quantified by HPLC. The hepatic extraction ratio and clearance of valerenic acid were very high in Wistar and TR^? rats (E: 0.983 ± 0.006 vs. 0.981 ± 0.004; Cl: 35.4 ± 0.21 mL/min vs. 35.3 ± 0.14 mL/min). However, biliary excretion and efflux of conjugates differed greatly in TR^? rats. While cumulative biliary excretion of unconjugated valerenic acid and the glucuronides M1–M7 dropped dramatically to 1–9%, their efflux into perfusate increased 1.5- to 10-fold. This indicates that valerenic acid and its glucuronides are eliminated into bile by Mrp2. In summary, valerenic acid was metabolized to several conjugates, whereby the canalicular transporter Mrp2 mediated biliary excretion of the parent drug and its glucuronides. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3839–3849, 2009