Distinct roles for luminal acidification in apical protein sorting and trafficking in zebrafish.

Epithelial cell physiology critically depends on the asymmetric distribution of channels and transporters. However, the mechanisms targeting membrane proteins to the apical surface are still poorly understood. Here, we performed a visual forward genetic screen in the zebrafish intestine and identified mutants with defective apical targeting of membrane proteins. One of these mutants, affecting the vacuolar H+-ATPase gene atp6ap1b, revealed specific requirements for luminal acidification in apical, but not basolateral, membrane protein sorting and transport. Using a low temperature block assay combined with genetic and pharmacologic perturbation of luminal pH, we monitored transport of newly synthesized membrane proteins from the TGN to apical membrane in live zebrafish. We show that vacuolar H+-ATPase activity regulates sorting of O-glycosylated proteins at the TGN, as well as Rab8-dependent post-Golgi trafficking of different classes of apical membrane proteins. Thus, luminal acidification plays distinct and specific roles in apical membrane biogenesis.

The evaluation of the distribution of CD133, CXCR1 and the tumor associated macrophages in different molecular subtypes of breast cancer.

Breast cancer has different molecular subtypes, which determine the prognosis and response to the treatment. CD133 is a marker for cancer stem cells in tumor microenvironment with diagnostic/therapeutic importance. The tumor associated macrophages (TAMs) interact with the cancer stem cells through the CXCR1 receptor. In this study, we wanted to investigate the expression of these markers in patients with different molecular subtypes, in order to detect pathophysiological mechanisms and new molecular targets for the prospective targeted therapies. In this study we hypothesized a difference in expression of these antigens among different subtypes. We investigated expression of antigens in breast cancer patients with luminal A (LA), luminal B (LB), HER2 overexpressing (HER2OE), triple negative (TN) subtypes (n=70) and control patients (n=10) without cancer diagnosis. We applied indirect immunohistochemistry and evaluated immunostaining. CD133 expression was at the periphery and CXCR1 expression was at the central area of the tumor. The cytoplasmic CXCR1, CD133 expressions and nuclear CD133 expression, which is prominent in the TN subtype, were observed in patients. There was a statistically significant difference between the groups for CD133 (p=0.004), CXCR1 (p=0.002) H-Score values and M2 macrophages/whole TAM ratios (p=0.022). Between the CD133 and CXCR1 H-scores, there was a weak positive correlation (r=0.249, p=0.035). This study showed the compartment specific expression of the CD133 and CXCR1 antigens in neoplastic cells. The use of CD133 as a stem cell marker may be limited to TN subtype, due to its heterogeneous expression.

Unearthing Regulatory Axes of Breast Cancer circRNAs Networks to Find Novel Targets and Fathom Pivotal Mechanisms.

Circular RNAs (circRNAs) possess valuable characteristics for both diagnosis and treatment of several human cancers including breast cancer (BC). In this study, we combined several systems, biology tools and approaches to identify influential BC circRNAs, miRNAs, and related mRNAs as the members of competing endogenous RNAs (ceRNAs) networks and related RNA binding proteins (RBPs) to study and decipher the BC-triggering biological processes and pathways. Rooting from the identified total of 25 co-differentially expressed circRNAs (DECs) between triple negative (TN) and luminal A subtypes of BC from microarray analysis, five hub DECs (hsa_circ_0003227, hsa_circ_0001955, hsa_circ_0020080, hsa_circ_0001666, and hsa_circ_0065173) and top eleven RBPs (AGO1, AGO2, EIF4A3, FMRP, HuR (ELAVL1), IGF2BP1, IGF2BP2, IGF2BP3, EWSR1, FUS, and PTB) were explored to form the upper stream regulatory elements. All the hub circRNAs were regarded as a super sponge having multiple miRNA response elements (MREs). Then, three BC leading miRNAs (hsa-miR-149, hsa-miR-182, and hsa-miR-383) were also introduced from merging several established ceRNAs networks. The predicted 7- and 8-mer MREs matches between hub circRNAs and leading miRNAs ensured their enduring regulatory capability. The mined downstream mRNAs of the circRNAs-miRNAs network then were presented to STRING database to form the PPI network and to decipher the issue from another point of view. The BC interconnected enriched pathways and processes guarantee the merits of the ceRNAs network's members as targetable therapeutic elements. This study suggested extensive panels of novel therapeutic targets that are in charge of BC progression, hence their impressive role cannot be excluded and needs deeper empirical laboratory designs.

A Physicochemically Optimized and Neuroconductive Biphasic Nerve Guidance Conduit for Peripheral Nerve Repair.

Clinically available hollow nerve guidance conduits (NGCs) have had limited success in treating large peripheral nerve injuries. This study aims to develop a biphasic NGC combining a physicochemically optimized collagen outer conduit to bridge the transected nerve, and a neuroconductive hyaluronic acid-based luminal filler to support regeneration. The outer conduit is mechanically optimized by manipulating crosslinking and collagen density, allowing the engineering of a high wall permeability to mitigate the risk of neuroma formation, while also maintaining physiologically relevant stiffness and enzymatic degradation tuned to coincide with regeneration rates. Freeze-drying is used to seamlessly integrate the luminal filler into the conduit, creating a longitudinally aligned pore microarchitecture. The luminal stiffness is modulated to support Schwann cells, with laminin incorporation further enhancing bioactivity by improving cell attachment and metabolic activity. Additionally, this biphasic NGC is shown to support neurogenesis and gliogenesis of neural progenitor cells and axonal outgrowth from dorsal root ganglia. These findings highlight the paradigm that a successful NGC requires the concerted optimization of both a mechanical support phase capable of bridging a nerve defect and a neuroconductive phase with an architecture capable of supporting both Schwann cells and neurons in order to achieve functional regenerative outcome.

High IKKα expression is associated with reduced time to recurrence and cancer specific survival in oestrogen receptor (ER)-positive breast cancer.

The aim of our study was to examine the relationship between tumour IKKα expression and breast cancer recurrence and survival. Immunohistochemistry was employed in a discovery and a validation tissue microarray to assess the association of tumour IKKα expression and clinico-pathological characteristics. After siRNA-mediated silencing of IKKα, cell viability and apoptosis were assessed in MCF7 and MDA-MB-231 breast cancer cells. In both the discovery and validation cohorts, associations observed between IKKα and clinical outcome measures were potentiated in oestrogen receptor (ER) positive Luminal A tumours. In the discovery cohort, cytoplasmic IKKα was associated with disease-free survival (p = 0.029) and recurrence-free survival on tamoxifen (p < 0.001) in Luminal A tumours. Nuclear IKKα and a combination of cytoplasmic and nuclear IKKα (total tumour cell IKKα) were associated with cancer-specific survival (p = 0.012 and p = 0.007, respectively) and recurrence-free survival on tamoxifen (p = 0.013 and p < 0.001, respectively) in Luminal A tumours. In the validation cohort, cytoplasmic IKKα was associated with cancer-specific survival (p = 0.023), disease-free survival (p = 0.002) and recurrence-free survival on tamoxifen (p = 0.009) in Luminal A tumours. Parallel experiment with breast cancer cells in vitro demonstrated the non-canonical NF-κB pathway was inducible by exposure to lymphotoxin in ER-positive MCF7 cells and not in ER-negative MDA-MB-231 cells. Reduction in IKKα expression by siRNA transfection increased levels of apoptosis and reduced cell viability in MCF7 but not in MDA-MB-231 cells. IKKα is an important determinant of poor outcome in patients with ER-positive invasive ductal breast cancer and thus may represent a potential therapeutic target.

Rab25 acts as an oncogene in luminal B breast cancer and is causally associated with Snail driven EMT.

The Rab GTPases regulate vesicular trafficking machinery that transports and delivers a diverse pool of cargo, including growth factor receptors, integrins, nutrient receptors and junction proteins to specific intracellular sites. The trafficking machinery is indeed a major posttranslational modifier and is critical for cellular homeostasis. Deregulation of this stringently controlled system leads to a wide spectrum of disorders including cancer. Herein we demonstrate that Rab25, a key GTPase, mostly decorating the apical recycling endosome, is a dichotomous variable in breast cancer cell lines with higher mRNA and protein expression in Estrogen Receptor positive (ER+ve) lines. Rab25 and its effector, Rab Coupling Protein (RCP) are frequently coamplified and coordinately elevated in ER+ve breast cancers. In contrast, Rab25 levels are decreased in basal-like and almost completely lost in claudin-low tumors. This dichotomy exists despite the presence of the 1q amplicon that hosts Rab25 across breast cancer subtypes and is likely due to differential methylation of the Rab25 promoter. Functionally, elevated levels of Rab25 drive major hallmarks of cancer including indefinite growth and metastasis but in case of luminal B breast cancer only. Importantly, in such ER+ve tumors, coexpression of Rab25 and its effector, RCP is significantly associated with a markedly worsened clinical outcome. Importantly, in claudin-low cell lines, exogenous Rab25 markedly inhibits cell migration. Similarly, during Snail-induced epithelial to mesenchymal transition (EMT) exogenous Rab25 potently reverses Snail-driven invasion. Overall, this study substantiates a striking context dependent role of Rab25 in breast cancer where Rab25 is amplified and enhances aggressiveness in luminal B cancers while in claudin-low tumors, Rab25 is lost indicating possible anti-tumor functions.

To Analyze the Amelioration of Phenobarbital Induced Oxidative Stress by Erucin, as Indicated by Biochemical and Histological Alterations.

PURPOSE: Phenobarbital is a commonly employed antidepressant and anti-epileptic drug. The cancer promoting activity of this genotoxic xenobiotic is often ignored. It is responsible for oxidative stress leading to modulation in xenobiotic and antioxidative enzymes. Glucosinolates and more specifically their hydrolytic products are known for their antioxidative and anticancer activities. The present study involves the analysis of hepatoprotective effect of erucin (isolated from Eruca sativa (Mill.) Thell.) against phenobarbital mediated hepatic damage in male wistar rats. METHODS: The liver homogenate was analyzed for oxidative stress (superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase, glutathione reductase and lactate dehydrogenase), other oxidative parameters (thiobarbituric acid reactive species, conjugated dienes and lipid hydroperoxide), phase I enzymes (NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase, cytochrome P420, cytochrome P450 and cytochrome b5), phase II enzymes (γ-glutamyl transpeptidase, DT-diaphorase and glutathione-S-transferase), serum parameters (alkaline phosphatase, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, direct bilirubin and total bilirubin) and certain histological parameters. RESULTS: Erucin accorded protection from phenobarbital induced hepatic damage by normalizing antioxidative enzymes, other oxidative parameters, phase I, II, and serum parameters. CONCLUSIONS: Erucin, an analogue of sulforaphane has the potential to act as an anticancer agent by regulating various biochemical parameters.

Luminal (Her2 negative) prognostic index and survival of breast cancer patients.

PURPOSE: The group of luminal (Her2 negative) is distinguished from other subtypes of breast cancer. We aimed to produce a prognostic index specific for luminal (Her2 negative) subtype breast cancer that could assist clinical treatment. METHODS: The test set comprised 406 consecutive luminal (Her2 negative) breast cancer patients. The relationship of 11 clinicopathologic factors including survivin with the 5-year disease-free survival was analyzed. RESULTS: In univariate analysis, TNM stage, surgery, tumor size, lymph node involvement, and survivin expression were prognostic factors. In multivariate analysis, tumor size [HR (95% CI): 1.98 (1.12-3.49), p=0.019], the number of lymph node metastasis [HR (95% CI): 1.75 (1.33-2.29), p<0.0001] and the expression of progesterone receptor [HR (95% CI): 0.58 (0.36-0.95), p=0.029] can independently predict prognosis. Prognostic index (PI) was calculated as 0.68×tumor size+0.56×the number of lymph node metastasis-0.54×PR. According to the PI, patients were categorized into three groups: low, middle, and high risk group with the 5-year disease-free survival rates of 91.91%, 84.97% and 70.47%, respectively (P<0.001). In the validation set, the luminal prognostic index (LPI) remained significant. CONCLUSION: The LPI may be a useful tool for evaluating the outcome of patients with luminal (Her-2 negative) breast cancer.

18F-FDG PET-CT imaging in the neoadjuvant setting for stages II-III breast cancer: association of loco­regional SUVmax with classical prognostic factors.

AIM: Classical prognostic and predictive factors serve to predict outcome and response to neoadjuvant treatment in stage II and III breast cancer. The purpose is to determine the relation between the SUVmax of the locoregional disease with these classical prognostic factors. METHODS: A prospective study including 43 stage II and III breast cancer patients was performed. In all the patients, two 18F-FDG PET-CT studies were performed before and after neoadjuvant chemotherapy. After this treatment, surgery and adjuvant treatment were carried out. To combine the information of the locoregional disease, the SUVmax of the lesion (tumor or abnormal lymphnodes) with the highest uptake was used. SUVmax and prognostic factors were studied with the Kruskal-Wallis non-parametric test and with the Mann-Whitney U. RESULTS: A statistically significant association between elevated SUVmax value and absence of estrogen receptors (ER) expression (16 vs.. 10; P<0.019) was found. Locorregional disease with positive HER2 phenotype had a statistically significant SUVmax value greater than Luminal A (estrogen and/or progesterone positive receptors with Ki67<15%) and B (estrogen and/or progesterone positive receptors with Ki67≥to 15%) (13.4 vs. 7.9 and 8.9; P<0.022 and P<0.024, respectively). Triple negative phenotype disease had higher SUVmax than Luminal A and B (15.4; P<0.030 and P<0.038). Positive correlation between the percentage of the Ki67 Proliferation Index and SUVmax (P<0.007) was demonstrated. High grade disease had a higher SUVmax than low grade (P<0.004). CONCLUSION: Locorregional SUVmax is associated to prognostic and predictive factors and reaffirms the utility of PET-CT as a tool in the common clinical practice.

Determinação simplificada de carbamazepina, fenitoína, fenobarbital e lamotrigina em plasma emonitoração terapêutica por HPLC/PDA / Simplified determination of carbamazepine, phenytoin,phenobarbital and lamotrigine in plasma and therapeuticmonitoring by HPLC/PDA

Carbamazepina, fenitoína, fenobarbital e lamotrigina são bem conhecidos como fármacos anticonvulsivantes usados para o tratamento da epilepsia. A utilização destes fármacos é associada a várias reações adversas, tornando necessário o controle terapêutico. Os ensaios foram realizados por meio de cromatografia líquida de alta eficiência e o método de extração utilizado foi o líquido-líquido. Os fármacos e o padrão interno de zolpidem foram separados por uma coluna de fase reversa (ACE5, C18150x 4,6 mm d.i.). A fase móvel constituída de acetonitrila (30%) e ácido cítrico/tampão fosfato pH 5,0 (70%) foi utilizada sob gradiente de fluxo de 0,7 a 1,2 mL/min e o comprimento de onda utilizado para a detecção dos analitos foi fixado em 210 nm. A técnica apresentou linearidade ao longo do intervalo de 0,5 a 20,0μg/mL para carbamazepina/lamotrigina e de 2,0 a 64,0 μg/mL, para fenitoina/fenobarbital com coeficiente de correlação linear maior que 0,98. As amostras de sangue foram obtidas de pacientes adultos com diagnóstico de epilepsia em acompanhamento no Ambulatório Municipal de Psiquiatria de Goiânia. A média de recuperação obtida foi de 96,8% a 108,5% para carbamazepina/lamotrigina e de 93,8% a 108,8% para a fenitoina/fenobarbital. Os limites de quantificação, precisão (CV< 15,0 %)e exatidão (E > 85,0 %) demonstraram estar em conformidade com as exigências da ANVISA. Nove pacientes foram avaliados para confirmar a validade do método.

Carbamazepine, phenytoin, phenobarbital and lamotrigine are well known anticonvulsant drugs used in the treatment of epilepsy. However, these medications are associated with side effects and therefore require therapeutic monitoring. Blood samples of adult outpatients diagnosed with epilepsy at the Goiânia Municipal Psychiatry Clinic (Brazil) were collected and analyzed using high-performance liquid chromatography. The analytes and internal standard (zolpidem) were extracted by liquid-liquid extraction. A reversed phase column (ACE 5, C18, 150 x 4.6 mm i.d.) was used. The mobile phase was composed of acetonitrile (30%) and citric acid/phosphate buffer, pH 5.0 (70%). The gradient flow rate was from 0.7 to 1.2 mL/min and the detection wavelength was set at 210 nm for all analytes. The analysis revealed a linear range from 0.5 to 20.0μg/mL for carbamazepine/lamotrigine and 2.0 to 64.0 μg/mL for phenytoin/phenobarbital. Mean recovery ranged from 96.8% to 108.5% for carbamazepine/lamotrigine and 93.8% to 108.8% for phenytoin/phenobarbital. The quantification limit, precision (CV < 15%) and accuracy (A >85%) proved to be in accordance with the requirements stipulated by the Brazilian National Health Surveillance Agency (ANVISA). Nine outpatients were evaluated to confirm the validity of the method.

Prediction of onset of crystallization in amorphous pharmaceutical systems: phenobarbital, nifedipine/PVP, and phenobarbital/PVP.

The aim of this work is to determine if a stability testing protocol based on the correlations between crystallization onset and relaxation time above the glass transition temperature (T(g)) can be used to predict the crystallization onsets in amorphous pharmaceutical systems well below their T(g). This procedure assumes that the coupling between crystallization onset and molecular mobility is the same above and below T(g). The stability testing protocol has been applied to phenobarbital, phenobarbital/polyvinylpyrrolidone (PVP) (95/5, w/w), and nifedipine/PVP (95/5, w/w). Crystallization onsets have been detected by polarized light microscopy examination of amorphous films; molecular mobility has been determined by dielectric relaxation spectroscopy above T(g) and by both isothermal calorimetry and modulated differential scanning calorimetry below T(g). We find that small amounts of PVP significantly retard re-crystallization. This dramatic effect of PVP is not related to mobility, so this approach applies, at best, to extrapolation of high temperature data on a given formulation to low temperatures. Variation in molecular mobility at these concentrations of PVP is not the dominant factor in determining variation in propensity for re-crystallization from glassy systems; we suggest surface interactions between PVP and nuclei and/or small crystals slowing growth control variation in crystallization kinetics between formulations.

Effects of ethanol and phenobarbital on hemoglobin adducts formation in rats exposed to benzidine and Direct Black 38.

The objective of this study was to evaluate the effects of pretreatment of ethanol (EtOH) and phenobarbital (PB), which are known to affect the metabolism of xenobiotics, in the formation of hemoglobin adducts in rats administered with benzidine (BZ) and Direct Black 38 (DB38). The experimental rats were divided into BZ and DB38 groups; each group was subdivided into control, EtOH, and PB groups. Blood samples were separated into hemoglobin and plasma immediately after obtaining and basic hydrolysis was done to convert the adducts into aromatic amines. Hydrolyzed BZ, monoacetylbenzidine (MABZ), and 4-aminobiphenyl (4ABP) were separated by reversed-phase liquid chromatography without derivatization. Then, quantitative analyses were performed using a high performance liquid chromatograph equipped with an electrochemical detector. The amount of metabolites was expressed in the hemoglobin binding index (HBI). As a result, the formations of hemoglobin in BZ-, MABZ-, and 4ABP-HBI of BZ-EtOH and BZ-PB groups were increased compared with those of BZ-control group. In DB38 group, all of HBIs except for BZ-HBI were increased more than those of DB38-control group regardless of the pretreatment. These results are attributable to the fact that EtOH and PB induced N-hydroxylation is related to the formation of hemoglobin adducts. They indicate that EtOH not only increases the adduct formation by inducing N-hydroxylation but also induces N-acetylation. PB induced N-hydroxylation and increased the adduct formation in BZ group, but decreased the adduct formation in DB38 group due to decreasing azo reduction. These results suggest that the effects of EtOH or PB should be considered in biochemical monitoring of BZ and DB38 for the assessment of intermittent exposure to BZ and DB38.

Correlation between molecular mobility and crystal growth of amorphous phenobarbital and phenobarbital with polyvinylpyrrolidone and L-proline.

The aim of the present study is to determine if the correlation between molecular mobility and crystallization growth rates exists over a broad temperature range from temperatures below the glass transition (T(g)) to temperatures above the glass transition. Phenobarbital and solid dispersions of phenobarbital with PVP and L-proline were studied in this research. Relaxation times below and above the T(g) were measured. Crystallization was followed in a hot-stage microscope and crystal growth rates were measured by observing radial growth of a single crystal. Arrhenius type temperature dependences were found both in relaxation times and crystal growth rates over studied temperature ranges, in all cases studied except in the case of pure phenobarbital, where a change of slope was observed for the crystal growth rate for the temperature range below T(g). For all cases, molecular mobility was correlated with crystal growth rate, for the temperature range studied, with a coupling coefficient of 0.38 for phenobarbital, and 0.23 and 0.28 for solid dispersions with PVP and proline respectively. By establishing the coupling between molecular mobility and crystal growth rate, predictive models can be created to estimate the stability of amorphous materials both, for pure form as well as for solid dispersions.

Miscibility of nifedipine and hydrophilic polymers as measured by (1)H-NMR spin-lattice relaxation.

The miscibility of a drug with excipients in solid dispersions is considered to be one of the most important factors for preparation of stable amorphous solid dispersions. The purpose of the present study was to elucidate the feasibility of (1)H-NMR spin-lattice relaxation measurements to assess the miscibility of a drug with excipients. Solid dispersions of nifedipine with the hydrophilic polymers poly(vinylpyrrolidone) (PVP), hydroxypropylmethylcellulose (HPMC) and alpha,beta-poly(N-5-hydroxypentyl)-L-aspartamide (PHPA) with various weight ratios were prepared by spray drying, and the spin-lattice relaxation decay of the solid dispersions in a laboratory frame (T(1) decay) and in a rotating frame (T(1rho) decay) were measured. T(1rho) decay of nifedipine-PVP solid dispersions (3 : 7, 5 : 5 and 7 : 3) was describable with a mono-exponential equation, whereas T(1rho) decay of nifedipine-PHPA solid dispersions (3 : 7, 4 : 6 and 5 : 5) was describable with a bi-exponential equation. Because a mono-exponential T(1rho) decay indicates that the domain sizes of nifedipine and polymer in solid dispersion are less than several nm, it is speculated that nifedipine is miscible with PVP but not miscible with PHPA. All the nifedipine-PVP solid dispersions studied showed a single glass transition temperature (T(g)), whereas two glass transitions were observed for the nifedipine-PHPA solid dispersion (3 : 7), thus supporting the above speculation. For nifedipine-HPMC solid dispersions (3 : 7 and 5 : 5), the miscibility of nifedipine and HPMC could not be determined by DSC measurements due to the lack of obviously evident T(g). In contrast, (1)H-NMR spin-lattice relaxation measurements showed that nifedipine and HPMC are miscible, since T(1rho) decay of the solid dispersions (3 : 7, 5 : 5 and 7 : 3) was describable with a mono-exponential equation. These results indicate that (1)H-NMR spin-lattice relaxation measurements are useful for assessing the miscibility of a drug and an excipient in solid dispersions.

Molecular mobility of nifedipine-PVP and phenobarbital-PVP solid dispersions as measured by 13C-NMR spin-lattice relaxation time.

Amorphous nifedipine-PVP and phenobarbital-PVP solid dispersions with various drug contents were prepared by melting and subsequent rapid cooling of mixtures of PVP and nifedipine, or phenobarbital. Chemical shifts and spin-lattice relaxation times (T(1)) of PVP, nifedipine, and phenobarbital carbons were determined by (13)C-CP/MAS NMR to elucidate drug-PVP interactions and the localized molecular mobility of drug and PVP in the solid dispersions. The chemical shift of the PVP carbonyl carbon increased as the drug content increased, appearing to reach a plateau at a molar ratio of drug to PVP monomer unit of approximately 1:1, suggesting hydrogen bond interactions between the PVP carbonyl group and the drugs. T(1) of the PVP carbonyl carbon in the solid dispersions increased as the drug content increased, indicating that the mobility of the PVP carbonyl carbon was decreased by hydrogen bond interactions. T(1) of the drug carbons increased as the PVP content increased, and this increase in T(1) became less obvious when the molar ratio of PVP monomer unit to drug exceeded approximately 1:1. These results suggest that the localized motion of the PVP pyrrolidone ring and the drug molecules is reduced by hydrogen bond interactions. Decreases in localized mobility appear to be one of the factors that stabilize the amorphous state of drugs.

Molecular mobility-based estimation of the crystallization rates of amorphous nifedipine and phenobarbital in poly(vinylpyrrolidone) solid dispersions.

The overall crystallization rates and mean relaxation times of amorphous nifedipine and phenobarbital in the presence of poly(vinylpyrrolidone) (PVP) were determined at various temperatures to gain further insight into the effect of molecular mobility on the crystallization rates of amorphous drugs and the possibility of predicting stability from their molecular mobility. Nifedipine-PVP (9:1 w/w) and phenobarbital-PVP (95:5 w/w) solid dispersions were prepared by melting and rapidly cooling mixtures of each drug and PVP. The amount of amorphous nifedipine remaining in the solid dispersion was calculated from the heat of crystallization,which was obtained by differential scanning calorimetry. The amount of amorphous phenobarbital remaining in the solid dispersion was estimated from the change in the heat capacity at its glass transition temperature (T(g)). The time required for the amount of amorphous drug remaining to fall to 90% (t(90)) was calculated from the profile of time versus the amount of amorphous drug remaining. The t(90) values for the solid dispersions studied were 100-1000 times longer than those of pure amorphous drugs when compared at the same temperature. Enthalpy relaxation of the amorphous drugs in the solid dispersions was reduced compared with that in the pure amorphous drugs, indicating that the molecular mobility of the amorphous drugs is reduced in the presence of PVP. The temperature dependence of mean relaxation time (tau) for the nifedipine-PVP solid dispersion was calculated using the Adam-Gibbs-Vogel equation. Parameters D and T(0) in this equation were estimated from the heating rate dependence of T(g). Similar temperature dependence was observed for t(90) and tau values of the solid dispersion, indicating that the information on the temperature dependence of the molecular mobility, along with the crystallization data obtained at around the T(g), are useful for estimating the t(90) of overall crystallization at temperatures below T(g) in the presence of excipients.

The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism.

Organisms encounter a wide range of foreign compounds--or 'xenobiotics'--with potentially harmful consequences. The cytochrome P450 (CYP) enzymes metabolize xenobiotics and thus are a primary defence against these compounds. Increased expression of specific CYP genes in response to particular xenobiotics is a central component of this defence, although such induction can also increase production of toxic metabolites. Here we show that the nuclear receptor CAR mediates the response evoked by a class of xenobiotics known as the 'phenobarbital-like inducers'. The strong activation of Cyp2b10 gene expression by phenobarbital, or by the more potent TCPOBOP, is absent in mice lacking the CAR gene. These animals also show decreased metabolism of the classic CYP substrate zoxazolamine and a complete loss of the liver hypertrophic and hyperplastic responses to these inducers. Cocaine causes acute hepatotoxicity in wild-type mice previously exposed to phenobarbital-like inducers and this toxicity is also absent in the CAR-deficient animals. Thus, loss of CAR function alters sensitivity to toxins, increasing or decreasing it depending on the compound. Modulation of CAR activity in humans may significantly affect metabolism of drugs and other xenobiotics.

Depressant and convulsant barbiturates both inhibit neuronal nicotinic acetylcholine receptors.

UNLABELLED: Neuronal nicotinic acetylcholine receptors (neuronal nAchRs) are sensitive to many anesthetics, including barbiturates, which suggests that these receptors are potential sites for anesthetic action. Subtle changes in molecular structures of the anesthetic barbiturates can produce compounds with potent convulsant activity. Whereas R(-) isomer of 1-methyl-5-phenyl-5-propyl barbituric acid (MPPB) exerts anesthetic action, S(+)MPPB exhibits pure excitatory effects, including convulsion. 5-(2-cyclohexilidene-ethyl)-5-ethyl barbituric acid is another example of a convulsant barbiturate. We compared the effects of depressant and convulsant barbiturates on the neuronal nAchR-mediated current to determine whether inhibition of neuronal nAchRs contributes to the anesthetic action of barbiturates. Whole cell nicotine-induced currents were recorded in PC12 derived from rat pheochromocytoma, using the conventional whole cell patch clamp technique in the presence and absence of barbiturates. Both depressant and convulsant barbiturates inhibited the nicotine-induced inward current reversibly and in a dose-dependent manner when co-applied with nicotine. All barbiturates accelerated the current decay. There was no significant difference between the concentrations for 50% inhibition for MPPB isomers. There was no correlation between inhibition of ganglionic nAchRs and anesthetic effects of the barbiturates. These results strongly oppose the idea that inhibition of neuronal nAchRs contributes to the anesthetic action of barbiturates. IMPLICATIONS: We found that both convulsant and depressant barbiturates inhibit the current mediated through ganglionic nicotinic acetylcholine receptors in PC12 cells. This finding suggests that the inhibition of neuronal nicotinic acetylcholine receptors does not contribute to the anesthetic action of barbiturates.

Ferrous iron-induced luminol chemiluminescence: a method for hydroxyl radical study.

We have investigated the chemiluminescence signal of the ferrous iron in the presence of the luminol and lucigenin. Ferrous, but not ferric, iron produced a transient signal in the presence of luminol, but not lucigenin. Ferrous iron-induced luminol chemiluminescence was significantly inhibited in a concentration-dependent manner by superoxide dismutase (SOD) and catalase. Specific hydroxyl radical scavengers, mannitol and dimethyl sulfoxide (DMSO), also markedly attenuated the ferrous iron-induced chemiluminescence. Additionally, antioxidants, urate, ascorbate, and methionine produced concentration-dependent significant inhibitions in this chemiluminescence. These results show that the hydroxyl radical generation is dependent on simultaneous formation of superoxide and hydrogen peroxide (H2O2). Ferrous iron does not generate a chemiluminescence signal in the presence of lucigenin suggesting that the formation of a hydroxyl radical is responsible for the luminol chemiluminescence. Thus, the present study has established a simple and inexpensive cell-free screening method for monitoring the scavenging effects of drugs on the hydroxyl radical.

Absence of DNA adduct formation by phenobarbital, polychlorinated biphenyls, and chlordane in mouse liver using the 32P-postlabeling assay.

Phenobarbital (PB), polychlorinated biphenyls (PCBs), and chlordane (CLD) increase liver tumor incidences in rodents, and all are tumor promoters. Most indirect tests for DNA reactivity, including mutagenicity and chromosomal damage, have been negative with these agents. Consequently, the modes of action for tumorigenesis by these compounds are not believed to involve direct DNA reactivity; however, only limited information from direct tests is available for the lack of DNA adduct formation. PB, PCBs, and CLD were tested for DNA adduct formation in the liver of male and female B6C3F1 mice after either single or 2-week dietary exposures. Single gavage dose levels were as follows: PB, 200 mg/kg; PCBs, 50 mg/kg; and CLD, 50 mg/kg. Dietary dose levels were as follows: PB, 1000 ppm; PCBs, 200 ppm and CLD, 200 ppm. Animals were killed 24 h following the end of test-substance administration. DNA was extracted from the liver, and DNA adduct concentrations were enriched using either 1-butanol extraction of adducted nucleotides or nuclease P1 digestion of unadducted nucleotides. Using this protocol, none of the three test compounds produced DNA adducts detected by 32P-postlabeling. Similar negative results were obtained for DNA from the livers of both male and female mice receiving either single or 2-week exposures. The two positive controls, benzidine for the 1-butanol extraction procedure and 2-acetylaminofluorene for the nuclease P1 procedure, showed the expected patterns of DNA adducts. These results support the conclusion that the carcinogenicity of PB, PCBs, and CLD in experimental animals is not the result of direct DNA reactivity, but involves epigenetic mechanisms.