Determination of sulpiride in pharmaceutical preparations and biological fluids using a Cr (III) enhanced chemiluminescence method

A highly sensitive and simple method for identifying sulpiride in pharmaceutical formulations and biological fluids is presented. The method is based on increased chemiluminescence (CL) intensity of a luminol–H₂O₂ system in response to the addition of Cr (III) under alkaline conditions. The CL intensity of the luminol–H₂O₂–Cr (III) system was greatly enhanced by the addition of sulpiride and the CL intensity was proportional to the concentration of sulpiride in a sample solution. Various parameters affecting the CL intensity were systematically investigated and optimized for determination of the sulpiride in a sample. Under the optimum conditions, the CL intensity was proportional to the concentration of sulpiride in the range of 0.068–4.0 µg/mL, with a good correlation coefficient of 0.997. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 8.50 × 10^‐6 µg/mL and 2.83 × 10^‐5 µg/mL, respectively. The method presented here produced good reproducibility with a relative standard deviation (RSD) of 2.70% (n = 7). The effects of common excipients and metal ions were studied for their interference effect. The method was validated statistically through recovery studies and successfully applied for the determination of sulpiride in pure form, pharmaceutical preparations and spiked human plasma samples. The percentage recoveries were found to range from 99.10 to 100.05% for pure form, 98.12 to 100.18% for pharmaceutical preparations and 97.9 to 101.4% for spiked human plasma. Copyright © 2012 John Wiley & Sons, Ltd.     

Correction for Irrelevant Absorption in Multicomponent Spectrophotometric Assay of Riboflavin, Formylmethylflavin, and Degradation Products: Kinetic Applications

In the spectrophotometric assay of multicomponent systems involved in drug degradation studies, some minor or unknown degradation products may be present. These products may interfere in the assay and thus invalidate the results due to their absorption in the range of analytical wavelengths. This interference may be eliminated by the application of an appropriate correction procedure to obtain reliable data for kinetic treatment. The present study is based on the application of linear and non-linear irrelevant absorption corrections in the multicomponent spectrophotometric assay of riboflavin and formylmethylflavin during the photolysis and hydrolysis studies. The correction procedures take into account the interference caused by minor or unknown products and have shown considerable improvement in the assay data in terms of the molar balance. The treatment of the corrected data has led to more accurate kinetic results in degradation studies.     

Multi-dimensional Co-separation Analysis Reveals Protein–Protein Interactions Defining Plasma Lipoprotein Subspecies

The distribution of circulating lipoprotein particles affects the risk for cardiovascular disease (CVD) in humans. Lipoproteins are historically defined by their density, with low-density lipoproteins positively and high-density lipoproteins (HDLs) negatively associated with CVD risk in large populations. However, these broad definitions tend to obscure the remarkable heterogeneity within each class. Evidence indicates that each class is composed of physically (size, density, charge) and compositionally (protein and lipid) distinct subclasses exhibiting unique functionalities and differing effects on disease. HDLs in particular contain upward of 85 proteins of widely varying function that are differentially distributed across a broad range of particle diameters. We hypothesized that the plasma lipoproteins, particularly HDL, represent a continuum of phospholipid platforms that facilitate specific protein–protein interactions. To test this idea, we separated normal human plasma using three techniques that exploit different lipoprotein physicochemical properties (gel filtration chromatography, ionic exchange chromatography, and preparative isoelectric focusing). We then tracked the co-separation of 76 lipid-associated proteins via mass spectrometry and applied a summed correlation analysis to identify protein pairs that may co-reside on individual lipoproteins. The analysis produced 2701 pairing scores, with the top hits representing previously known protein–protein interactions as well as numerous unknown pairings. A network analysis revealed clusters of proteins with related functions, particularly lipid transport and complement regulation. The specific co-separation of protein pairs or clusters suggests the existence of stable lipoprotein subspecies that may carry out distinct functions. Further characterization of the composition and function of these subspecies may point to better targeted therapeutics aimed at CVD or other diseases.Lipoproteins are circulating emulsions of protein and lipid that play important roles, both positive and negative, in cardiovascular disease (CVD).¹ Historically defined by their density as separated by ultracentrifugation, the major lipoprotein classes include the neutral lipid ester-rich very low-density and low-density lipoproteins (VLDLs and LDLs, respectively), which function to transport triglyceride and cholesterol from the liver to the peripheral tissues. Significant epidemiological evidence, in vitro studies, animal experiments, and human clinical trials have shown that high-LDL cholesterol is a bona fide causative factor in CVD (1). In contrast, protein- and phospholipid-rich high-density lipoproteins (HDLs) are thought to mediate the reverse transport of cholesterol from the periphery to the liver for catabolism and to perform anti-oxidative and anti-inflammatory functions (reviewed in Refs. 2 and 3). A host of human epidemiology and animal studies indicate that HDLs are atheroprotective (4). However, recent clinical trials of therapeutics that generically raise HDL, at least as measured by its cholesterol levels, have failed to confer the expected CVD protections (5–7).Although these traditional density-centric definitions have been used for nearly 40 years, accumulating evidence indicates that they are not particularly reflective of lipoprotein compositional and functional complexity. With respect to most physical traits (size, charge, lipid content, protein content, etc.), one can demonstrate significant heterogeneity within each density class. This suggests that particle subspecies exist with unique functions and effects on disease. For example, LDL can be resolved into large, buoyant and small, dense forms (8), with subjects carrying more cholesterol in the small, dense LDL exhibiting a greater CVD risk (9). HDL is particularly noted for heterogeneity, as it can be separated into numerous subfractions by density (10), diameter (11), charge (12), and major apolipoprotein content (13). Most strikingly, recent applications of soft-ionization mass spectrometry (MS) have identified upward of 85 HDL proteins with functions that go well beyond the structural apolipoproteins, lipid transport proteins, and lipid-modifying enzymes known from previous biochemical studies (14, 15). Many of these proteins imply functions as diverse as complement regulation, acute phase response, protease inhibition, and innate immunity (16). Individual HDL subspecies can apparently draw from this palette of proteins to produce distinct particles of distinct function. One well-defined HDL subfraction, termed trypanosome lytic factor, contains apolipoprotein apoA-I, haptoglobin-related protein, and apoL-I. Working together, these proteins enter the trypanosome brucei brucei and kill it via lysosomal disruption (17). There are numerous other instances of on-particle protein cooperation in HDL related to CVD (reviewed in Ref. 15). Furthermore, two-dimensional electrophoresis studies by Asztalos and colleagues (18), as well as our own work (11, 19), strongly support the concept that certain apolipoproteins segregate among different HDL particles. These observations present the intriguing possibility that the phospholipids of HDLs act as an organizing platform that facilitates the assembly of specific protein complexes (20). Such subspecies could have important functional implications in the context of CVD protection, inflammation, or even innate immune function. Furthermore, this subspeciation may explain why therapeutics that raise HDL cholesterol levels across the board have not yet shown promise with regard to CVD.To address this hypothesis, we began to think of lipoproteins as a continuum of phospholipid platforms that support the assembly of specific protein complexes analogous to those in cells that perform coordinated biological functions (i.e. ribosomes, centrosomes, etc.). Two common methods for characterizing protein complexes are tandem affinity purification (21) and immunoprecipitation. Both rely on the specific pull-down of a target protein (by either an introduced affinity tag or an antibody) followed by the identification of co-precipitated proteins via MS. Unfortunately, tandem affinity purification strategies are impractical in humans, and we have found that immunoprecipitation experiments with human plasma lipoproteins result in a high false-positive rate due to the low abundance of most of these proteins, particularly those in HDLs. Therefore, we took an alternative approach called co-separation analysis, a method based on the principle that stable protein complexes can be identified by tracking their co-migration as they undergo biochemical separation by multiple orthogonal approaches (22). Native proteins are analyzed in an unbiased manner without affinity tags or antibodies, and purification to homogeneity is not necessary for the identification of putative protein complexes.Most current studies of the lipoprotein proteome utilize samples isolated via density ultracentrifugation because contaminating lipid-unassociated lipoproteins, which can be highly abundant and obscure the identification of targeted lipid-associated proteins, are thus removed prior to the analysis. In previous work, we characterized the use of a calcium silica hydrate (CSH) resin that allowed the specific isolation of phospholipid-associated proteins and their subsequent MS identification without ultracentrifugation (11). This advance enabled the use of a variety of non-density-based separation methods for the study of plasma lipoproteins. Here, we take advantage of this to analyze the proteome of human plasma lipoproteins separated via three separation techniques that exploit different physicochemical properties: (i) gel filtration chromatography (size), (ii) anion exchange chromatography (charge interaction), and (iii) isoelectric focusing. By tracking the co-migration of specific proteins across these separations (Fig. 1), we identified a host of putative protein pairings, including the previously known trypanosome lytic factor HDL fraction, for further biochemical verification and characterization.Open in a separate windowFig. 1.Overview of the multi-dimensional separation co-migration analysis used in this study (see “Experimental Procedures” for details).     

Identification of interleukin-13 related biomarkers using peripheral blood mononuclear cells

Asthma is a chronic disorder characterized by airway inflammation, reversible bronchial obstruction, hyper-responsiveness and remodelling. Data from human in vitro studies and experimental in vivo models of asthma has implicated interleukin (IL)-13 in the asthma phenotype suggesting that a therapeutic agent against it could be effective in treating asthma. The role of biomarkers is becoming increasingly important in the clinical development of therapeutics. Here we describe the use of the GeneChip^® DNA microarray technology platform to explore and identify potential response to therapy biomarkers that are associated with the biology of IL-13. Peripheral blood mononuclear cells (PBMCs) from eight healthy donors were cultured in the presence of IL-13, IL-4, an anti-IL-13 monoclonal antibody (mAb) or an isotype control mAb, and RNA from the treated cells was subjected to microarray analysis. The results revealed a number of genes, such as CCL17 (TARC), CCL22 (MDC), CCL23 (MPIF-1), CCL26 (eotaxin 3) and WNT5A (human wingless-type MMTV integration site family member 5A), that showed increased expression in the IL-13 and IL-4 treatment groups. Real-time polymerase chain reaction (PCR) subsequently confirmed these results. A follow-up study in PBMCs from five additional healthy donors showed that the neutralization of IL-13 completely blocked IL-13-induced TARC, MDC and eotaxin 3 production at the protein level. These data suggest that TARC, MDC, eotaxin 3, CCL23 and WNT5A if validated could serve as potential biomarkers for anti-IL-13 therapeutics.     

Cigarette smoke metabolically promotes cancer,via autophagy and premature aging in the host stromal microenvironment

Cigarette smoke has been directly implicated in the disease pathogenesis of a plethora of different human cancer subtypes, including breast cancers. The prevailing view is that cigarette smoke acts as a mutagen and DNA damaging agent in normal epithelial cells, driving tumor initiation. However, its potential negative metabolic effects on the normal stromal microenvironment have been largely ignored. Here, we propose a new mechanism by which carcinogen-rich cigarette smoke may promote cancer growth, by metabolically “fertilizing” the host microenvironment. More specifically, we show that cigarette smoke exposure is indeed sufficient to drive the onset of the cancer-associated fibroblast phenotype via the induction of DNA damage, autophagy and mitophagy in the tumor stroma. In turn, cigarette smoke exposure induces premature aging and mitochondrial dysfunction in stromal fibroblasts, leading to the secretion of high-energy mitochondrial fuels, such as L-lactate and ketone bodies. Hence, cigarette smoke induces catabolism in the local microenvironment, directly fueling oxidative mitochondrial metabolism (OXPHOS) in neighboring epithelial cancer cells, actively promoting anabolic tumor growth. Remarkably, these autophagic-senescent fibroblasts increased breast cancer tumor growth in vivo by up to 4-fold. Importantly, we show that cigarette smoke-induced metabolic reprogramming of the fibroblastic stroma occurs independently of tumor neo-angiogenesis. We discuss the possible implications of our current findings for the prevention of aging-associated human diseases and, especially, common epithelial cancers, as we show that cigarette smoke can systemically accelerate aging in the host microenvironment. Finally, our current findings are consistent with the idea that cigarette smoke induces the “reverse Warburg effect,” thereby fueling “two-compartment tumor metabolism” and oxidative mitochondrial metabolism in epithelial cancer cells.     

Synaptic Metaplasticity Underlies Tetanic Potentiation in Lymnaea: A Novel Paradigm

We present a mathematical model that explains and interprets a novel form of short-term potentiation, which was found to be use-, but not time-dependent, in experiments done on Lymnaea neurons. The high degree of potentiation is explained using a model of synaptic metaplasticity, while the use-dependence (which is critically reliant on the presence of kinase in the experiment) is explained using a model of a stochastic and bistable biological switch.     

Chemoprevention of Skin Cancer with 1,1-Bis (3′-Indolyl)-1-(Aromatic) Methane Analog through Induction of the Orphan Nuclear Receptor,NR4A2 (Nurr1)

Background

The objective of this study was to demonstrate the anti-skin cancer and chemopreventive potential of 1,1-bis(3′-indolyl)-1-(p-chlorophenyl methane) (DIM-D) using an in vitro model.

Methods

In vitro cell cytotoxicity and viability assays were carried out in A431 human epidermoid carcinoma cell line and normal human epidermal keratinocytes (NHEK) respectively by crystal violet staining. Apoptosis induction in A431 cells (DIM-D treated) and NHEK cells pretreated with DIM-D (2 hr) prior to UVB irradiation, were assessed. The accumulation of reactive oxygen species (ROS) in DIM-D pretreated NHEK cells (2 hr) prior to UVB exposure was also determined. Immunocytochemistry and western blot analysis was performed to determine cleaved caspase 3 and DNA damage markers in DIM-D treated A431 cells and in DIM-D pretreated NHEK cells prior to UVB irradiation.

Results

The IC50 values of DIM-D were 68.7±7.3, 48.3±10.1 and 11.5±3.1 μM whilst for Epigallocatechin gallate (EGCG) were 419.1±8.3, 186.1±5.2 and 56.7±3.1 μM for 24, 48 and 72 hr treatments respectively. DIM-D exhibited a significantly (p<0.05) greater induction of DNA fragmentation in A431 cells compared to EGCG with percent cell death of 38.9. In addition, DIM-D induced higher expression in A431 cells compared to EGCG of cleaved caspase 3 (3.0-fold vs. 2.4-fold changes), Nurr1 (2.7-fold vs. 1.7-fold changes) and NFκB (1.3-fold vs. 1.1-fold changes). DIM-D also exhibited chemopreventive activity in UVB-irradiated NHEK cells by significantly (p<0.05) reducing UVB-induced ROS formation and apoptosis compared to EGCG. Additionally, DIM-D induced expression of Nurr1 but reduced expression of 8-OHdG significantly in UVB-irradiated NHEK cells compared to EGCG and UV only.

Conclusion

Our results suggest that DIM-D exhibits Nurr1-dependent transactivation in the induction of apoptosis in A431 cells and it protects NHEK cells against UVB-induced ROS formation and DNA damage.     

Conjugation of Benzylvanillin and Benzimidazole Structure Improves DNA Binding with Enhanced Antileukemic Properties

Benzyl-o-vanillin and benzimidazole nucleus serve as important pharmacophore in drug discovery. The benzyl vanillin (2-(benzyloxy)-3-methoxybenzaldehyde) compound shows anti-proliferative activity in HL60 leukemia cancer cells and can effect cell cycle progression at G2/M phase. Its apoptosis activity was due to disruption of mitochondrial functioning. In this study, we have studied a series of compounds consisting of benzyl vanillin and benzimidazole structures. We hypothesize that by fusing these two structures we can produce compounds that have better anticancer activity with improved specificity particularly towards the leukemia cell line. Here we explored the anticancer activity of three compounds namely 2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2MP, N-1-(2-benzyloxy-3-methoxybenzyl)-2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2XP, and (R) and (S)-1-(2-benzyloxy-3-methoxyphenyl)-2, 2, 2-trichloroethyl benzenesulfonate, 3BS and compared their activity to 2-benzyloxy-3-methoxybenzaldehyde, (Bn1), the parent compound. 2XP and 3BS induces cell death of U937 leukemic cell line through DNA fragmentation that lead to the intrinsic caspase 9 activation. DNA binding study primarily by the equilibrium binding titration assay followed by the Viscosity study reveal the DNA binding through groove region with intrinsic binding constant 7.39 µM/bp and 6.86 µM/bp for 3BS and 2XP respectively. 2XP and 3BS showed strong DNA binding activity by the UV titration method with the computational drug modeling showed that both 2XP and 3BS failed to form any electrostatic linkages except via hydrophobic interaction through the minor groove region of the nucleic acid. The benzylvanillin alone (Bn1) has weak anticancer activity even after it was combined with the benzimidazole (2MP), but after addition of another benzylvanillin structure (2XP), stronger activity was observed. Also, the combination of benzylvanillin with benzenesulfonate (3BS) significantly improved the anticancer activity of Bn1. The present study provides a new insight of benzyl vanillin derivatives as potential anti-leukemic agent.     

Prediction of Severe Disease in Children with Diarrhea in a Resource-Limited Setting

Objective

To investigate the accuracy of three clinical scales for predicting severe disease (severe dehydration or death) in children with diarrhea in a resource-limited setting.

Methods

Participants included 178 children admitted to three Rwandan hospitals with diarrhea. A local physician or nurse assessed each child on arrival using the World Health Organization (WHO) severe dehydration scale and the Centers for Disease Control (CDC) scale. Children were weighed on arrival and daily until they achieved a stable weight, with a 10% increase between admission weight and stable weight considered severe dehydration. The Clinical Dehydration Scale was then constructed post-hoc using the data collected for the other two scales. Receiver Operator Characteristic (ROC) curves were constructed for each scale compared to the composite outcome of severe dehydration or death.

Results

The WHO severe dehydration scale, CDC scale, and Clinical Dehydration Scale had areas under the ROC curves (AUCs) of 0.72 (95% CI 0.60, 0.85), 0.73 (95% CI 0.62, 0.84), and 0.80 (95% CI 0.71, 0.89), respectively, in the full cohort. Only the Clinical Dehydration Scale was a significant predictor of severe disease when used in infants, with an AUC of 0.77 (95% CI 0.61, 0.93), and when used by nurses, with an AUC of 0.78 (95% CI 0.63, 0.93).

Conclusions

While all three scales were moderate predictors of severe disease in children with diarrhea, scale accuracy varied based on provider training and age of the child. Future research should focus on developing or validating clinical tools that can be used accurately by nurses and other less-skilled providers to assess all children with diarrhea in resource-limited settings.