Current Pharmaceutical Biotechnology

The direct analysis of single biological molecules is getting increasingly important in basic as well as pharmaceutical research (e.g. for gene expression analysis). In particular single-molecule fluorescence detection provides exciting new opportunities to probe biochemical processes in unprecedented detail. Currently several academic and industrial research groups work on the development of single molecule detection based technologies in order to directly detect and analyze RNA and DNA molecules. As these developed methods are characterized as homogenous assays and obviate any amplification of the target or the signal, they provide clear advantages compared to methods like real-time PCR or DNA- arrays. In the following we describe a recently developed approach based on fluorescence correlation spectroscopy (FCS). This expression assay is based on gene-specific hybridization of two dye-labeled DNA probes to a selected target molecule (either DNA or RNA) in solution. The subsequent dual color cross-correlation analysis allows the quantification of the bio-molecule of interest in absolute numbers. Target concentrations of less than 10(-12) M can be easily monitored, covering the direct analysis of the expression levels of high, medium and low abundant genes.

Positron emission tomography (PET) is a molecular imaging modality that provides the opportunity to rapidly and non-invasively visualize tumors derived from multiple organs. In order to do so, PET utilizes radiotracers, such as 18F-FDG and 11C-acetate, whose uptake coincides with altered metabolic pathways within tumors. Increased expression and activity of enzymes in the fatty acid synthesis pathway is a frequent hallmark of cancer cells. As a result, this pathway has become a prime target for therapeutic intervention. Although multiple drugs have been developed that both directly and indirectly interfere with fatty acid synthesis, an optimal means to assess their efficacy is lacking. Given that 11Cacetate is directly linked to the fatty acid synthesis pathway, this probe provides a unique opportunity to monitor lipogenic tumors by PET. Herein, we review the relevance of the fatty acid synthesis pathway in cancer. Furthermore, we address the potential utility of 11C-acetate PET in imaging tumors, especially those that are not FDG-avid. Last, we discuss several therapeutic interventions that could benefit from 11C-acetate PET to monitor therapeutic response in patients with certain types of cancers.

Controlling elevated tissue-specific levels of cortisol may provide a novel therapeutic approach for treating metabolic syndrome. This concept has spurred large scale medicinal chemistry efforts in the pharmaceutical industry for the design of 11β-HSD1 inhibitors. High resolution X-ray crystal structures of inhibitors in complex with the enzyme have facilitated the structure-based design of diverse classes of molecules. A summary of binding modes, trends in structure-activity relationships, and the pharmacodynamic data of inhibitors from each class is presented.

Despite remarkable advances in cancer research, patients with malignant tumors such as high-grade glioma or advanced pancreatic carcinoma still face a poor prognosis. Because of the severe morbidity and mortality of such malignant tumor types, the identification of suitable molecular drug targets for causal treatment approaches is an important area of current research. Transforming growth factor-beta 2 (TGF-β2) is an attractive target because it regulates key mechanisms of carcinogenesis, in particular immunosuppression and metastasis, and is frequently overexpressed in malignant tumors. Here we describe the development of the antisense phosphorothioate oligodeoxynucleotide trabedersen (AP 12009) which was designed for the specific inhibition of TGF-β2 biosynthesis. In vitro and in vivo experiments confirmed the mode of action, efficacy and tolerability of trabedersen and paved the way for clinical studies. In patients with high-grade glioma, intratumoral treatment with trabedersen is currently evaluated in a pivotal, randomized and active-controlled phase III study. Intravenous application of trabedersen for the treatment of patients with advanced pancreatic carcinoma, metastasizing melanoma, or metastatic colorectal carcinoma is assessed in a currently ongoing phase I/II dose escalation study.

The binding of superquencher molecular beacon (SQMB) probes to human single-stranded cellular miRNA-122 targets was detected in various single live cells with femtosecond laser microscopy. For delivery of the SQMB-probes, 3D-nanoprocessing of single cells with sub-15 femtosecond 85 MHz near-infrared laser pulses was applied. Transient nanopores were formed by focusing the laser beam for some milliseconds on the membrane of a single cell in order to import of SQMB-probes into the cells. In single cells of the human liver cell lines Huh-7D12 and IHH that expressed miRNA-122, we measured target binding in the cytoplasm by two-photon fluorescence imaging. We found increased fluorescence with time in a nonlinear manner up to the point where steady state saturation was reached. We also studied the intracellular distribution of target SQMB and provide for the first time strong experimental evidence that cytoplasmic miRNA travels into the cell nucleus. To interpret nonlinear binding, a number of individual miRNA-122 positive cells (Huh-7D12 and IHH) and negative control cells, human VA13 fibroblasts and Caco-2 cells were analyzed. Our experimental data are consistent with the cytoplasmic assembly of nuclear miRNA and provide further mechanistic insight in the regulatory function of miRNAs in cellular physiology. An open issue in the regulation of gene expression by miRNA is whether miRNA can activate gene expression in addition to the well-known inhibitory effect. A first step for such a regulatory role could be the travelling of miRNA-RISC into the nucleus.

A number of molecular methods of gene expression analysis can approach genomic level. Among those, Serial Analysis of Gene Expression (SAGE) stands out. Unlike many other techniques, SAGE allows both qualitative and quantitative analysis of previously unknown transcripts. Over the course of the last 13 years, SAGE has became a recognized tool of large-scale gene expression profiling, being used extensively in human, animal, yeast and plant studies of various nature. A number of important adaptations was introduced both to the protocol of SAGE library construction and to the analytical algorithm employed. Moreover, some variations of the original protocol (MAGE, SADE, microSAGE, miniSAGE, longSAGE, superSAGE, deepSAGE, etc.) were derived to improve the utility of SAGE in certain conditions. Current review aims comparing the benefits and drawbacks of the techniques for high-throughput gene expression analysis (including SAGE) in a realistic, balanced manner. Issues related to modifications to the original protocol and further development of the SAGE are discussed.

The concept that the efficacy of all antipsychotic drugs (APDs) can be explained by their action on dopamine (DA) D2 receptors is most challenged by drugs such as clozapine which target serotonin (5-HT)2A receptors as an essential component of their efficacy and tolerability. The 5-HT2A receptor, along with 5-HT1A, 5-HT 2C, 5-HT 6 or 5-HT 7 receptors, all of which are components of the mechanism of action of clozapine, represent important targets for treating multiple aspects of schizophrenia, especially psychosis and cognitive impairment. The class of atypical antipsychotic drugs (APDs), of which clozapine is the prototype, share in common more effective 5-HT 2A receptor inverse agonism and weaker interference with D2 receptor stimulation, either through D2 receptor blockade or partial D2 receptor agonism. This has led to development of a selective 5-HT2A antagonist, ACP-103 (pimavanserin), which has been found to be effective as monotherapy in L-DOPA psychosis and has promise as an add-on agent for sub-effective doses of atypical APDs. We review here the extensive preclinical evidence to support the importance of 5-HT2A receptor inverse agonism to the action of clozapine and related atypical APDs, and evidence supporting the potential of selective 5-HT2A, 5-HT 6 , and 5-HT 7, antagonists, 5-HT1A partial agonists and 5-HT2C agonists for development of drugs which ameliorate psychosis or cognitive impairment.

The role of matrix metalloproteinases in disease has been investigated over the last two decades. A focus on this family of proteases is particularly emphasized in two major arthritides in humans, osteoarthritis and rheumatoid arthritis. Early work described the presence of multiple MMP family members in the joint of the disease state and recent advances in the development of new knockout mice and disease models have allowed investigators to directly test the role of the MMP proteases in arthritis. MMP-13 is expressed by chondrocytes and synovial cells in human OA and RA and is thought to play a critical role in cartilage destruction. The recent development of an MMP-13 knockout mouse has documented the important role for this enzyme in cartilage formation and further studies under disease conditions promise to reveal the function of this enzyme in disease pathology. This review describes a body of research that supports the development of novel selective MMP-13 inhibitors with the hope of developing these compounds in clinical trials for the treatment of arthritis.

Hyperpolarized (HP) (13)C labeled compounds can be used as MR contrast agents to investigate metabolic pathways in vivo in almost real time. To date, a high proportion of reported studies have utilized HP 1-(13)C pyruvate to investigate intracellular metabolism in tumors and other tissues. The long T(1) relaxation time of the carboxylate carbon enables the (13)C signal of the pyruvate to be followed for nearly 2 minutes following injection. During this time, pyruvate is rapidly metabolized to generate observable metabolites such as alanine and lactate. HP (13)C labeled compounds have, for example, also been used to non-invasively probe physiological parameters such as pH, which emphasizes the expanding potential of the technique. The commercial availability of dynamic nuclear polarization (DNP) systems to generate hyperpolarized material for injection has made the technique available to researchers worldwide. As a consequence, DNP (13)C MR has become a rapidly expanding area of research. The technique, with its specific strengths and weaknesses, has incredible potential coupled with inherent limitations, and this review aims to both present background to the technique and describe some of the necessary hardware and software essential to perform hyperpolarized (13)C studies. An overview of the current and future role of HP (13)C based molecular imaging is presented.

The 14-3-3 family of proteins was originally identified in 1967 as simply an abundant brain protein. However it took almost 25 years before the ubiquitous role of 14-3-3 in cell biology was recognized when it was found to interact with several signalling and proto-oncogene proteins. Subsequently 14-3-3 proteins were the first protein recognized to bind a discrete phosphoserine/threonine-binding motifs. In mammals the 14-3-3 protein family is comprised of seven homologous isoforms. The 14-3-3 family members are expressed in all eukaryotes and although no single conserved function of the 14-3-3s is apparent, their ability to bind other proteins seems a crucial characteristic. To date more than 300 binding partners have been identified, of which most are phosphoproteins. Consequently, it has become clear that 14-3-3 proteins are involved in the regulation of most cellular processes, including several metabolic pathways, redox-regulation, transcription, RNA processing, protein synthesis, protein folding and degradation, cell cycle, cytoskeletal organization and cellular trafficking. In this review we include recent reports on the regulation of 14-3-3 by phosphorylation, and discuss the possible functional significance of the existence of distinct 14-3-3 isoforms in light of recent proteomics studies. In addition we discuss 14-3-3 interaction as a possible drug target.

A nonapeptide, H-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2, corresponding to amino acids 6 to 14 of bombesin and its four analogues were synthesized by a solid-phase method and were tested for their comparative effect on reducing activity on food intake using male Wistar rats. The synthetic bombesin-fragment6-14 showed reducing effect on food intake using male Wistar rats. Of the synthetic analogues, [Phe(4F)13]bombesin-fragment6-14 exhibited the most potent effect. The reducing effect on food intake of [Sar11]bombesin-fragment6-14 was lower than that of our synthetic bombesin-fragment6-14, but the other two analogues, [betaAla11]bombesin-fragment6-14 and [1-Nal8]bombesin-fragment6-14 showed no reducing effect on food intake in rats.

The final proof about the specific mechanisms by which the different components of olive oil, the principal source of fat in a typical "Mediterranean diet", exert their potential protective effects on the promotion and progression of several human cancers requires further investigations. A recent discovery that dietary fatty acids can interact with the human genome by regulating the amount and/or activity of transcription factors has opened a whole new line of research aimed to molecularly corroborate the ant-cancer benefits of the olive oil-based Mediterranean diet and the underlying mechanisms. Our most recent findings reveal that oleic acid (OA; 18:1n-9), the main olive oil's monounsaturated fatty acid, can suppress the overexpression of HER2 (erbB-2), a well-characterized oncogene playing a key role in the etiology, invasive progression and metastasis in several human cancers. First, exogenous supplementation with physiological concentrations of OA significantly down-regulates HER2-coded p185(Her-2/neu) oncoprotein in human cancer cells naturally harboring amplification of the HER gene. Second, OA exposure specifically represses the transcriptional activity of the human HER2 gene promoter in tumor-derived cell lines naturally exhibiting HER2 gene amplification and p185(Her-2/neu) protein overexpression but not in cancer cells expressing physiological levels of HER2. Third, OA treatment induces the up-regulation of the Ets protein PEA3 (a transcriptional repressor of the HER2 gene promoter) solely in cancer cells naturally displaying HER2 gene amplification. Fourth, HER2 gene promoter bearing a PEA3 site-mutated sequence cannot be negatively regulated by OA, while treatment with OA fails to repress the expression of a human full-length HER2 cDNA controlled by a SV40 viral promoter. Fifth, OA-induced inhibition of HER2 promoter activity does not occur if HER2 gene-amplified cancer cells do no concomitantly exhibit high levels of Fatty Acid Synthase (FASN; Oncogenic antigen-519) as specific depletion of FASN, which itself similarly suppresses HER2 overexpression by inducing PEA3-dependent repression of HER2 gene promoter, strongly antagonizes the inhibitory effects of OA on HER2 gene promoter activity. Considering that OA treatment efficiently blocks FASN activity and down-regulates FASN protein expression, it is reasonable to suggest that an accumulation of supra-physiological concentrations of the FASN substrate malonyl-CoA, due to its reduced utilization by FASN in the presence of exogenous OA, appears to act as an indicator of "cell fuel" availability capable to suppress HER2 expression via formation of inhibitory "PEA3 protein-PEA3 DNA binding site" complexes on the endogenous HER2 promoter. Indeed, malonyl-CoA on its own dramatically decreases HER2 promoter activity, while OA or malonyl-CoA similarly up-regulates PEA3 gene promoter activity. This previously unrecognized ability of OA to directly affect the expression of a cluster of interrelated human cancer genes (i.e., HER2, FASN and PEA3) should open a new line of research aimed to explore the anti-cancer effects of OA. Certainly, an appropriate dietary intervention reproducing this prominent anti-oncogenic feature of the "Mediterranean diet" must be carried out in animal models and human pilot studies in the future. Only then we will know whether the old "Mediterranean dietary traditions" will become a new molecular approach in the management of cancer disease.

Positron emission tomography (PET) is a powerful and rapidly developing area of molecular imaging that is used to study and visualize human physiology by the detection of positron-emitting radiopharmaceuticals. Information about metabolism, receptor/enzyme function, and biochemical mechanisms in living tissue can be obtained directly from PET experiments. In particular, the interest in (18)F-labeled proteins remains high for both diagnoses and therapy monitoring purposes. The development of labeling strategies for the synthesis of new (18)F labeled protein is, however, not trivial. (18)F-containing prosthetic groups are often required for protein labeling to obtain high yield under mild labeling conditions and keep the bioactive character of the proteins. This Review highlights key aspects of protein (18)F-labeling method and discussed representative examples including (18)F-labeled human serum albumin, (18)F-labeled Annexin V, (18)F-labeled HER2 affibody, and (18)F-labeled low density lipoprotein.

Cancer is becoming the largest threat to human health. Apart from classical anti-cancer therapies such as surgery, chemotherapies, and radiotherapy, many new therapies are being developed or translated into clinical use. These therapies include various neoadjuvant chemotherapies, minimally invasive treatments, and molecular-targeted therapies. However, none of these methods benefit all patients because treatment should be personalized according to the response of each patient. A futile therapy makes a patient miss the optimum time for treatment and increases the medical burden to the society. Thus, a great challenge is encountered in monitoring such therapies. Classical methods based on anatomical changes such as computed tomography (CT) and magnetic resonance imaging (MRI) have well-known limitations in early response evaluation. Positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) as tracer is a promising method especially when integrated with CT or MRI in one system. This article reviews the current status of monitoring anti-cancer therapies, including the evolution of evaluation criteria from the World Health Organization to the Response Evaluation Criteria in Solid Tumor and the PET Response Criteria in Solid Tumor. The advantages of 18F-FDG PET/CT for response evaluation are analyzed in various malignant tumors, and the pertinent weaknesses are discussed. Finally, several future directions in monitoring anti-cancer therapies are prospected.

Apoptosis of cardiomyocytes induced by oxidative stress play a critical role in cardiac dysfunction associated with ventricular remodeling and heart failure. We recently reported that leonurine attenuated hypoxia-induced cardiomyocyte damage. In this study, we investigated the mechanism of leonurine (originally from Herba leonuri but we synthesized it chemically it as also called SCM-198) (H₂O₂)-induced rat embryonic heart-derived H9c2 cells from apoptosis. Exposing H9c2 cells to H₂O₂ significantly decreased cell viability, and this was attenuated by pretreatment with leonurine for 4 h in a concentration-dependent manner. Meanwhile, leonurine was found to reduce intracellular reactive oxygen species (ROS) generation in H₂O₂-stimulated cell. Moreover, H9c2 cells stimulated by H₂O₂ was accompanied with apparent apoptotic characteristics, including fragmentation of DNA, apoptotic body formation, release of cytochrome c, translocation of Bax to mitochondria, loss of mitochondrial membrane potential (ΔΨ(m)) and activation of caspase 3. Furthermore, H₂O₂ also induced rapid and significant phosphorylation of the c-Jun-N-terminal kinase 1/2 (JNK1/2), which was inhibited SP600125 (a JNK1/2 inhibitor). All of these events were attenuated by leonurine pretreatment. Taken together, these results demonstrated that leonurine could protect H9c2 cells from H₂O₂-induced apoptosis via modulation of mitochondrial dysfunction associated with blocking the activation of JNK1/2.

CYP1A1, an enzyme of the cytochrome P450 superfamily, is the most important xenobiotic-metabolizing enzyme of the placenta for which relevant inducible activity has been demonstrated throughout pregnancy. CYP1A1 metabolizes several drugs and compounds widely used in pharmacotherapy or present in diets. At the same time, this enzyme plays a key role in the bioactivation of procarcinogens and proteratogens, such as arylamines and polycyclic aromatic hydrocarbons (PAHs), which bind to placental and foetal DNA as DNA-adducts. The expression of CYP1A1 is transcriptionally up-regulated through the ligand-activated aryl hydrocarbon receptor (AhR). AhR plays an important role as mediator of an adaptive response to xenobiotics, as well as in normal physiology and embryonic development. Several exogenous AhR ligands, such as PAHs, polychlorinated biphenyls and halogenated dioxins, can be found in the constituents of numerous commercial products, including insulators and flame retardants, or as products of combustion processes, including chimney soot, charbroiled foods and cigarette smoke, or as the product of waste incineration. Exposure to these compounds subsequently affects cellular growth and differentiation, homeostasis, level of growth factors, reproduction function and hormonal regulation. Importantly, elevated CYP1A1 activity through activated AhR in placentas of women smokers has been associated with pregnancy complications, such as premature birth, intrauterine growth retardation (IUGR), structural abnormalities, foetal death or placenta abruption, risk of low birth weight, low birth length and low head circumference. We summarize the recent findings related to toxicological consequences of AhR activation and CYP1A1 induction in the human placenta during pregnancy.

Indirect effects of 3,4-methylenedioxy-N-methylamphetamine (MDMA) and metabolites on the cardiac cells are well-known, the mechanism(s) underlying direct MDMA-induced cardiotoxicity remaining to be clarified. To better understand the immuno-inflammatory phenomena accompanying the cardiac alterations during MDMA administration, we conducted a study in an in vivo animal model to evaluate the cellular morphological alterations related to the biological response between MDMA administration and inflammatory cytokines (tumor necrosis factor-alpha, IL-1beta, IL-6, 8, 10, and monocyte chemotactic protein-1). A total of 25 male rats were used. The effects were evaluated at 6, 16 and 24 hours after a single dose MDMA administered (20 mg/kg i.p.). We found high levels of the cardioinhibitory cytokines in rat heart after 3 and 6 hs from MDMA administration. Strongest reaction was observed at 24 hs for TNF-alpha, IL-1beta, IL-6, 8, 10 and for MCP-1. Furthermore, we still determined the presence of MDMA and MDA in the plasma of rats treated with MDMA intra-peritoneal single injection; it was present as early at 6 hs and still present 24 hs after treatment. Western blot analysis in cardiac samples demonstrated the IL-1beta and IL-6 reactions in rats died spontaneously at fourth hour. The rise of the selective cardioinhibitory cytokines may be interpreted as the adaptive response of jeopardized myocardium to the cardiac dysfunction resulting from MDMA injection.

Transplantation of pancreatic islets is a promising strategy for restoring insulin secretion in diabetes mellitus. To monitor transplanted islets, a method to evaluate the distribution in a non-invasive manner in vivo is needed. INS-1E, a stable differentiated insulin secreting cell line, and rodent islets were used to monitor cell transplantation by MRI. For labeling INS-1E cells in vitro, increasing concentrations of Resovist in culture medium were tested. For MR imaging in a clinical 3T scanner, we placed a layer of labeled INS-1E cells between two layers of 4% gelatin. Viability assay was performed. Cell function was evaluated by static incubation assay to assess insulin secretion. For in vivo imaging, iron labeled rodent islets were transplanted into the liver of streptozotocin induced diabetic rats and visualized by MRI. Blood sugar values were controlled and liver tissue was removed for histological analysis. SPIO labeled INS-1E cells did not show altered viability or reduced glucose stimulated insulin secretion in vitro. Double staining of labeled and unlabeled INS-1E cells showed no difference in the staining pattern. Labeling of rodent islets with SPIOs does not reduce their secretory activity or alter their viability. We visualized SPIO-labeled INS-1E cells and rat islets in vitro using a clinical 3T scanner. Diabetic rats transplanted with SPIO-labeled islets became normoglycemic. MR imaging successfully verified the distribution of labeled transplanted cells in vivo. Labeling INS-1E cells and rat islets with SPIOs does not alter their viability, while enabling MR imaging of labeled cells in vitro and within the living organism.

HIF-1α is regarded as a target for drug development in several diseases such as cancer. For high throughput screening of HIF-1α-targeted drug, we need to examine the activity quantitatively.In the present study, we proposed a method where stable expression system of HIF-1αwas combined with image correlation analysis. When the stable transformants were labeled with DRAQ5, we could detect Co(2+)-induced nuclear translocation by the use of cross-correlation analysis of the dual labeling images. In the case of high throughput screening for HIF-1α-targeted drug, we should use Pearson's correlation coefficient to judge nuclear translocation.

Objective: RA is one of autoimmune diseases, has drawn great attention of the world. Currently, the anti- IL-1β monoclonal antibody Canakinumab (ACZ885) for treatment of RA has entered into clinical trials. However, Full length antibody has large molecular weight, and is difficult to penetrate the tissue or the nidus. In contrast, scFv has low molecular weight and strong penetration ability, and is favorable to increase the drug concentration in the indus, hence improving the efficacy of the drug. The aim of this study is to obtain a neutralizing scFv antibody from a combinatorial scFv library against hIL-1β by the modified NLPA-based bacterial display system, for further development of the small molecule antibody drug for treatment of RA. Methods: The modified NIPA-based bacterial display system was used to construct the combinatorial scFv library derived from the spleen cDNA of immunized mice with hIL-1β. FACS was used to screen hIL- 1β -binding clones with FITC-labeled hIL-1β protein. Three clones were randomly selected from the third round of screening, and their nucleotide sequences were aligned with mouse immunoglobulin genes. The single chain antibody genes of the hIL-1β-binding clones were subcloned into the prokaryotic expression vector pET-27b for expression. The molecular mass of the purified anti-hIL-1β single chain antibody was about 28ku. The hIL-1β -binding ability of antibody were examined by ELISA and Western blot assays. Ability of the scFv antibody to neutralize hIL-1β was evaluated by the MTT test. Conclusions: In this study, it is the first time to use the NIPA-based bacteria display system to construct and screen the combinatorial scFv library. Three scFvs against hIL-1β were obtained from the scFv library of the immunized mice. Prokaryotically expressed and purified scFvs demonstrate binding ability with hIL-1β. Among the three clones, The MTT test suggests that scFv-20 is a neutralization antibody against hIL-1β. The study provides a lead candidate for further development of small molecule therapeutic antibodies for treatment of RA.

IL-1β and TNF-α play key roles in the inflammatory response. Their abnormal expression may cause the occurrence of various diseases, such as RA. Recently, treatments that target TNF-α and IL-1β have begun to be applied in the clinical setting. Although these biological agents can obtain mostly good results, they are ineffective in some patients. The reason for this result may be that these biological agents could not fully inhibit a variety of inflammatory cytokines in the inflammatory response. In the present study, a fusion protein gene which encoded human interleukin-1β scfv and soluble TNF receptor I (sTNFRI) was cloned. A number of in vitro assays demonstrated that anti-IL-1β scfv/TNFRI simultaneously bound to both targets. The bioactivity assay showed that the fusion protein could inhibit both the cytotoxicity of hTNF-α on L929 cells and hIL-1β-induced proliferation of L929 cells, indicating that the fusion protein has the ability to neutralize both hTNF-α and hIL-1β. In this study, we established the chicken type II collagen-induced rheumatoid arthritis model in Kunming mice, and evaluated the pharmacological effect of the fusion protein in vivo. Model mice were randomly divided into 8 groups (n=8): CIA model control group, DEX treatment group (1 mg/kg), intraperitoneal treatment group (high-dose: 5 mg/kg; medium-dose: 2 mg/kg; low-dose: 0.8 mg/kg), subcutaneous treatment group (high-dose: 5 mg/kg; medium-dose: 2 mg/kg; low-dose: 0.8 mg/kg), and healthy mice as control. The control group received the same volume of saline. The mice were administrated once every 2 days. Arthritis index, anti-CII antibody titers, cytokine levels, histopathological changes were examined. The results showed that anti-IL-1β scfv/TNFRI fusion protein could reduce the degree of joint swelling, inflammatory cell infiltration, synovial cell proliferation and the level of CII antibody in the sera. The Real-time PCR analysis showed that anti-IL-1β scfv/TNFRI had the ability to reduce the expression of IL-1β, TNF-α, IL-17A, MMP-3, IL-6 and improve the expression of IL-10 in a dose-dependent manner, suggesting that the fusion protein is the mediator for IL-1β and TNF-α involved in the RA process. Compared with DEX positive medicine control, anti-IL-1β scfv/TNFRI appeared more beneficial in treatment of CIA mice. The therapeutic effect of the anti-IL-1β scfv/TNFRI at 5mg/kg was significantly better than that of DEX treatment. So the anti-IL-1β scfv/TNFRI can become a candidate for treatment of RA.

The purpose of this study was to characterize the human term placental villous tissue explant culture model as a tool to study the formation and efflux of 1-chloro-2,4-dinitrobenzene (CDNB) conjugate 2,4-dinitrophenyl-S-glutathione (DNP-SG) as a model system for phase II metabolism and ATP-binding cassette (ABC) transporter-mediated cellular efflux. Placental tissue samples were obtained after cesarean section following normal pregnancies (n=9). Cultured villous tissue was monitored up to 48 h to study the effect of time in culture on biochemical parameters, formation and efflux of DNP-SG in the absence or presence of ATPase inhibitor sodium orthovanadate and the protein expression of ABC transporters - multidrug resistance associated protein 2 (MRP2), P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and enzyme glutathione-S-transferase isoform P1-1 (GSTP1-1). Villous tissue structure, tissue viability and expression of BCRP, GSTP1-1 remained unchanged, while expression of MRP2, P-gp and total tissue glutathione decreased with time in culture. Tissue integrity was unchanged up to 24 h but declined at 48 h. However, DNP-SG formation, DNP-SG efflux, and the extent of inhibition of DNP-SG efflux by sodium orthovanadate showed only minor changes through 48 h. Sodium orthovanadate decreased DNP-SG efflux, consistent with inhibition of apical ABC transporters. The results support the use of the cultured human term placental villous tissue explants model to study coordinated function of GSTP1-1 and apical ABC transporters in the formation and efflux of the model substrate DNP-SG.

The prime difference between generics and biosimilars is that while generics contain the exact active ingredient as in the originator product, biosimilars are only "similar" and not "identical" to the originator biological medicine. This difference appears due to the nature of the biopharmaceutical medicines which are extremely complex to manufacture (it is not possible to make an exact copy of a biotech medicine in the same way as a traditional chemical molecule can be copied). In fact, it is widely accepted that for biopharmaceuticals, the "process is the product". Minor changes during the manufacturing process can have critical consequences in the patients. The vast majority of the biopharmaceuticals on the market are produced by genetic engineering using various recombinant expression systems. Most of the recombinant proteins that have been granted marketing approval to date are produced either in E. Coli or in recombinant mammalian cell lines. Several approaches may be undertaken to determine biopharmaceuticals potency. Bioassays represent the most relevant potency-determining assay, as they directly assess the biological activity of the product. These assays involve applying a known quantity of the substance to be analyzed to a biological system which responds to this applied stimulus. The response is measured quantitatively, allowing an activity value to be assigned to the substance being assayed.

Psoriasis is a common (1-3% of the population worldwide), multifactorial, immune-mediated chronic skin disease. In psoriasis pathogenesis an over-reaction of local innate immune response initiates inflammation with subsequent involvement of adaptive immune response leading to the production of a panel of cytokines, chemokines and growth factors leading to epidermal hyperplasia. Recently, IL-21 has been involved in this process as this cytokine is overexpressed in psoriatic skin and can cause epidermal hyperplasia and inflammation when injected intradermally into mice. Moreover blockade of IL-21 with a human antibody against IL-21 reduces the epidermal thickness and the expression of Th1 and Th17 genes in the well-characterized model of human psoriasis-xenograft mouse. Therefore, the inhibition of this cytokine may be therapeutically effective in the treatment of psoriasis. Here we will review recent data on psoriasis pathogenesis focusing on the role of IL-21 as novel therapeutic target.

The accidental or intentional release of plutonium or americium can cause acute and long term adverse health effects if they enter the human body by ingestion, inhalation, or injection. These effects can be prevented by rapid removal of these radionuclides by chelators such as calcium or zinc diethylenetriaminepentaacetate (calcium or zinc DTPA). These compounds have been shown to be efficacious in enhancing the elimination of members of the actinide family particularly plutonium and americium when administered intravenously or by nebulizer. The efficacy and adverse effects profile depend on several factors that include the route of internalization of the actinide, the type, and route time of administration of the chelator, and whether the calcium or zinc salt of DTPA is used. Current and future research efforts should be directed at overcoming limitations associated with the use of these complex drugs by using innovative methods that can enhance their structural and therapeutic properties.

The highly polymorphic human cytochrome P450 2D6 (CYP2D6) metabolizes about 25% of currently used drugs. In this study, we have explored the interaction of a large number of substrates (n = 120) with wild-type and mutated CYP2D6 by molecular docking using the CDOCKER module. Before we conducted the molecular docking and virtual mutations, the pharmacophore and QSAR models of CYP2D6 substrates were developed and validated. Finally, we explored the interaction of a traditional Chinese herbal formula, Fangjifuling decoction, with CYP2D6 by virtual screening. The optimized pharmacophore model derived from 20 substrates of CYP2D6 contained two hydrophobic features and one hydrogen bond acceptor feature, giving a relevance ratio of 76% when a validation set of substrates were tested. However, our QSAR models gave poor prediction of the binding affinity of substrates. Our docking study demonstrated that 117 out of 120 substrates could be docked into the active site of CYP2D6. Forty one out of 117 substrates (35.04%) formed hydrogen bonds with various active site residues of CYP2D6 and 53 (45.30%) substrates formed a strong π-π interaction with Phe120 (53/54), with only carvedilol showing π-π interaction with Phe483. The active site residues involving hydrogen bond formation with substrates included Leu213, Lys214, Glu216, Ser217, Gln244, Asp301, Ser304, Ala305, Phe483, and Phe484. Furthermore, the CDOCKER algorithm was further applied to study the impact of mutations of 28 active site residues (mostly non-conserved) of CYP2D6 on substrate binding modes using five probe substrates including bufuralol, debrisoquine, dextromethorphan, sparteine, and tramadol. All mutations of the residues examined altered the hydrogen bond formation and/or aromatic interactions, depending on the probe used in molecular docking. Apparent changes of the binding modes have been observed with the Glu216Asp and Asp301Glu mutants. Overall, 60 compounds out of 130 from Fangjifuling decoction matched our pharmacophore model for CYP2D6 substrates. Fifty four out of these 60 compounds could be docked into the active site of CYP2D6 and 24 of 54 compounds formed hydrogen bonds with Glu216, Asp301, Ser304, and Ala305 in CYP2D6. These results have provided further insights into the factors that determining the binding modes of substrates to CYP2D6. Screening of high-affinity ligands for CYP2D6 from herbal formula using computational models is a useful approach to identify potential herb-drug interactions.

In this paper, the distribution and redistribution of the amphetamine derivative, 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) is brought into focus. Animal experimental data were compared with internationally reported MDMA-related human fatalities: in general, these turned out to be parallel with each other. Due to its inherent properties (e.g. significant volume of distribution), MDMA is liable to postmortem redistribution. Indeed, very high concentrations have been found in cardiac blood and tissues located centrally in the body (blood-rich organs such as lungs and liver in particular). This confirms that post-mortem redistribution due to diffusion from higher to lower concentration can easily take place, mainly at longer post-mortem intervals and when putrefaction occurs. Therefore, we can conclude that for post-mortem quantitation of amphetamine and derivatives, and MDMA in particular, peripheral blood sampling (e.g. femoral vein) remains compulsory. However, if the latter is impossible, MDMA quantification in a few alternative matrices such as vitreous humour and iliopsoas muscle may provide additional information to come to a reliable conclusion. Furthermore, it should be stressed that--at present--it is impossible to estimate the individual susceptibility to the various possible adverse effects of MDMA, which implies that it is impossible to provide a "safe" or "therapeutic" blood MDMA level. Therefore, in current forensic practice, the post-mortem pathological and toxicological findings should form an entity in order to draw a well-grounded conclusion.

Hepatocellular carcinoma (HCC) represents the third cause of cancer-related death. Because HCC is multi-centric with time, excluding the few transplanted patients, sooner or later it becomes untreatable with loco-regional therapies and, until some years ago, it was not responsive to systemic therapies. In 2005 a randomized trial indicated the efficacy of a product containing stem cell differentiation stage factors (SCDSF) taken from zebra fish embryos during the stage in which the totipotent stem cells are differentiating into the pluripotent adult stem cells. In such a trial the patients, with "intermediate" and "advanced" HCC according to BCLC/AASLD guidelines, presented benefit in terms of performance status (PS) and objective tumoral response, with some cases (2.4%) of complete response (CR). The aim of this cohort study is to report the experience of a tertiary referral center on the evidence of cases of CR in patients with "advanced" stage HCC treated with SCDSF as supportive care. CR was regarded as sustained disappearance of the neoplastic areas or blood supply therein, accompanied by normalization of AFP levels. Out of 49 patients consecutively recruited and retrospectively evaluated, 38 had "advanced" stage and 11 "terminal" stage. In 5 patients with "advanced" stage a sustained CR was reported (13.1%). Improvement on PS was obtained in 17 patients (34.6%). No side effects occurred. SCDSF treatment confirmed its efficacy in patients with "advanced" HCC, in terms of PS and tumoral response.

The protein-protein docking problem is one of the focal points of activity in computational structural biology. Adequate computational techniques for structural modeling of protein interactions are important because of the growing number of known protein structures, particularly in the context of structural genomics. The protein docking methodology offers tools for fundamental studies of protein interactions and provides structural basis for drug design. The paper presents a critical review of the existing protein-protein docking approaches in view of the fundamental principles of protein recognition.

Fluorescence Correlation Spectroscopy (FCS) has developed into a routine method to quantitatively study diffusion of molecules and kinetic processes. FCS has recently become popular to complement live cell imaging with biophysical information. The enabling technology has been commercially realised by combining laser scanning microscopes and fluorescence correlation spectrometers in one integrated platform. This article provides an overview of the Zeiss solution of such a combined instrument, the LSM 510 META / ConfoCor 2 system. We focus on the instrumental set up as well as technical advances and improvements in the software that controls FCS data acquisition and evaluation. In addition, we outline the calibration of the instrument and the work flow for data analysis with emphasis on in vivo pharmacological applications.

CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia). We investigated the efficacy of CNTO 530 in murine models of β-thalassemia and sickle cell anemia (Berkeley mice). β-thalassemic mice are deficient in expression of α -globin chain and heterozygous mice are characterized by a clinical syndrome similar to the human β-thalassemia intermedia. Berkeley mice are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Berkeley mice thus express human sickle hemoglobin A (HbS) and can also express human fetal hemoglobin. These mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. To test the effectiveness of CNTO 530, mice from both genotypes received a single subcutaneous (s.c.) dose of CNTO 530 or darbepoetin-α (as a comparator) at 10,000 U/kg, a dose shown to cause a similar increase in reticulocytes and hemoglobin in normal mice. Hematologic parameters were evaluated over time. CNTO 530, but not darbepoetin-α, increased reticulocytes, red blood cells and total hemoglobin in b-thalassemic mice. In Berkeley mice CNTO 530 showed an increase in reticulocytes, red blood cells, F-cells, total hemoglobin and fetal hemoglobin. In conclusion, CNTO 530 is effective in murine models of β-thalassemia and sickle cell anemia. These data suggest that CNTO 530 may have beneficial effects in patients with genetically mediated hemoglobinopathies.

Oncolytic viruses (OVs) are designed to replicate in, and subsequently lyse cancer cells. Numerous oncolytic virus platforms are currently in development. Here we review preclinical and clinical experience with JX-594, the lead candidate from the targeted and armed oncolytic poxvirus class. JX-594 is derived from a vaccinia vaccine strain that has been engineered for 1) enhanced cancer targeting and 2) has been "armed" with the therapeutic transgene granulocytemacrophage colony stimulating factor (GM-CSF) to stimulate anti-tumoral immunity. Poxviruses have many ideal features for use as oncolytic agents. The development of oncolytic vaccinia viruses is supported by a large safety database accumulated in the smallpox eradication program. In addition, poxviruses have evolved unique capabilities for systemic spread through the blood that can be harnessed for the treatment of metastatic disease. JX-594 demonstrates a high degree of cancer selectivity and systemic efficacy by multiple mechanisms-of-action (MOAs) in preclinical testing. Data from Phase 1 and 2 clinical trials has confirmed that these features result in potent and systemic efficacy in patients with treatment refractory metastatic cancers.

The role of oral chemotherapy has been getting expanded because of the potential advantage in patients' convenience and better quality of life as well as in cost-effectiveness as compared with intravenous chemotherapy. In this article, the history, mechanism of anti-tumor activity, and clinical use of oral chemotherapy using 5-fluorouracil (5-FU) derivative chemotherapeutic agents are reviewed. Pharmacological analysis has revealed that 5-FU, a basic chemotherapeutic agent widely used against a variety of malignant tumors, shows a time dependent anti-tumor activity, and that continuous maintenance of 5-FU concentration in blood is the optimal method in 5-FU administration. UFT, a combination drug of ftorafur (tetrahydrofuranyl-5-fluorouracil, tegafur, FT) and uracil, has been developed to have potent anti-tumor activity by maintaining higher 5-FU concentration in blood and tumor tissues for a long time. FT is a pro-drug that releases 5-FU continuously, and uracil is added to inhibit degradation of the released 5-FU. Clinically, oral administration of UFT has proved to be effective as an adjuvant therapy after surgery for some malignant tumors such as non-small cell lung cancer. Moreover, UFT has proved to be effective for inoperable advanced malignancies such as colorectal cancer, especially in combination with leucovorin or cisplatin. Recently, S-1, a more active oral 5-FU derivative chemotherapeutic agent has been developed in Japan. Several factors to affect anti-tumor effects and/or toxicities of 5-FU and the derivatives, such as thymidylate synthase activity, dehydropyrimidine dehydrogenase activity and p53 status, are also discussed in the article. In conclusion, oral administration of 5-FU derivatives such as UFT may have several clinical advantages over intravenous 5-FU administration.

Zevalin (ibritumomab tiuxetan, IDEC-Y2B8) is a murine IgG1 kappa monoclonal antibody conjugated to tiuxetan (MXDTPA) that chelates Yttrium or Indium and is directed against the CD 20 molecules of B lymphocytes. Phase I studies have determined the optimal dose of pretreatment rituximab to be 250 mg/m2 seven days prior and immediately prior to the administration of Zevalin. Phase I/II data have determined the dose of 0.4 mCi/kg to be the maximum tolerated dose (MTD) for patients with platelet counts > 150,000 and < 25% bone marrow involvement with NHL. The dose of 0.3 mCi/kg is the MTD in patients with platelet counts between 100,000-149,000. Toxicity is primarily hematologic, transient, and reversible. Dosimetry has been completed using 111In-2B8. Results to date demonstrate that, at the above doses, no patients exceeded the protocol-prescribed organ maximum dose of 2,000 cGy or red marrow maximum dose of 300 cGy. Therefore, future use will not require pretreatment dosimetry. Zevalin contains a pure beta-emitting isotope; no protective patient or staff isolation procedures are required. A randomized Phase III trial has been completed, comparing Zevalin with a standard dose of rituximab (375 mg/m2 q week for four weeks) in patients with relapsed indolent or follicular transformed NHL. The overall response rate (ORR) was 80% in the Zevalin arm compared to 56% (p = 0.002) in the rituximab arm. The CR was 30% vs. 16% (p=0.04). A nonrandomized trial in patients refractory to rituximab demonstrated an ORR of 74% and a CR rate of 15%. A Phase II study of a reduced dose of Zevalin in patients with mild thrombocytopenia demonstrated an ORR of 67% and a 33% CR rate. Zevalin is safe and effective in patients with relapsed or refractory NHL, even in patients refractory to prior rituximab therapy.

The ATP-binding cassette A1 (ABCA1) transporter plays a major role in the efflux of lipids by mediating the cellular transport of phospholipids and cholesterol to lipid-poor/lipid-free apolipoprotein A-I (apoA-I) particles and thereby exerting important anti-atherogenic effects. Although the mechanism whereby ABCA1 mediates cholesterol efflux is not completely understood, numerous studies have shown that, in addition to apoA-I, the expression level of the total or cell surface ABCA1 protein is a determining factor for the activity of ABCA1-mediated cholesterol efflux, and defects in ABCA1-mediated cholesterol efflux lead to various pathological conditions in different cells, including cardiovascular and metabolic disorders. It has been widely demonstrated that a growing list of natural and synthetic substances and metabolic regulators that modulate the expression of ABCA1 not only act directly on the ABCA1 gene promoter, but also occurs at the post-transcriptional level via micro-RNAs and post-translationally through the stabilization or localization of the protein. The complex regulatory network of ABCA1 results in promoting or suppressing cholesterol efflux from cells, therefore we speculate that the ABCA1 transporter is emerging as a novel therapeutic target for cardiovascular and metabolic disorders. Thus, ABCA1 is a key modulator of cellular cholesterol efflux and contributes to functional disorders in different types of cells.

The metabolic syndrome is defined as a cluster of disorders including visceral obesity, insulin resistance, hypertension, hypertriglyceridemia and low HDL. Patients have an increased risk to develop atherosclerotic disease characterized by excessive macrophage cholesterol deposition in the vascular wall. HDL removes excess cellular cholesterol which is subsequently transported to the liver for biliary excretion and thereby preserves cholesterol homeostasis. Circulating HDL levels are maintained by hepatic ATP-binding cassette transporter A1 (ABCA1) which also transports peripheral cell cholesterol to extracellular lipid acceptors. Lipid export activity of ABCA1 improves pancreatic -cell function and ameliorates insulin release. ABCA1 affects plasma membrane cholesterol distribution and formation of lipid rafts representing signalling platforms for diverse receptors including toll-like receptor 4 (TLR4). Pharmacological activation of ABCA1 pathways presumably progresses metabolic diseases, and current approaches demonstrate beneficial effects of small peptides mimicking ABCA1 ligands which stabilize ABCA1 and enhance lipid efflux similar to its physiological acceptor apolipoprotein A-I. Research of the last decade has resulted in the identification of several ABCA1 binding proteins influencing ABCA1 signalling, stability and activity. In the current review the proteins suggested to form a complex with ABCA1 are summarized and their up to now characterized features towards ABCA1 functions are described.

Gene therapy has become a clinical reality as demonstrated by remarkable benefits seen in Phase I/II clinical trials for hemophilia B, lipoprotein lipase deficiency and Leber's congenital amarousis. The choice of, and the improved understanding in vector characteristics have contributed significantly to this success. The adeno-associated virus (AAV) vectors used in these trials have been long known to be relatively safe and efficacious. However, certain factors, most notably host immunity to the vector, prevent their widespread use. In patients who have pre-existing antibodies to AAV, these vectors will be rapidly cleared. Administration of a relatively high initial dose of vector to achieve and sustain a higher margin of therapeutic benefit is limited by concerns of vector dose-dependent T cell response. Frequent vector administration necessitated by the non-integrating nature of the virus is difficult due to the variable, yet significant host immunological memory. Thus generation of AAV vectors that are immunologically inert is pivotal for the long-term success with this promising vector system. Several strategies, that aim targeted disruption of antigenic sites or those that chemically modify the vectors have been proposed for host immune evasion. While these approaches have been successful in the pre-clinical model systems, this continues to be a field of intense experimentation and constant improvisation due to limited information available on vector immunology or data from human studies. This review forms a comprehensive report on current strategies available to generate immunologically inert AAV vectors and their potential in mediating long-term gene transfer.

ATP-binding cassette (ABC) transporters are multispanning membrane proteins that utilize ATP to move a broad range of substrates across cellular membranes. ABC transporters are involved in a number of human disorders and diseases. Overexpression of a subset of the transporters has been closely linked to multidrug resistance in both bacteria and viruses and in cancer. A poorly understood and important aspect of ABC transporter biology is the role of phosphorylation as a mechanism to regulate transporter function. In this review, we summarize the current literature addressing the role of phosphorylation in regulating ABC transporter function. A comprehensive list of all the phosphorylation sites that have been identified for the human ABC transporters is presented, and we discuss the role of individual kinases in regulating transporter function. We address the potential pitfalls and difficulties associated with identifying phosphorylation sites and the corresponding kinase(s), and we discuss novel techniques that may circumvent these problems. We conclude by providing a brief perspective on studying ABC transporter phosphorylation.

In the present review we provide a summary of ATP-binding cassette (ABC) transporters in the central nervous system (CNS). Our review is focused on transporters of the ABC A, B, C, D, and G families that have been detected in the cells of the neurovascular unit/blood-brain barrier including brain capillary endothelial cells, pericytes, astrocytes, and neurons, as well as in other brain cells, such as microglia, oligodendrocytes, and choroid plexus epithelial cells. In this review, we provide an overview, organized by ABC family, of transporter expression, localization, and function. We summarize recent findings on ABC transporter regulation in the CNS and address the role of ABC transporters in CNS diseases including brain cancer, seizures/epilepsy, and Alzheimer's disease. Finally, we discuss new therapeutic strategies focused on ABC transporters in CNS disease.

The biliary secretion of bile acids is critical for multiple liver functions including digesting fatty nutrients and driving bile flow. When this process is impaired, the accumulating bile acids cause inflammatory liver injury. Multiple ABC transporters in the liver are key players to safeguard the hepatocyte and avoid toxicity due to bile acid over-accumulation. BSEP provides for efficient secretion of bile acids across the canalicular membrane against a steep concentration gradient. MDR3/Mdr2 and ABCG5/G8 secrete phosphatidylcholine and cholesterol, respectively, in coordination with BSEP-mediated bile acid secretion to mask the detergent/toxic effects of bile acids in the bile ductular space. Several lines of evidence indicate that when these critical steps are compromised, bile acid toxicity in vivo leads to inflammatory liver injury and liver cancer. In bsep-/- mice, liver cancer is rare. These mice display greatly increased expression of alternative bile acid transporters, such as Mdr1a/1b, Mrp3 and Mrp4. We believe these alternative transport systems provide an additional safeguard to avoid bile acid overload in liver. Such backup systems appear to be under-utilized in humans, as defects in BSEP and MDR3 lead to severe, often fatal childhood diseases. It is possible, therefore, that targeting ABC transporters and modulating the toxicity of the bile acid pool could be vital interventions to alleviate chronic inflammation and reduce the incidence of liver cancer in high-risk populations. The combination of an alternative ABC transporter with a novel substrate may prove an effective chemo-preventive or therapeutic strategy.

Genetic polymorphisms and mutations in drug metabolizing enzymes, transporters, receptors, and other drug targets (e.g., toxicity targets) are linked to inter-individual differences in the efficacy and toxicity of many medications as well as risk of genetic diseases. Validation of clinically important genetic polymorphisms and the development of new technologies to rapidly detect clinically important variants are critical issues for advancing personalized medicine. A key requirement for the advancing personalized medicine resides in the ability of rapidly and conveniently testing patients' genetic polymorphisms and/or mutations. We have recently developed a rapid and cost-effective method, named Smart Amplification Process 2 (SmartAmp2), which enables us to detect genetic polymorphisms or mutations in target genes within 30 to 45 min under isothermal conditions without DNA isolation and PCR amplification. Detection of mutations or single nucleotide polymorphisms (SNPs) in human ABC transporter genes is becoming more important, since their functional impairments are reportedly associated with inherited diseases. Thus, certain genetic polymorphisms of ABC transporters are considered important biomarkers for diagnosis of inherited diseases and/or risk of drug-induced adverse reactions. In this review article, we will present the new technology of the SmartAmp2 method and its clinical applications for detection of SNPs in human ABC transporter genes, i.e., ABCC4 and ABCC11.

ATP-Binding Cassette (ABC) transporters are important mediators of multidrug resistance (MDR) in patients with cancer. Although their role in MDR has been extensively studied in vitro, their value in predicting response to chemotherapy has yet to be fully determined. Establishing a molecular diagnostic assay dedicated to the quantitation of ABC transporter genes is therefore critical to investigate their involvement in clinical MDR. In this article, we provide an overview of the methodologies that have been applied to analyze the mRNA expression levels of ABC transporters, by describing the technology, its pros and cons, and the experimental protocols that have been followed. We also discuss recent studies performed in our laboratory that assess the ability of the currently available high-throughput gene expression profiling platforms to discriminate between highly homologous genes. This work led to the conclusion that high-throughput TaqMan-based qRT-PCR platforms provide standardized clinical assays for the molecular detection of ABC transporters and other families of highly homologous MDR-linked genes encoding, for example, the uptake transporters (solute carriers-SLCs) and the phase I and II metabolism enzymes.

The porphyrins (such as heme) are essential molecules within cells and have multiple roles in essential cellular processes such as: the mitochondrial electron transport chain, free-radical detoxification, and metabolism. The porphyrins need energy to traverse biological membranes. Our understanding of ABC transporters role in regulating intracellular porphyrin homeostasis is only now beginning to be understood. Two important contributors are members of the ABC transporter gene family: ABCB6 and ABCG2. ABCB6 is the first ABC transporter located in the outer mitochondrial membrane and oriented to facilitate porphyrin import. Consequently, ABCB6 can regulate and appropriately orchestrate porphyrin synthesis. This leads to an ability to regulate the amount of heme associated with heme requiring proteins. This ability can facilitate a cells protective response to an array of toxic insults. ABCG2 also binds and transports porphyrins, however its location at the plasma membrane provides a mechanism to remove excess porphyrins. Because ABCG2 is upregulated by hypoxia this provides a mechanism to export porphyrins, rebalance porphyrins and protect cells from porphyrin overaccumulation. Such a mechanism would be important to hypoxic cells which exhibit an increase in porphyrin synthesis under hypoxic conditions. Finally, we propose that these two transporters (ABCB6 and ABCG2) are coordinately regulated to modulate porphyrin concentrations under normal physiological and pathological conditions.

ABCG2, or breast cancer resistance protein (BCRP), is an ATP-binding cassette half transporter that has been shown to transport a wide range of substrates including chemotherapeutics, antivirals, antibiotics and flavonoids. Given its wide range of substrates, much work has been dedicated to developing ABCG2 as a clinical target. But where can we intervene clinically and how can we avoid the mistakes made in past clinical trials targeting P-glycoprotein? This review will summarize the normal tissue distribution, cancer tissue expression, substrates and inhibitors of ABCG2, and highlight the challenges presented in exploiting ABCG2 in the clinic. We discuss the possibility of inhibiting ABCG2, so as to increase oral bioavailability or increase drug penetration into sanctuary sites, especially the central nervous system; and at the other end of the spectrum, the possibility of improving ABCG2 function, in the case of gout caused by a single nucleotide polymphism. Together, these aspects of ABCG2/BCRP make the protein a target of continuing interest for oncologists, biologists, and pharmacologists.

The adenosine tri-phosphate binding cassette (ABC) transporters are one of the largest transmembrane gene families in humans. The ABC transporters are present in a number of tissues, providing protection against xenobiotics and certain endogenous molecules. Unfortunately, their presence produces suboptimal chemotherapeutic outcomes in cancer patient tumor cells. It is well established that they actively efflux antineoplastic agents from cancer cells, producing the multidrug resistance (MDR) phenotype. The inadequate response to chemotherapy and subsequent poor prognosis in cancer patients can be in part the result of the clinical overexpression of ABC transporters. In fact, one of the targeted approaches for overcoming MDR in cancer cells is that directed towards blocking or inhibiting ABC transporters. Indeed, for almost three decades, research has been conducted to overcome MDR through pharmacological inhibition of ABC transporters with limited clinical success. Therefore, contemporary strategies to identify or to synthesize selective "resensitizers" of ABC transporters with limited nonspecific toxicity have been undertaken. Innovative approaches en route to understanding specific biochemical role of ABC transporters in MDR and tumorigenesis will prove essential to direct our knowledge towards more effective targeted therapies. This review briefly discusses the current knowledge regarding the clinical involvement of ABC transporters in MDR to antineoplastic drugs and highlights approaches undertaken so far to overcome ABC transporter-mediated MDR in cancer.

Among the currently available options for the treatment of chronic myeloid leukemia (CML), ATP-competitive tyrosine kinases inhibitors (Gleevec®, Dasatinib® and Nilotinib®) represent one of the most promising therapeutic approaches developed in the last 10-15 years. However, the initial enthusiasm generated by the high response rate to these drugs has been dampened by the development of resistance. The T315I mutation at the gatekeeper residue is very frequent in advanced phases of the disease and is one of the main causes of resistance, disrupting important contact points between the inhibitors and the enzyme. Different strategies have been implemented to overcome this resistance, such as the recent development of more selective non-ATP competitive inhibitors targeting sites outside the ATP-binding cleft. Some of these allosteric inhibitors alter the kinase conformation, while others directly compete with the protein substrates. Another interesting family of allosteric inhibitors is represented by those compounds targeting the myristate-pocket of Bcr-Abl (myristate-pocket binders). The binding of these inhibitors blocks the Bcr-Abl kinase in the inactive conformation and provides an advantage in overcoming drug resistance due to kinase mutations. This review reports the last findings in the development of novel myristate-pocket binders of Bcr-Abl as promising anti-leukemia agents.

We have examined optical properties of a fluorene derivative with two positively charged substituents, 1,1'-diethyl-4,4'-(9,9-diethyl-2,7-fluorenediyl-2,1-ethanediyl)dipyridinium perchlorate (1), in water. The photoluminescence quantum yield of 1 was relatively high (35%) for use as a fluorescent probe in water. We also examined two-photon absorption (TPA) properties of 1 in methanol. The maximum value of the TPA cross-section (730 GM at 750 nm, 1 GM = 10(-50) cm(4) s photon(-1) molecule(-1)) was larger than that for most two-photon-excited fluorescent dyes including a classical mitochondria-selective fluorescent dye rhodamine 123. Preliminary fluorescence imaging experiments of the mitochondria in Paramecium caudatum and Hela living cells were carried out with 1. Bright green fluorescence was observed from the mitochondria in both living cells loaded 1 without toxicity effects. These our results indicate that water-soluble fluorene derivative 1 is a promising candidate as a two-photon-excited fluorescence probe for mitochondria in living cells.

This paper describes a novel molecular design strategy for obtaining efficient two-photon absorption (TPA) materials. The most popular strategy for enhancing the TPA cross-section (σ((2))) of a molecule is to enhance its transition dipole moment. However, this strategy also red shifts the one-photon absorption (OPA) band. Consequently, molecules with large transition dipole moments typically exhibit strong OPA at visible wavelengths, making it difficult to use such molecules for TPA-related applications in the visible wavelength region. Therefore, an alternative molecular design principle for TPA materials to enhance the transition dipole moment is strongly required. The present paper describes a novel molecular design strategy for reducing the detuning energy by incorporating an azulenyl moiety in a large, planar p-electron system. This strategy enhances σ((2)) without significantly red shifting the OPA band.

Discovery of small molecules able to induce several cellular self-killing mechanisms improved cancer therapy in the last years. Research focused on canonical apoptotic (mitochondria or death receptor related) pathways to induce cell death in several hematologic and solid malignancies, showing that treatment with different synthetic and natural compounds reactivates the cell death machinery previously silenced in resistant cancer cells. Besides the canonical apoptotic pathways, alternative pathways of cell death induction have recently been rediscovered as potential new targets for cancer therapy. Under certain conditions, protein folding can be disturbed causing an accumulation of unfolded proteins inside the endoplasmic reticulum (ER). This situation leads to stress ER, involving the transcriptional and translational machinery to induce the expression and post-transcriptional modifications of many factors involved in ER stress response mediated cell death. In this scenario, some apoptotic players like caspase 4 or caspase 12 start to control cell fate by inducing downstream cell death proteins. Recently, inhibitors of protein deacetylases have been demonstrated to potently induce this alternative cell death pathway and will be reviewed here.

3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) is a substituted amphetamine with potent central nervous stimulant effects. Increasing evidence suggests that one way of MDMA-induced toxicity involves the production of reactive oxygen and reactive nitrogen species and a subsequent production of oxidative/nitrosative stress. The free radicals can originate from several molecular pathways (oxidative deamination of monoamine, metabolic pathways, cathecolamines autoxidation, and hyperthermia) and their harmful effect causing potential biological damage such as lipoperoxidation and cellular death. The role of oxidative stress in mediating MDMA toxicity is illustrated by decreases in the activity of the endogenous enzymatic and non enzymatic antioxidants observed in cells in vitro and in animals model. This review examines the available evidence for the involvement of oxidative stress in the mechanisms of MDMA-induced cellular damage with the aim to contribute to the understanding of the cellular and molecular mechanisms involved in MDMA toxicity.