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1.  Direct inhibition of Retinoblastoma phosphorylation by Nimbolide causes cell cycle arrest and suppresses glioblastoma growth 
Purpose
Classical pharmacology allows the use and development of conventional phytomedicine faster and more economically than conventional drugs. This approach should be tested for their efficacy in terms of complementarity and disease control. The purpose of this study was to determine the molecular mechanisms by which nimbolide, a triterpenoid found in the well-known medicinal plant Azadirachta indica controls glioblastoma (GBM) growth.
Experimental Design
Using in vitro signaling, anchorage-independent growth, kinase assays, and xenograft models, we investigated the mechanisms of its growth inhibition in glioblastoma.
Results
We show that nimbolide or an ethanol soluble fraction of A. indica leaves (Azt) that contains nimbolide as the principal cytotoxic agent is highly cytotoxic against GBM in vitro and in vivo. Azt caused cell cycle arrest, most prominently at the G1-S stage in GBM cells expressing EGFRvIII, an oncogene present in about 20-25% of GBMs. Azt/nimbolide directly inhibited CDK4/CDK6 kinase activity leading to hypophosphorylation of the retinoblastoma (RB) protein, cell cycle arrest at G1-S and cell death. Independent of RB hypophosphorylation, Azt also significantly reduced proliferative and survival advantage of GBM cells in vitro and in tumor xenografts by downregulating Bcl2 and blocking growth factor induced phosphorylation of Akt, Erk1/2 and STAT3. These effects were specific since Azt did not affect mTOR or other cell cycle regulators. In vivo, Azt completely prevented initiation and inhibited progression of GBM growth.
Conclusions
Our preclinical findings demonstrate Nimbolide as a potent anti-glioma agent that blocks cell cycle and inhibits glioma growth in vitro and in vivo.
doi:10.1158/1078-0432.CCR-13-0762
PMCID: PMC3947304  PMID: 24170547
2.  Imatinib Analogs as Potential Agents for PET Imaging of Bcr-Abl/c-KIT Expression at a Kinase Level 
Bioorganic & medicinal chemistry  2013;22(1):623-632.
We synthesized two series of imatinib mesylate (STI-571) analogs to develop a Bcr-Abl and c-KIT receptor-specific labeling agent for positron emission tomography (PET) imaging to measure Bcr-Abl and c-KIT expression levels in a mouse model. The methods of molecular modeling, synthesis of STI-571 and its analogs, in vitro kinase assays, and radiolabeling are described. Molecular modeling revealed that these analogs bind the same Bcr-Abl and c-KIT binding sites as those bound by STI-571. The analogs potently inhibit the tyrosine kinase activity of Bcr-Abl and c-KIT, similarly to STI-571. [18F]-labeled STI-571 was prepared with high specific activity (75 GBq/μmol) by nucleophilic displacement and an average radiochemical yield of 12%. [131I]-labeled STI-571 was prepared with high purity (>95%) and an average radiochemical yield of 23%. The uptake rates of [18F]-STI-571 in K562 cells expressing Abl and in U87WT cells overexpressing c-KIT were significantly higher than those in the U87 cell and could be inhibited by STI-71 (confirming the specificity of uptake). PET scans of K562 and U87WT tumor-bearing mice with [18F]-STI-571 as a contrast agent showed visible tumor uptake and tumor-to-non-target contrast.
doi:10.1016/j.bmc.2013.10.040
PMCID: PMC4124913  PMID: 24280068
3.  A peptide probe for targeted brown adipose tissue imaging 
Nature communications  2013;4:10.1038/ncomms3472.
The presence of brown adipose tissue (BAT) responsible for thermogenic energy dissipation has been revealed in adult humans and has high clinical importance. Due to limitations of current methods for BAT detection, analyzing the abundance and localization of BAT in the body has remained challenging. Here, we screen a combinatorial peptide library in mice and characterize a peptide (with the sequence CPATAERPC) that selectively binds to the vascular endothelium of BAT, but not of intraperitoneal white adipose tissue (WAT). We show that in addition to BAT, this peptide probe also recognizes the vasculature of BAT-like depots of subcutaneous WAT. Our results indicate that the CPATAERPC peptide localizes to BAT even in the absence of sympathetic nervous system stimulation. Finally, we demonstrate that this probe can be used to identify BAT depots in mice by whole body near-infrared (NIR) fluorescence imaging.
doi:10.1038/ncomms3472
PMCID: PMC3806199  PMID: 24045463
4.  Dual-Labeling Strategies for Nuclear and Fluorescence Molecular Imaging: A Review and Analysis 
Molecular Imaging and Biology  2011;14(3):261-276.
Molecular imaging is used for the detection of biochemical processes through the development of target-specific contrast agents. Separately, modalities such as nuclear and near-infrared fluorescence (NIRF) imaging have been shown to non-invasively monitor disease. More recently, merging of these modalities has shown promise owing to their comparable detection sensitivity and benefited from the development of dual-labeled imaging agents. Dual-labeled agents hold promise for whole-body and intraoperative imaging and could bridge the gap between surgical planning and image-guided resection with a single, molecularly targeted agent. In this review, we summarized the literature for dual-labeled antibodies and peptides that have been developed and have highlighted key considerations for incorporating NIRF dyes into nuclear labeling strategies. We also summarized our findings on several commercially available NIRF dyes and offer perspectives for developing a toolkit to select the optimal NIRF dye and radiometal combination for multimodality imaging.
doi:10.1007/s11307-011-0528-9
PMCID: PMC3346941  PMID: 22160875
Dual-labeling; Multimodality; Radiochemistry; Positron emission tomography; Near-infrared fluorescence; Single-photon emission computed tomography; Medicine & Public Health; Imaging / Radiology
5.  Characterization of chemical, radiochemical and optical properties of a dual-labeled MMP-9 targeting peptide 
Bioorganic & medicinal chemistry  2011;19(12):3769-3776.
Optical imaging possesses similar sensitivity to nuclear imaging and has led to the emergence of multimodal approaches with dual-labeled nuclear/near-infrared (NIR) agents. The growing impact of 68Ga (t1/2 = 68 min) labeled peptides on preclinical and clinical research offers a promising opportunity to merge the high spatial resolution of NIR imaging with the clinically-accepted positron emission tomography (PET). Previously, dual-labeled agents have been prepared with longer-lived radiometals and showed no detrimental effects on optical properties as a result of radiolabeling. In this study, we selected a peptide (M2) that targets MMP-2/9 and is dual-labeled with IRDye 800CW and 68Ga. Since 68Ga chelation typically requires low pH (3.5–4) and elevated heating temperatures (95°C), we sought to evaluate the impact of 68Ga labeling on the optical properties of M2. An efficient method for preparation of 68Ga-M2 was developed and reaction conditions were optimized. Stability studies in PBS, DTPA, and serum were performed and high levels of intact agent were evident under each condition. The addition of multiple reporters to a targeting agent adds further complexity to the characterization and validation and thus requires not only testing to ensure the agent is stable chemically and radiochemically, but also optically. Therefore, fluorescence properties were evaluated using a spectrofluorometer as well as by fluorescence detection via HPLC. It was determined that 68Ga-labeling conditions did not impair the fluorescent properties of the agent. The agent was then used for in vivo imaging in a mouse model of heterotopic ossification (HO) with activated MMP-9 expression as an early biomarker which precedes mineralization. Although 68Ga-complexation greatly reduced binding affinity of the peptide and negated tracer uptake on PET, NIR imaging showed consistent fluorescent signal that correlated to MMP-9 expression. This attests to the feasibility of using 68Ga/NIR for dual-labeling of other peptides or small molecules for multimodality molecular imaging.
doi:10.1016/j.bmc.2011.04.054
PMCID: PMC3148023  PMID: 21612930
Dual-labeling; Near-infrared fluorescence; Gallium-68; MMP
6.  Albumin-Binding Domain Conjugate for Near-Infrared Fluorescence Lymphatic Imaging 
Molecular Imaging and Biology  2011;14(3):301-314.
Purpose
The aim of this study was to develop and characterize a novel peptide imaging agent for noninvasive near-infrared fluorescence imaging of protein transport by the lymphatics. An imaging agent consisting of a cyclic albumin-binding domain (cABD) peptide, with sequence, Arg-Leu-Ile-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp-Lys, was conjugated to a near-infrared fluorophore, IRDye800CW, allowing for enhanced vascular uptake, retention, and fluorescence imaging.
Procedure
Characterization of the cABD-IRDye800 peptide conjugate was performed using fluorescence spectroscopy to assess optical properties and SDS-PAGE and Biacore binding assays to determine binding affinity and specificity. Fluorescence imaging of normal C57BL/6 mice was conducted to monitor lymphatic uptake and retention.
Results
cABD-IRDye800 exhibited approximately six times greater fluorescent yield and greater stability than indocyanine green, an agent previously used in humans to image lymphatic vasculature. The agent exhibited affinity for albumin with IC50 and Kd in the nanomolar range and demonstrated superior retention characteristics within mouse lymphatics when compared with IRDye800CW.
Conclusions
cABD-IRDye800 has utility for assessing lymphatic function in mouse models of human lymphatic disease and the potential for use in clinical diagnostic imaging of the lymphatic vasculature.
Electronic supplementary material
The online version of this article (doi:10.1007/s11307-011-0499-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s11307-011-0499-x
PMCID: PMC3346932  PMID: 21688052
Near-infrared fluorescence; Lymphatic imaging; Albumin peptide conjugate; Medicine & Public Health; Imaging / Radiology
7.  Screening for suicidal thoughts in primary care: the views of patients and general practitioners 
Mental Health in Family Medicine  2008;5(4):229-235.
Background It has been argued that primary care practitioners have an important part to play in the prevention of suicide. However, levels of assessment of risk of suicide among patients treated in this setting are generally low.
Methods Cross-sectional survey of general practitioners (GPs) and people being treated in primary care who had signs of depression. The study combined open and closed questions on attitudes to screening or being screened for suicidal ideation.
Results One hundred and one of 132 patients took part in the survey and 103 of 300 GPs completed a questionnaire. A majority of both GPs and patients stated that people should be screened for suicidal ideation. However, an important minority of patients and GPs stated that asking or being asked such questions made them feel uncomfortable. Less than half of GPs had received formal training on the assessment of suicide risk. GPs told the researchers that barriers to screening included time pressures, culture and language, and concerns about the impact that screening could have on people's mental health. One-quarter of GPs and one-fifth of patients supported the notion that screening for suicidal ideation could induce a person to have thoughts of self-harm.
Conclusions GPs and family doctors should screen for suicidal risk among depressed patients and should receive training on how to do this as part of their general training in the assessment and management of mental disorders. Research should be conducted to examine what, if any, effect screening for suicidal ideation has on mental health.
PMCID: PMC2777583  PMID: 22477874
primary care; screening; suicide
8.  Effect of Sulfur Concentration on the Morphology of Carbon Nanofibers Produced from a Botanical Hydrocarbon 
Nanoscale Research Letters  2008;3(7):242-248.
Carbon nanofibers (CNF) with diameters of 20–130 nm with different morphologies were obtained from a botanical hydrocarbon: Turpentine oil, using ferrocene as catalyst source and sulfur as a promoter by simple spray pyrolysis method at 1,000 °C. The influence of sulfur concentration on the morphology of the carbon nanofibers was investigated. SEM, TEM, Raman, TGA/DTA, and BET surface area were employed to characterize the as-prepared samples. TEM analysis confirms that as-prepared CNFs have a very sharp tip, bamboo shape, open end, hemispherical cap, pipe like morphology, and metal particle trapped inside the wide hollow core. It is observed that sulfur plays an important role to promote or inhibit the CNF growth. Addition of sulfur to the solution of ferrocene and turpentine oil mixture was found to be very effective in promoting the growth of CNF. Without addition of sulfur, carbonaceous product was very less and mainly soot was formed. At high concentration of sulfur inhibit the growth of CNFs. Hence the yield of CNFs was optimized for a given sulfur concentration.
doi:10.1007/s11671-008-9143-3
PMCID: PMC3244866  PMID: 21816116
Carbon nanofiber; Spray pyrolysis method; Botanical hydrocarbon; Scanning electron microscopy; Transmission electron microscopy
9.  Interaction of 2-aminopyrimidine with dichloro-[1-alkyl-2-(naphthylazo) imidazole]palladium(II) complexes : Kinetic and mechanistic studies 
Background
The anticancer properties of cisplatin and palladium(II) complexes stem from the ability of the cis-MCl2 fragment to bind to DNA bases. However, cisplatin also interacts with non-cancer cells, mainly through bonding molecules containing -SH groups, resulting in nephrotoxicity. This has aroused interest in the design of palladium(II) complexes of improved activity and lower toxicity. The reaction of DNA bases with palladium(II) complexes with chelating N,N/donors of the cis-MCl2 configuration constitutes a model system that may help explore the mechanism of cisplatin's anticancer activity. Heterocyclic compounds are found widely in nature and are essential to many biochemical processes. Amongst these naturally occurring compounds, the most thoroughly studied is that of pyrimidine. This was one of the factors that encouraged this study into the kinetics and mechanism of the interaction of 2-aminopyrimidine (2-NH2-Pym) with dichloro-{1-alkyl-2-(α-naphthylazo)imidazole}palladium(II) [Pd(α-NaiR)Cl2, 1] and dichloro-{1-alkyl-2-(β-naphthylazo)imidazole}palladium(II) [Pd(β-NaiR)Cl2, 2] complexes where the alkyl R = Me (a), Et (b), or Bz (c).
Results
2-NH2-Pym reacts with 1a, 1b, and 1c to yield [{1-alkyl-2-(α-naphthylazo)imidazole}bis(2-aminopyrimidine)]palladium(II) (3a, 3b, 3c) dichloride and with 2a, 2b, and 2c to yield [{1-alkyl-2-(β-naphthylazo)imidazole}bis(2-aminopyrimidine)]palladium(II) (4a, 4b, 4c) dichloride in an acetonitrile (MeCN) medium. The products were characterized using spectroscopic techniques (FT-IR, UV-Vis, NMR). The ligand substitution reactions follow second order kinetics – first order dependence on the concentration of the Pd(II) complex and 2-NH2-Pym. Addition of LiCl to the reaction does not influence its rate. The thermodynamic parameters (standard enthalpy of activation, Δ‡H° and standard entropy of activation, Δ‡S°) were determined from variable temperature kinetic studies. The magnitude of the second order rate constant, k2, at 298 K, was shown to increase thus: b
Conclusion
The kinetics of the reaction between Pd(II) complexes (1 and 2) and 2-NH2-Pym were examined spectrophotometrically at 530 nm in MeCN under pseudo-first-order conditions. The reaction rate is largely influenced by the π-acidity of the chelating ligand, with substitution in the naphthyl azoimidazole backbone influencing the rate of the substitution process. The activation parameters, Δ‡H° and Δ‡S°, were determined and support the kinetic rate data.
doi:10.1186/1752-153X-1-23
PMCID: PMC2194761  PMID: 17939858
AAPS PharmSciTech  2006;7(3):E172-E177.
The main purpose of this work was to develop an oral microemulsion formulation for enhancing the bioavailability of acyclovir. A Labrafac-based microemulsion formulation with Labrasol as surfactant and Plurol Oleique as cosurfactant was developed for oral delivery of acyclovir. Phase behavior and solubilization capacity of the microemulsion system were characterized, and in vivo oral absorption of acyclovir from the microemulsion was investigated in rats. A single isotropic region, which was considered to be a bicontinuous microemulsion, was found in the pseudoternary phase diagrams developed at various Labrasol:Plurol Oleique:Labrafac ratios. With the increase of Labrasol concentration, the microemulsion region area and the amount of water and Labrafac solubilized into the microemulsion system increased; however, the increase of Plurol Oleique percentage produced opposite effects. The microemulsion system was also investigated in terms of other characteristics, such as interfacial tension, viscosity, pH, refractive index, diffusion, and bioavailability. Acyclovir, a poorly soluble drug, displayed high solubility in a microemulsion formulation using Labrafac (10%). Labrasol (32%), Plurol Oleique (8%), and water (50%). The in vitro intraduodenal diffusion and in vivo study revealed an increase of bioavailability (12.78 times) after oral administration of the microemulsion formulation as compared with the commercially available tablets.
doi:10.1208/pt070377
PMCID: PMC2750519  PMID: 17025257
Microemulsion; non-ionic surfactant; conductivity; interfacial tension; particle size
AAPS PharmSciTech  2006;7(3):E80-E86.
The purpose of the present study was to develop intranasal delivery systems of sumatriptan using thermoreversible polymer Pluronic F127 (PF 127) and mucoadhesive polymer Carbopol 934P (C934P). Formulations were modulated so as to have gelation temperature below 34°C to ensure gelation at physiological temperature after intranasal administration. Gelation temperature was determined by physical appearance as well as by rheological measurement. The gelation temperatures of the formulations decreased by addition of increasing concentrations of Carbopol (ie, from 29°C for 18% PF127 to 23.9°C for 18% PF127, 0.5% Carbopol). The mucoadhesive force in terms of detachment stress, determined using sheep nasal mucosal membrane, increased with increasing concentration of Carbopol. The results of in vitro drug permeation studies across sheep nasal mucosa indicate that effective permeation coefficient could be significantly increased by using in situ gelling formulation with Carbopol concentration 0.3% or greater. Finally, histopathological examination did not detect any damage during in vitro permeation studies. In conclusion, the PF 127 gel formulation of sumatriptan, with in situ gelling and mucoadhesive properties with increased permeation rate is promising for prolonging nasal residence time and thereby nasal absorption.
doi:10.1208/pt070367
PMCID: PMC2750509  PMID: 17025248
Carbopol; migraine; mucoadhesive; nasal; Pluronic F127

Results 1-11 (11)