Radioiodination and biological evaluation of nizatidine as a new highly selective radiotracer for peptic ulcer disorder detection

Nizatidine has been labeled using [¹²⁵I] with chloramine‐T as oxidizing agent. Factors such as the amount of oxidizing agent, amount of substrate, pH, reaction temperature, and reaction time have been systematically studied to optimize the iodination. Biodistribution studies indicate the suitability of radioiodinated nizatidine as a novel tracer to image stomach ulcer. Radioiodinated nizatidine may be considered a highly selective radiotracer for peptic ulcer imaging.     

Radioiodination of balsalazide,bioevaluation, and characterization as a highly selective radiotracer for imaging of ulcerative colitis in mice

This work focuses on tracking ulcerative colitis in mice. High labeling yield and radiochemical purity were achieved for the formation of a [^125/131I]balsalazide radiotracer at optimum conditions of oxidizing agent content (chloramines-T [Ch-T], 75 μg), substrate amount (100 μg), pH of reaction mixture (6), reaction time (30 min), and temperature (37°C), using radioactive iodine-125 (200–450 MBq). The radiolabeled compound, [^125/131I]balsalazide, was stable in serum and saline solution during 24 h. Balsalazide is acting as a peroxisome proliferator-activated receptor (PPARγ). Biodistribution studies were carried in normal and ulcerated colon mice. High uptake of 75 ± 1.90% injected dose/g organ (ID/g) observed in ulcerated mice confirmed the suitability of [¹³¹I]balsalazide as a novel radiotracer for ulcerative colitis imaging in mice.     

Non‐peptide‐based new class of platelet aggregation inhibitors: Design,synthesis, bioevaluation,SAR, and in silico studies

A series of 2‐oxo‐2‐phenylethylidene linked 2‐oxo‐benzo[1,4]oxazine analogues 17a–x and 18a–o , incorporated with a variety of electron‐withdrawing as well as electron‐donating groups at ring A and ring C, were synthesized under greener conditions in excellent yields (up to 98%). These analogues 17a–x and 18a–o were evaluated for their arachidonic acid (AA)‐induced platelet aggregation inhibitory activities in comparison with the standard reference aspirin (IC₅₀ = 21.34 ± 1.09 µg/mL). Among all the screened compounds, eight analogues, 17i , 17x , 18f , 18g , 18h , 18i , 18l , and 18o , were identified as promising platelet aggregation inhibitors as compared to aspirin. In addition, cytotoxic studies in 3T₃ fibroblast cell lines by MTT assay of the promising compounds ( 17i , 17x , 18f–18i , 18l , and 18o ) were also performed and the compounds were found to be non‐toxic in nature. Furthermore, the results on the AA‐induced platelet aggregation inhibitory activities of these compounds ( 17i , 17x , 18f–18i , 18l , and 18o ) were validated via in silico molecular docking simulation studies. To the best of our knowledge, this is the first report of the identification of non‐peptide‐based functionalized 2‐oxo‐benzo[1,4]oxazines as platelet aggregation inhibitors.

     

Targeting HIV-1 through molecular modeling and docking studies of CXCR4: leads for therapeutic development

The chemokine receptor CXCR4 is the receptor for several chemokines and major co-receptor for X4 human immunodeficiency virus type-1 strains entry into cell. A three-dimensional model of human CXCR4 was developed by homology modeling using the high-resolution bovine rhodopsin structure as template. Interactions between CXCR4 and flavonoids were investigated using in silico docking studies. The results underscore the potential of these compounds that they may become important new antiviral drugs to combat AIDS. It is worth mentioning also that apart from these existing flavonoids, there are many new compounds that may also be useful as topical agents to inactivate virus, or may act as adjuvants with other antiviral drugs.     

Densely substituted piperidines as a new class of elastase inhibitors: Synthesis and molecular modeling studies

Elastase is the only enzyme that has the capability to degrade elastin and collagen, the two proteins essential for skin and bones. The synthesis of some densely substituted piperidines functionalized with the trifluoromethyl group ( 4a‐j ) was carried out. The newly prepared compounds were subjected to elastase enzyme inhibitory potential and antioxidant activity assays. Among the series, 4i (IC₅₀ = 0.341 ± 0.001 μM) exhibited the maximum inhibition against elastase. Binding analysis delineated that the fluorine atom of ligand 4i showed hydrogen and hydrophobic bonds with Thr41 and Thr96, with bond distances of 3.84 and 5.631 Å, respectively. The obtained results indicate that these trifluoromethyl functionalized piperidine derivatives could be considered as potential candidates to treat skin disorders.     

Kinetic analysis of novel mono- and multivalent VHH-fragments and their application for molecular imaging of brain tumours

Background and purpose:

The overexpression of epidermal growth factor receptor (EGFR) and its mutated variant EGFRvIII occurs in 50% of glioblastoma multiforme. We developed antibody fragments against EGFR/EGFRvIII for molecular imaging and/or therapeutic targeting applications.

Experimental approach:

An anti–EGFR/EGFRvIII llama single-domain antibody (EG₂) and two higher valency format constructs, bivalent EG₂-hFc and pentavalent V2C-EG₂ sdAbs, were analysed in vitro for their binding affinities using surface plasmon resonance and cell binding studies, and in vivo using pharmacokinetic, biodistribution, optical imaging and fluorescent microscopy studies.

Key results:

Kinetic binding analyses by surface plasmon resonance revealed intrinsic affinities of 55 nM and 97 nM for the monovalent EG₂ to immobilized extracellular domains of EGFR and EGFRvIII, respectively, and a 10- to 600-fold increases in apparent affinities for the multivalent binders, V2C-EG₂ and EG₂-hFc, respectively. In vivo pharmacokinetic and biodistribution studies in mice revealed plasma half-lives for EG₂, V2C-EG₂ and EG₂-hFc of 41 min, 80 min and 12.5 h, respectively, as well as a significantly higher retention of EG₂-hFc compared to the other two constructs in EGFR/EGFRvIII-expressing orthotopic brain tumours, resulting in the highest signal in the tumour region in optical imaging studies. Time domain volumetric optical imaging fusion with high-resolution micro-computed tomography of microvascular brain network confirmed EG₂-hFc selective accumulation/retention in anatomically defined tumour regions.

Conclusions:

Single domain antibodies can be optimized for molecular imaging applications by methods that improve their apparent affinity and prolong plasma half-life and, at the same time, preserve their ability to penetrate tumour parenchyma.     

Rapid and widespread distribution of doxycycline in rat brain: a mass spectrometric imaging study

1. The penetration of tetracyclines into the brain has been widely documented. The aim of this work was to develop a matrix assisted laser desorption ionization-mass spectrometry imaging (MALDI MSI) method for the molecular histology of doxycycline (DOX) in the healthy rat brain.

2. The time-dependent distribution was investigated after an i.p. dose of 25?mg/kg at 0, 5, 30, 120, 240, 360 and 480?min postdose. LCMS/MS was used to quantify the drug in plasma and brain homogenates and MALDI MSI was used to determine the distribution of the analyte.

3. Within the first-hour postdose, the drug showed slow accumulation into the plasma and brain tissues. DOX brain concentration gradually increased and reached a peak (C_max) of 1034.9?ng/mL at 240?min postdose, resulting in a brain plasma ratio of 31%. The images acquired by MSI matched the quantification results and clearly showed drug distribution over the entire rat brain coronal section from 5?min and its slow elimination after 360-min postdose.

4. Our findings confirm that MALDI MSI provides an advanced, label-free and faster alternative technique for xenobiotic distribution such as DOX in tissues, making it an essential drug discovery tool for other possible neuroprotective agents.     

Synthesis,radiolabeling and evaluation of a new positively charged 99mTc‐labeled fatty acid derivative for myocardial imaging

¹²³I‐labeled fatty acids and ¹⁸F‐FDG are used as metabolic markers for detecting myocardial abnormalities. However, a ^99mTc‐based molecule may find wider application. In the present work, a new ^99mTc‐labeled, uni‐positively charged, 16‐carbon fatty acid has been prepared and evaluated in normal Swiss mice. The results are then compared with the neutral analogue reported earlier. A 16‐cysteinyl hexadecanoic acid conjugate was synthesized in a six‐step synthetic procedure starting with 16‐bromohexadecanoic acid. The ligand upon incubation with [^99mTcN(PNP6)]²⁺ core formed the required positively charged complex in ～85% yield. The complex, which was obtained as a mixture of syn‐anti isomers, was purified by HPLC and the major fraction was used for in vivo studies in Swiss mice. The biodistribution studies in Swiss mice showed initial uptake similar to ¹²⁵I‐IPPA followed by rapid clearance from the myocardium till 10 min p.i. Thereafter, the rate of clearance was significantly decreased, an observation reported earlier for positively charged fatty acid complexes. In terms of absolute uptake, the positively charged complex performed better than the neutral analogue reported earlier. The positively charged fatty acid complexes, prepared using [^99mTcN(PNP)]²⁺ core, seems to be better candidates for the development of myocardial metabolic tracers than their neutral counterparts. Copyright © 2010 John Wiley & Sons, Ltd.     

Preparation and biological evaluation of 99mTc–ropinirole as a novel radiopharmaceutical for brain imaging

Noninvasive brain imaging is a process that allows scientists and physicians to view and monitor the areas of the brain. The aim of this study was to formulate a novel radiopharmaceutical for the detection of brain disorders at early stages in susceptible patients. ^99mTc–ropinirole was prepared by the direct complexation of ropinirole with technetium‐99m. The results showed that the radiochemical yield ^99mTc–ropinirole was 92 ± 2.87% and the radiochemical yield was evaluated by paper chromatography and HPLC. In vitro studies showed that the formed complex was stable for up to 6 h. In vivo uptake of ^99mTc–ropinirole in the brain was 4.87 ± 0.15% injected dose/g organ at 30 min post‐injection, which cleared from the brain with time till it reaches 2.3% at 2 h post‐injection indicating that the brain uptake of ^99mTc–ropinirole is higher than that of the commercially available ^99mTc‐HMPAO, which is 2.25% at 30 min. Pre‐dosing mice with cold ropinirole reduced the brain uptake to 0.26 ± 0.01% injected dose/g organ, so this confirms the high specificity and selectivity of this radiotracer for the assessment of the dopamine receptors.     

The N-terminal region of CXCL11 as structural template for CXCR3 molecular recognition: synthesis, conformational analysis, and binding studies

The chemokines and their receptors play a key role in immune and inflammatory responses by promoting recruitment and activation of different subpopulations of leukocytes. The membrane receptor CXCR3 binds three chemokines, CXCL9, CXCL10, and CXCL11, and its involvement is recognized in many inflammatory diseases and cancers. Therefore, the inhibition of CXCR3 pathway through interactions with three ligands was indicated as putative therapeutic target for the treatment of these diseases, and some inhibitory compounds have already been described in the literature. Recently, we studied the interaction between CXCR3 and its three natural ligands and showed that three CXCR3 ligands bound the receptor mainly by their N-terminal regions using aromatic and electrostatic interactions, and, in particular, CXCL11 had the highest affinity for CXCR3. In light of these results, we focused our attention on what structural region(s) of CXCL11 interacted with CXCR3 and what were the structural features. Therefore, we have synthesized three peptides, corresponding to the N-terminal region of CXCL11, but with different aromatic amino acids, analyzed their conformations by circular dichroism, NMR, and molecular dynamics simulations, simulated their complexes with CXCR3 by docking methods, and validated these data by in vitro studies. The results showed that two peptides were able to bind CXCR3 and to mimic the molecular recognition of CXCL11 and demonstrated that N-terminal region of CXCL11 can be used as template and starting point to obtain new molecules by de novo design approaches.     

Neuroprotection by aminoguanidine after lateral fluid-percussive brain injury in rats: a combined magnetic resonance imaging,histopathologic and functional study

The present study examined the effects of a selective inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG), on neuronal cell survival and post-traumatic recovery in rats following a lateral fluid percussive brain injury. Daily treatment of AG at the dosage of 100 mg/kg or normal saline was given intraperitoneally into rats starting 2 h before or 30 min after brain injury. Treatment with AG significantly reduced lesion volumes in the brains of rats after injury, as evaluated by high-resolution magnetic resonance imaging (MRI). Immunohistochemical analysis showed a marked induction of iNOS expression in brain macrophages ipsilateral to the injury. Apoptotic neurons were observed in the ipsilateral cerebral cortex by in situ terminal transferase d-UTP nick-end labelling (TUNEL) and caspase-3 immunohistochemistry. In rats receiving prophylactic or post-injury treatment of AG, the number of degenerating neurons was markedly reduced in the cerebrum compared to those receiving saline injection. The location and extent of these pathologic changes correlated with MRI findings. Neurobehavioral studies showed that rotametric performance, grip-strength score, total and ambulatory locomotor responses and acoustic startle response were reduced in rats subjected to the injury but were significantly improved in AG-treated rats. It is suggested that inhibition of iNOS by AG may represent a potential therapeutic strategy for the treatment of traumatic brain injury.     

Identification and in vivo evaluation of a fluorine‐18 rolipram analogue, [18F]MNI‐617, as a radioligand for PDE4 imaging in mammalian brain

Phosphodiesterase (PDE) 4 is the most prevalent PDE in the central nervous system (CNS) and catalyzes hydrolysis of intracellular cAMP, a secondary messenger. By therapeutic inhibition of PDE4, intracellular cAMP levels can be stabilized, and the symptoms of psychiatric and neurodegenerative disorders including depression, memory loss and Parkinson's disease can be ameliorated. Radiotracers targeting PDE4 can be used to study PDE4 density and function, and evaluate new PDE4 therapeutics, in vivo in a non‐invasive way, as has been shown using the carbon‐11 labeled PDE4 inhibitor R‐(?)‐rolipram. Herein we describe a small series of rolipram analogs that contain fluoro‐ or iodo‐substituents that could be used as fluorine‐18 PET or iodine‐123 SPECT PDE4 radiotracers. This series was evaluated with an in vitro binding assay and a 4‐(fluoromethyl) derivative of rolipram, MNI‐617, was identified, with a five‐fold increase in affinity for PDE4 (K_d = 0.26 nM) over R‐(?)‐rolipram (K_d = 1.6 nM). A deutero‐analogue d₂‐[¹⁸F]MNI‐617 was radiolabeled and produced in 23% yield with high (>5 Ci/µmol) specific activity and evaluated in non‐human primate, where it rapidly entered the brain, with SUVs between 4 and 5, and with a distribution pattern consistent with that of PDE4.     

Efficient radiosynthesis and preclinical evaluation of [18F]FOMPyD as a positron emission tomography tracer candidate for TrkB/C receptor imaging

Herein we report an efficient radiolabeling of a ¹⁸F-fluorinated derivative of dual inhibitor GW2580, with its subsequent evaluation as a positron emission tomography (PET) tracer candidate for imaging of two neuroreceptor targets implicated in the pathophysiology of neurodegeneration: tropomyosin receptor kinases (TrkB/C) and colony stimulating factor receptor (CSF-1R). [¹⁸F]FOMPyD was synthesized from a boronic acid pinacolate precursor via copper-mediated ¹⁸F-fluorination concerted with thermal deprotection of the four Boc groups on a diaminopyrimidine moiety in an 8.7±2.8% radiochemical yield, a radiochemical purity >99%, and an effective molar activity of 187±93 GBq/μmol. [¹⁸F]FOMPyD showed moderate brain permeability in wild-type rats (SUV_max = 0.75) and a slow washout rate. The brain uptake was partially reduced (ΔAUC_40–90 = 11.6%) by administration of the nonradioactive FOMPyD (up to 30 μg/kg). In autoradiography, [¹⁸F]FOMPyD exhibits ubiquitous distribution in rat and human brain tissues with relatively high nonspecific binding revealed by self-blocking experiment. The binding was blocked by TrkB/C inhibitors, but not with a CSF-1R inhibitor, suggesting selective binding to the former receptor. Although an unfavorable pharmacokinetic profile will likely preclude application of [¹⁸F]FOMPyD as a PET tracer for brain imaging, the concomitant one-pot copper-mediated ¹⁸F-fluorination/Boc-deprotection is a practical technique for the automated radiosynthesis of acid-sensitive PET tracers.     

Evaluation of oxindole derivatives as a potential anticancer agent against breast carcinoma cells: In vitro,in silico,and molecular docking study

In this study we have performed the in vitro anticancer activity of spiro oxindole derivatives against MCF-7 (human Adreno carcinoma) and MDA-MB-231 (triple negative breast cancer) cell lines to propose a possible role of these derivatives in the treatment of cancer. Compound 6, which has an N-benzyl substitution with a chloro group on the indolin-2-one scaffold, had the most potent activity against MCF-7 (3.55 ± 0.49 μM) and MDA-MB-231 (4.40 ± 0.468 μM) of all the synthesized molecules. A normal mouse embryonic fibroblast (NIH/3 T3) cell line was used to test the cellular toxicity of these derivatives. The results showed that none of the compounds were cytotoxic to normal cells. In addition, pharmacokinetic (ADME) and toxicity study profiles were predicted in silico. All the synthesized derivatives (1 to 7) demonstrated the necessary physicochemical properties for bioavailability. Finally, in vitro results of promising compound 6 were validated using molecular docking and dynamic simulation studies, which revealed their binding affinities and conformational stability in the binding cavity. Thus, these derivatives may serve as lead structures for a new generation of anticancer agents.     

Molecular modeling and docking analysis of Entamoeba histolytica glyceraldehyde-3 phosphate dehydrogenase, a potential target enzyme for anti-protozoal drug development

The enzymes of the glycolytic pathway constitute an excellent target for the design of new anti-protozoal agents; glyceraldehyde-3 phosphate dehydrogenase catalyzes the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate (GAP) into d-glycerate 1,3-bisphosphate (1,3-diPG) in the presence of NAD+ and inorganic phosphate (P(i)). The prediction of three-dimensional structure of Entamoeba histolytica and the docking of the coumarins were performed using the crystal structure of glyceraldehyde-3 phosphate dehydrogenase from Leishmania mexicana as template. Energy minimization of the whole complex showed, as expected, that most of the template interactions are preserved in the Entamoeba structure, except for SER178, ARG220 and THR167. However, novel interactions involving ILE 193 and THR 173 were also observed.     

Conformational analysis of r207910, a new drug candidate for the treatment of tuberculosis, by a combined NMR and molecular modeling approach

R207910 is an enantiomeric compound from a new class of antimycobacterial agents, the diarylquinolines [Science; 307:223 (2005)]. As enantiospecific interaction is required for biologic activity, we have undertaken a combined nuclear magnetic resonance and molecular modeling study to gain new insights into its conformation in solution and its absolute configuration. A conformational analysis using a Monte-Carlo method has been performed on each of the four possible stereomers of this compound leading to the identification of their most stable conformation. Additional ab initio calculation was performed with emphasis on the strength of the observed intramolecular hydrogen bond. Simultaneously, a complete structural identification has been carried out by a set of monodimensional and bidimensional (1)H-(13)C-NMR experiments. Determination of inter-proton distances has been achieved by a series of (1)H-(1)H ROESY NMR experiments with different mixing times followed by a volume quantification of the correlations peaks. These experimental data were compared with the theoretical distances obtained from the conformational analysis. The remarkable match shows that R207910 adopts one of the low-energy conformations predicted by molecular modeling and belongs to the (RS, SR) couple of diastereoisomers. A posteriori validation of our approach has been performed by X-ray structure determination that concluded for the RS configuration.     

Drug targeting to brain: a systematic approach to study the factors,parameters and approaches for prediction of permeability of drugs across BBB

Introduction: Drug targeting to brain by circumventing the physiological barriers is a prerequisite for drugs acting on central nervous system (CNS) and therapeutic potential of many drugs can be improved by effectively targeting the drug(s) to brain.

Areas covered: Present review describes blood–brain barrier (BBB), drug transport mechanisms and factors affecting drug transportation across BBB along with in vitro BBB models; and the approaches for evaluation of permeability of drug across BBB.

Expert opinion: The development of a still awaited perfect in vitro model to mimic BBB is a challenging task. System biologist, network biologist and computational technologist should come together to integrate the role of transporters, physiological and pathophysiological complexity of BBB to replicate vascular properties of the brain microcapillaries as a suitable model to facilitate the high-throughput screening of CNS acting biomolecules.     

Synthesis,radiosynthesis and in vivo evaluation of [123I]‐4‐(2‐(bis(4‐fluorophenyl)methoxy)ethyl)‐1‐(4‐iodobenzyl)piperidine as a selective tracer for imaging the dopamine transporter

Dopamine transporter (DAT) neuroimaging is a useful tool in Parkinson's disease diagnosis, staging and follow‐up providing information on the integrity of the dopaminergic neurotransmitter system in vivo. 4‐(2‐(Bis(4‐fluorophenyl)methoxy)ethyl)‐1‐(4‐iodobenzyl)piperidine (7) has nanomolar affinity for DAT and better selectivity over the other monoamine transporters compared with the existing SPECT radioligands for DAT. The aim of this study was to synthesize and evaluate [¹²³I]‐7 as an in vivo tracer for DAT. The tributylstannyl precursor was synthesized with an overall yield of 25%. [¹²³I]‐7 was synthesized by electrophilic destannylation with a yield of 40±10%. Radiochemical purity appeared to be >98%, whereas specific activity was at least 667 GBq/µmol. Biodistribution studies in mice showed brain uptake of 0.96±0.53%ID/g at 30 s post injection (p.i.) and 0.26±0.02%ID/g at 3 h p.i. High blood activity was observed at all time points. Pretreatment with Cyclosporin A raised brain uptake indicating that [¹²³I]‐7 is transported by P‐glycoprotein (P‐gp) pumps. In rats, regional brain distribution of [¹²³I]‐7 was not in agreement with DAT distribution. These results indicate that [¹²³I]‐7 is not suitable for mapping DAT in vivo but could be a useful tracer for the P‐gp transporter. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of N‐(2‐diethylamino‐ethyl)‐4‐(4‐fluoro‐benzamido)‐2‐methoxybenzamide (desiodo‐MIP‐1145) by coupling technique and its radioiodination: a potential melanoma imaging agent

Radioiodinated MIP‐1145, which specifically targets melanin, is an ideal candidate for targeted therapy of melanoma. An analogue of MIP‐1145 lacking the iodo‐substituent (desiodo‐MIP‐1145) was synthesized as a labeling precursor in three simple steps. The radioiodination of desiodo‐MIP‐1145 by iodine‐125 was carried out via an electrophilic substitution reaction. An optimization study for the iodination reaction was carried out. The labeled compound was isolated and purified by means of electrophoresis and HPLC. The maximum radiochemical yield, 76%, was obtained with radiochemical purity greater than 99%. The log P value for [¹²⁵I]MIP‐1145 was measured as 4.5.     

Selective agonist binding of (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) and 2S-(2alpha,3beta,4beta)-2-carboxy-4-(1-methylethenyl)-3-pyrrolidineacetic acid (kainate) receptors: a molecular modeling study

Molecular models were constructed, using the published X-ray structure of rat glutamate receptor 2 (GluR2), for the ligand-binding domains of the human (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA)- and kainate-selective ionotropic glutamate receptors (iGluRs): GluR1-7 and KA1-2. Based on the analysis of the known X-ray structures of GluR2 in complex with glutamate, kainate, and AMPA, we have constructed binding motifs (relative positioning of a ligand in the binding site and the physico-chemical interactions that take place) for selected agonist ligands and found explanations for ligand-binding selectivity to homomeric receptors among the different iGluRs. Even a single sequence difference can explain significant differences in ligand-binding affinities between two receptors. In total, there are seven residues surrounding the binding cavity that affect agonist selectivity: in GluR2, these residues are Pro478, Thr480, Leu650, Ser654, Thr686, Tyr702, and Met708. Each of these seven positions has been shown, or is predicted, to influence the presence of one or more water molecules that, when present, may form bridging hydrogen bonds between particular ligands and receptors. By using this knowledge it should be possible to design new selective agonist ligands with high affinity for any AMPA/kainate receptor.