Puja Panwar Hazari

The role of ASIC1a in neuroprotection elicited by quercetin in focal cerebral ischemia

One of the major instigators to neuronal cell death and brain damage following cerebral ischemia is calcium dysregulation. The intracellular calcium overload resulting from glutamate excitotoxicity is considered a major determinant for neuronal loss during cerebral ischemia. Moreover, ASIC1a activation due to acidosis also promotes intracellular calcium overload during ischemic insult. Interestingly, ASIC1a was found to be inhibited by some flavonoids which carry an anti-inflammatory property particularly quercetin, which could be exploited in hypoxic conditions like cerebral ischemia. This encourages us to investigate the neuroprotective effect of quercetin besides its possible downstream signaling mechanism in focal cerebral ischemia. The treatment of quercetin 30min before ischemia and 4h after reperfusion shows significant protection from ischemic injury as noticed by reduction in cerebral infarct volume and neurobehavioral deficit. In addition to earlier calcium dependent rise in the levels of nitrite and MDA exhibited marked reduction (P<0.01) in their levels when given quercetin pretreatment in ischemic brain regions. The quercetin treatment also reduced the spectrin break down products (SBDP) caused by ischemic activation of calcium dependent protease calpain. In ex-vivo study, it was also observed that quercetin inhibited the acid mediated intracellular calcium levels in rat brain synaptoneurosomes. These studies suggest the neuroprotective role of quercetin in focal cerebral ischemia by regulation of ASIC1a.

Synthesis of Specific SPECT-Radiopharmaceutical for Tumor Imaging Based on Methionine: 99mTc-DTPA-bis(methionine)

Methionine-diethylenetriaminepentaaceticacid-methionine [DTPA-bis(Met)] was synthesized by covalently conjugating two molecules of methionine (Met) to DTPA and was labeled with (99m)Tc in high radiochemical purity and specific activity (166-296 MBq/micromol). Kinetic analysis showed K(m) of 12.95 +/- 3.8 nM and a maximal transport rate velocity (V(max)) of 80.35 +/- 0.42 pmol microg protein(-1) min(-1) of (99m)Tc-DTPA-bis(Met) in U-87MG cells. DTPA-bis(Met) had dissociation constants (K(d)) of 0.067 and 0.077 nM in U-87MG and BMG, respectively. (35)S-methionine efflux was trans-stimulated by (99m)Tc-labeled DTPA conjugate demonstrating concentrative transport. The blood kinetic studies showed fast clearance with t(1/2) (F) = 36 +/- 0.5 min and t(1/2) (S) = 5 h 55 min +/- 0.85 min. U-87MG and BMG tumors saturated at approximately 2000 +/- 280 nmol/kg of (99m)Tc-DTPA-bis(Met). Initial rate of transport of (99m)Tc-DTPA-bis(Met) in U-87MG tumor was found to be 4.68 x 10(-4) micromol/kg/min. The tumor (BMG cell line, malignant glioma) grafted in athymic mice were readily identifiable in the gamma images. Semiquantitative analysis from region of interest (ROI) placed over areas counting average counts per pixel with maximum radiotracer uptake on the tumor was found to be 11.05 +/- 3.99 and compared ROI with muscle (0.55 +/- 0.13). The tumor-to-contralateral muscle tissue ratio of (99m)Tc-DTPA-bis(Met) was found to be 23 +/- 3.3. Biodistribution revealed significant tumor uptake and good contrast in the U-87MG, BMG, and EAT tumor-bearing mice. In clinical trials, the sensitivity, specificity, and positive predictive values were found to be 87.8%, 92.8%, and 96.6%, respectively. (99m)Tc-DTPA-bis(Met) showed excellent tumor targeting and has promising utility as a SPECT-radiopharmaceutical for imaging methionine-dependent human tumors and to quantify the ratio of MET(+)/HCY(-).

Synthesis and evaluation of biodegradable PCL/PEG nanoparticles for neuroendocrine tumor targeted delivery of somatostatin analog

Purpose: Neuroendocrine tumors often present a diagnostic and therapeutic challenge. We have aimed to synthesize and develop biodegradable nanoparticles of somatostatin analogue, octreotide for targeted therapy of human neuroendocrine pancreatic tumor. Methods: Direct solid phase peptide synthesis of octreotide was done. Octreotide loaded PCL/PEG nanoparticles were prepared by solvent evaporation method and characterized for transmission electron microscopy, differential scanning calorimetery (DSC), Zeta potential measurement studies. The nanoparticles were evaluated in vitro for release studies and peptide content. For biological evaluations, receptor binding & cytotoxicity studies were done on BON-1 neuroendocrine tumor cell line. Biodistribution of radiolabeled peptide and nanoparticles, tumor regression studies were performed on tumor-bearing mouse models. Results: We have synthesized and purified octreotide with the purity of 99.96% in our laboratory. PEG/PCL nanoparticles with an average diameter of 130–195 nm having peptide loading efficiency of 66–84% with a negative surface charge were obtained with the formulation procedure. Octreotide nanoparticles have a negative action on the proliferation of BON-1 cells. In vivo biodistribution studies exhibited major accumulation of octreotide nanoparticles in tumor as compared to plain octreotide. Octreotide nanoparticles inhibited tumor growth more efficiently than free octreotide. Conclusions: Thus, it was concluded that the PCL/PEG nanoformulation of octreotide showed high tumor uptake due to the enhanced permeation and retention (EPR) effect and then peptide ligand imparts targetability to the sst2 receptor and there by showing increase tumor growth inhibition. Selective entry of nanoparticles to the tumor also give the reduce side effects both in vivo and in vitro.

Synthesis of oxovanadium(IV) Schiff base complexes derived from C-substituted diamines and pyridoxal-5-phosphate as antitumor agents.

Oxovanadium (IV) complexes of N,N′‐bispyridoxyl‐5, 5′‐bis (phosphate) ethylenediimine (L1) and N,N′‐bis(pyridoxyl)‐5,5′‐bis(phosphate)‐1′′‐(p‐nitrobenzyl)ethylenediimine (L2) were synthesized by condensation of optically active C‐substituted diamines and pyridoxal‐5‐phosphate. Oxovanadium (IV) complexes derived from L1 and L2 were evaluated as DNA cleavage agent (cleavage of supercoiled plasmid pBR322 DNA). Interestingly, both the oxovanadium (IV) complexes exhibited DNA nuclease activity, and the extent of oxidation of DNA by these vanadyl complexes was superior to VOSO4. The significant reduction in primary tumor and increased delay in tumor growth of 15 days was seen in the tumor regression analysis with oxovanadium (IV) complex of L1. With the preliminary studies performed with the pyridoxal‐5‐phosphate ‐based salen derivatives including the cytotoxicity and tumor regression, it is evident that the salen bifunctional chelating agent has obtained therapeutic potential if conjugated to a gene‐specific targeting molecule for the oxidation of guanine residue.

Preliminary evaluation of technetium-99m-labeled ceftriaxone: infection imaging agent for the clinical diagnosis of orthopedic infection

OBJECTIVE In this study we sought to assess the efficacy of a technetium-99m (Tc-99m)-labeled third-generation cephalosporin as an infection imaging agent in the accurate detection of the sites of bacterial infection in vivo. DESIGN Ceftriaxone (CRO) was formulated into a ready-to-use single-vial cold kit with a shelf-life of over 6 months and was successfully labeled with technetium. The radiolabeled drug, Tc-99m-CRO, was subjected to the following preclinical evaluations: radiochemical purity, in vitro and in vivo stability, bacterial binding assay, and pharmacokinetic studies in animals and in human patients. RESULTS The kit formulation exhibited excellent radiolabeling efficiency (∼99%) and high in vitro and in vivo stability. The radiolabeled drug exhibited slow blood clearance (12% at 4 h), and the high protein binding and excretion pattern of the labeled formulation mimics the reported pharmacokinetic profile of the drug alone. In the animal model, scintigraphy scans showed higher uptake of the radiopharmaceutical in infectious lesions, even at 1 h post-administration, in comparison to inflammatory lesions. The clinical evaluation of Tc-99m-labeled CRO showed a diagnostic accuracy of 83.3%, and a sensitivity and specificity of 85.2% and 77.8%, respectively. CONCLUSIONS This kit formulation has the potential for imaging bacterial infections with much higher sensitivity and specificity as compared to other Tc-99m-labeled antibiotics available as convenient ready-to-use kits in routine clinical practice.

A New SiF–Dipropargyl Glycerol Scaffold as a Versatile Prosthetic Group to Design Dimeric Radioligands: Synthesis of the [18F]BMPPSiF Tracer to Image Serotonin Receptors

A novel SiX–dipropargyl glycerol scaffold (X: H, F, or 18F) was developed as a versatile prosthetic group that provides technical advantages for the preparation of dimeric radioligands based on silicon fluoride acceptor pre‐ or post‐labeling with fluorine‐18. Rapid conjugation with the prosthetic group takes place in microwave‐assisted click conjugation under mild conditions. Thus, a bivalent homodimeric SiX–dipropargyl glycerol derivatized radioligand, [18F]BMPPSiF, with enhanced affinity was developed by using click conjugation. High uptake of the radioligand was demonstrated in 5‐HT1A receptor‐rich regions in the brain with positron emission tomography. Molecular docking studies (rigid protein–flexible ligand) of BMPPSiF and known antagonists (WAY‐100635, MPPF, and MefWAY) with monomeric, dimeric, and multimeric 5‐HT1A receptor models were performed, with the highest G score obtained for docked BMPPSiF: −6.766 as compared with all three antagonists on the monomeric model. Multimeric induced‐fit docking was also performed to visualize the comparable mode of binding under in vivo conditions, and a notably improved G score of −8.455 was observed for BMPPSiF. These data directly correlate the high binding potential of BMPPSiF with the bivalent binding mode obtained in the biological studies. The present study warrants wide application of the SiX–dipropargyl glycerol prosthetic group in the development of ligands for imaging with enhanced affinity markers for specific targeting based on peptides, nucleosides, and lipids.

Glioma Recurrence Versus Radiation Necrosis: Single-Session Multiparametric Approach Using Simultaneous O-(2-18F-Fluoroethyl)-L-Tyrosine PET/MRI.

Purpose This study aimed to investigate the potential of hybrid gadolinium (Gd)–enhanced 18F-fluoroethyl-L-tyrosine (18F-FET) PET/MRI in distinguishing recurrence from radiation necrosis using simultaneously acquired multiple structural and functional parameters. Methods Twenty-six patients (5 female and 21 male patients; mean ± SD age, 51.58 ± 15.97 years) with single or multiple contrast-enhancing brain lesions (n = 32) on MRI after surgery and radiation therapy were evaluated with simultaneously acquired Gd-enhanced 18F-FET PET/MRI. They were then followed up with resurgery and histopathological diagnosis (n = 9) and/or clinical/MRI– or PET/MRI–based imaging follow-up (n = 17). PET/MR images were analyzed using manually drawn regions of interest over areas of maximal contrast enhancement and/or FET uptake. Maximum target-to-background ratio (TBRmax), mean target-to-background ratio (TBRmean), and choline-to-creatine (Cho/Cr) ratios as well as normalized mean relative cerebral blood volume (rCBVmean) and mean apparent diffusion coefficient (ADCmean) were determined. The accuracy of each parameter individually and in various possible combinations for differentiating recurrence versus radiation necrosis was evaluated using 2-tailed independent samples Student t test, multivariate analysis of variance, and multivariate receiver operating characteristic analysis. Positive histopathological finding and long-term imaging/clinical follow-up suggestive of disease progression served as criterion standard. Results Of 26 patients, 19 were classified as recurrence, with 7 patients showing radiation necrosis. Individually, TBRmax, TBRmean, ADCmean, and Cho/Cr ratios as well as normalized rCBVmean was significant in differentiating recurrence from radiation necrosis, with an accuracy of 93.8% for TBRmax, 87.5% for TBRmean, 81.3% for ADCmean, 96.9% for Cho/Cr ratio, and 90.6% for normalized rCBVmean. The accuracy of both normalized rCBVmean and ADCmean was improved in combination with TBRmax or Cho/Cr ratio. However, TBRmax (or TBRmean) with Cho/Cr ratio yielded the highest accuracy, approaching up to 97%. Furthermore, maximum area under the curve is achieved with the combination of TBRmean, CBV, and Cho/Cr values. Conclusions Our findings suggest that FET uptake with Cho/Cr ratio and normalized rCBVmean could be most useful to distinguish primary glioma recurrence from radiation necrosis. Hybrid simultaneous multiparametric 18F-FET PET/MRI might play a significant role in the evaluation of patients with suspected glioma recurrence.

Solid phase synthesis, radiolabeling and biological evaluation of a 99mTc-labeled αVβ3 tripeptide (RGD) conjugated to DOTA as a tumor imaging agent

Solid phase synthesis of peptides-radiometal chelator can facilitate the creation of radioactive peptide libraries to be utilized in high throughput in in vivo screening of targeted molecular imaging agents. An αVβ3 tripeptide derivative DOTA-NH-Arg-Gly-Asp was synthesized by Fmoc solid phase peptide synthesis and analyzed by spectroscopic techniques. In order to radiolabel this RGD peptide with 99mTc-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was incorporated as a chelator. The DOTA-peptide conjugate binds to 99mTc with high efficiency at ambient temperature. The resulting conjugate is stable under physiological conditions for at least 24 h after radiocomplexation. The receptor binding studies of 99mTc-DOTA- αVβ3-tripeptide in established human tumor cell lines U-87MG and BMG revealed Kd values in the nM and μM range respectively. U-87MG tumors in athymic mice were accumulated in the γ-images and major accumulation of the radiotracer was observed in kidneys followed by liver and lungs. High tumor uptake was shown in the U-87MG tumor bearing athymic mice; tumor to muscle ratios reached 8.13 ± 2.18 and 35.09 ± 4.78 at 1 and 4 h after post injection respectively.

Synthesis, 68Ga-radiolabeling, and preliminary in vivo assessment of a depsipeptide-derived compound as a potential PET/CT infection imaging agent.

Noninvasive imaging is a powerful tool for early diagnosis and monitoring of various disease processes, such as infections. An alarming shortage of infection-selective radiopharmaceuticals exists for overcoming the diagnostic limitations with unspecific tracers such as 67/68Ga-citrate or 18F-FDG. We report here TBIA101, an antimicrobial peptide derivative that was conjugated to DOTA and radiolabeled with 68Ga for a subsequent in vitro assessment and in vivo infection imaging using Escherichia coli-bearing mice by targeting bacterial lipopolysaccharides with PET/CT. Following DOTA-conjugation, the compound was verified for its cytotoxic and bacterial binding behaviour and compound stability, followed by 68Gallium-radiolabeling. µPET/CT using 68Ga-DOTA-TBIA101 was employed to detect muscular E. coli-infection in BALB/c mice, as warranted by the in vitro results. 68Ga-DOTA-TBIA101-PET detected E. coli-infected muscle tissue (SUV = 1.3–2.4) > noninfected thighs (P = 0.322) > forearm muscles (P = 0.092) > background (P = 0.021) in the same animal. Normalization of the infected thigh muscle to reference tissue showed a ratio of 3.0 ± 0.8 and a ratio of 2.3 ± 0.6 compared to the identical healthy tissue. The majority of the activity was cleared by renal excretion. The latter findings warrant further preclinical imaging studies of greater depth, as the DOTA-conjugation did not compromise the TBIA101s capacity as targeting vector.

A homodimeric bivalent radioligand derived from 1-(2-methoxyphenyl)piperazine with high affinity for in vivo 5-HT1A receptor imaging

The bivalent ligand approach is very promising and can give rise to molecular probes with enhanced conformational flexibility for tissue selective modulation. In this study we describe the synthesis of a homodimeric 5-HT1A receptor ligand by incorporating two identical pharmacophores , 1-(2-methoxyphenyl)piperazine, linked through DTPA. This bivalent derivative was efficiently synthesized and characterized by 1H, 13C NMR and mass spectroscopy. 99mTc-labeling was performed with a high radiolabeling yield (>95%) and radiochemical purity (>98%) using very low ligand concentration. In vitro studies were carried out on hippocampal cultures and PC3 cell lines which express 5-HT1A receptors. The selectivity of the complex for 5-HT1A receptors is 1000 times more than it is for 5-HT2A receptors, with Kd in picomolar range. Hill coefficients between 1.9 and 2.3 were observed, indicating that the homodimeric binding pharmacophores appear to be essential for the cooperative binding mode. In vitro binding assays in rat hippocampal cultures demonstrated the high affinity of the complex for 5-HT1A receptors. Further studies include in vivo organ distribution and gamma scintigraphy carried out in rat and rabbit. Plasma clearance rate (CR) revealed a value of 4.86 μg mL−1 min−1 in normal rabbit. Dynamic imaging performed in rabbit showed the beginning of radiolabeled uptake in brain as early as 2 min. The tumor-to-contralateral muscle tissue ratio of 99mTc–DTPA–bis(MPBA) in athymic mice with PC3 xenograft was found to be 65 ± 3.3 at 1 h. Significant accumulation was seen in mice and rat brain at 10 min with 2.07 ± 0.76% ID g−1 and 2.81% ID g−1 respectively. The high uptake in hippocampus and cerebral cortex was accredited to the 5-HT1A receptor rich regions in post mortem rat brain. This imaging agent holds a promising future in imaging 5-HT1A receptors for the effective diagnosis of neuropathological disorders.