[en] The present work is on the radiolabeling and in-vitro evaluation of NODAGA-RGD (f)K and NODAGA-(RGD (y)K)₂ with ¹⁷⁷Lu³⁺ for targeting integrin α_vβ₃ receptors overexpression in angiogenetic tumor. Integrins belong to a family of α_vβ₃heterodimeric transmembrane glycoproteins involved in cell-cell and cell-matrix interactions. Theintegrin α_vβ₃ is known to play an important rolein tumour-induced angiogenesis, tumour proliferation, survival and metastasis. The Arg-Gly-Asp (RGD) sequence has been known to bind with the α_vβ₃ integrin that is expressed on the surface of angiogenic blood vessels or tumour cells. Thus, monomer and dimer derivatives of RGD linked with NODAGA chelator and radiolabeled with ¹⁷⁷Lu³⁺ have been evaluated as targeting agent for angiogenetic tumors. The NODAGA-RGD (f)K (piCHEM) and NODAGA-(RGD (y)K)₂ (ABX) were used at 1:4, 1:6, 1:8 µmol ratio (¹⁷⁷Lu:peptide) for labeling with ¹⁷⁷Lu obtained from BRIT (Specific activity (20 ± 5) mCi/jig). The synthesis was carried out aseptically in a laminar flow bench (manually) using 700 MBq of ¹⁷⁷Lu activity. Optimization of radiolabeling was carried out at varying molarity of acetate buffer (0.2-1.0 M), with and without gentisic acid (10 mg/ml and 20 mg/ml) at pH (5.0, 5.5 and 6.0) conditions. The reaction time and temperature also has been optimized. The product was purified by passing through pre activated C₁₈ column. Physicochemical QC studies which included pH, colour and RCP were carried out. Cell line uptake studies and the serum stability was determined up to 24 h at room temperature. Synthesis of ¹⁷⁷Lu³⁺-RGD is done with NODAGA-RGD monomer and dimer using 0.4 M molar acetate buffer at pH 5.5 with 10 mg/ml gentisic acid and without gentisic acid. The production yield was found to be maximum at 1:6 µmol ratio of ¹⁷⁷Lu:peptide bio molecule at temperature 95 ℃ (±3℃) for 35 min. RCP was found 99 % (± 3%) using ITLC-SG (0.1 M CB, product Rf = 0.0-0.1), Whatman-3 PC (50% ACN product Rf = 0.5-0.7) and (50% ethanol product Rf = 0.8-1.0). The product was found stable at room temperature for 24 hrs. There was no coloration found in the product in absence of gentisic acid up to 24 h which is produced in presence of gentisic acid. The product has shown increasing uptake up to 4 hours in selected cell line and found reducing afterward. The quality control data was found to be compliant with specifications for both the products. Laminar flow bench synthesis allows radiopharmaceutical production which is in compliance with the GMP/GRPP guidelines. Preclinical evaluation in suitable animal models are underway. (author)

[en] To optimise ^99mTc labeling of Folic acid and use ^99mTc-Folic acid as a diagnostic agent for Folate receptor expressing tumors in mouse model. Folic acid was radiolabeled with ^99mTc using Stannous Tartarate as a reducing agent. Radiolabeling was optimised by varying concentration of Folic acid, Stannous Tartarate, pH and incubation time. The RCP of ^99m Tc-Folic acid was checked by paper chromatography. The stability of ^99mTc- Folic acid was checked in human serum and with DTPA challenge test and was found to be stable. In-vitro uptake studies were done by incubating ^99mTc-Folic acid with Folate receptor expressing B16F10 murine Melanoma cells (1x10⁵). In-vivo biodistribution studies of ^99mTc- Folic acid were done in B16F10 Melanoma tumor model developed in-house in C57BL/6 mice. Gamma imaging studies were done post injection of ^99mTc- Folic acid in B16F10 Melanoma tumor model. The RCP of ^99m Tc-Folic was ≥90% which was stable in human serum and when subjected to DTPA challenge test. In-vitro uptake of ^99m Tc-Folic acid was 30 % at two hours when incubated with B16F10 murine Melanoma cells (1x10⁵) which decreased to 7% when cold Folic acid was added. In vivo biodistribution studies in B16F10 Melanoma tumor model showed tumor uptake of 8% at two hours post injection. Gamma imaging studies showed localisation of ^99m Tc-Folic acid in B16F10 Melanoma tumour developed in C57BL/6 mice. Folic acid was optimally radiolabeled with ^99mTc having RCP > 90%. Preliminary in-vivo studies in Folate receptor expressing tumor model showed tumor uptake of 8%, hence may be used in future as a diagnostic agent for detecting Folate receptor expressing tumors. In-vitro studies showed that cold Folic acid bound to the folate receptors competitively and inhibited the cell binding of radiolabelled folate in the cells. This shows that ^99mTc-FA is not interfering with the cell binding efficiency of Folic acid to the folate receptors. (author)

[en] Doxorubicin (DOX) is a most potent and anthracycline anticancerous drug which is widely used in clinical oncology for the treatment of various cancers. Silk fibroin (SF), a protein-based natural biomaterial, has emerged as a biocompatible biomaterial for drug delivery systems. Coating of SF nanoparticles with Tween-80 enhances the transportation of drug across the BBB. Technetium-99m (^99mTc) has been widely used for labeling radiopharmaceuticals, due to certain advantages like chemical and physical characteristics, large and easy availability, and low isotope cost. In this study, we describe the direct labeling of drug DOX, SF nanoparticles and Tween-80 coated SF nanoparticles with ^99mTc. Further, the quality and cell uptake have been evaluated for labeled products. The SF nanoparticles were prepared by nano precipitation method and characterized for different parameters, like surface morphology and vesicle shape, size, entrapment efficiency, and in vitro release pattern. The labeling of ^99mTc with DOX, SF nanoparticles, and Tween-80 coated SF nanoparticles were done by using a different concentration of reducing agent. Paper chromatography was used for performing the radiochemical purity of the product in acetone and saline. In-vitro stability of the ^99mTc labeled products in saline and fresh human serum was performed along with the transchelation with DTPA. The in-vitro cell uptake studies were performed on the C6 glioma and LN-229 cancer cell lines. The ^99mTc labeling efficiency with DOX and SF nanoparticles was found to be more than 90%. The maximum labeling efficiency was found to be at 20 µg stannous chloride, pH 5-6, and 200 µg of DOX at room temperature. The product found to be stable in serum and tranchelation study. Cellular uptake was higher in Tween-80 coated SF nanoparticles as compared to plain SF nanoparticles and free drug. DOX and nanoparticles were successfully labeled with ^99mTc and optimized for the different parameters showing high in-vitro stability in saline and fresh human serum. These SF nanoparticles could serve as a vector for the delivery of the drug to brain. (author)

[en] In recent time, ⁶⁴CuCl₂ is gaining prominence as a diagnostic PET radiotracer for applications in a wide variety of cancer, owing to its favorable nuclear characteristics (T_1/2: 12.7 h, EC (43.8%), β⁺ E_max: 651 keV (17.7%), β^- E_max: 578 keV (38.5%)). The applicability of ⁶⁴CuCl₂ as a PET radiopharmaceutical stems from the fact that Cu is an essential element which plays an important role in cell proliferation and angiogenesis. ⁶⁴Cu (II) is reduced to Cu (I) by reductases, followed by influx of copper by copper transporter 1 (hCTR1) in human cells. In the present study, efforts have been made to analyze the efficacy of ⁶⁴CuCl₂ through PET/ CT imaging in tumour bearing nude mice and normal rabbits and carrying out internal dosimetry. High specific activity no-carrier-added ⁶⁴Cu was produced by 64Zn (n, p) ⁶⁴Cu reaction in the APSARA-U reactor and radiochemical processing was carried out at Radiopharmaceuticals Division, BARC. Cell uptake and internalization potential was investigated using AR42J, B16F10 and MCF7 cell lines. PET/CT imaging and biodistribution studies were performed in mice with xenografted tumor generated using all the three cell lines. Carcinoma cell-line were grown in IMDM medium with 10% FBS in 5% CO₂ at 37℃. The nude mice were injected with carcinoma cell lines (2 x 106 cells/mice) at the proximal flank region of the mice and tumor was allowed to grow till 1 cm³ for experimentation. The ⁶⁴CuCl₂ (200 µCi/mice) was injected in xenografted mice through tail vein. PET imaging was performed using GEMINI-TF 16-Slice PET/ CT instrument with customized CT parameters of 90 V/100 mA with slice thickness of 2 mm and PET parameter of 10 minutes emission window/bed. Degree of receptor mediated uptake of the radiochemical was determined by biodistribution studies and SUV_max obtained from PET imaging study. The PET/CT imaging performed at intervals of 2 h, 4 h, 24 h, 48 h and 72 h post injection using ketamin/xylazine injection for anesthesia, showed incremental accumulation of ⁶⁴Cu in initial 4 h. Biodistribution analysis corroborated well with the PET analysis, which revealed maximum uptake in the tumour after 4 h post-injection and retention therein till 48 h. For rabbit PET/CT imaging, 1 mCi radionuclide injected through ear vein showed high uptake and long-term retention of radioactivity in liver as ⁶⁴CuCl₂ shows hepatobiliary clearance. Initial studies document the promising potential of NCA ⁶⁴CuCl₂ as a PET radiotracer in the form of a simple radiochemical. Preclinical development of this agent for subsequent clinical translation in patient are underway. (author)

[en] A novel hypoxia trace, 1-(2-(1"8"F) Fluoroethyl)-2-Nitroimidazole was developed in our lab. The accumulation of this tracer in the hypoxic region of B16F10 melanoma tumor (C57BL6 mice) section by pimonidazole based immunohistochemistry as well as autoradiography. This methodology will be useful in the assessment of newly developed. (author)

[en] Overexpression of COX-2 receptors is observed in a variety of tumours. Therefore, development of suitable ¹⁸F-labelled PET radiotracers of selective COX-2 inhibitors is an attractive option to target selective and specific inhibitors of COX-2. The binding free energy [ΔG(-KCal/mole)] of Ac- etaminophen and F-18 labelled derivative of Acetaminophen has been calculated by using AUTODOCK 4.2 and crosschecked using www.swissdock.ch against the PDB code 3LN1 and has been found to be comparable in both cases. This encouraging result provides the necessary impetus towards the development of F-18 labelled derivative of Acetaminophen based on its property of selective COX-2 inhibition in designing PET radiopharmaceutical for tumour imaging. Herein, we report the fully automated radiosynthesis of the novel F-18 Fluoroethylated paracetamol by direct radio-fluoroethylation of paracetamol and subsequent purification with SEP-PAK® cartridge purification. The evaluation of the PET radiotracer has been carried out by PET/CT imaging, bio-distribution in mice tumour model and histopathological studies. The fully automated radiosynthesis of ¹⁸F-Fluoroethylated paracetamol using general purpose synthesis module which, in principle, is similar to GE TRACERlab FXFDG, has been carried out in three steps: (i) Radiosynthesis of the fluoroethylating agent, [¹⁸F]Fluoroethyl tosylate (ii) Coupling of [¹⁸F]Fluoroethyl tosylate with paracetamol in DMSO solvent and (iii) purification by SPE using Sep Pak® Plus ALOX N cartridges. Pharmacokinetic studies were evaluated by PET/CT imaging study in healthy rabbit at two different time points. Biodistribution study was carried out in nude mice bearing tumour (MDA-MB-231). COX-2 overexpression in tumours was confirmed by histopathological studies. The non-decay corrected radiochemical yield is around (25±3) % (n = 3) within 60±2 mins (total synthesis time). The radiochemical purity is >95 % as confirmed by radio-TLC and radio-HPLC coupled with UV (λ=276 nm). Biodistribution study demonstrated significant tumour accumulation and retention over a period of two hours post injection. COX-2 overexpression in tumour was confirmed by histopathological studies using mouse anti- COX2 antibody. One-hour post injection PET/CT imaging study in healthy rabbit showed very fast clearance from liver and blood, however, with high accumulation of the tracer in highly proliferating regions like bone marrow and sub-mandibular jaws. The thick leg joints showed significant uptake which can be attributed to age related inflammation in aged rabbit and the well-known fact that COX-2 is overexpressed in inflammation. Both the kidneys as well as urinary bladder showed very high tracer accumulation indicating clearance via renal route. The PET/CT image of two-hours post injection showed complete blood clearance with elimination via renal route, however with bone marrow accumulation. No bone uptake other than the thick joints was observed throughout the period of PET/CT study confirming in vivo stability of the tracer. Thick joint uptake can be attributed to age-related inflammation of the aged rabbit and the well-known fact that COX-2 is overexpressed in inflammation. F-18 labelled Paracetamol has successfully been designed, developed and evaluated as a PET tracer for tumour imaging agent based on COX-2 overexpression in a variety of tumours.

[en] A fully automated radiosynthesis procedure of ("1"8F)Fluoroacetate by solid phase extraction purification was developed with a non-decay corrected radiochemical yield of 47.2 ± 3.0 % (n = 5) within a total synthesis time of 40 ± 1 min. Bio-distribution study in tumour (PC3: Human Prostate Cancer Cell Line) bearing nude mice showed good accumulation and retention of the tracer up to 180 min post injections. The localization of ("1"8F)Fluoroacetate in prostate tumour was further validated by autoradiography and histopathology. (author)

[en] Radioimmunotherapy (RIT) using ¹⁷⁷Lu-labeled monoclonal antibodies is increasingly gaining prominence in therapeutic nuclear medicine practices. Towards the use of these agents, the major challenges involved are preparation of the clinical dose and optimization of radiolabeling parameters of differently conjugated ¹⁷⁷Lu- labeled monoclonal antibodies (MoAbs) viz. ¹⁷⁷Lu-DOTA-Rituximab, ¹⁷⁷Lu-DOTA-Trastuzumab and ¹⁷⁷Lu-DOTA-Pertuzumab. Efficient radiolabeling of these MoAbs with varied molecular weight (143-148 KDa) depends on optimum pre-concentration, incubation-temperature, time and pH of conjugation and suitable purification of immunoconjugates. The present study merits the development of a single protocol which has been optimized for conjugation of rituximab, trastuzumab and pertuzumab with p-NCS-benzyl-DOTA and radiolabeling of these immunoconjugates using low specific activity (LSA) ¹⁷⁷LuCl₃. Commercial rituximab, trastuzumab, pertuzumab (5.0 mg/500 µL) preconcentrated to 80-100 µL using 30kDa MW cut-off ultra-centrifugal filtration-device at 3000 g for 17 minutes. Coupling of rituximab, trastuzumab, pertuzumab with p-NCS-benzyl-DOTA carried out at 1:10 molar ratio under incubation at 24℃ (2 h), followed by 4℃ (18 h) at pH~8.0. Reaction-mixtures purified using pre-conditioned PD-10 columns. All the three immunoconjugates eluted from PD-10 using 0.2M CH₃COONa-buffer (pH~5.5) and concentrations estimated by Bradford's-assay. The number of DOTA molecules per antibody determined by spectrophotometric-method using Pb (II)-Arsenazo (III) complex. Prior to radiolabeling, pH of ¹⁷⁷LuCl₃ (250-300 mCi, SA: 15-20 mCi/µg) adjusted to 6.5, using 0.2M CH₃COONa solution. ¹⁷⁷Lu-acetate incubated with all the three purified immunoconjugates at 37℃ for 90 minutes. Radiolabeled reaction-mixture purified using pre-conditioned PD-10 column. In-vitro stability of radioconjugates ascertained by adding ascorbic acid (60 mg). The RCP evaluated using TLC and HPLC both. Using 300 mCi of LSA ¹⁷⁷LuCl₃ (15 mCi/µg), all the three radioconjugates (80-85 mCi) could be prepared. All the three radioconjugates were clear and colorless, pH (5.5-6.0) and RAC (8-10 mCi/mL). The RCP of all the three radioconjugates estimated by TLC was >98% (R_f: 0.0-0.1), while RCP derived by HPLC was >98% (Rt: 11-13 minutes). The RLY of all the three radioconjugates were between 32-35%. The EL was <6 EU/mL and product was sterile with in-vitro stability upto 96 h (storage at -20℃) ℃with stabilizer. In all the three immunoconjugates, number of DOTA attached per antibody were ~3 and MoAbs concentration were ~2.8 mg/mL. A single, protocol could be successfully optimized for coupling of rituximab, trastuzumab, pertuzumab with p-NCS-benzyl-DOTA. The subsequent radiolabeling with 15-20 mCi/µg of ¹⁷⁷LuCl₃ yielded patient doses of the radioconjugates i.e ¹⁷⁷Lu-DOTA-Rituximab, ¹⁷⁷Lu-DOTA-Trastuzumab and ¹⁷⁷Lu-DOTA-Pertuzumab. Further studies towards clinical translation of all the three radiopharmaceuticals in patient are underway. (author)

[en] We present the results of our computation of the subregion complexity and also compare it with the entanglement entropy of a 2  +  1-dimensional holographic superconductor which has a fully backreacted gravity dual with a stable ground sate. We follow the ‘complexity equals volume’ or the CV conjecture. We find that there is only a single divergence for a strip entangling surface and the complexity grows linearly with the large strip width. During the normal phase the complexity increases with decreasing temperature, but during the superconducting phase it behaves differently depending on the order of phase transition. We also show that the universal term is finite and the phase transition occurs at the same critical temperature as obtained previously from the free energy computation of the system. In one case, we observe multivaluedness in the complexity in the form of an ‘S’ curve. (paper)

[en] Abundant expression of somatostatin receptors (SSTR) in differentiated neuroendocrine tumours (NET) serve as a potential target for designing agents for diagnostic imaging and treatment. In this study, the theranostic concept has been utilized by choice of SSTR-targeting compounds, Ga-68-DOTATATE and Lu-177/Y-90 DOTATATE towards diagnosis and treatment of NET. In-vitro and in-vivo evaluation of the efficacy of these NET specific diagnostic and therapeutic radiopharmaceutical pair was performed using SSTRII positive (AR42J) and negative (HCT116 and MCF7) cell lines. Y-90-DOTATATE was produced from Carrier free, clinical grade Y-90-Acetate, sourced from a two- stage Sr- 90/Y-90 generator system based on supported liquid membrane (SLM) technology. Lu-177- Chloride were produced in our research reactor using enriched Lutetium-177-oxide {Lu-176 (n,γ) Lu-177}. Ga-68-DOTATATE was synthesized using Gallium-68-Chloride obtained from Ge-68/Ga- 68 generator. The endotoxin limit was quantified by gel-clot BET assay and sterility test done by direct inoculation. Pancreatic carcinoma cell-line AR42J (expressing SSTRII), Human colon carcinoma cell-line HCT- 116 (with SSTRII expression) and MCF7 (with negative SSTR II expression) were used for the in vitro evaluation. The cells were grown in IMDM medium with 10% FBS in 5% CO2 at 37°C. In vitro cell-binding and receptor binding studies were performed by incubating AR42J cells and cell homogenate respectively with radioligand (~5x10– 12 mol DOTATATE) up to 120 min followed by washing with PBS. Non-specific binding was assessed by addition of cold DOTATATE (5x10–9 mol). The degree of receptor mediated uptake of the radio-conjugate was determined by biodistribution studies in tumour bearing athymic nude mice. The Lu-177/Y-90 DOTATATE (50μCi-100μCi/mice) was injected in xenografted mice through tail vein. Planar scintigraphy/ PETCT images were obtained for comparative analysis.