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ABSTRACT
Year : 2013  |  Volume : 28  |  Issue : 5  |  Page : 36-54  

Radiopharmacy


Date of Web Publication29-Nov-2013

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How to cite this article:
. Radiopharmacy. Indian J Nucl Med 2013;28, Suppl S1:36-54

How to cite this URL:
. Radiopharmacy. Indian J Nucl Med [serial online] 2013 [cited 2019 Dec 5];28, Suppl S1:36-54. Available from: http://www.ijnm.in/text.asp?2013/28/5/36/122403

RP-01

Fractionation of common cold kits as a cost-saving method in a low volume nuclear medicine department

Nisha Bhatia, Vandana Kumar Dhingra


Department of Nuclear Medicine, Cancer Research Institute, HIHT, Dehradun, Uttarakhand, India

Objective: Cold kits contain large amounts of reagent sufficient for labeling multiple doses of radiopharmaceutical and are wasted when preparing 1-2 doses with a single vial. We studied the feasibility of fractionation of cold kits and an innovative single dose preparation method. Materials and Methods: Common radiopharmaceuticals were prepared by fractionation and studies done were observed. Basic quality control for radiochemical purity was performed on samples thus prepared. 3 ml normal saline was added in the reagent vial (DTPA/MDP) aseptically. 4-5 mCi 99mTcO4 is taken in a syringe and 2-3 drops of DTPA is withdrawn from the vial. Similarly, MDP can be prepared in a vacuum vial with 1 ml reagent and 40-60 mCi of Tc99m each time. Precautions include change of needle, swabbing of rubber of vial and ensuring a negative pressure in the vial while keeping the vial upright constantly. The content should be mixed, stood for 10 min in room temperature and then be administered. The remaining vial can be stored in freezer compartment of the refrigerator for future use (after thawing) of upto 10 days. Results: Observations of 50 studies each by the above method were included for this study. Images of all studies done by radiopharmaceutical prepared by these methods were of acceptable quality with no incidence of free pertecnetate in soft tissues/salivary/thyroid gland/stomach. Radiochemical purity tests of formulated preparations were estimated with paper chromatography and labeling efficiency of >90 was noted for both radiopharmaceuticals prepared by this method. Conclusion: Fractionation of common cold kits can be practiced as a cost saving method if done with proper technique in low volume departments.

RP-02

A semi-quantitative evaluation of physiological organ distribution of 68Ga-DOTA-TATE in NET patients

Ankit Watts, Ravi Ranjan, Baljinder Singh, Jaya Shukla, Bhagwant Rai Mittal


Department of Nuclear Medicine and PET, PGIMER, Chandigarh, India

Objective: The aim of the present study was to derive the standardized uptake values from 68Ga-DOTA-TATE-PET imaging as a measure of physiological uptake range of the radiotracer in normal organs in NET patients. Materials and Methods: Thirty-one patients (12 M; 19 F; mean age = 41.5 years and range 14-67 years) who underwent 68Ga-DOTA-TATE PET/CT as a part of the diagnostic work-up of their NET disease status were included for semi-quantitative PET image analysis in the study. Briefly 37.0-189.0 MBq radioactivity of 68Ga-DOTA-TATE was injected intravenously and imaging was performed using discovery STE 16 (GE Healthcare, USA) PET/CT scanner with mean uptake time of 73.0 min (range 52.0-89.0 min). Reconstructed PET/CT images were visually analyzed on workstation (Adw 4.6). SUV-max values were calculated by drawing fixed ROI over brain, pituitary gland, thyroid, myocardium, salivary gland, aortic arch, lung, liver, adrenals, kidneys, uncinate process of pancreas and femur. None of these sites taken for semi-quantitative analysis had evidence of any metastatic NET disease. An appropriate statistical analysis was applied and the mean, median SUVmax values on each organ was calculated. Results: The highest SUV-max values (21.02 ± 8.57) for 68 Ga-DOTA-TATE were observed in spleen followed by kidney (12.56 ± 4.70), adrenals (10.11 ± 4.02), liver (6.76 ± 2.37), pituitary (5.85 ± 2.37), head of pancreas (4.1 ± 2.03), thyroid (3.1 ± 1.41) and salivary (1.9 ± 0.91) glands. A wide inter-patients' variation (range 1.2-9.82) in SUV-max values was observed for the head of pancreas and the focal uptake of the radiotracer was seen in only 7/31 patients. The SUV-max values on all the remaining sites were less than 1.0. A very negligible uptake was seen in brain. On visual scan interpretation, homogenous uptake pattern of 68Ga-DOTA-TATE was seen in spleen, thyroid and salivary glands, but the uptake was found to be inhomogeneous on the liver. A very sharp and focal uptake was noted on pituitary and adrenals. Conclusion: The uptake of 68Ga-DOTA-TATE in various organs reflects the pattern of somatostatin receptor density on these organs. The observed value of SUV-max of 68Ga DOTA TATE, in a patient and its comparison with the normal range in a given setting can be used to differentiate physiological and pathological sites. This normal database can also be used as a prognostic indicator following somatostatin receptor based targeted therapy in patients with metastatic NET disease on follow-up 68Ga PET. However, for the clinical validation of these results, the study needs to carried out in a larger cohort of patients.

RP-03

Kit formulation for the preparation of 68 Ga-DOTA-{NaI 3 }-Octreotide for PET imaging of somatostatin receptor positive cancers

Archana Mukherjee, Usha Pandey, Rubel Chakravarty, Haladhar Dev Sarma 1 , Grace Samuel, Ashutosh Dash


Isotope Applications and Radiopharmaceuticals Division, 1 Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India

Objective: Indigenous development of 68 Ge/ 68 Ga generator has resulted in easy availability of the PET radioisotope 68Ga independent of an on-site cyclotron. Availability of peptide formulations in ready to use kit form for facile radiolabeling with generator eluted 68 Ga is desired for wider application of 68 Ga radiopharmaceuticals. This study was aimed at the development of a kit formulation of somatostatin analogue, DOTA-{NaI3}-Octreotide (DOTANOC) for preparation of 68 Ga-DOTANOC in a clinical setting without automated synthetic modules, especially for countries like India. Materials and Methods: Single vial kits of DOTANOC were formulated using sodium acetate, freeze dried and tested for sterility and apyrogenicity. Sterile 0.1 N HCl was also prepared and provided with the kit for elution of 68 Ga from novel 68 Ge/ 68 Ga BARC generator. Optimization of radiolabeling with generator eluted 68 Ga, characterization of complex by PC and HPLC techniques, evaluation of stability of kits and bioevaluation studies in animals were carried out. Results: Four batches of DOTANOC kits were successfully formulated using sodium acetate. All the batches passed sterility and BET tests as per IP. Radiochemical yields exceeding 95% were observed when radiolabeled with 68 Ga (148-185 MBq) eluted from 68 Ge/ 68 Ga BARC generator. The 68 Ga-DOTANOC complex was stable in saline and human serum when studied upto 2 h. Consistency in 68 Ga labeling was achieved when tested up to 3 months. Biodistribution in Swiss mice revealed fast clearance of activity via renal route as reported for 68 Ga-DOTANOC peptide. Conclusion: Development of single vial DOTANOC kit for preparation of 68 Ga radiopharmaceutical was demonstrated. Deployment of kits for preparation of 68 Ga radiopharmaceuticals is expected to expand the clinical applications of newly developed molecular imaging agents.

RP-04

Kit formulation for the preparation of infection imaging 0 agent : 0 99m Tc-ubiquicidin (29-41)

Archana Mukherjee 1 , Chanda Arjun 2 , Doddathota Krishnaiah Ranganatha 2 , MeeraVenkatesh 1,2 , Grace Samuel 1,2


1
Isotope Applications and Radiopharmaceuticals Division, Bhabha Atomic Research Centre, 2 Quality Control, Radiopharmaceuticals, Board of Radiation and Isotope Technology, Navi Mumbai, Maharashtra, India

Objective: The aim of this study was to formulate indigenous Ubiquicidin, UBI (29-41) kit for preparation of infection imaging agent, 99m Tc-UBI (29-41). Single vial kit for UBI is not feasible as optimal radiolabelling of UBI is achieved under alkaline conditions at pH 9.0-10.Hence, two component kit was formulated. Materials and Methods: Two component kit was formulated and lyophilized under sterile conditions. First vial contained 50 μg UBI peptide and 30 μg SnCl 2 and second vial contained 0.1 N NaOH. Sterility (ST) and bacterial endotoxin test (BET) was carried out. Radiolabeling was carried out by addition of 1 mL of 99m TcO4- to the vial containing NaOH to achieve pH 9.0-10 and then entire content was transferred to the vial containing UBI. After incubation at RT, sterile saline was added. The complex was characterized by HPLC and ITLC. Stability of the radiolabeled preparation in saline and serum was tested. In vitro uptake of 99m Tc-UBI in S. aureus (ATCC 25923) and in vivo evaluation in rats bearing infection was also studied. Results: Kit passed ST and BET tests as per IP. The radiochemical yield of 99m Tc-UBI was >95% as determined by the various techniques. Complex showed Rt of 10.8-12 min in HPLC. 99m Tc-UBI was stable in saline and serum. Specific uptake of 99m Tc-UBI in bacteria was observed. In vivo studies in infection bearing animals showed uptake in infection site and clearance via kidneys. Conclusion: Two component kit for the preparation of infection imaging agent 99m Tc-UBI (29-41) was successfully formulated. Radiolabeling with 99m Tc, in-vitro and in vivo specificity of 99m Tc-UBI for infection detection was demonstrated.

RP-05

99m Tc carbonyl diethylenetriamine pentaacetic acid-avastin: Preparation and preliminary bioevaluation

Mythili Kameswaran, Usha Pandey, Haladhar Dev Sarma 1 , Grace Samuel


Isotope Applications and Radiopharmaceuticals Division, 1 Radiation Biology and Health Safety Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India

Objective: Vascular endothelial growth factor A (VEGF-A) is a protein that stimulates angiogenesis in various diseases, especially cancer. Bevacizumab (Avastin) is a humanized MoAb that inhibits VEGF-A and is currently approved by US FDA for metastatic cancers. The objective of the study was to explore the potential of 990 m Tc carbonyl labeled Avastin as a tumor imaging agent that would provide insights into the angiogenic stimulus while monitoring therapy. Materials and Methods: Avastin was conjugated with paraisothiocyanatobenzyl diethylenetriamine pentaacetic acid (p-NCS-Bz-DTPA) and subsequently radiolabeled with 99m Tc via the 99m Tc carbonyl synthon. Purification of 990 m Tc carbonyl DTPA-Avastin was carried out using PD-10 column and characterized by HPLC on a TSK GEL column. Cysteine challenge experiments were carried out to determine the in vitro stability of the radioconjugate. Biodistribution studies in melanoma tumor bearing animals were carried out to determine the uptake of the radioconjugate by the tumors. Results: The radiochemical purity of the radiolabeled antibody was >95%. In the standardized HPLC system, the radioconjugate had a retention time of 15.1 min while free 99m Tc carbonyl had Rt = 22.5 min. The radioconjugate exhibited good stability in the cysteine challenge experiments (upto 20 h at 4°C). Biodistribution studies in tumor bearing mice showed significant tumor uptake (~2.0% ID/g at 24h p.i.). Most the activity was cleared by the end of the study (24 h p.i.). Conclusions: The 99m Tc-carbonyl-DTPA-Avastin conjugate with a radiochemical purity of >95% exhibited excellent stability when stored at 4°C upto 24 h. Biodistribution studies in tumor mice showed good specificity for VEGF indicating its potential for further evaluation as a radioimmunoscintigraphy agent for various cancers.

RP-06

Enhanced brain targeting efficiency of intranasally administered radiolabelled antimigraine drug micelles with altered biodistribution

Swapna Nabar, Ratnesh Jain 1 , Tanuja Shet 2 , Uday Nayak, Vandana Patravale 1 , MGR Rajan


Radiation Medicine Centre (BARC), Tata Memorial Centre, Annexe Bldg, Parel, 1 Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Matunga, 2 Tata Memorial Hospital, Parel, Mumbai, Maharashtra, India

Objective: The investigation was aimed at designing and radiolabelling micellar nanocarrier for antimigraine drugs (zolmitriptan, sumatriptan) for nose-to-brain delivery. The objective was to improve brain targeting of these drugs and to investigate the regional brain distribution to identify the probable pathway of drug transport to the brain. Altered biodistribution of radiolabelled intranasal micellar drug was also studied. Material and Methods: Poloxamer 407, PEG 400, benzyl alcohol, Vit E TPGS, zolmitriptan, sumatriptan, stannous chloride dihydrate, sodium pertechnetate. Micellar nanocarriers suitable for brain targeting through olfactory mucosa was characterized for particle size by DLS, SANS and cryo-transmission electron microscopy. 99m Tc was used to radiolabel zolmitriptan and sumatritan for in-vivo studies in rats and rabbit. Brain uptake and localization of these antimigraine drug micelles were studied by doing biodistribution, autoradiography and nuclear imaging studies (rabbit). Results: Brain uptake via I.N route at 30 min, 60 min 120 min and 240 min of radiolabelled drug micelles was 4-5%, 5.5-6.8%, 4-5% and 3-3.6% I.D/g as compared to 0.8-1%, 0.7-1.5%, 0.7-1.6% and 0.7-0.9% I.D/g of radiolabelled drug solution. Brain localization and autoradiography studies demonstrated higher concentration of 99m Tc-drug micelles in olfactory and cerebellum regions. Autoradiography studies suggested invovement of intracellular, extracellular routes in nose-to-brain delivery of the drug. Imaging studies in rabbit with I.N radiolabelled drug micelles correlated with I.N (biodistribution, autoradiography) studies in rat. Rabbit images showed specific uptake in various regions of brain and spinal cord. Uptake by liver following intranasal administration of micellar drugs was negligible and significantly lower P < 0.05 as compared to I.V route. Conclusion: The investigation indicated the potential of micellar nanocarrier of drugs for improved migraine therapy via intranasal route. Also these drugs in micellar form could be given to patients via intranasal route with liver dysfunction.

RP-07

Modulation of pharmacokinetics through bi-functional chelators: Study on a group of 2-nitroimidazole- 99m Tc (CO) 3 complexes for detecting tumor hypoxia

Madhava B Mallia, Sweety Mittal, Haladhar D Sarma 1 , Sharmila Banerjee


Radiopharmaceuticals Division, 1 Radiation Biology and Health Science Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India

Objective: The aim of the study was to tune the pharmacokinetics of 2-nitroimidazole- 99m Tc(CO) 3 complex for detecting tumor hypoxia by modulating its lipophilicity using different bi-functional chelators (BFCA). Materials and Methods: Four 2-nitroimidazole- 99m Tc(CO) 3 complexes of different lipophilicities were synthesized and characterized. Corresponding rhenium analogues were also prepared at macroscopic level for structural elucidation. Results: Three 2-nitroimidazole complexes, each having different BFCA, viz. iminodiacetic acid (IDA), diethylene triamine (DETA) and aminoethyl glycine (AEG) were initially evaluated in fibrosarcoma tumor bearing Swiss mice. It was observed that tumor uptake of the complex is strongly dependent on the blood clearance of the radiotracer. The DETA and AEG complexes cleared very fast from blood and hence showed lower tumor uptake. The IDA complex showed good tumor uptake owing to its slow blood clearance, but resulted in poor target to non-target ratio. A fourth complex, a 2-nitroimidazole-BPA- 99m Tc(CO) 3 complex (BPA-bispicolamine), envisaged and synthesized to have a blood clearance pattern intermediate of that of IDA and DETA complex. As expected, upon evaluation in tumor-bearing animals, 2-nitroimidazole-BPA- 99m Tc(CO) 3 complex showed improvement in tumor uptake as well as target to non-target ratio. Conclusions: This study demonstrates that while designing agents for detecting hypoxia, modulation of in vivo pharmacokinetics of the radiopharmaceuticals can be achieved through careful tailoring of the ligand structure.

RP-08

Formulation and evaluation of freeze-dried kits of DOTA-TATE for the preparation of therapeutic dose of 177 Lu-DOTA-TATE for human administration

Tapas Das, Sharmila Banerjee, Ajit Shinto 1 , Koramadai Karuppusamy Kamaleshwaran 1 , Haladhar Dev Sarma 2

Isotope Applications and Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Trombay, 2 Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai, Maharashtra, 1 Department of Nuclear Medicine and PET, Kovai Medical Center and Hospital, Coimbatore, Tamil Nadu, India

Objective: The aim of the present work is to develop freeze-dried DOTA-TATE kits, which will enable the preparation of therapeutic dose of 177 Lu-DOTA-TATE at the hospital radiopharmacy, in a simple and convenient way. This kit is intended to be used for formulating the radiopharmaceutical prior to administration in patients suffering from neuroendocrine cancers with over-expression of somatostatin receptors. Materials and Methods: Several batches of freeze-dried DOTA-TATE kits, each comprising a lyophilized mixture of 200 μg of DOTA-TATE, 80 mg of gentisic acid and 13.9 mg of ammonium acetate, were prepared. For the preparation of 177 Lu-DOTA-TATE patient dose, the lyophilized powder of the kit vial was dissolved in water for injection and incubated with 177 LuCl 3 at 100°C for 45 min. Quality control studies were done by paper chromatography and HPLC. The pharmacokinetic behavior of the agent was studied by carrying out biodistribution studies in normal Wistar rats. Preliminary clinical evaluation of the kit was carried out by administering the preparation in patients suffering from neuroendocrine cancers. Results: The freeze-dried kits were used for the preparation of upto 200 mCi (7.4 GBq) therapeutic dose of 177 Lu-DOTA-TATE with a radiochemical purity of >99%. The developed kits have sufficiently long shelf-life (9 months) when stored at -4°C. Biological studies carried out in normal Wistar rats exhibited no significant accumulation of activity in any of the vital organs/tissue except in kidneys and non-accumulated activity showed major renal clearance. Clinical studies carried out in cancer patients exhibited accumulation of activity in the cancerous lesions and metastatic sites. Conclusion: The kit can be conveniently used at the hospital radiopharmacy for the preparation of therapeutic doses of 177 Lu-DOTA-TATE, suitable for human administration. The use of kit is expected to reduce the batch failure as well as radiation exposure to the working personnel.

RP-09

Microwave assisted synthesis of 131 I labeled phenyl acetic acid

Ravi Seshan, Kiran S Mehra, Richa Tiwari, Anand Gaurav, A Thulasidhasan, SS Sachdev


Radiopharmaceuticals Programme, Board of Radiation and Isotope Technology, BARC Vashi Complex, Navi Mumbai, Maharashtra, India

Objective: An antiproliferative and anti-differentiating effect of phenylacetate in hematological malignancies and in solid tumors has been reported. As the structure of 4-iodophenylacetic is close to that of phenylacetic acid, it is hypothesized that useful information on the biodistribution of the 131 I labelled iodophenyl acetic acid can be obtained from experimental animals, chiefly with regard to its uptake in neoplastic versus normal tissue. Most of the nucleophilic exchange reactions use high reaction temperature and long heating time. Under these reaction conditions, both radiochemical purity and labeling efficiency are reduced. The Cu + assisted nucleophilic exchange reaction is the mildest reaction condition for radioiodination of molecules. In our study we have used these reaction conditions to prepare 131 I labelled 4-iodophenylacetic acid. Microwave heating has been used to accelerate chemical reactions. Materials and Methods: Phenylacetic acid was iodinated with iodine in presence of a mixture of acetic acid and concentrated sulfuric acid which yielded an isomeric mixture. Separation of isomeric mixture was done by silica gel column chromatography which afforded pure 4-iodophenylacetic acid. The product was identified by TLC (silica gel, 10% methanol in chloroform v/v) and melting point. 4-iodophenylacetic acid was labeled with excess (1:2) of 131 I via nucleophilic isotopic exchange in presence of Cu + and heated using a microwave synthesizer for 3-4 min. Conclusion: The labeling efficiency was >97% and the radiochemical purity was >98%. Due to the shorter heating time, the microwave heating method had significantly reduced the radiochemical and chemical impurities.

RP-10

In vitro evaluation of 99m Tc-Ubiquicidin prepared using an indigenous kit

Chanda Arjun, Archana Mukherjee 1 , Grace Samuel, Meera Venkatesh 2

Quality Control Programme, Board of Radiation and Isotope Technology, 1 Isotope Application and Radiopharmaceuticals Division, Trombay, Mumbai, Maharashtra, India, 2 Division of Physical and Chemical Sciences, International Atomic Energy Agency, Vienna, Austria

Objective: To evaluate an indigenously formulated two component UBI kit for its physicochemical properties and bacterial binding properties. Ubiquicidin (UBI), a synthetic antimicrobial peptide, has been in recent times proposed as a 99m Tc-labelled tracer to distinguish bacterial infections from sterile inflammatory processes and to monitor the efficacy of antibiotic therapy. Materials and Methods: A two component UBI kit was prepared and lyophilized under optimum conditions. 99m Tc-UBI could be prepared by the addition of 1 mL of 99m TcO 4 - to the vial containing NaOH and the entire content transferred to the vial containing UBI. The final pH was 7.0-8.0. The complex was characterised for radiochemical purity using a C-18 Sep-Pak mini cartridge, reverse phase HPLC and ITLC. Serum stability and cysteine challenge studies were carried out to test the stability of the radiolabeled preparation. Bacterial binding studies were performed using 10 6 to 10 8 S. aureus (ATCC 25923) to test in vitro binding ability. Results: The radiochemical purity of the kit was found to be >95% as determined by the various techniques. Serum stability and cysteine challenge studies showed that the kit was stable to up to 4 h after preparation. Bacterial binding using 10 8 , 10 7 and 10 6 cells with 5 × 10 4 cpm of 99m Tc-UBI was found to be 37.58 ± 1.55%, 11.68 ± 1.20%, and 4.88 ± 0.27% respectively. In case of 10 8 cells, pre-incubation with 100-fold excess of cold UBI decreased the average binding of 99m Tc-UBI from 37.58 ± 1.55% to 14.83 ± 0.48% indicating that the 99m Tc-UBI binds to the bacteria specifically. Conclusion: The above data shows the potential of 99m Tc-UBI as an infection-imaging agent.

RP-11

Comparative study of biodistribution of PEG-modified radioiodinated poly (n-isopropyl acrylamide) with that of non-pegylated based thermoresponsive polymer for localized radiotherapy

Tarveen Karir, Haladhar Dev Sarma 1 , Puthusserical Abdul Rahman Hassan 2 , Pradeep Chaudhari 3 , Sandeepto Ghosh 3 , Dharmarajan Padmanabhan, Grace Samuel


Quality Control Programme, Board of Radiation and Isotope Technology, DAE, Vashi, 1 Radiation Biology and Health Sciences Division, 2 Chemistry Division, BARC, 3 Comparative Oncology and Small Animal Imaging Facility, ACTREC, TMC, Navi Mumbai, Maharashtra, India

Objective: PEGylation is one of several approaches that have been successfully applied to large molecules to improve their pharmacokinetics. In this study, we report the improved effect on the retention of the radiolabeled thermoresponsive polymer on intratumoral injection in Swiss mice by way of self-assembly of PEGylated- 125 I-Poly (N- Isopropyl acrylamide) (PNIPAM) polymer instead of chemical activation. This was done essentially to retain the retention property of the thermoresponsive polymer and enhance the radiotherapeutic effect of the radioiodinated polymer. Materials and Methods: Two groups of Swiss mice (6-8 weeks age) (n = 3) for each time point were induced fibrosarcoma tumor by injecting ~10 6 tumor cells in 150 μL sterile normal saline into the left lateral thigh muscles. The animals with tumor of ~1 cm diameter were selected for the study. Biodistribution as well as excretion patterns obtained by PEGylated- 125 I-PNIPAM polymer at 2 h, 1, 4, and 7 days post injection (p.i.) (n = 3) were compared with those obtained by non-PEGylated- 125 I-PNIPAM polymer. To ensure the in-vivo retention of PEGylated- 125 I-PNIPAM polymer, another set of Swiss mice (n = 3) bearing fibrosarcoma tumor were injected with PEGylated polymer intratumorally at multiple sites for obtaining planar images at 1 h, 3 h, 1, 3 and 5 days p.i. using preclinical small animal imaging gamma scanner. Results: The biodistribution of PEG modified radioiodinated polymer was found to be increased within the tumor (44 ± 1.23%) (n = 3) as compared to that of non-PEGylated- 125 I-PNIPAM polymer (32 ± 2.18%) (n = 3) at 2 h p.i. decreasing slowly to 10 ± 1.39% of the injected activity within 5 days p.i. No substantial organ-specific accumulation of the radioactivity released from the injected site, i.e. the tumor was observed. Most of the radioiodinated polymer was excreted through urine as compared to the non-PEGylated- 125 I-PNIPAM polymer. Planar imaging of mice bearing fibrosarcoma tumor (n = 3) when injected with PEGylated- 125 I-tyrosinamide PNIPAM intratumorally (~150 μCi) showed good retention at the injected site up to 5 days. Conclusion: The results indicate that PEGylation of the radioiodinated thermoresponsive PNIPAM polymer showed a considerable promise as an agent of choice for localized tumor radiotherapy.

RP-12

Rapid paper chromatography procedure for analysis of 99m Tc-DTPA

Sangeeta Haresh Joshi, Pramod Dodke, Saraswathy Padmanabhan, Grace Samuel


Quality Control Programme, Board of Radiation and Isotope Technology, DAE, Vashi, Navi Mumbai, Maharashtra, India

Objective: Radiochemical purity (RCP) is indicative of product efficacy, in quality control (QC) of radiopharmaceuticals. 99m Tc-labeled compounds are formulated in hospital radio pharmacy, using pre-certified cold kit and 99m Tc from 99 Mo - 99m Tc generators. QC analysis of final formulations is mandatory and needs to be carried out quickly at the hospital radiopharmacy prior to administration in patients. USP (2007) recommends electrophoresis (phosphate buffer, pH 6.8) whereas BP (2009) recommends TLC for RCP determination of 99m Tc-DTPA. At BRIT, existing practice uses paper chromatography in 85% methanol (RPC approved). This system is unable to detect the presence of reduced hydrolyzed technetium (RHT) in preparations. Hence, to improve the QC procedure for RCP analysis so as to accommodate the detection of colloidal impurities, short strip method akin to the BP TLC procedure was standardized. Materials and Methods: Seven batches of 99m Tc-DTPA were formulated and RCP was evaluated using new method developed at BRIT using both long (3 cm × 21 cm) and short strips and compared with RPC approved procedure, and BP method. The short strip paper chromatography was carried out using Whatman paper no. 3, 1 cm ×10 cm strips in duplicate and developed in two different solvents (MEK and Saline). Chromatogram was developed for 5 to 8 min. The distribution of radioactivity in the chromatogram was measured using radiochromatogram scanner. Pertechnetate impurity is detected in MEK (Rf 1) while RHT impurity is detected in saline (Rf 0). The % RCP is calculated by subtracting the sum of both impurities from 100. Results: RCP was in the range of 97-99 % in all the batches and showed excellent correlation to BP procedure with a variation of ±0.8%. Conclusion: The new method developed uses the easily available and inexpensive Whatman paper no. 3, shows excellent correlation to BP procedure, is superior to existing method in its ability to detect RHT and is hence recommended for adoption in a hospital radiopharmacy.

RP-13

Extension of shelf life of ethyl cysteinate dimer (TCK-42) kit of BRIT

Neelam Pilkhwal, Preeti Nair, Ravi Vanaja, Padmanabhan Saraswathy, Grace Samuel, Satbir Singh Sachdev


Quality Control Programme, Board of Radiation and Isotope Technology, BRIT-BARC Vashi Complex, Navi Mumbai, Maharashtra, India

Objective: BRIT has been supplying Kit for 99m Tc-Ethyl cysteinate dimer (ECD) injection (TCK-42), used for brain perfusion imaging, the shelf life of which is 4 months. The short useful shelf life of the kit necessitates frequent production and analysis of the product as well as inconvenience to users, in terms of having to place frequent orders for the kit and/or having to waste the vials which could not be used owing to its short shelf life. In order to address these problems it was decided to explore the possibility of increasing the shelf life of this kit, by carrying out physicochemical tests for six consecutive batches of the product for a period of 12 months. Materials and Methods : The tests were carried out at periodic intervals of 4, 6, 8, 10 and 12 months from date of production. ECD kit was labeled with upto 170 mCi of 99m Tc-Sodium Pertechnetate, derived from Geltech as well as Coltech Generator of BRIT. The parameters studied were appearance, pH and radiochemical purity (RCP) of the labeled product as per the RPC monograph. Appearance of reconstituted injection was observed and pH was tested with universal indicator pH paper. Radiochemical purity test was determined by ascending paper chromatography using Whatman 1 chromatography paper as support, 100% methanol as solvent and developing the chromatogram upto a solvent front of 15 cm. The distribution of radioactivity in the chromatogram was measured using a radiochromatogram scanner. Results: It was observed that the appearance of the reconstituted injections of all batches of the product at the various time intervals studied, was clear and colorless, pH was between 6 and 8, and RCP was above 90% for upto 12 months, indicating that the kit is stable upto 12 months. Conclusion: The shelf life of the kit can be extended to 10 months, after necessary regulatory approval.

RP-14

Studies on the effect of storage on disintegration of NA 131 I therapeutic capsules with high radioactivity

Richa Tiwari, Anand Gaurav, A Thulasidhasan, Kiran S Mehra, Ravi Seshan, SS Sachdev


Radiopharmaceuticals Programme, Board of Radiation and Isotope Technology, BARC Vashi Complex, Navi Mumbai, Maharashtra, India

Objective: This investigation was conducted to determine the effect of storage on disintegration of therapeutic capsules. {131 I}-Sodium iodide (capsule/solution form) has been the drug of choice for both diagnosis and therapy of thyroid cancer and hyperthyroid. As on date in our country therapeutic high doses of 131 I as Na 131 I, are administered through oral route as solution (liquid dosage form). Therapeutic capsule formulations provide an enhanced safety profile for nuclear medicine professionals and patients. Oral solutions raise the risk for spills during administration in addition to vomiting episodes by the patients creating large areas of high level radiation contamination. Pharmaceutical advantages of capsule over oral solution are accurate dose delivery, easy to swallow and masks the unpleasant taste and odor. Materials and Methods: Therapeutic doses of Na 131 I for oral administration in capsule form are being prepared and supplied by BRIT. These are made as 'capsule in capsule' which involves encapsulating a smaller capsule (of size "1") containing 131 I activity dispensed on a solid support (anhydrous sodium sulphate), with a larger size (size "0") capsule. Preparation of high doses capsules up to 5.55 GBq (150 mCi) of iodine-131 at the time of calibration are being planned by BRIT. These capsules generally have a shelf life of 30 days from the date of production. Conclusion: Various doses of capsule were prepared and assessed for disintegration time at different time intervals till the expiry date. It was observed that there is no significant change in the disintegration time on storage.

RP-15

Evaluation of 153 Sm-EDTMP injection suitable for clincal use at wider PH conditions

Yojana Singh, R Krishna Mohan, C Vrinda, G Prabhakar, Bikash Tiwari, SS Sachdev


Board of Radiation and Isotope Technology, BRIT-BARC, Vashi Complex, Navi Mumbai, Maharashtra, India

Objective: 153 Sm-EDTMP ( 153 Sm-Ethylene diaminetetramethylene phosphate) is a potential therapeutic radiopharmaceutical for palliative treatment. To obviate problems relating to the pH and have wider pH range (5-9), systematic radiochemical and biological studies were carried out to establish the stability of the final product for its clinical use. Standard operating protocol (SOP) for the formulation of 153 Sm-EDTMP involves reaction of radioactive 153 SmCl 3 ( 0.1 N HCl, pH 3-4) with alkaline solution of EDTMP ligand. This final formulation is suitably diluted to get final pH of 7-8, (narrow range). However, any deviation in pH of 153 SmCl 3 raw material used, results in non compliance of pH of the final product formulation. The adjustment of pH (7-8) during the production stage in hot cell facility results in additional dose (large batch size of 5-6 Ci) to the operating technicians. Materials and Methods: 153 Sm-EDTMP formulations at different pH ranges i.e. 5-6 and 8-9 were prepared using 0.05M HCl and 0.1 M NaOH, to obtain the desired complexes. Stability of the complexes was evaluated for radiochemical purity (RCP) by paper chromatography (PC) at different time points and post expiry date. Plans are in progress to carry out biodistribution (BD) studies in animals to ascertain the efficacy of formulations for aforementioned application. Results: Radiochemical purity (RCP) of the formulations at varying pH was not less than 95% throughout the stability study period of 7 days, post formulation. Conclusion: The known impurity present in 153 Sm-EDTMP radiopharmaceutical is 153 Sm colloid which in PC remains at point of spotting. Since the level of activity observed at the point of spotting for all formulations was within the permissible limits, hence studies established clinical utility of 153 Sm-EDTMP radiopharmaceutical over a wide range of pH (5-9). This, in turn allows the " 153 Sm" raw-material with wider pH fluctuations at the production site, for chelating with EDTMP.

RP-16

Performance evaluation of alumina sorbent based high activity 99 M0 o - 990 m T0 c 0 column (COLTECH) 0 generator in BRIT

Abhishek Sharma, TR Rajeswari, G Shunmugam, SK Sarkar, P Kumar, SS Sachdev


Board of Radiation and Isotope Technology, BARC Vashi Complex, Navi Mumbai, Mumbai, Maharashtra, India

Objective: Feasibility studies on production and supply of alumina based 99 Mo- 99m Tc generator (COLTECH) loaded with high activity of (n, f) 99 Mo for use in radio-pharmacies. BRIT has been supplying alumina column based 99 Mo- 99m Tc generators (COLTECH) to many nuclear medicine centres across India to avail 99m Tc for various radiopharmaceuticals formulations. The current weekly production and supply of COLTECH generators consists of 11.1 GBq (300 mCi), 18.5 GBq (500 mCi) and 22.2 GBq (600 mCi) generators calibrated for Monday. COLTECH generators supplied by BRIT have become very popular and are in high demand to customers. Thus there was a need to produce high activity 37 GBq (1000 mCi) COLTECH generators, study of the dose rate for supply and evaluate its performance to suit the need for radiopharmacies. Materials and Methods: High specific activity fission produced 99 Mo (10 3 -10 4 Ci/g of Mo) solution was obtained from IRE, Belgium and 'JSC, State Scientific Centre, Russia. It was diluted with 0.9% saline solution followed by the pH adjustment (pH 3-3.5) using dilute HNO 3 . Radioactive concentration (RAC) of diluted 99 Mo solution was assayed by measuring an aliquot of 100 μl using a dose calibrator inside the plant. 99 Mo solution containing 99 Mo activity of 1.3 Ci (48.1 GBq) to 2.5 Ci (92.5 GBq) on production day was loaded on to glass column (containing ~2 g of acidic alumina) followed by washing with 10-15 ml 0.9% NaCl solution under aseptic environment inside column generator production facility. The column was then assembled in a generator assembly using sterile needles, sterile air and solution filters etc. The same stock 99 Mo solution was used for production and supply of Coltech generators for customers. The generators were eluted with 0.9% NaCl solution after allowing growth of 99m Tc activity. Performance of the generators were evaluated in terms of 99m Tc yield, 99m Tc elution profile, 99 Mo breakthrough and others quality features of 99m Tc eluate. Results: Five high activity COLTECH generators (~1 Ci, 37 GBq) on calibration day were prepared in different batches and their performance evaluated. The average elution efficiency was observed to be 87% ±3% and 99 Mo breakthrough was found to be less than 8.8% ×10 -4 %. 99m Tc activity (upto 500 mCi) obtained from these generator were labelled with DTPA cold kit and labelling efficiency found to be satisfactory (>90%) which complies with RPC monograph. Dose rate on the outer package (TYPE A Package) of the generator were 50-60 mR/h on side of the package and 80-90 mR/h on the top of the package containing ~1.5 Ci 99 Mo activity which are far below permissible limit. Conclusion: High activity COLTECH generators (1 Ci) were produced, evaluated and feasibility to supply to users demonstrated.

RP-17

Feasibility studies on high dose labeling of BRIT's 0 kit for the preparation of 990 m T0 c -0 diethylenetriaminepentaacetic acid 0 injection

Archana Sunil Ghodke, Vanaja Ravi, Doddathota Krishnaiah Ranganatha, Satbir Singh Sachdev


Board of Radiation and Isotope Technology, BRIT-BARC Vashi Complex, Navi Mumbai, Maharashtra, India

Objective: Presently, kit for the preparation of 99m Tc- DTPA injection a single component kit supplied by BRIT contains 35 mg of DTPA with 2 mg of SnCl 2 . 2H 2 O in freeze dried form. Generally 50 mCi of Na 99m TcO 4 is used for the preparation of 99m Tc-DTPA injection. Many of the other commercial DTPA kit manufacturer claim that their kit can be used for labeling with much higher 99m Tc activities (upto 500 mCi) per kit vial. We have taken up this work to study the effect of labeling with higher activity (400-500 mCi/kit vial) on 99m Tc-DTPA kit supplied by BRIT. These preparations were evaluated for its radiochemical purity, stability and biodistribution (BD) studies in mice to ascertain the clinical utility of the formulation for renal studies. Materials and Methods: Several batches of 99m Tc-DTPA injection with higher 99m Tc activity (400-500 mCi) were prepared and tested for all quality control parameters. The radiochemical purity (RCP) was determined at 10 min, 4 h and 24 h post labeling in dual solvent system (85% methanol and saline) using paper chromatography technique. Also BD studies were done in mice to ascertain the products compliance with RPC monograph. Results: The radiochemical purity (RCP) of 99m Tc-DTPA injection prepared (with activity in the range of 400-500 mCi) was not less than 93%, indicating excellent radiochemical integrity of the complex throughout the study period up to 24 h post formulation. BD values in mice are satisfactory. The labeled product complies with all RPC specified parameters. Conclusion: Our studies established, single component DTPA kit supplied by BRIT is suitable for the preparation of 99m Tc-DTPA injection, using up to 500 mCi of 99m Tc for dynamic kidney imaging studies.

RP-18

Preliminary clinical investigations of 177 Lu-labelled Ha particles prepared using ready-to-use cold kits in the treatment of rheumatoid arthritis

Sudipta Chakraborty, Ajit Shinto 1 , KV Vimalnath, A Rajeswari, ER Radhakrishnan1, Ashutosh Dash


Isotope Application and Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Trombay, Mumbai, Maharashtra, 1 Nuclear Medicine and PET Services, Kovai Medical Centre and Hospital, Coimbatore, Tamil Nadu, India

Objective: Radiation synovectomy, which involves local intraarticular injection of suitable β-emitting radionuclides in the form of radiocolloids or radiolabeled particulates into the affected synovial joints, has been successfully employed in the treatment of arthritis of different types. Hydroxyapatite (HA) Ca 10 (PO4) 6 (OH) 2 is one of the preferred particulates for effective management of synovial inflammation owing to its excellent biocompatibility. The present paper describes the first clinical evaluation of 177 Lu-labeled hydroxyapatite (HA) particles of 1-10 μ size prepared at hospital radiopharmacy using ready-to-usecold kits. Materials and Methods: Hydroxyapatite particles (particle size range 1-10 μ) were synthesized and characterized by following the reported procedure. Cold kits (20 no.) were prepared by through mixing 5 mg of HA particles with a suitable buffering agent. For radiolabeling, 1 mL of normal saline was added to the kit vials followed by addition of ~740 GBq (10 mCi) of 177 Lu activity as 177 LuCl 3 solution. Reaction mixtures were incubated at room temperature for 30 min (pH 7-8) after thorough mixing using a vortex mixture. The prepared dose was injected into the synovial cavity of the knee joints of patients suffering from chronic rhematoid arthritis after quality control studies. Results: 177 Lu-labeled HA particles were prepared in excellent radiochemical purity (99.2 ± 0.3%) at the hospital radiopharmacy using the HA kit vials. The scintigraphic images of the patients recorded after administration of the radiolabled particulates showed near-complete retention of the activity in the synovium even after 30 d post-injection. The patients undergone treatment with 177 Lu-HA had reported significant simptomatic relief such as, reduced pain and increased joint mobility. Conclusion: The preliminary clinical investigations carried out with 177 Lu-HA indicate its potential for use as a viable and cost-effective agent for radiation synovectomy.

RP-19

Calculating specific activity of (n,
g) produced 177 Lu - is it simply 'activity produced divided by mass of the target irradiated'?

Vimalnath Nair, Sudipta Chakraborty, Ashutosh Dash


Isotope Application and Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Trombay, Mumbai, Maharashtra, India

Objective: Owing to its ideally suited decay properties for in vivo targeted therapy and favourable production logistics, 177 Lu {T½ =6.65 d; Eβ(max) = 497 keV (78.6%), 385 keV (9.1%), 176 keV (12.2%); Eγ = 208 keV (11.0%), 113 keV (6.4%)] has gadually emerged as one of the pivotal radionuclide for therapeutic nuclear medicine during the last decade. 177 Lu-labeled DOTA 0 -Tyr 3 -Octreotate (DOTA-TATE) has been very effective in peptide receptor radionuclide therapy (PRRT). A careful analysis of literature reveals that in order to achieve the desired therapeutic efficacy, the formulation of clinical dose of 177 Lu-DOTA-TATE requires 177 Lu to be available with a specific activity of at least 740 GBq/mg (20 Ci/mg). The present paper gives a theoretical analysis of determination of specific activity of 177 Lu produced via 176 Lu (n,γ) 177 Lu route. Materials and Methods: Since the neutron capture cross section of 176 Lu does not follow 1/v law, the yield of 177 Lu 'A' (in BQ) produced via 176 Lu (n,γ) 177 Lu at the end of irradiation is calculated using the formula



where, the symbols used have their usual significances. The specific activity (S) is calculated from the total activity (A) of 177 Lu using the equation



where, mo is the initial mass of lutetium in the target irradiated. However, radioactive decay of 177 Lu during the irradiation of 176 Lu target in the reactor leads to the formation of 177/178 Hf in a significant extent, thereby reducing the mass of lutetium. As a result, the actual specific activity of 177 Lu is significantly different, since the actual mass of lutetium present in the system post irradiation is different from the initial mass of the target irradiated. Using the burn-up correction, the actual specific activity S (Bq/mol) of 177 Lu can be expressed as



where, n 0 is the number of moles of the target isotope 176 Lu at the start of irradiation and n is the number of moles of other lutetium isotopes which do not lead to the formation of 177 Lu by (n,γ) process. Results: The theoritically calculated value of the actual or avavilable specific activity of 177 Lu after 21 d irradiation of enriched Lu target (82% 176 Lu) at thermal neutron flux of 1.2 × 10 14 n/cm 2 /s (30.87 mCi/μg, using k = 2.5) is significantly higher to that obtained without taking into account the transformation of 176 Lu target during irradiation (18.90 mCi/μg, using k = 2.5). Conclusion: Owing to the very high thermal neutron capture cross-section (σ =2090 b) of 176 Lu and the presence of strong resonance near thermal region, production of 177 Lu produced via 176 Lu (n,γ) 177 Lu route has some unique features. Unlike other (n,γ) produced medical radionuclides, the specific activity of 177 Lu can not be calculated by simply dividing the activity produced by mass of the target irradiated.

RP-20

Synthesis of novel DNA intercalating quinazoline analogue as tumor imaging agent for SPECT

Swati Aggarwal 1,2 , Anjani K Tiwari 1 , Gurmeet Singh 2 , Anil K Mishra 1


1
Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, 2 Department of Chemistry, University of Delhi, New Delhi, India

Objective: The synthesis of specific imaging agents is an important aspect in radiopharmaceutical development which improves the imaging of tumor tissues in different animal models and human. One of the vital characteristic for designing an agent for tumor, for planar molecules is the stimulation of the production of double stranded DNA breaks, hence, the intercalation property is significant requirement for the antitumor activity. The current study examines the DNA intercalative abilities of synthesised molecule through molecular docking and spectroscopic studies. Subsequently, in vitro and in vivo studies were performed with 99m Tc labelled molecule to check the efficacy of compound in tumor bearing mice. Materials and Methods: Structure base docking method was performed using X-ray crystal structure of duplex DNA from the Glide module of Schrodinger software. The synthesised compound was identified by NMR and mass spectroscopic techniques. Physico-chemical measurements were used to validate the intercalation property of the screened molecules. Biodistribution, blood kinetics and SPECT imaging was done of the 99m Tc radiolabeled molecule. Results: Molecular docking results led to the screening of molecules showing intercalation with the duplex DNA. The intercalation behaviour which is an important criterion for antitumor activity was further justified using absorbance, fluorescence and circular dichroism techniques done with CT-DNA. The complex formed with 99m Tc was found to be stable over a period of 24 h with high radiolabeling efficiency (98%). The bio-distribution studies and scintigraphic imaging show specific distribution pattern at the tumor site with excretion through hepatic and renal route. Conclusion: The preliminary studies show good results for this molecule as tumor imaging agent in specific animal model. Moreover the mechanism for mode of action for this compound is validated through computational and physico-chemical studies. Further longitudinal studies in other animal model may lead this compound for human clinical applications.

RP-21

Fully automated one-pot synthesis of 18F-Fluroethyl tyrosine using SPE method

N Lakshminarayanan, MGR Rajan


Radiation Medicine Center, Biomedical Group, Bhabha Atomic Research Centre, Mumbai Maharashtra, India

Objective: O-(2-{18F}Fluoroethyl)-L-tyrosine (18F-FET) is an aminoacid based PET radiotracer labeled with Fluorine-18 used for brain imaging. Our aim is to synthesize 18F-FET using 2-(O-tosyloxyethyl)-N-trityl-L-tyrosine-tert-butylester (TET) precursor and to develop procedure for production including purification and automation. Materials and Methods: Chemicals were procured from sigma. SPE resin was obtained from ABX, Germany. 18F-Fluoride was produced in GE-PETrace cyclotron. Radio-HPLC was done with Knauer HPLC-system using C-18 column (250 mm ×4 mm, 5 μm), UV-254 nm, solvent-2% ethanol. Radiochemistry was done in modified GE-TracerLab system (configured for {18F}FDG). Synthesis of 18F-FET was done by reaction of {18F}TBAF with TET in dry acetonitrile, followed by hydrolysis with trifluoroaceticacid/dichloromethane (1:3). Purification was done by loading reaction mixture onto H-RP and PS-HCO3 resins. The column was washed with water (20 mL) and 18F-FET was eluted with 30% ethanol. 18F-FET was analyzed using radio-TLC and HPLC with reference standard. Results: The fluorination was observed to be 90% ±2% (n = 5). The synthesis time including purification was 60 min. Reliably 33% ±3% (n = 5) (decay-uncorrected) yield of 18F-FET was obtained with synthesis time of 60 min. The synthesis was tested with varying level of radio-activity (3.7-20 GBq). 18F-FET was found to be apyrogenic, sterile with pH 6-7 and RCP >98%. The procedure can be adopted in a 18F-FDG module. 18F-FET was found to get eluted at 32 min in radio-HPLC and showed absence of chemical and radiochemical impurities. Conclusion: 18F-FET was synthesized successfully using TET-precursor. The labelling efficiency was 90%. A SPE procedure was developed for purification of the reaction mixture. The radiochemical yield was 33% (decay-uncorrected) and the radiochemical purity was >98%. The final product was found to be apyrogenic and sterile with pH 6-7. The whole procedure can be automated in a simple 18F-FDG module.

RP-22

Synthesis of Ni(II) complex based precursor for {18F}Fluoroethyltyrosine

N Lakshminarayanan, MGR Rajan


Radiation Medicine Centre, Biomedical Group, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India

Objective: The most preferable precursor in Fluorine-18 radiochemistry would be the one which can be adopted by a one-pot method, simpler approach and minimum sophistication for radio-synthesis. Based 18F]Fluroethyltyrosine {18F}FET can be synthesized starting with Ni(II)-(S)-BPB-Ty-CH2-CH2-OTs complex as precursor. The aim is to develop a simplified method for the synthesis of the {18F}FET precursor based on Ni(II)-(S)-BPB-Ty complex. Materials and Methods: Chemicals were procured from sigma. Radio-HPLC was done with spectra physics system using C-18 column (250 mm × 4 mm, 5 μm), UV-254 nm. {18F}Fluorine was produced in GE PETtrace medical cyclotron. Radiochemistry was done in modified GE-TracerLab system (configured for {18F}FDG). The precursor was synthesized in two steps. In the first step Ni-(S)-BPB-Tyr was synthesized by the reaction of (DL)tyrosine with (S)-BPB and Ni(NO3)2·6H2O in the presence of sodium methoxide in dry methanol. In the second step the above complex was reacted with ethyleneglycol ditosylate in the presence of 50% KOH to get Ni-(S)-BPB-Tyr-CH2-CH2-OTs precursor. Results: The precursor synthesis procedure gives a overall yield of 75% with 98% purity. The total process involves two steps. The synthesized precursor was characterized with 1H-NMR and 13C-NMR. 18F]FET was synthesized using the above precursor and the final product mixture was characterized by radio-TLC. The labeling efficiency was found to be 60%. Hydrolysis of the radio-labelled precursor with hydrochloric acid (0.5 M) was found to be 85% efficient leaving 15% of unhydrolyzed impurities. Conclusion: We have successfully synthesized Ni(II)-(S)-BPB-Tyr-CH2-CH2-OTs precursor. The final product and the intermediate were characterized by spectroscopy. The overall yield of the precursor was evaluated to be 75%. The precursor was labeled with Fluorine-18 and hydrolyzed to get final 18F-FET. The labeling efficiency was evaluated to be 60%.

RP-23

Radiosynthesis of 68 Ga-DOTA-ciprofloxacin for PET imaging of bacterial infections

Drishty Satpati, Sharmila Banerjee


Isotope Applications and Radiopharmaceuticals Division, Bhabha Atomic Research Center, Mumbai, India

Objective: Ciprofloxacin is a broad spectrum fluoroquinolone antibiotic that is active against gram-positive and gram-negative bacterial infections. The interaction of ciprofloxacin with bacterial DNA gyrase and DNA topoisomerase IV is the major mechanism leading to inhibition of DNA synthesis. Here we report the radiosynthesis of 68 Ga-DOTA-ciprofloxacin for PET imaging of infections. Materials and Methods: Ciprofloxacin was modified at C7 position so as to introduce the amine functionality suitable for conjugation with the bi-functional chelator, p-SCN-Bz-DOTA for radiolabeling with 68 Ga. 68 Ga was eluted from 68 Ge/ 68 Ga generator (Ithemba Labs, South Africa) with 5 mL of 0.1 N HCl, and the eluate was loaded onto a cation exchange column. 68 Ga was eluted with 500 μL mixture of acetone/HCl (97.6%/0.02 N). For radiolabeling, 68 Ga (37 MBq) eluted from 68 Ge/ 68 Ga generator was added to ciprofloxacin-DOTA conjugate (CP-DOTA) (100 μg) dissolved in water: Sodium acetate buffer (0.2 M, pH 4) (1:1, v/v) and incubated at 70°C for 15 min. The radiochemical yield of 68 Ga-DOTA-ciprofloxacin was determined using HPLC. Results: Ciprofloxacin-DOTA conjugate (CP-DOTA) was synthesized in a three-step reaction. Ciprofloxacin was first reacted with N-Boc-3-bromopropylamine which was then hydrolyzed with trifluoroacetic acid. The amine functionality thus introduced was then conjugated with p-SCN-Bz-DOTA. The product obtained was purified using semi-preparative HPLC, lyophilized and characterized by 1 H-NMR. 68 Ga-CP-DOTA was obtained in >95% radiochemical yield as determined by HPLC (retention times, 68 Ga-CP-DOTA: 16.2 min; 68 GaCl 3: 3.8 min). Radio-synthesis including elution, eluate purification and labeling could be completed within 30 min. The radiotracer was found to be stable in vitro for 3 h, as observed by no change in HPLC elution profile. Conclusion: Synthesis of CP-DOTA and radio-synthesis of 68 Ga-CP-DOTA was carried out efficiently in high yield. In vitro and in vivo biological evaluation studies for assessment of the prepared radiotracer as a potential bacteria-specific infection imaging agent is underway.

RP-24

Design, synthesis and biological evaluation of coumarin conjugated fused imidazo analogues as AChE imaging agent for SPECT

Pooja 1,2 , Vikas Kumar 1,3 , Anjani Kumar Tiwari 1 , Ramendra Pratap 2 , Krishna Chuttani 1 , Anil Kumar Mishra 1


1
Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, 2 Departments of Chemistry, 3 Zoology, University of Delhi, Delhi, India

Introduction: Alzheimer's disease (AD) is a progressive, degenerative disorder of the brain and is the most common form of dementia. AD is associated with a loss of cholinergic system with decreased levels of acetylcholine in the brain areas dealing with learning, memory, behavior and emotional responses. Loss of acetylcholine (ACh) in the brain is one of the reasons for AD, because of the inhibition by acetylcholinesterase (AChE) which is responsible for its hydrolysis and termination of active site. Coumarin are naturally occurring phytochemicals with wide range of biological activities, in this study imidazo fused heterocyclic system has been conjugated with coumarin and labeled with Technetium-99m ( 99m Tc) for their application as novel imaging agent for AChE. Materials and Methods: Two molecules were screened through computational methodology and their docking studies were performed for the two ligands named 3-(7-Hydroxy-benzo[d]imidazo {2, 1-b} thiazol-2-yl)-chromen-2-one (IBTOH) and 3-(8-Hydroxy-imidazo {1, 2-a} pyridin-2-yl)-chromen-2-one (IMPYOH). The computational studies were performed by modeling software, SCHRODINGERS with X-ray crystallographic protein AChE (PDB =1GQR). Identification of these two final products has been done by different spectroscopic techniques ( 1 H NMR, 13 C NMR and mass spectroscopy). The compound was labeled with 99m Tc by stannous chloride reduction method and analyzed in vitro and in vivo in mice. Ellman assay was performed to calculate IC 50 for AChE inhibition spectrophotometrically. Results: IBTOH and IMPY were labeled with 99m Tc to investigate their in-vivo radiopharmaceutical efficiency. Both these compounds forms stable complex having 96% and 97% labelling efficiency, with 98% radiochemical purity. In-vivo studies were carried out for 99m Tc- IBTO and 99m Tc- IMPYOH included blood kinetics showed a quick wash out from the circulation via hepatic and renal route and biodistribution revealed that maximum %ID/g was found in lungs at 2 h and scintigraphy images were taken in the healthy rabbit shows 3.35% and 3.36% brain uptake in specific regions for IBTO and IMPYOH respectively, with respect to whole body. AChE inhibition activity of IBTO and IMPYOH were assayed by the Ellmann's method and their IC 50 were calculated 5.01 μM and 31.62 μM respectively. Conclusion: Results indicate that IBTO is very good SPECT agent in comparison to IMPYOH as AChE imaging agent in brain. This may be due to appropriate lipophilicity of this compound which allows this to cross BBB. Further studies are required to prove the efficacy of these compounds in primates.

RP-25

Preparation of 131 I- lipiodol and fabrication of prototype semi-automated module

Aruna Korde, Archana Mukherjee, RN Ambade, Ghanashyam Shirke 1 , Rajendra Chavan 1 , A Dash, Grace Samuel


Isotope Application and Radiopharmaceuticals Division, BARC, Mumbai, 1 Texol Engineering Pvt. Ltd., Bavdhan, Pune, Maharashtra, India

Objective: Primary hepatocellular carcinoma (HCC) is the most common tumor with its highest incidence in the developing world. Lipiodol (Iodinated ester of poppy seed oil) exhibits longer retention in hepatoma compared to normal liver when injected directly into the hepatic artery. Intra-arterial injection of 131 I-Lipiodol delivers very high radiation dose to tumor with minimal damage to adjacent normal tissue making 131 I lipiodol an effective radio-therapeutic agent. The work was taken up with the aim to develop procedures and design and fabricate a semi-automated module suitable for synthesis of clinical grade 131 I lipiodol. Materials and Methods : 131 I lipiodol was prepared by isotopic exchange reaction of ionic 131 I and organic iodine. The radiolabeling parameters were standardized. The reaction was carried out by extracting 131 I in ethanol followed by iodine exchange in lipiodol. The effect of temperature was studied for labeling efficiency. Radiochemical evaluation was carried out using ITLC, using 85% methanol. The semi-automated synthesis module was designed based on optimized reaction parameters. The small sized module suitable to be placed in hot-cell or sufficiently shielded fume-hood is fabricated using reactant compatible solenoid valves and tubings. Results and Discussion: The radiolabeling yield was found to be dependent on drying conditions when ethanol was used as a solvent for exchange reaction. Consistent yield of around 60% was obtained under optimised conditions. The radiochemical purity of 131 I- Lipiodol was >95% as determined by ITLC. The product was stable for 1 week when stored in dark at ambient temperature. The fabricated semi-automated module will enable remote and precise liquid transfers as well as controlled heating with less than 5% variation. Pharmaceutically pure and safe product can be prepared from the module by terminal membrane filtration in sterile and pyrogen free product vial. Conclusion: 131 I-Lipiodol was successfully prepared in good yields and >95% purity with adequate stability. The indigenous fabrication of semi automated prototype module is an important contribution towards preparation of therapeutic radiopharmaceutical under GMP.

RP-26

{18 F}SIF-DPG site selective versatile synthon to prepare potential radioligands for targeting neuro-receptors with enhanced affinity

Puja Panwar Hazari, Jurgen Schulz 1 , Anil Kumar Mishra


Division of Cyclotron and Radiopharamceutical Sciences, Delhi, India, 1 University Bordeaux, INCIA, UMR 5287, F-33400 Talence, France

Objective: A new method for the fast production of "nonconventional" silicon containing precursor for 18 F-labeling of organic vectors is developed as a model to conjugate wide array of vector molecules. Approach envisaged is advent of attaching a suitable azide functionality linked to a bioactive molecule that reacts with the terminal SiH-dipropargyl glycerol prosthetic group in a site-selective manner within a time frame acceptable for PET-radiopharmaceutical production. Materials and Results: The stepwise synthesis of SiF conjugated building block involved a reaction of di-tert-butylchlorosilane with 4-bromophenol to yield 4-(di-tert-butylsilanyl) phenol. The silylated phenol was then treated with solketal to introduce glycerol scaffold in 63% yield. The resultant diol was obtained by deprotection step and treated with propargylbromide to render bivalent functionality for further conjugation. Rapid conjugation with the prosthetic group takes place in microwave assisted click conjugation under mild conditions.The optimum protocol for radiolabeling consisted of two parts. The extraction and entrapment of {18 F} - in the anion exchange resin (light quaternary methylamine cartridge) preconditioned with 0.5M K 2 CO 3 and water and eluted with a complex of Kryptofix 2.2.2. and K 2 CO 3 . After removing the solvents, the labelling synthon and glacial acetic acid (3 μL) in anhydrous DMSO (300 μL) was reacted with dried K222-K{18 F} at 80°C for 15 min. The formation of {18 F}SiF-conjugate was confirmed by radio-HPLC. In conclusion, the purpose of this study was to develop a valuable dual purpose prosthetic group which can be used in direct and indirect {18 F}-labeling methods to produce wide array of dimeric specific PET tracers. Thus, a bivalent homodimeric SiFA derivatized radioligand {18 F} BMPPSiF with enhanced affinity was developed using click chemistry approach that demonstrated preferential uptake in 5-HT1A receptor rich regions with positron emission tomography (PET).

RP-27

Design and synthesis of C-functionalized atridat to conjugate biomolecules for pre-targeted imaging: 68GA-ATRIDAT-BIOTIN

Surbhi Prakash, Puja Panwar Hazari, Ambika Parmar, Harleen Khurana, Surabhi Kirti Mishra, Virendra Kumar Meena, Anil Kumar Mishra


Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Delhi, India

Aim: The high specificity and strong affinity (K d =10 -15 M) of avidin for biotin plays a significant role in the two-step pretargeting protocol based on administration of the avidin antibody conjugate followed by the radioactive biotin chelate. Indigeneously synthesized tert-butyl 2,2'-(12-amino-11,13-dioxo-1, 4, 7,10-tetraazacyclotridecane-4,7-diyl)diacetate (ATRIDAT) is found to have a high stability constant with a wide range of metal ions. ATRIDAT was conjugated to biotin to obtain a specifically targeting molecule that binds avidin. Two step targeting could localize and image cancer cells in vivo which in turn can provide a high tumor targeting efficiency and modest signal amplification at the tumor site. Materials and Methods: Boc-protected diethyl amino malonate was conjugated with triethylene tetramine in high dilution method to give boc-protected macrocyclized product. This was then reacted with ter-butyl bromoacetate to increase the dentisity of the compound useful for metal chelation. After TFA cleavage, it was then reacted with NHS-activated biotin to give the desired product 2, 2'-(11,13-dioxo-12-(5-((3aS, 4S,6aR)-2-oxohexahydro-1H-thieno{3, 4-d}imidazol-4-yl)pentanamido)-1, 4, 7, 10-tetraaza-cyclotridecane-4,7-diyl)diacetic acid (ATRIDAT-Biotin). 68 Ga radiolabeling was performed and optimized to give more than 70% radiolabeling of chelate. Result and Conclusions: All intermediates and final compounds have been fully characterized by spectroscopic techniques, namely, 1H, 13C NMR and mass spectroscopy. ATRIDAT-Biotin was obtained in appreciable yield from condensation of boc-protected amino diethyl ester and triethylene tetramine and then its reaction with biotin after successive steps. Cold labeling of ATRIDAT-Biotin was done with GaCl3.3H2O in water under nitrogen atmosphere. Radiolabeling was done using generator eluted Gallium chloride ( 68 Ga, 74 MBq) which was then added to the conjugate after pH adjustment to ~4.5 using sodium acetate. More than 70% radiolabeling efficiency was obtained and the radioconjugate exhibited sufficient stability under physiological condition. Preliminary studies in vivo suggested appreciable uptake of 68 Ga-ATRIDAT-Biotin in tumor bearing mice. In conclusion a novel PET imaging agent based on two step pre-targeting with a new metal chelating system with biotin as biomarker has been developed which offers great possibilities in tumor imaging research applications for Ga-68 and Cu-64.

RP-28

Design and development of nucleolipid based fluorine labeled radiotracer: Synthesis based on 'click chemistry', theoretical insight and preliminary evaluation for imaging

Shubhra Chaturvedi, Ankur Kaul, Swastika Mishra, Bachcha Singh 1 , Anil Kumar Mishra


Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Delhi, 1 Department of Chemistry, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Objective: Nucleosides analogues are potential anti-cancer and anti-viral agents. Limited in-vivo stability and passive intracellular diffusion limits their utility. Cellular uptake of nucleoside analogues, especially sugar modified, depends primarily on passive diffusion, occasionally supplemented through nucleoside transporters. Amongst others, lipidic modification is a strategy for developing cellular penetrating nucleosides. Halogenated compounds particularly fluorine are important in imaging especially as PET and MRI tracer. In the present work we describe the synthesis and biological evaluation of fluorine labeled ketalised nucleoside, 1-{6-(2-{4-{2-(2-Fluoro-ethoxy)-ethoxymethyl}-2, 3-dihydro-{1, 2, 3}triazol-1-yl}-ethyl)-2, 2-dihexadecyl-tetrahydro-furo{3, 4-d}{1, 3}dioxol-4-yl]-1H-pyrimidine-2,4-dione (F-EG-DPU). The ketal linkage is of pharmacological interest, as it presents lipophilic prodrugs which can be catabolized in a slightly acidic medium. This presents potential drugs for acidic tumors. Materials and Methods: The syntheses involve conjugation of 16-hentriacontanone at 2' 3' position of the ribose ring of uridine. The 5' position is attached with an ether bearing linker through the triazole linkage. On the other side of the linker, tosyl which is a labile group that can be easily replaced by fluorine is attached. All the compounds and intermediates are characterised by 1 H, 13 C, 19 F NMR and mass spectrometry. The design of linker length has been optimized using docking studies with P-gp protein, an efflux protein. Results and Discussion: Cold fluorine labeling has been performed with an overall yield of 65%. Cytotoxicity studies using HEK and BMG cell lines indicate no appreciable toxicity. The tumor accumulation of the probe is studied using MRI in BALB/c mice. The results indicate enhanced uptake of the precursor as shown by the scans and cellular studies. Molecular modelling studies of the probe indicate poor substrate activity for P-gp. Other ADMET parameters and phase activity, predict the lipophilicity value 3.13 for the probe. Conclusion: Overall, results indicate that F-EG-DPU holds a promising future in imaging tumors and prodrug strategy for acidic tumors.

RP-29

Radiosynthesis of 68 Ga-labeled 5,10,15,20-tetra(Methyl-4-Pyridyl)-porphyrin for possible application as a pet radiotracer for tumor imaging

Mohini Bhadwal, Tapas Das, Sharmila Banerjee


Isotope Applications and Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Trombay, Mumbai, Maharashtra, India

Objective: Porphyrins are known to exhibit specific accumulation in neoplastic tissues. Cationic porphyrins are reported to intercalate in DNA helices. This property can be exploited in designing tumor-specific cationic porphyrin derivatives for radiolabeling with suitable radioisotopes. The present study, documents an attempt to prepare a 68 Ga-labeled cationic porphyrin derivative, namely 5,10,15,20-tetra(methyl-4-pyridyl)-porphyrin with the aim of assessing its potential as a PET radiotracer for tumor imaging. Materials and Methods: The porphyrin derivative was synthesized following a two-step reaction procedure. In the first step, 5,10,15,20-tetra(4-pyridyl)-porphyrin was prepared by refluxing an equimolar mixture of pyridine-4-carboxaldehyde and pyrrole in presence of nitrobenzene using propionic acid as solvent. Purification of the reaction mixture was carried out by silica gel column chromatography. In the second step, 5,10,15,20-tetra(4-pyridyl)-porphyrin was reacted with excess of CH 3 I for 2 days at room temperature. The methylated product was filtered and subsequently washed twice with chloroform to remove the starting material. Pure 5,10,15,20-tetra(methyl-4-pyridyl)-porphyrin derivative, thus obtained, was radiolabeled with 68 Ga, (eluted from a commercial 68 Ge/ 68 Ga generator and purified by cation exchange column using 20% 0.1N HCl in acetone as eluant) under reflux for 30 min in Na-acetate buffer (pH~4). The complex was further purified by using Sep-Pak® cartridges. The radiochemical purity of the complex was determined by paper chromatography and HPLC. Results: Characterisation of 5,10,15,20-tetra(methyl-4-pyridyl)-porphyrin was carried out by usual spectroscopic techniques. The radiolabeled porphyrin derivative could be prepared with >98% radiochemical purity under optimized reaction conditions. Experiments towards determining the bio- efficacy of the agent are presently underway. Conclusions: A charged cationic porphyrin derivative has been synthesized and radiolabeled with 68 Ga with high radiochemical purity. The total preparation time of the 68 Ga-radiotracer post elution of 68 Ga is 45 min. The applicability of the complex as a PET radiotracer for imaging of cancerous lesions is being evaluated.

RP-30

An overview of the use of radioactivity for nuclear medicine procedures in India

Pathak Subrata, Mallepula Madhu, Tandon Pankaj


Radiological Safety Division, Atomic Energy Regulatory Board, Mumbai, Maharashtra, India

Objective: The aim of this study is to comprehend the recent trend in the use of different radionuclides or radiotracers in diagnostic nuclear medicine (NM) procedures. It also includes state wise distribution of NM centres in India and number of popular scans carried out. Materials and Methods: In this study, all the data are compiled from Annual Safety Status Reports (ASSR) submitted by the NM centres. The data thus obtained are analysed using a global model developed by United Nations Scientific Committee on the Effects of Atomic Radiations (UNSCEAR). Results: The study shows that the 94% of scintigraphy is being performed using 99m Tc radiopharmaceuticals whereas about 5% is performed using 131 I radiopharmaceuticals. In case of PET imaging, 18 F labelled radio-tracers are the major contributor with 99% of PET scanning is being performed on 18 F radiopharmaceuticals. The details of the number of different scans carried out in the year 2012 are also analysed. The study revealed that during the last decade the annual number of diagnostics nuclear medicine procedures per 1,000 populations rises by a factor of two. There are about 190 functional NM centres catering to our country. Most of these centres are housed in the metro cities whereas there are states which do not have any. Conclusions: The data presented will provide a good platform to analyse the present status of NM health care in our country with global scenarios. This study though indicates a significant growth of NM, will also help the community to find out the sectors which demands improvement for inclusive growth of National NM healthcare status.

RP-31

Design, synthesis, characterization, radiolabeling of macrocyclic bifunctional chelating agent with technetium-99m and preclinical evaluation

Neelam Yadav, Krishna Chuttani 1 , Anil Kumar Mishra 1 , Bachcha Singh


Department of Chemistry, Centre of Advanced Studies, Faculty of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, 1 Division of Cyclotron and Radiopharmaceutical Sciences, INMAS, DRDO, Timarpur, Delhi, India

Objective: The target of this study was to develop a novel radiopharmaceutical for imaging and therapy of tumour cells. Materials and Methods: To achieve our objective, we have synthesized triaza methionine conjugated bifunctional chelator (MTPD). It was characterized by spectroscopic tools such as IR, NMR ( 1 H and 13 C) and ESI-mass spectrometry. Geometrical optimization of MTPD was done by DFT studies. The bifunctional chelating agent was radiolabeled with 99m Tc followed by preclinical studies. Molecular modeling and docking of MTPD have been also performed with potential enzymes involved in cholesterol biosynthetic pathway. Result: Labeling-efficiency of MTPD was more than 92% in vitro and in vivo both upto 24 h. Biodistribution studies show maximum uptake of the compound in liver at different time intervals. Blood-kinetics studies reveal maximum clearance of the radiolabeled compound in 6-7 h. Scintigraphy study of this bifunctional chelator shows significant uptake of the compound in tumor cells. Biological half life of 99m Tc-MTPD is found to be t 1/2 (F) 1 h 35 min and t 1/2 (S) 14 h 5 min. Conclusion: Labeling efficiency (92%), blood-clearance (>98%) and significant uptake in tumour cells indicated that MTPD may be used as tumour imaging agent.

RP-32

Theoretical insight into M 2+ /M 3+ -6-AMINO-(1H)-1, 4, 8, 11-tetraazacyclotridecane-5, 7-dione chelate: A bifunctional chelator as positron emission tomography tracers

Nidhi Chadha 1,2 , Anjani Kumar Tiwari 1 , Shubhra Chaturvedi 1 , Marilyn Daisy Milton 2 , Anil Kumar Mishra 1


1
Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, 2 Department of Chemistry, University of Delhi, Delhi, India

Objective: The interaction of metal ions with biomolecules and effect of coordination geometry of chelate as imaging agent can be a prudent aspect in developing probes for diagnostics. Metal complexes are widely used in all the imaging modalities. Thus in molecular diagnostics, molecular modeling plays an important role for design and analysis of structural, electronic parameters, metal-ligand complex structure, ligand selectivity, coordination number, lipophilicity, and thermodynamic stability. We have done molecular modeling studies of proposed bifunctional chelate (BFCs), 6-Amino-(1H)-1,4,8,11-tetraazacyclotridecane-5,7-dione, with metals namely M 2+ ; Cu-64/M 3+ ; Ga-68 that holds great potential for the development of molecular probes for PET. Materials and Methods: The methodology includes DFT analysis of ligands and transition metal complexes at ground state spin systems in vacuum and in solvent phase by Poisson Boltzmann Finite element method. Docking evaluation identifies binding metal-chelate complex with antibody which is an important aspect in case of site specific targeted therapeutic radiopharmaceuticals. Results: Geometric parameters for ligand were comparable with X-ray crystallographic structure in methanol. With the diagnosed Cu (II) and Ga (III) chelates, fundamental properties such as cavity size alon with proper denticity were observed to obtain thermochemical parameters. In case of Ga (III), due to similarity of ionic radii with high spin Fe (III) in-vivo, complexation properties with Fe (III) were also determined. The comparative studies resulted in modeling different possible spin orientations. The effect of different atomic orientations on energy and other electronic properties of the system were calculated. Also studied parameters include spin density and its distribution, nature of singly occupied molecular orbitals and symmetry-unrestrained optimization. Accurate spin density distributions are obtained by modification of the nuclear charge on metal ions. Interactions of these metals-chelates as molecular docking stations in imaging with antibodies were evaluated with and without metal loaded chelate. Conclusion: In this molecular modeling techniques are valuable in understanding behaviour and nature of Cu-64 and Ga-68 based BFCs when conjugated to peptide or monoclonal antibody for receptor of interest. Hence, theoretical studies play important role in the development of molecular imaging agents based on radiometal-BFC that requires evasion to excessive radiation damage to non-target cells under physiological conditions.

RP-33

Design, synthesis and in vitro evaluation of 18 F potential fluorinated molecules as pet tracers for Alzheimer's disease

Arun Bhusari, Mariam Degani, Harish Kundaikar, Machhindra Bochare, Harikesh Janmanchi, Mihir Khambete, MGR Rajan 1 , N Lakshminarayanan 1


Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Matunga (E), 1 Radiation Medicine Centre, Tata Memorial Hospital, BARC, Parel, Mumbai, Maharashtra, India

Introduction: Alzheimer's disease (AD) is a progressive irreversible neurological disorder and a leading cause of dementia. It is pathologically characterised by accumulation of β-amyloid plaques and numerous neurofibrillary tangles formed from filaments of highly phosphorylated tau proteins. Therefore monitoring or targeting of Aβ plaques or neurofibrillary tangles may be beneficial for the diagnosis of AD and assisting early therapeutic intervention. Presently, very few molecules like { 18 F} 2-Fluoro-2-deoxyglucose ({ 18 F} FDG) and Florbetapir ({ 18 F} AV-45) have been used for monitoring Aβ plaque by positron emission tomography (PET) scan. In the present study we are exploring the potential of a variety of scaffolds for Aβ plaque binding. Objectives: Design of scaffolds for interaction with the Aβ plaque with suitable physico-chemical properties. Synthesis of designed scaffolds, addition of 18 F with suitable linker, and their purification and spectroscopic characterization. Screening of fluorinated and non fluorinated scaffolds for binding with Aβ fibrils. Materials and Methods: The ligand based drug design study was carried out using phase, (version 2.0, Schrödinger, LLC, New York, NY, 2005). It was used to understand pharmacophoric features required for activity. The physico-chemical properties of scaffolds were predicted by Qikprop (version 2.5, Schrödinger, LLC, New York, NY, 2005). The designed fluorinated scaffolds were synthesized using cold fluorination and hot fluorination ( 18 F) methods and characterised by IR, NMR and MS. The radiolabelled scaffolds were purified by column chromatography. The Log P value of fluorinated scaffolds were determined using HPLC method on C-18 column (Kromasil, 15 cm, 4.6 mm, 3.5 μm) with PDA detector. Mobile phase was ACN-phosphate buffer (3:1), pH 7.4 at flow rate 1 ml/min. Spectrofluorometrically in vitro binding study of synthesized molecules with Aβ fibrils (500 nM) was carried out using Thioflavin T (32 μM) dye, considering curcumin (100 μM) as standard for Aβ fibrils binding at excitation-emission wavelength 440-480 nm respectively. Results and Discussion: The fluorinated and non fluorinated scaffolds showed similar alignment and similar pharmacophoric elements to that of reported Aβ plaque binding molecules. The experimentally calculated physico-chemical values were similar to the predicted values. The fluorinated and non fluorinated scaffolds showed promising binding to Aβ fibrils. Conclusion: Ligand based drug design and pharmacokinetic study assisted in the understanding of pharmacophoric elements in fluorinated and non fluorinated scaffolds necessary for the Aβ fibrils binding. Initial evaluation of synthesised molecules has led to the identification of scaffolds which can be optimised for diagnostic purpose.

RP-34

Design, synthesis and characterization of 1-(3-(4-(3 18 Fluoropropyl)-1H-1,2,3-triazol-1-yl)propyl)-4-(2-methoxyphenyl) piperazine as novel D 3 receptor ligand for PET Imaging

Sandhya Rangaswamy 1,2 , Raunak 1 , Swarndeep K Sethi 1,2 , Sandeep K Ghumman 2 , Anil K Mishra 1


1 Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, 2 Department of Chemistry, University of Delhi, New Delhi, India

Objective: The dopamine D 3 receptor subtype are potential neurochemical modulator of behavioral actions. The selective expression of D 3 receptors in limbic region has led a particular interest in this receptor as a potential mediator of some of the psychoaffective functions of dopamine neurotransmission. 1-(2-Methoxyphenyl) pipearazine(MPP) act as a potent antagonist of presynaptic and postsynaptic D 3 receptor. In an attempt to develop specific radiopharmaceuticals selective for D 3 receptors imaging, we have synthesized 1-(3-(4-(3 18 Fluoropropyl)-1H-1,2,3-triazol-1-yl)propyl)-4-(2-methoxyphenyl)piperazine. Materials and Methods: The secondary nitrogen of MPP was functionalized using 1-Bromo-3-chloroprpane to give alkylated MPP with the yield of 65%, which was further converted into its corresponding azide (98%).The 4-pentyn-1-ol was tosylated to give pent-4-ynyl-4-methylbenzenesulfonate with the yield of 97%. The tosylated alkyne and MPP azide were conjugated via a triazol ring by click chemistry to give 3-(1-(3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)propyl-4-methylbenzene sulphonate with the yield of 72%. The tosylated click analogue was radio fluorinated with K 18 F/Kryptofix at high temperature of 110°C in FX-N GE module to yield fluorinated ligand. Results: The synthesized ligand has been characterized by different spectroscopic technique (NMR and Mass). Radiolabelling efficiency was achieved more than 98% and the radio conjugate did not exhibit any dissociation under physiological condition. Further biological evaluation is under progress. Conclusion: We have successfully developed short-lived 18 F (t 1/2 = 110 min) in-house cyclotron and labelled MPP based derivative to yield 1-(3-(4-(3- 18 Fluoropropyl)-1H-1,2,3-Triazol-1-yl)propyl)-4-(2-methoxyphenyl)piperazine and fully characterized the ligand. The class of imaging agent holds a promising future in imaging D 3 receptor.

RP-35

Manual synthesis module: A cost effective and safe option for 68 Ga-labeled radiopharmaceuticals

Rakhee Vatsa, Priya, Nivedita Rana, Jaya Shukla, Bhagwant Rai Mittal


Department of Nuclear Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India

Introduction: Generator based PET tracers are cost effective than cyclotron produced. Ga-68 is versatile PET radionuclide having half-life of 68 minutes and can be eluted from 68 Ge/ 68 Ga Generator after every 4 h in a day. The exposure to the personnel involved in the synthesis of Ga-68 radiopharmaceuticals is quite high due to high positron energy (βmax =1.9 MeV). Though automated modules are available but the cost of these modules limits the use of Ga-68 radiopharmaceuticals. The availability of lead shielded manual modules extended the safe and cost effective radiosynthesis of Ga-68 radiopharmaceuticals. We illustrate of the utility of manual synthesis module in terms of exposure and synthesis time. Materials and Methods: Ga-68 dotatate was done using 30 mCi 68 Ge/ 68 Ga generator and manual chemistry module. Elution was done with 4 ml 0.05M HCl. Exposure levels were measured before, during and after production on surface of the module, at working surface and at 1 m distance using ionisation chamber based monitor (RAM GAM 1). The time period spent during the entire synthesis procedure was also recorded in order to estimate the exposure to the person during synthesis. Results and Conclusion: The time spent before elution in cleaning and preparation of module was 15 min, during elution and purification, 5 min and after production, 1 min. The mean exposure level at the surface of module before, during and after production was 2.52 μSv/h, 103.266 μSv/h and 24.866 μSv/h, respectively. The mean exposure level at working area and at 1 m distance before, during and after production was 1.513 μSv/h, 22.94 μSv/h, 6.546 μSv/h, respectively and 0.393 μSv/h,1.62 μSv/h, 0.853 μSv/h, respectively. Considering the duration of synthesis, mean radiation exposure measured during each production on surface, at work area and at 1 m distance was found to be 9.649 μSv, 2.399 μSv and 0.247 μSv, respectively. This translated to an annual exposure of 1.853 mSv, 0.460 mSv and 0.047 mSv for these three work location considering four productions per week. Hence, the total exposure at all working locations was found much less than the specified limits indicating that manual operation is not only a cost effective for the synthesis of Ga-68 radiopharmaceuticals but is also a safer option providing sufficient shielding that considerably reduces radiation exposure to personnel.

RP-36

Computational studies for optimization of organocatalysis in kryptofix free synthesis of 18 F radiotracers

Machhindra Dattatray Bochare, Mariam Sohel Degani, Harish Shashikant Kundaikar, Arun Manikrao Bhusari


Department of Pharmaceutical Science and Technology, Institute of Chemical Technology, Matunga (E), Mumbai, Maharashtra, India

Introduction: Kryptofix is widely and routinely used as a catalyst when metal fluorides are fluorinating agents in the synthesis of 18 F radiotracers for positron imaging tomography (PET). Ionic liquids; known as designer solvents are promising alternative organocatalysts with the features like low cost and easy separation. Computational studies were carried out to understand the role of ionic liquids as catalysts in nucleophilic fluorination. Objectives: (1) Study of nucleophilic fluorination reaction using computational methods (2) To understand role of catalyst in nucleophilic fluorination reactions (3) Study of properties of ionic liquids desirable for catalysis. Materials and Methods: Density functional theory (DFT) method (B3LYP) was employed to determine the reaction barriers. Full intrinsic reaction coordinate (IRC) analysis is carried out to confirm the reaction pathways. Single point energies and geometry optimizations were done using Jaguar (version 7.9, Schrφdinger, LLC, New York, NY, 2012). Results: Stable configuration of ionic liquids and the hydrogen bond studies correlate with physical properties of ionic liquids. Frontier molecular orbital interactions, electron density and charge transfer between anion and cation deepens the understanding of ionic liquids. Calculation of interaction energies between anion and cation of the ionic liquid catalyst, metal cation, fluoride anion and the leaving group gave insight about the role of ionic liquid as a catalyst. Changes in the structural features of the cation and anion affect the catalysis action which gives an understanding of the desirable properties of ionic liquid. Conclusion: With cognizance of the present computational studies, design and synthesis of 18 F radiolabeled diagnostic agents would be improved using optimized organocatalysis in the nucleophilic fluorination reactions.

RP-37

Biodistribution of 68 Ga-DOTA-TOC and 68 Ga-DOTA-TATE tracers in neuro-endocrine tumour patients: Characterization of uptake in normal organs

Nusrat Aland, Vikram Lele


Department of Nuclear Medicine and PET-CT, Jaslok Hospital and Research Center, Mumbai, Maharashtra, India

Introduction: The utility of 68 Ga-DOTA-TOC and 68 Ga-DOTA-TATE PET/CT is well established in neuroendocrine tumour diagnosis, staging, selecting patients for peptide receptor radionuclide therapy and response evaluation to therapy. However many a times primary neuroendocrine tumours are found at the physiological distribution sites of these tracers. In absence of morphological abnormality, uptake values are the only means to differentiate normal from diseased areas at these sites. Thus it is essential to establish a range for normal biodistribution of these tracers. Aim: (1) To define a range for the maximum standardized uptake values (SUVmax) for normal biodistribution of 68 Ga-DOTA-TOC and 68 Ga-DOTA-TATE tracers for differentiating between physiological uptake and tumor related somatostatin expression in the body. (2) To prove whether there is any difference in mean SUVmax values 68 Ga-DOTA-TOC and 68 Ga-DOTA-TATE tracers for each normal organ. Materials and Methods: 200 consecutive 68 Ga- DOTA-TOC and 68 Ga- DOTA-TATE PET-CT scans performed for clinically suspected and proven cases of neuroendocrine tumours from May 2009 to April 2010 were included in this study. 73 scans were performed with 68 Ga-DOTA-TOC and 127 were performed with 68 Ga-DOTA-TATE. Physiological tissue activity sites were noted defined by normal morphology, absence of surgical intervention and absence of metastatic spread during clinical follow-up and their SUVmax values were measured. Mean and normal range for the physiologic distribution for the two tracers was calculated and statistical tests were applied to find out whether there is any difference in the mean values for the two tracers in the region of physiological uptake. Results: Biodistribution of both tracers was similar and included pituitary gland, liver, spleen, adrenal glands, salivary glands, thyroid, uncinate process of pancreas and prostate. The normal range (mean ± standard deviation) for SUVmax values by body weight method for 68 Ga-DOTA-TOC tracer included: Pituitary (3.5 ± 1.88), salivary glands (4.2 ± 1.56), thyroid (3.9 ± 1.21), liver (10.9 ± 4.1), spleen (18.84 ± 6.3), uncinate process of pancreas (4.6 ± 3.9), adrenal glands (8.5 ± 3.1) and prostate (4.5 ± 1.6). For 68 Ga- DOTA-TATE tracer: Pituitary (4.9 ± 2.6), salivary glands (4.6 ± 1.3), thyroid (4.5 ± 1.38), liver (13.22 ± 4.6), spleen (21.25 ± 8.4), uncinate process of pancreas (6.25 ± 3.09), adrenal glands (10.5 ± 5.4) and prostate (6.9 ± 3.2). The SUVmax values for 68 Ga-DOTA-TATE tracer was statistically found to be higher than 68 Ga- DOTATOC at all the physiologic uptake sites. Conclusion: Calculation of normal range for physiologic biodistribution of both the tracers helped to differentiate normal from abnormal uptake of the tracer at physiological site in clinically suspected and proven cases of primary neuroendocrine tumours and their metastasis especially in those with no CT scan demonstrable lesion at that site.

RP-38

Radioactive alginate gel skin patch for the treatment of skin cancer

Jaya Shukla, Vikas Chaudhary, Priya Bhusari, Arun Reddy, Dipankar Day 1 , Bhagwant Rai Mittal 1


Departments of Nuclear Medicine and 1 Dematology, Post Graduate Institute of Medical Education and Research, Chandigarh, India

Objective: Basal cell carcinoma (BCC) is the most common skin cancer and originates from the lowest layer of epidermis. External radionuclide based radiation therapy with locally applied radioactive patch is an useful way to treat superficial skin cancers. We evaluated the utility of P-32 alginate gel patches, as an inert solid matrix, to immobilize radionuclide. Materials and Methods: Sodium alginate solutions (4%) was spread on parafilm and dipped into anhydrous CaCl 2 ( 0.2 M) solution. The patches were washed and air dried. Radioactive solution in the form of Tc-99m or P-32 colloid was spread on the gel to uniformly distribute the radioactivity on pre marked 1 cm 2 area and was left undisturbed to allow drying and avoid alteration in distribution. The image of alginate patches of Tc-99m colloid incorporated was acquired under gamma-camera, ROIs were drawn and counts were recorded to study radioactivity distribution. A teflon sheet of size bigger than patch (~2 cm) was placed over the outer surface over parafilm and exposure rate was observed. Dose delivered to the patient was calculated based on MIRD. Customized P-32 patch was applied for 3 h on a patient of biopsy proven basal cell carcinoma. The same patch was reapplied on 4 th and 7 th day. Clinical assessment, photographic recording and hematologic monitoring were done during the follow-ups. Results: Tc-99m colloid was used to study the distribution pattern of radioactivity on alginate film. The counts registered were almost similar. Radioactivity (P-32) was uniformly adsorbed on the gel layer. Exposure rate above the parafilm surface was reduced by covering the patch with teflon sheet. More than 20% relief of clinical symptoms was experienced by the patient after first application. On follow ups, significant symptomatic relief was observed. Conclusion: The alginate gel patch can be stable and suitable alternatives for radionuclide patch preparation for superficial skin cancers. Also our single patient observation demonstrated that P-32 patch therapy can be simple, non invasive and safe out-patient therapeutic procedure in treating patients of BCC.

RP-39

Labeling of trastuzumab with Tc-99m via DTPA for radioimmunoimaging of human epithelial receptor type 2/neu over-expressing breast cancer

Priya Bhusari, Jaya Shukla, Rakhee Vatsa, Gurpreet Singh, DK Dhawan, BR Mittal


Department of Nuclear Medicine and PET, PGIMER, Chandigarh, India

Introduction: Radioimmunoimaging has been an area of great interest since long. Monoclonal antibodies have been known as super targeting vehicles for imaging and therapy of various cancers. This study aimed to radiolabel Trastuzumab with Tc-99m via DTPA as a diagnostic radiopharmaceutical for Breast Cancer. Trastuzumab is a humanized monoclonal antibody against human epithelial receptor type 2 (HER-2) which over-expresses on 20-30% of all Breast Cancers. Materials and Methods: Trastuzumab, commonly known as Herceptin was procured from Zenentech, USA. It was purified from the mixture of amino-acids using Centricon 30 KDa tubes and the purified antibody was quantified spectrophotometrically. Conjugation was carried out by incubating antibody dissolved in 0.05 M bicarbonate buffer in DTPA-coated glass tubes (with Ab: DTPA ratio of 1:20-1:50) for 25-30 min. The conjugated mixture was purified to remove excess DTPA by Sephadex-G50 gel-column and again quantified spectrophotometrically. Radiolabeling was then performed by taking 100-150 ug of conjugated antibody; using 6-8 ul of SnCl 2 (4 mg/ml); 3-4 mCi of 99mTc04 and subsequently incubating the mixture for 25-30 min at neutral pH. Quality control was performed with ITLC using Acetone as mobile phase; Tc-99m-DTPA-Trastuzumab remained at bottom with Rf of 0.15 and free Tc-99m moved to the solvent front. Results and Conclusion: Labeling efficiency of Tc-99m-DTPA-Trastuzumab varied from 70% to 90% and pH did not considerably affect the radiolabeling. The study concluded that Tc-99m-DTPA-Trastuzumab can be used as a potential Radiopharmaceutical for diagnosis of Her2 positive Breast tumors.

RP-40

Technetium-99m eluate quality affects in vitro red blood cell labeling of healthy human volunteers blood: An university teaching hospital experience

Shivanand Bhushan, M Deeksha, Sibi Oommen


Department of Nuclear Medicine of SOAHS, Manipal University, Manipal, Karnataka, India

Objective: Purpose of the present study is to report and disseminate an initial experience gained with the 99m Technitium sodium pertechnetate eluate; obtained after second elution of the 99 Moly- 99m Tc column generator within 3-4 h on the same day; influences the labeling efficiency of 99m Tc labeled RBC while performing by in vitro method in the hospital pharmacy laboratory. Materials and Methods: Eluate from 99 Moly- 99m Tc column generator was used consisting radioactive concentration of 50 μCi in 1 ml of normal young healthy human volunteers (n = 27, male =14 and female =13) has not consumed any interfering synthetic chemicals/drugs blood collected in the heparinised syringe after taking informed consent duly approved by Institutional Ethics committee. SnCl 2 .2H 2 o used as reducing agent for 99m Tc of 2 μg/ml of concentration with mixing, incubating 20-30 min, separating packed red cells, washing with 0.9% saline and then counted in the calibrated Gamma ray Spectrophotometer. Percentage labeling efficiency of 99m Tc-RBC was calculated. Statistical variation Mean ± SD was calculated. Results: Labeling efficiency by in vitro method of RBC collected from human volunteers blood with the 99m Tc was showing poor percentage of 56.5 ± 3.8 as compared to the expected standard percentage binding of ≥90%. Result analysis reflecting that 99m Tc0 4 eluate used for RBC labeling may be containining more ground Technetium {99 Tc (ground)} that means ratio of 99m Tc to 99 Tc (g) may be very low, which has interfered with the 99m Tc (Gamma emitter 140 Kev). Limitation of the study is that 99 Tc (g) in the eluate from the column generator has to be quantified with the suitable and feasible method at different intervals to provide more evidence. Conclusion: Study concludes that 99m Tco 4 eluted for the red blood cell labeling should be fresh and should not be used if the generator has been eluted earlier within 3-4 h.

RP-41

99 Mo/ 99m Tc-TCM-autosolex generator: Physico-chemical and biological evaluation

Sankha Chattopadhyay, Luna Barua, Sujata Saha Das, Anirban De 1 , Umesh Kumar, Md Alam Neyar, Madhusmita, Arpit Mitra 3 , Bharat Ratna Sarkar 2 , Shantanu Ganguly 2 , Malay Kanti Das, MGR Rajan 3


1 Regional Centre, Board of Radiation and Isotope Technology, VECC, 2 Variable Energy Cyclotron Centre, 3 RRMC, VECC, Thakurpukur, Kolkata, 4 Radiation Medicine Centre, BARC/BRIT, Parel, Mumbai, India

Objective: Technetium-99m (t1/2 = 6.02 h; 140.51 keV (89%), principle γ-emission energy) is known to be the most useful radioisotope in diagnostic nuclear medicine. Worldwide crisis of fission 99 Mo based generator in recent past had put the Nuclear Medicine fraternity in very harsh situation. In order to have an indigenous solution of this problem, we developed a computer controlled semi automated 99 Mo/ 99m Tc generator using MEK solvent extraction technique, which utilizes abundantly available 99 Mo (n, γ) produced by (n, γ) reaction in BARC reactors. Materials and Methods: The TCM-AUTOSOLEX generator system was based on the selective extraction of pertechnetate ( 99m TcO4 ) in MEK from aqueous alkaline (n, γ) Na2 99 MoO4 solution, separation of the aqueous phase from the organic phase was controlled by an indigenously developed conductivity detector and subsequent purification of the organic phase by passing through an alumina column to remove traces of Mo, alkali, evaporation of the organic phase in a temperature controlled water bath and reconstitution in physiological saline to obtain pharmaceutical grade 99m Tc. The performance of the TCM-AUTOSOLEX module was evaluated by studying the recovery yield of 99m Tc, physico-chemical tests, sterility testing and BET assay studies. Image studies of rats with 99m Tc-MDP, 99m Tc-MIBI, 99m Tc-DTPA radiopharmaceuticals were also done. Results and Discussions: Different batches of 99 Mo-Moly (Batch size: 300 mCi or 500 mCi) were processed to recover pharmaceutical grade 99m Tc using TCM-AUTOSOLEX module. Average yield of separation of 99m Tc was above 95% and 99 Mo breakthrough in 99m Tc pertechnetate was <0.002% (n = 22). The pertechnetate obtained as above was checked for clarity, pH, radiochemical purity (RCP) and chemical purity. The efficacy of labeling specific compounds was assessed using standard radiopharmaceutical kits, such as 99m Tc-MDP, 99m Tc-DTPA and 99m Tc-MIBI and RCP was above 96% (n = 20). The product was sterile and pyrogen free. Conclusion: This module can provide the most economic 99m Tc-pertechnetate using indigenously produced 99 Mo from our research reactor.

RP-42

A simple purification method for the recovered 18 O-water for reuse in medical cyclotron to make 18 F

Amit Kumar, Shrinibas Nayak 1 , C Rajesh 1 , MGR Rajan 1


Medical Cyclotron Facility, Board of Radiation and Isotope Technology, 1 Radiation Medicine Centre, Bio-Medical Group, Bhabha Atomic Research Centre, Tata Memorial Hospital, Annexe, Parel, Mumbai, Maharashtra, India

18 F is the ideal PET radioisotope due to its half life, atomic size and the efficiency with which it can be incorporated into several molecules. Most of the 18 F PET radiopharmaceuticals are produced by nucleophilic bimolecular substitution (SN 2 ) reaction between 18 F (anion) and a suitable precursor with high labeling yields. 18 F is produced in the medical cyclotron by irradiating enriched {18 O} H 2 O with a proton beam via 18 O (p, n) 18 F nuclear reaction. During irradiation very negligible amount (few nanograms) of 18 O get converted to 18 F and hence {18 O] H 2 O is recovered after trapping the 18 F on an anion column. However the recovered {18 O} H 2 O gets contaminated with organic impurities like acetone and ethyl alcohol during the course of {18 F} radiopharmaceuticals synthesis. High cost of enriched {18 O} H 2 O and difficulties in its procurement due to the high global demand has forced us to find out a suitable method to purify and recycle after the first irradiation for the production of {18 F} radioisotope. In our study the recovered {18 O} H 2 O was purified by oxidation using alkaline KMnO4 and distillation. The purified {18 O} H 2 O was checked for organic impurities using gas chromatography and it was found that there was complete elimination of organic impurities after the treatment. The purified {18 O} H 2 O was used for 18 F-FDG production in our facility. The quality of 18 F-FDG was comparable to that when enriched {18 O} H 2 O was used. However, due to a nominal depletion in the {18 O} content, the irradiation period is increased to compensate for this.

RP-43

Synthesis of 177 Lu-DOTA-peptides in indigenously developed automated moduler system

HH Shimpi, YR Nitin, MGR Rajan


Radiation Medicine Centre, Bhabha Atomic Research Centre, TMH Annexe, Parel, Mumbai, Maharashtra, India

Objective: To develop a operational protocol for 177 Lu-DOTA-peptide synthesis in using our indigenously developed automated synthesis module operated through a laptop/PC based software. Materials and Methods: A microcontroller driven and HMI-software based automated synthesis module for 177 Lu-DOTA-peptide synthesis has been developed. The module was assembled using used solenoid valves and solid state relays. The software is programmed to carry out the synthesis in a timed sequence as follows: (1) Clean the system with 70% ethanol prior to synthesis. (2) Transfer the pH-adjusted ammonium acetate buffer with peptide and desired amount of 177 LuCl 3 to the reaction vial in pre-heated 95°C lead pot heater unit by using N 2 gas through membrane filter. (3) The reaction time is about 45-50 min. (4) Withdraw small amount of reaction mixture for quality control tests. (5) If RCP is ≥95% then no further purification is required and the reaction vial contents can be sent for direct terminal membrane filtration. (6) If RCP is <95% the step of purifying the 177 Lu-DOTA-peptide in the reaction mixture through C18 column is required. (7) Check the RCP of purified 177 Lu-DOTA-peptides and then terminal membrane filtration for patient use. Results: The HMI software is user friendly and it provides an alert message at the execution of each step. The graphics change according to the events to depict the flow of reactants and products making it easy to follow the progress of synthesis. So far, we have carried out a few cold runs in modular system with satisfactory results. Trial synthesis runs are planned to acertain radiological safety prior to regular use. Conclusion: Automation in the production of radiopharmaceuticals requiring multiple operations is known to ensure product reliability, reduce synthesis failures and reduce operating personnels' exposure to radiation.

RP-44

Development and radiolabeling of micellar nanocarriers of curcumin for nose to brain delivery

Swapnil Mohurle, Swapna Nabar 1 , Vandana Patravale, MGR Rajan 1


Institute of Chemical Technology, Matunga, 1 Radiation Medicine Centre, Bhabha Atomic Research Centre, Parel, Mumbai, Maharashtra, India

Objective: The study was aimed at designing radiolabeled micellar nanocarrier of curcumin for nose-to-brain delivery via olfactory pathways. Material & Methods: Curcumin, 99m Tc, Stannous chloride, Cremophor RH 40, Solutol HS 15, PEG 400, benzyl alcohol. The micellar nanocarriers were successfully formulated with the selected excipients and characterized for particle size and shape by DLS. Formulation comprising mixture of excipients having low particle size and polydispersity index (PDI) was selected. Drug excipient compatibility was evaluated with DSC studies. The stability of the curcumin in micellar formulation was tested at varied pH range (pH 6.0-8.0) with HPLC. Curcumin was radiolabeled with 99m Tc. Factors like pH (6.0-8.0), reaction time (10-120 min), reaction temperature (RT-37°C), SnCl2 concentration and reaction method were evaluated for optimal radiolabeling. RCP of radiolabeled curcumin/micelles was checked by paper chromatography in various solvents. The stability of radiolabeled drug/micelles was checked in simulated nasal fluid (SNF) and with DTPA challenge test. Results: Curcumin micelles with size of around 15 nm, were successfully formulated with the PDI of around 0.2. DSC studies showed the drug to be compatible with the excipients. The pH based stability studies of curcumin loaded micelles showed that >90% of the drug remained stable even upto pH 8 as opposed to plain drug solution in which <30% drug was found at pH 8. The RCP of curcumin/curcumin micelles was ≥96% which was stable upto 24 h (>90%). Radiolabeled curcumin/curcumin micelles were stable in SNF and when subjected to DTPA challenge test upto 24 h post radiolabeling. Conclusion: Curcumin was labeled with 99m Tc and the radiolabeled curcumin nanocarriers were found stable. They could be potentially used for imaging brain to access efficacy of intranasal brain targeted nasal drug delivery system.

RP-45

Comparison of elution efficiency of Mo99/Tc 99M radionuclide column generators supplied by three producers

Bhakti Shetye, Ashish Kumar Jha, M Priya, Sneha Mithun, P Mehajabeen, V Rangarajan


Department of Nuclear Medicine and Molecular Imaging, Tata Memorial Hospital, Parel, Mumbai, Maharashtra, India

Objective: 99mTc is known as a trade horse of nuclear medicine because it is readily available by the generator system. It has favourable chemical properties which are useful in its labelling with number of pharmaceuticals used to evaluate the functional parameters of various organ systems. Currently 99Mo/99mTc column generator is available from various suppliers in the market. All the suppliers claim excellent elution performance of their products. Hence we have performed this study to compare elution efficiency of three commercially available generators used in our department. Materials and Methods: In year 2010 and 2011 we received generators from three different suppliers: Isorad (Israel), Cis-Bio (France), Monrol (Turkey). Isorad and Monrol had supplied dry type generator while Cis-Bio had supplied wet type generator. We evaluated the performance of 10 Isorad generators, 10 Monrol generators and 10 Cis-Bio generators. We assessed elution history of all generators supplied by three suppliers to calculate elution efficiency. We calculated the elution efficiency of each generator for 5 consecutive days. Elution efficiency was calculated by the following formula. %Elution efficiency = (Measured eluted activity/Theoretical activity) *100. We calculated the average and standard deviation of elution efficiency over the period of 5 days for each generator separately and group of generator of each supplier independently. We have compared the average elution efficiency and standard deviation of generators supplied by three suppliers. Results: Average elution efficiency and standard deviation calculated for individual generator is tabulated and shown in [Table 1] and the average elution efficiency and standard deviation calculated for individual supplier is tabulated and shown in [Table 2]. Average elution efficiency for all the generator is more than 100% this might be because of the producer who have used the activity on higher side. Conclusions: In our study we have found excellent elution efficiency from all generators supplied by three suppliers. Sometimes in wet type generator activity may get trapped inside the column which results in low activity in the eluate. But in our study we have not found any significant difference in elution efficiency of dry and wet type generator.
Table 1: Percentage average elution efficiency and standard deviation for individual generator


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Table 2: Percentage average elution efficiency and standard deviation for individual suppliers


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RP-46

Application of structure based drug design for synthesis of pet radioligands for Alzheimer's disease

Harish S Kundaikar, Arun M Bhusari, Machhindra D Bochare, Mariam S Degani, N Lakshminarayanan 1 , MGR Rajan 1

Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, 1 Department of Atomic Energy, Board of Research and Nuclear Studies, Mumbai, Maharashtra, India

Introduction: Alzheimer's Disease (AD) is a progressive irreversible neurodegenerative disorder causing loss of reasoning and ability to care for self as seen in the affected older populations. There have been many efforts to rationalize drug design for AD based on knowledge of scaffolds, molecular dynamic simulations, and residue mutation analyses. Here we present a newer perspective of structure based drug design for AD. AD is characterized by deposition of senile plaques composed of neurotoxic β-amyloid (Aβ) peptides. Knowledge of binding sites on these peptides could help in the development of diagnostics for AD. X-ray crystal structures of these Aβ fibrils are unavailable, however high resolution NMR solution structure are reported. We have utilized these NMR structures of Aβ fibrils as well as PET tracers known for binding to Aβ peptides to decipher the binding site, using molecular modeling techniques. Materials and Methods: NMR structure and PET radiotracers known to bind Aβ peptide were obtained from literature. An array of molecular modeling methods were then used to logically identify a preferential binding site, including molecular and induced fit docking, site mapping, molecular mechanics and molecular dynamics. This site was further used to predict binding affinities of designed potential PET radioligands. Synthesis of cold-fluorinated ligands is being pursued using known or developed methods of fluorination. The synthesized molecules shall be evaluated using Thioflavin T binding studies. Results and Discussion: A binding site on Aβ fibrils for all the ligands used in study was found using different drug design methods. A small series of molecules was designed as fluorinated PET tracers. The designed molecules which were predicted to have good binding affinities to this site were taken up for synthesis. Validation studies of this site shall be presented. Conclusion: We propose a preferential binding site on Aβ peptides which could be used in the rational structure based drug design of PET radioligands for Alzheimer's Disease.

RP-47

Introducing 990 m Tc-0 norfloxacin complex as a potential infection imaging agent

RK Duggal, T Goel, P Johri, SS Sachdev 1 , AK Kohli 1


Regional Centre, Board of Radiation and Isotope Technology, C/O INMAS, Lucknow Road, Delhi, 1 Board of Radiation and Isotope Technology, Navi Mumbai, Maharashtra, India

Infection imaging has been a challenging area where there has not been any successful method to detect early infection areas in the body. Nuclear imaging methods using radiopharmaceuticals like 99m Tc-ciprofloxacin kits have provided a promising field by facilitating early detection of infection as well as distinguishing infection with inflammation in patients. Norfloxacin, a fluoroquinolone, is a broad spectrum antibiotic against both gram-positive and gram negative bacteria and is widely used for treating common as well as complicated infections. Previous studies have shown that 99m Tc-norfloxacin has better radiochemical purity and stability as compared with already established 99m Tc-ciprofloxacin. Here, 99m Tc labelled norfloxacin has shown labelling yield of >95% within 1 h of formulation. The stability of labelled norfloxacin is >6 h. Various compositions of norfloxacin, gentisic acid, stannous chloride, sodium chloride at different pH have been studied to optimize the labelling yield and stability of the 99m Tc-norfloxacin complex. Further studies related to its kit formulation as well as bio-distributions are under progress.

RP-48

Preclinical evaluation of anthraquinone based macrocyclic 68 Ga-DO3A-Act-AQ: A potential probe for theranostic application

Anupriya Adhikari, Kanchan Chauhan, K Ganesh Kadiyala, Abha Shukla, Krishna Chuttani, Anil K Mishra, Anupama Datta


Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, DRDO, Delhi, India

Aim: Positron emission tomography (PET) with positron emitter 68 Ga-based imaging probe is an upcoming field in molecular imaging as the radionuclide 68 Ga is instantaneously available via the 68 Ge/ 68 Ga radionuclide generator, have half-life of 67.7 min and a positron abundance of 89% making it suitable for PET imaging. Anthraquinone based compounds are known for their prominent position in cancer chemotherapy. They act at the duplex DNA level through stabilization of ternary complex with DNA topoisomerase ΙΙ. Their mode of action relates to their ability of damaging the nucleic acid thereby inducing cell death. It has been known that telomerase activity is high in tumor cells than other somatic cells, thus tumor cells have an almost infinite capacity to divide and hence can be immortalized. Many amino anthraquinone derivatives are reported of having telomerase inhibiting activity. Herein, the objective is to develop a new bi-functional chelating agent based on DO3A for labeling 68 Ga and conjugate it with anthraquinone derivative to synthesize DO3A based anthraquinone derivative for imaging with application in PET and therapeutic purpose. Materials and Methods: This work describes the potential of 68 Ga-(4,7-bis-carboxymethyl-10-{(9,10-dioxo-9,10-dihydro-anthracen-1-ylcarbamoyl)-methyl]-1, 4, 7,10tetraaza-cyclododec-1-yl)-acetic acid/DO3A-Act-AQ as diagnostic pharmaceutical by evaluating its deposition in cancerous tissues after the synthesis and radiolabeling with 68 Ga. 1,4,7,10-tetraazacyclododecane was converted to tris(t-butyl) ester of 1,4, 7-tris(carboxymethyl)-1, 4, 7, 10-tetraazacyclododecane. Fourth un-substituted arm was bound with 2-chloro-N-(9, 10-dioxo-9, 10-dihydro-anthracen-1-yl)-acetamide. The tertiary butyl groups were cleaved and the purified compound is radiolabeled with 68 Ga for biological evaluation studies like cytotoxicity, apoptosis induction study through FACS, DNA binding studies, blood kinetics and biodistribution. Results: (4,7-Bis-carboxymethyl-10-{(9, 10-dioxo-9, 10-dihydro-anthracen-1-ylcarbamoyl)-methyl}-1,4,7,10tetraaza-cyclododec-1-yl)-acetic acid was synthesized and characterized by NMR and mass spectroscopy. Initial radiolabeling studies showed high radiolabeling efficiency with 68 Ga >95%. Its biological potential as probe was checked by cytotoxicity on healthy and cancer cell lines (HEK and BMG-1). DNA binding and anticancer property of DO3A-Act-AQ was verified throughfluorescence, CD spectroscopy and flow cytometry. The clearance of radiocomplex from the blood checked through blood kinetics experimentwas within 40 min. Tumor uptake and biodistribution experiments using athymic nude mice xenografted with BMG-1 cell were performed. Conclusion: DO3A-Act-AQ is successfully synthesized, characterized and radiolabeled with 68 Ga. Initial studies suggests strong binding CT-DNA, anticancer property and rapid clearance from the blood. Further investigations like biodistribution characteristics and PET imaging confirmed high affinity towards tumor site in BMG-1 cells xenografted athymic micewhich further support its future as theranostic agent.

RP-49

Radiopharmaceutical Preparations in Indian Pharmacopoeia - An Update

AK Teotia, Ravendra Verma, Meenakshi Dahiya, Anuj Prakash, Robin Kumar, RM Singh, Sanyog Jain 1 , N Sivaprasad 2 , Aruna Korde 3 , NC Goomer 4 , MGR Rajan 5 , RK Sharma 6 , G.N. Singh


Indian Pharmacopoeia Commission Ministry of Health & Family Welfare, Govt. of India, Sec-23, Raj Nagar, Ghaziabad, 1 National Institute of Pharmaceutical Education & Research, (NIPER), Sector - 67, Phase X, SAS Nagar (Mohali), Punjab, 2 Board of Radiation & Isotope Technology, Department Atomic Energy, BRIT /BARC Vashi complex, Navi Mumbai, 3 Radiopharmaceutical Division, Bhabha Atomic Research Centre, Mumbai, 4 Board of Radiation and Isotope Technology, Department of Atomic Energy, Govt. of India, C/o Institute of Nuclear Medicine and Allied Sciences (INMAS) Brig. S.K. MazumdarMarg, Delhi, 5 Radiation Medicine Centre, Bhabha Atomic Research Centre, T MHlAnnexBuilding, Parel, Mumbai, 6 Institute of Nuclear Medicine and Allied Science (INMAS) Brig SK Mazumdar Marg, Delhi, India

Acknowledgement: Dr. Grace Samual, Dr. D. Padmanbhan, Shri VM Murhekar, DrSangeeta Joshi from Quality control of BRIT and members of RPC.

Radiopharmaceuticals (RPs) are pharmaceuticals or chemicals labeled with suitable radioisotopes used in nuclear medicine for diagnosis and therapy. The physical half-life of the radioisotopes used in radiopharmaceuticals are generally short and, are before administration to the patient.

Several RPs are listed in British Pharmacopoeia, European Pharmacopoeia, US Pharmacopoeia and WHO (International Pharmacopoeia). For the first time, monographs for 19 RPs and a General Chapter on Radiopharmaceutical Preparations have been included in the 7 th edition of the Indian Pharmacopoeia, the official pharmacopoeia of India. It will be updated from time to time and harmonized with other pharmacopoeias mentioned above.

The Indian Pharmacopoeia Commission has constituted a sub-committee with experts from reputed organizations and institutes in India drafting of monographs on commonly used RPs and the General Chapter on the subject.

The 19 RPs include 10 99m Tc-RPs containing 10 monographs, 5 Iodine labeled RPs, {18 F}Fludeoxyglucose, {18 F}Sodium Fluoride, {153 Sm}Samarium-EDTMP, and {32 P}Sodium Phosphate. Sixteen of the RPs are injectables, two are capsules and one is a solution for oral use.

In continuation of the first phase radiopharmaceutical monographs, IPC is now working on the second phase monographs on Radiopharmaceuticals for inclusion in the Indian Pharmacopoeia. The second phase monographs on Radiopharmaceuticals for Indian Pharmacopoeia are listed below:

  1. C-14, Urea Capsules
  2. 18-F DOPA (Fluorodopa)
  3. P-32 Samarium Phosphate Colloid
  4. F-18 FLT (Fluorothymidine)
  5. F-18 MISO (Fluoromisonidazole)
  6. Gallium {67 Ga} Citrate Injection
  7. Lutium-177 EDTMP
  8. Strontium Chloride ( 89 Sr) Injection
  9. Technetium ( 99m Tc) Labelledmacrosalb ( 99m Tc MAA) injection
  10. Technetium ( 99m Tc) Colloidal Rhenium Sulphide Injection
  11. Technetium ( 99m Tc) TetrofosminComplex Injection
  12. Technetium ( 99m Tc) Exametazimecomplex injection
  13. Technetium ( 99m Tc) Mertiatide injection
  14. Technetium ( 99m Tc) HSA Nanocolloids
  15. Technetium ( 99m Tc) Trodat
  16. Technetium ( 99m Tc) HYNIC TOC
  17. Thallous Chloride ( 201 T1) Injection


Members of the SNM-India and delegates of this SNM (I) conference are requested to go through the monographs on radiopharmaceuticals available in the 7 th edition of Indian Pharmacopoeia and give their valuable suggestions or send it to any of the authors above.



 
 
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