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ABSTRACT
Year : 2011  |  Volume : 26  |  Issue : 5  |  Page : 37-49  

Radiopharmacy


Date of Web Publication9-Dec-2011

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How to cite this article:
. Radiopharmacy. Indian J Nucl Med 2011;26, Suppl S1:37-49

How to cite this URL:
. Radiopharmacy. Indian J Nucl Med [serial online] 2011 [cited 2019 Nov 20];26, Suppl S1:37-49. Available from: http://www.ijnm.in/text.asp?2011/26/5/37/90733

RP-1

Effect of ethanol in BET Assay performed by kinetic chromogenic method for (18 F) Radiopharmaceuticals other than (18 F)FDG

A Mitra 1 , S Kulkarni 2 , C Rajesh 2 , MG R Rajan 1,2

1 MCF, BRIT, RMC, TMH Annexe, Parel, Mumbai, 2 RMC, MCF, BARC, TMH Annexe, Parel, Mumbai, India

In recent years, several (18 F) labeled radiopharmaceuticals other than 2-(18 F)fluoro-2-deoxy-D-glucose have become increasingly important for molecular imaging studies in oncology, and a rapid BET assay is essential to quantif y the bacterial endotoxin that may be present in such radiopharmaceutical preparations, prior to use in patients. The radiochemical synthesis of (18 F) labeled radiopharmaceuticals like 3'-Deoxy-3-(18 F)fluorothymidine ([18 F]FLT), (18 F)fluoroazomycinarabino side((18 F)FAZA) etc is based on use of SEP-PAK cartridge for isolation and purification instead of HPLC purification and requires 15% ethanolic water as elution solvent. BET assay for these radiopharmaceuticals can be routinely performed by portable test system (PTS) kinetic reader obtained from Endosafe Inc. The assay is based on kinetic chromogenic method and is known to be inhibited by presence of ethanol. Since all the above mentioned radiopharmaceuticals preparations contain ethanol, the aim of our study was to detect at what ppm/percentage level of ethanol, in final formulation, inhibition in BET assay is observed. The ppm/percentage levels of ethanol in final radiopharmaceutical formulations were detected by gas chromatography. In the present study, the BET Assay for (18 F)FDG and (18 F)FLT was performed at six different dilutions i.e 1:500. 1:200, 1:100, 1:50, 1:20 and 1:10 and all these dilutions were less than the maximum valid dilution (MVD). (18 F)FDG was chosen as control since the elution solven t for it is 100% water and for all the dilutions mentioned above, the spike recovery was between 50-200%. The CV of negative and positive control samples were found to be 0.0% and less than 10% respectively. However, in case of (18 F)FLT, for 1:20 and 1:10 dilutions, the spike recovery was below 50% but coefficient varian ce of negative and positive control sample were same as (18 F)FDG. These results indicate inhibition at 1:20 and 1:10 dilution due to presence of ethanol in (18 F)FLT, while at 1:200 dilution, BET Assay of (18 F)FLT by kinetic chromogenic method showed consistent result satisfying all the parameters.

RP-2

High-yielding fully-automated synthesis of (18 F)fluoromethylcholine using a GE TracerLab® MXFDG synthesizer

C Zankl, D Baumgart, S Hübner, S Jüttler, A Ruffani, M Müller


ABX Advanced Biochemical Compounds, GmbH, Heinrich-Glaeser-Str. 10-14, D-01454 Radeberg, Germany

Introduction: Increased cell membrane component metabolism is a distinctive characteristic of tumors. As a result of this elevated biosynthesis, tumors have an enhanced choline uptake which can be detected in vivo by positron emission tomography (PET). It has been shown that [11C]choline is particularly effective for imaging tumors i.a. localized in the brain, lung and prostate 1 in contrast to (18 F)FDG which shows less sensitivity and specificity. 2 Whilst proven to be an effective biomarker, [11C]choline has the drawback of a very short half-life (20 min). This can be overcome by using (18 F)fluoromethylcholine ((18 F)FMC) with a more favourable half-life of 110 minutes. Objective: Frequent usage of (18 F)FMC in clinical applications will depend on its steady availability. Several methods using the reactive intermediates (18 F)fluorobromomethane 3 or (18 F)fluorobromomethyl triflate 4 have been reported, with the drawback of low yields or complicate synthesis procedures due to a triflate oven. Our aim was to develop a reliable easy-to-use and high-yielding synthesis of (18 F) FMC by improving the destillation of (18 F)fluorobromomethane and its reaction with N, N-dimethylaminoethanol (DMAE). Materials and Methods: Synthesis of (18 F) FMC has been fully automated on a GE TracerLab® MXFDG module including the follwing steps: (a) 18 F-fluoride fixation, elution with TBA•HCO3 solution and azeotropic drying, (b) nucleophilic fluorination of dibromomethane, (c) destillation of (18 F) fluorobromomethane and reaction with DMAE, (d) purification by SPE using Sep-Pak C18 and Sep-Pak CM cartridges. Results: (18 F) FMC is usually synthesized by reacting (18 F) fluorobromomethane or (18 F) fluorobromomethyl triflate with neat DMAE. However, neat DMAE has not the best captivity to accumulate (18 F) fluorobromomethane. Several solvents were examined and the best results were obtained using a mixture of DMSO and DMAE. DMSO showed the best ability to concentrate (18 F) fluorobromomethane and additionally promotes the reaction with DMAE. Several runs for validation and quality control were performed and (18 F)FMC was obtained in constant yields of 27%±5% (non-decay corrected). Conclusion: We could establish an improved synthesis of (18 F) FMC with reproducible high yields using a disposable cassette system for GE TracerLab® MXFDG module. After purification on solid-phase exchange cartridges (18 F) FMC was obtained in 27%±5% (non-decay corrected) within 45 minutes. Furthermore, by changing reaction conditions the level of residual DMAE could be drastically reduced.

RP-3

Patient-specific syringe fractionate unit dose model: Practicable and economical approach of cold kit fractionation in hospital radio pharmacy

Shivanand Bhushan, Vasumathi Bhat, Oommen, Sibi, Indira Upadhya, Deeksha M Shetty, Rajesh Kumar, Nandini Pandit


Department of Nuclear Medicine, Manipal College of Allied Health Sciences, Manipal University, Manipal, India

Introduction: Cold Kits suitable for Tc99m labeling are readily available in multidosal vials from commercial manufacturers in freeze dried powdery form to assess organ function/ disease conditions. Several approaches has been made for fractionating multidosal full kit vial as cost reduction method. Fractionation of freeze dried cold kits reconstituted with inactive diluents and fractionate in vials kept in proper storage condition for subsequent uses have been documented practices in radiopharmacy since last three decades. The fractionated vials are considered as "vial fractionate model" in this study. The proposed study is to find suitable approach to use the fractionate in the sterilized and disposable clinical syringe following the "closed procedure" in routine practices. The present study provides practice based evidences to develop the "Patient- Specific Syringe Fractionate Unit Dose Model". Objective: The purpose of the present study is to develop and find out the feasibility for the preparation of patient specific unit dose of Tc99m-labelled Radiopharmaceuticals in a Sterlised disposable clinical syringe over the fractionated vials for clinical evaluation. Materials and Methods: Cold kits having only single vial preparation protocol, purchased from Board of Radiation and Isotope Technology (BRIT) Mumbai were used for the study between June 2003 to September 2011. Preparation of Tc99m-DTPA (TCK-7), Tc99m-MDP (TCK-30) and Tc99m-DMSA(TCK-35) used for renogram / gastro esophageal reflux, bone scan and renal cortical scan respectively. Fractionate of 1:5 is cold kit contents reconstituted with 1-2ml of sterile 0.9% NaCl to ensure complete dissolution. 1:5 of fractionate was withdrawn into a sterile clinical disposable syringe preloaded with Tc99mO4 - calculated for the specific patient. Immediately the contents were mixed well and the air bubbles present if any were removed. Patient- specific dose ready for administration, after the incubation period stated by the manufacturer. Results: More than 3000 of Tc99m-DTPA, Tc99m- MDP and Tc99m-DMSA(III) altogether were prepared between June 2003 to September 2011 as "Patient Specific Syringe Fractionate Unit Doses" following close to close procedure over full vials and fractionated vials. It is observed that withdrawing the fractionate from vacuumed full vial require technical skill and practice. Patients need not to be waitlisted for pooling the investigations. Patient in urgency can walk-in for investigation during available gamma camera time which leads to high level patient-satisfaction. Reduces loss of pertechnetate compare to "Vial Fractionate Model". Reduces manpower work load for preparation and maintenance of fractionate vials in radio pharmacy, cuts down the cost per study significantly to more than eighty percent. Patient specific Syringe unit dose model is feasible, easy, and practicable in routine practice for the hospital radio pharmacists. Retrospective analysis reveals that no significant alteration in biodistribution of radiopharmaceuticals were reported and served as post-hoc quality control check. Although practice based experiences confirms its feasibility for any radio pharmacy laboratory, but the study is having non-compliance of checking the pH and percentage radiochemical purity (% RCP) of individual dose before administration to patients. The model must be checked to see the effects of the volume and quality of eluates, diluents used and ligand to stannous concentration per unit dose and get validated. This model needs further modification for those kits which requires complex compounding and heating. Conclusion: Study concludes that the developed "Patient Specific Syringe Fractionate Unit Dose Model" is an easy, cost effective, and feasible for any hospital radio pharmacy laboratory. It improves the prompt nuclear medicine Service quality by increasing Patients Satisfaction. Further prospective study required to validate the model for completing the laboratory as well as Clinical evaluation following the Good Hospital Radio pharmacy Practices.

RP-4

Design and synthesis of neuro-catecholamine analogue and their efficiency as radiopharmaceutical

Pooja, Anjani K Tiwari, Anil K Mishra

Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Brig. S. K. Mazumdar Road, Delhi, India

Introduction: Multidentate aminocarboxylate ligands such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) are versatile platforms for the presentation of biomolecular recognition units in the design and investigation of biologically functional molecules. In this study, we propose to synthesize and evaluate a catecholamine analogue pendented with EDTA, which is also applicable for optical imaging agent when bind with some lanthanide metal ion. Materials and Methods: Designing of the ligand EDTA-bis(tyramine) have been performed by the molecular modeling software, SCHRODINGERS. Various parameters like pKa, energy calculations and different force parameters along with the all varieties of descriptors have been calculated by quik prop application followed by synthesis and characterization. The compounds was characterized by different spectroscopic techniques (FTIR, NMR and mass spectroscopy) and later radiolabelled with Tc99m. Results: The novel compounds was labeled with Tc99m by direct labeling method using stannous chloride as reducing agent at optimized conditions of pH, stannous ion concentration and incubation time to achieve the maximum labeling efficiency (>95%).It forms stable complex with Tc99m with high radiochemical purity(98%) and showed significant accumulation in brain. Blood kinetic study showed a quick wash out from the circulation. Excellent quality radio images confirms that this compound is potential candidate for brain imaging. Conclusion: The preliminary results indicates the usefulness of this modified EDTA-bis (tyramine) in imaging.

RP-5

In vitro bacterial binding assay of Scintibact (Tc99m-CAX) in isolates of Staphylococcus aureus

0Ankur Kaul, AK Babbar, Asha Attri, Rashi Mathur, AK Mishra


Institute of Nuclear Medicine and Allied Sciences, Brig SK Majumdar Marg, Delhi, India

In vitro studies of bacterial binding assay could possibly answer the occurrence of inaccuracy in specificity to bacteria in experimental models of human disease and clinical trials in humans. The study was carried out to assess the false positive outcome of some of the clinical studies and the method involved evaluation of the binding ability of the Scintibact to viable isolates of taphylococcus aureus in vitro in comparison to non-specific control. Four groups (in triplicate) were defined as: Blank control with Tc-99m-CAX(A); live bacteria with Tc-99m-CAX(B); Heat killed bacteria with Tc-99m-CAX(C); and live bacteria with Tc-99m-DTPA(D). To each group, 0.1 ml of radiopharmaceutical (final conc.: 2μg/ml of CAX ;600μg/ml of DTPA; 50μCi/ml of Tc99m) was added and the samples were incubated for 15 mins at 37° C and the pellet so formed, after each centrifugation, was washed with PBS, this process was carried out twice and the percentage of binding assay in each set was calculated. No Significantly different values of binding assay were observed in case of group- B(3.02 to 5.15%)in comparison to group-C (2.54 to 4.24%) While the non specific control: Tc-99m-DTPA (group-D) showed negligible activity(0.42 to 0.73%) in the pellet. The in vitro experimental data of this study justifies the uptake of Tc-99m-CAX at the sites with no active bacterial infections in the clinical data. However, the evaluation of binding of Tc-99m-CAX to different bacterial species at different time points are being planned to further augment these findings.

RP-6

Computational study of novel bis (thiosemicarbazone) analogues: Potential hypoxia imaging agents

Nidhi Chadha 1,2 , Anjani K Tiwari 1 , MD Milton 1 , Anil K Mishra 1

1 Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Brig. S. K. Mazumdar Road, 2 Department of Chemistry, University of Delhi, Delhi, India

Introduction: Bis(thiosemicarbazones) ligands and their metal complexes with copper are known to be good agents as hypoxia imaging agents. These complexes are known to show important properties such as SOD like activites, anti cancer activites in chemotherapy and in radiolablled form as non-selective blood perfusion tracer. Hence, study and development of bis(thiosemicarbazone) as a potential hypoxic agents are important in this regard. our objective is to design new ligands in series of known bis(thiosemicarbazones) derivatives by molecular modelling techniques and to determine their properties via QM/MM approaches since their redox potential, electronic properties are known to effect their activity in vivo in tumor cells. Materials and Methods: For this work molecular modelling software SCHRODINGER was used. 10 test molecules and 10 known molecules were taken to study. Their geometrical optimization, single point energy calculations were done. 3D QSAR and 2D QSAR studies were done after knowing their physiochemical properties such as blood brain barrier, lipophilicity, solubility, human serum albumin binding. Hence, structure-activity relationship was determined. Ligands for which best results were obtained by statistical studies were analysed further. Metal complexs of these ligands with copper were studied by DFT theory, an QUANTUM MECHANICAL approaches. After this, their redox potential was determined which is required important parameter for these bis(thiosemicarbazones) ligands to be an potential hypoxia imaging agents. Results and Conclusion: After evaluation, redox potential and other electronic properties were determined which are important parameters for their activity in vivo for hypoxia cells. Similarly QM studies confirms the property of this compounds for Tc99m based complexes. Further, these ligands and their metal complexes will be evaluated for NM applications through PET and SPECT.

RP-7

Design and synthesis of anti-integrin mAb conjugated Bismacrocyclic system for SPECT signal amplification to target β1 and β3 integrins

Surbhi Prakash, Puja Panwar Hazari 1 , Niraj Singh, Harleen Khurana, Raunak, Anil Kumar Mishra 1


Division of Cyclotron and Radiopharmaceutical Sciences Institute of Nuclear Medicine and Allied Sciences, 1 Brig. S. K. Mazumdar Road, Delhi, India

Aim: The Monoclonal antibodies against abundantly expressed integrin could prove to be effective tracer molecules for radioimmunodiagnosis. The primary objective of the current study was to design a bismacrocyclic system for amplification of SPECT signal for in vivo imaging of integrins. Materials and Methods: 5-Nitroisopthalatediethylester was covalently linked to two molecules of bromoethylamine which was then functionalized with DO3A.The nitro group in the resultant bismacrocycle was reduced to amino group and attached for in vivo imaging using anti integrin antibody. Conjugation of anti integrin monoclonal antibody was performed by adding 10 mM chelate solution to 300 mL of a solution containing 2 mg of antibody in 0.1 M sodium phosphate, pH 7. Saturated trisodium phosphate solution(40 mL) was added to make the pH 8.5. The reaction mixturewas incubated at 37° C for 60 min and then subjected tocentrifuged column gel chromatography, which removed the unreacted chelate, and the buffer changed to 0.1 M sodiumacetate, pH 5.5. UV absorbance at 280 nm for the centrifuged column effluent was used to determine the antibody concentration and 57 Co binding assay used to obtain the bound chelate concentration. Results and Conclusion: All intermediates and finalcompounds have been fully characterized by spectroscopic techniques, namely, 1H, 13C NMR and mass spectroscopy The preparation of bis macrocycle was achieved in high yields starting from 5-Nitroisopthalatediethylester followed by reduction. It was then conjugated with two antibodies- integrin β1and β3 antibody. Complexation of the bismacrocycle with cold Ga(III)chloride was carried out in water under nitrogen atmosphere. Radiolabeling was done using generator eluted Gallium chloride ( 68 Ga, 74 MBq) which was then added to the conjugate. The pH of the reactionmixture was adjusted to 6.5 with ammonium acetate solution(0.5 M) to get optimum radiolabeling yield.Biological evaluation was carried ou in human tumor cell line (U-87MG) malignant glioma which are known to express integrins. This antigen has been used successfully as a target for imaging and therapy because of its overexpression in tumors. These receptors proteins are highly expressed in metastatic malignancies and tumors providing high degree of invasiveness to the cancerous cells which in turn confer a degree of targeting specificity. Therefore, the tumor cells can proportionally bind more radiolabeled mAbs. In conclusion, the preliminary studies with this novel bis-macrocycle are encouraging to carry out further in vivo experiments for targeted imaging of human tumors in animal models.

RP-8

Development of Tc99m-labeled EDTA-bis(Glutathione) for imaging GSH efflux in metastatic tumors

Harleen Khurana, Puja Panwar Hazari 1 , Viren K Meena, Surbhi Prakash, Niraj, Ambika Parmar, Anil K Mishra 1


Division of Cyclotron and Radiopharmaceutical Sciences Institute of Nuclear Medicine and Allied Sciences, 1 Brig. S. K. Mazumdar Road, Delhi, India

Introduction: Reactive oxygen species (ROS) and oxidative stress has been associated within cancer cells. Glutathione (GSH) is a antioxidant, preventing damage to important cellular components caused by reactive oxygen species such as free radicals and peroxides. Highly metastatic cancer cellas have higher GSH content and acceleration of glutathione efflux facilitates selective GSH depletion in metastatic cells thereby sensitization of metastatic cells to therapy. We have developed a novel drug Tc99m labeled EDTA-bis GSH as a tumor imaging agent and have evaluated the potential of Tc99m EDTA-Bis-Glutathione(EDTA bis GSH) in U 87MG tumor xenograft in athymic mice nu/nu mice. Materials and Methods: Successful synthesis of EDTA-bis(GSH) was done by conjugating Glutathione to ethylenediaminetetraaceticacid bisanhydride and confirmed using NMR spectra and mass spectroscopy. The compound was labeled with Tc99m with labeling efficiency of 95-99%. The compound was found to be stable in saline up to 24 hrs. It was also found that in serum the drug interacts with other proteins. Efflux studies were carried out in malignant glioma cells. In U-87 Malignant glioma bearing mice model, in vivo imaging and biodistribution study were performed. Results: Tc99m-EDTAbis (GSH) showed high labeling efficiency up to 98% in vivo and vitro condition. High tumor to non-tumor ratio was observed in scintigraphy in tumor xenograft. High uptake was also seen in kidneys and bladder. High uptake in kidneys represented the excretion via renal system. Moderate uptake of liver and intestine may have been caused by the clearance of EDTAbis (GSH) through hepatobiliary route of excretion. Other organs, such as lung, spleen, stomach and heart showed minimal uptake. Conclusion: Tc99m- EDTA-bis(GSH) showed excellent tumor targeting and has promising utility as a SPECT-radiopharmaceutical for imaging human tumors.

RP-9

An antisense oligonucleotide conjugated to PET/SPECT contrast agent for in vivo imaging of 5HT1A receptor gene expression

Virendra K Meena, Puja Panwar Hazari 1 , Harleen Khurana, Surbhi Prakash, Raunak Anil Kumar Mishra 1


Division of Cyclotron and Radiopharmaceutical Sciences Institute of Nuclear Medicine and Allied Sciences, 1 Brig. S. K. Mazumdar Road, Delhi, India

Introduction: 5HT1A is a potential marker to diagnosis of Alzheimer's and Parkinson's disease. So it could be employed to detect the patients having these neurological disorders. Tc99m / 68 Ga, a positron-emitting radionuclides (half-life, 6 hours) and (half-life, 68 min) respectively, along with a macrocyclic chelating agent, Tri-substituted cyclen was used for labeling of antisense oligonucleotides targeting 5HT1A receptor mRNA. A marked reduction in the expression of 5HT1A receptors could be seen in the patients of these disorders. Objective: So the main focus of this study is to develop imaging system to diagnose the Alzheimer's and Parkinson's disease. Materials and Methods: For that we employed antisense oligonucleotide for 5HT1A receptors which are highly expressed in normal hippocampal neurons and marked reduction in expression in both the disease conditions, to serve this purpose the antisense oligonucleotide was modified at 5' terminal Azide and conjugated to a macrocyclic chelating agent (i.e. Tri-substituted cyclen) through terminal Azide using click chemistry approach. The conjugate was coordinated to 68 Ga /Tc99m at the 5' position and cell penetrating peptide at the 3' terminus of this oligonucleotide to target the mRNA of 5HT1A receptors. The oligonucleotide conjugate was evaluated for the stability with 68 Ga /Tc99m in saline and further serum protein binding affinity. Further studies of biodistribution and biokinetic studies are evaluated in BALB/c mice. Cellular uptake studies were done by Immunofluorescence and electron microscopy in hippocampal neurons. The study demonstrated gene expression in the brain in vivo could be imaged with antisense radiopharmaceuticals that are conjugated to a brain mRNA-targeting system. Results: The oligonucleotides can be stably labelled with 68 Ga /Tc99m and DOTA chelate. Intravenously injected 68 Ga /Tc99m-oligonucleotides revealed high-quality PET/SPECT images, allowing quantification of the biokinetics in brain tissues and major organs. The biodistribution and biokinetics of intravenously administered 68 Ga /Tc99m-oligonucleotide varied considerably with the nature of the oligonucleotide backbone. Conclusion: We conclude that 68 Ga /Tc99m labelling of oligonucleotides is a feasible and convenient approach for in-vivo imaging and quantification of oligonucleotide biokinetics in living animals with PET/SPECT.

RP-10

Preparation of 68 Ga-labeld cyclic RGD peptide dimer as a potential PET radiotracer for tumor imaging using a novel 68 Ge/ 68 Ga generator developed in house

S Chakraborty, R Chakravarty, HD Sarma 1 , A Dash, S Banerjee, MRA Pillai


R0 adiopharmaceuticals Division, 1 Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai, India

Introduction: Integrin αvβ3 plays a significant role in angiogenesis during tumor growth and metastasis, and is a receptor for the extracellular matrix proteins with the exposed arginine(R)-glycine(G)-aspartic acid(D) tripeptide sequence. The overexpression of integrin αvβ3 during tumor growth and metastasis presents an interesting molecular target for both early detection and treatment of rapidly growing solid tumors. The recent introduction of 68 Ga-PET imaging into clinical practice has made a significant impact in the field of PET imaging which does not require the availability of a cyclotron. 68 Ga is a positron emitter with a short half-life of 68 min, which is suitable for the pharmacokinetics of many peptides and other small molecules owing to their quick blood clearance and rapid target localization. In the present study the cyclic RGD peptide dimer E[c(RGDfK)] 2 (E=Glutamic acid, f=phenyl alanine, K=lysine) coupled to the bifunctional chelator DOTA is chosen as the targeting biomolecule owing to the improved tumor uptake and retention of the dimer compared to its monomeric counterpart. Taking these factors into consideration, an attempt has been made to prepare 68 Ga labeled DOTA-E[c(RGDfK)] 2 as a potential PET radiotracer for tumor imaging. Materials and Methods: DOTA-E[c(RGDfK)] 2 was custom synthesized by M/s. ABX Advance Biomedical Compounds, Germany. The conjugate was characterized by MALDI mass spectrometry. 68 Ga was eluted form a 68 Ge/ 68 Ga generator developed in house using nanoceria-polyacrylonitrile (PAN) composite sorbent as 68 GaCl 3 and used for radiolabeling without further purification. The 68 Ga labeling of DOTA-E[c(RGDfK)] 2 was achieved by incubating 25 μg of the conjugate with 68 GaCl 3 (74-111 MBq) in acetate buffer (pH 3.5) at 90°C for 15 min. The radiolabeled conjugate was characterized by HPLC and its biological efficacy was studied in Swiss mice bearing fibrosarcoma tumors. Results: The 68 Ga complex of DOTA-E[c(RGDfK)] 2 was prepared with >98% radiochemical purity and the complex exhibited excellent in vitro stability. The results of the biodistribution studies revealed significant tumor uptake within 10 min p.i. (4.14±0.54%IA/g). The activity accumulated in the tumor was observed to increase further (4.61±0.31%IA/g) at 30 min p.i. Initial accumulation of activity was observed in various non-target organs viz. liver, GIT, kindey, lungs etc. However, with the progress of time, the uptake in non-target organs was observed to reduce gradually. The tumor to blood ratio was observed to increase from 1.75±0.42 at 10 min p.i. to 2.25±0.20 at 60 min p.i., while the tumor to liver and tumor to muscle ratio increased from 2.71±0.76 to 3.31±0.84 and 5.37±1.08 to 8.97±1.32, respectively, between the same time points. The radiolabeled conjugate exhibited predominant urinary excretion, as more than 80% of the injected activity cleared via renal pathway within 60 min p.i. Conclusion: These preliminary studies indicate the potential of the developed agent for possible use in early detection of tumor by PET imaging. However, further studies are warranted in animal models to assess the potential applicability of the agent.

RP-11

Synthesis and biological evaluation of chalcone derivatives with Tc99m as potential probe for detection of β-amyloid plaques

K Ganesh Kadiyala, Kanchan Chauhan, Anupriya Adhikari, Neeraj, Anupama Datta, Anil K Mishra


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

Aim: In SPECT imaging, Tc-99m is a radionuclide of choice for diagnostic imaging due to its favourable decay properties, 6 h half life, low cost and easy availability. Current interest is focused on developing new generation of highly selective, target specific radiopharmaceuticals consisting of a metal complex conjugated to an organic molecule with high binding for a receptor. So far many radiolabeled probes based on the core structure of Congo Red and Thioflavin T have been developed as imaging agents specific for β-amyloid plaques. However flavones are the new class of pharmacaphores which also show high binding affinity to Aβ-aggregates. Chalcone is categorized as a member of flavonoids containing flavones having a chemical structure in which the ether linkage is removed from the flavone structure. It is also reported that chalcone consists of a structurally similar moiety to curcumin which has favorable binding affinity to β-amyloid plaques and sufficient brain permeability. Herein, the objective is to develop a new bifunctional chelating agent based on picolinic acid for Tc99m labelling and conjugate it with chalcone derivative to synthesize a chalcone based picolinic acid derivative for imaging with application in SPECT. Materials and Methods: This work describes the potential of Tc99m-Pic 2 -N-Chal (6,6'-(2-(bis(2-(4-((E)-3-(4-(dimethylamino)phenyl)acryloyl)phenoxy)ethyl)amino)ethylazanediyl)bis(methylene)dipicolinic acid) as diagnostic pharmaceutical by evaluating its deposition in cancerous tissues after the synthesis and radiolabeling with Tc99m. Pyridine-2,6-dicarbonyl dichloride was used as the precursor for the synthesis of the chelating agent diethyl-6,6'-(2-aminoethylazanediyl)bis(methylene)dipicolinate (Pic 2 -N). Finally, Pic 2 -N was conjugated to chalcone conjugated with dibromopropane to give Pic 2 -N-Chal. The compound is radiolabeled with Tc99m for scintigraphic studies. Results: Pic 2 -N-Chal was synthesized and characterized by NMR and mass spectroscopy. Initial radiolabeling studies show high radiolabeling efficiency with Tc99m>95% and the stability in serum indicated that Tc99m remained bound to Pic 2 -N-Chal up to 24h. Its biological potential as probes by testing it affinity for Aβ aggregates and β-amyloid plaques in sections of brain tissue from mice and their uptake by and clearance from the brain in biodistribution experiments using normal mice will be evaluated. Conclusion: The agent, Pic 2 -N-Chal shows strong binding with Tc99m, even after 24h the binding was >90%. Further investigations on the biodistribution characteristics and affinity towards Aβ aggregates and β-amyloid plaques will be carried out.

RP-12

Synthesis and biological evaluation of thiocarbamate derivatized estradiol as SPECT agent for Tc99m

Kanchan Chauhan, K Ganesh Kadiyala, Anupriya Adhikari, Neeraj Katiyar, Anupama Datta, Anil K Mishra


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

Aim: Estrogen receptor (ER) is an attractive target for molecular imaging and therapy due to its upregulation in osteoporosis, ovarian cancer, and breast cancer. Ability to use molecular imaging in-vivo for monitoring expression of ER offers a mean for differentiating primary tumors, detecting metastases, guiding optimal choice of therapy, and following the impact of novel interventions based on targeting ER. Some of the known compounds having potential as PET and SPECT agent for determining the status of estrogen receptor are 16α-(18 F)-fluoro-17β-estradiol and, cis-methoxy-[123 I]-iodovinylestradiol, respectively. It is also reported that a high affinity to ER is retained upon the introduction of the bulky substituents at the 17α position of E2 (Estradiol). Substituting the hydrogen atom at the 17α position gives rise to the ligand which is more selective to ER. Herein, we delineate the synthesis of target specific estradiol derivative Est-NH-CS 2 for SPECT application. This derivative contains dithiocarbamate group conjugated with the estradiol derivative via linker and thus are capable of binding to ER with high affinity. Widely used concept of click chemistry was applied during synthesis. Materials and Methods: Synthesis was performed using estrogen derivative, 17α-ethynylestradiol as precursor. The 17α-ethynyl-estradiol was reacted with 2-azidoethanamine using click chemistry with proper stoichiometry to give 15-(1-(2-aminoethyl)-1H-1,2,3-triazol-4-yl)-14-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-2,15-diol (Est- NH 2 ). Its conjugation with chelate dithiocarbamate by using carbon disulfide in NaOH solution gave Est-NH-CS 2 . For radiolabeling with Tc99m. Synthesis was confirmed by 1 H NMR, 13 C NMR and ESI-MS spectroscopy. The ER binding affinity was checked by Tc99m radiolabeled Est-NH-CS 2 . Estrogen receptor ability was evaluated in human MCF7 cells. Scintigraphic and biodistribution studies were carried out on estrogen receptor-positive MCF7 tumor bearing athymic mice. Results: Synthesized compounds were characterized by NMR and Mass spectrometry. Radiolabeling efficiency with Tc99m was found to be >97%, serum stability indicated that Tc99m remained bound to Est-NH-CS 2 up to 24h. Initial investigation of blood clearance showed quick washout from the circulation via renal route. Conclusion: Tc99m- Est-NH-CS 2 can be prepared in high radiochemical yield. Radiolabeling efficiency with Tc99m being >97%. Its scintigraphic studies and biodistribution characteristics towards estrogen receptor cell lines (MCF cells) are to be evaluated. Predication of ADME properties of synthesized molecule by using Schrodinger Maestro Software is to be done.

RP-13

Development of estradiol conjugate with macrocyclic chelate (DO3A-Act-Est) as SPECT/PET radiopharmaceutical for tumor imaging

Anupriya Adhikari, Kanchan Chauhan, K Ganesh Kadiyala, Sunil Pal, Anupama Datta, Anil K Mishra


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

Aim: Single Photon Emission Computed Tomography (SPECT) has become a powerful scientific and clinical tool for probing biochemical processes in the human body. 80% of radiopharmaceuticals used in nuclear medicine are Tc99m labeled because of its favorable physical and radiation characteristics. Targeting the delivery of hormones and drugs with a specific molecular recognition site and simultaneously monitoring their distribution and localization in the body is an important goal in diagnostic molecular imaging. One such example is to target organs and tissues using a selective estrogen receptor modulator (SERM) which is responsive to estrogen and can be monitored in vivo. ERα and ERβ are the two estrogen receptors (ER) which are very similar proteins but their expression distribution is different in various tissues. Action of estrogens in tissues like uterus and mammary gland is mediated by ERα whereas role of ERβ is established in cardiovascular system, brain, ovary and several animal models of inflammation. Also, ERα is an important marker and traditional target in breast cancer diagnosis. Some of the known examples to image ER are 18 F- or 123I-labeled ER ligands for PET or SPECT respectively. The scope of MRI experiments is increased by the potential target-directed contrast agents developed recently. Towards the same direction, we have designed target specific estradiol tert-butyl 2,2',2''-(10-(3-(2-(4-((14S,15R)-2,15-dihydroxy-14-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-15-yl)-1H-1,2,3-triazol-1-yl)ethylamino)propyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (DO3A-N 3 -Est) having application in SPECT and PET. Materials and Methods: This work describes the potential of DO3A-N 3 -Est as diagnostic pharmaceutical by evaluating its deposition in cancerous tissues after the synthesis and radiolabeling with Tc99m, 68Ga and 64Cu. 1,4,7,10-Tetraazacyclododecane was converted to tris(t-butyl) ester of 1, 4, 7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane. Fourth unsubstituted arm was bound with a linker N-(2-azidoethyl)-3-bromopropan-1-amine conjugated with 17α-ethynylestradiol by utilizing the wide application of click chemistry to give the compound DO3A-N 3 -Est. Finally, the compound is radiolabeled with Tc99m for scintigraphic studies. Results: DO3A-N 3 -Est was synthesized and characterized by NMR and Mass spectroscopy. Radiolabeling efficiency with Tc99m was found to be >97% and the stability in serum indicated that Tc99m remained bound to DO3A-N 3 -Est up to 24h. Initial investigation of blood clearance showed a quick wash out from the circulation and biological half life were determined. Biodistribution characteristics of Tc99m- DO3A-N 3 -Est are to be examined. Conclusion: Tc99m-DO3A-N3-Est can be prepared in high radiochemical yield. Its biodistribution studies are to be examined. Further investigation on the BMG glioblastoma will be carried out. Attempts are also being made to use the cavity of 1,4,7,10-tetraazacyclododecane to introduce 68Ga and 64Cu to determine the application of DO3A-N 3 -Est in PET imaging.

RP-14

Production of 68 Ga DOTA-Peptide using Manual Labeling system and 68 Ge/ 68 Ga Generator -Initial Indian Experience of 45 Runs

Atul Gada, Giri Prasad, Y Mahita, Praveen, N Rao, Swaroopa, N Kavitha Reddy, Jyotsna Rao, M Mansoor, Kalyani, Prathyusha


Apollo Gleneagles PET-CT and Cyclotron Centre, Hyderabad, Andhra Pradesh, India

Introduction: Recently 68 Ga labeled Tracers have major clinical impact compared to Tc99m and In111 labeled radiopharmaceuticals in imaging of neuroendocrine tumors. providing high quality PET images, which can be produced onsite, independent of cyclotron. Objective: To Establish and Standardize 68 Ga-DOTA-Peptide synthesis and Asses Effectiveness of fluidic system module integrated to generator. Material and Methods: First Peptide and 0.05M sod acetate buffer are sent in Preheated Reactor followed by elution of generator in 1 MNT using 0.05M HCL into reactor. 68 Ga from generator eluate binds to DOTA chelator which is coupled to peptide. After incubation time (10 MNT) the reaction mixture is passed over silica cartridge to hold and separate labeled 68Ga-peptide. Non labeled ions viz. 68 Ga, 68 Ge passed over cartridge and are washed to waste. After separation labeled product is eluted from reverse phase cartridge with ethanol and sent for purification via sterile filter. PH and Radiochemical purities were checked (TLC). Time-Activity audit was carried out and yields were calculated. Results and Conclusion: Manual system setup is efficient and reliable in synthesis with Over all Yield of about 65-70% in average run time of 35 minutes. RCP >97% and average PH=4.3. PH plays important role in labeling and should be maintained in a narrow range. It provides for built in feature of pre-purification and concentration of 68 Ga eluate. Existing integrated Design features provides for reduced loss due to sticking of reagents and precious peptide, radiation shielding, compact size, ease of handling, maintenance and troublesoothing.

RP-15

Experience with 68 Ga DOTATOC synthesis module and imaging: Technologist perspective

Philip Blessy, Parab Anil, Khan Umer, Patil Kashinath, Lele V


Department of Nuclear Medicine and PET/CT, Jaslok Hospital and Research Centre, Mumbai, India

Introduction: Somatostatin receptor imaging using 68 Ga DOTATATE is a valuable imaging modality in management of neuroendocrine tumor patients. We started 68 Ga DOTATATE synthesis and 68 Ga DOTATATE PET/CT imaging in May 2009 and till date we have done more than 200 scans. Objective: To describe technologist's perspective of working with 68 Ga DOTATATE synthesis module and PET/CT imaging. Materials and Methods: 68 Ga I seluted from 68 Ge with 0.1Mhcl. the eluted 68 Ga activity is then fixed onto strata XC cartridge and the remainder of hcl is pushed out from the cartridge by passing air through ot. The 68 Ga activity is then eluted into the reaction vial by the eluent (N2). During the reaction time of 400sec at 85deg C 68 Ga is incorporated to the peptide. This solution is then cooled and diluted by adding water and the crude product is the transferred to C 18 light cartridge. The product is then transferred to the product vial by ethanol and it is further diluted with saline. The yield per elution is greater than 80%. QC of the Rp: HPLC QC was performed and RCP purity was more than 95%. Dose administered: Varying dose of 500microCi -3Mci. Injection to imaging time: 40mins - 1hr



We have observed that even with a less activity of 600microCi we have got good quality images by reducing the injection to imaging time to 40 mins and increasing the time per bed during pet acquisition to 3.0 mins. Results: We have scanned around 200 patients since 2009 with varying activity of 600 microCi - 4 mci. We have observed that even with a less activity of 600 micro Ci we have got good quality images by reducing the injection to imaging time to 40mins and increasing the time per bed during pet accqusiton to 3.0 mins. Conclusion: Our experience with 68 Ga DOTATATE synthesis module has been satisfactory. Imaging protocol of PET/CT can be modified when less radiotracer is injected and satisfactory images can be acquired.

RP-16

Preparation of 18 F-Fluoroethyltyrosine: Preliminary studies

Lakshminarayanan N, Arjun G, Rajan MGR


Medical Cyclotron Facility, BRIT/BARC, Radiation Medicine Centre, BARC, TMH Annexe, Parel, Mumbai, India

Introduction: (F-18)FET is an analogue of the amino acid tyrosine, labeled with F-18. It is reported to have advantages over (F-18)FDG in differentiating inflammation from tumors and with a potential for grading tumors - particularly gliomas (1). A synthesis procedure for (F-18)FET using semi-prep HPLC purification has been reported by Hamacher, et al (2). In our opinion, the HPLC purification step introduces some uncertainity and lengthens the synthesis time besides requiring a higher degree of automation and maintenance. We have prepared (F-18)FET in a GETracer Lab system (configured for making FDG) using 2-(O-tosyloxyethyl)-N-trityl-L-tyrosine-tert-butyl ester (TET) as precursor. Following radiofluorination and hydrolysis, purification was carried out by Solid Phase Extraction, using a neutral alumina (3.5g in 1cm ID Column). The species in the reaction mixture were identified by radio TLC using different mobile phases. The labeling efficiency was found to be >90%. Materials and Methods: TET-Precursor, resins, Ethanol, WFI, Acetonitrile were obtained from ABX, Germany, 3.5 gm of neutral alumina, from the Chromabond® Set IV column procured from ABX was repacked and conditioned with water for injection (WFI). All reagents were prepared in WFI, conductivity less than 0.1μS/cm. TLC plates (1cm x 13cm) of silica gel from Merck, India, were used. Propanol and butanol of AR Grade were from SD Fine Chemicals, Mumbai. 18 F-Fluoride was produced by 18 O(p,n) 18 F reaction in the cyclotron. The (F-18) fluoride was trapped in 45mg PS-HCO 3 column. (F-18)Fluoride was eluted with 0.5mL of 75mM Tetrabutylammonium bicarbonate solution to obtain (F-18)Fluoride in the form of 18 F-TBAF in the reactor vessel. Then to this, 1mL of dry acetonitrile was added and azeotropic distillation was done to remove the acetonitrile. Drying was aided by flushing the reaction vessel with a stream of He-gas. The precursor in acetonitrile (8mg/800uL) was added and heated to 85°C and maintained at this temperature for 5 min. The reaction was then cooled to 40°C with He drying continued inside the reactor. To this 30% TFA in 1,2 dichloroethane was added. After 2 min, the temperature was raised to 70°C and maintained for 7 min. Results and Discussion: The labelling efficiency of 18 F, i.e. the incorporation of 18 F into the precursor was >90%. Post hydrolysis, the reaction mixture was characterized by radio-TLC and four bands were identified - colloidal, free (F-18)fluoride, labelled (F-18)-FET and un-hydrolyzed (F-18)-precursor. 18 F-FET was identified by co-spotting a reference standard of (F-19)FET. 18 F-Fluoride was identified by its R f in the solvent system. It was also observed that freshly opened TFA gave a better yield, rather than using stored TFA. The reaction medium was diluted with 2mL of WFI, and loaded onto the alumina column. Further elution with 10mL of WFI was carried out and the species eluted out were identified as (F-18)Fluoride and a radio-active impurity, presumably a colloid, by TLC in various mobile phases i.e. water, butanol, isopropanol systems. In an attempt to separate the (F-18)- FET and unhydrolysed. FET, eluting agents of increasing non-polarity viz. different compositions of EtOH iPOH and BuOH were tried out. The un-hydrolysed species can be reduced by optimizing the hydrolysis conditions. It was observed that solution of isopropyl alcohol: Water (7:3) could elute out the trapped (18 F)-FET. Conclusion: 18 F-FET was synthesized in a modified GETracer Lab using the TET-precursor. The labelling efficiency of 18 F i.e. the incorporation of 18 F into the precursor was >90%. The various species in the reaction mixture were identified. The SPE method shows potential for a simple purification system. Further studies are in progress to validate and use the (F-18)FET in animal bio-distribution studies.

RP-17

Radio synthesis and biological evaluation of HYNIC-Tyr 3 Octreotide


Ashok Behera, Indranil Banerjee, Kakali De, Sankha Chattopadhayay 1 , Mridula Misra

Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, West Bengal, 1 Radiopharmaceuticals Laboratory, Regional Centre, Board of Radiation and Isotope Technology, Variable Energy Cyclotron Centre, 1/AF, Bidhan Nagar, Kolkata, India

Introduction: Somatostatin-receptor (SDTr) scintigraphy using 111 In-DTPA-D-Phe1-octreotide has been shown to be clinically valuable for the localization of primary and metastatic sstr expressing neuroendocrine (NE) tumors. But the use of 111 In as a radioisotope results in high cost, long half life (67 h), suboptimal image quality and a high radiation burden to the patient. So these disadvantages could be overcome by the use of Tc99m labelled somatostatin analogs. Due to the wide availability and cost-effectiveness of Tc99m, it is of major importance for routine clinical applications. On the other hand, Tc99m has superior nuclear characteristics (half-life of 6 h and monoenergetic gamma radiation of 141 keV), which minimise the radiation exposure of the patient while providing high-resolution images with currently available instrumentation. Objective: This work reports on the synthesis, radiolabelling and biological evaluation of [Tc99m] HYNIC-Tyr 3 -Octreotide with potential use in the visualization of somatostatin receptor-positive tumours and their metastases. Materials and Methods: All chemicals and solvents were of either HPLC or analytical grade and were used without further purification. O-t-butylthreoninol-2-chloro-trityl resin, Fmoc-D-Phe, Fmoc-Cys (ACM), Fmoc-Lys (Boc)-OH, Fmoc-D-Trp (BOC)-OH, Fmoc-Thr (tbu)-OH, Fmoc-Tyr (tBu)-OH and TBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate), were obtained from NovaBiochem (Switzerland). All other chemicals and solvents were either obtained from MERCK, Germany or from SRL, India. HYNIC-Tyr 3 -Octreotide was semi-automatically synthesized on a peptide synthesizer. Radiolabelling with Tc99m was performed by SnCl 2 method. The radiochemical purity assessment and quality control was achieved by RP-HPLC and ITLC method. In vitro serum stability study of Tc99m-peptide was performed post preparation at different time intervals. Bio-distribution study was performed on Sprauge Dawley rats at different time periods. Renal clearance, plasma protein binding and metabolic study analysis (rat kidney and urine) was also performed on Sprauge Dawley rats. Cell culture and Internalisation study were performed on AR42J and C6 glioma cell line (obtained from NCCS Pune). All culture media were supplied by Gibco, Invitrogen. Animal experiments were performed in compliance with the regulations of our Institutional Animal Ethics Committee (Indian Institute of Chemical Biology). In-house male Wistar rats (200±10 g) were used for the preparation of cortex membranes. Protein concentration of the samples was determined according to Bradford assay method using bovine serum albumin as the standard. Results and Conclusion: HYNIC-Tyr 3 -Octreotide was successfully synthesised, purified and charecterised by HPLC chromatogram, and MALDI-MS spectra. The radiolabelling efficiency was more than 98%, when determined by ITLC and HPLC. In the biodistribution study, we found high uptake of Tc99m-peptide in kidney than any other organs. The blood clearance was rapid with rapid excretion through kidneys and relatively low uptake in liver. In conclusion, the new radioligand reported herein, combines the advantages of high affinity, rapid target localisation and fast body clearance.

RP-18

Synthesis, radiolabelling and biological evaluation of new somatostatin receptor positive tumour imaging agent

Ashok Behera, Indranil Banerjee, Kakali De, Sankha Chattopadhayay 1 , Mridula Misra


Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, West Bengal, 1 Radiopharmaceuticals Laboratory, Regional Centre, Board of Radiation and Isotope Technology, Variable Energy Cyclotron Centre, 1/AF, Bidhan Nagar, Kolkata, India

Introduction: Somatostatin receptors are mostly overexpressed by primary and metastatic malignant disease, mainly of neuroendocrine origin and have prompted a worldwide search for radiolabelled somatostatin analogues for use in somatostatin receptor mediated tumour imaging (SRI). Synthesis, purification and radiolabelling of a novel peptide with Tc99m is reported here, along with results of experiments with cell preparations and animal models demonstrating the very promising characteristics of [Tc99m] HYNIC-HIS 3 -TATE for SRI applications. Objective: The main objective of our current work is the development of new somatostatin analogs that would retain the general characteristics of [Tyr 3 ] octreotide while showing potential for clinical application. This work reports on the synthesis and evaluation of a novel Tc99m-based somatostatin analogue [Tc99m] HYNIC-His 3 TATE with potential use in the visualization of somatostatin receptor-positive tumours and their metastases. Materials and Methods: All chemicals and solvents were of either HPLC or analytical grade and were used without further purification. Resin, protected amino acids and coupling agents were obtained from NovaBiochem (Switzerland). Di-isopropyl ethylamine (DIPEA) was obtained from SRL, India. All other chemicals and solvents were either obtained from Sigma Aldrich, MERCK Germany or from SRL, India. The new somatostatin analog was semi-automatically synthesized on a peptide synthesizer. Radiolabelling with Tc99m was performed by SnCl 2 method. The radiochemical purity assessment and quality control was achieved by RP-HPLC and ITLC method. In-vitro serum stability study of Tc99m-peptide was performed post preparation at different time intervals. Bio-distribution study was performed on Sprauge Dawley rats at different time periods. Renal clearance, plasma protein binding and metabolic study analysis (rat kidney and urine) was also performed on Sprauge Dawley rats. Cell culture and Internalisation study was performed on C6 glioma cell line (obtained from NCCS Pune). All culture media were supplied by Gibco, Invitrogen. Animal experiments were performed in compliance with the regulations of our Institutional Animal Ethics Committee (Indian Institute of Chemical Biology). In-house male Wistar rats (200±10 g) were used for the preparation of cortex membranes. Protein concentration of the samples was determined according to Bradford assay method using bovine serum albumin as the standard. Results and Conclusion: The new somatostatin analog (HYNIC-HIS 3 -TATE) was successfully synthesised, purified and charecterised by HPLC chromatogram and MALDI-MS spectra. The radiolabelling efficiency was more than 98%, when determined by ITLC and HPLC. In the biodistribution study by invasive method, we found high uptake of Tc99m-peptide in kidney than any other organs. The blood clearance was rapid with rapid excretion through kidneys and relatively low uptake in liver. In conclusion, the new radioligand reported herein, combines the advantages of high affinity, rapid target localisation and fast body clearance.

RP-19

Synthesis and biological evaluation of D 2 -receptor based ligands labeled with SPECT radionuclide for neuroendocrine tumor

Swarndeep Kaur Sethi 1,2 , Raunak 1 , R Sandhya 1 , MD Milton 2 , Anil K Mishra 1


1
Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Brig. S.K. Mazumdar Road, 2 Department of Chemistry, University of Delhi, New Delhi, India

Introduction: Dopamine type 2 receptors (D 2 ) are primarily located in the basal ganglia of the mammalian brain but also occur in other parts of the brain, such as the cortex and play a critical role in the cell signaling process responsible for information transfer through neurons in the nervous system. The receptors, which are located at the neuronal membrane, belong to the monoamine subclass of the G-protein-coupled seven transmembrane receptors (GPCRs). Spiperone is a psychoactive drug and a well known D 2 receptor antagonist. The triazaspirodecanone moeity of spiperone is a major determinant of its D 2 receptor binding affinity and selectivity. The preservation of specific binding towards dopamine D 2 receptor even after large chemical modification performed make the molecule a good and potential SPECT agent. Materials and Methods: The above D 2 -receptor analogue was synthesized by reacting bromoethylamine hydrobromide with sodium azide followed by alkylation of primary amine. The alkylated moiety was first click conjugated with propargylated 1-phenyl-1,3,8-triazaspiro-(4,5)-decan-4-one and followed by covalent attachment with trisubstituted cyclen. The D 2 -receptor binds Tc99m with high efficiency and the radiolabeling efficiency was found to be >95% and the stability in serum indicated that Tc99m remained bound to the drug upto 24 hours. Results: All the intermediates and final compound were characterized by 1 H, 13 C NMR and Mass Spectroscopy. The in vivo biodistribution and blood kinetics studies exhibited rapid clearance of the radiolabeled complex and excretion through the hepatobilliary and renal route. The tumor (IMR-32and PC-12) grafted in athymic mice were readily identifiable in the gamma images. Biodistribution revealed significant tumor uptake in the (IMR-32and PC-12) tumor bearing mice. Conclusion: We have synthesized Tc99m- based DOTA-triazole-1-phenyl-1,3,8-triazaspiro-(4,5)-decan-4-one conjugate which shows excellent tumor targeting and has promising utility as SPECT-radiopharmaceutical for imaging.

RP-20

Design and synthesis of DTPA-βamyloid conjugate for SPECT imaging

Nisha Saini 1,2 , Raunak Varshney 1 , AK Tiwari 1 , MPS Ishar 2 , Sachin Soni 1 , AK Mishra 1

1 Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Brig S K Majumdar Marg, New Delhi, 2 Department of Pharmaceutical sciences, Guru Nanak Dev University, Amritsar, India

Introduction: Alzheimer's disease (AD) is a neurodegenerative disorder that is characteristic of amyloid deposition in the forms of plaques. Development of probes for in vivo labeling and detection of β-amyloid (Aβ) plaques in patients of Alzheimer's disease (AD) is of significant scientific interest and it assist in the development of drugs, targeting Aβ plaques for treatment of AD. The aim of this study is to develop potential technecium 99m (Tc99m) labeled diagnostic imaging agents for Aβ plaques. Materials and Methods: We have synthesized Benzothiazole derivative conjugated with DTPA-dianhydride. Arylbenzothiazole is prepared from conjugation of aminobenzoic acid and aminothiaphenol which is conjugated to DTPA-dianhydride. All intermediates and final compound was fully characterized by 1 HNMR, 13 C NMR and ESI-MS. Results: The synthesised compound displayed good binding affinity for synthetic Aβ aggregates and used for in-vivo imaging of Aβ plaques with SPECT technique. This work is under process. The radiolabeling efficiency of drug is >95% and serum stability up to 6hr indicates drug bound to Tc99m successfully. Radiolabeled compound is excreted by hepatobiliary as well as by renal route and also showed rapid blood clearance. Biodisribution study in normal mice showed reasonable penetration through blood brain barrier. Conclusion: We have successfully synthesized Aβ plaques imaging agent which showed good serum stability, rapid blood clearance, radiolabeling efficiency and binding affinity for plaques,

RP-21

Patient-specific syringe fractionate unit dose model: Practicable and economical approach of cold kit fractionation in hospital radio pharmacy

Bhushan Shivanand, Bhat Vasumathi, Oommen, Sibi, Upadhya Indira, Shetty Deeksha M, Kumar Rajesh, Pandit Nandini


Department of Nuclear Medicine, Manipal College of Allied Health Sciences, Manipal University, Manipal, India

Introduction: Cold kits suitable for 99m Technitium labeling are readily available in multidosal vials from commercial manufacturers in freeze dried powdery form to assess organ function/ disease conditions. Several approaches has been made for fractionating multidosal full kit vial as cost reduction method. Fractionation of freeze dried cold kits reconstituted with inactive diluents and fractionate in vials kept in proper storage condition for subsequent uses have been documented practices in radiopharmacy since last three decades. The fractionated vials are considered as "vial fractionate model" in this study. The proposed study is to find suitable approach to use the fractionate in the sterilized and disposable clinical syringe following the "closed procedure" in routine practices. The present study provides practice based evidences to develop the "Patient- Specific Syringe fractionate Unit Dose Model". Objective: The purpose of the present study is to develop and find out the feasibility for the preparation of Patient specific Unit dose of Tc99m-labelled Radiopharmaceuticals in a Sterlised disposable clinical syringe over the fractionated vials for clinical evaluation. Materials and Methods: Cold kits having only single vial preparation protocol, purchased from Board of Radiation and Isotope Technology (BRIT) Mumbai were used for the study between June 2003 to September 2011. Preparation of Tc99m-DTPA (TCK-7), Tc99m-MDP (TCK-30) and Tc99m-DMSA(TCK-35) used for renogram / gastro esophageal reflux, bone scan and renal cortical scan respectively. Fractionate of 1:5 is Cold kit contents reconstituted with 1-2ml of sterile 0.9% NaCl to ensure complete dissolution. 1:5 of fractionate was withdrawn into a sterile clinical disposable syringe preloaded with Tc99mO4 - calculated for the specific patient. Immediately the contents were mixed well and the air bubbles present if any were removed. Patient- specific dose ready for administration, after the incubation period stated by the manufacturer. Results: More than 3000 of Tc99m-DTPA, Tc99m- MDP and Tc99m-DMSA(III) altogether were prepared between June 2003 to September 2011 as "Patient Specific Syringe Fractionate Unit Doses" following close to close procedure over full vials and fractionated vials. It is observed that withdrawing the fractionate from vacuumed full vial require technical skill and practice. Patients need not to be waitlisted for pooling the investigations. Patient in urgency can walk-in for investigation during available gamma camera time which leads to high level patient -satisfaction. Reduces loss of pertechnetate compare to "Vial Fractionate Model". Reduces manpower work load for preparation and maintenance of fractionate vials in radio pharmacy, cuts down the cost per study significantly to more than eighty percent. Patient specific Syringe unit dose model is feasible, easy, and practicable in routine practice for the hospital radio pharmacists. Retrospective analysis reveals that no significant alteration in biodistribution of radiopharmaceuticals were reported and served as post-hoc quality control check. Although practice based experiences confirms its feasibility for any radio pharmacy laboratory, but the study is having non-compliance of checking the pH and percentage radiochemical purity (% RCP) of individual dose before administration to patients. The model must be checked to see the effects of the volume and quality of eluates, diluents used and ligand to stannous concentration per unit dose and get validated. This model needs further modification for those kits which requires complex compounding and heating. Conclusion: Study concludes that the developed "Patient Specific Syringe Fractionate Unit Dose Model" is an easy, cost effective, and feasible for any hospital radio pharmacy laboratory. It improves the prompt nuclear medicine service quality by increasing Patients satisfaction. Further prospective study required to validate the model for completing the laboratory as well as clinical evaluation following the good hospital radio pharmacy practices.

RP-22

Design and synthesis of Tc99m -anthraquinone based analogues for nuclear medicine applications

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, Brig. S. K. Mazumdar Road, 2 Department of Chemistry, University of Delhi, New Delhi, India

Introduction: Catecholamines contain catechol or 3,4-dihydroxybenzene group. These do not cross the blood brain barrier. Therefore, it is necessary to design such related analgues which can cross BB barrier and to device a mechanism for their transport into the CNS. Here, an anthraquinone based tyramine analogue was synthesized to obtain a system which can be active in the central nervous system. Materials and Methods: Designing of the compound includes the prediction of physico-chemical escriptors and ADME properties were calculated using molecular modeling software, Maestro. Synthesis and characterization was done by various spectrometry techniques(NMR,Mass). Results: The 1 H, 13 C spectra's of the compound "N-(2-methylanthraquinone)-4-(2-aminoethyl)phenol" shows the characteristic peaks and the molecular ion peak was observed at m/z 357 from mass spectrometry. It was found from the ADME properties that it mimics clebopeide which is a dopamine antagonist drug with antiemtic and prokinetic properties. Also, it forms stable complex with Tc99m with high radiochemical purity(95%). Conclusion: The prepared anthraquinone analogue can act as a radiopharmaceutical and an efficient fluorophore.

RP-23

In vitro bacterial binding assay of Scintibact (Tc99m-CAX) in isolates of Staphylococcus aureus

Ankur Kaul, AK Babbar, Asha Attri, Rashi Mathur, AK Mishra


Institute of Nuclear Medicine and Allied Sciences, Brig SK Majumdar Marg, Delhi, India

In vitro
studies of bacterial binding assay could possibly answer the occurrence of inaccuracy in specificity to bacteria in experimental models of human disease and clinical trials in humans. The study was carried out to assess the false positive outcome of some of the clinical studies and the method involved evaluation of the binding ability of the Scintibact to viable isolates of Staphylococcus aureus in vitro in comparison to non-specific control. Four groups (in triplicate) were defined as: Blank control with Tc-99m-CAX(A); live bacteria with Tc-99m-CAX(B); Heat killed bacteria with Tc-99m-CAX(C); and live bacteria with Tc-99m-DTPA(D). To each group, 0.1 ml of radiopharmaceutical (final conc.: 2μg/ml of CAX ;600μg/ml of DTPA; 50μCi/ml of Tc99m) was added and the samples were incubated for 15 mins at 37° C and the pellet so formed, after each centrifugation, was washed with PBS, this process was carried out twice and the percentage of binding assay in each set was calculated. No Significantly different values of binding assay were observed in case of group- B(3.02 to 5.15%)in comparison to group-C (2.54 to 4.24%) While the non specific control: Tc-99m-DTPA(group-D) showed negligible activity(0.42 to 0.73%) in the pellet. The in vitro experimental data of this study justifies the uptake of Tc-99m-CAX at the sites with no active bacterial infections in the clinical data. However, the evaluation of binding of Tc-99m-CAX to different bacterial species at different time points are being planned to further augment these findings.

RP-24

Optimization of dry distillation method for production of 131 I at Pilot Scale

RN Ambade, MSA Khan, SN Shinde, Viju Chirayil and PV Joshi 1


Radiopharmaceuticals Division, Bhabha Atomic Research Centre

Introduction: 131 I is extensively used in nuclear medicine for last five decades for both diagnosis and therapy. Iodine-131 has been regularly produced in BARC and supplied to BRIT for necessary formulation and subsequent supply to various users. Currently it is produced by wet distillation method involving oxidative dissolution of irradiated tellurium metal with subsequent reduction and distillation of 131 I. Though reliable, this method has its drawbacks. Annually ~150 liter of radioactive acidic liquid waste (Dose rate >500 R/Hr) is generated for the production of ~55 TBq (~1.5 kCi) of 131 I activity. Efforts to replace this method with a dry distillation process are in the final stages. The advantage of dry distillation is reduction in volume of radioactive waste and a faster process with better control of radioactivity concentration. It has been demonstrated at pilot scale and the experiences are reported in the current paper. Objective: To optimize the process parameters for the production of high purity 131 I by dry distillation technique at pilot scale. Materials and Methods: High purity Tellurium dioxide powder was used for target material. TeO 2 (5-20 g) was irradiated for one week in Dhruva reactor at a neutron flux of 8.3 x 10 13 n 1 cm -2 s -1 and was mixed with inactive TeO 2 powder with a view to simulate full production batch of 50 Ci. The simulated target in a quartz crucible is loaded into a specially designed quartz assembly -with the provision to purge gas- in an induction furnace (M/s. Lectroheat Induction, Mumbai) using specially designed semiautomatic gadgets. This is connected in series to a glassware system consisting of the tellurium trap (Glass Coil) and iodine trap (0.25 mg/ml Na 2 SO 3 solution). All reagents used were AR grade. Commercial grade Argon (carrier gas) was passed through the leak tight system with a fixed flow rate. Tellurium dioxide is heated using an inconel cylinder as a susceptor. Distillation yield was estimated by determining 131 I Activity in the target and in the trap using an HPGE-MCA system. RC purity was determined by paper chromatography. Te content was estimated by a spot test method. Results and Conclusion: A number of experiments were conducted at pilot scale (upto 148 GBq) to optimize process parameters like distillation temperature, carrier gas flow rate, TeO 2 trap etc. Distillation temperature is optimized at 740 ° C for three hours to obtain a uniform melting of the target. Carrier gas flow rate was optimized at 15±5 mL/min. Increase in the flow rate above 100 mL/min considerably increased carryover of TeO 2 . Tellurium trapping was studied using a glass coil trap. It was maintained at 100 0 C to reduce loss of 131 I. The trap controlled Te content below detection limit (<5 μg/mL) in the product. The optimized process has a distillation yield of 90±5%. The product 131 I as sodium iodide passes all the quality control tests and the entire process is optimized to reduce exposure to operating personnel and can be used for regular production of high purity 131 I as sodium iodide with minimum processing time, minimum waste volume, higher yield and higher radioactive concentration suitable for radiolabeling of the medically important bio-molecules.

RP-25

Synthesis and radiolabeling of rifampicin chelate conjugate: A possible SPECT imaging agent for infection

Ravi Hegde, Vijaya Raj KK, Ritika Mukherjee, Joseph Arukuwe 1 , Peter Iveson 2, Afsal K Mohammed


Medical Diagnostics R & D, GE Healthcare, Bangalore, India; 1 Medicinal Chemistry R & D, GE Healthcare, Oslo, Norway; 2 Medical Diagnostics R & D, GE Healthcare, Amersham, UK

Infectious diseases have historically been more prevalent and among the leading causes of death in developing nations, with the big three being respiratory infections, tuberculosis, and HIV/AIDS. (1) With rise in global awareness of the increased risk of co-infections (e.g. HIV-TB), multi-drug resistance and emerging infectious diseases such as avian influenza, there have been renewed research efforts on diagnostics, screening and therapeutics. Early and accurate diagnosis and localization of infection allow prompt and successful treatment and decrease associated morbidity. Currently, radiopharmaceuticals specially developed for discrimination between bacterial infections and sterile inflammatory processes are of great interest. (2) Radiolabeled leukocytes are still the radiopharmaceutical agents of choice used in the diagnosis of focal bacterial infection and inflammation. However, this technique is time-consuming, needs a sterile environment, and has risk associated with handling of potentially contaminated blood. There is still great interest in the development of new radiopharmaceuticals for infection imaging. Rifampicin (RIF; also known as rifampin) is a bactericidal antibiotic drug of the rifamycin group and is the most popular broad-spectrum antibacterial agent in this class. RIF inhibits bacterial ribonucleic acid (RNA) synthesis by binding to RNA polymerase. The RIF-RNA polymerase complex is extremely stable and as such an in vivo imaging based on RIF and retaining this property would have value in the diagnosis of a range of infections. Recently, Shah et al. (3) reported direct labeling of RIF with Technetium (Tc99m) for methicillin-resistant staphylococcus aureus (MRSA) infection imaging. This direct labeling of RIF may lead to reduced pharmacological activity and also structure of the Tc- complex is not well characterized by the authors. Moreover, their study was only restricted to infection caused by MRSA. Based on these observations we have decided to label RIF through a linker. Initially, RIF was converted to Schiff's base by reacting RIF aldehyde with N-Amino piperazine and later this was conjugated to a chelate followed by complexation with Tc99m. All the structures were confirmed by spectral analysis and performed initial antibacterial activity testing. Based on the in vitro results and radiolabeling results it is concluded that RIF-chelate-Tc99m could be a potential infection SPECT imaging agent. Further studies are in progress.

RP-26

Liver tumor Imaging using a Tc-99m labeled RGD peptide in a mouse model

Ritika Uppal Mukherjee, Sandipto Ghosh 1 , Vishakha Mangale, Vijaya Raj KK, Matthew Morrison 2 , Pradip Chaudhari 1 , Sudeshna Adak


GE Healthcare, Medical Diagnostics, Bangalore, 1 Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Center, Kharghar, Navi Mumbai, India, 2 GE Healthcare, Medical Diagnostics, Amersham, UK

Introduction: Targeting the molecular pathways of angiogenesis offers great potential in detecting disease pathology using in vivo imaging technologies such as MRI, SPECT and PET. Angiogenesis or the formation of new blood vessels is associated with a wide range of diseases, such as cancer, atherosclerosis, rheumatoid arthritis and chronic inflammation etc. The increased expression of two integrins, αvβ3 and αvβ5, has been associated with angiogenesis. A number of molecular imaging agents specific to αvβ3 have been developed. Of these imaging agents, radiolabeled probes based on the tri-peptide RGD have shown high affinity for αvβ3 integrin. The agent we have developed, Maraciclatide or NC100692, contains the RGD tri-peptide in a cyclic conformation with a disulfide bond and a thioether bridge (structure given below). The amino side chain of the lysine residue is used for the attachment of a chelate for technetium. Cyclic RGD peptides such as this have already proven their specificity for integrins. The increased uptake and retention of Maraciclatide has been demonstrated in animal models of ischemia and myocardial infarction. Clinical trials with this agent have also demonstrated its efficacy in targeting malignant tumors in breast cancer patients. Objective: Our study investigates the efficacy of this agent to image liver tumors in HT-29 xenograft mouse models. Based on our results we can evaluate the potential of this agent in assisting the prognosis, diagnosis and treatment monitoring of hepatic tumors. Materials and Methods: Maraciclatide was developed as an experimental kit at GE Healthcare, Oslo and was radiolabeled with Tc-99m. The vial containing the ligand was reconstituted with the sodium pertechnetate and allowed to stand for 20 min at room temperature. The radiochemical purity was evaluated by running ITLC using saline as the eluting solvent and was found to be >95%. Micro SPECT/CT protocol: All animals (naïve Swiss mice or SCID mice with subcutaneous tumors) were injected intravenously with >20 MBq of Tc-99m labeled Maraciclatide in a volume not more than 0.15 mL. The SPECT imaging was performed using the GE FLEX Triumph system. For the first 30 min, dynamic study was performed. This was followed by SPECT/CT imaging at 30 min, 60 min and 120 min post-injection. Immediately after the CT scan, the animals were sacrificed and blood, urine, brain, liver, lungs, kidneys, heart, spleen, intestine, muscle and bone were collected and weighed. Each tissue was counted at the dose calibrator and the%ID/g was determined. Results: Our preliminary results confirm previous studies and demonstrate that in control mice, Maraciclatide has a rapid clearance from blood and key background tissues. The predominate route of excretion for Marciclatide is via the kidneys and bladder, with some retention seen in liver tissue which is likely related to normal liver integrin expression and some non-specific binding. Due to the relatively low background uptake of Maraciclatide we will next explore the tracer's utility for the detection of tumors in the liver.

RP-27

Preparation and preliminary biological evaluation of 177 Lu-labeled porphyrin-DOTA conjugate as possible tumor targeting agent

Mohini Bhadwal, Sweety Singhal, Tapas Das, Sudipta Chakraborty, Haladhar Dev Sarma 1 , Sharmila Banerjee


Radiopharmaceuticals Division, 1 Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India

Introduction: Porphyrins and its derivatives exhibit inherent affinity for localization in tumors and hence they can behave as carrier ligands for the active transport of radionuclides into cancer cells. Therefore radiolabeled porphyrin derivatives can be envisaged as potential agents for tumor diagnosis and targeted tumor therapy. Keeping this in mind, an attempt was made to radiolabel a newly synthesized water soluble unsymmetrical porphyrin-DOTA conjugate with 177 Lu, for exploring its potential in targeted tumor therapy. 177 Lu was chosen as radionuclide owing to its suitable nuclear decay characteristics [T 1/2 =6.65 d, Eβ(max) =0.49 MeV, Eγ=208 keV (11%)] and ease of its large-scale production with adequately high specific activity and excellent radionuclidic purity using the medium flux research reactors available in our country. Materials and Methods: 177 Lu was produced by irradiation of enriched Lu 2 O 3 (74% in 176 Lu) at a thermal neutron flux of 1×10 14 n/cm 2 .s for 14 d. A water soluble unsymmetrical porphyrin namely, 5-(4-(3-amino)-n-propyloxyphenyl)-10,15,20-tris-(4-carboxymethyleneoxyphenyl)porphyrin was coupled with p-isothiocyanato-benzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-NCS-benzyl-DOTA) and the conjugate was radiolabeled with 177 Lu. The radiolabeling was carried out by incubating 100 μg of porphyrin-DOTA conjugate with 20 μL of 177 LuCl 3 (25 mCi/μg) in 200 μL of 0.1 M NH 4 OAc buffer at 60-70 0 C for 1 h, maintaining the pH between 4 and 5. The radiolabeling yield was determined by paper chromatography (50% aqueous acetonitrile as developing solvent) and reverse phase HPLC employing gradient elution technique (water and acetonitrile mixtures containing 0.1% trifluoroacetic acid as the mobile phase). The biological efficacy of the conjugate was studied in Swiss mice bearing fibrosarcoma tumor by biodistribution and scintigraphic imaging. Results: Porphyrin-DOTA conjugate was obtained in ~80% yield and its formation was confirmed from the satisfactory UV-Vis and 1 H-NMR spectra 177 Lu was obtained with a specific activity of ~925 TBq/g and radionuclidic purity of 99.98%. 177 Lu-DOTA-porphyrin conjugate was obtained in 80% radiolabeling yield as observed by paper chromatography and HPLC studies. Biodistribution studies revealed good tumor uptake (4.12% ID/g, ID: Injected Dose)) within 30 min p.i. which was observed to be retained till 48 h p.i. (4.5% ID/g), upto which the studies were continued. Tumor/muscle ratio was found to be 3.2 at 30 min p.i., which significantly increased to 6.56 at 48 h p.i. Fast clearance of the activity was observed from non-target tissue/organs including blood and muscles. Scintigraphy studies carried out in tumor bearing mice showed the accumulation of significant amount of activity in tumor which was retained till 3 d p.i. up to which the imaging studies were carried out. Conclusion: The radiolabeled 177 Lu-DOTA-porphyrin conjugate exhibited good tumor uptake and retention of activity in tumor with significant tumor to blood and tumor to muscle ratios. The scintigraphy studies carried out also showed similar findings. The encouraging results in preliminary biological studies indicate the possible potential of further development of 177 Lu-labeled porphyrin as an agent for targeted tumor therapy.

RP-28

177 Lu -DOTA-TATE synthesis: In house experience at RMC

Shimpi HH, Nabar SJ, Sonwane GA, Rajan MGR, Nayak UN


Radiation Medicine Centre, BARC, TMH Annexe, Parel, Mumbai, India

In the past few years, 177 Lu-DOTA-TATE has been extensively used in India for radio-receptor-therapy of various endocrine and other tumors. Recently, Radiation Medicine Centre has also started therapy with 177 Lu-DOTA-TATE. 177 Lu is a radio-lanthanide with half-life similar to 131 I, but with a lower energy gamma, more suited for scintigraphy and radiotherapy. 177 LuCl 3 - was supplied by Radiopharmacy Division (RPhD) BARC / BRIT with specific activity of about 20 - 40 mCi / μg to the various Centres and labeling of DOTA-TATE done at the individual centers. The radio-labeling of 177 Lu-DOTA-TATE for therapy was performed as per the protocol given by RPhD with high yield and purity. In this presentation, we wish to highlight a few modifications to the synthesis procedure, carried out at RMC, which are simple and easy to adopt and implement. The modifications fulfill radiation safety criteria such as distance, time and shielding in the synthesis procedure. The RPhD technique entails the additions of required volume of aqueous DOTA-TATE in a calculated volume of 0.1M-ammonium acetate buffer and gentisic acid followed by the addition of required volume of 177 LuCl 3 radioactivity. The pH of the resultant solution was adjusted to 4.0 - 4.5 by drop wise addition of NaOH solution. The reaction mixture incubated at 85° -90° C for about 60 min and after quality control assay the product was sterilized by 0.22μ membrane filtration. The RMC-modified procedure consists mainly of adjusting pH of the buffer solution itself to 4.0 - 4.5, instead of adjusting the pH in reaction vial, in the presence of radioactivity. Incubation of reaction mixture was carried out in a thermostatically controlled heater attached to a hot lead pot (no water used) in place of hot water bath. This reduced the time for complete synthesis by almost half and also the radiation exposure. Trials of this procedure were carried out at RMC, BARC, and examined with respect to its performance, quality, and radiation safety. It was found that the 177 Lu - DOTA - TATE synthesis, by the modified procedure also gave high purity and good yield. So far by using modified method some 15 patient doses were prepared. The simple to adopt modifications may be very helpful for those who routinely synthesize the 177 Lu - DOTA - TATE at their Centres.

RP-29

Evaluation of Tc99m-microspheres for lung deposition studies using gamma camera

Shimpi HH, Hazare Shruti 1 , Menon MD 1


Radiation Medicine Centre, BARC, Tata Memorial Centre, Annexe, Parel, Mumbai, 1 Bombay College of Pharmacy, Kalina, Mumbai, India

Gamma-Scintigraphy has been widely employed to study the pattern of deposition and clearance of radiolabeled particles from the lungs, which can provide a better idea of whether the microspheres are reaching to the site of tumor metastasis in lung. Reactive oxygen species (ROS) play various roles in the process of tumor metastasis and many cases the lungs are the first organs to be encountered by tumor cells, and this makes it major site for tumor metastasis. Localized antioxidant enzyme delivery to the lungs via inhalation can be a means to treat lung cancer. The objective of this work is to evaluate the deposition of Tc99m-antioxidant enzymes loaded microspheres and their retention in the animal's lungs. Catalase microspheres were formulated with Tc99m using stannous pyrophosphate in non-aqueous medium. Microspheres (20-80 mg) were suspended with stannous pyrophosphate in MEK solution and vortexed for 2-3 min. Tc99mO 4 (3-4 mCi) in MEK was then added to the above solution, mixed well and incubated for (5, 10 and 15 min) with intermittent shaking. Different parameters such as amount of microspheres, incubation time, stability of the complex, and particle size were studied for its effect on labeling efficiency. In vitro stability of labeled complex was also checked for 2 hrs. In-vivo deposition study of radiolabelled microspheres was carried in New Zealand white male rabbits. Rabbits weighing 1.5-3.0 kg and about 8-9 months of age. were placed in specially fabricated acrylic-plastic chamber. 40 mg of labeled microspheres (approximately 3-4 mCi) was aerosolized in the chamber using nebulizer accessory for 5 min. Rabbits were allowed to breathe normally in the chamber. Suitable precautions were taken to prevent oral ingestion of the activity. Images of rabbit's lung were taken at periodic intervals viz., 1 hrs, 2 hrs, 4hrs, and 24 hrs using gamma camera. Gamma camera images of Tc99m-labeled microspheres showed consistent lungs uptake and retention of the radioactivity in lungs for 24 hrs. This concludes that the antioxidant enzyme delivery system is specifically retaining in the lungs and it will help to treat the lungs metastasis or cancer cells in lungs.

RP-30

Radiolabeling and gamma scintigraphy of gastro-retentive nano-particles

HH Shimpi, NM Dand 1 , SS Shidhaye 1 , PC Chaudhury 2 , R Gaikwad 3 , A Samad 3 , V Kadam 3


Radiation Medicine Centre, BARC, TMH Annexe, Parel, Mumbai, 1 Bharati Vidyapeeth's College of Pharmacy, Sec - 8, C. B. D. Belapur, Navi Mumbai, 2 Advanced Centre for Treatment, Research and Education on Cancer, Sec - 22, Kharghar, Navi Mumbai, 3 Bombay Veterinary College, Parel, Mumbai, India

Cinnarizine (CIN) is an anti-histaminic drug, also used as a labyrinthine sedative and as a peripheral anti-vasoconstrictor agent in the treatment and prophylaxis of motion sickness. This drug is sparingly soluble in water and has a short half-life. Thus gastro retention combined with prolonged release of the drug is gaining importance in the drug-formulation due to its advantage of improved bioavailability. The use of gamma scintigraphy in clinical trials to evaluate the gastrointestinal transit and performance of oral pharmaceutical solid dosage forms is well established. Thus nano-particles of Cinnarizine were made with an aim of gastro-retention by muco-adhesion and assessed by using radiolabeling and gamma scintigraphy for its in vivo gastro retention studies. The optimized method for preparation of nano-particles of Cinnarizine (CIN) includes sodium alginate as the polymer of choice. A solution of CIN was mixed with 1% solution of Poloxamer 188 and this suspension was stirred till solvent completely evaporated. In this mixture, an aqueous solution of calcium chloride and sodium alginate was added dropwise under constant stirring. Finally 1% solution of soya lecithin was added and stirred. This gave nanoparicals of Cinnarizine after spray drying. The radiolabeling of this Cinnarizine was performed by using stannous chloride as reducing agent in the presence of Tc99mO 4 . Nanoparticles of CIN were suspended in water and incubated with 150 μg of SnCl 2 in dilute HCl. (Tc99m) Sodium pertechnetate was added, mixed well and incubated for 15 min. This suspension was centrifuged at 5000 rpm for 5 min. Supernatant was decanted and labeled particles were collected. The study of stability of the labeled particlals were performed by keeping this suspension in 0.1 M HCl upto 6 hr. and radioactiviy was measured at different time intervals. The gamma imaging studies were performed in 6 New Zealand white rabbits. Animals were subjected to over-night fasting and 2-3 mCi Tc99m-nanoparticles administered orally. Rabbits were placed in sitting position under camera and whole body images were taken at different time intervals (10 min, 0.5, 1, 2, 3, 4, 5 and 6 hrs) for a period of 6 hrs. The radioactivity retained was calculated and the gastro retention was assessed. The stability results of labeling nanoparticles showed about 99% activity retained with complex upto 6hrs, and scintigraphic images demonstrated specific uptakes in gastric region. We conclude that the labeled nanoparticles showed good labeling with longer stability. Gamma camera images showed accumulation activity in the gastric area only upto 24 hrs, which confirms the aim of establishing the concept of gastro retention of the formulation.

RP-31

Comparison of Tc-99m labeled methionine and C-11 methionine radiotracer in the detection of breast carcinomas

Sonia Mahajan, Madhavi Tripathi, Abhinav Jaimini, Maria M D'Souza, Krishna Chouttani 1 , Santosh Pandey, Harish Rawat, Dinesh Singh, AK Mishra 1 , Rajnish Sharma, Anupam Mondal


Department of PET Imaging, Institute of Nuclear Medicine and Allied Sciences (INMAS), New Delhi, 1 DCRS, INMAS, New Delhi, India

Introduction: The cost effectiveness and non-availability of Cyclotron in underdeveloped and developing countries is a basic problem. Therefore studies were undertaken after labeling Methionine with generator produced Tc-99m for its possible use in breast cancer imaging. Objective: To compare C-11 methionine (MET) and Tc-99m methionine radiopharmaceuticals in primary staging of breast carcinomas. Materials and Methods: 12 patients with biopsy proven carcinoma breast have been included in the study, so far. All patients underwent C-11 MET PET/CT and Tc99m labeled methionine SPECT-CT scans. Scans were done on two different days. 20mCi of C-11 MET was injected intravenously and images were acquired on PET/CT scanner after 20 min. Next day, 20mCi of Tc99m MET was injected intravenously and planar/SPECT images were acquired on gamma camera after 30 min, 1 hr and 2 hrs. Results: Accumulation of C-11 methionine was assessed visually and by calculating SUV values. Assessment of Tc-99m methionine was done visually and by calculating tumor to background ratio and the results were compared. Both Tc99m methionine and C-11 methionine show increased accumulation at the primary site. Apart from this, increased C-11 MET accumulation is noted in the malignant ipsilateral axillary lymph nodes. However, there is limited concentration of Tc99m MET in the same anatomical region. Conclusion: Our preliminary study reveals that technetium methionine has high potential to be used as an alternative tracer to C-11 methionine in patients of breast cancer. Further clinical trials shall be conducted to evaluate the role of Tc99m methionine in patients of breast cancer especially nodal and metastatic involvement.

RP-32

Design of DTPA-(bis amide) as AChE inhibitors for quantifying nerve gases activity

Vikas S Rathore 1,2 , Anjani K Tiwari 1 , Neeta Sehgal 2 , Anil K Mishra 1


1
Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Brig. S. K. Mazumdar Road, Delhi, 2 Department of Zoology (Center for Advanced Studies), University of Delhi, Delhi, India

Introduction: DTPA can be easily and efficiently labelled with radionuclide such as Tc99m, and 111 In with high radiochemical purity and stability. However, its passive and nonspecific distribution in vivo limits its utility. Tc99m-DTPA-bis (amide) derivatives have been successfully used as target specific radiopharmaceutical. Some of the conjugates of DTPA-Amino acids exhibited the properties of native amino acids as well as show strong binding affinity in selective cells specifically in nerve region. Molecular Modelling Study: To gain insight into the molecular determinants that modulate the inhibitory activity, a molecular modeling study was performed to explore the possible ligand- AChE (acetylcholinesterase) interaction. Docking studies of Diethylene triamine penta acetic acid (DTPA)- amino acid derivatives on the active site of TcAChE inhibited by Rivastigmine (PDB: 1GQR) revealed their mode of interaction, structural and positional requirements for potential AChE inhibition. The synthesized compounds was characterized by different spectroscopic techniques (FTIR, NMR and mass spectroscopy) and later radiolabelled with Tc99m. Results: Induced fit docking of 1GQR with the 10 different DTPA-(bis amide) compounds (a- j) show the binding with a GLIDE (grid based ligand docking with energetic) Score of-16.035, -13.951, -13.455, -12.684, -12.298, -11.275, -10.540, -9.901, -8.840 and -4.460 (more negative better the fit) respectively. However, the nerve gasses (Soman, Sarin, DFP, DCP and Tabun) show comparatively far less GLIDE G Scores -6.947, -5.813, -3.860, -3.754 and -3.430. That suggested the better binding affinity of DTPA-(bis amide) analogues with the target AChE than their competitor nerve gases. Through interaction analysis, it was found that Asp72, Asn85, Tyr121, Tyr130, Trp279, Phe330 and Hie440 were important anchoring residues for the DTPA compounds and is the main contributors to the inhibitor interaction. It forms stable complex with Tc99m with high radiochemical purity (97%) and showed significant accumulation in brain as well as in vitro validation with brain homogenate for AChE assay. Conclusion: Our goal of this study was to investigate newly designed candidates as AChE inhibitors against nerve gasses.




 

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