Mythili Kameswaran

Detection of antibodies to defined M. tuberculosis antigen (38 kDa) in cerebrospinal fluids of patients with tuberculous meningitis.

Antibodies to the 38 kDa antigen of M. tuberculosis which is serospecific to the tuberculosis complex group of organisms was studied in CSF samples of patients with tuberculosis meningitis. Patients were classified into four groups, viz. post-mortem-proved, culture-proved, clinically suspected and tuberculoma. Anti-38 kDa antibody was detected by ELISA and was positive in 60%, 80% 62.5% and 0%, respectively in the four groups. Controls showed a false-positive detection of 5%. Follow-up of patients was done up to 6 weeks and antibody levels dropped in all the patient groups.

99mTc carbonyl DTPA–Rituximab: Preparation and preliminary bioevaluation

The anti CD20 antibody Rituximab was conjugated with para isothiocyanato benzyl diethylene triamine penta acetic acid (p-NCS-Bz-DTPA) and subsequent radiolabeling with (99m)Tc was carried out via the (99m)Tc carbonyl synthon. The (99m)Tc labeled antibody conjugate exhibited >95% radiochemical purity after purification and retained good in vitro stability when studied up to 24h at room temperature. In vitro cell binding studies carried out in Raji cells expressing CD20 antigen validated the biological efficacy of the preparation.

A Freeze-Dried Kit for the Preparation of 188Re-HEDP for Bone Pain Palliation: Preparation and Preliminary Clinical Evaluation

(188)Re-HEDP is an established radiopharmaceutical used for pain palliation in patients with osseous metastasis. Considering commercial availability of (188)W/(188)Re generator, the accessibility to a lyophilized kit would make preparation of this radiopharmaceutical feasible at the hospital radiopharmacy having access to a generator. A protocol for the preparation of a single-vial lyophilized hydroxyethane 1,1-diphosphonic acid (HEDP) kit was developed and its consistency was checked by preparing six batches. Each sterile lyophilized kit prepared as per the protocol contained 9 mg of HEDP, 3 mg of gentisic acid, and 4 mg of SnCl2.2H2O. Randomly selected kits from all six batches were subjected to thorough quality control tests that were passed by all batches. (188)Re-HEDP could be prepared by addition of 1 mL of freshly eluted Na(188)ReO4 (up to 3700 MBq) containing 1 μmol of carrier ReO4(-) (perrhenate) and heating at 100°C for 15 minutes. (188)Re-HEDP with >95% radiochemical purity could be consistently prepared using the lyophilized kits. Sterile (188)Re-HEDP prepared using the lyophilized kit was evaluated in patients with osseous metastasis. Post-therapy images of the patient were compared with (99m)Tc-MDP bone scan and found to be satisfactory. The bone-to-background as well as tumor-to-normal bone uptake ratio was found to be significant. All patients who received therapy reported significant pain relief within a week to 10 days post-administration of (188)Re-HEDP.

Stereoselective synthesis of an iodinated resveratrol analog: preliminary bioevaluation studies of the radioiodinated species.

Stereoselective synthesis of an E-hydroxystilbene has been carried out using the McMurry reaction. Synthesis of a monoiodinated hydroxystilbene has been carried out by a McMurry cross-coupling reaction. For the purpose of biological evaluation, the facile electrophilic substitution route has been attempted to radioiodinate it with (125)I. The HPLC pattern of the radioiodinated hydroxystilbene, which could be obtained in >90% radiochemical purity, was found to be identical to that of its non-radioactive analog that has been independently prepared using the McMurry cross-coupling route. In vitro cell uptake studies were carried out in breast cancer cells MCF7, overexpressing estrogen receptors. In vivo biodistribution studies in female Swiss mice show a uterine uptake of 0.85±0.4% ID/g at 3h.p.i. with a uterus to muscle ratio of 2.83. Uptake in the thyroid was insignificant indicating good in vivo stability of the radioiodinated hydroxystilbene.

Clinical scale preparation and evaluation of 131I-Rituximab for Non-Hodgkin's Lymphoma

Abstract Radioimmunotherapy (RIT) with anti CD20 MoAb conjugated to a β− emitting radioisotope like 131I or 90Y has the added advantage of delivering radiation not only to tumor cells that bind the antibody but also due to a crossfire effect, to neighboring tumor cells inaccessible to the antibody. In order to make available an indigenous radioimmunotherapeutic agent for Non Hodgkins Lymphoma (NHL), radioiodinated Rituximab has been prepared and evaluated at a clinical scale. Radioiodination of Rituximab was performed by the conventional Chloramine T method using 7.4 GBq Na131I in a lead shielded plant. Six batches of radioiodination were prepared and characterized by electrophoresis and HPLC to evaluate the reproducibility of the product. The product remained stable retaining the radiochemical purity > 95% upto 5 days after radioiodination. In vitro cell binding studies and biodistribution studies in normal Swiss mice have indicated the potential of this molecule as a radioimmunotherapeutic agent for NHL.

Preparation & in vitro evaluation of 90 Y-DOTA-rituximab

Background & objectives: Radioimmunotherapy is extensively being used for the treatment of non-Hodgkins lymphoma (NHL). Use of rituximab, a chimeric anti-CD20 antibody directed against the CD20 antigen in combination with suitable beta emitters is expected to result in good treatment response by its cross-fire and bystander effects. The present work involves the conjugation of p-isothiocyanatobenzyl DOTA (p-SCN-Bn-DOTA) to rituximab, its radiolabelling with 90Y and in vitro and in vivo evaluation to determine its potential as a radioimmunotherapeutic agent. Methods: Rituximab was conjugated with p-SCN-Bn-DOTA at 1:1 antibody: DOTA molar ratio. The number of DOTA molecules linked to one molecule of rituximab was determined by radioassay and spectroscopic assay. Radiolabelling of rituximab with 90Y was carried out and its in vitro stability was evaluated. In vitro cell binding studies were carried out in Raji cells expressing CD20 antigen. Biodistribution studies were carried out in normal Swiss mice. Results: Using both radioassay and spectroscopic method, it was determined that about five molecules of DOTA were linked to rituximab. Radiolabelling of the rituximab conjugate with 90Y and subsequent purification on PD-10 column gave a product with radiochemical purity (RCP) > 98 per cent which was retained at > 90 per cent up to 72 h when stored at 37°C. In vitro cell binding experiments of 90Y-DOTA-rituximab with Raji cells exhibited specific binding of 20.7 ± 0.1 per cent with 90Y-DOTA-rituximab which reduced to 15.5 ± 0.2 per cent when incubated with cold rituximab. The equilibrium constant Kd for 90Y-DOTA-Rituximab was determined to be 3.38 nM. Radiolabelled antibody showed clearance via hepatobiliary and renal routes and activity in tibia was found to be quite low indicating in vivo stability of 90Y-DOTA-rituximab. Interpretation & conclusions: p-SCN-Bn-DOTA was conjugated with rituximab and radiolabelling with 90Y was carried out. In vitro studies carried out in Raji cells showed the specificity of the radiolabelled conjugate suggesting the potential uitability of the formulation as a radiopharmaceutical for therapy of NHL.

68Ga labeled Erlotinib: A novel PET probe for imaging EGFR over-expressing tumors

Molecular imaging using radiolabeled Tyrosine Kinase Inhibitors (TKI) is a promising strategy for detection and staging of EGFR-positive cancers. A novel analogue of one such TKI, Erlotinib has been developed for PET imaging by derivatizing the parent Erlotinib molecule for conjugation with the bifunctional chelator p-SCN-Bn-NOTA towards radiolabeling with 68Ga. NOTA-Erlotinib conjugate was synthesized and characterized by NMR and ESI-MS techniques. The conjugate was radiolabeled with 68Ga in 95±2% yield, as evidenced by HPLC characterization. The logP value of 68Ga-NOTA-Erlotinib was - (0.6±0.1). The 68Ga-NOTA-Erlotinib conjugate was characterized using its natGa-NOTA-Erlotinib surrogate. Cell viability studies showed that the NOTA-Erlotinib conjugate retained the biological efficacy of the parent Erlotinib molecule. Further, 68Ga-NOTA-Erlotinib exhibited an uptake of 9.8±0.4% in A431 cells which was inhibited by 55.1±0.2% on addition of cold Erlotinib (10µg) confirming the specificity of the radioconjugate for EGFR expressing cells. In the biodistribution studies carried out in tumor bearing SCID mice, 68Ga-NOTA-Erlotinib conjugate showed moderate tumor accumulation (1.5±0.1% ID/g at 30minp.i.; 0.7±0.2% ID/g at 1hp.i.). Hepatobiliary clearance of the radioconjugate was observed. The 68Ga-NOTA-Erlotinib conjugate was found to have high in vivo stability as determined by the metabolite analysis study using urine sample of the Swiss mice injected with the preparation. The overall properties of 68Ga-NOTA-Erlotinib are promising and merit further exploration. To the best of our knowledge, this is the first report on the design of a 68Ga labeled Erlotinib for PET imaging of EGFR and opens avenues for the successful development of 68Ga labeled TKI for imaging of EGFR over-expressing tumors.

Single vial cold kits optimized for preparation of gastrin releasing peptide receptor (GRPR)-radioantagonist 68Ga-RM2 using three different 68Ge/68Ga generators

&NA; 68Ga‐RM2 is a gastrin releasing peptide receptor (GRPR) antagonist PET (positron emission tomography) radiotracer which is being investigated in clinical trials as a potential prostate cancer imaging agent. Simple, one‐step kit formulation of 68Ga‐RM2 would facilitate multicentre trials and allow easier integration in hospital radiopharmacy. Herein we report development of three sets of single‐vial RM2 cold kits validated for formulation with three respective 68Ge/68Ga generators eluted in 0.6 M, 0.1 M and 0.05 M HCl (hydrochloric acid). Cold kits of varied pH (2, 3, 4 and 5) were prepared using 2 M sodium acetate for three different 68Ge/68Ga generators to determine influence of pH on the radiochemical yield of 68Ga‐RM2. Buffer content was optimized with respect to volume of 68GaCl3 eluate to be added (1 mL/2 mL/ 5 mL). Sterility, apyrogenicity and long term stability of cold kits; in vitro and serum stability of 68Ga‐RM2 were investigated. In vitro cellular uptake and inhibition studies were performed to demonstrate the specificity of kit‐formulated 68Ga‐RM2. The radiochemical yield of 68Ga‐RM2 formulated from three different generators was observed to be maximum at pH 3 (99 ± 0.5%). Cold kits stored for 6 months at 0 °C also resulted in high radiochemical yield. 68Ga‐RM2 exhibited excellent in vitro stability (1 h) and serum stability (1 h). In vitro cellular uptake of 5 ± 0.8% in PC3 cells with >85% inhibition was observed for the 68Ga‐RM2 radiotracer indicating its specificity towards GRPR expression. These simple, robust kits shall allow hospitals with different generators to participate in clinical studies of 68Ga‐RM2 for screening of GRPR‐expressing prostate tumors.

Preclinical evaluation of 131I-Bevacizumab – A prospective agent for radioimmunotherapy in VEGF expressing cancers

This study focuses on preparation and evaluation of 131I-bevacizumab by Iodogen method for targeting VEGF over-expressing cancers for therapy. 131I-Bevacizumab exhibited radiochemical purity of 98.0±0.7%. In vitro stability of 131I-Bevacizumab was retained at >85% in both saline and serum at 37°C upto 5 days post iodination. In vitro cell studies showed good immunoreactivity and uptake by VEGF expressing tumor cells. Uptake and retention of 131I-Bevacizumab in tumor with reduction in uptake in presence of cold Bevacizumab confirmed its specificity to VEGF.

Preliminary evaluation of the potential of 99mTc carbonyl–DTPA–Rituximab as a tracer for sentinel lymph node detection

Preliminary work with (99m)Tc carbonyl-DTPA-Rituximab was attempted to test its feasibility as a sentinel lymph node (SLN) tracer for patients with breast cancer. (99m)Tc labeling of DTPA-Rituximab conjugate was carried out via (99m)Tc carbonyl synthon which exhibited >95% radiochemical purity and good in vitro stability. In vitro studies of (99m)Tc carbonyl-DTPA-Rituximab in normal and malignant B cells showed higher binding in malignant cells. In vivo distribution of (99m)Tc carbonyl-DTPA-Rituximab in Wistar rat footpad model indicated good retention by B-cells present in the sentinel lymph node.