Analysis of absorbed dose in radioimmunotherapy with 177Lu-trastuzumab using two different imaging scenarios: a pilot study.

OBJECTIVES: Internal organ dosimetry is an important procedure to demonstrate the reliable application of 177Lu-trastuzumab radioimmunotherapy for human epidermal growth factor receptor-positive metastatic breast cancers. We are reporting the first human dosimetry study for 177Lu-trastuzumab. Another objective of our study was to calculate and compare the absorbed doses for normal organs and tumor lesions in patients before radioimmunotherapy with 177Lu-trastuzumab using two different imaging scenarios. METHODS: Eleven patients (48.27 ± 8.95 years) with a history of metastatic breast cancer were included in the study. Postadministration of 177Lu-trastuzumab (351.09 ± 23.89 MBq/2 mg), acquisition was performed using planar and hybrid imaging scenarios at 4, 24, 72 and 168 h. Single-photon emission computed tomography/computed tomography imaging was performed at 72 h postinjection. Acquired images were processed using Dosimetry Toolkit software for the estimation of normalized cumulated activity in organs and tumor lesions. OLINDA/EXM 2.0 software was used for absorbed dose calculation in both scenarios. RESULTS: Significant difference in normalized cumulated activity and the absorbed dose is noted between two imaging scenarios for the organs and tumor lesions (P < 0.05). Mean absorbed dose (mGy/MBq) estimated from heart, lungs, liver, spleen, kidney, adrenal, pancreas and colon using planar and hybrid scenarios were 0.81 ± 0.19 and 0.63 ± 0.17; 0.75 ± 0.13 and 0.32 ± 0.06; 1.26 ± 0.25 and 1.01 ± 0.17; 0.68 ± 0.22 and 0.53 ± 0.16; 0.91 ± 0.3 and 0.69 ± 0.24; 0.18 ± 0.04 and 0.11 ± 0.02; 0.25 ± 0.22 and 0.09 ± 0.02 and 0.75 ± 0.61 and 0.44 ± 0.28, respectively. CONCLUSIONS: On the basis of our dosimetric evaluation, we concluded that radioimmunotherapy with 177Lu-trastuzumab is well tolerated to be implemented in routine clinical practice against HER2 positive metastatic breast cancer. Liver is the main critical organ at risk. Hybrid scenario demonstrated significantly lower absorbed doses in organs and tumors compared to the multiplanar method.

Critical APJ receptor residues in extracellular domains that influence effector selectivity.

Human APJ receptor/apelin receptor (APJR), activated by apelin peptide isoforms, regulates a wide range of physiological processes. The role of extracellular loop (ECL) domain residues of APJR in ligand binding and receptor activation has not been established yet. Based on multiple sequence alignment of APJ receptor from various organisms, we identified conserved residues in the extracellular domains. Alanine substitutions of specific residues were characterized to evaluate their ligand binding efficiency and Gq -, Gi -, and ß-arrestin-mediated signaling. Mutation-dependent variation in ligand binding and signaling was observed. W197 A in ECL2 and L276 L277 W279 -AAA in ECL3 were deficient in Gi and ß-arrestin signaling pathways with relatively preserved Gq -mediated signaling. T169 T170 -AA, Y182 A, and T190 A mutants in ECL2 showed impaired ß-arrestin-dependent cell signaling while maintaining G protein- mediated signaling. Structural comparison with angiotensin II type I receptor revealed the importance of ECL2 and ECL3 residues in APJR ligand binding and signaling. Our results unequivocally confirm the specific role of these ECL residues in ligand binding and in orchestrating receptor conformations that are involved in preferential/biased signaling functions.

Dose-dependent cell cycle arrest and apoptosis in HER2 breast cancer cells by177Lu-CHX-A"-DTPA-Trastuzumab.

BACKGROUND: Trastuzumab is a Food and Drug Administration-approved humanized monoclonal antibody which targets the extracellular domain of human epidermal growth factor receptor 2 (HER2) receptor overexpressed on HER2-positive breast cancer cells. The combination of Lutetium-177 (177 Lu) (t½= 6.7 days, Eßmax497 keV (78.6%) and trastuzumab makes it a suitable targeting agent for radioimmunotherapy. In preclinical and clinical studies,177 Lu-Trastuzumab has proven to be effective for the treatment of HER2-positive malignancies such as breast and ovarian cancer. OBJECTIVES: In this study, we report the mechanism of action of177 Lu-CHX-A"-diethylenetriaminepentaacetic acid (DTPA)-trastuzumab at the cellular and molecular level by performing various in vitro assays in HER2-positive MDA-MB-453 breast cancer cells. MATERIALS AND METHODS: Trastuzumab was conjugated to the bifunctional chelating agent (BFCA) para-isothiocyanatobenzyl-DTPA and radiolabeled with177 Lu. In vitro cell binding studies were carried out in MDA-MB-453 cells to confirm the specificity of the complex toward the receptor. Cellular toxicity, cell cycle, and cell death analysis were also performed for exploring the potential of the radioimmunoconjugate at cellular and molecular level. RESULTS: In vitro cell binding studies showed a maximum binding of 10.7 ± 0.1% which reduced to 2.9 ± 0.1% on coincubation with unlabeled antibody. Our study revealed that the cellular toxicity was dose dependent, and mode of cell death was predominantly by apoptosis. The radioimmunoconjugate retarded the cell in the S phase of cell cycle with two-fold increase in G2/M arrest which justifies the enhanced apoptosis at higher doses. CONCLUSIONS: The study revealed that the formulation can execute a dose-dependent cellular toxicity through induction of apoptosis.

Induction of different cellular arrest and molecular responses in low EGFR expressing A549 and high EGFR expressing A431 tumor cells treated with various doses of 177Lu-Nimotuzumab.

INTRODUCTION: Radioimmunotherapy (RIT) is a major anti-cancer therapy in cancer management multimodalities. 177Lu-Nimotuzumab has been in the use for radioimmunotherapy of EGFR expressing tumors. This study focuses on understanding the differential cellular and molecular mechanisms of anti-tumor effects of 177Lu-Nimotuzumab on low EGFR expressing A549 and high EGFR expressing A431 tumor cells. MATERIALS AND METHODS: Nimotuzumab labeled with 177Lu was characterized by SE-HPLC. Specificity of 177Lu-Nimotuzumab to EGFR expressed on A549 and A431 cells was confirmed by competitive assay using increasing amounts of unlabeled Nimotuzumab. Cellular responses of A549 (low EGFR) and A431 (high EGFR) in response to different doses of 177Lu-Nimotuzumab were determined by Viable count assay for cellular viability, cell-cycle analysis by DNA staining, apoptotic assay for cell death, and CFSE dilution assay for cellular proliferation capacity. The number of DNA DSBs formed was determined using γ-H2AX assay with PI staining. Transcription of genes involved in DNA damage response and repair (DRR) pathways was monitored by RT-qPCR. RESULTS: 177Lu-Nimotuzumab characterized by SE-HPLC exhibited a radiochemical purity of 99.1 ± 0.6%. Cell binding competition studies with 177Lu-Nimotuzumab showed specific binding of 34.3 ± 1.7% with A431 cells and 18.4 ± 1.9% with A549 cells which decreased when co-incubated with unlabeled Nimotuzumab. Cytotoxicity and DNA damage (DNA DSBs) increased with an increase in the dose of 177Lu-Nimotuzumab. A549 displayed G2/M arrest while A431 showed G1 arrest. Apoptotic death was determined to be one of the modes of death of arrested A549 and A431 cells which increases with the increase in the dose of 177Lu-Nimotuzumab. Loss of proliferation capacity was higher in A431 showed by CFSE staining at different doses of 177Lu-Nimotuzumab. Transcription profile of most DRR genes in A431 and A549 showed a decrease in the transcription at 4 h followed by recovery at 16 h post-treatment. The degree of transcription of most DRR genes was similar, irrespective of 177Lu-Nimotuzumab dose. CONCLUSION: 177Lu-Nimotuzumab induces different cellular arrest and molecular responses in low EGFR expressing A549 and high EGFR expressing A431 tumor cells. This study would enable the development of integrative novel treatment strategies for radioimmunotherapy in low and high EGFR expressing tumors by 177Lu-Nimotuzumab with therapeutic gains.

Comparative In Vitro Cytotoxicity Studies of 177Lu-CHX-A″-DTPA-Trastuzumab and 177Lu-CHX-A″-DTPA-F(ab')2-Trastuzumab in HER2-Positive Cancer Cell Lines.

Background: Human epidermal growth factor receptor 2 (HER2) is found to be amplified in ∼15%-20% of breast cancers. In this study, the authors report the synthesis and comparative in vitro therapeutic efficacy of 177Lu-CHX-A″-DTPA-trastuzumab and 177Lu-CHX-A″-DTPA-F(ab')2-trastuzumab to determine their potential as theranostic agents for patients with breast cancer. Materials and Methods: Bivalent F(ab')2-trastuzumab was produced by enzymatic digestion of trastuzumab, conjugated with p-SCN-Bn-CHX-A″-DTPA and subsequently radiolabeled with 177Lu. Cell viability, membrane toxicity assays, and apoptosis analysis were carried out with 177Lu-CHX-A″-DTPA-trastuzumab and 177Lu-CHX-A″-DTPA-F(ab')2-trastuzumab in HER2-positive ovarian (SK-OV-3) and breast cancer (SK-BR-3 and MDA-MB-453) cells. Results: In vitro cell binding studies showed ∼20%-25% binding of 177Lu-CHX-A″-DTPA-trastuzumab and 177Lu-CHX-A″-DTPA-F(ab')2-trastuzumab to SK-OV-3, SK-BR-3, and MDA-MB-453 cells. The cells exhibited similar degree of membrane integrity and cellular toxicity when treated with same amount (activity) of 177Lu-CHX-A″-DTPA-F(ab')2-trastuzumab and 177Lu-CHX-A″-DTPA-trastuzumab, and the toxicity was dose dependent. The mode of cell death was predominantly by apoptosis and necrosis with both the radioimmunoconjugates. Conclusions: The results indicated that the efficacy of both the radioimmunoconjugates, in terms of inducing cell death, was similar thereby ascertaining their potential as good therapeutic agents for patients with breast cancer.

Preparation and preliminary bioevaluation studies of 68 Ga-NOTA-rituximab fragments as radioimmunoscintigraphic agents for non-Hodgkin lymphoma.

Rituximab is used for the treatment of non-Hodgkin lymphoma (NHL). This study focuses on development of 68 Ga-labeled rituximab fragments, (68 Ga-NOTA-F (ab')-rituximab and 68 Ga-NOTA-F (ab')2 -rituximab, as PET-imaging agents for NHL. Rituximab was digested with immobilized pepsin and papain to yield F (ab')2 and Fab fragments respectively that were characterized by size exclusion HPLC (SE-HPLC) and SDS-PAGE. They were conjugated with p-SCN-Bn-NOTA, labeled with 68 Ga and characterized by SE-HPLC. Intact rituximab was labeled with gallium-68 for comparison. Specificity of 68 Ga-labeled immunoconjugates was ascertained by immunoreactivity and cell binding studies in Raji cells, while biodistribution studies were performed in normal Swiss mice. Gradient SDS-PAGE under nonreducing condition showed molecular weights of F (ab')2 -rituximab and F (ab')-rituximab as approximately 100 and 40 Kd, respectively. Radiochemical purity (RCP) of 68 Ga-NOTA-F (ab')2 -rituximab and 68 Ga-NOTA-F (ab')-rituximab were 98.2 ± 0.5% and 98.8 ± 0.2% respectively with retention times of 17.1 ± 0.1 min and 19.3 ± 0.1 min in SE-HPLC. 68 Ga-labeled rituximab fragments were stable in saline and serum up to 2-hour post preparation and exhibited specificity to CD20 antigen. Immunoreactivity of 68 Ga-labeled immunoconjugates was greater than 80%. Clearance of the fragmented radioimmunoconjugates was predominantly through renal route. Preliminary results from this study demonstrate the potential of 68 Ga- NOTA-F (ab')2 -rituximab and 68 Ga-NOTA-F (ab')-rituximab as PET imaging agents for NHL.

Synthesis, radiolabeling, and evaluation of gastrin releasing peptide receptor antagonist 68 Ga-HBED-CC-RM26.

The acyclic chelator HBED-CC has attained huge clinical significance owing to high thermodynamic and kinetic stability of 68 Ga-HBED-CC chelate. It provides an excellent platform for quick preparation of 68 Ga-based radiotracers in high yield. Thus, the present study aimed at conjugation of gastrin releasing peptide receptor (GRPr) antagonist, RM26, with HBED-CC chelator for 68 Ga-labeling. In vitro and vivo behavior of the peptide tracer, 68 Ga-HBED-CC-PEG2 -RM26, was assessed and compared with 68 Ga-NODAGA-PEG2 -RM26. The peptide tracers, 68 Ga-HBED-CC-PEG2 -RM26 and 68 Ga-NODAGA-PEG2 -RM26, prepared either by wet chemistry or formulated using freeze-dried kits exhibited excellent radiochemical yield and in vitro stability. The two peptide tracers cleared rapidly from the blood. Biodistribution studies in normal mice demonstrated slightly higher or comparable uptake of 68 Ga-HBED-CC-PEG2 -RM26 in GRPr-expressing organs pancreas, stomach, and intestine. The preliminary studies suggest high potential of 68 Ga-HBED-CC-PEG2 -RM26 for further investigation as a GRPr imaging agent and the wide scope of HBED-CC chelator in development of 68 Ga-based peptide tracers.

Synthesis and preliminary evaluation of 99mTc-Hynic-fragments [F(ab')2 and F(ab')] of Rituximab as radioimmunoscintigraphic agents for patients with Non-Hodgkin's lymphoma.

This study was to evaluate the potential of 99mTc-Hynic-fragments of Rituximab as radioimmunoscintigraphic agents for diagnosis of patients with Non-Hodgkin's Lymphoma (NHL). Rituximab was digested with immobilized pepsin and papain to yield F(ab')2-Rituximab and F(ab')-Rituximab fragments respectively. Purified fragments were characterized by SE-HPLC and SDS-PAGE and subsequently radiolabeled with technetium-99m using Hynic as bifunctional chelator. The 99mTc-Hynic-F(ab')2-Rituximab and 99mTc-Hynic-F(ab')-Rituximab exhibited good in-vitro stability and specificity to Raji cells. Biodistribution studies demonstrated rapid pharmacokinetics and clearance predominantly through renal route.

Design, synthesis, and comparative evaluation of 99m Tc(CO)3 -labeled N-terminal and C-terminal modified asparagine-glycine-arginine peptide constructs.

The present study describes modification of asparagine-glycine-arginine (NGR) peptide at N-terminally and C-terminally by introduction of a tridentate chelating scaffold via click chemistry reaction. The N-terminal and C-terminal modified peptides were radiometalated with [99m Tc(CO)3 ]+ precursor. The influence of these moieties at the two termini on the targeting properties of NGR peptide was determined by in vitro cell uptake studies and in vivo biodistribution studies. The two radiolabeled constructs did not exhibit any significant variation in uptake in murine melanoma B16F10 cells during in vitro studies. In vivo studies revealed nearly similar tumor uptake of N-terminally modified peptide construct 5 and C-terminally construct 6 at 2 h p.i. (1.9 ± 0.1 vs 2.4 ± 0.2% ID/g, respectively). The tumor-to-blood (T/B) and tumor-to-liver (T/L) ratios of the two radiometalated peptides were also quite similar. The two constructs cleared from all the major organs (heart, lungs, spleen, stomach, and blood) at 4 h p.i. (<1% ID/g). Blocking studies carried out by coinjection of cCNGRC peptide led to approximately 50% reduction in the tumor uptake at 2 h p.i. This work thus illustrates the possibility of convenient modification/radiometalation of NGR peptide at either N- or C-terminus without hampering tumor targeting and pharmacokinetics.

Preparation of 177Lu-Trastuzumab injection for treatment of breast cancer.

The objective of this study was the facile preparation of 177Lu-CHX-A''-DTPA-Trastuzumab injection for breast cancer therapy. Trastuzumab conjugated with CHX-A''-DTPA-NCS was radiolabeled with 177Lu in >95% radiochemical purity. In vitro studies in SKBR3 and MDA-MB-453 cells confirmed specificity of 177Lu-CHX-A''-DTPA-Trastuzumab to HER2 positive cells. The radioimmunoconjugate showed good immunoreactivity, in vitro stability in saline and Kd of 1.01 ±â€¯0.13 nM in SKBR3 cells. Clearance of 177Lu-CHX-A''-DTPA-Trastuzumab in Swiss mice was predominantly through the hepatobiliary route with minimal bone uptake.

Molecular determinants on extracellular loop domains that dictate interaction between ß-arrestin and human APJ receptor.

The human APJ receptor (APJR), activated by apelin isoforms, regulates cardiovascular functions and fluid homeostasis. Understanding its structure-function relationship is crucial for a comprehensive knowledge of signalling aberrations that cause several physiological disorders. Here, we demonstrate the influence of extracellular loop (ECL) domains in the mechanism of ß-arrestin-mediated signalling from human APJR: Apelin system. Alanine mutations of evolutionarily conserved residues were characterized using receptor internalization, ß-arrestin pull-down, Akt phosphorylation and cell migration assay. C281A and 268 KTL270 -AAA in ECL3 were deficient in all assays, whereas 183 MDYS186 -AAAA mutant in ECL2 showed impaired ß-arrestin-mediated signalling but demonstrated Gi -dependent cell migration. Our findings establish that conserved residues in the extracellular domain play a prominent role in modulating receptor interactions with the ß-arrestin signalling cascade.

Preparation of 177 Lu-labeled Nimotuzumab for radioimmunotherapy of EGFR-positive cancers: Comparison of DOTA and CHX-A″-DTPA as bifunctional chelators.

This study was aimed at evaluating the role of bifunctional chelators DOTA-NCS and CHX-A″-DTPA-NCS used for conjugating 177 Lu with Nimotuzumab on the radiochemical yields, purity, in vitro stability, and specificity of the radioimmunoconjugates to EGFR. Two immunoconjugates were prepared wherein Nimotuzumab was conjugated with the acyclic ligand p-NCS-Bn-CHX-A″-DTPA and macrocyclic ligand p-NCS-Bn-DOTA. These were radiolabeled with 177 Lu, purified on PD-10 column, and characterized by SE-HPLC. In vitro stability was determined up to 4 days post preparation. Specificity of the radioimmunoconjugates was ascertained by in vitro studies in A431 cells while the biodistribution patterns were studied in normal Swiss mice up to 96 hours post injection. Four to five molecules of CHX-A″-DTPA/DOTA were attached to one molecule of Nimotuzumab. Radiochemical purity of both 177 Lu-CHX-A″-DTPA-Nimotuzumab and 177 Lu-DOTA-Nimotuzumab was determined to be greater than 98%. Both the radioimmunoconjugates exhibited good in vitro stability at 37°C up to 4 days post preparation in saline, and their clearance was largely by the hepatobiliary route. The DOTA- and CHX-A″-DTPA-based radioimmunoconjugates could be prepared with good radiochemical purity, in vitro stability, and specificity to EGFR. Further studies in EGFR-positive cancers would pave way for them for use in the clinics.

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

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.

Clinical utility of 188Rhenium-hydroxyethylidene-1,1-diphosphonate as a bone pain palliative in multiple malignancies.

188Rhenium-hydroxyethylidene-1,1-diphosphonate (188Re-HEDP) is a clinically established radiopharmaceutical for bone pain palliation of patients with metastatic bone cancer. Herein, the effectiveness of 188Re-HEDP for the palliation of painful bone metastases was investigated in an uncontrolled initial trial in 48 patients with different types of advanced cancers. A group of 48 patients with painful bone metastases of lung, prostate, breast, renal, and bladder cancer was treated with 2.96-4.44 GBq of 188Re-HEDP. The overall response rate in this group of patients was 89.5%, and their mean visual analog scale score showed a reduction from 9.1 to 5.3 (P < 0.003) after 1 week posttherapy. The patients did not report serious adverse effects either during intravenous administration or within 24 h postadministration of 188Re-HEDP. Flare reaction was observed in 54.2% of patients between day 1 and day 3. There was no correlation between flare reaction and response to therapy (P < 0.05). Although bone marrow suppression was observed in patients receiving higher doses of 188Re-HEDP, it did not result in any significant clinical problems. The present study confirmed the clinical utility and cost-effectiveness of 188Re-HEDP for palliation of painful bone metastases from various types of cancer in developing countries.

Rhenium-188 Hydroxyethane 1,1-Diphosphonic Acid (HEDP) for Bone Pain Palliation Using BARC-HEDP Kits versus Pars-HEDP Kits: A Comparison on Preparation and Performance Aspects at Hospital Radiopharmacy.

PURPOSE OF THE STUDY: Rhenium-188 hydroxyethane 1,1-diphosphonic acid (HEDP) is a clinically established radiopharmaceutical for palliation of bone pain due to osseous metastases. Recently, the Bhabha Atomic Research Centre (BARC) had developed a freeze-dried kit for the preparation of rhenium-188 HEDP. The present study compares the radiochemistry aspects of indigenous BARC-HEDP kits with commercially available HEDP kits from Pars Isotope Company, Iran. MATERIALS AND METHODS: Freeze-dried HEDP kits were obtained from Radiopharmaceuticals Division, BARC, and Pars Isotope Company, Iran. Following recommended procedures, rhenium-188 HEDP was prepared using freeze-dried kits from both sources using freshly eluted rhenium-188 sodium perrhenate obtained from a commercial tungsten-188/rhenium-188 generator. RESULTS: Both kits could be used for the preparation of rhenium-188 HEDP in >95% radiochemical purity (RCP). Rhenium-188 HEDP prepared from both kits showed comparable in vitro stability as well as pharmacokinetic properties. The normal bone-to-soft tissue ratio observed for rhenium-188 HEDP prepared using BARC-HEDP kit and Pars-HEDP kit was 1.993 and 1.416, respectively. CONCLUSIONS: Both HEDP kits provided a user-friendly solution for the preparation of rhenium-188 HEDP. While Pars-HEDP-kit permits the addition of only 2 mL of rhenium-188 perrhenate solution per kit vial, BARC-HEDP-kit allows up to 5 mL. This feature permits the preparation of patient dose of rhenium-188 HEDP even with older generators providing rhenium-188 perrhenate having a low radioactive concentration (activity/mL). In addition, availability of an indigenous product is always preferable over imported options.

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.

In Vitro Evaluation of 188Re-HEDP: A Mechanistic View of Bone Pain Palliations.

Skeletal metastasis is common in advanced stages of various cancers, particularly of the prostate and breast carcinoma. 188Re-HEDP (1-hydroxyethane 1, 1-diphosphonic acid) is a clinically established radiopharmaceutical for bone pain palliation of osseous metastasis, and it takes advantage of high bone affinity. The present work aims at elucidating the possible mechanisms of cell killing by 188Re-HEDP in osteosarcoma cells and biodistribution studies in mice.188Re-HEDP complex was prepared by using lyophilized HEDP kits prepared in-house. In vitro cellular uptake in mineralized bone matrix was found to be 13.41% ± 0.46% (at 2 hours), which was reduced to 2.44% ± 0.12% in the presence of excess amounts of unlabeled HEDP ligand. Uptake of 188Re-HEDP in bones of normal Swiss mice in vivo and mineralized bone in vitro indicated its affinity toward the bone matrix. The study also revealed that cellular toxicity and G2/M cell cycle arrest were dose dependent. At higher doses, G2/M cell cycle arrest was observed, which might be the major cause of cell death and a possible mechanism of bone pain relief.

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.

Preparation and preclinical evaluation of 131 I-trastuzumab for breast cancer.

Trastuzumab that targets the human epidermal growth factor receptor type 2 (HER2) is known to benefit patients with HER2+ metastatic breast cancer. The objective was to explore the potential of 131 I-trastuzumab for treatment of breast cancers. Radioiodination of trastuzumab was carried out by chloramine-T method, purified by using PD-10 column, and characterized by size exclusion high-performance liquid chromatography on a gel column. In vitro studies were carried out in HER2+ cells to determine the specificity of the radioimmunoconjugate. Uptake and retention of 131 I-trastuzumab were determined by biodistribution studies in tumor-bearing non-obese diabetic/severe combined immunodeficiency and normal severe combined immunodeficiency mice. The radiochemical purity (RCP) of 131 I-trastuzumab was 98 ± 0.4% with retention time of 17 minutes by high-performance liquid chromatography. In vitro stability studies exhibited RCP of more than 90% in serum at 37°C after 120 hours of radioiodination. In vitro cell binding with 131 I-trastuzumab in HER2+ cells showed binding of 28% to 35% which was inhibited significantly, with unlabeled trastuzumab confirming its specificity. Kd value of 131 I-trastuzumab was 0.5 nM, while its immunoreactivity was more than 80%. Uptake of more than 12% and retention were observed in the tumors up to 120 hours p.i. 131 I-trastuzumab prepared in-house-exhibited RCP of more than 98%, excellent immunoreactivity, affinity to HER2+ cell lines and good tumor uptake thereby indicating its potential for further evaluation in HER2+ breast cancers.

Evaluation of (177)Lu-CHX-A''-DTPA-Bevacizumab as a radioimmunotherapy agent targeting VEGF expressing cancers.

This study aimed at the preparation and evaluation of (177)Lu-CHX-A''-DTPA-Bevacizumab for targeting VEGF over-expressing cancers. Bevacizumab conjugated to p-NCS-Bn-CHX-A''-DTPA was radiolabeled with (177)Lu. The radioimmunoconjugate characterized by SE-HPLC exhibited radiochemical purity of 98.0±0.6%. In vitro stability was retained upto 4 days at 37°C. In vitro cell binding studies showed good uptake by VEGF expressing U937 tumor cells. Biodistribution studies in melanoma model showed significant uptake and retention of (177)Lu-CHX-A''-DTPA-Bevacizumab in tumor with reduction in uptake in presence of cold Bevacizumab confirming its specificity to VEGF.