Ubiquicidin-Derived Peptides Selectively Interact with the Anionic Phospholipid Membrane.

Ubiquicidin (UBI)/ribosomal protein S30 (RS30) is an intracellular protein with antimicrobial activities against various pathogens. UBI (29-41) and UBI (31-38) are two crucial peptides derived from Ubiquicidin, which have shown potential as infection imaging probes. Here, we report the interactions of UBI-derived peptides with anionic and zwitterionic phospholipid membranes. Our isothermal titration calorimetry results show that both peptides selectively interact with the anionic phospholipid membrane (a model bacterial membrane) and reside mainly on the membrane surface. The interaction of UBI-derived peptides with the anionic phospholipid membrane is exothermic and driven by both enthalpy (ΔH) and entropy (ΔS), with the entropic term TΔS being greater than ΔH. This large entropic term can be a result of the aggregation of the anionic vesicles, which is confirmed by dynamic light scattering (DLS) measurements. DLS data show that vesicle aggregation is enhanced with increasing peptide-to-lipid molar ratios (P/L) and is found to be more pronounced in the case of UBI (29-41). DLS results are found to be consistent with independent transmission measurements. To study the effects of UBI-derived peptides on the microscopic dynamics of the model bacterial membrane, quasielastic neutron scattering (QENS) measurements have been carried out. The QENS results show that both peptides restrict the lateral motion of the lipid within the leaflet. UBI (29-41) acts as a stronger stiffening agent, hindering the lateral diffusion of lipids more efficiently than UBI (31-38). To our knowledge, this is the first report illustrating the mechanism of interaction of UBI-derived peptides with model membranes. This study also has implications for the improvement and design of antimicrobial peptide-based infection imaging probes.

Multifunctional Core-Shell Glyconanoparticles for Galectin-3-Targeted, Trigger-Responsive Combination Chemotherapy.

Galectin-3 (gal-3) plays a crucial role in various cellular events associated to tumor metastasis and progression. In this direction, gal-3 binding core-shell glyconanoparticles based on citrus pectin (CP) have been designed for targeted, trigger-responsive combination drug delivery. Depolymerization via periodate oxidation in heterogeneous medium yielded low-molecular weight dialdehyde oligomers (CPDA) of CP with a gal-3 binding property (Kd = 160.90 µM). CPDA-based core-shell nanoparticles prepared to enhance the gal-3 binding specificity via a multivalent ligand presentation have shown to reduce homotypic cellular aggregation, tumor cell binding with endothelial cells, and endothelial tube formation, the major steps involved in the progression of cancer. Immune-fluorescence and flow cytometric analysis confirmed significant reduction in gal-3 expression on MDA-MB 231 cancer cells upon incubation with nanoparticles. An on-demand tumor microenvironment-responsive release of drugs at low pH and high concentrations of glucose and glutathione prevailing in tumor milieu was achieved by introducing a cleavable Schiff's base, a boronate ester, and disulfide linkages within the shell of the nanoparticles. Nanoparticles with encapsulated sulindac in the core and doxorubicin (DOX) in the shell demonstrated target specificity and enhanced internalization with synergistic cytotoxic effects with a 30-fold reduction in IC50 in DOX-resistant, triple-negative MDA-MB 231 breast cancer cells. Nanoparticles were radiolabeled with 131I radioisotopes with ≥80% efficiency while retaining its gal-3 binding property. Biodistribution studies of radiolabeled placebo nanoparticles and drug-loaded CPDA nanoparticles demonstrated proof of concept of gal-3 targeting seen as preferential accumulation in the gal-3-expressing tissues of the gastric tract. The CPDA core-shell nanoparticles are thus promising platforms for gal-3 targeting and inhibition of gal-3-mediated processes involved in cancer progression with a potential of radiolabeling for in vivo monitoring or delivering therapeutic doses of radiation and on-demand triggered, target-specific drug release.

Synthesis of aryl ethers of carbohydrates via reaction with arynes: selective O-arylation of trans-vicinal dihydroxyl groups in carbohydrates.

A new method for the O-arylation of carbohydrates under metal-free conditions using arynes as an aryl source has been developed. This approach works well with mono, di and trihydroxy compounds. Preferential O-arylation takes place at primary over secondary and equatorial over axial. Site-selective O-arylation was achieved with the substrate having trans vicinal diequatorial hydroxyls.

68 Ga-labeling of internalizing RGD (iRGD) peptide functionalized with DOTAGA and NODAGA chelators.

The dual interaction with integrins and neuropilin-1 receptor is the peculiar feature of iRGD peptide. Hence, in the present study, two iRGD peptide analogs were synthesized with DOTAGA and NODAGA as bifunctional chelator and aminohexanoic acid as a spacer for radiometalation with 68 GaCl3 . Negatively charged 68 Ga-DOTAGA-iRGD and neutral 68 Ga-NODAGA-iRGD radiotracers were investigated through in vitro cell uptake studies and in vivo biodistribution studies. Significant internalization of radiotracers in murine melanoma B16F10 cells was observed during in vitro studies. During in vivo studies, tumor uptake was higher for neutral 68 Ga-NODAGA-iRGD, but 68 Ga-DOTAGA-iRGD exhibited better tumor-to-blood ratio due to faster blood clearance. High kidney uptake of the two radiotracers was the limitation, which needs to be resolved through modification either in the peptide backbone or spacer/chelator.

Green chemistry appended synthesis, metabolic stability and pharmacokinetic assessment of medicinally important chromene dihydropyrimidinones.

A green chemistry approach has been developed for the synthesis of chromene dihydropyrimidinone (CDHPM) using recyclable Fe/Al pillared clay catalyst. Pharmacokinetic parameters like aqueous solubility, lipophilicity, P-glycoprotein (P-gp) ATPase activity, permeability, plasma protein binding, red blood cell (RBC) partitioning, metabolic stability in liver microsomes and in silico computations have been studied for the most potent anticancer chromene dihydropyrimidinone hybrid 1. This compound exhibited low solubility, optimum lipophilicity, no P-gp inhibitory activity, intermediate permeability, high plasma protein binding, low RBC partitioning, acceptable metabolic stability in rat liver microsomes (RLM) as well as human liver microsomes (HLM) with transitional hepatic extraction ratio.

Pharmacokinetic evaluation of medicinally important synthetic N,N' diindolylmethane glucoside: Improved synthesis and metabolic stability.

An improved route for the synthesis of N,N'-diindolyl methane (DIM) glycosides has been developed by using Fe/Al pillared clay catalyst. In-silico pharmacokinetics followed by in-vitro studies like aqueous solubility, lipophilicity, P-glycoprotein (P-gp) dependent ATPase activity, permeability, plasma protein binding, RBC partitioning, metabolic stability in different liver microsomes and its in-vitro-in-vivo extrapolation were conducted for the most potent derivative namely NGD16. The compound was found to have low solubility, optimum lipophilicity, no P-gp inhibitory activity, intermediate permeability, high plasma protein binding, low RBC partitioning, acceptable metabolic stability in rat liver microsomes (RLM) as well as human liver microsomes (HLM) with intermediate hepatic extraction ratio.

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 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.

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.

Ce-141-labeled DOTMP: A theranostic option in management of pain due to skeletal metastases.

Owing to its favorable radioactive decay characteristics (T1/2  = 32.51 d, Eß [max] = 434.6 keV [70.5%] and 580.0 keV [29.5%], Eγ  = 145.4 keV [48.5%]), 141 Ce could be envisaged as a theranostic radionuclide for use in nuclear medicine. The present article reports synthesis and evaluation of 141 Ce complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylenephosphonic acid (DOTMP) as a potent theranostic agent targeting metastatic skeletal lesions. Ce-141 was produced with 314 ± 29 MBq/mg (n = 6) specific activity and >99.9% radionuclidic purity (n = 6). Around 185 MBq dose of [141 Ce]Ce-DOTMP was synthesized with 98.6 ± 0.5% (n = 4) radiochemical yield under optimized conditions of reaction, and the preparation showed adequately high in vitro stability. Biodistribution studies in normal Wistar rats demonstrated significant skeletal localization and retention of injected activity (2.73 ± 0.28% and 2.63 ± 0.22% of injected activity per gram in femur at 3 hours and 14 days post-injection, respectively) with rapid clearance from non-target organs. The results of biodistribution studies were corroborated by serial scintigraphic imaging studies. These results demonstrate the potential utility of 141 Ce-DOTMP as a theranostic molecule for personalized patient care of cancer patients suffering from painful metastatic skeletal lesions.

An improved kit formulation for one-pot synthesis of [99m Tc]Tc-HYNIC-E[c(RGDfK)]2 for routine clinical use in cancer imaging.

Radiolabeled Arg-Gly-Asp (RGD) peptide derivatives have immense potential for non-invasive monitoring of malignancies overexpressing integrin αv ß3 receptors. Easy availability of suitable radiotracers would augment the utility of this class of molecular imaging agents. Towards this, the present article describes the development of an improved lyophilized kit for the routine clinical formulation of [99m Tc]Tc complex of HYNIC-conjugated dimeric cyclic RGD peptide derivative E-[c(RGDfK)]2 (E = glutamic acid, f = phenyl alanine, K = lysine) without using Sn2+ and systematic evaluation of its efficacy. Five batches of the kits were prepared, and [99m Tc]Tc-HYNIC-E[c(RGDfK)]2 radiotracer was synthesized with high radiochemical purity (98.6 ± 0.5%) and specific activity (124.8 GBq/µmol) using the kits. Biodistribution studies in C57BL/6 mice bearing melanoma tumor exhibited significant accumulation of the radiotracer in tumor (5.32 ± 0.56 %ID/g at 60 min p.i.), and this uptake was also found to be receptor-specific by blocking studies. Preliminary human clinical investigations carried out in 10 breast cancer patients revealed high radiotracer uptake in the tumor along with good tumor-to-background contrast. The developed kit formulation showed an exceptionally high shelf-life of at least 18 months. These results demonstrated promising attributes of the developed kit formulation and warrant more extensive clinical investigations.

Preparation and preliminary in vivo evaluation of 166 Ho-labeled microspheres for possible use in radioembolic therapy of liver cancer.

Hepatocellular carcinoma (HCC) or liver cancer is an increasingly prevalent and highly morbid disease with critical significance in the Asian and African subcontinents. Among the various therapies currently used in the clinic to combat the global menace of HCC, radioembolization with suitable therapeutic isotopes is an effective targeted approach. In the Indian context, the significant cost and logistical disadvantage of imported radioembolic formulations for HCC therapy make it essential to develop more feasible indigenous alternatives-using locally available radioisotopes and microspheric carriers-that can serve the nuclear medicine community. With this aim Ho-166 was produced with good specific activity (>13 GBq mg-1 ) and purity (>99%) by reactor irradiation. Various commercially available microspheres were labeled with this therapeutic radioisotope, characterized for yield and stability of the radiolabeling, and tested for their in vivo retention and stability in Wistar rat model by viable surgery. Under the optimized reaction conditions, 166 Ho-labeled microspheres were prepared with high yield (>94%-99%) and in vitro stability (>95%) in saline and serum. Retention studies in animal model showed that 166 Ho-labeled microspheres remained stable in vivo and showed excellent retention in the site of interest (~95% at 72-hour p.i.). The study indicates good potential and warrants further investigation for application of these indigenous radiolabeled microspheres for HCC therapy. The successful application of this technology in the clinic would lead to logistically advantageous and cost-effective indigenous alternatives to expensive imported therapeutic solutions.

177 Lu-DOTMP induces G2/M cell cycle arrest and apoptosis in MG63 cell line.

Bone pain is the major manifestation of skeletal metastases. Although various treatment modalities are available for bone pain palliation, use of radiolabeled phosphonates is documented to be more effective. Among radionuclides available for this purpose, lutetium-177 is gaining popularity due to its moderate beta energy, theranostic capability, favorable half-life and convenient production logistics. 177 Lu-DOTMP has shown considerable promise as a metastatic bone pain palliating agent in preliminary evaluations and recent clinical studies. Therefore, an attempt was made to elucidate the possible mechanism of in vitro cell death induced by 177 Lu-DOTMP in MG63 cells. 177 Lu-DOTMP binding studies were carried out in mineralized bone of MG63 cells and around 50% binding was observed. Skeletons of Wistar rats showed 1.78 ± 0.5% IA/g at a 3 h time period which was almost constant up to 7 days. MG63 cells were incubated with 3.7 and 37 MBq of 177 Lu-DOTMP for 48 h prior to perform assays. An increase in the magnitude of cell toxicity and apoptotic DNA fragmentation was observed. Enhancement of G2/M phase cell cycle arrest and apoptosis were documented which were dose-dependent. Thus, 177 Lu-DOTMP induced apoptotic cell death in MG63 cells, which might be one of the primary causes of pain relief in osseous metastases.

Synthesis and biodistribution studies of 99m Tc labeled fatty acid derivatives prepared via "Click approach" for potential use in cardiac imaging.

123 I-Iodophenylpentadecanoic acid (IPPA) is a metabolic agent used in nuclear medicine for diagnosis of myocardial defects. Efforts are underway worldwide to develop a 99m Tc substitute of the above radiopharmaceutical for the aforementioned application. Herein, we report synthesis and biodistribution studies of 99m Tc labeled fatty acids (8, 11, and 15 carbons) obtained via "click chemistry" for its potential use in myocardial imaging. ω-Bromo fatty acids (8C/11C/15C) were synthetically modified at bromo terminal to introduce a heterocyclic triazole with glycine sidearm in a five step procedure. Modified fatty acids were subsequently radiolabeled with preformed [99m Tc(CO)3 ]+ synthon to yield the desired fatty acid complexes which were evaluated in Swiss mice. All the radiolabeled complexes were obtained with radiochemical purities >80%, as characterized by HPLC. Biodistribution studies of all three complexes in Swiss mice showed myocardial uptake of ~6-9% ID/g at 2 minutes post-injection, close to* I-IPPA (~9% ID/g). Complexes exhibited significant retention in the myocardium up to 30 minutes (~1% ID/g) but were lower to the standard agent (~7% ID/g). Similar uptake of activity in myocardium for the newly synthesized complexes in comparison to 125 I-IPPA along with favorable in vivo pharmacokinetics merits potential for the present "click" design of complexes for myocardial imaging.

Barium titanate microparticles as potential carrier platform for lanthanide radionuclides for their use in the treatment of arthritis.

Since the inception of radiation synovectomy, a host of radioactive colloids and microparticles incorporating suitable therapeutic radionuclides have been proposed for the treatment of arthritis. The present article reports the synthesis and evaluation of barium titanate microparticles as an innovative and effective carrier platform for lanthanide radionuclides in the preparation of therapeutic agents for treatment of arthritis. The material was synthesized by mechanochemical route and characterized by X-ray diffraction, scanning electron microscopy, surface area, and particle size distribution analyses. Loading of lanthanide radionuclides (166 Ho, 153 Sm, 177 Lu, and 169 Er) on the microparticles was achieved in high yield (> 95%) resulting in the formulation of loaded particulates with excellent radiochemical purities (> 99%). Radiolanthanide-loaded microparticles exhibited excellent in vitro stability in human serum. In vitro diethylene triamine pentaacetic acid challenge study indicated fairly strong chemical association of lanthanides with barium titanate microparticles. Long-term biodistribution studies carried out after administration of 177 Lu-loaded microparticles into one of the knee joints of normal Wistar rats revealed near-complete retention of the formulation (> 96% of the administered radioactivity) within the joint cavity even 14 days post-administration. The excellent localization of the loaded microparticles was further confirmed by sequential whole-body radio-luminescence imaging studies carried out using 166 Ho-loaded microparticles.

The potential of radiolabeled chemotherapeutics in tumor diagnosis: Preliminary investigations with 68 Ga-gemcitabine.

Preclinical Research & Development Gemcitabine, a nucleoside analog, is a well-known chemotherapeutic drug that is used either alone or with other agents to treat a wide variety of cancers. The aim of the present work was to evaluate the potential of 68 Ga-labeled gemcitabine for its application in positron emission tomography (PET) imaging of tumorous lesions. Gemcitabine was coupled with p-NCS-benzyl-DOTA in order to facilitate radiolabeling with 68 Ga. The gemcitabine-p-NCS-benzyl-DOTA was radiolabeled with 68 Ga, obtained from a 68 Ge/68 Ga radionuclide generator. The radiolabeled product was characterized by high performance liquid chromatography (HPLC) and its tumor specificity was evaluated by biodistribution studies in Swiss mice bearing fibrosarcoma tumors. Preliminary bioevaluation study showed good tumor uptake within 1 hr post-administration [2.5% Injected Activity (IA) per g of tumor] with rapid renal clearance (>90% IA) and a high tumor to muscle ratio. 68 Ga-gemcitabine may have potential as a PET agent for tumor imaging.

Radiolabeled inorganic nanoparticles for positron emission tomography imaging of cancer: an overview.

Over the last few years, a plethora of radiolabeled inorganic nanoparticles have been developed and evaluated for their potential use as probes in positron emission tomography (PET) imaging of a wide variety of cancers. Inorganic nanoparticles represent an emerging paradigm in molecular imaging probe design, allowing the incorporation of various imaging modalities, targeting ligands, and therapeutic payloads into a single vector. A major challenge in this endeavor is to develop disease-specific nanoparticles with facile and robust radiolabeling strategies. Also, the radiolabeled nanoparticles should demonstrate adequate in vitro and in vivo stability, enhanced sensitivity for detection of disease at an early stage, optimized in vivo pharmacokinetics for reduced non-specific organ uptake, and improved targeting for achieving high efficacy. Owing to these challenges and other technological and regulatory issues, only a single radiolabeled nanoparticle formulation, namely "C-dots" (Cornell dots), has found its way into clinical trials thus far. This review describes the available options for radiolabeling of nanoparticles and summarizes the recent developments in PET imaging of cancer in preclinical and clinical settings using radiolabeled nanoparticles as probes. The key considerations toward clinical translation of these novel PET imaging probes are discussed, which will be beneficial for advancement of the field.

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.

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.