A “mix‐and‐use” approach for formulation of human clinical doses of 177Lu‐DOTMP at hospital radiopharmacy for management of pain arising from skeletal metastases

Use of bone‐seeking radiopharmaceuticals is an established modality in the palliative care of pain due to skeletal metastases. ¹⁷⁷Lu‐DOTMP is a promising radiopharmaceutical for this application owing to the ideally suited decay properties of ¹⁷⁷Lu and excellent thermodynamic stability and kinetic rigidity of the macrocyclic complex. The aim of the present study is to develop a robust and easily adaptable protocol for formulation of clinical doses of ¹⁷⁷Lu‐DOTMP at hospital radiopharmacy. After extensive radiochemical studies, an optimized strategy for formulation of clinical doses of ¹⁷⁷Lu‐DOTMP was developed, which involves simple mixing of approximately 3.7 GBq of ¹⁷⁷Lu activity as ¹⁷⁷LuCl₃ solution to an aqueous solution containing 5 mg of DOTMP and 8 mg of NaHCO₃. The proposed protocol yielded ¹⁷⁷Lu‐DOTMP with >98% radiochemical purity, and the resultant formulation showed excellent in vitro stability and desired pharmacokinetic properties in animal model. Preliminary clinical investigations in 5 patients showed specific skeletal accumulation with preferential localization in the osteoblastic lesion sites and almost no uptake in soft tissue or any other major nontarget organ. The developed “mix‐and‐use” strategy would be useful for large number of nuclear medicine centers having access to ¹⁷⁷Lu activity and would thereby accelerate the clinical translation of ¹⁷⁷Lu‐DOTMP.     

Synthesis and biological evaluation of morpholines linked coumarin–triazole hybrids as anticancer agents

A series of novel morpholines linked coumarin–triazole hybrids ( 6a–6v ) has been synthesized and evaluated for their anti‐proliferative potential on a panel of five human cancer cell lines, namely bone (MG‐63), lung (A549), breast (MDA‐MB‐231), colon (HCT‐15) and liver (HepG2), using MTT assay. Among all, the compound 6n {7‐((1‐(2,4‐dichlorobenzyl)‐1H‐1,2,3‐triazol‐4‐yl) methoxy)‐4‐((2,6‐dimethylmorpholino) methyl)‐2H‐chromen‐2‐one} showed significant growth inhibition against MG‐63 cells with an IC₅₀ value of 0.80 ± 0.22 μM. Further, induction of apoptosis by 6n of MG‐63 cells confirmed as a result of morphological changes, the sub‐G1 phase arrest, increased percentage of apoptotic cells, and decrease in mitochondrial membrane potential and increase in reactive oxygen species levels. The in vitro Gal‐1 expression in cell culture supernatant of MG‐63 cells treated with compound 6n showed dose‐dependent reduction. The binding constant (K_a) of 6n with Gal‐1 was calculated from the intercept value which was observed as 3.0 × 10⁵ M^?1 by fluorescence spectroscopy. Surface plasmon resonance showed that 6n binds to Gal‐1 with binding constant (K_a) of 1.29E+04 1/Ms and equilibrium constant KD value of 7.54E?07 M, respectively. Molecular docking studies revealed the binding interactions of 6n with Gal‐1.     

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

Owing to its favorable radioactive decay characteristics (T_1/2 = 32.51 d, E_β [max] = 434.6 keV [70.5%] and 580.0 keV [29.5%], E_γ = 145.4 keV [48.5%]), ¹⁴¹Ce could be envisaged as a theranostic radionuclide for use in nuclear medicine. The present article reports synthesis and evaluation of ¹⁴¹Ce 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 [¹⁴¹Ce]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 ¹⁴¹Ce‐DOTMP as a theranostic molecule for personalized patient care of cancer patients suffering from painful metastatic skeletal lesions.     

Ketoconazole-induced JNK phosphorylation and subsequent cell death via apoptosis in human osteosarcoma cells

This study examined the effect of ketoconazole on viability, apoptosis, mitogen-activated protein kinases (MAPKs) and Ca²⁺ levels in MG63 osteosarcoma cells. Ketoconazole at 20–200 μM decreased cell viability via apoptosis as demonstrated by propidium iodide staining and activation of caspase-3. Immunoblotting suggested that ketoconazole induced phosphorylation of ERK and JNK, but not p38, MAPKs. Ketoconazole-induced cell death and apoptosis were partially reversed by the selective JNK inhibitor SP600125, but not by the selective ERK inhibitor PD98059, suggesting that ketoconazole’s cytotoxic action was via JNK, but not via ERK and p38 MAPKs. Ketoconazole at a concentration of 100 μM induced [Ca²⁺]_i increases. Chelation of intracellular Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) totally inhibited ketoconazole-induced [Ca²⁺]_i increases without reversing ketoconazole-induced cell death. Collectively, in MG63 cells, ketoconazole induced cell death and apoptosis via evoking JNK phosphorylation in a Ca²⁺-independent manner.     

Production,quality control,and determination of human absorbed dose of no carrier added 177Lu‐risedronate for bone pain palliation therapy

In this study, the radiocomplexation of risedronic acid, a potent bisphosphonate with a no carrier added (NCA) ¹⁷⁷Lu, was investigated and followed by quality control studies, biodistribution evaluation, and dosimetry study for human based on biodistribution data in Wistar rats. The moderate energy β⁻ emitter, ¹⁷⁷Lu (T _½ = 6.7 days, E _βmax = 497 keV), has been considered as a potential agent for development of bone‐seeking radiopharmaceuticals. Because the specific activity of the radiolabeled carrier molecules should be high, the NCA radionuclides have an effective role in nuclear medicine. Many researchers illustrated an NCA ¹⁷⁷Lu production; among these separation techniques, extraction chromatography has been considered more capable than other methods. The NCA ¹⁷⁷Lu was produced with specific activity of 48 Ci/mg and radionuclidic purity of 99.99% by the irradiation of enriched ¹⁷⁶Yb target in thermal neutron flux of 4 × 10¹³ n·cm⁻²·s⁻¹ for 14 days. The NCA ¹⁷⁷Lu was mixed to a desired amount of sodium risedronate (15 mg/mL, 200 μL) and incubated with stirring at 95°C for 30 minutes. The radiochemical purity of ¹⁷⁷Lu‐risedronate was determined by radio thin‐layer chromatography, and high radiochemical purities (>97%) were obtained under optimized reaction conditions . The complex was injected to Wistar rats, and complex biodistribution was performed 4 hours to 7 days postinjections showing high bone uptake (9.8% ± 0.24% ID/g at 48 hours postinjection). Also, modeling the radiation dose delivery by RADAR software for the absorbed dose evaluation of each human organ showed a major accumulation of the radiocomplex in bone tissue.     

Danazol has potential to cause PKC translocation,cell cycle dysregulation,and apoptosis in breast cancer cells

Danazol, the established clinical drug, has given promising therapeutic results in a series of clinical trials with breast cancer patients. Danazol shares structural similarities with several known PKC agonists and fits well into the C1 domain. Danazol binds to the C1b domain of PKC with K_d of 5.64 ± 1.27 μm . MD simulation studies further support that the PKC–danazol molecular model is stable and showing minimum distortion to the structure during the simulation period. Immunofluorescence and Western blotting studies indicate that MDAMB‐231 cells stimulated with danazol exhibit translocation of PKCα to the plasma membrane. Cells stimulated with danazol causes appearance of several phosphorylated proteins in lysate and plasma membrane. In addition, danazol affects carcinogenic molecule (PMA)‐induced intracellular signaling in cancer cells. It halted the cancer cells in the G1 phase of the cell cycle and reduced the viability of ER^+ve and triple‐negative breast cancer cells with an IC₅₀ of 31 ± 2.63 and 65 ± 4.27 μg/ml, respectively. DNA fragmentation and flow cytometry experiments revealed that the cell death follows the apoptotic pathway. It affects mitochondrial membrane potentials and releases cytochrome‐C from mitochondria to induce downstream apoptosis in breast cancer cells. Hence, the current study may help clinicians to re‐design their treatment strategy to optimize therapeutic potentials of the molecule.     

Syntheses and evaluation of 68Ga‐ and 153Sm‐labeled DOTA‐conjugated bisphosphonate ligand for potential use in detection of skeletal metastases and management of pain arising from skeletal metastases

This article reports the syntheses and evaluation of ⁶⁸Ga‐ and ¹⁵³Sm‐complexes of a new DOTA (1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid)‐conjugated geminal bisphosphonate, DOTA‐Bn‐SCN‐BP, for their potential uses in the early detection of skeletal metastases by imaging and palliation of pain arising from skeletal metastases, respectively. The conjugate was synthesized in high purity following an easily adaptable three‐step reaction scheme. Gallium‐68‐ and ¹⁵³Sm‐complexes were prepared in high yield (>98%) and showed excellent in vitro stability in phosphate‐buffered saline (PBS) and human serum. Both the complexes showed high affinity for hydroxyapatite particles in in vitro binding study. In biodistribution studies carried out in normal Wistar rats, both the complexes exhibited rapid skeletal accumulation with almost no retention in any other major organ. The newly synthesized molecule DOTA‐Bn‐SCN‐BP would therefore be a promising targeting ligand for the development of radiopharmaceuticals for both imaging skeletal metastases and palliation of pain arising out of it in patients with cancer when radiolabeled with ⁶⁸Ga and ¹⁵³Sm, respectively. A systematic comparative evaluation, however, showed that there was no significant improvement of skeletal accumulation of the ¹⁵³Sm‐DOTA‐Bn‐SCN‐BP complex over ¹⁵³Sm‐DOTMP (1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetramethylenephosphonic acid) as the later itself demonstrated optimal properties required for an agent for bone pain palliation.     

Acrylamide-induced apoptosis in rat primary astrocytes and human astrocytoma cell lines

This study aimed to evaluate the acrylamide (ACR)-induced apoptotic effects on rat primary astrocytes and three human astrocytoma-derived cell lines (U-1240 MG, U-87 MG, and U-251 MG). As determined through the MTT assay, treatment with 1 and 2 mM ACR for 24–72 h resulted in decreased cell viability in all cells. Decreases in cell viability could be blocked in all cells with the exception of U-251 MG cells by Z-DEVD FMK. ACR-induced dose-dependent apoptotic effects were also demonstrated by increases in the sub-G₁ phase cell population in all cells. The decreased expressions of pro-caspase 3, 8, and 9 and the interruption of the mitochondrial membrane potential were observed in all cells. Exposure to 2 mM ACR for 48 h resulted in increased Bax/Bcl-2 ratios in primary astrocytes and U-87 MG cells, whereas the overexpression of Bcl-2 was observed in U-1240 MG and U-251 MG cells. The ACR-induced increases in the levels of p53 and pp53 in primary astrocytes could be attenuated by caffeine. These results suggest the existence of a common apoptotic pathway among all cell types and that U-87 MG cells may be a suitable substitute in vitro model for primary astrocytes in future studies on ACR-induced neurotoxicity.     

αvβ3 mediated tumor imaging using 99mTc labeled NAD/monosaccharide coated ferrihydrite nanoparticles

This study describes the synthesis of highly water‐soluble, non‐toxic, and biocompatible nicotinamide adenine dinucleotide (NAD)/glucosamine (=Nga1Fh) and NAD/glucosamine/gluconic acid coated ferrihydrite nanoparticles (=Nga2Fh) and their possible uses to target tumors in living animals via ^99mTc and ¹²⁵I radioisotope labeling. The structural properties were investigated using DLS, zeta potential, TEM, FT‐IR, XRD, and Raman spectroscopy. The cell toxicity in CT26 cancer cells and in vivo tumor targetability in U87MG and CT26 tumor‐bearing mice was further evaluated using cRGDyK‐tagged and cRGDfK‐tagged ferrihydrite nanoparticles. The average diameters of the resulting Nga1Fh and Nga2Fh nanoparticles were <5 to 7 and <3 nm, respectively. The Nga2Fh nanoparticles did not show cell toxicity until 0.1 mg/mL. Using gamma camera imaging, ^99mTc‐cRGDfK‐Nga2Fh showed the highest tumor uptake in a U87MG tumor‐bearing mouse when compared with that of ^99mTc‐cRGDyK‐Nga2Fh and ^99mTc‐Nga2Fh. The image‐based tumor‐to‐muscle ratio by time for ^99mTc‐cRGDfK‐Nga2Fh was 3.8 ± 1.7, 4.2 ± 2.0, 7 ± 1.5, 13 ± 2.0, 8 ± 3.7, and 2 ± 1.6 at 5 and 30 minutes, 1, 2, 4, and 24 hours, respectively. Although further studies are needed, the NAD/monosaccharide coated ferrihydrite nanoparticles could be presented as an interesting material for a drug delivery system.     

Radiolabeling,quality control,biodistribution, and imaging studies of 177Lu‐ibandronate

The purposes of this study were as follows: (1) to radiolabel ibandronic acid (IBA, a third‐generation bisphosphonate) with ¹⁷⁷Lu, investigating optimal labeling conditions, and (2) to analyze biodistribution and imaging properties of intravenous ¹⁷⁷Lu‐ibandronate (¹⁷⁷Lu‐IBA) administered in animals. ¹⁷⁷Lu‐labeled methylene diphosphonate (¹⁷⁷Lu‐MDP) served as a comparator agent. Differing proportions of IBA solution and ¹⁷⁷LuCl₃ solution were combined to determine an optimal ratio for radiolabeling purposes, varying pH, temperature, and time to establish ideal reactivity conditions. Radiochemical purity of the labeled compounds was then assessed by paper chromatography. In vitro and in vivo stabilities were also measured at specific time intervals. In Kunming mice, biodistributions of ¹⁷⁷Lu‐IBA and ¹⁷⁷Lu‐MDP and respective agent activities in various organs were monitored by gamma counter, and we performed single photon computed tomography/computed tomography (SPECT/CT) imaging of ¹⁷⁷Lu‐IBA in normal New Zealand White rabbits. Radiolabeling yields for ¹⁷⁷Lu‐IBA proved to be >97% within 30 minutes at 90°C, and its radiochemical purity ensured stability in vitro and in vivo. Furthermore, we found that ¹⁷⁷Lu‐IBA is readily soluble in water, showing higher skeletal uptake than ¹⁷⁷Lu‐MDP but lower uptake by liver and spleen. The image quality of ¹⁷⁷Lu‐IBA was so clear that even after 6 days, analysis was still feasible.     

Proteasome inhibitor MG132 reduces growth of As4.1 juxtaglomerular cells via caspase-independent apoptosis

The proteasome inhibitor MG132 has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). Here, we investigated the molecular mechanisms of MG132 in As4.1 juxtaglomerular cell death in relation to apoptosis and levels of ROS and glutathione (GSH). MG132 inhibited the growth of As4.1 cells with an IC₅₀ of approximately 0.3–0.4 μM at 48 h and induced cell death, accompanied by the loss of mitochondrial membrane potential (MMP; ∆Ψ_m), Bcl-2 decrease, activations of caspase-3 and caspase-8, and PARP cleavage. MG132 increased intracellular ROS levels and GSH-depleted cell numbers. However, caspase inhibitors, especially Z-VAD (pan-caspase inhibitor) intensified cell growth inhibition, cell death, MMP (∆Ψ_m) loss, and Bcl-2 decrease in MG132-treated As4.1 cells. Z-VAD also slightly intensified increases in ROS levels and GSH depletion in MG132-treated As4.1 cells. In conclusion, MG132 reduced the growth of As4.1 cells via caspase-independent apoptosis. The changes in ROS and GSH levels by MG132 and caspase inhibitors partially influenced the growth inhibition and death of As4.1 cells.     

Pirarubicin inhibits multidrug-resistant osteosarcoma cell proliferation through induction of G2/M phase cell cycle arrest

Aim:

Pirarubicin (THP) is recently found to be effective in treating patients with advanced, relapsed or recurrent high-grade osteosarcoma. In this study, the effects of THP on the multidrug-resistant (MDR) osteosarcoma cells were assessed, and the underlying mechanisms for the disruption of cell cycle kinetics by THP were explored.

Methods:

Human osteosarcoma cell line MG63 and human MDR osteosarcoma cell line MG63/DOX were tested. The cytotoxicity of drugs was examined using a cell proliferation assay with the Cell Counting Kit-8 (CCK-8). The distribution of cells across the cell cycle was determined with flow cytometry. The expression of cell cycle-regulated genes cyclin B1 and Cdc2 (CDK1), and the phosphorylated Cdc2 and Cdc25C was examined using Western blot analyses.

Results:

MG63/DOX cells were highly resistant to doxorubicin (ADM) and gemcitabine (GEM), but were sensitive or lowly resistant to THP, methotrexate (MTX) and cisplatin (DDP). Treatment of MG63/DOX cells with THP (200–1000 ng/mL) inhibited the cell proliferation in time- and concentration-dependent manners. THP (50–500 ng/mL) induced MG63/DOX cell cycle arrest at the G₂/M phase in time- and concentration-dependent manners. Furthermore, the treatment of MG63/DOX cells with THP (200–1000 ng/mL) downregulated cyclin B1 expression, and decreased the phosphorylated Cdc2 at Thr¹⁶¹. Conversely, the treatment increased the phosphorylated Cdc2 at Thr¹⁴/Tyr¹⁵ and Cdc25C at Ser²¹⁶, which led to a decrease in Cdc2-cyclin B1 activity.

Conclusion:

The cytotoxicity of THP to MG63/DOX cells may be in part due to its ability to arrest cell cycle progression at the G₂/M phase, which supports the use of THP for managing patients with MDR osteosarcoma.     

Preparation of clinical‐scale 177Lu‐Rituximab: Optimization of protocols for conjugation,radiolabeling, and freeze‐dried kit formulation

Rituximab is a monoclonal chimeric antibody, which has been approved by the US Food and Drug Administration for immunotherapy of non–Hodgkin lymphoma. Bexxar and Zevalin are the two other approved radiolabeled antibodies for radioimmunotherapy of non–Hodgkin lymphoma; however, they are of murine origin that reduces their treatment efficacy. So as to circumvent this, efforts have been made to radiolabel Rituximab with various therapeutic radioisotopes. In the present study, an effort has been made to optimize the conjugation (bifunctional chelating agent and antibody) and radiolabeling procedures for the preparation of clinical‐scale ¹⁷⁷Lu‐labeled Rituximab. An attempt was also made to prepare the freeze‐dried Rituximab kit for the easy and convenient clinical translation of the agent. Clinical‐scale ¹⁷⁷Lu‐Rituximab (40 mCi, 1.48 GBq) was prepared with >95% radiochemical purity using the kit. Biological evaluation of ¹⁷⁷Lu‐Rituximab was performed by in vitro cell binding studies in Raji cell lines, which showed satisfactory binding at 4°C and 37°C. Pharmacokinetic behavior of the agent, evaluated by biodistribution studies in normal Swiss mice, revealed high blood and liver uptake at the initial time points, although it exhibited slow and gradual clearance with time. The study indicates that clinical‐scale ¹⁷⁷Lu‐Rituximab could be conveniently formulated using the methodology described in the present article.     

Eudesmol Isomers Induce Caspase‐Mediated Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells

Eudesmols are naturally occurring sesquiterpenoid alcohols that present cytotoxic effect to cancer cells. Herein, all eudesmol isomers displayed cytotoxicity to different tumour cell lines. α‐Eudesmol showed IC₅₀ values ranging from 5.38 ± 1.10 to 10.60 ± 1.33 μg/mL for B16‐F10 and K562 cell lines, β‐eudesmol showed IC₅₀ values ranging from 16.51 ± 1.21 to 24.57 ± 2.75 μg/mL for B16‐F10 and HepG2 cell lines, and γ‐eudesmol showed IC₅₀ values ranging from 8.86 ± 1.27 to 15.15 ± 1.06 μg/mL for B16‐F10 and K562 cell lines, respectively. In addition, in this work, we studied the mechanisms of cytotoxic action of eudesmol isomers (α‐, β‐ and γ‐eudesmol) in human hepatocellular carcinoma HepG2 cells. After 24‐hr incubation, HepG2 cells treated with eudesmol isomers presented typical hallmarks of apoptosis, as observed by morphological analysis in cells stained with haematoxylin–eosin and acridine orange/ethidium bromide. None of eudesmol isomers caused membrane disruption at any concentration tested. Moreover, eudesmol isomers induced loss of mitochondrial membrane potential and an increase in caspase‐3 activation in HepG2 cells, suggesting the induction of caspase‐mediated apoptotic cell death. In conclusion, the eudesmol isomers herein investigated are able to reduce cell proliferation and to induce tumour cell death by caspase‐mediated apoptosis pathways.     

Radiolabeling,quality control,and biological characterization of 177Lu‐labeled kanamycin

Kanamycin is an antibiotic, isolated from Streptomyces kanamyceticus, which is used to treat serious bacterial infections. The fact that the present radioligand ^99mTc‐kanamycin used for diagnosis is short‐lived, raised a need to label and study kanamycin with one of the most important beta (β) radiation emitting isotope ¹⁷⁷Lu. Labeling yield of ¹⁷⁷Lu‐kanamycin was confirmed by different chromatography techniques such as paper chromatography, TLC, HPLC. Several experiments were performed to optimize labeling with changing reaction conditions such as pH, temperature, amount of ligand, and reaction time. In vitro stability analysis was performed incubation with human serum. Electrophoresis analysis was also conducted to determine the charge on ¹⁷⁷Lu‐kanamycin. The biodistribution and scintigraphy were performed in normal mice and rabbit, respectively, at different time intervals of postinjection. ¹⁷⁷Lu‐kanamycin was prepared with very high yield (~100%), with excellent stability in vivo and in vitro (>99% 6 hr postprep.), at pH 7. Maximum labeling was achieved at less reaction time (15 min), with maximum conjugation of the ligand (12.5 mg) with ¹⁷⁷Lu. Electrophoresis analysis showed net neutral charge. The radioligand showed rapid clearance from body in biodistribution and scintigraphy studies. The preparation ¹⁷⁷Lu‐kanamycin could be used as a radio‐pharmaceutical for infection imaging purpose, especially when transporting the radioligand to long‐range distances.     

177Lu‐labeled monomeric,dimeric and multimeric RGD peptides for the therapy of tumors expressing α(ν)β(3) integrins

The conjugation of peptides to gold nanoparticles (AuNPs) produces biocompatible and stable multimeric systems with target‐specific molecular recognition. Peptides based on the cyclic Arg‐Gly‐Asp (RGD) sequence have been reported as high‐affinity agents for the α(ν)β(3) integrin. The aim of this research was to prepare a multimeric system of ¹⁷⁷Lu‐labeled gold nanoparticles conjugated to c(RGDfK)C (cyclo(Arg‐Gly‐Asp‐Phe‐Lys)Cys) and to compare the radiation‐absorbed dose with that of ¹⁷⁷Lu‐labeled monomeric and dimeric RGD peptides to α(ν)β(3) integrin‐positive U87MG tumors in mice. DOTA‐GGC (1,4,7,10‐tetraazacyclododecane‐N‐N′,N″,N?‐tetraacetic acid‐Gly‐Gly‐Cys) and c(RGDfK)C peptides were synthesized and conjugated to AuNPs by a spontaneous reaction of the thiol groups. Transmission electron microscopy, ultraviolet–visible, X‐ray photoelectron spectroscopy, Raman and far‐infrared spectroscopy techniques demonstrated that AuNPs were functionalized with the peptides. For the ¹⁷⁷Lu‐AuNP‐c(RGDfK)C to be obtained, the ¹⁷⁷Lu‐DOTA‐GGC radiopeptide was first prepared and added to a solution of AuNPs followed by c(RGDfK)C (25 µl, 5 µ m ) at 18 °C for 15 min. ¹⁷⁷Lu‐DOTA‐GGC, ¹⁷⁷Lu‐DOTA‐cRGDfK and ¹⁷⁷Lu‐DOTA‐E‐c(RGDfK)₂ were prepared by adding ¹⁷⁷LuCl₃ (370 MBq) to 5 µl (1 mg/ml) of the DOTA derivative diluted with 50 µl of 1 m acetate buffer pH 5. The mixture was incubated at 90 °C in a block heater for 30 min. Radiochemical purity was determined by ultrafiltration and HPLC analyses. Biokinetic studies were accomplished in athymic mice with U87MG‐induced tumors. The radiochemical purity for all ¹⁷⁷Lu‐RGD derivatives was 96 ± 2%. ¹⁷⁷Lu‐absorbed doses per injected activity delivered to U87MG tumors were 0.357 ± 0.052 Gy/MBq (multimer), 0.252 ± 0.027 Gy/MBq (dimer) and 0.102 ± 0.018 Gy/MBq (monomer). ¹⁷⁷Lu‐labeled dimeric and multimeric RGD peptides demonstrated properties suitable for targeted radionuclide therapy of tumors expressing α(ν)β(3) integrins. Copyright © 2012 John Wiley & Sons, Ltd.     

Multifunctional targeted therapy system based on 99mTc/177Lu‐labeled gold nanoparticles‐Tat(49–57)‐Lys3‐bombesin internalized in nuclei of prostate cancer cells

Radiolabeled gold nanoparticles may function simultaneously as radiotherapy and thermal ablation systems. The gastrin‐releasing peptide receptor (GRP‐r) is overexpressed in prostate cancer, and Lys³‐bombesin is a peptide that binds with high affinity to the GRP‐r. HIV Tat(49–57) is a cell‐penetrating peptide that reaches the DNA. In cancer cells, ¹⁷⁷Lu shows efficient crossfire effect, whereas ^99mTc that is internalized in the cancer cell nuclei acts as an effective system of targeted radiotherapy because of the biological Auger effect. The aim of this research was to evaluate the in vitro potential of ^99mTc‐labeled and ¹⁷⁷Lu‐labeled gold nanoparticles conjugated to Tat(49–57)‐Lys³‐bombesin peptides (^99mTc/¹⁷⁷Lu‐AuNP‐Tat‐BN) as a plasmonic photothermal therapy and targeted radiotherapy system in PC3 prostate cancer cells. Peptides were conjugated to AuNPs (5 nm) by spontaneous reaction with the thiol group of cysteine (Cys). The effect on PC3 cell viability after laser heating of the AuNP‐Tat‐BN incubated with the cancer cells was conducted using an Nd:YAG laser pulsed for 5 ns at 532 nm (0.65 W/cm²). For the ^99mTc/¹⁷⁷Lu‐AuNP‐Tat‐BN to be obtained, the ¹⁷⁷Lu‐DOTA‐Gly‐Gly‐Cys and ^99mTc‐HYNIC‐octreotide radiopeptides were first prepared and added simultaneously to a solution of AuNP‐Tat‐BN. ^99mTc/¹⁷⁷Lu‐AuNP‐Tat‐BN (20 Bq/cell) was incubated with PC3 cells, and the effect on the cell proliferation was evaluated after 3 days. Fluorescence images of ^99mTc/¹⁷⁷Lu‐AuNP‐Tat‐BN internalized in nuclei of PC3 were also obtained. After laser irradiation, the presence of AuNP‐Tat‐BN caused a significant increase in the temperature of the medium (46.4 vs 39.5 °C of that without AuNP) resulting in a significant decrease in PC3 cell viability down to 1.3%. After treatment with ^99mTc/¹⁷⁷Lu‐AuNP‐Tat‐BN, the PC3 cell proliferation was inhibited. The nanosystem exhibited properties suitable for plasmonic photothermal therapy and targeted radiotherapy in the treatment of prostate cancer.     

Extracts of marine sponge <Emphasis Type="Italic">Polymastia janeirensis</Emphasis> induce oxidative cell death through a caspase-9 apoptotic pathway in human U138MG glioma cell line

We have studied the apoptotic pathway activated in response to marine sponge extracts of Polymastia janeirensis. The effect on intracellular ROS production was also examined. Exposure of U138MG glioma cell line to doses higher than 5 μg/mL has decreased glioma cell viability, with an IC₅₀ <15 μg/mL for both aqueous and organic extracts. However, extracts at higher doses (50 and 100 μg/mL) have stronger cytotoxic effects, decreasing more than 90% of glioma cell viability. The antioxidant Trolox? (100 μM) reversed the cell death percentage induced by extracts at 10 and 25 μg/mL. The type of cell death induced by such high doses was predominantly necrosis, while a high percentage of apoptotic glioma cells was found at 10 μg/mL. Moreover, inhibition of caspase-8 with Z-IETD (a caspase-8 inhibitor) had no effect on the amount of apoptosis induced by 10 μg/mL, but inhibition of caspase-9 with Z-LEHD (a caspase-9 inhibitor) decreased apoptosis. We also observed a dose-dependent increase in ROS production, and similarly to effects observed on viability of glioma cells, and on cell death, higher doses also had more severe effects. Co-treatment with Trolox? significantly reduced ROS production by extracts at doses lower than 50 μg/mL. This is a first report demonstrating that marine sponge extracts of P. janeirensis induce oxidative cell death through a caspase-9 apoptotic pathway.     

Preparation of 177Lu‐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 ¹⁷⁷Lu 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 ¹⁷⁷Lu, 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 ¹⁷⁷Lu‐CHX‐A″‐DTPA‐Nimotuzumab and ¹⁷⁷Lu‐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.