99mTc-HYNIC-Annexin V detection of apoptosis in the rabbit model of liver cancer.

To investigate the feasibility of detection of apoptosis in vivo by 99mTc-HYNIC-Annexin V, Annexin V was labeled with 99mTc through HYNIC. 18 New Zealand rabbits implanted VX-2 were randomly divided into control (n = 8) and paclitaxel (PAC, n = 10) groups, given 2 mL/kg of normal saline or 2.4 mg/kg of PAC intravenously. The liver tumor imaging was detected by SPECT through intravenous injection of 99mTc-HYNIC-Annexin V before treatment, 24 hours and 48 hours after treatment respectively. Tumor radioactive count proportion to non-tumor sites was calculated. When the last imaging was finished, the rabbits were sacrificed. The tumor was taken out and divided into two pieces, one for TUNEL immunohistochemical analysis and the other for flow cytometry (FCM). We found that the rate of Annexin V labeled with 99mTc through HYNIC was more than 95%, and radiochemical purity was above 95%. The SPECT showed that two groups had no significant tumor imaging before the treatment. There is no significant tumor imaging in control group, while the PAC group 24 h and 48 h after treatment showed significant accumulation. The Tumor/non-Tumor (T/NT) in PAC group at 24 h and 48 h after chemotherapy was significantly different from that in the control group and PAC group prior to treatment. There was no significant difference between 24 h and 48 h in PAC group. The TUNEL-positive cells detected by immunohistochemistry and apoptotic rate detected by FCM in PAC group were significant different from those in control group. The T/NT was significantly correlated to TUNEL-positive cells and apoptotic rate of the tumor. PAC can induce apoptosis of rabbit VX-2 liver cancer cells. 24-48 h after paclitaxel chemotherapy is a window time for apoptosis detection. Apoptotic cells in vivo can be detected by SPECT through 99mTc-HYNIC-Annexin V.

Peptide derived from plant defensins: A promising 68Ga radiolabelled agent for diagnostic of infection foci in PET.

The development of new radiopharmaceuticals for the detection of hidden infection foci has great relevance for early detection and the selection of the correct treatment, particularly in immunosuppressed patients. In that sense, the labelling of antimicrobial peptides (AMPs) that are capable of binding specifically to the pathogenic microorganism which causes the infection, should provide a sufficiently specific agent, able to distinguish an infection from a sterile inflammation. Defensins are particularly interesting molecules with antimicrobial activity, the EcgDf1 defensin was identified from the genome of a Uruguayan native plant, Erythrina crista-galli, the 'Ceibo' tree. Our group has previously reported a synthetic biologically active short analogue EcgDf21 (ERFTGGHCRGFRRRCFCTKHC) successfully labelled with 99mTc. Herein we present a shorter analogue which also preserves the γ-core domain, as a pharmacophore for a potential infection detection agent. This peptide was derivatized with the bifunctional chelating agent 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) through a lysine linker in the amino-terminal group (NOTA-KGHCRGFRRRC) and radiolabelled with 68Ga ([68Ga]Ga-NOTA-K-EcgDf1(10)). The [68Ga]Ga-NOTA-K-EcgDf1(10) labelling procedure rendered a product with high radiochemical purity and stability in the labelling milieu. The Log P value indicated that the complex has a hydrophilic behaviour, confirmed by the biodistribution profile. The [68Ga]Ga-NOTA-K-EcgDf1(10) complex demonstrated specific binding to cultures of Candida albicans and Aspergillus niger. Its biodistribution showed renal elimination and low accumulation in the rest of the body. It was possible to successfully differentiate sterile inflammation from infection by PET images in nude mice with a target/non-target ratio of 3.3 for C. albicans and 3.7 for A. niger, respectively.

Ligand-free 99mTc-polyurea dendrimer complexes: nanoradiotheranostics targeting ovarian cancer.

A folic acid-targeted polyurea (PURE) dendrimer was easily radiolabelled with Technetium-99m (99mTc-PUREG4-FA2) avoiding the use of additional ligands and bioconjugation chemistry. This straightforward strategy is enabled in PURE dendrimers due to their favourable surface terminal groups configuration, showing coordination capabilities and turning these biodendrimers into attractive platforms for nanoradiotheranostics.

Rational Design and Comparison of Novel 99mTc-Labeled FAPI Dimers for Visualization of Multiple Tumor Types.

Recognizing the significance of SPECT in nuclear medicine and the pivotal role of fibroblast activation protein (FAP) in cancer diagnosis and therapy, this study focuses on the development of 99mTc-labeled dimeric HF2 with high tumor uptake and image contrast. The dimeric HF2 was synthesized and radiolabeled with 99mTc in one pot using various coligands (tricine, TPPTS, EDDA, and TPPMS) to yield [99mTc]Tc-TPPTS-HF2, [99mTc]Tc-EDDA-HF2, and [99mTc]Tc-TPPMS-HF2 dimers. SPECT imaging results indicated that [99mTc]Tc-TPPTS-HF2 exhibited higher tumor uptake and tumor-to-normal tissue (T/NT) ratio than [99mTc]Tc-EDDA-HF2 and [99mTc]Tc-TPPMS-HF2. Notably, [99mTc]Tc-TPPTS-HF2 exhibited remarkable tumor accumulation and retention in HT-1080-FAP and U87-MG tumor-bearing mice, thereby surpassing the monomeric [99mTc]Tc-TPPTS-HF. Moreover, [99mTc]Tc-TPPTS-HF2 achieved acceptable T/NT ratios in the hepatocellular carcinoma patient-derived xenograft (HCC-PDX) model, which provided identifiable contrast and imaging quality. In conclusion, this study presents proof-of-concept research on 99mTc-labeled FAP inhibitor dimers for the visualization of multiple tumor types. Among these candidate compounds, [99mTc]Tc-TPPTS-HF2 showed excellent clinical potential, thereby enriching the SPECT tracer toolbox.

Validation of HPLC and TLC analytical methods to determine radiochemical purity of 99mTc-cAbVCAM1-5, a new experimental radiotracer.

Cardiovascular diseases, including fatal myocardial infarctions from atheromatous plaques, are the primary global mortality cause. Detecting stenotic atheromatous plaques is possible through coronary angiography, but vulnerable plaques with eccentric remodeling are undetectable with current diagnostic methods. Addressing this challenge, our group developed a radiopharmaceutical drug targeting vascular cell adhesion molecule 1 (VCAM-1), radiolabeled with technetium-99m. Given the absence of a monograph in the European Pharmacopoeia, and in order to draft the investigational medicinal product documentation, analytical methods had to be validated by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) to determine the radiochemical purity (RCP) of 99mTc-cAbVCAM1-5. This study therefore presents the results of the validation of analytical methods obtained in this context. The method validation followed the European Association of Nuclear Medicine (EANM) recommendations adapted from ICH Q2(R1), ensuring conformity with specificity, accuracy, repeatability and intermediate precision, linearity, robustness, quantification limit (LoQ), and range criteria. Regarding the results of specificity, both HPLC and TLC methods demonstrated excellent separation of 99mTc-cAbVCAM1-5 from impurities 99mTcO4-. Accuracy results indicated recovery percentages within the range of 99.52-101.40% for the HPLC and 99.51-101.97% for TLC, ensuring reliable measurements for each concentration of 99mTcO4-. Precision of the methods was validated by assessing repeatability and intermediate precision. Linearity was determined over the usual concentrations range and the correlation coefficient was greater than 0.99 for both methods. The limit of quantification was measured by diluting the 99mTcO4- to obtain a signal-to-noise ratio of around 10:1. Under these conditions, we obtained an LOQ of 2.10 MBq/mL for HPLC and 2Mbq/mL for TLC. In conclusion, the analytical methods developed in this study comply with EANM recommendations. This therefore allows us to correctly assess the radiochemical purity of 99mTc-cAbVCAM1-5, a new radiotracer targeting inflammation in vulnerable plaques.

Pre-clinical evaluation of 99mTc-labeled chalcone derivative for amyloid-ß imaging post-head trauma.

Aß42 plaque formation is one of the preliminary pathologic events that occur post traumatic brain injury (TBI) which is also among the most noteworthy hallmarks of AD. Their pre symptomatic detection is therefore vital for better disease management. Chalcone-picolinic acid chelator derivative, 6-({[(6-carboxypyridin-2-yl)methyl](2-{4-[(2E)-3-[4-(dimethyl amino)phenyl]prop-2-enoyl]phenoxy}ethyl)amino}methyl)pyridine-2-carboxylic acid, Py-chal was synthesized to selectively identify amyloid plaques formed post head trauma using SPECT imaging by stable complexation to 99mTc with > 97% efficiency without compromising amyloid specificity. The binding potential of the Py-chal ligand to amyloid plaques remained high as confirmed by in vitro binding assay and photophysical spectra. Further, the Py-chal complex stained amyloid aggregates in the brain sections of rmTBI mice model. In vivo scintigraphy in TBI mice model displayed high uptake followed by high retention while the healthy rabbits displayed higher brain uptake followed by a rapid washout attributed to absence of amyloid plaques. Higher uptake in brain of TBI model was also confirmed by ex vivo biodistribution analysis wherein brain uptake of 3.38 ± 0.2% ID/g at 2 min p.i. was observed for TBI mice model. This was followed by prolonged retention and more than twofold higher activity as compared to sham mice brain. This preliminary data suggests the specificity of the radiotracer for amyloid detection post head trauma and applicability of 99mTc labeled Py-chal complex for TBI-induced ß-amyloid SPECT imaging.

Preparation and bioevaluation of a novel 99mTc-labelled propylene amine oxime (PnAO) containing two 4-methyl-2-nitroimidazole groups as a promising tumor hypoxia imaging agent.

Hypoxia is a common phenomenon in solid tumors, and its presence inhibits the efficacy of tumor chemotherapy and radiotherapy. Accurate measurement of hypoxia before tumor treatment is essential. Three propylene amine oxime (PnAO) derivatives with different substituents attached to 2-nitroimidazole were synthesized in the work, they are 3,3,9,9-tetramethyl-1,11-bis(4-bromo-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dione dioxime (Br2P2), 3,3,9,9-tetramethyl-1,11-bis(4-methyl-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dione dioxime (Me2P2) and 3,3,9,9-tetramethyl-1,11-bis(4,5-dimethyl-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dione dioxime (2Me2P2). The three compounds were radiolabeled with 99mTc to give three complexes([99mTc]Tc-Br2P2, [99mTc]Tc-Me2P2 and [99mTc]Tc-2Me2P2) with good in vitro stability. [99mTc]Tc-Me2P2 with a more suitable reduction potential had the highest hypoxic cellular uptake, compared with [99mTc]Tc-2P2 that have been previously reported, [99mTc]Tc-Br2P2 and [99mTc]Tc-2Me2P2. Biodistribution results in S180 tumor-bearing mice demonstrated that [99mTc]Tc-Me2P2 had the highest tumor-to-muscle (T/M) ratio (12.37 ± 1.16) at 2 h in the four complexes. Autoradiography and immunohistochemical staining results revealed that [99mTc]Tc-Me2P2 specifically targeted tumor hypoxic regions. The SPECT/CT imaging results showed that [99mTc]Tc-Me2P2 could target the tumor site. [99mTc]Tc-Me2P2 may become a potential hypoxia imaging agent.

[99mTc]Tc-labeled HYNIC conjugated chlorambucil as a tumor targeting Agent: Synthesis, characterization and ex-vivo evaluation.

Chlorambucil is an alkylating drug that finds application towards chemotherapy of different types of cancers. In order to explore the possibility of utilization of this drug as an imaging agent for early diagnosis of solid tumors, attempt was made to synthesize a 99mTc complex of chlorambucil and evaluate its potential in tumor bearing small animal model. HYNIC-chlorambucil was synthesized by conjugation of HYNIC with chlorambucil via an ethylenediamine linker. All the intermediates and final product were purified and characterized by standard spectroscopic techniques viz. FT-IR, 1H/13C-NMR as well as by mass spectrometry. HYNIC-chlorambucil conjugate was radiolabeled with [99mTc]Tc and found to be formed with > 95 % radiochemical purity via RP-HPLC studies. The partition coefficient (Log10Po/w) of the synthesized complex was found to be -0.78 ± 0.25 which indicated the moderate hydrophilic nature for the complex. Biological behaviour of [99mTc]Tc-HYNIC-chlorambucil, studied in fibrosarcoma bearing Swiss mice, revealed a tumor uptake of about 4.16 ± 1.52 %IA/g at 30 min post-administration, which declined to 1.91 ± 0.13 % IA/g and 1.42 ± 0.14 %IA/g at 1 h and 2 h post-administration, respectively. A comparison of different [99mTc]Tc-chlorambucil derivatives (reported in the contemporary literature) formulated using different methodologies revealed that tumor uptake and pharmacokinetics exhibited by these agents strongly depend on the lipophilicity/hydrophilicity of such agents, which in turn is dependent on the bifunctional chelators used for formulating the radiolabeled chlorambucils.

Preparation, Characterization, and Radiolabeling of Anti-HER2 scFv With Technetium Tricarbonyl and Stability Studies.

Breast cancer is the most common diagnosed cancer, and the second cause of cancer death among women, worldwide. HER2 overexpression occurred in approximately 15% to 20% of breast cancers. Invasive biopsy method has been used for detection of HER2 overexpression. HER2-targeted imaging via an appropriate radionuclide is a promising method for sensitive and accurate identification of HER2+ primary and metastatic lesions. 99mTc-anti-HER2 scFv can specifically target malignancies and be used for diagnosis of the cancer type and metastasis as well as treatment of breast cancer. We radiolabeled anti-HER2 scFv that was expressed in Escherichia coli and purified through Ni-NTA resin under native condition with 99mTc-tricarbonyl formed from boranocarbonate. HER2-based ELISA, BCA, TLC, and HPLC were used in this study. In the current study, anti-HER2 scFv was lyophilized before radiolabeling. It was found that freeze-drying did not change the binding activity of anti-HER2 scFv to HER2. Results demonstrated direct anti-HER2 scFv radiolabeling by 99mTc-tricarbonyl to hexahistidine sequence (His-tag) without any changes in biological activity and radiochemical purity of around 98%. Stability analysis revealed that 99mTc-anti-HER2 scFv is stable for at least 24 h in PBS buffer, normal saline, human plasma proteins, and histidine solution.

Exploring the potential of radiolabeled duramycin as an infection imaging probe.

The continuous pursuit of designing an ideal infection imaging agent is a crucial and ongoing endeavor in the field of biomedical research. Duramycin, an antimicrobial peptide exerts its antimicrobial action on bacteria by specific recognition of phosphatidylethanolamine (PE) moiety present on most bacterial membranes, particularly Escherichia coli (E. coli). E. coli membranes contain more than 60% PE. Therefore, duramycin is an attractive candidate for the formulation of probes for in situ visualization of E. coli driven focal infections. The aim of the present study is to develop 99m Tc labeled duramycin as a single-photon emission computed tomography (SPECT)-based agent to image such infections. Duramycin was successfully conjugated with a bifunctional chelator, hydrazinonicotinamide (HYNIC). PE specificity of HYNIC-duramycin was confirmed by a dye release assay on PE-containing model membranes. Radiolabeling of HYNIC-duramycin with 99m Tc was performed with consistently high radiochemical yield (>90%) and radiochemical purity (>90%). [99m Tc]Tc-HYNIC-duramycin retained its specificity for E. coli, in vitro. SPECT and biodistribution studies showed that the tracer could specifically identify E. coli driven infection at 3 h post injection. While 99m Tc-labeled duramycin is employed for monitoring early response to cancer therapy and cardiotoxicity, the current studies have confirmed, for the first time, the potential of utilizing 99m Tc labeled duramycin as an imaging agent for detecting bacteria. Its application in imaging PE-positive bacteria represents a novel and promising advancement.

Formulation and Preclinical Testing of Tc-99m-Labeled HYNIC-Glc-FAPT as a FAP-Targeting Tumor Radiotracer.

Molecular imaging and targeted radiotherapy with radiolabeled fibroblast activation protein inhibitor (FAPI) targeting peptide probes hold great potential for enhancing the clinical management of patients with FAP-expressing cancers. However, the high cost of PET probes has prompted us to search for new FAP-targeting single-photon imaging agents. In this study, HYNIC-Glc-FAPT is synthesized and radiolabeled with technetium-99m using tricine/EDDA or dimer tricine as coligands to produce [99mTc]Tc-tricine/EDDA-HYNIC-Glc-FAPT and [99mTc]Tc-tricine(2)-HYNIC-Glc-FAPT. Both [99mTc]Tc-tricine/EDDA-HYNIC-Glc-FAPT and [99mTc]Tc-tricine(2)-HYNIC-Glc-FAPT were effectively synthesized with an excellent radiochemistry yield (both >97%, n = 6) in a single-step technique, and their stability in PBS and human serum was satisfactory. Compared to [99mTc]Tc-tricine(2)-HYNIC-Glc-FAPT, [99mTc]Tc-tricine/EDDA-HYNIC-Glc-FAPT exhibited a more hydrophilic nature with a log P of -3.53 ± 0.12. In vitro cellular uptake and blocking assays, internalization, efflux experiments, and affinity experiments all suggested a mechanism with high FAP-specificity and affinity. SPECT imaging and biodistribution of [99mTc]Tc-tricine/EDDA-HYNIC-Glc-FAPT demonstrated sustained high tumor uptake in BALB/c nude mice bearing U87MG tumors for 6 h. It demonstrated a long-range retention characteristic and more rapid clearance ability from nontarget organs. Collectively, we successfully synthesized [99mTc]Tc-tricine/EDDA-HYNIC-Glc-FAPT and [99mTc]Tc-tricine(2)-HYNIC-Glc-FAPT, and the excellent targeting properties of [99mTc]Tc-tricine/EDDA-HYNIC-Glc-FAPT suggest a potential diagnostic value in future clinical studies for advanced-stage FAP-expressing malignancies, especially in prognostic evaluation of tumors for it low price and convenient source.

Vimentin-targeted radiopeptide 99m Tc-HYNIC-(tricine/EDDA)-VNTANST: a promising drug for pulmonary fibrosis imaging.

OBJECTIVE: Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by the accumulation of extracellular matrix. Because there is no effective treatment for advanced IPF to date, its early diagnosis can be critical. Vimentin is a cytoplasmic intermediate filament that is significantly up-regulated at the surface of fibrotic foci with a crucial role in fibrotic morphological changes. METHODS: In the present study, VNTANST sequence as a known vimentin-targeting peptide was conjugated to hydrazinonicotinic acid (HYNIC) and labeled with 99m Tc. The stability test in saline and human plasma and log P determination were performed. Next, the biodistribution study and single photon emission computed tomography (SPECT) integrated with computed tomography (CT) scanning were performed in healthy and bleomycin-induced fibrosis mice models. RESULTS: The 99m Tc-HYNIC-(tricine/EDDA)-VNTANST showed a hydrophilic nature (log P  = -2.20 ±â€…0.38) and high radiochemical purity > 97% and specific activity (336 Ci/mmol). The radiopeptide was approximately 93% and 86% intact in saline and human plasma within 6 h, respectively. The radiopeptide was substantially accumulated in the pulmonary fibrotic lesions (test vs. control = 4.08 ±â€…0.08% injected dose per gram (ID/g) vs. 0.36 ±â€…0.01% ID/g at 90 min postinjection). SPECT-CT images in fibrosis-bearing mice also indicated the fibrotic foci and kidneys. CONCLUSION: Because there is no available drug for the treatment of advanced pulmonary fibrosis, early diagnosis is the only chance. The 99m Tc-HYNIC-(tricine/EDDA)-VNTANST could be a potential tracer for SPECT imaging of pulmonary fibrosis.

Synthesis and evaluation of 99mTc-labeled 1-(2-Pyridyl)piperazine derivatives as radioligands for 5HT7 receptors.

Glioblastoma multiform (GBM) is one of the most aggressive tumors of the central nervous system in humans. GBM overexpresses serotonin-7 receptors (5-HT7Rs); hence, this study aims to develop 5-HT7R targeted radiotracers. Aryl piperazine derivatives can act as ligands for 5-HT7R. Therefore, compounds 6 and 7 as 1-(3-nitropyridin-2-yl)piperazine derivatives were synthesized and radiolabeled with 99mTcN2+ core. Radiolabeled 6 and 7 (99mTcN-[6] and 99mTcN-[7]) were prepared with high radiochemical purity (RCP > 96%). They displayed high affinity toward U-87 MG cell line 5-HT7R. The calculated Ki for 99mTcN-[7] was lower than that of 99mTcN-[6] (14.85 ± 0.32 vs 22.57 ± 0.73 nM) which indicates the higher affinity of 99mTcN-[7] toward 5-HT7R. A molecular docking study also confirmed the binding of these radiotracers to 5-HT7R. The biodistribution study in normal mice revealed that 99mTcN-[7] has the highest brain accumulation at 30 min post-injection (0.54 ± 0.12 %ID/g) while the uptake of 99mTcN-[6] is much lower (0.14 ± 0.02 %ID/g). The biodistribution study in the xenograft model confirms that the radiotracers recognize the tumor site. 99mTcN-[6], and 99mTcN-[7] showed the highest tumor uptake at 1-hour post-injection (5.44 ± 0.58 vs 4.94 ± 1.65 %ID/g) and tumor-to-muscle ratios were (4.61 vs. 5.61). The injection of pimozide blocks the receptors and significantly reduces the tumor-to-muscle ratios at 1-hour post-injection to 0.81 and 0.31, respectively. In correlation with in vitro study, 99mTcN-[6] and 99mTcN-[7] visualize the tumor site in U-87 MG glioma xenografted nude mice and display the tumor-to-muscle ratios of 7.05 and 6.03.

Copper-free click bioconjugation of technetium-99m complexes using strained cyclononyne derivatives.

Click chemistry, in particular copper-free click reactions, has gained growing interest for radiolabelling purposes in the field of radiopharmaceutical sciences. [99mTc][Tc(CO)3(H2O)3]+ works as an excellent starting complex for the radiolabelling of biomolecules under mild conditions. A new chelator, investigated for the copper-free strain-promoted cycloaddition (SPAAC), was synthesised containing the 2,2'-dipicolylamine (DPA) moiety for the 99mTc-tricarbonyl core and compared with a DPA chelator based on activated esters for conventional radiolabelling. For the copper-free click labelling procedure, a DPA containing 4,8-diazacyclononyne moiety was prepared from a sulfonyl-modified diamide (four steps, 64% yield) followed by the Nicholas reaction with butyne-1,3-diol. The 99mTc-DPA-DACN-complex was prepared with a radiochemical conversion (RCC) of 89% after 30 min. The following SPAAC reaction with an azide-functionalised PSMA molecule was performed within 4-5 hours at 100 °C to obtain the PSMA (prostate-specific membrane antigen) targeting 99mTc-complex with 79% RCC and without side products. For comparison, a second DPA-chelator based on a tetrafluorophenyl (TFP) ester was prepared (three steps, 64% yield) and was successfully radiolabelled with [[99mTc]Tc(CO)3(H2O)3]+ with 89% RCC after 20 min and >99% radiochemical purity after separation using an RP18 cartridge. The subsequent conjugation of an amine-functionalised PSMA targeting molecule was performed with 23% RCC after 150 min. Two other unknown side products were observed indicating the decomposition of the TFP ester during the labelling. All nonradioactive Re(CO)3 complexes were synthesised from (Et4N)2[ReBr3(CO)3] (91% yield for the natRe-DPA-TFP ester, 76% yield for natRe-DPA-DACN) and characterised to confirm the identity of the 99mTc-complexes.

Improving tumor/muscle and tumor/blood ratios of 99mTc-labeled nitroimidazole propylene amine oxime (PnAO) complexes with ethylene glycol linkers.

Three nitroimidazole propylene amine oxime (PnAO) derivatives with different lengths of ethylene glycol chain were synthesized and radiolabeled with 99mTc. The radiochemical purities of three 99mTc-labeled complexes, oxo[[6,6,12,12-tetramethyl-1,17-bis(2-nitro-1H-imidazol-1-yl)-3,15-dioxa-7,11-diazaheptadecane-5, 13-dione dioximato] (3-)-N,N',N'',N''']-technetium-99m (99mTc-2P2O1), oxo[[9,9,15,15-tetramethyl-1,23-bis(2-nitro-1H-imidazol-1-yl)-3,6,18,21-tetraoxa-10, 14-diazatricosane-8,16-dione dioximato] (3-)-N,N',N'',N''']-technetium-99m (99mTc-2P2O2) and oxo[[15,15,21,21-tetramethyl-1,35-bis(2-nitro-1H-imidazol-1-yl)-3,6,9,12,24,27,30,33-octaoxa-16,20-diazapentatriacontane-14,22-dione dioximato] (3-)-N,N',N'',N''']-technetium-99m (99mTc-2P2O4), were above 90%, and they were all stable both in vitro and in vivo. The hypoxia/normoxia uptake ratios of the three complexes were 2.92 ± 0.61, 2.63 ± 0.64 and 2.29 ± 0.67 in S180 cellular uptake assay (4 h). All of these complexes presented good hypoxia selectivity. The results of biodistribution studies in S180 tumor-bearing mice revealed that the tumor/muscle (T/M) ratios (7.20 ± 2.37, 7.19 ± 1.75, 5.56 ± 1.10) and tumor/blood (T/B) ratios (1.66 ± 0.34, 1.73 ± 0.25, 2.13 ± 0.19) at 4 h of three complexes were significantly higher than those of 99mTc-2P2 (3.24 ± 0.65, 0.81 ± 0.34) without the ethylene glycol chains. Among them, 99mTc-2P2O4 had the best T/B ratio. The new complexes have higher tumor/blood and tumor/muscle ratios by adding suitable length of ethylene glycol chain. It is helpful for the design and optimization of hypoxic imaging agents.

Development and Evaluation of 99mTc Tricarbonyl Complexes Derived from Flutamide with Affinity for Androgen Receptor.

With the objective to develop a potential 99mTc radiopharmaceutical for imaging the androgen receptor (AR) in prostate cancer, four ligands bearing the same pharmacophore derived from the AR antagonist flutamide were prepared, labeled with 99mTc, and their structures corroborated via comparison with the corresponding stable rhenium analogs. All complexes were obtained with high radiochemical purity. Three of the complexes were highly stable, and, due to their favorable physicochemical properties, were further evaluated using AR-positive and AR-negative cells in culture. All complexes exhibited considerable uptake in AR-positive cells, which could be blocked by an excess of flutamide. The efflux from the cells was moderate. They also showed significantly lower uptakes in AR-negative cells, indicating interactions with the AR receptor. However, the binding affinities were considerably reduced by the coordination to 99mTc, and the complex that exhibited the best biological behavior did not show sufficient specificity towards AR-positive cells.

PEGylated and Non-PEGylated TCP-1 Probes for Imaging of Colorectal Cancer.

PURPOSE: Previous studies indicate that 99mTc- and fluorescent-labeled c[Cys-Thr-Pro-Ser-Pro-Phe-Ser-His-Cys]OH (TCP-1) peptides were able to detect colorectal cancer (CRC) and tumor-associated vasculature. This study was designed to characterize the targeting properties of PEGylated and non-PEGylated TCP-1 peptides for CRC imaging. PROCEDURES: Cell uptake of cyanine 7 (Cy7)-labeled TCP-1 probes (Cy7-PEG4-TCP-1 and Cy7-TCP-1) was investigated in three CRC cell lines (human, HCT116 and HT29; mouse, CT26). Xenograft and orthotopic CRC tumor models with HCT116 and CT26 cells were used to characterize biodistribution and CRC tumor-targeting properties of TCP-1 fluorescence and radioligand with and without PEGylation, [99mTc]Tc-HYNIC-PEG4-TCP-1 vs. [99mTc]Tc-HYNIC-TCP-1. RESULTS: Fluorescence images showed that TCP-1 probes were distributed in the cytoplasm and nucleus of CRC cells. When CT26 cells were treated with unlabeled TCP-1 peptide prior to the cell incubation with Cy7-PEG4-TCP-1, cell fluorescent signals were significantly reduced relative to the cells without blockade. Relative to Cy7-TCP-1, superior brilliance and visibility of fluorescence was observed in the tumor with Cy7-PEG4-TCP-1 and maintained up to 18 h post-injection. [99mTc]Tc-HYNIC-PEG4-TCP-1 images in xenograft and orthotopic CRC models demonstrated that TCP-1 PEGylation preserved tumor-targeting capability of TCP-1, but its distribution (%ID/g) in the liver and intestine was higher than that of [99mTc]Tc-HYNIC-TCP-1 (1.51 ± 0.29 vs 0.53 ± 0.12, P < 0.01). Better tumor visualization by [99mTc]Tc-HYNIC-TCP-1 was observed in the orthotopic CRC model due to lower intestinal radioactivity. CONCLUSIONS: TCP-1-based probes undergo endocytosis and localize in the cytoplasm and nucleus of human and mouse CRC cells. Tumor detectability of fluorescent TCP-1 peptide with a PEG4 spacer is promising due to its enhanced tumor binding affinity and rapid clearance kinetics from nontumor tissues. Non-PEGylated [99mTc]Tc-HYNIC-TCP-1 exhibits lower nonspecific accumulation in the liver and gastrointestinal tract and might have better capability for detecting CRC lesions in clinical sites. TCP-1 may represent an innovative targeting molecule for detecting CRC noninvasively.

Novel 99mTc labeled complexes with bisphosphonate isocyanide: Radiosynthesis and evaluation as potential bone-seeking agents.

In order to develop 99mTc-labeled complexes with bisphosphonate isocyanide as novel bone imaging agents, two bisphosphonate isocyanide derivatives (CNALN and CNPAM) were synthesized and radiolabeling was performed for preparing the corresponding [99mTc]Tc(I) complexes. [99mTc]Tc-CNALN and [99mTc]Tc-CNPAM were obtained with high radiochemical purity and showed good in vitro stability. Both of them were hydrophilic and had high affinity to hydroxyapatite. The biodistribution studies in mice revealed [99mTc]Tc-CNALN showed higher bone/background ratios at 60 min post-injection. In single photon emission computed tomography (SPECT) imaging study, [99mTc]Tc-CNALN had an obvious accumulation in bone, suggesting it would be a promising bone-seeking agent.

The preparation, biodistribution, and human's absorbed dose evaluation of Radio-Scandium-HYNIC-TOC for somatostatin-receptor-positive neuroendocrine tumors therapy by animal study.

BACKGROUND: Most of the neuroendocrine tumors (NETs) express Somatostatin receptors (SSTr), which are the main bases for the development of several radiopharmaceuticals for therapy and imaging of these types of tumors. In this study, 46 Scandium nuclide was used to label a peptide compound via hydrazinonicotinyl-Tyr3-Octreotide (HYNIC-TOC) and researched further for somatostatin-receptor NETs treatment. METHODS AND MATERIALS: The labeling procedure was conducted at 95°C for 10 min. The compound stability was tested in the environment of human serum at 37°C. The biodistribution of compound was investigated in balb/c normal mice and mice bearing AR4-2J tumor. Absorbed Doses of Human Organs were estimated by extrapolation of the biokinetics data of compound in mice to human's organs and then the absorbed doses were estimated by application of MATLAB and MIRDOSE software. RESULTS: Labeling yield was more than 90% with 555 MBq/mg specific activity. The radio-labeled compound expressed well consistency in human serum. The tumor uptake reached 3.831 ID/g% until 4 h post-injection and increased to 5.564%ID/g until 24 h post-injection. CONCLUSION: The main achievement of this study was high tumor uptake of 46 Sc-HYNIC-TOC which may be therapeutically valuable for the therapy of NETs. The estimation of the absorbed dose of human from 47 Scandium-HYNIC-TOC showed low absorbed doses in critical organs and the elimination of the radiopharmaceutical was through the gastrointestinal tract.

Impact of Different [Tc(N)PNP]-Scaffolds on the Biological Properties of the Small cRGDfK Peptide: Synthesis, In Vitro and In Vivo Evaluations.

Background: The [99mTc][Tc(N)(PNP)] system, where PNP is a bisphosphinoamine, is an interesting platform for the development of tumor 'receptor-specific' agents. Here, we compared the reactivity and impact of three [Tc(N)(PNP)] frameworks on the stability, receptor targeting properties, biodistribution, and metabolism of the corresponding [99mTc][Tc(N)(PNP)]-tagged cRGDfK peptide to determine the best performing agent and to select the framework useful for the preparation of [99mTc][Tc(N)(PNP)]-housing molecular targeting agents. Methods: cRGDfK pentapeptide was conjugated to Cys and labeled with each [Tc(N)(PNP)] framework. Radioconjugates were assessed for their lipophilicity, stability, in vitro and in vivo targeting properties, and performance. Results: All compounds were equally synthetically accessible and easy to purify (RCY ≥ 95%). The main influences of the synthon on the targeting peptide were observed in in vitro cell binding and in vivo. Conclusions: The variation in the substituents on the phosphorus atoms of the PNP enables a fine tuning of the biological features of the radioconjugates. ws[99mTc][Tc(N)(PNP3OH)]- and [99mTc][Tc(N)(PNP3)]- are better performing synthons in terms of labeling efficiency and in vivo performance than the [99mTc][Tc(N)(PNP43)] framework and are therefore more suitable for further radiopharmaceutical purposes. Furthermore, the good labeling properties of the ws[99mTc][Tc(N)(PNP3OH)]- framework can be exploited to extend this technology to the labeling of temperature-sensitive biomolecules suitable for SPECT imaging.