Optimization of the radiosynthesis of [68Ga]Ga-PSMA-11 using a Trasis MiniAiO synthesizer: do we need to heat and purify?

INTRODUTION:: [Ga]Ga-prostate specific membrane antigen (PSMA)-11 showed a clear gain in sensitivity for lesion detection in the biological recurrence of prostate cancer as compared to the standard [F]fluorocholine radiopharmaceutical. To meet the strong demand for [Ga]Ga-PSMA-11, we aimed to optimize an automated radiolabeling process by evaluating the influence of different key parameters on radiochemical purity and radiochemical yield. METHODS: The radiosynthesis of [Ga]Ga PSMA-11 was performed using a Trasis MiniAio synthesizer and a Ge/Ga GalliaPharm generator supplied by Eckert & Ziegler, Berlin, Germany. Optimized labeling parameters were evaluated by variation of sodium acetate concentrations and temperature of radiolabeling as well as the purification process. RESULTS: For each condition tested, radiochemical purity was higher than 99% in the final vial without batch failure, indicating a robust and fast radiosynthesis process. Radiosynthesis without the solid phase extraction purification process at room temperature in less than 5 min resulted in a radiolabeling efficiency of over 99% and remained stable at least 4 h without manual processing to limit operator radiation exposure. CONCLUSION: The procedure was completely automated and provided a high radiochemical yield. It can be performed several times a day, facilitating the clinical demand of this radiopharmaceutical.

Enhancing capacity and synthesis of [68Ga]68-Ga-PSMA-HBED-CC with the lyophilized ready-to-use kit for nuclear pharmacy applications.

INTRODUCTION: The authors describe the newly proposed synthesis technique for the gallium-68 (Ga-68)-labeled tracer ([Ga]Ga-PSMA-HBED-CC) for imaging expression of the prostate-specific membrane antigen (PSMA). An effort was applied to design the lyophilized cold kit (isoPROtrace-11) as a time-saving technique resulting in increased radiochemical yields. PROCEDURES: The initial material for labeling was obtained from a Ge/Ga-generator. For labeling with the lyophilized cold kit isoPROtrace-11, 2.5 ml 0.1 M HCl of the middle Ga-68 elution fraction were added to the kit, shook for dissolving the vial's contents and kept for 5 minutes at room temperature. A systematic comparison was carried out between results obtained with the cold kit technique and with previously used Modular-Lab module concerning the radiochemical yield, purity, and the time of producing. RESULTS: Automated module-involved synthesis of [Ga]Ga-PSMA-HBED- CC resulted in a radiochemical yield of 84.2 ± 6.3% and purity of >95% after 25 minutes. The room temperature cold kit gave a radiochemical yield of >98% and purity of >95% after 5 minutes. CONCLUSION: Using the kit method reduced the labeling time. The cold kit method increased production efficiency because less of the eluted Ga-68 was wasted.

Synthesis, characterization and evaluation of 68Ga labelled monomeric and dimeric quinazoline derivatives of the HBED-CC chelator targeting the epidermal growth factor receptor.

Tyrosine kinase (TK) receptors including epidermal growth factor receptors (EGFRs) are known to be overexpressed in a wide variety of solid tumors associated with poor prognosis. The HBED-CC chelator N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid 1 was coupled via one or both its propionic acid moieties with the quinazoline EGFR-TK inhibiting pharmacophore 4-amino-N-(4-((3-bromophenyl)amino)quinazolin-6-yl)butanamide 3 resulting in either a monomeric 4 or a dimeric 5 species. Ligands 4 and 5 reacted with Ga3+ generating the corresponding complexes Ga4 and Ga5. Both ligands and complexes were characterized with mass spectrometry and NMR spectroscopy and evaluated in vitro with MTT assays in A431 cells, where they showed IC50 values in the range 51.6 to 68.8 µM. Labeling of ligands 4 and 5 with the PET radionuclide 68Ga was quantitative and resulted in tracers [68Ga]Ga4 and [68Ga]Ga5 with radiochemical purities greater than 98%, which were also characterised by comparative RP-HPLC studies with Ga4 and Ga5 respectively. Radiotracers [68Ga]Ga4 and [68Ga]Ga5 were stable (intact tracer over 98%) in the reaction mixture (120 min) and in human serum (30 min). Both tracers were evaluated in vivo with biodistribution experiments in SCID mice bearing A431 tumors presenting tumor uptake of 1.34 for [68Ga]Ga4 and 1.01 %ID/g for [68Ga]Ga5 at 5 min, which was slightly decreased at 60 min p.i. and then remained stable until 120 min p.i. To the best of our knowledge, this is the first report of monomeric and dimeric quinazoline conjugates with the chelator HBED-CC, which can serve as a basis for further development of EGFR-TKI targeting tracers.

Synthesis, Labeling and Preclinical Evaluation of a Squaric Acid Containing PSMA Inhibitor Labeled with 68 Ga: A Comparison with PSMA-11 and PSMA-617.

The L-lysine urea-L-glutamate (KuE) represents a key motif in recent diagnostic and therapeutic radiopharmaceuticals targeting the prostate specific membrane antigen (PSMA). Using a squaric acid moiety for coupling of KuE with a radioactive label, the squaric acid as a linker in the PSMA ligand seems to mimic the aromatic structure of the naphthylalanine unit on PSMA-617. In this work, we investigate the influence of squaric acid moiety on the biological activity of the compound carrying a KuE motif and three typical chelates. The derivatives TRAM.SA.KuE, DOTAGA.SA.KuE and NODAGA.SA.KuE were all synthesized in straightforward organic reactions and purified by HPLC afterward. Different amounts of tracer were labeled at different temperatures with 68 Ga. PET examinations were performed on NMRInu/nu nude mice with an LNCaP tumor on the right hind leg including ex vivo investigations of the organs. For comparison, 68 Ga-derivatives of PSMA-11 and PSMA-617, the derivatives most commonly used in clinics, were investigated in the same animal model.

Contribution of 5th minute and 2nd hour images to standard imaging in (68Ga)PSMA 11 PET/CT.

OBJECTIVE: To investigate the superiority or contribution of 5th minute pelvic and 2nd hour whole body Gallium68-prostate-specific membrane antigen-HBED-CC [(68Ga)PSMA 11] Positron Emission Tomography/Computed Tomography (PET/CT) images to 1st hour imaging in patients with prostate cancer (PCa). MATERIALS AND METHODS: A total of 63 patients diagnosed with PCa who underwent (68Ga)PSMA 11 PET/CT between April 2019 and June 2019 and who had 5th minute and 1st and 2nd hour images were included in the study. Early (5th minute) pelvic region and 1st and 2nd hour full body images were obtained from all patients. The regions of interest (ROI) were drawn from the background tissues and the physiological uptake sites in a way to include the same lesions from primary and metastatic lesions in all three imagings, and SUVmax values, and tumor-background ratio (TBR) were calculated. RESULTS: The mean age of the patients was 69.81 ± 8.78 (min/max: 51/91) years. In the 5th minute images, prostate gland and bed were easier to evaluate, because of low bladder activity. However, lymph node evaluation was more difficult due to high vascular activity. In the prostate gland, lymph nodes and bone metastases, both SUVmax values and TBR rates increased with time from the 5th minute (p < 0.001). At the 2nd hour, some lesions became more visible, while decreased activity was observed in some lesions. However, none of the patients required a change in the stage or treatment. CONCLUSION: In conclusion, the 5th minute pelvic images in (68Ga)PSMA 11 PET/CT were helpful in visual evaluation of the prostate gland and bed, while 2nd hour images showed high SUVmax and TBR rates in malignant lesions. As the SUVmax values of benign lesions were found to be lower in the 2nd hour, when compared to the 1st hour, it was thought that the 2nd hour imaging could be used in the additional imaging for suspicious lesions without the need for very long waiting times.

Evaluation of an Automated Module Synthesis and a Sterile Cold Kit-Based Preparation of 68Ga-PSMA-11 in Patients with Prostate Cancer.

68Ga-labeled urea-based inhibitors of the prostate-specific membrane antigen (PSMA), such as 68Ga-PSMA-11, are promising small molecules for targeting prostate cancer (PCa). Although this radiopharmaceutical was produced mostly by means of manual synthesis and automated synthesis modules, a sterile cold kit was recently introduced. The aim of our study was to evaluate the image quality of 68Ga-PSMA-11 PET/CT (PSMA-PET) in a population of PCa patients after the injection of comparable activities of 68Ga-PSMA-11 obtained with the 2 different synthetic procedures. A secondary aim was to identify secondary factors that may have an impact on image quality and, thus, final interpretation. Methods: Two different groups of 100 consecutive PCa patients who underwent PSMA-PET were included in the study. The first group of patients was imaged with 68Ga-PSMA-11 obtained using synthesis modules, whereas the second group's tracer activity was synthesized using a sterile cold kit. All PET images were independently reviewed by 2 nuclear medicine diagnosticians with at least 2 y of experience in PSMA-based imaging and unaware of the patients' clinical history. The 2 reviewers independently rated the quality of each PSMA-PET scan using a 3-point Likert-type scale. In cases of discordance, the operators together reviewed the images and reached a consensus. Performance was evaluated on the basis of the expected biodistribution, lesion detection rate, and physiologic background uptake. Results: Overall, 104 of 200 (52%) PSMA-PET scans were positive for PCa-related findings. No significant differences in image quality between cold kits and synthesis modules were found (P = 0.13), although a higher proportion of images was rated as excellent by the observers for kits than for modules (45% vs. 34%). Furthermore, after image quality had been dichotomized as excellent or not excellent, multivariate regression analysis found several factors to be significantly associated with a not-excellent quality: an increase in patient age (+5 y: odds ratio [OR], 1.40; 95% confidence interval [CI], 1.12-1.75), an increase in patient weight (+5 kg: OR, 1.89; 95% CI, 1.53-2.32), an increase in 68Ga-PSMA-11 uptake time (+10 min: OR, 1.45; 95% CI, 1.08-1.96), and a decrease in injected activity (-10 MBq: OR, 1.28; 95% CI, 1.07-1.52). Conclusion: No significant differences were identified between the 2 groups of patients undergoing PSMA-PET; therefore, we were not able to ascertain any significant influences of tracer production methodology on final scan quality. However, increased patient age, increased patient weight, decreased injected activity, and increased 68Ga-PSMA-11 uptake time were significantly associated with an overall poorer image quality.

Amphiphilic ligand modified gold nanocarriers to amplify lanthanide loading for ultrasensitive DELFIA detection of Cronobacter.

Conventional dissociation-enhanced lanthanide (Ln3+) fluoroimmunoassays (DELFIAs) using Ln3+ chelate-labeled antibodies as molecular probes exhibit limited sensitivity because of their relatively low Ln3+ labeling ratio per biomolecule. Herein, we applied gold nanoflowers (AuNFs) as amplified nanocarriers to increase the Ln3+ labeling ratio in a single molecular binding event for improving the sensitivity of traditional DELFIA. Two thiolated amphiphilic ligands (thiolated ethylenediaminetetraacetic acid (EDTA) and thiolated acylhydrazine-terminated ligands), consisting of a hydrophobic alkane chain, oligo(ethylene glycol) unit, and functional terminal of the EDTA or acylhydrazine group, were designed for the surface modification of AuNFs. The resultant ligand-coated AuNFs exhibited dual functions of Ln3+ chelation via the EDTA group and oriented attachment of antibodies via the acylhydrazine group. By utilizing 80 nm AuNFs as amplified carriers, we demonstrated that the maximum Eu3+ loading amount reached 1.07 × 104 Eu3+ ions per AuNF, which is approximately two to three orders of magnitude higher than that of traditional molecular probes, thereby amplifying the luminescence signal and enhancing the sensitivity of DELFIA. By combining a magnetic-mediated sandwich-type DELFIA method, the designed amplified AuNF nanoprobes achieved an ultrasensitive luminescence detection of Cronobacter muytjensii with a limit of detection (LOD) of 1.2 × 102 cfu mL-1 in a powdered infant formula. This LOD value was ca. 230-fold lower than that of the traditional colorimetric immunoassay. The designed signal amplification strategy using bifunctional ligand-modified AuNFs as enhanced Ln3+ nanocarriers provided a huge potential for building various ultrasensitive luminescence immunoassays for in vitro biodetection.

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.

Automated production of [68 Ga]Ga-DOTANOC and [68 Ga]Ga-PSMA-11 using a TRACERlab FXFN synthesis module.

The interest in gallium-68 labelled positron-emission tomography probes continues to increase around the world. However, one of the barriers for routine clinical use is the cost of the automated synthesis units for relatively simple labelling procedures. Herein, we describe the adaptation of a TRACERlab FXFN synthesis module for the automated production of gallium-68 radiopharmaceuticals using a cation-exchange cartridge for postprocessing of the 68 Ge/68 Ga generator eluate. The recovery of activity from the cartridge was 95.6% to 98.9% using solutions of acidified sodium chloride (5 M with pH = 1-3). The radiosyntheses of [68 Ga]Ga-DOTANOC and [68 Ga]Ga-PSMA-11 were performed using acetate sodium buffer or 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, with a total duration of 21 and 23 minutes, respectively, including generator elution and radiopharmaceutical dispensing. Activity yields were 77% ± 2% for [68 Ga]Ga-PSMA-11 and 68% ± 3% for [68 Ga]Ga-DOTANOC (n > 100). The labelled peptides had a radiochemical purity exceeding 97%, and all quality control parameters were in conformity with the limits prescribed by the European Pharmacopoeia.

Fe-HBED Analogs: A Promising Class of Iron-Chelate Contrast Agents for Magnetic Resonance Imaging.

Contrast-enhanced magnetic resonance imaging is an essential tool for disease diagnosis and management; all marketed clinical magnetic resonance imaging (MRI) contrast agents (CAs) are gadolinium (Gd) chelates and most are extracellular fluid (ECF) agents. After intravenous injection, these agents rapidly distribute to the extracellular space and are also characterized by low serum protein binding and predominant renal clearance. Gd is an abiotic element with no biological recycling processes; low levels of Gd have been detected in the central nervous system and bone long after administration. These observations have prompted interest in the development of new MRI contrast agents based on biotic elements such as iron (Fe); Fe-HBED (HBED = N,N'-bis(2-hydroxyphenyl)ethylenediamine-N,N'-diacetic acid), a coordinatively saturated iron chelate, is an attractive MRI CA platform suitable for modification to adjust relaxivity and biodistribution. Compared to the parent Fe-HBED, the Fe-HBED analogs reported here have lower serum protein binding and higher relaxivity as well as lower relative liver enhancement in mice, comparable to that of a representative gadolinium-based contrast agent (GBCA). Fe-HBED analogs are therefore a promising class of non-Gd ECF MRI CA.

68Ga-PSMA PET/CT in prostate cancer. / La PET/TC con 68Ga-PSMA en el cáncer de próstata.

Positron emission tomography/computed tomography (PET/CT) with 68Ga-PSMA is a non-invasive diagnostic technique to image prostate cancer with increased prostate-specific membrane antigen (PSMA) expression. PSMA is a transmembrane protein present in all prostatic tissues. Increased PSMA expression is seen in several malignancies, although prostate cancer is the tumour where it presents higher concentrations. Almost all prostate adenocarcinomas show PSMA expression in most of lesions, primary and metastatic. Immunohistochemistry has demonstrated that the expression of PSMA increases in patients with de-differentiated, metastatic or hormone-refractory tumours. Moreover, the expression level of PSMA has a prognostic value for disease outcome. PET measures the three-dimensional distribution of 68Ga-PSMA, producing semi-quantitative images that allow for non-invasive assessment of PSMA expression.

One-pot synthesis of trifunctional chitosan-EDTA-ß-cyclodextrin polymer for simultaneous removal of metals and organic micropollutants.

The global contamination of water resources with inorganic and organic micropollutants, such as metals and pharmaceuticals, poses a critical threat to the environment and human health. Herein, we report on a bio-derived chitosan-EDTA-ß-cyclodextrin (CS-ED-CD) trifunctional adsorbent fabricated via a facile and green one-pot synthesis method using EDTA as a cross-linker, for the adsorption of toxic metals and organic micropollutants from wastewater. In this system, chitosan chain is considered as the backbone, and the immobilized cyclodextrin cavities capture the organic compounds via host-guest inclusion complexation, while EDTA-groups complex metals. The thoroughly characterized CS-ED-CD was employed for batch adsorption experiments. The adsorbent displayed a monolayer adsorption capacity of 0.803, 1.258 mmol g-1 for Pb(II) and Cd(II) respectively, while a heterogeneous sorption capacity of 0.177, 0.142, 0.203, 0.149 mmol g-1 for bisphenol-S, ciprofloxacin, procaine, and imipramine, respectively. The adsorption mechanism was verified by FT-IR and elemental mapping. Importantly, the adsorbent perform is effective in the simultaneous removal of metals and organic pollutants at environmentally relevant concentrations. All these findings demonstrate the promise of CS-ED-CD for practical applications in the treatment of micropollutants. This work adds a new insight to design and preparation of efficient trifunctional adsorbents from sustainable materials for water purification.

Influência do ressecamento da superfície radicular, do tratamento com EDTA e ácido Hialurônico na viabilidade e expressão de citocinas inflamatórias de fibroblastos do ligamento periodontal / Influence of root surface dryness and treatment with EDTA and Hyaluronic acid on the viability and expression of inflammatory cytokines of periodontal ligament fibroblasts

O momento ideal para o reimplante de um dente avulsionado é imediatamente após a avulsão, no entanto, isso nem sempre é possível. Uma série de fatores influenciam na viabilidade das células do ligamento periodontal (PDL) contribuindo para acelerar ou minimizar a ocorrência da reabsorção radicular ou anquilose, consequências mais frequentes dos reimplantes. Um destes fatores é o período extra-oral e o tratamento da superfície radicular. O EDTA (ácido etilenodiamino tetracético) a 17% e o ácido hialurônico (AH) são utilizados para tratar a superfície radicular, visando menor ocorrência de reabsorção inflamatória e por substituição. Sendo assim, os objetivos deste estudo foram: a) avaliar a viabilidade de fibroblastos do ligamento periodontal (PDLF) em contato com discos radiculares submetidos ou não ao ressecamento de superfície por diferentes tempos e tratados com EDTA e/ou AH; b) quantificar as citocinas inflamatórias IL-6, IL-8, IL-1ß e TNF-α expressas por PDLF; c) observar a adesão de PDLF na superfície do discos radiculares tratados. Foram obtidos 108 discos de dentina e cemento da superfície radicular de dentes bovinos, com 4,5 mm de diâmetro, que foram previamente submetidos a dissolução do ligamento periodontal em solução de hipoclorito de sódio 1% por 15 min. Em seguida os discos foram esterilizados em concentrações decrescentes de álcool (100%,90%,80% e 70%). Os espécimes foram submetidos ou não ao ressecamento radicular por 1h ou 24h e tratados com EDTA associado ou não ao ácido hialurônico, colocados em placas de 96 poços onde foram semeadas células de culturas primárias de PDLF, que ficaram em contato com os discos por 48 h. A viabilidade celular na superfície dos discos foi avaliada através do ensaio de XTT. A microscopia eletrônica de varredura foi utilizada para verificar a adesão de PDLF à superfície dos discos. A detecção e quantificação das citocinas foi realizada pelo teste ELISA. Os dados foram submetidos à análise estatística pelo teste ANOVA e Tukey (p < 0,05). A maior média foi apresentada no grupo sem ressecamento para o tratamento EDTA+AH (148,39), que diferiu significativamente dos grupos controle e EDTA. No grupo ressecamento 1 h EDTA+AH (144,91) foi diferente dos demais. Para ressecamento 24h, verificou-se que o grupo EDTA+AH diferiu do grupo controle. Não houve modificações na expressão das citocinas IL-6, IL-8, IL-1ß e TNF-α quando foi acrescentado os tratamentos propostos. A IL-6 mostrou uma diminuição quando em contato com AH no periodo de 24h. Foi observada pelo MEV adesão de PDLF na superfície de todos os discos tratados e em todos os períodos analisados. Conclui-se que o ácido hialurônico é uma alternativa de tratamento para casos de dentes avulsionados já que mostrou seu papel promovendo adesão e aumento da viabilidade(AU)

The ideal time for reimplantation of an avulsed tooth is immediately after avulsion, however, this is not always possible. A number of factors influence the viability of the cells of the periodontal ligament (PDL) contributing to accelerate or minimize the occurrence of root resorption or ankylosis, more frequent consequences of reimplantation. One of these factors is the extra-oral period and root surface treatment. EDTA (17% ethylenediaminetetraacetic acid) and hyaluronic acid (HA) are used to treat the root surface, aiming for a lower occurrence of inflammatory resorption and replacement. Thus, the objectives of this study were: a) to evaluate the viability of periodontal ligament fibroblasts (PDLF) in contact with root disks submitted to surface dryness at different times and treated with EDTA and / or AH; b) quantify the inflammatory cytokines IL-6, IL-8, IL-1ß and TNF-α expressed by PDLF; c) observe the adhesion of PDLF on the surface of the treated root discs. 108 dentin and cementum disks were obtained from the root surface of bovine teeth, 4.5 mm in diameter, which were previously submitted to dissolution of the periodontal ligament in 1% sodium hypochlorite solution for 15 min. Then the disks were sterilized in decreasing concentrations of alcohol (100%, 90%, 80% and 70%). The specimens were submitted to root resection for 1 h or 24 h and treated with EDTA, whether or not associated with hyaluronic acid, placed in 96-well plates where cells from PDLF primary cultures were seeded and left in contact with the discs for 48 h. Cell viability at disc surfaces was assessed by the XTT assay. Scanning electron microscopy was used to verify the adhesion of PDLF to the disc surface. Detection and quantification of cytokines was performed by ELISA. Data were submitted to statistical analysis by the ANOVA and Tukey test (p <0.05). The highest mean was presented in the non-dry group for EDTA + AH treatment (148,39), which differed significantly from the control and EDTA groups. In the dryness group 1 h EDTA + AH (144.91) was different from the others. For dryness 24 h, it was found that the EDTA + AH group differed from the control group. There were no changes in the expression of cytokines IL-6, IL-8, IL-1ß and TNFα when the proposed treatments were added. IL-6 showed a decrease when in contact with HA in the 24-hour period. It was observed by the MEV adhesion of PDLF on the surface of all treated discs and in all periods analyzed. It is concluded that hyaluronic acid is an alternative treatment for cases of avulsed teeth since it showed its role promoting adhesion and increased viability(AU)

A Double Prodrug with Improved Membrane Permeability over the Parent Chelator HBED Provides Superior Cytoprotection against Hydrogen Peroxide.

The clinical use of N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) has been hindered by its lack of bioavailability. N,N'-bis(2-boronic pinacol ester benzyl)ethylenediamine-N,N'-diacetic acid methyl, ethyl, and isopropyl esters 7 a-c, respectively, and their dimesylate salts 8 a-c, are double prodrugs that mask the two phenolate and two carboxylate donors of HBED as boronic esters and carboxylate esters, respectively. Their activation by chemical hydrolysis and oxidation, their passive diffusivity, and their cytoprotective capabilities have been investigated here. 8 a-c hydrolyzed in minimum essential medium at 37 °C with half-lives of 0.69, 0.81, and 2.28 h, respectively. The intermediate formed, 9 [N,N'-bis(2-boronic acid benzyl)ethylenediamine-N,N'-diacetic acid], then underwent oxidative deboronation by H2 O2 to give HBED (k=1.82 m(-1) min(-1) ). Solubility measurements in mineral oil and in phosphate buffer indicated that 7 a had a better balance between lipid and aqueous solubilities than did HBED. 7 a was also able to passively diffuse across a lipid-like silicone membrane (log flux=-0.36), whereas HBED-HCl was not. 8 c provided better protection to retinal cells than did HBED against a lethal dose of H2 O2 (84 % vs. 28 % protection, respectively, at 44 µm). These results suggest that the double prodrugs have better membrane permeability than does HBED, and therefore could be therapeutically useful for improving the delivery of HBED.

Novel double prodrugs of the iron chelator N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED): Synthesis, characterization, and investigation of activation by chemical hydrolysis and oxidation.

The development of iron chelators suitable for the chronic treatment of diseases where iron accumulation and subsequent oxidative stress are implicated in disease pathogenesis is an active area of research. The clinical use of the strong chelator N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) and its alkyl ester prodrugs has been hindered by poor oral bioavailability and lack of conversion to the parent chelator, respectively. Here, we present novel double prodrugs of HBED that have the carboxylate and phenolate donors of HBED masked with carboxylate esters and boronic acids/esters, respectively. These double prodrugs were successfully synthesized as free bases (7a-f) or as dimesylate salts (8a-c,e), and were characterized by (1)H, (13)C, and (11)B NMR; MP; MS; and elemental analysis. The crystal structure of 8a was solved. Three of the double prodrugs (8a-c) were selected for further investigation into their abilities to convert to HBED by stepwise hydrolysis and H2O2 oxidation. The serial hydrolysis of the pinacol and methyl esters of N,N'-bis(2-boronic acid pinacol ester benzyl)ethylenediamine-N,N'-diacetic acid methyl ester dimesylate (8a) was verified by LC-MS. The macro half-lives for the hydrolyses of 8a-c, measured by UV, ranged from 3.8 to 26.3 h at 37 °C in pH 7.5 phosphate buffer containing 50% MeOH. 9, the product of hydrolysis of 8a-c and the intermediate in the conversion pathway, showed little-to-no affinity for iron or copper in UV competition experiments. 9 underwent a serial oxidative deboronation by H2O2 in N-methylmorpholine buffer to generate HBED (k = 10.3 M(-1) min(-1)). The requirement of this second step, oxidation, before conversion to the active chelator is complete may confer site specificity when only localized iron chelation is needed. Overall, these results provide proof of principle for the activation of the double prodrugs by chemical hydrolysis and H2O2 oxidation, and merit further investigation into the protective capabilities of the prodrugs against H2O2-induced cell death.

Comprehensive Quality Control of the ITG 68Ge/68Ga Generator and Synthesis of 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC for Clinical Imaging.

UNLABELLED: A good-manufacturing-practices (GMP) (68)Ge/(68)Ga generator that uses modified dodecyl-3,4,5-trihydroxybenzoate hydrophobically bound to a octadecyl silica resin (C-18) as an adsorbent has been developed that allows for dilute HCl (0.05N) to efficiently elute metal-impurity-free (68)Ga(3+) ready for peptide labeling. We characterized the performance of this generator system over a year in conjunction with the production of (68)Ga-labeled DOTATOC and Glu-NH-CO-NH-Lys(Ahx)-HBED-CC (PSMA-HBED-CC) intended for clinical studies and established protocols for batch release. METHODS: A 2,040-MBq self-shielded (68)Ge/(68)Ga generator provided metal-free (68)GaCl3 ready for peptide labeling in the fluidic labeling module after elution with 4 mL of 0.05N HCl. The compact system was readily housed in a laminar flow cabinet allowing an ISO class-5 environment. (68)Ga labeling of peptides using GMP kits was performed in 15-20 min, and the total production time was 45-50 min. Batch release quality control specifications were established to meet investigational new drug submission and institutional review board approval standards. RESULTS: Over a period of 12 mo, (68)Ga elution yields from the generator averaged 80% (range, 72.0%-95.1%), and (68)Ge breakthrough was less than 0.006%, initially decreasing with time to 0.001% (expressed as percentage of (68)Ge activity present in the generator at the time of elution), a unique characteristic of this generator. The radiochemical purity of both (68)Ga-DOTATOC and (68)Ga-PSMA-HBED-CC determined by high-performance liquid chromatography analysis was greater than 98%, with a minimum specific activity of 12.6 and 42 GBq/µmol, respectively. The radionuclidic ((68)Ge) impurity was 0.00001% or less (under the detection limit). Final sterile, pyrogen-free formulation was provided in physiologic saline with 5%-7% ethanol. CONCLUSION: The GMP-certified (68)Ge/(68)Ga generator system was studied for a year. The generator system is contained within the fluidic labeling module, and it is compact, self-shielded, and easy to operate using simple manual techniques. The system provides radiolabeled peptides with high (>98%) radiochemical purity and greater than 80% radiochemical yield. The (68)Ge levels in the final drug products were under the detection limits at all times. (68)Ga-DOTATOC and (68)Ga-PSMA-HBED-CC investigational radiopharmaceuticals are currently being studied clinically under investigational new drug (IND) applications submitted to the U.S. Food and Drug Administration.

68Ga-PSMA-HBED-CC PET for Differential Diagnosis of Suggestive Lung Lesions in Patients with Prostate Cancer.

UNLABELLED: In prostate cancer (PC) patients, the differentiation between lung metastases and lesions of different origin, for example, primary lung cancer, is a common clinical question. Herein, we investigated the use of Glu-NH-CO-NH-Lys(Ahx)-HBED-CC ((68)Ga-PSMA-HBED-CC) for this purpose. METHODS: PC patients (n = 1,889) undergoing (68)Ga-PSMA PET/CT or PET/MR scans were evaluated retrospectively for suggestive lung lesions. For up to 5 lesions per patient, location, CT diameter, CT morphology, and SUVmax were determined. The standard for classification was either histopathologic evaluation or, in the case of PC metastases, responsivity to antihormone therapy. A comparison of the different classes was executed by Student t test. Prostate-specific antigen and prostate-specific membrane antigen (PSMA) immunohistochemistry were performed if histologic samples were available; (68)Ga-PSMA autoradiography was performed on an exemplary case of PET-positive lung cancer. RESULTS: Eighty-nine lesions in 45 patients were identified, of which 76 were classified as PC (39 proven, 37 highly probable), 7 as primary lung cancer, and 2 as activated tuberculosis; 4 lesions remained unclear. The mean SUVmax was 4.4 ± 3.9 for PC metastases and 5.6 ± 1.6 for primary lung cancer (P = 0.408). Additionally, substantial differences in SUVmax intraindividually were detected. The 2 tuberculous lesions showed an SUVmax of 7.8 and 2.5. Using immunohistochemistry, we could demonstrate PSMA expression in the neovasculature of several PSMA PET-positive lung cancers as well as in tuberculous lesions from our histologic database. CONCLUSION: Quantitative (SUV) analysis of (68)Ga-PSMA PET was not able to discriminate reliably between pulmonary metastases and primary lung cancer in PC patients. The reason for the unexpectedly high tracer uptake in non-PC lesions is not completely clear. PSMA expression in neovasculature provides a possible explanation for this finding; however, other contributing factors, such as tracer binding to proteins other than PSMA, cannot be excluded at present.

A Novel Europium Chelate Coated Nanosphere for Time-Resolved Fluorescence Immunoassay.

A novel europium ligand 2,2',2'',2'''-(4,7-diphenyl-1,10-phenanthroline-2,9-diyl) bis (methylene) bis (azanetriyl) tetra acetic acid (BC-EDTA) was synthesized and characterized. It shows an emission spectrum peak at 610 nm when it is excited at 360 nm, with a large Stock shift (250 nm). It is covalently coated on the surface of a bare silica nanosphere containi free amino groups, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-Hydroxysuccinimide. We also observed an interesting phenomenon that when BC-EDTA is labeled with a silica nanosphere, the chelate shows different excitation spectrum peaks of about 295 nm. We speculate that the carboxyl has a significant influence on its excitation spectrum. The BC-EDTA/Eu3+coated nanosphere could be used as a fluorescent probe for time-resolved fluorescence immunoassay. We labeled the antibody with the fluorescent nanosphere to develop a nanosphere based hepatitis B surface antigen as a time-resolved fluorescence immunoassay reagent, which is very easy to operate and eliminates potential contamination of Eu3+ contained in the environment. The analytical and functional sensitivities are 0.0037 µg/L and 0.08 µg/L (S/N≥2.0) respectively. The detection range is 0.08-166.67 µg/L, which is much wider than that of ELISA (0.2-5 µg/L). It is comparable to the commercial dissociation-enhanced lanthanide fluoro-immunoassay system (DELFIA) reagents (0.2-145 µg/L). We propose that it can fulfill clinical applications.

Star-shaped tetraspermine enhances cellular uptake and cytotoxicity of T-oligo in prostate cancer cells.

PURPOSE: An oligonucleotide termed 'T-oligo' having sequence homology with telomere overhang has shown cytotoxicity in multiple cancers. We have demonstrated that T-oligo can induce apoptosis in androgen independent prostate cancer cell line DU-145. In this report, we evaluate the use of star-shaped tetraspermine (SSTS) for delivery of T-oligo. METHODS: SSTS was synthesized from spermine and its intrinsic cytotoxicity towards DU-145 cells was compared with spermine and branched polyethyleneimine (bPEI). Atomistic molecular dynamic (MD) simulations were conducted to understand binding and complexation of spermine and SSTS with T-oligo. Complexation was also determined using gel electrophoresis and SYBR gold assay. Complexes were characterized for size, cellular uptake and antiproliferative effect. RESULTS: SSTS exhibited significantly lower toxicity than spermine and bPEI. Its affinity towards T-oligo was significantly higher than spermine as determined by experimental studies and confirmed by MD simulations and it formed stable complexes (TONPs) with T-oligo. TONPs facilitated cellular uptake and nuclear accumulation of T-oligo and their cytotoxic potential was observed at concentration several folds lower than that required for T-oligo alone. CONCLUSION: SSTS significantly enhanced therapeutic benefits associated with the use of T-oligo and can be developed as a delivery vehicle for its in-vivo therapeutic applications.