Automated production of [68Ga]Ga‐DOTANOC and [68Ga]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 FX_FN synthesis module for the automated production of gallium‐68 radiopharmaceuticals using a cation‐exchange cartridge for postprocessing of the ⁶⁸Ge/⁶⁸Ga 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 [⁶⁸Ga]Ga‐DOTANOC and [⁶⁸Ga]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 [⁶⁸Ga]Ga‐PSMA‐11 and 68% ± 3% for [⁶⁸Ga]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.     

Synthesis and biological evaluation of 68Ga‐labeled Pteroyl‐Lys conjugates for folate receptor‐targeted tumor imaging

In order to develop novel ⁶⁸Ga‐labeled PET tracers for folate receptor imaging, two DOTA‐conjugated Pteroyl‐Lys derivatives, Pteroyl‐Lys‐DOTA and Pteroyl‐Lys‐DAV‐DOTA, were designed, synthesized and radiolabeled with ⁶⁸Ga. Biological evaluations of the two radiotracers were performed with FR‐positive KB cell line and athymic nude mice bearing KB tumors. Both ⁶⁸Ga‐DOTA‐Lys‐Pteroyl and ⁶⁸Ga‐DOTA‐DAV‐Lys‐Pteroyl exhibited receptor specific binding in KB cells in vitro. The tumor uptake values of ⁶⁸Ga‐DOTA‐Lys‐Pteroyl and ⁶⁸Ga‐DOTA‐DAV‐Lys‐Pteroy were 10.06 ± 0.59%ID/g and 11.05 ± 0.60%ID/g at 2 h post‐injection, respectively. Flank KB tumor was clearly visualized with ⁶⁸Ga‐DOTA‐DAV‐Lys‐Pteroyl by Micro‐PET imaging at 2 h post‐injection, suggesting the feasibility of using ⁶⁸Ga‐labeled Pteroyl‐Lys conjugates as a novel class of FR targeted probes.     

A simple method for preparation of pure 68Ga‐acetate precursor for formulation of radiopharmaceuticals: Physicochemical characteristics of the 68Ga eluate of the SnO2 based‐68Ge/68Ga column generator

Gallium‐68 radioisotope is an excellent source in clinical positron emission tomography application due to its ease of availability from germanium‐68 (⁶⁸Ge)/gallium‐68 (⁶⁸Ga) generator having a shelf life of 1 year. In this paper, a modified method for purification of the primary eluate of ⁶⁸Ge‐⁶⁸Ga generator by using a small cation exchange resin (Dowex‐50) column has been described. The breakthrough of ⁶⁸Ge before and after purification of ⁶⁸Ga eluate was 0.014% and 0.00027%, respectively. The average recovery yield of ⁶⁸Ga after purification was 84% ± 8.6% (SD, n  = 335). The results of the physiochemical studies confirmed that the ⁶⁸Ga‐acetate obtained is suitable for labeling of radiopharmaceuticals.     

Microwave accelerated 68Ga‐labelling of oligonucleotides

Oligonucleotides are extensively used for characterization of gene expression in vitro and have now been studied as inhibitors of gene expression in vivo in various diseases. Labelled antisense oligonucleotides are therefore of potential interest for possible in vivo imaging of gene expression, considering the biology of tumors and applications in designing novel molecule‐targeted therapies. In the present work a method of microwave accelerated ⁶⁸Ga‐labelling of oligonucleotides and analysis of the resulting tracers are described. Four modified and functionalized 17‐mer oligonucleotides with a hexylamine group in the 3′‐ or 5′‐position were studied. The oligonucleotides were conjugated to the bifunctional chelator, 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA), and then labelled with ⁶⁸Ga(T_1/2=68 min) using microwave activation. The isolated decay‐corrected radiochemical yields ranged from 30 to 52%. Labelled products were stable in water and ethanol for more than 4 h. The impact of the labelling procedure on the oligonucleotide probes was investigated using hybridization to a complementary 17‐mer sense oligonucleotide in solution. Chemical modification did not influence either the labelling or hybridization ability of the oligonucleotides. The radiolabelled oligonucleotides will be used for the further in vitro and in vivo biology studies. Copyright © 2003 John Wiley & Sons, Ltd.     

Preclinical evaluation of potential infection‐imaging probe [68Ga]Ga‐DOTA‐K‐A9 in sterile and infectious inflammation

The development of bacteria‐specific infection radiotracers is of considerable interest to improve diagnostic accuracy and enabling therapy monitoring. The aim of this study was to determine if the previously reported radiolabelled 1,4,7,10‐tetraazacyclododecane‐N,N′,N″,N?‐tetraacetic acid (DOTA) conjugated peptide [⁶⁸Ga]Ga‐DOTA‐K‐A9 could detect a staphylococcal infection in vivo and distinguish it from aseptic inflammation. An optimized [⁶⁸Ga]Ga‐DOTA‐K‐A9 synthesis omitting the use of acetone was developed, yielding 93 ± 0.9% radiochemical purity. The in vivo infection binding specificity of [⁶⁸Ga]Ga‐DOTA‐K‐A9 was evaluated by micro positron emission tomography/magnetic resonance imaging of 15 mice with either subcutaneous Staphylococcus aureus infection or turpentine‐induced inflammation and compared with 2‐deoxy‐2‐[¹⁸F]fluoro‐D‐glucose ([¹⁸F]FDG). The scans showed that [⁶⁸Ga]Ga‐DOTA‐K‐A9 accumulated in all the infected mice at injected doses ≥3.6 MBq. However, the tracer was not found to be selective towards infection, since the [⁶⁸Ga]Ga‐DOTA‐K‐A9 also accumulated in mice with inflammation. In a concurrent in vitro binding evaluation performed with a 5‐carboxytetramethylrhodamine (TAMRA) fluorescence analogue of the peptide, TAMRA‐K‐A9, the microscopy results suggested that TAMRA‐K‐A9 bound to an intracellular epitope and therefore preferentially targeted dead bacteria. Thus, the [⁶⁸Ga]Ga‐DOTA‐K‐A9 uptake observed in vivo is presumably a combination of local hyperemia, vascular leakiness and/or binding to an epitope present in dead bacteria.     

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

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

Development of 68Ga‐labeled fatty acids for their potential use in cardiac metabolic imaging

While [¹¹C]palmitate continues to be a promising tracer for cardiovascular Positron Emission Tomography (PET) imaging, unfavourable logistics due to the short half‐life of ¹¹C (20 min) and cumbersome labeling methodologies are the major impediments that limit its widespread use. In order to circumvent such limitations, an attempt has been made to explore the potential of ⁶⁸Ga‐labeled fatty acid analogs for metabolic imaging owing to the availability of ⁶⁸Ga through a ⁶⁸Ge/⁶⁸Ga generator on an on‐demand basis. In this study, two fatty acid conjugates were synthesized by conjugation of p‐SCN‐benzyl NOTA with the ω‐amino group of 11‐amino undecanoic acid and 12‐amino dodecanoic acid, respectively, under alkaline conditions. Both derivatives were radiolabeled in high yields with ⁶⁸Ga obtained from an in‐house ⁶⁸Ge/⁶⁸Ga generator. Biodistribution studies in Swiss mice showed reasonable myocardial uptake at 2 min for both derivatives (7.4 ± 2.8% ID/g for 11‐carbon fatty acid‐NOTA conjugate and 6.4 ± 2.1% ID/g for 12‐carbon fatty acid‐NOTA conjugate), which cleared rapidly over 30 min. However, significant activity was found in blood for both tracers, with heart/blood ratios observed to be below 0.5 at all time points, diminishing the potential of the synthesized complexes for cardiac imaging. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparative evaluation of 68Ga‐labeled NODAGA,DOTAGA, and HBED‐CC‐conjugated cNGR peptide chelates as tumor‐targeted molecular imaging probes

The biological behavior of ⁶⁸Ga‐based radiopharmaceuticals can be significantly affected by the chelators’ attributes (size, charge, lipophilicity). Thus, this study aimed at examining the influence of three different chelators, DOTAGA, NODAGA, and HBED‐CC on the distribution pattern of ⁶⁸Ga‐labeled NGR peptides targeting CD13 receptors. ⁶⁸Ga‐DOTAGA‐c(NGR), ⁶⁸Ga‐NODAGA‐c(NGR), and ⁶⁸Ga‐HBED‐CC‐c(NGR) were observed to be hydrophilic with respective log p values being −3.5 ± 0.2, −3.3 ± 0.08, and −2.8 ± 0.14. The three radiotracers exhibited nearly similar uptake in human fibrosarcoma HT‐1080 tumor cells with 86%, 63%, and 33% reduction during blocking studies with unlabeled cNGR peptide for ⁶⁸Ga‐DOTAGA‐c(NGR), ⁶⁸Ga‐NODAGA‐c(NGR), and ⁶⁸Ga‐HBED‐CC‐c(NGR), respectively, indicating higher receptor specificity of the first two radiotracers. The neutral radiotracer ⁶⁸Ga‐NODAGA‐c(NGR) demonstrated better target‐to‐non‐target ratios during in vivo studies compared to its negatively charged counterparts, ⁶⁸Ga‐DOTAGA‐c(NGR) and ⁶⁸Ga‐HBED‐CC‐c(NGR). The three radiotracers had similar HT‐1080 tumor uptake and being hydrophilic exhibited renal excretion with minimal uptake in non‐target organs. Significant reduction (p < .005) in HT‐1080 tumor uptake of the radiotracers was observed during blocking studies. It may be inferred from these studies that the three radiotracers are promising probes for in vivo imaging of CD13 receptor expressing cancer sites; however, ⁶⁸Ga‐NODAGA‐c(NGR) is a better candidate.     

Preclinical assessment of 68Ga‐PSMA‐617 entrapped in a microemulsion delivery system for applications in prostate cancer PET/CT imaging

It has in recent years been reported that microemulsion (ME) delivery systems provide an opportunity to improve the efficacy of a therapeutic agent whilst minimising side effects and also offer the advantage of favourable treatment regimens. The prostate‐specific membrane antigen (PSMA) targeting agents PSMA‐11 and PSMA‐617, which accumulate in prostate tumours, allow for [⁶⁸Ga]Ga³⁺‐radiolabelling and positron emission tomography/computed tomography (PET) imaging of PSMA expression in vivo. We herein report the formulation of [⁶⁸Ga]Ga‐PSMA‐617 into a ME ≤40 nm including its evaluation for improved cellular toxicity and in vivo biodistribution. The [⁶⁸Ga]Ga‐PSMA‐617‐ME was tested in vitro for its cytotoxicity to HEK293 and PC3 cells. [⁶⁸Ga]Ga‐PSMA‐617‐ME was administered intravenously in BALB/c mice followed by microPET/computed tomography (CT) imaging and ex vivo biodistribution determination. [⁶⁸Ga]Ga‐PSMA‐617‐ME indicated negligible cellular toxicity at different concentrations. A statistically higher tolerance towards the [⁶⁸Ga]Ga‐PSMA‐617‐ME occurred at 0.125 mg/mL by HEK293 cells compared with PC3 cells. The biodistribution in wild‐type BALB/C mice showed the highest amounts of radioactivity (%ID/g) presented in the kidneys (31%) followed by the small intestine (10%) and stomach (9%); the lowest uptake was seen in the brain (0.5%). The incorporation of [⁶⁸Ga]Ga‐PSMA‐617 into ME was successfully demonstrated and resulted in a stable nontoxic formulation as evaluated by in vitro and in vivo means.     

Synthesis and evaluation of 68Ga‐HBED‐CC‐EDBE‐folate for positron‐emission tomography imaging of overexpressed folate receptors on CT26 tumor cells

The ⁶⁸Ga is a positron‐emitting radionuclide that can be combined with bifunctional chelating agents and bioactive substances for use as positron‐emission tomography (PET) diagnostic agents. The HBED‐CC is an acyclic chelating agent that is rapidly labeled with ⁶⁸Ga under mild conditions. To target cancer cells, bioactive substances can be conjugated to the carboxyl terminus of HBED‐CC. Because folic acid strongly binds to folate receptors that are overexpressed on the surfaces of many types of cancer cells, it was coupled with HBED‐CC through a small polyethylene glycol‐based linker (EDBE) to generate an active, receptor‐selective targeting system. The HBED‐CC‐EDBE‐folate (HCEF) precursor was readily labeled with ⁶⁸Ga in 5 minutes at room temperature (98% radiochemical yield; 99% radiochemical purity after isolation). In cellular uptake tests, higher uptakes of ⁶⁸Ga‐HCEF were observed for the CT26 and KB cell lines (which express folate receptors) than for the A549 cell line (which does not). Finally, in vivo micro‐PET measurements over 2 hours of binding in BALB/c mice into which CT26 tumors had been transplanted showed the selective accumulation of ⁶⁸Ga‐HCEF in the folate receptor‐expressing CT26 tumors. These results confirmed the potential of ⁶⁸Ga‐HCEF as a PET diagnostic agent for tumors that express folate receptors.     

Single vial kit formulation of DOTATATE for preparation of 177Lu‐labeled therapeutic radiopharmaceutical at hospital radiopharmacy

The clinical applications of radiolabeled somatostatin analogue ¹⁷⁷Lu‐DOTA‐Tyr³‐Thr⁸‐Octreotide (¹⁷⁷Lu‐DOTATATE) constitute a promising treatment option for patients with disseminated and inoperable neuroendocrine (NET) tumors. Formulation of ¹⁷⁷Lu‐DOTATATE in hospital radiopharmacy under aseptic conditions in a safe and reliable manner is a major constraint for its extensive use. The present work was intended to develop a kit for the safe preparation of the therapeutic radiopharmaceutical, viz. ¹⁷⁷Lu‐DOTATATE of high quality that can be easily adapted at conventional hospital radiopharmacies. Single vial kits of DOTATATE were formulated and evaluated for suitability for radiolabeling as well as stability on its storage. Patient dose of ¹⁷⁷Lu‐DOTATATE (7.4 GBq) could be successfully prepared using semi‐automated in‐house setup that assures safe handling and high yields of product of pharmaceutical purity suitable for clinical use. Fast clearance of activity via renal route was observed in preclinical biodistribution studies of ¹⁷⁷Lu‐DOTATATE carried out in normal Swiss mice. Deployment of in‐house produced ¹⁷⁷LuCl₃, cold kits and easy adaptability of synthesis setup at hospital radiopharmacy for preparation is likely to expand applications of peptide receptor radionuclide therapy.     

Exploring the radiosynthesis and in vitro characteristics of [68Ga]Ga‐DOTA‐Siglec‐9

Vascular adhesion protein 1 is a leukocyte homing‐associated glycoprotein, which upon inflammation rapidly translocates from intracellular sources to the endothelial cell surface. It has been discovered that the cyclic peptide residues 283–297 of sialic acid‐binding IgG‐like lectin 9 (Siglec‐9) “CARLSLSWRGLTLCPSK” bind to vascular adhesion protein 1 and hence makes the radioactive analogues of this compound ([⁶⁸Ga]Ga‐DOTA‐Siglec‐9) interesting as a noninvasive visualizing marker of inflammation. Three different approaches to the radiosynthesis of [⁶⁸Ga]Ga‐DOTA‐Siglec‐9 are presented and compared with previously published methods. A simple, robust radiosynthesis of [⁶⁸Ga]Ga‐DOTA‐Siglec‐9 with a yield of 62% (non decay‐corrected) was identified, and it had a radiochemical purity >98% and a specific radioactivity of 35 MBq/nmol. Furthermore, the protein binding and stability of [⁶⁸Ga]Ga‐DOTA‐Siglec‐9 were analyzed in vitro in mouse, rat, rabbit, pig, and human plasma and compared with in vivo pig results. The plasma in vitro protein binding of [⁶⁸Ga]Ga‐DOTA‐Siglec‐9 was the lowest in the pig followed by rabbit, human, rat, and mouse. It was considerably higher in the in vivo pig experiments. The in vivo stability in pigs was lower than the in vitro stability. Despite considerable species differences, the observed characteristics of [⁶⁸Ga]Ga‐DOTA‐Siglec‐9 are suitable as a positron emission tomography tracer.     

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

The acyclic chelator HBED‐CC has attained huge clinical significance owing to high thermodynamic and kinetic stability of ⁶⁸Ga‐HBED‐CC chelate. It provides an excellent platform for quick preparation of ⁶⁸Ga‐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 ⁶⁸Ga‐labeling. In vitro and vivo behavior of the peptide tracer, ⁶⁸Ga‐HBED‐CC‐PEG₂‐RM26, was assessed and compared with ⁶⁸Ga‐NODAGA‐PEG₂‐RM26. The peptide tracers, ⁶⁸Ga‐HBED‐CC‐PEG₂‐RM26 and ⁶⁸Ga‐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 ⁶⁸Ga‐HBED‐CC‐PEG₂‐RM26 in GRPr‐expressing organs pancreas, stomach, and intestine. The preliminary studies suggest high potential of ⁶⁸Ga‐HBED‐CC‐PEG₂‐RM26 for further investigation as a GRPr imaging agent and the wide scope of HBED‐CC chelator in development of ⁶⁸Ga‐based peptide tracers.     

Synthesis and in vivo evaluation of gallium‐68‐labeled glycine and hippurate conjugates for positron emission tomography renography

The objective of this study was to evaluate four new ⁶⁸Ga‐labeled 1,4,7,10‐cyclododeca‐1,4,7,10‐tetraacetic acid (DOTA)/1,4,7‐triazacyclononane‐1,4,7‐triacetic acid derived (NODAGA)‐glycine/hippurate conjugates and select a lead candidate for potential application in positron emission tomography (PET) renography. The non‐metallated conjugates were synthesized by a solid phase peptide synthesis method. The ⁶⁸Ga labeling was achieved by reacting an excess of the non‐metallated conjugate with ⁶⁸GaCl₄^? at pH ?4.5 and 10‐min incubation either at room temperature for NODAGA or 90 °C for DOTA. Radiochemical purity of all ⁶⁸Ga conjugates was found to be >98%. ⁶⁸Ga‐NODAGA‐glycine displayed the lowest serum protein binding (0.4%) in vitro among the four ⁶⁸Ga conjugates. Biodistribution of ⁶⁸Ga conjugates in healthy Sprague Dawley rats at 1‐h post‐injection revealed an efficient clearance from circulation primarily through the renal–urinary pathway with <0.2% of injected dose per gram remaining in the blood. The kidney/blood and kidney/muscle ratios of ⁶⁸Ga‐NODAGA‐glycine were significantly higher than other ⁶⁸Ga conjugates. On the basis of these results, ⁶⁸Ga‐NODAGA‐glycine was selected as the lead candidate. ⁶⁸Ga‐NODAGA‐glycine PET renograms obtained in healthy rats suggest ⁶⁸Ga‐NODAGA‐glycine as a PET alternate of ^99mTc‐Diethylenetriaminepentaacetic acid (DTPA).     

68Ga‐labeled Ciprofloxacin Conjugates as Radiotracers for Targeting Bacterial Infection

With an aim of developing a bacteria‐specific molecular imaging agent, ciprofloxacin has been modified with a propylamine spacer and linked to two common bifunctional chelators, p‐SCN‐Bz‐DOTA and p‐SCN‐Bz‐NOTA. The two ciprofloxacin conjugates, CP‐PA‐SCN‐Bz‐DOTA ( 1 ) and CP‐PA‐SCN‐Bz‐NOTA ( 2 ), were radiolabeled with ⁶⁸Ga in >90% radiochemical yield and were moderately stable in vitro for 4 h. The efficacy of ⁶⁸Ga‐ 1 and ⁶⁸Ga‐ 2 has been investigated in vitro in Staphylococcus aureus cells where bacterial binding of the radiotracers (0.9–1.0% for ⁶⁸Ga‐ 1 and 1.6–2.3% for ⁶⁸Ga‐ 2 ) could not be blocked in the presence of excess amount of unlabeled ciprofloxacin. However, uptake of radiotracers in live bacterial cells was significantly higher (p < 0.01) than that in non‐viable bacterial cells. Bacterial infection targeting efficacy of ⁶⁸Ga‐ 1 and ⁶⁸Ga‐ 2 was tested in vivo in rats where the infected muscle‐to‐inflamed muscle (⁶⁸Ga‐ 1 : 2 ± 0.2, ⁶⁸Ga‐ 2 : 3 ± 0.5) and infected muscle‐to‐normal muscle ratios (⁶⁸Ga‐ 1 : 3 ± 0.4, ⁶⁸Ga‐ 2 : 6.6 ± 0.8) were found to improve at 120 min p.i. Fast blood clearance and renal excretion was observed for both the radiotracers. The two ⁶⁸Ga‐labeled infection targeting radiotracers could discriminate between bacterial infection and inflammation in vivo and are worthy of further detailed investigation as infection imaging agents at the clinical level.     

Preparation of 68Ga‐Mg‐Ca‐phytate colloid and its evaluation as a liver imaging agent

The objective of this study was to investigate the radiosynthesis of ⁶⁸Ga‐Mg‐Ca‐phytate colloid and then characterise the formulation for radiochemical purity (RCP), radioactive particle size distribution, and biodistribution in normal rats. This radiocolloid was prepared by mixing an aqueous solution of phytic acid, ⁶⁸Ga³⁺ ions, a dispersant, Mg²⁺ and Ca²⁺ ions, and then heating the contents at 100°C for 5 minutes. After cooling the vial to 5°C, the solution was basified to pH 5 and stored in the cold. The resulting product contained 92±3% RCP ⁶⁸Ga‐colloidal particles and a low level (8±3%) of soluble ⁶⁸Ga‐Mg‐Ca‐phytate. Particle size experiments defined the radioactive particle population was 6±4% <20 nm, 90±6% 20 to 200 nm, and 4% were >200 nm in diameter. Intravenous injection of the ⁶⁸Ga‐colloid dispersion to rats resulted in 93% uptake by the liver plus spleen, 1% lungs, 1% total blood, and 6% in the carcass after 20 minutes. This optimal formulation remained stable at 5°C for 1½ hours in vitro, and it resulted in the same biodistribution as the formulation prepared at t  = 0 hours. The preclinical data so far indicate that ⁶⁸Ga‐Mg‐Ca‐colloid has excellent potential as a liver imaging agent.     

Efficient gallium‐68 radiolabeling reaction of DOTA derivatives using a resonant‐type microwave reactor

Gallium‐68 (⁶⁸Ga, t_1/2 = 68 min) can be easily obtained from a ⁶⁸Ge/⁶⁸Ga generator, and several such systems are commercially available. The use of positron emission tomography (PET) imaging using ⁶⁸Ga‐labeled radiopharmaceuticals is expected to increase in both preclinical and clinical settings. However, the chelation between a ⁶⁸Ga cation and the bifunctional macrocyclic chelates that are used for labeling bioactive substances, such as 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA), requires a relatively long reaction time and high temperature to achieve a high radiochemical yield. Previously, we reported on a novel resonant‐type microwave reactor that can be used for radiosynthesis and the usefulness of this reactor in the PET radiosynthesis of ¹⁸F. In the present study, the usefulness of this resonant‐type microwave reactor was evaluated for the radiolabeling of model macrocyclic chelates with ⁶⁸Ga. As a result, microwave heating of resonant‐type microwave reactor notably improved the rate of the ⁶⁸Ga labeling chelate reaction in a short time period of 2 minutes, compared with the use of a conventional heating method. Additionally, it was found that the use of this reactor made it possible to decrease the amount of precursors required in the reaction and to improve the molar activity of the labeled compounds.     

Synthesis of 68Ga‐labeled NOTA‐RGD‐GE11 heterodimeric peptide for dual integrin and epidermal growth factor receptor‐targeted tumor imaging

Radiolabeled Arg‐Gly‐Asp (RGD) peptide analogs have been extensively studied for α_vβ₃ integrin‐targeted angiogenesis imaging. According to recently presented evidence, the dodecapeptide GE11 has high affinity to the epidermal growth factor receptor (EGFR), which is overexpressed in many types of cancer. Dual‐receptor molecular imaging probes with two different heterodimeric peptides exhibit improved cancer targeting efficacy. In the present study, the design and synthesis of a new RGD‐GE11 peptide heterodimer for dual α_vβ₃ integrin/EGFR‐targeted cancer imaging are described. The RGD‐GE11 heterodimer was linked with 6‐aminohexanoic acid (6‐Ahx) and cysteine and conjugated with 1,4,7‐triazacyclononane‐N,N′,N″‐triacetic acid (NOTA) to form NOTA‐RGD‐cys‐6‐Ahx‐GE11. The monomeric peptides, NOTA‐cys‐6‐Ahx‐GE11 and c(RGDyK), were formed by a peptide synthesizer. The peptide heterodimer NOTA‐RGD‐GE11 was obtained by NOTA‐cys‐6‐Ahx‐GE11 and maleimidopropyl‐c(RGDyK) conjugation with a thioether linkage. The NOTA peptide conjugate was labeled with freshly eluted ⁶⁸Ga and purified using reversed‐phase high‐performance liquid chromatography. The ⁶⁸Ga‐NOTA‐RGD‐cys‐6‐Ahx‐GE11 was successfully prepared, in this study, with a radiochemical yield of 85% and a radiochemical purity of >98%. These results warrant further investigation of this heterodimeric peptide's binding affinity to the receptors.     

Synthesis and characterization of scVEGF‐PEG‐[68Ga]NOTA and scVEGF‐PEG‐[68Ga]DOTA PET tracers

Vascular endothelial growth factor (VEGF) signaling via vascular endothelial growth factor receptor 2 (VEGFR‐2) on tumor endothelial cells is a critical driver of tumor angiogenesis. Novel anti‐angiogenic drugs target VEGF/VEGFR‐2 signaling and induce changes in VEGFR‐2 prevalence. To monitor VEGFR‐2 prevalence in the course of treatment, we are evaluating ⁶⁸Ga positron emission tomography imaging agents based on macrocyclic chelators, site‐specifically conjugated via polyethylene glycol (PEG) linkers to engineered VEGFR‐2 ligand, single‐chain (sc) VEGF. The ⁶⁸Ga‐labeling was performed at room temperature with NOTA (2,2′,2′′‐(1,4,7‐triazonane‐1,4,7‐triyl) triacetic acid) conjugates or at 90 °C by using either conventional or microwave heating with NOTA and DOTA (2,2′,2′′,2′′′‐(1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetrayl) tetraacetic acid) conjugates. The fastest (~2 min) and the highest incorporation (>90%) of ⁶⁸Ga into conjugate that resulted in the highest specific radioactivity (~400 MBq/nmol) was obtained with microwave heating of the conjugates. The bioactivity of the NOTA‐ and DOTA‐containing tracers was validated in 3‐D tissue culture model of 293/KDR cells engineered to express high levels of VEGFR‐2. The NOTA‐containing tracer also displayed a rapid accumulation (~ 20 s after intravenous injection) to steady‐state level in xenograft tumor models. A combination of high specific radioactivity and maintenance of functional activity suggests that scVEGF‐PEG‐[⁶⁸ Ga]NOTA and scVEGF‐PEG‐[⁶⁸ Ga]DOTA might be promising tracers for monitoring VEGFR‐2 prevalence and should be further explored. Copyright © 2011 John Wiley & Sons, Ltd.