Ga Generator Research Articles

Improving mixing efficiency in a closed circuit water flow rig for radiotracer studies

Investigations were conducted on a laboratory scale water flow rig to determine the flow characteristics for improving mixing efficiency in the tanks for radiotracer studies. Approximately 20 mCi eluates of 68 Ga from 68 Ge/ 68 Ga generator were injected into the fluid at 1.0 and 1.2 minutes pulses in a second and third generation system. At measured mean residence time ( τ m ) per tank of 1.68 - 2.04 minutes in the second generation, mixing efficiency was in the range 27.91 - 33.88%. Mixing efficiency of 81.57 - 90.98 % showed improvement for the modified rig with τ m per tank between 7.80 and 8.70 minutes. The cumulative residence time distribution data for the last tanks in the two generations were fitted by perfect mixers in series model using program tools in Microsoft Excel. The volumetric flow rates of 4.55 and 8.0 L/min for the two rigs conformed to the values measured by radiotracer pulse velocity method, indicating that the flow meters functioned correctly. The modified rig with scaled-up mixing techniques could serve as platform for training in evaluating mixing vessels and flow meters in industrial process plants.

68Ga-labeling and in vivo evaluation of a uPAR binding DOTA- and NODAGA-conjugated peptide for PET imaging of invasive cancers

The urokinase-type plasminogen activator receptor (uPAR) is a well-established biomarker for tumor aggressiveness and metastatic potential. DOTA-AE105 and DOTA-AE105-NH(2) labeled with (64)Cu have previously been demonstrated to be able to noninvasively monitor uPAR expression using positron emission tomography (PET) in human cancer xenograft mice models. Here we introduce (68)Ga-DOTA-AE105-NH(2) and (68)Ga-NODAGA-AE105-NH(2) and evaluate their imaging properties using small-animal PET. Synthesis of DOTA-AE105-NH(2) and NODAGA-AE105-NH(2) was based on solid-phase peptide synthesis protocols using the Fmoc strategy. (68)GaCl(3) was eluted from a (68)Ge/(68)Ga generator. The eluate was either concentrated on a cation-exchange column or fractionated and used directly for labeling. For in vitro characterization of both tracers, partition coefficient, buffer and plasma stability, uPAR binding affinity and cell uptake were determined. To characterize the in vivo properties, dynamic microPET imaging was carried out in nude mice bearing human glioma U87MG tumor xenograft. In vitro experiments revealed uPAR binding affinities in the lower nM range for both conjugated peptides and identical to AE105. Labeling of DOTA-AE105-NH(2) and NODAGA-AE105-NH(2) with (68)Ga was done at 95°C and room temperature, respectively. The highest radiochemical yield and purity were obtained using fractionated elution, whereas a negative effect of acetone on labeling efficiency for NODAGA-AE105-NH(2) was observed. Good stability in phosphate-buffered saline and mouse plasma was observed. High cell uptake was found for both tracers in U87MG tumor cells. Dynamic microPET imaging demonstrated good tumor-to-background ratio for both tracers. Tumor uptake was 2.1% ID/g and 1.3% ID/g 30 min postinjection and 2.0% ID/g and 1.1% ID/g 60 min postinjection for (68)Ga-NODAGA-AE105-NH(2) and (68)Ga-DOTA-AE105-NH(2), respectively. A significantly higher tumor-to-muscle ratio (P<.05) was found for (68)Ga-NODAGA-AE105-NH(2) 60 min postinjection. The use of (68)Ga-DOTA-AE105-NH(2) and (68)Ga-NODAGA-AE105-NH(2) as the first gallium-68 labeled uPAR radiotracers for noninvasive PET imaging is reported, which combine versatility with good imaging properties. These new tracers thus constitute an interesting alternative to the (64)Cu-labeled version ((64)Cu-DOTA-AE105 and 64Cu-DOTA-AE105-NH(2)) for detecting uPAR expression in tumor tissue. In our hands, the fractionated elution approach was superior for labeling of peptides, and (68)Ga-NODAGA-AE105-NH(2) is the favored tracer as it provides the highest tumor-to-background ratio.

A novel PET tracer for the imaging of αvβ3 and αvβ5 integrins in experimental breast cancer bone metastases

The aim of this study was the evaluation of (68)Ga-DOTA-E-[c(RGDfK)](2) as a novel PET tracer to image αvβ3 and αvβ5 integrins. For this purpose, DOTA-E-[c(RGDfK)](2) was labeled with (68)Ga, which was obtained from a (68)Ge/(68)Ga generator, purified by solid-phase extraction and the radiochemical purity analyzed by radio-RP-HPLC. (68) Ga-DOTA-E-[c(RGDfK)](2) was obtained reproducibly in radiochemical yields of 60 ± 6% and with an excellent radiochemical purity of >99%. In nude rats bearing bone metastases after injection of MDA-MB-231 human breast cancer cells, biodistribution studies were performed to evaluate the accumulation of the radiotracer in selected organs, blood and bone metastases 0.5, 1, 2 and 3 h post injection. A rapid uptake into the bone metastases and rapid blood clearance was observed, resulting in tumor-blood ratios of up to 26.6 (3 h post injection) and tumor-muscle ratios of up to 7.9 (3 h post injection). A blocking experiment with coinjected αvβ3/αvβ5 antagonist showed the tumor uptake to be receptor-specific. In an initial in vivo micro PET evaluation of the tracer using the same animal model, the bone metastasis was clearly visualized. These results suggest that (68)Ga-DOTA-E-[c(RGDfK)](2) is a promising PET tracer suitable for the imaging of αvβ3 and αvβ5 integrins in bone metastases. This novel PET tracer should be further evaluated concerning its usefulness for early detection of bone metastases and monitoring treatment response of these lesions.

Scaled-up radiolabelling of DOTATATE with 68Ga eluted from a SnO2-based 68Ge/68Ga generator

A scaled-up radiolabelling and improved post-labelling purification procedure for [(68)Ga]DOTATATE is reported, using a more than 1 year old SnO(2)-based 1850MBq (68)Ge/(68)Ga generator (initially double-loaded with 3700MBq (68)Ge) as a source of ionic (68)Ga. The elution method of choice comprised elution with 0.6M HCl in a single 4mL fraction, containing up to 95% of the total eluted (68)Ga activity. The unpurified fraction was directly used for labelling after pH adjustment with 2.5M sodium acetate. Labelling efficiencies were determined at 90-95°C at various reaction times and reaction volumes of up to 5.7mL, using either 30μg or 50μg DOTATATE. Only the latter amount resulted in consistently high labelling efficiency in excess of 95%. Post-labelling purification, carried out on Sep-Pak C18, showed that 50% ethanol in saline was a superior desorption eluant than 100% ethanol. The highest and most consistent decay-corrected radiochemical yields (89%) were obtained using 50μg DOTATATE and a 20min reaction time.

Synthesis and evaluation of a novel 68Ga-chelate-conjugated bisphosphonate as a bone-seeking agent for PET imaging

(68)Ga is a positron-emitting nuclide that has significant imaging potential given that, unlike cyclotron-produced (18)F, the isotope can be produced on-site utilizing a (68)Ge/(68)Ga generator. We recently synthesized a novel bone-seeking agent by coupling a bisphosphonate with the (68)Ga chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). This study presents a first report on the potential of this (68)Ga bone-seeking radiopharmaceutical in the detection of bone metastases. 4-Amino-1-hydroxybutylidene-1,1-bisphosphonate was conjugated with 2-[4,7-di(carboxymethyl)-1,4,7-triazonan-1-yl]pentanedioic acid, yielding 2-[4,7-di(carboxymethyl)-1,4,7-triazonan-1-yl]-5-[(4-hydroxy-4,4-diphosphonobutyl)amino]-5-oxopentanoic acid (NOTA-BP). (68)Ga-labeled NOTA-BP ([(68)Ga]NOTA-BP) was prepared by complexation of NOTA-BP with [(68)Ga] gallium chloride and evaluated in in vitro experiments, biodistribution experiments and micro-positron emission tomography (PET) imaging experiments. The labeling of NOTA-BP with (68)Ga was completed by heating for 10 min. [(68)Ga]NOTA-BP was determined to have a radiochemical purity of over 95%, a high affinity for hydroxyapatite and a high stability in plasma. In in vivo biodistribution experiments, [(68)Ga]NOTA-BP demonstrated high bone uptake potential. Compared with (99m)Tc-labeled methylene diphosphonate ([(99m)Tc]MDP) and [(18)F]fluoride, [(68)Ga]NOTA-BP exhibited faster blood clearance and a higher bone-to-blood ratio. In addition, mouse model bone metastasis was detected by micro-PET imaging at 1 h postinjection of [(68)Ga]NOTA-BP. We have developed a novel (68)Ga-radiolabeled bone-seeking agent. This [(68)Ga]NOTA-BP complex was found to have a high bone affinity and rapid blood clearance, and may thus prove to be useful as a bone-seeking agent for clinical PET.

Improved column-based radiochemical processing of the generator produced 68Ga

An improved chemical strategy for processing of the generator produced (68)Ga was developed based on processing of the original (68)Ge/(68)Ga generator eluate on a micro-column. Direct pre-concentration and purification of the eluted (68)Ga is performed on a cation-exchange resin in hydrochloric acid/acetone media. A supplementary step based on a second micro-column filled with a second resin allows direct re-adsorption of (68)Ga eluted from the cation exchanger. (68)Ga is finally striped from the second resin with a small volume of pure water. For this purpose a strong anion exchanger and a novel extraction chromatographic resin based on tetraalkyldiglycolamides are characterized. The strategy allows online pre-concentration and purification of (68)Ga from the original generator eluate. The supplementary column allows transferring (68)Ga with high radionuclide and chemical quality in the aqueous solution with small volume and low acidity useful for direct radiolabeling reactions.

Development of a nano-zirconia based 68Ge/68Ga generator for biomedical applications

Most of the commercially available (68)Ge/(68)Ga generator systems are not optimally designed for direct applications in a clinical context. We have developed a nano-zirconia based (68)Ge/(68)Ga generator system for accessing (68)Ga amenable for the preparation of radiopharmaceuticals. Nano-zirconia was synthesized by the in situ reaction of zirconyl chloride with ammonium hydroxide in alkaline medium. The physical characteristics of the material were studied by various analytical techniques. A 740 MBq (20 mCi) (68)Ge/(68)Ga generator was developed using this sorbent and its performance was evaluated for a period of 1 year. The suitability of (68)Ga for labeling biomolecules was ascertained by labeling DOTA-TATE with (68)Ga. The material synthesized was nanocrystalline with average particle size of ~7 nm, pore-size of ~4 Å and a high surface area of 340±10 m(2) g(-1). (68)Ga could be regularly eluted from this generator in 0.01N HCl medium with an overall radiochemical yield >80% and with high radionuclidic (<10(-5)% of (68)Ge impurity) and chemical purity (<0.1 ppm of Zr, Fe and Mn ions). The compatibility of the product for preparation of (68)Ga-labeled DOTA-TATE under the optimized reaction conditions was found to be satisfactory in terms of high labeling yields (>99%). The generator gave a consistent performance with respect to the elution yield and purity of (68)Ga over a period of 1 year. The feasibility of preparing an efficient (68)Ge/(68)Ga generator which can directly be used for biomedical applications has been demonstrated.

Characteristics of SnO2-based 68Ge/68Ga generator and aspects of radiolabelling DOTA-peptides

PET scintigraphy with (68)Ga-labelled analogs is of increasing interest in Nuclear Medicine and performed all over the world. Here we report the characteristics of the eluate of SnO(2)-based (68)Ge/(68)Ga generators prepared by iThemba LABS (Somerset West, South Africa). Three purification and concentration techniques of the eluate for labelling DOTA-TATE and concordant SPE purifications were investigated. Characteristics of 4 SnO(2)-based generators (range 0.4-1 GBq (68)Ga in the eluate) and several concentration techniques of the eluate (HCl) were evaluated. The elution profiles of SnO(2)-based (68)Ge/(68)Ga generators were monitored, while [HCl] of the eluens was varied from 0.3-1.0 M. Metal ions and sterility of the eluate were determined by ICP. Fractionated elution and concentration of the (68)Ga eluate were performed using anion and cation exchange. Concentrated (68)Ga eluate, using all three concentration techniques, was used for labelling of DOTA-TATE. (68)Ga-DOTA-TATE-containing solution was purified and RNP increased by SPE, therefore also 11 commercially available SPE columns were investigated. The amount of elutable (68)Ga activity varies when the concentration of the eluens, HCl, was varied, while (68)Ge activity remains virtually constant. SnO(2)-based (68)Ge/(68)Ga generator elutes at 0.6 M HCl >100% of the (68)Ga activity at calibration time and ±75% after 300 days. Eluate at discharge was sterile and Endotoxins were <0.5 EU/mL, RNP was always <0.01%. Metal ions in the eluate were <10 ppm (in total). Highest desorption for anion purification was obtained with the 30 mg Oasis WAX column (>80%). Highest desorption for cation purification was obtained using a solution containing 90% acetone at increasing molarity of HCl, resulted in a (68)Ga desorption of 68±8%. With all (68)Ge/(68)Ga generators and for all 3 purification methods a SA up to 50 MBq/nmol with >95% incorporation (ITLC) and RCP (radiochemical purity) by HPLC ±90% could be achieved. Purification and concentration of the eluate with anion exchange has the benefit of more elutable (68)Ga with 1 M HCl as eluens. The additional washing step of the anion column with NaCl and ethanol, resulted in a lower and less variable [H(+)] in the eluate, and, as a result the pH in the reaction vial is better controlled, more constant, and less addition of buffer is required and concordant smaller reaction volumes. Desorption of (68)Ga-DOTA-TATE of SPE columns varied, highest desorption was obtained with Baker C(18) 100 mg (84%). Purification of (68)Ga-DOTA-TATE by SPE resulted in an RNP of <10(-4)%. Eluate of SnO(2)-based (68)Ge/(68)Ga generator, either by fractionated elution as by ion exchange can be used for labelling DOTA-peptides with (68)Ga at a SA of 50 MBq/nmol at >95% incorporation and a RCP of ±90%. SPE columns are very effective to increase RNP.

Labelling of commercially available human serum albumin kits with 68Ga as surrogates for 99mTc-MAA microspheres

An alternative method of labelling different commercially available human serum albumin kits with generator produced (68)Ga as possible surrogate markers for the investigation of the biodistribution, and to exclude a possible lung shunt in patients with solid primary tumours and liver metastases by positron emission-tomography (PET) and effective substitute for (99m)Tc HSA as a PET perfusion tracer is described. The radioactive labelling with (68)Ga was based on the elution of a commercial (68)Ge/(68)Ga generator system, which uses a modified tin dioxide column to absorb (68)Ga. We obtained (68)Ga-MAA and (68)Ga-HSA in radiochemical yields of 79 ± 5% with less than 5% free (68)Ga. The whole procedure takes a total of less than 30 min from elution to the final product.

Automated synthesis, characterization and biological evaluation of [68Ga]Ga-AMBA, and the synthesis and characterization of natGa-AMBA and [67Ga]Ga-AMBA

Ga-AMBA (Ga-DO3A-CH(2)CO-G-[4-aminobenzoyl]-QWAVGHLM-NH(2)) is a bombesin-like agonist with high affinity for gastrin releasing peptide receptors (GRP-R). Syntheses for (nat)Ga-AMBA, [(67)Ga]Ga-AMBA and [(68)Ga]Ga-AMBA were developed. The preparation of HPLC-purified and Sep-Pak purified [(68)Ga]Ga-AMBA were fully automated, using the built-in radiodetector of the Tracerlab FX F-N synthesizer to monitor fractionated (68)Ge/(68)Ga generator elution and purification. The total synthesis time, including the fractional elution of the generator, was 20 min for Sep-Pak purified material and 40 min for HPLC-purified [(68)Ga]Ga-AMBA. Both [(67)Ga]Ga-AMBA and [(177)Lu]Lu-AMBA showed comparable high affinity for GRP-R in the human prostate cancer cell line PC-3 in vitro (k(D)=0.46+/-0.07; 0.44+/-0.08 nM), high internalization (78; 77%) and low efflux from cells at 2 h (2.4+/-0.7; 2.9+/-1.8%). Biodistribution results in PC-3 tumor-bearing male nude mice showed comparable uptake for [(177)Lu]Lu-, [(111)In]In-, [(67)Ga]Ga- and [(68)Ga]Ga-AMBA.

Nanoceria-PAN Composite-Based Advanced Sorbent Material: A Major Step Forward in the Field of Clinical-Grade 68Ge/68Ga Generator

The (68)Ge/(68)Ga generator has high potential for clinical positron emission tomography (PET) imaging. However, because of the unavailability of a suitable sorbent material, the commercially available (68)Ge/(68)Ga generators are not directly adaptable for the preparation of (68)Ga-labeled radiopharmaceuticals. In view of this, a new nanoceria-polyacrylonitrile (PAN) composite sorbent has been synthesized by decomposition of a cerium oxalate precursor to cerium oxide and its subsequent incorporation in PAN matrix for the development of a clinical grade (68)Ge/(68)Ga generator. The X-ray diffraction (XRD) studies and BET nitrogen adsorption technique revealed that nanocrystalline ceria had an average particle size of approximately 10 nm, surface area of 72 +/- 3 m(2)/g and an average pore size of 3.8 +/- 0.1 A. Investigation of the distribution ratio (K(d)) values for the prepared sorbent in 0.01 N HCl medium revealed the suitability of the sorbent for the quantitative retention of (68)Ge and efficient elution of clinical grade (68)Ga. A 370 MBq (10 mCi) (68)Ge/(68)Ga chromatographic generator was developed using this sorbent. (68)Ga could be regularly eluted from this generator with >80% elution yield. The eluted (68)Ga possess high radionuclidic purity (<1 x 10(-5)% of (68)Ge impurity), chemical purity (<0.1 ppm of Ce, Fe and Mn ions) and was amenable for the preparation of (68)Ga-labeled radiopharmaceuticals. The generator gave a consistent performance with respect to the elution yield and purity of (68)Ga over an extended period of 7 months.

A Triazacyclononane‐Based Bifunctional Phosphinate Ligand for the Preparation of Multimeric 68Ga Tracers for Positron Emission Tomography

For application in positron emission tomography (PET), PrP9, a N,N',N''-trisubstituted triazacyclononane with methyl(2-carboxyethyl)phosphinic acid pendant arms, was developed as (68)Ga(3+) complexing agent. The synthesis is short and inexpensive. Ga(III) and Fe(III) complexes of PrP9 were characterized by single-crystal X-ray diffraction. Stepwise protonation constants and thermodynamic stabilities of metal complexes were determined by potentiometry. The Ga(III) complex possesses a high thermodynamic stability (log K([GaL])=26.24) and a high degree of kinetic inertness. (68)Ga labeling of PrP9 is possible at ambient temperature and in a wide pH range, also at pH values as low as 1. This means that for the first time, the neat eluate of a TiO(2)-based (68)Ge/(68)Ga generator (typically consisting of 0.1 M HCl) can be directly used for labeling purposes. The rate of (68)Ga activity incorporation at pH 3.3 and 20 degrees C is higher than for the established chelators DOTA and NOTA. Tris-amides of PrP9 with amino acid esters were synthesized to act as models for multimeric peptide conjugates. These conjugates exhibit radiolabeling properties similar to those of unsubstituted PrP9.

Computer controlled 68Ga milking and concentration system

Here we describe an automated (68)Ge/(68)Ga generator milking and concentration procedure. It is especially suited for reproducible preparation of (68)Ga in volumes of approximately 200 microl with optimum activity for subsequent labelling of small amounts of peptides or proteins ideal for microPET applications.

Acceptance Tests and Quality Control of Ge/ Ga Generators

In this paper we describe test methods useful to evaluate the performance of Ge/ Ga generators and to assess radionuclidic purity of the eluates. In our tests on the elution profile of the generator, we found that less than 8.0 ml are sufficient to recover > 98 % of the activity. Elution yield was conforming the manufacturer specifications, being initially > 85 % and still > 64 % after 7 months of use. 68Ge breakthrough in the eluates was limited; even if radionuclidic purity was > 99.99 % , further analyses, in addition to physical tests, is recommended in order to assess chemical impurities that could determine interferences in labelling procedures. Keywords: Ga/, Ge generator, radionuclidic purity, quality assurance

Molekulare Bildgebung neuroendokriner Tumoren mit68Ga-markierten Peptiden (Somatostatinrezeptor-PET/CT)

Die Rezeptor-PET/CT mit 68Ga-markierten Somatostatinanaloga (DOTA-NOC, DOTA-TOC oder DOTA-TATE) ermöglicht die hochsensitive molekulare Bildgebung neuroendokriner Tumoren (NET) aufgrund der Expression von Somatostatinrezeptoren und die Erfassung verschiedener Rezeptorsubtypen. Unsere Ergebnisse bei über 3 000 Untersuchungen zeigen, dass die Rezeptor-PET/CT eine erheblich höhere Nachweisgenauigkeit hat, als die konventionelle Szintigrafie (auch wenn diese in SPECT/CT-Technik erfolgt), und dass eine sehr exakte topografische Zuordnung der Tumorherde möglich ist. Mit der Berechnung von Standardized Uptake Values (SUV) stehen reproduzierbare und untersucherunabhängige Messwerte zur Verfügung, die es ermöglichen, Patienten für die Radiorezeptortherapie zu selektionieren und den Therapieverlauf zu überwachen. Die Rezeptor-PET/CT ist das sensitivstes Verfahren zum Nachweis von unbekannten Primärtumoren (CUP-Syndrom), was insbesondere für die Detektion von Pankreas- und Dünndarm-NET gilt; sie ermöglicht eine Ganzkörperuntersuchung („one stop shop”) zum Staging sowie die Selektion von Patienten zur Radiorezeptortherapie und die Tumornachsorge mit einem patientenfreundlichen Verfahren, das schnell (innerhalb einer Stunde) durchführbar ist und mit geringerer Strahlenexposition als die einer konventionellen Ganzkörper-CT erfolgen kann. Die Verwendung des 68Ge/68Ga-Generators hat sich unter klinischen Bedingungen über mehrere Jahre zuverlässig bewährt. Der effektive Kostenaufwand für 68Ga-markierte Somatostatinanaloga dürfte bei entsprechenden Untersuchungszahlen geringer sein, als der für die bisherige szintigrafische Diagnostik. Die Entwicklung anderer tumorspezifischer Peptide sowie weiterer DOTA- oder NOTA-gekoppelter Radiopharmazeutika eröffnet für die Zukunft neue Perspektiven, die letztendlich dazu führen könnten, dass der 68Ga-Generator für die PET/CT künftig eine ähnlich bedeutende Rolle spielen wird, wie dies für den 99mTc-Generator und die konventionelle Gammakamera seit Jahrzenten der Fall ist.

Nuclear Medicine and Biology 2009

article i nfo Purpose: Bone metastases are a serious aggravation for patients suffering from cancer. Therefore, early recognition of bone metastases is of great interest for further treatment of patients. Bisphosphonates are widely used for scintigraphy of bone lesions with 99m Tc. Using the 68 Ge/ 68 Ga generator together with a macroyclic bisphosphonate a comparable PET-tracer comes into focus. Procedures: The bisphosphonate DOTA-conjugated ligand BPAMD was labelled with 68 Ga. ( 68 Ga)BPAMD was evaluated in vitro concerning binding to hydroxyapatite and stability. The tracer's in vivo accumulation was determined on healthy rats and bone metastases bearing animals by μ-PET. Results: BPAMD was labelled efficiently with 68

Validation of 68Ge/68Ga generator processing by chemical purification for routine clinical application of 68Ga-DOTATOC

Imaging of somatostatin receptor expressing tumours has been greatly enhanced by the use of (68)Ga-DOTATOC and PET/CT. In this work, a purification method for the (68)Ge/(68)Ga generator eluate and a method to produce (68)Ga-DOTATOC suitable for clinical use were evaluated. The generator eluate was purified and concentrated on a cation-exchange cartridge in HCl/acetone media. The efficacy of this procedure in eliminating metal impurities from the (68)Ga solution was investigated by ICP-MS. The radiotracer quality was evaluated by radio-TLC, GC and gamma-ray spectrometry. (68)Ga-DOTATOC preparations (n=33) were carried out with a mean synthesis yield of 59.3+/-2.8% (not corrected for decay) and a batch activity ranging from 555 to 296 MBq. The radiochemical and radionuclidic purity were >98% and 99.9999%, respectively. With this purification process, >95% of the Fe(III), Zn(II) and Mn(II) were eliminated from the solution. (68)Ga-DOTATOC produced with this method can be efficiently used in nuclear medicine departments for PET evaluations.

Preparation of a Promising Angiogenesis PET Imaging Agent: 68Ga-Labeled c(RGDyK)–Isothiocyanatobenzyl-1,4,7-Triazacyclononane-1,4,7-Triacetic Acid and Feasibility Studies in Mice

Arg-Gly-Asp (RGD) derivatives have been labeled with various radioisotopes for the imaging of angiogenesis in ischemic tissue, in which alpha(v)beta(3) integrin plays an important role. In this study, cyclic Arg-Gly-Asp-D-Tyr-Lys [c(RGDyK)] was conjugated with 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bz-NOTA) and then labeled with (68)Ga. The labeled RGD so produced was subjected to an in vitro binding assay and in vivo biodistribution and PET studies. A mixture of SCN-Bz-NOTA (660 nmol) and c(RGDyK) (600 nmol) in 0.1 M sodium carbonate buffer (pH 9.5) was allowed to react for 20 h at room temperature in the dark for thiourea bond formation. The conjugate obtained was purified by semipreparative high-performance liquid chromatography (HPLC). The purified c(RGDyK)-SCN-Bz-NOTA (NOTA-RGD) was then labeled with (68)Ga from a (68)Ge/(68)Ga generator and purified by semipreparative HPLC. A competitive binding assay for c(RGDyK) and NOTA-RGD was performed with (125)I-c(RGDyK) as a radioligand and alpha(v)beta(3) integrin-coated plates as a solid phase. (68)Ga-NOTA-RGD (0.222 MBq/100 microL) was injected, through a tail vein, into mice with hind limb ischemia and into mice bearing human colon cancer SNU-C4 xenografts. Biodistribution and imaging studies were performed at 1 and 2 h after injection. The labeling of NOTA-RGD with (68)Ga was straightforward. The K(i) values of c(RGDyK) and NOTA-RGD were 1.3 and 1.9 nM, respectively. In the biodistribution study, the mean +/- SD uptake of (68)Ga-NOTA-RGD by ischemic muscles was 1.6+/-0.2 percentage injected dose per gram (%ID/g); this uptake was significantly blocked by cold c(RGDyK) to 0.6+/-0.3 %ID/g (P<0.01). Tumor uptake was 5.1+/-1.0 %ID/g, and the tumor-to-blood ratio was 10.3+/-4.8. Small-animal PET revealed rapid excretion through the urine and high levels of tumor and kidney uptake. Stable (68)Ga-NOTA-RGD was obtained in a straightforward manner at a high yield and showed a high affinity for alpha(v)beta(3) integrin, specific uptake by angiogenic muscles, a high level of uptake by tumors, and rapid renal excretion. (68)Ga-NOTA-RGD was found to be a promising radioligand for the imaging of angiogenesis.

68Ga‐PET: a powerful generator‐based alternative to cyclotron‐based PET radiopharmaceuticals

PET (positron emission tomography) is a powerful diagnostic and imaging technique which requires short-lived positron emitting isotopes. The most commonly used are accelerator-produced (11)C and (18)F. An alternative is the use of metallic positron emitters. Among them (68)Ga deserves special attention because of its availability from long-lived (68)Ge/(68)Ga generator systems which render (68)Ga radiopharmacy independent of an onsite cyclotron. The coordination chemistry of Ga(3+) is dominated by its hard acid character. A variety of mono- and bifunctional chelators have been developed which allow the formation of stable (68)Ga(3+)complexes and convenient coupling to biomolecules. (68)Ga coupling to small biomolecules is potentially an alternative to (18)F- and (11)C-based radiopharmacy. In particular, peptides targeting G-protein coupled receptors overexpressed on human tumour cells have shown preclinically and clinically high and specific tumour uptake. Kit-formulated precursors along with the generator may be provided, similar to the (99)Mo/(99m)Tc-based radiopharmacy, still the mainstay of nuclear medicine.

A fully automated synthesis for the preparation of 68Ga-labelled peptides

Generator-produced Ga has attracted increasing interest for radiolabelling peptides used in PET applications. So far, the synthesis of Ga-peptide radiopharmaceuticals is mainly based on semi-automated systems. Here we describe a fully automated approach for the synthesis of Ga-labelled peptides. A commercially available Ga generator was eluted with 0.1 mol . l HCl. Reaction parameters such as buffer conditions, pH range, reaction temperature and time, volume of reaction solution and generator fraction were optimized for labelling DOTA-Tyr-octreotide (DOTATOC). Reaction yields, pH, radiochemical purity, sterility, endotoxins, breakthrough of Ge and final Ge content were determined. A fully automated radiopharmaceutical synthesis device based on a modular concept for remote-controlled processing was developed and evaluated for a number of DOTA-derivatized peptides. DOTATOC could be labelled in almost quantitative yields by heating 10-50 nmol peptide at pH 3.5-4.0 for 5 min at 95 degrees C in 1.5 ml. Purification using a reversed-phase cartridge was required to avoid any potential Ge breakthrough: final activities of Ge were below 100 Bq . ml. Automated synthesis resulted in overall decay-corrected reaction yields of about 60% within 10 min. Even after 1 year using a 1110 MBq generator more than 130 MBq Ga-DOTATOC could be obtained. Moreover, it was demonstrated that a variety of DOTA-derivatized peptides can be labelled using identical reaction conditions with high yields. The system described allows the fully automated, efficient and rapid preparation of Ga-DOTA-derivatized peptides. It has been used successfully and reliably for routine preparations in clinical studies.