Radiochemical and analytical aspects of inter-institutional quality control measurements on radiopharmaceuticals.

BACKGROUND: Clinically applied radiopharmaceuticals have to meet quality release criteria like a high radiochemical yield and radiochemical purity. Many radiopharmaceuticals do not have marketing authorization and have no dedicated monograph within the European pharmacopeia, therefore general monographs on quality control have to be applied for clinical applications. These criteria require standardization and validation in labeling and preparation, including QC measurements according to well-defined standard operation procedures. QC measurements however, are often based on detection techniques specific for a certain LC-system. Multi-institutional research and development of new radiopharmaceuticals lead to an increase in multicenter trials. Although all institutes' radiopharmacies are using the same standardized labeling and operation procedures, they often use different LC and radiodetection systems. Here we present a comparison of QC assessments for 3 radiopharmaceuticals with focus on the interpretation of chromatograms, data-output and potential differences in local practical performances of QC on (U)HPLC. METHODS: QC assessments for [111In]In-CCK, [68Ga]Ga-Bombesin and [177Lu]Lu-PSMA analogs were compared. Two of the radiopharmaceutical QC assessments were also applied in other institutes using their own HPLC-systems and concordant software. Data from the HPLC-injections and measurements is processed and summarized in chromatograms, based on a variety of smoothing algorithms for which different software programs are applied. Described radiopeptides were labeled and analyzed according their standardized labeling and operation procedures. RESULTS: Integration of main peaks on chromatograms resulted in a range of RCP, depending on the smoothing algorithm used. [111In]In-CCK(A), 68Ga-Bombesin(B) and [177Lu]Lu-PSMA(C) analogs had a RCP range of 88%-96%(A), 89-95%(B) and 92-99%(C) respectively. Important factors affecting final RCP value were site specific background radiation-levels, intrinsic system properties such as noise and sensitivity, personal interpretation e.g. peak-tailing and smoothing algorithms. CONCLUSION: Measurement of RCP shows a strong method- and system-dependency, even when parameters are validated, standardized and SOP are followed. Release criteria are frequently based on RCP data from one central location. The lack of inter inter institutional validation and standardization in RCP determination makes the results therefore rather arbitrary. For multicenter trials, we recommend to compare locally determined RCP under validated and standardized conditions of in-line activity detection between institutes for each radiopharmaceutical.

Semi-automated system for concentrating 68Ga-eluate to obtain high molar and volume concentration of 68Ga-Radiopharmaca for preclinical applications.

INTRODUCTION: 68Ga-radiopharmaceuticals are common in the field of Nuclear Medicine to visualize receptor-mediated processes. In contrast to straightforward labeling procedures for clinical applications, preclinical in vitro and in vivo applications are hampered for reasons like e.g. volume restriction, activity concentration, molar activity and osmolality. Therefore, we developed a semi-automatic system specifically to overcome these problems. A difficulty appeared unexpectedly, as intrinsic trace metals derived from eluate (Zn, Fe and Cu) are concentrated as well in amounts that influence radiochemical yield and thus lower molar activity. METHODS: To purify Gallium-68 and to reduce the high elution volume of a 68Ga-generator, a NaCl-based method using a column containing PS-H+ was implemented in a low volume PEEK system. Influence on reducing osmolality, acidity and the amount of PS-H+ resin (15-50 mg) was investigated. [68Ga]Ga was desorbed from the PS-H+ resin with acidified 2-5 M NaCl (containing 0.05 M of HCl) and 68Ga-activity was collected. DOTA-TATE was used as a peptide model. All buffers and additives used for labeling were mixed with Chelex 100 (~1 g/50 mL) for >144 h and eventually filtered using a 0.22 µm filter (Millipore). Quantification of metals was performed after labeling by HPLC (UV). RESULTS: Gallium-68 activity could be desorbed from PS-H+ cation column with 3 M NaCl, and >60% (120-180 MBq) of [68Ga]Ga was collected in <0.3 mL. Taking into account the used amount of 68Ga-eluate, buffer and other excipients, the overall amount of trace metal per labeling was <1.5 nmol. DOTA-TATE could be labeled with [68Ga]Ga with high radiochemical yield, >99% (ITLC), and a radiochemical purity of >95% (HPLC). CONCLUSION: With the here described concentration system and metal purification technique, a low activity containing 68Ga-generator can be used to label DOTA-peptide in preclinical applicable amounts >60 MBq/nmol (40-60 MBq/0.1 mL) and within 20 min.

Optimizing labelling conditions of 213Bi-DOTATATE for preclinical applications of peptide receptor targeted alpha therapy.

BACKGROUND: 213Bismuth (213Bi, T1/2 = 45.6 min) is one of the most frequently used α-emitters in cancer research. High specific activity radioligands are required for peptide receptor radionuclide therapy. The use of generators containing less than 222 MBq 225Ac (actinium), due to limited availability and the high cost to produce large-scale 225Ac/213Bi generators, might complicate in vitro and in vivo applications though.Here we present optimized labelling conditions of a DOTA-peptide with an 225Ac/213Bi generator (< 222 MBq) for preclinical applications using DOTA-Tyr3-octreotate (DOTATATE), a somatostatin analogue. The following labelling conditions of DOTATATE with 213Bi were investigated; peptide mass was varied from 1.7 to 7.0 nmol, concentration of TRIS buffer from 0.15 mol.L-1 to 0.34 mol.L-1, and ascorbic acid from 0 to 71 mmol.L-1 in 800 µL. All reactions were performed at 95 °C for 5 min. After incubation, DTPA (50 nmol) was added to stop the labelling reaction. Besides optimizing the labelling conditions, incorporation yield was determined by ITLC-SG and radiochemical purity (RCP) was monitored by RP-HPLC up to 120 min after labelling. Dosimetry studies in the reaction vial were performed using Monte Carlo and in vitro clonogenic assay was performed with a rat pancreatic tumour cell line, CA20948. RESULTS: At least 3.5 nmol DOTATATE was required to obtain incorporation ≥ 99 % with 100 MBq 213Bi (at optimized pH conditions, pH 8.3 with 0.15 mol.L-1 TRIS) in a reaction volume of 800 µL. The cumulative absorbed dose in the reaction vial was 230 Gy/100 MBq in 30 min. A minimal final concentration of 0.9 mmol.L-1 ascorbic acid was required for ~100 MBq (t = 0) to minimize radiation damage of DOTATATE. The osmolarity was decreased to 0.45 Osmol/L.Under optimized labelling conditions, 213Bi-DOTATATE remained stable up to 2 h after labelling, RCP was ≥ 85 %. In vitro showed a negative correlation between ascorbic acid concentration and cell survival. CONCLUSION: 213Bismuth-DOTA-peptide labelling conditions including peptide amount, quencher and pH were optimized to meet the requirements needed for preclinical applications in peptide receptor radionuclide therapy.

Radiolabeling of DOTA-like conjugated peptides with generator-produced (68)Ga and using NaCl-based cationic elution method.

Gallium-68 ((68)Ga) is a generator-produced radionuclide with a short half-life (t½ = 68 min) that is particularly well suited for molecular imaging by positron emission tomography (PET). Methods have been developed to synthesize (68)Ga-labeled imaging agents possessing certain drawbacks, such as longer synthesis time because of a required final purification step, the use of organic solvents or concentrated hydrochloric acid (HCl). In our manuscript, we provide a detailed protocol for the use of an advantageous sodium chloride (NaCl)-based method for radiolabeling of chelator-modified peptides for molecular imaging. By working in a lead-shielded hot-cell system,(68)Ga(3+) of the generator eluate is trapped on a cation exchanger cartridge (100 mg, ∼8 mm long and 5 mm diameter) and then eluted with acidified 5 M NaCl solution directly into a sodium acetate-buffered solution containing a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or DOTA-like chelator-modified peptide. The main advantages of this procedure are the high efficiency and the absence of organic solvents. It can be applied to a variety of peptides, which are stable in 1 M NaCl solution at a pH value of 3-4 during reaction. After labeling, neutralization, sterile filtration and quality control (instant thin-layer chromatography (iTLC), HPLC and pH), the radiopharmaceutical can be directly administered to patients, without determination of organic solvents, which reduces the overall synthesis-to-release time. This procedure has been adapted easily to automated synthesis modules, which leads to a rapid preparation of (68)Ga radiopharmaceuticals (12-16 min).

Overview of Development and Formulation of ¹77Lu-DOTA-TATE for PRRT.

Peptide receptor radionuclide therapy (PRRT) using radiolabeled somatostatin analogs has become an established procedure for the treatment of patients suffering from inoperable neuroendocrine cancers over-expressing somatostatin receptors. Success of PRRT depends on the availability of the radiolabeled peptide with adequately high specific activity, so that required therapeutic efficacy can be achieved without saturating the limited number of receptors available on the target lesions. Specific activity of the radionuclide and the radiolabeled somatostatin analog are therefore an important parameters. Although these analogs have been investigated and improved, and successfully applied for PRRT for more than 15 years, there are still many possibilities for further improvements that fully exploit PRRT with 177Lu-DOTA-TATE. The here summarized data presented herein on increased knowledge of the components of 177Lu-DOTA-TATE (especially the purity of 177Lu and specific activity of 177Lu) and the reaction kinetics during labeling 177Lu-DOTA-TATE clearly show that the peptide dose and dose in GBq can be varied. Here we present an overview of the development, formulation and optimisation of 177Lu-DOTA-TATE, mainly addressing radiochemical parameters.

Accurate assessment of whole-body retention for PRRT with (177)Lu using paired measurements with external detectors.

The aim of this study was to assess the accuracy of the results of whole-body measurements by comparison with the urine collection method in the PRRT with (177)Lu and furthermore to develop a more accurate method of paired measurements. Excreted samples were collected at given intervals and activities were measured by a dose calibrator. Traditionally, whole-body activities during subsequent measurements are normalized individually to the administered activity. In order to correct for the effects of the activity in the bladder during the baseline measurement before the first voiding and activity redistributions in the patient body during subsequent measurements, a series of paired measurements before and after each voiding were carried out. Time-dependent detector responses at given times were derived and time-activity retentions were then determined. Compared to the results of the urine collection, whole-body activities by traditional whole-body measurements were overestimated by ca. 14% at 1 h after administration and randomly varied from -29% to 49% at 24 h. Measurement uncertainties of whole-body activities were from ± 4% (the coverage factor k=2) at 1 h to >± 20% at 24 h by the urine collection and ± 7% by paired measurements, respectively. Whole-body activities at 1 h by paired measurements were validated using the results by measurements of the collected first urine. The new method of paired measurements has an equivalent measurement accuracy and even better during the later measurements with respect to the urine collection method and therefore can replace urine approach for assessing the time-activity remaining in the patient body.

Alternative method to determine Specific Activity of (177)Lu by HPLC.

UNLABELLED: Peptide Receptor Radionuclide Therapy (PRRT) with (177)Lu-DOTA-peptides requires (177)Lu with high specific activity (SA) and values >740 GBq (177)Lu per mg Lu to maximise the atom% of (177)Lu over total Lu. Vendors provide SA values which are based on activity and mass of the target, whereas due to "burn-up" of target, these SA values are not accurate. For a radiochemist the SA of (177)Lu is of interest prior to radiolabeling. An alternative method to determine SA was developed by HPLC, which includes a metal titration of a known amount of DOTA-peptide with a known amount of activity ((177)Lu), and a unknown amount of metal ((177+nat)Lu). Based on an HPLC separation of radiometal-DOTA-peptide and DOTA-peptide, and the concordant ratio of these components the metal content ((177+nat)Lu) can be calculated, and eventually the SA of (177)Lu can be accurately determined. These experimentally determined SA values exceeded the estimated values provided by vendors by 27 ± 16%, (range 6-73 %). The deviation of SA values for samples from the same Lu batch was <2% (n ≥ 10). IN CONCLUSION: the SA of (177)Lu is apparently often higher as stated by vendors in comparison to the experimentally determined actual values. For this reason, the SA of (177)Lu-DOTA-TATE and other Lu-DOTA-peptides could be increased accordingly.

Application of single-vial ready-for-use formulation of 111In- or 177Lu-labelled somatostatin analogs.

For the sake of safety it would be desirable to store and transport the ready-for-use liquid formulation (diagnostics and therapeutics) of radiolabelled peptides. The use of ethanol, in combination with a mixture of gentisic- and ascorbic acid, has superior effects on stabilizing radiolabelled somatostatin analogs. As a consequence, (111)In- and (177)Lu-labelled somatostatin analogs can be stored and transported in a single-vial ready-for-use liquid formulation up to 7 days after radiolabelling.

Reversed phase free ion selective radiotracer extraction (RP-FISRE): a new tool to assess the dynamic stabilities of metal (-organic) complexes, for complex half-lives spanning six orders of magnitude.

This paper introduces reversed phase free ion selective radiotracer extraction (RP-FISRE) as a new tool to assess the stability of metal complexes, as illustrated by the assessment of the stability of [(177)Lu]Lu-DOTA-octreotate. To this end, the TUDelft-developed FISRE, where the released metal is column-retained and the complex eluted, was changed into RP-FISRE, where the complex is column-retained and the released metal is eluted. This change in the approach allows for studies to be performed with high stability complexes. This paper presents RP-FISRE, the strength of the radiotracer approach, and the first-ever kd data on the release of (177)Lu from [(177)Lu]Lu-DOTA-octreotate.

The untapped potential of Gallium 68-PET: the next wave of 68Ga-agents.

(68)Gallium-PET ((68)Ga-PET) agents have significant clinical promise. The radionuclide can be produced from a (68)Ge/(68)Ga generator on site and is a convenient alternative to cyclotron-based PET isotopes. The short half-life of (68)Ga permits imaging applications with sufficient radioactivity while maintaining patient dose to an acceptable level. Furthermore, due to superior resolution, (68)Ga-PET agents have the ability to replace current SPECT agents in many applications. This article outlines the upcoming agents and challenges faced during the translational development of (68)Ga agents.

A new automated NaCl based robust method for routine production of gallium-68 labeled peptides.

A new NaCl based method for preparation of gallium-68 labeled radiopharmaceuticals has been adapted for use with an automated gallium-68 generator system. The method was evaluated based on 56 preparations of [(68)Ga]DOTATOC and compared to a similar acetone-based approach. Advantages of the new NaCl approach include reduced preparation time (<15 min) and removal of organic solvents. The method produces high peptide-bound % (>97%), and specific activity (>40 MBq nmole(-1) [(68)Ga]DOTATOC) and is well-suited for clinical production of radiopharmaceuticals.

Simplified NaCl based (68)Ga concentration and labeling procedure for rapid synthesis of (68)Ga radiopharmaceuticals in high radiochemical purity.

A simple sodium chloride (NaCl) based (68)Ga eluate concentration and labeling method that enables rapid, high-efficiency labeling of DOTA conjugated peptides in high radiochemical purity is described. The method utilizes relatively few reagents and comprises minimal procedural steps. It is particularly well-suited for routine automated synthesis of clinical radiopharmaceuticals. For the (68)Ga generator eluate concentration step, commercially available cation-exchange cartridges and (68)Ga generators were used. The (68)Ga generator eluate was collected by use of a strong cation exchange cartridge. 98% of the total activity of (68)Ga was then eluted from the cation exchange cartridge with 0.5 mL of 5 M NaCl solution containing a small amount of 5.5 M HCl. After buffering with ammonium acetate, the eluate was used directly for radiolabeling of DOTATOC and DOTATATE. The (68)Ga-labeled peptides were obtained in higher radiochemical purity compared to other commonly used procedures, with radiochemical yields greater than 80%. The presence of (68)Ge could not be detected in the final product. The new method obviates the need for organic solvents, which eliminates the required quality control of the final product by gas chromatography, thereby reducing postsynthesis analytical effort significantly. The (68)Ga-labeled products were used directly, with no subsequent purification steps, such as solid-phase extraction. The NaCl method was further evaluated using an automated fluid handling system and it routinely facilitates radiochemical yields in excess of 65% in less than 15 min, with radiochemical purity consistently greater than 99% for the preparation of (68)Ga-DOTATOC.

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.

Iodination and stability of somatostatin analogues: comparison of iodination techniques. A practical overview.

For iodination ((125/127)I) of tyrosine-containing peptides, chloramin-T, Pre-Coated Iodo-Gen(®) tubes and Iodo-Beads(®) (Pierce) are commonly used for in vitro radioligand investigations and there have been reliant vendors hereof for decades. However, commercial availability of these radio-iodinated peptides is decreasing. For continuation of our research in this field we investigated and optimized (radio-)iodination of somatostatin analogues. In literature, radioiodination using here described somatostatin analogues and iodination techniques are described separately. Here we present an overview, including High Performance Liquid Chromatography (HPLC) separation and characterisation by mass spectrometry, to obtain mono- and di-iodinated analogues. Reaction kinetics of (125/127)I iodinated somatostatin analogues were investigated as function of reaction time and concentration of reactants, including somatostatin analogues, iodine and oxidizing agent. To our knowledge, for the here described somatostatin analogues, no (127)I iodination and optimization are described. (Radio-)iodinated somatostatin analogues could be preserved with a >90% radiochemical purity for 1 month after reversed phase HPLC-purification.

Effectiveness of quenchers to reduce radiolysis of (111)In- or (177)Lu-labelled methionine-containing regulatory peptides. Maintaining radiochemical purity as measured by HPLC.

An overview how to measure and to quantify radiolysis by the addition of quenchers and to maintain Radio-Chemical Purity (RCP) of vulnerable methionine-containing regulatory peptides is presented. High RCP was only achieved with a combination of quenchers. However, quantification of RCP is not standardized, and therefore comparison of radiolabelling and RCP of regulatory peptides between different HPLC-systems and between laboratories is cumbersome. Therefore we suggest a set of standardized requirements to quantify RCP by HPLC for radiolabelled DTPA- or DOTA-peptides. Moreover, a dosimetry model was developed to calculate the doses in the reaction vials during radiolabelling and storage of the radiopeptides, and to predict RCP in the presence and absence of quenchers. RCP was measured by HPLC, and a relation between radiation dose and radiolysis of RCP was established. The here described quenchers are tested individually as ƒ(concentration) to investigate efficacy to reduce radiolysis of radiolabelled methionine-containing regulatory peptides.

(68)Ga-labeled DOTA-peptides and (68)Ga-labeled radiopharmaceuticals for positron emission tomography: current status of research, clinical applications, and future perspectives.

In this review we give an overview of current knowledge of (68)Ga-labeled pharmaceuticals, with focus on imaging receptor-mediated processes. A major advantage of a (68)Ge/(68)Ga generator is its continuous source of (68)Ga, independently from an on-site cyclotron. The increase in knowledge of purification and concentration of the eluate and the complex ligand chemistry has led to (68)Ga-labeled pharmaceuticals with major clinical impact. (68)Ga-labeled pharmaceuticals have the potential to cover all today's clinical options with (99m)Tc, with the concordant higher resolution of positron emission tomography (PET) in comparison with single photon emission computed tomography. (68)Ga-labeled analogs of octreotide, such as DOTATOC, DOTANOC, and DOTA-TATE, are in clinical application in nuclear medicine, and these analogs are now the most frequently applied of all (68)Ga-labeled pharmaceuticals. All the above-mentioned items in favor of successful application of (68)Ga-labeled radiopharmaceuticals for imaging in patients are strong arguments for the development of a (68)Ge/(68)Ga generator with Marketing Authorization and thus to provide pharmaceutical grade eluate. Moreover, now not one United States Food and Drug Administration-approved or European Medicines Agency-approved (68)Ga-radiopharmaceutical is available. As soon as these are achieved, a whole new radiopharmacy providing PET radiopharmaceuticals might develop.

Comparison of the binding and internalization properties of 12 DOTA-coupled and ¹¹¹In-labelled CCK2/gastrin receptor binding peptides: a collaborative project under COST Action BM0607.

PURPOSE: Specific overexpression of cholecystokinin 2 (CCK2)/gastrin receptors has been demonstrated in several tumours of neuroendocrine origin. In some of these cancer types, such as medullary thyroid cancer (MTC), a sensitive diagnostic modality is still unavailable and therapeutic options for inoperable lesions are needed. Peptide receptor radionuclide therapy (PRRT) may be a viable therapeutic strategy in the management of these patients. Several CCK2R-targeted radiopharmaceuticals have been described in recent years. As part of the European Union COST Action BM0607 we studied the in vitro and in vivo characteristics of 12 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated CCK2R binding peptides. In the present study, we analysed binding and internalization characteristics. Stability, biodistribution and imaging studies have been performed in parallel by other centres involved in the project. METHODS: Determination of IC(50) values was performed using autoradiography, with DOTA-peptides displacing (125)I-CCK from receptors on tissue sections from human tumours. Saturation binding and internalization experiments were performed using (111)In-labelled peptides. The rat AR42J cell line and the human A431-CCK2R transfected cell line were utilized for in vitro experiments; dissociation constants (K(d)) and apparent number of binding sites (B(max)) were determined. Internalization was determined in receptor-expressing cells by incubating with tracer amounts of peptide at 37 and 4°C for different times up to 120 min. Surface-bound peptide was then stripped either by acid wash or subsequent incubation with 1 µM unlabelled peptide at 4°C. RESULTS: All peptides showed high receptor affinity with IC(50) values ranging from 0.2 to 3.4 nM. Saturation experiments also showed high affinity with K(d) values in the 10(-9)-10(-8) M range. B(max) values estimated in A431-CCK2R cells ranged from 0.6 to 2.2 × 10(6) per cell. All peptides showed high levels of internalization when incubated at 37°C. CONCLUSION: All DOTA-conjugated peptides showed high receptor binding and internalization properties and appear suitable for further characterization, as described in other articles of this issue.

Reduction of 68Ge activity containing liquid waste from 68Ga PET chemistry in nuclear medicine and radiopharmacy by solidification.

PET with 68Ga from the TiO2- or SnO2- based 68Ge/68Ga generators is of increasing interest for PET imaging in nuclear medicine. In general, radionuclidic purity (68Ge vs. 68Ga activity) of the eluate of these generators varies between 0.01 and 0.001%. Liquid waste containing low amounts of 68Ge activity is produced by eluting the 68Ge/68Ga generators and residues from PET chemistry. Since clearance level of 68Ge activity in waste may not exceed 10 Bq/g, as stated by European Directive 96/29/EURATOM, our purpose was to reduce 68Ge activity in solution from >10 kBq/g to <10 Bq/g; which implies the solution can be discarded as regular waste. Most efficient method to reduce the 68Ge activity is by sorption of TiO2 or Fe2O3 and subsequent centrifugation. The required 10 Bq per mL level of 68Ge activity in waste was reached by Fe2O3 logarithmically, whereas with TiO2 asymptotically. The procedure with Fe2O3 eliminates ≥90% of the 68Ge activity per treatment. Eventually, to simplify the processing a recirculation system was used to investigate 68Ge activity sorption on TiO2, Fe2O3 or Zeolite. Zeolite was introduced for its high sorption at low pH, therefore 68Ge activity containing waste could directly be used without further interventions. 68Ge activity containing liquid waste at different HCl concentrations (0.05-1.0 M HCl), was recirculated at 1 mL/min. With Zeolite in the recirculation system, 68Ge activity showed highest sorption.

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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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)%. CONCLUSIONS: 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.

Optimised labeling, preclinical and initial clinical aspects of CCK-2 receptor-targeting with 3 radiolabeled peptides.

Medullary thyroid carcinoma (MTC) expresses CCK-2 receptors. (111)In-labeled DOTA-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH(2) (DOTA-MG11), DOTA-DAsp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH(2) (DOTA-CCK), and (99m)Tc-labeled N(4)-Gly-DGlu-(Glu)(5)-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH(2) ((99m)Tc-Demogastrin 2) are analogs developed for CCK-2 receptor-targeted scintigraphy. All 3 radiolabeled analogs were selected on the basis of their high CCK-2 receptor affinity and their good in vitro serum stability, with in vitro serum t(1/2) values of several hours. Radiolabeling of DOTA-peptides with (111)In requires a heating procedure, typically in the range of 80 degrees -100 degrees C up to 30 min. Following this procedure with DOTA-MG11 resulted in a >98 % incorporation of (111)In, however, with a radiochemical purity (RCP) of <50 %. The decrease in RCP was found to be due to oxidation of the methionine residue in the molecule. Moreover, this oxidized compound lost its CCK-2 receptor affinity. Therefore, conditions during radiolabeling were optimised: labeling of DOTA-MG11 and DOTA-CCK with (111)In involved 5 min heating at 80 degrees C and led to an incorporation of (111)In of >98 %. In addition, all analogs were radiolabeled in the presence of quenchers to prevent radiolysis and oxidation resulting in a RCP of >90 %. All 3 radiolabeled analogs were i.v. administered to 6 MTC patients: radioactivity cleared rapidly by the kidneys, with no significant differences in the excretion pattern of the 3 radiotracers. All 3 radiolabeled analogs exhibited a low in vivo stability in patients, as revealed during analysis of blood samples, with the respective t(1/2) found in the order of minutes. In patient blood, the rank of radiopeptide in vivo stability was: (99m)Tc-Demogastrin 2 (t(1/2) 10-15 min)>(111)In-DOTA-CCK (t(1/2) approximately 5-10 min)>(111)In-DOTA-MG11 (t(1/2)<5 min).