Breeman, W.A.P.

Scaled-up radiolabelling of DOTATATE with 68Ga eluted from a SnO 2-based 68Ge/ 68Ga generator (Article)

2012-01-01T00:00:00Z

A scaled-up radiolabelling and improved post-labelling purification procedure for [68Ga]DOTATATE is reported, using a more than 1 year old SnO2-based 1850MBq68Ge/68Ga generator (initially double-loaded with 3700MBq68Ge) as a source of ionic68Ga. The elution method of choice comprised elution with 0.6M HCl in a single 4mL fraction, containing up to 95% of the total eluted68Ga 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.

Speciation of no-carrier-added 68Ga prior to its labeling for PET imaging (Article)

2011-10-19T00:00:00Z

The present article describes the probable speciation of68Ga radionuclide just before labeling to DOTA peptides for PET imaging. The68Ga eluted from an anion exchange column after its purification was analyzed for its elemental composition and pH at several stages. Neutron activation analysis of the eluted fractions yields the concentrations of Na and Cl, pH measurements indicate the concentration of free H+ions in the medium and specific activity calculations indicate the concentration of68Ga in the solution. Using all these information we get the idea of speciation of no carrier added Ga in the eluted fractions from CHEAQS programme. The estimations indicate that Ga is mostly present as GaCl2+in the total MiliQ eluate. However, just before labeling of DOTA the pH of the Ga-containing eluate is adjusted to ~3.5 using HEPES buffer and at that condition Ga remains as Ga3+species which is responsible for a successful and efficient labeling. The MilliQ eluate collected before actual labeling was estimated for trace elements using inductively coupled plasma atomic emission spectrometry was found to contain a few ppb of Al, Co, Pd and Pt that did not interfere in the actual labeling. A clear idea about the prerequisite of68Ga species before labeling to a peptide might be of special interest for its judicious application as a radiopharmaceutical.

Comparison of biological stability and metabolism of CCK2 receptor targeting peptides, a collaborative project under COST BM0607 (Article)

2011-08-01T00:00:00Z

Purpose Stability of radiolabelled cholecystokinin 2 (CCK2) receptor targeting peptides has been a major limitation in the use of such radiopharmaceuticals especially for targeted radionuclide therapy applications, e.g. for treatment of medullary thyroid carcinoma (MTC). The purpose of this study was to compare the in vitro stability of a series of peptides binding to the CCK2 receptor [selected as part of the COST Action on Targeted Radionuclide Therapy (BM0607)] and to identify major cleavage sites. Methods Twelve different 1,4,7,10-tetraazacyclododecane- N,N',N'',N'''-tetraacetic acid (DOTA)-minigastrin/CCK conjugates were provided within an European COST Action (BM0607) by different laboratories and radiolabelled with177Lu. Their in vitro stabilities were tested in fresh human serum. Radiochemical yields (RCY) and intact radioligands for half-life calculations were determined by radio-HPLC. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis of metabolites was performed to identify cleavage products using conjugates labelled with excess stablenatLu, incubated in serum at 37°C. Urine metabolite analysis after injection in normal mice was performed by radio-HPLC analysis.Results Variable stability in human serum was found for the different peptides with calculated half-lives between 4.5± 0.1 h and 198±0.1 h (n=2). In urine of normal mice only metabolised peptide fragments were detected even at short times after injection for all peptides. MALDI-TOF MS revealed a major cleavage site of all minigastrin derivatives between Asp and Phe-NH2 at the C-terminal end. Conclusion Development of CCK2 receptor ligands especially for therapeutic purposes in patients with MTC or small cell lung cancer (SCLC) is still ongoing in different laboratories. This comparative study provided valuable insight into the importance of biological stability especially in the context of other results of this comparative trial within the COST Action BM0607.

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 (Article)

2011-08-01T00:00:00Z

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 COSTAction 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 presentstudy, 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 IC50values was performed using autoradiography, with DOTA-peptides displacing125I-CCK from receptors on tissue sections from human tumours. Saturation binding and internalization experiments were performed using111In-labelled peptides. The rat AR42J cell line and the human A431-CCK2R transfected cell line were utilized for in vitro experiments; dissociation constants (Kd) and apparent number of binding sites (Bmax) 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 IC50values ranging from 0.2 to 3.4 nM. Saturation experiments also showed high affinity with Kdvalues in the 10-9-10-8M range. Bmaxvalues estimated in A431- CCK2R cells ranged from 0.6 to 2.2×106per 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.

Gastrin-releasing peptide receptor-based targeting using bombesin analogues is superior to metabolism-based targeting using choline for in vivo imaging of human prostate cancer xenografts (Article)

2011-07-01T00:00:00Z

Purpose: Prostate cancer (PC) is a major health problem. Overexpression of the gastrin-releasing peptide receptor (GRPR) in PC, but not in the hyperplastic prostate, provides a promising target for staging and monitoring of PC. Based on the assumption that cancer cells have increased metabolic activity, metabolism-based tracers are also being used for PC imaging. We compared GRPR-based targeting using the68Ga-labelled bombesin analogue AMBA with metabolism-based targeting using18F-methylcholine (18F-FCH) in nude mice bearing human prostate VCaP xenografts. Methods: PET and biodistribution studies were performed with both68Ga-AMBA and18F-FCH in all VCaP tumour-bearing mice, with PC-3 tumour-bearing mice as reference. Scanning started immediately after injection. Dynamic PET scans were reconstructed and analysed quantitatively. Biodistribution of tracers and tissue uptake was expressed as percent of injected dose per gram tissue (%ID/g). Results: All tumours were clearly visualized using68Ga-AMBA.18F-FCH showed significantly less contrast due to poor tumour-to-background ratios. Quantitative PET analyses showed fast tumour uptake and high retention for both tracers. VCaP tumour uptake values determined from PET at steady-state were 6.7±1.4%ID/g (20-30 min after injection, N=8) for68Ga-AMBA and 1.6±0.5%ID/g (10-20 min after injection, N=8) for18F-FCH, which were significantly different (p<0.001). The results in PC-3 tumour-bearing mice were comparable. Biodistribution data were in accordance with the PET results showing VCaP tumour uptake values of 9.5±4.8%ID/g (N=8) for68Ga-AMBA and 2.1±0.4%ID/g (N=8) for18F-FCH. Apart from the GRPR-expressing organs, uptake in all organs was lower for68Ga-AMBA than for18F-FCH. Conclusion: Tumour uptake of68Ga-AMBA was higher while overall background activity was lower than observed for18F-FCH in the same PC-bearing mice. These results suggest that peptide receptor-based targeting using the bombesin analogue AMBA is superior to metabolism-based targeting using choline for scintigraphy of PC.

68Ga-labeled DOTA-peptides and 68Ga-labeled radiopharmaceuticals for positron emission tomography: Current status of research, clinical applications, and future Perspectives (Article)

2011-07-01T00:00:00Z

In this review we give an overview of current knowledge of68Ga-labeled pharmaceuticals, with focus on imaging receptor-mediated processes. A major advantage of a68Ge/68Ga generator is its continuous source of68Ga, independently from an on-site cyclotron. The increase in knowledge of purification and concentration of the eluate and the complex ligand chemistry has led to68Ga-labeled pharmaceuticals with major clinical impact.68Ga-labeled pharmaceuticals have the potential to cover all today's clinical options with99mTc, with the concordant higher resolution of positron emission tomography (PET) in comparison with single photon emission computed tomography.68Ga-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 all68Ga-labeled pharmaceuticals. All the above-mentioned items in favor of successful application of68Ga-labeled radiopharmaceuticals for imaging in patients are strong arguments for the development of a68Ge/68Ga 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-approved68Ga-radiopharmaceutical is available. As soon as these are achieved, a whole new radiopharmacy providing PET radiopharmaceuticals might develop.

Effect of interferon-a treatment on [68Ga-DOTA,Tyr3,Thre8] octreotide uptake in CA20948 tumors: A small-animal PET study (Article)

2011-04-01T00:00:00Z

In peptide receptor radionuclide therapy of neuroendocrine tumors, improvements have been made by increasing the affinity for receptors and by protecting critical organs (e.g., kidneys). However, tumor parameters involved in radiopeptide uptake are still under investigation. Interferon-a (IFNa) is used as biotherapy for neuroendocrine tumors. Several mechanisms of action are described, but the potential effect of IFNa on tumor uptake of labeled peptide has not been studied in vivo yet. Methods: Twenty-six male CA20948 tumor-bearing Lewis rats were imaged before and during IFNa treatment using quantitative small-animal PET with [68Ga-DOTA,Tyr3,Thre8]octreotide. Imaging was performed at days 0, 3, and 7. Animals were divided into 3 groups according to the treatment: Control (injected daily with saline), half (4 d of IFNa treatment from day 0 to day 3, then saline), and full (7 d of IFNa). A daily dose of IFNa (1.5 mIU) was administered subcutaneously. Quantitative PET results are expressed as percentage injected dose per cm3 and normalized to baseline (day 0) values. Tumor size was monitored by PET and caliper measurements. Results: Gross tumor uptake and tumor volumes increased in all groups over the 7-d period. On day 3, mean±SD ratios to day 0 were 1.2±0.2, 1.3±0.5, and 1.2±0.4, respectively, for control, half, and full groups. On day 7, respective values were 1.1±0.2, 1.3±0.6, and 1.5±0.4. At day 3, a comparison among groups showed no statistically significant difference. At day 7, the full group showed a significantly higher ratio in activity concentration than the control group (P = 0.021). A good correlation was found between tumor volumes assessed by small-animal PET and caliper measurements (R = 0.89, P<0.0001). Conclusion: As expected, over a period of 7 d, both tumor volumes and radiopeptide uptake increased in all animals. However, the activity concentration increased significantly more at day 7 in animals treated for 7 d with IFNα, compared with controls. This is the first, to our knowledge, in vivo indication that IFNa is able to increase tumor uptake of the labeled analog in a small-animal model of neuroendocrine tumors. The mechanisms underlying this effect (flow, vascular permeability, receptor upregulation) remain unknown and need to be further investigated. Copyright

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

2011-04-01T00:00:00Z

PET with68Ga from the TiO2- or SnO2- based68Ge/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 of68Ge activity is produced by eluting the68Ge/68Ga generators and residues from PET chemistry. Since clearance level of68Ge activity in waste may not exceed 10 Bq/g, as stated by European Directive 96/29/EURATOM, our purpose was to reduce68Ge 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 the68Ge activity is by sorption of TiO2or Fe2O3and subsequent centrifugation. The required 10 Bq per mL level of68Ge activity in waste was reached by Fe2O3logarithmically, whereas with TiO2asymptotically. The procedure with Fe2O3eliminates â‰¥ 90% of the68Ge activity per treatment. Eventually, to simplify the processing a recirculation system was used to investigate68Ge activity sorption on TiO2, Fe2O3or Zeolite. Zeolite was introduced for its high sorption at low pH, therefore68Ge 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 (Article)

2011-02-01T00:00:00Z

Objectives: PET scintigraphy with68Ga-labelled analogs is of increasing interest in Nuclear Medicine and performed all over the world. Here we report the characteristics of the eluate of SnO2-based68Ge/68Ga 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 SnO2-based generators (range 0.4-1GBq68Ga in the eluate) and several concentration techniques of the eluate (HCl) were evaluated. The elution profiles of SnO2-based68Ge/68Ga generators were monitored, while [HCl] of the eluens was varied from 0.3-1.0M. Metal ions and sterility of the eluate were determined by ICP. Fractionated elution and concentration of the68Ga eluate were performed using anion and cation exchange. Concentrated68Ga eluate, using all three concentration techniques, was used for labelling of DOTA-TATE.68Ga-DOTA-TATE-containing solution was purified and RNP increased by SPE, therefore also 11 commercially available SPE columns were investigated. Results: The amount of elutable68Ga activity varies when the concentration of the eluens, HCl, was varied, while68Ge activity remains virtually constant. SnO2-based68Ge/68Ga generator elutes at 0.6M HCl >100% of the68Ga activity at calibration time and ±75% after 300 days. Eluate at discharge was sterile and Endotoxins were <0.5EU/mL, RNP was always <0.01%. Metal ions in the eluate were <10ppm (in total). Highest desorption for anion purification was obtained with the 30mg Oasis WAX column (>80%). Highest desorption for cation purification was obtained using a solution containing 90% acetone at increasing molarity of HCl, resulted in a68Ga desorption of 68±8%. With all68Ge/68Ga generators and for all 3 purification methods a SA up to 50MBq/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 elutable68Ga with 1M 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 of68Ga-DOTA-TATE of SPE columns varied, highest desorption was obtained with Baker C18100mg (84%). Purification of68Ga-DOTA-TATE by SPE resulted in an RNP of <10-4%. Conclusions: Eluate of SnO2-based68Ge/68Ga generator, either by fractionated elution as by ion exchange can be used for labelling DOTA-peptides with68Ga at a SA of 50MBq/nmol at >95% incorporation and a RCP of ±90%. SPE columns are very effective to increase RNP.

A standardised study to compare prostate cancer targeting efficacy of five radiolabelled bombesin analogues (Article)

2010-07-01T00:00:00Z

Purpose: Prostate-specific antigen (PSA)-based screening for prostate cancer (PC) has dramatically increased early diagnosis. Current imaging techniques are not optimal to stage early PC adequately. A promising alternative to PC imaging is peptide-based scintigraphy using radiolabelled bombesin (BN) analogues that bind to gastrin-releasing peptide receptors (GRPR) being overexpressed in PC. When labelled to appropriate radionuclides BN targeting of GRPRs may also provide applications for peptide radionuclide receptor therapy (PRRT). Assessment studies under identical experimental conditions allowing a reliable comparison of the potential of such analogues are lacking. This study was performed to evaluate and directly compare five promising radiolabelled BN analogues for their targeting efficacy for PC under standardised conditions. Methods: The BN agonists [111In]DOTA-PESIN, [111In]AMBA, [111In]MP2346 and [111In]MP2653 and one antagonist [99mTc]Demobesin-1 were evaluated in GRPR-overexpressing human PC-3 tumour-bearing mice to determine peptide stability in vivo, biodistribution and GRPR targeting potential by animal SPECT/CT imaging and ex vivo autoradiography. Results: HPLC analysis of blood showed intact Demobesin-1 at 5 and 15 min after injection (64.1 ± 1.6% and 41.0 ± 01%, respectively) being much less for the other compounds. AMBA, the second most stable analogue, showed 36.1 ± 2.7% and 9.8 ± 1.1% intact peptide after 5 and 15 min. PC-3 tumour uptake at 1 h was comparable for Demobesin-1, AMBA, PESIN and MP2346 (3.0 ± 0.4, 2.7 ± 0.5, 2.3 ± 0.5 and 2.1 ± 0.9%ID/g, respectively), but very low for MP2653 (0.9 ± 0.2%ID/g). In addition, MP2346 showed undesirably high uptake in the kidneys (7.9 ± 1.9%ID/g) being significantly less for the other analogues. AMBA, MP2346 and PESIN revealed favourable increases in tumour to blood ratios over time while changes in tumour to kidney and pancreas ratios for Demobesin-1 from 1 to 24 h after injection were significantly better than for the other analogues. All analogues visualised PC-3 tumours by SPECT/CT and autoradiography. Conclusion: In the present study the BN antagonist Demobesin-1 was the best performing analogue showing superior in vivo stability, highest tumour uptake and retention while pancreatic and renal clearance were rapid. PESIN and AMBA were the best GRP agonists with sufficient in vivo stabilities as well as high tumour uptake and retention. Based on these results all three analogues deserve further evaluation for clinical use in PC patients.

Androgen-regulated gastrin-releasing peptide receptor expression in androgen-dependent human prostate tumor xenografts (Article)

2010-06-15T00:00:00Z

Human prostate cancer (PC) overexpresses the gastrin-releasing peptide receptor (GRPR). Radiolabeled GRPR-targeting analogs of bombesin (BN) have successfully been introduced as potential tracers for visualization and treatment of GRPR-overexpressing tumors. A previous study showed GRPR-mediated binding of radiolabeled BN analogs in androgen-dependent but not in androgen-independent xenografts representing the more advanced stages of PC. We have further investigated the effect of androgen modulation on GRPR-expression in three androgen-dependent human PC-bearing xenografts: PC295, PC310 and PC82 using the androgen-independent PC3-model as a reference. Effects of androgen regulation on GRPR expression were initially studied on tumors obtained from our biorepository of xenograft tissues performing reverse transcriptase polymerase chain reaction (RT-PCR) and autoradiography (125I-universal-BN). A prospective biodistribution study (111In-MP2653) and subsequent autoradiography (125I-GRP and111In-MP2248) was than performed in castrated and testosterone resupplemented tumor-bearing mice. For all androgen-dependent xenografts, tumor uptake and binding decreased drastically after 7 days of castration. Resupplementation of testosterone to castrated animals restored GRPR expression extensively. Similar findings were concluded from the initial autoradiography and RT-PCR studies. Results from RT-PCR, for which human specific primers are used, indicate that variations in GRPR expression can be ascribed to mRNA downregulation and not to castration-induced reduction in the epithelial fraction of the xenograft tumor tissue. In conclusion, expression of human GRPR in androgendependent PC xenografts is reduced by androgen ablation and is reversed by restoring the hormonal status of the animals. This knowledge suggests that hormonal therapy may affect GRPR expression in PC tissue making GRPR-based imaging and therapy especially suitable for non-hormonally treated PC patients.

Tumor uptake of 68Ga-DOTA-Tyr3-octreotate: animal PET studies of tumor flow and acute somatostatin receptor modulation in the CA20948 rat model (Article)

2010-02-01T00:00:00Z

Introduction: Factors determining the in vivo uptake of radiolabeled somatostatin analogs by neuroendocrine tumors are poorly known. The aim is to evaluate in vivo tumor perfusion and regulation of somatostatin receptors (sstr) following acute exposure to octreotide, in an animal model of neuroendocrine tumor. Methods: H215O flow studies were performed in 8 CA20948 tumor-bearing rats and another 36 rats underwent three [68Ga]-DOTA-Tyr3-octreotate imaging sessions at 24-h intervals. After baseline (Day 0) imaging, scanning was repeated on Day 1 after octreotide injection (175 μg/kg), with a variable delay: no injection (controls, n=7), coinjection (n=6), and octreotide injection 20 min (n=7), 2 h (n=8) and 4 h (n=8) before imaging. Repeat images without octreotide was performed at Day 2 followed by sacrifice and tumor counting. Results: H215O studies failed to measure quantitative tumor perfusion in this model. On Day 1, ratio of tumor uptake to Day 0 was 1.2±0.3 in controls; 0.6±0.2 in the coinjection group; 0.9±0.2, 1.1±0.1 and 1.2±0.2 in the other groups, respectively. Uptake in the coinjection group showed a statistically significant reduction of tumor uptake (P<.0001). All groups showed increased uptake on Day 2, without statistical differences between groups. In vivo tumor counts showed good correlation with ex vivo countings (R2=0.946). Conclusion: Acute exposure to unlabeled octreotide in this neuroendocrine tumor model results in a rapid recycling or resynthesis of sstr. Positron emission tomography (PET) allowed to reliably assess quantitative uptake of [68Ga]-DOTA-Tyr3-octreotate over time in the same animal, but failed in this model to measure tumor perfusion.

Comparison of three radiolabelled peptide analogues for CCK-2 receptor scintigraphy in medullary thyroid carcinoma (Article)

2009-03-06T00:00:00Z

Purpose: Cholecystokinin 2 (CCK-2) receptor overexpression has been demonstrated in a high percentage of medullary thyroid carcinomas (MTC). Analogous to somatostatin receptors, CCK-2 receptors might be viable targets for radionuclide scintigraphy and/or radionuclide therapy. Several CCK-2 receptor-binding radiopeptides have been developed, and some have been carried through into clinical studies. However, these studies are mostly limited and difficult to compare. The aim of this study was to evaluate the diagnostic and therapeutic potential of three promising CCK-2 receptor-binding radiopeptides in patients with MTC. Methods: 111In-DOTA-(D)Asp-Tyr-Nle-Gly-Trp-Nle- Asp-Phe-NH2 (111In-DOTA-CCK), a CCK analogue, and the gastrin-based ligands 99mTc-N4-Gly-(D)Glu-(Glu) 5-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (99mTc- demogastrin 2) and 111In-DOTA-(D)Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe- NH2 (111In-DOTA-MG11) were each administered to the same group of six patients. Planar images made at 3-5, 7 and 24 h p.i. were used for comparison of tumour visualisation and renal uptake. Results: 99mTc-demogastrin 2 scintigraphy visualised all known lesions and new lesions in four of six patients. 111In-DOTA-CCK and 111In-DOTA-MG11 on the other hand missed several lesions; tumour uptake of these two radiopharmaceuticals was quite low. Comparison of retention of renal activity showed no major differences between the three radiopeptides. Conclusion: 99mTc-demogastrin 2 scintigraphy appeared most promising as a diagnostic tool in patients with MTC. Further studies are required to evaluate its value in patient management. Direct comparisons of the compounds studied strongly suggests that 111In-DOTA-CCK and 111In-DOTA-MG11 have less potential as imaging agents than 99mTc-demogastrin 2. These DOTA-linked compounds are considered unlikely to be useful for radionuclide therapy because of low tumour uptake.

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

2008-11-01T00:00:00Z

Medullary thyroid carcinoma (MTC) expresses CCK-2 receptors.111In-labeled DOTA-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2(DOTA-MG11), DOTA-DAsp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2(DOTA-CCK), and99mTc-labeled N4-Gly-DGlu-(Glu)5-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2(99mTc-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 t1/2values of several hours. Radiolabeling of DOTA-peptides with111In requires a heating procedure, typically in the range of 80°-100°C up to 30 min. Following this procedure with DOTA-MG11 resulted in a >98 % incorporation of111In, 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 with111In involved 5 min heating at 80°C and led to an incorporation of111In 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 t1/2found in the order of minutes. In patient blood, the rank of radiopeptide in vivo stability was:99mTc-Demogastrin 2 (t1/210-15 min)>111In-DOTA-CCK (t1/2≈5-10 min)>111In-DOTA-MG11 (t1/2<5 min).

Somatostatin receptor gene therapy combined with targeted therapy with radiolabeled octreotide: a new treatment for liver metastases. (Article)

2002-01-01T00:00:00Z

OBJECTIVE: To evaluate the effect of peptide receptor radionuclide therapy (PRRT) on somatostatin receptor (SSR)-transfected colon carcinoma cells in a rat liver metastases model.SUMMARY BACKGROUND DATA: Previously the authors have shown highly effective therapy with PRRT of SSR-positive tumors. This treatment is SSR-mediated; successful treatment is seen only in SSR-positive tumors, with no effect in SSR-negative tumors. As many tumors lack this receptor, the idea arose to transfect SSR-negative tumor cells with an SSR gene to apply PRRT on these SSR-transfected tumor cells. METHODS: CC531 colon carcinoma cells (SSR-negative) were transfected in vitro with an SSR (subtype 2) gene (CC2B). Liver metastases were produced after intraportal injection of these tumor cells in rats. On day 7, animals were treated with 185 or 370 MBq [177 Lu-DOTA0, Tyr3 ]octreotate. After 21 days rats were killed and liver metastases were counted. RESULTS: Treatment with 370 MBq [177 Lu-DOTA0, Tyr3 ]octreotate showed a significant antitumor response in rats with CC2B liver metastases (SSR-positive) in comparison with controls. No significant antitumor effect was seen in PRRT-treated rats with CC531 liver metastases (SSR-negative). Also, a dose-dependent tumor response was seen in rats with CC2B liver metastases treated with 185 MBq [ 177Lu-DOTA0, Tyr3 ]octreotate compared with controls. In addition, rats with mixed liver metastases treated with 185 MBq [177 Lu-DOTA0, Tyr3 ]octreotate had significantly fewer metastases compared with controls. CONCLUSIONS: The authors showed an impressive antitumor effect of SSR (subtype 2)-transfected colon carcinoma cells with PRRT in a rat liver metastasis model. Moreover, rats with mixed liver metastases had significantly fewer liver metastases compared with control rats, which may be due to a radiologic bystander effect of [177 Lu-DOTA0, Tyr3 ]octreotate. This phenomenon is beneficial in the concept of in vivo gene therapy.

Aspects of receptor scintigraphy with neuropeptides (Doctoral Thesis)

1995-04-20T00:00:00Z

Internalization of the radioiodinated somatostatin analog [125I-Tyr3]octreotide by mouse and human pituitary tumor cells: increase by unlabeled octreotide (Article)

1995-01-01T00:00:00Z

Recently, we developed a technique that allows the in vivo visualization in man of somatostatin receptor-positive neuroendocrine tumors after i.v. injection of [125I-Tyr3]octreotide or [111In-DTPA-D-Phe1]octreotide. Radiotherapy of such tumors using somatostatin analogs coupled to alpha- or beta-emitting radionuclides has been proposed as an application for radiolabeled somatostatin analogs. To develop this concept further, it is of importance to know whether the above-mentioned radiolabeled somatostatin analogs are internalized by the tumor cells, and whether it might be possible to manipulate the degree of internalization. In the present study we investigated the internalization of a stable somatostatin analog, [125I-Tyr3]octreotide, by mouse AtT20/D16V pituitary tumor cells and primary cultures of human GH-secreting pituitary tumor cells. Treatment of the cells with low pH was used to distinguish between membrane-bound (acid-releasable) and internalize (acid-resistant) radioligand. [125I-Tyr3]octreotide showed a time-dependent increasing accumulation in AtT20 cells; after 4 h of incubation, values up to 6-8% of the dose of radioligand added were obtained. Binding and internalization of [125I-Tyr3]octreotide were temperature dependent and inhibited by pertussis toxin. Inhibitors of lysosomal degradation did not increase the amount of internalized radioligand. After 4 h of incubation, 88% of the radioactivity present in the cells was still peptide bound, suggesting a low intracellular breakdown of this radioligand. Six of seven human GH-secreting adenoma cell cultures also internalized [125I-Tyr3]octreotide (variation between 0.24-4.98% of the dose radioligand added). Displacement of binding and internalization of [125I-Tyr3]octreotide by unlabeled octreotide showed a bell-shaped curve in AtT20 cells. At low concentrations (0.1 and 1 nM), binding and internalization were increased, whereas at higher concentrations, saturation occurred. In contrast to this, binding of [125I-Tyr3]octreotide to a broken cell preparation of AtT20 cells was displaced in a dose-dependent manner by unlabeled octreotide, with an IC50 of 0.1 nM. Similar observations were made in the human GH-secreting adenoma cell cultures. In conclusion, a high amount of [125I-Tyr3]octreotide is internalized in a specific-, time-, temperature-, and pertussis toxin-sensitive GTP-binding protein-dependent manner by mouse AtT20 and human GH-secreting pituitary tumor cells. In the presence of a low concentration of unlabeled octreotide, a rapid increase in the amount of [125I-Tyr3]octreotide internalized by AtT20 cells and by the majority of the human GH-secreting adenoma cell cultures was found.(ABSTRACT TRUNCATED AT 400 WORDS)