Abstract: Introduction: Siderophores are low-molecular-mass iron chelatorsserving asiron transporters foralmost all bacteria, fungi andsome plants.
Ironisanessentialelementformajorityoforganismsandplaysanimportantroleinvirulenceofpathogenicorganisms. 68Gaisapositronemitter
withcomplexingpropertiescomparabletothoseofFe(III)andreadilyavailablefromagenerator.Initialstudieswith 68Ga-triacetylfusarinineC
(TAFC)showedexcellenttargetingpropertiesinaratinfectionmodel.Wereporthereontheinvitroandinvivoevaluationofothersiderophores
radiolabelled with 68Ga as potential radiopharmaceuticals for infection imaging.
Methods: 68Ga labelling was performed using acetate buffer. Stability, log P and protein binding values were determined. In vitro uptake
was tested using iron-deficient and iron-sufficient Aspergillus fumigatus (A.f.) cultures. Biodistribution of 68Ga-siderophores was studied in
Balb/c mice.
Results: Significant differences among studied siderophores were observed in labelling efficiency, stability and protein binding. Uptake in
A.f. cultures was highly dependent on iron load and type of the siderophore. In mice, 68Ga-TAFC and 68Ga-ferrioxamine E (FOXE)
showed rapid renal excretion and low blood values even at a short period after injection; in contrast, 68Ga-ferricrocin and 68Ga-ferrichrome
revealed high retention in blood and 68Ga-fusarinine C showed very high kidney retention.
Conclusions: Some of the studied siderophores bind 68Ga with high affinity and stability, especially 68Ga-TAFC and 68Ga-FOXE. Low
values of protein binding, high and specific uptake in A.f., and excellent in vivo biodistribution make them favourable agents for Aspergillus
infection imaging.
Abstract: PURPOSE: A molecular target involved in the angiogenic process is the α(v)β(3) integrin. It has been demonstrated in preclinical as well as in clinical studies that radiolabelled RGD peptides and positron emission tomography (PET) allow noninvasive monitoring of α(v)β(3) expression. Here we introduce a (68)Ga-labelled NOTA-conjugated RGD peptide ([(68)Ga]NODAGA-RGD) and compare its imaging properties with [(68)Ga]DOTA-RGD using small animal PET.
METHODS: Synthesis of c(RGDfK(NODAGA)) was based on solid phase peptide synthesis protocols using the Fmoc strategy. The (68)Ga labelling protocol was optimized concerning temperature, peptide concentration and reaction time. For in vitro characterization, partition coefficient, protein binding properties, serum stability, α(v)β(3) binding affinity and cell uptake were determined. To characterize the in vivo properties, biodistribution studies and microPET imaging were carried out. For both in vitro and in vivo evaluation, α(v)β(3)-positive human melanoma M21 and α(v)β(3)-negative M21-L cells were used.
RESULTS: [(68)Ga]NODAGA-RGD can be produced within 5 min at room temperature with high radiochemical yield and purity (> 96%). In vitro evaluation showed high α(v)β(3) binding affinity (IC(50)â=â4.7â±â1.6 nM) and receptor-specific uptake. The radiotracer was stable in phosphate-buffered saline, pH 7.4, FeCl(3) solution, and human serum. Protein-bound activity after 180 min incubation was found to be 12-fold lower than for [(68)Ga]DOTA-RGD. Biodistribution data 60 min post-injection confirmed receptor-specific tumour accumulation. The activity concentration of [(68)Ga]NODAGA-RGD was lower than [(68)Ga]DOTA-RGD in all organs and tissues investigated, leading to an improved tumour to blood ratio ([(68)Ga]NODAGA-RGD: 11, [(68)Ga]DOTA-RGD: 4). MicroPET imaging confirmed the improved imaging properties of [(68)Ga]NODAGA-RGD compared to [(68)Ga]DOTA-RGD.
CONCLUSION: The introduced [(68)Ga]NODAGA-RGD combines easy accessibility with high stability and good imaging properties making it an interesting alternative to the (18)F-labelled RGD peptides currently used for imaging α(v)β(3) expression.
Abstract: OBJECTIVES:
Radiolabelled somatostatin analogues have found wide clinical use in nuclear medicine for both diagnostic and therapeutic applications. Here, we describe the development of a fully automated synthesis system allowing radiolabelling of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-derivatized peptides with Ga/In/Lu and Y, meeting radiation safety and pharmaceutical requirements.
MATERIALS AND METHODS:
The system consists of a syringe pump, a holder for insertion of a single use multivalve cassette, a heater and a removable radiation shielding. Ga labelling was performed in acetate buffer and Lu, Y and In labelling in ascorbate buffer, respectively, followed by purification on a C18 cartridge and final sterile filtration. Cross-contamination was prevented by using disposable cassettes and also by ensuring pharmaceutical standards. Radiochemical purity (RCP) was determined by instant thin-layer chromatography on silica gel impregnated glass fibres and reversed-phase high performance liquid chromatography.
RESULTS:
Ga-DOTA-peptides were prepared with high RCP (>91%) and radiochemical yields (RCY>80% decay corrected) and Ge content was less than 0.0001% in all cases. Synthesis time did not exceed 30 min. In, Lu and Y labelling of DOTA-peptides resulted again in high yields (approximately 90%) and RCP (approximately 95%) and total synthesis time of less than 45 min. Radiation dose to fingers was considerably reduced when compared with manual labelling procedures.
CONCLUSION:
The described system allows fully automated, aseptic preparation of DOTA-peptides radiolabelled with different radionuclides in high radiochemical yields and pharmaceutical quality suitable for clinical application.
Abstract: The diagnosis of invasive pulmonary aspergillosis (IPA) is difficult
and lacks specificity and sensitivity. In the pathophysiology of
Aspergillus fumigatus, iron plays an essential role as a nutrient
during infection. A. fumigatus uses a specific and highly efficient
iron uptake mechanism based on iron-complexing ferric ion
Fe(III) siderophores, which are a requirement for A. fumigatus virulence.
We aimed to evaluate the potential of siderophores
radiolabeled with 68Ga, a positron emitter with complexing properties
comparable to those of Fe(III), as a radiopharmaceutical for
imaging IPA. Methods: 68Ga radiolabeling of the A. fumigatus
siderophores desferri-triacetylfusarinine C (TAFC) and desferriferricrocin
(FC) was performed at high specific activity. Stability,
protein binding, and log P values were determined. In vitro uptake
in A. fumigatus cultures was tested under varying conditions.
Biodistribution was studied in healthy noninfected BALB/c
mice, and uptake was studied in a model of A. fumigatus infection
using immunosuppressed Lewis rats. Results: High-specific-
activity 68Ga labeling could be achieved, and resulting
complexes were stable in serum, toward diethylenetriaminepentaacetic
acid and Fe(III) challenge. Both siderophores showed
hydrophilic properties (68Ga-TAFC, log P 5 22.59; 68Ga-FC,
log P 5 23.17) with low values of protein binding for 68Ga-
TAFC (,2%). Uptake of both siderophores was highly dependent
on the mycelial iron load and could be blocked with an
excess (10 mM) of siderophore or NaN3, indicating specific, energy-
dependent uptake. In noninfected mice, 68Ga-TAFC showed
rapid renal excretion and lowblood values (1.660.37 percentage
injected dose per gram [%ID/g] at 30 min); in urine only intact
68Ga-TAFC was detected. In contrast, 68Ga-FC revealed high retention
in blood (16.1 6 1.07 %ID/g at 90 min) and rapid metabolism.
In the rat IPA model, lung uptake of 68Ga-TAFC was
dependent on the severity of infection, with less than 0.04
%ID/g in control rats (n 5 5) and 0.29 6 0.11 %ID/g in mildly
infected (n 5 3) and 0.95 6 0.37 %ID/g in severely infected
(n 5 4) rats. PET showed focal accumulation in infected lung
tissue. Conclusion: Both siderophores bound 68Ga with high
affinity, and 68Ga-TAFC, especially, showed high stability. 68Ga-
TAFC displayed highly selective accumulation by A. fumigatus
subspecies in vitro and in vivo. The high and specific uptake by
A. fumigatus proves the potential of 68Ga-labeled siderophores
for the specific detection of A. fumigatus during infection. They
hold promise as new PET agents for IPA.
Abstract: Here we describe a fully automated approach for the synthesis of 68Ga-labelled DOTA-peptides based on pre-concentration and purification of the generator eluate by using a cation exchange-cartridge and its comparison with fully automated direct labelling applying fractionated elution. Pre-concentration of the eluate on a cation exchange cartridge both using a resin-based and a disposable cation-exchange cartridge efficiently removed 68Ge as well as major metal contaminations with Fe and Zn. This resulted in a high labelling efficiency of DOTA-peptides at high specific activity (SA) with short synthesis times.
Abstract: Three different procedures for the labeling of hyaluronan (HA) with 111In, 125I and 14C radionuclides were compared, and the kinetic stability of radiolabeled HA under different conditions (saline, artificial gastric juice and plasma) was established. Modification of HA structure with bifunctional chelating agents (DTPA) or with the prosthetic group (tyramine or tyrosine) was essential prior 111In and 125I labeling. These chemical labeling techniques were fast, simple and inexpensive, and labeled agents with a high specific activity were obtained. The only disadvantage of these methods was the occurrence of unknown functional groups in the HA molecule requiring further characterization of the compound. Conversely, HA labeling with 14C by biotechnological synthesis was found to be rather expensive and time-consuming process. Although, the final product 14C-HA was identical to natural HA its low specific activity presents certain limitation for its application in biological experiments. Stability studies showed that 14C-HA and 125I-Tm-HA were stable in all studied mediums. In the case of 125I-Trs-HA, stability slightly decreased in rat plasma and in artificial gastric juice with increasing time. The least stable was 111In-DTPA-HA, which degraded completely after 48 h in artificial gastric juice. Kinetic stability studies may provide primary information concerning the properties of radiolabeled HA in vitro, which is essential for the use and explanation of its behavior in biological experiments.
Abstract: In this study, the complexation rates of two new phosphinate H4dota (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) analogs, H5do3apPrA and H4do3apABn, and H4dota itself were compared under identical conditions (H5do3apPrA=10-{[(2-carboxyethyl)hydroxyphosphoryl]methyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid; H4do3apABn=10-{[(4-aminophenyl)hydroxyphosphoryl]methyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid). The biodistribution of their 111In and 90Y complexes in healthy rats was also studied. Unlike the observation obtained under âchemicalâ conditions where differences between the ligands were observed no such differences in complexation rates were found under radiochemical conditions. The ligands bind the radiometals similarly to H4dota. So, âchemicalâ formation kinetic data should be transferred into radiochemical conditions only with high care and âradiochemicalâ complexation experiments should be run as part of standard in vitro studies with new ligands considered as potential radiopharmaceuticals. Pharmacokinetic studies in rats showed similar distribution characteristics for both phosphinate H4dota analogs radiolabelled with 111In and 90Y when compared with that of the 111InâH4dota complex. No specific uptake in any organ and tissue of rats was determined. The phosphinate complexes are not accumulated in calcified tissues.
Abstract: Background: In this study, some important biological characteristics of two radiolabelled somatostatin analogues 111In-DOTA-1-Nal3-octreotide (DOTA-NOC) and 111In-DOTA-Tyr3-octreotate (DOTA-TATE) were compared. Materials and Methods: Rats were used for in vivo biodistribution experiments and in vitro cell models (OK and AR42J cell lines) were used for simulating the internalization in the kidney and in subtype 2 somatostatin receptor (SSTR2)-positive tissues, respectively. Results: Significantly higher radioactivity concentrations in rat organs with high density of somatostatin receptors after 111In-DOTA-NOC administration in comparison with 111In-DOTA-TATE were observed. The predominant urine excretion was associated with accumulation of the radioactivity in the kidney, where higher retention of 111In-DOTA-TATE compared to 111In-DOTA-NOC was detected. In the OK cell line the opposite results were found. No significant differences in the in vitro internalization and externalization of radioactivity to AR42J cell line were found for either peptide suggesting their same affinity for SSTR2. Conclusion: Preclinical experiments indicated that 111In-DOTA-NOC is a very promising peptide for somatostatin receptor-positive tumour visualization. The conflict between the in vitro and in vivo kidney handling showed that the transfer of results from in vitro to in vivo conditions and their interpretation should be performed very carefully because both types of experiments can be affected by different factors, making their simple comparison difficult.
Abstract: OBJECTIVE: Radiolabeled receptor-specific somatostatin analogs labeled with gamma- or beta-emitting radionuclides are useful for scintigraphic imaging and/or therapy of selected neuroendocrine tumors. However, significant renal uptake may result in radiotoxicological injury of the kidney and can limit clinical application of the agents. The aim of the study was to analyze renal handling, rate, and mechanism of renal accumulation of two somatostatin receptor-targeted peptides, [DOTA(0), Tyr(3), Thr(8)]-octreotide (DOTA-TATE) and [DOTA(0), 1-Nal(3)]-octreotide (DOTA-NOC), labeled with indium-111 using in vitro methods.
METHODS: The perfused rat kidney and freshly isolated rat renal cells were used as experimental models. The perfusion was performed in a recirculation regimen at constant pressure with solution containing bovine albumin, erythrocytes, and a mixture of essential substrates. The renal cells were isolated from rat kidneys using two-phase collagenase perfusion. Accumulation studies were used to evaluate the renal uptake of the peptides and to compare their accumulation with that of passively or actively transported model drugs. The influence of selected inhibitors of receptor-mediated endocytosis and the inhibition of energy-dependent transport processes on the uptake were also investigated using isolated renal cells.
RESULTS: The renal clearance of (111)In-DOTA-NOC in the perfused rat kidney was significantly lower than that of (111)In-DOTA-TATE. Reverse situation was found in the case of renal retention. Pretreatment of the perfused kidney with maleate markedly decreased the renal retention. (111)In-DOTA-NOC was accumulated in the isolated renal cells at a higher rate than (111)In-DOTA-TATE (ratio 3: 1). The uptake of the radiopeptides in renal cells was higher than the uptake of not only the passively transported sucrose but also actively transported and accumulated methylglucose. The rank order of potency to inhibit the uptake by active endocytosis was approximately aprotinin > maleate > lysine. The uptake of the radiopeptides in the renal cells was temperature dependent.
CONCLUSIONS: Both in vitro methods showed a higher renal accumulation of (111)In-DOTA-NOC in comparison with (111)In-DOTA-TATE. The renal uptake was partly decreased by inhibitors of receptor-mediated endocytosis and by a block of energy-dependent processes. A significant participation of active transport processes in renal accumulation of the studied peptides was confirmed.
Abstract: Background: Somatostatin analogues labelled with radiometals or radiohalogens are useful for the imaging and treatment of somatostatin receptor-containing tumours. In this study, the procedures for the radioiodination of glucose-Tyr3-octreotate (gluc-Tyr3-tate) and radiolabelling of DOTA-Tyr3-octreotate (DOTA-Tyr3-tate) with 111In, 177Lu and 125I were compared and their metabolism in rats was analyzed. The usefulness of high performance liquid chromatography (HPLC) analysis and instant thin-layer chromatography on silica gel (ITLC-SG) for both radiochemical purity determination and analysis of metabolism in urine was investigated. Materials and Methods: For labelling with radiometals, the formation of a complex with the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) functionality of the peptide was employed. Radioiodination was performed by the chloramime-T method. The radiochemical purity of radiolabelled peptides and the analyses of rat urine were determined by HPLC and/or ITLC-SG methods. Male Wistar rats were used in the elimination studies. Results: DOTA-Tyr3-tate was simply radiolabelled with radiometals with high yield and high radiochemical purity. Stopping of the reaction was a critical step for radioiodination, therefore labelling of gluc-Tyr3-tate and DOTA-Tyr3-tate with 125I was not so simple and the reaction product had to be purified by preparative HPLC analysis. Whereas 111In-DOTA-Tyr3-tate and 177Lu-DOTA-Tyr3-tate were eliminated in rat urine in a practically unchanged form, a significant proportion of metabolites was observed with radioiodinated peptides, particularly at longer time intervals. Conclusion: Labelling of DOTA-Tyr3-tate with radiometals is simple and the radiochemical purity of prepared compounds is very high, while iodination of the peptides demands purification of the product by preparative HPLC. The analysis of rat urine showed that excretion of radioiodinated peptides included a significant proportion of metabolites.
Abstract: Background: Due to their high CCK-2/gastrin receptor selectivity, high affinity, and rapid background clearance, radiolabeled minigastrins (MG) are emerging as promising new tools in the diagnosis and therapy of CCK-2/gastrin receptor-positive tumors. In this study, the pharmacokinetic profile, particularly the excretion mode, of two 111In-labeled minigastrins was compared in rats. The first tracer, 111In-MG-0 is based on (D)Glu1-MG, while the second, 111In-MG-11, is its des-(Glu)5-derivative, expected to be less retained in renal tissue. Materials and Methods: The fate of 111In-MG-0 and 111In-MG-11 in the body of rats was investigated during biodistribution and bioelimination experiments, while the respective elimination parameters were determined in perfused rat liver and kidney models. Results: During biodistribution both compounds were rapidly cleared from the blood and most non-target organs whereas activity levels in the bowel and stomach declined slowly. The overall contribution of hepatobiliary excretion of 111In-MG-0 and 111In-MG-11 was relatively small. In the perfused rat liver their elimination into the bile was negligible. In contrast, renal excretion was the major excretion pathway for both analogs, mainly via glomerular filtration. However, kidney levels were substantially higher and retention was more prolonged in the case of 111In-MG-0 as compared to 111In-MG-11. Conclusion: The presence of the (Glu)5-chain in 111In-MG-0 appears to be implicated in the prolonged radioactivity retention in the kidney of rats.
Abstract: The complex formation of the bifunctional monophosphinic acid DOTA analogue DOSAPÎÎn (1,4,7,10-tetraazacyclododecane-4,7,10-triacetic-l-{methyl[(4-aminophenyl)methyl] phosphinic acid}) with 111In at a tracer level was analyzed. Formation of a complex between 111In and DO3APABn was very rapid even at room temperature and high radiolabeling yields were achieved. As introducing the methylphosphinic acid arm to the DOTA structure generated a chiral centre, more than one peak (probably corresponding to various diastereoisomers) of 111In-DO3APABn were separated on HPLC. Four peaks were separated by HPLC; they probably correspond to four diastereoisomers of 111In-DO3APABn originating from combination of chirality of complexes of DOTA-like ligands with chirality of coordinated phosphorus atom. Studies in rats showed rapid elimination of radioactivity from the blood and other organs and tissues. The results indicate that DOSAPABn represents a promising ligand for radiolabeling of target-specific biomolecules with radiometals.
