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Ga-Based PET-Radiopharmaceuticals at Room
I. Velikyan,*,† H. Maecke,‡ and B. Langstrom†,§ Uppsala Applied Science Lab, GEMS PET Systems AB, GE Healthcare, SE-752 29 Uppsala, Sweden, Division of Radiological Chemistry, University Hospital, CH-4031 Basel, Switzerland, and Department of Biochemistry and Organic Chemistry, BMC, Uppsala University, SE-75124 Uppsala, Sweden. Received September 5, 2007; Revised Manuscript Received November 13, 2007
A straightforward labeling using generator produced positron emitting 68Ga, which provides high quality images, may result in kit type production of PET radiopharmaceuticals and make PET examinations possible also at centers lacking accelerators. The introduction of macrocyclic bifunctional chelators that would provide fast 68Gacomplexation at room temperature would simplify even further tracer preparation and open wide possibilities for 68 Ga-labeling of fragile and potent macromolecules. Gallium-68 has the potential to facilitate development of clinically practical PET and to promote PET technique for individualized medicine. The macrocyclic chelator, 1,4,7-triazacyclononanetriacetic acid (NOTA), and its derivative coupled to an eight amino acid residue peptide (NODAGA-TATE, [NODAGA0, Tyr3]Octreotate) were labeled with 68Ge/68Ga-generator produced positron emitting 68Ga. Formation kinetics of 68Ga-NOTA was studied as a function of pH and formation kinetics of 68 Ga-NODAGA-TATE was studied as a function of the bioconjugate concentration. The nearly quantitative radioactivity incorporation (RAI > 95%) for 68Ga-NOTA was achieved within less than 10 min at room temperature and pH 3.5. The concentrations of NODAGA-TATE required for RAI of >90% and >95% were, respectively, 2–5 and 10 µM. In both cases the purification of the 68Ga-labeled products was not necessary since the radiochemical purity was >95% and the preparation buffer, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) is suitable for human use. In order to confirm the identity of the products, complexes comprising natGa were synthesized and analyzed by mass spectrometry. The complex was found to be stable in the reaction mixture, phosphate buffer, and human plasma during 4.5 h incubation. Free and peptide conjugated NOTA formed stable complexes with 68Ga at room temperature within 10 min. This might be of special interest for the labeling of fragile and potent macromolecules and allow for kit type preparation of 68Ga-based radiopharmaceuticals.
INTRODUCTION 68
The long shelf life generator produced positron-emitting Ga (T1/2 ) 68 min) is of potential interest for clinical PET. It is readily available on demand and it decays by 89% through positron emission resulting in images with high resolution and potential accurate quantification. Its half-life of 68 min might be sufficient for production and application of tracers and it minimizes the radiation dose to the patient. It also allows repetitive examinations. The only stable chemical form in solution at physiological conditions is Ga (III) cation that can form stable complexes with chelators, naked or conjugated with macromolecules or small organic molecules. It is established that macrocyclic chelator, 1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA), either free or coupled to macromolecules forms very stable complexes with 68Ga. However, the complex formation with DOTA derivatives requires elevated temperature (1, 2). A fast, reliable method comprising preconcentration and purification of the 68Ge/68Ga generator eluate and microwave heating was developed for automated production of peptide based tracers with higher specific radioactivity (2–4). The introduction of macrocyclic chelators that would provide fast 68Ga-complexation at room temperature would simplify * Address for correspondence: Irina Velikyan, Uppsala Applied Science Lab, GEMS PET Systems AB, GE Healthcare, Husbyborg, SE-75229 Uppsala, Sweden, Phone: +46 18 180917, Fax: +46 18 180912, E-mail:
[email protected]. † GE Healthcare. ‡ University Hospital. § Uppsala University.
even further the tracer preparation and allow for kit type labeling analogous to one carried out with the SPECT isotope 99mTc in addition–offering the sensitivity and quantification advantages of the PET technique. The fast 68Ga-labeling reaction at room temperature might also become an attractive tool when producing fragile temperature sensitive macromolecular tracers. The recognized potential of PET technique and 68Ga-based radiopharmaceuticals for use in oncology, cardiology, neurology, and infection diseases strongly motivates the development of such a production procedure (5–10). The worldwide interest toward 68 Ga is increasing quickly and, for example, in Europe at least 40 centers have already introduced the generator. Moreover, the struggle for more efficient authorization of radiopharmaceuticals by legislative institutions is progressing steadily (11). The introduction of 68Ga-based radiopharmaceutical kit production that would not require cyclotron and qualified chemists would make the PET radiopharmaceuticals readily available and might accelerate the expansion of nuclear medicine and promote PET examinations for personalized medicine worldwide. Macrocyclic chelators that form very stable complexes with metal cations are of paramount interest for radiopharmaceutical design. Gallium complexes with NOTA (12–14) and its various derivatives, namely, 1,4,7-tris(2-mercaptoethyl)-1,4,7-triazacyclononane (TACN-TM) (15), 1,4,7-triazacyclononane-1-succinic acid-4,7-deacetic acid (NODASA) (16), 1,4,7-triazacyclononaneN,N′,N″-tris(methylenephosphonic) acid (NOTP) (17, 18), 1,4,7triazacyclononane-N,N′,N′′-tris(methylenephosphonate-monoethylester) (NOTPME) (17, 18) have demonstrated octahedral coordination geometry, high thermodynamic stability and similar plasma and in ViVo stability. It has been reported that the
10.1021/bc700341x CCC: $40.75 2008 American Chemical Society Published on Web 01/19/2008
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biodistribution and clearance pattern of 67Ga-NOTA phosphonate analogues depend on the overall charge of the complexes (17). The thermodynamic stability constant (log K) of Ga(III)DOTA (19) and Ga(III)-NOTA (12, 14) complexes was determined, respectively, to be 21.33 and 30.98. It is the compactness of the triazanonane ring and the steric efficiency of the pendant acetate groups that lead to the formation of complexes of unusually high stability and selectivity for the Ga(III) (14). However, it should be stressed that the high stability of macrocyclic complexes is provided by the extremely slow dissociation reactions. Typical dissociation constant values are 105 to 107 times lower than those of open-chain analogues. The hole-size effects influence both thermodynamics and kinetics of macrocyclic complexes. This is because a chelator in its minimum energy metal-binding conformation will be optimized for a particular size of a metal ion, and when other metal ions are bound, the chelator conformational energy will rise with a resultant decrease in stability of the complex. Thus, macrocycles are selective for metal ions (20–22). For a given ionic size the stability of the complex increases with increasing charge. Another factor which influences the complexation rate and the stability is the presence of pendant arms, which help to achieve the full coordination number of the metal ion. The abovementioned characteristics make NOTA very attractive for radiopharmaceutical development. In this work we investigated the potential of 68Ga in combination with NOTA for a simple kit type production of radiopharmaceuticals at room temperature.
EXPERIMENTAL PROCEDURES Materials. HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), sodium acetate, and double distilled hydrochloric acid (Riedel de Haën) were obtained from Sigma-Aldrich Sweden (Stockholm, Sweden). Sodium dihydrogen phosphate, disodium hydrogen phosphate, and trifluoroacetic acid (TFA) were obtained from Merck (Darmstadt, Germany). The purchased chemicals were used without further purification. Deionized water (18.2 MΩ), produced with a Purelab Maxima Elga system (Bucks, UK) was used in all reactions. 68 Ga Production. 68Ga (T1/2 ) 68 min, β+ ) 89%, and EC ) 11%) was available from a 68Ge/68Ga-generator-system (Cyclotron Co., Ltd., Obninsk, Russia) where 68Ge (T1/2 ) 270.8 d) was attached to a column of an inorganic matrix based on titanium dioxide. The 68Ga was eluted with 6 mL of 0.1 M hydrochloric acid. 68 Ga-Labeling of NOTA. HEPES (7–14 mg) or sodium acetate (16 mg) buffering agents was added to 200 µL of 68Ga and the pH was adjusted with HCl and NaOH to give pH values between 2 and 7. NOTA (50 nmol, synthesized at Grove Centre, GB) was added and the reaction mixture was incubated at room temperature. The reaction mixture was analyzed by thin layer chromatography (TLC) applying the analyte to a polyethyleneimine cellulose plate (PEI-Cellulose F, Merck, Germany) and using 0.4 M NaH2PO4 (pH ) 3.5) as running buffer. Autoradiography was employed to image the TLC strips. A phosphor storage plate (Molecular Dynamics, Amersham Biosciences, U.K.) was placed on top of the strips. The plate was scanned with PhosphorImager (PI) SI unit (Molecular Dynamics, Amersham Biosciences, U.K.) and analyzed using ImageQuant 5.1 software. The nonincorporated (free) 68Ga stayed at the origin and RF of the 68Ga-complex was 0.9. 68 Ga-Labeling of NODAGA-TATE. The pH of the 68Ge/ 68 Ga-generator eluate was adjusted to 3.5–5.0 by adding either HEPES to give finally a 1.0 M solution with regard to HEPES or sodium acetate to give finally a 0.4 M solution with regard to sodium acetate. Then 0.2–20 nmol of NODAGA-TATE (received as lyophilized powder from Helmut Maecke, Division of Radiological Chemistry, University Hospital, Basel, Switzerland, and dissolved in deionized water to give stock solutions
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of 0.1 mM and 1 mM concentration) were added and the reaction mixture was incubated at room temperature. The reaction mixture was analyzed on an HPLC system from Beckman (Fullerton, CA) consisting of a 126 pump, a 166 UV detector, and a radiation detector coupled in series. Data acquisition and handling was performed using the Beckman System Gold Nouveau Chromatography software package. The column used was a Vydac RP 300 Å HPLC column (Vydac, USA) with the dimensions 150 mm × 4.6 mm, 5 µm particle size. The applied gradient elution was as follows: A ) 10 mM TFA; B ) 70% acetonitrile (MeCN), 30% H2O, 10 mM TFA with UV-detection at 220 nm; flow was 1.2 mL/min; 0–2 min isocratic 20% B, 20–90% B linear gradient 8 min, 90–20% B linear gradient 2 min. LC-ESI-MS Analysis. Electrospray ionization mass spectrometry (ESI-MS) was performed using a Fisons Platform (Micromass, Manchester, UK) with positive mode scanning and detecting [M + 1H]1+, [M + 2H]2+, [M + 3H]3+, and [M + NH4]+ species. NOTA was detected at m/z ) 304 for [M + 1H]1+. The analysis of 69,71Ga-NOTA was performed by direct infusion. Two different systems, namely, formic acid and ammonium acetate, were used resulting in the detection of m/z ) 370.23, m/z ) 372.1 for [M + H]+ and m/z ) 387.16, m/z ) 389.26 for [M + NH4]+, respectively. NODAGA-TATE was detected at m/z ) 704 for [M + 2H]2+ and 470 for [M + 3H]3+, and 69,71Ga-NODAGA-TATE was detected at m/z ) 738 for [M + 2H]2+. The 69,71Ga-NOTA and 69,71Ga-NODAGA-TATE were synthesized under conditions indentical to radiolabeling and were used for the identification of the radio-HPLC chromatogram signals. The coupled HPLC system was from Alliance (Waters 2695, UK) with photodiode array UV detector. The column used was an Atlantis, dC 18, HPLC column with the dimensions 100 mm × 2.1 mm, 3 µm particle size. Isocratic elution was applied with the following parameters: A ) 10 mM formic acid; B ) 100% acetonitrile (MeCN), with UV-detection at 210–400 nm; flow was 0.3 mL/min, A/B ) 80/20. Stability in Human Plasma. The reaction mixture of 68GaNOTA (RAI > 95%, 50 µL) was added to the prewarmed (37 °C) plasma (500 µL) and incubated for 270 min. The samples (100 µL) were taken at 10, 30, 60, 120, and 270 min time points and acetonitrile (400 µL) was added to sediment the proteins present. Then the samples were centrifuged at 14 000 rpm for 10 min at +4 °C (Allegra X-22R Centrifuge, Beckman Coulter, USA). The supernatant was collected and filtered through a 0.2 µm nylon membrane at 13 200 rpm for 1 min at +4 °C. The radioactivity of the pellets, supernatant, and filters was measured in a well counter and ionization chamber. The data was used to calculate the extraction efficiency. The supernatant was analyzed by TLC applying the analyte to a polyethyleneimine cellulose plate (PEI-Cellulose F, Merck, Germany) and using 0.4 M NaH2PO4 (pH ) 3.5) as running buffer. Autoradiography was employed to image the TLC strips. Stability in Reaction Mixture and PBS. The stability of the 68Ga-NOTA and 68Ga-NODAGA-TATE complexes was monitored in PBS and reaction mixture. The samples (5–20 µL) were taken at 10, 30, 60, 120, and 240 min time points. 68GaNOTA was analyzed by PEI-cellulose F TLC followed by autoradiography imaging of the strips. 68Ga-NODAGA-TATE was analyzed by UV-radio-HPLC.
RESULTS AND DISCUSSION The general structure of NOTA derivative chelators is given in Figure 1 and consists of three macrocyclic amine groups and three carboxylic groups for coordination to Ga(III) and an additional functional group (Y1) such that the chelate can be conjugated to a vector, e.g., preferably alkylamine, alkylsulfide, alkoxy, alkyl carboxylate, arylamine, aryl sulfide, or R-halo-
Technical Notes
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Figure 1
Figure 2. Reaction scheme for the complexation of 68Ga(III) with NOTA (Y3dH) and NODAGA-TATE (Y3 ) CH2CH2C(O)R, where R ) Tyr3-octreotate).
acetyl; Y2 and Y3 can be H or contain one or more functional moieties that would on one hand improve the complexation depending on a particular metal cation and on the other hand change the overall charge and hydrophilicity of the complex in order to modify the pharmacokinetics and blood clearance rates, e.g., alkylamine, alkoxy, alkyl carboxylate, phenol, hydroxamate, aryl sulfide, and alkyl. In this particular work, 68Ga-labeling of 1,4,7-triazacyclononanetriacetic acid (NOTA) and its derivative coupled to an eight amino acid residue peptide (NODAGA-TATE, [NODAGA0, Tyr3] octreotate) was performed at room temperature considering the possibility for a simple kit type tracer production as well as preparation of tracers based on temperature sensitive fragile macromolecules (Figure 2). Formation kinetics of 68Ga-NOTA was studied during 1 h at room temperature as a function of pH values covering the range from 2 to 7. The aliquots of the reaction mixture sampled at 1, 3, 10, 30, and 60 min time points were analyzed by thin layer chromatography (TLC) both directly after the aspiration and later after the reaction was first quenched in water. The radioactivity incorporation at early time points was slightly higher in the case of the quenched samples, indicating most probably that the complexation reaction continued even after 200–1000-fold dilution of the reaction mixture. In addition, the reproducibility of the direct analysis was higher with relative standard deviation (RSD) values of ∼1% and ∼4%, respectively, for the direct and quenched cases. Thus the TLC analysis performed directly after the aspiration of the aliquots was preferred. The nonincorporated (free) 68Ga stayed at the origin and RF of the 68Ga-complex was 0.9. The time course of 68Ga complexation reaction with NOTA at room temperature for varied buffer and pH conditions is presented in Figure 3A. Radioactivity incorporation reached a plateau within 10 min independent of the pH. The radioactivity incorporation (RAI > 95%) for 68Ga-NOTA was achieved within less than 10 min at room temperature and pH 3.5 using HEPES buffer (Figure 3B). In order to confirm the identity of the product, the complex comprising stable gallium isotopes (69,71Ga) was synthesized and analyzed by mass spectrometry (ESI-MS). 69,71Ga-NOTA was synthesized under the same conditions as its radioactive counterpart, but using a mixture of 68Ga and 69,71Ga cations. The aim of the use of a mixture of radioactive and stable gallium
isotopes was twofold: (1) to create a reaction condition identical to the labeling procedure; (2) to allow following the reaction. The formed neutral complex was expected to be difficult to protonate in order to observe it by ESI-MS. However, it was possible to optimize the analysis conditions and confirm the formation of 69,71Ga-NOTA using two different systems, namely, formic acid and ammonium acetate and detecting respectively m/z ) 370.23, m/z ) 372.1 for [M + H]+ and m/z ) 387.16, m/z ) 389.26 for [M + NH4]+. The production of the complex was also indirectly verified by the consumption of NOTA and the drastic decrease of the corresponding signal at m/z ) 304 for [M + 1H]1+. The stability of the 68Ga-NOTA complex was monitored in PBS, reaction mixture, and human plasma and was found stable and in agreement with literature data (12–15, 18, 23). The complex extraction efficiency from the plasma was 65 ( 4% at 10, 30, 60, 120, and 270 min time points, and the only intact complex was detected after the incubation at 37 °C during 4.5 h. The choice of the time frame was dictated by the half-life of 68Ga and consequent useful tracer application time (4 × T1/2). Formation kinetics of 68Ga-NODAGA-TATE was studied for varied concentration of the bioconjugate. The graph (Figure 4) presents the time course of 68Ga complexation reaction with NOTA conjugated peptide at room temperature in sodium acetate buffer for varied concentration of the bioconjugate. Radioactivity incorporation reached a plateau within 10 min. The nearly quantitative radioactivity incorporation (>95%) required 10 µM and higher concentration of the bioconjugate. This concentration exceeded 4× the one required when using microwave heating (2). The formation of 68Ga-NODAGA-TATE was monitored by HPLC with UV- and Radio-detection (Figure 5). The authentic reference substance (69,71Ga-NODAGA-TATE) was synthesized under the same conditions as its radioactive counterpart, but using a mixture of 68Ga and 69,71Ga cations for the same reason as described above for 69,71Ga-NOTA. The identity of the authentic reference substance (69,71GaNODAGA-TATE) was confirmed by LC-ESI-MS detecting m/z ) 704 for [M + 2H]2+, m/z ) 470 for [M + 3H]3+ of NODAGA-TATE, and m/z ) 738 for [M + 2H]2+ of 69,71GaNODAGA-TATE. The stability of the 68Ga-NODAGA-TATE was monitored in the reaction mixture and PBS. The UV and radio signals corresponded to the tracer and no additional signals were detected indicating the stability of the molecule. In both cases of 68Ga-NOTA and 68Ga-NODAGA-TATE the purification of the product was not necessary since the radiochemical purity was >95% and the preparation buffer, 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) is suitable for intravenous injections. For example, HEPES is currently used as an ingredient of indium(In-111)oxin GE Healthcare radiopharmaceutical (manufacturer, GE Healthcare Limited; registration number, 800820; approval date, 1990–06–21; Medical Products Agency, Sweden). Previously, the high similarity of the geometry of gallium complexes with NOTA and NODASA independent of the pendant arm modification has been demonstrated by X-ray
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Figure 3. (A) Time course of 68Ga complexation reaction with NOTA at room temperature and varied pH conditions (N ) 2–3, the error bars are omitted for simplicity). (B) Radioactivity incorporation after 10 min incubation at room temperature as a function of pH.
Figure 4. (A) Time course of 68Ga complexation reaction at room temperature for varied concentration of NODAGA-TATE (N ) 2–3, the error bars are omitted for simplicity). (B) Radioactivity incorporation as a function of the bioconjugate concentration (10 min incubation, RT).
Figure 5. A typical example of an UV- and radio-HPLC chromatograms, respectively, for 69,71Ga-NODAGA-TATE and 68Ga-NODAGA-TATE. The reaction mixture was spiked with the stable reference. (The UV-signals at Rt of 12.66 and 13.33 are systematic and not related to the analyte.)
crystallography (12, 13, 16). In this study the complexation yields and conditions were similar for NOTA and NODAGATATE indicating that most probably the labeling is affected only by the macrocyclic chelator and consequently the latter might be coupled to a number of biologically active vectors resulting in a simple production of wide range of PET radiopharmaceuticals. The fast and nearly quantitative labeling results in higher specific radioactivity which might be crucial for the accurate quantification of PET examinations as well as in the application of expensive and highly potent macromolecular tracers. The labeling at room temperature allows the production of tracers comprising temperature sensitive and fragile macromolecules like antibodies. The nearly quantitative radioactivity incorpora-
tion at room temperature and reaction solution constitution compatible with human use promise a very simple kit type production.
CONCLUSIONS The complexation of 68Ga with NODAGA-TATE and NOTA was observed at RT and pH 3.5 within 10 min with the yield of >95%. The product purification was not necessary since the incorporation was nearly quantitative and the HEPES buffer is suitable for human use. Formation of 68Ga-NOTA and 68GaNODAGA-TATE was confirmed by the synthesis and subsequent ESI-LC-MS analysis of the counterparts comprising stable gallium isotopes.
Technical Notes 68
Ga-NOTA was confirmed to be stable in human plasma at 37 °C during 4.5 h. The suggested 68Ga and NOTA based labeling procedure might be of special interest for the labeling of fragile and potent macromolecules and might allow for a simple kit type preparation of 68Ga-based radiopharmaceuticals.
ACKNOWLEDGMENT Professor Vaughan Griffiths and Dr. Philip Duncanson (Queen Mary, University of London) and Dr. Peter Iveson (GE Healthcare) are acknowledged for the provision with NOTA and Medical Diagnostics, GE Healthcare, for partial financial support.
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