The authors have no acknowledgements.

1. Preparation of EOB-DTPA and Ga[EOB-DTPA]
Caution: Please consult all relevant material safety data sheets (MSDS) of the used organic solvents, acids and alkalines before use. Perform all steps in a fume hood and use personal protective equipment (safety glasses, gloves, lab coat).
<ol>
	<li>Isolation of EOB-DTPA from gadoxetic acid
	<ol>
		<li>Put 3 ml of 0.25 M gadoxetic acid injectable solution into a flask. Add 500 mg (5.6 mmol) of oxalic acid to the stirred solution.</li>
		<li>After stirring for 1 hr, filter the suspension through a frit using reduced pressure. Wash the residue three times with 3 ml of water, respectively.</li>
		<li>Combine the aqueous filtrates and equip the solution with a pH electrode. Add 12 M hydrochloric acid to the filtrate until the pH is about -0.1.</li>
		<li>Remove the solvent in vacuo to yield a colorless residue. Store under inert gas.</li>
		<li>Wash the residue thoroughly (at least three times) with ethyl acetate to remove the excess of oxalic acid. Dry the residue in vacuo.</li>
		<li>Redissolve the residue in 2 ml of water at room temperature and then cool the solution in an ice bath. Without removing the ice bath, add 0.5 M aqueous sodium hydroxide solution dropwise until the formation of a colorless gluey solid is observed.</li>
		<li>Remove the water by decantation. Wash the solid two more times with 1 ml of cold water. Dry the solid in vacuo to yield the first product fraction.</li>
		<li>Isolate a second product fraction from the combined fractions of decanted water via column chromatography (silica, methanol/water 4/1).24 Remove the solvent in vacuo.</li>
		<li>If the thus obtained solid is not pure white, redissolve it in 1 ml of water, add 10 ml of ethanol and subsequently 10 ml of diethyl ether to precipitate the product. Filter through a frit using reduced pressure and dry in vacuo.</li>
		<li>Combine both solid fractions of EOB-DTPA and perform NMR spectroscopic,25 mass spectrometric26 and elemental27 analyses.</li>
	</ol>
	</li>
	<li>Synthesis of Ga[EOB-DTPA] 
	CAUTION: Store solid Ga(III) chloride under a dry inert atmosphere, since upon contact with air, moisture or grease decomposition takes place, resulting in corrosive fumes and formation of yellow, brown or black impurities.
	<ol>
		<li>Prepare a 0.11 M stock solution by dissolving 1.94 g (11.0 mmol) of Ga(III) chloride in 100 ml of water. Dilute 1 ml of 25% aqueous ammonia solution with 4 ml of water.</li>
		<li>Dissolve 80 mg (0.15 mmol) of EOB-DTPA in a flask in 10 ml of water. If necessary, heat the solvent to achieve complete dissolution.</li>
		<li>Add 1.4 ml (0.15 mmol) of the Ga(III) chloride stock solution. Equip the flask with a stirrer and pH-electrode. Add diluted aqueous ammonia solution dropwise until the pH of the solution is approximately 4.1. Stir at room temperature for 30 min.</li>
		<li>Remove the solvent in vacuo. Place the residue in a flask, equipped with a stillhead with a central and parallel side neck. Equip the central neck with a cooling finger and the side neck with a vacuum pump outlet</li>
		<li>Heat the residue under reduced pressure (125 &#176;C, 0.6 mbar). Periodically remove sublimated ammonium chloride (visible as white coating of the glass surface) from the cooling finger and still head, as well as from the upper parts of the flask with a slightly wet cloth. Continue the process until there is no visible formation of new sublimate.</li>
		<li>To remove final traces of ammonium chloride wash the residue three times with 0.5 ml of hot methanol, respectively. Dry the colorless residue in vacuo. Perform NMR spectroscopic,25 mass spectrometric26 and elemental27 analyses.</li>
	</ol>
	</li>
</ol>
2. General Labeling Procedure
CAUTION: All experiments including direct or indirect contact with radioactive substances must be undertaken by trained personnel only. Please use appropriate shielding equipment. Collect any radioactive waste separately and store and dispose in accordance with valid regulations.
<ol>
	<li>Elution of the generator 
	Note: A 40 mCi 68Ge/68Ga generator with the mother nuclide bound as oxide on dodecyl-3,4,5-trihydroxybenzoate silica was used. Elution and purification may be performed manually or, as was the case in this procedure, as a combined automated process using a peristaltic pump and dispenser unit.
	<ol>
		<li>Prepare solutions of 5.5 M, 1.0 M and 0.05 M hydrochloric acid. Prepare a solution of 5.0 M sodium chloride containing 25 &#181;l of 5.5 M hydrochloric acid per ml. Prepare a buffer solution of pH 4.6 by combining 4.1 g sodium acetate, 1 ml HCl (30%) and 2.5 ml glacial acetic acid and diluting the mixture with water to 50 ml.</li>
		<li>Precondition the PS-H+ cartridge by flushing it slowly with 1 ml of 1.0 M hydrochloric acid and subsequently 5 ml of water.</li>
		<li>Elute the silica column of the generator with 4 ml 0.05 M HCl.12 Load the 68Ga eluate onto the PS-H+ cartridge.</li>
		<li>Flush the cartridge with 5 ml of water and subsequently dry it with 5 ml of air. Elute the 68Ga from the cartridge with 1 ml 5.0 M acidified sodium chloride solution.28</li>
	</ol>
	</li>
	<li>Labeling of EOB-DTPA with 68Ga
	<ol>
		<li>Dissolve 1 mg (1.9 &#181;mol) of EOB-DTPA in 1 ml of water. From this solution take 100 &#181;l (0.19 &#181;mol) and dilute them with 9.9 ml of water to prepare a 19 &#181;M (10 &#181;g/ml) stock solution of EOB-DTPA.</li>
		<li>Remove 50 &#181;l (equaling 22-29 MBq) of the solution containing 68Ga and put into a vial. Add 50 &#181;l (0.5 &#181;g) of a 19 mM stock solution of EOB-DTPA and 300 &#181;l of buffer to raise the pH to 4.0. Shake briefly and incubate the solution at room temperature for 5 min. Remove an aliquot of 1-5 &#181;l and put to HPLC or TLC analysis.</li>
		<li>Perform radio HPLC analysis on a reversed phase (RP) C18 column.29 Use the following mobile phase: A - water/trifluoroacetic acid (99.9%/0.1%), B - acetonitrile/trifluoroacetic acid (99.9%/0.1%), gradient: 06 min 80% A &#8594; 0% A (0.5 ml/min), 610 min 0% A (0.5 ml/min).</li>
		<li>Determine the peak intensities of the radio HPLC signals as area under curve. Calculate the labeling yield as radiochemical purity (RCP) of the tracer as follows: 
		RCP = AGa-EOB-DTPA/(AGa + AGa-EOB-DTPA) &#8729; 100% 
		AGa-EOB-DTPA: area under curve of 68Ga[EOB-DTPA] 
		AGa: area under curve of free 68Ga</li>
	</ol>
	</li>
</ol>
3. Labeling Efficiency
<ol>
	<li>Perform labeling procedures as described in section 2. Use a consistent range of starting activity of 68Ga eluate, e.g., 22-29 MBq (40-140 &#181;l, depending on the freshness of the eluate).</li>
	<li>Add the required amount of buffer solution to adjust the pH to 3.8-4.0 (40-190 &#181;l, depending on the volume of 68Ga eluate). Add the required amount of ligand stock solution (10-70 &#181;l of a 19 mM solution).</li>
	<li>Add the required amounts of water to adjust the overall volume of each labeling probe to 1.75 ml. Mix thoroughly and let the sample stand for 5 min at room temperature. Perform HPLC analysis as described in section 2 to determine the labeling yield.</li>
	<li>Perform labeling procedures with amounts of ligand between 0.1 &#181;g and 0.7 &#181;g in steps of 0.1 &#181;g. Perform experiments in triplicates for each ligand concentration. Calculate the mean yield and standard deviation.</li>
</ol>
4. In Vitro Stability
<ol>
	<li>General procedure and preparations
	<ol>
		<li>Dissolve a tablet of phosphate buffered saline (PBS) in 200 ml of deionized water to prepare a PBS stock solution with a phosphate concentration of 10 mM.</li>
		<li>Perform labeling of 22-29 MBq 68Ga with 0.5 &#181;l of EOB-DTPA stock solution, as described in section 2. Depending on the volume of the 68Ga eluate, adjust the amount of buffer, as described in section 3. Withdraw samples of labeling solution containing 6-12 MBq of tracer to perform stability measurements.</li>
		<li>Perform radio TLC analysis on 80 mm silica gel coated aluminum plates using 0.1 M aqueous sodium citrate as eluent and analyze the plates with a TLC radioactivity scanner.30 Determine the intensities of the TLC signals as area under curve. Calculate the RCP of the tracer as follows: 
		RCP = AGa-EOB-DTPA/(AGa-free + AGa-EOB-DTPA + AGa-colloidal) &#8729; 100% 
		AGa-EOB-DTPA: area under curve of 68Ga[EOB-DTPA] 
		AGa-free: area under curve of free 68Ga 
		AGa-colloidal: area under curve of colloidal 68Ga</li>
		<li>Calculate RCPt/RCP0 for every time point. Plot the thus standardized RCP vs. time difference since the starting point t = 0 min. 
		RCPt = RCP of 68Ga[EOB-DTPA] at time point t. 
		RCP0 = RCP of 68Ga[EOB-DTPA] at t = 0 min.</li>
	</ol>
	</li>
	<li>Stability in phosphate buffered saline (A)
	<ol>
		<li>To 65 &#181;l of labeling solution add 150 &#181;l of PBS stock solution and 60 &#181;l of sodium hydroxide solution (0.1 M) to raise the pH to 7.4. Mix thoroughly.</li>
		<li>Remove an aliquot of 1-5 &#181;l to perform TLC analysis (&#39;starting point&#39;). Immediately store the solution in an incubator at 37 &#176;C and remove aliquots to perform TLC analysis at representative time points over 3 hr.</li>
	</ol>
	</li>
	<li>Stability towards excess of apo-transferrin in PBS (B)
	<ol>
		<li>To 120 &#181;l of labeling solution add 50 &#181;l of PBS stock solution and 430 &#181;l of sodium hydroxide solution (0.1 M) to raise the pH to 7.4. Add 40 &#181;l of a solution of apo-transferrin (25 mg/ml). Mix thoroughly.</li>
		<li>Remove an aliquot of 1-5 &#181;l to perform TLC analysis (&#39;starting point&#39;). Immediately store the solution in an incubator at 37 &#176;C and remove aliquots to perform TLC analysis at representative time points over 3 hr.</li>
	</ol>
	</li>
	<li>Stability in human serum (C)
	<ol>
		<li>To 500 &#181;l of human serum add 25 &#181;l of labeling solution and 45 &#181;l of sodium hydroxide solution (0.1 M) to raise the pH to 7.4. Mix thoroughly.</li>
		<li>Remove an aliquot of 1-5 &#181;l to perform TLC analysis (&#39;starting point&#39;). Immediately store the solution in an incubator at 37 &#176;C and remove aliquots to perform TLC analysis at representative time points over 3 hr.</li>
	</ol>
	</li>
</ol>
5. Determination of Distribution Coefficient LogD
<ol>
	<li>Perform labeling procedures as described in section 2. To 50 &#181;l of labeling solution add 20 &#181;l of PBS stock solution and 170 &#181;l of sodium hydroxide solution (0.1 M) to raise the pH to 7.4.</li>
	<li>Withdraw 200 &#181;l from that solution and put it into a plastic V-vial. Add 200 &#181;l of n-octanol. Close the vial and vortex for 2 min. Then centrifuge the sample at 1,600 x g for 5 min.</li>
	<li>Remove triplicates of 40 &#181;l from the n-octanol phase and the aqueous phase each and put them in separate V-vials. Be careful not to mix up the layers.</li>
	<li>Measure the activity of each sample in a gamma well counter for 30 sec. For each sample immediately repeat the measurement twice and thereof calculate the mean activity &#8121;t in counts per minute (cpm). List the thus gained &#8121;t,W1, &#8121;t,W2 and &#8121;t,W3 (activities in aqueous samples) and &#8121;t,O1, &#8121;t,O2, &#8121;t,O3 (activities in n-octanol) along with the respective time point t of their determination.</li>
	<li>Define the time point of the measurement of the last sample as t0. Determine and list &#916;t in min by calculating &#916;t = t-t0. Perform decay correction of &#8121;t, using the following formula: 
	&#8121;0 = &#8121;t &#183; 2(&#916;t/68 min).</li>
	<li>Calculate &#8121;0,W as the mean of &#8121;0,W1, &#8121;0,W2 and &#8121;0,W3 as well as &#8121;0,O as the mean of &#8121;0,O1, &#8121;0,O2 and &#8121;0,O3. Calculate logD using the following formula: 
	logD = log[(&#8121;0,O &#183; 40 &#181;g)/(&#8121;0,W &#183; 33 &#181;g)].</li>
	<li>Perform the entire experiment in triplicates and calculate the mean logD along with its standard deviation.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone-seeking+TRAP+conjugates:+surprising+observations+and+their+implications+on+the+development+of+gallium-68-labeled+bisphosphonates.">Bone-seeking TRAP conjugates: surprising observations and their implications on the development of gallium-68-labeled bisphosphonates.</a> EJNMMI Res. 2, 13 (2012).</li><li>Schmitt-Willich, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+Physicochemical+Characterization+of+a+New+Gadolinium+Chelate:+The+Liver-Specific+Magnetic+Resonance+Imaging+Contrast+Agent+Gd-EOB-DTPA.">Synthesis and Physicochemical Characterization of a New Gadolinium Chelate: The Liver-Specific Magnetic Resonance Imaging Contrast Agent Gd-EOB-DTPA.</a> Inorg. Chem. 38, 1134-1144 (1999).</li><li>Zhernosekov, K., Nikula, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=68Ga+generator+for+positron+emission+tomography.">68Ga generator for positron emission tomography.</a> , (2012).</li><li>Simecek, J., Hermann, P., Wester, H. J., Notni, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+is+68Ga+Labeling+of+Macrocyclic+Chelators+Influenced+by+Metal+Ion+Contaminants+in+68Ge/68Ga+Generator+Eluates?.">How is 68Ga Labeling of Macrocyclic Chelators Influenced by Metal Ion Contaminants in 68Ge/68Ga Generator Eluates?.</a> ChemMedChem. 8, 95-103 (2013).</li><li>Baur, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+Radiolabelling+and+In+Vitro+Characterization+of+the+Gallium-68-,+Yttrium-90-+and+Lutetium-177-Labelled+PSMA+Ligand,+CHX-A''-DTPA-DUPA-Pep.">Synthesis, Radiolabelling and In Vitro Characterization of the Gallium-68-, Yttrium-90- and Lutetium-177-Labelled PSMA Ligand, CHX-A''-DTPA-DUPA-Pep.</a> Pharmaceuticals (Basel). 7, 517-529 (2014).</li><li>Boros, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGD+conjugates+of+the+H2dedpa+scaffold:+synthesis,+labeling+and+imaging+with+68Ga.">RGD conjugates of the H2dedpa scaffold: synthesis, labeling and imaging with 68Ga.</a> Nucl. Med. Biol. 39, 785-794 (2012).</li><li>Beck, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hematology. , (1998).</li><li>Patel, V., Morrissey, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical and Professional Clinical Skills. , (2001).</li><li>Bartke, A., Constanti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic Endocrinology. , (1998).</li><li>Bernstein, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Therapeutic+Activity+for+Gallium.">Mechanisms of Therapeutic Activity for Gallium.</a> Pharmacol. Rev. 50, 665-682 (1998).</li><li>Clausen, J., Edeling, C. J., Fogh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=67Ga+Binding+to+Human+Serum+Proteins+and+Tumor+Components.">67Ga Binding to Human Serum Proteins and Tumor Components.</a> Cancer Res. 34, 1931-1937 (1974).</li><li>Dumont, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+64Cu-+and+68Ga-Labeled+RGD+conjugates+show+improved+PET+imaging+of+&#945;v&#946;3+integrin+expression+and+facile+radiosynthesis+[Erratum+to+document+cited+in+CA156:116856.">Novel 64Cu- and 68Ga-Labeled RGD conjugates show improved PET imaging of &#945;v&#946;3 integrin expression and facile radiosynthesis [Erratum to document cited in CA156:116856.</a> J. Nucl. Med. 52, 1498 (2011).</li><li>Pohle, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=68Ga-NODAGA-RGD+is+a+suitable+substitute+for+18F-Galacto-RGD+and+can+be+produced+with+high+specific+activity+in+a+cGMP/GRP+compliant+automated+process.">68Ga-NODAGA-RGD is a suitable substitute for 18F-Galacto-RGD and can be produced with high specific activity in a cGMP/GRP compliant automated process.</a> Nucl. Med. Biol. 39, 777-784 (2012).</li><li>Notni, J., Pohle, K., Wester, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Be+spoilt+for+choice+with+radiolabelled+RGD+peptides:+Preclinical+evaluation+of+68+Ga-TRAP(RGD)3.">Be spoilt for choice with radiolabelled RGD peptides: Preclinical evaluation of 68 Ga-TRAP(RGD)3.</a> Nucl. Med. Biol. 40, 33-41 (2013).</li></ol>

The authors have nothing to disclose.

EOB-DTPA is accessible through a multi-step synthesis33 but may just as well be isolated from available contrast agents containing gadoxetic acid. For this purpose, the central Gd(III) ion can be precipitated with an excess of oxalic acid. After removing Gd(III) oxalate and oxalic acid the ligand can be isolated by precipitation in cold water at pH 1.5. However, in order to enhance yields column chromatography of the filtrate can be performed instead or as a follow-up procedure. Either method yields the analytically pure ligand in total yields of 70% (Figures 1-3, Table 1).
We found that in order to isolate Ga[EOB-DTPA] adjusting the pH with ammonia solution is advantageous compared to the use of sodium hydroxide, since the by-product ammonium chloride may be removed from the very hydrophilic residue via sublimation. Under the aforementioned conditions this process takes place slowly. Since non-negligible amounts of chloride were still detectable after five days, the remaining salt was washed out with methanol. Although this work-up procedure results in partial loss of Ga[EOB-DTPA], the product was obtained in analytical purity with an overall yield of 46% (Figures 4-6, Table 1). For the isolation of both EOB-DTPA and its Ga(III) complex, the use of reversed phase chromatography should be considered as an alternative method of purification, especially since decomposition of silica gel is likely when using highly polar solvents.
The labeling process of EOB-DTPA required the use of highly pure solvents, chemicals and metal-free equipment to avoid the presence of competing metal ions, due to 68Ga being present in nanomolar amounts (2 MBq of 68Ga in a 1.75 ml sample equal a nuclide concentration of 0.14 nM). Labeling of EOB-DTPA to 68Ga occurs at pH 3.8-4.0 within five minutes at room temperature. Investigations on the 68Ga-labeling efficiency require determination of the labeling yield while keeping the reaction conditions pH, temperature and reaction time as well as starting activity of 68Ga constant or in a justifiable range. For each data point (i.e., ligand concentration) the experiment should be performed at least three times to provide a reasonable confidence level, since the concentrations of both ligand and 68Ga are very low and the labeling yield therefore sensitive to even slight deviations of the reaction conditions. For example, as the 68Ga eluate ages, aliquots of increasing volume need to be withdrawn to provide a constant starting activity, thereby requiring increasing volumes of buffer. Furthermore, aging of the eluate results in increasing concentrations of the decay product 68Zn, which itself might act as a competitor for 68Ga, thus negatively affecting labeling efficiency.13,34,35 Practically quantitative labeling of 22-29 MBq 68Ga is achieved under the aforementioned conditions with amounts of EOB-DTPA &#8805; 0.7 &#181;g (Figure 9), with contents of free 68Ga &#8804; 2% and about 5% of colloidal 68Ga present in samples.
While HPLC provided superior baseline separation of free 68Ga and 68Ga[EOB-DTPA], it is not suited to detect colloidal 68Ga. We therefore chose TLC to determine the RCP during stability measurements, wherein the quantification of transferrin or protein-bound 68Ga was required. We found baseline separation acceptable for this purpose (Figure 10); however, the use of size exclusion chromatography or filtration methods15,36 to remove colloidal fractions, followed by HPLC analysis, might be considered as alternatives. The 68Ga complex exhibits a stronger retention on TLC plates (Rf = 0.3) if the sample is withdrawn directly from labeling solution as opposed to samples at physiological pH (Rf = 0.5). We suggest this observation might be explained by different protonation states of the complex.
In vitro stability determinations of 68Ga tracers are usually performed in PBS15,17 or alternative buffer systems mimicking the physiological pH37, as well as in solutions containing apo-transferrin37, which is the main competitor for 68Ga in blood, or in human serum15,17. In our experiments the addition of 0.1 M sodium hydroxide solution to PBS was required to adjust the pH of the samples to 7.4. We could not assert that the phosphate concentration influences the rate of degradation, since stability experiments in solutions of varying phosphate concentration (0.8 mM and 5.5 mM (A)) yielded non-reproducible results. However, we found that a solution B, containing apo-transferrin (1.6 mg/ml, which is within range of the normal plasma content38) and 0.8 mM phosphate (human blood usually exhibits a phosphate level of 0.8&#8211;1.5 mM39,40), causes decomposition at a rate comparable to that observable in human serum (C). In solutions AC, after 185 min the content of colloidal 68Ga had increased by about 24%, while the content of free 68Ga had increased by 11% in solution A, 17% in solution B and 27% in solution C (Table 2). The fact that 68Ga formed by tracer decomposition is predominantly present as free 68Ga as opposed to colloidal or protein-bound 68Ga in B and C might be due to transferrin saturation or comparably slow transferrin binding rates.41&#160; The observed overall low stability (Figure 11) of 68Ga[EOB-DTPA] is comparable to tracers featuring similar DTPA derived chelators.15,16,18 Usually, information on the early arterial and venous perfusion phase of the liver are gained by performing MRI scans within the first 3 minutes4,21 after administering Gd[EOB-DTPA], while the hepatocyte presence is detected in the delayed phase 20 minutes3,4,23 up to several hours21,22 after injection. After 20 minutes in human serum 93% of 68Ga[EOB-DTPA] remain intact. Expectedly, the signal-to-noise ratio by that time would have deteriorated due to increasing amounts of 68Ga-transferrin, which is present in plasma and tissue expressing transferrin receptors, as well as free 68Ga gallate.41,42
For predicting a tracers tissue distribution n-octanol/water partition coefficients logP or distribution coefficients logD can be determined as ratio of activity concentrations in the two phases. By definition, the logD parameter does not differentiate between multiple species present in a medium, which makes it suitable for our experiments due to the possibility of different protonation states of the tracer as well as its decomposition in the aqueous phase. To determine logD by extraction the aqueous medium is usually buffered with PBS to mimic blood conditions.17,43-45 For aforementioned reasons we used diluted PBS, exhibiting a phosphate concentration of 0.8 mM and physiological pH. Following extraction with n-octanol and centrifugation, the removal of several aliquots from the same phase allows for inaccuracies caused by pipetting to be reduced. Due to the very low activity concentrations in n-octanol one should be careful to avoid cross-contamination with the aqueous phase and to ensure quantitative transfer into a separate vial. Distribution coefficients determined by this procedure were reproducible, and while they allow for a rough estimation of lipophilicity, a direct comparison to a logP of Gd[EOB-DTPA] is not possible. Due to the specificity of Gd[EOB-DTPA] resulting not primarily from lipophilicity but rather its hepatobiliary uptake additional experiments in living subjects or cells would be necessary to provide more extensive information on the biodistribution as well as stability in vivo of 68Ga[EOB-DTPA]. Altogether, an application as imaging agent for the perfusion and early hepatobiliary phase is imaginable.

Gadoxetic acid, a common name for the Gd(III) complex of the ligand EOB-DTPA1, is a frequently used contrast agent in hepatobiliary magnetic resonance imaging (MRI).2,3 Due to its specific uptake by liver hepatocytes and high percentage of hepatobiliary excretion it enables the localization of focal lesions and hepatic tumors.2-5 However, certain limitations of the MRI technique (e.g., toxicity of the contrast agents, limited applicability in patients with claustrophobia or metal implants) call for an alternative diagnostic tool.
Positron emission tomography (PET) is a molecular imaging method, wherein a small amount of a radioactive substance (tracer) is administered, upon which its distribution in the body is recorded by a PET scanner.6 PET is a dynamic method that allows for high spatial and temporal resolution of images as well as quantification of the results, without having to deal with the side-effects of MRI contrast agents. The informative value of the obtained metabolic information can be further increased by combination with anatomical data received from additional imaging methods, as most commonly achieved by hybrid imaging with computed tomography (CT) in PET/CT scanners.
The chemical structure of a tracer suitable for PET must include a radioactive isotope serving as positron emitter. Positrons have a short life-span since they almost immediately annihilate with electrons of the atom shells of surrounding tissue. By annihilation two 511 keV gamma photons with opposite direction of movement are emitted, which are recorded by the PET scanner.7,8 To form a tracer, PET nuclides may be bound covalently to a molecule, as is the case in 2-deoxy-2-[18F]fluoroglucose (FDG), the most extensively used PET tracer.7 However, a nuclide may also form coordinative bonds to one or several ligands (e.g., [68Ga]-DOTATOC9,10) or be applied as dissolved inorganic salts (e.g., [18F] sodium fluoride11). Altogether, the structure of the tracer is crucial as it determines its biodistribution, metabolism and excretion behavior.
A suitable PET nuclide should combine favorable characteristics like convenient positron energy and availability as well as a half-life adequate for the intended investigation. The 68Ga nuclide has become an essential force in the field of PET over the last two decades.12,13 This is mainly due to its availability through a generator system, which allows on-site labeling independently from the vicinity of a cyclotron. In a generator, the mother nuclide 68Ge is absorbed on a column from which the daughter nuclide 68Ga is eluted and subsequently labeled to a suitable chelator.6,14 Since the 68Ga nuclide exists as a trivalent cation just like Gd(III)10,13, chelating EOB-DTPA with 68Ga instead would yield a complex with the same overall negative charge as gadoxetic acid. Accordingly, that 68Ga tracer might combine a similar characteristic liver specificity with the suitability for PET imaging. Although gadoxetic acid is purchased and administered as disodium salt, in the following context we will refer to it as Gd[EOB-DTPA] and to the non-radioactive Ga(III) complex as Ga[EOB-DTPA], or 68Ga[EOB-DTPA] in case of the radiolabeled component for the sake of convenience.
To evaluate their applicability as tracers for PET, radioactive metal complexes need to be examined extensively in in vitro, in vivo or ex vivo experiments first. To determine the suitability for a respective medical problem, various tracer characteristics like biodistribution behavior and clearance profile, stability, organ specificity and cell or tissue uptake need to be investigated. Due to their non-invasive character, in vitro determinations are often performed prior to in vivo experiments. It is generally acknowledged that DTPA and its derivatives are of limited suitability as chelators for 68Ga due to these complexes lacking kinetic inertness, resulting in comparably fast decomposition when administered in vivo.14-20 This is primarily caused by apo-transferrin acting as a competitor for 68Ga in plasma. Nevertheless, we investigated this new tracer concerning its possible application in hepatobiliary imaging, wherein diagnostic information may be provided within minutes post-injection3,4,21-23, thereby not necessarily requiring long-term tracer stability. For this purpose we isolated EOB-DTPA from gadoxetic acid and initially performed the complexation with natural Ga(III), which exists as mixture of two stable isotopes, 69Ga and 71Ga. The complex thus obtained served as non-radioactive standard for the following chelation of 68Ga. We used established methods and simultaneously evaluated their suitability for determining the 68Galabeling efficiency of EOB-DTPA and to investigate the lipophilicity of the new 68Ga tracer and its stability in different media.

<table><tbody><tr><td>primovist</td><td>Bayer</td><td>-</td><td>0.25 M</td></tr><tr><td>gallium(III) chloride</td><td>Sigma-Aldrich Co.</td><td>450898</td><td></td></tr><tr><td>water (deionized)</td><td>-</td><td>-</td><td>tap water deionizing equipment by Auma-Tec GmbH</td></tr><tr><td>hydrochloric acid 12 M</td><td>VWR</td><td>20252.29</td><td></td></tr><tr><td>sodium hydroxide</td><td>Polskie Odczynniki Chemiczne S.A.</td><td>810925429</td><td></td></tr><tr><td>oxalic acid</td><td>Sigma-Aldrich Co.</td><td>75688</td><td></td></tr><tr><td>ethyl acetate</td><td>Brenntag GmbH</td><td>10010447</td><td></td></tr><tr><td>silica gel</td><td>Merck KGaA</td><td>1.10832.9025</td><td>Geduran Si 60 0.063-0.2&amp;nbsp;mm</td></tr><tr><td>TLC silica gel 60 F254</td><td>Merck KGaA</td><td>1.16834.0001</td><td></td></tr><tr><td>methanol</td><td>VWR</td><td>20903.55</td><td></td></tr><tr><td>ethanol</td><td>Brenntag GmbH</td><td>10018366</td><td></td></tr><tr><td>eiethylether</td><td>VWR</td><td>23807.468</td><td>stored over KOH plates</td></tr><tr><td>ammonia solution (25%)</td><td>VWR</td><td>1133.1</td><td></td></tr><tr><td>pH electrode</td><td>VWR</td><td>662-1657</td><td></td></tr><tr><td>stirring and heating unit</td><td>Heidolph</td><td>505-20000-00</td><td></td></tr><tr><td>pump</td><td>Ilmvac GmbH</td><td>322002</td><td></td></tr><tr><td>frit</td><td>-</td><td>custom design</td><td></td></tr><tr><td>NMR spectrometer</td><td>Bruker Coorporation</td><td>-</td><td>Ultra Shield 400</td></tr><tr><td>mass spectrometer</td><td>Thermo Fisher Scientific Inc.</td><td>-</td><td></td></tr><tr><td>elemental analyser</td><td>Hekatech GmbH Analysentechnik</td><td>-</td><td>EuroVector EA 3000 CHNS</td></tr><tr><td>deuterated water D&lt;sub&gt;2&lt;/sub&gt;O</td><td>euriso-top</td><td>D214</td><td>99.90% D</td></tr><tr><td colspan="4">&lt;strong&gt;Material/Equipment required for labeling procedures&lt;/strong&gt;</td></tr><tr><td>&lt;sup&gt;68&lt;/sup&gt;Ge/&lt;sup&gt;68&lt;/sup&gt;Ga generator</td><td>ITG Isotope Technologies Garching GmbH</td><td>A150</td><td></td></tr><tr><td>pump and dispenser system</td><td>Scintomics GmbH</td><td>-</td><td>Variosystem</td></tr><tr><td>hydrochloric acid 30% (suprapur)</td><td>Merck KGaA</td><td>1.00318.1000</td><td></td></tr><tr><td>water (ultrapur)</td><td>Merck KGaA</td><td>1.01262.1000</td><td></td></tr><tr><td>sodium chloride (suprapur)</td><td>Merck KGaA</td><td>1.06406.0500</td><td></td></tr><tr><td>sodium acetate (suprapur)</td><td>Merck KGaA</td><td>1.06264.0050</td><td></td></tr><tr><td>glacial acetic acid (suprapur)</td><td>Merck KGaA</td><td>1.00066.0250</td><td></td></tr><tr><td>sodium citrate dihydrate</td><td>VEB Laborchemie Apolda</td><td>10782</td><td>&gt;98.5%</td></tr><tr><td>PS-H&lt;sup&gt;+&lt;/sup&gt; Cartridge (S)</td><td>Macherey-Nagel</td><td>731867</td><td>Chromafix</td></tr><tr><td>&lt;em&gt;apo&lt;/em&gt;-Transferrin</td><td>Sigma-Aldrich Co.</td><td>T2036</td><td></td></tr><tr><td>PBS buffer (tablets)</td><td>Sigma-Aldrich Co.</td><td>79382</td><td></td></tr><tr><td>human serum</td><td>Sigma-Aldrich Co.</td><td>H4522</td><td>from human male AB plasma</td></tr><tr><td>flasks, columns, &lt;em&gt;etc.&lt;/em&gt;</td><td></td><td>custom design</td><td></td></tr><tr><td>pH electrode</td><td>Knick Elektronische Messger&amp;auml;te GmbH &amp;amp; Co. KG</td><td>765-Set</td><td></td></tr><tr><td>binary pump (HPLC)</td><td>Hewlett-Packard</td><td>G1312A (HP 1100)</td><td></td></tr><tr><td>UV Vis detector (HPLC)</td><td>Hewlett-Packard</td><td>G1315A (HP 1100)</td><td></td></tr><tr><td>radioactive detector (HPLC)</td><td>EGRC Berthold</td><td></td><td></td></tr><tr><td>HPLC C-18-PFP column</td><td>Advanced Chromatography Technologies Ltd.</td><td>ACE-1110-1503/A100528</td><td></td></tr><tr><td>HPLC glass vials</td><td>GTG Glastechnik Graefenroda GmbH</td><td>8004-HP-H/i3&amp;micro;</td><td></td></tr><tr><td>pipette</td><td>Eppendorf</td><td>-</td><td></td></tr><tr><td>plastic vials</td><td>Sarstedt AG &amp;amp; Co.</td><td>6542.007</td><td></td></tr><tr><td>plastic vials</td><td>Greiner Bio-One International GmbH</td><td>717201</td><td></td></tr><tr><td>activimeter</td><td>MED Nuklear-Medizintechnik Dresden GmbH</td><td>-</td><td>Isomed 2010</td></tr><tr><td>tweezers</td><td></td><td>custom design</td><td></td></tr><tr><td>incubator</td><td>Heraeus Instruments GmbH</td><td>51008815</td><td></td></tr><tr><td>vortex mixer</td><td>Fisons</td><td>-</td><td>Whirlimixer</td></tr><tr><td>centrifuge</td><td>Heraeus Instruments GmbH</td><td>75003360</td><td></td></tr><tr><td>gamma well counter</td><td>MED Nuklear-Medizintechnik Dresden GmbH</td><td>-</td><td>Isomed 2100</td></tr><tr><td>water for chromatography</td><td>Merck KGaA</td><td>1.15333.2500</td><td></td></tr><tr><td>acetonitrile for chromatography</td><td>Merck KGaA</td><td>1.00030.2500</td><td></td></tr><tr><td>trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>91707</td><td></td></tr><tr><td>TLC radioactivity scanner</td><td>raytest Isotopenmessger&amp;auml;te GmbH</td><td>B00003875</td><td>equipped with beta plastic detector</td></tr></tbody></table>

investigations on the ga(iii) complex of eob-dtpa and its 68ga radiolabeled analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

The ligand EOB-DTPA and the non-radioactive Ga(III) complex were analyzed via 1H and 13C{1H} NMR spectroscopy, mass spectrometry and elemental analysis. The results listed in Table 1 and depicted in Figures 1-6 verify the purity of the substances.
Elution of the 68Ge/68Ga generator yielded solutions of 400-600 MBq 68Ga. The described labeling procedure results in the formation of the desired tracer 68Ga[EOB-DTPA], indicated as radio HPLC peak exhibiting a retention time of 2.8 min (Figure 7). Comparison with the retention time of the Ga[EOB-DTPA] standard in the UV-vis detector at 220 nm (2.7 min, Figure 8) confirms successful labeling. Uncoordinated 68Ga is detected as radio peak at 2.1 min (Figure 7). The 68Ga-labeling efficiency of EO-BDTPA was investigated by determining the labeling yield as a function of the ligand concentration via HPLC (Figure 9). The yields were determined in triplicate and standard deviations were calculated.
Depending on the pH and the concentration of anions present in solution, uncoordinated or non-labeled 68Ga may exist in various species, e.g., gallates or insoluble hydroxide.31 The generalized term &#34;free 68Ga&#34;32 is used for all non-labeled species in solution except the hydroxide, which is generally referred to as &#34;colloidal 68Ga&#34;. Under the described analysis conditions, free 68Ga moves with the solvent front (Rf = 1.0) on a TLC plate. Colloidal 68Ga cannot be detected via HPLC, while on a TLC plate it appears as activity at the origin (Rf = 0). A representative chromatogram of a TLC plate analyzed with a TLC radioactivity scanner is shown in Figure 10. The tracer exhibits different retention behavior, depending on whether a sample of labeling solution (pH 3.8-4.0, Rf = 0.3) or a sample of physiological pH (Rf = 0.5) was analyzed.
To investigate the stability of the tracer, freshly labeled 68Ga[EOB-DTPA] was added to samples of physiological pH, containing diluted PBS (phosphate concentration 5.5 mM, A), excess of apo-transferrin (1.6 mg/ml in diluted PBS with a phosphate concentration of 0.8 mM, B) and human serum (C), respectively. Over time, the radiochemical purity (RCPt) of tracer in the samples was determined via TLC. The percentage of intact tracer was calculated as the ratio of RCPt at the respective time points and RCP0 at the starting point (Table 2). This was necessary due to the labeling solutions containing tracer of differing RCP0 (93-96%). The thus standardized percentage of intact tracer is depicted as a function of time in Figure 11.
For the determination of logD aqueous samples of tracer in a diluted PBS solution were prepared. The samples were mixed with n-octanol, centrifuged and subsequently aliquots were removed to determine the activity concentration in both phases. Activity values and subsequent calculation of logD thereof are depicted in Table 3. The mean logD value is 3.54 &#177; 0.08.
<img alt="Figure 1" src="/files/ftp_upload/54334/54334fig1.jpg" /> 
Figure 1. 1H-NMR spectrum of EOB-DTPA. The spectrum was recorded in D2O at 400.1 MHz. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54334/54334fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54334/54334fig2.jpg" /> 
Figure 2. 13C{1H}-NMR spectrum of EOB-DTPA. The spectrum was recorded in D2O at 100.6 MHz. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54334/54334fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54334/54334fig3.jpg" /> 
Figure 3. MS of EOB-DTPA (electrospray ionization (ESI), methanol, negative mode). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54334/54334fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54334/54334fig4.jpg" /> 
Figure 4. 1H-NMR spectrum of Ga[EOB-DTPA]. The spectrum was recorded in D2O at 400.1 MHz. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54334/54334fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54334/54334fig5.jpg" /> 
Figure 5. 13C{1H}-NMR spectrum of Ga[EOB-DTPA]. The spectrum was recorded in D2O at 100.6 MHz. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54334/54334fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54334/54334fig6.jpg" /> 
Figure 6. MS of Ga[EOB-DTPA] (ESI, methanol, negative mode), along with a detailed depiction of the isotope pattern of the molecular peak. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54334/54334fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54334/54334fig7.jpg" /> 
Figure 7. Representative HPLC chromatogram of a sample of 68Ga[EOB-DTPA] containing in parts uncoordinated 68Ga, as recorded by the radioactivity detector. Uncoordinated 68Ga exhibits a retention time of 2.1 min, while the tracer is detected at 2.8 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54334/54334fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/54334/54334fig8.jpg" /> 
Figure 8. Representative HPLC chromatogram of the standard substance Ga[EOB-DTPA], as detected in the UV-vis channel at 220 nm. The retention time of the cold standard is 2.7 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54334/54334fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" src="/files/ftp_upload/54334/54334fig9.jpg" /> 
Figure 9. Depiction of 68Ga-labeling efficiency of EOB-DTPA. The labeling yield as determined via HPLC is plotted as a function of the concentration of EOB-DTPA (22-29 MBq starting activity, pH 3.8-4.0, 5 min, RT). The standard deviation is depicted by error bars. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54334/54334fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" src="/files/ftp_upload/54334/54334fig10.jpg" /> 
Figure 10. Representative TLC chromatogram revealing different 68Ga species. A sample of 68Ga[EOB-DTPA] in diluted PBS (phosphate concentration 5.5 mM, pH = 7.4) was analyzed after 110 minutes of incubation. Exemplary distribution of colloidal 68Ga (Rf = 0), 68Ga[EOB-DTPA] (Rf = 0.5) and free 68Ga (Rf = 1.0) on a 70 mm TLC plate as detected by a TLC radioactivity scanner is presented. Counts are decay corrected. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54334/54334fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" src="/files/ftp_upload/54334/54334fig11.jpg" /> 
Figure 11. Stability determinations of 68Ga[EOB-DTPA] in different media. The decay corrected, standardized percentage of intact tracer as determined via TLC, is depicted as a function of time. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54334/54334fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/54334/54334table1.jpg" /> 
Table 1. Results of NMR spectroscopic, MS and elemental analyses performed for EOB-DTPA and Ga[EOB-DTPA]. Relative MS peak intensities are given in %, assignment to peaks are given in square brackets. Elemental CHN values were calculated for C23H33N3O11&#183;H2O (EOB-DTPA) and (NH4)0.75H1.25[C23H28GaN3O11]&#183;2H2O (Ga[EOB-DTPA]).
<img alt="Table 2" src="/files/ftp_upload/54334/54334table2.jpg" /> 
Table 2. Stability determination of 68Ga[EOB-DTPA] in different media. The RCP of 68Ga[EOB-DTPA] in media A, B and C was determined via TLC at given time points. The composition of the samples is given as percentages in % of tracer / free 68Ga / colloidal 68Ga. The percentage of intact tracer is standardized as ratio of RCPt/RCP0. RCP0 is the respective RCP of the tracer at t = 0 min.
<img alt="Table 3" src="/files/ftp_upload/54334/54334table3.jpg" /> 
Table 3. Determination of logD. Decay corrected values &#8121;0,X of three aliquots (x = 1, 2, 3) removed from each phase (W: aqueous, O: n-octanol) of a sample. All activities are given in cpm. LogD is calculated as described in section 5 of the protocol. The experiment was repeated twice.

Watch this Scientific Journal Video about Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue at JoVE.com

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

investigations-on-ga-iii-complex-eob-dtpa-its-68ga-radiolabeled

Nanyang Technological University

Research

JoVE Journal

Chemistry

9.9K Views.  University Hospital Jena. A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

Video: Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. Although heterogeneous in presentation, a common denominator among these tumors is the overexpression of somatostatin receptors. 68Ga-DOTATATE is a somatostatin analog labeled with the positron emitter gallium-68 (68Ga). For well-differentiated neuroendocrine tumors, 68Ga-DOTATATE positron emission tomography (PET)/computed tomography (CT) imaging is used for diagnosis, determination of disease burden, and therapy selection.
This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiolabeling of 68Ga-DOTATATE is performed with a fully automated labeling module coupled to a germanium-68 (68Ge)/68Ga generator. Quality control of the final product evaluates radiochemical purity with instant thin-layer chromatography and solid-phase chromatography, and pH prior to patient injection. Periodic quality control is performed to determine 68Ge breakthrough, sterility, and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) content. Patient preparation includes patient instructions, a protocol for 68Ga-DOTATATE during treatment with somatostatin analogs, and intravenous administration of the radiopharmaceutical. For PET/CT imaging, the acquisition and reconstruction settings are described. For each step, radiation safety will be highlighted, as well as time constrictions due to the short half-life of 68Ga.
Fully automated in-house production and quality control of 68Ga-DOTATATE leads to very high success rates (95%) and produces two to four patient dosages per batch, depending on the yield of the generator. In conclusion, 68Ga-DOTATATE PET/CT imaging is a noninvasive and fast method of providing information on the tumor burden of neuroendocrine tumors (NETs) while also assisting in diagnosis and therapy selection.

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Well-differentiated neuroendocrine tumors overexpress somatostatin receptors which can be utilized for diagnostic imaging with the radiolabeled somatostatin analog 68Ga-DOTATATE. This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiation safety and time constrictions due to the short half-life of 68Ga are taken into account.

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. ...

Cancer Research

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA &#8211; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

Bioengineering

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized cancer therapies in recent years. However, only a fraction of patients responds to PD-1/PD-L1 inhibitors, owing to the heterogeneous expression of PD-L1 in tumor cells. This heterogeneity presents a challenge in the precise detection of tumor cells by the commonly used immunohistochemistry (IHC) approach. This situation calls for better methods to stratify patients who will benefit from immune checkpoint blockade therapy, to improve treatment efficacy. Positron emission tomography (PET) enables real-time visualization of the whole-body PD-L1 expression in a noninvasive way. Therefore, there is a need for the development of radiolabeled tracers to detect PD-L1 distribution in tumors through PET imaging.
Compared to their L-counterparts, dextrorotary (D)-peptides have properties such as proteolytic resistance and remarkably prolonged metabolic half-lives. This study designed a new method to detect PD-L1 expression based on PET imaging of 68Ga-labeled PD-L1-targeted D-peptide, a D-dodecapeptide antagonist (DPA), in tumor-bearing mice. The results showed that the [68Ga]DPA can specifically bind to PD-L1-overexpressing tumors in vivo, and showed favorable stability as well as excellent imaging ability, suggesting that [68Ga]DPA-PET is a promising approach for the assessment of PD-L1 status in tumors.

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

This study developed a noninvasive and real-time method to evaluate the distribution of programmed death-ligand 1 in the whole body, based on positron emission tomographic imaging of [68Ga] D-dodecapeptide antagonist. This technique has advantages over conventional immunohistochemistry and improves the efficiency of identifying appropriate patients who will benefit from immune checkpoint blockade therapy.

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized ...

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

Automated Production of [68Ga]Ga-FAPI-46 on the iPHASE MultiSyn Synthesizer

An Automated Radiosynthesis of [68Ga]Ga-FAPI-46 for Routine Clinical Use

[68Ga]Ga-FAPI-46 is a promising new tracer for the imaging of fibroblast activation protein (FAP) by positron emission tomography (PET). Labeled FAP inhibitors (FAPIs) have demonstrated uptake in various types of cancers, including breast, lung, prostate, pancreatic and colorectal cancer. FAPI-PET also possesses a practical advantage over FDG-PET as fasting and resting are not required. [68Ga]Ga-FAPI-46 exhibits enhanced pharmacokinetic properties, improved tumor retention, and higher contrast images than the earlier presented [68Ga]Ga-FAPI-02 and [68Ga]Ga-FAPI-04. Although a manual synthesis protocol for [68Ga]Ga-FAPI-46 was initially described, in recent years, automated methods using different commercial synthesizers have been reported.
In this work, we describe the development of the automated synthesis of [68Ga]Ga-FAPI-46 using the iPHASE MultiSyn synthesizer for clinical applications. Initially, optimization of the reaction time and comparison of the performance of four different solid phase extraction (SPE) cartridges for final product purification were investigated. Then, the development and validation of the production of 0.6-1.7 GBq of [68Ga]Ga-FAPI-46 were conducted using these optimized parameters. The product was synthesized in 89.8 &#177; 4.8% decay corrected yield (n = 6) over 25 min. The final product met all recommended quality control specifications and was stable up to 3 h post synthesis.

This work describes the automated production of up to 1.7 GBq of [68Ga]Ga-FAPI-46 on the iPHASE MultiSyn synthesizer for PET imaging of fibroblast activation protein.

[68Ga]Ga-FAPI-46 is a promising new tracer for the imaging of fibroblast activation protein (FAP) by positron emission tomography (PET). Labeled FAP ...

Automated Preparation of [68Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe targeting the fibroblast activation protein for positron emission tomography imaging of the tumor microenvironment. The reaction conditions involved acetate buffer (final concentration: 0.1 M), methionine as an anti-radiolysis agent (final concentration: 5.4 mg/mL), and 30 &#181;g of 3BP-3940, with heating for 8 min at 98 &#176;C. A final purification step on a C18 cartridge was necessary to obtain a radiolabeled product of high purity. In contrast, the generator-produced 68Ga was used directly without a concentration step on a cation exchange cartridge. The production of three validation batches confirmed the method&#39;s reliability, allowing the synthesis of [68Ga]Ga-3BP-3940 in 22.3 &#177; 0.6 min with high radiochemical purity (RCP), as determined by both radio-HPLC (99.1% &#177; 0.1%) and radio-TLC (99.2% &#177; 0.1%). The average radiochemical yield, based on RCP values measured by radio-HPLC, was 74.4% &#177; 3.3%. The stability of the radiolabeled product was demonstrated for up to 4 h after preparation. This protocol provides a reliable, rapid, and efficient methodology for the preparation of [68Ga]Ga-3BP-3940, which can easily be transposed to a clinical setting.

This research describes the automated process for [68Ga]Ga-3BP-3940 production with the GAIA V2 synthesizer, for PET imaging of fibroblast activation protein. The results of quality control tests performed on three test batches are also presented.

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe ...

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for magnetic resonance imaging (MRI). Many commercially available ligands are especially useful because they contain 
functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive 
activated esters and isothiocyanate groups or thiol-reactive maleimides. While metalation of these ligands is considered common 
knowledge in the field of bioconjugation chemistry, subtle differences in metalation procedures must be taken into account when 
selecting metal starting materials. Furthermore, multiple options for purification and characterization exist, and selection of the most 
effective procedure partially depends on the selection of starting materials. These subtle differences are often neglected in published 
protocols. Here, our goal is to demonstrate common methods for metalation, purification, and characterization of lanthanide complexes 
that can be used as contrast agents for MRI (Figure 1). We expect that this publication will enable biomedical scientists to incorporate 
lanthanide complexation reactions into their repertoire of commonly used reactions by easing the selection of starting materials and 
purification methods.

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

Biology

Preparing a 68Ga-labeled Arginine Glycine Aspartate (RGD)-peptide for Angiogenesis

The &#945;v&#946;3 integrin is a heterodimeric adhesion molecule involved in tumor cell migration and angiogenesis. The integrin is overexpressed in angiogenic tumor endothelial cells, where it typically has a low concentration. This specific expression of &#945;v&#946;3 makes it a valid biomarker for antiangiogenic and imaging drugs. As a functional imaging modality, positron emission tomography (PET) provides information about biochemical and physiological changes in vivo, due to its unique high sensitivity at the nanomolar scale. Hence, radiometal-based PET radiopharmaceuticals have received great attention for the non-invasive quantification of tumor angiogenesis. This paper provides a systemic protocol to prepare a new radiometal-labeled peptide for the evaluation of angiogenesis. This protocol contains information about radiochemical reliability, lipophilicity, cell uptake, serum stability, and pharmacokinetic properties. The 68Ga-RGD-peptide is one of the representative PET ligands toward &#945;v&#946;3 integrin. Here, we introduce a protocol to prepare a 68Ga-RGD-peptide and the evaluation of its biological efficacy.

Preparing a 68Ga-labeled Arginine Glycine Aspartate RGD-peptide for Angiogenesis

The &#945;v&#946;3 integrin is a type of adhesion protein that is highly expressed on activated endothelial cells undergoing angiogenesis. Thus, evaluating the integrity of the integrin is of great interest in oncology. Here, we introduce a method to prepare 68Ga-labeled radiopeptides and a method to assess its biological effectiveness.

The &#945;v&#946;3 integrin is a heterodimeric adhesion molecule involved in tumor cell migration and angiogenesis. The integrin is overexpressed in ...

Investigations on the Ga(III) Complex of EOB-DTPA and Its ⁶⁸Ga Radiolabeled Analogue

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Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

Development of a ⁶⁸Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of ⁶⁸Ga-Labelled Nano Molecules

An Automated Radiosynthesis of [⁶⁸Ga]Ga-FAPI-46 for Routine Clinical Use

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