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

Podsumowanie

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.

Streszczenie

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 3.8–4.0 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' 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.

Wprowadzenie

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]-DOTATOC^9,10) 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-injection^3,4,21-23, 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.

Protokół

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).

1. Isolation of EOB-DTPA from gadoxetic acid
   1. 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.
   2. 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.
   3. 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.
   4. Remove the solvent in vacuo to yield a colorless residue. Store under inert gas.
   5. Wash the residue thoroughly (at least three times) with ethyl acetate to remove the excess of oxalic acid. Dry the residue in vacuo.
   6. 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.
   7. 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.
   8. 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.
   9. 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.
   10. Combine both solid fractions of EOB-DTPA and perform NMR spectroscopic,²⁵ mass spectrometric²⁶ and elemental²⁷ analyses.
2. 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.
   1. 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.
   2. 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.
   3. 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.
   4. 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
   5. Heat the residue under reduced pressure (125 °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.
   6. 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.

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.

1. 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.
   1. 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 µl 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.
   2. Precondition the PS-H⁺ cartridge by flushing it slowly with 1 ml of 1.0 M hydrochloric acid and subsequently 5 ml of water.
   3. Elute the silica column of the generator with 4 ml 0.05 M HCl.¹² Load the ⁶⁸Ga eluate onto the PS-H⁺ cartridge.
   4. 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
2. Labeling of EOB-DTPA with ⁶⁸Ga
   1. Dissolve 1 mg (1.9 µmol) of EOB-DTPA in 1 ml of water. From this solution take 100 µl (0.19 µmol) and dilute them with 9.9 ml of water to prepare a 19 µM (10 µg/ml) stock solution of EOB-DTPA.
   2. Remove 50 µl (equaling 22-29 MBq) of the solution containing ⁶⁸Ga and put into a vial. Add 50 µl (0.5 µg) of a 19 mM stock solution of EOB-DTPA and 300 µl 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 µl and put to HPLC or TLC analysis.
   3. 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 → 0% A (0.5 ml/min), 610 min 0% A (0.5 ml/min).
   4. 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 = A_Ga-EOB-DTPA/(A_Ga + A_Ga-EOB-DTPA) ∙ 100%
      A_Ga-EOB-DTPA: area under curve of ⁶⁸Ga[EOB-DTPA]
      A_Ga: area under curve of free ⁶⁸Ga

3. Labeling Efficiency

1. 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 µl, depending on the freshness of the eluate).
2. Add the required amount of buffer solution to adjust the pH to 3.8-4.0 (40-190 µl, depending on the volume of ⁶⁸Ga eluate). Add the required amount of ligand stock solution (10-70 µl of a 19 mM solution).
3. 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.
4. Perform labeling procedures with amounts of ligand between 0.1 µg and 0.7 µg in steps of 0.1 µg. Perform experiments in triplicates for each ligand concentration. Calculate the mean yield and standard deviation.

4. In Vitro Stability

1. General procedure and preparations
   1. 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.
   2. Perform labeling of 22-29 MBq ⁶⁸Ga with 0.5 µl 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.
   3. 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 = A_Ga-EOB-DTPA/(A_Ga-free + A_Ga-EOB-DTPA + A_Ga-colloidal) ∙ 100%
      A_Ga-EOB-DTPA: area under curve of ⁶⁸Ga[EOB-DTPA]
      A_Ga-free: area under curve of free ⁶⁸Ga
      A_Ga-colloidal: area under curve of colloidal ⁶⁸Ga
   4. Calculate RCP_t/RCP₀ for every time point. Plot the thus standardized RCP vs. time difference since the starting point t = 0 min.
      RCP_t = RCP of ⁶⁸Ga[EOB-DTPA] at time point t.
      RCP₀ = RCP of ⁶⁸Ga[EOB-DTPA] at t = 0 min.
2. Stability in phosphate buffered saline (A)
   1. To 65 µl of labeling solution add 150 µl of PBS stock solution and 60 µl of sodium hydroxide solution (0.1 M) to raise the pH to 7.4. Mix thoroughly.
   2. Remove an aliquot of 1-5 µl to perform TLC analysis ('starting point'). Immediately store the solution in an incubator at 37 °C and remove aliquots to perform TLC analysis at representative time points over 3 hr.
3. Stability towards excess of apo-transferrin in PBS (B)
   1. To 120 µl of labeling solution add 50 µl of PBS stock solution and 430 µl of sodium hydroxide solution (0.1 M) to raise the pH to 7.4. Add 40 µl of a solution of apo-transferrin (25 mg/ml). Mix thoroughly.
   2. Remove an aliquot of 1-5 µl to perform TLC analysis ('starting point'). Immediately store the solution in an incubator at 37 °C and remove aliquots to perform TLC analysis at representative time points over 3 hr.
4. Stability in human serum (C)
   1. To 500 µl of human serum add 25 µl of labeling solution and 45 µl of sodium hydroxide solution (0.1 M) to raise the pH to 7.4. Mix thoroughly.
   2. Remove an aliquot of 1-5 µl to perform TLC analysis ('starting point'). Immediately store the solution in an incubator at 37 °C and remove aliquots to perform TLC analysis at representative time points over 3 hr.

5. Determination of Distribution Coefficient LogD

1. Perform labeling procedures as described in section 2. To 50 µl of labeling solution add 20 µl of PBS stock solution and 170 µl of sodium hydroxide solution (0.1 M) to raise the pH to 7.4.
2. Withdraw 200 µl from that solution and put it into a plastic V-vial. Add 200 µl of n-octanol. Close the vial and vortex for 2 min. Then centrifuge the sample at 1,600 x g for 5 min.
3. Remove triplicates of 40 µl 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.
4. 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 Ᾱ_t in counts per minute (cpm). List the thus gained Ᾱ_t,W1, Ᾱ_t,W2 and Ᾱ_t,W3 (activities in aqueous samples) and Ᾱ_t,O1, Ᾱ_t,O2, Ᾱ_t,O3 (activities in n-octanol) along with the respective time point t of their determination.
5. Define the time point of the measurement of the last sample as t₀. Determine and list Δt in min by calculating Δt = t-t₀. Perform decay correction of Ᾱ_t, using the following formula:
   Ᾱ₀ = Ᾱ_t · 2^{(Δt/68 min)}.
6. Calculate Ᾱ_0,W as the mean of Ᾱ_0,W1, Ᾱ_0,W2 and Ᾱ_0,W3 as well as Ᾱ_0,O as the mean of Ᾱ_0,O1, Ᾱ_0,O2 and Ᾱ_0,O3. Calculate logD using the following formula:
   logD = log[(Ᾱ_0,O · 40 µg)/(Ᾱ_0,W · 33 µg)].
7. Perform the entire experiment in triplicates and calculate the mean logD along with its standard deviation.

Wyniki

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. T...

Dyskusje

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 analyt...

Materiały

Name	Company	Catalog Number	Comments
primovist	Bayer	-	0.25 M
gallium(III) chloride	Sigma-Aldrich Co.	450898
water (deionized)	-	-	tap water deionizing equipment by Auma-Tec GmbH
hydrochloric acid 12 M	VWR	20252.29
sodium hydroxide	Polskie Odczynniki Chemiczne S.A.	810925429
oxalic acid	Sigma-Aldrich Co.	75688
ethyl acetate	Brenntag GmbH	10010447
silica gel	Merck KGaA	1.10832.9025	Geduran Si 60 0.063-0.2 mm
TLC silica gel 60 F254	Merck KGaA	1.16834.0001
methanol	VWR	20903.55
ethanol	Brenntag GmbH	10018366
eiethylether	VWR	23807.468	stored over KOH plates
ammonia solution (25%)	VWR	1133.1
pH electrode	VWR	662-1657
stirring and heating unit	Heidolph	505-20000-00
pump	Ilmvac GmbH	322002
frit	-	custom design
NMR spectrometer	Bruker Coorporation	-	Ultra Shield 400
mass spectrometer	Thermo Fisher Scientific Inc.	-
elemental analyser	Hekatech GmbH Analysentechnik	-	EuroVector EA 3000 CHNS
deuterated water D2O	euriso-top	D214	99.90% D
Material/Equipment required for labeling procedures
68Ge/68Ga generator	ITG Isotope Technologies Garching GmbH	A150
pump and dispenser system	Scintomics GmbH	-	Variosystem
hydrochloric acid 30% (suprapur)	Merck KGaA	1.00318.1000
water (ultrapur)	Merck KGaA	1.01262.1000
sodium chloride (suprapur)	Merck KGaA	1.06406.0500
sodium acetate (suprapur)	Merck KGaA	1.06264.0050
glacial acetic acid (suprapur)	Merck KGaA	1.00066.0250
sodium citrate dihydrate	VEB Laborchemie Apolda	10782	>98.5%
PS-H+ Cartridge (S)	Macherey-Nagel	731867	Chromafix
apo-Transferrin	Sigma-Aldrich Co.	T2036
PBS buffer (tablets)	Sigma-Aldrich Co.	79382
human serum	Sigma-Aldrich Co.	H4522	from human male AB plasma
flasks, columns, etc.		custom design
pH electrode	Knick Elektronische Messgeräte GmbH & Co. KG	765-Set
binary pump (HPLC)	Hewlett-Packard	G1312A (HP 1100)
UV Vis detector (HPLC)	Hewlett-Packard	G1315A (HP 1100)
radioactive detector (HPLC)	EGRC Berthold
HPLC C-18-PFP column	Advanced Chromatography Technologies Ltd.	ACE-1110-1503/A100528
HPLC glass vials	GTG Glastechnik Graefenroda GmbH	8004-HP-H/i3µ
pipette	Eppendorf	-
plastic vials	Sarstedt AG & Co.	6542.007
plastic vials	Greiner Bio-One International GmbH	717201
activimeter	MED Nuklear-Medizintechnik Dresden GmbH	-	Isomed 2010
tweezers		custom design
incubator	Heraeus Instruments GmbH	51008815
vortex mixer	Fisons	-	Whirlimixer
centrifuge	Heraeus Instruments GmbH	75003360
gamma well counter	MED Nuklear-Medizintechnik Dresden GmbH	-	Isomed 2100
water for chromatography	Merck KGaA	1.15333.2500
acetonitrile for chromatography	Merck KGaA	1.00030.2500
trifluoroacetic acid	Sigma-Aldrich	91707
TLC radioactivity scanner	raytest Isotopenmessgeräte GmbH	B00003875	equipped with beta plastic detector