3’-Deoxy-3’-[ 18 F]Fluorothymidine—[ 18 F]FLT is a Positron Emission Tomography (PET) tracer which has been used for noninvasive assessment of proliferation activity in several types of cancer. During the past few years, some novel approaches for [18F]FLT synthesis have been developed, mainly focused on optimization of reaction conditions and purification methods. The present study reports the use of two different eluents in the final step of [18F]FLT production and the evaluation of its effect on radiochemical yield and product quality. The first eluent evaluated was water: ethanol (90:10, v/v), commercially available, and the second was NaCl 0.9% (saline): ethanol (92:8, v/v). The mean of the corrected radiochemical yields corresponded to 27% ± 7% for elution with water and ethanol and to 23% ± 3% for elution with saline and ethanol, which could indicate that the eluent solutions have similar elution strength. Besides, quality control results were in accordance with the requirements and demonstrated that there was no significant difference between both formulations. Considering that pharmaceutical preparations containing ethanol should be preferentially diluted with saline to avoid hemolysis, the eluent saline:ethanol (92:8, v/v) was chosen for [18F]FLT extraction and final formulation.
The usefulness of Positron Emission Tomography/Computed Tomography (PET/CT) imaging has been established for diagnosis and response assessment of various diseases, especially in oncology [
Efforts have been applied to develop new PET imaging agents to quantify specific metabolic processes such as cell proliferation, receptor density and function, and other characteristics [
[18F]FLT stable analogue ([19F]3’-deoxy-3’-fluorothymidine, FLT) was developed for the first time in 1969 intending to be an antitumoral agent [
The [18F]FLT formulations generally are constituted by an ethanolic solution containing up to 10% of this solvent [
However, whenever possible, it is desirable that have radiopharmaceutical formulations containing as low ethanol content as possible to reduce the risk of hemolysis. Another reason is that children, women during pregnancy and lactation, persons with alcoholism liver disease and/or epilepsy, could experience some toxic effects due to injection of higher ethanol concentrations [
This article presents a comparative study between two different eluents: water:etha- nol (90:10, v/v), commercially available, and saline:ethanol (92:8, v/v) solution. Both were compared in order to determine the effect on the radiochemical yield and final formulation properties.
The reagents kits and sterile cassettes used for production of [18F]FLT were purchased from ABX Advanced Biochemical Compounds (Radeberg, Germany). Fluorothymidine standard was also supplied by ABX. Enriched 18O water was purchased from Center of Molecular Research (Moscow, Russia). The reference standards of stavudine and 3’- chloro-3’-deoxy-thymidine (chlorothymidine) were acquired from U. S. Pharmacopeia (Rockville, USA). Other chemicals and solvents were of analytical grade and were obtained from conventional chemical suppliers. PTSTM cartridges were supplied by Charles River Laboratories (Wilmington, USA). Soybean-Casein digest and fluid thio- glycolate media were acquired from Newprov (Paraná, Brazil).
SepPak® Light Accell Plus QMA, Oasis® WAX, Oasis® HLB, SepPak® Alumina N and Chromabond® PS-H+ cartridges were conditioned before use: QMA with 0.5 M carbonate solution (10 mL), water (15 mL) and dried with air; HLB, Alumina and WAX with ethanol (5 mL), water (10 mL) and dried with air; and PS-H+ with ethanol (2 mL), water (4 mL) and dried with air.
The water:ethanol (90:10, v/v) solution was used without any modifications from ABX (Radeberg, Germany). Ethanol acquired from J.T. Baker (Phillipsburg, USA) was used to prepare the 8% (v/v) solution of ethanol in 0.9% sodium chloride for injection (saline) which was purchased from Sanobiol (Pouso Alegre, Brazil).
[18F]Fluoride (11 to 135 GBq) was produced on a 16.5 MeV Cyclotron PETtrace® (GE Healthcare Technologies, Waukesha, USA) via the 18O(p,n)18F nuclear reaction. The solution containing [18F]fluoride was delivered from the cyclotron to a TracerLab® MXFDG module (GE Healthcare, USA), adapted for the production of [18F]FLT as shown in
Light Accell Plus QMA). Then, it was eluted with 0.75 mL of 0.075 M tetrabutylammonium bicarbonate to the reaction vessel where was dried by azeotropic distillation at 95˚C using three successive additions of anhydrous acetonitrile (80 μL) and low flow of nitrogen (40 mL/min). Once [18F]fluoride-tetrabutylammonium was obtained, 25 mg of the [18F]FLT precursor (3-N-BOC-5-O-DMTr-3-O-nosyl-lyxothymidine) solubilized in anhydrous acetonitrile was added to the dried residue in the reactor vial. Nucleophilic substitution was carried out at 100˚C for 5 minutes, followed by hydrolysis step with 2 M HCl (1.5 mL) at 85˚C for another 5 minutes. The mixture was allowed to evaporate at the same temperature. Then, it was cooled and neutralized with 1 M NaOH (2.4 mL) and passed through PS-H+, WAX and HLB cartridges for purification. Cartridges were washed with ethanol 2% (v/v) (approximately 90 mL). [18F]FLT was eluted from the HLB cartridge with a saline:ethanol (92:8, v/v) or a water:ethanol (90:10, v/v) solution (both 14 mL). The product was passed through an Alumina N short cartridge and sterilized through a 0.22 μm membrane filter. Total synthesis time was 54 minutes.
The pH, radionuclidic identity and purity, residual solvent (acetonitrile), ethanol determination, radiochemical identity and purity, chemical purity (tetrabutylammonium, thymine, thymidine, stavudine, chlorothymidine), bacterial endotoxins, and sterility of each final formulation of [18F]FLT were assessed. The pH was measured using micro pHmeter (Mettler Toledo, USA). Radionuclidic identity was confirmed by measuring the half-life of the final product in a CRC 25R dose calibrator (Capintec, USA) at 3 time points. Radionuclidic purity of [18F]FLT was determined on a gamma-ray spectrometer with an ultrapure Ge detector (Canberra Multichannel Analyzer, USA).
The amount of ethanol and residual solvent (acetonitrile) in [18F]FLT was determined by gas chromatography. Analyses were carried out in a Varian 3900 GC equipment (Santa Clara, USA), equipped with split/splitless injector inlet and a flame ionization. An Agilent CP-Wax 52 CB column (30 m × 0.53 mm; 1 μm) was used as the stationary phase. Helium at a flow rate of 1.5 mL/min was employed as the carrier gas. The GC oven temperature was programmed as follows. Initially the temperature was maintained at 45˚C for 4 minutes. Then the temperature was increased to 80˚C at a rate of 30˚C/min, and maintained at 80˚C for 0.83 min. The injector port and detector were maintained at 140˚C and 250˚C, respectively. The injector was operated in a split mode of 70:1.
Both products were tested for chemical and radiochemical purity by analytical HPLC. Analyses were performed using an Agilent 1200 Liquid Chromatograph (Santa Clara, USA), equipped with a UV detector (adjusted at 267 nm) and a Raytest radioactive detector (Straubenhardt, Germany). A Supelcosil LC-18 column (250 × 4.6 mm; 5 μm) was used for separation. The mobile phase A was ethanol 10% in water, while mobile phase B was ethanol 100%, used as a gradient according to
Time (minutes) | % mobile phase A | % mobile phase B | Condition |
---|---|---|---|
0 to 10 | 100 | 0 | Isocratic |
10 to 13 | 90 | 10 | Gradient |
13 to 18 | 90 | 10 | Isocratic |
18 to 21 | 100 | 0 | Gradient |
A spot test was used to evaluate tetrabutylammonium cation in [18F]FLT [
Bacterial endotoxins were quantified by LAL kinetic chromogenic method, using the Endosafe® - Portable Test System (Charles River, USA). Sterility test was performed by direct inoculation of [18F]FLT into soybean-casein digest and fluid thioglycolate media.
Although various precursors have been proposed for the radiosynthesis of [18F]FLT, in this work, 5’-O-DMTr-3’-O-nosyl-lyxothymidine was used, as several reports indicated high incorporation of [18F]fluoride. A wide range of radiochemical yield and chemical purity for the synthesis of [18F]FLT have been described in literature, mainly because it may be affected by many factors, such as: phase-transfer agent, precursor amount, synthesis module, purification method, etc. Although semipreparative HPLC purification is described by some authors [
Quality control tests, product specifications and results obtained for [18F]FLT are presented in
A syringe with 20 mL of water:ethanol (90:10, v/v) is included in the reagent kit used in this work. Tests using different volumes of eluent solution were performed and the results (not shown) indicated that the yield was the same when 14 mL was used. Smaller volumes make feasible further dilutions, reducing ethanol final concentrations.
Since no brazilian pharmacopoeic monograph is current available for [18F]FLT, the quality control parameters were established according the European Pharmacopoeia 8.0 and related literature [
Quality Control Tests | Ph.Eur 8.0 | Ethanol 10% in water for injections (n = 6) | Ethanol 8% in saline (n = 11) | |
---|---|---|---|---|
Corrected yield (%) | Not applicable | 27± 7 | 23 ± 3 | |
Specific activity in the end of synthesis (GBq/μmol) | Not applicable | >117.1 | >178.6 | |
pH | 4.5 to 8.5 | 4.54 to 7.03 | 4.57 to 7.06 | |
Residual Solvents (Acetonitrile) (%V/V) | <0.04 | 0.00012 to 0.00193 | 0.00096 to 0.0067 | |
Ethanol Determination (%V/V) | <10% or 2.5 g per administration | 6.15 to 8.99 | 5.81 to 8.07 | |
Radiochemical Purity (%) | >95% | 98.43 to 99.40 | 98.43 to 99.19 | |
Radionuclidic Identity (min) | 105 to 115 | 109.56 to 110.70 | 109.30 to 113.08 | |
Radionuclidic Purity (%)* | >99.9% | 99.86 to 99.99 | 99.70 to 100.00 | |
Chemical Purity | Tetrabutylammonium (μg/mL) | <2.6 mg/V** | <50 | <50 |
Thymine (μg/mL) | Any other impurities, for each one < 0.1 mg/V** | 0.089 to 0.898 | 0.081 to 0.146 | |
Thymidine (μg/mL) | 0.129 to 0.176 | 0.096 to 0.176 | ||
Chlorothymidine (μg/mL) | 0.132 to 0.532 | 0.148 to 0.425 | ||
Stavudine (μg/mL) | <0.1 mg/V** | 0.023 to 0.061 | 0.019 to 0.040 | |
Fluorothymidine (μg/mL) | <0.1 mg/V** | 0.178 to 0.466 | 0.057 to 0.509 | |
Bacterial Endotoxin (EU/mL) | <175/V** | <5.00 | <5.00 | |
Sterility | Sterile | Pass | Pass |
*Radionuclidic Purity, according to the Alovudine’s monograph, should present results higher than 99.9%. However, 18F based radiopharmaceuticals monographs, like USP (>99.5% [
tion were assessed and both formulations meet the requirements established in European Pharmacopoeia 8.0.
It is known that thymidine and thymine are endogenous substances. Their concentrations in the formulation (
As thymidine, fluorothymidine and chlorothymidine differs only in the carbon 3 ligand (hidroxide, fluoride and chloride, respectively) in their molecular structure, it is hard to believe that chlorothymidine will not affect [18F]FLT cellular’s uptake, considering its atomic configuration and chemical properties. As far as we know, only Pascali and co-workers reports its presence in the final product [
Stavudine, which is used as an anti-retroviral agent, is often prescribed 40 mg orally twice a day to generate a maximal therapeutic plasma concentration of 0.5 ± 0.2 μg/mL [
A typical HPLC chromatogram obtained for the [18F]FLT obtained by elution with saline:ethanol (92:8, v/v) is shown in
The GC analysis of [18F]FLT revealed that it contains a maximum of 8.75% ethanol and 0.00236% of residual acetonitrile (
[18F]FLT was successfully produced using the synthesizer TracerLab® MXFDG. The final
step of the synthesis of [18F]FLT using two different eluent solutions led to similar final radiopharmaceuticals formulations. Saline:ethanol (92:8, v/v) was chosen as our standard, considering that satisfactory yields were achieved and quality control results were in accordance with the requirements. Finally, this radiopharmaceutical can be consistently produced and controlled at Centro de Desenvolvimento da Tecnologia Nuclear.
This work was supported by Centro de Desenvolvimento da Tecnologia Nuclear (CDTN, CNEN), Fundação de Amparo à Pesquisa (FAPEMIG APQ-04740-10), INCT de Medicina Molecular (CNPq 573646/2008-2; CBB-APQ-00075-09) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 573646/2008-2). The authors would like to thank the whole group of the Unidade de Pesquisa e Produção de Radiofármacos of CDTN for technical support on production and quality control. We are also grateful to Fundação Ezequiel Dias (FUNED).
The authors declare that there is no conflict of interests.
Nascimento, L.T.C., Silveira, M.B., Ferreira, S.M.Z.M.D. and Silva, J.B. (2017) Comparison between Two Ethanolic Solutions for 3’-Deoxy-3’-[18F] Fluorothymidine Elution. Advances in Che- mical Engineering and Science, 7, 23-33. http://dx.doi.org/10.4236/aces.2017.71003