Comparison between Two Ethanolic Solutions for 3 ’-Deoxy-3 ’-[ 18 F ] Fluorothymidine Elution

3’-Deoxy-3’-[F]Fluorothymidine—[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 [F]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 [F]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 [F]FLT extraction and final formulation.


Introduction
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 [1].[ 18 F]Fluorodeoxyglucose ([ 18 F]FDG) is the most commonly used PET radiopharmaceutical for tumor imaging with a wide field of application.
However, it has some limitations for evaluating malignancies in organs with physiological uptake such as central nervous system tissues, heart and bladder [2] [3].Besides, [ 18 F]FDG is not a highly selective tracer for tumor imaging and cannot distinguish be-tween inflammatory processes and cancer, for example [4].
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 [5].3'-deoxy-3'-[ 18 F]fluorothymidine ([ 18 F]FLT) is a radioactive analogue of the nucleoside thymidine which has been proposed for imaging of cellular proliferation [6].Clinical studies have demonstrated that this radiopharmaceutical is a promising radiotracer for detection and therapy response assessment in several types of cancer, including breast, lung, colorectal and brain cancers [7] [8] [9], or other disturbs such as inflammation in arthritis [10].
[ 18 F]FLT stable analogue ([ 19 F]3'-deoxy-3'-fluorothymidine, FLT) was developed for the first time in 1969 intending to be an antitumoral agent [11].Later, in the 90's it was used as the anti-retroviral agent named alovudine [12].In both cases, it showed toxicity incompatible with therapeutic applications [13] [14].The 18 F-labelled thymidine was synthesized for the first time in 1991 to study the anti-retroviral mechanism [15].The molecule was used for PET for the first time in 1996 and from this year to now, different synthesis routes were investigated to improve its radiochemical yield and radiochemical purity [16] [17] [18].
The [ 18 F]FLT formulations generally are constituted by an ethanolic solution containing up to 10% of this solvent [19] [20] [21] [22].The use of ethanol is required in the purification step when high yields are desired.According to the International Conference on Harmonization [23], the concentration of ethanol should be lower than 0.5% for human use as it is a class 3 solvent, unless specifically justified.However, commercially available drugs, as paricalcitrol, may contain up to 20% of ethanol [24].Radiopharmaceuticals such as [ 18 F]fluoromisonidazole and [ 18 F]fluoroethyltyrosine also contain a high concentration of ethanol (up to 10% and 20% respectively) in their final formulation [25].
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 [26].
This article presents a comparative study between two different eluents: water:ethanol (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.

General
The reagents kits and sterile cassettes used for production of [ 18 F]FLT were purchased from ABX Advanced Biochemical Compounds (Radeberg, Germany).Fluorothymidine standard was also supplied by ABX.Enriched 18 O 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 ob-tained from conventional chemical suppliers.PTS TM cartridges were supplied by Charles River Laboratories (Wilmington, USA).Soybean-Casein digest and fluid thioglycolate media were acquired from Newprov (Paraná, Brazil).
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).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).

Automated Synthesis of [ 18 F]FLT
[ 18 F]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.

Quality Control of [ 18 F]FLT
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 [ 18 F]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 [ 18 F]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 [ 18 F]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 Table 1.The mobile phase flow rate was 1.2 mL/min.The presence of non-radioactive impurities (thymine, thymidine, stavudine and chlorothymidine) was also checked under the same HPLC conditions.The method was validated in terms of quantification/detection limits, precision, specificity, accuracy, robustness and linearity.

Results and Discussion
Although various precursors have been proposed for the radiosynthesis of [ 18 F]FLT, in this work, 5'-O-DMTr-3'-O-nosyl-lyxothymidine was used, as several reports indicated high incorporation of [ 18 F]fluoride.A wide range of radiochemical yield and chemical purity for the synthesis of [ 18 F]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 [22] [28] [29], it has been reported as time consuming.In the present study, purification was performed by means of solid-phase extraction cartridges, a simple, rapid and efficient purification method suitable for the TracerLab MX FDG synthesis module [20].
Quality control tests, product specifications and results obtained for [ 18 F]FLT are presented in Table 2.All parameters were in agreement to quality criteria confirming that [ 18 F]FLT, eluted with both solution, was suitable for use.A solution without ethanol was previously evaluated (data not shown), as proposed by Pascali and co-workers [20].However, the uncorrected radiochemical yields were lower than 7% and the concentration of impurities such as thymidine was higher than 100 μg/mL.
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 [ 18 F]FLT, the quality control parameters were established according the European Pharmacopoeia 8.0 and related literature [20] [30].The impurities (thymine, thymidine, stavudine, chlorothymidine and unknown ones), must be found in concentrations lower than 6.7 μg/mL, considering the maximum volume (V) of 15 mL.In this work their concentra- It is known that thymidine and thymine are endogenous substances.Their concentrations in the formulation (Table 2) are meaningless for toxicity purposes.However, as thymidine is a [ 18 F]FLT competitor for the intracellular accumulation [33] [34], it should have the lower concentration as possible in order to avoid lack of efficiency.
Viertl and co-workers discovered that pretreatment with a low-dose of 5-fluoro-2'deoxyuridine, a thymidine synthesis inhibitor, increased uptake of [ 18 F]FLT in different tumor xenografts [35], being a promising tool to increase [ 18 F]FLT's sensitivity in humans.
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 [ 18 F]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 [20].
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 [36].As small amount of stavudine was detected in both [ 18 F]FLT formulations, any toxic effect due to its presence is very unlikely.
A typical HPLC chromatogram obtained for the [ 18 F]FLT obtained by elution with saline:ethanol (92:8, v/v) is shown in Figure 2. It was possible to verify the presence of already described [20] and unknown impurities.The peak corresponding to [ 18 F]FLT was visualized using the radioactive detector (Figure 2 The GC analysis of [ 18 F]FLT revealed that it contains a maximum of 8.75% ethanol and 0.00236% of residual acetonitrile (Figure 3) for the saline:ethanol (92:8, v/v) eluent and a maximum of 8.99% of ethanol and 0.00193% of acetonitrile in the water:ethanol (90:10, v/v) eluent, both in accordance with the acceptance criteria.In [ 18 F]FLT final product, the ethanol is not a residual solvent, unlike [ 18 F]FDG, being added purposely to have better yields.

[ 18 F
Figure 1.TracerLab MX FDG configuration, accessories and reagents used for the production of [ 18 F]FLT.
(b)).Radioactivity detection also revealed three minor peaks corresponding to unidentified radiochemical impurities; however, their sum never exceeded 2%.The retention time of the main radioactive peak observed in the chromatogram of [ 18 F]FLT test solution was similar to that of the fluorothymidine reference standard.

Figure 3 .
Figure 3. GC chromatogram of saline:ethanol (92:8, v/v) [ 18 F]FLT's eluent.Acetonitrile's peak is magnified on the upper right side of the figure.Conditions: Agilent CP-Wax 52 CB column (30 m × 0.53 mm; 1 μm) eluted with Helium at a flow rate of 1.5 mL/min.Injector port and flame ionizing detector at 140˚C and 250˚C respectively.Oven was submitted to a temperature gradient, varying from 45˚C to 80˚C.RT = retention time.uV = microvolts.

Table 1 .
Mobile phase composition in HPLC analysis.
® -Portable Test System (Charles River, USA).Sterility test was performed by direct inoculation of [ 18 F]FLT into soybean-casein digest and fluid thioglycolate media.