1 Introduction
One of the most common radioisotopes is 188Re (T1/2 = 16.9 h). It is appropriate for both radiotherapy and radiodiagnosis as it emits beta-particles (Emax = 2.12 MeV) and gamma-rays (Eγ = 155 keV, Iγ = 15%). In several studies, two radionuclides have been combined for theranostic applications, such as in the case of DOTA-rituximab labeled with 111In and 90Y, where these isotopes are used for imaging and radiotherapy, respectively [1]. In addition to the similarity of its chemical nature to that of technetium, Rhenium-188 is an effective radioisotope for in vivo applications [2-4]. 188W (T1/2 = 69.4 d) is produced by double-neutron capture on natural tungsten targets, whereas 186W (28.43%) occurs in low abundance; 186W (n, γ) and 187W have a cross-section for thermal neutron capture of 38b and integral resonance of 419b [5-6]. Similar features are exhibited by 187W (n, γ) and 188W, with respective values of 64 and 2760 b for the cross-section for thermal capture and integrated resonance [7]. Open access to carrier-free 188Re-perrhenate is a big benefit of using the 188W/188Re generator, where elution produces about 50% 188Re in 24 h. The 188W/188Re generator employs a number of matrixes, such as the ZrSiW matrix [8], which has been used as an outstanding matrix in the 113Sn/113mIn generator [9]. The production of 188Re requires a high-performance generator that provides great versatility in the production of a variety of therapeutic agents labeled with 188Re and generators having a long shelf-life exceeding six months. A few high-flux reactors are available for producing 188W [10]. The use of low-cost reusable tandem concentration units [11] offers high-specific 188Re volume solutions (i.e. > 700 mCi/ml for 1 Ci generator). The 188W/188Re generator is particularly important for providing a reliable source of this versatile therapeutic radioisotope, especially in developing regions where the isotope must be transported over long distances and the distribution costs are high [12].
The novel anti-epileptic drug lacosamide (LCM) operates by a dual mode of action through its interactions with both the sodium voltage-gated channel and the collapsin response mediator protein 2 (CRMP2), which promote neurite outgrowth. LCM is able to reduce neuronal excitability by a voltage-gated response. Several studies point to the binding of LCM to CRMP2, as well as the ability of LCM to directly affect the function of CRMP2 [13]. CRMPs are strongly expressed in adults and in the developing nervous system [14-16]. All CRMP proteins (CRMP 1–5) are associated with the cytoskeleton [17,18] and are important for the metastasis and invasion of cancer cells [19]. CRMP proteins have recently been implicated in a set of human cancers [20-30]. It has been reported that the neuronal autoantibody of CRMP5 is linked to patients at risk of lung carcinoma [30-32]. The role of other CRMP proteins in cancer growth still needs to be clarified. Although there is evidence that CRMP proteins play a vital role in cancer development, further exploration of the underlying mechanisms is needed.
The present work focuses on the separation of 188Re based on the use of ZrSiW gel as a new 188W/188Re generator to produce 188Re with high radiochemical yield and radionuclidic purity. LCM, a new anti-epileptic medication, is labeled with 188Re, where the 188Re-LCM complex is anticipated to be relevant as a radiotheranostic agent, the action of which exploits the relationship between the presence of CRMP proteins in cancer cells and progression of the disease. The chemical structure of LCM is shown in Fig. 1.
2 Methods
2.1 Materials
All chemicals and reagents used in this work were of high purity. All experiments were carried out using doubly distilled water. LCM (M.W. = 250,294 g/mol) was purchased from Aldrich Chemical Company. Stannous chloride dihydrate (SnCl2·2H2O, M.W. = 225.64 g/mol), was purchased from Sigma Chemical Company, USA. Albino mice weighing 30‒35 g were used for the experiments.
2.2 Preparation of 188W/188Re generator
Tungsten oxide (WO3; >99.9%) was irradiated in the water-cooled Second Egyptian Research Reactor (ERR-2) using a thermal neutron flux of 1‒5 × 1014 n cm−2 s−1 for 2 d, then cooled for 10 d to allow the decay of short-lived radioisotopes (ETR-2; 22 MW). The 188W radioactivity was around 5 mCi (185 MBq). The irradiated WO3 (0.56 g) was dissolved in 10 ml of 5 M NaOH and then diluted with 40 ml of distilled water to obtain 50 ml of 0.05 M 188W solution. A 50 ml aliquot of 0.3 M silicate solution was prepared by dissolving 1.8 g of Na2SiO3 in 50 ml of water at room temperature, and the 188W solution was then added to the silicate solution. The 188W-silicate mixture solution was then added dropwise to 50 ml of warm 0.05 M Zr(IV) solution (0.8 g Zr OCl2.8H2O dissolved in 50 ml of 0.2 M HCl, at 50 °C) over 30 min, with constant stirring. The pH of the solution was adjusted from 12.8 to 8 by adding a few drops of concentrated HCl, to form a white precipitate. The gel substance was left overnight for the precipitate to settle. Thereafter, the precipitate was separated and washed with doubly distilled water until a constant pH was reached, then dried in an electric furnace for 24 h at 50 °C [8].
2.3 Quality control of rhenium-188
2.3.1 Chemical stability
ZrSi188W gel (100 mg) was combined with 10 ml of various concentrations of HCl or NaOH and left overnight at room temperature. The ZrSi188W gel was separated by filtration and the concentrations of Zr(IV), 188W(VI), and Si(IV) in the aqueous phase were determined. Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) was used to determine the concentration of the studied elements (Zr, Si, and W) in the aqueous solutions after ensuring decay of the radioisotopes of tungsten.
2.3.2 Radiochemical purity
The 188Re chemical species in the eluted solution were identified using ascending paper chromatography (Whatman No. 3, 15 cm long and 1 cm wide) with acetone as a developing solvent. The Rf (retardation factor) was determined as follows:
2.3.3 Radionuclidic and chemical purity
The radionuclides in the eluted solution were determined immediately and after 15 days of elution to assess the 185W, 187W, and 188W breakthrough using a HPGe detector. Direct detection of 185W (T1/2 = 75.1 d) and 188W (T1/2 = 69.4 d) in the presence of 188Re is difficult, as they emit very low-intensity gamma-lines (Eγ = 125 and 290.7 keV) (Iγ = 0.019 and 0.4%, respectively), making it difficult to distinguish 185W and 188W due to the high relative gamma-intensity for 188Re (Eγ = 155 keV, Iγ = 15%) due to the Compton background. 187W (T1/2 = 23.72 h; Eγ = 134.2, 479.5 and 685.8 keV; Iγ = 8.85, 21, and 27.3%, respectively) could be identified easily and instantly with the separated 188Re.
2.3.4 Labeling procedure of LCM
A certain concentration of LCM was placed into a 10 ml clean penicillin vial with nitrogen-purged doubly distilled water. The vial was closed with an aluminum cap under positive pressure of nitrogen gas, and the necessary quantity of a newly prepared deoxygenated stannous chloride dihydrate solution was added. Finally, the eluted 188Re (37 MBq) was added to the above mixture in order to obtain a maximum radiochemical yield at the appropriate temperature for the recommended time. The various factors affecting the radiochemical yield of 188Re-LCM were investigated.
The radiochemical yield of 188Re-LCM was estimated by varying the labeling factors. This was achieved by adding 250 μl of newly prepared deoxygenated stannous chloride dihydrate (25‒350 μg; 0.45‒6.2 mM), 50 μl lacosamide (50 μg; 4 mM) dissolved in water, 50 μl of different buffer solutions from pH 2 to pH 11, and varying the reaction temperature from 25 to 100 °C and reaction time from 5 to 60 min. The highest radiochemical yield of 188Re-LCM was achieved by adding 50 μl LCM (50 μg; 4 mM), 50 μl (pH 4) citrate buffer, 250 μl of freshly prepared stannous chloride solution (250 μg; 4.4 mM), and 188Re (100 μl, 37 MBq) at 25 °C within 30 min.
2.4 Radiochemical Analysis
The labeling yield of the 188Re-LCM complex was estimated as outlined below.
2.4.1 Electrophoresis analysis
The electrophoresis technique was used to determine the type of charge carried by the 188Re-LCM complex using a Whatman No. 3 paper sheet (40 cm length × 2.5 cm width); 5 μl of the reaction mixture was placed 8 cm away from the cathode and then spontaneously evaporated. Electrophoresis was performed with phosphate buffer (0.05 M, pH 7) as the electrolyte for 90 min at a voltage of 300 V. When the mixture reached the solvent front, the paper was removed, dried and cut into strips, each strip being 1 cm long, and the strips were then counted in a well-type NaI (T1) crystal connected to the gamma-counter. 188Re-LCM remained at the spotting point and did not migrate due to the charge and ion mobility.
2.4.2 TLC analysis
The reaction mixture was filtered through a 0.22 mm Millipore filter under sufficient pressure to separate and determine the proportion of hydrolyzed 188Re and stannous hydroxide colloids, as shown in the following equation:
Secondly, the TLC silica gel sheets were marked with a line 2 cm from the bottom using a blunt pencil and cut into segments of 1‒13 cm each. After the filtration process, 5 μl of the filtered solution was spotted on the sheet and a saline solution of 0.9% NaCl was used for development in a closed jar. The sheet was removed, dried, and cut into small pieces, each piece having a width of 1 cm, and counted in a gamma-counter. 188ReO4‒ moved with the solvent front (RF = 1), while the 188Re-LCM complex remained at the baseline (RF = 0). The radiochemical yield of 188Re-LCM was calculated as follows:
2.5 Determination of Partition Coefficient
The partition coefficient was determined to evaluate the ability of radiopharmaceuticals to pass through the blood brain barrier (BBB) by in vitro screening. This experiment was carried out by mixing 188Re-LCM in a centrifuge tube with the same amounts of phosphate buffer and 1-octanol (0.025 M at pH 7.4). The mixture was shaken at room temperature for 1 min, and then centrifuged at 5000 rpm for 5 min. A 100 μl aliquot from each phase was drawn into test tubes and counted. The partition coefficient was identified as the ratio of the organic to aqueous phase. Measurements were carried out in triplicate according to the following equation; the partition coefficient is expressed as log p [33].
2.6 HPLC analysis
The radiochemical purity of 188Re-LCM was determined by direct injection of 5‒10 μl of the reaction solution into the chromatographic column (RP18 – 250 × 4 mm, 5 μm, Lischrosorb) using the HPLC Shimadzu instrument consisting of LC-9A pumps with a Rheohydron injector and UV detector (SPD-6A). Detection was performed at 210 nm. Sodium dihydrogenphosphate monohydrate at pH 3 in acetonitrile (700:300 v/v) was used at a flow rate of 1.0 ml/min, with a column temperature of 40 °C [34]. A fraction collector was used to collect the labeled complex, and the activity of the complex was measured using a NaI (Tl) well-type crystal connected to a single-channel analyzer.
2.7 Molecular mass analysis
The molecular mass of Re-LCM was measured using a Shimadzu GCMS-QP-1000EX mass spectrometer (Research and Development Center for Drug Discovery, Ain Shams University, Cairo, Egypt). The optimum conditions for preparing Re-LCM were achieved by using inactive perrhenate (ReO4‒) instead of 188ReO4‒ with the same molar ratio of LCM to perrhenate to stannous chloride (1:1:5) at pH 4. The reaction was performed for 30 min at room temperature.
2.8 Biodistribution Studies
The animal research was carried out under the supervision of the governmental research authority. The mice comprised one group of normal albino mice. The experiment was carried out by diluting the purified 188Re-LCM neutral solution with 1 ml of saline to filter the solution using a 0.22 μm Millipore filter, after which the animals were injected with the solution for the biodistribution study. Healthy albino mice, each weighing approximately 30‒35 g, were injected in the tail vein using 200 μl of the solution with a specific activity of 30 MBq 188Re per 0.002 mmol LCM. The mice were kept in a metabolic cage on ordinary diet, then sacrificed 10, 60, 120, and 240 min after injection. Fresh blood, bone, and muscle samples were collected in pre-weighed vials and counted. The various organs were separated and counted. The mean percentage of the delivered dose per organ was calculated. Blood, bone, and muscles were assumed to be 7, 10, and 40% of the total body weight, respectively [35].
3 Results and discussion
3.1 188W/188Re generator
The ZrSi188W gel was prepared for the 188W/188Re generator with a high content of tungsten (393.3 mg W/g gel) [8]. 188Re was eluted using a saline solution at a flow rate of 0.5 ml/min (0.9% NaCl); substantial removal (>90%) was obtained in the first 4 ml. The elution yield of 188Re and the 188W breakthrough were found to be 75±3 and 10‒3±2 × 10‒4%, respectively.
The ascending paper chromatography technique employing a Whatman No. 3 paper and acetone as a developing solvent was used to determine the purity of 188Re. Figure 2(a) shows a single peak corresponding to 98±0.06% radiochemical purity at Rf = ~1, related to 188ReO4‒ [8].
Gamma-ray spectrometry and radioactive decay measurements were performed using an HPGe detector linked to a multi-channel analyzer to confirm the separation of 188Re, free of any radionuclides, as shown in Fig. 2(b).
3.2 Preparation of 188Re-LCM
3.2.1 Effect of stannous chloride concentration
Figure 3(a) shows the effect of the amount of stannous chloride on the radiochemical yield of 188Re-LCM. The labeling yield of 188Re-LCM increased from 12.3±0.2 to 87.5±1.8% when the quantity of stannous chloride was increased from 25 to 250 μg. Stannous chloride exceeding 250 µg led to a decrease in the 188Re-LCM yield from 57.7±0.6 at 300 µg to 39.9±0.4% at 350 µg. The decrease in the 188Re-LCM yield with low amounts of stannous chloride may be due to insufficient stannous chloride to reduce all the rhenium(VII) (ReO4‒) to the lower oxidation states [36,37]. Most of the ligand molecules were involved in complex formation; thus, perrhenate was reduced to insoluble rhenium(IV) (ReO2.xH2O) in the absence of the ligand at high concentrations of stannous chloride [38]. In addition, the reduction reaction rate increased to produce a colloid at high concentration of Sn(II), where tin appears to be more competitive with regard to the complexation reaction, thereby reducing the labeling yield of 188Re-LCM.
3.3 Effect of pH
Figure 3(b) presents the results obtained when 188Re-LCM was prepared by varying the pH of the reaction medium. In acidic medium (pH = 2), hydrated 188ReO2 may be formed [39], which reacts with other reagents, such as SnO, to form a 188ReO2 complex during the perrhenate reduction reactions using stannous chloride [40]; this reduced the radiochemical yield of the 188Re-LCM complex to 73±1.1%. Upon increasing the pH from 2 to 4, the radiochemical yield of 188Re-LCM reached a maximum value of 87.5±1.8%. At pH greater than 8, the yield of 188Re-LCM dropped dramatically, which may be due to stannous hydroxide (Sn(OH)3) formation [36-37]. Moreover, the low yield of 188Re-LCM may be due to the fact that LCM is more readily hydrolyzed in alkaline solutions compared to acidic solutions [41].
3.4 Effect of reaction temperature
The highest labeling yield of 188Re-LCM (87.5±1.8%) was obtained at room temperature (25±3 °C), followed by a gradual decrease to 35±0.2% when the temperature of the reaction was increased to 100 °C (Fig. 3(c)). The results demonstrate that room temperature (25±3 °C) is the most appropriate temperature for formation of the 188Re-LCM complex. Although LCM is stable and does not decompose up to 70 °C for 7 d when it is exposed to dry heat [41], the radiochemical yield of 188Re-LCM decreased when the temperature was raised. This may be due to disintegration of the 188Re-LCM complex, without any effect on LCM up to 70 °C.
3.5 Effect of reaction time
The influence of the reaction time (5, 15, 30, 45, and 60 min) on the labeling yield of 188Re-LCM is shown in Fig. 3(d). The yield of 188Re-LCM increased from 72.1±1.1 to 87.5±1.8% and achieved equilibrium when the reaction time was increased from 5 to 30 min at room temperature.
3.6 Fragmentation of Re-LCM
Figure 4(a) shows the full-scan mass spectrum of Re-LCM in the range of 100–800 m/z. The MS data for Re-LCM were acquired in positive ion mode and displayed peaks at m/z values of 456/453, corresponding to the [M+H]+ ions. The fragmentation of Re-LCM produced an ion peak at m/z 439 due to the loss of H2O from the parent ion. The removal of a methoxyl and water group from the parent ion formed the fragment ion at m/z 408, which was further fragmented to generate the ion at m/z 256.
3.7 Reaction mechanism
Mass spectral analysis indicated that Re-LCM was successfully formed on the basis of the molecular mass of the parent ion (m/z = 456), and confirmed that one molecule of LCM binds with the Re(III) oxo core to form a complex. Herein, Re-LCM was prepared using a 1:1:5 molar ratio of LCM:perrhnate:stannous chloride at pH 4, at room temperature, within 30 min. This ratio was used in order to reduce Re from (VII) to (III), to facilitate reaction between one molecule of LCM and the Re(III) oxo core to form the suggested complex (Fig. 4 (b)) according to the following equations:
3.8 Stability of 188Re-LCM
The stability of 188Re-LCM was studied at room temperature (25±3°C) to determine the appropriate time for injection to avoid the formation of unwanted products, because these unwanted radioactive products can accumulate in non-target organs. Table 1 shows the stability of 188Re-LCM up to 24 h.
| Time post labeling (h) | 188Re-Lacosamide,%at room temperatureMean±S.D. |
|---|---|
| 1 | 87.5±0.7 |
| 2 | 85.1±1.3 |
| 4 | 83.8±0.9 |
| 8 | 82.4±1.6 |
| 12 | 82.3±1.1 |
| 24 | 82.0±0.7 |
3.9 In vitro stability of 188Re-LCM in serum
The in vitro stability of the 188Re-LCM complex was also studied in blood serum. In this experiment, 5 ml of blood was taken and centrifuged for 5 min at 3000 rpm; the blood serum was then separated from the blood cells. A 1.5 ml aliquot of serum was taken in a clean vial and 0.5 ml of 188Re-LCM was added. The vial was incubated at 37 °C, and the radiochemical purity of the labeled complex was checked at various time intervals (1–8 h), as shown in Table 2.
| Time post labeling (h) | 188Re-Lacosamide, % in serum at 37 °CMean±S.D. |
|---|---|
| 1 | 87.5±0.9 |
| 2 | 87.4±0.5 |
| 4 | 87.1±1.2 |
| 8 | 86.7±0.7 |
3.10 Lipophilicity
The 188Re-LCM partition coefficient was found to be 2.31±0.02, which indicates that the brain blood barrier (BBB) could be easily crossed by the target.
3.11 HPLC Analyses
Figure 4(c) displays the UV absorbance of LCM and the radio-chromatogram of 188Re-LCM. The species in the radio-labeling reaction mixture were elucidated separately at specific retention times (Rt). LCM, 188Re-LCM, and the inorganic 188Re-complex were separated at retention times of 3, 5, and 7 min, respectively. The retention time of the inorganic 188Re-complex on the RP-18 column is longer than that of 188Re-LCM, which may be because the molecular weight of the inorganic 188Re-complex is greater than the molecular weight of 188Re-LCM. The inorganic 188Re-complex may also interfere with the 188Re-LCM in vivo study. Thus, HPLC must be used to separate the undesired inorganic 188Re-complex from 188Re-LCM. The fractions containing the 188Re-LCM complex were collected and evaporated to remove the solvent under vacuum (reduced pressure). The residue was redissolved in physiological saline and filtered by using a 0.22 μm Millipore filter for sterilization. The resulting 188Re-LCM preparation was suitable for the biodistribution studies.
3.12 Biodistribution study
The in vivo behavior of 188Re-LCM was determined in mice at 10, 60, 120, and 240 min after intravenous injection. Table 3 presents the bio-evolution of the radiolabeled complex in the most relevant organs. Bio-evolution of 188Re-LCM in normal mice showed that the complex had high initial blood, muscle, and bone uptake at 10 min post injection. The clearance of 188Re-LCM from the blood was high, as the proportion reached 6.1% at 240 min after injection, which may be due to low plasma protein binding of 188Re-LCM. The present biodistribution data are consistent with the data presented by De Biase et al. [42] and Wong and Sountain [43] who reported low binding of plasma protein with LCM. 188Re-LCM is capable of crossing the blood brain barrier, resulting in significant initial brain uptake (1.9% at 10 min), maximum brain localization (5.2% at 120 min), and good retention (4.3% at 240 min post injection) in mice.
| Organs or body fluids | Time post injection (min) | |||
|---|---|---|---|---|
| 10 | 60 | 120 | 240 | |
| Brain | 1.9±0.5 | 3.8±0.3 | 5.6±0.2 | 4.3±0.1 |
| Blood | 20.3±1.0 | 13.9±0.7 | 9.0±0.6 | 6.1±0.2 |
| Liver | 15.8±0.8 | 8.3±0.6 | 7.3±0.4 | 7.5±0.3 |
| Intestine | 10.9±0.4 | 14.2±0.9 | 11.4±1.1 | 9.2±1.3 |
| Stomach | 5.1±0.4 | 7.0±0.7 | 10.3±1.0 | 9.0±0.5 |
| Lungs | 1.2±0.2 | 1.4±0.2 | 1.5±0.1 | 1.7±0.1 |
| Heart | 1.3±0.1 | 1.4±0.1 | 0.8±0.1 | 0.6±0.1 |
| Kidneys | 13.7±0.3 | 10.3±0.4 | 11.6±0.4 | 13.1±0.2 |
| Spleen | 1.1±0.1 | 0.9±0.1 | 0.8±0.1 | 0.3±0.1 |
| Muscles | 5.1±0.3 | 4.9±0.4 | 3.7±0.2 | 3.0±0.2 |
| Bones | 3.3±0.3 | 3.9±0.2 | 5.1±0.1 | 5.7±0.1 |
| Thyroid | 0.3±0.1 | 0.7±0.1 | 0.9±0.1 | 1.1±0.1 |
| Urine | 18.0±0.8 | 28.3±0.6 | 30.2±1.2 | 37.1±2.4 |
| Brain* | 4.8±0.5 | 9.5±0.3 | 14.0±0.2 | 10.8±0.1 |
| Blood* | 7.3±1.0 | 5.0±0.7 | 3.2±0.6 | 2.1±0.2 |
| (Br/Bl)* | 0.66 | 1.9 | 4.38 | 5.1 |
The maximum brain localization of 188Re-LCM is higher than that of the commercially available 99mTc-HMPAO and 99mTc-ECD, and the retention time is better than that of the recently research-proven brain imaging radiopharmacetials 99mTc-Trazadone and 99mTc-Ropinirole [44-47].
188Re-LCM was removed from circulation mainly through the urine (approximately 37.1% at 240 min after 188Re-LCM injection) [42,43,48]. With respect to the radioactivity delivered to the brain per gram tissue in the blood (target to non-target ratio), it was found that the ratio increased as time passed, reaching a maximum value of 5.1 at 240 min after injection. 188Re-LCM was mainly removed from the circulation, with low wash out from brain tissue, indicating the ability and efficiency of 188Re-LCM to localize in the brain due to its binding with CRMP2, as well as its ability to directly impact the role of CRMP2 [13]. A site of interaction between LCM and its target CRMP2 protein has not been identified; molecular modeling revealed five pockets capable of binding LCM [49]. The phenyl ring of LCM is ensconced in a cavity of CRMP2; therefore, 188Re incorporation did not affect the pharmacological properties of LCM.
4 Conclusion
188Re-LCM as a new radiolabeled agent for CRMP2 targeted cancer theranostics could be produced in a yield of 87.5±1.8% under the optimal reaction conditions using 50 μl LCM (4 mM), 250 μl stannous chloride (4.4 mM), at pH 4, with 100 μl 188Re (37 MBq), at room temperature, within 30 min. This agent is stable both in vitro and in vivo. Biodistribution study of 188Re-LCM in normal mice and calculation of the target to non-target ratio (T/NT) showed that both high uptake and long retention in the brain were achieved. Further studies on the potential of 188Re-LCM as a therapeutic agent for treating brain cancer cells are warranted in the future.
Ethical approval: All international, national, and/or institutional guidelines applicable were pursued in the care and use of animals.
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