Abstract
The aim of this study is to find the optimum conditions of labeling luteolin flavonoid compounds with technetium-99m (99mTc) to meet the purity requirements stated in the United States Pharmacopeia. This compound is expected to be a potential radiotracer compound for diagnosing cancer. The optimization method in labeling luteolin with technetium determines the parameters such as pH, SnCl2.2H2O, genistein concentration, and incubation time. Optimization results of Technetium-99m-luteolin labeling obtained optimum pH conditions 8, the amount of SnCl2.2H2O as a reducing agent 60 μL, the optimum amount of luteolin 6 mg/ml, and the optimum incubation time is 30 min. This optimum condition obtained a 99mTc-Luteolin radiochemical purity yield of 94.15%. The radiochemical purity percentage of the 99mTc-Luteolin compound has fulfilled the requirements listed at United States Pharmacopeia, which is ≥90%.
Keywords: Luteolin, radiochemical purity, radiotracer, technetium-99m, technetium-99m-genistein
INTRODUCTION
Cancer is a term that describes a disease with a condition where there is uncontrolled cell growth that goes beyond its habits and can spread and attack to other organs. In 2018, cancer is estimated to be the second leading cause of death in the world with 9.6 million cases.[1]
Luteolin is a compound conjugate acid of a 2-(3,4-dihydroxyphenyl)-5-hydroxy-4-oxo-4H-chromen-7-olate luteolin-7-olate (1-). Luteolin is one of the potential flavonoids that has been proven to be efficacious as an antioxidant, anti-inflammatory, and for cancer treatment.[2,3] Luteolin is reported to be able to inhibit the catalytic activity of topoisomerase 1 which is a cancer-inhibiting mechanism.[4]
Radiopharmaceuticals are radioisotopes conjugated with biological molecules capable of targeting organs or cells in specific tissues. This radioactive drug can be used for diagnosis and increasingly, for the treatment of diseases. The most widely used radioisotope in diagnostic nuclear medicine is technetium-99m (99mTc). This radioisotope can bind to certain molecules, allowing the diagnosis of many diseases, including certain types of cancer. 99mTc is a radioisotope that can emit pure gamma rays with energy of 140.5 keV, which has a short half-life of about 6 h, does not emit charged particle radiation, can be obtained in the form of free carrier, and can bind to many compounds.[5]
The formation of compounds labeled between radioisotopes and ligands for cancer diagnosis purposes must have the specificity to recognize a receptor. Luteolin is an isoflavone compound that has been shown to have anticancer activity through the inhibitory mechanism of topoisomerase I catalytic activity. In humans, the levels of topoisomerase I have been shown to be elevated in colorectal tumors compared to normal colon mucosa.[6]
The purpose of this research was to determine the optimization parameters for the synthesis of 99mTc-genestein compounds which have the potential as radiotracer compounds for cancer.
MATERIALS AND METHODS
The tools used in this study were a set of paper chromatography, dose calibrator (Victoreen®), micropipette (Eppendorf®), analytic balance (Mettler Toledo® Type AL204), oven (Memmert®), single channel analyzer (SCA) (ORTEC®), and syringe (Terumo®).
The materials used were luteolin (Sigma-Aldrich®), acetone (Merck®), aquabidestilata (IKA Pharma®), DMSO, HCl 0.1 N, Na. 99mTcO4− (PT. Ansto), Physiological NaCl (IKA Pharma®), NaOH 0.1 N, universal pH indicator (Merck®), KLT SGF-254 (Merck®) plate, instant thin layer chromatography-silica gel (ITLC-SG) (Agilent Technologies®), and SnCl2.2H2O plates (Sigma-Aldrich®).
Optimization of pH
Determination of the optimum pH conditions on the 99mTcO4− labeling method was carried out using five variations of pH 6, 7, 8, 9, and 10. The composition of each formula is shown in Table 1. Luteolin solution was added with 60 mL of SnCl2.2H2O solution, then the pH of the solution was adjusted by 0.1N NaOH or 0.1N HCl. After the pH was reached, 99mTcO4− the solution was added, and the solution was incubated for 30 min. The solution formed was tested for the purity of the complex formed by dropping it on the thin layer chromatography (TLC) plate SGF-254 and ITLC-SG.[7]
Table 1.
The optimum pH and radiochemical purity of 99mTc luteolin labeled compounds
| pH | Luteolin (μL) | NaOH 0.1N (μL) | TcO4− (μL) | Physiological NaCl (μL) | Polluter | Radiochemical purity (%) | Description | |
|---|---|---|---|---|---|---|---|---|
| 99mTcO2 | 99mTcO4− | |||||||
| 6 | 100 | 5 | 500 | 335 | 2.29 | 1.25 | 96.46 | Cloudy |
| 7 | 100 | 10 | 500 | 330 | 2.70 | 0.16 | 97.15 | Cloudy |
| 8 | 100 | 15 | 500 | 325 | 5.56 | 2.63 | 91.81 | Clear |
| 9 | 100 | 20 | 500 | 320 | 4.19 | 6.41 | 89.40 | Clear |
| 10 | 100 | 25 | 500 | 315 | 0.37 | 91.92 | 7.72 | Clear |
Optimization of SnCl2.2H2O
In determining the optimum conditions for SnCl2H2O, there is an additional treatment in the form of a vial vacuum. It aims to prevent oxidation by O2 before reacting with 99mTcO4−. Determination of the amount of SnCl2.2H2O as a reductor is done using six variations of the amount of SnCl2, namely 40, 50, 60, 70, 80, and 90 μL with the formula as shown in Table 2 and Figure 1. The test is carried out at the optimum pH, which is pH 8.[8]
Table 2.
Composition of formulas for optimization reducing agent of SnCl2.H2O
| Luteolin (μL) | NaOH 0.1N (μL) | SnCl2 (μL) | TcO4− (μL) | Polluter | Radiochemical purity (%) | Description | |
|---|---|---|---|---|---|---|---|
| 99mTcO2 (%) | 99mTcO4− (%) | ||||||
| 100 | 15 | 40 | 500 | 3.61 | 8.42 | 87.97 | Clear |
| 100 | 15 | 50 | 500 | 2.03 | 1.11 | 96.86 | Clear |
| 100 | 15 | 60 | 500 | 2.51 | 0.69 | 96.80 | Clear |
| 100 | 20 | 70 | 500 | 3.65 | 1.33 | 95.02 | Clear |
| 100 | 20 | 80 | 500 | 4.09 | 1.51 | 94.40 | Clear |
| 100 | 20 | 90 | 500 | 3.66 | 8.54 | 87.91 | Clear |
Figure 1.
The optimum of SnCl2.2H2O and radiochemical purity of Technetium-99m Luteolin
Optimization of concentration luteolin
The optimization conditions of the luteolin using five variations of concentration: 3, 4, 5, 6, and 7 mg/mL.
The SnCl2.2H2O reducing agent was added, and 99mTcO4- amount of 500 μL. Two milliliters of physiological NaCl was added to each solution and then incubated for 20 min. Evaluation of the 99mTc-Genistein complex formed is evaluated by measuring the radiochemical purity by dripping each solution on the KLT SGF-254 and ITLC-SG plates.[7]
Optimization of incubation time
The procedure for determining the optimum incubation time for luteolin marking with 99mTcO4− used five variations of incubation time, namely 0, 15, 30, 45, and 60 min. Vials that already contain 100 μL luteolin solution and SnCl2.H2O solution are then adjusted to the optimum pH, which is pH 7. The determination of the purity of 99mTc-Luteolin was tested with TLC SGF-254 and ITLC-SG plates.[7]
Calculation of technetium-99m-luteolin purity percentage
The method for testing the purity of compounds of 99mTc-Luteolin uses TLC and is measured by SCA. The method consists of a stationary phase TLC SGF-254 and ITLC-SG plates. The mobile phase is ethanol: water: ammonia (2: 5: 1) and NaCl physiological solution. This mobile phase solution is called C1.[7]
The equation for calculating the purity of 99mTc-Luteolin compounds is as follows.[9]
Calculation of labeled compounds technetium-99m-Luteolin
%99mTc-Luteolin = 100% − (%99mTcO2+ %99mTcO4−).
RESULTS AND DISCUSSION
The 99mTc-Luteolin compound is formed from a coordinated covalent bond between 99mTc as a metal and luteolin as a marker or ligand compound. The 99mTc-Luteolin complex formed through the following reactions:
Luteolin + Sn2++99mTc(VII)O4
→99mTc(IV) Luteolin + Sn4+ +99mTcO4
In this reaction, the complex compound 99mTc-Luteolin is produced as the main product, and also 99mTcO2 and 99mTcO4 − compounds which are radiochemical impurities.[9]
Labeling of luteolin using radioactivity makes it possible to monitor luteolin compounds in the body using a gamma camera that can read radiation exposure provided by technetium. Luteolin is known as a good antioxidant and induces apoptosis in tumor cells and has a valuable effect in cancer prevention and therapy. Luteolin can inhibit the formation of superoxide, namely by inhibiting the activity of xanthine oxidase. In addition, luteolin can also provide antioxidant effects by blocking or producing more endogenous antioxidants such as catalase, glutathione-S-transferase, and glutathione reductase. Therefore, this compound is expected to function as a potential radical scavenging radiotracer.[10] The formation of the 99mTc-genestein complex involves electron donors. The structure of luteolin is shown in Figure 2.
Figure 2.
Chemical structure of luteolin[11]
The radiochemical purity test of a 99mTc-Genistein can be measured by the thin layer chromatography method. The stationary phase used is TLC SGF-254 with the mobile phase of physiological NaCl solution. The separation of 99mTcO4−, 99m TcO4− impurity will move toward the peak, while 99mTc-genistein will remain at the spot point, ITLC-SG stationary phase with C1 mobile phase will separate 99mTcO2. The 99mTcO2 impurity will remain at the spot point, and 99mTc-genistein will move toward the peak.[9]
Test results on pH optimization
pH conditions are very influential in the formation of the Tc-Luteolin complex. In addition, the pH conditions will determine the optimum conditions of the SnCl2.2H2O reducing agent.[7] The results for pH optimization testing are shown in Table 1 and Figure 3.
Figure 3.
The optimum pH and radiochemical purity of technetium-99m-luteolin labeled compounds
The optimum pH was obtained at pH 7, where the highest radiochemical purity was obtained, which was 97.15%. Under an alkaline pH condition of 8, more TcO4 impurities will be produced. At high pH conditions, Sn (II) will be hydrolyzed, and hence that the ability as a reducing agent decreases. In acidic pH conditions, more impurities will be produced 99mTcO2 because the reducing agent SnCl2.2H2O will reduce more strongly in acidic conditions.[7,8,12]
Test for optimum concentration of SnCl2.2H2O
The amount of SnCl2 reducing agent used must be sufficient for the reaction to run well. The amount of impurities TcO4− and TcO2− will affect the purity parameters of 99mTc-Luteolin. The amount of SnCl2 that is too little cannot reduce 99mTc7 + well and hence that it will produce a lot of TcO4−. Meanwhile, if the amount of SnCl2 is excessive, it can produce a lot of 99mTc4 + which will increase the amount of TcO2− impurity.[13] The results for the optimization of SnCl2.2H2O solutions are shown in Table 2 and Figure 1.
Optimization conditions of SnCl2.2H2O solutions were 30 μl with a purity of 90.84% ± 2.38%, with impurities of 99mTcO2 (5.06% ± 1.13%), and 99mTcO4− (4.10% ± 0.94%). If the amount of the solution of SnCl2.2H2O is more partial, hydrolyzed SnCl2 forms its hydroxide, and binds with 99mTc-reduced to form 99mTcO2 colloid, so that the amount of impurity will be more 99mTcO2.
Test for the optimum concentration of luteolin
The number of ligands (Luteolin) that are not optimal will cause an increase in the number of impurities and reduce the percentage purity of compounds marked 99mTc-luteolin. In testing this parameter, five variations of the amount of luteolin are used: 3, 4, 5, 6, and 7 mg/mL and with the formula shown solutions are in Table 3 and Figure 4.
Table 3.
Composition of formulas for optimization of concentration of luteolin
| Luteolin (mg/mL) | pH | SnCl2 (μL) | NaOH 0.1 N (μL) | Polluter | Radiochemical purity (%) | Description | |
|---|---|---|---|---|---|---|---|
| 99mTcO2 (%) | 99mTcO4− (%) | ||||||
| 3 | 8 | 60 | 10 | 8.14 | 1.99 | 89.88 | Clear |
| 4 | 8 | 60 | 10 | 7.12 | 1.92 | 90.96 | Clear |
| 5 | 8 | 60 | 10 | 5.63 | 0.80 | 93.58 | Clear |
| 6 | 8 | 60 | 15 | 5.64 | 0.21 | 94.15 | Clear |
| 7 | 8 | 60 | 15 | 3.27 | 32.54 | 64.14 | Clear |
Figure 4.
The optimum of luteolin and radiochemical purity of technetium-99m luteolin labeled compounds
Optimization of the amount of luteolin with the highest percentage of purity is found in the amount of luteolin 6 mg/mL with a percentage of 94.15%. The use of luteolin amounts < 6 mg/mL will reduce the percentage purity of compounds marked 99mTc-Luteolin as the amount of TcO2− impurities increases.[9,12]
Test for optimum incubation time
The duration of incubation time can affect the optimum formation of compounds marked 99mTc-luteolin. Adequate contact time can affect the reaction between SnCl2, 99mTc and luteolin. Variations of incubation time used in this study were 0, 15, 30, 45, and 60 min.[12] The formula is shown in Table 4 and Figure 5.
Table 4.
Composition of formulas for determining optimization of incubation time
| Incubation time (min) | pH | SnCl2 (μL) | NaOH 0.1 N (μL) | Polluter | Radiochemical purity (%) | Description | |
|---|---|---|---|---|---|---|---|
| 99mTcO2 (%) | 99mTcO4− (%) | ||||||
| 0 | 8 | 60 | 10 | 3.31 | 0.88 | 95.81 | Clear |
| 15 | 8 | 60 | 10 | 6.60 | 2.73 | 91.84 | Clear |
| 30 | 8 | 60 | 10 | 5.09 | 1.07 | 93.84 | Clear |
| 45 | 8 | 60 | 15 | 5.23 | 1.38 | 93.40 | Clear |
| 60 | 8 | 60 | 15 | 4.20 | 1.40 | 94.41 | Clear |
Figure 5.
The optimum of incubation time and radiochemical purity of technetium-99m luteolin labeled compounds
Optimization of incubation time was obtained at 30 min, with a purity percentage of 93.84%. This is because at the incubation time of 30 min is obtained at least from the amount of TcO4− impurities.
CONCLUSIONS
Optimization of the conditions in the labeling of the 99mTc-Luteolin compound obtained a 94.15% radiochemical purity, where this condition fulfills USP requirements, which must be ≥90%.[14]
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgments
The author also expresses the deepest gratitude and appreciation thanks to Fairuz Nabilah Syafarina and Chair of the Center for Applied Nuclear Science and Technology (PSTNT), National Nuclear Energy Agency, Bandung, for collaboration in this research.
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