99mTc labeled genistein kits: development methods and quality control for breast cancer radiotracer applications


 Objective.

Selective estrogen receptor modulators (SERMs) have been widely used to treat breast cancer, osteoporosis, and postmenopausal symptoms. SERMs have an affinity for estrogen receptors (ER) in target tissues and resist stimulation of the breast, bone, and endometrium. Genistein as an isoflavone compound that has a high affinity for ERβ targets makes it a potential target or prognostic marker for breast cancer. This study was carried out to develop 99mTc-genistein kit that can be used to detect breast cancer.
Methods.

The synthesis process and quality control were investigated to obtain the optimal formula for the ratio of a substance, reducing agent, optimal conditions of the synthesis reaction, physicochemical properties of the kit, and its stability to meet the requirements of radiochemical purity.
Results.

The radiochemical purity in the development of the radiopharmaceutical kit was 93.25% ± 0.30%. The physicochemical properties of the kit preparations showed hydrophilic properties, good plasma protein binding, no electrical charge, and were stable at storage temperatures.
Conclusions.

The radiochemical purity of the radiopharmaceutical kits meets the requirements of the United State Pharmacopeia and has good physicochemical properties to be developed into kits.


Introduction
The death rate caused by breast cancer globally in 2020 is 684,996 and this is predicted to increase every year 1 . Early detection of breast cancer, when it is small and has not spread, will be easier to treat successfully 2 . In addition, the sensitive method of detecting micrometastatic conditions allows therapy to be scaled up in a higher risk subset of patients, and avoids patients from potential unnecessary side effects of treatment 3 .
Genistein is an iso avones compound that is abundantly found in soybean seeds with the chemical name [5,7-dihyroxy-3-(-4-hydroxyphenyl)-4H-1-benzopyran-4-one], shown to be potentially speci c in the treatment of certain types of breast tumors 8 . Genistein shows a strong a nity for human estrogen receptor beta 9 .
The development of new radiopharmaceutical imaging kits is important for the early diagnosis of breast cancer and patient survival. Currently, more than 80% of nuclear medicine uses single-photon emission computerized tomography (SPECT) to detect cancer with radiopharmaceuticals 10 . Technetium-99m is the most preferred gamma-emitting radioisotope in the preparation of SPECT radiopharmaceuticals because it has ideal properties such as 6.02 h physical half-life, low cost, and γ-radiation energy (0.1405 meV), and ready availability 11-13 . This study aims to develop a 99m Tc-genistein kit for the early detection of breast cancer patients speci cally and accurately. In addition, we offer a less laborious method with direct labeling, of simple components with quali ed radiochemical purity. Stability of 99m Tc-genistein. The in vitro stability of the 99mTc-genistein kit was determined at room temperature. Measurements will be observed the percentage of radiochemical purity by TLC every 1 hour for 5 hours of observation time and its physical appearance.

Materials
Electric charge measurement. Determination of the charge of the 99m Tc genistein kit was carried out by electrophoresis method using Whatman no.1 paper (GE Healthcare). Whatman paper (37cm x 1cm) is marked every 1 cm apart, with 0 in the middle. On the right, the scale of 18 is written for positive values, and the anode is placed, while the scale is 18 on the left for negative values and the cathode is placed. At the zero point, the 99m Tc genistein kit is spotted and the paper was immersed in 0.02 N phosphate buffer solution, pH 7.5. The electrophoresis device was supplied with a voltage of 350 Volts, 11-16 mA. After 1 hour, Whatman paper pieces were measured by SCA.
Determination of Lipophilicity. The test was conducted by adding 2 mL of 1-octanol solution and 2 mL of 0.9% NaCl (saline solution) pH 7.4 into centrifuge tubes and adding 10-50 μL of 99m Tc-genistein solution. The solution was homogenized by vortexing for 1 minute and centrifuged at 3000 rpm for 10 minutes.
Each 100μL of the 1-octanol fraction and saline solution was counted for radioactivity by SCA. The partition coe cient was determined by calculating the radioactivity ratio of 1-octanol and saline solution.
Measurement of plasma protein binding. Determination of plasma protein binding was carried out by precipitation method using TCA solution. A total of 500 μL of blood samples were added to 50 μL of radiopharmaceutical 99m Tc genistein and homogenized by vortex for 1 minute. The mixture was incubated for 10 minutes at 37°C, then 1 mL of 0.9% NaCl solution and 1 mL of 5% TCA solution were added. This solution was centrifuged at 3000 rpm for 15 minutes. The precipitate formed is then separated from the supernatant. The supernatant solution was added again with 1 mL of TCA solution, the process of precipitation and separation was repeated. The precipitate fraction was washed again with the addition of 1 mL of 0.9% NaCl solution, centrifuged and separated again. Radioactivity of the precipitate fraction (a) and the supernatant (b) will be measured by SCA and the plasma protein binding value is calculated by the following equation.

Results And Discussion
The process of labeling genistein with 99m Tc was carried out by extracting 99m Tc from a standard generator with a solution of 0.9% NaCl solution. The chemical form of 99m Tc in the generator is Na 99m TcO 4 , which has a valence value of 7 + . At this valence, technetium cannot bind to genistein, so a reducing agent is needed. There are many studies in the synthesis of 99m Tc radiopharmaceuticals using stannous chloride as a reducing agent, because it has good reducing power and is non-toxic. In addition, stannous chloride effectively facilitates the reaction during the radiolabeling process [17][18] . The reaction of decreasing the valence number of 99m Tc with the reducing agent SnCl 2 can be explained by the following reaction 19 . The decrease in the valence of 99m Tc becomes more reactive so that it can form a 99m Tc genistein complex.
The preparation process of 99m Tc genistein contained impurities TcO 4− and TcO 2 , which could be identi ed by a simple TLC method. The TLC method was carried out with 2 plates and 2 different mobile phases, aiming to overcome radiochemical purity calculations errors due to the presence of TcO 2 and TcO 4 − impurities. The scheme for synthesizing of 99m Tc genistein compounds using the direct labeling method can be seen in Figure 1.
The optimum conditions for the labeling process of 99m Tc genistein with a concentration of SnCl 2 .2H 2 O solutions were 15 mg/mL, genistein concentration 10 mg/mL, optimum pH at 8, and incubation time of 10 minutes and obtained RCP of 93.25% ± 0.30%. These results indicate that the radiopharmaceutical formulation has met the requirements of the RCP, with a clear, colorless solution. Furthermore, this RCP value is predicted to be able to reach the target organ well so that it can emit photons to be detected on a gamma camera.
The stability test at room temperature. This test will obtain information on physicochemical changes and the RCP value of the preparation. The assay is carried out for 5 hours after incubation time, it is based on the half-life of 99m Tc 6.01 hours. Therefore, the RCP value was calculated every 1 hour, for 5 hours of observation time. The stability results showed that the 99m Tc genistein preparation met the requirements as a radiopharmaceutical kit with an RCP > 90% for 2 hours and a clear physical appearance until the 5th hour (Table 1 and gure 2). Table 1 The stability test of 99m Tc genistein at room temperature. results also showed that the 99m Tc genistein kit had the appearance of a clear, colorless, and particle-free solution for up to 5 hours of observation.
Lipophilicity value. Provides predictive information on drug permeability in cell/organ membranes and the compound`s polarity being tested to determine its pharmacokinetic and pharmacodynamic pro le.
Lipophilicity as log P according to Lipinski rules of 5 (Ro5) with a value of log P < 5 21 . The results of the lipophilicity of the 99m Tc genistein kit had a log P value of -0.77134 ± 0.12. These values indicate that this radiopharmaceutical kit is hydrophilic, can pass through the absorption mechanism well, with less active penetration into cells. Therefore, this log P result still complies with Lipinski's rule.
Electric charge. The charge of a compound can be determined by its separation and movement in an electric eld. This charge can provide information on transport mechanisms in the body. Electrophoresis showed that the 99m Tc genistein kit did not migrate and remained at the starting point (scale 0). 99m Tc genistein kits are neutral, so there is no migration of the sample. In contrast, the 99m Tc movement from cathode to anode is due has a negative charge originating from TcO 4-( gure 2).
Plasma protein binding. The amount of 99m Tc genistein bound to plasma proteins was determined by the precipitation method. This method uses a 5% TCA solution as a protein precipitation agent. The result of plasma protein binding of the 99m Tc genistein preparation was 75.29% ± 2.59%, indicating that this preparation was strongly bound to plasma proteins.