Technetium-99m-labeled genistein as a potential radical scavenging agent.

The purpose of this study was to determine the optimum conditions for labeling genistein compounds with technetium-99m (99mTc) radionuclides and the percentage of purity obtained in accordance with the requirements of the United State Pharmacopeia. The method used is optimization of several parameters including pH, SnCl2.2H2O as reducing agents, genistein concentration, and incubation time. The results showed that the optimum conditions for labeling 99mTc-Genistein were obtained under conditions of pH 8, the amount of SnCl2.2H2O reducing agents was 30 μg, 0.5 mg genistein, and in 10 min. The optimization of this condition resulted in radiochemical purity in the labeling of 99mTc-Genistein compounds at 95.43% ± 0.85%. The radiochemical purity of the labeling of 99mTc-Genistein compounds has met the requirements of the United State Pharmacopeia as a compound marked for diagnosis of more than 90%.

INTRODUCTION

Cancer is the main disease that causes the biggest death in the world and caused deaths of around 8.2 million in 2012. The biggest cause of cancer deaths every year is caused by lung cancer, breast cancer, liver cancer, stomach cancer, and colorectal cancer.[1]

In Indonesia, about 65% of people with breast cancer come to the doctor at an advanced stage; this indicates the patient is late to detect breast cancer.[2]

Radiopharmaceuticals are compounds containing radioactive elements used for diagnostic purposes and therapeutic treatments for human diseases. Radiopharmaceuticals currently used are more than 95% more for diagnostic purposes and about 5% are used for therapeutic treatment. The application of the use of a compound labeled Technetium-99m (99mTc) in nuclear medicine is very dominant, which is more than 80%.[3]

99mTc for diagnostic purposes because it can emit pure gamma rays (140.5 keV) and has a short half-life time of around 6 h. Short half-life time is expected how the radiation emitted by 99mTc can be used up immediately after the diagnosis process is complete so that the impact of radionuclide exposure can be minimized.[4]

The main requirement of radionuclides and ligands for the diagnosis of cancer is that they can bind to the receptors to form complex compounds. One of the ligands that can be used is genistein. Genistein is an isoflavone compound that has pharmacological effects as anticancer and is able to bind to estrogen receptors with the nature of the selective estrogen receptor modulators.[5]

MATERIALS AND METHODS

Paper chromatography, dose calibrator (Victoreen®), 5 μL micropipette, 10–100 μL, and 100–1000 μL (Eppendorf®), analytic balance (Mettler Toledo® Type AL 204), oven (Memmert®), Single Channel Analyzer (SCA) (ORTEC®), syringe (Terumo®), 10 mL glass vial.

The materials used are genistein (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

The determination of the optimum pH used in labeling genistein with 99mTcO4- used is pH 3; 4; 5; 6; 7; 7.5; 8; 9; and 10. Stock genistein solution is added with a solution of SnCl2·2H2O, and the pH is adjusted by adding NaOH or HCl. After pH is obtained, then a 99mTcO4- solution is added. The solution was formed, then it was dropped on the KLT SGF-254 and ITLC-SG plates to determine the purity of the 99mTcO4- Genistein complex.[6]

Optimization of concentration SnCl2.2H2O solution

The determination of the optimum concentrations of SnCl2.2H2O solution was used five variations in the concentration of SnCl2.H2O: 10, 20, 30, 40, and 50 μL. After adding 100 μL of genistein solution, each solution is adjusted to optimum pH. After that, each solution was added with a solution of 99mTcO4- as much as 500 μL, then, the volume of the vial was equated with the addition of NaCl physiological solution to 2 mL and incubated for 30 min. The solution was formed, then dropped on the KLT SGF-254 and ITLC-SG plates to determine the purity of the 99mTc-Genistein complex.[6]

Optimization of concentration genistein

The determination of optimum genistein concentrations used nine variations in genistein: 1; 2; 3; 4; 5; 6; 7; 7.5; and 10 mg/mL. Each solution was added with a solution of SnCl2.2H2O, and also added 99mTcO4- as much as 500 μL. After that, NaCl physiological solution was added to 2 mL and incubated for 30 min. Checking the purity of 99mTc-Genistein complexes was done by dripping each solution on the KLT SGF-254 and ITLC-SG plates.

Optimization of incubation time

Determination of the optimum incubation time on marking genistein with 99mTcO4- used five variations of incubation time, namely 5, 10, 15, 30, and 45 min. Each solution of genistein added a solution of SnCl2.2H2O in accordance with the optimum results. The pH conditions are adjusted according to the optimum pH that has been obtained. After that, each solution was added with a solution of 99mTcO4- and NaCl physiological solution. Checking the purity of 99mTc-Genistein complexes was done by dripping each solution on the KLT SGF-254 and ITLC-SG plates.[6]

The purity percentage of 99mTc-genistein compounds

The purity of the compound marked 99mTc-Genistein was determined using the TLC method which was then analyzed using SCA. The stationary phase used is the KLT SGF-254 and ITLC-SG plates. For the mobile phase, two solvents are used, namely C1 solution consisting of ethanol: water: ammonia (2: 5: 1) and NaCl physiological solution.[7]

The purity percentage of a compound labeled 99mTc-Genistein is calculated based on the percentage of 99mTcO4- and 99mTcO2(impurity) using the following equation.[8]

Calculation of labeled compounds 99mTc-Genistein:

%99mTc-Genistein = 100% − (%99mTcO2+ %99mTcO4-)

RESULTS

Testing of optimum pH conditions

The pH factor is important in the stability of compounds marked and can affect the reduction power of the reducing agent used, namely SnCl2.2H2O.[5] The results for pH optimization testing and radiochemical purity of 99mTc-genistein labeled compounds are shown in Table 1 and Figure 1.

Table 1

The Optimum pH and Radiochemical Purity of 99mTc Genistein Labeled Compounds

pH	Average Radiochemical Purity (%)			Description
	Polluter 99mTcO2	Polluter 99mTcO4-	99mTc-Genistein
3	13.23±0.46	3.38±1.74	83.40±2.08	cloudy
4	13.88±0.77	1.46±0.07	84.66±0.89	cloudy
5	10.77±0.66	3.83±1.18	85.40±1.56	cloudy
6	10.45±1.20	3.59±0.78	85.96±1.65	cloudy
7	9.28±0.58	2.09±0.79	88.64±1.13	cloudy
7,5	7.19±1.94	2.98±1.73	89.83±3.01	cloudy
8	3.05±0.45	4.98±1.16	91.97±1.43	clear, yellowish
9	7.59±0.96	11.47±1.16	80.94±1.74	clear, yellowish
10	9.72±0.40	9.90±1.15	80.38±1.40	clear, yellowish

Figure 1

The optimum pH and radiochemical purity of 99mTc genistein labeled compounds

Test for optimum concentration of SnCl2.2H2O

SnCl2 is reducing agent when in the form of Sn2+ Sn2+ is often used to reduce the level of reduction in medium with a low level of toxicity.[9] The results for the optimization of SnCl2.2H2O solutions and radiochemical purity of 99mTcgenistein labeled compounds are shown in Table 2 and Figure 2.

Table 2

The Optimum Concentration of SnCl2.2H2O and Radiochemical Purity of 99mTc genistein labeled compounds

SnCl2·2H2O solution	Average Radiochemical Purity (%)			Description
	Polluter 99mTcO2	Polluter 99mTcO4-	99mTc-Genistein
10 μl	7.02±1.15	5.36±1.37	87.62±2.06	clear, yellowish
20 μl	8.08±1.71	5.05±1.74	86.87±2.82	clear, yellowish
30 μl	5.06±1.13	4.10±0.94	90.84±2.38	clear, yellowish
40 μl	8.76±2.11	3.25±0.94	87.99±2.67	clear, yellowish
50 μl	15.51±1.63	4.26±0.69	80.22±2.05	clear, yellowish

Figure 2

The optimum SnCl2 solution and radiochemical purity of 99mTc genistein labeled compounds

Optimum concentration of genistein

Determination of the third parameter is the concentration of genistein solution, the concentration used is 1; 2; 3; 4; 5; 6; 7; 7.5; and 10 mg/mL. Ligands used to make radiopharmaceutical kits, should have a purity of 99% for other marked compounds derived from ligand impurities.[910] The result for optimization concentration of genestein and radiochemical purity of 99mTc-genistein labeled compounds are shown in Table 3 and Figure 3.

Table 3

The Optimum Concentration of Genistein Solution and Radiochemical Purity of 99mTc-Genistein Labeled compounds

Concentration of Genestein	Average Radiochemical Purity (%)			Description
	Polluter 99mTcO2	Polluter 99mTcO4-	99mTc-Genistein
1 mg/mL	3.49±0.39	27.00±0.37	69.51±0.62	clear, yellowish
2 mg/mL	6.28±0.19	13.99±0.05	79.73±0.23	clear, yellowish
3 mg/mL	4.39±2.52	10.13±2.03	85.48±3.74	clear, yellowish
4 mg/mL	7.35±0.58	4.71±0.91	87.94±1.25	clear, yellowish
5 mg/mL	4.07±1.28	3.74±0.97	92.19±1.86	clear, yellowish
6 mg/mL	7.39±0.58	2.67±0.25	89.94±0.73	cloudy
7 mg/mL	8.86±0.16	2.36±0.75	88.78±0.88	cloudy
7.5 mg/mL	0.73±0.52	5.34±0.72	93.92±1.03	cloudy
10 mg/mL	0.83±0.70	5.15±0.70	94.02±1.15	cloudy

Figure 3

The optimum concentration of genestein and radiochemical purity of 99mTc genistein labeled compounds

The ideal incubation condition is at room temperature because to facilitate the preparation of compounds marked. The incubation time variations carried out were 5, 10, 15, 30, and 45 min. Variation in incubation time is needed to determine the optimum time for Sn2+ to reduce 99mTcO4- to 99mTcO2. The result for optimization of incubation time and radiochemical purity of 99mTc-genistein labeled compounds are shown in Table 4 and Figure 4.

Table 4

The Optimum Incubation Time of Genistein Solution and Radiochemical Purity of 99mTc-Genistein Labeled Compounds

Incubation time (min)	Average Radiochemical Purity (%)			Organoleptis
	Polluter 99mTcO2	Polluter 99mTcO4-	99mTc-Genistein
5	1.30±0.71	4.39±1.17	94.31±1.58	clear, yellowish
10	0.57±0.14	4.00±0.72	95.43±0.85	clear, yellowish
15	0.94±0.41	4.90±1.14	94.16±1.39	clear, yellowish
30	1.08±0.45	4.34±1.34	94.58±1.64	clear, yellowish
45	1.22±0.42	5.88±1.39	92.90±1.67	clear, yellowish

Figure 4

The optimum incubation time and radiochemical purity of 99mTc genistein labeled compounds

DISCUSSIONS

99mTc radionuclides can form complex compounds with other elements into radiopharmaceutical preparations. The radiopharmaceutical preparation can be used for diagnosis. After being given to patients, radiopharmaceutical preparations can localize certain organs so that imaging can be carried out in the body based on cell function and physiology.[4] Tc can bind to the ligand to form a complex bond that has an electron donor group. The structure of genestein can be seen in Figure 5.[6]

Figure 5

Structure of genestein

The purity test of a 99mTc-Genistein compound can be analyzed by the TLC method. The stationary phase used is thin-layer chromatography TLC SG F254 with the mobile phase of physiological NaCl solution to separate the form of 99mTcO4-, 99mTcO4- impurity will move toward the peak while 99mTc-Genistein will remain at the bottling point and ITLC-SG stationary phase with C1 mobile phase to separate 99mTcO2. The 99mTcO2 impurity will remain at the bottling point, and 99mTc-Genistein will move towards the peak.[11]

The results at pH 8 showed that the optimum purity of 99mTc-Genistein was 91.97% ± 1.43% with impurity of 99mTcO2 3.05% ± 0.45% and impurity 99mTcO4- 4.98% ± 1.16%. At a pH which is more basic than 8, it produces a higher 99mTcO4- impurity, because at a higher pH, Sn (II) will be hydrolyzed to stano hydroxide which is no longer functioning as a reducing agent. Whereas in more acidic pH conditions, it will increase the amount of impurity 99mTcO2 because the reducing agent SnCl2.2H2O will reduce more strongly in acidic conditions.[91012]

The results obtained on the optimum number of SnCl2.2H2O solutions were 30 μl with a purity of 90.84% ± 2.38% with impurities of 99mTcO2 5.06% ± 1.13% and impurities 99mTcO4- 4.10% ± 0.94%. If the amount of the solution of SnCl2.2H2O is more partial, SnCl2 is hydrolyzed to form its hydroxide which will then bind to 99mTc-reduced to form 99mTcO2 colloid, so that the amount of impurity will be more 99mTcO2.[13]

The optimization of genistein concentration was obtained at 10 mg/mL, but the solution produced was cloudy, as well as at 6, 7, and 7.5 mg/mL. Requirements for intravenous injection preparation, the solution produced must be clear. Therefore, the optimum concentration used was a concentration of 5 mg/mL, with a purity of 92.19% ± 1.86%.

The incubation time is strongly related to the preparation of a radiopharmaceutical preparation to be used. This is related to the effectiveness of the half-life of radionuclide used, and the purpose of therapy or diagnosis. The incubation time obtained is ideal for the application of 10 min. In the next minute, there was a relatively low decrease in purity and was not significant. The optimization of incubation time of 99mTc-Genistein was obtained 10 min with optimum purity of 99mTc-Genistein, which was 95.43% ± 0.85% with impurity of 99mTcO2 0.57% ± 0.14% and impurity 99mTcO4- 4.00% ± 0.72%.

CONCLUSIONS

The optimum condition for marking 99mTc-Genistein produced radiochemical purity of 95.43% ± 0.85%. This fulfills the radiochemical purity requirements set by the United State Pharmacopeia as marked compounds for diagnosis of more than 90%.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.