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ORIGINAL ARTICLE
Year : 2020  |  Volume : 11  |  Issue : 2  |  Page : 59-63  

The optimization method for synthesis of technetium-99m-luteolin as radiotracer in the development of cancer drugs from flavonoid


1 Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Padjadjaran University, Sumedang, West Java, Indonesia
2 Applied Nuclear Science and Technology Center (PSTNT), National Atomic Energy Agency (BATAN), Bandung, West Java, Indonesia

Date of Submission29-Oct-2019
Date of Decision30-Dec-2019
Date of Acceptance22-Jan-2020
Date of Web Publication22-Apr-2020

Correspondence Address:
Mr. Danni Ramdhani
Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Padjadjaran University, Sumedang 45363, West Java
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/japtr.JAPTR_161_19

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  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 a99mTc-Luteolin radiochemical purity yield of 94.15%. The radiochemical purity percentage of the99mTc-Luteolin compound has fulfilled the requirements listed at United States Pharmacopeia, which is ≥90%.

Keywords: Luteolin, radiochemical purity, radiotracer, technetium-99m, technetium-99m-genistein


How to cite this article:
Ramdhani D, Sriyani ME, Nabila S F. The optimization method for synthesis of technetium-99m-luteolin as radiotracer in the development of cancer drugs from flavonoid. J Adv Pharm Technol Res 2020;11:59-63

How to cite this URL:
Ramdhani D, Sriyani ME, Nabila S F. The optimization method for synthesis of technetium-99m-luteolin as radiotracer in the development of cancer drugs from flavonoid. J Adv Pharm Technol Res [serial online] 2020 [cited 2020 Sep 29];11:59-63. Available from: http://www.japtr.org/text.asp?2020/11/2/59/283073




  Introduction Top


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 (99m Tc). This radioisotope can bind to certain molecules, allowing the diagnosis of many diseases, including certain types of cancer.99m Tc 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 of99m Tc-genestein compounds which have the potential as radiotracer compounds for cancer.


  Materials and Methods Top


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. 99m TcO4 (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 the99m TcO4 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,99m TcO4 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 of99mTc luteolin labeled compounds

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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 with99m TcO4. 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

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Figure 1: The optimum of SnCl2.2H2O and radiochemical purity of Technetium-99m Luteolin

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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, and99m TcO4- amount of 500 μL. Two milliliters of physiological NaCl was added to each solution and then incubated for 20 min. Evaluation of the99m Tc-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 with99m TcO4 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 of99m Tc-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 of99m Tc-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 of99m Tc-Luteolin compounds is as follows.[9]





Calculation of labeled compounds technetium-99m-Luteolin

%99m Tc-Luteolin = 100% − (%99m TcO2+ %99m TcO4).


  Results and Discussion Top


The99m Tc-Luteolin compound is formed from a coordinated covalent bond between99m Tc as a metal and luteolin as a marker or ligand compound. The99m Tc-Luteolin complex formed through the following reactions:

Luteolin + Sn2++99mTC(VII)O4

99mTC(IV)Luteolin + Sn4+ +99mTcO4

In this reaction, the complex compound99m Tc-Luteolin is produced as the main product, and also99m TcO2 and99m TcO4 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 the99m Tc-genestein complex involves electron donors. The structure of luteolin is shown in [Figure 2].
Figure 2: Chemical structure of luteolin[11]

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The radiochemical purity test of a99m Tc-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 of99m TcO4−, 99m TcO4 impurity will move toward the peak, while99m Tc-genistein will remain at the spot point, ITLC-SG stationary phase with C1 mobile phase will separate99m TcO2. The99m TcO2 impurity will remain at the spot point, and99m Tc-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

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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 produced99m TcO2 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 of99m Tc-Luteolin. The amount of SnCl2 that is too little cannot reduce99m Tc7 + well and hence that it will produce a lot of TcO4. Meanwhile, if the amount of SnCl2 is excessive, it can produce a lot of99m Tc4 + 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 of99m TcO2 (5.06% ± 1.13%), and99m TcO4 (4.10% ± 0.94%). If the amount of the solution of SnCl2.2H2O is more partial, hydrolyzed SnCl2 forms its hydroxide, and binds with99m Tc-reduced to form99m TcO2 colloid, so that the amount of impurity will be more99m TcO2.

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 marked99m Tc-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

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Figure 4: The optimum of luteolin and radiochemical purity of technetium-99m luteolin labeled compounds

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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 marked99m Tc-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 marked99m Tc-luteolin. Adequate contact time can affect the reaction between SnCl2,99m Tc 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

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Figure 5: The optimum of incubation time and radiochemical purity of technetium-99m luteolin labeled compounds

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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 Top


Optimization of the conditions in the labeling of the99m Tc-Luteolin compound obtained a 94.15% radiochemical purity, where this condition fulfills USP requirements, which must be ≥90%.[14]

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.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
World Health Organisation: International Agency for Research on Cancer. Latest Global Cancer Data: Cancer Burden Rises to 18.1 Million New Cases and 9.6 Million Cancer Deaths in 2018, The United Nations; 2018. Available from: https://who.int/cancer/prglobocanfinal.pdf. [Last accessed on 2019 Oct 30].  Back to cited text no. 1
    
2.
Wu CC, Fang CY, Hsu HY, Chuang HY, Cheng YJ, Chen YJ, et al. EBV reactivation as a target of luteolin to repress NPC tumorigenesis. Oncotarget 2016;7:18999-9017.  Back to cited text no. 2
    
3.
Jang S, Kelley KW, Johnson RW. Luteolin reduces IL-6 production in microglia by inhibiting JNK phosphorylation and activation of AP-1. Proc Natl Acad Sci U S A 2008;105:7534-9.  Back to cited text no. 3
    
4.
Chowdhury AR, Sharma S, Mandal S, Goswami A, Mukhopadhyay S, Majumder HK. Luteolin, an emerging anti-cancer flavonoid, poisons eukaryotic DNA topoisomerase I. Biochem J 2002;366:653-61.  Back to cited text no. 4
    
5.
IAEA. Technetium-99m Radiopharmaceuticals: Status and Trends. Viena: IAEA; 2009.  Back to cited text no. 5
    
6.
Phaniendra A, Jestadi DB, Periyasamy L. Free radicals: Properties, sources, targets, and their implication in various diseases. Indian J Clin Biochem 2015;30:11-26.  Back to cited text no. 6
    
7.
Komárek P, Kleisner I, Komárková I, Konopková M. The use of redox polymers in labelling procedures of proteins and peptides with 99mTc. II. Technique of preparation of kits for protein labelling by 99mTc and its effect on the stability and radiochemical purity. Nucl Med Rev Cent East Eur 2000;3:69-72.  Back to cited text no. 7
    
8.
Misyetti. Instability study of radiopharmaceutical dry kits signed99m Tc viewed from chemical and physical aspects. Indonesia J Nucl Sci Technol 2006;7:65-81.  Back to cited text no. 8
    
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Owunwanne A, Patel M, Sadek S. The Handbook of Radiopharmaceuticals. London: Chapman and Hall Medical; 2012.  Back to cited text no. 9
    
10.
Bandyopadhyay M. Incorporation of herbs into Sandesh, an Indian sweet dairy product, as a source of natural antioxidants. Int J Dairy Technol 2007;60:228-33.  Back to cited text no. 10
    
11.
Lin Y, Shi R, Wang X, Shen HM. Luteolin, a flavonoid with potential for cancer prevention and therapy. Curr Cancer Drug Targets 2008;8:634-46.  Back to cited text no. 11
    
12.
Sayed HJ, Amirhossein A, Reza T. Preparation and Biodistribution Study of Technetium-99m-Labeled Quersetin as a Potential Radical Scavenging Agent. Budapest, Hungary: Springer Science; 2006.  Back to cited text no. 12
    
13.
Gopal SB. Fundamental of Nuclear Pharmacy. 5th ed. New York: Springer; 2003. p. 83, 113, 114, 288.  Back to cited text no. 13
    
14.
United States Pharmacopeial Convention. Official Monographs: USP 28. Technetium 99m Tc-Pertechnetate Injection (Sodium). United States Pharmacopeia (USP) 28-National Formulary (NF) 23, Rockville, Marland, USA., 2005. p. 1861.  Back to cited text no. 14
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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