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

doi:10.21203/rs.3.rs-1009713/v1

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Research Article

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

https://doi.org/10.21203/rs.3.rs-1009713/v1

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posted 04 Nov, 2021

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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.

Cancer Biology

Clinical Pharmacology

Technetium-99m

genistein

kit

breast cancer

estrogen receptors β

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 successfully². 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 treatment³.

Selection of alternative treatments and predictive factors for breast cancer prognosis that are currently widely used include estrogen receptor-positive (ER+), carcinoembryonic antigen (CEA), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1). The evaluation of clinical variables, such as nodal involvement, tumor size, histological type, tumor grade, and surgical margins ^[4–6]. The active ER signal stimulates cell proliferation, and accounts for 75% of all diagnosed breast cancers⁷.

Genistein is an isoflavones 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 specific in the treatment of certain types of breast tumors⁸. Genistein shows a strong affinity for human estrogen receptor beta⁹.

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 radiopharmaceuticals¹⁰. 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 ^99mTc-genistein kit for the early detection of breast cancer patients specifically and accurately. In addition, we offer a less laborious method with direct labeling, of simple components with qualified radiochemical purity.

Materials

Genistein and SnCl₂·2H₂O were purchased from Sigma-Aldrich, and the solvents used were purchased from Merck.

Instant thin layer chromatography‑silica gel (ITLC‑SG) (Agilent, Technologies), dose calibrator (Victoreen), Single Channel Analyzer (ORTEC). The Technetium-99m radioisotope was obtained from the Enviro Technetium-99m Generator, manufactured by Enviro Korea Co., Ltd.

Methods

Method for preparation of ^99mTc genistein kit. The preparation of^99mTc genistein compound was carried out by optimizing the formulation of all parameters: genistein solution as ligand, SnCl₂.H₂O solution as reducing agent, pH value, and incubation time. In addition, sodium pertechnetate solution [^99mTc] with an activity 7.4 - 17 MBq was added in the process of forming ^99mTc genistein. The radiochemical purity (RCP) of the complex formation is monitored by TLC and must fulfill the USP requirement of > 90% ^14-15.

Thin‑layer chromatography (TLC) procedure. Determination of the radiochemical purity of ^99mTc genistein from impurities ^99mTcO₂ and ^99mTcO₄^- using the TLC method. This quality control method is simple and practical to use. The stationary phase used is TLC SGF 254 with a mobile phase of NaCl solution. This TLC system aims to separate TcO^4- impurities, where TcO^4- will migrate and ^99mTc genistein compound will remain at the starting point.

The separation of ^99mTcO₂ was carried out with the stationary phase ITLC‑SG and the mobile phase a mixture of C₂H₅OH : H₂O : NH₄OH (2:5:1). As a result, the impurity of ^99mTcO2 will migrate carried away by the mobile phase onto the TLC plate, and the ^99mTc genistein compound will remain at the starting point. The strips were measured for their radioactive activity using SCA (Single Channel Analyzer), and the calculation of RCP of ^99mTc genistein is based on the equation:

% ^99mTc-Genistein = 100 % - (% ^99mTcO₂ + % ^99mTcO₄^-) ¹⁶

Stability of ^99mTc-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.

Electric charge measurement. Determination of the charge of the ^99mTc 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 ^99mTc 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 ^99mTc-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 coefficient 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 ^99mTc 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.

The process of labeling genistein with ^99mTc was carried out by extracting ^99mTc from a standard generator with a solution of 0.9% NaCl solution. The chemical form of ^99mTc in the generator is Na^99mTcO₄, 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 ^99mTc 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 ^99mTc with the reducing agent SnCl₂ can be explained by the following reaction¹⁹. The decrease in the valence of ^99mTc becomes more reactive so that it can form a ^99mTc genistein complex.

The preparation process of ^99mTc genistein contained impurities TcO⁴⁻ and TcO₂, which could be identified 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 TcO₂ and TcO₄⁻ impurities. The scheme for synthesizing of ^99mTc genistein compounds using the direct labeling method can be seen in Figure 1.

The optimum conditions for the labeling process of ^99mTc genistein with a concentration of SnCl₂.2H₂O 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 ^99mTc 6.01 hours. Therefore, the RCP value was calculated every 1 hour, for 5 hours of observation time. The stability results showed that the ^99mTc 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 figure 2).

Table 1

The stability test of ^99mTc genistein at room temperature.
Time (min)	Impurities (%)		RCP (%) ^99mTc genistein	Appearance
Time (min)	^99mTcO₂	^99mTcO₄	RCP (%) ^99mTc genistein	Appearance
10 60 120 180 240 300	1.33 ± 0.3 1.70 ± 0.5 2.55 ± 0.42 4.51 ± 1.96 8.76 ± 2.37 7.05 ± 2.94	5.66 ± 0.4 6.28 ± 0.12 6.90 ± 0.16 8.67± 1.84 10.48 ± 2.58 17.98 ± 2.08	93.01 92.02 90.55 86.82 80.76 74.97	clear, colorless clear, colorless clear, colorless clear, colorless clear, colorless clear, colorless

The room temperature stability of the ^99mTc genistein kit showed RCP values > 90% up to 2 hours. However, after 2 hours of storage at room temperature, the RCP value of the kit decreased due to the increase in impurity values of TcO₄^- and TcO₂. This could be due to the reduced ^99mTc, which has not been bound to genistein, can return to its original form because the reaction is irreversible²⁰. The stability results also showed that the ^99mTc 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 profile. 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 ^99mTc 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 field. This charge can provide information on transport mechanisms in the body. Electrophoresis showed that the ^99mTc genistein kit did not migrate and remained at the starting point (scale 0). ^99mTc genistein kits are neutral, so there is no migration of the sample. In contrast, the ^99mTc movement from cathode to anode is due has a negative charge originating from TcO^4- (figure 2).

Plasma protein binding. The amount of ^99mTc 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 ^99mTc genistein preparation was 75.29% ± 2.59%, indicating that this preparation was strongly bound to plasma proteins.

The development of the synthesis of ^99mTc genistein kit was performed by direct labeling method, and simple quality control by TLC method by obtaining radiochemical purity that met the requirements. Determination of physicochemical properties showed that the ^99mTc genistein compound has good properties and is potentially developed into a radiotracer kit for breast cancer.

World Health Organization (WHO). Global Health Estimates 2020: Deaths by Cause, Age, Sex, by Country and by Region, 2000-2019. WHO; 2020. Accessed December 11, 2020. who.int/ data/gho/data/themes/mortality-andglobal-health-estimates/ghe-leadingcauses-of-death.
World Health Organization. Guide to Early Cancer Diagnosis. 2017.
Heather A. Parsons, Justin Rhoades, Sarah C. Reed, Gregory Gydush, Priyanka Ram, Pedro Exman, et al. Sensitive Detection of Minimal Residual Disease in Patients Treated for Early-Stage Breast Cancer. 2020. Clin Cancer Res; 26(11): 2556-64
Cheang MC, Chia SK, Voduc D, Gao D, Leung S, Snider J, et al. Ki67 index, HER2 status, and prognosis of patients with luminal B breast cancer. Journal of the National Cancer Institute. 2009; 101: 736-50.
Vallejos CS, Gomez HL, Cruz WR, Pinto JA, Dyer RR, Velarde R, et al. Breast Cancer Classification According to Immunohistochemistry Markers: Subtypes and Association With Clinicopathologic Variables in a Peruvian Hospital Database. Clinical breast cancer. 2010; 10: 294-300.
Maric P, Ozreti_c P, Levanat S, Oreskovi_c S, Antunac K, Beketi_c-Oreskovi_c L. Tumor markers in breast cancereevaluation of their clinical usefulness. Coll Antropol. 2011;35(1):241-247.
Perou, C. M., Sorlie, T., Eisen, M. B., van de Rijn, M., Jeffrey, S. S., Rees, C. A., et al. Molecular portraits of human breast tumours. Nature. 2000, 406: 747–752.
Ronis, M. J. Effects of soy containing diet and isoflavones on cytochrome P450 enzyme expression and activity. Drug Metab. Rev. 2016, 48: 331–341.
Hua Jiang, Jingjing Fan, Lin Cheng, Pan Hu, Renbin Liu., The anticancer activity of genistein is increased in estrogen receptor beta l-positive breast cancer cells. Onco Targets and Therapy. 2018, 11: 8153-63.
Ting, G.; Chang, C.-H.; Wang, H.-E. Cancer Nanotargeted Radiopharmaceuticals for Tumor Imaging and Therapy. Anticancer Res. 2009, 29: 4107-18.
Ilem-Ozdemir, D.; Asikoglu, M.; Ozkilic, H.; Yilmaz, F.; Hosgor-Limoncu, M.; Ayhan, S. Gamma Scintigraphy and Biodistribution of ^99mTc-Cefotaxime Sodium in Preclinical Models of Bacterial Infection and Sterile Inflammation. J. Labelled Comp. Radiopharm. 2016, 59, 109−116.
Brooks, G. F.; Carroll, K. C.; Butel, J. S.; Morse, S. A.; Mietzner, T. Pathogenesis of Bacterial Infection. Jawetz, Melnick and Adelberg’s Medical Microbiology; Appleton-Lange: Stanford, 1998; pp. 134−144.
Rennen, H. J. J. M.; Boerman, O. C.; Oyen, W. J. G.; Corstens, F. H. M. Imaging Infection/Inflammation in the New Millennium. European Journal of Nuclear Medicine 2001, 28: 241−252.
Ramdhani D, Sriyani M.E., Ayu F, Technetium-99m-labeled genistein as a potential radical scavenging agent. Journal of Advanced Pharmaceutical Technology & Research. 2019; 10: 112-6.
United States Pharmacopeial Convention. Official Monographs: USP 28. Technetium ^99mTc‑Pertechnetate Injection (Sodium). United States Pharmacopeia (USP) 28‑National Formulary; 2005.
Owunwanne A, Patel M, Sadek S. The Handbook of Radiopharmaceuticals. London: Chapman and Hall Medical; 2012.
Khan, N.-U.-H.; Naqvi, S. A. R.; Sohail, H.; Roohi, S.; Jamal, M. A. Technetium-99m Labeled Ibuprofen: Development and Biological Evaluation Using Sterile Inflammation Induced Animal Models. Mol. Biol. Rep. 2019, 46, 3093.
Vladimir Sadkin, Viktor Skuridin, Evgeny Nesterov, Elena Stasyuk, Alexander Rogov, et. al, ^99mTc-labeled nanocolloid drugs: development methods. Scientific Reports. 2020. 10:14013
Saha, G. B., Fundamentals of Nuclear Pharmacy. 5th Edition penyunt. New York: Springer-Verlag. 2010.
Zolle, I. Technetium-99m Pharmaceuticals: Preparation and Quality Control. New York: Springer Berlin Heidelberg New York. 2007.
Waring, M.J. Lipophilicity In Drug Discovery. Expert Opinion on Drug Discovery. 2010. 5(3): 235-248.

No competing interests reported.

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Version 1

posted 04 Nov, 2021

You are reading this latest preprint version

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

Status:

Version 1

Abstract

Objective.

Methods.

Results.

Conclusions.

Figures

Introduction

Materials And Methods

Results And Discussion

Conclusions

References

Additional Declarations

Status:

Version 1