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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 7  |  Issue : 4  |  Page : 112-116

Evaluation of radioiodinated curcumin for its potential as a tumor-targeting radiopharmaceutical


Bhabha Atomic Research Centre, Radiopharmaceuticals Division, Mumbai, Maharashtra, India

Date of Web Publication1-Feb-2017

Correspondence Address:
Chandan Kumar
Bhabha Atomic Research Centre, Radiopharmaceuticals Division, Mumbai, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-0168.199309

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  Abstract 

Introduction: Curcumin, a component of the spice turmeric has widely reported anticancer properties in several types of cancer. The differential accumulation and mechanism of its action in normal and cancer cells have proven its potential in targeting tumor. Therefore, it was of interest to label curcumin with a suitable radionuclide and explore its potential for use in nuclear medicine.
Materials and Methods: Curcumin was labeled with 125I by iodogen method. The radiochemical purity was analyzed by paper electrophoresis and high-performance liquid chromatography (HPLC) method. Cell binding was carried out in murine lymphoma and melanoma cell lines. Bioevaluation and pharmacokinetics of radioiodinated curcumin was carried out in lymphoma-bearing mice for various time points (1, 3, 24, and 48 h).
Results: The efficiency of labeling was >75% and the radiochemical purity postpurification was >95% The maximum uptake (~7% at 2 h, 37°C using 5 × 105 cells) was observed in EL4 cells. Significant tumor uptake in lymphoma-bearing mice was observed at 180 min (3.3 ± 0.76% ID/g). In addition, pharmacokinetics of radioiodinated curcumin is fast, with the majority of the preparation out of the bloodstream in 3 h.
Conclusion: The results of these studies suggest that curcumin has the potential for targeting lymphomas, which may be used as diagnostic/therapeutic agent by labeling with other radionuclides.

Keywords: 125I-curcumin, anticancer, curcumin, lymphoma, radiopharmaceutical


How to cite this article:
Kumar C, Subramanian S, Samuel G. Evaluation of radioiodinated curcumin for its potential as a tumor-targeting radiopharmaceutical. J Radiat Cancer Res 2016;7:112-6

How to cite this URL:
Kumar C, Subramanian S, Samuel G. Evaluation of radioiodinated curcumin for its potential as a tumor-targeting radiopharmaceutical. J Radiat Cancer Res [serial online] 2016 [cited 2020 Aug 15];7:112-6. Available from: http://www.journalrcr.org/text.asp?2016/7/4/112/199309


  Introduction Top


Curcumin, the yellow pigmented, non-nutritive major food flavoring agent in the Indian diet is a naturally occurring polyphenolic phytochemical isolated from the rhizome of turmeric (Curcuma longa L). Since centuries, curcuminoids have been consumed as dietary spices at up to 100 mg/day by people in India and certain other countries however, regarded as pharmacologically safe at those levels. Curcumin has been reported to possess a wide range of pharmacological actions including anti-inflammatory [1] antimicrobial,[2],[3] antioxidant,[4] and anticancer agents.[3],[5],[6],[7],[8] The extract of C. longa contains three different curcuminoids, namely, curcumin, demethoxycurcumin, and bisdemethoxycurcumin, together accounting for 2%–5% of the total extract and widely believed to be responsible for the biological activity. Curcumin has been shown to have differential uptake in normal splenocytes and cancer cells [9],[10] and also the different mechanism of action in normal and tumor cells.[11] Hence, it was of interest to study the potential of radioiodinated curcumin for use in nuclear medicine for cancer management. There have been earlier reports of curcumin and its derivatives labeled with 99m Tc and 18 F for imaging β-amyloid plaques of Alzheimer's disease and its biodistribution in normal animals has also been studied.[12],[13],[14],[15],[16],[17],[18] Herein, we report the uptake of radioiodinated curcumin in murine lymphoma cell line in vitro and in vivo using a tumor-bearing animal model. Curcumin was labeled with 125 I, using iodogen method. Since the chemical behavior of 131 I,125 I, and 123 I may be regarded identical, the information obtained from the in vitro and in vivo studies of 125 I-curcumin will serve as basic information for carrying out further work with radioiodinated curcumin toward application in nuclear medicine.


  Materials and Methods Top


Materials

All chemicals were procured from M/s Sigma unless otherwise mentioned in the text. Iodine-125 (as Na 125 I) was obtained from Radiochemicals Section, Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai, India. All solvents used were of high-performance liquid chromatography (HPLC) grade, procured from M/s Merck India Ltd.

Cell lines

EL4 (murine lymphoma) and B16-F10 (murine melanoma) cell lines were procured from the National Centre for Cell Science, India. EL4 was cultured in RPMI 1640 medium while B16-F10 in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum (Invitrogen, CA) and antibiotic/antimycotic solution. All cultures were maintained at 37°C in a humidified 5% CO2 incubator.

Radiolabeling (iodination) of curcumin with 125 I

Curcumin was dissolved in dimethyl sulfoxide (DMSO) (10 mM) and aliquots were stored at −20°C for later use. Curcumin was radioiodinated with 125 I using the Iodogen method.[19] Briefly, 50 μg iodogen was coated in a glass test tube by addition of 100 μl of iodogen solution in chloroform (0.5 mg/ml) which was dried with nitrogen purging. 1 μg of curcumin (in 0.1% DMSO) was added to the iodogen coated tube along with 30 μl phosphate buffer (0.5 M, pH 7.5) and 37 MBq of 125 I. The reaction vial was shaken intermittently and after 10 min, the reaction was stopped by transferring the mixture to another test tube. The labeled curcumin was separated from free iodide by extraction with chloroform. Chloroform was removed under nitrogen and the product was dissolved in minimum amount of dimethyl sulfoxide (0.1%).

Characterization of 125 I-curcumin

The radiolabeling yield and radiochemical purity of the 125 I-curcumin were estimated using paper electrophoresis and HPLC.

Paper electrophoresis

For carrying out paper electrophoresis, Whatman 3 MM chromatography paper strip (25 × 1.0 cm) was soaked in 0.025 M phosphate buffer pH 7.5 and allowed to stand in air for a few minutes to evaporate excess buffer so that the sample can be applied on the paper without spreading. The samples (reaction mixture and purified fraction) were spotted on separate Whatman chromatography strip and electrophoresis was carried out in phosphate buffer (0.025 M, pH 7.5) at ~10 V/cm for 1 h. After completion of electrophoresis, the strip was air dried and scanned on the TLC scanner (Raytest Mini-Gita TLC Scanner Model BGO-V detector). PdCl2 was used to ascertain the position of migration of iodide species toward the anode on the paper strip.

High performance liquid chromatography

HPLC analysis was carried out for the curcumin, Na 125 I, reaction mixture and purified fraction of 125 I-curcumin on a JASCO PU 2080 plus dual pump HPLC system, Japan, with a JASCO 2075 Plus tuneable absorption detector and a Raytest (Model Socket 81030043) radiometric detector system, using a C18 reverse phase HiQ Sil column to measure the radioactivity. Gradient HPLC was performed using water and methanol as the mobile phase (0–4 min 100% H2O, 8–12 min 95% H2O, 15–25 min 5% H2O, and 30 min 100% H2O) at a flow rate of 1 ml/min. The radioactive peaks were monitored using radioactivity detector. 10 µl of curcumin, Na 125 I, reaction mixture and purified fraction of 125 I-curcumin were injected separately, and the HPLC patterns were recorded. The curcumin peak in the ultraviolet (UV) chromatogram was used for identification of the radiolabeled curcumin and to ascertain the purity of 125 I-curcumin. The radioactivity peak for Na 125 I was used to identify the unlabeled free iodide (Na 125 I) in the reaction mixture. The labeling yield was calculated as the percentage of radioactivity in the 125 I-curcumin peak in the HPLC profile of the reaction mixture.

Lipophilicity of the labeled product

Lipophilicity of the labeled product was estimated by octanol-water partition.[20] 100 μl of purified radioiodinated curcumin along with 900 μl of distilled water was mixed with 1000 μl of octanol. This mixture was vortexed vigorously for 5 min and centrifuged to separate the two phases. From each phase, 5 μl aliquots were taken and measured for radioactivity. Again 800 μl of the organic phase was mixed vigorously with equal volume of water and the partition procedure was repeated till constant ratio of partition between the two phases was obtained. Partition coefficient (pow) was calculated as logarithm of the ratio of count in organic phase to the count in aqueous phase.

In vitro cell uptake of 125 I-curcumin in murine tumor cells

In vitro cell uptake studies were carried out in EL4 and B16-F10 cell lines. 5 × 105 cells per reaction tube were incubated with different concentrations of the 125 I-curcumin for 2 h at 37 °C. For inhibition studies, a separate set of tubes was prepared similar to the above but containing additionally an excess of unlabeled curcumin (100 times). After incubation, cells were washed thrice with ice-cold phosphate buffer saline and radioactivity associated with the cell pellet was measured in NaI (Tl) gamma detector. Cell uptake of radioiodinated curcumin was calculated as the percentage of total radioactivity associated with the cell pellet.

In vivo distribution and pharmacokinetics of 125 I-curcumin in tumor-bearing mice

In vivo studies were performed in compliance with the institutional animal ethics committee laws governing the conduct of animal experiments. Murine lymphoma was raised in C57BL/6 mice as a localized subcutaneous tumor transplanted on the dorsum. The tumors were allowed to grow to a maximum diameter of 10 mm. Radiolabeled curcumin (3.7 MBq) was administered intravenously to each animal. Different sets of animals (3 animals per se t) were used for different time points (1, 3, 24, and 48 h). At the end of each time point, the corresponding animals were sacrificed and the relevant organs excised for measurement of retained radioactivity in flat-bed NaI (Tl) gamma detector. Distribution of the radioactivity in relevant organs was compared in terms of percentage of injected dose (% ID) retained per gram of specific tissue.


  Results Top


Estimation of radiolabeling and purity analysis of 125 I-curcumin by paper electrophoresis

The labeling yield and radiochemical purity of 125 I-curcumin was determined by paper electrophoresis [Figure 1]. It was found that ~75% labeling yield was obtained. However, after purification with chloroform, the radiochemical purity was achieved up to 95%. In paper electrophoresis,125 I-curcumin remained near the point of spotting (Rf0–0.3) while free iodide migrated toward anode with the applied voltage.
Figure 1: Paper electrophoresis of reaction mixture and purified fraction of 125I-curcumin

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Determination of radiolabeling and purity analysis of 125 I-curcumin by high-performance liquid chromatography

The labeling yield and radiochemical purity of 125 I-curcumin was also estimated by HPLC [Figure 2] wherein the percentages of yield and purity were found to be similar to that observed in paper electrophoresis system. In HPLC, the peak at 2.8 min corresponds to (free Na 125 I) iodide whereas radioiodinated curcumin elutes out between 22 and 24 min, which was confirmed by the UV peak of unlabeled curcumin.
Figure 2: High performance liquid chromatography pattern of curcumin, Na125I, reaction mixture and purified fraction of 125I-curcumin (directed from top to bottom is the profile of unlabeled curcumin, free radio iodide, reaction mixture and purified fraction of 125I-curcumin)

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Characterization of lipophilicity of 125 I-curcumin

The octanol partition coefficients indicated the nature of compounds. The value of pow for the 125 I-curcumin was 1.02, indicating that the 125 I-curcumin was highly lipophilic in nature.

in vitro cell binding study

In vitro studies were performed in two different murine cell lines. The results of the in vitro cell uptake studies are illustrated in [Figure 3]. The highest uptake of 125 I-curcumin in the case of EL4 murine lymphoma cells was 6.8 ± 0.5% at 9.65 nM, while in the presence of excess amount of curcumin was 1.2 ± 0.38% at 9.65 nM. Whereas in the case of B16-F10 murine melanoma cells uptake of 125 I-curcumin was below 1% in the presence and absence of excess of curcumin.
Figure 3: Uptake of 125I-curcumin in EL4 lymphoma and B16-F10 melanoma cell lines (cell binding value is the mean ± standard deviation value of three independent experiments)

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In vivo tumor uptake study

The pattern of in vivo distribution/clearance from major organs in localized lymphoma tumor bearing C57BL/6 mice is given in [Figure 4] (% ID/g). It is found that tumor uptake of 125 I-curcumin in mice remains constant in 60–180 min (~3.3% ID/g) and is gradually washed out in 48 h. The critical ratios of tumor/blood (ID/g) and tumor/muscle (ID/g) are 0.82 ± 0.24 and 2.73 ± 0.66, respectively at 3 h pi [Table 1]. Most of the radioactivity gets washed out within 24 h from the major organs however, thyroid uptake increases significantly with time.
Figure 4: Percent injected dose per gram of 125I-curcumin in major organs in lymphoma-bearing C57BL/6 mice (where n = 3 and values are mean ± standard deviation)

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Table 1: Ratios of 125I-curcumin uptake in tumor to blood and muscle (from percentage of injected dose/g values) at different time points postinjection in lymphoma-bearing C57BL/6 mice

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  Discussions Top


The widely reported anticancer activities of curcumin make it a candidate of interest for the development of novel radiolabeled derivatives for use in cancer nuclear medicine. Its polyphenolic structure suggests the feasibility of labeling with iodine.[14],[16] Curcumin has been derivatized for its labeling with PET radionuclides such as 18 F for possible use in diagnosis of β-amyloid plaque imaging [13],[16],[18] and tumor.[17] Clinically, relevant radioactive isotopes of iodine include 123 I (T1/2 = 13.1 h, Eγ = 159 keV), which is relevant in diagnostic imaging and 131 I (T1/2 = 8.04 d, Eβ= 606 keV), which is a therapeutic isotope.125 I (T1/2 = 60.14 d, Eγ = 35 keV) with its convenient half-life and emission properties makes it easy to prepare and evaluate radioiodinated derivatives, whose in vitro uptake and in vivo localization data may provide useful information on the performance of 123 I and 131 I labeled curcumin. Labeling yield of curcumin with 125 I was reasonably high ~75%.In vitro studies indicated appreciable uptake of the labeled preparation in murine lymphoma cells. The diversity of uptake results suggests that the curcumin uptake may vary depending on the type of cell, indicating different efficacies of curcumin-based treatment for different tumor types.[9],[10] However, in vivo studies with murine lymphoma tumor model showed an increase of tumor to muscle ratios up to 3 h and decreased to 48 h. The activity in blood did not clear quickly, which possibly is due to high lipophilicity of 125 I-curcumin. However, it washed out over time, leading to low tumor/blood ratios which increases up to 3 h and subsequently reduces. High intestinal activity in the early stages of the experiment may be due to the fast clearance from the gastrointestinal tract within 3 h. The gradual increase in the thyroid activity up to ~8.5% (ID/organ) over a period of 24 h indicates a small percentage of in vivo deiodination of the labeled product with time.


  Conclusion Top


The in vitro and in vivo studies indicated that radioiodinated curcumin has a potential for specific targeting of lymphomas for diagnostic and therapeutic applications. However, further modifications in the lead structure needs to be carried by introducing polar linkers/pharmacokinetic modifiers so as to modify the high lipophilic nature of 125 I-curcumin and improve the in vivo pharmacokinetics.

Acknowledgments

The authors would like to thank Dr. R.B. Manolkar and Mr. P.V. Joshi of the Radiopharmaceuticals Division, BARC for providing in-house Na125I to carry out these studies. Authors would also thank Dr. Meera Venkatesh former head Radiopharmaceuticals Division, BARC to allow carrying out this work.

Financial support and sponsorship

Nil.

Conflicts oF interest

There are no conflicts of interest.

 
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