99mTc-Luteolin: Radiolabeling, In Silico ADMET and Biological Evaluation as a Natural Tracer Tumor imaging

ABSTRACT The focus of the staged work was radiolabelled 5,7,3ʹ, 4ʹ tetra-hydroxy flavone (Luteolin), isolated from Plantago lanceolata L., (F. Plantaginaceae), with 99mtechnetium (99mTc) to produce a natural tracer 99mTc-Luteolin (99mTc-Lut), and to evaluate the biological distribution in silico and in vivo. Labeling was carried out by reduction reaction using SnCl2 as reducing agent giving a maximum radiochemical yield (RCY) up to 97% at 40°C, pH4, for 30 min., with concentration of 99mTc and Luteolin of 150 μL. The tracer (99mTc-Lut) showed 12 h., in vitro stability. In silico ADMET screening of 99mTc-Lut indicated its high bioavailability in all body organs. The bio-distribution profile indicating the highly uptake of the tracer 99mTc-Lut, in tumor mice target/non-target ratio (T/NT) up to two-fold compared to normal mice, whereas the clearance from mice was observed via both renal and feces pathway. 99mTc-Lut, could be valued as a probable natural radiopharmaceutical tracer for tumor appearance.

Luteolin is a broadly distributed flavones in the plant kingdom, which is raised mainly in the form of aglycone, glycoside (O-, or C-glycoside), or methylated derivatives (Elmaidomy et al., 2019;Souleles & Laskaris, 1988). Luteolin and its glycosides derivatives, reported to have anti-inflammatory, and antioxidant activities (Cai et al., 1997;Chen et al., 2007). Several in vivo investigations hint that luteolin had also cancer chemopreventive activity, with different mechanism (Elangovan et al., 1994;Manju & Nalini, 2007, 2005. As luteolin has a broad field of in vitro biological activities, its bioavailability and metabolism must be studied to be significant in an in vivo environment. Radiopharmaceuticals are molecules that contain a radionuclide, and are used for diagnosis or therapy in clinical setting. Consequently, this radioactive drug could be utilized for the investigation and/or the healing of diseases (Rennie, 1999). The main group of these compounds was the radiotracers, which was a synthetic compound, in which one or more atoms were changed by a radionuclide. So by depending on its radioactive decay; it could be utilized to examine the chemical reactions mechanism by tracking the line that the radioisotope followed, from reactants to products (Rennie, 1999). Radiolabeling or radiotracing was thus the radioactive form of an isotopic labeling. Radioisotopes of phosphorus, carbon, hydrogen, technetium, iodine, and sulfur, have applied broadly to track the passage of the biochemical reactions. A radioactive tracer could still be handled to line the circulation of a material within a natural system as a tissue or cell (Rennie, 1999).
In this study, luteolin was isolated from P. lanceolata, labeled with 99m Tc through reduction reaction in the presence of SnCl 2 . In silico and In vivo tissue distribution comparative study of 99m Tc-Lut, in healthy and solidtumor-bearing, male Swiss albino mice, was investigated.

Plant materials
The plant under investigation was collected and identified. Time of collection and the detailed identification method of P. lanceolata aerial parts were mentioned in supplementary data Page (S2).

Chemicals and reagents
Details of chemicals and reagents used in this experiment were mentioned in supplementary data page (S2-3).

Apparatus
The apparatus details used in this experiment were mentioned in supplementary data page (S3).

Extraction and fractionation of the plant material
The plant under investigation was powdered, extracted and fractionation steps used in this experiment were mentioned in supplementary data page (S3-4).

Isolation and purification of compound
The major isolated compound was isolated from P. lanceolate and identified as luteolin using 1 H-and 13 C-NMR and physicochemical properties all the detailed experiment were mentioned in supplementary data page (S4).

In silico ADMET Properties of luteolin
The details for in silico ADMET Properties of luteolin were mentioned in supplementary data page (S5-6).

Labeling of Luteolin with 99m Tc
Details of labeling Luteolin with 99m Tc were mentioned in supplementary data page (S6).

Radiochemical Purity of 99m Tc-luteolin
Identification of radiochemical Purity of 99m Tc-Lut, in this experiment was mentioned in supplementary data page (S6).

Animal treatment
The animals used, was treated and kept in suitable environmental conditions as mentioned in supplementary data page (S7).

Animal Ethical Statement
The study was tested in compliance with rules determined by animal ethics committee of Egyptian atomic energy authority.

Induction of tumor in mice
Steps for tumor induction in mice were mentioned in supplementary data page (S7).

In vivo tissue distribution study of 99m Tc-Luteolin in mice
In vivo tissue distribution study of 99m Tc-Lut., in mice steps were mentioned in supplementary data page (S7).

Statistical analysis
Details of statistical analysis used in this experiment were mentioned in supplementary data page (S8).

In silico ADMET Properties of luteolin
In silico ADMET profiling of luteolin, utilizing PreADMET program version 2.0, indicated that, this flavones had a middle absorption to BBB (0.367582), a high absorption to HIA (79.427233), strong bounding to PPB (99.717233), moderate SP (−4.28017), middle Caco-2 (4.53973), MDCK permeability (36.5205). Also, luteolin inhibited CYP 2 C 9 , CYP 2 C 19 , and CYP 3 A 4 and with no effect in Pgp. Toxicity screening results using PreADMET, showed mutagenicity against AMES test with a positive mode with TA100_NA, and potential rat and rodent carcinogenicity. Also, hERG inhibition was of a medium risk. The results of the in silico screening showed that luteoiln theoretically was potentially high bioactive. These features were showed by its low plasma concentrations, due to its extensive cellular uptake (Singh, 2006). Tight control of the compound plasma concentrations was achieved through binding to serum albumin (Fitzpatrick & Wynalda, 1981), which lowered plasma concentrations of the unbound bioactive compound; while giving a storage system for prolonged release into the plasma. Moreover, albumin-bound compounds were more safe against oxygen-dependent degradation, resulting in prolonging their biological availability, and further extending their plasma half-life. This reply would protect target tissues from high-level uptake of compound, which might produce a toxic reaction in the cells (Fitzpatrick & Wynalda, 1981). Indeed, delayed release of this compound from plasma proteins would establish a more constant rate of cellular uptake . Besides that, the ADME properties for this compound increased its toxicity and carcinogenicity at long-term use.

Labeling of luteolin with 99m Tc 99m
Tc was used for labeling luteolin using SnCl 2 .2H 2 O (Spies & Pietzsch, 2007). Labeling of different anticancer drugs take an interest in last century to act as a tracer for imaging and treating cancer as 99m Tc-zolmitriptan as lung cancer imaging (Rashed et al., 2016), 125 I-sibutramine for brain imaging (Motaleb et al., 2011), The different parameters which change the radiochemical yield of 99m Tc -Lut, were studied as follow

Effect of pH of the reaction
According to this study, pH found to have a great change on the labeling yield of 99m Tc -Lut. Where, a maximum yield (87%) obtained at pH 4. While, pH at neutral or alkaline region found to decrease the yield to 27, 50%, respectively ( Figure 2). This finding suggested that, luteolin work best in mild acidic medium.

Effect of reaction time
According to this study, short reaction time was poor to form the complex, leading to a low yield of 99m Tc -Lut, (Figure 3) (Essa et al., 2015). On the other hand, increasing the reaction time to 30 min., showed significant increase in the radiochemical yield of 99m Tc -Lut, up to 92.5%. While, further increasing of the reaction time over 30 min., has almost no effect (Figure 3).

Effect of temperature
This study showed that, the maximum radiochemical yield of 99m Tc -Lut, (97%) (Figure 4), was obtained at 40°C. Increasing temperature above the previous value around 50-80°C, decreased 99m Tc -Lut., yield, as the rate of side reaction, increased (Amin et al., 2009).

Effect of 99m Tc content
This study showed that 99m Tc-Lut, yield increased to 97% by increasing 99m Tc concentration to 150 μL, at 40°C for 30 min ( Figure 5). And quite the opposite, increasing 99m Tc concentration ˃150 μL, decreased gradually 99m Tc-Lut, yield through the colloid increasing (Ibrahim et al., 2011).

Effect of luteolin amount
This study showed that, at low concentration; 50μL 99m Tc-Lut, yield was poor (60%), and this finding suggested insufficient ligand amount to complex with all reduced 99m Tc (Rashed et al., 2016).While, increasing the concentration of luteolin up to 150μL, 99m Tc-Lut, yield reached to maximum value (97%), further increasing don't affect (Figure 6 and 7).

Effect of reaction temperature with time
The relation between the reaction time and the yield of 99m Tc-Lut, at various temperatures was studied.     Radiochemical yield of 99m Tc-Lut, was significantly increased from 77.5 to 93.8%, with increasing reaction time from 5 min to 30 min., extending the reaction time ˃30 min does not affect the radiochemical yield in room temperature. While, reaction at 40°C showed no significantly increasing in 99m Tc-Lut, yield. In case of 60 °C, the radiochemical yield was lower comparing to 40 °C, and this finding suggested side reactions (Amin et al., 2009). Consequently, the maximum yield of 99m Tc-Lut, was obtained at 40 °C for 15 min.

In-vitro stability of 99m Tc-Luteolin
The tracer was stable up to 12 h., at room temperature (25°C). It is significant to know the proper time for injection to reduce the development of the undesired products that arise from the radiolysis of the labeled compound as illustrated in Table 2.

Radiochemical Purity of 99m Tc-luteolin
The radiochemical purity of 99m Tc-Lut, was determined using electrophoresis analysis . Samples investigation from the reaction mixture produced two peaks ( Figure  8). One was corresponding to the free technetium, which migrated toward the anode, with 11 cm distance, while 99m Tc-Lut, at the stage of spotting, depending on their charge and ionic mobility (Ibrahim et al., 2011). It gave radiochemical yield equal to 97%.    Figure 8. Electrophoresis of 99m Tc luteolin.

Biodistribution of 99m Tc-Luteolin in mice
In healthy group, it was noticed that the blood uptake at 30 min., was about 14.7% decreased to 4.6% after 180 minutes, nine luteolin metabolites was spotted in mice plasma and bile by liquid chromatographytandem mass spectrometry, where luteolin-3ʹglucuronide revealed the highest systemic exposure among them . Luteolin was picked up in the rat plasma as two metabolites; the glucuronidate or sulfate forms of the O-methylate conjugate (Sarawek et al., 2008). Data from the present investigation indicated that 99m Tc-Lut, experienced high blood to tissue perfusion after only 30 min., (Table  3). Data also showed 99m Tc-Lut, continued to leak from the blood to the peripheral tissue over 180 min., and there was different tissue distribution kinetics, with more affinity of 99m Tc-Lut, to stomach, GIT and liver tissues, about 25.7%, 15.7% and 16.3% respectively, at 30 min., post injection (Table 3). This uptake seemed to be stable or somewhat increased at 180 min., where absorption of flavone takes place in the digestive path while microorganisms participate in their hydrolysis (Makarova, 2011). Luteolin and its metabolites dispense in the gastro-intestine, kidney liver, and lung; while biliary excretion influenced the elimination pathways of conjugated luteolin (Deng et al., 2017). Kinetics studies recommended two pathways in rat liver participated in the metabolic mood of luteolin, the methylation and glucuronidation which improve for each other, and the glucuronidation was the predominant one . Luteolin undergoes Enterohepatic recirculation (Sarawek et al., 2008). At 180 min., post injection, the activity taken by the gathered urine was about 3.6%, the urinary elimination of luteolin appeared to be not the main excretion route (Walle et al., 2001). Shimoi et al. (1998), reported an excretory recovery of unmodified luteolin in rat urine of 4%. Muscarinic acetylcholine receptors were reported to be involved in the renal effects of luteolin (Boeing et al., 2017). Excretion via feces; may be the chief route of elimination of luteolin, and its metabolites (Walle et al., 2001). In our data feces, uptake showed 2.8% after 30 min., till reaching about 8% after 180 min. Ying et al. (2008) established the RP-HPLC method, which showed that the total accumulative excretion of luteolin was 37% (11% in urine, 26% in feces and bile). In the present study, the muscle uptake was about 10.2% at 30 min., post injection and decreased to 5.5% at 180 min., (Table 3). 99m Tc-Lut, showed low brain uptake, about 0.1%, throughout the 180 min.
The bio-distribution of 99m Tc-Lut, tracer in solid tumor-bearing mice was investigated (Table 3). Solid tumor was induced into the right thigh muscle and the left thigh muscle was selected as control. It was found that the uptake by the tumor muscle was 10.6% at 30 min., post-injection, reaching to 9.6% at 180 min., while the uptake of the control muscle was 5.1% at 30 min., post-injection decreased to 3.8% through the 180 min. Data showed that 99m Tc-Lut, tended to accumulate in tumor muscle versus healthy muscle, by a comparative increase (Table 3).

Conclusion
Luteolin was isolated from P. lanceolata, labeled with 99m Tc through reduction reaction in the presence of SnCl 2 at 40°C (pH4) within 15 min., to obtain the labeling 99m Tc-Lut, with maximum yield of 97%. The tracer 99m Tc-Lut, shows 12 hr in-vitro stability. In silico ADMET screening of 99m Tc-Lut, indicated its high bioavailability. In vivo tissue distribution comparative study of 99m Tc-Lut, in healthy and solid-tumor-bearing, male Swiss albino mice, showed the highly uptake of the tracer 99m Tc-Lut, in tumor mice T/NT ratio up to twofold normal mice whereas the clearance from mice established by RP-HPLC method to proceed via both renal and feces pathway. 99m Tc-Lut, shows promise as a natural radiopharmaceutical tracer.