Introduction

Diagnostic radiopharmaceuticals contain γ-emitters whose radiation readily penetrates the body, thus permitting external detection and measurement. The pattern of radiation biodistribution allows a physician to evaluate both the morphology and functioning of organs [1, 2]. Technetium-99m is the most important radionuclide in diagnostic nuclear medicine. The preferential use of 99mTc-radiopharmaceuticals reflects the ideal nuclear properties of the isotope (T 1/2 = 6 h, 140 keV γ-emitter), as well as its low cost and convenient availability from commercial generators [3–5]. In 99mTc-radiopharmaceuticals, the metal is bound to a transporting moiety that delivers the radioactivity to a specific site in the body determined by the properties of the transporter [6]. Current research is aimed to formulate new radiopharmaceuticals for hepatobiliary imaging studies. For human use, there are two different classes of 99mTc-complexes for hepatobiliary studies. These two classes are iminodiacetic acid and pyridoxalamino acid derivatives [7]. All the 99mTc-radiopharmaceuticals used for hepatobiliary imaging show similar pharmacokinetic properties in animals and human. They are effectively extracted from the blood by the liver and excreted into the bile. Furthermore, they assess disease of hepatocytic function and the functional status of the cystic duct and gallbladder. Biliary duct patency and hepatic diseases can be assessed by the scintigraphic procedure termed cholescintigraphy [8]. The majority of 99mTc hephatobiliary agents are iminodiacetic acid derivatives including 99mTc-disofenin (DISIDA) [9], 99mTc-mebrofenin [10–15], 99mTc-EHIDA [9], 99mTc-lidofenin [16, 17], 99mTc-JODIDA [11], 99mTc-IOIDA [8], 99mTc-BPIDA [18] and 99mTc-IIODIDA [19].

Ursodeoxycholic acid [3α,7β-dihydroxy-5β-cholan-24-oic acid] (UDCA) (Fig. 1) is one of the secondary bile acids, which are metabolic byproducts of intestinal bacteria. Primary bile acids are produced by the liver and stored in the gall bladder. When secreted into the intestine, primary bile acids can be metabolized into secondary bile acids by intestinal bacteria. Primary and secondary bile acids help the body digest to fats. UDCA helps to regulate cholesterol by reducing the rate at which the intestine absorbs cholesterol molecules while breaking up micelles containing cholesterol. Because of this property, UDCA is used to treat (cholesterol) gallstones non-surgically. While some bile acids are known to be colon tumor promoters (e.g. deoxycholic acid), others such UDCA are thought to be chemopreventive, perhaps by inducing cellular differentiation and/or cellular senescence in colon epithelial cells [20]. It is believed to inhibit apoptosis [21]. It is the only FDA approved drug to treat primary biliary cirrhosis [22].

Fig. 1
figure 1

Chemical structure of UDCA

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In this study, a simple method for 99mTc-labeling of UDCA has been investigated. The certain reaction parameters affecting the rate of the reaction including the function of the initial solvent, hydrogen ion concentration, substrate and reducing agent amounts and reaction time have been investigated. The method afforded a high radiochemical yield of pure 99mTc-UDCA.

Experimental

Materials

Drugs and chemicals

Technetium-99m was eluted as 99mTcO4 − from a 99Mo/99mTc generator (radionuclidic and radiochemical purity 99.99 %, 1 Ci, Elutec, Brussels, Belgium). UDCA was obtained as a gift from Sigma Pharmaceutical Company-Egypt, Tin chloride was purchased from Sigma Chemical Company, USA. And all other chemicals were purchased from Merck and they were reactive grade reagent.

Animals

Swiss Albino mice weighing 20–30 g were purchased from the Institute of Eye Research Cairo, Egypt. The animals were kept at constant environmental and nutritional conditions throughout the experimental period and kept at room temperature (22 ± 2) °C with a 12 h on/off light schedule. Animals were kept with free access to food and water all over the experiment.

Methods

Labeling procedure

Accurately weighed 2 mg UDCA was transferred to an evacuated penicillin vial. Exactly 50 μg SnCl2 solution was added and the pH of the mixture was adjusted to 8 using 0.1N NaOH and phosphate buffer then the volume of the mixture was adjusted to 1 ml by N2-purged distilled water. 1 ml of freshly eluted 99mTcO4 − (~400 MBq) was added to the above mixture. The reaction mixture was vigorously shaken and allowed to react at room temperature for 30 min. to complete the reaction [23, 24].

Quality control

The radiochemical yield and purity of 99mTc-complexes were determined by Paper chromatographic method (PC) and electrophoresis.

Paper chromatography of 99mTc-UDCA

Radiochemical yield of 99mTc-UDCA was determined by paper chromatography in which, the reaction mixture was spotted on ascending paper chromatography strips (10 × 1.5 cm). Free 99mTcO4 − in the preparation was determined using acetone as the mobile phase. Reduced hydrolyzed technetium was determined by using 2N HCl as a mobile phase to differentiate between reduced colloids which persist near the point of spotting and both complex and free, which move towards the front of chromatogram. After complete development, the strips were dried then cut into 0.5 cm pieces and their radioactivities counted in a well type Gamma counter: Scalar Ratemeter SR7, Nuclear enterprises LTD. USA.

Electrophoresis conditions

Electrophoresis was done with EC-3000 p-series programmable (E.C.apparatus corporation) power and chamber supply units using cellulose acetate strips. The strips were moistened with 0,05 M phosphate buffer pH 7.2 ± 0.2 and then were introduced in the chamber. Samples (5 μl) were applied at a distance of 10 cm from the cathode. The applied voltages were 300 v and standing time for one and half hours then the radioactivity values were continued. Developed strips were dried and cut into 1 cm segments and counted by a well-type NaI scintillation counter. The radiochemical yield was calculated as the ratio of the radioactivity of the labeled product to the total radioactivity.

Factors affecting % labeling yield

This experiment was conducted to study the different factors that affect labeling yield such as tin content as (SnCl2·2H2O), substrate content, pH of the reaction, and reaction time. The experiment was repeated by keeping all variables constant except the factor under study, till the optimum conditions are achieved.

Biodistribution of the labeled 99mTc-UDCA

In-vivo biodistribution studies were performed using 9 mice divided into three groups of three mice each. Each animal was injected in the tail vein with 0.2 ml solution containing 5–10 kBq of 99mTc-UDCA. The mice were kept in metabolic cages for the required time. Mice were killed by cervical dislocation at various time intervals (30, 60 and 120 min) after injection and the organs or tissues of interest were removed, washed with saline, weighted and their radioactivity were counted. Correction was made for background radiation and physical decay during the experiment [25, 26]. The weights of blood, bone and muscles were assumed to be 7, 10 and 40 % of the total body weight, respectively [27]. Differences in the data were evaluated with the Student t test. Results for p using the 2-tailed test are reported and all the results are given as mean ± SEM. The level of significance was set at P < 0.05.

Results and discussion

Paper chromatography

Radiochemical purity and stability of 99mTc-UDCA complex were assessed by ascending paper chromatographic method and electrophoresis condition. In case of ascending paper chromatographic method acetone was used as the developing solvent, free 99mTcO4 − moved with the solvent front (R f = 1), while 99mTc-UDCA and reduced hydrolyzed technetium remained at the point of spotting. Reduced hydrolyzed technetium was determined by using 2N HCl as a mobile phase as where reduced hydrolyzed technetium remains at the origin (R f = 0) while other species migrate with the solvent front (R f = 1). The radiochemical purity was determined by subtracting the sum of the percent of reduced hydrolyzed technetium and free pertechnetate from 100 %. The radiochemical yield is the mean value of three experiments.

Electrophoresis

The paper electrophoresis pattern revealed that 99mTc-UDCA complex moved towards the cathode, indicating the cationic nature of this complex. But 99mTcO4 − moved considerably toward the anode, suggesting that it has a high negative charge.

Factors affecting labeling yield

Effect of tin chloride content

The majority of 99mTc-radiopharmaceuticals are prepared using SnCl2·2H2O, [Sn(II)],for reduction of 99mTc from heptavalent to lower valence state, which facilitates its chelation by compounds of diagnostic importance. The effect of tin chloride as a reducing agent on the labeling of UDCA with 99mTc is illustrated in Fig. 2 at low amount of Sn(II), the radiochemical yield of 99mTc-UDCA complex was low (74.5 % at 25 μg) with the appearance of free pertechnetate (22.5 %) due to insufficient Sn(II) to reduce all pertechnetate present in the reaction mixture. Increasing the amount of Sn(II) to 50 μg led to an increase in labeling yield to 97.5 ± 0.5 %. By increasing the amount of reducing agent above 50 μg, until (125 μg), the labeling yield decreased again to 57.6 % due to colloid formation (37.4 %) [28].

Fig. 2
figure 2

Effect of SnCl2·2H2O concentration on the labeling yield of 99mTc-UDCA; reaction conditions: 2 mg UDCA, 25–125 μg of SnCl2·2H2O, 0.5 ml (~400 MBq) of 99mTcO4 at pH 8, the reaction mixture was kept at room temperature for 30 min

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Effect of substrate amount

The influence of UDCA amount as a substrate on the labeling yield using 50 μg of SnCl2·2H2O (tin chloride) was shown in Fig. 3. The increase of the amount of UDCA was accompanied by a significant increase in the labeling yield, where it reached above 79 % at 1 mg of UDCA. Increasing the amount of UDCA from 1 to 2 mg produced significant increase in the labeling yield (97.5 %). Increasing the amount of starting material is usually increases the total amount of incorporated 99mTc since there is a minimum limit to the volume used [27]. 2 mg of UDCA was required to obtain maximum labeling yield. Below this amount there will be a significant decrease in the yield apparently because of insufficient amount of the ligand to bind all the reduced technetium and the amount of colloid increased. On the other hand, using higher amount did not significantly affect labeling yield.

Fig. 3
figure 3

Percent labeling yield of 99mTc-UDCA as a function of substrate amount; reaction conditions: 1–5 mg UDCA, 50 μg of SnCl2·2H2O, 0.5 ml (~400 MBq) of 99mTcO4 at pH 8, the reaction mixture was kept at room temperature for 30 min

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Effect of pH

In order to reach the suitable pH value for maximum radiochemical yield, labeling of UDCA with 99mTc was carried out at different pH ranging from 7 to 11 as shown in Fig. 4, it is observed that 99mTc-UDCA is considered negligible from 2 to 6 due to its degradation in acidic medium. The test was performed using 2 mg of UDCA, 0.5 m1 of each buffer at different pH values at 30 min reaction time. As shown in Fig. 4, pH 8 is the optimum pH at which the maximum yield was obtained (97.5 %). At pH 10 and 11 the yield was 68.5, 60.4 %, respectively.

Fig. 4
figure 4

Effect of pH on the labeling yield of 99mTc-UDCA, 2 mg of UDCA, 50 μg of SnCl2·2H2O, 0.5 ml (~400 MBq) of 99mTcO4 at 7-11 pH, the reaction mixture was kept at room temperature for 30 min

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Effect of reaction time

Figure 5 shows the relationship between the reaction time and the yield of 99mTc-UDCA. Radiochemical yield was significantly increased from 80 to 97.5 % with increasing reaction time from 1 to 30 min, respectively. Extending the reaction time to 60 min produced no significant change of the radiochemical yield.

Fig. 5
figure 5

99mTc-UDCA yields vs. reaction time, 2 mg UDCA, 50 μg of SnCl2·2H2O, 0.5 ml (~400 MBq) of 99mTcO4 at pH 8, the reaction mixture was kept at room temperature at different time post labeling

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In vitro stability of 99mTc-UDCA

In the present experiment, a slight decrease in the stability of 99mTc-UDCA from 97.0 to 96 % at 2 and 6 h post labeling was observed. Further significant decrease in the yield was observed at 12 and 24 h post labeling, as the yield was 94.8 % (Table 1).

Table 1 Effect of time on the Stability of 99mTc-UDCA
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Biodistribution of 99mTc-UDCA in mice

Biodistribution study of 99mTc-UDCA in normal mice showed that 99mTc-UDCA was distributed rapidly in blood, kidney, liver and intestine at 30 min post injection. After 1 h, 99mTc-UDCA uptake was significantly increased in blood, stomach, intestine and bone while, it decreased in liver, heart and muscle. At 2 h post injection, the majority of tissues and organs showed significant decrease in 99mTc-UDCA uptake. On the other hand, lung, intestine and stomach showed significant increase in 99mTc-UDCA uptake (Table 2). The uptake in liver was 15.37, 12.0 and 3.0 % at 30 min, 1 and 2 h respectively. This uptake may be useful for radioimaging of the liver.

Table 2 Biodistribution of 99mTc-UDCA in mice
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Conclusion

99mTc-UDCA was prepared easily at pH 8 using 50 μg SnCl2·2H2O as a reducing agent with a labeling yield of 97.5 ± 0.3 %. 99mTc-UDCA complex was formed once the addition of 99mTc to the reaction mixture and the formed complex was stable up to 6 h, which shows high stability time. The data obtained from the biodistribution of 99mTc-UDCA reflect the rapid uptake in the liver which was enough to give an imaging picture.