Effects of Ursodeoxycholic Acid on the Biodistribution and Excretion of Various Technetium-99m Radiopharmaceuticals in Rat

In previous studies, ursodeoxycholic acid (UDCA) accelerated the biliary excretion of technetium-99m (Tc-99m) hepatobiliary radiopharmaceutical and increased F-18 uoro-deoxy-glucose (FDG) excretion from various organs. As an extension of the previous studies, the present study investigated the effect of oral administration of UDCA on the biodistribution and excretion of various Tc-99m labeled radiopharmaceuticals.


Abstract Background
In previous studies, ursodeoxycholic acid (UDCA) accelerated the biliary excretion of technetium-99m (Tc-99m) hepatobiliary radiopharmaceutical and increased F-18 uoro-deoxy-glucose (FDG) excretion from various organs. As an extension of the previous studies, the present study investigated the effect of oral administration of UDCA on the biodistribution and excretion of various Tc-99m labeled radiopharmaceuticals.

Materials
Sixty rats were randomly assigned to the Control or UDCA groups. Tc-99m HDP, Tc-99m pertechnetate, and Tc-99m DMSA were injected via the tail vein. Thirty min after the injection, the Control group was administered normal saline and the UDCA group was given 5 mg or 10 mg of UDCA orally. Thirty min, and 1, 2, 3, and 4 h after administration of saline or UDCA, images were acquired using a gamma camera.
Images were analyzed by drawing a region of interest (ROI) in the relevant organs depending on the type of radiopharmaceutical used. Four imaging parameters, Radioactivity, Rate of change (%), Rate of change by section (%), and Radioactivity/net injection dose (Radioactivity/ID (%)) were compared between groups. To investigate the action of UDCA on radiotracer excretion in the kidneys and from the soft tissues, Tc-99m MAG 3 dynamic imaging was performed and Radioactivity and Radioactivity/total body activity (Radioactivity/TBA (%)) of the soft tissue, both kidneys, and the bladder were compared.

Results
In the image analysis of Tc-99m HDP with 10 mg of UDCA, Radioactivity, and Radioactivity/ID (%) of the buttock was lower in the UDCA group at 4 h (P = 0.019, P = 0.031). Similar effects were also found in the Tc-99m HDP experiments with 5 mg of UDCA. Rate of change (%) of buttock Radioactivity was signi cantly different at 3 h − 30 min and 4 h − 30 min and buttock Radioactivity in the UDCA group decreased more substantially (P = 0.008, P = 0.024). In the image analysis of Tc-99m pertechnetate, Radioactivity of the buttock was higher in the Control group at 1, 3, and 4 h. Rates of change (%) in the thyroid gland and the buttock were different at 1 h -30 min, 3 h -30 min, and 4 h − 30 min and Radioactivity in the UDCA group decreased more substantially. Radioactivity/ID (%) was statistically different in the buttock at 4 h (P = 0.041). In the image analysis of Tc-99m DMSA, Radioactivity of the kidney increased in the UDCA group at 1 h -30 min, while that in the Control group showed little change.
In the analysis of the Tc-99m MAG 3 dynamic images, Radioactivity and Radioactivity/TBA (%) of the kidney were signi cantly higher in the UDCA group at 2 min (P = 0.014, P = 0.035). At 5 and 10 min, Radioactivity/TBA (%) of the soft tissue in the UDCA group was lower than that of the Control group (P = 0.001, P = 0.022). At 20, 25, and 30 min, there was no difference in Radioactivity and Radioactivity/TBA (%) of soft tissue, both kidneys, and the bladder.

Conclusion
The present study demonstrated that administration of UDCA increases renal excretion and soft tissue clearance of Tc-99m labeled radiopharmaceuticals. This investigation could contribute to broadening of the eld of pharmacologic application of UDCA.

Background
Technetium-99m (Tc-99m) scintigraphy is widely-used less-invasive imaging modality that has been used to evaluate the functions of various organs and diagnose diseases. Tc-99m diphosphonate bone scintigraphy is one of the most commonly used imaging tests in the nuclear medicine department to survey bone metastasis or to characterize various bone lesions. Tc-99m pertechnetate scintigraphy is also widely performed to evaluate thyroid and salivary gland function and to detect ectopic gastric mucosa in Meckel's diverticulum. Tc-99m dimercaptosuccinic acid (DMSA) scintigraphy, which is very sensitive for detecting renal cortical abnormalities, is often used to detect pyelonephritis in patients suspected of urinary tract infection.
Ursodeoxycholic acid (UDCA), the 7-β hydroxy epimer of chenodeoxycholic acid (Fig. 1), is normally present in humans in a concentration of about 3% of the bile acid pool [1] and has various hepatoprotective effects that involve increasing the hydrophilicity of bile juice. It modi es the bile acid component by decreasing the levels of hydrophobic bile acids while increasing the proportion of nontoxic hydrophilic bile acids. It also has a choleretic effect, facilitating hepatocellular bile acid excretion, as well as having cytoprotective, membrane stabilizing, and immunomodulatory properties [2][3][4]. UDCA is a Food and Drug Administration-approved medicine for cholesterol gall stone dissolution and primary biliary cirrhosis [5]. Although UDCA was evaluated as an investigational medicine in a wide swath of hepatic and extrahepatic disorders, studies applying UDCA to the molecular imaging eld are rare.
In a previous clinical study with young, healthy volunteers (mean age: 26.3 ± 2.1 years), hepatobiliary scintigraphy was performed twice per volunteer within three days, for the control and the UDCA-treated studies [6]. When the subjects were orally administered a single dose of UDCA (200 mg) 15 min before intravenous injection of Tc-99m diisopropyliminodiacetic acid (DISIDA), the time until visualization of the gallbladder (GB) was shortened from 14.2 ± 6.6 to 9.1 ± 2.8 min, and the maximum activity of the GB was markedly increased from 83.8 ± 38.4 to 132.3 ± 59.8 [6]. With UDCA pretreatment, the GB was clearly visualized in an earlier phase of hepatobiliary scintigraphy. Consequently, UDCA shortened the total imaging time and increased the speci city of hepatobiliary scintigraphy for assessing functional obstruction of the cystic duct.
Furthermore, based on a report that UDCA increases lipase activity, which breaks down triglycerides [7], we investigated the effects of oral administration of milk and UDCA on the excretion of 2-deoxy-2-[ 18 F] uoro-d-glucose (F-18 FDG) from various organs of rats [8]. Administration of milk and UDCA enhanced F-18 FDG e ux from the brain, liver, and small intestine. There was a signi cant increase in glucose-6-phosphatase (G6Pase) and a decrease in hexokinase 2 (HK2) expression in the organs in the milk + UDCA group compared to those of the Control group [8]. This suggested that F-18 FDG-6phosphate was dephosphorylated by G6Pase and transformed into F-18 FDG which is a chemical structure that is easily e uxed from cells. In addition, decreased HK2 expression was also considered to contribute to the back diffusion of F-18 FDG from the organs. Since HK2 phosphorylated F-18 FDG to F-18 FDG-6-phosphate, which is retained in the cytoplasm and hardly diffuses from cells, decreased HK2 expression was thought to increase the portion of F-18 FDG compared to F-18 FDG-6-phosphate, resulting in reduced radioactivity of the organs.
As an extension of the previous studies, the current study aimed to investigate the effect of oral administration of UDCA on the biodistribution and excretion of three Tc-99m labeled radiopharmaceuticals, Tc-99m hydroxy-methylene-diphosphonate (HDP), Tc-99m pertechnetate, and Tc-99m DMSA, in rats. Furthermore, this study tried to identify possible mechanisms underpinning the phenomenon with a Tc-99m mercapto-acetyl-triglycine (MAG 3 ) experiment.

Experimental protocols
Sixty rats were randomly assigned to the Control or UDCA groups. About 300 µL (74 MBq, 2 mCi) of Tc-99m HDP, Tc-99m pertechnetate, or Tc-99m DMSA were injected via tail vein of the rats under anesthesia by inhalation of iso urane (2%). Injection time, dose of the radiotracer prepared, and the dose that remained in the syringe after injection were recorded to calculate the net injection dose.
Just after radiotracer injection, total body radioactivities of the rat were measured by placing the rat into a well-type dose calibrator (Capintec Inc., USA, 2012. Figure 2a). UDCA was prepared by grinding the tablets into a ne powder using a mortar and pestle and dissolving the powder in normal saline. Thirty min after radiotracer injection, the Control group was administered normal saline (500 µL, 0.9%) and the UDCA group was given 5 mg or 10 mg of UDCA (500 µL) orally using a exible oral zonde needle (Φ 1.7 × 90 mm, polyethylene tube, Duksan General Science, Seoul, Korea, Fig. 2b). The amounts of UDCA were calculated based on the practice guide for dose conversion between animals and humans and previous experiments [8,9].
At each time points (30 min and, 1, 2, 3, and 4 h) after administration of normal saline or UDCA, images were sequentially acquired for 2 min using a gamma camera (Symbia T16, Siemens Medical Solutions, USA, 2011) with an energy peak setting of 140 keV ± 7.5% for Tc-99m. One detector with a low-energy high-resolution collimator was used with at a distance of 7 cm between the detector and the table. During image acquisition, a standard point source of radioactivity of about 1.11 MBq (30 µCi) of the same radiopharmaceutical in 1 mL of normal saline was placed at the upper left corner of the image eld for image analysis. After all 5 time points, total body activity of the rat was measured once again using a dose calibrator.

Image analysis
Images were analyzed using PMOD software (version 3.7) by drawing a region of interest (ROI) over the relevant organs depending on the type of radiotracer used. For analysis of the Tc-99m HDP image, the right shoulder, lumbar spine, liver, and right buttock were chosen. The ROI over the right shoulder was a 10.0 × 10.0 mm circle, while that over the lumbar spine was an 8.0 × 16.0 mm rectangle. Those over the liver, and right buttock were 12.0 × 12.0 mm circles (Fig. 3a). For analysis of the Tc-99m pertechnetate image, ROIs were drawn over the thyroid gland, stomach, and right buttock. The ROI over the thyroid gland was a 13.0 × 13.0 mm circle. That over the stomach was a 30.0 × 25.0 mm oval and that over the right buttock was a 15.0 × 15.0 mm circle (Fig. 3b). For analysis of the Tc-99m DMSA image, ROIs were drawn over the right kidney, left kidney, and right buttock. The ROI over each kidney was a 22.0 × 29.0 mm oval and that over the buttock was a 15.0 × 15.0 mm circle (Fig. 3c). The ROI over the standard point source was drawn as a 27.2 × 52.4 mm rectangle in all radiotracer images.
Four imaging parameters, Radioactivity, Rate of change (%), Rate of change by section (%), Radioactivity/time-corrected net injection dose (Radioactivity/ID (%)), were compared. The rst parameter, Radioactivity (kBq) was calculated from counts in the ROI using a correction factor deduced from standard point source information. The second parameter, Rate of change (%) was calculated as (radioactivity at each time point -radioactivity of 30 min image)/radioactivity of 30 min image, which re ects the rate of change from time 30 min to each speci c time point. The third parameter, Rate of change by section (%), was calculated as (radioactivity at each time point -radioactivity at the time point just before)/radioactivity of the time point just before. The last parameter, Radioactivity/time-corrected net injection dose (Radioactivity/ID (%)), was calculated by dividing Radioactivity of the ROI by timecorrected net injection dose.
Tc-99m MAG 3 experiment Tc-99m MAG 3 was used to elucidate the mechanism of action of UDCA on radiotracer excretion in the kidneys and from the soft tissues using dynamic image acquisition. Thirteen rats were randomly distributed to the Control (n = 6) and UDCA (n = 7) groups.
Images were obtained 10 min after oral administration of normal saline (500 µL, Control group) or UDCA (500 µL, 10 mg, UDCA group). Before acquiring the scans, rats were anesthetized and a 27-gauge infusion set with a 30 cm tube lled with saline supplemented with heparin (50 international units (IU)/mL) was inserted into a tail vein [10]. Just after the start of the dynamic image acquisition, about 300 µL (74 MBq) of Tc-99m MAG 3 was infused, followed by ushing with 700 µL of normal saline. Dynamic images were acquired for 30 min at a rate of 1 frame per second, resulting in a total of 1800 scans.
Images were analyzed by drawing ROIs over the total body, both kidneys, and the bladder. The ROI of the total body was 110.7 × 211.2 mm and that of the standard point source was a 27.2 × 52.4 mm rectangle. The ROIs over both kidneys and the bladder were drawn along the outlines of the organs (Fig. 3d).
Soft tissue radioactivity was calculated by subtracting the radioactivity of both kidneys and the bladder from total body radioactivity. Since there might be functional differences between the left and right kidneys, the sum of both kidneys' counts was used for image analysis and for calculating the time to peak renal activity (T max ) and half-time of renal activity (T 1/2 ). Radioactivity and Radioactivity/total body activity (radioactivity/TBA (%)) of the soft tissue, both kidneys, and the bladder were compared.

Statistical analysis
All data are presented as mean ± standard deviation (SD). All image parameters and total body activity of both groups were compared using the Mann-Whitney U test (IBM, SPSS Statistics 23 USA). A P value of less than 0.05 was considered statistically signi cant. Net injection dose and total body radioactivity measured by dose calibrator Net injection dose (Net ID) was calculated by subtracting the radioactivity that remained in the syringe after injection from the radioactivity of the radiotracer as prepared. Time correction was applied based on the injection time to calculate the net injection dose. Net ID was not statistically different between the Control and the UDCA groups in all radiopharmaceutical experiements (Table 1). Total body radioactivity (TBA) measured just after radiotracer injection was similar between the Control and the UDCA groups in all experiments. The TBAs after all 5 image acquisition time points were generally lower in the UDCA group, except in the Tc-99m DMSA experiment. However, there were no signi cant differences between the 2 groups ( Fig. 7 and Table 1). Time-activity curves of radioactivity in the kidneys in the 2 groups were drawn and compared. Because renal function may differ between the 2 kidneys in the same animals, whole-kidney radioactivity was used to draw the time-activity curve. Time to peak renal activity (T max ) and half-time of renal activity (T 1/2 ) were shorter in the UDCA group (T max = 3.430 min; T 1/2 = 17.432 min) than in the Control group (T max = 4.134 min; T 1/2 = 23.321 min). T max and T 1/2 are shown on the kidney time-activity curve (Fig. 8).
At the 15 min time point, Radioactivity/TBA (%) of the soft tissue in the Control group was higher than that of the UDCA group, however, the P-value was 0.051 (15 min, Control : UDCA = 32.793 ± 3.986% : 29.532 ± 2.793%, P = 0.051).
At the 20, 25, and 30 min time points, there was no difference between the 2 groups in Radioactivity and Radioactivity/TBA of soft tissue, both kidneys, and the bladder. Figure 9 demonstrates Radioactivity/TBA (%) curves of the soft tissue and bladder. An asterisk (*) indicates statistical signi cance.
Net ID and peak radioactivity of the kidney did not differ between the 2 groups (net ID, Control : UDCA = 51.282 ± 1.

Discussion
UDCA, 3α,7β-dihydroxy-5β-cholan-24-oic acid, is normally present in the human bile acid pool at concentration up to 4%; it is formed by 7β-epimerization of chenodeoxycholic acid in the colon by a bacterial enzymatic reaction [10]. UDCA is the only drug approved by the Food and Drug Administration for treatment of primary biliary cirrhosis. It is also approved for cholesterol gall stone dissolution. UDCA reportedly increases the hydrophilicity of the bile acid pool and bile ow as well as exerting immunesuppressive effects [11][12][13].
After oral administration of UDCA, about 90% of a therapeutic dose is absorbed in the small bowel and enters the portal circulation [14]. Subsequently, it undergoes e cient extraction from portal blood by the liver. First-pass extraction from the portal blood ranges from 50-70% [15]. In the liver, UDCA is conjugated with either glycine or taurine and excreted into the bile [15]. UDCA in bile juice accumulates in the gallbladder and is expelled into the duodenum. In the small intestine, some conjugated UDCA is deconjugated and reabsorbed in the terminal ileum [15]. Small quantities of UDCA appear in the systemic circulation due to its high rst-pass metabolism [16].
The biologic half-life of orally administered UDCA in humans is quite long, estimated at 3.5 to 5.8 days, due to enterohepatic circulation [15]. After oral administration of a 500 mg tablet of UDCA to healthy volunteers, T max of plasma concentrations occurs at 60 min and a second peak concentration occurs at 180 min [15]. In the present study, most Tc-99m radiopharmaceuticals remaining in the soft tissue were observed to be signi cantly lower in the UDCA group at the 3 and 4 h time points. The long time course of UDCA action is considered due to be its long biologic half-life related to effective enterohepatic circulation.
UDCA is known to act mainly in the hepatobiliary system. In the current study, I measured liver Radioactivity with Tc-99m HDP, and as expected, liver Radioactivity in the UDCA group showed greater excretion than was seen in the Control group at the 2 h -30 min and 4 h -30 min.
UDCA is generally well tolerated with few side effects. Diarrhea is the main reported side effect. The incidence of diarrhea in controlled human studies was up to 3% [15]. In the current study, only a few rats showed loose stool after taking UDCA and no rat suffered from watery diarrhea.
During clinical imaging tests, various pharmacologic interventions have been attempted to aid diagnosis. In the nuclear medicine eld, when performing scintigraphy for Meckel's diverticulum, glucagon can be used to increase the intake of Tc-99m pertechnetate, and cimetidine can be administered to suppress excretion from the gastric mucosa, with the aim of reaching a higher lesion-to-background ratio with high sensitivity. When performing a hepatobiliary scan to check for acute cholecystitis, if the gallbladder does not show for up to 1 h, instead of obtaining a delayed image at 4 h, morphine can be administered to increase biliary tract pressure by contracting the sphincter of Oddi.
The mechanisms of uptake and excretion pathways of various radiopharmaceuticals are thought to be one factor affecting the action of UDCA. Tc-99m HDP, one type of Tc-99m diphosphonate radiopharmaceutical, is taken up by hydroxyapatite crystals in the surface of bone by chemisorption. This strong adsorption may make it di cult for UDCA to release Tc-99m HDP from the bones and joints. In the experiments with Tc-99m HDP in the normal rat and fracture model, the 4 imaging parameters of ROIs in the lumbar spine and the shoulder were not signi cantly different between the 2 groups. On the other hand, on day #1 after fracture, the amount of Radioactivity at the tibia fracture site decreased more in the UDCA group at 2 h -30 min, 3 h -30 min, and 4 h -30 min. It is presumed that UDCA can act more freely in tissue that has been loosened by a fracture. This effect of UDCA was not seen at the tibia fracture site on day #6 after fracture. Unlike Radioactivity of the fracture site, that of the buttock, representing soft tissue, was found to be lower in the UDCA group in both normal rats and fracture models mostly at the 3 or 4 h time points.
Tc-99m pertechnetate scintigraphy is a widely performed nuclear imaging modality that is used to evaluate thyroid and salivary gland function and to detect ectopic gastric mucosa in Meckel's diverticulum. Tc-99m pertechnetate is transported by the sodium iodide symporter; therefore, Tc-99m pertechnetate scintigraphy is a powerful imaging modality for assessing sodium iodide symporter activity in various organs [16,17].
Tc-99m DMSA scintigraphy is used to gain information on renal cortical morphology. Two mechanisms of Tc-99m DMSA tubular uptake are peritubular extraction from the blood into the proximal tubular cell of the pars recta, where the last straight part of the proximal tubule and glomerular ltration, followed by tubular reabsorption [18][19][20][21].
Tc-99m MAG 3 , another popular renal radiopharmaceutical, is more speci c to renal excretory function.
The most important mechanism of uptake of Tc-99m MAG 3 is extraction from blood in efferent arterioles by basolateral uptake into cells lining the proximal tubules, which mediated by an active anion transport system and mostly occurs in the rst convoluted portion of the proximal tubules [22]. Various factors can affect the absorption and excretion of radiopharmaceuticals, such as the patient's clinical condition, the radiopharmaceutical preparation method, and the effects of other medicines which the patient is taking. Given the results of the current study, UDCA is thought to be an example of a medicine that affects uptake and excretion of various radiopharmaceuticals. The locations of UDCA's impact and the substances on which UDCA acts should be identi ed at the cellular and molecular biological level in further studies.
TBA measured by a dose calibrator after image acquisition was also generally lower in the UDCA group, except for in the Tc-99m DMSA experiment. T max and T 1/2 obtained in the Tc-99m MAG 3 experiment were also shorter in the UDCA group (T max : 3.430 min, T 1/2 : 17.432 min) than in the Control group (T max : 4.134 min, T 1/2 : 23.321 min). However, the differences were not statistically signi cant. The reason for statistical insigni cance might be the small number of rats used. A further larger-scale experiment is necessary.
This study has several limitations. First, since investigating the effect of UDCA on various radiopharmaceuticals, the number of rats in each radiopharmaceutical group was not large. Second, the dose-effect of UDCA was con rmed only in the Tc-99m HDP experiment using 5 and 10 mg of UDCA.
Studies with more varied doses should be conducted to determine the optimal dose. Third, in the renal excretion experiment using Tc-99m MAG 3 , because the time that rats could be maintained under anesthesia was limited to 30 min due to safety concerns, the pharmacological effects of UDCA after 30 min could not be investigated. Besides, because this study con rmed the effects of UDCA only by animal experiments using rats, additional research at the cellular and molecular biological scale is necessary to identify the mechanisms of action of UDCA on various Tc-99m radiopharmaceuticals. Nevertheless, to our knowledge, it is the rst study to evaluate the effect of UDCA on the biodistribution and renal excretion of various Tc-99m radiopharmaceuticals.

Conclusions
In sum, various parameters in the buttock of the UDCA group were lower in all 3 Tc-99m radiopharmaceutical experiments. Liver activity in the Tc-99m HDP experiment and thyroid activity in the Tc-99m pertechnetate experiment were also lower in the UDCA group than the Control group. However, UDCA did not affect any of the 4 parameters in the lumbar spine or shoulder joint. Interestingly, in the Tc-99m DMSA experiment, earlier accumulation of the radiotracer into the kidneys was found in the UDCA T