It is considered, lipophilic compounds labeled with radionuclides are used for liver imaging to evaluate the functional status of the hepatocytes and the patency of the biliary duct [1]. Most hepatobiliary agents labeled with ^99mTc are iminodiacetic acid (IDA) derivatives, including ^99mTcdisofenin[N-(2,6-diisopropylacetanilide) iminodiacetic acid, DISIDA] [2], ^99mTc-mebrofenin [N-(3-bromo- 2,4,6-trimethylacetanilide)iminodiacetic acid] [3-6], ^99mTc-EHIDA[N- (2,6-diethylacetanilide)iminodiaceticacid], ^99mTclidofen[N-(2,6- dimethylacetanilide) iminodiaceticacid] [7,8], ^99mTc-IODIDA [N-(3- iodo-2,4-diethylacetanilide)iminodiaceticacid] [9], and ^99mTc-IOTIDA [N-(3-iodo-2,4,6-trimethyl acetanilide)iminodiacetic acid] [10,11]. In nuclear medicine, ^99mTc is the most widely radionuclide due to its suitable half-life (T1/2=6.01 h), γ-ray energy (140 keV, 89.4%) convenient for SPECT, and very low abundance of β-emission [12-15]. The erstwhile reported complexes of ^99mTc used in nuclear medicine procedures make extensive use of the technetium oxo [^99mTcO]₃+ core where in ^99mTc is in the +V oxidation state. In the development of receptor specific radiopharmaceuticals, a major drawback encountered in the use of [^99mTcO]₃+ complexes is the low specific activity of the resulting radiolabeled product due to requirement of high concentration of ligand for stabilization of the (+V) oxidation state of ^99mTc. In the recent past, the advent of the novel organometallic tricarbonyl core of technetium viz. [^99mTc(CO)₃(H₂O)₃]+ wherein Tc research efforts directed towards the preparation of high specific activity complexes of ^99mTc. The use of this core has been adequately demonstrated in the development of ^99mTc-based CNS receptor ligands [16,17]. The tricarbonyl core possesses a low spin d6 Tc(I) center which is kinetically inert and hence offers more ligand flexibility in terms of size, charge and lipophilicity without influencing the thermodynamic stability. Here, Ursodeoxycholic acid [3α,7β-dihydroxy-5β-cholan-24- oic acid] (UDCA) 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. ^99mTc-tricarbonyl UDCA can be prepared with high radiochemical yield and study its stability with time in human serum, then biological distribution experiments can be studied.

Materials

The materials utilized for the experiments are given below.

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), Ursodeoxycholic acid was obtained as a gift from Sigma Pharmaceutical Company-Egypt, and all other chemicals were purchased from Merck and they were reactive grade reagent.

Apparatus: A well-type NaI scintillation γ-Counter model Scalar Ratemeter SR7 (Nuclear Enterprises Ltd., USA) was used for radioactive measurement; Electrophoresis apparatus model EC- 3000 p-series programmable (E.C.apparatus corporation) power and chamber supply units using cellulose acetate strips was used.

Animals: Swiss Albino mice weighing 20-30 gm 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 hr on/off light schedule. Animals were kept with free access to food and water all over the experiment.

Methods

The methods implemented are described below.

Synthesis of ^99mTc-tricarbonyl precursor: [^99mTc(CO)₃(H₂O)₃]+ ion was prepared by the addition of 1 ml of ^99mTc-pertechnetate (20– 100 mCi ^99mTcO4) to a penicillin vial with 7.15 mg sodium carbonate, 4.5 mg sodium boranocarbonate, 2.85 mg sodium tetraborate and 8.5 mg sodium tartrate. After heating for 30 min in a boiling water bath and cooling, the basic solution (pH ≈ 11) to room temperature. The labeling yield and stability of the ^99mTc-tricarbonyl precursor were determined using reversed phase high-performance liquid chromatography (HPLC). The ^99mTc-tricarbonyl precursor was successfully prepared with a high radiochemical yields (>96%) [9].

Radiolabeling with ^99mTc-tricarbonyl precursor: Labeling was performed by adding 1 ml of the prepared ^99mTc-tricarbonyl precursor to the 3 mg UDCA, at room temperature. Next, the reaction vial was heated at 100°C for 30 min. After cooling to room temperature, labeling yields were checked by radio-HPLC [10].

Quality control: The radiochemical yield and purity of ^99mTctricarbonyl UDCA was determined electrophoresis condition and HPLC.

Electrophoresis Conditions: Paper 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 0.05M phosphate buffer pH 7.2 ± 0.2 or saline 0.9% and then were introduced in the chamber. Samples (5 μl) were applied at a distance of 10-12 cm from the cathode. Standing time and applied voltage were continued for one and half hours to 3 hrs. Developed strips were dried and cut into1 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.

Radioactivity yield (%) = Peak activity of the ^99mTc-tricarbonyl UDCA ×100/Total activity

HPLC analysis for ^99mTc-tricarbonyl: Reversed phase-HPLC method which consists of pumps LC-9A, Rheodyne injector and UV spectrophotometer detector (SPD-6A) operated at a wavelength of 276 nm was developed a reversed phase column Lichrosorb RP18 (250 mm × 3 mm, 5 μm) column. The mobile phase consisted of methanol (solvent B) and 0.05M TEAP (solvent A). The HPLC gradient was made up of an isocratic elution (100% A) for the first 0 ~ 5 min; a linear gradient of 75% A/25% B to 100% A/0% B for 5 ~ 8 min; a linear gradient of 66% A/34% B to 75%A/25% B for 8 ~ 11 min; a linear gradient of 0% A/100% B to 66% A/34% B for 11~22 min; and an isocratic elution (100% B) for 22~25 min. The flow rate was 0.6 ml/min and a 10 μl aliquot was injected into the column [11,12].

Biodistribution of the labeled ^99mTc-tricarbonyl 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-tricarbonyl UDCA. The mice were kept in metabolic cages for the required time. Mice were sacrificed 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 counted. Correction was made for background radiation and physical decay during the experiment [13].The weights of blood, bone and muscles were assumed to be 7, 10 and 40% of the total body weight, respectively [14]. 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.

Drug inhibition study of ^99mTc-tricarbonyl UDCA: To confirm that the ^99mTc-tricarbonyl UDCA was accumulated specifically with high affinity binding located in liver, different concentration of cold UDCA (50 μg/kg of mouse) was injected at 5 min prior to the injection of ^99mTc-tricarbonyl UDCA [15-17].

Statistical analysis: Data were evaluated with one way analysis of variance test. Results for p are reported, and all the results are given as mean ± SEM. The level of significance was set at p<0.05.

Electrophoresis

The paper electrophoresis pattern revealed that ^99mTc-tricarbonyl UDCA complex moved towards the anode at optimum conditions pH 9, indicating the cationic nature of this complex.

In vitro stability test of ^99mTc-tricarbonyl UDCA: In-vitro stability of ^99mTc-tricarbonyl UDCA was studied in order to determine the suitable time for injection to avoid the formation of the undesired products that result from the radiolysis of complex [18]. These undesired radioactive products might be accumulated in non-target organs. The results of stability showed that the ^99mTc-tricarbonyl UDCA is considered stable for 2 hours at 37°C (n=5 experiments) which remains stable ~98 ± 0.2% by RP-HPLC.

Stability in serum: The stability of ^99mTc-tricarbonyl Lev can be determined by RP-HPLC as in that indicated this complex remained stable during 6 h in normal serum at 37°C resulted in a small release of radioactivity (n=5 experiments) which decreased from >98% to 90% at 12 h [19].

Optimization: This complex was optimized at pH 9 than 11, 10, or 8 as in with 3 mg substrate and remain stable up to 6 h where was ppt. in acidic medium

Drug inhibition study of ^99mTc-tricarbonyl UDCA: By the intravenous injection of cold UDCA result in extensive decrease in the accumulation of radioactivity of ^99mTc-tricarbonyl UDCA within the liver as it dropped from 25.04 ± 0.19 to 5 ± 0.12% ID/g at 60 min post injection by the injection of 0.25. 0.5, 0.75 and 1 μg of the cold UDCA. These intensive decreases confirm the selectivity and high binding affinity of ^99mTc-tricarbonyl UDCA to liver.

Biodistribution of ^99mTc-tricarbonyl UDCA in mice: Biodistribution study of ^99mTc-tricarbonyl UDCA in normal mice showed that it was distributed rapidly in blood, kidney, liver and intestine at 30 min post injection. After 1 h, ^99mTc-tricarbonyl UDCA uptake was significantly decreased in blood, stomach and bone while, it increased in liver, intestine and kidneys. At 2 h post injection, the majority of tissues and organs showed significant decreased in ^99mTctricarbonyl UDCA uptake. On the other hand, lung and intestine showed significant increase in ^99mTc-tricarbonyl UDCA uptake [20]. There were gradual increase of liver and intestine uptakes through experiment time points, which indicated that the clearance mechanisms of these complexes were through the renal and hepatobiliary path way where the urine was increase from13.5 ± 0.005 at 30 min into 45.57 ± 0.33 at 2 hr. The uptake in liver was 22.50, 25.04 and 10.44% at 30 min, 1 h and 2 hr respectively that considered more than ^99mTc-UDCA which gives 15.37 ± 0.19 at 30 min post injection. This uptake may be useful for radioimaging of the liver.

Limitations of hepatobiliary imaging: Atomic medication techniques can be drawn out. It can take a few hours to days for the radiotracer to aggregate in the body piece of interest and imaging may take up to a few hours to perform, however now and again, more up to date gear is accessible that can considerably abbreviate the methodology time. The determination of structures of the body with atomic solution may not be as high as with other imaging procedures, for example, CT or MRI. Be that as it may, atomic medication sweeps are more touchy than different systems for a mixed bag of signs, and the utilitarian data picked up from atomic drug exams is regularly ridiculous by other imaging procedures.

A ^99mTc-tricarbonyl UDCA was prepared under 30 min heating at 100ºC, labeling yield and stability were analyzed by high performance liquid chromatography (HPLC) with stability for 6 h at pH 9. ^99mTctricarbonyl UDCA was considered more specific and located in liver than ^99mTc- UDCA as previous before. The data obtained from the biodistribution of ^99mTc-tricarbonyl UDCA reflect the rapid uptake in the liver which was enough to give an imaging picture. As a result, ^99mTctricarbonyl UDCA has high accumulation in liver and is considered a novel radiopharmaceutical for liver imaging.

The financial support of research Center of the college of pharmacy, and Dean of scientific Research at King Saudi University is greatly appreciated.