Development and biodistrubition modeling of 99 m Tc-DTPA

Purpose: In this study, the team modeled the biodistribution and the efficiency of two 99m-technetium diethylene triamine penta acetate (99mTc-DTPA) based radiopharmaceuticals. Methods: The first radiopharmaceutical (DTPA-CNESTEN) is developed at the laboratories of the radiopharmaceutical production unit of the National Center for Nuclear Energy, Sciences and Technologies (CNESTEN-Morocco), and the second one is the commercial DTPA (DTPA-ref). Freeze-dried kits were successfully radiolabeled (radiochemical purity >95%) with the 99m Tc. Then drugs were injected to male BALB/c mice. In each 2 min, 5 min, 15 min, 1 h and 2 h time points after injections we evaluate tissue’s distributions characteristics. At the end, an automatic modeling of the data were recorded from thyroid, blood and urinary excretion kinetics and biodistribution in mice using both DTPA kits. The study aimed to extract the parameters of the function used to fit the recorded data. Results and Conclusion: the team concluded that the biodistribution of 99mTc-DTPA can be modeled using a combination of two exponential parts. Moreover, the resultant plots showed that there is strong correlation between the formula found in literature and the one derived on the basis of the fit of data sets in this study. In addition, it was found that the biodistribution behaviors of the developed kit and the commercial one were very close. The obtained results suggest that the developed DTPA has practically the same kinetics as the commercial one.


Introduction
Radiopharmaceuticals are radioactive compounds used for imaging and diagnosis of human diseases. Some of the radioactive are used for cancer therapy (about 5%). 1, 2 A radiopharmaceutical has two components: a radionuclide and an agent (the pharmaceutical), which directs the radionuclide to a receptor antigen, ionic pump, or other site of interest within the body. Some radiopharmaceuticals are simple, such as the ionic form of the radionuclide, while most radiopharmaceuticals have a complex chemical structure where the radionuclide provides a signal, indicating the site of localization of the carrier molecule. 2,3 Diethylene triamine penta acetic acid (DTPA) is one of pharmaceuticals. It used in different complexes for different objective, for examples: [111In-DTPA-DPhe1] octreotide is used for diagnostic stomastatin sintigraphy 4,5,6,7 , Diethylene triamine penta acetic acid neolactosyl human serum albumin labeled with technetium-99m ( 99m Tc-DTPA-LSA kit) afford the opportunity of hepatic receptor imaging. 8 Diethylene triamine penta acetate galactosyl human serum albumin (GSA) is used as human hepatic asialoglycoprotein receptorbinding radiopharmaceutical in Japan since 1992. 8 In constant, DTPA is an efficient candidate when it is radiolabeled with a therapeutic radioisotope such as 177 Lu or 90 Y. 9,10,11,12 Although this molecule has a negligible bridging capacity. 13 It is used as a simple radiopharmaceutical (technetiated radiopharmaceutical). 99m Tc-DTPA is the most commonly used radiopharmaceutical for renography. 14 The commercial DTPA kits are usually made of pentasodium or calcium trisodium salt of DTPA (diethylene triamine penta acetate. The freeze-dry kitcontains appropriate amounts of stannous chloride dehydrate in lyophilized form. Radiolabeling is performed by adding oxidant-free 99m TcO4 -to the kit vial and mixing. In this study, attempt was made to fit the obtained experimental data in order to establish the developed DTPA biodistribution mathematical model. Also, the team had compared the behaviors of the developed kit (DTPA-CNESTEN) and the commercial one (DTPA-ref).

Gamma counter
The gamma counter used is NaI detector type COBRA 5002 from Packard.

Radiolabeling
Radiolabeling of DTPA-CNESTEN and the DTPA-ref, l, were performed by reconstituting the freeze-dried kit using 5 mL of freshly eluted 99m TcO4 -solutions. The 99m TcO4 -solutions were eluted from 99m Mo/ 99m Tc generator containing a maximum of 40 mCi of activity. The 99m Tc-DTPA labeled in this manner should be stable for over 4 hours after labeling. 15, 16

Animals
To evaluate tissue's distributions characteristics of DTPA-CNESTEN and DTPA-ref, biodistribution studies were performed using male BALB/c mice, average weight about 50-60 g. The animals were carried from Pasteur Institute-Casablanca. During this study, they were reared in our laboratory and they had free access to food and water at all times. The room temperature was about 20 °C.

Radiochemical purity
Radiochemical purity (RCP) is an important quality parameter for radiopharmaceuticals, as their radiochemical form determines their biodistribution. 14,17 For this reason, the use of radiopharmaceutical in vivo needs RCP testing to be carried out just before administration to the patient. 16,18,19 To determine radiochemical purity labeling efficiency of the product, European Pharmacopeia's chromatographic method, which is fast and easy, has been used. 14 This method involves ascending instant thin layer chromatography (ITLC). The ITLC strips were utilized as the stationary phase and two different solvent systems as indexed in European Pharmacopeia 2008. The procedure involved spotting about 2 µl sample of 99m Tc DTPA onto chromatography strips 10 cm in length. After developing in the solvent, the strips were cut into two portions and the activity in each portion was measured in the form of counts using a gamma counter. The percentage activity at the origin and the front were determined. The complex 99m Tc DTPA remained at the origin and free technetium traveled with the solvent (methyl-ethyl-ketone MEK) front (Rf = 0.9 -1.0). The percentage of colloid was determined using 0.9% solution of sodium chloride as the mobile phase (the 99m Tc-DTPA and TcO4 -have about the same Rf in this system). Complexes were successfully labeled (RCP > 95%) and stable about 24 hours.

Biodistribution study
Biodistribution studies regroup almost all of studies using radiopharmaceuticals. It is an important analytical step in preclinical studies. 20 After successful operations of radiolabeling, dilution (with 0.9% NaCl solution) to obtain a volumetric activity about 400 µCi / 5.4 mL and RCP was above 95%. Mice were weighed and injected with 0.1 mL of the radiolabeled compound in the tail vein. After injections, the animals were kept in separately numbered beakers. And their urine was collected.
The injected activity is calculated by taking the difference between the weight of the syringe before and after the injection. At the end of 2 min, 5 min, 15 min, 1 h and 2 h time points, the animals were killed. They were dissected and blood sample was first taken by heart puncture. Then the other organs of interest were carefully dissected, placed in individual disposable plastic tubes and accurately weighed. The focus of this study was thyroid, blood, and urine. At least three animals were studied at each time point. After killing each mouse, the tail was removed and kept separately. The activity in the organs and the tail were measured in the gamma counter.
The total retained dose (%TRD) estimated as follows: % TRD(organ) = × 100 Where, "A" denotes the activity or counts in the organ of interest and "B" represents the activity or counts in all organs and the carcass except for the tail.
To accurately estimate the activity and to account for decay corrections in the 99m Tc activity, standard solutions of the radiopharmaceuticals were prepared.

Data Modeling
Radiopharmaceuticals administered to humans disappear from the biological system through fecal or urinary excretion. This biological disappearance follows a bi-exponential law 21,22 , expressed as: where, "t" designate time, "a" and "c" the intercepts and "b" and "d" are the slopes of the fast and the slow components of the plasma disappearance curve.
In this work, the team used the Open Curve Fitting Tool 3.3.1, incorporated under the MATLAB R2013b platform 23 , to compute the best values of the exponential parameters a, b, c and d with 95% confidence bounds. The optimal values of these parameters correspond to the minimum of the mean square error, which can be defined as the differences between the original data y (response value) and the predicted response value ŷ at each predictor value.
For both sets of data, the mean values of the parameters defined above were calculated. The Table 1 below shows the obtained values of the model parameters; whereas Figure 1 illustrates the plot of the experimental data and the corresponding mathematical model.  The comparison of the results obtained for the developed DTPA at the CNESTEN and the commercial one are illustrated in the Figure 2 below. This figure shows that the developed medicine has the same behavior as the commercial one.

Conclusion
In this study, the team concluded that the biodistribution of DTPA developed at the CNESTEN, can be modeled using a combination of two exponential parts. In addition, the graphics representations of the modeled data show that the drug evolution in both thyroid and blood has an opposite sense with its evolution in the urinary excretion. However, the obtained results proved that the developed DTPA has practically the same kinetics as the commercial one. International Journal of Cancer Therapy and Oncology 5 www.ijcto.org