In Vitro and In Vivo Behaviour of 111In Complexes of TTHA, TTHA-Bis(Butylamide) and TTHA-Bis(Glucamide): Stability, Biodistribution and Excretion Studied by Gamma Imaging

Aiming at radiopharmaceutical application, 111In3+ complexes of the polyaminocarboxylates TTHA, TTHA-bis(butylamide) and TTHA-bis(glucamide) were investigated. The in vitro stability of 111In(TTHA)3− and 111In(TTHA-bis(butylamide)- was evaluated by measuring the exchange of 111In3+ from the complexes to transferrin and the results were compared with those for 111In(DTPA)2−. We also performed biodistribution studies of the three 111In3+ complexes by gamma-imaging in Wistar rats and by measuring the radioactivity in their organs. TTHA and its derivatives seem to have similar in vivo biodistribution with prevailing renal excretion.


Introduction
Nuclides of indium have found widespread use in nuclear medicine. ln, a /-emitting isotope, has nearly ideal physical characteristics (it decays by electron capture emitting 2 usable gamma photons of 173 KeV and 247 KeV with a 184% photon yield [1]). ln has a half-life of 2.8 days which allows studies over several days with small activities administered radioactivity [2]. It also presents suitable chemical properties for in vivo applications: only the 3+ oxidation state occurs in water and its aqueous chemistry is dominated by its strong Lewis acidity and oxophilicity [3].
Among the chelating agents which have found applications in medicine, DTPA (diethylenetriaminepentaacetic acid) remains one of the most used [2], because it forms stable complexes with many cations and allows the preparation of bifunctional chelating derivatives; it can easily be linked to high molecular weight compounds. 4In(DTPA) has been described as an ideal agent for scintigraphic studies of the cerebrospinal fluid pathway [4,5].
Functionalization may be important to increase the selectivity of a radiotracer and to modulate its hydrophilic/lipophilic character. Since the biodistribution and excretion of substances injected into the blood stream are influenced by factors like molecular size, molecular weight, charge and hydrophilicity of the complex [6], we study in this work the in vitro and in vivo behaviour of ln complexes of the DTPA analogue TTHA (triethylenetetraaminehexaacetic acid) and of two of its derivatives, TTHA-bis(butylamide) and TTHA-bis(glucamide) (Fig.l)  high capacity for binding other metal ions, namely Ga and In [9]. When In s njected n the form of lnCI3, the metal ion is transchelated by transferrin and the radioisotope is then found in areas of high iron uptake such as bone marrow, liver and spleen [10]. Consequently, before molecules or biomolecules labelled with ln can be considered as valid radiotracers in vivo, it must be shown that the chelates are thermodynamically stable or kinetically inert towards transmetallation with transferrin.
In Vitro and In Vivo Behaviour of 11  Therefore we studied the in vitro stability of 1111n(TTHA) 3and bis(butylamide)] and compared it with that of In(DTPA) [11]. 11In[TTHA-

In vitro stability
The in vitro stability .of In(TTHA)and In(TTHABuA)was evaluated by measuring the transchelation of ln to transferrin in blood serum as a function of time. This study was performed by gel filtration, after appropriate time intervals, of the solutions containing transferrin and the ln / complexes, using a method described in the literature [10]. A carrier free solution of 150 1 of lnCl in 0.1 N HCI (370 MBq/g In) was mixed with 2 ml of 0.05 M sodium citrate (pH 6.5). This solution was mixed with 10 1 of a ligand solution, in such a way that a 1:1 ligand-metal ratio was obtained. A 200 1 aliquot of this mixture was added to 3 ml of human serum (final solution activity 1.5 mCi) and was subjected to gel filtration (Sephadex G-25, lx15 cm column). The column was eluted with 0.01 M PBS buffer (pH 7.4) at a flow rate of ca. 38 ml/h. Samples were taken after a dead volume of 4 rain. Activity in the samples was detected with a /well-counter. The same procedure has been used with a transferrin solution (2x10 M). Before mixing this solution with the complex, a NaHCOz solution was added (final HCO concentration 5 mM [11]). A 200 1 aliquot of this mixture was added to 10 t1 of an In(TTHABuA)solution. At appropriate time intervals, 25 !1 of this mixture was subjected to gel filtration as described above.

Gamma imaging
A gamma camera-computer system (GE 400 AC\STARPORT) was used for data acquisition and pre-processing. Data processing and display were performed with a CityDesk software developed for these experiments. IBM AT compatible computer usingln3._ Gamma images for the three complexes studied in this work and for In(DTPA)as comparison, were obtained using 300-400 g Wistar rats (groups of four animals with (50 mg/ml) ! chlorpromazine (2.5%) :3) d a. 150 lCi of. In complexes were injected into the femoral vein (previously catheterised with an heparinised abocat 26G) or in the tail vein. The animals were then positioned in dorsal or ventral decubitus over the detector. Image acquisition was initiated immediately after radiotracer injection. Sequences of 180 images (360 in the cases of In(TTHA) and In(TTHABuA)), of ten seconds each, were acquired to 64x64 matrices. Blood samples were taken during the dynamic acquisition and subsequently counted in a y well-counter.
The efficiency of labelling of the ligands with ln 3/ was checked at 24 h intervals by chromatography. This study was performed with an ITLC-SG/butanone system, analysing 10 !1 of each solution of the complex. Gamma images of the chromatograms were obtained to 128xt28 matrices with a total acquisition time of 15 min. For all the complexes the percentage of bound ln / was nearly 100%.
To analyse the transport of radiotracer over time, three regions of interest were drawn on the image files, corresponding to the thorax, liver and left kidney. From these regions, timeactivity curves were obtained using home-made software.
In addition, static data were acquired at 24, 48 and 72 h after the radiotracer injection.

Biodistribution experiments
Two groups of four animals were injected with ca. 100 lCi of In(.DTPA) 2 and In(TTHAGluA2)-and sacrificed 2 h later. The majors organs were removed, weighted and counted in a y well-counter. Similar biodistribution studies were also performed with the rats used in the gamma experiments referred in the previous section sacrificed at 72 h after injection with all the ln chelates studied in this work.

In vitro stability
The preliminary results on the in vitro stability of In(TTHA) z and in(TTHABuA2)-in blood serum and in a transferrin solution are summarised in Table 1. Literature data on In(DTPA) [12] are included for comparison. The presently studied complexes dissociate more rapidly than In(DTPA)-. This is in agreement with published thermodynamic stability  [7,13], which reflect the structures of the chelates in solution [7,14]. 3.2. Images and biodistribution data Fig.2 represents the averaged time-activity curves, obtained from the dynamic acquisitions for each region of interest. The curves were smoothed and normalised for the maximum activity of each one. The complexes studied undergo an early retention, both in kidneys and in liver and spleen. These results contrast with the time-activity curves obtained for the In(DTPA) complex, where the liver-spleen curve is similar to the thorax curve, corresponding to blood activity. The thorax curves for the In(TTHA) , In(TTHABuA)-and In(TTHAGluA) chelates also correspond only to blood activity.
The scintigraphic images at 30 minutes, 24 h and 72 h after ln chelates injection in rats are illustrated in Fig.3. In the early images, and for all the complexes, the activity is preferentially Iocalised in kidneys. In some of these images the injection site is also observed. After 24 h the activity was spread out and was then Iocalised mainly in the abdominal region and in the kidneys. The 72 h images show the same behaviour, but it is particularly noticeable that In(TTHAGluA) has higher uptake by the liver-spleen region. The biodistribution results (in percent of injected dose per gram of organ) obtained at 2 h and 72 h are shown in Fig. 4 and agree with the gamma-imaging. It can be noticed that in contrast to In(DTPA)-,In(TTHAGluA)-has low tissue specificity and undergoes both renal and hepatobiliary clearance. The biodistribution results obtained at 72 h (Fig.4) show that for all the complexes the activity is preferentially Iocalised in the kidneys. This indicates renal excretion of the chelates, which is consistent with their structure, molecular weight and hydrophilicity. The high late retention of the 111n complexes of TTHA, TTHABuA2 and TTHAGluA2 by the reticulo-endothelial system may be related with the formation of colloidal particles of indium hydroxide associated with partial demetallation of the chelates [15], in addition to the natural occurrence of the complexes. High radioactivity levels in blood after 30 minutes suggest that the complexes may bind to serum proteins, perhaps albumin, but further in vitro studies are necessary to validate this hypothesis. None of the complexes passes through the blood-brain barrier, as expected for high molecular weight and non-lipophilic complexes. There is no evidence of bone marrow accumulation, which is seen when the indium-transferrin complex is formed [16].
In conclusion TTHA and its derivatives have similar in vivo behaviour and the linkage of the lipophilic side chains in the case of TTHABuA2 and TTHAGluA2 does not seem to influence the biodistribution and clearance of these complexes.