Synthesis of 4-Amino-1-Hydroxy-Butane-1,1-Diphosphonate (AHBDP) - Stannous Complexes for the Preparation of Ahbdp-Sn(II)-Tc and its Biodistribution in Rats

The new potential tracer of bone imaging, AHBDP-Sn(II)-TcO.3H2O was synthesized by reducing the TcO4− to TcO2+ in the presence of AHBDP and Sn(ll)’s reducing agent. We found that tin rapidly forms a stable complex with AHBDP, giving AHBDP-Sn(II).3H2O. In the excess of AHBDP-Sn(ll).3H2O, the AHBDP-Sn(II).3H2O coordinates with TcO2+ to give AHBDP-Sn(II)-TcO.3H2O which could polymerise or oligomerise to give hydrophobic species. The overall process appears as a first-order reaction (K= 0.67 ± 0.005s−1). In rats, the fixation of AHBDP-Sn(II)-99mTcO. 3H2O on bone is homogeneous and the scintigraphic images have the same quality as those of 1-hydroxymethane-1,1-diphosphonate-Technetium (HMDP-99mTc). The activity in non-target organs was neglible.

The subsequent reduction of TcO4(') from +7 to a lower level of oxidation in the presence of various ligands is essential for radiopharmaceuticals. Stannous chloride (SnCI2) is frequently used as a reducing agent for TcO4('). Unfortunately, in aqueous solution, Sn(ll) is easily hydrolyzed and.
oxidized. Its solution is stable only in the presence of hydrochloric acid 5-9. However, it has been reported that a large excess of SnCI 2 in labelling systems affects not only the quality, purity and stability of the radiopharmaceuticals, but also the biological behaviour of 99mTc-complexes.
Sn(ll) forms chelates with many substrates so that the labelling procedures generally yield a mixture of tin and technetium chelates [10][11][12] Deutsch proposed that tin can and does bind to T complexes 1. Vol. 2, No. 4, 1995 Synthesis of 4-Amino-l-Hydroxy-Butane-l,l-Diphosphonate Huigen et al. showed that the HEDP-Sn(II)-Tc has a molecular charge between -4 and -9 at pH 7 and that in an acidic pH, the charge increases 13 .Tji et al showed that the diphosphonatetechnetium complexes adsorb on calcium phosphate, as a model of bone adsorption. For the -Tc(lll), -Tc(IV), and HEDP-Tc(V) complexes, the adsorption on calcium phosphate increases in the following order Tc(V) < Tc(IV) < Tc(lll) 3. It was observed that the chemical structure of complexes affects the biological behavior. According to its chemical structure, we can predict its biological function. However, the chemical structure of the technetium-complexes was not clearly shown. In this study, we want to report the synthesis and physico-chemical characterisation of the AHBDP-Sn(II)-Tc by using AHBDP-Sn(II).3H20 as a chelating and reducing agent for the technetium. We have also investigated its biodistribution in rats. The AHBDP is dissolved in water and its solution is stable for many hours. SnCI 2 was dissolved in a 0,1 N HCI solution. The dissociated constant, k a, is equal to 5.7 10 -3 M'l.s "1 6.The Sn(ll) concentration is measured spectrophotometrically by complexation with cocathiline, using 523 2773 M'l.cm"1.
Potentiometric titrations were done with a CG-837 pH meter at 22C, using the Gerat( Schott glass electrode, model N 52A. A solution (25 ml) containing 0:1, 1:1 and 1:3 metal to ligand molar S. Mankhetkorn, C. Blanehot, M. Duran-Cordobes Metal-Based Drugs D. El Manouni, Y. Leroux and J.L. Moretti ratios was introduced into the titration cell, and the proton ion concentration was determined by a number of successive readings after addition of small quantity of standard 0.01 N NaOH.
The tissue distribution was performed at 30 minutes, 1, 2 and 3 hours post injection of 74 MBq per rat. After dissection of rat, organs sample activities were counted by NaI(TI) scintillator (Packard COBRA 5010).

I. Complexation of AHBDP-Sn(II)
The kinetic studies and final product analysis were performed for several concentration ratios of AHBDP and Sn(ll). The pH value of the solutions varied between to 11.

Spectrophotometric studies:
The initial AHBDP and Sn(ll) concentrations varied from 10 -4 to 10 -2 M. The solution of AHBDP did not absorb UV-Vis light. At pH 3, the mixture solution of AHBDP and Sn(ll) gave an absorption band at 224 nm. In alkaline solution, the absorbance at this wavelength decreased while a new band appeared at 276 nm. The solutions were stable for less than 5 hours.The reaction was done in less than 1 minute.
For the study of the complexes, two series of experiments were performed. In one series, the concentration of AHBDP was varied in the presence of a constant excess of Sn(ll) ([Sn(ll)] =10 -2 M). However, in the other, the Sn(ll) concentration was variable and the AHBDP constant; [AHBDP] 10 -2 and 5 x 10 -2 M. The pH of the solutions was adjusted to 3, using 1.0 N HCI. The formula of the complexes was further investigated by spectrophotometrical measurements16. The absorption band at 224 nm increased as the molar ratios of Sn(II):AHBDP increased. This absorbance was stable when the molar ratio was equal to 1. In these conditions, the reaction was done with stoichiometry 1:1.

!.2. Potentiometric titrations of AHBDP-Sn(II)
The titration curves are illustrated in Figure for AHBDP chelate of Sn(ll) at 1:0, 1:1 and 1:3 ratios of metal to ligand. There is evidence of complex formation between AHBDP and Sn(ll). Titrations of equimolar amounts of AHBDP and Sn(ll) resulted in a steep rise at [NaOH] 0.002 M, corresponding to the formation of a 1:1 complex. A 1:3 molar ratio resulted in steep rise at [NaOH] 0.0014 M. This indicates that AHBDP-Sn(II) exists as 1:1 complex.
In purified water, AHBDP is present in the zwitterionic form. The PKa of AHBDP were determined, pk al, Pka2 and Pka3 are equal to 3, 6.8 and 10.
In order to identify the products of the reaction, two series of experiments were carried out in the following conditions" [AHBDP] [Sn(ll)] =10 -2 M. In the first series the pH was equal to 2, while in the second, it was adjusted to 11 with NaOH. After the completion of the reaction, the solutions were lyophilized and the products were analyzed by 31p.NMR. It should be noted that the lyophilized powder at pH 11 was instable in the ambient conditions; its color changed from white to black and became insoluble in water. On the other hand, the lyophilized powder at pH 2 was stable and soluble in water. The 31 p. NMR spectrum was very different from the one that obtained from the pure AHBDP (The pure AHBDP 5 19.5 ppm; In complex solutions this peak has disappeared). This indicated that there is a modified electronic environment of phosphorus and all of AHBDP is complexed.
2. Complexation of AHBDP-Sn(II)-Tc We have verified that AHBDP-Sn(II) solutions were stable for several hours and we always used fresh solutions in our experiments.

Spectmphotometric studies
In this series of experiments, the concentration of AHBDP-Sn(II) varie from 10 "6 to 2 x 10 -3 M and the concentration of TcO 4(') varied from 10 -6 to 10 "2 M. The pH varied from 1.5 to 11. Immediately after the mixture of AHBDP-Sn(II) and TcO 4(') the solution became yellow. For the following series of solutions with equimolar amounts of AHBDP-Sn(II) and TcO4(') and the excess of the TcO 4('), the solutions became opaque and were composed of a brown precipitate.
For the excess solutions of AHBDP-Sn(II), [AHBDP-Sn(II)] 10[TCO4(')], the evolution of the absorption spectra was investigated. The absorbance at 224 nm immediately increased after the addition of TcO4(') and then decreased before stabilising. There was a new absorption band at 400 nm. The evolutions of the absorbance at 400 nm are reported in figure 2. A pseudo plateau was reached by absorbance, within 25 minutes; however, the absorption spectra changed slowly and a yellow precipitate appeared over 72 hours. It should be noted that the pseudo plateau was attained independently of the initial concentrations of the reagent, as it can be seen in the inset of Figure 2. In these conditions of stoichiometry 1:1 the composition of the solution at the pseudo plateau was thus independent of the reagent concentrations and the average extinction for the complexes could be calculated using the slope of the straight line of inset of Figure 2 2190 M"l.cm "1 All curves, such as the one in figure 2, could be analysed according to a firstorder kinetic law, with a rate constant of 0.67 + 0.005 s " 1 We have verified that the formation of the complex was independent of pH and that complex was stable as a function of dilution.

Potentiometric titrations of AHBDP-Sn(II)-Tc
The titration curves are illustrated in figure 3 for AHBDP-Sn(II) chelates Tc for 1:0, 1:10 and 1:25 ratios of metal to ligand. In steep inflection, the three curves were identical, there were no protons released from the complex for pH < 6. It is possible that after the reduction of the TcO4(') to TcO(2+), the AHBDP-Sn(II) chelates TcO (2+) forming a complex as shown in scheme 2. The . . could either polymerise or oligomerise to give the formation of the hydrophobic species, indicated by the yellow precipitate in the solution after 72 hours.

Biodistribution of AHBDP-Sn(II)-99mTc in rat 3.1 Preparation of the AHBDP-Sn(II)-99mTc
Paper chromatography of AHBDP-Sn(II)-99mTc (prepared under the conditions described above) showed that all the activity was Iocalised at the origin. These results showed that the AHBDP-Sn(II)-99mTc was almost free from Na99mTco4 The yield of labelling was about 96 % and the complexes was stable for at least two hours.

Tissue distribution
Uptake of the AHBDP in various organs of rats at 0.5,1, 2 and 3 hours post injection, indicated that the greater part of the complex was distributed in the rats' bones, kidneys and urinary bladders. Lungs, spleen, liver, heart and muscle showed negligible activity. Per gram organ uptake, as shown in table 1, was found to be highest in bone as compared to the other organs.
Studies on blood clearance of the AHBDP showed that during the initial post injection period, there was a rapid loss of activity in the blood and it was reduced to only 6% of the injection dose Vol. 2, No. 4, 1995 Synthesis of 4-Amino-l-Hydroxy-Butane-l,l-Diphosphonate within 30 minutes. Thereafter, further clearance was lower at hour post injection, the blood still showed 2% of the injected activity.  The experimental data indicated that Sn(ll) has formed a stable 1:1 complex with AHBDP. Its oxidation state is +2. This is in agreement with the previous work performed by  We propose, for the overall reaction, the mechanism shown in scheme 1, at pH 3, where the AHBDP is in the predominating species 2= and the Sn(ll) is solvated. The first step could be the exchanging of H20 by oxygen anions leading to the hydrated complexes. Next, the coordination of Sn(ll) is stabilized by the atom donor, nitrogen atom of the amine group, with the elimination of one molecule of water, corresponding to 4.

Complexation of AHBDP-Sn(II)-99Tc
The study of spectrophometric and potentiometric titrations shows that the technetium has formed a complex, chelated by AHBDP-Sn(II).3H20 with hydroxyl groups, giving AHBDP-Sn(II)-TcO.3H2Oo This is in agreement with the previous works reported by Tji et al. and Deutsch et al.o It was observed that for equimolar amounts of AHBDP-Sn(II) and TcO4(') and the excess of the TcO4('), the solutions are almost formed with the brown precipitated (as mentioned above) and we propose that the technetium remains at the degree of oxidation +4. The Tc(IV) formed the complex with AHBDP-Sn(II).2H20 and/or hydrolyzed to give TcO2.2H20 that precipitated. For the excess of ligand, the reaction was done in several steps. The first step was the reduction of TcO 4(')to TcO (2+) by AHBDP-Sn(II).2H20. The second step was the chelation of TcO (2+) by BHADP-Sn(II).2H20 that we propose the mechanism in scheme 2. The first step o the reaction was the exchange of two molecules of water by the hydroxyl function of the phosphonic groups, including the addition of the atom donor, Sn(ll) to Tc, giving the hydrated complexes. The product had intramolecular rearrangement, with the elimination of one molecule of water to give The second step was the polymerization or oligomerization of the 5 to give the hydrophobic species. Either the first or the second step of the mechanism could have given the constant rate of 0.67 +_ 0.005 s "1

Biodistribution
It is clear that AHBDP-Sn(II)-99mTcO.3H20 and HMDp-99mTc has a high affinity to bone. The kinetics and saturation time of the uptake to bone was not significantly different in either complex.
AHBDpoSn(II)-99mTcO.3H2 O is eliminated more rapidly than HMDp-99mTc. The signal/background ratio of AHBDP-Sn(II)-99mTcO.3H20 was lower than that of HMDp-99mTc, assessed by tibia and vertibra versus blood (figure 4) and muscle ( Figure 5). Accumulated activities in non-target organs was negligible. The biological behavior of AHBDpoSn(II)-99mTcO.3H2 O was closed to other diphosphonate compounds; HMDP,HEDP, etc. We found that the AHBDP-Sn(II)-TcO. 3H20 complex was stable, with the variation of pH and dilution. In rats, it fixed homogeneously on bone and the scintigraphic images had the same quality as that of HMDp-99mTc image. We propose to consider the AHBDP-Sn(II)-99mTcO.3H20 as a good tracer for bone imaging.