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Novel [99mTcIII(PS)2(Ln)] mixed-ligand compounds (PS= phosphinothiolate; L= dithiocarbamate) useful in design and development of TcIIIbased agents: synthesis, in vitro and ex vivo biodistribution studies Nicola Salvarese, Nicolò Morellato, Antonio Rosato, Laura Meléndez-Alafort, Fiorenzo Refosco, and Cristina Bolzati J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501088w • Publication Date (Web): 21 Oct 2014 Downloaded from http://pubs.acs.org on October 28, 2014

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Journal of Medicinal Chemistry

Novel [99mTcIII(PS)2(Ln)] mixed-ligand compounds (PS= phosphino-thiolate; L= dithiocarbamate) useful in design and development of TcIII-based agents: synthesis, in vitro and ex vivo biodistribution studies

Nicola Salvarese,a* Nicolò Morellato,b Antonio Rosato,a,c Laura Meléndez-Alafort,a Fiorenzo Refosco, c Cristina Bolzati c*

a

Dipartimento di Scienze Chirurgiche, Oncologiche e Gastroenterologiche, Università degli

Studi di Padova, via Gattamelata, 64, 35138 Padua, Italy; b Dipartimento di Scienze del Farmaco, Università degli Studi di Padova, Via Marzolo 5, 35131 Padua, Italy. c Istituto Oncologico Veneto, via Gattamelata, 64, 35138 Padua, Italy. d IENI-CNR, Corso Stati Uniti, 4, 35127 Padua, Italy.

ACS Paragon Plus Environment

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KEYWORDS. Technetium, Rhenium, Phosphine, Dithiocarbamate, Imaging, SPECT. ABSTRACT A general procedure for the preparation of a new class of neutral six-coordinated mixed ligand [99mTcIII(PS)2(Ln)] compounds (PS = trisalkyl-phosphino-thiolate; Ln = dithiocarbamate) is reported as well as their in vitro stability and the ex-vivo tissues distribution studies. [99mTc(PS)2(Ln)] complexes were prepared in high yield in nearly physiologic conditions following a one-pot procedure. For instance, the chemical identity of [99mTc(PSiso)2(L1)] (PSiso = 2-(diisopropylphosphino)ethanethiol; L1 = pyrrolidine dithiocarbamate) was determined by HPLC comparison with the corresponding 99gTc-complex. All complexes comprise the stable [99mTcIII(PS)2]+ moiety, where the remaining two coordination positions are saturated by a dithiocarbamate chelate, also carrying bioactive molecules (e.g. 2-methoxyphenilpiperazine). [99mTc(PS)2(Ln)] complexes were inert toward ligand exchange reactions. No significant in vitro and in vivo biotransformation were observed underlining their remarkable thermodynamic stability and kinetic inertness. These results could be conveniently utilized to devise a novel class of 99mTcIII-based compounds useful in radiopharmaceutical applications.

INTRODUCTION In the last few years, the global interruption of 99Mo supply has made the situation particularly problematic from a medical standpoint, underlining the importance of 99mTc in Nuclear Medicine practices 1, 2 and the need for a reliable supply network comprising alternative production options of this radionuclide 3-5, which unquestionably remains the best atom for SPECT imaging, owing to its optimal nuclear properties (t1/2 = 6.02 h; Eγ = 141 keV), low cost, and easy availability.


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Actually, it is used in different chemical forms in more than 85 % of clinical SPECT practices and it is indispensable for the estimated 40 million medical imaging procedures that take place per year around the world6. In the last decade, the technetium chemistry has been dominated by the development of diverse asymmetrical mixed-ligand complexes. In this context, many of the most successful advances have centered on Tc(I) and Tc(V), particularly, on complexes based on the kinetically inert [Tc(CO)3]+ and [Tc(N)(PNP)]+ building blocks, proposed as suitable platforms in the design of potential 99mTc ‘essential’ and ‘target-specific’ radiopharmaceuticals7. One of the advantages resulting from the use of this type of complexes lies in their asymmetrical nature. In this connection, chelation systems can be carefully chosen in order to strike a suitable balance between stability and lipophilicity thus improving their pharmacokinetic profile and making possible the modulation of their biological properties7. The trivalent state is one of the most common and stable oxidation states of technetium; notwithstanding, none of the radiopharmaceuticals currently in clinical use contains the metal in this oxidation state and only a restricted number of studies concerning the use of MIII (99mTc/188Re) are described. Nevertheless, examples of 99mTcIII-based radiopharmaceuticals have been already described in the past years and one remarkable example being

99m

Tc-terboroxime,

Cardiotec®, approved by FDA as myocardial imaging agent. Other models are given by: ì) a class of six-coordinated monocationic compounds of general formula [99mTc(diphos)2X2]+ (where diphos is a diphosphine ligand of the type R2P-CH2CH2-PR2 and X is Cl, Br)

8-13

, and a

class of monocationic complexes of general formula [99mTc(SB)(PR3)2]+, abbreviated as Qn (where SB is a dianionic tetradentate Schiff base ligand and PR3 is a tertiary monophosphine ligand)

14

, both investigated as potential myocardial imaging agents; ìì) a series of neutral,


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trigonal bipyramidal, mixed-ligand TcIII complexes of the type [99g/99mTc(NS3)(CNR)] [where NS3 is the tripodal ligand 2,2',2''- nitrilotris(ethanethiol) and CNR is a isocyanide with an organic functional group], recently described as a platform for the development of MIII-agents15-18. A common reason to explain the low number of MIII-complexes produced at micromolar level (10-6-10-7 M) lies in the lack of simply available methods for preparing these compounds in aqueous solutions12, 14, 15, 17. At millimolar level (10-3 M) the preparations of these compounds are performed in organic solvents, using labile pre-reduced M(III,V)-complexes as starting materials, in presence of equimolar or exceeding amounts of ligands. These reaction conditions are not apt for the production of the corresponding complexes at micromolar level, where more strict reaction condition are employed to form TcIII-compound from perthecnetate. At micromolar level, indeed, permetalate anions, in the form of NaMO4 (M=

99m

Tc/188Re), are used as starting

materials and sterile and pyrogen-free physiological conditions are required for human administration. In addition, for a clinical routine application the labelling must be simple, rapid, safe and high yielding (radiochemical yield > 90%), consequently suitable for a kit formulation19, 20

. Hence, the chemistry of TcIII and ReIII complexes as applied to nuclear medicine can still be

considered as largely unexplored. Phosphino-thiols and dithiocarbamates represent classes of efficient chelate ligands whose reactivity toward several metal ions is well documented21,

22

. Their peculiar coordination

properties allow the stabilization of metals in different oxidation states. Illustrative examples are the chemistries exhibited by Group 7 elements, Tc and Re7, 21-26. In the past years our group has been reported a systematic investigation on the reactivity of these two bidentate ligands toward different

99/99m

Tc precursors including [99g/99mTcO4]- and

[99g/99mTc(N)]2+. These studies led to the development of different class of bis-substitute


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symmetrical or asymmetrical [99g/99mTc(N)]-complexes along with a series of tris-substituted TcIII-compounds adopting either five- or six-coordinate geometry27-35. The physical-chemical properties of such compounds has been conveniently exploit to develop different Tc-based system for radiopharmaceutical applications. In our ongoing efforts to investigate the coordination chemistry of

99g

Tc and Re with

phosphine-thiolate and dithiocarbamate ligands, we recently described the synthesis and the characterization of a new class of six-coordinated mixed ligand complexes of the type [MIII(PS)2(Ln)], (where M = Re,

99g

Tc; PS = 2-(diphenylphosphino)ethanethiol or 2-

(diisopropylphosphino)ethanethiol; Ln = dithiocarbamate36. These compounds display a distorted trigonal-prismatic geometry, with a P2S4 coordination donor set, where the two π-acceptor phosphorus atoms are in a reciprocal trans position, whereas the sulfur atoms of the phosphinothiolate donors and of the dithiocarbamate ligand are in a reciprocal cis configuration (Figure1). In this configuration, the two identical phosphinothiolate ligands appear to be tightly bound to the metal center, defining a stable [MIII(PS)2]+ moiety, in which the particular arrangement of the coordinating atoms induces an elongation of the bond distances between the metal and donor atoms of the dithiocarbamate ligand. However, this sort of labilizing effect is counteracted by the coordination ability of the dithiocarbamate, which upon coordination makes possible an extensive π delocalization, generating a neutral and stable species.

Figure 1 here

These complexes possess some main features, which make them particularly interesting for the development of novel

99m

TcIII- and

188

ReIII-agents for theranostic applications. The structural


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asymmetry, which potentially permits a fine chemical modulation of the physicochemical properties of the complexes by an independent variation of both the substituents at phosphinothiolate and at dithiocarbamate ligand thus improving their pharmacologic and/or pharmacokinetic profile. The frame [MIII(PS)2]+ (Tc, Re) resembles a substitution-inert buildingblock, which might allow a combinatorial metal-fragment strategy

7, 19, 37

to the preparation of

[MIII(PS)2]+-based compounds libraries. Aim of this study is to verify two basic requirements in support of the actual usefulness of this new class of TcIII-complexes in the radiopharmaceutical applications. The first is related to the possibility of efficiently prepare the compounds through a simple labelling approach, performed in physiological media through a one-step preparation easily transferable to a lyophilized cold-kit apt, after reconstitution with Na[99mTcO4], for human administration. The second is connected with the stability of the 99mTcIIIP2S4 coordination sphere in the biological environments. Within we report the synthesis, the characterization and the biological evaluation of a series of mixed

99m

TcIII-complexes, as templates for the development of new potential [99mTcIII(PS)2]-

based SPECT imaging agents. The chemical structures of the obtained complexes are outlined in Figure 2.

Figure 2 here

The in vitro stability and the transchelation toward an excess of GSH, Cys and EDTA of all 99m

Tc-compounds were evaluated. Tissue distributions and metabolism in healthy rats of the

complexes are also reported.


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RESULTS AND DISCUSSION Synthesis of [99mTc(PS)2(Ln)] complexes 99m

TcIII-complexes were efficiently prepared starting from [99mTcO4]-, by ligand-exchange or

redox-substitution reactions following the pathways of synthesis sketched in Scheme 1. The labeling procedures were carried out at autogenous pH, using either a two-step (Method 1) or a one-step (Method 2 and 3) approach. The RCYs were optimized considering reaction parameters such as reagents order of addition, ligands concentration, temperature and reaction time. In all cases, the reaction progress was followed by chromatographic techniques.

Scheme 1 here

According to the two-step procedure complexes were prepared in hydro-alcoholic solution in which pertechnetate was converted into a reactive Tc-intermediate that in a separate step undergo ligand exchange to afford the final complex. Thoroughly, the first step involved the preliminary conversion of the starting [99mTcO4]- into a mixture of intermediate compounds originated by the interaction of prereduced metal ion with the phoshino-thiolate ligand. Reaction was completed after 30 min at 75 °C. At this step, HPLC analysis revealed the presence of different products (Scheme 1; chromatogram a). Among them, the product with Rt 14.90 min was identified as the five-coordinated [99mTc(PSiso)2(S∩Piso)] species by HPLC comparison with the corresponding Re-analogues. The latter was previously prepared at macroscopic level by reacting the precursor [ReCl3(MeCN)(PPh3)2] with a slight excess of PSisoH ligand in toluene/ethanol solution, and characterized by ESI(+) MS analysis (m/z [Re(PSiso)2(S∩Piso)] = 599)36.


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In the second step the preparation was completed by addition of the selected dithiocarbamate ligand (Ln) to afford the final mixed complex in high RCY (Scheme 1, chromatograms b-e). Coordination of Ln was immediately achieved at autogenous pH and at RT. HPLC profile revealed the presence of a single peak. Despite the unfeasibility in identifying all species generated in the first step, the mixture was easily and quantitatively converted into the corresponding final [99mTc(PSiso)2(Ln)] compound by reaction with the appropriate dithiocarbamate ligand. This behavior suggest that all compounds of the intermediate mixture possess the same [99mTcIII(PSiso)2]+ moiety. Alternatively, [99mTc(PSiso)2(Ln)] compounds were quickly and quantitatively prepared following a one-step procedure (Method 2, 3) which required the addition of a Na99mTcO4 saline solution, freshly eluted from the generator, to the reaction vial containing SnCl2 (as reducing agent) and the two bidentate ligands, PSisoH and Ln. In this situation, ligand substitution takes place almost simultaneously with the reduction of the pertechnetate. The reaction was conducted both in hydroalcoholic solution (EtOH/saline, 80/20; Method 2) and in saline solution (Method 3). In this latter condition, the use of co-solubilizing agent, such as cyclodextrin, is required to improve the solubility of both PSisoH and [99mTc(PSiso)2(Ln)], as well as the stability of the PSisoH ligand toward oxidation. Therefore, a saline solution of γ-cyclodextrin (10 mg/mL) was used as indicated in the Experimental section (Method 3). HPLC chromatogram of the reaction mixture clearly showed the presence of a single peak that was exactly coincident with the peak product obtained by the two-step preparation (Scheme 1, chromatograms b-e).


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Table 1 reports the chromatographic properties and the RCY values of the various [99mTc(PSiso)2(Ln)] complexes prepared with Method 2 and Method 3 along with their log P values. Table 1 here No significant differences in Rt values were observed. As expected, all the compounds were found to be lipophilic, with log P values in the range of 3.18 – 1.52. Complex 1 is the most lipophilic compound of the series (log P = 3.18 ± 0.05); however, modification of the dithiocarbamate backbones yielded a modification of the lipophilic character of the final complex. Thus, the introduction of functional or pharmacophore groups such as ester (L2) or 2methoxyphenilpiperazine (L4) is responsible for the enhanced hydrophilic character of the complex. As an example, the chemical nature of the selected [99mTc(PSiso)2(L1)] complex was confirmed by comparison of its HPLC behavior with that of the corresponding Tc-99g complex, prepared at macroscopic level and full characterized by elemental analysis, mass spectroscopy, and

31

P and

proton NMR spectra36. Figure 3 shows the overlap of the chromatographic profiles (UV- Radio) of the [99m/99gTc(PSiso)2(L1)] complexes. Figure 3 here Following the one pot procedure (Method 2), the most suitable reaction conditions were determined through a careful balance of the relative amounts of PSisoH and Ln used in preparations. Variation of the temperature was also considered. A fixed amount of the initial activity (185 MBq) of freshly eluted [99mTcO4]- was used in each preparation and the final volume of the reaction mixture was 1.650 mL. The reaction time was fixed to 30 min.


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For exemplificative purpose, Figure 4A illustrates the influence of the molar stoichiometric ratio between PSisoH and L1 ligands and of the temperature on the radiochemical yield of 1. Figure 4 here Data analysis revealed that, for a given amount of PSisoH, the best RCY was achieved by heating the reaction mixture at 75 °C and when the molar ratio PSisoH/Ln did not exceed 1. In these conditions, 1 was obtained with a radiochemical yield of 96.03% ± 0.70. When the reaction was carried out using an insufficient amount of dithiocarbamate, the fivecoordinated [99mTc(PSiso)2(S∩Piso)] complex along with the other byproducts (vide supra), generated by the interaction of the prereduced metal ion with the phoshino-thiolate ligand, were observed. This fact showed that the [99mTc(PSiso)2(S∩Piso)] formation competed with the quantitative production of the [99mTc(PSiso)2(Ln)] complex. Plausible, the formation of the stable [99mTcIII(PSiso)2]+ moiety was straightforward, but the remaining coordinating positions were subject to an exchange equilibrium where PSisoH and Ln were in competition. Then, the radiolabeling efficiency of the ligands has been assessed: ligands concentrations were gradually decreased (in the range 3.4 – 0.042 mM) following the standardized conditions (PSiso/Ln concentration ratio equal to 1; Temperature: 75 °C; reaction time: 30 min). For instance, the dependence of RCY (%) on the concentration of PSisoH and NH4L1 in the preparation of 1 is illustrated in Figure 4B. Highest RCYs (>90%) were achieved using a ligand concentrations between 3.4 and 0.17 mM, which correspond to an amount of PSisoH in the range of 1 – 0.05 mg and of NH4L1 in the range 0.92 – 0.046 mg. Similar results were observed for the other compounds.


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Tc-centered one pot synthesis is often critical for the successful development of new radiotracers20. It is worth mentioning that for

99m

Tc-based agents, radiosynthesis must be

performed in aqueous solution under sterile, pyrogen-free conditions and should be completed within 30 min. The yield of the radiotracer must be ≥ 90% with high solution stability (possibly ≥ 6h) because the injection of a mixture of

99m

Tc-species decreased the organ specificity. In

addition, this requirement essentially eliminates any chromatographic purification of the desired tracer20. Acquired data indicate that the adopted pathways of synthesis allow overcoming the relatively complicated labelling conditions generally employed to form

99m

TcIII-complexes from

pertechnetate, which required multistep reactions and organic solvents12,14,15,17, perfectly meeting the basic principle of the one pot synthesis and the requirements for Nuclear Medicine practice application. Actually, the

99m

Tc-labelings are straightforward, all complexes were efficiently prepared in

nearly physiological condition by one pot procedure simply mixing the starting [99mTcO4]- with the two bidentate PSH and Ln ligands in the presence of the reducing agent. No labile intermediate exchange complexes, such as

99m

Tc-EDTA or

99m

TcIII

99m

Tc-gluconate, were necessary.

Moreover, in these preparations no detectable formation of the corresponding tris-substituted complexes, comprising identical bidentate ligands, in a tbpy or in octahedral environment, was observed in agreement with the fact that MIIIP2S4 arrangement is the most kinetically and thermodynamically stable combination of atoms. As previously observed at macroscopic level 36, these results clearly suggest that the formation of [99mTc(PSiso)2(Ln)] complexes is promoted by the chemical properties of the [99mTcIII(PS)2]+


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moiety, which can be considered the key intermediate for the preparation of these TcIII based mixed ligand compounds. The particular arrangement of atoms around the metal center permits, when reacted with the selected dithiocarbamate ligands, the formation of the final [99mTc(PS)2(Ln)] complex, in high yield, regardeless the adopted pathway of synthesis. This fact is the driving force for the synthesis process. Actually, these reactions were highly selective; indeed when a combination of phosphino-thiol and dithiocarbamate ligands co-exist in the reaction mixture in the right stoichiometric ratio the almost quantitative formation of the mixed six-coordinated [99mTc(PSiso)2(Ln)] compound was detected.

In vitro stability and metabolism studies Stability studies were performed on the HPLC-purified compounds, evaluating the variation of their RCP (expressed as percentage) over the time. [99mTc(PSiso)2(Ln)] complexes remained stable in their reaction mixture for 24 h after labeling. Likewise, the compounds were stable for 3 h of incubation at 37 °C, after purification, in ethanol solution or in PBS solution containing 10% ethanol (SI Figure S1). Challenge experiments with Cys, GSH and EDTA were conducted incubating the HPLCpurified complexes for 24 h at 37 °C with an excess of exchanging ligands (10 mM). No significant changes of RCPs of the [99mTc(PSiso)2(Ln)] complexes were observed within 3 h of incubation. After 24 h, a tolerable reduction of the RCPs was detected. HPLC profiles clearly show the formation of an additional hydrophilic compound, as consequence of a transchelation reaction between Ln and the challenge agent. Nevertheless, at this time point, RCPs were never less than 60% (SI Figure S1).


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In vitro biotransformation of the radio-complexes was assessed by incubating the selected purified compound at 37 °C in human and rat sera as well as in rat liver homogenate to achieve a prediction of their in vivo stability and resistance to the degradation process. Compounds were found to be adequately stable after incubation, at 37 °C for 4h, in rat and human sera. Data clearly indicate that no significant biotransformation of the native compounds and no significant interaction with the serum proteins were observed. HPLC profile of the complexes collected after sera incubation display that most of the injected activity perfectly matched the initial product (SI Figure S2); small amounts of hydrophilic species were detected after 30 min of incubation both in human and rat sera. Likewise, the complexes were also found stable after homogenate exposure. The acquired data revealed that the radiolabeled compound remains intact from prolonged incubation (SI Figure S3).

Biodistribution studies All studies involving animal testing were carried out in accordance with the ethical guidelines for animal research adopted by the University of Padua, acknowledging the Italian regulation and European Directive 86/609/EEC as to the animal welfare and protection and the related codes of practice. Biodistribution studies of all asymmetrical

99m

TcIII-complexes were carried out in male

Sprague Dawley rats. Before the injection, the radiolabeled compounds were purified following the procedure described in the Experimental section. Purification of the radiolabeled compound before i.v. injection was necessary in order to remove excess of cold ligands and any radiochemical impurity formed during the labeling procedure in order to appreciate the


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distribution profile and the metabolism of the sole radioactive complex. The radiochemical purity and stability of the purified compounds were verified with HPLC prior to in vivo administration, as well as during the utilized period, to guarantee >90% purity of the injected radiolabeled compounds. The RCP and the stability of the purified complexes were evaluated prior to in vivo administration and for the succeeding 6 h. The tissue uptake of the

99m

TcIII-

complexes are summarized in Table 2.

Table 2 here

In general, all the 99mTcIII-complexes do not appear to possess favorable biological properties. The distribution profiles of the complexes were connected to the physical-chemical properties of the compounds. In particular, the collected data show that the substituents at the dithiocarbamate ligand influence the biodistribution pattern and differences were observed in blood, liver, and kidney accumulations. With exception for [99mTc(PS)2(L4)] these compounds show a rapid blood clearance. The high liver and intestine uptake suggests that the hepatobiliary system is the major route of excretion of the administered agents. Indeed, the kidneys uptake was quite low, as well as the renal clearance. For all complexes, a high spleen uptake was detected followed by slow wash out, at 2 h p.i the %ID was found to decrease by approximately 50-60%. This occurrence may attributable to the formation of large aggregates that would localize in organs such liver, spleen and lungs. The stomach activity was low and any increase of the %ID in this organ during all the investigation time was found, to indicate that this new class of complexes are stable in vivo. No significant uptake in target organs such as heart or brain were observed.


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Notable the behavior of complex 1 in the myocardial tissue, which was found to increase over the time (0.74±0.02 %ID at 20 min p.i vs 1.14±0.02 %ID at 60 min p.i), probably as result of a redistribution of the activity from the liver. Among the tested compounds, 3 seems to show a most favorable pharmacokinetic profile characterized by quick enough blood clearance (1.50±0.03 %ID at 2 min p.i vs 0.76 ±0.01 %ID at 20 min p.i), lower lungs, spleen and liver accumulation followed by wash-out. At 2 h p.i the activity in the liver was approximately half of the initial value, meanwhile in the spleen the activity was 1/3 of its initial amount. The kidney uptake was higher with respect to 1, 2 and 4, but the wash out was slow and the activity excreted in the urine low, similarly to the other compounds. Nevertheless, in all cases at 2 h p.i the urine were collected and analyzed by HPLC. The HPLC radiometric profile invariably showed a single peak, which overlapped the native compounds, indicating that any metabolic transformation occurred (Figure 5).

Figure 5 here

Considering that for all these

99m

Tc-compounds the hepatobiliary system is the main route of

elimination metabolism study was performed on the selected [99mTc(PS)2(L1)] (1) complex in order to investigate the in vivo stability of both the [99mTc(PS)2]+ moiety and the P2S4 coordination sphere. The in vivo stability and metabolism of 1 were evaluated comparing the chromatographic profiles of the injected compound with that of the activity extracted from the liver and the endoluminal content of the small intestine from animals administrated 20 and 120 min earlier 38, 39.


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Approximately 85% of the initial activity localized in the liver or in the liquids was taken out and analyzed. The chromatographic profiles evidenced the presence of a single peak which perfectly overlapped the control compound indicating that any metabolic transformation occurred (Figure 6).

Figure 6 here

These results point to the remarkable in vitro and in vivo stability of these complexes, indicating that the [99mTcIII(PS)2]+ scaffold and the metal-dithiocarbamate bond are kinetically stable toward challenge reaction and in biological environments. This property is strictly connected to the structural features of these complexes generated by the balance between the MIII electronic requirements and the π-acceptor and π-donor properties of the ligands bound the metal center. This is an appealing aspect suitable for the development of novel

99m

TcIII-based

radiopharmaceuticals. However, structural modifications are required to improve the pharmacokinetic profile of these compounds. Considering the asymmetrical nature of these compounds, better pharmacokinetic profile may be obtain by varying the substituents to the phosphine-thiolate and/or dithiocarbamate ligand. In this perspective, dithiocarbamate ligands have shown to be of greatly useful in radiopharmaceutical design thank to a wide variety of substituents that can be incorporated in the ligand backbone including bioactive molecules such as pharmacophore groups and small peptides

40, 41

. Thus, dithiocarbamate can be considered a good chelating system for

incorporating a target vector into the 99mTcIII-asymmetrical complex.


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This occurrence, joined to the high stability of the [99mTc(PS)2(Ln)] complexes here established, represent a hint for the future development of a new class of [99mTc(PS)2]-based target-specific tracers.

CONCLUSIONS This study devises a general procedure for the preparation of a new class of mixed ligand 99m

TcIII-based complexes containing different bidentate ligands bound to the same metal center,

which could be used to design new SPECT imaging agents. [99mTc(PS)2(Ln)] complexes were efficiently prepared following a one-pot procedure in which reduction of the pertechnetate and the ligand substitution take simultaneously place. The applied procedure perfectly meets the basic requirements for Nuclear Medicine practice application. The process is supported by the chemical properties of the [99mTcIII(PS)2]+ moiety that represents the key intermediate for the development of this new class of neutral mixed ligand complexes characterized by the substitution inert [MIII(PS)2]+ moiety and by a mono-negative chelate (Ln), which complete the metal coordination sphere. This latter can be conveniently use to carry bioactive molecule (e.g. 2-methoxyphenilpiperazine) for receptor targeting application. Complexes were found to be stable in vitro and in vivo, underlining their remarkable thermodynamic stability and kinetic inertness. The flexibility of this system, due to the possibility of changing the substituents at the P atoms and/or at dtithiocarbamate ligand, without affecting the P2S4 coordinating sphere, may be utilized to design a wide range of mixed compounds and to modulate their biological properties. In a wider perspective, the current study provides important basic informations concerning the applicability of the [99mTcIII(PS)2]+ moiety as suitable building block for the development of new


17


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Page 18 of 46

potential [99mTcIII(PS)2]-agents. Study to assess the reactivity of different mono-negative chelate toward this new building block are currently under investigation.

EXPERIMENTAL SECTION Materials All common chemicals were purchased from Sigma Aldrich (Milan, Italy). Prolabo® HPLCgrade acetonitrile was purchased from VWR International and used without further purification. 4-(ethoxycarbonyl)piperidinedithiocarbamate, (EtOOCPipL2),

sodium

4-(ethoxycarbonyl)piperidinium

N-adamantyldithiocarbamate

(NaL3)

and

sodium

salt 4-(2-

methoxyphenyl)piperazine-1-dithiocarbamate (NaL4) were synthesized as previously described 19, 40

. 2-(diisopropylphosphino)ethanethiol (PSisoH) was purchased from Argus Chemicals

(Prato, Italy). Owing to the tendency of the phosphinothiol ligands to oxidize, all the solvents used in the reactions with alkyl-phosphino thiol ligands were previously degassed to remove traces of dissolved dioxygen. Technetium-99m, as sodium pertechnetate-99m (Na[99mTcO4]), was eluted from a 99Mo/99mTc generator (Elumatic III, IBA CIS bio, France) using 0.9% saline.

Analysis Thin-Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) analyses were used to evaluate the radiochemical yields (RCY) and the stability as radiochemical purity (RCP), both expressed as percentage, of the compounds. RCYs were in the range of 92%97%.


18

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TLC analysis was performed on SiO2 plates (Merck, Milano, Italy) eluted with CH2Cl2 or a mixture of CH2Cl2/CH3OH (90/10). The activity on the plates was detected and measured using a Cyclone Instrument equipped with a phosphorus imaging screen and OptiQuant image analysis software (Packard, Meridian, CT). Rf values were in the range 0.4-0.7. HPLC was performed on a Beckman System Gold instrument equipped with a programmable solvent Model 126, a scanning detector Module 166 (λ = 216 nm) and a radioisotope detector Model 3200 Bioscan, by using a Waters SymmetryShield RP C8 Guard Column (5.0 µm, 100 Å, 3.9 × 20 mm) and a Waters SymmetryShield RP C8 Column (5.0 µm, 100 Å, 4.6 × 250 mm). Solvent A: aqueous ammonium acetate 0.01 M (pH 7), solvent B: acetonitrile; isocratic: 0 – 30 min B = 90%; flow rate = 1 mL/min.

Radiochemical synthesis Synthesis of [99mTc(PSiso)2(Ln)] complexes. Complexes were prepared following different way of synthesis involving multi- or one-pot procedures. The amounts of ligands used in preparations were: PSisoH, 5.6 x 10-3 ‒ 2.8 x 10-4 mmol (1 ‒ 0.5 mg) dissolved in 0.1 mL of ethanol; Ln as salt form, 5.6 x 10-3 ‒ 2.8 x 10-4 mmol dissolved in 0.2 mL of degassed ethanol, (NH4L1: 0.92 ‒ 0.046 mg; EtOOCPipL2: 3.65 ‒ 0.165 mg; NaL3:1.4 ‒ 0.07 mg; NaL4: 3.24 – 0.162 mg).

Method 1 (two steps reaction, in hydroalcoholic solution). In a capped and di-nitrogensaturated Pyrex® vial containing ethanol (1 mL), tin(II) chloride (SnCl2, 0.1 mg in 0.1 mL of saline) and PSisoH a freshly eluted Na[99mTcO4] saline solution (50.0 – 800 MBq in 0.250 mL) was added. The reaction mixture was vortexed and heated at 75 °C for 30 min. Then, the vial


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Page 20 of 46

was allowed to cool to room temperature (RT) and the salt form of L was added. After 5 min of incubation at RT, RCYs of the complexes evaluated by HPLC were in the range of 92%-97%. Method 2 (one-pot reaction, in hydroalcoholic solution). In a capped and di-nitrogen-saturated Pyrex® vial containing ethanol (1 mL), tin(II) chloride (SnCl2, 0.1 mg in 0.1 mL of saline), PSisoH and Ln, a freshly eluted Na[99mTcO4] saline solution (50.0 – 800 MBq in 0.250 ml) was added. The reaction mixture was vortexed and heated at 75 °C for 30 min. RCYs of the complexes evaluated by HPLC were exactly comparable to those achieved by Method 1. Method 3 (one-pot reaction, in saline). In a capped and di-nitrogen-saturated Pyrex® vial containing γ-cyclodextrin saline solution (10 mg/mL; 0.6 mL), SnCl2 (0.1 mg in 0.1 mL of saline), PSisoH (dissolved in 0.5 mL of γ-cyclodextrin saline solution) and Ln a freshly eluted Na[99mTcO4] saline solution (50.0 – 800 MBq in 0.250 mL) was added. The reaction mixture was vortexed and heated at 75 °C for 30 min. RCYs of the complexes evaluated by HPLC were comparable to those achieved by Method 1 and 2. Dose Preparation. HPLC purification of the Tc-compounds was performed to eliminate the excess of free ligands. Thus, a fraction of the reaction mixture (containing 37 – 185 MBq) was withdrawn, concentrated to a maximum volume of 0.1 ml by di-nitrogen stream, and injected in the HPLC instrument; the fraction containing the product was collected in a glass vial and the solvent was completely evaporated by di-nitrogen stream. The complex was dissolved in the minimum amount of ethanol. The solution was diluted with saline to obtain an aqueous solution with 10% of ethanol and utilized for in vitro and in vivo studies.

Radiolabeling efficiency


20

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The radiolabeling efficiency of the ligands was determined following the standardized labelling condition reported in the Method 2. The concentrations of ligands were progressively decreased, in the range 5.6 x 10-3 ‒ 2.8 x 10-4 mmol for both PSisoH an Ln ligands. The RCYs were determined by HPLC after 30 min of incubation at 75°C.

Determination of LogP values n-Octanol/water partition coefficients were determined by vortex mixing (20 min) 3 mL of noctanol, 3 mL of PBS (pH = 7.4), and 100 µl of the HPLC-purified radiolabeled compound. After centrifugation (3,000 g for 10 min), aliquots (100 µl) of both the organic and the aqueous phases were collected and counted with a γ-counter. Partition coefficients were expressed as (activity concentration in n-octanol)/(activity concentration in water).

In vitro Stability Studies The in vitro stability of the complexes was evaluated by monitoring via HPLC the radiochemical purity (RCP) at different time points using the following procedures. Solution stability of the

99m

TcIII-complexes. Aliquots of each reaction mixture containing the

relevant 99mTcIII-complex were analysed by HPLC at 1 and 12 h post-labelling. After HPLC purification the stability of the

99m

TcIII-complexes was assessed in physiological

buffered pH media such as saline and PBS. Thus, 50 µL of the selected [99mTc(PSiso)2(Ln)] complex was added to a propylene test tube containing saline (450 µL) or PBS (450 µL; pH 7.4). The mixture was vortexed and incubated at 37 °C for 24 h. At 0.5, 1, 2, 3 and 24 h aliquots were withdrawn and analysed by HPLC.


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Page 22 of 46

Challenge with cysteine (Cys), glutathione (GSH) and ethylenediaminotetraacetic acid (EDTA). Challenge experiments were carried out on the purified complexes using an excess of Cys, GSH, or EDTA. 50 µl of the selected HPLC-purified [99mTc(PSiso)2(Ln)] complex was added to a propylene test tube containing Cys hydrochloride (50 µl of an aqueous 10 mM solution) and PBS (400 µl; pH 7.4). The mixture was vortexed and incubated at 37 °C for 24 h. At 0.5, 1, 2, 3 and 24 h aliquots of the reaction mixture were withdrawn and analysed by HPLC. A similar procedure was applied using GSH (50 µL, 10 mM) and EDTA (50 µL, 10 mM) as challenge ligand. A blank reaction containing an equal volume of water instead of the challenge ligand was studied in parallel as control. Stability in rat and human sera. The in vitro stability in rat serum and human serum of the complexes was evaluated by monitoring the RCP at different time points using the following procedures. In a propylene test tube, 50 µL of HPLC-purified [99mTc(PSiso)2(Ln)] complex was added to: a) 450 µL of human serum, b) 450 µL of rat serum. The resulting mixtures were incubated at 37 °C for 240 min. After 15, 30, 60, 120 and 240 min of incubation, 50 µL samples were removed and 200 µL of ethanol added to each to precipitate the proteins. The sample was centrifuged (5 min at 12000 rpm) until the precipitated proteins formed a pellet. The supernatant was collected and the pellet was washed with 200 µL ethanol and centrifuged again. For each sample, 100 µL of the combined supernatants were equilibrated with 200 µL of PBS, and the resulting solutions were analysed by HPLC to evaluate the serum stability of the complex. The experiments were performed in duplicate. Metabolism. For incubation in liver homogenates, the organ freshly excised from a male rat was rapidly rinsed and homogenized in 20 mM HEPES buffer (pH 7.3) with an Ultra-Turrax T18 basic homogenator (IKAWorks, Inc.) for 2 min at 4 °C.


22

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The in vitro metabolism of 1 was evaluated by monitoring the radiochemical purity at different time points using the following procedure. In a test tube, 100 µL of the purified

99m

Tc-complex

(100 MBq) were added to 900 µL of fresh 30% liver homogenate and 900 µL HEPES buffer as a blank. The samples were incubated at 37 °C for 24 h. Aliquots (100 µL) of each solution were withdrawn and added to 200 µL of acetonitrile to precipitate the proteins. The sample was centrifuged (5 min at 3000 rpm) until the precipitated proteins formed a pellet. The supernatant was collected and the pellet was washed with 200 µL acetonitrile and centrifuged again (5 min at 14000 rpm). For each sample, 100 µL of the combined supernatants were analysed by TLC and HPLC to evaluate the stability of the complex. The experiments were performed in duplicate.

Animal Studies Animal experiments were carried out in compliance with the relevant national laws relating to the conduct of animal experimentation. Biodistribution. Ex vivo biodistribution studies were carried out in male Sprague-Dawley rats. Before the injection

99m

Tc-complexes 1 – 4 were purified by HPLC as previously described.

Rats weighing 180–200 g were anesthetized with an intraperitoneal injection of Zoletil 100 (40 mg/kg). A jugular vein was surgically exposed and 0.1 mL (300–370 kBq) of the solution containing the radioactive complex was injected. The rats (n = 3) were sacrificed by cervical dislocation at different time points (2, 20, 60, 120 min for complexes 1 and 3; 2, 20, 60, 120 min for complexes 2 and 4) after injection. The blood was withdrawn from the heart with a syringe immediately after death and quantified. Organs were excised, rinsed in saline, weighed, and the


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Page 24 of 46

activity was counted in a NaI well counter (Cobra II 5002, PerkinElmer) . The results were expressed as the percentage of injected dose per gram (%ID/g). Metabolites in the urine. At 120 min post injection (p.i), the urine was collected from the bladder and directly analyzed by HPLC.

In Vivo Stability and Metabolism. 100 µL of the solution containing the radioactive complex 1 (370-500 MBq) were injected into the jugular vein of anesthetized rats (180-200 g). At 20 and 120 min p.i the animals were sacrificed. Extraction from the liver. The extraction of the complex from the liver was performed following the previously reported procedures 38. Metabolites in the intestine. The intestinal lumen was rinsed with 2 mL of water; 80% of the total activity was collected in the endo-luminal content. The liquid fraction was separated from the intestinal content by centrifugation (3.000 x g for 10 min) and counted. 50% of the activity was found in the first liquid fraction (Fraction I). Exhaustive extraction of the residual activity from the solid fraction was performed by using acetonitrile (2 mL) followed by centrifugation (3.000 x g for 10 min) (Fraction II). An aliquot (100 µL) of each fraction was analyzed by TLC and HPLC.


24

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Figure 1. [MIII(PS)2(L)] complexes


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Page 26 of 46

Figure 2 List of complexes studied in the present work.


26

12 .90

4.0

3.5

3.0

2.5

Volts

2.0

1.5

1.0

7

6 .1

5

10 .67

0.5 3 .9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


12 .60

Page 27 of 46

0.0 0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

Minutes

Figure 3. HPLC profile of complex 1. Comparison of [99mTc(PSiso)2(L1)] (solid trace) with the corresponding [99gTc(PSiso)2(L1)] (dash trace)


27


A 100 95 90

RCY (%)

85 80 75 70 65 60 1/0.5; RT

1/1; RT

1/0.5; 75°C 1/0.8; 75°C 1/1; 75°C 1/1.5; 75°C 1/2; 75°C

PSisoH / NH4L1; Temp.

B 100 95 90 85

RCY (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 46

80 75 70 65 60 3.4 mM

1.7 mM

0.34 mM

0.17 mM

0.085 mM

0.042 mM

[Ligands] Figure 4. A: Variation of RCY (expressed as percentage) of the formation of complex 1 as a function of the ligand molar stoichiometric ratio (PSisoH/NH4L1) and the temperature (temp.).


28

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RCY (%) was evaluated by HPLC (peak integration) after 30 min. B: Variation of RCY (expressed as percentage) of the formation of complex 1 as a function of the ligands concentration ([Ligands], expressed as millimolar). RCY (%) was evaluated by HPLC (peak integration) after 30 min at 75 °C. PSisoH/NH4L1 stoichiomolar ratio was 1. Values are means ± standard deviation (n = 3).


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Page 30 of 46

Figure 6. HPLC profiles of [99mTc(PSiso)2(L1)] 1 after tissue extraction at 120 min after injection. Peaks represent control (A); activity extracted with MeCN from liver (B); endoluminal content (C). Similar results were observed at 20 min after injection.


30

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Figure 7. HPLC profiles of the rat urines collected at 120 min post-injection. a) Complex 1. b) Complex 2. c) Complex 3. d) Complex 4.


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Page 32 of 46


32

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


Scheme 1. Schematic drawing of the two synthetic methods and of the resulting [99mTc(PSiso)2(Ln)] complexes along with the corresponding HPLC profiles. Method 1 two-step procedure conducted in hydro-alcoholic solution (saline/EtOH; 20/80). Method 2 one-pot procedure, ì = hydro-alcoholic solution (saline/EtOH; 20/80). Method 3 one-pot procedure, ì = saline solution of γcyclodextrin (10 mg/mL). HPLC profiles of: the intermediate mixture (a); of complex 1 (b), 2 (c), 3 (d) and 4 (e); the chromatographic conditions

are

explicated

in

experimental

section.


33


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Complexes

HPLC, Rt 99g

Page 34 of 46

RCY (%)

Log P

99m

Method 2

Method 3

13.04±0.29

96.03±0.70

96.09±0.29

3.18±0.05

[Tc(PSiso)2(L2)] 2

11.26±0.30

94.14±2.69

91.23±0.13

2.27±0.08

[Tc(PSiso)2(L3)] 3

11.23±0.21

93.40±0.89

91.24±0.47

2.20±0.20

[Tc(PSiso)2(L4)] 4

12.89±0.53

94.51 ±0.79

93.2 ±0.54

1.50±0.14

[Tc(PSiso)2(L1)] 1

Tc

12.65±0.05

Tc

Table 1. HPLC data for the obtained 99mTcIII- complexes. Values are means ± standard deviation (n ≥ 3).


34

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[99mTc(PSiso)2(L1)]

[99mTc(PSiso)2(L2)]

Organ

2 min

20 min

60 min

120 min

2 min

20 min

60 min

120 min

Blood

1.74 ± 0.40

0.73 ± 0.17

0.81 ± 0.17

0.48 ± 0.11

1.88 ± 0.36

0.55 ± 0.02

0.49 ± 0.16

0.40 ± 0.22

Brain

0.10 ± 0.03

0.05 ± 0.01

0.04 ± 0.01

0.03 ± 0.00

0.08 ± 0.01

0.05 ± 0.00

0.04 ± 0.01

0.05 ± 0.00

Heart

0.88 ± 0.21

0.74 ± 0.02

1.14 ± 0.02

1.15 ± 0.18

0.73 ± 0.15

0.51 ± 0.08

0.65 ± 0.09

0.50 ± 0.00

Lungs

4.19 ± 1.12

2.25 ± 0.56

2.64 ± 0.18

2.05 ± 0.42

2.31 ± 0.06

1.43 ± 0.09

1.33 ± 0.01

1.09 ± 0.10

Liver

9.43 ± 2.56

9.41 ± 0.37

9.54 ± 0.07

8.83 ± 0.42

8.91 ± 0.13

8.12 ± 0.06

8.02 ± 0.07

7.70 ± 0.35

Spleen

12.22 ± 3.16

10.34 ± 3.72

10.28 ± 2.12

6.30 ± 0.41

9.36 ± 4.16

7.27 ± 2.41

5.47 ± 2.77

3.77 ± 0.81

Kidney

1.28 ± 0.25

0.96 ± 0.00

1.55 ± 0.07

1.47 ± 0.00

0.54 ± 0.09

0.54 ± 0.09

0.65 ± 0.11

0.93 ± 0.01

Stomach

0.20 ± 0.02

0.34 ± 0.09

0.39 ± 0.09

0.82 ± 0.53

0.14 ± 0.01

0.30 ± 0.17

0.28 ± 0.01

0.27 ± 0.09

Intestine

0.45 ± 0.14

3.62 ± 0.31

8.92 ± 1.64

13.05 ± 1.03

0.15 ± 0.06

0.98 ± 0.03

3.11 ± 0.13

2.52 ± 1.11

Pancreas

0.27 ± 0.06

0.30 ± 0.01

0.34 ± 0.09

0.45 ± 0.03

0.21 ± 0.09

0.23 ± 0.04

0.31 ± 0.01

0.28 ± 0.03

Muscle

0.08 ± 0.00

0.12 ± 0.00

0.19 ± 0.01

0.21 ± 0.06

0.05 ± 0.01

0.08 ± 0.01

0.11 ± 0.00

0.13 ± 0.02

Bone

0.21 ± 0.00

0.22 ± 0.00

0.37 ± 0.03

0.34 ± 0.02

0.21 ± 0.04

0.22 ± 0.02

0.23 ± 0.02

0.20 ± 0.02

Urine

0.00 ± 0.00

0.00 ± 0.00

0.18 ± 0.23

0.24 ± 0.27

0.00 ± 0.00

0.02 ± 0.02

0.04 ± 0.01

0.25 ± 0.02

[99mTc(PSiso)2(L3)]

Organ

2 min

20 min

60 min

[99mTc(PSiso)2(L4)]

120 min

2 min

20 min

60 min

120 min


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Page 36 of 46

Blood

1.50 ± 0.03

0.76 ± 0.01

0.71 ± 0.02

2.02 ± 2.27

0.96 ± 0.17

0.88 ± 0.20

0.98 ± 0.11

1.02 ± 0.44

Brain

0.08 ± 0.00

0.07 ± 0.00

0.05 ± 0.00

0.04 ± 0.01

0.05 ± 0.01

0.07 ± 0.00

0.06 ± 0.00

0.06 ± 0.01

Heart

1.13 ± 0.10

1.02 ± 0.08

1.25 ± 0.09

0.88 ± 0.00

0.25 ± 0.02

0.29 ± 0.03

0.41 ± 0.03

0.46 ± 0.09

Lungs

1.88 ± 0.37

1.52 ± 0.16

1.61 ± 0.19

1.01 ± 0.03

1.03 ± 0.20

1.01 ± 0.23

1.07 ± 0.11

1.18 ± 0.27

Liver

7.02 ± 1.02

6.57 ± 0.09

6.18 ± 0.70

4.01 ± 0.03

10.01 ± 0.36

10.10 ± 0.74

8.59 ± 0.11

8.75 ± 3.24

Spleen

7.43 ± 3.05

7.72 ± 1.23

3.68 ± 0.07

2.68 ± 0.93

11.21 ± 3.18

10.33 ± 2.64

5.56 ± 0.42

6.34 ± 1.06

Kidney

2.90 ± 0.14

2.56 ± 0.20

2.75 ± 0.01

2.20 ± 0.04

0.50 ± 0.01

0.55 ± 0.02

0.65 ± 0.06

0.96 ± 0.38

Stomach

0.37 ± 0.01

0.56 ± 0.06

0.92 ± 0.36

0.76 ± 0.28

0.16 ± 0.05

0.19 ± 0.01

0.40 ± 0.01

0.37 ± 0.03

Intestine

0.62 ± 0.10

1.40 ± 0.73

6.65 ± 3.41

8.25 ± 3.57

0.10 ± 0.00

0.66 ± 0.02

1.60 ± 1.20

2.86 ± 0.92

Pancreas

0.47 ± 0.05

0.49 ± 0.11

0.44 ± 0.02

0.42 ± 0.01

0.13 ± 0.01

0.13 ± 0.00

0.17 ± 0.01

0.29 ± 0.19

Muscle

0.15 ± 0.03

0.16 ± 0.02

0.16 ± 0.01

0.15 ± 0.01

0.05 ± 0.00

0.05 ± 0.00

0.07 ± 0.01

0.08 ± 0.02

Bone

0.36 ± 0.03

0.38 ± 0.03

0.41 ± 0.08

0.31 ± 0.01

0.26 ± 0.01

0.23 ± 0.03

0.26 ± 0.01

0.36 ± 0.10

Urine

0.00 ± 0.00

0.01 ± 0.01

0.71 ± 0.22

0.15 ± 0.05

0.00 ± 0.00

0.06 ± 0.08

0.14 ± 0.00

0.16 ± 0.16

Table 2. Ex vivo biodistribution data of [99mTc(PSiso)2(Ln)] complexes (1- 4) in male Sprague– Dawley rats.Values are expressed as mean %ID/g ± standard deviation (n = 3). ■ ASSOCIATED CONTENT *S Supporting Information


36

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Stability of [99mTc(PSiso)2(L)] complexes after incubation in GSH, Cys, EDTA (10 mM) and PBS. Stability of [99mTc(PSiso)2(L2)] complex after incubation in rat serum (15, 30, 60, 12 and 240 minutes). HPLC profiles of [99mTc(PSiso)2(L1)] 1 incubated at 37 °C for 4 h in rat liver homogenate.

This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author. *Cristina Bolzati, Ph.D. Phone +39 049 8275352. Fax +39 049 8275366. E-mail: [email protected]. Working Addresses. Dipartimento di Scienze del Farmaco, Università degli Studi di Padova, Via Marzolo 5, 35131 Padua, Italy. *Nicola Salvarese. Ph.D. Phone: +39 049 8275352; Fax +39 049 8275366. E-mail: [email protected]. Working Addresses. Dipartimento di Scienze del Farmaco, Università degli Studi di Padova, Via Marzolo 5, 35131 Padua, Italy. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes


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The authors declare no competing ﬁnancial interest. ACKNOWLEDGMENT This research was supported by MIUR through PRIN 20097FJHPZ-004, FIRB RBAP114AMK “RINAME” and PON 01_02388, and by the Italian Association for Cancer Research (AIRC, IG 13121). REFERENCES (1) Mariani, G.; Bruselli, L.; Duatti, A. Is PET always an advantage versus planar and SPECT imaging? Eur J Nucl. Med. Mol. Imaging. 2008, 35,1560-1565. (2) Zimmermann, R. G. Why are investors not interested in my radiotracer? The industrial and regulatory constraints in the development of radiopharmaceuticals. Nucl. Med. Biol. 2013, 40, 155-166. (3)

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Table of Contents graphic. An efficient general procedure for the preparation of a series of Tc(III) six-coordinated mixed ligand [99mTc(PS)2(Ln)] compounds, (PS = trisalkylphosphino-thiolate; Ln = dithiocarbamate) useful in radiopharmaceutical applications is presented, along with their in vitro and in vivo evaluation.


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Mixed-Ligand Compounds - American Chemical Society

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